Virtual Library

Abstract
The Driving Force of Social Networking According to Facebook executives, the social network reports 1.06 billion monthly active users. This number has been steadily increasing with a 25% increase in monthly users from last year and a rise of 28% in daily users. Now that we know the numbers what is happening in the world of patients. Though the premise exists that Facebook is strictly for social media networking, I wouldn’t hesitate to guess that patients, especially those who are home bound, spend an enormous amount of time on the web. Patients with rare diseases, in particular, have found Facebook to be a useful tool in finding others facing similar circumstances. PatientsLikeMe.com, launched in 2004, uses a number of tools to collect data and derives a hefty profit from selling this data to pharmaceutical companies as well as research institutions. It reports over 200K users on the site covering 1800 diseases. It uses sophisticated questionnaires to gather data about its users. Another active social media network, 23andme.com promotes the selling of an at-home saliva kit to map out genetic codes. They provide users with interactive tools meant “to shed new light on your distant ancestors, your close family and most of all, yourself.” In 2013, this company spent up to $5 million in advertising, further demonstrating that healthcare on the web is a lucrative field. Law firms representing mesothelioma patients spent over $50 million in goggle keyword advertising in 2012, making mesothelioma the most expensive word in Google advertising. Many of the larger firms representing victims of asbestos have now also launched Facebook pages and groups. These are usually marketed as patient support and advocacy sites providing patients with support and referrals to both medical and legal professionals. To the unsuspecting patient, these sites appear to be either VA sponsored or true advocacy sites which could not be further from the truth. Do you really want your patients to receive medical advice and referrals from representatives of legal entities or other for-profit operations with secondary motives? Nurses are the most trusted professionals valued by the public according to numerous surveys. Who is better equipped to engage with patients in social media and help them to understand the “rules of engagement”? The median age for the diagnosis of lung cancer is 72 and for mesothelioma 70. People in their 70s are less likely to be knowledgeable about the risks vs. the benefits of engaging in social media networking. We are all too familiar with the patient or family members who present to the office armed with paperwork obtained during web searches. Some of the information is valuable, but much is not applicable to their current situation and some is entirely misleading. An inordinate amount of time is consumed by those affected by the disease and the practitioner sorting through and commenting on relevancy of such information. I would suggest that if a nurse could guide their navigation of the web and provide accurate medical information and accurate interpretation of this information, the patient and their representatives as well as the provider could have a more focused discussion. Social media provides a unique opportunity to capture large numbers of patients and their advocates which can be used as a tool for both education and support. It would be the role of the nurse to explain the workings of the platform and to set guidelines to assist in protecting privacy to the degree possible when engaging on these sites. Patients need to understand the potential consequences of engaging in online health-related discussions and must be willing to accept the risks associated with membership in a group. It is fairly common for potential employers to peruse Facebook pages to gather additional background information on future employees. It could certainly be feasible that health insurance companies, especially if a dispute arises, might also turn to social media for information. Patients should be encouraged to read the privacy policy on these sites and an open dialogue about privacy within the group should be an ongoing. Patients themselves are the driving force in this healthcare-related online movement. As a result, hundreds of groups run by patients are available for others to join and this is where this trend can become problematic. Patients with the loudest voice on Facebook, or perhaps the miracle responders, can sway a captive patient group into potential risky decisions with the best of intentions. Groups starting off as support groups can quickly and unexpectedly have their conversations shift toward sharing of medical information and practice. As nurses, we know that patients need to be fully informed without bias to be able to practice good decision-making. Nurse-led groups can promote support and can help to guide the conversation to avoid misinformation or “cyber opinion bullying” by the strongest patient leader. Nurses can designate a peer group leader but promote their role within the group as the medical monitor. This provides an opportunity to gently correct misinformation and present new medical information to the group thus maintaining professionalism and the integrity of the group. Patients with rare diseases are often isolated and local support groups may not be applicable to their particular situation. Patients in these groups often express relief that they are able to connect with others in similar circumstances. However, connecting with others in this manner can be bittersweet. The group can celebrate the victories but will also mourn setbacks and death of group members. Having a nursing professional in the group provides access to a trained individual who can assist in emotional healing as well as recognize and refer if group members need one on one counseling. In cancer, less than 10% of all patients enroll in clinical trials. As nurses we can educate patients about clinical trials and promote participation in such trials. Cancer patients are seeking knowledge on the web, and as educators, it is our role to see that this need is met. T

Abstract not provided

Abstract
Malignant Pleural Mesothelioma is a global challenge for heath professionals, and the role of surgery is key to that challenge. Surgery has a palliative function and a curative function. The palliative function is to drain the fluid, take a biopsy, optimise lung re-expansion, and prevent fluid recurrence by pleurodesis. Video assisted thoracoscopy (VAT) is the palliative operation of choice because it is less painful, has a reduced hospital stay, and the surgeon can assess the chest cavity for suitability for more aggressive treatment. An important predictor for a successful VAT pleurodesis is lung re-expansion after initial pleural drainage assessed by radiology and a patient response of “I can breath now.” If pleurodesis is ineffective and the fluid recurs, or if lung entrapment prevents lung re-expansion, then open surgery via thoracotomy with partial or subtotal pleurectomy / decortication should be considered, providing the patient is medically fit and has a reasonable life expectancy. Partial pleurectomy /decortication is more invasive, has an increased potential for prolonged air-leak, is more painful and has a longer recovery period, however the patient may benefit from an improved quality of life, living without symptoms related to lung entrapment. Brancatisano (1991) reported our experience of 50 patients having partial pleurectomy / decortication. The median survival was 16 months with a range of 3 to 54 months and 21% of patients survived more than two years. The curative role is less defined. Surgical resection is the curative treatment option for most solid malignant tumours and this ideal underpins the practice of surgeons treating MPM. Cytoreductive surgery, to reduce the burden of disease and prolong disease free living by complete macroscopic surgical resection is offered to patients with early disease in some centers. Here lies the key challenge. There are differing operative techniques aiming for the same outcome: extrapleural pneumonectomy, EPP, and lung sparing pleurectomy/decortication, EPD EPP, offered since the 1970s, involves complete resection en bloc of the lung, parietal pleura, ipsilateral pericardium, and ipsilateral hemi-diaphragm, along with complete excision of thoracic lymph nodes. Defects of the pericardium and hemi-diaphragm are repaired with Gortex mesh to prevent cardiac and visceral herniation. An early publication reported an unacceptable morbidity and mortality of 45% and 31%respecively (Butchart, 1976). David Sugarbaker, determined to improve patient survival, continued offering EPP but high recurrence rates proved surgery alone provided little benefit to survival. With persistence, his team offered EPP in combination with chemotherapy (prior to pemetrexed) and radiotherapy. In 1991 Sugarbaker reported survival rates of 70% at one year and 48% at two years. Morbidity and mortality rates were 19% and 6% respectively. This multimodality treatment required surgery to reduce tumour bulk, while chemotherapy and radiotherapy treated micro-metastatic disease. In 1996, Sugarbaker again reported similar results but added that epithelial cell types had better survival rates compared to sarcomatoid or mixed histology tumours. Other centres around the world reported individual surgical series each contributing to a collection of experiences. Weder (2007) reported on 45 EPP trimodality patients and found epithelial cell type had better survival. Cao (2010), attempted to evaluate the safety and efficacy of EPP found an overall survival of 13 – 23.9 months and concluded/confirmed that selected patients might benefit from EPP especially when combined with neoadjuvant chemo and adjuvant radiotherapy. Controversy about the ability of EPP to affect a cure or contribute to improved survival was to be sorted by the UK Mesothelioma and Radical Surgery feasibility study known as the MARS trial. This randomised control trial concluded that EPP offers no benefit and possibly harms patients. Surgeons, skilled at performing EPP and who had reported their results vehemently opposed this conclusion. The only centre in Australia offering trimodality therapy reported 70 patients having EPP between 1994 and 2008, having a median survival of 20 months, and morbidity and mortality of 37% and 5.7% respectively (Yan, 2009). Pleurectomy / decortication or P/D was the other preferred operation as some surgeons questioned the morbidity and mortality of EPP. Valarie Rusch (1993) pioneered P/D in order remove all visible and palpable tumours and achieve macroscopic complete resection. Other surgeons varied this procedure making comparing results difficult so specific terminology was used to describe 3 approaches. 1) Extended or radical pleurectomy/decortication (EPD); parietal and visceral pleura is resected along WITH diaphragm and pericardium. 2) Pleurectomy/decortication (PD); parietal and visceral pleura is resected WITHOUT diaphragm and pericardium. 3) Partial pleurectomy/visceral decortication as a palliative procedure. Individual cancer centers have reported case series. Flores (2008) compared EPP to pleurectomy / decortication. Nerangi-Miandoab (2008) reported epithelial cell type was a predictor of survival in patients having pleurectomy / decortication compared to no surgery. Nakas (2008) concluded patients not suitable for EPP should be offered radical P/D. Zahid (2011) found P/D may improve survival but at the expense of increased morbidity and is best offered to patients enrolled in prospective trials. Global differences related to the role of surgery in cytoreductive therapy relate to what radical surgical procedure provides the best chance of extended disease free survival. Is it EPP, lung sparing EPD, or P/D? What is certain is that EPP and EPD should be offered as part of trimodality therapy. Furthermore, the procedure must be performed by an experienced surgeon, and supported by a team with proven expertise and experience to care for these patients. Rusch (2012) reminds us that the role of surgery in the management of MPM remains controversial but it also remains a treatment option because of the limited benefits of radiotherapy and chemotherapy. Surgery is offered either as a palliative procedure or with curative intent. There is little need for debate about the palliative option but debate about curative intent continues. Finally, while global differences are important, what is learnt from collective experiences is more important. There is an urgent need to identify patients with favourable prognostic factors, suitable for cytoreductive therapy. We need to provide support, hopeful encouragement, and equitable access to multi modality treatment because there is a growing number of well living long-term survivors.

Abstract not provided

Background
The main challenge in computed tomography (CT) screening for lung cancer is the high prevalence of pulmonary nodules and the relatively low incidence of lung cancer. Thresholds for nodule size and growth rate, which determine which nodules require additional diagnostic procedures, should be based on the lung cancer probability of the individual.

Methods
Diameter, volume and volume-doubling time (VDT) of 9,681 non-calcified nodules detected by CT screening in 7,155 subjects were used to quantify lung cancer probability. Complete coverage on all lung cancer diagnoses was obtained by linkages with the national cancer registry. The nodule management algorithm recommended by the ACCP was evaluated and an improved algorithm, based on lung cancer probability, was proposed.

Results
Lung cancer probability was low in subjects with a nodule volume <100mm³ (≤0.7%) or maximum transverse diameter <5mm (≤0.6%) Moreover, probability in these subjects was not significantly different from that in subjects without nodules (0.4%). Lung cancer probability was 0.9-5.8% for nodules with a volume 100-300mm³ or a diameter 5-10mm; the VDT further stratified the probability: 0.0-0.9% for VDTs>600days, 4.0% for VDTs 400-600days and 6.7-25.0% for VDTs<400days. Lung cancer probability was high for participants with nodule volumes ≥300mm³ (8.9-26.1%) or diameters ≥10mm (11.1-26.2%), even with long VDTs. Finally, raising the thresholds for nodule size recommended by the ACCP for an indeterminate result from 4mm to 5mm and for a positive result from 8mm to 10mm, would yield fewer follow-up CT examinations (from 29.8% to 22.2%) and fewer additional diagnostic procedures (from 8.9% to 5.3%) while maintaining the sensitivity at 94.2%.

Conclusion
Small nodules (volume <100mm³ or diameter <5mm) are not predictive for lung cancer. Immediate diagnostic evaluation is necessary for subjects with large nodules (volume ≥300mm³ or diameter ≥10mm) and only for subjects with nodules of intermediate size is VDT assessment advocated.

Abstract not provided

Background
Malignant pleural mesothelioma (MPM) incidence is increasing and has no known cure. Non randomised studies suggest that video assisted thoracoscopic (VAT) pleurectomy is effective in controlling pleural effusion and may be associated with increased survival compared to talc pleurodesis.

Methods
A multicentre randomised controlled trial of VAT pleurectomy versus talc pleurodesis was undertaken for patients > 18 years with any sub-type confirmed or suspected MPM with a pleural effusion who were fit enough to undergo VAT pleurectomy. Exclusion criteria included previous pleurodesis by any approach. Previous malignancy was permitted if there was no evidence of active disease and MPM had been confirmed. Participants were risk stratified using a modified EORTC prognostic scoring system. Talc pleurodesis was performed via tube thoracostomy or by poudrage at thoracoscopy. VAT pleurectomy involved partial parietal pleurectomy and decortication of the visceral pleura, where appropriate, to achieve lung re-expansion. A total of 196 patients was required to show a survival difference at 1 year of 59% (VAT pleurectomy) versus 37% (talc pleurodesis). Ethical approval was granted by Huntingdon, Cambridge (UK) Research Ethics committee: H02/809; ISRCTN: 34321019; ClinicalTrials.gov NCT00821860.

Results
Between 2003 and 2012, 196 patients (120 confirmed, 76 suspected) were randomised across 9 UK centres. 21 cases suspected MPM were subsequently found not to have MPM and excluded (pre-planned in protocol), leaving 87 VAT pleurectomy and 88 talc pleurodesis for the main analysis. Baseline characteristics were similar between the two groups; overall mean age 69 years, 86% men and 75% had known asbestos exposure. Eighty four per cent showed epithelioid disease, 78% were IMIG stage 3/4 and 49% were high risk as per EORTC criteria. The allocated procedure was completed for 73 (83%) talc and 78 (90%) VAT pleurectomy patients. One year survival rates (primary outcome measure) were 57% for the talc group and 52% in the pleurectomy group (hazard ratio 1.03 (95% CI: 0.76, 1.42), p=0.83). Of the secondary outcome measures, pleural effusion was controlled in 37% of talc and 59% pleurectomy patients at one month (p=0.008) and in 57% of talc and 76% pleurectomy patients at 6 months (p=0.04). At 9 and 12 months control of pleural effusion was similar between groups. Median hospital stay was longer in pleurectomy patients (8 days (range 1-31) vs. 6 (range 1-15), p<0.001) and this group had significantly more complications, predominantly prolonged air leak (26% vs. 8%, p=0.009). Based on patients with complete data there was a significant benefit in EQ5D quality of life at 6 months (mean difference 0.08 (95%CI 0.003,0.16), p=0.042) and 12 months (mean difference 0.19 (95%CI 0.05,0.32), p=0.006) in favour of the pleurectomy group. Adjusting for bias due to missing data prior to death reduced the difference in 12 month EQ5D to 0.09 (95%CI -0.04,0.22), p=0.16.

Conclusion
MesoVATS showed that VAT pleurectomy significantly improved control of pleural effusion versus talc pleurodesis and improved quality of life. However, overall survival was not increased and the pleurectomy group experienced more complications. Subgroup analyses will investigate which patients benefit most from which intervention. Funded by the BUPA Foundation

Abstract not provided

Background
The two primary objectives of RTOG 0617 were to compare the overall survival(OS) differences of 1) standard-dose(SD)(60Gy) versus high-dose(HD)(74Gy) radiotherapy (RT) with concurrent chemotherapy(CT); and 2) the addition of cetux to standard CRT. Cetux is a monoclonal Ab targeting EGFR with activity when combined with CT in metastatic NSCLC and head and neck cancer (HNC), and with RT in locally advanced HNC.

Methods
This Phase III Intergroup trial randomized pts in a 2 x 2 factorial design. Concurrent CRT included weekly paclitaxel(45 mg/m2) & carboplatin(AUC=2). Pts randomized to cetux received a 400 mg/m2 loading dose on Day 1 followed by weekly doses of 250 mg/m2. All pts were to receive 2 cycles of consolidation CT. This is the initial report of survival outcome based on cetux. The trial was designed for 450 evaluable patients with 80% power and a 1-sided alpha of 0.0125 to detect a 29% reduction in OS failure for each comparison (RT and cetux).

Results
544 pts were accrued, and 419 and 465 are eligible for RT and cetux analyses. Median follow up is 18.7 months. Cetux delivery was acceptable in both the concurrent and consolidation phases. Therapy related ≥Grade 3 non-hematologic toxicity was higher in the cetux group; 70.5% vs 50.7% (p<.0001). Grade 4 and 5 events were 35.8% and 28.2%, respectively. Median survival was 23.1 vs 23.5 months, & 18-month OS rates were 60.8% vs 60.2% on the cetux vs non-cetux arms, respectively (p=0.484, HR=0.99), which crossed a protocol-specified futility boundary for early reporting. As previously reported, median survival times and 18-month OS rates for SD and HD arms were 28.7 vs 19.5 months, and 66.9% vs 53.9% respectively (p=0.0007, HR=1.56). There was no significant interaction between RT dose and the use of cetux. The OS rates for the 4 arms of this trial are shown in Table. An H-score analysis, a measure EFGR positivity, is forthcoming.

Table: Overall Survival Rates with 95% CI (pts accrued while all 4 arms were open)

Time	60 Gy	74 Gy	60 Gy + Cetux	74 Gy + Cetux
12m	78.4% (68.9, 85.4)	62.6% (51.7, 71.6)	80.0% (70.8, 86.6)	74.7% (64.9, 82.2)
18m	67.9% (57.6, 76.2)	52.3% (41.5, 62.0)	67.1% (56.8, 75.5)	58.0% (47.6, 67.1)

Conclusion
In pts receiving CRT for Stage III NSCLC, 74 Gy is not superior to and may be worse than 60 Gy in terms of OS. Cetux provides no survival benefit in the setting of CRT for Stage III NSCLC.

Abstract not provided

Background
Detecting and targeting the oncogenic drivers EGFR and ALK have transformed the care of patients with lung adenocarcinomas. The LCMC was established to use multiplexed assays to test tumors for alterations in 10 genes and provide the results to clinicians to select treatments and clinical trials matched to the driver detected.

Methods
Fourteen LCMC sites enrolled patients with metastatic lung adenocarcinomas and tested their tumors in CLIA laboratories for activating mutations in 10 oncogenic driver genes.

Results
Tumors were tested from 1,007 patients for at least one gene and 733 for all 10 genes. An oncogenic driver was found in 466 (64%) of fully-genotyped cases. Among these 733 tumors, drivers found were: KRAS 182 (25%), sensitizing EGFR 122 (17%), ALK rearrangements 57 (8%), “other” EGFR 29 (4%), two genes 24 (3%), HER2 19 (3%), BRAF 16 (2%), PIK3CA 6 (1%), MET amplification 5 (1%), NRAS 5 (1%), MEK1 1 (<1%), AKT1 0. For cases with any genotyping, we used results to select a targeted therapy or trial in 275 (28%). Among 938 patients with follow-up, the median survivals were 3.5 years for the 264 with an oncogenic driver treated with genotype-directed therapy, 2.4 years for the 318 with an oncogenic driver with no genotype-directed therapy, and 2.1 years for the 360 with no driver identified (p<0.0001).

Conclusion
Individuals with lung cancers with oncogenic drivers receiving a corresponding targeted agent lived longer than similar patients who did not. An actionable driver was detected in 64% of tumors from patients with lung adenocarcinomas; more than one was present in 3%. Multiplexed testing aided physicians in choosing therapies and targeted trials in 28% of patients. This paradigm for care and research will expand as genotyping becomes more efficient with Next-Gen platforms, additional drivers are identified (i.e.ROS1 and RET), and more targeted drugs become available in the pharmacy and through clinical trials. Supported by HSS NIH NCI 1RC2CA148394-01. Trial Registered with Clinicaltrials.gov: NCT01014286.

Abstract not provided

Background
Serum levels of the potent immune-modulatory cytokine interleukin (IL)-6, which signals via the shared gp130 co-receptor, are elevated in smokers, and are higher still in those with lung cancer, the leading cause of cancer death worldwide. However, the precise role that IL-6 plays in the pathogenesis of lung cancer remains to be elucidated. To utilize in vivo molecular dissection of gp130 signaling to identify specific molecular components of the IL-6 cytokine family network which facilitate tobacco-related lung carcinogenesis.

Methods
Nicotine-derived Nitrosamine Ketone (NNK) is a potent tobacco carcinogen and reliably induces lung tumours in mice. We therefore examined the effect of deregulated IL-6 signaling on NNK-induced lung carcinogenesis by using gp130[F/F] mice which carry a knock-in mutation in the endogenous gene for gp130, the critical co-receptor for the IL-6 cytokine family.

Results
The lungs of these mice display IL-6-induced hyperactivation of the latent transcription factor Stat3 via gp130 in the absence of gp130-mediated PI3K/Akt and Mapk signaling. The gp130[F/F] and control gp130[+/+] (wild-type) mice were injected with NNK or PBS and observed over 16 weeks prior to the evaluation of lung tumourigenesis at the cellular (immunohistochemistry, histology) and molecular (qPCR) levels. In response to NNK, the absolute number of lung lesions in gp130[F/F] mice (3.81 ± 0.84; mean ± SEM) was significantly reduced compared to gp130[+/+ ](6.43 ± 1.72; p<0.05) mice. In addition, we also observed a significant reduction in the size of lesions in gp130[F/F] compared to gp130[+/+] mice (1.00 ± 0.05mm vs 0.69 ± 0.05; p<0.0001). Notably, the number and size of lesions in the lungs of NNK-treated gp130[F/F]:Stat3[-/+] mice displaying genetically-reduced Stat3 hyperactivation were comparable to gp130[F/F] mice.

Conclusion
Collectively, our data suggest that IL-6/gp130-mediated activation of the PI3K/Akt and/or Mapk pathways, but not Stat3, plays an important role in promoting NNK-induced lung carcinogenesis.

Background
Development of new methods for selective chemosensitization and radiosensitization of human lung tumors is acutely needed. We propose to combine inhibitors of the chaperone activity along with inhibitors of chaperone expression to treat lung cancer. Our approach is based on such facts: (i) cancer cells are addicted to the chaperone activity of heat-shock protein 90 (Hsp90) because it is required for their survival and proliferation, (ii) inhibitors of the Hsp90 chaperone activity (e.g. 17AAG) may have a 100-fold higher affinity to Hsp90 in malignant cells than in normal cells and (iii) these Hsp90 inhibitors cause undesirable induction of cytoprotective chaperones Hsp70 and Hsp27 that can impair antitumor effects of the Hsp90 inhibition.

Methods
Cultured cells of human lung cancer cell lines SQ-5, H460 and A549, and, for comparison, human normal lung-derived alveolar epithelium or fibroblasts were subjected to 17AAG and known inhibitors of the Hsp induction (quercetin, triptolide, NZ28) and conventional drugs (carboplatin, doxorubicin, paclitaxel) or clinically relevant doses (3-5 Gy) of gamma-photon irradiation. Inhibition of the intracellular Hsp90 chaperone activity was detected on a delay in the Hsp90-dependent reactivation of thermally denatured luciferase in the heat-stressed transfectants. The cell death or survival was assessed in annexin V-staining and clonogenic assays. The angiogenic potential of lung cancer cells was tested in cell-proliferation assays with cultured human vascular endothelial cells (EC). The cellular levels of cell survival- and angiogenesis-related proteins were analyzed by Western blotting.

Results
It was found that 10-100 nM 17AAG significantly retarded proliferation in all the three lung cancer cell lines studied. As biomarkers of the Hsp90 activity inhibition, the specific depletion of Akt, survivin and HIF-1alpha as well as up-regulation of Hsp70 and Hsp27 were revealed in those cell samples. In parallel, co-treatment with 10-30 uM quercetin or 2-4 nM triptolide, or 0.3-1 uM NZ28 fully prevented the Hsp up-regulation in the 17AAG-treated lung cancer cells and rendered them much more sensitive to carboplatin, doxorubicin and paclitaxel, and to gamma-radiation exposure. Importantly, no enhanced cytotoxicity was observed in cultures of normal human fibroblasts and epithelial cells co-treated by the same way. The enhanced chemo- and radiosensitization of lung cancer cells appears to be due to simultaneous blockade of both the Hsp90-dependent antiapoptotic pathways and the 17AAG-induced up-regulation of cytoprotective Hsp70 and Hsp27. Besides the sensitizing effects on the lung cancer cells, the double-inhibitor combination considerably reduced their angiogenicity that was manifested in down-regulation of the HIF-1alpha and VEGF expression levels, and also in the clearly impaired proliferative response of EC stimulated with lung cancer cell culture-conditioned growth medium.

Conclusion
Combined use of the Hsp90 chaperone activity inhibitors and blockers of the concomitant induction of inducible chaperones Hsp70 and Hsp27 enables to selectively sensitize human lung cancer cells to chemo- and radiotherapy, and to suppress the lung cancer-associated angiogenesis. In perspective, such an approach may be developed into a new, effectual strategy for lung cancer treatment.

Background
Non-small-cell lung cancers (NSCLCs) constitute about 85% of lung cancers and have been associated with a poor prognosis, despite the introduction in clinical practice of targeted therapies. The majority of patients are still treated in first-line with a cisplatin containing doublet. New molecular predictors should be discovered. Our attention has been focused on KRAS. Mutations in the KRAS gene have been found in 17% of NSCLC patients mostly at codon 12. It has been observed that a pool of mutations, responsible for different aminoacid substitutions, occured at this position. In NSCLC these mutations are present at a different frequency compared to colon and pancreatic cancer. It has been supposed that different aminoacids in the same position of KRAS protein could differently impact on tumor progression and drug resistance. To test this hypothesis, KRAS overexpressing clones with the three most common mutations found in NSCLC patients (G12C, G12D and G12V) have been generated in NCI-H1299 cell line. The clones showed a different response in vitro to cisplatin (Garassino MC et al, Annals of Oncology, 2011).

Methods
The clones were xenografted in nude mice to determine the antitumor activity of cisplatin in vivo. The effect of cisplatin on signalling pathways and DNA damage response was determined by western blotting, immunofluorescence and Real-Time PCR. Platinum adducts on DNA were revealed by DRC-ICP-MS.

Results
The resistance induced by the presence of G12C KRAS was still present compared to wild-type KRAS clone when these clones were transplanted in nude mice and treated with cisplatin. Some of the logic pathways functionally linked to KRAS, such as MAPK and PI3K, did not seem to account for the different cisplatin sensitivity observed. Factors previously reported to be associated to cisplatin resistance, such as the levels of cisplatin transporter CTR-1, of GSH and GST enzyme, and the total intracellular platinum levels were comparable among the clones. Several concordant results seem to indicate that, with a similar ability to enter the cells and to reach the DNA, an increased removal of platinum bound to DNA might account for the resistance of G12C clone: i) the cell cycle perturbation induced by cisplatin indicated that a reduced G2/M cell cycle phase block was observed in the G12C clone compared to all the others; ii) H2AX foci and ATM phosphorylations following treatment were barely detectable in the resistant clone; iii) the levels of platinum bound to DNA were much faster declining in G12C cells. The involvement in the resistance of G12C clone of Nucleotide Excision Repair and Fanconi Anemia repair mechanisms, which have been associated to the removal of adducts from DNA, was excluded by our experiments.

Conclusion
Altogether these data reveal the possibility that the presence of G12C KRAS mutation stimulates a DNA repair mechanism, not yet identified, able to faster remove platinum from DNA before the formation of double strand cross-links. Studies are ongoing to specifically address this point. Understanding why the different KRAS mutations influence the response to cisplatin could be useful in the clinical setting to tailor therapies for patients.

Background
p53 mutations have been categorized as type I (contact) and type II (conformational) mutations. Differential effects of type I vs. type II p53 mutations in spontaneous lung tumor formation, and their relationship with secondary genetic alterations have not been previously reported. .

Methods
We evaluated the potential of two common type I (273H) and type II (175H) mutations under the transcriptional control of the human surfactant protein C (SP-C) promoter to induce lung tumors in transgenic mice. Necropsies of 138 non-transgenic, 207 SP-C-p53-273H and 171 SPC-p53-175H transgenic mice in progressive age cohorts were performed.

Results
Ninety-one tumors, all adenocarcinomas, were observed; 8 (5.8%), 37 (17.9%) and 46 (26.9%) in non-transgenics, p53-273H and p53-175H, respectively (non-transgenic vs. 273H, p=0.010; non-transgenic vs. 175H, p= 0.0003; logistic regression). Type II p53 mutants had an earlier onset of tumors; 23 of 98 p53-175H mice developed tumors before the age of 13 months, compared to 7 of 108 p53-273H mice (p=0.012, logistic regression). K-ras mutations occurred in a substantial proportion (21 of 50, 42%) of murine lung tumors sequenced. For both the non-transgenic and the p53-273H transgenics, tumor K-ras codon 12-13 mutations occurred after 13 months with a peak incidence at 16–18 months. However, for the p53-175H transgenics K-ras codon 12-13 mutations were observed as early as six months, with a peak incidence between the ages of 10-12 months. Codons 12-13 were the predominant location in p53-175H transgenics (6 of 7), whereas codon 61 (6 of 10) was more common in p53-273H transgenics.

Conclusion
The observation of accelerated tumor onset, early appearance and high frequency of K-ras codon 12-13 mutations in type II p53-175H mice confirms the enhanced oncogenic function of conformational p53 mutations, and the gains in early genetic instability for tumors containing these mutations compared to contact mutations. These data would suggest that not only the presence, but also the type of p53 mutations in human lung cancer should be considered when evaluating prognosis and developing treatment strategies for this malignancy. These mice develop a single-lung tumor that is easy to follow with CT imaging. Thus, these animal models provide a framework for evaluation of the effects of these mutations on response to standard and novel anticancer treatment interventions.

Background
Background: Detection of homozygous deletion of the 9p21 locus, the site of the CDKN2A/p16 gene, is useful in differentiating reactive mesothelial hyperplasia (RMH) from malignant pleural mesothelioma (MPM) in pleural effusion cytology, biopsy specimens, and surgical specimens. However, the cutoff value for 9p21 FISH varies. Furthermore, 9p21 FISH reveals not only homozygous deletion, but also heterozygous deletion, in MPM tissue samples, and only two studies have evaluated heterozygous deletion. The aim of this study was to establish a FISH cutoff value for 9p21 homozygous deletion and apply it to a clinical diagnosis. In addition, we investigated the significance of 9p21 heterozygous deletion in MPM.

Methods
Design: Ninety-five histopathological (49 MPM + 47 RMH) and 35 cytological (15 MPM + 20 RMH) cases were studied. Cutoff values were evaluated as mean + 3SD based on the results of RMH 9p21 FISH. The copy number of the p16 gene was analyzed in 22 MPM tissue samples and 17 RMH by real-time PCR, using RSP6 as a control gene, and samples were divided into three groups (1.0: no deletion, 0.5: one allele deletion, and 0: two allele deletions). Methylation-specific PCR (MSP) was performed in two heterozygous deletion dominant cases. Overall survival was evaluated using Kaplan-Meier survival analysis and log-rank test.

Results
Results: In MPM, 44/49 (90%) cases were homozygous deletion-positive (>10% cutoff value), and 12/49 (24%) cases were heterozygous deletion-positive (>46.6% cutoff value) by p16 FISH. None of RMH cases were deletion-positive in tissue samples. Real-time PCR was performed for 22 cases of MPM and 17 cases of RMH. The p16 copy number in RMH was 0.83-1.28 (average 1.06). In MPM, 16/22 (73%) revealed a two-allele deletion pattern for p16. Two cases of homozygous deletion-negative MPM by FISH also had no deletion pattern by real-time PCR. One case of heterozygous deletion-dominant MPM (over 55%) revealed a two-allele deletion of p16 by real-time PCR, and two cases showed a one-allele deletion of p16. These cases had no methylation by MSP. In 25 cases of MPM for which follow-up was available, the overall survival rate was significantly lower in homozygous deletion-positive MPM (21/25 cases) than homozygous deletion-negative MPM (4/25 cases) (p= 0.01).

Conclusion
Conclusion: 9p21 FISH was useful for differentiating MPM from RMH, and p16 homozygous deletion in MPM has the potential to be a prognostic factor. Moreover, a combination of p16 FISH, p16 real-time PCR and MSP is useful for discriminating heterozygous deletion from homozygous deletion in MPM. The presence of heterozygous deletion suggest the possibility of a p16-independent tumorgenesis pathway in MPM.

Background
Platinum-based combination chemotherapy is the first-line treatment for advanced non-small cell lung cancer, but tumor recurrence occurs in most of patients who have received this treatment. Recent evidences suggested existence of CD133+ cancer stem cells is the cause of drug resistance and tumor recurrence. However, there is no efficient way to eliminate CD133+ cancer stem cells. Accordingly, the goal of this study is to identify novel compounds that targeting CD133+ CSCs of non-small cell lung cancer.

Methods
A hCD133 promoter-driven GFP reporter construct was used to identify CD133+ cells. CD133+ cells were FACS sorted from H460 cell line. The stemness and drug resistance of CD133+ cells were characterized according to their sphere formation, differentiation, gene expression, and chemo-response. IC~50~ of each novel compound was determined using MTT assay.

Results
CD133+ lung cancer cells identified by a human CD133 promoter- driven GFP reporter exhibited drug resistance and expressed stem-cell characteristics. Treatment of lung cancer cells with cisplatin was sufficient to result in enrichment of CD133+ cells, to induce DNA damage responses, to up-regulate levels of ABCG2 and ABCB1 which therefore increased resistance to doxorubicin and paclitaxel. We further found that cisplatin-induced enrichment of CD133+ cells was mediated by activation of notch signaling as judge by increased levels of NICD. Pre-treatment of cells with g-secretase inhibitor, DAPT, remarkably reduced cisplatin-induced enrichment of CD133+ cells and increased the sensitivity to doxorubicin and paclitaxol. Ectopic expression of NICD reversed the function of DAPT on drug sensitivity. The similar effect was also observed in tumor-engraft experiments as cisplatin treatment significantly increased the number of CD133+ cells in tumor-engraft in nude mice. In contrast, intra-tumor injection of DAPT together cisplatin was able to significant decrease the number of CD133+ cells in the tumor-engraft of mice. To identify novel compounds that may target CSC and cancer cells, we tested the cytotoxic effect 30 synthesized small compounds. Among of these compounds, 4 compounds, NCKU-44, NCKU-291, NCKU487 and NCKU-570, effectively killed both CD133+ CSCs and parental cancer cells. Three compounds, NCKU-46, NCKU-mo and NCKU-ts, specifically targeted to CD133+ CSCs.

Conclusion
In our study, we developed a drug-screening platform of CD133+ CSC of non-small cell lung cancer. At least 7 novel compounds were identified as potential CSC-targeting agents, which could be used in the future pre-clinical trials.

Background
We previously reported that SCLC distinct characteristics by primary tumor location (central type or peripheral type) and TTF-1 expression. Peripheral type and/or TTF-1 expression were predictive factors of poor prognosis in SCLC. In this study, we examined whether or not SCLC distinct characteristics of gene expression by primary tumor location, using microarray analysis of snap frozen tissue samples.

Methods
Fourteen SCLC, diagnosed from biopsies and/or surgical materials, for which snap frozen tissue samples were available, were collected during 2004-2011 from Japanese patients. We evaluated the location of primary tumor (central or peripheral) by CT at diagnosis. Total RNAs from the snap frozen fresh tissue sample of the surgically resected SCLC (n=14, central type:3, peripheral type:11) were used to prepare cDNAs, which were hybridized to the Whole genome Human 60K Microarray chips (Agilent Technologies Inc, Tokyo, Japan). Data normalization, log transformation, statistical analysis, gene ontology (GO) analysis, and pattern study were performed with GeneSpring GX12 (Agilent Technologies Inc, Tokyo, Japan) and Ingenuity Pathway Analysis software, with the stringency of P < 0.01 and a < 2-fold change by moderated t-test corrected with Bonferroni multiple testing.

Results
There were a total of 31551 genes scored as differentially expressed between groups. A total of 833 genes were identified that differed significantly in expression: 424 genes were upregulated and 409 genes were downregulated in peripheral type compared with central type. Interestingly, upregulated gene analysis revealed that peripheral type SCLC had increased levels of neuroendocrine genes such as NCAM, Synaptophysin (SYN), Chromogranin A (CGA), Gastrin-releasing peptide (GRP), ASCL1, and TTF-1. Subsequently, GO and network analysis revealed that most of upregulated genes in peripheral group were related to neuron functions. Hierarchical cluster analysis clearly distinguished between central type and peripheral type.

Conclusion
SCLC had a distinct gene expression profile from tumor location and had higher expression of genes associated with neural function in peripheral SCLC. Defining gene expression profiles by tumor location may help elucidate distinct characteristics of SCLC between central type and peripheral type.

Background
Cancer-associated fibroblasts (CAFs) contribute to cancer progression through multiple pathways.

Methods
Here we comprehensively compared gene expression in lung cancer cells cultured with conditioned medium from lung CAFs and normal lung fibroblasts by cDNA microarray analysis.

Results
The expression of many genes was up-regulated in cancer cells by CAFs’ conditioned medium, particularly cell adhesion molecules integrins and anti-apoptotic protein Bcl-2. Additional analysis indicated that the expression of integrins seems to be upstream of that of Bcl-2. We identified transforming growth factor-β as a candidate factor that induces the expression of those genes in cancer cells. Immunohistochemical studies on clinical lung cancer tissues revealed that integrins and Bcl-2 are more highly expressed in the leading cells, but not the following cells, at the invasive front of cancer nests, which are adjacent to or in proximity to the stroma. Furthermore, the expression of integrins and Bcl-2 in the leading cells was correlated with the clinical stages of cancer progression including lymph node metastasis.

Conclusion
In conclusion, our results suggest that CAFs promote lung cancer progression partly through the direct regulation of gene expression in the leading cells of invasive cancer nests.

Background
Many early-stage NSCLCs in spite of treating with surgical resection and adjuvant therapy relapse, occur metastasis and become fatal. Therefore there is an urgent need for research the key molecular events driving lung cancer invasion and metastasis that could lead to achieve effective prevention, reliable diagnosis, preparing useful prognostic indicator and treatment. Atypical protein kinase C lambda/iota (aPKCλ/℩), Partitioning defective-3 (Par3), Partitioning defective-6 (Par6) and Drosophila Lethal giant larvae mammalian homologue (Lgl2) are implicated in cell growth and apoptosis, and the maintenance of epithelial cell polarity. Loss of apical-basal polarity in epithelial cells is one of the hallmarks of aggressive and invasive cancer. This study was designed to investigate the correlation of expression of aPKCλ/℩ and Lgl2 with the clinicopathological characteristics and prognosis of lung adenocarcinoma, and to analyze the molecular mechanisms of invasion and metastasis of the tumor.

Methods
This study included 107 patients who underwent resection of lung tumor and diagnosed as lung adenocarcinoma at the Tokai University Hospital between 2002 and 2005. The expression of aPKCλ/℩ and Lgl2 was examined by immunohistochemistry. E-cadherin-mediated cell-cell adhesion is considered a suppressor of cancer cell invasion. Low E-cadherin expression in NSCLC tumors has been reported in several studies to be associated with a more aggressive behavior of tumor cells and a worse prognosis. In this study, the colocalization of aPKCλ/℩ and E-cadherin was observed by double-immunohistochemistry. Furthermore, we also analyzed the interaction of aPKCλ/℩, Par3 and Lgl2 by immunoprecipitation-Western blot assays.

Results
Immunohistochemical analysis showed that aPKCλ/℩ and Lgl2 were expressed in lung adenocarcinoma and correlated with lymphatic invasion and metastasis. Kaplan-Meier regression analysis showed that patients with　increased aPKCλ/℩　expression had significantly shorter overall survival than those with lower aPKCλ/℩ expression. Double-immunohistochemistry showed E-cadherin decreased in highly expressed aPKCλ/℩ of tumor cells. Furthermore, immunoprecipitation-Western blot assays showed that aPKCλ/℩-Lgl2-Par6 complex and　aPKCλ/℩-Par3-Par6 complex increased in lung adenocarcinoma tissue.

Conclusion
These results suggested that aberrant formation of both aPKCλ/℩-Lgl2-Par6 complex and aPKCλ/℩-Par3-Par6 complex could induce lung cancer progression by loss of cell-cell adhesion in lung adenocarcinoma.

Background
Acute inflammation is usually a rapid and self-limiting process, however it does not always resolve. This leads to the establishment of a chronic inflammatory state and provides the perfect environment for the transformation process. It has been estimated that approximately 25% of all malignancies are initiated or exacerbated by inflammation caused by infectious agents. Furthermore, inflammation is linked to all of the six hallmarks of cancer (evasion of apoptosis, insensitivity to anti-growth signals, unlimited replicative potential, angiogenesis, increase in survival factors and invasion and metastasis). It is thought that inflammation may play a critical role in lung carcinogenesis given that individuals with inflammatory lung conditions have an increased risk of lung cancer development. This study focuses on inflammation as a contributory factor in non small cell lung cancer (NSCLC), concentrating primarily on the pathological involvement of the pro-inflammatory cytokines, TNF-α, IL-1β, and hypoxia.

Methods
A normal bronchial epithelial cell line (HBEC4) was modified to stably and functionally over-express TNF-α and IL-1β (alone or in combination) and were continuously cultured for three months under normoxic or hypoxic (Invivo~2~ Hypoxia Workstation - 0.5% oxygen) conditions. Subsequently a range of experimental assays were carried out to assess functional cell change. These included: transformation assay (soft agar), proliferation (BrdU ELISA), invasion (Cell Invasion assay), migration (Scratch assay) and angiogenesis (Endothelial tube formation). Gene expression changes were also assessed using qPCR Cancer PathwayFinder arrays.

Results
Although transformation was not evident by soft agar, the adhesion potential (ICAM and VCAM levels) of normoxic and hypoxic clones has amplified and the growth rate has increased over time. Under normoxia the IL-1β and the TNF-α/IL-1β clones displayed an increased invasive capacity compared with the empty vector control (p<0.05). Differences were also detected in the gene expression profile implicated in the pathways involved in the hallmarks of cancer - cell signalling (FOS, JUN), apoptosis (BAD, BAX, BCL2), angiogenesis (CXCL8), adhesion (ITGB3), invasion (S100A4, TIMP1) and cell cycle regulation (p53, c-myc). A number of these targets are currently undergoing validation.

Conclusion
This study provides a valuable isogenic cell line model in which to study the effect of prolonged chronic inflammation. Although the cells have not developed anchorage independent growth as assessed by soft agar, there are distinct indications that phenotypic changes occurred within the three month time frame. As pro-longed chronic exposure to inflammation is a pre-requisite for many disease states, these results warrant extended growth studies to further delineate the complex roles of TNF-α, IL-1β and hypoxia in the process of carcinogenesis. This will assist in the development of novel cancer target therapeutics and chemo-preventive agents in lung cancer.

Background
IL-23A is a member of the IL-6 super-family and is composed of covalently bound IL-12p40 and IL-23p19 (IL-23A) subunits. The functional IL-23R is composed of the IL-23R and IL-12Rβ1 subunits and once activated can promote the STAT3 signalling pathway. IL-23A plays a key role in chronic inflammation through the promotion of IL-17 production by T helper 17 cells (Th 17). Stimulation of IL-23A associated pathways produces pro and anti oncogenic effects which are cell type dependant. We have previously identified IL-23A axis as deregulated in NSCLC and sought to examine the family in greater detail.

Methods
The expression of IL-23A/IL-23R was examined in a series of resected fresh frozen NSCLC tumours and in a panel of normal (HBEC) and NSCLC cell lines. Epigenetic regulation of IL-23A/IL-23R was determined in NSCLC cell lines using histone deacetylase (HDi) and DNA methyltransferase (DNMTi) inhibitors. A ChIP assay was performed to investigate the direct effect of Trichostatin A (TSA) on the IL-23A/IL-23R promoter regions. Additionally, the effect of Gemcitabine on IL-23A/IL-23R expression was examined. Finally, the effect of recombinant IL-23 treatment and Apilimod (STA 5326) (a small molecule which blocks the expression of IL-23 and IL-12) on NSCLC cell line proliferation was examined. IL-23 expression was also studied in a panel of NSCLC cisplatin sensitive and resistant cell lines. Furthermore due to its role in immune regulation, we are currently studying the effect of IL-23 on innate immune cell function.

Results
IL-23A expression was significantly elevated in a cohort of NSCLC patient tumour samples (p<0.05). Differential IL-23R expression was evident in a panel of NSCLC normal/tumour matched pairs (n=20), with no expression in 25%, reduced levels in 20% and increased levels in 55% of tumour samples compared with matched normal. In addition, IL-23A/IL-23R was found to be epigenetically regulated through histone post-translational modifications and DNA CpG residue methylation in the A549 cell line (p<0.05), with associated chromatin remodelling at both promoters. Gemcitabine, also induced IL-23A/IL-23R expression in this cell line (p<0.05). Furthermore, treatment with recombinant IL-23 resulted in increased cellular proliferation in the A549 cell line, while Apilimod reduced cellular proliferation. Preliminary results indicate that IL-23 can potentially affect the function of innate γδ T cells. In a panel of cisplatin resistant and sensitive cell lines, IL-23A was up-regulated in 3/5 resistant cell lines. Work is ongoing to assess the IL-23R expression in CisR and Parent cells lines and the effect of co-treating cells with HDi and Apilimod and to determine whether these compounds can re-sensitise cells to cisplatin therapy.

Conclusion
Based on our results the IL-23A/IL-23R axis is dysregulated in NSCLC. The IL-23 family is subject to epigenetic regulation through HDAC modifications and CpG island methylation. Gemcitabine treatment also affected the expression of this family. Recombinant IL-23 was pro-proliferative in NSCLC and blocking IL-23 with Apilimod reduced the proliferative capacity of the cells. Levels of IL-23A are differentially expressed between cisplatin sensitive and resistant cell lines. Apilimod may be a novel therapeutic drug in the treatment of NSCLC.

Background
Pequi (Caryocar brasiliense Camb) is a Brazilian fruit that has high concentrations of antioxidants, such as vitamin C and E, carotenoids, phenolic compounds and essential oils. Consumption of foods high in antioxidants, particularly carotenoids and phenolic compounds have been associated in the prevention of oxidative damage caused by reactive species (ROS), including DNA damage, and can reduce the risk of cancer, atherosclerosis and other degenerative diseases. The aim of this study was to estimate the antioxidant enzyme activity of catalase, superoxide dismutase and glutathione peroxidase and to evaluate the antioxidant activity of the pequi oil, measuring lipid peroxidation, DNA damage and nitric oxid synthases expression.

Methods
The study was performed in 40 male BALB/c mices: 35 animals were submitted to two doses of 1,5g/kg intraperitoneal of urethane (U=5), 10 of these mices received by gavage 15μL of pequi pulp oil (U+O), 10 animals received by gavage 15μL of ethanolic extract of pequi pulp (U+E=10) and the other 10 animals received by gavage 3μg/kg of betacarotene (U+B). 5 mices didn’t receive the urethane doses neither the gavage (C=5). After 60 days, the groups were sacrificed. The enzymatic antioxidant defense was measured by biochemical test. The antioxidant activity of pequi oil was evaluated in the lung tissues by the biochemical TBARS test (Thiobarbituric acid-reactive substances) and the DNA damage by the comet test method. Nitric Synthase expression was analyzed by imunohistochemestry.

Results
The lung parenchyma from the Urethane groups that received and didn’t receive gavage, showed neoplasic formations induced by the chemical carcinogenesis in contrast with control group (C). The results of the TBARS test showed a significant decreased of the lipid peroxidation in the groups that were treated by gavage (U+O)(U+E)(U+B), when compared with Urethane group (U) (p<0,05). In the same way, the image analysis of the comet assay showed a statistical significant decreased of the DNA damage cells in the groups that received treatment by gavage (U+O)(U+E)(U+B) when compared with urethane group (p<0.001). The nitric oxid synthases expression was higher in the urethane group (U), but showed a significant decrease in groups that received the gavage (p<0,05). The catalase, glutathione peroxidase and superoxide dismutase test, as well as the ratio between then, didn’t show a significant difference.

Conclusion
We conclude that because of its composition, high in antioxidants, pequi fruit is efficient to diminish the oxidative stress status (DNA damage and lipid peroxidation) and the nitric oxid synthases expression in chemical carcinogenesis induced by urethane. Our results also suggest that the antioxidant enzyme defense are not envolved in this process, suggesting that antioxidants present in pequi fruit may have a greater impact in lung cancer treatment.

Background
Lung adenocarcinomas can be classified into two major subtypes, terminal respiratory unit (TRU) type and non-TRU type. We previously reported that non-TRU type adenocarcinoma has unique clinical and morphological features compared with TRU type adenocarcinoma. In brief, among 36 cases of non-terminal respiratory unit type adenocarcinoma, 24 cases revealed mucous columnar cell changes that were continuous with bronchial ciliated columnar cells. The mucous columnar cells became dysplastic showing loss of cilia, disorientation, and enlarged nuclei. Adenocarcinoma arose from these dysplastic mucous columnar cells and, characteristically, this type of adenocarcinoma showed acute inflammation, and honeycombing changes in the background. TTF-1 immunostaining was consistently negative. In a case study with 14 males and 10 females, including 12 smokers or ex-smokers, EGFR and KRAS mutations were detected in 3 and 6 patients, respectively. We think that this kind of adenocarcinoma arising through mucous columnar cell change belongs to non-terminal respiratory unit type adenocarcinoma, and mucous columnar cell change is a precursor lesion of pulmonary adenocarcinoma. We investigated whether microRNA (miRNA) expression profiles can differentiate between non-TRU type and TRU type lung adenocarcinoma.

Methods
MiRNA expression in lung adenocarcinoma with non-TRU and TRU type was analyzed by microarray analysis with 1205 human and 144 human viral miRNAs.

Results
Eight pairs of lung adenocarcinomas and corresponding noncancerous lung tissues (4 for non-TRU type and 4 for TRU type) were analyzed by microarray analysis. Initially, 44 miRNAs showed statistical differences in expression between lung cancer tissues and corresponding noncancerous tissues. Comparison analyses between non-TRU type and TRU type revealed 30 miRNAs with statistically different expression. Particularly, hsa-mir-494 and ebv-mir-BART19 were upregulated with more than 5-fold difference as compared with TRU type adenocarcinoma. Similarly, hsa-mir-551b was downregulated at more than 4-fold as compared with TRU type adenocarcinoma. Only 2 miRNAs (hsa-mir-1 and hsa-mir-133b) were shared in both non-TRU and TRU type of adenocarcinoma.

Conclusion
We demonstrated that non-TRU type adenocarcinoma showed different miRNAs expression patterns compared with TRU type adenocarcinoma, suggesting different carcinogenic pathways for these tumors. Further studies are required for validation the results from microarray analysis.

Background
Cancer stem-like cells (CSCs) / Cancer-initiating cells (CICs) have been described as a small population that have (i) tumor initiating ability, (ii) differentiation ability and (iii) self-renewal ability and regarded as major causes of cancer recurrence, distant metastasis and treatment resistance. CSCs/CICs have been thought to have similar molecular mechanisms to normal stem cells and keep undifferentiated state. And we thought that Lung CSCs/CICs constantly exchange the state of differentiation and dedifferentiation. Previously we showed that SOX2 is overexpressed in stem-like cells of human lung adenocarcinoma cell lines. And so, we think SOX2 is important for the sustention of CSCs/CICs. In this study, we examined the differentiation and dedifferentiation of Lung CSCs/CICs in single-cell level and investigated the regulation of SOX2 expression in Lung CSCs/CICs.

Methods
Lung adenocarcinoma cell lines LHK2 were stained with Hoechst33342 dye and CSCs/CICs were isolated as Side population (SP) cells and non-CSCs/CICs were isolated as Main population (MP) cells using a FACS Aria II cell sorter. Many single cell clones (SP clones, MP clones) were established from SP cells and MP cells respectively. SOX2 expression was addressed by qPCR. LHK2 were treated with HDAC inhibitor Trichostatin A (TSA).

Results
SP cells and MP cells were generated from each SP clones and MP clones. The SOX2 expression of SP clones were higher than that of MP clones. TSA treatment enhanced the expression of SOX2 and increased the rate of SP cells.

Conclusion
It was indicated that the differentiated Lung carcinoma fractions were dedifferentiated into CSCs/CICs and SOX2 expression was important for the mechanisms. It was considered that the differentiation and dedifferentiation occurred with comparative ease in Lung carcinoma, so we thought that the mechanisms related to the epigenetic regulation. In this study, it was indicated that SOX2 expression in Lung cancer cells were regulated by histone acetylation. Therefore, Lung CSCs/CICs phenotype might be regulated by epigenetic mechanisms.

Background
The MAGE-A3 and PRAME tumor antigens are potential targets for cancer immunotherapy. In patients with non-small cell lung cancer (NSCLC), specific mutations of the epidermal growth factor receptor (EGFR) gene are associated with improved therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs); mutations in the KRAS gene confer resistance to EGFR-TKIs. In NSCLC, EGFR and KRAS mutations are predominantly found in adenocarcinomas. The relationship between MAGE-A3 or PRAME expression and EGFR/KRAS mutational status is unknown. The presence of specific EGFR mutations determines the selection of patients for EGFR-TKIs therapy. With cancer immunotherapeutics and EGFR-TKIs potentially moving on to the same treatment stages, it is important to determine whether patients eligible for EGFR-TKI therapies might be also eligible for treatment with MAGE-A3 or PRAME cancer immunotherapeutics. We have previously reported that MAGE-A3 and PRAME were expressed in 36% and 66% of NSCLC tumor samples, and in 27% and 44% of adenocarcinoma samples, respectively (Linder et al. ASCO 2012). Here we report the MAGE-A3 and PRAME expression rates and mutational status of EGFR and KRAS in NSCLC adenocarcinoma.

Methods
This was a single-center, non-interventional, retrospective study conducted at the Lungenklinik Hemer (Germany). MAGE-A3 and PRAME mRNA expression patterns in patients with pathologically proven stage I, II or III NSCLC were determined by quantitative real-time PCR on formalin-fixed paraffin-embedded (FFPE) tumor samples. EGFR and KRAS mutational status was analyzed on DNA extracted from FFPE samples using laboratory-developed PCR-based assays. Samples from patients with adenocarcinoma i.e. adenocarcinoma, bronchoalveolar adenocarcinoma, adenosquamous carcinoma, for which MAGE-A3 and/or PRAME expression results were available were selected for this analysis.

Results
Out of 311 samples, 277 and 287 produced valid results for EGFR and KRAS mutational status, respectively. EGFR mutations were found in 10.1%, and KRAS mutations in 33.8% of samples. The association between MAGE-A3 or PRAME expression and EGFR mutational status is shown (table). Figure 1 EGFR mutational status was not associated with MAGE-A3 expression but was significantly associated with PRAME expression (table). There was no association between KRAS mutational status and MAGE-A3 (P=0.6109) or PRAME (P=0.8983) expression.

Conclusion
In this study, tumor samples expressing PRAME tended to be associated with wild-type EGFR status. Thus, among patients with PRAME-positive tumors, only very few would be eligible for EGFR-TKI therapy. This indicates a potentially straightforward treatment choice for patients and physicians, should EGFR-TKIs and PRAME cancer immunotherapeutics become available for similar indications. Funding: GlaxoSmithKline Biologicals SA.

Background
Non-small cell lung cancer (NSCLC) is the leading cause of cancer morbidity and mortality in the Western world with a poor overall 5 year survival of <15%. The most effective systemic chemotherapy for NSCLC is cisplatin-based combination treatment. However, chemoresistance is a major therapeutic problem and understanding the mechanisms involved is critical to the development of new therapeutic intervention strategies. The PI3K pathway plays an important role in NSCLC and we and others have shown increased PI3K signaling to be associated with a more aggressive disease with poor prognosis. Several proteins in this pathway have been indicated as potential mediators of cisplatin resistance in other cancers, and our group has previously identified the PI3K-activated transcription factor NFκB as a key player in this setting. In this study, targeted inhibition of three strategic points of the PI3K pathway was carried out with the aim of overcoming acquired resistance to cisplatin in these cell lines.

Methods
A panel of cisplatin resistant cell lines was previously generated in our laboratory through prolonged exposure to the drug. Expression of PI3K pathway related genes was compared between H460 parent (H460PT) and H460 cisplatin resistant (H460CR) cells using a PI3K pathway SABiosciences RTPCR array. Identified genes of interested were further investigated via PCR and Western blot in these cells as well as A549 parent (A549PT) and A549 cisplatin resistant (A549CR) cells. Three strategic points of the pathway were inhibited using GDC-0980, a dual PI3K-mTOR inhibitor currently in Phase II clinical trials in NSCLC, and DHMEQ, an inhibitor of NFkB translocation which has been used extensively both in vitro and in vivo. Effects of the two inhibitors on the parent & cisplatin resistant cell lines both with and without cisplatin were assessed by BrdU proliferation assay and multiparameter apoptosis assay (High Content Analysis).

Results
One of the most notable targets to emerge from the PI3K pathway RTPCR array screen was NFKBIA; the gene which codes for NFκB inhibitor IκBα. This gene was shown to be 12 fold overexpressed in H460CR compared to H460PT. This finding was validated at both the RNA and protein level by PCR and Western blot. NFκB was also found to be overexpressed in cisplatin resistant cells compared to their respective parent cells. Inhibition of NFκB by DHMEQ led to significantly improved inhibition of proliferation and induction of apoptosis in cisplatin resistant cells compared to parent cells. Preliminary data indicates that inhibition of PI3K and mTOR by GDC-0980 did not offer as significant a benefit as inhibition of NFκB in the cisplatin resistance setting, though further data from combination studies will be presented.

Conclusion
We conclude that the PI3K pathway plays an important role in resistance to cisplatin in NSCLC, particularly when signaling proceeds through the transcription factor NFκB. Targeting this pathway may be of benefit in re-sensitizing cisplatin resistant tumours to the drug.

Background
The MAGE-A3 tumor antigen is expressed in non-small cell lung cancer (NSCLC), and is a potential target for cancer immunotherapy. NSCLC patients may also have specific epidermal growth factor receptor (EGFR) gene mutations, which are responsible for sensitivity to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in metastatic NSCLC patients. In Asian populations, a relatively low MAGE-A3 expression (Therasse et al. ASCO 2012) and a relatively high frequency of EGFR mutations (An et al. 2012) have been reported compared to Caucasian populations. Three phase III studies, one with MAGE-A3 cancer immunotherapeutic and two with EGFR-TKIs, are ongoing in patients with early-stage, resected NSCLC. However, to date, a direct association between MAGE-A3 expression pattern and EGFR mutational status has not been investigated. Here, we evaluate a potential link between MAGE-A3 expression and frequency of EGFR mutations in Chinese NSCLC patients.

Methods
This single-center, non-interventional, retrospective study was conducted at Guandong Lung Cancer Institute, China. Adenocarcinoma and squamous cell carcinoma (SCC) fresh frozen (FF) stage IB, II or IIIA NSCLC tumor samples were collected. MAGE-A3 transcript levels were determined using quantitative real-time PCR. The presence of EGFR mutations was detected using bidirectional Sanger sequencing on genomic DNA extracted from the same FF tumor samples.

Results
MAGE-A3 was expressed in 15.6% of adenocarcinoma (N=96) and 31.6% of SCC (N=95) samples. EGFR mutations were found in 43.8% of adenocarcinoma and 5.3% of SCC samples. MAGE-A3 expression rates and EGFR mutational status are shown (table). Figure 1

Conclusion
In this study, mutated EGFR status was observed in more than half of MAGE-A3-positive adenocarcinoma samples. However, due to a small number of samples analyzed, no statistical association could be concluded. Additional larger studies, especially in Asian patients with adenocarcinoma, are needed to confirm the findings. This study was funded by GlaxoSmithKline Biologicals SA.

Background
The new lifestyle adaptations in the developing countries altered the environment and these contribute in the development of various respiratory diseases like asthma, COPD, etc. Though it is well known that COPD may lead to development of lung cancer, the effect of asthma on lung cancer development is not well studied. Lung cancer is a leading cause of mortality worldwide as more than a million people die every year. Few recent meta-analytic studies showed a positive correlation between asthma and lung cancer. But, there are no reports to demonstrate the status of DNA damage in asthma.

Methods
8-hydroxydeoxyguanosine, DNA damage marker, was measured in BAL fluids and sera samples of human asthmatics. To see the gene expression related to DNA damage response, we performed the quantitative Real Time PCR array in human normal and asthmatic lungs. Significantly altered genes were validated at mRNA as well as in protein levels by using western blotting and immunohistochemistry.

Results
Increased levels of 8-hydroxydeoxyguanosine was observed in BAL fluids and sera of human asthmatics. Human and murine asthmatic lungs show an increase in the expression of 8-hydroxy-2'-deoxyguanosine. In comparison to normal, human asthmatics showed a decrease in the expression of key DNA damage response proteins like gamma-H2AX, Atm, chk2, Mre11 and DNA-pkcs.

Conclusion
For the first time, we have shown that there may be defective DNA damage response in asthmatics which might link asthma to lung cancer. As various meta-analysis studies indicate a positive correlation between asthma and lung cancer, it is necessary to delineate the mechanisms underlying this.

Background
Epithelial growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) show incredible response rate up to around 80 % to non-small cell lung cancer patients with EGFR sensitive mutations, and also contribute to improve overall survival time of such patients. However, it is no doubt that almost all patients have recurrences in time. Some genetic aberrant changes such as the secondary mutation T790M in exon 20 of EGFR are known to have influence on drug resistance, but most mechanisms of drug resistance are still unclear. Recently, Some studies suggested that heterogeneous distribution of EGFR mutations seemed to have correlations with treatment failure of EGFR-TKIs, but others reported that heterogeneity was extremely rare. Heterogeneity of EGFR is still remains to be controversial. In this study, we reported one rare case that resected lung after chemotherapy with gefitinib showed heterogeneous distribution of L858R point mutations and it’s correlation with effects of EGFR-TKIs.

Methods
A 68-year-old woman was diagnosed as lung adenocarcinoma (T3N1M0 stageⅢa) with the L858R point mutation. Unilateral Pulmonary Artery Occlusion test before operation showed a high probability that patient’s oxygenation would decrease to near the tolerance limit by right peumonectomy. For the purpose of reducing the tumor volume to avoid the pneumonectomy, she received gefinitib (250mg/body/day) as neo-adjuvant chemotherapy for three months and succeeded. Continuously, she underwent right middle and lower lobectomy and lymph node dissection (ND2a). Pathological examinations of resected lung specimens were done using H&E staining and immunohistochemistry with L858R Mutant Specific Rabbit monoclonal antibody for EGF Receptor. Two different tissue parts were respectively macrodissected from Formalin-Fixed Paraffin-Embedded tissue slides and analyzed genetically by direct sequencing analysis.

Results
H&E and immunohistochemical staining specimens showed that this resected identical tumor was divided into two areas. One area consisted of necrotizing and degenerating cell population and was stained with L858R specific antibody. The other area consisted of viable cell population with relatively low nuclear grade and was not stained with L858R specific antibody. Additionally, direct sequencing analysis of each extracted DNA from these two different areas revealed that the former area contained only wild type base sequence in exon 21 of EGFR, but the latter contained a mixture of point mutation of L858R and wild type base sequence in exon21. Secondary T790M mutations were not detected in both areas. The result of direct sequencing analysis supported heterogeneous distribution of L858R point mutations which was found on immunohistchemical staining. Comparing with pathological finding of HE staining, it was implied that therapeutic effect of gefitinib was limited only to the tissue area having L858R point mutation and cells of non-mutated tissue area survived.

Conclusion
This study suggested that there may be intratumoral heterogeneity of EGFR mutation and therapeutic effect of EGFR-TKIs could be limited only to mutant cells. As a result, wild-type cells would survive and develop drug resistance against EGFR-TKIs, even if tumors had no secondary T790M mutations in exon 20 of EGFR.

Background
Somatostatin receptors (SSTRs) have been shown to be overexpressed in 80-90% of all neuroendocrine tumors, including SCLCs. Previous reports have shown that SSTR2 activation modulates cell proliferation by acting through the ERK signaling cascade. We hypothesized that down regulation of SSTR2 will inhibit cell proliferation in lung cancer cell lines that overexpresses this receptor.

Methods
SSTR2 expression was assessed by western blot analysis in 28 lung cancer cell lines. H520 (SCC) and H727 (Carcinoid) were used as an in vitro model for studying the effects of targeted down-regulation of SSTR2 by shRNA. Several SSTR2 null-colonies of these cell lines were generated by antibiotic selection. Cell proliferation was measured at four different time points using MTS metabolic assay. In addition, we evaluated the effect of SSTR2 down regulation in colony formation potential of our lung cancer cell lines.

Results
Western blot analysis of 28 lung cancer cell lines showed expression of SSTR2 in 17/18 (94%) of SCLCs including carcinoids, 3/6 (50%) of NSCLCs, and 0/4 (0%) of non-immortalized human bronchial cells. Post transfection analysis by western blot revealed a significant decrease in SSTR2 protein expression in multiple SSTR2 null-colonies. We observed a 37% decrease in cell growth in H520 and a 38% decrease in H727 by day 6 with p values of 0.007 and 0.002 respectively. In addition, down regulation of SSTR2 resulted in reduction in the number of colonies formed by both cell lines.

Conclusion
These results demonstrate that that knocking down SSTR2 in lung cancer cells induces a marked decrease in cell proliferation. Ongoing studies are focused on understanding the effect of knocking down SSTR2 on oncogenic traits of lung cancer cells including cell survival, apoptosis, migration and invasion and the cellular mechanisms underpinning our original observation.

Background
The present study investigated adenosine-induced apoptosis in human malignant pleural mesothelioma cells.

Methods
MTT assay, TUNEL staining, flow cytometry using propidium iodide and annexin V-FITC, real-time RT-PCR, Western blotting, and assay of caspase-3, -8, and -9 activities were carried out using malignant pleural mesothelioma cell lines such as NCI-H28, NCI-H2052, NCI-H2452, and MSTO-211H cells, and P53 or A3 adenosine receptor was knocked-down by transfecting each siRNA into cells.

Results
Adenosine induced apoptosis in all the malignant pleural mesothelioma cells used here, independently of caspase activation. The adenosine effect was prevented by the adenosine transporter inhibitor dipyridamole, the adenosine kinase inhibitor ABT-702, or the A3 adenosine receptor inhibitor MRS1191. Adenosine upregulated expression of the p53 mRNA and protein, that is abolished by ABT-702, but not by knocking-down A3 adenosine receptor. Adenosine-induced apoptosis in NCI-H28 cells was significantly inhibited by knocking-down p53 and in part by knocking-down A3 adenosine receptor.

Conclusion
The results of the present study show that AMP converted from intracellularly transported adenosine upregulates p53 expression to induce caspase-independent apoptosis in malignant pleural mesothelioma cells and that A3 adenosine receptor also participates partially in the apoptosis by the different mechanism.

Background
Microvasculature and microenvironment play important roles in proliferation, invasion, metastasis and prognosis in non-small cell lung cancer (NSCLC), which might be altered by many anti-angiogenic drugs. Epigallocatechin-3-gallate (EGCG), a natural anti-angiogenesis agent refined from green tea, was defined to have multiple effects on angiogenesis factors, such as endothelial growth factor (VEGF) and angiopoietins (ANGs). Hypothesizing that EGCG might regulate microvasculature and microenvironment in NSCLC, the effects of EGCG on microvessel density (MVD), expression of Ang-1 and Ang-2, interstitial fluid pressure (IFP), tumor hypoxia, and chemotherapy sensitivity were examined.

Methods
Build nude mice xenograft tumor model(A549 cell line). Treat them with EGCG(i.p.), Counting MVD labeled by CD31, observing Ang-1 and Ang-2 expressions detected by quantum dots double-label immunofluorescence assessment. interstitial fluid pressure (IFP) was measured by the Wink-in-needle method, while hypoxia was assessed by polarographic electrode and pimonidazole (PIMO) immunohistochemistry. Assuming that these changes would increase response to chemotherapy, tumor growth studies were performed in nude mice with xenografts, which were then treated with EGCG and the chemotherapeutic agent.

Results
EGCG treatment led to a significant decrease of MVD, and of Ang-2 expression, while Ang-1 decreased with no significance. Both IFP and hypoxia were decreased. Tumor growth studies show that EGCG therapy combined with cisplatin led to synergistic inhibition of tumor growth, compared with administration of each treatment separately (P < 0.001). According to linear regression analysis, IFP was positively correlated with PIMO staining (R2 = 0.618, P = 0.002), Ang-2 was correlated with MVD (R2 = 0.423, P = 0.022), IFP (R2 = 0.663, P = 0.01) and PIMO staining (R2 = 0.694, P = 0.01).

Conclusion
IFP and delivery of oxygen might be improved by rebalance of Ang-1/Ang-2 under the treatment of EGCG in NSCLC, which also acts as a sensitizer of chemotherapy. These studies established a new mechanism for using EGCG as an adjuvant chemotherapy agent through modifying microvasculature and microenvironment.

Background
Lung cancer remains the most common cause of cancer-related death in the world for which novel systemic treatments are urgently needed. Protein homeostasis that regulates protein levels and their fold is critical for cancer cell proliferation and survival. A complex network of cellular organelles and signaling cascades is involved in control of protein homeostasis including endoplasmatic reticulum (ER). Thus, proteins in control of ER homeostasis are increasingly recognized as potential therapeutic targets. Molecular chaperone heat shock protein 90 (Hsp90) and histone deacetylase (HDAC) play an important role in ER homeostasis. Previous studies demonstrate that Hsp90 and HDAC inhibitors are individually functional against lung cancer. In this work we suggested that combined Hsp90 and HDAC inhibitors may elevate ER stress thereby enhancing the anti non small lung cancer (NSCLC) activity.

Methods
NSCLC cell lines (A549, H1299, H460) were treated with 17-DMAG (Hsp90 inhibitor), PTACH (HDAC inhibitor) and both drugs simultaneously for 24 hours. Cells were harvested and analyzed for ER stress markers (Immunoblot), viability (WST1 assay), motility (Scratch assay), and death (Flow cytometer).

Results
Using an in vitro cell line model we demonstrated that 17-DMAG co-administration with PTACH caused elevated ER stress (more than 110%, p<0.05) accompanied by apoptotic cell death (Annexin V) (7-21%, p<0.05). Moreover, 17-DMAG/PTACH treated cells lost the ability to migrate (57-85% of scratch closure, p<0.05).

Conclusion
Our findings provide proof-of-concept that targeting ER homeostasis is therapeutically beneficial in lung cancer cell lines. Indeed, the elevated ER stress caused by 17-DMAG/PTACH combined treatment leads to increased cell death of NSCLC cell lines compared to the application of the drugs separately.

Background
To evaluate and compare quantitatively aberrant methylation in the promoter area of EGFR gene in patients (p) with in non small cell lung cancer (NSCLC) lung tumor tissue (TT), lung normal tissue (NT), blood (B), bronchial aspirate (BA) and sputum (S). Correlate these methylation patterns with clinical variables.

Methods
DNA was extracted from samples of B, S and BA from patients with NSCLC prior to surgery and resected TT and NT. Methylation patterns were analyzed by the bisulfite conversion and subsequent pyrosequencing (QIagen PyroMark System). We analyzed three CpG islands in the promoter region of EGFR gene.

Results
43p were included, median age 66 years (range 46-79), 35 men and 8 women. Histology: 26p adenocarcinoma, 14 squamous and 3 others. 2p had EGFR mutation. Smoking status: 4p never smokers, 21p former smokers, 18p smokers. Significant differences in methylation percentage (%Mth) were observed between TT and S in the following islands: CpG2 (11.8vs7.1, p = 0.008), CpG3 (10vs7.4, p = 0.037) and in overall promoter area (10.6vs7. 6, p = 0.034). The %Mth was higher in women vs men in TT CpG1 island (16.2vs24.9, p = 0.042) and TT CpG2 island (15.8vs26.5, p = 0.013). The %Mth was higher in squamous histology compared to adenocarcinoma in NT CpG2 island (22.6vs14.1, p = 0.017) and S CpG1 island (13.7vs8.2, p = 0.047). However, the %Mth was higher in the overall promoter area of adenocarcinoma respect to squamous histology (19.7vs12.8 TT, p = 0.042). The %Mth was higher in overall promoter area of NT respect Stage I vs II vs III (22.3 vs 13 vs 17.5, p = 0.03). The %Mth was higher in former smokers compared to current smokers and non smokers in NT CpG3 (22.8 vs 13 vs 14.1, p = 0.032). The %Mth in NT CpG3 was higher in never smokers and former smokers > 5 years, respect to current smokers and former smokers <5 years, (23.3 vs 15.8, p = 0.05).

Conclusion
EGFR aberrant hypermethylation was higher in tumor tissue respect to sputum, with no correlation with EGFR mutation. The %Mth in normal tissue was higher in never smoker and former smokers > 5 years patients. * This study was funded by the Carlos III Health Institute (PS09/00308), Spain

Background
Four neuroendocrine subtypes are distinguishable which differ in the extent of differentiation and grade of biological aggressiveness. Therefore the differentiation of atypical from typical carcinoids or large cell neuroendocrine carcinomas and small cell carcinomas is essential for treatment options and prognosis. However, for pathologists it is often a challenge to establish a faithful differential diagnosis with an accurate prognosis which is restricted in terms of limited specificity of immunohistochemical markers and possible artifacts. Thus we investigated two types of miRNAs as an additional tool for differential diagnosis and possible molecular targets.

Methods
A collective of 38 patients suffering from well to poorly differentiated neuroendocrine lung tumours were examined. Two different miRNAs (miR-21, miR-34a) were investigated in four distinct subtypes of pulmonary neuroendocrine tumours by comparative gene expression analysis.

Results
miRNA 21 was upregulated in G3-neuroendocrine tumors (Mean rank: 26.8; 28.75) as compared to carcinoids (mean rank: 12.33; 12.07) with a significance of 0.00033. High-expression level of miRNA 34a was associated with atypical carcinoids (p = 0.010).

Conclusion
miRNAs are suitable candidates for differential neuroendocrine lung cancer diagnosis. A close association is implicated between the elevated miR-21 in high-grade and miR-34a in low grade atypical neuroendocrine lung carcinomas which could potentially be exploited as practical supportive markers for differential lung cancer diagnosis in routine. However, some additional research and validation studies are needed to utilize them as routine markers or potential molecular targets for personalized medicine.

Background
Molecular targeted therapy based on TKIs, directed at the Epidural growth factor receptor (EGFR) is one of the recent option for the management of advanced Non-Small Cell Lung Cancer (NSCLCs). EGFR gene mutations, exon 19 deletions (E19 del (LREA deletions)) and exon 21(L858R) are most common and good predictions of response to EGFR-TKI treatment. EGFR gene mutations are found in approx. 10% of Caucasian patients and up to 50% of Asian populations[(1)] .Recent studies have endorsed these incidences in European and Asian populations. The frequency of EGFR mutation is higher in non-smokers and in women[(2,3) ].The aim of the study was to determine the frequency of EGFR gene mutation in advanced non small cell lung cancer patients presenting to SKMCH & RC.

Methods
Between Sep 2011 to Sep 2012, we have sent EGFR Mutation testing for 15 patients with proven histology of TTF-1 positive adenocarcinoma lung. EGFR gene mutations in exons 18, 19,20 and 21 were carried out by National University Hospital Singapore and Singapore General Hospital. Data was collected and analysed. Frequencies were determined.

Results
10 (66.6 %) patients were male and 5 (33.3 %) were females. Eight (53.3 %) were smokers and 7 (46.6 %) were non smokers.Three (20 %) patients were having EGFR mutation +ve . One with +ve mutation on exon 19, one with +ve mutation on exon 21 and one with +ve EGFR mutation on exon 20. Two of these three( 66.6%) patients were smoker and one ( 33.3%)out of them was non smoker. All three patients were male. One (6.6%) patient has insufficient biopsy material to carry out the test. While 8 (53.3 %) patients have –ve EGFR mutation on all exons and 3 (20%) patients have unsatisfactory results on few exons and –ve mutation on other exons.

Conclusion
The frequency of EGFR mutation in our institution was 20 % and all these patients were male, two third patients were smokers. This trend is not in accordance with literature, needs more number of patients to find out the real trend.

Background
MED12 negatively regulates TGF-b receptor signaling. Loss of MED12 induces an EMT-like phenotype associated with chemotherapy resistance. IDO and IL6 activate the JAK2/STAT3 signaling pathway, which – together with NFkB (RelA) signaling – is often altered in lung cancer. BIM could influence response to chemotherapy. We have examined these components and KRAS mutations in NSCLC tumor samples and correlated results with progression-free survival (PFS).

Methods
The mRNA expression of MED12, IDO, JAK2, STAT3, RelA and BIM was examined in microdissected tumor samples from p with stage IV NSCLC. mRNA levels were assessed by RT-PCR and categorized by terciles (high vs low/intermediate). KRAS mutations were assessed by high resolution melting.

Results
A total of 55 p with performance status (PS) 0-1, treated with platinum plus either gemcitabine or pemetrexed: median age, 62 years; 27.6% females; 84.2% smokers; 66% adenocarcinoma; 16% with KRAS mutations. There was no correlation between gene expression levels and KRAS mutation status. In the multivariate analysis, including gene expression levels, histology and PS, only MED12 and STAT3 were associated with PFS (low MED12: HR=11.6, P=0.005; high STAT3: HR=6.5, P=0.01). HR for low BIM expression was 2.4 (P=0.16). None of the markers were associated with overall survival.

Conclusion
To the best of our knowledge, this is the first time that low expression of MED12 with significantly shorter PFS in NSCLC p receiving platinum-based chemotherapy. MED12 could be a new biomarker of chemoresistance and inhibition of TGF-bR signaling can restore chemotherapy response in patients with low MED12.

Background
Adenocarcinoma in situ (AIS) of the lung shows a very favorable prognosis, with a 5-year survival rate of 100%. However, early but invasive adenocarcinoma (eIA) sometimes has a fatal outcome. In order to elucidate the key molecule that affects the malignant progression of adenocarcinoma at an early stage, we previously compared the expression profiles of AIS with those of eIA, and found that stratifin (SFN, 14-3-3 sigma) is one of the differentially expressed genes related to tumor progression (Aya Shiba-Ishii, IJC. 2011). Immunohistochemistry (IHC) with anti-SFN antibody revealed that more than 95% of eIAs were SFN-positive, in comparison with only 13% of AISs. We also found that promoter demethylation triggered aberrant SFN overexpression in eIAs (Aya Shiba-Ishii, AJP. 2012). Here, we performed functional analysis of SFN and identification of SFN binding protein (SBP) to clarify how SFN affects the progression of lung adenocarcinoma.

Methods
For in vitro functional analysis, we performed RNA interference and expression vector transfection analyses and subsequent cell proliferation assays or cell cycle phase assay using a lung adenocarcinoma cell line (A549). An in vivo animal study was also performed using siSFN-transfected A549. Additionally, in order to identify SBP, SFN vector-transfected A549 was subjected to pull-down assay and subsequent LC-MS analysis. Interaction of SFN and SBP was examined using co-IP and western western blotting.

Results
Suppression of SFN expression by siSFN significantly reduced cell proliferation activity and the S-phase subpopulation. Transfection of the SFN expression vector led to a significant increase in cell proliferation. A549 treated with siSFN showed reduced tumor development relative to the controls. Pull-down assay and LC-MS analysis revealed that SKP1 is one of the SBPs. SKP1 is a component of the SKP1-Cullin1-F-box containing complex (SCF complex). We found that Fbw7, one of the substrate recognition subunits of SCF complex, also binds to the SFN-SKP1 complex, resulting in up-regulation of cyclin E1 phosphorylation by SFN.

Conclusion
Although SFN was originally identified as a negative regulator of the cell cycle, especially in response to p53-sensitive DNA damage, subsequent reports indicated that it is a positive mediator of cell proliferation. In breast cancer, SFN induces G1/S progression by increasing the expression of cyclin D1. Here, we demonstrated that SFN enhanced the proliferative capacity of lung adenocarcinoma cells both in vitro and in vivo. We also revealed that SFN down-regulated expression of the SCF[Fbw7] complex. As cyclin E1 is a common target of SCF[Fbw7] ubiquitination, we predict that SCF[Fbw7] down-regulation by SFN might induce stabilization of cyclin E1. Since we also found that the S-phase subpopulation was decreased after siSFN treatment, SFN might induce G1/S progression through an increase of cyclin E1 phosphorylation in lung adenocarcinoma. In conclusion, SFN facilitates cell proliferation and malignant progression of lung adenocarcinoma, and its mechanism of action is thought to be associated with inhibition of SCF[Fbw7] ubiquitination and degradation.

Background
Lung cancer occurs in a significant number of never-smokers. Epigenetic changes in lung cancer potentially represent important diagnostic, prognostic and therapeutic targets. Accordingly, we sought to determine if there are differences in DNA methylation between lung adenocarcinomas and adjacent non-malignant lung tissue in never-smokers.

Methods
Using the Illumina Infinium HumanMethylation27 BeadChip we compared the DNA methylation profiles of 28 adenocarcinomas of the lung of never-smokers with their paired adjacent non-malignant lung tissue. The β value represents each methylation data point as the ratio of fluorescent signals between the methylated sites and the sum of methylated and unmethylated sites, which ranges continuously from 0 (unmethylated) to 1 (fully methylated). We used the Mann Whitney U test to compare β values between groups using JMP (SAS Institute Inc. Cary, NC USA). Then, using the Illumina Human WG DASL beadchip, we correlated differential methylation changes with gene expression changes from the same 28 samples. We validated our findings with 24 samples of lung adenocarcinomas and paired non-malignant tissue from The Cancer Genome Atlas (TCGA). Database for Annotation, Visualization, and Integrated Discovery was used to determine gene enrichment.

Results
We observed a distinct separation in methylation profiles between tumor and adjacent nonmalignant lung tissue using principal component analysis. Tumors were generally hypomethylated (β=0.15) when compared to adjacent non-malignant tissue (β=0.17; p=0.02). There were 1906 differentially methylated (Bonferroni corrected p value <0.05) CpG sites between tumor and adjacent non-malignant lung tissue. Of these sites, 1198 were within classically defined CpG islands where tumors (median β=0.38) were hypermethylated compared to adjacent non-malignant tissue (median β=0.22; p<0.0001), and 708 sites were outside of CpG islands where tumors (β=0.49) were hypomethylated compared to adjacent non-malignant tissue (β=0.56; p<0.0001). When compared to a dataset of 24 lung adenocarcinomas and paired non-malignant tissue from TCGA, 1841 of these 1906 (96.6%) sites were also differentially methylated with the same direction of change between tumor and non-tumor lung tissue. We matched 1483 genes with the differentially methylated CpG sites and found that these genes were enriched with the terms glycoprotein, signal, plasma membrane part, homeobox in addition to a few other terms. There were significant differences in expression of 376 genes (Bonferroni corrected p<0.05) of the 1483 (25.4%) differentially methylated CpG sites. There was an inverse correlation between methylation and gene expression in 80% of these genes. Genes that were not significantly differentially expressed and were hypermethylated within CpG sites were enriched for homeobox genes (false discovery rate = 1.2E-27).

Conclusion
The methylation profiles of lung adenocarcinomas of never-smokers and their adjacent non-malignant lung tissue are significantly different. Differential methylation of a CpG site was associated with altered gene expression in approximately 25% of cases. Despite the differential methylation of homeobox genes, no significant changes in expression of these genes were detected.

Background
Neuroendocrine differentiation (NED) has been observed in approximately 25-33% of all lung tumors. Essentially all small cell lung cancer (SCLC) and carcinoid tumors show distinct histological morphology and stain positive to neuroendocrine markers such as neuroamines, neuropeptides, dense core secretory granules, chromogranin A, neuroendocrine specific protein (NSP), and neuron-specific enolase (NSE). It was later recognized that NED was not limited to SCLC and carcinoid, and it could involve non-small cell lung cancer (NSCLC) such as large cell neuroendocrine carcinoma (LCNEC). Subsequently, it was found that about 10-20% of NSCLC, including adenocarcinomas and squamous cell carcinomas, also exhibit some neuroendocrine properties despite being considered non-neuroendocrine types. It has been postulated that NED of NSCLC represents an intermediary transition between SCLC and NSCLC, or that NED may be a predictor of the response to chemotherapy or radiotherapy, as well as a predictor of shorter survival in patients with stage I adenocarcinoma of the lung. Irrespective of these observations, the clinical significance of NED of NSCLC remains unclear.

Methods
We hypothesize that the investigation of the intracellular signal transduction pathway involved with NED of NSCLC cells may reveal potential molecular targets for the treatment of NSCLC with NED. We explore the potential molecular pathway involved in NED of NSCLC and its clinical association, through the induction of NED of a NSCLC cell line NCI –H157 in vitro. We further confirmed our findings using activators vs. inhibitors to these signal transduction pathways in vitro. We conducted immunochemical stains for phopsphorylated ERK1/2 expression of lung tumors know to have NED. ERK1/2 positivity was defined by 5% or more tumor cells stained positive with phospho-Erk½.

Results
We discovered that NED of NSCLC was associated with the activation of Erk1/2-MAPK signal transduction pathway, and the inhibition of the Akt signal transduction pathway. Using specific activator (Pb[2+]) and inhibitors (siRNA-Erk1/2, and U0126) to the Erk1/2 /MAP-kinase pathway, as well as the inhibitor (LY294002) to the Akt pathway, we found that Erk1/2/MAP-kinase activation was essential for NED of NCI-H157 cells. Staining of Erk1/2/MAP-kinase pathway reveals a high rate of positivity in NSCLC tumors with NED when compared with other neuroendocrine lung tumors. The positive rate of IHC was 0/10 (0%) for typical carcinoid; 2/10 (20%) for atypical carcinoid; 4/9 (44.4%) for small cell lung carcinoma; and was 7/10 (70%) for large cell neuroendocrine lung carcinoma.The ERK1/2 activation was statistically different (**p<0.01 by two tailed Fisher exact test) comparing all carcinoid tumors (2/20, typical plus atypical) with high-grade neuroendocrine carcinoma (11/19, SCLC plus NSCLC) .

Conclusion
Through the in vitro NED induction of NSCLC cell line NCI-H157, and in vivo correlative investigations in lung tumors with NEC, we discovered that NED of NSCLC was associated with the activation of Erk1/2-MAPK signal transduction pathway. To our knowledge, our findings are the first to describe the potential involvement of Erk/MAPK signal transduction pathway of NSCLC in the association with NED. Further investigation of the Erk/MAPK signal transduction pathway of NSCLC may yield discoveries in identifying specific molecular targets for the treatment of NSCLC with NED.

Background
Fibroblast growth factor (FGF) family consists of at least 23 polypeptides which have important functions in embryonic development, tissue repair, and tumorigenesis. Recent studies have shown that the activation of FGF9 is associated with pathogenesis of several cancers. In the lungs, FGF9 was highly expressed in adenocarcinoma by immunohistochemistry, and disturbing FGF9 function reduced the proliferation of lung adenocarcinoma. However, its clinicopathological and biological significance in non-small cell lung cancer (NSCLC) is unclear. The purpose of this study is to clarify the characteristics of FGF9-expressing NSCLC.

Methods
Using a cDNA microarray data set for 90 surgically resected NSCLC and corresponding non-tumorous lung tissue samples, we analyzed the relationship between the expression of FGF9 and their clinicopathological characterisitics. Also, we validated FGF9 expression by quantitative RT-PCR, and immunohistochemistry at protein level. Associations between FGF9 expression and clinicopathological factors were assessed by the χ2 test and Mann-Whitney U-test. Log-rank test was applied for survival analysis, and Kaplan-Meyer curve (Fig.1) was drawn. Multivariate analyses of the influence of variables on overall survival were performed with using Cox proportional hazards model.

Results
Nine out of 90 (10%) NSCLC had “high” FGF9 expression compared with corresponding non-cancerous lung tissues. Histologically, 5 out of 9 FGF9-high NSCLC were adenocarcinoma, and there was no squamous cell carcinoma. The correlations between FGF9 expression and sex, smoking history or clinical stage were not observed. On the other hand, postoperative recurrence rates and 3-year survival rates were 56% vs. 36% (p=0.033) and 44% vs. 88% (p=0.001) for FGF9-high vs. -low NSCLC patients, respectively. The overall survival of the patients with high-FGF9 expression was significantly worse compared that with FGF9-low NSCLC patients (p<0.001). At protein level, FGF9 expression (immunohistochemistry) was significantly higher in FGF9-high mRNA group compared with FGF9-low group. FGF9 was confirmed to be expressed in cancer cells in these resected NSCLC tissues, and localized in cytoplasm of the cells. Figure 1

Conclusion
FGF9 is highly expressed in a subset of lung adenocarcinoma, and is associated with poor prognosis.

Background
Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) are response predictive markers for the treatment of lung adenocarcinoma by using EGFR tyrosine kinase inhibitors. The stability of EGFR is dependent on its interaction with heat shock protein 90 (Hsp90), and Hsp90 inhibition may induce ubiquitin-mediated degradation of EGFR proteins. However, the mechanism of EGFR downregulation by HSP90 inhibition remains unclear. Here, we report a novel interaction between EGFR mutants and the E3 ubiquitin ligase C-terminus of Hsp70-interacting protein (CHIP), which can ubiquitinate and destabilize EGFR mutants

Methods
Cell culture, transfection and drug treatments NSCLC (A549, H358, H827, and PC9 cells) and Chinese hamster ovarian (CHO) cell lines used in the study were obtained from ATCC. Transfections of different DNA constructs were performed using Lipofectamine 2000 or PolyJet Western blot and immunoprecipitation Cells were then chilled on ice, washed twice with ice-cold PBS, and lysed in a buffer. Protein concentrations were determined using a Bradford assay kit. Equal amounts of protein (20 μg) in cell lysates were separated by SDS-PAGE, transferred to membranes, immunoblotted with specific primary and secondary antibodies, and detected by chemiluminescence with the enhanced chemiluminescence detection reagents. Cell viability assay After trasnfection with or without CHIP, cell metabolic activity was determined every 24 hours by using the CCK-8 (Dojindo, Gaithersburg, MD) colorimetric assay according to the manufacturer’s instructions. Cell cycle analysis by FACS Cells were seeded in 6-well plate and incubated overnight before transfection. After harvest at 48 h following transfection, cells were washed twice with pre-chilled PBS and resuspended in PBS (100 μL) at a concentration of 1 × 106 cells/mL. Cell cycle analysis was performed using the Coulter DNA PrepTM Reagents Kit. Xenograft studies A549 and PC9 cells were cultured as monolayers, trypsinized and resuspended in an equal volume of PBS at 5 × 107 per ml, respectively. BALB/c female nu/nu mice (5 weeks of age) were given bilateral subcutaneous injections of 5 × 106 (0.1 ml). Tumor growth was monitored twice each week by measuring the tumor size using calipers

Results
In CHO cells expressing either WT or mutant EGFR, the expression of the EGFR mutants L858R, G719S, and L747_E749del A750P was inhibited following overexpression of CHIP, whereas WT EGFR expression was not diminished in CHIP transfected cells. In a pull-down assay, CHIP interacted with G719S and del L747_E749del A750P, but not L858R, and simultaneously induced the ubiquitination and proteasomal degradation of its proteins. Similarly, the expression of mutant EGFR proteins in the non-small cell lung cancer cell line PC9 was also diminished by CHIP-mediated ubiquitination and degradation relative to WT EGFR. In EGFR mutant cell lines, CHIP inhibited cell proliferation as well as the depletion of phosphorylated Akt. In addition, CHIP inhibited the tumor growth of PC9 cells in xenografts of CHIP-overexpressing stable cell lines.

Conclusion
These data suggest that CHIP-induced ubiquitination of EGFR mutants may be responsible for EGFR regulation in lung adenocarcinoma and provide evidence that CHIP could be a novel E3 ligase for EGFR mutants.

Background
Our recent studies revealed that hepatoma-derived growth factor (HDGF) was highly expressed in non-small cell lung cancer (NSCLC) cells, when HDGF targeted-silenced by siRNA strategy, anchorage-independent growth of NSCLC cells can be significantly inhibited, the ability of NSCLC cells to invade across BD Matrigel membrane barrier can also be significantly inhibited, clearly suggested HDGF’s important role in cell growth, invasion and metastasis of NSCLC. However, the molecular mechanism is still undiscovered. Here, we reported that HDGF-ADAM9…may be the novel pathway for HDGF promotes growth and invasion of NSCLC cells. The expression of HDGF, ADAM9 in human resected NSCLC tissues will be detected, and the co-relationship, coordination of HDGF and ADAM9 will be evaluated to provide evidence, to elucidate the logic possibility of the HDGF-ADAM9…pathway.

Methods
siRNAs targeting HDGF were designed, used for specifically silencing HDGF in NSCLC cells. In vitro and in vivo cell growth and invasion assay were conducted. cDNA microarray and Western blot were used to explore the novel pathway, the possible molecular mechanism, by which HDGF promotes growth and invasion of NSCLC cells. Immunohistochemical SP method was used to detect the expression of HDGF and down-stream modulated genes in NSCLC tissues. Multivariate analysis and survival analysis were conducted to evaluate the clinical significance, the co-relationship, the possible coordination of HDGF and down-stream genes.

Results
Western blot revealed that HDGF protein expression in NSCLC cells were down-regulated more than 90% after silenced by targeted siRNA; anchorage-independent growth of A549 and H226 cells were inhibited significantly (P=0.000, 0.003, respectively); the ability of invading across BD Matrigel membrane barrier were inhibited significantly (P=0.004, 0.000, respectively). cDNA microarray revealed that a panel of genes, including AXL, GLO1, and ADAM9, were significantly down-regulated when HDGF was silenced by siRNA, suggested the possible pathways in which HDGF was involved. The expression of HDGF and ADAM9 were Immunohistochemically detected in 63 cases of completely resected stage Ⅰ NSCLC, found highly expressed in NSCLC when compared with normal control lung tissues (P=0.003, 0.001, respectively); highly expressed HDGF and ADAM9 were found correlated with significantly declined 5-year survival rates (P=0.009, 0.015, respectively). HDGF expression was revealed correlated positively and significantly with ADAM9 expression in these resected stage Ⅰ NSCLC (Pearson r=0.547, P=0.000).

Conclusion
These results clearly revealed that HDGF may promote cell growth and invasion of NSCLC cells via ADAM9 pathway, HDGF-ADAM9…should be a novel pathway of lung cancer invasion and metastasis. High expression of HDGF and ADAM9 correlate with shortened survival time, predict lower 5-year survival rates, suggesting that HDGF and ADAM9 are novel biomarkers for predicting prognosis in resected stage Ⅰ NSCLC, revealing their significance as novel molecular staging biomarkers. HDGF and ADAM9 may also become useful predictive biomarkers for the selection of adjuvant chemotherapy treatment of NSCLC to improve personalized postoperative treatment in resected stage Ⅰ non-small cell lung cancer. (This study was partly supported by grant from the Nature Science Foundation of Liaoning Province, China, No.20102285; and the Fund for Scientific Research of The First Hospital of China Medical University, No.FSFH1210).

Background
Microsatellite instability (MSI) can be analyzed by microsatellite markers consisting of mono-, di- and trinucleotide repeat sequences. In primary lung cancer, chromosomal instability including loss of heterozygosity (LOH) has been thought to play an important role in carcinogenesis. Although MSI was thought to be unrelated to lung carcinogenesis, recent findings suggest the role of tetranucleotide repeat sequence instability in prostate, skin, bladder and lung cancers. Unlike Bethesda panel markers (D17S250, D2S123, D5S346, BAT25 and BAT26) for Lynch syndrome, these tetranucleotide markers are not unified. Moreover, there are frequent elevated microsatellite alterations at selected tetranucleotides (EMAST) that are distinct from traditional MSI in several tumor types. EMAST in lung cancer has not been reported to date. We investigated EMAST in non-small cell lung cancers (NSCLCs) using selected tetranucleotide repeat markers (EMAST markers: D8S321, D20S82, UT5037, D8S348, D2S443, D21S1436, D9S747, D9S303, D9S304 and MYCL1) based on previous reports and analyzed their correlation to clinicopathological factors.

Methods
Sixty-five NSCLC tissue samples (19 squamous cell carcinomas, 39 adenocarcinomas, one adenosquamous cell carcinoma and six large cell carcinomas) without lymph node metastasis and preoperative chemotherapy or radiation therapy were obtained after resection from Yokohama City University Medical Center. Tumorous DNA was extracted by laser captured microdissection, and paired normal DNA was extracted from non-tumorous tissue or normal lymph nodes. Genotyping for EMAST determination was carried out using ten tetranucleotide repeat markers and five Bethesda panel markers.

Results
Using the ten tetranucleotide repeat markers, MSI was detected in 42 of 65 (64.9%) of the tissue samples. There was a higher rate of MSI at selected tetranucleotide markers than at Bethesda panel markers in the tissue samples (12.3%). The high EMAST group (MSI found at two or more markers) was significantly correlated to the presence of multiple malignant neoplasms (p=0.021) compared to the low EMAST group (MSI at none or one marker). We also examined a representative malignant neoplasm (renal pelvic carcinoma) complicated with NSCLC using EMAST markers. The tumor showed EMAST in the two markers (D2S443 and D21S1436).

Conclusion
Our results suggest that EMAST could participate in carcinogenesis of NSCLCs and lead to other malignant neoplasms.

Background
CADM1, a member of the immunoglobulin superfamily cell adhesion molecules, acts as a tumor suppressor in a variety of human cancers. CADM1 expression is frequently lost in various cancers and CADM1 inactivation by promoter methylation was observed in 44% of non-small cell lung cancer (NSCLC). In contrast, CADM1 is ectopically expressed in adult T-cell leukemia (ATL), conferring an invasive phenotype characteristic to ATL. Therefore, CADM1 plays dual roles in human oncogenesis: as a tumor suppressor in epithelial cancers and as an oncoprotein that promotes invasion in ATL cells. Here, we investigate the roles of CADM1 in small cell lung cancer (SCLC).

Methods
We studied splicing variant of CADM1 in 5 primary NSCLC tumors and a primary SCLC tumor, as well as 16 SCLC and 10 NSCLC cell lines. Expresssion and splicing variant of CADM1 was examined by RT-PCR, Western blotting, immunohistochemistry, and SSCP, respectively.

Results
Immunohistochemistry demonstrates that 10 of 35 (29%) primary SCLC tumors express CADM1 protein. Western blotting and RT-PCR analyses have revealed that CADM1 is significantly expressed in 11 of 14 SCLC cells growing in suspension cultures but in neither of 2 SCLC cells showing attached growth to plastic dishes, suggesting that CADM1 is involved in anchorage-independent growth in SCLC. Then, we demonstrate that SCLC expresses a unique splicing variant of CADM1 (variant 8/9) containing additional extracellular fragments corresponding to exon 9 in addition to variant 8, a common isoform in epithelia. Variant 8/9 of CADM1 is almost exclusively observed in SCLC and testis, although this variant protein localizes along the membrane and shows similar cell aggregation activity to variant 8. Interestingly, both variant 8/9 and variant 8 of CADM1 show enhanced tumorigenicity in nude mice when transfected into SBC5, a SCLC cell lacking CADM1. Inversely, suppression of CADM1 expression by shRNA reduced spheroid-like cell aggregation of NCI-H69, a SCLC cell expressing a high amount of CADM1. These findings suggest that CADM1 enhances the malignant features of SCLC, as is observed in ATL.

Conclusion
CADM1 could provide a novel molecular target for the diagnosis and growth suppression of SCLC cells.

Background
Lung carcinoma is the fifth common cancer affecting Saudi men. Recently, translocation of the anaplastic lymphoma kinase (ALK) gene is found to play a predictive role in adenocarcinoma tumor genesis. ALK gene rearrangement can identify patients with adenocarcinoma who are sensitive to ALK inhibitors. However, no data are available on the prevalence of ALK rearrangements changes in Middle Eastern population. Therefore, we carried out this study to evaluate the prevalence of ALK rearrangements in lung adenocarcinoma of Saudi patients.

Methods
ALK gene rearrangements were studied using fluorescence in situ hybridization (FISH) on 97 adenocarcinoma samples utilizing tissue microarray format. ALK gene translocations identified by BAC clone RP11-328L16 were studied by the break part probe from Vysis (Abott Molecular, Il, USA).

Results
Ninety seven (97) lung adenocarcinoma cases were evaluated. There were 3 cases exhibited ALK gene rearrangement (3%). All of these 3 cases was moderately differentiated adenocarcinoma. None of our cases showed signet cells or abundant intracellular mucin.

Conclusion
This is the first study that reveals frequency of ALK translocation in a ethically unique cohort of Saudi lung cancer patients. The findings of this study show that incidence of ALK adenocarcinoma is similar to the published western data and these patients can benefit from targeted therapy like Crizotinib- a dual ALK and MET inhibitor that has shown promising results in clinical trials.

Background
Epidermal growth factor receptor (EGFR) activating mutation is an important predictive biomarker of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC), while family history of cancer also plays an important role in the neoplasia of lung cancer. This study aimed to investigate the association between family history of cancer and EGFR mutation status in NSCLC population.

Methods
From February 2008 to May 2012, 538 consecutive NSCLC patients with known EGFR mutation status were included into this study. Amplification refractory mutation system (ARMS) method was used to detect EGFR mutation. The associations between EGFR mutation and family history of cancer were evaluated using logistic regression models.

Results
EGFR activating mutation was found in 220 patients and 117 patients had family cancer histories among first-degree relatives. EGFR mutation was more frequently detected in adenocarcinoma patients (p<0.001), never-smoker (p<0.001) and with family history of cancer (p=0.031), especially who had family history of lung cancer (p=0.008). In multivariate analysis, the association of EGFR mutation with family history of cancer also existed (p=0.027).

Conclusion
NSCLC Patients with family history of cancer, especially family history of lung cancer, might have a significantly higher incidence of EGFR activating mutation.

Background
Limited information exists regarding long non-coding RNAs (lncRNAs) which regulate gene expression during initiation and progression of human lung cancers. The present study was undertaken to utilize a novel in vitro model system to examine long non-coding RNA alterations mediated by cigarette smoke in normal respiratory epithelia and lung cancer cells.

Methods
Normal human small airway epithelial cells (SAEC) and cdk-4/h-TERT immortalized human bronchial epithelial cells (HBEC), as well as Calu-6 and H-841 lung cancer cells were cultured in the presence or absence of cigarette smoke condensate (CSC). Array and qRT-PCR techniques were used to assess lncRNA and gene expression profiles in CSC-treated or control cells. qRT-PCR techniques were used to evaluate expression levels of BC070487 and its target gene, ZNFX1 in primary lung cancers relative to paired normal lung tissues. Chromatin immunoprecipitation (ChIP), RNA-crosslink immunoprecipitation (CLIP), and methyl DNA immunoprecipitation (MeDIP) techniques were used to evaluate chromatin alterations within the ZNFX1 promoter region mediated by BC070487. MTS, invasion, and murine xenograft experiments were performed to examine proliferation, invasion and tumorigenicity of lung cancer cells following manipulation of BC070487 or ZNFX1 expression.

Results
Under relevant exposure conditions, CSC mediated a 4-7 fold up-regulation of BC070487, with a 4-8 fold down-regulation of ZNFX1 in normal respiratory epithelia and lung cancer cells. BC070487 epression correlated inversely with expression of ZNFX1 (BC070487: 1.38-10.31 fold up vs ZNFX1: 2.26-26.29 fold down) in primary lung cancers relative to adjacent normal lung tissues. Over-expression or depletion of BC070487 inhibited or enhanced expression, respectively, of ZNFX1 in normal respiratory epithelia and lung cancer cells. CSC as well as BC070487-mediated repression of ZNFX1 coincided with increased occupancy of EZH2, SUZ12 and BMI1, as well as increased H3K27Me3 and decreased H3K4Me3 levels within the ZNFX1 promoter region. CSC or over-expression of BC070487 enhanced binding of BC070487 with EZH2, SUZ12 and BMI1 proteins in SAEC and Calu-6 cells. CSC exposure mediated a significant increase in DNA methylation in one of two CpG islands spanning the ZNFX1 regulatory region in SAEC and Calu-6 cells; this phenomenon coincided with enhanced binding of BC070487 with DNMT3a and 3b, and recruitment of these de-novo DNA methyltransferases to the ZNFX1 regulatory region in these cells. Over-expression of BC070487 increased proliferation and invasion potential of lung cancer cells in-vitro, and enhanced growth of lung cancer xenografts in nude mice. Consistent with these findings, ZNFX1 depletion enhanced, whereas constitutive over-expression of ZNFX1 inhibited growth and tumorigenicity of lung cancer cells.

Conclusion
Cigarette smoke induces expression of LncRNA BC070487, which in turn represses ZNFX1 in normal respiratory epithelia and lung cancer cells. These findings provide a novel mechanism by which cigarette smoke mediates initiation and progression of lung cancers, and highlight the potential implications of smoking status of lung cancer patients at diagnosis or during therapy.

Background
In non small cell lung cancer (NSCLC) it is of vital importance to develop biomarkers that allow us to assign a more accurate prognosis for patients in early stages, so we can apply the right treatment at the right time, preventing that the cancer reaches advanced stages when the cure rate is close to zero.

Methods
In this study we have selected a set of 9 genes related to immunoregulation and inflammation processes to analyze their relative expression on mRNA by qPCR in lung tissue samples from patients with NSCLC and resectable stage. CCL2 and CD1c genes showed a significantly lower expression in tumor tissue versus healthy tissue. After that, we analyzed the correlation between expression levels of these genes and relevant clinicopathological variables in the patients. Poorly differentiated tumors showed higher expression levels of CD209, CCL2, LGALS1 and LGALS2, whereas in smokers an increased expression of galectin LGALS1 was documented. Finally, survival analysis was performed using the Kaplan-Meier method to assess the utility of genes as prognostic biomarkers in this disease.

Results
In the subgroup of patients with squamous cell histology, those with lower levels of expression of CCL22 showed a significant increase in overall survival (OS) and progression-free survival (PFS). In our cohort of patients with resectable NSCLC, survival analyses showed that elevated levels of galectin LGALS2 and CCL2 chemokine are associated with a better prognosis in overall survival (OS) and progression-free survival (PFS).

Conclusion
The data obtained in our study provide further evidence about the necessity of tumors to manipulate the microenvironment around them as a prelude to progress and invade tissues.

Background
Endobronchial dysplasia is a premalignant lesion commonly found in current and former smokers. Identifying and treating these lesions before they progress to lung cancer may improve survival. The iloprost chemoprevention trial demonstrated that supplementation with the prostacyclin analog, iloprost, reduced histologic dysplasia in former smokers (Keith et al. Cancer Prev Res. 2011). However, accurate detection of dysplasia requires invasive bronchoscopy to collect multiple endobronchial biopsy samples. This study is the first step in generating blood-based markers of dysplasia to identify individuals at high risk for developing lung cancer and who could benefit from chemoprevention treatment.

Methods
Baseline serum samples (n=70) collected from current and former smokers enrolled in the iloprost chemoprevention trial were analyzed with the SOMAscan proteomic platform, which measures 1129 proteins with a median limit of detection of 40 fM and 5% CV. To characterize dysplasia, 6 standardized endobronchial sites, as well as any others that appeared suspicious by either white light or autofluorescence visualization, were biopsied from each study participant and scored by expert pathologists. Samples were stratified by worst biopsy score (Max) for proteomic analysis. Biomarkers correlating with Max pathology score were identified using principal component analysis (PCA), a multivariate technique to identify correlated variables, and the univariate, non-parametric Kolmogorov-Smirnov test (KS test). Serum proteins correlating with pulmonary function were also analyzed.

Results
Six proteins correlated with the progression of Max pathology. The change in serum level of these proteins ranged from 14-50% when comparing the lowest (n=16) and highest (n=39) Max pathology score groups. The proteins function in neoplastic progression, cell adhesion, inflammation and metabolic regulation. The protein with the most significant change (FDR correct p value = 0.05) regulates plasma clearance of steroid hormones. The serum protein most strongly correlated with lung function in our study was VEGFR2, which mediates VEGF induced endothelial proliferation and is known to be reduced in the lungs of smokers and patients with COPD and emphysema.

Conclusion
Our preliminary results of serum biomarkers associated with preneoplastic dysplasia warrant further study. If validated, this serum-based test to identify individuals who may benefit from chemopreventive intervention could impact lung cancer survival. This work was supported by NCI grants CA 58187 and CA165780.

Background
Proteomic analysis of blood and tissue can reveal essential connections between the biochemical pathways altered in malignancy and tools for cancer diagnosis and treatment. The two major histologic subtypes of non-small cell lung cancer (NSCLC), adenocarcinoma (AD) and squamous cell carcinoma (SQ) differ in prognosis and optimal treatment. Targeting molecular pathways that drive malignancy has led to a paradigm shift in the development of specific treatments for patients based on their tumor mutation profile. We have conducted a comparative proteomic analysis of lung tumor histologic and driver mutation subsets to reveal biomarkers that link critical pathways for cell growth and survival to specific tumor phenotypes and genotypes.

Methods
We analyzed 68 NSCLC tumor and matched non-tumor tissue lysates (2 ug total protein/sample) with the SOMAscan proteomic platform, which measures 1129 proteins with a median limit of detection of 40 fM and 5% CV. The study consisted of 49 AD and 19 SQ tumors, 88% of which were Stage I or II. Somatic driver mutations were identified with multiplex PCR (SnapShot genotyping). Pairwise proteomic comparisons of tumor/non-tumor or AD/SQ tissue samples were performed using the Mann-Whitney test. The non-parametric Kruskal-Wallis test was used to discover differences among multiple pairwise driver mutation comparisons. Dependency network analysis was used to explore correlations enriched in tumor tissue vs non-tumor tissue. The statistical significance of the results was adjusted for multiple comparisons using false discovery rate (FDR) correction.

Results
Differences between tumor and non-tumor tissue were dominated by inflammatory, apoptotic and cell proliferation proteins. A total of 79 proteins were significantly different between AD and SQ at a 15% FDR. When compared to non-tumor levels, these proteins divided into 3 phenotypes: AD only (9 proteins), SQ only (19 proteins) or Both (51 proteins). Both refers to proteins that are tumor biomarkers in both AD and SQ and the protein levels are different between AD and SQ. The most common pattern was a progression in protein levels from non-tumor to AD to SQ, whether the pattern was higher or lower in tumor tissue. These proteins are members of cell proliferation and inflammatory pathways. This observation is consistent with the SQ only proteins, which are enriched for angiogenesis, cell proliferation and cell adhesion proteins. Driver mutation analysis revealed 5 inflammatory proteins that were higher in KRAS vs EGFR mutations and a TNF-alpha antagonist that was suppressed in EGFR mutants.

Conclusion
Unexpected findings that the AD proteome is closer to non-tumor lung tissue than SQ were revealed through broad proteomic profiling. Alteration in cell proliferation and inflammation pathways discovered in this study may lead to new insights in tumor biology and targeted therapeutics. This work was supported by a grant from the LUNGevity Foundation, NCI grant CA 58187 and Cancer Center Support Grant (P30CA046934).

Background
PRMT5 is an arginine methyltransferase that regulates cellular events by methylation of Arginine residues on histone and non-histone proteins. PRMT5 cooperates with chromatin remodelers and co-repressors to induce epigenetic silencing, and is overexpressed in several human cancers. It also has impact on cell growth and transformation pathways by modulation of E2F1, p53, EGFR and CRAF. We investigated the role of PRMT5 in lung cancer.

Methods
The expression pattern of PRMT5 using immunohistochemistry from resection specimens obtained with an IRB approved protocol. Immortalized lung cancer cells (A549, H719, H520 and H1299) were obtained from ATCC and normal bronchial airway cells (HPAEpiC, HBEpiC) were obtained from ScienCell. Western immunoblot and cell cycle analysis by flow cytometry was performed using standard techniques. A novel and specific inhibitor of PRMT5 developed by colleagues at OSU was applied to in vitro culture systems.

Results
The expression of PRMT5 was analyzed in 9 lung cancer resection specimens (3 adenocarcinoma, 3 squamous, 2 small cell and 1 large cell neuroendocrine cancer). All 9 showed diffuse cytoplasmic and variable nuclear PRMT5 expression. PRMT5 was also seen in reactive type 2 pneumocytes and respiratory epithelium adjacent to the tumors but not in alveolar parenchyma, fibroblasts or endothelial cells. Using Western immunoblot, PRMT5 is highly expressed in A549, H719, H1299 and H520 cells compared with normal cells such as HPAEpiC and HBEpiC. We knockdowned the expression of PRMT5 by lentiviral shRNAs and identified several clones with effective PRMT5 inhibition. Inhibition of PRMT5 was associated with slow cell growth. The cell proliferation decreased 58.4% and 62.3% in H1299 and A549 knockdown cells respectively. Interestingly, while H4R3 methylation was decreased with PRMT5 knockdown in A549 it was not in H1299 cells. We further analyzed the role of PRMT5 by using a specific inhibitor developed by researchers at OSU, CPD5. One of the PRMT5 specific marks of histone H4R3 methylation was inhibited and significant cell cycle changes were observed in A549 and H1299 cells treated for 24 hr and 48 hr. At 24 hours, the percentage of cells in G0/G1 was 57.1% (control) compared with 66.4% in CPD5 treated A549 cells, and 52.5% (control) compared with 70.9% in CPD5 treated H1299 cells. The expression of p21 was increased while cyclin E1 was decreased in A549 cells treated with CPD5. In contrast, the expression of cell cycle related proteins were not found in PRMT5 knock-down cells. Immunoprecipitation and other protein interaction techniques are in process to identify new PRMT5 targets.

Conclusion
In summary, PRMT5 is highly expressed in lung cancer cells compared to normal lung. A novel PRMT5 inhibitor and shRNAs can inhibit cell proliferation. Although PRMT5 inhibitor could induce cell death by cell cycle regulation and apoptosis, different pathways may be involved in PRMT5 knock-down cells and speaks to the complexity regulating mechanisms in different histological lung cancer patients. The differences in H4R3 methylation suggest that epigenetic repression may be dominant in some cancers, but that non-epigenetic mechanisms may be relevant in others. Further exploring of PRMT5 targets are ongoing.

Background
Although p53 alterations have been studied in pulmonary neuroendocrine tumors, these studies have only tested immunochemistry. Typical (TC) and atypical (AC) carcinoids manifest a heterogeneous Rb-protein pattern. Other markers (c-kit, c-met, and PDGFa/b) have unknown functions on pulmonary carcinoids.

Methods
We have evaluated 60 consecutive lung carcinoid samples, referred between 1989 to 2011 for surgical treatment with respect to assessed the profile markers involved in lung carcinoids (AC and TC) using qRT-PCR.

Results
All genes studied were positives in both TC and AC samples. When we compared TC and AC results, we found differences as follow: p53 was positive in 72.72% AC and 50% TC; Rb positivity was found in 63.64% AC and 70.45% TC; 81.82% AC and 59.1% TC were positive for c-met; c-kit was positive in 27.27% AC and 22.73% TC; 36.36% AC and 25% were positive for PDGFa; PDGFb was positive in 72.73% AC and 50% TC.Figure 1

Conclusion
From these results, it can be hypothetized that a progressive higher degree of malignancy from TC to AC is paralleled by alterations in profile markers of proliferation, apoptosis, and angiogenesis. The regarding markers (c-met, c-kit, and PDGFa/b) have unknown functions on lung carcinoids. Thus, our results are added to the evidence that the expression of neuroendocrine differentiation in pulmonary carcinoid tumors is controlled by multiple genetic determinants.

Background
Neuroendocrine (NE) lung tumors display a broad spectrum of morphological types, ranging from typical carcinoids (TC), with low malignant potential, to small cell lung carcinoma (SCLC) with the most rapid and disseminated growth. Two new additional NE forms are recognized: the intermediate grade, atypical carcinoid (AC), and high-grade large cell neuroendocrine carcinoma (LCNEC). It is believed that lung carcinoid tumors are derived from endocrine cells (PNECs/NEBs), regulated by ASCL-1, of the airway tract. Active Notch-1 signaling in the developing endoderm inhibits endocrine differentiation via supression of ASCL-1. The aim of this study was to evaluate expression of developmental transcription factors and Notch signaling components in human lung carcinoids samples.

Methods
Sixty formalin-fixed paraffin embedded human lung carcinoid tumor (typical and atypical) samples were analyzed by IHQ and qRT-PCR. Tissue arrays were used in order to perform the IHQ. We assessed the profile of markers involved in lung carcinoid tumors using qRT-PCR with SYBR Green.

Results
In the present work we found that Notch pathway components have variable levels when we compare typical vs atypical histology in a set of human lung carcinoids. The heatmap (Figure) shows the presence of a group of genes with low expression by IHQ in almost all samples analyzed (NOTCH1, NOTCH4, DLL3 and DLL4). The remaining genes (NOTCH2, NOTCH3, JAG1, DLL1 and HES5) show clear differences in expression between samples.Figure 1

Conclusion
The molecular studies on human lung carcinoid samples indicate that the Notch signaling pathway, and the basic Helix-Loop-Helix (bHLH) transcription factors, including ASCL1, might regulate the neuroendocrine phenotype in human lung carcinoids. The Notch signaling pathway components and their transcription factors may be significant regulators of neuroendocrine indifferentiation in human lung carcinoid tumors.

Background
Although p53 alterations have been studied in pulmonary neuroendocrine tumors, these studies have only tested immunochemistry. Typical (TC) and atypical (AC) carcinoids manifest a heterogeneous Rb-protein pattern. Other markers (c-kit, c-met, and PDGFa/b) have unknown functions on pulmonary carcinoids.

Methods
We have evaluated 60 consecutive lung carcinoid samples, referred between 1989 to 2011 for surgical treatment with respect to assessed the profile markers involved in lung carcinoids (AC and TC) using qRT-PCR.

Results
All genes studied were positives in both TC and AC samples. When we compared TC and AC results, we found differences as follow: p53 was positive in 72.72% AC and 50% TC; Rb positivity was found in 63.64% AC and 70.45% TC; 81.82% AC and 59.1% TC were positive for c-met; c-kit was positive in 27.27% AC and 22.73% TC; 36.36% AC and 25% were positive for PDGFa; PDGFb was positive in 72.73% AC and 50% TC.Figure 1

Conclusion
From these results, it can be hypothetized that a progressive higher degree of malignancy from TC to AC is paralleled by alterations in profile markers of proliferation, apoptosis, and angiogenesis. The regarding markers (c-met, c-kit, and PDGFa/b) have unknown functions on lung carcinoids. Thus, our results are added to the evidence that the expression of neuroendocrine differentiation in pulmonary carcinoid tumors is controlled by multiple genetic determinants.

Background
Lung cancer is the leading cause of cancer-related deaths worldwide, with poor survival largely attributed to late stage of disease at diagnosis and frequent metastasis. Typically, tumor cells colonize the regional lymph nodes before spreading to distant sites, significantly dampening prognosis. The presence and number of affected nodes can be detected by routine imaging procedures, but occasionally, additional affected nodes are not detected until time of surgery, which impacts staging. The identification of biomarkers that stratify node positive (NP) and node negative (NN) patients could improve detection of aggressive disease, while a deeper understanding of the underlying biology of NP lung cancer could lead to the design of novel therapeutic interventions. MicroRNAs (miRNAs) are major regulators of gene expression that control a wide range of cellular processes, and have been shown to be excellent biomarker candidates due to their stability in biofluids. The role of miRNA deregulation in NP lung cancer is poorly understood; therefore, we sought to compare miRNA expression levels of lung adenocarcinoma (AC) cases with and without nodal involvement in order to identify miRNAs associated with tumor aggressiveness. We hypothesize that a subset of miRNAs are specifically deregulated in NP AC, representing potential biomarkers, and that identification of miRNA-mRNA interaction networks will shed light on the biological mechanisms of tumor spread that may be therapeutically exploited.

Methods
A panel of 45 NN and 28 NP primary AC tumors were collected along with paired adjacent non-malignant tissues. All samples underwent expression profiling of miRNA (Illumina GAIIx small RNA sequencing) and mRNA (Illumina microarray). NN and NP groups were analyzed separately as follows: matched tumor and normal miRNA normalized read count comparisons were performed (Wilcoxon Signed-Rank test, Bejamini-Hochberg corrected p<0.05), and miRNAs that were significantly deregulated in tumors were further investigated. miRNA fold changes (FC) were calculated on a case by case basis, and FC values were then compared between groups (Mann Whitney U Test p<0.05). miRNAs that (i) displayed a minimum 2-fold frequency of disruption >50% of NP cases, (ii) had a median FC>2 in the NP group, (iii) had a Signed-Rank corrected p<0.05, and (iv) had a MWU p<0.05 were considered to be NP-specific. Target prediction analysis of these miRNAs was then performed, and mRNA expression data was utilized to ensure predicted target expression was anti-correlated with miRNA expression. Candidate targets were input into Ingenuity Pathway Analysis to determine pathways implicated in NP AC.

Results
Fourteen miRNAs were NP-specific according to our criteria. These miRNAs included those previously associated with biology and metastasis of epithelial cancers, as well as miRNAs novel to lung AC. Target analysis implicated several pathways and functions previously associated with a metastatic phenotype such as the TGFβ pathway.

Conclusion
These results indicate that miRNA expression differs between NP and NN lung AC, further demonstrating the involvement of miRNAs in the regulation of the metastatic process. The role of these miRNAs as prognostic markers and serum biomarkers, as well as the mechanism of action of these miRNAs in AC biology must be further explored.

Background
CXCR4 is a chemokine receptor that activates signaling pathways responsible for trafficking of cells to sites of inflammation, retention of stem cells in the bone marrow and plays a role in migration and metastatic processes in tumour cells. Previous work by the Bebb lab demonstrated that female stage IV NSCLC patients with high CXCR4 expression have significantly worse survival compared to their low CXCR4 expressing counterparts. Recently, a positive regulatory loop was discovered linking CXCR4 and ER signaling pathways leading to increased gene expression of SDF-1 and other ER dependent genes. We explored the relationship between CXCR4 and ER/PR signaling in NSCLC cell lines, and assessed the expression of ER/PR in stage IV tissue samples to determine if ER/PR could be contributing to the observed gender dependent difference in clinical outcome seen in CXCR4 high expressers.

Methods
Two male and two female NSCLC cell lines were characterized for ER and CXCR4 by western blot. Activity of the CXCR4 and ER pathways in each cell line after stimulation with SDF-1 and estradiol (E2), respectively, were assessed by western blot of downstream signaling targets ERK, AKT, phospho-ER and PR. Cross activity of the CXCR4 and ER pathways was examined by stimulating cells with E2 and quantifying SDF-1 and PR mRNA by rtPCR. Tumor specimens from stage IV NSCLC patients (Glans-Look Lung Cancer Database) previously assessed for CXCR4 were analyzed for ERα and PR expression by quantitative immunohistochemistry (IHC) using the HistoRx AQUA® platform. Correlation between CXCR4 and ER/PR expression was assessed using Pearson’s rank correlation, and associations between marker expression and clinical factors/overall survival were assessed using a Cox proportional hazards model.

Results
All cell lines expressed varying levels of CXCR4, ERα and ERβ, and were responsive to both SDF-1 and E2 stimulation. Preliminary data suggests that activation of AKT and ERK after stimulation with SDF-1 occurs more rapidly in the male cell lines than in the female cell lines, and that there were differences in SDF-1 gene expression after E2 stimulation between the male and female lines. Other experiments are ongoing and full data will be presented. ER and PR AQUA scores were obtained for 131 patients (63 females and 68 males). There were weak positive correlations between both ER/CXCR4 (r = 0.153) and PR/CXCR4 (r = 0.278), however, only the correlation between CXCR4 and PR was significant (p = 0.002). Similarly, only PR expression was significantly associated with overall survival in the model (p = 0.03)(HR = 1.46). More detailed sub-group analyses further exploring the association with gender is ongoing, and will be presented.

Conclusion
The CXCR4 and ER pathways are active in NSCLC cell lines. There appears to be differences in the responsiveness of male and female NSCLC cell lines to CXCR4 and ER pathway activation. ERs and PRs are present in stage IV NSCLC tissue samples, and are associated with both CXCR4 expression and overall survival in our patient cohort. The full implication of these observations is still being investigated and further analyses will be presented.

Background
Tumor microenvironment plays an important role in regulating cell growth and metastasis. Recently, we developed an ex vivo lung cancer model (4D) that forms perfusable tumor nodules on tissue that mimics human lung cancer histopathology and protease secretion patterns better than tumor cells grown on a petri dish (2D). In this study, we aim to determine if the gene expression profile of cells grown in the model (4D) is a better predictor of survival in lung cancer patients compared to the gene expression profile of cells grown on a matrigel (3D).

Methods
We compared the gene expression profile (Human OneArray v5 chip) of A549 cells, human lung cancer cell line, grown on petri dish (2D) against the same cells grown in the tissue of our ex vivo model (4D) and performed gene ontology (GO) analysis. We obtained gene expression data of A549 cells grown on petri dish (2D) and matrigel (3D) from GEO Accession No. GSE17347 and compared the gene expression profiles. We analyzed the differential gene signature for 4D and 3D in human lung cancer database for survival.

Results
Expression array analysis showed that there were 2954 gene probes differentially expressed between 2D and 4D. The analysis showed up-regulation of genes associated with mesenchymal cells (CDH2 and VIM). GO analysis showed up-regulation of several genes associated with extracellular matrix (MMP1, MMP9, MMP10, COL4A1, COL5A1), polarity (DLX2, GLI2, HOXD10, HOXD11), and cell fate and development (PPM1A, SALL1, SOX4, ZEB2, JAG1, SOX2, TP63). Moreover, expression array analysis of the 2D and 3D showed 1006 genes that were differentially expressed, with only 36 genes (4%) having the same expression pattern as differential expression between 2D and 4D. There was no difference in expression of genes associated with mesenchymal features (CDH2 and VIM) between 2D and 3D. Finally, the differential gene expression signature between 2D and 4D correlated with poor survival in patients with lung cancer (n = 1492, Log rank p = 1.5 x 10[-7]) while the differential gene expression signature between 2D and 3D correlated with good survival in patients with lung cancer (n = 1492, Log rank p = 1.7 x 10[-9]).

Conclusion
The genes necessary to form a perfusable nodule in the 4D model are the genes that are important in cell-matrix interaction, polarity and cell fate, which are lacking in a 2D model. The gene expression signature in the 4D model correlates with poor survival in lung cancer patients, which may be due to presence of more cells with mesenchymal features in the 4D model compared to 2D culture. On the other hand, the tumor cells grown on 3D model show the genes important in tumor cell interaction with matrix without difference in genes identify cells with mesenchymal features. Thus, there may be fewer cells with invasive properties, which may explain the good survival in lung cancer patients. The 4D ex vivo lung cancer model may be a better mimic of the natural progression of tumor growth in lung cancer patients compared to 3D model.

Background
Circulating tumor cells are tumor cells found in the vasculature or lymphatics of cancer patients that are thought to be responsible for metastasis. Recently, we developed an ex vivo 4D lung cancer model that forms perfusable tumor nodules. We have found that there are live tumor cells in the circulation in the model (CTCs). In this study, we aim to determine (i) the characteristics of these cells in vivo and (ii) whether the gene expression profile signature of these cells predicts survival in patients with lung cancer.

Methods
We used 393P and 344SQ mouse lung cancer cells for the in vivo experiment. The 393P cells that were grown on petri dish (2D) did not form metastatic lesions when injected into the flank of 129sv mice while 344SQ 2D cells did form metastatic lesions. Both cells were grown in the 4D model. We measured the size of the tumors and the number of CTCs for 14 days. We analyzed the mesenchymal characterisitics of 2D vs CTCs. We injected the CTCs in the tail vein of the 129sv mice and determined if they formed metastasis. We used A549 cells for the gene expression experiment. We placed the A549 2D cells in the 4D model and measured the size of the tumors and the number of CTCs. We compared the gene expression profile (Human OneArray v5 chip) of A549 2D against the A549 CTCs. We analyzed the differential gene signature in human lung cancer database for survival.

Results
All of the cell lines formed perfusable lung nodules in the 4D model by day 2 and formed circulating tumor cells starting day 5. The 344SQ cells grew faster (p = 0.01) and formed more circulating tumor cells (p = 0.8) compared to the 393P cells initially but by day 14 there was no significant difference in tumor size (p = 0.8) and number of circulating tumor cells (p = 0.7). The 393P CTCs were less mesenchymal (lower CDH2 (p < 0.0001), ZEB1 (p = 0.001) and VIM (p < 0.0001)) compared to 393P 2D while 344SQ CTCs were more mesenchymal (higher CDH2 (p < 0.0001), ZEB1 (p < 0.0001) and VIM (p < 0.0001)) compared to 344SQ 2D. When 393P CTCs were injected into the tail vein of 129sv mice, they did not form metastatic lesions while 344SQ CTCs formed metastatic lesions in the lung. Gene expression analysis of A549 2D compared to A549 CTCs showed that there were 2504 gene probes that were differentially expressed. This signature correlated with poor survival in patients with lung cancer (n = 1492, Log rank p = 2.6 x 10[-5]).

Conclusion
The circulating tumor cells that were isolated from the ex vivo 4D lung cancer model mimic the pattern of metastasis in vivo. Moreover, the circulating tumor cell signature correlates with poor survival in patients with lung cancer. The circulating tumor cells that are isolated in the ex vivo 4D lung cancer model may be the cells responsible for metastasis.

Background
We previously reported that lung adenocarcinoma cell lines could be classified into two groups according to gene expression patterns (Matsubara et al. Am J Pathol 2010). Group 1 cell lines expressed bronchial epithelial markers (CK7, MUC1, TTF-1) at high levels, while Group 2 cell lines showed epithelial-mesenchymal transition (EMT) phenotype (low-E-cadherin and high vimentin) and reduced expression of the bronchial epithelial markers. All cell lines mutated for EGFR, MET, HER2, and a cell line, LC-2/ad, recently found to harbor CCDC6-RET fusion (Matsubata, et al. J Thorac Oncol 2012), belonged to Group 1, and expressed EGFR, MET, HER3 at high levels, while KRAS-mutated cells lines were equally distributed in the two groups. Also, these two groups showed distinct sensitivity for gefitinib and cisplatin, respectively (Matsubara et al. J Thorac Oncol 2010, 2012). Here we tested the relevance of this classification for morphology and cancer-stromal interaction in 3 dimensional culture and mouse xenograft.

Methods
The 40 lung carcinoma cell lines consisted of 35 adenocarcinomas, 4 large cell carcinomas, and 1 adenosquamous carcinoma. These cells were plated onto Matrigel with or without lung-derived fibroblasts. Cell lines were also allowed to form spheroid in suspension culture, and then plated into collagen gel with or without lung-derived fibroblasts. To form xenograft tumors, cell lines were injected subcutaneously into NOD/SCID mice and allowed to form tumors, and resulting tumors were subjected to histologic analysis at 4-8 weeks.

Results
We demonstrated that on Matrigel essentially all Group 1 cell lines formed round spheroid, while Group 2 cell lines formed either round, stellate, or grape-like spheroid. Round spheroid forming cells remained non-invasive in monoculture, but upon co-culture with fibroblast, they acquired the ability to invade into Matrigel or collagen gels. In contrast, Groups 2 cell lines that formed stellate or grape-like spheroid showed invasive growth in monoculture, and co-culture with fibroblast seemed to enhance the invasive growth in some, but not all Groups 2 cell lines. Thus, Group 1 cell line required stromal fibroblast for invasion, while most Group 2 cell lines are endowed the ability to invade the stroma on their own. Consistent with this notion, when subcutaneously injected into immunocompromized mice, essentially all tumors derived from Group 1 cell lines were enriched with stromal fibroblast, while most tumors derived from Group 2 cell lines exhibited medullary histology with scanty stromal fibroblast.

Conclusion
Collectively, the data suggest that our two-way classification based on gene expression pattern has relevance for genetic, morphologic, and biologic properties of lung adenocarcinomas.

Background
The molecular screening is a key step to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy. We investigated the clinical relevance of targeted next generation sequencing (NGS) as a molecular screening for EGFR-TKI therapy in never-smoking lung adenocarcinoma (NSLA).

Methods
We obtained DNA from 48 NSLA received gefitinib or erlotinib for their recurrent disease after surgery. The Sanger sequencing and peptide nucleic acid clamp polymerase chain reaction (PCR) were used to analyze EGFR, KRAS, BRAF and PIK3CA mutations. We analyzed ALK, RET and ROS1 rearrangements by fluorescent in situ hybridization or reverse transcriptase-PCR and quantitative real-time PCR. Finally, Ion Torrent NGS was performed in 31 cases harboring only EGFR exon 19 deletions (19DEL) or L858R mutation or none of above mutations.

Results
After excluding mutations other than EGFR 19DEL or L858R, samples were divided into 4 groups; 1) responders to EGFR-TKIs with only 19DEL or L858R (n=15); 2) primary resistance to EGFR-TKI with only 19DEL or L858R (n=4); 3) primary resistance to EGFR-TKI without any mutations (n=8); 4) responders to EGFR-TKI without any mutations (n=4). All conventionally detected mutations were confirmed with NGS. Additionally uncovered predictive mutations include; one PIK3CA E542K and one BRAF in group 2; two KRAS (G12V and G12D), one PIK3CA E542K and one concomitant PIK3CA and EGFR L858R in group 3; one EGFR 19DEL in group 4. Newly detected mutations were validated by Sanger sequencing.

Conclusion
Targeted NGS provided more accurate and clinically useful molecular classification of NSLA. It may improve personalized therapy for individual patients.

Background
Figure 1During thoracoscopic surgery, rounded cotton-tip dissectors (Blunt Cherry Dissectors[®]) have been used to maintain the position of the lungs. However, moistening of cotton tips due to blood or pleural effusion often makes it difficult for surgeons to maintain the lung position when using this equipment. Therefore, we developed a polygonal-shaped tip dissector using porous polypropylene, called the Rotary Dissector, in order to achieve a greater amount of frictional force against the lung. In the present study, we assessed the difference in the rotational frictional force between the Blunt Cherry Dissector and Rotary Dissector, as well as the usefulness of the Rotary Dissector in thoracic surgery.

Methods
The rotational frictional force of the Rotary Dissector and the Blunt Cherry Dissector was estimated using a gel. We measured the weight and volume of gel that was scooped and pushed out in one direction by each dissector. Furthermore, we used the newly developed Rotary Dissectors for video-assisted thoracic surgery (VATS), and assessed the usefulness of this equipment.

Results
The weight and volume of gel pushed out was 1.14 g and 948.4 mm[3] with the Rotary Dissector and 0.34 g and 270.6 mm[3] with the Cherry Dissector, respectively. Moreover, the Rotary Dissector had 3 times the frictional force compared to that of the Cherry Dissector. During VATS, we used the Rotary Dissector and successfully detached the pleural adhesion without causing any lung injuries and could maintain the lung position to obtain a clear surgical field.

Conclusion
Thus, we noted that Rotary Dissectors can yield a greater amount of frictional force as compared to conventional dissectors, and can be safely used in VATS.

Background
Next-generation sequencing (NGS) studies in cancer are often limited by the amount, quality and purity of tissue samples obtained from patients. In this situation, primary xenografts have proven useful in providing preclinical models. Although xenograft lines are maintained in immunodeficient mice, we and others have shown that they retain important characteristics that are irreversibly lost in cell culture. Since the stromal component of xenograft tumors is derived from the host, the presence of mouse DNA and RNA has the potential to limit the use of these models for next-generation sequencing (NGS) analysis.

Methods
We prospectively addressed this question in an established primary xenograft model of small cell lung cancer (SCLC), a malignancy that is almost always diagnosed using small biopsies or needle aspiration cytology. We first developed an in-silico strategy that separates human and mouse reads with at least 97% accuracy. We then compared NGS data from a series of primary xenograft models with clonally derived, stroma-free cell lines, and with published datasets derived from the same models.

Results
Starting with the NCI-H209 cell line as a reference sample, we show that low coverage whole genome analysis demonstrated remarkable concordance between published genome data and internal controls, despite the presence of mouse genomic DNA. NGS analysis of exon-capture DNA revealed that this enrichment procedure was highly species-specific, with less than 4% of reads aligning to the mouse genome. Human-specific expression profiling with RNA-Seq replicated array-based gene signatures, whereas mouse- transcript profiles correlated with published datasets from human cancer stroma.

Conclusion
Primary xenograft models may therefore be a useful NGS platform for cancers where tissue samples are limiting.

Background
Smoking-mediated lung cancer will cause at least 134,000 deaths in the USA this year. Even when detected at the earliest stages (stage IA and IB), five year survival is just slightly more than 50%. Chronic pulmonary inflammation is an established risk factor for lung cancer. Increased lung tumor macrophage infiltration is associated with poor prognosis, and murine lung tumor models exhibit increased macrophage infiltration. We hypothesize that chronic cigarette smoke exposure causes changes in inflammatory cell numbers and phenotypes creating a permissive environment for tumor formation. Chemopreventive agents such as the prostacyclin analog iloprost, may mitigate these changes.

Methods
FVB mice were given control AIN-76A chow or AIN-76A with 0.015% pioglitazone two weeks prior to smoke exposure. Mice were exposed to 70-90 mg/m[3] particulate level cigarette smoke for 6 hours/day, 5 days/week. Lungs were harvested 1 and 22 weeks after smoke exposure and after 22 weeks smoke/20 weeks ambient air (standard protocol for tumor induction). Bronchoalveolar cells were collected for qRT-PCR analysis of macrophage markers. Tumors were harvested and dissected from uninvolved tissue. Uninvolved lung tissue was digested and immune cell content analyzed by flow cytometry.

Results
Cigarette smoke exposure induced changes in macrophage phenotype after 1 week as indicated by CD11b exression (flow cytometry) and phenotypic markers (qRT-PCR). Macrophage infiltration increased in 22 week smoke/20 week ambient air exposed mice. Neutrophil numbers were increased at one week of smoke exposure. CD4 and CD8 positive T cell numbers decreased during smoke exposure but returned to control levels after smoking cessation. Smoke exposure differentially affected macrophage programming at each time point.

Conclusion
Cigarette smoke exposure affects pulmonary inflammatory cell numbers and function as early as 1 week after smoking initiation. Some of these changes are transient (increases in neutrophil numbers), while others are consistent but return to normal after smoking cessation (decreases in T cell populations). Smoke exposure resulted in early functional effects on macrophages and alveolar macrophage infiltration was still increased 20 weeks after smoking cessation, indicating that the effects of cigarette smoking on innate immunity are long lasting.

Background
Background: recent evidence shows a role for immunotherapy via checkpoint antibodies in a subgroup of patients with non-small cell lung cancer (NSCLC). We have recently published the complex interplay between tumors and the immune system[1]. The modulating effect of the tumor on the immune system, mainly immunosuppressive, is challenging. MDSC are described to play a major role in this tumor associated immune suppression by inhibiting cytotoxic Tcell function. The aim of the present study is to relate the number of MDSC to two well-known prognostic factors in NSCLC: stage of the disease and WHO performance status.

Methods
Methods: MDSC were determined in peripheral blood mononucleair cell fractions of 195 treatment-naive NSCLC patients (185 stage IV (aNSQ), 10 stage I-III(eNSQ), and 20 healthy controls (HC)). WHO performance status was determined by experienced investigators prior to the start of treatment. In this abstract the relation between clinical data and polynucleair MDSC are presented. Flowcytometric analysis was performed within 5 hours after the blood was drawn. MDSC were characterized as CD16low,CD11b+,CD14-,HLA-DR-,CD15+, and CD33+.

Results
Results: A large variation in the number of MDSC was observed in patients with NSCLC, also in the same stage of the disease and WHO performance status. In a subgroup of patients with eNSQ high MDSC levels were found, but also in patients with aNSQ low levels of MDSC were found. For the group as a whole, however, an increased number of MDSC was found in patients with aNSQ compared to HC and eNSQ (p<.001, in both comparisons). Comparison of the limited number of patients with of eNSQ and HC no statistical difference was found but there is a tendency for an increase in the number of MDSC per stage of NSCLC. The number of MDSC increased with worse WHO performance status ( p=0.05 between 0 and 2). Whether the number of MDSC has prognostic or predictive value to response to chemotherapy in all patients is subject of current research. Follow up data in all patients with response to chemotherapy, progression free survival and overall survival are at present collected.

Conclusion
Conclusion: Peripheral blood MDSC levels are increased in patients with aNSQ compared to eNSQ and HC. MDSC are increased in patients with worse WHO performance status. The number of MDSC shows large variation between patients with similar stage of the disease, showing the complex interplay between tumor and immune system. 1 Aerts JG, Hegmans JP, Cancer Research 2013

Background
Immune modulatory strategies directed against CTLA4 (Cytotoxic T-Lymphocyte Antigen 4), PD1(Programmed cell Death 1) and PD-L1 (Programmed cell Death 1 Ligand 1) either in monotherapy or in combination with chemotherapy and radiotherapy offer great promise as novel treatments against lung cancer. Its outcome and therapeutic ratio may depend on the expression of the target molecules in tumours and the surrounding non-malignant lung tissue. We here report the expression of CTLA4, PD1 and PD-L1 in primary resected NSCLC and in the surrounding non-malignant lung.

Methods
RNA sequencing was performed on tumor tissue and normal lung tissue from resection specimens of NSCLC patients. The association between CTLA4, PD1 and PD-L1 expression levels and histology, gender, age, smoking status (current smoker vs. smoking cessation of at least one year), COPD and CRP blood levels was calculated both in the primary tumour and the normal lung. Results are expressed as mean +/- SD and range. Means were compared using Wilcoxon’s signed rank test for the distributions were non-parametrical. P-values <0.05 were considered significant.

Results
Fourteen patients were studied, 11 males and 3 females, with a mean age of 64.6 +/- 10.0 years (41-79). All patients had a smoking history: 6 were current smokers, 8 former smokers. COPD status: 8 no COPD, 1 GOLD class I, 3 GOLD class II, 2 GOLD class III. Pre-operative CRP (mg/dL): 15.0 +/- 2.9 (0.20-54.7). Seven patients had squamous cell cancer, 7 adenocarcinoma. PD-L1 expression in the lung was 3833 +/- 787 (372-5072), PD-L1 in the tumour 2595 +/- 1657 (788-6689), with a tumour/lung ratio of 0.67 +/- 0.37 (0.21-1.32). PD-L1 squamous vs. adenocarcinoma was 1840 +/- 1012 vs. 3350 +/- 1896, p<0.001. PD-L1 in the lung of ex-smokers vs. current smokers: 4136 +/- 811 vs. 3430 ± 591, p<0.001. These associations were not found for CTLA4 and PD1 receptor: CTLA4 tumour squamous vs. adenocarcinoma: 100.9 +/- 46.3 vs. 95.3 +/- 73.6, p=0.86; CTLA4 lung smokers vs. ex-smokers: 78.4 +/- 41.3 vs. 107.0 +/- 75.1, p=0.75. PD1 receptor tumour squamous vs. adenocarcinoma: 591 +/- 244 vs. 689 +/- 263, p=0.31 PD1 receptor in the lung of smokers vs. ex-smokers: 599 +/- 278 vs. 695 +/-216, p=0.46. No associations between CTLA4, PD1 and PD-L1 in the tumour or the lung and gender, age, COPD and CRP blood levels were found.

Conclusion
RNA sequencing is a useful method to dissect relevant molecules in the tumour and the lungs. Our results show more PD-L1 expression in adenocarcinoma than in squamous cell cancer, more PD-L1 expression in the non-malignant lung of ex-smokers than in current smokers and reduced PD-L1 in tumours compared to adjacent lung tissue.

Background
Cancer metastasis is the main cause of cancer-related deaths. Metastatic cancer cells spread through blood vessels where they constantly interact with platelets and leukocytes, forming tumor microemboli and thereby protected from otherwise rapid elimination from host immune defense cells such as NK cells. Platelets are known to be prometastatic and pro-angiogenic. They release platelet-derived growth factors, chemokines, various adhesion molecules and coagulation factors that effectively promote cancer spreading and metastasis. We previously demonstrated that many metastatic cancer cells acquire immune-resistance by aberrant expression of KIRs on their surface. We showed that cancer cells with aberrant expression or with forced ectopic expression of KIRs are more resistant to NK killing than those with null or low KIR expression. Pre-blocking with anti-KIR antibodies effectively reverses their sensitivity to NK killing. Here we report that KIR-expressing cancer cells interact strongly with platelets leading to increase NK tolerance compared to cancer cells with null or low KIR expression. These data support a novel mechanism of immune-tolerance due to aberrant KIR expression on metastatic cancer cells.

Methods
DNA microarrays were performed on metastatic lung cancer cells re-derived from orthotopic human metastatic lung tumors in athymic nude rats. Aberrant expression of KIR genes was detected and verified with immunohistochemistry and flow cytometry. For ectopic expression, lung adenocarcinoma parental cells (H2122-GFP) were transfected with KIR2DL1 (LL454) and/or KIR3DL1 (LL456) plasmids. Stable transformants were enriched by cell sorting. Binding of these cancer cells with differential KIR expression with human platelets, pre-labeled with anti-CD41-APC antibodies, were analyzed by flow cytometry. NK killing of GFP-tagged cancer cells with differential KIR expressions in the presence and absence of human platelets was accessed with fluorescence intensity in a fluorescent plate reader.

Results
Using in vitro cytotoxic assays, we found that KIR expression or platelet coating on cancers clearly increased their resistance to NK cell killing when compared with parental cells. Interestingly, we found that platelet coating on those metastatic cancer cells with high aberrant KIR expression increased their IC50 values by 6 to 14 folds respectively when compared with parental cells, while platelet coating on those cells with forced KIR expression also increased their IC50 values by 9 to 12 folds respectively. Our observation shows that NK tolerance correlates positively with platelet coating and the levels of KIR expression on the cancer cells (with correlation coefficient = 0.9 to 0.98), and that the NK resistance is the highest when both KIR aberrant expression and platelet coating are present on the cancer cells.

Conclusion
We discovered a novel mechanism of immune resistance to NK cells due to aberrant expression of KIRs on cancer cells. Aberrant KIR expression on cancer cells enhanced their interaction with platelets leading to further increase in NK tolerance than those cells with null or low KIR expression. This observation suggests that anti-platelet antagonists and anti-KIR antibodies may have clinical potential for the treatment or prevention of metastatic and immune resistant cells.

Background
Malignant pleural mesothelioma (MPM) is an aggressive tumour linked to chronic inhalation of asbestos fibers, with poor prognosis and increasing incidence in industrialized countries. Currently available chemotherapeutic regimens achieve a median progression free and overall survival of 6 and 12 months respectively. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family, which induces cancer cell death through extrinsic apoptotic pathway, while sparing normal cells. The aim of this study was to investigate the antitumor activity of recombinant human Apo2L/TRAIL (Dulanermin, Amgen;Genentech) in combination with antifolate-based chemotherapy in MPM cell lines and in vivo preclinical mouse model.

Methods
In vitro apoptosis assay was performed using Annexin-V-Fluos staining kit (Roche) according to the manufacturer’s instructions. Epithelioid (ZL55) and sarcomatoid (ZL34) cell lines were treated with carboplatin plus pemetrexed (CP), dulanermin (D) or CPD and then Annexin V positive cells were detected by flow cytometry using a FACSCalibur apparatus and CellQuest software (BD Biosciences). p53 protein expression level was detected by western blot analysis using a specific antibody. TRAIL death receptors (DR4 and DR5) and decoy receptors (DcR1 and DcR2) expression levels were assessed by flow cytometric analysis. In vivo experiments were performed in 30 SCID male mice, implanted subcutaneously in the right flank with 2x10[6 ]ZL55 cells suspended in 0.1 ml volume of RPMI, aged 6 weeks. When tumor volume reached 50-150 mm[3], the mice were randomized in 4 treatment groups: 1) not treated (NT); 2) C (75 mg/Kg day 1) plus P(100 mg/Kg day 1); 3) D (60 mg/Kg days 1 to 3); 4) CPD. Tumor volumes were recorded every second day, and mice suppressed at the 21[th] day or when tumor volume reached 500 mm[3].

Results
We observed a significant increase of specific cell death in epithelioid and sarcomatoid cell lines treated with CPD compared to those receiving CP or D as single agent (p<0.001). We then observed p53 activation in both cell lines after chemotherapy, and a subsequent significant increase of DR4/5 expression levels (p<0.005) without upregulation of decoy receptors. We finally assessed antitumor activity of CP and/or D in a ZL55 mouse model. We observed a statistically significant reduction of tumor volume at every time point in the three treatment groups compared to not treated; moreover, tumor volume was significantly reduced in mice treated with CPD (mean volume 58 mm[3]) compared to CP (mean volume 175 mm[3]) or D (mean volume 109 mm[3]) as single agent at the 21th day (p= 0.005). Finally, no difference in tumor growth was observed between mice treated with D compared to CP.

Conclusion
CP sensitizes MPM cell lines to TRAIL-dependent apoptosis in vitro, probably through p53 activation and subsequent upregulation of death receptors. CPD significantly reduces tumor volume in epithelioid mesothelioma mouse model compared to chemotherapy alone or dulanermin as a single agent; furthermore antitumor activity of dulanermin was comparable to that reported with chemotherapy. In vivo experiments in a sarcomatoid mouse model are currently ongoing.

Background
Lung cancer is the leading cause of cancer-related mortality worldwide and there are more than one million new cases reported worldwide each year. Small cell lung cancer (SCLC) makes up 15% to 20% of all lung cancers with a 5-year survival rate of 5% to 10%. Bcl-2 is a central regulator of cell survival that is overexpressed in the majority of SCLC and contributes to both malignant transformation and therapeutic resistance. The emergence of BH3 mimetics that modulate Bcl-2 pathway by occupying the BH3 groove represents a rational approach for the treatment of this neoplasm. S1 is a BH3 mimetic that depends on Bax/Bak completely. The purpose of this work was to study the key factors that determine the sensitivity of SCLC cells to S1 and the mechanism underlying the resistance of BH3 mimetics.

Methods
Western bolt was used to evaluate the contribution of Bcl-2 family members to the cellular response of 11 SCLC cell lines to a BH3 mimetic S1. Viable cells were determined using MTS assay. To study the potential mechanism of resistance to S1, we derived resistant lines from initially sensitive H1688 cells. Quantitative PCR was performed to investigate Bcl-2 up-regulation. Gene silencing and MEK/ERK inhibitor PD98059 were used to demonstrate the involvement of ERK1/2 signaling in S1-induced Bcl-2 expression.

Results
Results showed relatively higher levels of Bcl-2 and phosphorylated Bcl-2 (pBcl-2) characterized naïve SCLC cell lines that were de novo resistant to S1. Likewise, a progressive increase in the relative levels of Bcl-2 and pBcl-2 characterized the increased acquired resistance of H1688 cells following chronic exposure to S1. Furthermore, acute treatment of S1 induced Bcl-2 expression and phosphorylation. We showed that BH3 mimetics including S1 and ABT-737 induced ER stress and then activated MEK/ERK pathway. The dual function of MEK/ERK pathway in defining BH3 mimetics was illustrated: ERK1/2 activation leaded to Bcl-2 transcriptionally up-regulation and sustain phosphorylation in naïve and acquired resistant SCLC cells. pBcl-2 played a key role in creating resistance of S1 and ABT-737 not only by sequestrating pro-apoptotic proteins, but also a positive feedback to promote ERK1/2 activation.Figure 1

Conclusion
These results provide significant novel insights into the molecular mechanisms for crosstalk between ER stress and endogenous apoptotic pathways in SCLC following BH3 mimetics treatment.

Background
Seven Sirtuin family members (SIRT1-7), comprising a family of NAD+-dependent protein deacetylases and ADP-ribosyltransferases, are key proteins that regulate multiple physiological processes. SIRT2 was recently reported to play an important role in carcinogenesis. However, its role in non-small cell lung cancer (NSCLC) has not yet been investigated.

Methods
In this study, we analyzed the expression pattern of SIRT2 in NSCLC tissues from clinical patients and in cell lines

Results
We found that SIRT2 was significantly down-regulated at both the mRNA and protein levels in tumor than non-tumor tissues or cells, which were corroborated by the NSCLC tissue microarray results. Overexpression of SIRT2 in A549 and H1299 cells caused cell proliferation inhibition, cell apoptosis induction and cell cycle arrest. Further analysis showed that SIRT2 overexpression increased the ROS (reactive oxygen species) production and P27 levels. Moreover, up-regulation of SIRT2 by Resveratrol in NSCLC cells increased the sensitivity to cisplatin treatment. .

Conclusion
Taken together, our results implied that down-regulation of SIRT2 was associated with NSCLC, and regulation of SIRT2 might be an important target for NSCLC therapy

Background
Erlotinib (ERL), a specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, benefits patients with non-small cell lung cancer (NSCLC), especially if the cancer harbors EGFR active mutations (mtEGFR). However, those who initially respond eventually develop progressive disease through acquired resistance. Bevacizumab (BEV), a humanized anti-vascular endothelial cell growth factor monoclonal antibody, has been demonstrated to be effective in combination with standard chemotherapies for advanced NSCLC patients. We hypothesized that the combination of the two agents may be more effective because they work through different modes of action. In the present study, we examined the antitumor activity of BEV in combination with ERL in human NSCLC xenograft models harboring mtEGFR.

Methods
Mice (BALB-nu/nu) were subcutaneously inoculated with NSCLC cell lines harboring mtEGFR (exon19 deletion); HCC827 and B901L. BEV (5 mg/kg) was intraperitoneally administered once a week for 3 weeks, and ERL was orally given daily for 21 days at doses of 5 and 40 mg/kg for HCC827 and B901L, respectively. Antitumor activity was evaluated by tumor volume (TV; mm[3]) on day 22. In order to examine the prolonged antitumor effect of the combination of BEV with ERL, the B901L xenograft model was used. BEV (5 mg/kg) was intraperitoneally administered once a week for 13 weeks and ERL (60 mg/kg; maximum effective dose) was orally given daily for 91 days. The antitumor activity was evaluated on day 92, with an interim evaluation on day 22. The microvessel density (MVD) of tumor tissues was evaluated by CD31 immunohistochemistry of specimens obtained on day 2 from mice treated with BEV (5 mg/kg) and ERL (40 mg/kg). Statistical analysis was performed using the Wilcoxon test.

Results
In the HCC827 model, the TV (mean±SD) of control, BEV, ERL, and BEV+ERL was 1300±424, 686±84, 615±185, and 194±29, respectively. In the B901L model, the TV of control, BEV, ERL, and BEV+ERL was 1739±761, 819±293, 294±198, and 57±19, respectively. The antitumor activity of BEV+ERL was significantly better than that of BEV or ERL monotherapy (p≤0.05) in both models. In the prolonged treatment experiment using B901L, on day 22, BEV (1096±298) showed significantly higher (p≤0.05) tumor growth inhibition than control (2452±731), but the ERL (61±31) and BEV+ERL (53±30) showed even better (p≤0.05) tumor regression effects than BEV. However, during further treatment, tumor regrowth was observed in the ERL group, even though ERL was consecutively given, and the TV on day 92 (1105±1001) was significantly greater than that on day 22 (61±31). Tumor regrowth was also observed in the BEV+ERL group; however, it was much slower compared to the ERL group and there was no significant difference between TV on day 92 (79±70) and that on day 22 (53±30). The MVD was significantly decreased to the same level in the BEV and BEV+ERL groups.

Conclusion
Compared to ERL monotherapy, the combination of BEV and ERL demonstrated promising efficacy in mouse models of NSCLC harboring EGFR activating mutations. The encouraging preclinical results warrant further investigation in a clinical setting.

Background
Bevacizumab (BEV), a humanized anti-vascular endothelial cell growth factor monoclonal antibody, is used in combination with chemotherapy for patients with non-small cell lung cancer (NSCLC). The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) benefit patients with NSCLC, especially when this cancer harbors EGFR mutations (mtEGFR); however, those who initially respond eventually develop resistance. To treat the patients with EGFR-TKI acquired resistance, a combination of BEV and docetaxel (DTX) may be a possible treatment modality based on different mechanisms of action. In the present study, we investigated the antitumor activity of BEV in combination with DTX in EGFR-TKI-resistant NSCLC xenograft models.

Methods
Mice (BALB-nu/nu) were subcutaneously inoculated with NSCLC cell lines; NCI-H1993 (MET amplification) and NCI-H1975 (mtEGFR/T790M). The treatment was started (day 1) after tumor growth was confirmed. BEV (5 mg/kg) was administered intraperitoneally on days 1, 8, 15 and DTX was administered intravenously on day 1 at a dose of 60 or 20 mg/kg as a maximum effective dose in NCI-H1993 or NCI-H1975 xenografts, respectively. The antitumor activity was evaluated by tumor volume (TV; mm[3]) on day 46 in the NCI-H1993 model and on day 50 (with an interim evaluation on day 19) in the NCI-H1975 model. Tumor cells in the proliferation phase were immunohistochemically evaluated using Ki-67 as an index (count/1000 tumor cells) on day 8 in both tumors. Statistical analysis was performed using the Wilcoxon test.

Results
In the NCI-H1993 model, the TV (mean±SD) of control, BEV, DTX, BEV+DTX on day 46 was 1630±492, 670±116, 204±85, 49±34, respectively. The antitumor activity of BEV+DTX was significantly better than that of DTX monotherapy (p≤0.05). In the NCI-H1975 model, TV of control, BEV, DTX, BEV+DTX was 4631±2384, 1581±572, 18±19, 16±10, respectively, on day 19. The BEV+DTX combination and DTX monotherapy showed similar effects on tumor regression at this point. However, after that, tumor regrowth was observed in the DTX group, and increased beyond the initial TV in 5 out of 6 mice, whereas no tumor regrowth was observed in BEV+DTX group at day 50. The TV of DTX and BEV+DTX groups on day 50 was 2631±2391 and <10 respectively. The number of Ki-67 positive cells (mean±SD) in tumor tissue taken from control, BEV, DTX, BEV+DTX on day 8 was, respectively, as follows: NCI-H1993: 672±50, 638±25, 559±38, 459±37; NCI-H1975: 533±28, 401±57, 381±49, 290±34. The number of Ki-67 positive cells in tumors treated with BEV+DTX was significantly lower than in those treated with DTX (p≤0.05) in both models, although there were no significant differences in TV between the two groups on day 8.

Conclusion
Combining BEV with DTX can decrease the tumor cells in a proliferation phase more effectively than DTX alone in the early stage of the therapy. This may provoke a stronger antitumor effect later on and delay tumor regrowth. These data are encouraging, and the combination of BEV+DTX may be a promising treatment modality for patients with NSCLC after acquiring a resistance to EGFR-TKIs.

Background
Bevacizumab (BEV) is a humanized monoclonal antibody against vascular endothelial cell growth factor (VEGF) that inhibits VEGF-mediated angiogenesis in many types of tumors. OS and PFS were significantly prolonged in patients with advanced non-small cell lung cancer (NSCLC) who received BEV maintenance after induction with BEV+chemotherapy (ECOG4599 study). The maintenance study AVAPERL demonstrated that, after induction with pemetrexed (PEM)+BEV+cisplatin therapy, BEV+PEM combination significantly prolonged PFS compared to BEV monotherapy. However, the comparative efficacy of BEV+PEM to PEM monotherapy is not clear. In the present study, we investigated the antitumor effect of the BEV+PEM combination compared to BEV or PEM monotherapy as continuation maintenance after BEV+PEM treatment in a human NSCLC xenograft model.

Methods
SCID mice were subcutaneously inoculated with human NSCLC cell line NCI-H2228. As an induction treatment, BEV (5 mg/kg; maximum effective dose) and PEM (400 mg/kg; maximum effective dose) were administered intraperitoneally on days 1, 8, and 15 after randomization by tumor volume (TV). On day 22, the mice treated with BEV+PEM were re-randomized and BEV and/or PEM were administered intraperitoneally weekly until day 85. The antitumor activity was evaluated by TV on day 85. Microvessel density (MVD) and thymidylate synthase (TS) expression in tumor tissues were evaluated using specimens taken on day 85. MVD was analyzed by CD31 immunohistochemistry and evaluated in terms of the ratio of stained area to observed area. TS expression was evaluated by Western blot analysis. Statistical analysis was performed using the Wilcoxon test.

Results
TV (mm[3]; mean±SD) of BEV+PEM group on days 1 and 22 was 335±72 and 329±59, respectively, whereas TV of control group on day 22 was 671±67, indicating that BEV+PEM completely inhibited tumor growth during induction treatment. TV of control, BEV, PEM, and BEV+PEM groups on day 85 was 2748±1334, 586±320, 883±271, and 348±48, respectively. Each treatment group showed a significantly higher antitumor activity (p < 0.001) compared to control. Specifically, BEV+PEM completely inhibited tumor growth throughout the experiment, and showed a significantly higher antitumor activity than BEV (p < 0.05) or PEM (p < 0.001) monotherapy. MVD (%) was significantly lower (p < 0.01) in BEV (1.40±0.36) and BEV+PEM (1.24±0.49) groups than in control (6.63±1.89) and PEM (4.62±0.71) groups, indicating that the decrease in MVD caused by BEV treatment was not affected by the combination with PEM. TS expression level, which is inversely correlated with the effect of PEM, was lower in BEV+PEM group than in PEM group.

Conclusion
Compared to PEM monotherapy, the combination of BEV with PEM showed better antitumor effect as a maintenance therapy in a xenograft model. Decreases in MVD and TS expression may contribute to the effect of combination therapy.

Background
Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, shows notable effects against non-small cell lung cancers (NSCLCs) harboring EGFR-activating mutations such as exon 19 deletion or L858R mutation. However, almost all patients eventually acquire resistance to erlotinib. In this study, we investigated whether the level of exposure to erlotinib affects incidence of acquired resistance in EGFR-mutated NSCLC cell lines and examined their resistant mechanisms.

Methods
Human NSCLC cells B901L and PC-9 harboring exon 19 deletion and II-18 cells with L858R mutation were continuously exposed to 0.5 or 5 µM of erlotinib in 96-well plates for several months. The upper dose was chosen because the recommended dose of erlotinib (150 mg) has been reported to achieve maximum plasma concentration (Cmax) of about 5 µM. Cell growth inhibition was determined by a crystal violet assay. EGFR mutation status was determined by melting curve analysis. MET copy number was determined by real-time PCR analysis. Phosphorylations of EGFR, HER2 and ERK were measured by Western blot analysis.

Results
Resistant B901L, PC-9 and II-18 cells were emerged in 24, 8 and 7 wells by exposing to 0.5 µM erlotinib, and in 13, 2 and 0 wells by exposing to 5 µM erlotinib, respectively. No alterations in EGFR mutation status including T790M mutation nor acquisition of MET amplification were detected in any resistant cells. In parent cells, treatment with erlotinib markedly inhibited phosphorylation of EGFR, HER2 and ERK. In the parent cells, treatment with erlotinib markedly inhibited EGFR phosphorylation but not HER2 and ERK phosphorylation, compared with the parent cells.

Conclusion
In present in vitro analysis, it was suggested that exposure to the recommended dose of erlotinib would avoid acquired resistance in NSCLC with exon19 deletion or L858R mutation of EGFR. Pathways activated by phosphorylated HER2 may affect acquired resistance to erlotinib.

Background
Despite rapid strategy improvments of advanced lung cancer , traditional chemotherapy or targeted therapies, especially EGFR-TKI (epidermal growth factor receptor) will emerge treatment resistant eventually. The main cause for treatment failure of advanced lung cancer is drug resistance, and it’s molecule mechanism could be key to improve theraputic effect. The transcription factors Oct4 and Nanog were reported to be involved in drug resistance of many malignant tumors, which are closely related with the prognosis of patients. These factors are expected to be potential drug targets. The present study is designed to explore potential mechanisms about stem cell transcription factors’ regulation of epithelial mesenchymal transition (EMT) invlolved in drug resistance of lung cancer.

Methods
MTT，immunohistochemistry, immunofluorescence, Western blot, RT-PCR experiments in vitro and in vivo were employed. Clinical samples (104 postoperation and 94 advance stage samples) were collected to assess the clinical prognostic value of stem transcription factors( Oct4 and Nanog), （CD133 and ABCG2）and epithelial mesenchymal transition (EMT) markers（E-Cadherin and N-Cadherin）with clinical features. Down-regulation of Nanog by RNA interference were used to find the change of chemosensitivity and EGFR-TKI sensitivity. Combined with the specific mechanisms in vitro and clinical samples to interpret the regulation of EMT involved in drug resistance.

Results
The immunocytochemistry experiments showed that expression pattern of A549 cell line are CD133 (+), ABCG2 (+ +), Oct4 (-) and Nanog (-); A549/DDP cell line are CD133 (+ + +), ABCG2 (+ +), Oct4 (+ +) and Nanog (+ + +). There are significant differences in expression of CD133, Oct4 and Nanog between the two cell lines. Cell immunofluorescence experiments showed that stem cell transcription factors expressed in cell nucleus of A549 cell line, and stem cell surface marker CD133 located on the cell membrane. RT-PCR and Western blot were used to the examination of A549 and A549/DDP cell lines for CD133, ABCG2 and Oct4, Nanog’ expression. The results showed that the expression of these markers in cisplatin resistance cell line. Examination by RT-PCR of the samples from 40 paired cases of lung cancer and adjacent tissue, showed that Oct4 and Nanog in lung cancer group was higher than those in paracancerous tissues, suggesting that Oct4 and Nanog might play an important role in lung cancer initiation and malignant growth process. The expression of these transcription factors in 104 samples from lung cancer patients, analysed by immunohistochemical method, showed the expression level of these factors were significantly higher in stage III and IV patients than in stage I and II patients. It also showed that the higher the Oct4 and Nanog expressed, the poorer prognosis were (54.3 vs 68.5 months, P=0.053) and (52.7vs.72.1months, P= 0.022). The patient with negative expression of CD133 or ABCG2 survived longer than those with positive expression (72.8 vs. 50.2 month, P = 0.021) and (76.5 vs.47.7 months, P=0.000). Patients with positive expression of E-Cadherin survived longer than those with negative markers (67.6 vs 54.1 months, P=0.035), While N-Cadherin+ patients survived shorter than N-Cadherin- patients (55.1 vs. 77.1 months, P=0.025). In overall survival between patients with and those without chemotherapy, there is no significant difference (63.8 vs. 60.6 months, P=0.851). But subgroup analysis revealed patients undergoing chemotherapy with positive expression of CD133 enjoyed significantly longer survival (53.8 vs. 32.3 months). A subgroup analysis also showed that patients with negative expression of Nanog with chemotherapy survived longer than those without chemotherapy (69.3 vs. 76.4 months), while patients with positive Nanog treated with chemotherapy could survive longer than those without chemotherapy (57.0 vs. 38.7 months). In the 74 patients received chemotherapy after progression, we found that many patients with PR are Nanog- (82.1% vs. 17.9%), while patients with Nanog+ tend to have progression diease (61.4% vs. 38.6%). K-M survival analysis indicated that patients with Nanog (-) N-Cadherin (-) CD133 (-) survived longer than those with Nanog (+) N-Cadherin (+) CD133 (+), Nanog (+) N-Cadherin (-) CD133 (-), Nanog (+) N-Cadherin (-) CD133 (+) and Nanog (-) N-Cadherin (-) CD1 33 (-) patients (101.9 vs. 60.0, 101.9 vs. 54.6, 101.9 vs. 38.2 months). Multivariate regression analysis showed that stage of lung cancer, CD133, N-Cadherin and Nanog were independent prognostic factors. Downregulation of Nanog could partially restore the cell sensitivity to cisplatin in cisplatin-resistant cell line under RNA interference. In the EGFR primary drug resistance experiment, we found that CD133 and ABCG2 were higherly expressed in H23 (EGFR primary resistance cell line) than PC-9 (EGFR sensitive cell line). Detection of CD133, ABCG2 and Oct4, Nanog expression by RT-PCR and western blot in PC-9 and H23 cell lines showed that these markers expressed significantly higher in H23; Using immunohistochemical method, we analysed EGFR, C-met and CD133, ABCG2 and Oct4，Nanog in 104 cases of postoperative samples and analysed the relationship between their expression and prognosis. The results showed that C-met expressed in patients in advanced stage (P=0.0860), adenocarcinoma(P=0.0355) and non-smoking patients. K-M analysis showed that C-met expressed in patients with wild type EGFR(P= 0.0436). We further tested stem cell factors in 94 cases of advanced stage who received ERFG-TKI and analysed relationship between drug efficacy and prognosis. The results suggest that Nanog tend to express in smokers (39.4%vs.14.8%, P=0.0071), and Oct4 in adenocarcinoma (38% vs.8.3%, p=0.0170). Patients with negative Oct4 expression have longer PFS (time to progression of disease) (11 vs.7.0 m, P=0.041), patients with negative expression of Nanog have significantly longer PFS than patients with higher expression (13.5 vs.3.8m, P=0.000). Patients with high E-cadherin expression have longer PFS than patients with lower expression (13.5m vs 6.8., P=0.015), Patients with lower N-cadherin expression have longer PFS than patients with high expression (12.3 vs.7.4m, P=0.020). Multiple regression analysis suggested that Nanog, ECOG score and RR are independent prognostic factor. RNA interference results showed that down-regulation of Nanog expression can increase sensitivity to EGFR-TKI in H23.

Conclusion
This study will reveal the mechanisms of drug resistance in lung cancer from a new point of view. To provide the evidence of stem cell factor and EMT related pathway as a new target for reverse the drug resistance in the future.

Background
Bronchoscopic ablation of lung cancer has to date been limited to carcinoma in-situ located in the central airway or, using high-power lasers, to palliation in advanced obstructive disease . Bronchoscopic ablation of peripheral lung cancer is still under development. We are developing a new technology platform for localization (by fluorescence) and enhanced photothermal therapy (PTT) of peripheral lung lesions, based on porphysomes, which are novel, multi-functional, all-organic porphyrin-lipid nanoparticles. Even without active targeting, porphysomes accumulate within tumor through the enhanced permeability and retention (EPR) effect. In parallel, we have developed a prototype fluorescent endoscope system that allows visualization of peripheral lesions by the porphyrin fluorescence. Using this combination of novel instrumentation and nanoparticles, endoscopic PTT of lung cancer is demonstrated in preclinical lung cancer animal models in vivo.

Methods
The in vivo porphysome biodistribution was evaluated in both orthotopic lung tumors (A549, H460, H520) in mice and in VX2 tumors implanted directly in rabbit lung. Porphysomes were administered intravenously at a dose of 20 mg/kg, and the tumor fluorescence was imaged in vivo daily for 3 days (excised lung in the mouse model and endoscopically in the rabbit model). Ex vivo VX2 tissue was illuminated using a 670 nm diode laser to determine the optimized treatment parameters for PTT. Subsequently, in vivo bronchoscopic visualization of VX2 fluorescence and PTT was demonstrated in the rabbit model. The extent of ablation was histologically evaluated by NADH metabolic activity staining.

Results
The highest tumor-to-normal lung fluorescence ratio was achieved at 48 h post-injection of porphysomes(Rabbit VX2: n=4). The same trend was confirmed in the 3 kinds of orthotopic lung cancer mouse Xenografts (n=8). The ex vivo study revealed that 250 mW and 10 min of 670 nm laser irradiation raised the tumor tissue temperature by more than 20[o]C, so that this setting was used also in vivo and achieved thermal coagulation zones within the tumor of up to 13 mm diameter. In comparison, laser treatment alone without porphysomes caused minimal ablation zones of < 1.5 mm diameter.

Conclusion
Systemically administrated porphysomes accumulated in the lung cancer tissue with a high enough concentration to enable marked photothermal coagulation. The combination of systemically-administrated porphysomes with endoscopic near-infrared laser irradiation is a promising strategy for minimally-invasive bronchoscopic interventional therapy for peripherally-located lung cancer.

Background
Lung adenocarcinomas with EGFR mutation initially show good responses to EGFR- tyrosine kinase inhibitors (TKIs). However, emergence of acquired resistance is almost inevitable. To analyze molecular mechanisms underlying acquired resistance, we established a cell line from a patient who acquired resistance to gefitinib/erlotinib.

Methods
A 64-year-old woman underwent a pulmonary resection for lung adenocarcinoma in February 2009 (pT2aN0M0, EGFR L858R mutation). In April 2010, pulmonary metastases, mediastinal lymph node metastases, and right pleural effusion were identified. Gefitinib was started and this led to a complete response. However, despite continuous treatment with gefitinib, pleural effusion and serum carcinoembryonic antigen (CEA) levels gradually increased. In December 2010, gefitinib was switched to erlotinib but the serum CEA level continued to increase, and erlotinib was stopped on March, 2011. Even though she received no treatment after erlotinib withdrawal, interestingly, the serum CEA level was decreased significantly (from 89.5ng/ml on March 2011 to 19.2ng/ml on April 2011), and erlotinib was re-started on April 2011. The serum CEA level increased after re-administration of erlotinib (55.7ng/ml on May 2011). Therefore the regimen was switched to cisplatin/pemetrexed and she responded to this combination chemotherapy (CEA 9.1ng/ml on July 2011). This clinical experience may be an actual case of “drug addiction phenomenon” that we and others have observed in preclinical acquired resistance models (Suda K, et al. Lung Cancer 2012; Das Thakur M, et al. Nature 2013). We established a cell line from her pleural effusion obtained on March 2011 in drug-free condition (designated as ACC-GR1 cells). We analyzed this cell line to evaluate the efficacy of erlotinib and dacomitinib, an irreversible EGFR-TKI. In addition, we examined phosphorylation status of 42 receptor tyrosine kinase (RTK) of ACC-GR1 cells using Human Phospho-RTK Array Kit (R&D Systems) with/without erlotinib or dacomitinib. We also examined PC9 lung adenocarcinoma cell line for phosphorylation status of RTKs with/without erlotinib.

Results
ACC-GR1 cells harbored the T790M mutation, in addition to the original L858R mutation in the EGFR gene. ACC-GR1 cells do not have amplification of MET proto-oncogene. IC~50~ values for erlotinib and dacomitinib were 2.9 uM and 0.05 uM, respectively. Phospho-RTK array analysis revealed marked activation of EGFR and MET, in addition, activation of ERBB2, ERBB3, RET, and AXL in a culture condition without EGFR-TKI. Treatment with 1 uM of erlotinib led to mild inhibition of EGFR and MET phosphorylation (71% and 58%, respectively, compared with control), and phosphorylation of other RTKs fell below detectable limits. Treatment with 1 uM of dacomitinib led to further inhibition of EGFR phosphorylation (35% compared with control). In PC9 cells, phosphorylation of EGFR and MET were also observed in drug-free condition, and remarkably inhibited by 1 uM erlotinib treatment (10% and 64%, respectively, compared with control).

Conclusion
A cell line model established from pleural effusion of a patient who acquired resistance to gefitinib/erlotinib harbored T790M mutation and responded to dacomitinib in vitro. The acquired resistant cells showed activation of several RTKs in drug free condition, and these are remarkably inhibited by EGFR-TKI treatment.

Background
Malignant mesothelioma is an aggressive cancer with a low response to current therapies and consequently a poor prognosis. There is an urgent need to identify novel and more effective treatments to improve survival and quality of life for patients with mesothelioma. While a number of novel therapeutic agents have recently been examined in clinical trials, to date none of these has resulted in changes to standard management. This is due in part to a lack of robustness and relevance of the pre-clinical models used to assess new treatments. Therefore validated and biologically relevant pre-clinical models that demonstrate the clinical behaviour and drug sensitivity of mesothelioma, as well as typical resistance mechanisms, are necessary to improve the discovery of new treatments. Here we describe the generation and evaluation of chemotherapy resistant pre-clinical models of mesothelioma derived from the previously utilized syngeneic II-45 rat mesothelioma model.

Methods
Cell lines resistant to the current standard of care agents: cisplatin, pemetrexed gemcitabine, vinorelbine, and cisplatin and pemetrexed in combination, were generated by 15 rounds of exposure to the respective agent. Normal rat mesothelial cells (4/4 RM.4), the parental II-45 and the 5 resulting chemo-resistant mesothelioma cell lines were characterised for resistance to these and other agents. Tumours arising from syngeneic pleural engraftment of these cell lines were assessed by size, morphology, immunohistochemical markers of human malignancy, chromosomal changes and expression of genes involved in drug resistance and metabolism. Engrafted rats were assessed for survival, and circulating haematological, cytokine and biomarker profiles were generated and compared.

Results
Five different II-45 cell lines with approximately 2-fold resistance to the chemotherapeutic agent they were repeatedly exposed to, were established (cisplatin, p < 0.05; pemetrexed, gemcitabine, vinorelbine, p < 0.001). Cross resistance to other classes of anti-cancer agents also developed indicating potential multi-drug resistant phenotypes. Tumours derived from both the parental and chemo-resistant cell lines were immunohistochemically indistinct from human mesothelioma with positive labeling for WT1, calretinin, HBME-1, cytokeratin and popoplanin and negative labeling for two carcinoma related markers TTF-1 and CD15. Homozygous deletion of p16[INK4A]/p14[ARF], and increased expression of several members of the ATP-binding cassette transporter superfamily and Androgen receptor (AR) were demonstrated, consistent with findings in human mesothelioma. Corresponding with biomarkers studies in human disease, increased levels of osteopontin (p < 0.01), mesothelin (p < 0.01), vascular endothelial growth factor (p < 0.001) and neutrophil to lymphocyte ratio were identified relative to control bloods. Further, the acquisition of chemo-resistance resulted in changes to tumour morphology with tumours ranging from essentially epithelioid in the gemcitabine resistant tumours to entirely sarcomatoid in the combination (cisplatin and pemetrexed) resistant tumours. Overall survival was also affected by the acquisition of resistance with rats with pemetrexed resistant tumours having decreased survival.

Conclusion
These models display many features corresponding with the human disease, and thus provide powerful and robust pre-clinical platforms for in vivo mesothelioma studies.

Background
Malignant pleural mesothelioma (MPM) is an aggressive asbestos-related malignancy characterized by frequent resistance to chemo- and radiotherapy. Signals induced by Fibroblast growth factors (FGF) and their high-affinity receptors (FGFR) have been identified as important drivers for malignant growth in several tumor entities including thoracic malignancies. We have recently demonstrated that FGFR signals stimulate growth and migration in MPM cell models, whereas inhibition of FGFR1 reduced tumor growth and had an antagonistic effect on malignant behavior of MPM cells. In several cell models of biphasic MPM, FGF2 induced phenotypical changes reminiscent of epithelial-mesenchymal-transition (EMT). In this study we analyzed these FGF-induced morphological and functional alterations and the associated signal transduction mechanisms in more detail.

Methods
Cells were stimulated with recombinant FGF2 and analyzed by microscopy and ImageJ software. The specific inhibitors PD166866, UO126, MK2206, LY294002 and SB431542 were used to block FGFR1, MEK, AKT, PI3K and TGFbeta receptors, respectively. Alterations in gene expression were determined via whole-genome expression arrays and further evaluated by immunofluorescence. Downstream signaling was investigated by immunoblotting.

Results
In M38K and SPC212 cells FGF2 induced morphological alterations that were characterized by a more spindle-shaped appearance and reduced contacts with adjacent cells. This correlated with increased cell migration. With respect to signal transduction, the effects of FGF2 could be blocked by inhibition of FGFR1 or MEK, whereas inhibition of AKT, PI3K or receptors of the TGFbeta/activin family had no effect. Expression arrays of both cell models indicated regulation of several matrix metalloproteinases (MMP1, MMP4), the integrin subunit ITGA6 and the two TGFbeta family-related proteins INHBB and Smad7. Since the phenotypical changes were similar to EMT, genes previously connected to EMT were analyzed. Whereas slug/SNAI2 and ZEB1 were increased, other mesenchymal genes such as vimentin or N-cadherin were already expressed at high levels in the untreated cells, likely due to the mesodermal origin of mesothelial cells. In M38K, E-cadherin was decreased whereas in the more “fibroblastoid-type” SPC212 E-cadherin was generally expressed at very low levels.

Conclusion
Our data suggest that FGF2 induces morphological changes that result in a more sarcomatoid cell morphology and expression of some markers connected to EMT. These effects depend on the MAPK pathway and are connected to more aggressive cell behavior, paralleling the higher aggressiveness and worse prognosis of the sarcomatoid and biphasic compared to the epithelioid histological subtype of MPM.

Background
Lung cancer is the most common cause of cancer death. Despite advances in treatment of other cancers, overall 5-year survival rates for advanced non-small cell lung cancer (NSCLC) are dismal. Expression of βIII-tubulin (encoded by TUBB3 gene) is associated with resistance to tubulin-binding agents in a range of tumor types including NSCLC[1]. We identified a multifactorial role for βIII-tubulin in chemosensitivity by showing that it can mediate response not only to tubulin-binding agents, but also DNA damaging agents in NSCLC[2, 3], validating βIII-tubulin as a therapeutic target in NSCLC. We hypothesized that in vivo targeting of the TUBB3 gene would sensitise NSCLC tumours to chemotherapy.

Methods
DNA-directed RNA interference (ddRNAi) is a potent gene silencing approach that can achieve prolonged suppression of target genes. We developed a triple cassette shRNA vector containing three unique targets against the TUBB3 gene (βIII~TRP~) and a control vector containing three non-targeting sequences (Cont~TRP~). Gene and protein analysis was achieved using RT-qPCR and western blotting respectively. An orthotopic mouse xenograft model of NSCLC was used to evaluate gene ddRNAi delivery, efficacy and chemo sensitisation. Mouse tumour growth was monitored using non-invasive xenogen imaging. Statistical analysis included Mann–Whitney test and Log rank (Mantel-Cox).

Results
Transient transfection of NSCLC H460 cells with the βIII~TRP~ vector resulted in >40% decrease of TUBB3 gene expression and a concomitant decrease in βIII-tubulin protein level compared to the Cont~TRP~ vector. This led to increased in vitro sensitivity to both paclitaxel and cisplatin. Using a clinically relevant orthotopic model of NSCLC we treated H460 tumour-bearing mice with the βIII~TRP~ vector encapsulated in a lipophilic delivery agent and this resulted in a potent decrease (71.6%) in TUBB3 expression in tumours compared to Cont~TRP~ vector (P=0.03). Median survival increased by 6 days in the Cont~TRP~~+ cisplatin~ versus Cont~TRP~ groups although this increase was not significant. In contrast, median survival increased by 21 days in the βIII~TRP + cisplatin~ versus βIII~TRP+ vehicle~ groups (P=0.003). Importantly, in vivo suppression of TUBB3 combined with cisplatin treatment led to a significant increase in overall survival compared to βIII~TRP + vehicle~ control mice (P=0.0174).

Conclusion
This study provides the first evidence that a vector-based gene suppression approach can potently silence TUBB3 expression and increase chemosensitivity in vivo, highlighting this as a promising treatment strategy for drug refractory NSCLC. References 1. Kavallaris M. Microtubules and resistance to tubulin-binding agents. Nat Rev Cancer. 2010;10:194-204. 2. McCarroll JA, Gan PP, Liu M, Kavallaris M. betaIII-tubulin is a multifunctional protein involved in drug sensitivity and tumorigenesis in non-small cell lung cancer. Cancer Res. 2010;70:4995-5003. 3. Gan PP, Pasquier E, Kavallaris M. Class III beta-tubulin mediates sensitivity to chemotherapeutic drugs in non small cell lung cancer. Cancer Res. 2007;67:9356-63.

Background
Lung cancer is the leading cause of cancer deaths worldwide. Despite advances and progresses in surgery, chemotherapy, and radiotherapy over the last decades, the death rate from lung cancer has remained largely unchanged, which is mainly due to metastatic disease and multi drug resistance. Because of the overall poor prognosis, new treatment strategies for lung cancer patients are urgently needed. The aim of this study was to investigate the interactions between pemetrexed and vinorelbine for human adenocarcinoma via various chemotherapy schedules.

Methods
HCC cells and cisplatin resistant HCC cells (HCC-res) were treated with different schedules of combination of cisplatin (Cis), vinorelbine (Vin), and pemetrexed (Pem) respectively, more specific as Cis, Vin, Pem, Cis+Vin, Cis+Pem, Vin+Pem, Vin→Pem, Pem→Vin, Cis+Vin+Pem, Cis→Pem→Vin, and Cis→Vin→Pem. Cell growth inhibition was determined by cell viability analysis. Cell apoptosis was analyzed via annexin V staining and FACS analysis. And cytoplasm Ca[2+] was measured with Ca[2+] indicator dye Fura-2 AM.

Results
Vin and Pem caused a strong dose-dependent cytotoxic effect in both HCC and HCC-res cells. The IC50 values of Vin against HCC and HCC-res cells were 10.34 ± 1.12 nM and 9.98 ±2.12 nM, respectively. The IC50 values of Pem against these cells were 110.77 ± 17.28 nM and 118.89 ±18.77 nM respectively. The application of different therapy schedules induced a significant time dependent cell growth inhibition on HCC naïve and cisplatin resistant cells. The therapy scheme of Cis→Pem→Vin showed the strongest inhibitory effect on both HCC and HCC-res cells. The application of different therapy schedules on HCC and HCC-res cells increased the percentage of cells undergoing apoptosis, except the application of Vin alone. In both HCC and HCC-res cells, Cis→Pem→Vin was found the most effective to induce apoptosis. The application of different therapy schedules on HCC and HCC-res cells increased [Ca[2+]]~c~. Only the application of Vin alone failed to increase [Ca[2+]]~c~ in HCC cells. The most elevated [Ca[2+]]~c ~was found in the cells treated with Cis→Pem→Vin in both HCC and HCC-res cells

Conclusion
We demonstrated that the sequential application of Cis, Vin and Pem has a synergistic effect in cell growth inhibition, apoptosis induction, and [Ca[2+]]~c~ elevation in HCC and HCC-res cells. The Ca[2+ ]overload could lead to apoptosis, which was related to the cell growth inhibitory effect of chemotherapeutics in lung cancer cells. It might cast a light to develop chemotherapy schedules for patients, and to overcome cisplatin resistance in lung cancer.

Background
Chromosome Region Maintenance 1 (CRM1) is a nuclear exporter which transports certain proteins from the nucleus to the cytoplasm, including tumor suppressor proteins (TSPs) and other modulators of proliferation. Overexpression of CRM1 correlates with cancer progression in several human cancers, suggesting that CRM1 could serve as a novel target for treatment of cancers. NSCLC is an aggressive carcinoma which is not yet curable. The aim of our study was to explore the therapeutic efficiency of novel drug-like CRM1 inhibitors in NSCLC in vitro and in vivo, and to investigate the cytotoxic mechanisms of CRM1 inhibitors in NSCLC cell lines with EGFR-TKI resistance mutation.

Methods
KPT-185 and KPT-276 are selective inhibitors of nuclear export (SINE) that block CRM1. Cell viability, apoptosis and cell cycle were evaluated in 6 NSCLC cell lines (H1975, H1650, A549, H2228, HCC827, H1650 Gefitinib Resistance (H1650GR)) after treated with KPT-185; expression level of CRM1 in NSCLC cell lines was detected after exposed to KPT-185; TSPs were detected by western blot to explore the possible mechanisms of KPT-185 inducing NSCLC cells growth inhibition and apoptosis. NOD-SCID mice bearing H1975 (epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistant) tumors were treated orally with KPT-276 (similar structure to KPT-185, but improved animal pharmacokinetics) to examine the efficacy and side-effects of KPT-276 in vivo.

Results
In 6 NSCLC cell lines, growth inhibition showed in a time- and dose-dependent way after treated with KPT-185. The EGFR TKI resistant cell lines H1975 and H150GR were sensitive to KPT-185. Cell apoptosis analysis showed that KPT-185 induced NSCLC cells apoptosis in a dose-dependent manner. Also, KPT-185 induced cell cycle arrest at the G1/S checkpoint in NSCLC cell lines. CRM1 protein expression of 6 NSCLC cell lines was down regulated when treated with KPT-185, which could be completely abolished by bortezomib. CRM1 inhibition by KPT-185 up-regulated the expression of proteins involved in apoptosis in NSCLC cell lines, and down-regulated the expression of EGFR and survivin. In the xenograft H1975 model, tumor growth was significantly inhibited in KPT-276 oral treatment group compared with vehicle control group and EGFR-TKI treatment group (P<0.01), and there was no significant loss in body weight or side-effects in KPT-276 treatment group.

Conclusion
SINE CRM1 inhibitors showed anti-tumor activity in NSCLC both in vitro and in vivo, especially in EGFR-TKI resistance cell lines, it could inhibit the growth of NSCLC, arrest cell cycle, reduce expression of CRM1 protein, and the anti-tumor activity of SINE is mainly through inducing cell apoptosis. SINE is a novel CRM1 inhibitor and a promising clinical candidate.

Background
This study was performed to validate a newly developed sentinel lymph node (SLN) targeting tracer, indocyanine green–neomannosyl human serum albumin (ICG:MSA), and a thoracoscopic version of the intraoperative color and fluorescence imaging system (ICFIS) for lung cancer SLN mapping.

Methods
5 μg/kg ICG concentrations of ICG alone or ICG:MSA were injected into rat thigh and the results were compared. The fluorescence signal-to-background ratio (SBR) of SLNs were recorded and evaluated over a 2-h period using ICFIS. In addition, a SLN biopsy by video-assisted thoracoscopic surgery (VATS) was performed using ICG:MSA in porcine lung by using thoracoscopic ICFIS.

Results
The newly developed ICG:MSA showed a significantly improved SBR compared to ICG alone throughout the trials. All SLNs were identified in both rat (10 SLNs in 10 rat thighs) and pig (10 SLNs in 10 porcine lungs) under in vivo conditions. All SLNs were dissected successfully using VATS with help of thoracoscopic ICFIS.

Conclusion
ICG:MSA is accumulated in the SLN by uptake and retention through the mannose-specific receptors on a macrophage. Thoracoscopic ICFIS successfully assisted SLN mapping despite low near-infrared (NIR) light transmission in the commercial thoracoscope. Based on the results of the thoracoscopic SLN mapping, we expect that the ICG:MSA and thoracoscopic ICFIS can be translated to clinical trials in the near future.

Background
Background: Small cell lung cancer (SCLC) has an overall 5-year survival rate of < 5% despite initial response rates to first line chemotherapy of 70-90%. MYC family amplification occurs in 30% of SCLC and MYC amplified tumors have been shown to be dependent on aurora kinase B for their survival. Aurora B kinase (AURKB) is a chromosomal passenger protein that plays a critical role in mitosis by regulating chromosome alignment, accurate segregation, and cytokinesis during the mitotic stages. We evaluated the effects of the selective aurora B kinase inhibitor AZD1152-HQPA (active drug) in a panel of 15-SCLC lines.

Methods
Methods: Nine lines had MYC-family amplification (MYC-5 lines, MYCL1-2 lines, MYCN-2 lines) and 6-lines had no MYC-family amplification. Growth inhibition by AZD1152-HQPA was assessed by tetrazolium based assays. Apoptosis was determined using the DNA binding dyes YOPRO and propidium iodine (PI) and analysis by flow cytomery. The induction of polyploidy was evaluated by flow cytometry following PI staining of the DNA. The effect of AZD1152-HQPA on anchorage independent growth was determined by soft agar colony forming assays. Finally, we evaluated the in vivo efficacy of AZD1152 (prodrug) on SCLC xenografts in nude mice.

Results
Results: AZD1152-HQPA GI50 values for the 4 most sensitive SCLC lines were 11-30 nM and < 25% of untreated control growth was observed at 60 nM. Five lines had GI50 values of 30-60 nM but at 600 nM AZD1152-HQPA cell viability remained at 49-55% of untreated control growth. In the remaining 6-lines cell viability at 600 nM was 60-93% of control growth. Growth inhibition marginally correlated with MYC amplification (p=0.044) and was strongly correlated with MYC + MYCL1 + MYCN amplification (p=0.011). Apoptosis (20-28%) was induced by 30 nM AZD1152-HQPA at 24-48 hours in the most sensitive cell lines. Marked increases in DNA ploidy (8N) was observed after 24 hour exposure to AZD1152-HQPA (30nM) in the most sensitive cell lines and at 48 hours in the 5 intermediate lines and in 2 of the resistant lines. In vivo AZD1152 (i.p. 100 mg/kg/M-F for two weeks) induced tumor regression in nude mice implanted with a sensitive cell line. AZD1152 at 50mg/kg/day inhibited tumor growth; however these tumors began growing following treatment cessation. A resistant line was also implanted into nude mice and AZD1152 at 50 and 100 mg/kg/day caused tumor regression. Polyploidy was induced in this cell line at 48 hours post 30 nM AZD1152-HQPA. Anchorage independent growth in this resistant line was also completely inhibited by AZD1152-HQPA 25 nM. The doses of AZD1152-HQPA used in this study are within the range reported to be clinically achievable.

Conclusion
Conclusions: AZD1152-HQPA growth inhibition significantly correlated with MYC-family amplification in SCLC lines but some SCLC lines without MYC-family amplification were also inhibited. Additional biomarkers are needed to identify SCLC patients most likely to respond to AZD1152.

Background
Neovascularization has a critical role in the growth and metastatic spread of tumors, and involves recruitment of circulating endothelial progenitor cells (EPCs) from bone marrow. In this study, we examined whether EPCs could promote tumor angiogenesis, and found that the tumor growth was enhanced by the administration of EPCs.

Methods
To test the hypothesis that genetically modified bone marrow-derived EPCs can be effective carriers of therapeutic agents to tumor sites, we conducted human interferon-beta (HuIFN- b) gene transfection of EPCs with a virus vector in vitro .

Results
When HuIFN-b was applied in the ex vivo culture of EPCs, HuIFN- β-transduced EPCs achieved efficient killing of the total population of SPC-A1 cells, indicating a bystander effect was elicited by HuIFN- b-transduced EPCs in vitro . When SCP-A1 cancer cells were coimplanted along with ex vivo cultivated EPCs subcutaneous injection in nude mice, the tumor growth was increased. However, the anti-tumor effect of interferon-beta (IFN- β) offset the tumor-progressive character of EPCs and the tumor growth, and the vascular density of tumor tissues increased by coimplanted EPCs were decreased upon IFN- b treatment. In addition, overall expression levels of vascular endothelial growth factor in tumor tissues were decreased upon IFN-b treatment.

Conclusion
Our results suggest that gene-transfected EPCs could be useful as a tumor-specific drug delivery system.

Background
Mutant Kras was observed in more than 20% of non-small cells lung cancers (NSCLC) and represented one of the most dominant oncogene during cancer progression. However, there were fewer effective inhibitors which specific targeting of mutant Kras in clinical trials. The immunotherapeutic effects of Kras DNA vaccine in lung cancer were not well understood.

Methods
In this study, we investigated anti-tumor efficacy of mutant Kras DNA vaccine in spontaneous mouse lung tumor model driven by Kras[G12D]. TetO-Kras[G12D] and Scgb1a1-rtTA bitransgenic mice were treated with doxycycline to induce lung tumor for 5 months. To evaluate therapeutic effects of Kras DNA vaccine, bitransgenic mice with lung tumor were treated with wild-type Kras or mutant Kras[G12D] plasmids by skin administration.

Results
In this model, we found that skin administration of mutant Kras DNA vaccine had better therapeutic effects compared to control group. Additionally, dominant negative Kras (N17) DNA vaccine significantly decreased tumor nodules on lung in transgenic mice expressed mutant Kras. Th-1 immune response was dramatically enhanced in vaccine-treated mice compared with in control mice.

Conclusion
Overall, our results indicated that Kras DNA vaccine produced an effective anti-tumor response. Furthermore, skin administration of Kras DNA vaccine is effective as a potential approach for lung cancer therapy.

Background
Complete surgical resection of solid malignancies is often not achieved due to growth of occult tumour cells, distant metastases, or the invasiveness of the tumour mass. Hence there is a need for adjuvant therapies that may be administered following surgery to target residual tumour. Using a murine mesothelioma model we have previously shown that combining gemcitabine with anti-CTLA4 is effective at generating an anti-tumour immune response, leading to tumour regression and improved survival. We sought to use this treatment regimen to improve the post-surgical outcome of debulking surgery.

Methods
The murine mesothelioma line generated in our lab, AB1-HA, was inoculated subcutaneously into the flanks of Balb/c mice. Established tumours (<50mm[2]) were debulked by surgical removal of 75% of the tumour mass. Treatment with gemcitabine and anti-CTLA4 were commenced on the day of surgery, and mice were monitored for residual tumour outgrowth and survival. Size-matched controls were treated in parallel.

Results
Dual administration of gemcitabine with anti-CTLA4 was effective at causing regression of remaining tumour and improving survival following partial debulking surgery, over either therapy alone. The outcome of this treatment when administered to debulked tumours was comparable to that achieved against smaller, size-matched tumours.

Conclusion
We have shown that larger tumours can be successfully partially debulked and the remaining tumour treated using a combination of gemcitabine and anti-CLTA4. Thus, combined chemo-immunotherapy is a feasible option for post-surgical treatment option to remove residual tumours.

Background
Asbestos-induced mesothelioma in MexTAg transgenic mice closely resembles the key features of human disease. This model is highly suited to testing new chemotherapies and cancer prevention strategies. The resultant mesothelioma responds to cytotoxic chemotherapy with efficacy of the same order of responsiveness as human mesothelioma. The transgene, large T Antigen is an oncogene encoded by Simian Virus 40, and the role of this viral oncogene in tumourigenesis has been widely studied. Here we investigate the gene expression differences between TAg tumours and wild type tumours and examine overall similarity to human mesothelioma at the molecular level.

Methods
not applicable

Results
We found TAg expressing mouse tumours were 90% identical to wild type mouse tumours. The key pathway that was affected by these gene differences was cell cycle. Gene set enrichment analysis showed that the TAg:wild type tumour differences were homolgous to the Rb null genotype in a TAg dose dependent manner. To address whether transgenic mouse tumours were a good representation of human mesothelioma when compared to wild type mouse tumours, we showed that of genes differentially expressed between i) TAg or ii) wild type mouse tumours and mouse mesothelial cells, there were the same number of gene similarities with the set of genes that differentiate between human mesothelioma and human mesothelial cells. Human mesotheliomas commonly have a deletion of the cdkN2 locus, encoding the tumour suppressor genes p16 and p15. We found that while wild type mouse tumours contained the p16 deletion, TAg tumours did not.

Conclusion
In summary, the asbestos-induced mesotheliomas in MexTAg mice are comparable to human mesothelioma at the molecular level. We hypothesize that TAg expressing mice develop tumours in a more uniform way than wild type mice following asbestos exposure and are not dependent on deletion of p16 for tumourigenesis.

Background
Background: As a result of the recent deep molecular profiling of NSCLC samples, FGF receptor inhibition has emerged as a promising strategy for targeting a sub-set of squamous NSCLC (Sq NSCLC) tumours that carry FGFR gene amplifications, mutations and fusions. AZD4547 is a potent, orally available and selective inhibitor of FGFR 1, 2 and 3 and is currently in phase II clinical development.

Methods
not applicable

Results
Results: In pre-clinical patient derived models of FGFR1 amplified Sq NSCLC, AZD4547 is able to induce dose dependent tumour growth inhibition and tumour regression and this is correlated to FGFR1 protein expression and inhibition of signalling pathways downstream of FGFR1. Since the FGFR2 mutations described in Sq NSCLC do not present as clear codon hot-spots we also investigated a sub-set of the FGFR2 mutations using inducible expression in non-transformed cells. We found these mutations to be constitutively active and capable of inducing 3D colony formation in non-transformed cells. Both FGFR signalling and 3D colony growth were inhibited potently by AZD4547 treatment. We have developed a number of biomarker assays that enable both patient selection and exploratory analysis of patient samples. We found that FGFR1 gene amplified samples are enriched for those expressing both FGFR1 mRNA and protein. AZD4547 is currently being tested as a monotherapy in Sq NSCLC patients whose tumours carry FGFR1 amplification and we will describe preliminary observations from this trial including a comprehensive molecular profile of the tumour from a patient who experienced a durable partial response following AZD4547 treatment.

Conclusion
Conclusion: In view of the strong and emerging platform of evidence that implicates dis-regulated FGFR signalling in Sq NSCLC and the early evidence of clinical activity, FGFR inhibition warrants continued clinical investigation in patients whose tumours carry FGFR genetic lesions including amplification, mutation and gene fusions.

Background
Resistance to apoptosis is a hallmark of cancer that can be reversed by several chemotherapy drugs and targeted therapies, including cisplatin and erlotinib. In vivo imaging of apoptosis would be of great interest to study molecular mechanisms of drug activity and resistance. The aim of this study is to investigate whether in vivo micro-imaging of apoptosis could be used for early assessment of treatment response in a murine xenograft model of human lung cancer.

Methods
A549 (EGFR wild type), H1650 (carrying EGFR exon 19 deletion that confers sensitivity to Erlotinib) and H1975 (carrying EGFR mutations L858R and T790M, resistant to Erlotinib) cell lines were used to induce subcutaneous tumors in Nude mice. In vivo micro-imaging of apoptosis was performed using fibered confocal fluorescence microscopy (FCFM) after intra-venous injection of a fluorogenic caspase 3 substrate. Tumors were treated by cisplatin (10mg/kg) (5 mice, A549 xenografts), erlotinib (25mg/kg) (6 mice, 2 A549, 2 H1650, 2 H1975), or vehicle (6 mice, 2 A549, 2 H1650, 2 H1975).

Results
In A549 xenografts treated by cisplatin, apoptosis was detected in vivo at 24h post-treatment. Fluorescence intensity ratio (FIR) was significantly higher than in untreated tumors (16.9+/-3.1 vs 4.8+/-2.1 respectively, p<0.001). 24h after treatment by erlotinib, FIR in H1650 erlotinib sensitive tumors was significantly higher than in A549 tumors, and than in H1975 erlotinib resistant tumors (12.2+/-2.4 vs 6.1+/-1.2 vs 1.9+/-0.7 respectively, p<0.01, Figures 1 and 2). Results were confirmed ex vivo on harvested tumor xenografts by TUNEL assay and immunohistochemistry for activated caspase 3, and on cell lines in vitro by flow cytometry and fluorescence microscopy. Figure 1 Figure 2

Conclusion
Early in vivo micro-imaging of apoptosis using FCFM makes it possible to differentiate sensitive from resistant tumors to Erlotinib in a murine xenograft model of human lung adenocarcinoma.

Background
In second line setting there is no standard treatment for patients with mesothelioma, since all agents tested in clinical trials failed to improve survival. An individual patient however, may actually benefit from second line treatment, considering the patient population is heterogeneous. Yet, there are no predictive markers to identify those patients likely to respond to a certain drug. We developed protocols for in vitro drug sensitivity testing with several cytotoxic agents using primary tumor cells derived from pleural fluid of our patients. Recently, we implemented a personalized second line treatment protocol. Here we will report the outcome of the pilot study.

Methods
Cells were isolated from pleural fluid, drawn from patients with mesothelioma for symptom relief. Diagnosis of each mesothelioma patient was confirmed by regular pathological staining. Cells were plated and incubated with a five point concentration range of 5 single drugs and 2 two-drug combinations for 72 hours. Cell viability was determined by a metabolic assay. Each concentration point was measured in triplo and a biological duplo experiment was performed to check reproducibility. The drugs used in this screen were all previously tested in clinical mesothelioma trials. Patients with pleural fluid that were fit and progressed after standard first line treatment were considered for second line chemotherapy. Choice of second line treatment was based on screening results.

Results
Thirty-nine mesothelioma patients had pleural fluid drawn for culture of primary tumor cells and subsequent drug sensitivity screening. Drug screens were successful in 22 patients (56%). Drug sensitivity profiles were available within two weeks after isolation of tumor cells, which is appropriate for clinical decision making. Agents tested for were cisplatin or carboplatin, pemetrexed, gemcitabine, vinorelbine, oxaliplatin, a combination of cisplatin and pemetrexed and of oxaliplatin and gemcitabine. Individual dose-response curves showed different sensitivity to the various cytotoxic agents. Ten patients were chemo-naive at the time of the drug sensitivity screen. Five of them received first line chemotherapy (cisplatin and pemetrexed). Two of them had progressive disease. Both demonstrated evident resistance to cisplatin and pemetrexed in their drug sensitivity profile. Four patients received second line treatment based on the drug sensitivity profile of their primary tumor cells. To date, treatment response is evaluated for two patients. Both patients received a combination of oxaliplatin and vinorelbine. The first patient had a clinical response. For the second patient, oxaliplatin/vinorelbine was the best option, although her in vitro profile suggested a rather resistant tumor. She was progressive and showed an unusual large amount of necrosis on repeated CT imaging. Patient three and four are currently treated with oxaliplatin and gemcitabine and gemcitabine monotherapy, respectively. Their treatment responses have to be awaited.

Conclusion
Personalized drug screening using primary tumor cells is feasible within a clinically relevant time period and may yield new treatment combinations with better responses.

Background
Lung cancer ranks among the most commonly occurring malignancies, and is the leading cause of cancer-related mortality worldwide. Chemotherapy for lung cancer can be made more specific to tumor cells, and less toxic to normal tissues, through the use of ligand-mediated drug delivery systems.

Methods
For the screening of targeting peptides, a phage-displayed peptide library underwent affinity selection with lung cancer cells. Targeting phage clones were selected by ELISA, immunofluorescence, flow cytometry and an in vivo homing assays. Targeting peptides can be used to develop ligand-mediated targeted therapy or targeting imaging probe. For preclinical study, we evaluated therapeutic efficacy of targeting liposomes using xenograft, orthotopic- and metastatic-tumor animal models

Results
In this study, we investigated the targeting mechanism of the ligand-mediated drug delivery system using lung cancer targeting peptides. Conjugation of lung cancer targeting peptides to liposomes enhanced the amount of drug delivered directly into NSCLC cells, through receptor-mediated endocytosis. Accumulation of peptide-conjugated liposomal doxorubicin (SP-LD) in tumor tissues was 11.2-fold higher than that of free doxorubicin, and the area under the concentration-time curve (AUC0-72 hours) was increased 159.2-fold. Furthermore, SP-LD enhanced therapeutic efficacy and increased the survival rate of tumor-bearing mice in syngenic, metastatic and orthotopic animal models.

Conclusion
The current study suggests that tumor-specific peptides may be used to create chemotherapies specifically targeting tumor cells in the treatment of NSCLC. The use of lung cancer targeting peptide-conjugated liposomes enhances pharmacokinetic properties, improves efficacy and safety profiles, and allows for controlled biodistribution and drug release.

Background
Lung cancer is the major cause of cancer death in the world while non small cell lung cancer (NSCLC) accounts approximately 85% of all lung cancer diagnosis. EGFR mutations, found in 10–30% of patients with NSCLC characterize a subpopulation with exquisite sensitivity to EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, the clinical benefits of first-generation TKIs (like gefitinib or erlotinib) can be further improved because of the development of drug-acquired resistance within 10–14 months in patients who initially respond to the treatment. Therefore, there is a need to discover next generation medicines as EGFR-TKIs for NSCLC patients.

Methods
Our EGFR program was first started to screen 20,000 in-house compounds for EGFR (wild type, 32D- EGFRWild-Type) activity in EGFR-transfected 32D cells and further performed knowledge-based design. More than 300 analogues were synthesized in this series and US and ROC patents were granted in 2013 and PCT patent application is under examination. We had identified DBPR112 as a potent EGFR-TKI with oral in vivo activity in a mouse model for lung adenocarcinoma.

Results
DBPR112 showed IC50 of 2 nM in HCC827 cells and potent EGFRWild-Type (IC50: 10 nM) and EGFRL858R/T790M (IC50: 70 nM) kinase inhibition which are better than gefitinib and similar to that of BIBW2992 (Afatinib, developed by Boehringer Ingelheim) that is currently under phase III clinical trial. DBPR112 was orally (F = 49%) administered against the growth of human lung HCC827 tumors subcutaneously xenografted in nude mice. A dramatic reduction of the tumor size was noted in the tumors treated with DBPR112 without significant loss of body weights in the nude mice. In addition, the pharmacokinetics properties of DBPR112 are superior to those of Afatinib.

Conclusion
Considering the fact that EGFR kinase plays an important role in lung adenocarcinoma with drug-sensitive mutations in EGFR, our goal is to develop novel and potent EGFR kinase inhibitors for the treatment of lung cancer, either as a single agent or in combination with existing cancer treatment. DBPR112 is a highly potent small-molecule against various forms of EGFR kinase. The non-clinical profile of DBPR112 showed promising in vivo efficacy. We, therefore, propose to design a preclinical program to assess the developability of DBPR112 and conduct comprehensive pre-clinical studies. We hope to make DBPR112 an investigational new drug for the treatment of lung cancer in the near future.

Background
Patients with non-small cell lung cancer (NSCLC) who have activating epidermal growth factor receptor (EGFR) mutations derive remarkable benefit from treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKIs). But drug-resistant problem appears sequentially and novel therapeutic strategies should be explored to overcome EGFR-TKI resistance. As the first EGFR-TKI in China and the third in world, icotinib shows good efficacy and tolerability in patients with advanced NSCLC. This study aims to establish icotinib resistant cell line-HCC827/IR and analyze it’s biological character for further study of EGFR-TKIs resistance.

Methods
HCC827 is a cell line with a deletion in the exon 19 of EGFR gene. HCC827 cells were exposed to increasing concentrations of icotinib (10nM to 20uM). Cells with the ability to grow in 20uM of icotinib were obtained 8 months after the initial drug exposure. The cell proliferation, viability, distribution of cell cycle, EGFR gene sequence (exon 18, 19, 20 and 21), EGFR-TKIs cross-resistance and the response to a histone deacetylases inhibitor (Chidamide, CS055) were evaluated after allowing the cells to grow in drug-free conditions for 2 months.

Results
Population doubling time (PDT) of HCC827/IR was not different from HCC827 (32.3±6.0h vs. 36.3±2.4h, P=0.198). In the cell cycle distributions of HCC827/IR, the cell number in G0/G1 phase were decreased (P=0.035), but the cell number in S and G2/M phase had no significant change compared with parent cells (P=0.388 and P=0.205, respectively). The resistance index (RI=HCC827IR~IC50~/ HCC827~IC50~) of HCC827/IR to icotinib was (1.98±0.15)×10[3]. And HCC827/IR cells also showed high resistance to the other two EGFR-TKIs (gefitinib and erlotinib), the RI of gefitinib and erlotinib was (2.36±0.082)×10[3 ]and (1.069±0.004)×10[3], respectively. But, the sequence of EGFR gene did not changed before and after resistance to EGFR-TKIs. In addition, HCC827/IR was sensitive to CS055 (IC~50~=254±32nM).

Conclusion
This study successfully established icotinib resistant NSCLC cell line-HCC827/IR. HCC827/IR cells had some different biological characters compared with parent cells and showed high cross-resistance to other EGFR-TKIs, but it was sensitive to CS055. The results could provide experimental reference for clinical application of TKIs and provide cell line model for further study of TKI-resistance.

Background
ROS1 is a receptor tyrosine kinase that regulates pathway signaling related to cell proliferation, growth and survival. Chromosomal rearrangements involving ROS1 have recently been identified as potential driver mutations in non-small-cell lung cancers (NSCLC) and preclinical and clinical studies indicate a significant response to crizotinib in tumor cells harboring this mutation. We developed an RT-PCR assay for all ROS1 fusion gene transcripts described in NSCLC to date which is suitable for use with FFPE tissue.

Methods
Primers and probes have been specifically designed to detect 14 known fusion transcripts from seven different fusion partners with the ROS1 gene using six multiplexed reactions. These fusion gene pairs include: CD74-ROS1, SLC34A2-ROS1, GOPC (FIG)-ROS1, SDC4-ROS1, EZR-ROS1, TPM3-ROS1, and LRIG3-ROS1. The 14 different ROS1 fusion products were represented by specifically designed and manufactured pCR 2.1-TOPO and pZErO 2.1 plasmids. After screening and identifying ROS1 rearrangements in clinical samples, the specific variants were further differentiated by gel electrophoresis and sequencing.

Results
All 14 ROS1 fusion genes could be detected using this RT-PCR assay. We screened 578 FFPE lung cancer samples that were negative for EGFR, KRAS and ALK mutations and detected 12 ROS1 rearrangements in this cohort (2.08%). All positive tumors were adenocarcinomas of the lung, 75% of the patients were female and median age was 53 years. The ROS1 rearrangements that were identified are: SLC34A2 (Exon 13)-ROS1 (Exon 32 and 34), CD74 (Exon 6)-ROS1 (Exon 34), EZR (Exon 10)-ROS1 (Exon 34) and SDC4 (Exon 2)-ROS1 (Exon 32). Messenger RNA expression of ROS1 was significantly higher in ROS1 positive samples than wild-type samples (median 5.2 vs.1.0; p<0.001). An inter-assay comparison of 31 samples containing four ROS1 positives using RT-PCR and FISH showed 100% concordance.

Conclusion
The clinical relevance of ROS1 testing in NSCLC and potentially other solid tumors as a predictive biomarker for sensitivity to crizotinib is rapidly increasing. We developed a multiplexed RT-PCR assay that enables the detection of ROS1 fusion genes using FFPE tissue and provide a rapid and cost effective screening tool.

Background
With existing therapeutic efforts, patients with lung cancer have a poor prognosis. Assessment of tumor size, lymph node status and presence of metastases is currently applied for determining prognosis and treatment modality, but predicted and real outcomes can vary significantly. Biomarkers with reliable prognostic significance are therefore of utmost importance but due to a lack of immediate correlation between levels of protein and their corresponding mRNA, a screen based on the kinase activity become a promising option to circumvent this limitation with the tremendeous advantage of focusing on therapeutically targetable enzymatic activities. Several protein tyrosine kinase inhibitors already clinically approved for the treatment of lung cancer are targeting some of the 400 types of DNA signatures described in silico in the human genome. The aim of this study is to clarify the following hypothesis: Is the in vitro multiplexed tyrosine phosphorylation of substrates a possible approach to molecularly classify the kinome of early stage lung adenocarcinoma biopsies and obtain a diagnostic /prognostic signature correlating with the survival of patients?

Methods
We have built a tumor bank of frozen malignant and non-neoplastic lung surgically obtained specimen and recorded all clinical interventions, follow up treatments and outcome for each of our patients. We incubated TNM stage 1 and 2 lung adenocarcinoma kinomes on PamChip®4 microarrays and followed the kinetics of the multiplexed tyrosine phosphorylation for 144 peptides substrates. Image quantification, quality control, statistical analysis and interpretation of data were performed with the BionavigatoR software.

Results
We screened 84 paired malignant TNM stage 1 and 2 lung adenocarcinoma and non-neoplastic lung biopsies for the multiplex tyrosine phosphorylation of substrates immobilized on a PamChip®4microarrays. Based on a 76-point ‘response-signature’ we obtained 73 % of correct prediction with a 10 fold cross validation PLS-DA analysis in TNM stage 1 lung adenocarcinoma biopsies. Moreover, we detected 26 peptides substrates significantly more inhibited in kinomes of long-term survivors than in kinomes of the short-term survivors.

Conclusion
In frozen biopsies of TNM stage I adenocarcinoma and with a PLS-DA analysis applied to a 76-point ‘response-signature’ we present the feasibility to discriminate between long-term and short-term survivors. Furthermore, the found differences in enzymatic activities in lung biopsies may result in the identification of new targets in future anti lung cancer therapy efforts.

Background
In this study, we attempted to detect EGFR and KRAS mutations in plasma/pleural effusion of patients with advanced NSCLC by pyrosequencing, to investigate whether plasma and pleural effusion DNA could be used as a substitute for tumor tissues for detecting gene mutations, and to explore the correlation of EGFR/KRAS mutations with the efficacy of the epithelial growth factor receptor-tyrosine kinase inhihitor (EGFR-TKI) as well as their correlation with survival in TKI-treated patients.

Methods
Blood, pleural effusion and tumor tissues were obtained from 146, 64 and 63 patients with advanced NSCLC, of whom there were 40 matched tissue and plasma samples, 24 matched tissue and pleural effusion samples. The exons 19, 20 and 21 of EGFR was amplified by mutant-enriched PCR using selective restriction enzyme digestion, and exon 2 of KRAS in plasma were amplified by nested-PCR. Then mutations were detected by pyrosequencing. The association between mutations and the patients’ survival was analyzed using Kaplan-Meier.

Results
EGFR mutations were detected in 34.38% tissues (22/64), 24.24% plasma samples (24/96) and 30.16% pleural effusion samples(19/63). KRAS mutations were detected in 4.69% tissues (3/64), 6.16% plasma samples (9/146) and 7.93% pleural effusion samples. No statistical significance was found in EGFR/KRAS mutations between plasma/pleural effusion and tumor tissues (p>0.05). The same EGFR genes were observed in plasma and matched tissue in 34 patients (consistency: 85%).The sensitivity of detecting EGFR mutations is 73.33% and the specificity is 92 %.There was only 1 KRAS mutation detected in the 40 tissues , but no mutation in the matched plasma (consistency: 97.5%). Same EGFR/KRAS genes were observed in 21, 23 pleural effusion and matched tissue respectively (consistency: 87.5%, 95,83%). The sensitivity of detection of EGFR/KRAS mutations is 77.78% and 66.67%. The specificity is 93.33 % and 100%. Significant correlation existed between EGFR mutations in tumor tissue/pleural effusion from patients with advanced NSCLC, and a smoking history, and histopathologic type (p<0.05). Among the 38 TKI-treated patients, the disease control rate (DCR) and objective response rate (ORR) were 90% and 60%, respectively, in patients with EGFR mutation in plasma, and 53.57% and 17.86%, respectively, in patients with wide-type EGFR (DCR, p=0.059; ORR, p=0.019).The DCR and ORR were 66.67% and 33.33%, respectively, in patients with wide-type KRAS in plasma, and 40% and 0%, respectively, in patients with KRAS mutation. Patients with EGFR activating mutations in plasma had a favoring median PFS of 10.5 months, significantly longer than the patients with wild-type EGFR (5.0 months) (p=0.228). The median PFS was 2.5 months for patients with KRAS mutation and 9 months for patients with wild-type KRAS, respectively (p=0.000).

Conclusion
A high consistency exists between EGFR/KRAS mutation detection in plasma/pleural effusion and tumor tissues. Plasma/pleural effusion could be used as a substitute for tumor tissues for detecting gene mutations. EGFR and KRAS mutations in plasma are highly associated with the treatment response and prognosis of TKI-treated patients. It is feasible to detect EGFR and KRAS mutations in plasma/pleural effusion by pyrosequencing. The detection sensibility of EGFR mutations can be increased by ME-PCR, facilitating choosing patients for TKI treatment.

Background
The elevated serum level of MUC1 (KL-6, CA15.3) in lung cancer patients is considered to be a marker for advanced adenocarcinoma and a prognostic factor for disease progression after treatment. The aim of this study was to evaluate the serum MUC1 levels in primary lung cancer patients in the Russian Federation, a large portion of which are patients with squamous cell lung cancer (SCC).

Methods
Serum samples were obtained from 346 patients at admission. Two hundred ninety three patients were diagnosed with a malignant lung tumor (MLT), i.e. SCC (n=136); adenocarcinoma, AC (n=89); adenosquamous cancer, ASqC (n=21); bronchioloalveolar carcinoma, BAC (n=12); large cell carcinoma, LCC (n=3); small cell cancer (n=4); anaplastic cancer (n=8); carcinoid (n=17); sarcoma (n=3). A non-malignant lung disease (NMLD) was confirmed histologically in 53 patients: benign tumors (n=10), pneumofibrosis (n=13), tuberculosis (n=9), a resolution stage of pneumonia (n=21). Anti-MUC1 ICO25 monoclonal antibody-based and validated inhibition enzyme-linked immunosorbent assay was used (normal range 9 - 38 U/ml, median 24 U/ml).

Results
An occurrence of the elevated serum MUC1/ICO25 levels (³40 U/ml) in patients with MLTs was significantly higher than in patients with NMLDs (151 [51.5%] vs. 7 [13.2%] cases, correspondingly; р<0.001), and it positively correlated with clinically aggressive tumor histology: AC, 61.0%; ASqC, 61.9%; SqCC, 44.9%; BAC, 25.0%; LCC and small cell cancer, 100%; anaplastic cancer, 50.0%; carcinoid, 23.5%; sarcoma, 0%. The significantly elevated serum MUC1/ICO25 levels (³60 U/ml) were found in 81 (27.6%) patients with MLTs and 1 (1.9%) patient with NMLDs. In patients with nonsquamous non-small cell lung cancers (AC, BAC, LCC) and ASqC the serum MUC1/ICO25 ³60 U/ml were found in 44.0% (55/125) of cases. The rate of these cases positively correlated with the advanced TNM stage and depended on the patients’ age: statistically significant difference between stages I-II and stages IIIB-IV was found in patients aged 65 years and older (15.8% [3/19] vs. 84.2% [16/19]; p<0.001), but not in patients aged less than 65 years (28.6% [8/28] vs. 48.6% [17/35]; p=0.127). In patients with SCC the overall rate of serum MUC1/ICO25 ³60 U/ml was 16.9% (23/136), and the reversed age-related dependence of those levels on the tumor stage was bserved. In patients aged less than 65 years the higher rate of the serum MUC1/ICO25 ³60 U/ml correlated with more advanced TNM stage: 0% (0/13), 13.8% (4/29),28.6% (6/21) and 52.9% (9/17) for stages I, II, IIIA and IIIB-IV, respectively (I-II vs. IIIB-IV, p<0.001). In patients with SCC aged 65 years or older the serum MUC1/ICO25 ³60 U/ml was found in 0% (0/19), 30.0% (3/10), 7.7% (1/13) and 0% (0/14) of cases for stages I, II, IIIA and IIIB-IV, respectively. In the entire group of patients the rate of the serum MUC1/ICO25 ³60 U/ml did not depend on the tumor histological grade, local tumor extension, location of distant metastases or comorbidities.

Conclusion
The abnormal MUC1/ICO25 serum levels correlate with unfavorable prognostic clinical characteristics of the lung cancer, and its potential clinical significance is not restricted to the cases of lung adenocarcinoma.

Background
Recent advances in molecular profiling of non-small cell lung cancer (NSCLC) have led to the replacement of platinum-based chemotherapy with targeted therapies for certain genetic subsets of NSCLC (ALK rearrangements, some EGFR activating mutations). It is also known that myriad pathways can drive resistance, the unfortunate norm for most patients. A greater understanding of the overlap across multiple biomarker subsets, including activating mutations, signal transduction pathways, and immune system markers, might aid in prognostic assessment, predictive biomarker development and the design of combination or sequential treatment regimens.

Methods
The prevalence and prognostic significance of nine biomarkers (TTF1, p63, EGFR mutation, KRAS mutation, MET immunohistochemistry [IHC], PDL1 IHC, PTEN IHC, NaPi2B IHC, ECDH IHC) across two independent sample sets (Set 1, n=561; Set 2, n=300) were tested. With the exception of ECDH, all assays were IVD or companion diagnostics. Set 1 was collected from patients who were eligible for surgery with curative intent from 2003–2005 at MD Anderson Cancer Center in the USA. Samples from Set 2 were part of a collaboration between the University of Colorado Cancer Center, USA and The Norwegian Radium Hospital, and contained surgically-resected NSCLC tissues collected from 2006–2011.

Results
The prevalence of each biomarker varied significantly by histology. For adenocarcinoma samples, the prevalence of each biomarker was: EGFR mutation (13%), KRAS mutation (29%), TTF1 IHC (83%), p63 IHC (7%), MET IHC (50%), PDL1 IHC (45%), PTEN loss IHC (11%), NaPi2B IHC (76%), EGFR IHC (FLEX cut-off, 11%). In squamous-cell carcinoma, the prevalence of each biomarker was: TTF1 IHC (2%), p63 IHC (87%), MET IHC (13%), PDL1 IHC (50%), PTEN loss IHC (13%), NaPi2B IHC (3%), EGFR IHC (FLEX cut-off, 40%). In addition, more than 67% of patients were positive for more than one biomarker and >33% were positive for at least three biomarkers. The diagnostic criteria for each biomarker and correlations with patient characteristics will be described in further detail. Figure 1. Biomarker Overlap in Adenocarcinoma in Set 1 (n=337) Figure 1

Conclusion
Collectively, these data suggest that the biomarker landscape in NSCLC is complex and will be increasingly dynamic as more experimental agents approach pivotal testing. Grant support: this study was partially funded by UT Lung Specialized Programs of Research Excellence grant (P50CA70907; IIW)

Background
Apoptotic pathways are controlled by activation or inhibition of the intracellular caspases. Inhibitor of apoptosis proteins (IAPs) are endogenous inhibitors of caspase activity. We aimed to investigate the relationship of the apoptosis regulators X-linked inhibitor of apoptosis (XIAP) and ubiquitin specific protease 8 (USP8) with clinical parameters, survival and response to chemotherapy in advanced stages of non-small cell lung cancer (NSCLC).

Methods
A total of 34 NSCLC patients (28 males, 6 females) and 44 healthy volunteers were included in the study. Most of the patients had squamous cell histology (62%), while others had adenocarcinoma (29%) and unclassified (8%) types. All patients had locally advanced stage IIIA-IIIB (50%) or metastatic (50%) disease. XIAP and USP8 levels were measured by ELISA method.

Results
The median serum XIAP levels were similar in patients and the controls (p=NS). USP8 level was higher in patients compared to the control group (p<0.0001). In univariate analysis, there was no significant relationship between XIAP and USP8 serum levels and age, sex, performance status, weight loss, stage of disease, histopatological type and response to chemotherapy. In the low and high XIAP and USP8 groups, there was no significant difference in progression-free survival (PFS) (p=0.432 and p=0.50, respectively) and overall survival (OS) (p=0.989 and p=0.90, respectively).

Conclusion
No relationship was found with serum XIAP and USP8 levels and clinical parameters, response to chemotherapy, PFS and OS in patients with advanced stages of NSCLC.

Background
The prognostic role of signal transducer and activator of transcription 3 (STAT3) and phosphorylated STAT3(p-STAT3) in non-small-cell lung cancer (NSCLC) remains controversial. To clarify its impact on survival, we performed a meta-analysis to quantitatively assess STAT3 and p-STAT3 expression on prognosis of NSCLC.

Methods
Published studies were identified using a systematic and thorough literature search.To be eligible, a study had to investigate STAT3 or p-STAT3 expression rates of NSCLC patients in different characteristics and survival data of STAT3 or p-STAT3 expression.

Results
A total of 17 retrospective trials were chosen for meta-analysis, including 1793 patients.We showed that the estimated pooled HR (0.67, 95%CI: 0.57-0.77) of 9 trials (STAT3: HR 0.71, 95%CI 0.38-1.04; p-STAT3: HR 0.67 ,95%CI 0.56-0.77) for NSCLC was statistically significant (P<0.0001), suggesting that high STAT3 or p-STAT3 expression is a strong predictor of poor prognosis among patients with NSCLC.For the risk factors, pooled analysis of patients with STAT3 positivity, demonstrated a statistically significant OR (3.82, 95%CI: 2.37-6.16) between poorly differentiated carcinoma and well-moderately, OR( 5.68, 95%CI: 3.16-10.21)between patients in stage III-IV and patients in stage I-II, and OR (3.41, 95%CI: 2.12-5.49 ) between patients with lymph node metastasis and patients without lymph node metastasis. However, pooled analysis of patients with p-STAT3 positivity, only demonstrated a statistically significant OR (4.51, 95%CI: 1.57-12.96) between poorly differentiated carcinoma and well-moderately(P<0.05).

Conclusion
High STAT3 or p-STAT3 expression is a strong predictor of poor prognosis among patients with NSCLC. The conclusion should be confirmed by large prospective studies with long-term follow-up.

Background
EGFR-activating mutations are predictive of high response rates and overall survival gains in patients (pts) with pulmonary adenocarcinoma, treated with EGFR- tyrosine-kinase inhibitors (EGFR-TKIs), as erlotinib. Mutations in the EGFR gene, especially in the exon 20 (T790M), are related to resistance to EGFR-TKIs. We investigated if a polymorphic DNA sequence in exon 20 (Q787Q, NCBI database 162093G>A, SNP ID: rs1050171) was associated with clinical outcomes in pulmonary adenocarcinomas, treated with erlotinib.

Methods
It is a prospective, observational study on all consecutively pts whose tumors were genotyped for EGFR-activating mutations. Tumor samples were formalin-fixed and paraffin-embedded. Tumor areas were selected and macrodissected, followed by whole DNA extraction and amplification by PCR. EGFR genotyping was performed through DNA sequencing (exons 18, 19, 20 and 21) by Sanger´s methodology. Pts with adenocarcinomas harbouring EGFR-activating mutations were treated with erlotinib.

Results
191 pts had tumor samples genotyped between Aug/2011 and Apr/2013. Median age was 64 y (17-90), 106 (56%) female. According to ethnicity, 154 pts were Caucasian (81%), 26 African-American (14%) and 11 Asian (6%). Seventy pts were classified as never-smokers (37%), 23 (12%) as light-smokers (≤ 10 p.y.) and 95 as current smokers (51%). EGFR activating mutations could be identified in 54 out of 191 samples (28%): 35 were exon 19 deletions (65%), 15 were L858R mutation in exon 21 (30%), and three were rare mutations (G719S and G719A in exon 18, and V774M in exon 20). Polymorphism Q787Q in EGFR gene (exon 20) was detected in 108 samples (56.5%). The polymorphic status did not correlate with gender (p=0.324), smoking status (p=0.810) or EGFR mutational status (p=0.238), but it was more frequently detected in Caucasian pts (p=0.0002). Considering all 191 studied pts, no difference in median overall survival was detected according to polymorphic status (19.6 mo. vs. 24.3 mo., HR 0.86; 95% CI 0.54-1.38, p=0.541). There was no difference in response rate to erlotinib according to the polymorphic status (p=0.248). In addition, no difference in median overall survival was detected according to polymorphic status among the 38 pts treated with erlotinib and presenting EGFR-activating mutations (not reached in both groups, HR 2.44; 95% CI 0.31-16.01, p=0.425).

Conclusion
Polymorphism Q787Q in EGFR gene (exon 20) was commonly detected in pulmonary adenocarcinomas (56.5%), being more frequent in Caucasian pts. The presence of polymorphic status was neither related to sensitivity to erlotinib, nor to survival outcomes in our pts.

Background
Identification of actionable driver mutations in non-small cell lung carcinoma (NSCLC) has become increasingly important for the prioritisation of targeted therapies. Mutational analysis of formalin-fixed paraffin embedded (FFPE) tissues presents several challenges including generally limited and fragmented DNA, the need to identify a range of biologically significant mutations and a pressing need for a fast turn around time at a cost-effective way. Our aim was to determine optimal methods for quantification of DNA for mutational analysis in NSCLC and to develop a new custom assay that could perform multigene mutational analysis on the limited quantity of DNA available in the small NSCLC samples frequently submitted for testing.

Methods
DNA was extracted from FFPE tissues including cytology specimens. Spectrophotometry quantification was compared with Qubit 2 Fluorometer measurements and the Sequenom SampleID assay for accurate and meaningful assessment of extracted DNA for diagnostic mutational profiling. We have previously established a diagnostic protocol for somatic mutation profiling in NSCLC using a commercial DNA mass spectrometry kit (Oncocarta v1.0) and compared it with a new custom kit “OncoFocus” developed in collaboration with Sequenom. These assays utilise target amplicons of small sizes for efficient amplification in fragmented DNA and simultaneously profile a range of actionable mutations in EGFR, KRAS, BRAF and NRAS. Preliminary verification of the “OncoFocus” assay was performed in 27 NSCLC samples, 3 lung cancer cell lines and 2 control genomic DNA samples.

Results
We found spectrophotometry significantly overestimated DNA quantity particularly at low concentrations. We also studied the correlation of DNA quantities with estimated copies of DNA templates as determined by SampleID. The results suggested that a minimum of 300 ng DNA is needed to achieve the required 300 – 500 amplifiable genomic copies per reaction for the OncoCarta analysis, which remains difficult to achieve for many diagnostic NSCLC samples. We developed a more focused diagnostic panel “OncoFocus” which could be performed reliably with less DNA but which includes key actionable mutations in 159 hotspots in EGFR (n=109), KRAS (n=17), BRAF (n=15) and NRAS (n=18) requiring only 150 ng of DNA. Somatic mutations were identified in 23 samples and 3 cell lines including EGFR (n=22), KRAS (n=6) and BRAF (n=1). No false positive results were observed in 4 FFPE and 2 control samples. The whole process from the receipt of FFPE samples to issuing a report can be completed within 5 working days and the “OncoFocus” panel has increased our capacity per chip (iPLEX II) from 15 to 31 samples. The “OncoFocus” panel also results in decreased per sample testing costs.

Conclusion
The Qubit fluorometer is a more reliable and accurate method to quantify DNA derived from FFPE for mutational analysis than spectrophotometry. We also conclude that DNA mass spectrometric analysis using a new custom “OncoFocus” panel is an effective and efficient test that simultaneously detects 159 mutational hotspots, in the generally lower quantity of DNA obtained from routine FFPE NSCLC samples.

Background
Patients’ EGFR mutation status prior to treatment impacts outcomes and, EGFR testing has been developed as a companion diagnostic; this relationship between therapeutic and diagnostic agents is known as personalized healthcare. Recently, it was reported that about half of patients may acquire resistance to EGFR-TKIs following therapy, mainly by appearance of EGFR mutations, such as T790M. Thus, it is important to assess EGFR mutation status before and during treatment to determine the most appropriate treatment regimens for patients. A number of PCR-based techniques are used in the assessment of EGFR mutations. In Japan, the “Scorpion-ARMS” therascreen® EGFR Rotor-Gene Q PCR Kitis (therascreen EGFR assay) the only available in vitro diagnostic (IVD) test. The cobas® EGFR Mutation Test (cobas EGFR assay) is the only FDA-approved kit for IVD testing in the US.In this study, we compared the performance of the cobas EGFR assay and the therascreen® EGFR assay using FFPE tissue specimens from NSCLC patients.

Methods
We extracted DNA from 149 FFPE tissues of NSCLC, according to the manufacturer’s instructions and performed a comparative study of cobas EGFR and therascreen EGFR methods.

Results
EGFR mutations were identified in 63 NSCLC specimens (42.3%) using the cobas EGFR assay and 61 samples (41.2%) using the therascreen EGFR assay. The concordance rate between the cobas EGFR assay and therascreen EGFR assays was 145/149 (97.3%). Only three discordants between these EGFR assays were observed. One T790M mutation in combination with an L858R mutation was identified by the cobas EGFR assay. No invalid assay results occurred with the cobas EGFR assay.

Conclusion
The cobas EGFR assay has two advantages. One is that the process is easily performed by stable methods. It takes only 8 hours, from tumor specimen to results generated using the semi-automated system. Thus, patients assessed using the cobas EGFR assay can begin the most appropriate treatment quickly. The other advantage is that only a very small amount of DNA (150 ng) is required to detect mutation status using the cobas EGFR assay. Our results show a high concordance rate (97.3%) of cobas EGFR with an existing IVD product, the therascreen EGFR assay. In this study, one double mutant, T790M in combination with L858R, was only identified by the cobas EGFR assay. The cobas EGFR assay appears to give the most accurate and appropriate results for NSCLC patients.

Background
The 5-year survival for stage IB non-small cell lung cancer (NSCLC) is only 55%, but the benefit of adjuvant chemotherapy in this setting remains equivocal. Numerous prognostic markers have been examined, but none to date have moved into clinical practice. There is an urgent need to identify novel molecular markers that can select high risk patients, who may potentially benefit from adjuvant chemotherapy.

Methods
We identified 471 consecutive patients with stage IB primary NSCLC according to the American Joint Commission on Cancer, (AJCC) 6[th] edition tumour-node-metastasis staging system, who underwent surgical resection between 1990 and 2008. Patients who received neoadjuvant or adjuvant treatments were excluded. Pathology reports were reviewed and pathologic characteristics were extracted. Expression of phosphorylated Akt (pAkt) in both cytoplasmic and nuclear locations was assessed by immunohistochemistry, and clinicopathologic factors were analyzed against 10-year overall survival using Kaplan-Meier and Cox proportional hazards model.

Results
455 (96.6%) cancers were adequate for pAkt immunohistochemical analysis. The prevalence of pAkt expression in the cytoplasm and nucleus of the cancers was 60.7% and 43.7% respectively. Patients, whose cancers expressed higher levels of pAkt in the cytoplasm, had a trend towards longer overall survival than those with lower levels of cytoplasmic pAkt (p=0.06). Conversely, patients whose cancers expressed higher levels of pAkt in the nucleus had a poorer prognosis than those with lower levels of nuclear pAkt expression (p=0.02). Combined low cytoplasmic/high nuclear expression of pAkt was an independent predictor of overall survival [HR=2.86 (95% CI:1.35-6.04); p=0.006] when modeled with age [HR= 1.05 (95% CI: 1.03-1.07); p<0.001], extent of operation [HR= 2.11 (95% CI: 1.48-3.01); p<0.001], visceral pleural invasion [HR=1.63 (95% CI: 1.24-2.15); p<0.001], gender, tumour size, histopathologic type and grade (p>0.05).

Conclusion
Levels of expression of pAkt in the cytoplasm and nucleus and visceral pleural invasion are independent prognostic factors that can help to select patients with high risk disease, who may potentially benefit from adjuvant chemotherapy.

Background
Activating EGFR mutations confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs) in patients with NSCLC, but responses are transient, with delay in disease progression but no impact on survival. Concomitant genetic alterations could account for these incomplete clinical responses. Erlotinib-treated EGFR-mutant NSCLC patients harboring the EGFR T790M mutation had shorter PFS than those without the mutation (12 vs 18 months [m]). Low BIM levels were associated with gefitinib resistance in EGFR-mutant NSCLC.

Methods
The efficacy results of the EURTAC trial were updated at January 24, 2013. We have evaluated the frequency and potential impact of pretreatment EGFR T790M mutations and BIM mRNA expression in 95 patients with EGFR-mutant NSCLC included in the EURTAC trial.

Results
T790M mutations were detected in 65.26% of patients. PFS to erlotinib was 9.7 m for those with T790M mutations and 15.8 m for those without, while among patients receiving chemotherapy, it was 6 and 5.1 m, respectively (P<0.0001). BIM expression was successfully analyzed in 83 patients. PFS to erlotinib was 12.9 m for those with high BIM levels and 7.2 m for those with low/intermediate BIM levels, while among chemotherapy-treated patients, it was 5.8 and 5.5 m, respectively (P=0.0003). OS was 28.6 m for patients with high BIM expression and 22.1 m for those with low/intermediate BIM expression (P=0.0364). The multivariate analyses showed that erlotinib was a marker of longer PFS (HR, 0.35; P=0.0003), while high BIM expression was a marker of longer PFS (HR, 0.49; P=0.0122) and OS (HR, 0.53; P=0.0323).

Conclusion
BIM mRNA expression is a biomarker of PFS and OS in EGFR-mutant NSCLC. T790M mutations and BIM mRNA expression can potentially be used for designing combination therapeutic strategies for use in lieu of EGFR TKI monotherapy.

Background
In 3% of cases, lung cancer is diagnosed in patients younger than 45 years. The epidemiology, biology and clinical history of young lung cancer patients is generally different from the adult counterpart: the higher percentage of mutations has the potential to influence both tolerability and response to treatment with consequent impact on quality of life and survival. The biomolecular characterization of the disease in this subgroup will allow the design of clinical studies dedicated to young patients, that will lead to the identification of specific items that are not deducible from trials opened to the general adult population. In this study, Next-Generation Sequencing (NGS) technology has been applied to archival tissue samples to enhance tumor-specific genomic profile knowledge in this selected cohort of young patients (pts).

Methods
A retrospective analysis has been performed at the Thoracic Unit of San Luigi Hospital from January 2007 to March 2013, collecting 13 lung cancer-diagnosed pts (10 completely sequenced; in 3 cases the analysis is ongoing), aged between 15-39 years. Genomic DNA was extracted by microdissected formalin-fixed and paraffin embedded (FFPE) tumor samples of all pts and by lymphocytes of three healthy controls (ctrl). Amplicons NGS libraries for 46 oncogenes included in the Ion Torrent Cancer Panel were generated, following manufacture guidelines, and sequenced in Personal Genome Machine (PGM) Ion Torrent instrument. Variant Caller included in Torrent Suite Software was used to identify mutations.

Results
Twenty-two non-synonymous, 3 frameshifts, 3 stop-gain and 55 synonymous somatic sequence variations were found in 10 young adult patients (allelic frequency ≥ 10%). Excluding synonymous mutations, the most frequently altered genes in patients were TP53 (7 mutations; 25%), followed by EGFR and KDR (5 mutations; 18%), PI3K (3 mutations; 11%), KIT (7 mutations; 14%), FGFR3-ABL1-MET-ATM-RB1-SMO (1 mutation; 3.6%). Furthermore, 14 of these mutations are annotated in SIFT or in PolyPhen databases as “mutations that could damage affected protein”. Overall, we identified 28 mutations annotated in COSMIC database, among which the most frequent were COSM149673, COSM28026 and COSM6223-COSM22413 with 5,4 and 2 counts, respectively. We also found 93 SNPs in our cohort, including the most frequent rs7688609, rs1873778 and rs1050171 with 13 counts (10 pts; 3 ctrl) followed by rs1800861 (9 pts; 3ctrl); rs41115 (8 pts; 2ctrl); rs1870377 (4 pts; 1ctrl); rs3822214 (2 pts; 2ctrl); rs3729687 (2 pts; 1ctrl); rs2228230 (2 pts) and rs3730358-rs3135898 (1 pt; 1ctrl).

Conclusion
The development of new biological techniques, such as the next-generation sequencing, could allow to collect a wide number of mutations. From these preliminary results, some interesting data have been discovered concerning SNPs or mutations. This pivotal retrospective analysis is the basis for the ongoing prospective collection. A better definition of molecular-genetic pattern in this selected young population of patients could increase the knowledge about the lung cancer etiology and suggest age-related new trials design.

Background
It is reported that abundance of EGFR mutations is related with efficacy of EGFR TKI in advanced NSCLC patients with mutant EGFR. This study was designed to investigate influence of EGFR mutations and their abundance on efficacy of EGFR TKI by a quantitative method.

Methods
190 NSCLC treated with EGFR TKI and available tissue for EGFR mutations were enrolled into the study; 113 were FFPE specimen, and 62 were fresh tissue. EGFR mutation was detected with the kit of AmoyDx ARMS and percentage of mutant EGFR was tested with the method of an Allele Specific PCR with Competitive Blocker (ASB-PCR). In this assay, copies of EGFR mutants were calibrated by standard curve, and the mutation rates were estimated through normalizing by copies of a conserved sequence in EGFR exon2. The relationship between abundance of EGFR mutations and efficacy of EGFR TKI was analyzed.

Results
Of 190 samples, 15 were censored due to EGFR exon2 copies less than 100; finally, 175 enrolled into data analysis. Mutant percentage less than 0.1% was defined as wild-type, 0.1%~2% as low abundance, 2%~20% as moderate abundance, and more than 20% as high abundance. The mutant rate was 56.6% and 62.3% by using AmoyDx ARMS and ASB-PCR methods, respectively. The accordance rate of EGFR mutations was 89.7% by two methods. Of 175 samples, 20, 27 and 62 harbored low, moderate and high abundances of mutant EGFR, respectively; 66 were wild-type EGFR. Median progress free survival (mPFS) was 4.9 (95% CI, 3.4 to 6.4), 8.3 (95% CI, 3.3 to 13.3) and 16.0 months (95% CI, 10.4 to 21.7) in patients with low, moderate and high abundances of mutant EGFR (p =0.012). The mPFS of low abundance was not longer than that of those patients with wild-type EGFR (2.0 months, 95% CI, 0.2 to 4.1; p=0.261). Objective response rate (ORR) was 67.7%, 44.4%, 25.0% and 19.7%, and disease control rate (DCR) was 90.3%, 81.5%, 55.0% and 45.5% in patients with high, moderate, low abundance and wild-type EGFR, respectively (p<0.001). But ORR and DCR were no difference between low abundance and WT groups (p=0.754; p=0.610, respectively).

Conclusion
The abundance of EGFR mutations could affect the efficacy of EGFR-TKI, and quantitation of mutant EGFR could better predict for efficacy of EGFR TKI in advanced NSCLC.

Background
The search for novel biomarkers to define more successful and individual treatment approaches represent an important challenge for those involved in the care for patients with malignant pleural mesothelioma (MPM). In this exploratory study, we have systematically investigated the proteins present in plasma of MPM patients and correlated their levels with disease outcomes.

Methods
Plasma samples from twelve MPM patients (6 ‘short-’ and 6 ‘long-term’ survivors from parallel phase II studies investigating thalidomide) were used for proteomic analyses. Our series included samples from 9 patients with epithelial MPM and 3 patients with biphasic MPM. Plasma samples were immuno-depleted of the 14 most abundant proteins prior to labelling for isobaric tag for relative and absolute quantitation (iTRAQ) analysis using mass spectrometry. The most promising candidates and mesothelin were chosen for selected reaction monitoring mass spectroscopy (SRM-MS) quantification and enzyme-linked immunosorbent assay (ELISA) validation. Statistical analyses using T-Test of peak areas were used to identify proteins that were differentially expressed between the short- and long-term survivor groups.

Results
Median survival of short- and long-term survivors (1.2 and 38.3 months, respectively) differed significantly (p = 0.001). This was also the case for the neutrophil-to-lymphocyte ratio (NLR) that was significantly higher in the group of short-term survivors (p=0.03). Other baseline characteristics did not reveal major differences between the short- and long-term survivors. The total number of proteins identified was 226 (1% false discovery rate) in iTRAQ. A number of those were found to be differentially expressed between short- and long-term survivors (≥1.2-fold change; p≤0.05) by iTRAQ: selenoprotein P; tetranectin; insulin-like growth factor-binding protein 2 (IBP2); osteonectin (SPARC); platelet basic protein (CXCL7); and attractin. Mesothelin was assessed to validate the proteomic methodology: SRM-MS quantification was highly correlated with the MESOMARK ELISA values with a Pearson correlation of 0.82 (p=0.001). SRM-MS quantification revealed that the concentrations of attractin (p=0.02), tetranectin (p=0.003) and selenoprotein P (p=0.001) were higher in long-term survivors. In contrast, there was a trend for an increase in the concentration of SPARC (p=0.32), IBP2 (p=0.12) and CXCL7 (p=0.19) to be correlated with shorter survival. Furthermore, quantification by ELISA demonstrated an association between long survival and low concentration of SPARC (p=0.07) as well as high tetranectin (p=0.13).

Conclusion
We have demonstrated the feasibility of using the iTRAQ and SRM-MS proteomic techniques to investigate potential prognostic protein markers in plasma of MPM patients. Potential prognostic biomarkers worthy of further studies include SPARC and tetranectin and we plan to validate these in a larger clinical cohort.

Background
Small cell lung is a rapidly proliferating disease. Because of its high proliferation index, cancer cells rely on glycolysis, rather than oxidative phosphorylation, for ATP generation. Furthermore SCLC tumours often contain regions of hypoxia which switches tumour cells to a glycolytic phenotype. Increased glycolysis leads to increased lactate production which is effluxed from the cell in order to prevent reduced intracellular pH or inhibition of metabolic pathways via the monocarboxylate transporter (MCT) proteins MCT1 and MCT4. Inhibition of these transporters has been proposed as a method of selectively targeting highly glycolytic cancer cells. AZD3965 is an orally bioavailable MCT1 specific inhibitor currently under evaluation in phase I clinical trials. In an in vitro model of SCLC we have recently shown that in hypoxic conditions resistance to AZD3965 is associated with increased MCT4 levels (Polanski et al. submitted). Aim: To examined the expression and clinical significance of MCT1 and MCT4 in SCLC.

Methods
Archival SCLC biopsy specimens and clinical data from 78 patients presenting to the University Hospital of South Manchester and the Christie Hospital between 1994 and 2005 were analyzed. Nine representative cores from the tumour specimens were used to generate three TMAs. Sections were then stained for the markers MCT1, MCT4 and for the hypoxic marker CAIX. Staining was evaluated by two independent scorers and extent and intensity of the staining were estimated. A combined score for each case was calculated as the mean product of extent and intensity for all the cores in a case. The association between MCT1, MCT4 or CAIX and known prognostic factors was evaluated by Fisher’s exact test. Kaplan-Meier analysis was used to assess overall survival rates and Log Rank test was used for the comparison of the survival distributions.

Results
The proportion of tumours with any expression of MCT1, MCT4 or CAIX was 99%, 99%, 90% respectively. Higher levels of expression (intensity x extent) were observed for MCT1 (median=8.17) compared to MCT4 (median=2.21; p<0.001). A positive correlation was observed for CAIX expression and MCT4 expression. Tumours with CAIX expression, high MCT1 expression (>median) and low MCT4 expression ( 10 x 109/L, platelets < 150 x 109/L, Na < 135 mmol/L; LDH > 550 IU/L). However, high MCT1 expression score was associated with worse survival (14 vs. 32 months; p=0.019). Neither MCT4 nor CAIX expression was prognostic. Of the known prognostic factors assessed, extensive stage was significantly associated with shorter overall survival (6 vs. 27 months; p<0.001).

Conclusion
MCT1 and MCT4 are often expressed in SCLC and 21% cases in this series express a pattern associated with potential sensitivity to MCT1 inhibition. Higher expression of MCT1 is an adverse prognostic factor in univariate analysis reinforcing further evaluation of MCT1 inhibition in this disease.

Background
Malignant Pleural Mesothelioma (MPM) is an aggressive disease, often diagnosed at an advanced stage. It is characterized by a long latency period and prior asbestos exposure. Currently accurate diagnosis of MPM is difficult due to the lack of sensitive biomarkers, and despite minor improvements in treatment, median survival rates rarely exceed 12 months. Accumulating evidence suggests that aberrant expression of long non-coding RNAs (lncRNAs) plays an important functional role in cancer biology. LncRNAs are a class of recently discovered non-protein coding RNAs >200 nucleotides in length with a role in regulating transcription. The aims of this study were to characterize the expression and function of these lncRNAs in MPM.

Methods
To identify novel lncRNAs involved in MPM, microarray profiling was performed on five cell lines - the immortalized normal mesothelial cell line (MeT-5A) and four MPM lines (two epithelioid H28 and H226 and two biphasic MM05 and MSTO) using Invitrogen’s NCode lncRNA microarrays. These allow simultaneous assessment of mRNA and lncRNA content. High priority candidate lncRNAs were selected on the basis of statistical (P<0.05) and biological (>3-fold difference) significance. Expression of high priority candidates were technically validated using RT-qPCR, and biologically validated in three independent test sets. Pathway analyses were performed to interrogate the relationship between lncRNA and mRNA expression. Cell proliferation and colony formation assays were used to investigate lncRNA function.

Results
Microarray profiling and real-time qPCR validation identified 9 lncRNA candidates with significant differential expression in MPM compared with normal mesothelial cells Validation in three independent test sets by RT-qPCR analysis demonstrated consistent up-regulation of four of these lncRNAs. Receiver Operating Curve analysis showed that two of these candidates were able to separate benign pleura and MPM with high sensitivity and specificity. In addition, high expression of AK054908 was associated with nodal metastases with lower levels of AK130275 and AF268386 observed in patients receiving induction chemotherapy. Cases with higher EF177379 levels also demonstrated a trend to improved survival. The majority of mRNAs co-expressed with candidate lncRNAs were associated with cellular and metabolic processes including cell cycle, cell death and apoptosis. In functional studies, siRNA knockdown of AK130275 showed suppression of cell growth and colony formation in MPM cells with moderate changes observed following knockdown of EF177379.

Conclusion
To our knowledge this is the first systematic study of lncRNA expression profiles in MPM. We have found that lncRNA expression profiles can distinguish malignant mesothelium from normal pleural tissue, and that some lncRNAs are associated with nodal metastasis and long term survival. We also demonstrate that lncRNAs have potential prognostic and diagnostic utility with functional roles in regulating cell growth. Further work is required to evaluate whether these lncRNAs are capable of differentiating mesothelioma from lung cancer and benign asbestos-related diseases, and to reveal their specific functions in MPM pathogenesis.

Background
There has been a focus on the use of thymidylate synthase (TS) as a biomarker for the long term administration of metabolic antagonists for lung cancer. TS mRNA levels have been measured by real time RT-PCR in tumors collected using a micro-dissection method; however, this method has not been applicable for general use and its clinical use has been problematic. Therefore, this study investigated the correlation of TS mRNA expression level and a commonly used semiquantitative immunohistochemical method.

Methods
Resected lung specimens were collected from 47 patients with Stage IA (T1b) and IIB primary non-small-cell lung cancer who had undergone lobectomy between 2006 and 2012 at our Hospital. Levels of mRNA expression were measured using the Danenberg Tumor Profile method while TS immunostaining with anti-TS mouse IgG MoAB was graded into 4 levels.

Results
Of 47 patients, 24 patients were male and 23 patients were female. Thirty six people had adenocarcinoma while 11 patients exhibited squamous cell carcinoma. The number of patients at each IA, IB, IIA and IIB stage were 20, 20, 5, and 2, respectively. TS mRNA expression levels of these patients were between 0.69 and 12.68 (a median value of 2.47). Immunostaining indicated that Grade 0, 1, 2, and 3 were observed in 3, 17, 15 and 12, respectively. A moderate positive correlation was observed at a correlation coefficient of r=0.4880 between mRNA expression levels and immunostain levels (A). Moreover, a significant correlation between mRNA expression levels and immunostain levels was observed when mRNA expression levels were divided into two groups, one with higher than median values and the other with lower than median values, where Grade 0 and 1 were assigned as negatively stained and Grade 2 and 3 were assigned as positively stained (B).Figure 1

Conclusion
Immunostaining is a useful method for measuring TS level when TS is being considered as an appropriate biomarker for postoperative adjuvant chemotherapy with 5-FU delivertives.

Background
The diagnosis and treatment of non-small cell lung cancer (NSCLC) was revolutionised with the discovery of epidermal growth factor receptor (EGFR) activating mutations and the corresponding treatment of mutation-positive cancers with EGFR tyrosine kinase inhibitors (EGFR-TKI, i.e. gefitinib, erlotinib). While EGFR-TKI’s are highly effective in this setting, patients invariably relapse due to acquired resistance to the targeted therapy. In approximately 50% of the cases, resistance to gefitinib and erlotinib is associated with a T790M mutation that renders the EGFR tumor molecule unresponsive to these inhibitors. A number of 2[nd] and 3[rd] generation inhibitors are currently in development to address the primary T790M resistance mechanisms (afatinib, AZD9291, Clovis 1686). Identifying the relevant patient population for such drugs may rely on diagnostic analysis of re-biopsy material to determine the mechanism of acquired resistance. However, such invasive surgical procedures may present a significant challenge in a proportion of patients with EGFR TKI-resistant disease. Availability of a reliable, minimally invasive method to assess the EGFR mutation status of NSCLC patients could have major clinical benefits for patients and support access to novel targeted therapies. Recently, several highly sensitive technologies have been described for detection of EGFR mutations in the small amount of tumor DNA that is shed into the circulating plasma (ctDNA). ctDNA has potential as a minimally-invasive alternative to surgical biopsies and as such, holds promise for disease diagnosis and early assessment of disease progression.

Methods
We undertook a comprehensive cross-technology comparison of three technology platforms for detection of T790M mutation in ctDNA extracted from patient plasma: 1) ARMS-based detection using the Roche cobas® EGFR mutation detection kit; 2) digital droplet PCR using the BioRad ddPCR instrument (by MolecularMD); and 3) bead-based digital PCR using the Inostics BEAMing technology. In total, 140 frozen plasma samples (approximately 80 EGFR mutation +ve and 60 EGFR mutation -ve), obtained from EGFR-TKI resistant patients enrolled on two AstraZeneca clinical studies were used to assess mutation prevalence, false negative, and false positive rates. Each individual patient plasma sample was split and evaluated across multiple platforms. ctDNA was extracted and tested by operators blinded to the EGFR tumor mutation result for 3 common mutations (Exon 19 deletion E746_A750delELREA, L858R and T790M). The EGFR mutations status of patient-matched tumor biopsies was available for all plasma samples to allow for ctDNA-tumor concordance testing.

Results
Overall, concordance of EGFR mutation status was high between all 3 methods. In addition, the false positive rate in the known EGFR mutation -ve cases was low for all 3 methods. However, differences were seen in the rate of false negative results (assay sensitivity) between methods, with digital PCR showing increased assay sensitivity compared with the ARMS based method. A comprehensive comparative analysis for all 3 technologies will be presented.

Conclusion
Digital droplet and BEAMing PCR platforms both provide sensitive detection of EGFR mutations in ctDNA isolated from circulating plasma. Importantly, both platforms also provide results in relation to wild-type EGFR molecules, allowing for quantification EGFR mutation load

Background
Thymidylate synthase (TS) is a potential predictive marker for efficacy of treatment with pemetrexed. The current study aimed at investigating whether TS expression changes during non-pemetrexed chemotherapy of non-small cell lung cancer (NSCLC) thus making rebiopsy necessary for deciding on pemetrexed second line treatment.

Methods
TS immunohistochemístry was performed on biopsies and available resection specimens from 65 NSCLC patients stage T1-3N0-2 treated with preoperative carboplatin and paclitaxel (NAC-group) and 53 NSCLC patients stage T1-4N0-1 treated with surgery without preceding chemotherapy (OP-group) served as controls. The diagnostic biopsies and subsequent resection samples were compared in order to evaluate for concordance in TS expression in groups with and without preoperative chemotherapy.

Results
No statistically significant change in TS expression was observed between diagnostic biopsies and subsequent surgical resections of primary tumors in either the OP-group (p=0.186) or the NAC-group (p=0.542). Primary tumors were discordant between diagnostic biopsies and resection specimens when TS expression was dichotomized into high (H-score>150) and low (H-score ≤150), in 45% and 33% in the OP-group and NAC-group, respectively (p=0. 288).

Conclusion
The discordance observed between paired serial samples likely reflects intratumoral heterogeneity of TS expression and highlights the need of sufficient representative material for TS expression analysis if this biomarker is to be used for treatment selection.. TS expression in primary tumors remained unchanged and new biopsies for deciding on 2nd line pemetrexed do not seem warranted based on the current results.

Background
In a recently published study our group proposes a possible predictive impact of immunohistochemically detected RRM1 on vinorelbine efficacy in advanced NSCLC. This thesis was based on results from a randomized phase III trial comparing triplet-chemotherapy (paclitaxel, cisplatin, gemcitabine) to standard doublet-therapy (cisplatin, vinorelbine). We found that increased expression of RRM1 was associated with significantly decreased progression-free survival (PFS) and overall survival (OS) only in the patients receiving cisplatin-vinorelbine therapy. These findings were suprising since the overexpression of RRM1 is conventionally associated with resistance towards the chemotherapeutic agent gemcitabine, which is a potent inhibitor of ribonucleotide reductase (RNR). RNR is an essential enzyme for DNA synthesis that converts ribonucleoside di-phosphates into deoxyribonucleoside di-phosphates. The enzyme consists of a large sub-unit (RRM1) and a small sub-unit (RRM2). Vinorelbine is a spindle-poison and resistance towards this agent has never been associated with RRM1 over-expression. It has however been shown that vinorelbine can reduce the repair of radiotherapy-induced DNA damage in small-cell lung-cancer cell-lines, pointing to a possible interaction between vinorelbine and the DNA repair system. This study aimed at further exploring the possible predictive value of immunohistochemically detected RRM1 in patients receiving vinorelbine therapy. For this purpose we chose a cohort of malignant pleural mesothelioma patients treated with cisplatin and vinorelbine in a phase II trial.

Methods
Fifty-four consecutive patients with MPM, were enrolled between February 2003 and September 2006 into a phase II trial with cisplatin and vinorelbine. The formalin-fixed paraffin-embedded bioptic tumor specimens from these MPM patients were retrospectively evaluated for RRM1 expression by immunohistochemistry (IHC) using an H-score. The cut-off point was chosen as the upper quartile value of the H-scores to separate positive (H-score ≥upper quartile) from negative (H-score

Results
Sixty-six patients had enough tumor tissue for IHC. The upper quartile H-score was 3 yielding 15 positive and 34 (69%) negative tumors in the cisplatin-vinorelbine treated group. In the carboplatin-pemetrexed treated group there were 6 positive and 11 (64%) negative tumors. There was a significant overall survival advantage only in the RRM1-negative patients treated with cisplatin and vinorelbine (log rank p= 0.002). This group had a two-year survival rate of 47% opposed to 13 % for the RRM1-positive tumors treated with this combination. In the carboplatin-pemetrexed-treated group there were no differences in overall survival according to marker status, with two-year survival rates of 0% and 9% in the RRM1-positive and –negative group respectively.

Conclusion
Patients with RRM1-negative tumors treated with cisplatin-vinorelbine combination therapy had a prolonged overall survival. There was no survival advantage in the RRM1-negative group when patients were treated with carboplatin and pemetrexed. Our study suggest a possible role of RRM1 in predicting efficacy of cisplatin-vinorelbine combination chemotherapy ,also in MPM, which supports the similar finding from our group in NSCLC patients. Thus, RRM1 may be a biomarker for vinorelbine though the results require further validation.

Background
Variety of molecular methods for somatic mutations detection in NSCLC demonstrate discrepancies in sensitivity, cost and performance. Presence of activating mutation in TK domain of EGFR gene qualifies NSCLC patients to targeted therapy with TK inhibitors. Sanger sequencing is still a 'gold standard' for EGFR point mutation analysis and FISH is the primary method for detecting copy number of specific genes in tissue sections. Alternative methods like qRT-PCR, CISH or MLPA are more and more commonly used to detect EGFR point mutations or MET or HER2 amplification, yet results on their accuracy remain inconclusive.

Methods
We compared EGFR mutation analysis performed on 278 adenocarcinoma ( NSCLC FFPE and cytology) samples using Real-Time PCR technology with modified taqman probes to detect 29 EGFR mutations in exon 18, 19, 20 and 21 and Multiplex Ligation-dependent Probe Amplification (MLPA) to detect the most common deletion in exon 19, substitutions in exon 21 (L858R), 20 (T790M) and 18 (G719X), and insertion in exon 20 (S768I). Secondly, we evaluated MET and HER2 amplification in all 278 samples by MLPA only.

Results
EGFR mutation analysis demonstrated identical results in 97.5% cases between qRT-PCR and MLPA; 2.5% of discrepancy came from limit detection between those two methods (1% Limit of Detection for qRT-PCR; and 10-15% for MLPA). The fluorescent signals to cross the threshold for those 2.5% cases were detected in late cycles and positively correlated with small percentage of tumour cells in the sample. Both methods are more sensitive than traditional Sanger Sequencing which limit of detection is in a range 15%-25%. Additional MET and HER2 amplification analysis indicated that 10% of NSCLC samples carried MET and 1.8% - HER2 amplification.

Conclusion
Both Real-Time PCR and MLPA are accurate tools to detect point mutations or chromosomal aberration, respectively - as an alternative method to time consuming Sanger sequencing and FISH analysis for NSCLC biomarker detection. As MET amplification is known negative prognostic factor, in particular for patient treated with TK inhibitors, we suggest to use both technologies in NSCLC diagnostics: qRT-PCR for EGFR somatic mutation analysis and MLPA to detect MET amplification.

Background
Genomic profiling of SqCC has identified somatic alterations in NRF2 or its negative regulators (NFE2L2 mutations/amplifications, KEAP1 or CUL3 mutations/deletions) in ~1/3 of tumors. These alterations result in activation of the NRF2 transcriptional program, but the clinical significance of this pathway in lung SqCC patients is unknown. We hypothesize that a gene expression signature that reflects somatic NRF2-activating alterations may be identified and correlated with NRF2 protein over-expression. Furthermore, such gene expression or its immunohistochemical correlates may have prognostic significance and/or may be predictive of adjuvant chemotherapy benefit in early stage resectable lung SqCC patients.

Methods
Logistic regression (LR) and SAM analysis were applied independently to 104 SqCC cases from The Cancer Genome Atlas (TCGA) that had both microarray gene expression and mutation data to identify genes associated with NRF2 pathway mutational status. Overlapping genes were used to define the signature, which was then tested in 3 independent SqCC microarray datasets to evaluate its prognostic value. Correlation of the signature with NRF2 and KEAP1 mutations and with NRF2 and KEAP1 immunoreactive protein expression by immunohistochemistry (IHC) was evaluated. We also tested the gene expression signature as a potential predictor of adjuvant chemotherapy benefit in a subset of NCIC JBR.10 adjuvant chemotherapy trial patients with microarray data.

Results
A 28-gene signature that distinguished SqCC with or without aberration of the NRF2 pathway genes (NFE2L2/KEAP1/CUL3) in the TCGA dataset was identified. This gene signature that putatively represents NRF2 pathway activation status separates consistently SqCC into 2 groups in independent datasets. Both NRF2/KEAP1 mutation and NRF2 protein expression by IHC were significantly correlated with the NRF2 pathway activation signature (p<0.001 for each comparison). KEAP1 protein expression was not associated with the gene expression signature. No prognostic effect of the activated signature was observed in three independent datasets. In the JBR.10 patient cohort, a trend toward improved survival with adjuvant chemotherapy was observed in patients with the NRF2 “wild type” signature (HR 0.32, 95%CI 0.065-1.6 p=0.16), but not in patients with the “activated” signature (HR 2.28, 95%CI 0.24–22, p=0.48; interaction p=0.15).

Conclusion
A gene expression signature based on mutational activation of the NRF2 pathway may be predictive of benefit from adjuvant cisplatin/vinorelbine in SqCC. Patients with NRF2 pathway activating somatic alterations may have reduced benefit from this therapy. NRF2 immunohistochemistry could potentially be useful to identify NRF2-activated lung SqCC patients who may not benefit from adjuvant chemotherapy but this requires further validation.

Background
Oncogenic mutations of KRAS play important roles in cancer progression and resistance to chemotherapy in non-small cell lung cancer (NSCLC). Ras-related proteins, RALA and RALB, are members of the RAS superfamily of small GTPases and act as downstream effectors of KRAS. Therefore RAL proteins may be involved in cancer cell survival, invasion and metastasis. In this study, authors investigated the prognostic role of RALA and RALB overexpression in non-small cell lung cancer (NSCLC) patients with KRAS mutations.

Methods
Expression status of RALA and RALB in the tumor tissue of NSCLC patients whose tumor harbors KRAS mutations were evaluated by immunohistochemistry(n=12). In addition, we investigated 11 metastatic lung tumor tissue samples from KRAS mutant colorectal cancer patients. Predictive factors for survival were calculated using Kaplan-Meier estimator and Cox proportional hazards model.

Results
There was no statistically significant relation between RALA/RALB overexpression and KRAS mutation subtypes. However, RALB was frequently overexpressed in the tumor of advanced stageof NSCLC with KRAS mutation(p=0.015, χ[2]-test). Furthermore, the overall survival of the patients with RALB overexpression was significantly shorter than that of the patients without RALB overexpression (2.7 vs. 7.3 months, P<0.001; Mann-Whitney U test). In metastatic lung tumor tissues from KRAS mutant colorectal cancer patients, RALB protein overexpression showed only a trend toward shortened disease free survival.

Conclusion
RALB overexpression might be related to rapid disease progression and poor prognosis in NSCLC patients with KRAS mutations.

Background
The chemokine CXCL16 and its receptor CXCR6 are expressed on a variety of immune cells, and have been shown to influence angiogenesis. Hence, these markers are potentially interesting markers in NSCLC treatment. The expression of CXCR6 and CXCL16 has been examined in a variety of human cancers; however no studies have investigated their influence on prognosis in non-small cell lung cancer (NSCLC). We therefore wanted to explore their prognostic significance in NSCLC, in addition to examining associations with our previously investigated immunologic and angiogenic markers.

Methods
Resected tumor tissue from consecutive unselected stage I-IIIA NSCLC patients (1990-2005) were collected in two different cohorts; Nordland Central Hospital, NCH (n = 176), and University Hospital of North Norway, UNN (n = 159). Tissue microarrays were constructed from duplicate cores of neoplastic epithelial and stromal areas of each specimen. Immunohistochemistry was used to evaluate the expression of CXCR6 and CXCL16. Correlation analyses with well-known adaptive immunological (CD4, CD8, CD20), innate immunological (CD68, CD56, CD1A) and angiogenic (vascular endothelial growth factors and receptors, platelet derived growth factors and receptors and fibroblast growth factor-2 and receptor-1) markers were done. Disease-specific survival was used as endpoint, and survival analyses were performed in each cohort separately and in the combined total population.

Results
In univariate analysis, ↑stromal cell CXCL16 expression was a significant positive prognostic factor in the combined patient cohort (P = 0.016), supporting the similar trends in each cohort separately (NCH, P = 0.045; UNN, P = 0.137). Interestingly, the combinations (Figure 1) increased (↑stromal and ↑cancer cell), mixed (↑↓ and ↓↑) and reduced (↓stromal ↓cancer cell) CXCL16 expression patterns showed 5-year survival rates of 71%, 58% and 48%, respectively (P = 0.016). CXCR6 was expressed in cancer cells, but did not show any significant prognostic impact. There were no strong correlations between CXCR6/CXCL16 and immunological or angiogenic markers. In the multivariate analysis, combined ↓cancer and ↓stromal cell CXCL16 expression was an independent negative prognostic factor in the total cohort when compared to ↑stromal and ↑cancer cell expression (hazard ratio: 2.41; 95% confidence interval: 1.14 – 5.12, P = 0.022). Figure 1

Conclusion
We have shown that combined ↑cancer and ↑stromal cell CXCL16 expression is an independent positive prognostic factor in NSCLC. Further studies are warranted to elucidate the biological mechanism underlying this finding.

Background
Cancer-Testis antigens (CTAs) are immunogenic molecules exclusively expressed in normal testes but with aberrant expression in up to 30% of NSCLC. NY-ESO-1, is a CTA whose expression is associated with poorer survival but improved response to chemotherapy.[1] The promoter regions for many CTA genes contain CpG islands which are predominantly methylated in most tissues. In cancer, hypomethylation of CTA gene promoters hsvr been shown to be determining factor for the frequent re-expression of these genes. Herein we sought to correlate NY-ESO-1 promoter methylation (PM) with protein expression in lung cancers and test whether methylation status can be used as a predictive and prognostic marker in NSCLC.

Methods
We reviewed the clinicopathological data for patients with pathological N2 NSCLC treated with surgical resection followed by either observation or adjuvant chemotherapy, if treated after 2004. Genomic DNA from formalin fixed paraffin embedded (FFPE) samples were purified and bisulfite converted according to the manufacturer’s (QIAGEN) instructions. Mutational status was determined in genomic DNA using Sequenom's Oncocarta panel v1.0. Tumour samples were cut and stained with NY-ESO-1 antibody (E978). Previously described protocols for methylation-specific NY-ESO-1 primers[2] and beta-actin[3] control probes were used. Using real-time quantitative methylation-specific polymerase chain reaction (qMS-PCR), the level of NY-ESO-1 methylation was determined and expressed as the ratio of methylated to unmethylated molecules (M/UM). We used a cut-off fold-difference of 5 (Log2, M/UM) to separate tumour samples into hypomethylated and hypermethylated groups. Survival estimates were obtained using the Kaplan-Meier method. Comparison across groups was performed using the Chi-squared or Fisher's test.

Results
NY-ESO-1 PM MS-PCR was successful in 92/100 (92%) of specimens. NY-ESO-1 was hypermethylated in 86% (79/92) of samples. Age, smoking history, gender, histology and oncogenic mutations were not associated with presence of NY-ESO-1 PM. NY-ESO-1 hypomethylation was inversely associated with protein expression (p<0.0001). In multivariate analysis, hypomethylation of the NY-ESO-1 promoter was an independent poorer prognostic marker in patients not treated with chemotherapy (HR 2.97, 95%CI 1.06-8.29, p=0.038), after adjusting for age, gender, stage IIIA/B and histology. Conversely, in patients treated with chemotherapy there were no differences in survival associated with NY-ESO-1 PM, suggesting hypomethylated tumours remain chemosensitive.

Conclusion
NY-ESO-1 hypomethylation was an independent adverse prognostic marker in patients not treated with chemotherapy. Given their poorer outcome, association with chemosensitivity and known immunogenicity, CTAs may make attractive targets for immunotherapy, demethylating agents and immune checkpoint inhibition. References 1.John et al. PLOS One, In press. 2.James et al. Oncogene 2006. 3.Eads et al. Cancer Res 2001. .

Background
Intra-tumor heterogeneity can confound mutational status in oncodriver genes and challenge targeted cancer therapy strategies. Ultra-deep sequencing can detect low-frequency and expanded clonal mutations in primary tumors to better inform treatment decisions

Methods
KRAS coding exons in 61 treatment-naïve colorectal cancer (CRC) tumors were sequenced, along with KRAS, EGFR, ALK, and MET in lung tumors from 3 Chinese non-small cell lung cancer (NSCLC) patients

Results
Forty-two percent of CRC patients (28/61) harbored mutations in the KRAS active domain, 12 patients harbored >1 mutation, and eleven patients (18%), of which 5 had frequencies <10%, were not detected by Sanger sequencing. Low frequency KRAS active (G12R) and EGFR kinase domain mutations (G719A) were identified in one NSCLC patient. Multiple low frequency mutations in KRAS, EGFR, and MET and ALK gene copy number increases were found in a second NSCLC patient; a third NSCLC patient had EML4-ALK fusion with ALK, EGFR, and MET mutations. Multiple low frequency mutations occurred within individual gene in all three patients.

Conclusion
A complex pattern of intrinsic low frequency driver mutations in well-known tumor oncogenes may exist prior to treatment, potentially resulting in resistance to targeted therapies. Ultra-deep sequencing can characterize intra-tumor heterogeneity and identify such mutations which could ultimately impact treatment decisions.

Background
In a previous report of EGFR WT advanced NSCLC patients treated with first-line platinum-based chemotherapy we observed a worse clinical outcome for KRAS-mutants compared with KRAS WT patients (Metro et al. ESMO 2012). Here, we assessed whether this phenomenon could be due to different levels of ERCC1 expression.

Methods
From a prospectively maintained database of EGFR WT advanced NSCLC patients diagnosed at a single Institution between January 2006 and November 2012, we identified the individuals who had a known KRAS mutation status and tissue available for assessment of ERCC1 mRNA expression. Total RNA was isolated from paraffin-embedded tumor specimens using RNeasy Mini kit and automatically purified by QiaCube instrument (Qiagen). Quantification of mRNA expression levels of ERCC1 was analyzed by real-time one-step RT-PCR using QuantiFast technology by RotorGeneQ instrument (Qiagen), and the results were compared considering β-actin as the internal reference gene.

Results
One hundred and eleven patients were evaluable, 60 of which were KRAS-mutants. Among KRAS-mutants, the rate of codon 12/13/61 mutations were 80%/13.3%/6.7% respectively. Baseline patients characteristics were as follows: median age was 62 years (35-84), 36.9% were male, 63.9% were stage IV, 78.3% were PS 0 or 1, 87.3% were ever-smokers, and 71.1% had received a first-line platinum-based chemotherapy. More ever-smokers were present in the KRAS-mutant subgroup compared with WTs (90% versus 76.5%, respectively, P = 0.08). ERCC1 average scores ranged from 0.1 to 26.7, the values being not normally distributed (Kolmogorov-Smirnov test, P<0.0001). Median and mean overall ERCC1 values for all patients were 1.3 and 2.2 [standard deviation (SD) 3.4], respectively. There was no statistically significant difference in terms of ERCC1 median values betwen KRAS-mutants and KRAS WTs (1.4 vs. 1.3, respectively, P = 0.27). Nevertheless, mean ERCC1 expression levels were found to be significantly higher in KRAS-mutants compared with KRAS WTs [2.9 (SD 4.5) vs. 1.4 (SD 0.8), respectively, P = 0.02]. This finding was due to 7 KRAS-mutant patients (ERCC1 high) coming out with ERCC1 levels higher than 5.0, thus notably incresing mean ERCC1 values. In the group of patients treated with first-line platinum-based chemotherapy (n = 79), median progression-free survival was 1.9 months for KRAS-mutant, ERCC1 high patients (n = 6), 5.1 months for KRAS-mutant, ERCC1 low patients (n = 38), and 7.1 months for KRAS WT patients (n = 35) (P = 0.003).

Conclusion
KRAS-mutant NSCLCs may express higher levels of ERCC1 compared with KRAS WTs, which could translate into poor sensitivity to first-line platinum-based chemotherapy. Combination strategies of platinum-based chemotherapy plus KRAS-targeting agents may represent an appealing upfront strategy for KRAS-mutants advanced NSCLCs, particularly in presence of concomitant expression of high ERCC1 levels.

Background
Metabolomics is an approach that is increasingly used in research of different types of cancer as it reflects the most proximate deviations in metabolism of cells and tissues. It has been shown before that untargeted metabolomics can effectively distinguish diseased tissue samples from healthy ones and diseased individuals from healthy ones regarding the general metabolite pattern of the samples.

Methods
In this study lung cancer tissue and macroscopically non-cancer tissue were obtained from lung cancer patients (n=45) and analysed using liquid chromatography combined with tandem mass spectrometry to acquire full spectra of metabolites between mass-to-charge ratio (m/z) 50-1000 extracted in hydrophobic and hydrophilic phase in positive and negative ionisation mode. Principal component analysis (PCA), sparse partial least squares discriminant analysis (SPLS-DA), paired t-test were used for data analysis. Fragmentation and comparison of fragment spectra to public and in-house databases were used for metabolite identification.

Results
PCA and SPLS-DA indicated the existence of systematic differences in between the two types of tissue based on the data matrix extracted from mass-spectra. All together 496 signals (13% of all measured signals) were found to have significant (p<0.001) alteration by t-test between cancer and non-cancer spectra. Eleven most prominent of them were selected for fragmentation. Their mass-to-charge ratios (m/z) were in hydrophilic phase as follows: +86, -147, +168, -171, -201, and in hydrophobic phase +735, +736, -774, -775, -888 and -889. Signals with m/z of -171, +735, +736, -774 and -775 were found more abundant in non-cancer tissue and the signals with m/z of +86, +168, -147, -201, -888 and -889 in the cancer tissue compared to non-cancer tissue. From hydrophobic phase signals +735 and +736 were identified as unspecified species of phosphatidylcholines. M/z -888 was identified as phosphatidylinositol 18:0-20:3 (stearic acid and Mead acid as lipid acid residues) having on average two-fold stronger signal in the spectra of cancer tissue compared to non-cancer tissue. The phosphatidylinositols are, in addition to structural purposes in membranes, known to be used as a depot for PUFA-s, such as arachidonic acid (AA; 20:4). As the increase of free Mead acid in blood has been linked to the deficiency of essential fatty acids (as AA) in food, we suggest our finding indicates that lung cancer cells have significantly increased demand for inflammatory mediators synthesized from AA.

Conclusion
Cancer tissue is clearly distinguishable from non-cancer tissue with mass spectrometry. From all low-molecular weight metabolites 13% are differing in cancerous and non-cancer tissue. Most striking changes were assigned to phospholipids and in particular to Mead acid containing phosphatidylinositols, which are upregulated in cancer tissues by 100%. The results imply differential regulation of polyunsaturated fatty acids in lung cancer, which improves our knowledge of molecular mechanisms of cancer and opens new ways for accurate prognostic markers and personalized approach to postoperative treatment.

Background
Radiation-induced lung damage (RILD) is a dose-limiting toxicity of lung radiotherapy. Individual sensitivity can be measured by changes in Hounsfield Units over time (delta HU) on CT scans (De Ruysscher et al. Acta Oncol 2013). This endpoint is specific for lung damage and does not correlate with dyspnoea, which is multi-factorial. In this study, we investigated the association between density changes over time and SNPs aiming at finding individual sensitivity for RILD.

Methods
Delta HU/Gy and delta HU/Gy x MLD (Mean Lung Dose), the latter to take into account a volume factor for RILD, were correlated with 314 SNPs related to fibrosis and inflammation. The outcome variables were square root transformed because both were not normally distributed. Univariate ANOVA analyses were performed for comparisons of means. P-values of less than 0.01 were considered to be significant.

Results
Eighty-nine lung cancer patients were studied, 63 men and 26 females. Twenty patients were treated with radiotherapy alone, 31 with sequential chemo-RT and 38 with concurrent chemo-RT. Twenty percent of the patients developed grade 2 or more clinical dyspnoea after treatment. Three SNPs were significantly correlated with delta HU/Gy: rs2252070 (p=0.006, MMP13), rs2230588 (p=0.009, JAK1) and rs12901071 (p=0.009, SMAD3) [Table 1A]. For delta HU/Gy x MLD, significant associations were found for rs3819122 (p=0.008, SMAD4), rs2230529 (p=0.009, ITGB2) and rs2230588 (p=0.009, JAK1) [Table 1B]. Figure 1

Conclusion
Quantification of CT density changes due to radiotherapy, measured as HU changes over time as a specific and quantitative endpoint for RILD correlates with specific SNPs in genes involved in signal transduction of cytokines (SMAD3/4, JAK1), in the extracellular matrix (MMP13) and in cell adhesion (ITGB2). External validation will follow.

Background
Mutational profiling for targetable oncogenic drivers is systematically recommended in the pretherapeutic workup of metastatic lung adenocarcinoma. Pathological and molecular diagnoses may be performed on specimens from metastatic lesions, when the primary pulmonary tumor is not accessible, either because of proximal location increasing the risk of transthoracic procedures, or when the material collected is insufficient in size. The feasibility of performing molecular diagnoses on small specimens from bone metastases has been questioned over time. To prospectively study patients with bone metastases from lung cancer, we set up a multidisciplinary group at the Hospices Civils de Lyon, including rheumatologists, pulmonologists, oncologists, interventional radiologists, pathologists and molecular biologists.

Methods
POUMOS was a prospective observational study aiming at evaluating the feasibility of routine percutaneous biopsy of synchronous bone metastases from lung adenocarcinoma, to perform pathological diagnosis and mutational profiling on the bone lesion. Results were correlated with that obtained on specimens from the primary tumor, if available. Technically, bone metastasis specimens, usually multiple, were sent fresh for immediate formalin-fixation, and, if possible, snap-freezing. Decalcification of bone was performed using EDTA for a better preservation of cell morphology and DNA. Mutational profiling of EGFR, KRAS, HER2, BRAF, and PIK3CA, as well as testing for ALK rearrangements, was conducted as recommended by the French National Cancer Institute (INCa) for all adenocarcinoma cases. DNA extraction was performed after laser microdissection of cancer cells; genotyping was based on direct sequencing and/or SNaPshot. Complete description of the process will be presented at the meeting.

Results
Starting April 2011, 46 patients with lung adenocarcinoma and synchronous bone metastasis were enrolled. No grade 3-4 adverse effects were reported after the bone biopsy. Mutational profiling was obtained in 45 (98%) cases; one specimen did not provide with sufficiently good quality DNA for the analysis. EGFR mutation was observed in 6 (13%) patients, KRAS mutation in 14 (30%) patients, HER2, BRAF and PIK3CA mutations in 1 (2%) patient each. Updated results, especially correlations between the mutational profiles of primary lung tumors and bone metastases, will be reported at the meeting.

Conclusion
Our data demonstrate the feasibility of percutaneous biopsy of synchronous bone metastasis from lung adenocarcinoma to conduct mutational profiling for common oncogenic alterations. The establishment of multidisciplinary teams to ensure the coordination between clinicians, radiologists and pathologists, makes routine pathological and molecular diagnosis on bone metastasis specimens a fast, reliable and safe procedure.

Background
KRAS mutations are common driver mutations in 30% of non-small cell lung cancer. However, treatment for lung cancer patients with KRAS mutations remains unestablished. Therefore, to screen other targetable receptor tyrosine kinases (RTKs) in lung cancers with KRAS mutations, we performed phospho-RTK array analyses using 6 KRAS mutated lung cancer cell lines.

Methods
Sixteen NSCLC cell lines were analyzed using Human-Phospho-RTK Array Kit (R&D Systems, MN) and relative phosphorylation levels of 42 RTKs were examined. Phosphorylation levels of RTKs were quantified relative to the average of positive controls using image analysis software JustTLC (Sweday, Lund, Sweden). We also examined growth inhibitory effect of small interfering RNA (siRNA) and erlotinib in KRAS mutant lung cancer cell lines. In addition, we developed erlotinib-resistant cell lines from erlotinib-sensitive H358 cells by chronic drug exposure for 3 months.

Results
In the phospho-RTK array analysis, phosphorylation levels of EGFR were lowest in the cell lines with KRAS mutation (Table1). Three of six cell lines with KRAS mutation showed IGF-1R phosphorylation and the difference was statistically significant compared with 0 of ten cell lines without KRAS mutation (P=0.03, Fisher's Exact test). KRAS siRNA did not induce apoptosis in 5 cell lines with KRAS mutation but mild apoptosis in H358 cells. Moreover, only H358 showed mild sensitivity to erlotinib (IC~50~ 120nmol/L). Analysis for erlotinib-resistant H358 cells identified increased phosphorylation of IGF-1R compared with H358 parent cells (18.0 times).Figure 1

Conclusion
These results suggest IGF-1R may be a candidate molecular target in lung cancers with KRAS mutation.

Background
Epidermal growth factor receptor (EGFR) gene variation is one common driver mutation in lung cancer. In lung adenocarcinoma, EGFR mutations are the powerful predictors to the efficacy of EGFR-TKIs. However, the phenomenon seemed to be inconsistent in lung squamous carcinoma. The present study aims to investigate the sensitivity of lung squamous cell carcinoma (SCC) harboring EGFR mutation to EGFR-TKIs, and explore the likely molecular mechanisms through constructing EGFR mutant lung SCC cell lines and analyzing the results transcriptomics.

Methods
This study retrospectively enrolled 91 patients with advanced lung SCC who received EGFR-TKIs in Beijing University Cancer Hospital. Denaturing high-performance liquid chromatography (DHPLC) and immunohistochemistry (IHC) were used for the detection of EGFR mutations and protein expressions of P63 and TTF-1, respectively. Stable cell lines were constructed through lipofectamine and confirmed by western-blot, soft agar cloning formation and tumoregeneity in nude mice. Cell titer Glo was used for cell number counting. RNASeq was used for the analysis of transcriptomics and related signaling pathways were screened in KEGG website. SPSS (17.0 version) was used for statistics.

Results
The frequency of EGFR mutation was 16.2% (64/396) in lung SCC. In lung cancer patients with EGFR mutation, lung SCC patients accounted for 7% (64/863), commonly in male (71.9%), smoker (59.4%) and EGFR exon 19DEL (64.1%). In total 91 patients who received EGFR-TKIs, the objective response rate (ORR) and the disease control rate (DCR) were 11.0% (10/91) and 56.0% (51/91) respectively and the progression-free survival (PFS) and the overall survival (OS) were 2.9 months and 16.3 months. In terms of 60 patients who provided EGFR mutated status, 29 patients harbored EGFR mutation and the other 31 without EGFR mutation. The PFS of the subgroup with EGFR mutation after EGFR-TKIs showed prolonged trend compared with that of the subgroup without EGFR mutation, however, the statistics failed to amount to significant difference (2.4 months vs. 1.8 months, p=0.071). No statistical differences were observed in PFS ORR, DCR and OS between the two subgroups. IHC results demonstrated that most of lung SCC patients harboring EGFR mutation showed P63 positive and TTF-1 negative (15/18). We then constructed lung SCC cell lines with EGFR mutation (Beas-2B/EGFR 19DEL cell line and Beas-2B/EGFR 21L858 cell line), which showed resistant to erlotinib with the IC50 of 3.43±0.56μM和11.9±1.18μM. After the treatment of erlotinib, the mainly up-regulated signaling pathways included TGFβ pathway and hedgehog pathway, and RNA levels of some mesenchymal genes were also up-regulated.

Conclusion
Lung SCCs with EGFR mutation were not uncommon, who showed insensitivity and even resistance to EGFR-TKIs. The likely molecular mechanisms included TGFβ related epithelial-mesenchymal transition (EMT) and tumor stem cell like differentiation, and the upregulation of bone morphogenetic protein 4 (BMP4).

Background
Acquired resistance in NSCLC with activating EGFR mutations has been defined by Jackman et al. The molecular mechanisms have been studied in small series of patients: in about 50% of cases, T790M mutation confers acquired resistance, in 25% c-Met overexpression detected by IHC. Other as of yet unknown mechanisms might also play a role. Here we present the acquired resistance mechanisms in a cohort of 12 patients (out of 33 pts.) with activating EGFR mutations treated with TKI and identified at our center (see Halbfass et al.). Treatment methods and outcome are also described

Methods
159 consecutive patients (s. abstract Halbfass et al.) were molecularly studied for EGFR mutations in exons 18-21. EGFR mutation analysis was performed by Sanger Sequencing or by hybridization based COBAS methodology after microdissection of tumor tissue to ascertain a high percentage of tumor tissue. c-Met IHC was performed automatized by standard procedure (BondMax, Menarini). ALK rearrangement was studied using a break apart FISH-Probe (Abbott). Remission was measured by RECIST 1.1 criteria.

Results
Of 159 patients tested, 33 had an EGFR mutation, of which 3 had primary resistance mutations (T790M 1 case, Exon 20 insertions 2 cases). Of these 30 pts., 20 had acquired resistance and 12 were rebiopsied. 8 were not rebiopsied for various reasons, the most common being progress in the CNS. Of the 12 rebiopsied pts, c-Met was successfully studied in 8, T790M in 12 and ALK in 6. In 5/8 pts., c-Met was amplified, in 4/12 pts. the T790M mutation was found and in 1/6 pts., an ALK rearrangement was observed. 4 pts. received afatinib, 2 pts. chemotherapy, 2 pts. afatinib and cetuximab, 1 pt. afatinib and crizotinib and 2 pts. other treatments. Detailed treatment results will be presented at the meeting.

Conclusion
The understanding of resistance mechanisms in acquired TKI resistance is of academic interest and might be important for subsequent treatment options. Therefore, rebiopsy at progress while on TKI therapy should be discussed with patients. As targeted treatment options are available for c-Met, ALK as well as T790M, resistance mechanisms potentially may guide therapeutic strategies in EGFR mutated patients with acquired resistance.

Background
The interactions between radiation dose, toxicity and systemic responses are poorly understood during radiotherapy of patients with NSCLC). The purpose of this study was to monitor DNA damage using the gamma-H2AX assay in tissues inside and outside irradiated volumes of patients with NSCLC.

Methods
This prospective ethics approved study assessed 12 patients receiving radiotherapy was planned to 60 Gy in 30 fractions over 6 weeks. Six patients receive concurrent platinum doublet chemotherapy (chemoRT), and six patients had radiotherapy alone. The number, distribution and kinetics of gamma-H2AX foci were compared with the irradiated volume. Lymphocytes and eyebrow hairs were processed for gamma-H2AX staining and microscopy at each of 5 time-points; baseline, 1 and 24 hours post-first fraction, 4 weeks into radiotherapy, and 3 months after treatment completion.

Results
The mean irradiated target volume was 384 cm[3] (range 87-1137 cm[3]). We observed the presence of a small subpopulation of lymphocytes with multiple (>5) gamma-H2AX foci at 1-hour post-first fraction. There was no difference in this subpopulation between patients receiving chemoradiotherapy or radiotherapy alone at baseline (p=0.26) nor at 1-hour (p=0.24) There was a strong correlation between the size of this subpopulation and irradiated volume (r=0.84, p<0.01), indicating direct radiation exposure. This suggests potential utility of the gamma-H2AX assay as a human in-vivo biodosimeter. This subpopulation was not detected at 24 hours due to DNA damage repair. A trend for reduction of this subpopulation and the average number of foci in lymphocytes analysed at 4 weeks of radiotherapy suggests an impaired radiation response after multiple radiotherapy fractions. By contrast, the mean number of observed hair follicle gamma-H2AX foci was not different from baseline to 1-hour post first-fraction (p=0.42), elevated at 24 hrs (p=0.10) and 4 weeks (p=<0.01) but was not different from baseline at 3 months (p=0.31). The scattered dose at the eyebrow was recorded at <0.01 Gy per fraction and was insufficient to directly induce the observed gamma-H2AX signal Figure 1 Figure 1 - a brisk DNA damage response in out-of-field eyebrow hair 24-hours post radiation (gamma-H2AX foci in green)

Conclusion
The Gamma-H2AX assay on peripheral blood at 1-hour may be a useful as a human in-vivo biodosimeter. To our knowledge, this study is the first report of abscopal DNA damage response in hair follicles associated with radiotherapy in cancer patients. Further validation of our findings on a larger patient cohort is warranted.

Background
Rash, liver dysfunction, diarrhea and pneumonitis are known as adverse events of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Transforming growth factor-β1 (TGF-β1) is a cytokine and acts as an anti-proliferative factor in normal epithelial cells and epithelial-mesenchymal transition (EMT) with invasion and metastasis in cancer cells. TGF-β1 mediated Smad activation caused EMT, and activation of the EGFR. TGF-β1-mediated EGFR activation was abolished by EGFR suppression or extracellular EGF depletion pathway. TGF-β1 genotypes are associated with serum level of TGF-β1. It has been hypothesized that TGF-β1 genotypes may be implicated in clinical outcomes of EGFR-TKIs.

Methods
Patients were identified through a query of patient information for subjects enrolled in the Medical Information System in Osaka City University Hospital between January 1999 and February 2012. The associations between three genetic polymorphisms of TGF-β1 (C-509T, T869C, and G915C) and adverse events of erlotinib and gefitinib have been studied. Genomic DNA was extracted from peripheral blood or formalin-fixed and paraffin-embedded tissues. TGF-β1 genotypes were determined using RT-PCR method. The primers were designed to amplify the target fragments of TGF-β1 rs1800469, rs1800471, and rs1982073, respectively. In order to identify the risk factors for the adverse events, gender, age, stage and three genetic polymorphisms of TGF-β1 were selected and estimated for their potential confounding effects on rash, diarrhea, and liver dysfunction by multivariate analysis. Unconditional logistic regressions were used to compute the odds ratios (ORs) and their 95% confidence intervals (CIs). All analyses were two-sided, and p values of less than 0.05 were considered statistically significant. This study was approved by the ethics committee of Osaka City University (approval number, 1700).

Results
A total of 255 patients received gefitinib, and 75 patients received erlotinib. In the gefitinib group, the rates of rash, diarrhea, and liver dysfunction of grade 1 or more were 66.7%, 26.0% and 48.5%, respectively. In the erlotinib treatment group, the rates of rash diarrhea, and liver dysfunction of grade 1 or more were 84.1%, 43.5% and 33.3%, respectively. The C-509T consisted of TT in 26.6%, CT or CC in 73.3% of patients (n=289), respectively. The T869C consisted of CC in 26.1%, TC or TT in 73.9% of patients (n=272), respectively. The G915C consisted of GG in 100% of (n=313) patients. TT of the C-509T and CC of the T869C were significantly associated diarrhea of grade 1 or more in erlotinib group (OR 0.21, 95% confidence interval [CI] 0.054-0.72, p < 0.001, and OR 0.21, 95% CI 0.05-0.73, p = 0.014, respectively). No associations were observed between TGF-β1 genotypes and any adverse events in gefitinib group.

Conclusion
Minor alleles of TT of the C-509T and CC of the T869C were associated with erlotinib induced diarrhea. These alleles are generally associated with high level of TGF-β1 in serum. The increase level of TGF-β1 may be a risk factor for mucosal damage of the gastrointestinal tract in patients treated with erlotinib.

Background
Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Although some progress has been made in the development of its treatment, most patients are diagnosed at advanced stage and have a short overall survival rate. Just as specific genes have to be analyzed before selecting a targeted therapy, microRNAs (miRNAs) are emerging as biomarkers for NSCLC diagnosis and prognosis. miRNAs are small oligonucleotides that regulate target mRNAs in the post-transcriptional step driving the expression of some genes involved in the tumorigenesis of NSCLC. Due to their resistance to nuclease digestion, miRNAs can be detected both in lung tissue samples and in body fluids as circulating factors. Our aim was to identify specific non-invasive cancer biomarkers involved in NSCLC tumorigenesis regulation.

Methods
33 NSCLC patients, 19 male and 14 female, were enrolled. The mean age was 63.3 and 66% were smokers. 88% of patients had advanced stage (IIIb-IV) tumors of which 23 were adenocarcinoma and 10 squamous cell carcinoma. We collected serum before chemotherapy and, when available, tissue samples from biopsy. MiRNA expression profiling of 33 serum and 10 tissue paired samples and 10 serum samples from normal volunteers were investigated by Affymetrix GeneChip miRNA Array. meV software was used for statistical analysis, and target genes identified by deregulated miRNAs were analyzed through the miRWalk database.

Results
Statistical analysis revealed 47 miRNAs differentially expressed in NSCLC serum samples compared to control serum (p<0.05) and 29 miRNAs deregulated in NSCLC tissue samples compared to their normal counterparts. 326 miRNAs resulted differentially expressed between the serum and the tissue of NSCLC patients. Among these deregulated miRNAs, the Venn diagram comparing tumor/normal serum, tumor/normal tissue and tumor serum/tissue samples showed that the serum of NSCLC patients was characterized by 22 miRNAs, while 10 miRNAs identified the tumor tissue of NSCLC patients. Only one miRNA, miR-133a, was detected both in NSCLC serum samples and in tissue ones. Focusing on miRNAs involved in NSCLC pathogenesis, of 22 miRNAs miR-486-5p had a lower mean expression ratio (MER) in tumor vs normal serum (8.70±3.28 vs 10.78±1.35), as did let7b which had a MER of 5.28±2.63 in tumor serum vs 7.14±1.8 in normal serum samples. Of 10 miRNAs, miR-200c had the lowest MER in tumor vs normal tissue (1.25±0.10 vs 2.37±0.09 respectively) as did miR-29c* which had a MER of 2.18±0.08 in tumor tissue samples vs 2.34±0.11 in normal tissues. miR-133a had a comparable MER in tumor serum vs tumor tissue (2.41±0.17 vs 2.46±0.17 respectively). These miRNAs are able to target PIK3CA and PTEN genes, according to the miRWalk database, which are involved in the EGFR-related pathway.

Conclusion
Our results highlighted specific miRNA expression profiles, both in the serum and in the tissue of NSCLC patients, able to identify circulating miRNAs that could be used as non-invasive biomarkers for early diagnosis, or to predict prognosis in NSCLC patients thus improving personalized therapy. These data are preliminary to a prospective clinical validation in a multicentric trial.

Background
Epidermal growth factor receptor (EGFR) is widely distributed in human epithelial cell membrane, including esophageal squamous cell carcinoma (ESCC). The purpose of this study was to evaluate the relationship between malignant biological behavior and EGFR status in ESCC.

Methods
We investigated tumor specimens from 56 patients with surgically resected ESCC from 2004 through 2008. Immunohistochemistry (IHC) was performed to analyze the expression of EGFR. Fluorescence in situ hybridization (FISH) was performed to assess the EGFR gene amplification. EGFR mutations in exons 19-21 were detected by pyrosequencing technology. A chi-square test or Fisher exact test for independence was used to examine the correlation among the status of EGFR protein, gene and the several clinicopathological factors. Overall survival and disease-free survival were constructed using the Kaplan-Meier method, and the log-rank test was used to evaluate the statistical significance of differences. Multivariate analysis was performed using the Cox proportional hazard method.

Results
EGFR was overexpressed in 30 of 56 ESCC (53.6%) and was correlated with tumor differentiation (p=0.047) (Figure 1.). EGFR gene amplification was found in 13(23.2%) cases and was correlated with the presence of lymph node metastasis (p=0.001) and higher pathological tumor-node-metastasis (pTNM) stage (p=0.042). There was no EGFR mutation in the clinical samples of 56 patients with ESCC. In univariate analysis, there was no correlation between the prognosis and EGFR protein expression, but EGFR gene amplification was a significant predictor of better prognosis (p=0.031). The multivariate analysis revealed that EGFR gene amplification was significantly correlated with better disease-free survival (p=0.037) and overall survival (OS) (p=0.026). However, patients with EGFR gene amplification did more likely receive aggressive treatment in clinical practice.Figure 1

Conclusion
EGFR protein overexpression and gene amplification in ESCC were correlated with the malignant biological behavior, including tumor differentiation and lymph node metastasis. Further studies should be conducted to analyze the prognostic value of EGFR protein overexpression and gene amplification in patients with ESCC.

Background
Trametinib is a reversible and highly selective allosteric inhibitor of MEK1/MEK2. In a 2:1 randomized, open-label phase 2 study, trametinib showed an overall response rate of 12% but did not show improvement in progression-free survival (PFS) over docetaxel as second-line treatment in patients with KRAS-mutant NSCLC. Median overall survival (OS) was 8 months in the trametinib arm and has not been reached in the docetaxel arm (ASCO 2013 abstract #8029). Several CAF markers were identified as prognostic and predictive of clinical benefit in patients with renal cell carcinoma (Tran, Lancet 2012), and of tumor shrinkage in patients with NSCLC (Nikolinakos, Cancer Research 2010), after receiving targeted therapy.

Methods
Of 134 patients randomized, plasma samples (n = 116 baseline [113 KRAS-mutant], n = 89 day 22) from patients who consented for this optional study were analyzed for 38 CAFs using SearchLight multiplex assays in a CLIA-certified laboratory. Baseline CAF levels were tested for association with PFS using proportional hazards regression within and between arms. Correlation between baseline CAFs and baseline tumor burden was assessed using the Spearman rank correlation test. Change from baseline was assessed using Wilcoxon signed rank tests. P < .01 was considered significant; for treatment arm by CAF-level interaction, P < 0.05 was considered significant.

Results
Lower baseline levels of IL-6 and OPN and higher baseline levels of TRAIL were associated with longer PFS in patients treated with trametinib but not those treated with docetaxel. Interaction between CAFs and trametinib or docetaxel treatment was significant for IGFBP-1, MMP-9, E-selectin, and VEGF. There was a positive correlation between IL-6 levels and baseline tumor burden. At day 22, decreases (trametinib: ANG-2, IGF-1, IGFBP-3, IL-10, IL-2R, TIMP-1; docetaxel: IL-6, MIP-1A, MIP-1B, SDF-1) and increases (trametinib: IGFBP-1, IL-6, MMP-2, PIGF, VEGF) in CAF levels were observed. Additional results (pairwise correlation, CAF levels and OS, change from baseline CAF levels and PFS and OS) will be reported.

Conclusion
Circulating baseline CAF levels may be predictive of PFS for patients treated with trametinib or docetaxel. CAF levels are modulated by each treatment. The impact of circulating baseline CAFs on PFS warrants additional investigation in well-designed clinical trials. Plasma CAF profiling may aid in the prognostic evaluation of patients and determine potential therapeutic response to trametinib treatment in patients with NSCLC.

Background
Lung cancer is the leading cause of death among malignant tumors worldwide therefore it is important to develop novel diagnostic techniques.

Methods
This is a prospective case control study including two groups of patients: Group I: healthy volunteers as control .Group II: Patients with advanced non-small cell lung cancer (NSCLC). Plasma VEGF 165 levels were measured at baseline by ELISA before first line Gemcitabine-Cisplatin regimen. High VEGF 165 cutoff taken was >703 pg/ml .Primary end point was comparison of plasma VEGF 165 levels in cases and controls. Secondary endpoint was correlation between High VEGF 165 and clinical response (CR), Progression free survival (PFS) and overall survival (OS) in advanced NSCLC patients.

Results
35 patients with advanced NSCLC and 34 age and sex matched normal subjects as control were included and followed up during the period from 10/2009 to 10/2012 with median follow up of 10.5 months. Median plasma VEGF 165 level was 707 pg./ml in cases versus 48 pg./ml in controls, (p < 0.001).No significant correlation was found between plasma VEGF 165 level and clinical response(p < 0.5).No significant correlation was found between plasma VEGF 165 level and Median PFS and OS (p=1 and 0.7 respectively.

Conclusion
Plasma VEGF 165 is a potential diagnostic marker in advanced NSCLS.

Background
Although a high level of thymidylate synthase (TS) expression in malignant tumours has been suggested to be related to a reduced sensitivity to the antifolate drug pemetrexed, no direct evidence for such an association has been demonstrated in routine clinical samples from patients treated with this drug. The purpose of this study was to evaluate the impact of quantitative TS expression in tumor cells as predictor of the efficacy in patients with advanced non-small cell lung cancer, small cell lung cancer (SCLC) and mesothelioma treated with pemetrexed in our institution.

Methods
54 patients were included in this study: 40 stage IV NSCLC (26 adenocarcinomas, 11 large cell, and 3 squamous cell carcinoma), 3 SCLC and 11 mesothelioma. 21 patients received platins-pemetrexed as first line NSCLC, 20 pemetrexed in monotherapy as second and further lines and 3 carboplatin-pemetrexed fo extensive disease SCLC. Total RNA was isolated by RNeasy FFPE procedure (Qiagen). The expression of TS was analyzed by RT-qPCR using appropriate mRNA specific primers and probes in LightCycler 480II platform at 45 cycles. TS levels was calibrated to expression in normal tissue.

Results
From 54 cases, TS expression data were available in 32 cases, detecting overexpression in 23 (71.8%) and low expression in 9 (28.2%) patients. The response rate for patients with low TS expression was 0.63 compared with 0.15 in patients with overexpression (p=0.015). A significant benefit in time to progression was observed in patients with low expression (median TTP 12 vs. 2 months respectively, p= 0.002), whereas did not impact on overall survival (median OS 20 vs. 19 months respectively, p= 0.595).

Conclusion
TS overexpression in tumor cells correlated with a reduced response to pemetrexed-containing chemotherapy and might be used as a predictive biomarker in advanced lung and mesothelioma cancer patients.

Background
The EGFR mutated status becomes a very important factor for NSCLC patients considering of the treatment, but the mechanism of the mutation is still unknown.Our study aimed to detect the correlations among EGFR mutations and polymorphisms of EGFR and CYP1A1 genes and their associations with clinical outcome of NSCLC patients treated with EGFR-TKI.

Methods
We evaluated the EGFR mutations, the genotypes for EGFR Intron1 (CA) n, R497K and CYP1A1 *2A, *2C polymorphisms in 70 Chinese patients with NSCLC. Genetic polymorphisms were correlated to EGFR mutations. As to subgroup of 36 patients who accepted the EGFR-TKI treatment and had systemic 5 years follow up data, the associations among the somatic EGFR mutations, the genomic polymorphisms of EGFR and CYP1A1 and clinical outcome of the EGFR-TKI were analyzed.

Results
The data show that EGFR Intron1 (CA) n and CYP1A1*2A, *2C polymorphisms were correlated with EGFR mutations (P=0.006, P=0.001, and P=0.008, respectively) and all the three polymorphisms were also associated with EGFR 19 exon delection (P=0.007, P=0.033, and P=0.006, respectively); whereas the multivariate analysis demonstrated that only CYP1A1*2A polymorphism was associated with EGFR somatic mutations (P=0.021). For 36 patients treated with EGFR-TKI, the EGFR mutation and CYP1A1*2A polymorphism showed correlation with clinical response of EGFR-TKI(P=0.001, and P=0.011, respectively); However, the multivariate analysis confirmed that the EGFR mutation is still the most effective predictive factor (P=0.006) ; Either the log-rank test and Cox regression analysis demonstrated that the CYP1A1*2A polymorphism is independent prognostic factor for patients’ overall survival treated with EGFR-TKI( P=0.000 for both statistical analysis).

Conclusion
The results demonstrate that the CYP1A1*2A polymorphism is correlated with EGFR somatic mutation; for advanced NSCLC patients with EGFR-TKI therapy, the EGFR mutation status is still most effective predictor for clinical response of EGFR-TKI, whereas the CYP1A1*2A polymorphism is an independent prognostic factor. The inner mechanisms deserve thorough study.

Background
A large number of studies have evaluated the impact of TP53 mutation on the prognosis of patients with non-small cell lung cancer (NSCLC); however, the results of these studies are still controversial. Recently, considerable intratumor heterogeneity for genetic alterations has been demonstrated in various human cancers, including lung cancer.

Methods
In the present study, we evaluated TP53 mutations in NSCLCs by direct sequencing and observed remarkable variation in the values of the relative intensity (RI, the height of the peak of mutated allele/the height of the peak of non-mutated allele) of TP53 mutation. We also examined whether the RI values were associated with intratumor heterogeneity of TP53 mutation. In addition, we evaluated the relationship between TP53 mutation and survival outcome.

Results
The patients with TP53 mutation did not have significantly worse survival compared to those without the mutation. However, when tumors with TP53 mutation were categorized into two groups, those with a low and those with a high RI, the latter group had significantly worse survival compared to those with wild-type TP53 (adjusted hazard ratio = 2.58, 95% confidence interval = 1.21-5.48, P = 0.01), whereas the former group did not.

Conclusion
These results suggest that intratumor genetic heterogeneity may be an important factor in determining the role of TP53 mutation on the prognosis of NSCLC patients.

Background
Met is a receptor tyrosine kinase that is encoded by c-Met a proto-oncogne. Abnormal c-met expression has been described in several solid tumors most notably in non small cell lung carcinoma and gastric carcinoma where tumor growth and survival is driven by met signaling. Furthermore, met is also a resistance pathway for EGFR tyrosine kinase inhibitors. Met-targeted therapies are currently being tested in clinical trials and met IHC is being used as one of the methods to evaluate met expression. Malignant mesothelioma is an aggressive tumor with limited treatment options and short survival. Met-based therapies could be considered in mesotheliomas with deregulated c-Met expression.

Methods
The study cohort included 39 malignant mesothelioma (Age 15-91 years; Sex: 11 female and 28 male; Histologic type: 1 sarcomatoid, 7 biphasic and 21 epithelioid, primary location: 8 peritoneal and 31 pleural). Immunohistochemistry was performed using an anti-total c-Met rabbit monoclonal antibody (clone SP-44, Ventana Medical Systems, Tucson, AZ, USA) using 4 micron sections of large biopsies or resection specimens and an automated platform (Ventana Benchmark Ultra, Ventana Medical Systems, Tucson, AZ, USA). The staining was evaluated for intensity (none, week, moderate, strong) and extent (percent of tumor staining) and a score was assigned to each tumor on a scale from 0 to 3+ (0: no staining, 1+: any staining in less than 50% of tumor, 2+: moderate to strong staining in >50% of tumor, 3+: strong staining in >50% of tumor). Tumors with and IHC score of 3+ and 2+ were considered positive.

Results
Twenty (20) tumors were scored negative and 19 tumors positive. Strong (3+) staining was seen in 3 epithelioid mesotheliomas (all pleural), 2+ staining was seen in 1 biphasic and 15 epithelioid mesotheliomas, while the remaining 6 biphasic, 1 sarcomatoid and 13 epithelioid mesotheliomas were negative (1+: 19; 0: 1).

Conclusion
c-Met positivity (3+ or 2+) is seen in the majority of epithelioid mesothelomas (18 of 21) and in the minority of biphasic mesotheliomas (1 of 7), while the single sarcomatoid mesothelioma was negative. Since the majority of malignant mesotheliomas are of the epithelioid subtype we expect the majority of malignant mesotheliomas be considered met positive as determined by immunohistochemistry and potentially amenable to met-targeted therapy. Correlation with met amplification and activating c-met mutations needs to be investigated to better understand the mechanisms of c-met over expression in malignant mesotheliomas.

Background
The discovery of various genetic alterations that underlie lung cancer has opened-up a new era in the development of specifically targeted therapies employing specific alteration-dependent inhibitors. In the vast majority of NSCLC patients genetic alterations occur in EGFR (20-25%), BRAF (3%), and ALK (3-10%), seemingly in a predominant mutual exclusive manner. Hence, patient stratification for EGFR, KRAS, BRAF, and ALK alterations is becoming common practice among oncologists. The mutational status of NSCLC patients is considered dynamic and can change in due course of the disease and selection in response to therapy. Longitudinal monitoring of the mutational status of NSCLC patients is of crucial importance to tailor targeted therapy by adapting treatment regimens according to mutational status. However, technical challenges associated with serial tumor biopsy constitute a major challenge in longitudinal molecular monitoring and targeted treatment of NSCLC patients. Blood-based assays may overcome such limitations and allow for frequent assessment of the mutational status.

Methods
Here, tumor tissue, platelets, and/or plasma of NSCLC patients were collected and analyzed for the presence of translocated ALK using FISH, IHC, and/or RT-PCR. Monitoring of EML4-ALK in platelets and plasma was performed by RT-PCR on EML4-ALK positive patients treated with crizotinib and correlated to the clinical response as measured by serial radiographic CT.

Results
From a multicenter cohort of NSCLC patients we identified EML4-ALK positive patients by FISH, IHC, and RT-PCR, for blood platelet and/or plasma isolation. In blood platelets of ALK positive NSCLC patients (n=24) and ALK negative control subjects (n=54) we demonstrated an EML4-ALK test sensitivity of 70-80% and specificity of 100%. We detected EML4-ALK in plasma of ALK positive NSCLC patients (n=22), however with inferior sensitivity of 20-30%. In addition, longitudinal EML4-ALK monitoring in blood platelets of an ALK positive NSCLC patient correlated with the crizotinib response.

Conclusion
We demonstrate here that blood platelets of NSCLC patients are a biosource for the detection of the EML4-ALK translocation and may proof useful for longitudinal monitoring of ALK inhibitors in NSCLC patients, with superior outcome over plasma.

Background
Epidermal growth factor receptor (EGFR) antagonists are therapeutic agents that can be effective in colorectal cancer (CRC) treatment. It has been shown that 40% of CRC tumors have activating KRAS exon 2 codon 12 and 13 mutations and that these mutations are associated with a poor response to EGFR antagonists. Recent studies have shown that mutations in KRAS exons 3 and 4 as well as NRAS exons 2, 3, and 4 also predict poor response to EGFR agonists such panitumumab. Sensitive detection (down to 1%) with Sanger sequencing of such diagnostic biomarkers is necessary to determine the presence or emergence of drug resistant tumor cell populations. Locked Nucleic Acid (LNA) containing oligonucleotides (oligos) have been used in microRNA (sample preparation), RNA (in situ hybridization) and DNA (SNP detection using allele-specific PCR) analysis applications. Incorporation of LNA into oligos has the advantage of increasing the melting temperature of the LNA/complementary template duplex after hybridization as compared to duplexes using standard oligos.

Methods
To improve the mutation detection limits of Sanger sequencing, an LNA-based approach has been developed to cycle sequence the mutant allele selectively in the presence of the wild-type allele. During cycle sequencing, an additional annealing step is added to hybridize the LNA-containing oligo (BLOCker-oligo) to the template DNA. A denaturing step is then performed at a temperature at which the BLOCker-oligo remains annealed to the wild-type sequence while the LNA oligo denatures from the mutant sequence. The sequencing primer then anneals to the mutant sequence and is subsequently extended. Both forward and reverse strand BLOCker oligos can be developed enabling sensitive enrichment for bidirectional sequencing.

Results
To show applicability of this methodology in CRC samples, the limit of detection in both the forward and reverse sequencing directions for multiple codon 12 and codon 13 KRAS exon 2 mutations with and without the addition of the KRAS exon 2 wild-type specific BLOCker-oligo will be demonstrated. To date, the increase in sensitivity is ~10 fold. In addition, a series of FFPE samples will be analyzed for mutations in NRAS and KRAS exons 2 – 4 with and without BLOCker-oligos to show the increase sensitivity of this approach.

Conclusion
BLOCker sequencing is an efficient methodology for the detection of any mutation present in a sample using standard laboratory equipment and Sanger sequencing. The assays being developed at Transgenomic will be offered as CE IVD kits for the detection of all TKI-response associated mutations in KRAS and NRAS Exons 2, 3 and 4.

Background
Malignant pleural mesothelioma (MPM) is an aggressive tumor in the pleural lining of the lung often resulting from prior exposure to asbestos. Median overall survival following frontline chemotherapy with pemetrexed/cisplatin is approximately 12 months. There is no established second line treatment. New therapeutic modalities are urgently needed to improve the prognosis of MPM patients. Approximately 50% of MPM patients exhibit homozygous disruption of the NF2 tumor suppressor gene by mutation and/or deletion resulting in lack of expression of functional merlin protein. VS-6063 is a potent, orally active, inhibitor of focal adhesion kinase (FAK).

Methods
Proliferation of drug-treated mesothelioma cell lines in 3-dimensional (3D) Matrigel culture was assessed by MTS, and orthotopic MPM xenograft growth in the lungs was measured in vehicle- vs. VS-6063-treated SCID mice. Since absence of merlin expression can theoretically result from several mechanisms including NF2 mutation and chromosome 22 abnormalities, we assessed NF2 gene deletion by FISH and merlin protein levels by IHC in the same human mesothelioma tumor samples.

Results
Among a panel of mesothelioma cell lines in 3D culture, MPM lines lacking expression of merlin protein were found to be especially sensitive to the selective FAK inhibitor VS-6063. In contrast, MPM cell lines with wild-type merlin were less sensitive with EC~50~ values greater than 1 μM. Accordingly, oral dosing of VS-6063 induced significant tumor growth inhibition in a merlin-negative mesothelioma model pre-implanted in the lungs of mice. To enable the planned stratification of MPM patients by merlin status, an immunohistochemistry (IHC) assay has been optimized to quantify merlin protein levels. A merlin IHC H-score below the defined cutoff was associated with loss of at least one copy of chromosome 22, indicating that chromosomal deletion is an important mechanism of merlin loss in mesothelioma patients.

Conclusion
These data support the clinical development of VS-6063 for treatment of malignant pleural mesothelioma patients stratified by merlin/NF2 status. VS-6063 is being studied in a registration-directed mesothelioma trial.

Background
Erlotinib (E) is currently indicated as first line therapy in patients with EGFR mutations, and as second and third line treatment for patients with advanced metastatic NSCLC. In Cyprus comprehensive EGFR testing started in January 2011. In this study we reviewed all the patients that received E between 2007-2010, without having undergone EGFR mutation testing, looking at predictive factors of response to E. In view of evidence suggesting that EGFR mutations are found in predominantly TTF1 +ve tumours, TTF1 status as well as clinical factors (i.e. adenocarcinoma histology, female sex and non smoking status) were assessed as potential predictive factors of response to E.

Methods
100 consecutive patients are included. 3 patients received this as 1[st] line, 49 patients as 2[nd] line and 48 patients as 3rd line and beyond. Previous treatments included gemcitabine platinum doublet as first line, and either pemetrexed or docetaxel as 2[nd] line therapy. Patients had regular Chest x-rays (every 2-3 cycles) and CT scans (every 3-4 cycles) and were assessed clinically on a monthly basis. Survival outcomes for both progression free survival (PFS) and overall survival (OS) were calculated with Kaplan Meier. Subset analyses using smoking status, sex, histology type and TTF1 and finally Cox regression was undertaken.

Results
40 female and 60 male were treated with E. Median age is 66 years (range 32-79). 45 patients were WHO performance status (PS) 0-1, 47 PS 2-3. For 8 patients PS was not recorded. Median progression-free survival (PFS) for all patients was 95 days (95% CI 68-118). Median overall survival (OS) from starting E was 144 days (95% CI 103-185). Toxicity was predominantly grade 0-2. Ten (10) patients had a partial response and 34 patients had stable disease. Subset analyses were undertaken based on smoking status, sex, histology type and TTF1. Univariate analysis for PFS using KM plots showed a statistically significant difference for sex, smoking and TTF1. On Cox regression only gender and TTF1 were statistically significant (0.007 and 0.006). There was a striking difference in median OS between patients with TTF1-ve tumours (33 days) and TTF1 +ve tumours (149 days). See table underneath.

	Median PFS (days)	95% CI (days)	Log Rank	Median OS (days)	95% CI (days)	Log Rank
All patients	95	68-118	-------	144	103-185	-------
Female	165	46-284	0.0137	236	45-427	0.1470
Male	80	40-120		104	60-148
< 10 Pack Yrs	203	149-257	0.0009	291	193-389	0.0034
> 10 Pack Yrs	54	15-93		84	53-115
TTF1 – ve	28	11-45	0.0002	33	28-38	0.0001
TTF1 +ve	109	34-184		149	35-263

Conclusion
TTF1 is a better predictor of benefit to E than histology, sex and smoking status. The very low median PFS and OS for patients with TTF1 –ve tumours would suggest that such patients derive no benefit from E, hence TTF1-ve status acts as a negative predictor of benefit to E.

Background
Recent advances in next generation sequencing provide us with the ability to simultaneously analyze both mutation status and copy number changes across a large number of cancer related genes. Here we discuss our experience in developing, validating and applying a 47 gene panel across our lung tumor patient population in a clinical laboratory.

Methods
Genomic DNA was extracted from 20 Formalin Fixed Paraffin embedded (FFPE) tissue samples from lung tumors for validation and over 80 patient samples upon clinical implementation. Extracted DNA was analyzed for quality and amplifiable quantity with suitable performance criteria established during validation. Between 10 and 250 ng of total genomic DNA was then subjected to the Truseq Amplicon (Illumina) assay for enrichment of the target regions across 47 different genes. Libraries were then sequenced on the Miseq (Illumina) to an average depth of coverage between 2000 and 4000x with 186 basepair, paired end reads. Data was then analyzed using analysis pipelines composed of various in house and open source tools, to detect insertions, deletions, amplifications and single nucleotide variants to an allele frequency of 5%.

Results
Across the clinical sample set, 67% of samples were found to have at least one disease associated mutation, 8% had only an unclear variant, 13% were ‘normal’, while 12% had DNA that was not adequate for testing. The average number of mutations seen in samples with disease associated changes was 1.6, with a range from 1 to 4. Half of the samples analyzed were found to have Tp53 mutations, followed by EGFR changes seen in 11%. While only 16 patients had EGFR changes, nearly half (7) of these had co-mutations, including changes at positions 709, 719, 768 and 790. Many of the EGFR changes were complicated insertion/deletions found in exons 19 and 20, which were missed by standard bio-informatic algorithms and only captured by in house tools. KRAS changes were seen in 21% of patients followed by PTEN, MET, STK11 and APC mutations seen in at least 2 patients. Other known somatic mutations were also identified in ATM, BRAF, CTNNB1, FBXW7, FLT3, GNAS, HRAS, NRAS, PIK3CA and ALK in at least one patient. This included the hotspot mutation F1174L in ALK, which is seen with high frequency in neuroblastoma.

Conclusion
The mutation status of many clinically relevant genes can be reliably detected in FFPE samples using a single molecular assay followed by high throughput sequencing. Using this approach known somatic mutations seen frequently in other tumor types can be readily identified in lung tumors, and highlights the future benefits of tumor profiling.

Background
The use of biomarkers for selecting patients with non-small cell lung cancer (NSCLC) for treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is essential for achieving a satisfactory therapeutic outcome. EGFR mutations have been found to be predictive of response to EGFR-TKIs. The aim of this study was to explore whether biomarkers which can be identified in plasma, such as EGFR mutations, circulating free DNA, and levels of expressed cytokines are predictive for response to EGFR-TKIs and patient survival time.

Methods
Formalin-fixed and paraffin-embedded biopsies from tumor tissues and paired plasma samples were collected from 134 patients with advanced NSCLC, EGFR mutations in both types of specimens were assessed by an ARMS/Scorpion assay using real-time PCR. Expression levels of transforming growth factor-alpha and beta one (TGF-α and TGF-β1) were assessed using an enzyme-linked immunosorbent assay (ELISA). Concentrations of circulating free DNA were detected in both NSCLC patients and healthy subjects by a colorimetric assay using ultraviolet spectrometry. The clinical significance of EGFR mutations, levels of cytokines, and circulating free DNA was assessed in advanced NSCLC patients treated with EGFR-TKIs.

Results
EGFR somatic mutations were detected in the tumors from 68 of 134 (50.7%) advanced NSCLC patients, and EGFR mutations were detected in the plasma samples from 17 (12.7%) NSCLC patients. Also, the concentrations of circulating free DNA were higher in NSCLC patients than in healthy subjects (P<0.01). EGFR-TKI treatment produced significant effects on progression-free survival (PFS) and overall survival (OS) that were related to the presence of EGFR mutations detected in the tumor tissues (P<0.01).Patients with high levels of TGF-β1 showed shorter OS and worse response to EGFR-TKI treatment than patients with low TGF-β1 levels (P<0.01); however, patients with different expression levels of TGF-α showed no difference in either PFS or OS (P>0.05). Multivariate analysis showed that younger age, adenocarcinoma, never smoking and EGFR somatic mutation were associated with a longer PFS time, and adenocarcinoma, never smoking, low performance status (PS) score, EGFR somatic mutation and low levels of TGF-β1 were associated with greater OS (P<0.05).

Conclusion
Plasma levels of TGF-β1 may be a marker for predicting response to EGFR-TKIs and survival time in NSCLC patients, and levels of circulating free DNA could be a biomarker for differentiating between NSCLC patients and healthy individuals.

Background
Long term health related quality of life (HRQoL) among patients operated for non-small cell lung cancer (NSCLC) has not been extensively studied, as most studies has end-point within 24 months following surgery. Endpoints ike progression-free survival and overall survival, postoperative HRQoL could be more important to the patient.

Methods
A total of 586 patients were operated for NSCLC in the Department of Cardiothoracic surgery of the Helsinki University Hospital between January 2000 and June 2009. Two validated quality of life "questionnaires, the 15D and the EORTC QLQ-C30 with its lung cancer specific module QLQ-LC13, were sent to patients alive in June 2011. Results of the 15D were compared with those of an age- and gender-standardized general population. Patient and treatment features predicting higher or lower long-term HRQoL were identified.

Results
Of the 276 patients who were sent the questionnaires, 230 (83.33%) answered. The median follow-up time was 4.85 years. When compared with the general population (picture), our patient group had significantly lower scores in the 15D total score, representing the total HRQoL of the patients, and in the dimensions Mobility, Breathing, Usual activities, Depression, Distress and Vitality. The patient group scored significantly higher in the dimensions Hearing and Mental function. Features predicting lower long-term HRQoL were comorbidity, measured with the Charlson comorbidity index (CCI), post-operative complications and pre-operative FEV~1~% 70% of the predicted value. Adjuvant-therapy was observed to predict higher long-term HRQoL.Figure 1

Conclusion
NSCLC patients suffer from permanently reduced long-term HRQoL compared to the age- and gender-matched normal population. Factors associated with reduced HRQoL were presence of comorbidity, postoperative complications and reduced FEV1-status preoperatively. The occurrence of complications was also associated with a significantly reduced survival rate. Adjuvant-therapy was associated with a higher HRQoL. Age and gender were not associated with significant differences in the total 15D-score or the QLQ-C30 global health score.

Background
Systematic mediastinal lymphadenectomy is still essential for an adequate postoperative staging of NSCLC. We tried to investigate the controversial role of sentinel node biopsy (SNB) in early stage non small cell lung cancer (NSCLC) surgery

Methods
A total of 52 patients with clinical T1N0MO NSCLC underwent SN navigation lobectomy using Tc-99 labeled tin colloid followed by systematic mediastinal lymphadenectomy (SML) in two years time period (2010-2012). Mapping of the mediastinal lymph nodes by their number and station followed by histopathological evaluation was performed. Patients data were statistically analyzed.

Results
Intraoperative SN was identified in 45 (87%) of these patients with 92% of accuracy. We found lobe specific skip nodal metastases in 5 (10%) patients resulting in upstaging. The incidence of ML metastases seemed to be more often in adenocarcinoma patients (p<0.05), but skip nodal metastases showed higher rate in squamous cell carcinoma patients. Intraoperative frozen section was not confirmed accurate for detecting micrometastases in two (4%) patients. Operative time was prolonged for 10 (8-25) minutes showing no difference in complication rate.

Conclusion
Procedure showed absolute safety and high accuracy. Our results indicated that SN identification could reduce mediastinal lymph node dissection in early stage NSCLC. Further clinical studies should be carried out in order to prove that minimally invasive surgical procedures could be curative for T1N0MO NSCLC.

Background
The extent of mediastinal lymph node assessment during surgery for non-small cell cancer remains controversial. Different techniques are used, ranging from simple visual inspection of the unopened mediastinum to an extended bilateral lymph node dissection. Furthermore, there are different terms to define these techniques: Sampling is the removal of one or more lymph nodes guided by preoperative finding. Systematic (full) nodal dissection is the removal of all mediastinal tissue containing the lymph nodes systematically within anatomical landmarks.

Methods
A Medline search was conducted to identify articles in English, addressing the role of mediastinal lymph nodes resection in the treatment of NSCLC

Results
Opinions favoring full lymphatic dissection include complete resection, improved nodal staging and better local control due to resection of undetected micrometastasis. Arguments against routine full lymphatic dissection are increased morbidity, increased operative time and lack of evidence of improved survival. For complete resection of non-small cell lung cancer a systematic nodal dissection is recommended for many authors, as the standard approach during surgery: it ascertains both adequate nodal staging and completeness of resection.

Conclusion
Whether extending the lymph node dissection influences survival or recurrence rate remains to be determined. There are valuable arguments in favor of not only an improved local control but also an improved long-term survival. However, the impact of lymph node dissection in long-term survival should be further assessed by large-scale multicenter randomized trials.

Background
The use of resections lesser than lobectomy as definitive management of a stage I non-small cell lung carcinoma (NSCLC) is a topic that creates controversy in the global medical community. To describe the current conclusions concerning the relative indications of each type of resection in the surgical treatment of stage I NSCLC, as well as the international results from their application concerning the local recurrence, disease-free survival, and five-year survival rates.

Methods
Thirty four prospective and retrospective studies registered in PubMed and Scopus electronic databases during the last twenty five years were reviewed. Bibliographies and handsearching of journals were used to identify trials. Studies’ authors, citations, objectives, and results were extracted. No meta-analysis was used. Validation of results was discussed.

Results
Segmentectomies were superior to wedge resections in terms of local recurrence and cancer-related survival rates. Sublobar resections were superior to lobectomy concerning preservation of pulmonary parenchyma. It was recommended that high-risk patients undergo segmentectomy. Lobectomies were superior to segmentectomies only for tumors >2 cm (T2bN0M0) as regarding disease-free and overall 5-year survival. There was no significant difference for tumors <2 cm in most studies. Free surgical margins were crucial for local control rates. Systematic lymphadenectomy was mandatory regardless of type of resection. In cases of pure bronchoalveolar carcinoma, segmentectomy was recommended. Shorter hospital stay was achieved with sublobar resections.

Conclusion
The choice of type of resection for T1aN0M0 tumors should rely on specific patient and tumor characteristics. Patient age and tumor size are the most important factors. Further prospective randomized trials are needed to determine minimal resections in early lung cancer patients.

Background
Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS) has been gradually application in clinic, but its value in staging of lung cancer and dignosing of mediastinal mass need to be evaluated. We intends to study comparatively the clinical value of EBUS and mediastinoscopy, and, want to provide a basis for the rational choice applications.

Methods
Between July 2009 and December 2012, 361 patients accepted mediastinoscopy, including 199 cases of lung cancer and 162 of mediastinal mass. During the same period, 348 patients accepted EBUS , including 216 cases of lung cancer and 132 of mediastinal mass. All of the biopsy samples were tested in the pathological department of Shanghai chest hospital. Analyzing the data of two groups and comparing the clinical value of mediastinoscopy and EBUS both on preoperative staging of lung cancer and diagnosing of mediastinal mass.

Results
With the pathological results as a gold diagnosis standard, the accuracy, sensitivity and specificity of mediastinoscopy diagnostic efficacy are 98.28%, 98.03% and 100%, respectively on lung cancer, and, 98.11%, 97.62% and 100%, respectively on mediastinal mass. For EBUS, the accuracy, sensitivity and specificity of diagnostic efficacy are 95.69%, 94.74% and 100%, respectively on lung cancer, and, 82.64%, 77.42% and 100%, respectively on mediastinal mass. Two techniques have not significant difference on diagnosing and staging of lung cancer (P>0.05), but have statistically difference on mediastinal mass (P<0.05), especially on sarcoidosis (P=0.0109), tuberculosis(P=0.0135) and lymphoma(P=0.0036).

Conclusion
Mediastinoscopy and EBUS have a similar clinical value in diagnosing and staging on lung cancer, but mediastinoscopy is superior to EBUS in diagnosing on mediastinal mass.

Background
To determine factors predicting early and long term mortality and complications in patients who underwent a thoracotomy because of primary lung cancer.

Methods
Data of patients who underwent a thoracotomy in the Catharina Hospital Eindhoven between January 1995 and January 2011 have been collected retrospectively from the medical files. Early mortality was defined as mortality <30 days after surgery. Last date of follow up was January 1, 2013. Patients were divided in three groups according to date of surgery (1: 1995-1999, 2: 2000-2004 and 3: 2005-2010). Complications were divided into major and minor complications. Predicting factors were assessed with uni- and multivariate logistic regression analysis. For long-term mortality and survival predicting factors were assessed using the Cox proportional hazards model.

Results
In total 501 patients underwent a thoracotomy due to primary lung cancer. Overall 30 day mortality (early mortality) was 5.6% (n=28). Multivariate analysis showed that age over 70 (p=0.002), pneumonectomy (p=0.008) and a pre-operative VO2max of <15 ml/kg/min (p=0.02) were significant predictors of early mortality. With respect to long term survival, 308 (61%) patients had died at the end of the follow-up period. Median survival time was 44 months, with an overall 5- and 10- year survival of 45% and 27%, respectively. The 5-year survival for stage I, II and III-IV was 61%, 46% and 16%, respectively (p<0.0001, log rank test). Furthermore Cox regression analysis showed that stage (stage I, stage II compared to stage III-IV), FEV1% ≤70%, a history of cerebrovascular disease (CVD) and surgery in an earlier time period (1, 2 compared to 3) were significant predictors of long term mortality. Finally, the only significant predictor for major postoperative complications was a history of COPD (OR 2.32; 95% CI 1.38-3.91).

Conclusion
In this cohort, mortality and complication rates in lung cancer patients after thoracotomy were in line with literature. Significant predictors of early mortality were age, pneumonectomy and pre-operative VO2max. Significant predictors of long term mortality were disease stage, FEV1%, a history of CVD and surgery in an earlier time period.

Background
The incidence of lung cancer in the older population is increasing. In case of resectable primary lung cancer, surgery remains the best treatment for cure, independent of age. However, the prevalence of co-morbidities among elderly lung cancer patients is significantly higher. A presumed fear of increased postoperative morbidity and mortality in the elderly patients has resulted in the delivery of sub-optimal cancer surgery. We believe all elderly cancer patients should be offered optimal treatment depending on their functional status not on their age; a major step in ensuring this would be to establish the appropriate surgical management protocol for elderly cancer patients. Thus it is important to determine whether the elderly would indeed benefit from the same management standards as their younger counterparts. This study aimed to determine the suitable operative procedures for elderly patients with primary lung cancer.

Methods
Between January 2006 and December 2012, 98 patients aged over 75 years with primary lung cancer received lobectomies in our hospital. We divided the patients into group A (75-79 years old) and group B (over80 years old) and analyzed their relapse-free survival and postoperative complication incidence.

Results
The patients in group A (46 men and 17 women) had a mean age of 76.8 years, 36 patients had adenocarcinomas, 24 had squamous cell carcinomas, and 3 had other tumors. A mediastinal lymph node dissection (MLND) was performed in 52 patients but not in 11 patients. The patients’ pathological stages were 1A (21), 1B (13), 2A (5), 2B (12), 3A (11), and 4 (1). The 3-year relapse-free survival was 66.3 % (MLND-positive, 62.6 %; MLND-negative, 90.9 %). Postoperative complications occurred in 30.8 % of the MLND-positive patients and 18.2 % of the MLND-negative patients. The patients in group B (26 men and 9 women) had a mean age of 82.4 years, 17 patients had adenocarcinomas, 12 had squamous cell carcinomas, and 6 had other tumors. An MLND was performed in 24 patients but not in 11 patients. The patients’ pathological stages were 1A (12), 1B (10), 2A (5), 2B (3), 3A (1), and 3B (4). The 3-year relapse-free survival was 73.3 % (MLND-positive, 75.7 %; MLND-negative, 68.2 %). Postoperative complications occurred in 16.7 % of the MLND-positive patients and 9.1 % of the MLND-negative patients.

Conclusion
This study showed a survival benefit in elderly lung cancer patients who underwent lobectomies. In clinically well-documented early nodal stage disease (N0 patients and N1 patients with limited hilar disease), an MLND did not have a therapeutic effect and thus may not be necessary. Omitting MLNDs in elderly lung cancer patients also provided fewer postoperative complications. In this study, we proved that elderly patients with resectable lung cancer who received lobectomies without MLNDs could achieve long-term survival and be a safe procedure.

Background
The term "ground glass opacity (GGO)" on high-resolution computed tomography (HRCT) is defined as “hazy increased attenuation in the lung that does not obliterate the bronchial and vascular margins” by Fleischner Society. The identification of small lung nodules of GGO on HRCT often implies lung cancer, especially well differentiated adenocarcinoma or atypical adenomatous hyperplasia; however, there is no objective definition of GGO, such as the computed tomography number.

Methods
A single institutional retrospective study. To access the correlation between radiological and pathological diagnosis of the patients with small pure GGO on HRCT. Thirty-nine consecutive surgically resected patients with pure GGO less than 30 mm detected by HRCT between July 2008 and March 2013 in our department were retrospectively examined. The median follow-up of these patients was 28.7 (1.9 - 92.7) months.

Results
The median age of the patients was 64 (range 42-82) years old, 19 patients were male and 20 were female. The median size of major axis of lung nodules was 11 (range 5-25) mm, and 29 (74.4%) were less than 15 mm and 10 were between 15 and 30 mm in diameter. Twenty-eight (71.8%) patients had a single nodule, whereas 11 patients had multiple nodules. Six of the 39 patients had a previous history of malignancy (three lung cancers and three other cancers). During the follow-up period, 22 patients had nodules that were stable in size or appearance, and five patients had nodules that either became enlarged or in which the opacity increased, as determined by HRCT. The other twelve patients were operated based on the findings of their first HRCT, basically by the attending surgeons’ decision. Partial resection was performed in seven patients, segmentectomy in 11 patients and lobectomy was performed in 21 patients. Histologically, thirty-seven patients had adenocarcinoma, one had small cell carcinoma and one had a benign tumor. Among the 37 patients with adenocarcinoma, 14 were adenocarcinoma in situ, five tumors were minimally invasive and 18 were invasive according to the IASLC/ATS/ERS classification. There was no postoperative recurrence during the follow-up period.

Conclusion
Even if the small pulmonary nodules present as pure GGO, they may still be adenocarcinoma with an invasive nature. The timing of surgery should be considered carefully so that a chance to achieve a cure of such patients is not missed.

Background
Superior sulcus tumors (SST) comprise a subgroup of non-small-cell lung cancers that arise near the pulmonary apex or superior sulcus. They generally invade the chest wall and brachial plexus, and occasionally the subclavian vessels. Induction chemoradiation therapy followed by surgery is the recommended treatment for SST. However, surgical approaches for SST remain controversial, partly because of their infrequent use. Several approaches to resecting these tumors have been described, depending on the precise localization and involvement of the surrounding organs. These include posterolateral thoracotomy, hemi-clamshell and transmanubrial osteomuscular-sparing approaches. It is necessary to establish the appropriate multimodality therapy for SST, including the optimal surgical approaches.

Methods
We retrospectively analyzed the clinical courses of patients with SST treated with surgery at our institution. A total of 2765 patients with non-small-cell lung cancer were treated surgically at Osaka City General Hospital, Japan, from January 1995 to December 2012. Among these, 34 patients with SST were investigated in this study.

Results
The mean age of the patients was 62 years (range, 42–90 years). There were 32 men and two women. Seventeen patients had squamous cell carcinoma, 12 had adenocarcinoma, and five patients had tumors of other histological types. There were 21 patients with stage 2B, 10 with stage 3A, and three patients with stage 3B disease. Two patients received induction chemotherapy, and 22 patients received induction chemoradiotherapy. Posterolateral thoracotomy was performed in 11 patients and anterior thoracotomy (hemi-clamshell, transmanubrial osteomuscular-sparing approaches) in 22 patients. A combination of anterior and lateral thoracotomies was applied in one patient. Pulmonary lobectomy was performed in 25 patients, segmentectomy in one patient, and pulmonary partial resection in nine patients. The resected surrounding organs, other than the chest wall, were the subclavian artery in two patients, the superior vena cava in two, and the aortic arch and vertebral body in two patients each. The median follow-up period was 16 months (range, 3–154 months). Postoperative 1-, 3- and 5-year survival rates were 72%, 46%, and 34%, respectively. Investigation of clinicopathological factors with potential impacts on postoperative outcome identified pathological nodal extension as the only significant factor indicating poor prognosis (p < 0.01). Tumor markers, surgical approach, type of pulmonary resection, and type of resected surrounding organ had no effect on postoperative outcome. No viable tumor was observed in seven of 22 patients treated with induction chemoradiotherapy, and the postoperative 5-year survival rate in these seven patients was 86%. Recurrent disease was observed in 17 patients during the postoperative follow-up period. Local recurrence was observed in five patients and recurrence in distant organs was observed in 13 patients.

Conclusion
Patients with node-positive SST have a poor prognosis, and surgical indications should be investigated fully in these patients. Induction chemoradiotherapy is necessary to treat SST. The major sites of recurrence are in distant organs, and the type of pulmonary resection does not affect postoperative outcome. Partial resection may be an acceptable option in patients with no detectable viable tumor after induction chemoradiotherapy.

Background
Recently, multiple primary lung cancer is inreasing with progress of imaging diagnostic technology and improvement in treatment results. We assesed the selection of the type of pulmonary resection, operative morbidity, mortality and the outcome of our cases which enforced surgical treatment for multiple primary lung cancer.

Methods
Of 840 patients who underwent operation for primary lung cancer between January 2001 and May 2013.We consider 57(6.7%) to have a multiple primary lung cancer. We use the criteria of Martini and Melamed as a diagnosis of multiple primary lung cancer.

Results
The group comprised 27 men and 30 women. Median age was 66.7 years (48-80). It occured synchronous in 21 cases and metachronous in 36 cases. Cell type combination was adenocarcinoma-adenocarcinoma in 37 cases, adenocarcinoma-squamous cell carcinoma in 10 cases, squamous cell carcinoma-squamous cell carcinoma in 6 cases, adenocarcinoma-large cell carcinoma in 3 cases and large cell carcinoma-large cell carcinoma in 1 case. Operative procedures was lobectomy-lobectomy in 13 cases, lobectomy-segmentectomy in 12 cases, lobectomy-edge resectionin 19 cases, edge resection-edge resection in 5 cases and lobectomy/edge resection-radio therapy in 3 cases. Pathological stage for the first cancer was IA/IB/IIA/IIB/III = 35/14/4/0/4. The 5 year survival rate after first surgery was 81.7%, and second surgery was 72.2%.

Conclusion
With this result, we consider that surgical resection may prove beneficial in cases which postoperative good survival can be expected by the aggressive surgical approach, despite it is difficult to distinguish multiple primary lung cancers and metastatic cancers preoperatively.

Background
Postoperative acute respiratory distress syndrome (ARDS) is a recognized complication of pulmonary resection. ARDS following lung resection has a miserable prognosis, with overall hospital mortality rates over 25%. Previous studies demonstrated that there were risk factors of ARDS after pulmonary resection including age, chronic obstructive pulmonary disease (COPD), interstitial pneumonia, concurrent cardiac disease, prior therapy, remaining lung perfusion, duration of operation, increased blood loss and so on. Neutrophils and neutrophil elastase (NE) are believed to play a key role in the endothelial injury and increased vascular permeability characteristic of ARDS. Sivelestat sodium hydrate is a selective NE inhibitor and has been shown to improve respiratory status in cases of ARDS. It has not been well known whether or not NE inhibitors are beneficial for prevention of ARDS after lung resection.

Methods
We conducted a pilot study to investigate the efficacy of perioperative administration of sivelestat sodium hydrate to prevent postoperative ARDS in 34 non-small cell lung cancer (NSCLC) patients who had the various preoperative risk factors of the incidence of ARDS after pulmonary resection in Chiba University between 2009 and 2011. They received sivelestat sodium hydrate (5mg/kg/day) intravenously for 7 days starting at the beginning of operation.

Results
The patient demographics were as follows: median age, 68 years of age (range 47 to 83 years), male/female ratio, 31/3, clinical stage I/II/III, 9/6/19. The histology was adenocarcinoma (n =19), squamous cell carcinoma (n=10) and others (n =5). Risk factors of ARDS included induction chemotherapy (n=3), induction chemoradiotherapy (n=17), interstitial pneumonia (n=10), COPD (n=3) and medical history of ARDS (n=1). All 34 patients underwent complete resection. The operations included 2 partial resections, 31 lobectomies, and 1 pneumonectomy. Of the 31 patients who received lobectomy, bronchial or arterial plasty was performed in 9 patients. The postoperative mortality rate was 2.9%. One patient died of heart failure on the nineth postoperative day. There was no incidence of ARDS after pulmonary resection in all patients.

Conclusion
Perioperative administration of sivelestat sodium hydrate can be beneficial to prevent postoperative ARDS in NSCLC patients. Prospective studies are required.

Background
In 2005, we reported the study of intentional limited resection for small peripheral lung cancer based on intraoperative pathologic exploration. At that time, only 14 patients with a small adenocarcinoma showing ground-glass opacity (GGO) had undergone limited resection. After that, we have continued limited resection and follow-up. The median follow-up time from the operation has reached 80 months, so we analyze the long-term results of this procedure.

Methods
Between 1996 and 2013, we enrolled 56 patients in this study. Entry criteria were: 1) cT1aN0M0 peripheral adenocarcinoma, 2) High resolution computed tomography (HRCT) findings suspected of having a Noguchi type A or B adenocarcinoma, and 3) pulmonary function adequate to permit lobectomy. When the tumor consisted of GGO only or GGO with a solid component that accounted for less than 50% of the surface area on HRCT, the tumor was suspected to be Noguchi type A or B adenocarcinoma. Wedge resection or segmentectomy was performed, and was followed by an intraoperative pathologic exploration. After confirming the diagnosis of Noguchi type A or B by intraoperative pathologic exploration, operation was completed. No systematic lymph node dissection or sampling was performed. If the lesion was not Noguchi type A or B, extended segmentectomy or lobectomy with systematic lymph node dissection was performed instead.

Results
Between 1996 and 2013, we enrolled 56 patients in this study. Limited resection was performed in all patients, wedge resection in 52, and segmentectomy in 4. Intraoperative pathologic exploration revealed that the lesion was not Noguchi type A or B in 11 patients. In these 11patients, we underwent extended segmentectomy in 2 and lobectomy in 9 with systematic lymph node dissection. Intentional limited resection was completed in 45 patients. Of these, 7 had Noguchi type A tumors, and 38 had Noguchi Type B tumors based on intraoperative pathologic exploration. Postoperative pathologic examination revealed 10 patients with Noguchi type A, 31 patients with Noguchi type B, and 4 patients with Noguchi type C. We recommended reoperation to 4 patients with Noguchi type C, but all refused reoperation and has been carefully followed at 3-month intervals. There was no postoperative and 30-day mortality or in-hospital mortality. There were no morbidities. All patients but one are alive without recurrence of lung cancer at the time of writing. Only one patient died of malignant lymphoma without recurrence. The follow-up periods have ranged from 6 to 195 months, and median follow-up period is 80 months. The overall 5- and 10-year survival rates were 96% and 96%, respectively. The 5- and 10-year recurrence –free proportions were 100% and 100%, respectively.

Conclusion
When patients are carefully selected by preoperative HRCT and intraoperative pathologic exploration, limited resection can be an acceptable option for the treatment of T1aN0M0 adenocarcinoma showing GGO lesion.

Background
N1 disease is a subset of non-small cell lung cancer (NSCLC) with a different prognosis than other subsets. Although the NCCN guideline recommends adjuvant chemotherapy alone in completely resected N1 disease, locoregional failure, which could have been prevented by postoperative radiotherapy (PORT), may not be uncommon. Using PORT with modern techniques has resulted in significantly higher rates of local control, disease-free survival, and overall survival. Therefore, this study aimed to evaluate the actuarial rates of locoregional failure in patients with pathologic N1 NSCLC and to identify the risk factors associated with an increased risk of locoregional failure, which could have been potentially prevented by PORT after complete resection.

Methods
Between 2003 and 2010, we enrolled 136 patients who underwent complete resection with pathologically confirmed N1 disease through the prospective lung cancer database of Seoul National University Bundang Hospital. Patients who underwent neoadjuvant therapy, adjuvant radiotherapy, or operative mortality were excluded. Multiple factors potentially related to outcomes including patient-related factors, surgery-related factors, and pathologic factors were extensively evaluated. Locoregional failure, which could have been potentially prevented by PORT, was defined as recurrence at either a bronchial stump, or a resected margin of the lung, hilum, and mediastinum. Other failures were ipsilateral lung recurrence, pleural seeding, and metastasis of distant organs. Univariate analysis by a log rank test and multivariate analysis by the Cox proportional hazards model were performed to identify risk factors independently associated with a higher risk of locoregional failure.

Results
The median follow-up duration was 45 months (6-114) and recurrence developed in 54 (40%) patients. The actuarial 5-year rates of disease-free survival and overall survival were 56% and 66%, respectively. From the perspective of first site recurrence, 23 (17%) locoregional failures, which could have been potentially prevented by PORT, included recurrence in the mediastinum in 10, bronchial stump in 5, regional lymph nodes in 4, and mediastinum + others in 4. The 31 (23%) other failures included a distant organ in 17, ipsilateral lung in 12, and pleural seeding in 2. The median survival time from locoregional failure and other failures was 41 and 57 months, respectively; however, there was no significant difference. Risk factors of locoregional failure were squamous cell carcinoma, number of involved node (>1), pathologic stage (IIIA), interlobar node involvement, more than 2 node stations of involvement, and a lymph node ratio greater than 10% by univariate analysis. Pathologic stage (HR=4.768, 95% CI=1.641-13.859, p=0.01), interlobar node involvement (HR=2.783, 95% CI=1.057-7.327, p=0.04), and squamous cell carcinoma (HR=2.449, 95% CI=0.929-6.454, p=0.07) were independent risk factors by multivariate analysis.

Conclusion
Locoregional failure was more common than expected, and pathologic stage, interlobar node involvement, and cell type were independent risk factors for locoregional failure after complete resection of N1 NSCLC. A prospective clinical trial may be necessary to evaluate the effectiveness of adjuvant radiotherapy in patients with these risk factors.

Background
Malignant pleural effusion or pleural seeding is detected in advanced non-small cell lung cancer (NSCLC) patients, and they are generally associated with poor prognosis. Systemic chemotherapy is the mainstay modality in these patients. However, it is not enough to improve the survival. Intrapleural perfusion hyperthermic chemotherapy (IPHC) provides direct effect to pleural seeding cancer cells. This study attempted to evaluate the efficacy and safety of IPHC.

Methods
From 2003 to 2012, 41 patients who underwent IPHC for malignant pleural effusion or pleural seeding for NSCLC in our institute. The IPHC was performed with cisplatin (dose:150-200mg/m[2]) for 90 minutes after resection of primary tumor. Efficacy was determined by computed tomography and Positron Emission Tomographic (PET) standardized uptake value (SUV) postoperatively.

Results
The IPHC group consisted of 25 males and 16 females. The mean age was 60.98±9.80 year ranging from 33 to 78. Preoperative pleural mean SUV was 1.46±1.88 (R: 0-6) and postoperative pleural mean SUV was 1.58±1.72 (R: 0-5) (p= 0.933). Sixteen patients were received adjuvant systemic chemotherapy. The 2-year and 5-year survival rate were 82.1% and 44.9%, respectively. Major post-IPHC complications are acute renal insufficiency (n=4, 9.76%) and arrhythmia (n=2, 4.88%). There were no difference in sex, age, adjuvant systemic chemotherapy, tumor size, nodal statues between the patients who survived more than 2 years and less than 2 years. There was no difference in SUV of preoperative main mass and pleura between the patients who survived more than 2 years and less than 2 years. However, the SUV of postoperative pleura in the patients who survived more than 2 years (SUV: 2.92±1.98) was less than that of the patients who survived less than 2 years (SUV: 1.16±1.45) (p=0.031).

Conclusion
IPHC would be safety procedure for malignant pleural effusion or pleural seeding. IPHC may provide better survival compared with the systemic chemotherapy only in the highly selected patients. Low post-IPHC SUV uptake would be provide longer survivor.

Background
In Japan, an aging society, the number of surgical operations performed in the elderly has been increasing. In this study, to investigate whether the surgical resection of non-small cell lung cancer benefits extremely elder patients (85-year-old or over), we analyzed the clinical outcome of our patients.

Methods
Sixteen consecutive patients aged 85 years or older who underwent surgical resection of primary non-small cell lung cancer in our hospital from May 2002 to September 2012 were enrolled in this study. The patients’ operative procedure, respiratory function before operation, histological type, tumor size, clinical stage, comorbidity, surgery-related complications, prognosis, and recurrence were retrospectively reviewed.

Results
There were 11 males and 5 females. Their mean age was 86.6 years (range: 85-93 years). The mean follow-up period after operation was 1290 days (range: 249-4029 days). Operative procedures include lobectomy (N=4), segmentectomy (N=6), and wedge resection (N=6). Among them, thoracoscopic surgeries were performed in 14 patients. Patients treated by segmentectomy had poorer pulmonary function than others in terms of the forced expiratory volume in 1 second (FEV1.0) (mean±SD: 1.43±0.40 vs 1.94±0.39 L, p<0.05) and vital capacity (VC) (mean±SD: 2.02±0.51 vs 2.55±0.53 L, p=0.06). Histological type include adenocarcinoma (N=11) and squamous cell carcinoma (N=5). The tumor size ranged 11 mm to 48 mm. All 16 patients were negative for lymph node metastasis. Pathological stage was IA in 11 patients, IB in 4 patients, and IIB in 1 patient. Seven patients had comorbidity (e.g. COPD, post-bypass grafting for coronary aretery, and others). Seven patients were smokers. Mortality rate was 0% and morbidity rate was 25% (e.g. pulmonary fistula treated with pleurodesis in 1 patient, atrial fibrillation in 2 patients, heart failure in 1 patient, and delirium in 1 patient). Fourteen patients are alive, whereas 2 patients died from other diseases than lung cancer. The 2 patients died after 1950 days and 1344 days after operation, respectively. No patients had recurrence during the follow-up period.

Conclusion
No patients died from surgery or post-surgical complications. Our study indicates that the surgical resection of non-small cell lung cancer benefits patients, even with extremely elder age (85-year-old or over). For our institution’s principal indications for pulmonary surgery, patients with elder age (75-years-old or older) have to (i) be able to undergo pulmonary physiotherapy, (ii) have good performance status (0-1 in ECOG scale), and (iii) be free from psychological disorder such as dementia. Additionally, in cases with extremely older age (85-year-old or over), we consider patients with clinical stage I or patients without lymph node metastasis are good candidates. Lobectomy can be performed in patients with better pulmonary function, whereas patients with worse pulmonary function should undergo segmentectomy. Wedge resection is selected only if the lesion is judged to be completely resectable. We consider these inclusion criteria and the manner of selecting operative procedures will provide a good prognosis, even though the patient is 85 years or older.

Background
Induction chemoradiotherapy (CRT) followed by surgery (iCRT) is one of treatment strategies for locally advanced non-small cell lung cancers (NSCLCs). We have previously reported its feasibility and good clinical outcome with approximately 60% of 5-year overall survival rate. .Perioperative nutritional status,is considered as one of important factors for improved clinical outcome after surgery and other treatments. Here, we investigated the perioperative nutritional status in NSCLC patients treated by iCRT (CRT group) to evaluate the influence of nutritional variables on clinical outcome by comparing that in NSCLC patients with simple pulmonary resection without CRT (non-CRT group) .

Methods
Thirty-three consecutive patients with locally adcanced NSCLC who underwent iCRT from January 1, 2009, until December 31, 2011 at our institute were included in this study. The regimen of CRT was two cycles of docetaxel (40 mg/m[2]) plus cisplatin (40 mg/m[2]) with concurrent radiotherapy (46 gray) and the surgery was performed within 6 weeks of completing induction CRT. We compared nutrition-related factors and clinical outcome in 33 iCRT patients with those in 58 consecutive NSCLC patients who underwent lobectomy during January 1 to December 31, 2011 at out institute. .As for blood nutritional factors, total lymphocyte count (TLC), albumin (Alb), total cholesterol (T-cho), choline esterase (ChE), were examined. The prognostic nutritional index (PNI) was also calculated by Alb and TLC. Each nutrition-relatd factors were examined 1) before CRT, 2) before surgery and 3) one month after surgery.

Results
Median age of CRT group (61 years old) was significantly younger than that of non-CRT group (69 years old). Twenty-one males and 12 females and 44 males and 14 females were enrolled in CRT and non-CRT groups, respectively. Before any treatment, no significant difference was observed in body mass index and any blood nutritional factors in both groups. After induction CRT, TLC was significantly decreased, and additionally, Alb, T-cho, and ChE were significantly decreased after surgery comparing with those before surgery (after CRT). As for preoperative status in both groups, TLC, Alb and PNI were significantly lower in CRT group than in non-CRT group. Regard with surgery, extended surgery, operating time, and blood loss was significantly heavier in CRT group than in non-CRT group. Perioperative mortality rate was 0% in both groups and the frequency of post-operative complication was similar in both groups (51% and 41% in CRT and non-CRT groups, respectively). The length of hospital stay after surgery was significantly longer in CRT group (median 23 days) than in non-CRT group (median 14 days). Among CRT group, patients with loiw PNI index could not administrate adjuvant chemotherapy.

Conclusion
Perioperative nutritional status, especially TLC, is suppressed after CRT and moreover after surgery. Suppression of nutritional status continued one month after surgery with induction CRT and severe suppression of nutritional status disturbs further treatment such as adjuvant chemotherapy.

Background
Patients with cerebro- and cardio-vascular comorbidities (CCVC) who undergo surgery represent a high-risk group and require careful perioperative management. In the present study, we aimed to retrospectively analyze the postoperative complications (POC) of patients with CCVC who had undergone pulmonary resection for lung cancer. Patients with cerebro- and cardio-vascular comorbidities (CCVC) who undergo surgery represent a high-risk group and require careful perioperative management. In the present study, we aimed to retrospectively analyze the postoperative complications (POC) of patients with CCVC who had undergone pulmonary resection for lung cancer.

Methods
Among 288 patients who underwent pulmonary resection at our institution from January 2009 to December 2011, we examined the records of 51 patients with CCVC (17.7%) to identify the risk factors for developing POC. Among the analyzed patients, we noted the presence of 34 POC, including tachyarrhythmia in 9, prolonged pulmonary fistula in 9, pyothorax in 2, cerebral infarction in 2, requirement of long-term oxygen therapy in 2, interstitial pneumonia in 2, delirium in 2, and other POC in 4. Several patients had multiple POC.

Results
We examined 43 male patients (84.3%); the median age was 72 years and the median preoperative forced expired volume in 1s (FEV~1~) was 2200 mL (range, 1120–3420). The patients with CCVC included 12 with cerebral infarction, 2 with transient cerebral ischemic attacks, 2 with cerebral hemorrhage, 1 with subarachnoid hemorrhage, 4 with cerebral aneurysm, 10 with arrhythmia, 17 with ischemic heart disease, 1 with valvular heart disease, 8 with aortic aneurysm/dissection, 11 with peripheral arterial disease, and 1 with a left atrial myxoma; several of these patients had multiple CCVC. Moreover, 2 patients underwent pneumonectomy, 37 underwent lobectomy, 3 underwent segmentectomy, and 9 underwent wedge resection. Postoperative morbidity rates were 21.4% in cerebrovascular comorbidity patients (p = 0.015), 53.5% in the cardiovascular comorbidity patients (p < 0.0001), 71.4% in CCVC patients (p = 0.0028), and 12.3% in patients without CCVC. No operative or in-hospital mortality was noted. Gender, age, smoking status, and smoking index were not found to be significantly related to the incidence of POC. However, patients with an FEV~1~ < 2200 mL were found to be significantly more likely to develop POC (p = 0.036).

Conclusion
We noted that patients with CCVC and low FEV~1 ~were more likely to develop POC.

Background
Multidisciplinary team (MDT) meetings have developed to enhance disease-specific interdisciplinary communication towards accelerating the initiation of appropriate therapy and improving both patient care and survival. They have long been a mainstay in the management of patients with cancer. This study aims to compare resection rates, surgical cure (based on post-resection pathological status) and co-morbidity status in consecutive lung cancer cases (stages IA-IIB, IASLC TNM staging 6[th] or 7[th] ed.), as presented at our institution’s Lung Cancer MDT meeting, compared with a similar contemporaneous control group not presented at the MDT meeting.

Methods
Data for this study was drawn from the Clinical Cancer Registry Project (ClinCR) database in NSW, which captures all cases of lung cancer managed in the public health system as well as databases used by the Lung Cancer MDT and Cardiothoracic Surgery. Cases of non-small cell lung cancer (NSCLC) appropriate for curative surgery based on pre-operative stage IA-IIB were analysed for MDT presentation, resection rate, surgical cure rate and for pre-operative Charlson co-morbidity score.

Results
Data were analysed from the time period 2006-2011. During this time 755 cases of NSCLC were diagnosed, 377 were presented at MDT (49.9%) and 378 were not (50.1%). A total of 138 cases of early stage disease (stages IA-IIB) were identified, 90 of which were presented at MDT (65.2%) and 48 were not (34.8%). Significantly more cases of early stage disease presented at MDT, 33/90 (36.7%) had resection than cases of early stage disease not presented at MDT 13/48 (27.1%) (p=0.015). Overall resection rate in early stage disease (MDT and non-MDT) was 46/138 (33.3%). In resected cases the surgical cure rate (according to surgical margins) did not differ significantly between the two groups with 30/33 (90.9%) in the MDT group and 12/13 (92.3%) in the non-MDT group achieving surgical cure (p=0.75). For cases of early stage NSCLC presented at MDT the odds ratio for surgical resection was 2.5. Further analysis of Charlson co-morbidity score will clarify associated co-morbidities, which may help explain this difference.

Conclusion
Surgical resection rates are statistically higher in early stage NSCLC cases presented at MDT than in contemporaneous cases not presented at MDT in our study population. Surgical cure rates for resected patients appear high in both groups. Further data on co-morbidities and identifiable referral patterns, including patients managed outside the public hospital system, may clarify reasons for the disparity in resection rates and enable identification of potentially reversible barriers to treatment.

Background
Although large-cell neuroendocrine carcinoma (LCNEC) was categorized as a variant of large cell carcinoma on the WHO histologic classification of lung carcinomas, the clinical and biological features of LCNEC resemble those of small cell lung carcinoma. Therefore, there is no consensus on the treatment strategy for LCNEC, and an indication of surgical treatment for LCNEC is still controversial. Even though preoperative accurate diagnosis of LCNEC is difficult, the aim of this study was investigating patients with pulmonary LCNEC in whom better postoperative outcome is expected.

Methods
We retrospectively reviewed patients with pulmonary LCNEC on permanent pathologic diagnosis who underwent pulmonary resection at the 3 institutions between 1999 and 2011. We reviewed the medical records of each patient for demographic, clinical, and pathologic data including age, sex, smoking status, preoperative serum CEA, radiologic tumor size, c-stage, surgical procedure, extent of lymphadenectomy, p-stage, lymph node metastasis, visceral pleural invasion, lymphatic permeation, vascular invasion, and adjuvant chemotherapy. Disease-free survival (DFS) was calculated using the Kaplan-Meier method, and factors associated with DFS were analyzed with the log-rank test.

Results
Of the 18 patients eligible for this study, 14 were male and 4 were female. The median age was 74 years (range, 53 to 85). According to the current TNM classification, 12 patients had c-stage I disease, 4 had c-stage II disease, and 2 had c-stage IIIA disease. The majority of patients (13 patients, 72%) underwent lobectomy, 1 underwent pneumonectomy, 1 underwent bilobectomy, and 3 underwent wedge resection. On pathologic diagnosis, 8 patients had p-stage I disease, 5 had p-stage II disease, and 5 had p-stage IIIA disease. Following surgical treatment, cisplatin-based adjuvant chemotherapy was applied for 3 patients. The 1-year and 2-year DFS were 39% and 39%, respectively, with the median follow-up period of 9 months (range, 2 to 80). During the follow-up period, 10 patients (56%) developed recurrence, and the recurrence was identified within the first year post-resection in all the 10 patients. By the log-rank test, smoking status (non- or former, vs. current) and surgical procedure (lobectomy or greater, vs. limited resection) were identified as significant factors associated with DFS.Figure 1

Conclusion
Of patients with pulmonary LCNEC undergoing surgical treatment, a long-term prognosis might be expected if no recurrence is identified within the first year post-resection. If diagnosis of LCNEC is preoperatively obtained, surgical treatment is recommended for patients without current smoking status, and lobectomy or greater resection should be the surgical procedure of first choice.

Background
Although positron emission tomography/computed tomography (PET/CT) seems to be able to provide accurate information of lymph nodes status in non-small cell lung cancer (NSCLC) patients, we sometimes experience surgical cases with unexpected lymph nodes metastasis. The objective of this study is to demonstrate clinicopathological characteristics of patients with unexpected pathological (p) N1 and N2 NSCLC.

Methods
All patients with lung cancer underwent enhanced CT of the chest and PET/CT preoperatively for evaluating of lymph node status. Mediastinoscopy or EBUS-TBNA was not routinely performed. Unexpected pN1 and pN2 diseases were defined as surgical cases which were proved to have hilar or mediastinal lymph nodes metastasis postoperatively in spite of negative 18-fluoro-2-deoxy-D-glucose (FDG) uptake in hilar or mediastinal lymph nodes on preoperative PET/CT. We retrospectively reviewed clinical features of these patients and analyze predictive factors for these unexpected diseases.

Results
Between January 2007 and December 2012, 533 patients with lung cancer underwent　surgical resection at our institution. Among them, we reviewed 182 patients who had available preoperative PET/CT data and underwent a curative-intent operation with systematic or selective lymph node dissection. One hundred fifty-one patients (83%) had lung cancer with expected lymph node status, whereas, 31 patients (17%) were found to have lung cancer with unexpected lymph nodes metastasis, consisting of 12 (39%) unexpected pN1 and 19 (61%) unexpected pN2 diseases. There were 16 men and 15 women with a median age of 67 years. Seventeen patients were current- or past- smokers, and 14 were never-smokers. Tumor size ranged from 12 to 52 mm, and pathological T factor was pT1a in 3, pT1b in 5, pT2a in 19, pT2b in 3, and pT3 in 1. Histological type of the primary tumor were adenocarcinoma in 28 (90%), squamous cell carcinoma in 2, and large cell carcinoma in 1. Among 28 adenocarcinomas, the most common predominant subtype was papillary (20 patients; 71%), followed by acinar (5 patients), solid with mucin, micropapillary, and invasive mucinous (one patient, respectively). Of these patients, 7 patients had micropapillary component in varying proportions in their tumors. EGFR gene mutation status was available in 22 patients, and of these, 12 patients (55%) had tumors with EGFR gene mutation, consisting of exon 19 deletion in 5, exon 21 point mutation in 5, and other minor mutation in 1. In univariate analysis, tumor size (> 3cm), pleural, and lymphatic invasion were significant predictive factors for unexpected pN1 and pN2 diseases. Only in 151 adenocarcinomas, papillary predomiant tumor and having micropapillary component were significant predictive factors for unexpected lymph node metastasis.

Conclusion
We should take care that false-negative rate is relatively high on preoperative PET/CT for lymph node status in NSCLC. Histological findings of the primary tumor are often important because they can provide predictive information for lymph nodes status even if there is no FDG uptake in regional lymph nodes on preoperative PET/CT. We hope new accurate imaging modality which can reflect tumor histology and lymph nodes micrometastasis.

Background
The average age of the general population is increasing in the worldwide. Therefore, there is also an increasing number of elderly patients presenting with potentially-resectable lung cancer. We retrospectively reviewed the outcomes of octogenarians or over who underwent pulmonary resection for primary non-small cell lung cancer (NSCLC) to identify the independent factor of overall survival.

Methods
We conducted a retrospective single-institution review of patients aged 80 years or over who underwent pulmonary resection for primary NSCLC from 1990 to 2012 at Nagasaki university hospital. The various clinicopathological data, including gender, histological type, body mass index, comorbidity, clinical stage, surgical procedure, extent of lymph node dissection, and pathological stage were analyzed.

Results
119 octogenarians or over underwent pulmonary resection. The median age was 82 years (range 80-92 years). Of the total patient number, 56 (47.1%) had respiratory and 44 (33.6%) had cardiovascular comorbidity diagnosed preoperatively. The clinical stage was I in 97 (81.5%) patients, II in 13, III in 6, IV in 3. Operations included 82 (68.9%) lobectomies, 2 (1.7%) bilobectomies, 15 (12.6%) segmentectomies, and 19 (16.0%) partial resections. Only 31 (26.1%) were performed mediastinal lymph node dissection. The pathological stage was I in 79 (81.5 down to 66.4 %) patients, II in 16 (13.4%), III in 21 (17.7%), IV in 3 (2.5%). 26 (21.8%) patients presented with postoperative respiratory complications, and 11 (9.2%) were cardiovascular, and the operative mortality was 1 (0.8%). The 5-year survival rates were 46.0% for all patients, 60.8% for stage I patients. The disease specific 5-year survival rates were 60.1% for all patients, 79.5% for stage I patients, respectively. In univariate analysis, female (p<0.04), clinical stage (p<0.002), and pathological stage (p<0.000) was independent and cardiovascular comorbidity was marginally (p<0.05) factor for overall survival. In multivariate analysis, only advanced pathologic stage (stage II, more) was independent predictor of overall survival [p<0.0001, Hazard ratio: 3.17, 95% confidence interval 1.76-5.73]. Figure 1

Conclusion
Surgical treatment for selected patients aged 80 years or over or with primary NSCLC can be performed safely with low morbidity and mortality in this study. We recommend that limited operation might be the best surgical treatment, especially for stage I NSCLC. In the future, establishment of accurate clinical staging as well as early detection for lung cancer, and the appropriate treatment for advanced stage NSCLC for aged people should be studied for the upcoming aged society.

Background
In order to clarify clinical implication and therapeutic strategies managing minute malignant pleural effusion unexpectedly found at thoracotomy, we analyzed diagnostic results of cytodiagnosis of pleural effusion found at thoracotomy and prognosis of the patients treated in our institute retrospectively.

Methods
From 2004 to 2013, 502 non-small cell lung cancer patients were surgically treated in our institute. At the time of thoracotomy pleural effusion cytology (PEC) was performed in the cases that small amount of subclinical, minute pleural effusion was found unexpectedly. In some cases pleural lavage cytology (PLC) was performed simultaneously.

Results
In 254 patients (50.6%) out of 502, minute pleural effusion was detected and PEC was performed. In 25 patients (9.8%), malignant pleural effusion was demonstrated. PLC was performed in 191 cases, and positive results were obtained in 16 cases (8.3%). In these 16 cases, PEC was also performed in 12 cases and positive results were obtained in 11 cases. And in 4 cases no pleural effusion was found but PLC was positive for malignancy. Post operative 5 yrs survival of malignant pleural effusion group was 43.7%. In p-N0 cases, post operative survival of PEC positive cases was tend to poorer than surgically treated PEC negative cases (p=0.13), but in p-N1, N2 cases, post operative survival was almost equal between these groups (p=0.66).

Conclusion
Cytodiagnosis of minute pleural effusion found at thoracotomy was performed in 51% of surgical cases of NSCLC. Subclinical malignant pleural effusion found at thoracotomy may be a prognostic factor in clinical stage one case. PLC at the time of thoracotomy may become a complementary examination of PEC.

Background
An extended sleeve lobectomy is a useful procedure so as to spare the lung parenchyma. However, the resection of the bronchus can cause an increment in the tension at the site of the anastomosis and mismatches in the size of the bronchial orifices. Induction chemoradiotherapy (CRT) followed by surgery is a therapeutic option for locally advanced non-small cell lung cancer (NSCLC). Induction CRT, especially radiotherapy, has a negative effect on bronchial healing in the bronchial stump or anastomosis in a pulmonary resection.

Methods
The medical records were reviewed for nine NSCLC patients who underwent extended sleeve lobectomy after CRT between December 2007 and January 2013. Disease stage was evaluated with imaging analyses, including enhanced chest computed tomography (CT) scan, brain magnetic resonance imaging, positron emission tomography-CT scan and bronchoscopy. Induction CRT was performed for eight cases using cisplatin and docetaxel with concurrent thoracic radiation. For one patient who had synchronous laryngeal cancer, 5-fluorouracil and nedaplatin were used as chemotherapy. The radiation dose was 46 or 40 Gy using a conventional fractionation (2 Gy/day). Patients without progressive disease or good general condition underwent surgery. The bronchial anastomosis was basically wrapped with an omental pedicled flap or pericardial fat pad with prophylactic intent. The pre- and postoperative first-second forced expiratory volume was measured. The overall survival (OS) and the disease-free survival (DFS) were calculated from the date of initialing induction CRT until the date of death or the last follow-up for OS and until confirmed death of any cause or recurrence at local or distant site for DFS. The survival curve was calculated by the Kaplan-Meier method.

Results
The median patient age was 60 years (range, 50 to 73 years). There were seven men and two women. The histological subtype was squamous cell carcinoma in six patients and adenocarcinoma in three patients. Five patients had clinical stage (c-stage) IIIA, two patients had c-stage IIIB, and two patients had c-stage IIB. The radiation dose was 46 Gy in seven patients and 40 Gy in two patients. An extended sleeve lobectomy was performed for the left lingular division and the lower lobe in four patients, the right upper lobe and trachea in one patient, the right upper lobe, carina and trachea in one patient, the right middle and lower lobe in one patient, the right upper and middle lobe and the carina in one patient, and the right upper lobe and superior segment of the lower lobe in one patient. While no postoperative 90-day deaths occurred in this series, one case developed a bronchopleural fistula on postoperative day (POD) 25 and one case developed a bronchovascular fistula on POD 163. No cases of local recurrence occurred. The first-second forced expiratory volume before surgery was 2.52 ± 0.58 L (mean ± standard deviation), while that after surgery was 1.80 ± 0.66 L. The 2-year overall survival and disease-free survival rates were 63.5% and 47.6%, respectively.

Conclusion
Our experience suggests that an extended sleeve lobectomy after induction CRT is feasible, but careful patient selection and perioperative management is mandatory.

Background
Oschner and DeBakey in 1940, reviewed the world literature of 191 esophageal resections with a 72% mortality rate. Later on with advent of better preoperative, intaoperative and postoperative management advances, the risk of mortality had decreased to less than 10%. Various radical resections were described both in resection of primary (Eg: Transhiatal esophagectomy(THE), transthoracic esophagectomy (TTE),enbloc esophagectomy,etc..,) and lymph node dissection (two field, three field , etc.,). The role of radical surgery is still controversial and its becomes necessary to decide which patient can tolerate a radical procedure in the preoperative setting itself.

Methods
We retrospectively analyzed surgical and medical records of 154 patients of carcinoma esophagus operated in a single unit at our center to assess the predictive factors for postoperative morbidity for the period 2006-2012.

Results

Table 1: Demographic profile of the Patients: Characteristics Number of patients ( Total N = 154) Age Mean Range 55.857 ± 10.3989 20-79 years Sex Male Female 92 62 Histopathological Examination Squamous cell carcinoma Adenocarcinoma Adenosquamous carcinoma 124 28 2 Site of lesion Middle third esophagus Lower third thoracic esophagus and Gastroesophageal tumors 36 118 Duration of symptoms ( Mean ± S.D) Preoperative albumin (gm/dl)( Mean ± S.D) Comorbidities Neoadjuvant treatment 2.481± 1.7684 (Range 1 -12) 3.938 ± 0.3684 (Range 3-4.9) 25 ( COPD-12, Diabetes Mellitus-10, Hypertension-8, Ischemic heart disease-3; Hypothyroidism-3, Previous history of pulmonary Koch’s-3) 10 (6 patients - NACTRT, 4 patients- NACT) Surgery performed Transhiatal esophagectomy Transthoracic esophagectomy 140 14 Complications Pulmonary complications Anastamotic leak Mortality Abdominal infection Chyle leak 18 11 12 ( 7 had pulmonary complication also) 3( All 3 had pulmonary complication ) 2 1( also had pulmonary complication)

NACTRT- Neoadjuvant chemoradiotherapy, NACT-Neoadjuvant chemotherapy Of 154 patients who underwent esophagectomy at our center, 140 patients underwent THE and 14 underwent TTE. Mean duration of dysphagia was 2.4 months. One hundred and twenty four patients had squamous cell carcinoma and 30 patients had other histological types. Twenty five patients had comorbidities as described in Table.1. Eighteen patients developed postoperative complication of which three died - one case due to massive pulmonary embolism and two cases due topulmonary infection and septicaemia). Twelve patients had anastamotic leak (7.7%), all were managed conservatively. When multivariate regression analysis was performed for the predicted risk factors and development of complication, preop albumin ( less than 4gm/dl) and histological type (non squamous cell carcinoma) were associated significantly with increased post operative complications (Table.2). Abnormal pulmonary function tests though showed increased risk of complications it didn’t attain statistical significance. Table 2: Multivariate logistic regression analysis of predictive factors for early postoperative complications:

Independent variables Coefficient Standard Error t P Albumin (Less than 4 gm/dl) -0.1505 0.07519 -2.001 0.0472*(significant) Comorbidity (Yes) 0.1012 0.06716 1.507 0.1339 Duration of dysphagia (Absolute dysphagia) 0.004364 0.01576 0.277 0.7823 Histopathological examination ( Non squamous cell carcinoma) -0.1877 0.07332 -2.559 0.0115*(Significant) Neoadjuvant treatment ( Yes) 0.1043 0.1147 0.910 0.3645 Site of lesion ( Middle 3rd) 0.06701 0.06641 1.009 0.3146 Pulmonary function tests (Abnormal) 0.04156 0.02232 1.862 0.0646

Conclusion
We achieved a 2% mortality rate during the study period. Preoperative serum albumin and histological subtype were associated with increased postoperative mortality. Neoadjuvant therapy was not associated with increased complication rate.

Background
A new lung adenocarcinoma classification is being proposed by the International Association for the Study of Lung Cancer, American Thoracic Society, and European Respiratory Society (IASLC/ATS/ERS), which was based on histomorphologic subtype and had recently been validated in a North American series of 514 stage I lung adenocarcinomas. However, its distribution of patient number was biased, especially adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), and non-mucinous lepidic predominant groups, compared with in Japan. We evaluated an appropriateness of new classification in a series in Japan and whether the classification could be useful for selecting limited cases undergoing sublobar resection.

Methods
We retrospectively reviewed clinical records of all patients operated on for non-small cell lung cancer from 1997 to 2011 (n=825). 292 patients (35.4%) had pathological stage IA adenocarcinoma of the lung classified according to the Union for International Cancer Control/American Joint Committee on Cancer 7th Edition. Some pathologists, blinded to patient outcome, performed histopathologic subtyping according to the proposed new IASLC/ATS/ERS classification. Statistical analyses were made including Kaplan–Meier and Cox regression analyses.

Results
There were 160 females (54.8%) and 132 males (45.2%) with a median age of 67 years (29–84 years) and 212 pT1a and 80 pT1b patients (tumor size: 16.7±7.1 [2-30] mm). Three overall prognostic groups were identified: low grade: AIS (n=103, 35.3%) and MIA (n=24, 8.2%) had 97.1% and 95.1% of disease-free survival at 5 years (DFS, median follow-up was 54 months); intermediate grade: non-mucinous lepidic predominant (n=61, 20.9%), acinar predominant (n=38, 13.0%), and papillary predominant (n=48, 16.4%), with 80.1%, 86.5%, and 62.4% of DFS; and high grade: invasive mucinous adenocarcinoma (n=5, 1.7%), solid predominant (n=12, 4.1%) and micropapillary predominant (n=1, 0.3%), with 88.9% of DFS. DFS in low grade was significant better than in other two grades (P<0.005), however, there was no significant difference between in intermediate and high grade groups due to lower DFS in papillary predominant or insufficient patient number in high grade group. Preoperative imaging examinations such as consolidation/tumor (C/T) ratio on high resolution CT and maximum standardized uptake value (SUVmax) by FDG-PET were correlated with histopathologic grade according to new classification (P<0.01). Moreover, sublobar resection was undergone for 144 cases (49.3%), more cases had been identified small tumor, low C/T ration, low SUVmax, and low grade subtypes, and DFS in sublobar resection was 96.4% which was significant better than in lobectomy (78.6%, P=0.0002).

Conclusion
In this study, we can evaluate with enough number of patients classified to AIS, MIA, or non-mucinous lepidic predominant according to the new IASLC/ATS/ERS classification. Most of subtypes correlated with DFS, except of papillary predominant and subtypes in high grade clinical aggressiveness, which may need more clinical investigation. Patients in low grade subtypes who underwent sublobar resection had better DFS, which can be predicted using tumor size and preoperative imaging examinations such as C/T ratio and SUVmax. So, the new classification has advantages for better selection of limited cases undergoing sublobar resection as a curative surgery.

Background
Postoperative complications are common after major lung resections for cancer. Although there have been important advances in operative technique and perioperative care, lung resection for cancer is still a major surgery with higher incidence of complications in centres with a low case volume. We undertook a retrospective analysis of a prospectively maintained surgical database to ascertain whether the grade of the operating surgeon influenced the occurrence of postoperative complications and mortality in a tertiary level teaching cancer centre.

Methods
Data from a prospectively maintained database (Aug 2004 to May 2013) was analysed and the following parameters were retrieved: age, sex, type of surgery, grade of surgeon (Consultant vs trainee), postoperative major complications including pulmonary complications, air leak, bronchopleural fistula and mortality. ICU and hospital stay were also compared between surgeries performed by consultants and trainees. Categorical variables were analysed by the Chi square or Fischer’s exact test and numerical variables using the unpaired Student t test. P values less than 0.05 were considered statistically significant.

Results
A total of 654 patients (494 male, 160 female; mean age 54.4, range 14 to 83 years) underwent lung resection for primary lung tumors in the study period. The overall major morbidity and postoperative mortality was 10.6% and 1.7% respectively. Consultant thoracic surgeons performed 532 surgeries while trainees performed 122 procedures. Major morbidity was 11.2% and 8.2% (p=0.336) and postoperative mortality was 1.9% and 0.8% (p=0.628) when lung resection was performed by consultants and trainees respectively. The incidence of pulmonary complications (10.2% vs 9.9%, p=0.916) and bronchopleural fistula (2.2% vs 1.8%, p=0.749), median ICU (both 0 days) and hospital stay (both 5 days) were also similar in the two groups.

Conclusion
Early postoperative outcomes after major lung resection for primary lung tumors are independent of the level of training of the operating surgeon. These results are qualified by the fact that operations performed by trainees were closely supervised and assisted by experienced consultant thoracic surgeons in a teaching tertiary level cancer centre.

Background
The treatment of choice for early-stage lung cancer is surgical resection. Over the years, there has been an increase in minimally-invasive surgical (MIS) options for lung resection, including video-assisted thoracoscopic (VATS) lobectomy and robotic-assisted thoracoscopic (RATS) lobectomy. These MIS approaches have reformed lobectomies, decreased overall morbidity as well as decreased post-operative pain and hospital length of stay (LOS) when compared to traditional thoracotomy lobectomies. In spite of the recent rapid surgical expansion of MIS lobectomy, little is known about the learning curve of RATS lobectomy. In order to move forward in this new surgical technologic era while enhancing patient safety, determination of this learning curve is crucial.

Methods
We retrospectively analyzed the perioperative outcomes of 236 consecutive patients who underwent robotic lobectomy at our institution between September 2010 and June 2013. Patients were grouped chronologically into four quartiles, with 59 patients in each quartile. A comparison was performed between quartiles with respect to operative times, intraoperative estimated blood loss (EBL), hospital LOS, and in-hospital mortality. Statistical analysis was undertaken using analysis of variance (ANOVA), linear regressions, and t-tests. Significance was set at p-value <0.05.

Results
A total of 236 patients with a mean age of 67 ± 10 years underwent RATS lobectomy between September 2010 and June 2013. Each of the four quartiles had overall conversion-to-thoracotomy rates of ≤10% and emergency conversion rates of ≤5%. Although intraoperative EBL and hospital LOS were not significantly different among the quartiles, both EBL and hospital LOS showed a decreasing trend over each chronologic quartile. Operative times increased during the 2nd quartile, but also showed a decreasing trend with subsequent quartiles. There was an overall pathologic upstaging in 39% of the RATS lobectomy patients. Lastly, the in-hospital mortality rate was 5.1% for the 1st quartile and 0% for each subsequent quartile.

Conclusion
In contrast to the steep learning curves associated with other MIS procedures, RATS lobectomy may result in only a modest learning curve for surgeons with extensive VATS lobectomy experience, as is suggested from the unchanging and low hospital LOS, intraoperative EBL, and conversion to open lobectomy rates across all quartiles. Additionally, the increase in operative time in the 2nd quartile is most likely associated to the extension of the RATS approach to more difficult lobectomy cases after relative comfort with RATS lobectomy has been achieved. The high percentage of overall pathologic upstaging is likely due to more complete mediastinal lymph node dissection than would be achieved with either deliberate lymph node sampling only or else inadequate lymph node dissection due to limitations in visualization, instrumentation, or technical experience with VATS lobectomy. The decrease in mortality rate from the 1st to subsequent quartiles can be attributed to increased proficiency in RATS lobectomy. Thus, a bi-phasic learning curve is demonstrated by increased operative times after establishment of comfort with RATS lobectomy after initial success and subsequent extension of RATS to more complicated patients (1[st] phase), while operative times, EBL, hospital LOS, and mortality decrease with improved patient selection and more RATS experience (2nd phase).

Background
Lung cancer remains by far the biggest cause of cancer deaths worldwide, exceeding the next four cancers combined. Meanwhile, health systems struggle with ballooning costs and an unclear relationship between cost and outcomes. Within this context, there exists a great need for reliable, goal-oriented, and cost-effective care. ProvenCare[®] Lung Cancer is a national, multi-institutional care improvement collaborative that identifies and prescribes best practice elements of care in order to improve outcomes of pulmonary resection for lung cancer.

Methods
Incorporating principles of reliability science, the ProvenCare[® ]group established a protocol to promote adherence to and completion of best practice elements of care for patients undergoing pulmonary resection for lung cancer. Thirty-eight care elements span the continuum from preoperative assessment through intraoperative and inpatient postoperative care, and on to post-discharge follow-up. An original 6 thoracic surgical teams, subsequently expanded to twelve institutions, prospectively measured adherence to the prescribed elements of care in an “all or none” measure of success (adherence to all 38 elements is required for “compliance”). These data are to be compared both in a pre- and post-ProvenCare[®] involvement within participating sites, as well as to contemporaneous data within the Society of Thoracic Surgeons (STS) General Thoracic Database, to compare operative morbidity and mortality between the two groups. Figure 1

Results
To date, nearly 900 patients have been enrolled at the twelve Collaborative sites. Certain elements have proven more difficult than others to accomplish reliably, namely adherence to postoperative pain control and pulmonary toilet regimens. Even so, the redesign of the complex care required by the lung resection patient to embed evidence-based best practices into everyday patient flow, has resulted in high adherence to the prescribed elements of care, ranging from 92 to 100%. A comparison of the ProvenCare[®] study group to the STS Database control will be undertaken once an additional 200 patients are enrolled.

Conclusion
Quality and reliability represent frontiers of cancer care, and have the potential to improve patient outcomes as much as blockbuster medications or innovative radiation or surgical techniques, and perhaps more cost-effectively. We present an example of a national, collaborative, multidisciplinary model of reliable care that represents the first attempt to test patient care outcomes in this context.

Background
The incidence of a second primary lung cancer has been reported with 1-2% per patient year. Still, relatively few data has been published about this selected group of patients with sometimes conflicting results. Most data has been included low patient numbers that derived either from multiple institutions or from a long time period that may have rendered conclusions difficult caused by varying diagnostic procedures and therapeutic developments. Moreover, data regarding clinical characteristics is lacking for patients with first primary lung cancer who might be at risk for developing second primary lung cancer

Methods
From January 1999 to December 2008, 65 patients with second primary lung cancer, classified by the criteria proposed by Martini and Melamed, were treated at our Institution. We had 34 patients with a synchronous tumour and 31 with metachronous. As second treatment, we performed 11 lobectomies and 54 segmentectomies and widewedge resections. Histology was adenocarcinoma in 58, squamous in 4, adenosquamous in 8, large cells in 2 and pleomorphic in 1.

Results
Overall 5-year survival from second surgery was 69%; overall operative mortality was 0.65% (1 patient). Regarding the interval of surgery, the second operation had performed later than 2 years group showed a better 5-year survival than within 2 years group (80.6% and 69.2%, respectively, P = 0.008). Compared with lobectomies, segmentectomy showed a no significantly changes in survival rate(69 and 60%, respectively, P >0.051).

Conclusion
From our experience, lobectomy should still be considered as the treatment of choice in the management of second primary lung cancer, but sublobar resection remains a valid option in high-risk patients with limited pulmonary function.

Background
The clinical impact of mediastinal lymph node dissection (MLND) during pulmonary metastasectomy remains controversial. Especially the prognostic contribution of MLND on the prevention of tumor recurrence in patients with no evidence of mediastinal lymph node metastasis has not been clearly defined. We aimed to clarify the role of MLND during pulmonary metastasectomy in this population.

Methods
We retrospectively reviewed 632 patients who underwent pulmonary metastasectomy from January 2006 to December 2010 in Asan Medical Center. Among them, two hundred nine patients were identified to meet the following criteria and comprised the current study population: the presence of preoperative computed tomography (CT) and positron emission tomography (PET) within 2 months before pulmonary metastasectomy, definite control of the primary tumor, and no evidence of mediastinal lymph node metastasis. Of 209 patients, sixty-seven patients underwent MLND during pulmonary metastasectomy (MLND group), whereas 142 patients underwent pulmonary resection only (non-MLND group). Between-group recurrence-free survival was compared, and risk factors for tumor recurrence were evaluated. The data on tumor recurrence were obtained through a median follow-up duration of 42 months (range 2-83 months).

Results
The study population was composed of 119 male and 90 female, and the age at the first pulmonary metastasectomy ranged from 13 to 82 years (median, 56 years). Primary tumor pathologies included colorectal cancer (n=104, 49.8%), hepatobiliary cancer (n=38, 18.2%), kidney cancer (n=17, 8.1%), sarcoma (n=14, 6.7%), and the others (n=36, 17.2%). Disease-free interval from initial primary tumor treatment to the first metastasis ranged from 1 to 94 months (median, 25 months). Overall 5 year recurrence-free survival rate was 30.1%. There was no difference in recurrence rates between the MLND group and the non-MLND group (5 year recurrence-free survival: 30.0% vs. 24.5%, p=0.927). On multivariable analysis, primary tumor histopathology (p<0.001), disease-free interval (p=0.016), and the number of nodules (p<0.001) emerged as significant and independent prognostic factors for recurrence. After adjustment by these three significant variables, mediastinal lymph node dissection did not affect recurrence-free survival (hazard ratio, 0.924; 95% confidence interval, 0.641-1.333; p=0.672).

Conclusion
Tumor recurrence after pulmonary metastasectomy was affected by the histopathology of the primary tumor, disease-free interval, and the number of metastatic nodules. However, the role of mediastinal lymph node dissection as a part of pulmonary metastasectomy is obscure in patients with no evidence of mediastinal lymph node metastasis based on CT and PET.

Background
Lung cancer generally presents in an advanced stage and tuberculosis(TB) is a common disease in the subcontinent. This study describes locally advanced lung cancer showing low nodal involvement by cancer and concomitant TB.

Methods
Retrospective analysis of lung cancer database of Department of Surgical Oncology , BRA IRCH , AIIMS (2012 -2013 ) was performed and 28 cases were identified who underwent surgeries for lung mass. Only primary lung cancers were included. The clinical features , histopathology and management of these patients were analyzed.

Results
A total of 1293 cancer patients were operated between 2012 and 2013 in the department of surgical oncology. Out of which 28 patients were diagnosed to have lung mass with an incidence of 2.1%. Lung cancer was common in fifth decade. Predominant in males [M:F - 3.6:1]. Equal incidence of left and right lung. Five had old Koch's disease. Eighteen were smokers and majority were diagnosed to have lung cancer by computed tomography guided tissue diagnosis. Bronchoscopy detected 9 central tumors. All underwent R 0 resection except one case which was unresectable. Majority were in stage III [ 18 cases]but only 2 patients had nodal invovlement. Chest wall was resected in 4 patients with an average of three ribs resected. Final histopathology showed majority of adenocarcinoma [ 12 cases] followed by squamous cell carcinoma [ 8 cases]. Five patients showed features of concomitant TB.

Conclusion
Due to high prevalence of TB in this subcontinent , nodal staging in pre-operative imaging assessment might be fallacious. Imaging and positron emission tomography results should be interpreted more cautiously while making surgical decisions regarding operability.