Virtual Library

Background:
Molecular genetics have allowed us to categorize non-small cell lung cancer (NSCLC) based on their genetic profile. KRAS mutations occur in 25-30% of NSCLCs. KRAS regulates cellular function in response to growth factors and their receptors. When mutated, KRAS is constitutively active and is responsible for driving tumor oncogenesis. Direct inhibition of KRAS has not been a successful clinical strategy. The strategy of synthetic lethality (targeting a non-lethal defect in cancer cells combined with a second defect, that together make the cancer cell more susceptible to treatment) has gained traction in recent years. Several synthetic lethal targets have been identified with KRAS. We have previously shown that MET plays an important role in the oncogenic addiction observed in KRAS mutated NSCLC and contributes to both tumor growth and metastasis. However, the development of resistance in MET targeting due to upregulation of RON, a related receptor tyrosine kinase, is also evident. Our hypothesis is that dual targeting of MET and RON may be synthetic lethal to KRAS mutated NSCLC and studies to investigate this as a potential therapeutic strategy are warranted.

Methods:
MET- and RON-specific siRNAs (small molecule inhibitors), crizotinib, and the ligand for MET (hepatocyte growth factor), were used in in vitro assays. Immunoblotting, cell viability, and cell migration assays were carried out in a panel of KRAS mutated as well as KRAS wild type NSCLC cells. In addition, human bronchial epithelial cells (HBECs) that were rendered tumorigenic with sequential mutations in CDk4, hTERT, p53, and KRAS genes were also used.

Results:
Our analysis of a panel of NSCLC cells showed that most KRAS mutant cell lines express both MET and RON, and stimulation with HGF activated KRAS effector pathways such as MAPK, AKT and S6RP. When we silenced MET expression with siRNA, it led to upregulation of RON, indicating the interaction between MET and RON. Cell viability assays using crizotinib showed that KRAS mutant cell lines (A549 and H460) are three-fold more sensitive than KRAS wild type cells (H1975 and H1437), and cells with MET amplification (H1993) showed the highest response. Preliminary data with the KRAS-transformed HBECs also showed that they are more sensitive to crizotinib inhibition than the non-transformed control HBECs. Wound healing assays with these same cells showed a similar trend in MET specific inhibition of cell migration in KRAS-mutated cells compared to wild type cells.

Conclusion:
These data highlight the potential therapeutic benefit of targeting MET and RON simultaneously in a subpopulation of KRAS mutated NSCLC patients who may have MET overexpression or amplification. Based on KRAS oncogenic addiction to MET, we propose that NSCLC cells that are MET amplified and KRAS mutated are potentially synthetic lethal and will benefit from dual MET/RON treatment

Background:
Despite the numerous efforts made to target KRAS directly, this protein is still undruggable. A number of therapeutics that target linked KRAS pathway members have been tested, but their efficacy in KRAS mutant lung adenocarcinoma is still controversial. Understanding the biochemically linked protein signaling network associated with a KRAS mutation may lead to the identification of therapeutic targets to identify patients that may benefit from a therapeutic agent targeting KRAS downstream substrates.

Methods:
Thirty-four archived samples from surgically-treated KRAS mutant adenocarcinomas were included in this study. Samples were collected at the H.Lee Moffitt Cancer Center & Research Institute (Tampa, FL) and at the Santa Maria della Misericordia Hospital (Perugia, Italy). Pure cancer epithelial cell subpopulations were isolated using Laser Capture Microdissection. The expression/activation level of 155 proteins was then measured by Reverse Phase Protein Microarray, a high-throughput semi-quantitative platform.

Results:
The protein activation level of ERK (as measured by phosphorylation of T202/Y204), a direct downstream substrate of KRAS activity, was highly variable across KRAS mutant samples. While a subgroup of patients showed, as expected, high activation of ERK, approximately 2/3 of the patients had a comparable ERK activation level to the wild-type counterpart previously analyzed. The activation level of the remaining protein signaling analytes was then compared between samples with high and low ERK activation. Tumors with high levels of ERK activation showed a significant increase in the signaling network of: 1) the MAPK proliferative pathway including Ras-GRF1 S916, Mek 1/2 S217/221, MSK1 S360, p38MAPKinase T180/Y182 (p=0.03, p<0.01, p=0.04, p<0.01 respectively), 2) the AKT-mTOR pathway including Akt S473, AMPKα1 S485, ATP Citrate Lyase S454, LKB1 S428, mTOR S2448, p70S6K T389, p70S6K T412, 4E-BP1 S65 (p<0.01, p<0.01, p<0.01, p<0.01, p<0.01, p<0.01, p=0.02, p=0.03 respectively).

Conclusion:
This analysis suggests that the signaling network of KRAS mutant lung adenocarcinomas, while manifesting expected ERK activation as a group, is highly variable. In fact a majority of KRAS mutant tumors had the same range of MEK-ERK activation as KRAS WT tumors. Analysis of high and low ERK activation in the KRAS mutant tumors revealed druggable protein signaling activation of a number of important targets. If validated in a larger study set, these data may have important clinical implication for the allocation of patients toward more effective and specific targeted treatments.

Background:
While KRAS mutation is a negative predictive marker for EGFR tyrosine kinase inhibitor therapy, there is limited data available regarding the influence of KRAS mutation on the organ specificity of lung adenocarcinoma metastases.

Methods:
In our retrospective, single center study, 820 lung adenocarcinoma patients with KRAS mutation analyses were included. At the time of diagnosis, 462 patients had metastatic disease. These cases were analyzed for the potential association between KRAS status and metastatic site and clinical outcome. Patients with known EGFR mutations were excluded from the study.

Results:
534 (65.3%) KRAS wild-type and 284 (34.7%) KRAS-mutant cases were identified. There was no difference in the KRAS mutation prevalence between the metastatic (35.7%) and non-metastatic cases (33.4%). The most frequent metastatic sites included bone (29%), contralateral lung (24.8%), ipsilateral lung (19.7%), brain (17.3%), adrenal gland (15.6%), pleura (12.8%) and liver (11.7%). Patients with multiple metastases tended to have inferior median overall survival (OS) compared to those with single-organ metastasis (6.3 vs. 8.2 months, respectively; p=0.09) and, moreover, showed a slight but non-significant increase in the prevalence of KRAS mutations (38.5%, p=0.35). Importantly, patients with brain (35.8%), bone (33.1%) or adrenal gland (35.2%) metastases demonstrated similar KRAS mutation frequencies. However, both ipsilateral and contralateral intrapulmonary metastatic cases demonstrated increased KRAS mutation frequency when compared to those with extrapulmonary metastases (42.2% and 42.5%, p=0.014). In contrast, pleural dissemination and liver metastasis were associated with decreased KRAS mutation prevalence (25.4% and 24.1%, respectively; p=0.007). We found no difference in the median OS between KRAS-mutant and WT cases in any metastatic site-specific analysis.

Conclusion:
Lung adenocarcinoma patients with KRAS-mutant tumors more often present with intrapulmonary metastases. KRAS mutation prevalence, however, lacks to provide prognostic information. Further studies are required to determine if KRAS status can be used to risk stratify patients for the onset of pulmonary metastasis.

Background:
Kras mutations are one of the most common driver mutations in NSCLC, which promote lung tumorigenesis. And many patients harboring Kras mutation fail to benefit from chemotherapy. Treatment of Kras-mutant lung cancer is still a challenge. It is reported that telomere shorting inhibits tumor formation and prolongs life span in a KrasG12D mouse lung cancer model. Telomerase inhibitors show a trend toward improving survival of patients with advanced NSCLC with short telomere. However, the roles of telomerase inhibition have not been defined in Kras-mutant NSCLC.

Methods:
KrasG12D was lentivirally transduced into normal human lung cell line (BEAS-2B) and lung cancer cell lines (Calu-3 and H1299) for stable expression. The cells were transfected with TERTshRNA or treated with the telomerase inhibitor BIBR1532 to suppress the telomerase activity. Telomerase activity and telomere length were examined by the telomeric repeat amplification protocol (TRAP) assay and the Southern blot analysis of terminal restriction fragment lengths. Cell proliferation, colony formation and migration were analyzed by cell growth curves, soft agar assay and transwell migration assay. Calu-3- KrasG12D xenograft mice models were used to validate the effects of telomerase inhibition on cell growth and chemosensitivity in vivo.

Results:
We found that continuing inhibition of telomerase shorted telomere length and inhibited mutant KrasG12D-induced cell migration, colony formation, long-term proliferative capability and activation of Kras signaling pathway in both normal human lung and lung cancer cells. In addition, decreasing telomerase activity increased cells sensitivity to chemotherapeutic agents in Calu-3 and H1299 cells with KrasG12D overexpression. Notably, the effects of telomerase inhibition on Kras-mutant cells were P53 independent. In vivo experiments also confirmed treatment with telomerase inhibitor significantly enhanced tumor growth inhibition and the antitumor efficacy of chemotherapy in Calu-3-KrasG12D tumor-bearing mice.

Conclusion:
Our data suggest that Kras mutation-induced lung carcinogenesis and chemoresistance are attenuated by telomerase inhibition. Targeting telomerase/telomere may be a promising therapeutic strategy for Kras mutant NSCLC.

Background:
KRAS represents the most commonly mutated oncogene in non-small cell lung cancer (20-30%). Multiple studies have suggested mutations of KRAS, epidermal growth factor receptor (EGFR), and anaplastic lymphoma kinase (ALK) to be mutually exclusive[i], though there are few case studies showing coexisting EGFR and KRAS mutations[ii].

Methods:
We reviewed clinical genotyping data from 118 patients with stage I – IV KRAS mutated NSCLC. We investigated prevalence of concomitant EGFR and ALK mutations and evaluated clinical behavior in regards to overall survival (OS) and response to tyrosine kinase inhibitor therapy.

Results:
Among these 118 samples with KRAS alterations (codon 12 =98, 13 = 8, 61 = 3, 146 = 2, 189 =1, amplification = 6), median OS was 61.97 months (Graph 1). Concomitant EGFR mutations were noted in 6 subjects (5.0%) and ALK mutations were noted in 2 subjects (1.7%). One patient was found to have mutations of KRAS, EGFR, and ALK. These patients’ stage at diagnosis, response to TKI therapy (if utilized), and OS is documented in Table 1. Figure 1 Figure 2





Conclusion:
This analysis demonstrates it is possible for KRAS mutations to occur concurrently with EGFR and ALK missense mutations (not translocation) and emphasizes that a complete molecular analysis should be performed on all NSCLC patients. Further data is needed to more firmly elucidate how these concurrent mutations affect clinical behavior. Citations [I] Gainor JF, Varghese AM, Ou SH, et al. ALK rearrangements are mutually exclusive with mutations in EGFR and KRAS in non-small cell lung cancer. Clin Cancer Res. 2013 Aug 1; 19(15): 4273-81. [II] Zhu CQ, Sants GC, Ding K, et al. Role of KRAS and EGFR as biomarkers of response to erlotinib in National Cancer Institute of Canada Clinical Trials Group study BR.21. Journal of Clinical Oncology. 2008 Sep 10; 26(26): 4268-4275.

Background:
KRAS is the most frequently mutated oncogene in lung adenocarcinoma patients. The prognostic rolo of mutatn-KRAS in lung adenocarcinoma is controversial, especially in non-asian populations. Studies also suggest the potencial predictive role of mutant-KRAS in the context of chemosensitivity of NSCLC. The aim of our study is to analyze the role of KRAS mutations as prognostic factor in advanced NSCLC, and their value as predictive biomarker of chemotherapy efficacy

Methods:
Retrospective study of patients with advanced NS-NSCLC in our institution between january-2013 and december-2014. Mutation analysis for KRAS was performed an the relation with overall survival was assessed. Secondary endpoints were its relation with progression-free survival and response to chemotherapy.

Results:
A total of 42 patients met inclusion criteria. Median age was 61.5 years. Thirty-three male (78.6%), 27 ECOG-PS 0-1 (64.3%), and 40 (95.2%) adenocarcinoma. Twenty-six patients (61.9%) received chemotherapy as first line treatment, 4 (9.5%) anti-EGFR treatment and 12 supportive care. Nine patients (21.4%) harboured KRAS mutations, all of them at exon 12. There were no differences in age, performance status or smoking history between patients with KRAS mutants vs those with wild-type tumours; instead KRASmut patients presented a higher rate of brain metastases (55.5 vs 20%; p=0.05) and higher number of methastatic locations at diagnosis (77.7 vs 41.3% of patients with more than one site of metastases). Median Overall Survival was superior for patients with wild type tumours (19 vs 10 months, p =0.22). There were no differences in response rate in patients treated with platinum doblet chemotherapy (Wild-type vs KRAS mut: 44.4 vs 33.3%, p=0.5), but progression free survival and overall survival were superior for wild-type tumour patients (PFS: 3 vs 15 months, p=0.001; OS: 10 vs NR, p=0.06)

Conclusion:
With the limitation of small numbers, our data suggest that KRASmut patients are a subgroup with poorer prognostic. Moreover, they seem to benefit less from standard chemotherapy based in platinum doublets.

Background:
Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. The most commonly mutated oncogene in NSCLC encodes for KRAS and its mutations are usually associated to a poor prognosis. The aim of this study was to evaluate the prevalence and the prognosis of these mutations in a Portuguese cohort of patients with NSCLC, EGFR wild-type.

Methods:
We included 201 patients diagnosed with NSCLC, EGFR wild-type, followed in a Lung Cancer Unit. KRAS mutations in exon 12 and 13 were screened. Demographic and clinical data were analyzed. Overall survival, objective response to first-line chemotherapy and time to progression was evaluated in patients staged IIIB or IV at diagnosis.

Results:
173 (81.1%) were male, mean age 67±12 years, 40.1% smokers and 42.6% ex-smokers. At diagnosis, 9.1% were stage IA, 4.6% IB, 3% IIA, 2.5% IIB, 13.7% IIIA, 11.7% IIIB, 54.8% IV. 68.2% were adenocarcinoma and 21.4% squamous tumors. 79.5% was performance status 0-1. KRAS mutations were found in 46 (22.9%) patients and in 4.5% results were not valid. The most common mutations were G12C (41.8%) and G12V (26.1%). There was a statistically significant association between KRAS mutations and non-squamous histology (93.5% in KRAS mutated patients vs. 74.1% in KRAS wild-type patients, p=0.020) and a history of past or current smoking (93.4% vs. 78.4%, p=0.032). No statistical differences were found regarding age, gender, performance status or cancer stage at diagnosis. With respect to patients staged IIIB or IV at diagnosis, overall survival tended to be inferior in patients with KRAS mutations (median survival: 5 vs. 9 months, p=0.127). There was no statistical difference between groups regarding response to first-line chemotherapy and time to progression after first-line chemotherapy.

Conclusion:
The prevalence of KRAS mutation in this Portuguese cohort is consistent with results of similar studies in other countries (20-25%). KRAS mutations were associated to adenocarcinoma histology and smoking habits. Despite overall survival tending to be half in KRAS mutated IIIB/IV patients, this study showed little relevance as a prognostic marker. Thus, it is important to pursue the search for other molecular biomarkers that could be used in prognosis and even as therapy targets.

Background:
Cell-extracellular matrix interactions participate in several steps required for tumor cell invasion and because of that, a group of glycosaminoglycans have been targeted as potentially useful tumor markers. Hyaluronan has shown promise, but still there is uncertainty about its localization in tumor tissue and its relationship with histological types and angiogenesis. Regarding that, we evaluated the association between HA and degree of malignancy through its expression in lung tumor tissue and association with angiogenesis.

Methods:
Forty-six lung specimens were evaluated. Hyaluronan and microvessel (CD34) quantification in situ was done in FFPE sections of nonneoplastic cells, lung cancer cells, and tumor stroma. Colocation was evaluated in tumor stroma using confocal microscopy. Cox proportional hazards model, MantelHaenszel test and Pearson’s x2 were used to evaluate the hyaluronan and microvessel staining inferences and the relationship between them.

Results:
Squamous cell carcinoma showed abundant hyaluronan on the cancer cell-stroma interface coincident with prominent microvessel staining and identical colocalization at confocal microscopy. Strong hyaluronan staining associated with cancer cells was significant in 32.1% of squamous cell carcinoma compared to 17.9% of adenocarcinoma and 0.0% in large cell carcinoma (P<0.001). Adenocarcinomas revealed strong stromal hyaluronan staining in contrast with the hyaluronan-poor tumor cells. The foci of hyaluronan stromal staining was coincident with foci of microvessel and colocalization. Furthermore, adenocarcinoma more often showed a lower percentage of hyaluronan-positive cancer cells (35.7% of cases) than large cell carcinoma (14.3% of cases) or squamous cell carcinoma (0% of cases; P<0.001). For large cell carcinoma, the hyaluronan signal in tumor cells was very poor and contrasted with the foci of staining in stroma, coincident with focal microvessel density and colocalization. All these results are shown in Figure 1. A significant direct association was found between tumors with a high percentage of HA and MVD in tumor stroma (R=0.6; P=0.02). Similarly significant was the direct association between tumors at the N1 stage and high levels of hyaluronan in cancer cells (R=0.31; P=0.05). In addition, tumors in the T4 stage presented positive association with a high percentage of hyaluronan-positive cancer cells (R=0.80; P=0.01).Figure 1



Conclusion:
Our findings showed that an elevated hyaluronan signal in tumor cells was associated with poor prognosis and its localization relationship with histological types and angiogenesis was related to malignancy of lung cancer. To realize these findings a greater larger scale study in a randomized trial will be required.

Background:
According to the International Association for the Study of Lung Cancer , American Thoracic Society, and European Respiratory Society (IASLC/ATS/ERS) classification, lepidic predominant pattern in pT1 lung adenocarcinoma is divided into adenocarcinoma in situ　(AIS),　minimally invasive adenocarcinoma (MIA), and lepidic predominant invasive adenocarcinoma (LPIA) by using new diagnostic criteria. However the new criteria have many item to diagnose MIA. So we simply classified the pT1 lepidic predominant adenocarcinoma by using only collapse and fibrosis below 5cm as invasive component, and we evaluated prognosis of MIA.

Methods:
A total of 231 patients treated for pT1 lepidic predominant lung adenocarcinoma by complete resection at National cancer center hospital east, Chiba, Japan from January 2003 to December 2010 were assessed. We excluded multiple tumor and mucinous invasive adenocarcinoma from the analysis. We classified 187 patients into AIS, MIA, LPIA, according to the IASLC/ATS/ERS classification. The MIA was defined as group A. In the LPIA, we defined invasive component as collapse and fibrosis 5 cm below, and reclassified into MIA and LPIA. Reclassified MIA and LPIA were defined as Group B and C respectively. We analyzed the prognosis of these patients retrospectively.

Results:
AIS, Group A, Group B, Group C were 52　(22.5%), 29 (12.5%), 39　(16.9), 111 (48.1%) respectively. Positive lymphatic invasion and, or vascular invasion and, or pleural invasion in Group A, Group B, Group C were 0 (0%), 4 (1.2%), 24 (21.6%) respectively. There are significant difference in 5-year recurrence free survival (5y-RFS) between Group A and B (5y-RFS rate 100% versus 88.1%; p = 0.022), and Group A and C (5y-RFS rate 100% versus 88.1%: p = 0.046).

Conclusion:
Max collapse and fibrosis below 5 cm correlated with the prognosis of pT1 lepidic predominant adenocarcinoma. Max collapse and fibrosis below 5cm is more simpl and easy method to measure invasive component than the new IASLC/ATS/ERS classification. This method may have potential to diagnose MIA instead of the IASLC/ATS/ERS classification.

Background:
The distinction between separate primary lung cancers (SPLC) or intrapulmonary metastases (IM) is of great clinical importance because of the substantial staging and prognostic implications. With the broad implementation of CT screening for lung cancer, the recognition of multiple tumor nodules is increasingly common. Currently, similarities and differences in histology between two tumors provide the most definitive distinction between SPT and IM. However, the level of agreement among pathologists regarding this question has not been tested. The IASLC Pathology Committee and the Multidisciplinary SPT Working Group has addressed this issue through a pilot online survey. This study assesses the feasibility and reports preliminary results of a web-based survey to determine interobserver variation in distinguishing SPT and IM.

Methods:
A pilot study was conducted to test whether multiple observers could assess a collection of 50 cases of multiple tumors through a digital web-based system. Five pairs of resected nodules were assembled from the University of Colorado and scanned into an image database using an Aperio AT2 slide scanner (Leica Biosystems) with a 40X objective. Reviewers were asked to review slide images, to provide a histological diagnosis according to WHO criteria, to answer questions regarding specific histological details related to each nodule and to determine whether the multiple nodules were SPT and IM. Combined results were evaluated for level of concordance on the central question of primary or metastatic status. Results were also correlated with EGFR, KRAS, ALK and TP53 mutational status.

Results:
A total 21 pulmonary pathology subspecialists completed the survey, evaluating 10 nodules from 5 patients. Ten of the reviewers were from the US, 3 from Japan, 2 from the UK, and one each from Canada, France, Germany, the Netherlands, Korea and Sweden. On the question of SPLC vs IM, 10 reviewers agreed on all cases and these determinations were regarded the histological consensus. There was 85% overall concordance with the consensus diagnosis. Most of dissenting opinions related to a single case. In all but one instance, tumors from the same individual with different histological diagnoses were designated SPLC. However, in 30% of the cases, tumors from the same individual with identical histological diagnoses were determined to be SPLC. The histological attributes regardless of WHO diagnostic category that significantly (each p>0.0001) contributed to this conclusion included lepidic growth, cell size, nuclear pleomorphism and nucleolar prominence. The mutational status of these cases was in complete agreement with the histological consensus. Mutations that distinguished SPT included KRAS, EGFR or TP53 mutation in only one member of a tumor pair or different EGFR mutations in each member of a pair. In IM, identical KRAS mutation was found in both members of a tumor pair.

Conclusion:
In this pilot study a high level of consensus was achieved in separating SPLC vs or IM. A large minority (30%) of tumor pairs with identical histological diagnoses were determined to be SPLC suggesting that histological features beyond those used for WHO classification are taken into account when determining SPT status.

Background:
Nodal micrometastasis in non-small cell lung cancer (NSCLC) is associated with a poorer survival rate than node-negative disease. Furthermore, lymph node micrometastasis often cannot be detected using conventional hematoxylin and eosin staining of frozen sections; detection requires additional time-consuming immunohistochemical (IHC) analysis of paraffin-embedded tissue. We developed a novel ultrarapid immunohistochemical staining method in which an AC electric field is used to facilitate detection of tumor cells. This method allows detection of tumor cells in frozen sections in less than 20 min, and could be a useful tool for frozen diagnosis. We previously reported IHC analysis for NSCLC in detection of lymph node micrometastasis without misdiagnosis using the rapid-IHC protocol developed at our institute. This technology, which has been patented, was released in May 2014 as "Histotech-R-IHC[R]". The purpose of rapid-IHC analysis during surgery for NSCLC is the utility of intraoperative diagnosis of lymph node metastasis.

Methods:
Thirty-four patients with NSCLC were enrolled in the study between June 2014 and March 2015 after obtaining signed informed consent. Surgery was performed at Akita University School of Medicine and University Hospital. The patients were taken to an operating room, and the standard preparations were made for a thoracotomy and lung resection such as lobectomy with systematic/selective nodal dissection or segmentectomy. Dissected lymph nodes from each patient were used in this study. Intraoperative samples from dissected lymph nodes were sectioned, conventionally stained with HE, and immunohistochemically labeled with anti-CK (AE1/AE3) antibody using the rapid-IHC procedures, after which they were examined by a pathologist.

Results:
IHC analyses were completed within 20 min, and the diagnosis was made by the pathologist within about 30 min. Two patients were diagnosed as positive on the basis of conventional histological examination, and the same two patients were deemed positive on the basis of CK detection using rapid-IHC. There were no micrometastases in this study. All patients diagnosed as negative based on CK detection using rapid-IHC were pathologically N0. Twenty-one patients underwent lobectomy, and 13 patients received segmentectomy. Twenty-eight patients underwent lymph node dissection of hilar and mediastinal (ND2a) nodes, and six patients underwent lymph node dissection of hilar nodes only (ND1).

Conclusion:
The rapid-IHC device is useful for intraoperative diagnosis of lymph node metastasis in lung cancer surgery. We want to apply this method to the minimally invasive surgery selection such as segmentectomy and selective mediastinal lymph node dissection. Further investigation in multicenter studies will be needed to confirm the utility of this method.

Background:
The purpose of this study is to assess the histology of malignant pleural mesothelioma using computed tomography (CT) based imaging.

Methods:
28 patients with malignant pleural mesothelioma were used (histologies: 17 epithelioid, 11 biphasic). A CT scan was acquired for each patient prior to surgical resection of the tumor. A radiologist identified and outlined the tumor boundary on each CT section that demonstrated tumor. These outlines were analyzed to determine the total volume of disease present, the mean volume of disease per outlined section, and the distribution of Hounsfield Unit (HU) values throughout the outlined tumor. These parameters were used to differentiate tumors of epithelioid and biphasic histologies. For each parameter, cutoffs were determined to maximize the extraction of biphasic cases from the entire cohort, while minimizing the extraction of epithelioid cases.

Results:
Discernable differences were extracted from the images of the two different histologies of the disease. Figure 1 shows the mean HU value, the standard deviation and skew in the distribution in the HU values, and the volume of tumor represented on each CT section demonstrating disease. With regard to HU distribution, the biphasic cases generally had a higher mean HU value. For example, 73% of the biphasic cases had a mean value greater than 30, compared to only 29% of the epithelioid cases. Biphasic cases also tend to have a more negative skew in their HU distribution; 73% of biphasic cases had a skew value less than -1, compared to 35% of epithelioid cases. It was also seen that biphasic cases also tended to have a higher volume of tumor present throughout their disease presenting CT sections. There were promising results from extracting the biphasic cases by using optimized cutoffs from gathered data. The criteria used were as follows: Cases that exhibited more than 9 mL of tumor per outlined CT section, or exhibited a mean HU value greater than 10 as well as a skew in HU values less than -1 were extracted from the cohort and identified as biphasic. Of the cases that match these criteria, 10 were actually biphasic while 6 were actually epithelioid. These results are 91% specific, missing only one biphasic case, and 65% specific, correctly excluding 11 of the 17 epithelioid cases. Figure 1 Figure 1 – Comparison of Epithlioid and Biphasic cell types.



Conclusion:
This study demonstrates that CT-based imaging may be a useful tool for the assessment of tumor histology through image analysis.

Background:
Combined pulmonary fibrosis and emphysema (CPFE) is a clinical syndrome that is diagnosed with computed tomography (CT), sometimes in conjunction with histopathology. Mostly all patients with CPFE are smokers, and thus, they are at high risk of developing lung cancer (LC). The histological and clinical characteristics of coexisting LC and CPFE syndrome remain unclear. Therefore, we conducted a retrospective study to explore the clinicopathological characteristics of LC along with CPFE (LC-CPFE).

Methods:
We retrospectively reviewed the data of 1647 patients who underwent lung resection for pulmonary masses at Shinshu University Hospital between December 1995 and December 2013. After excluding patients without CT images, patients with metastatic tumors, and patients without sufficient clinical and histological information, the remaining patients were divided into four groups based on chest CT findings: LC-CPFE, LC along with pulmonary fibrosis (LC-PF), LC along with emphysema (LC-Emp), and LC in normal lungs (LC-Norm). The clinicopathological characteristics of patients with LC-CPFE were compared to those of the patients in the other groups.

Results:
After excluding patients for the reasons described above, 985 patients were enrolled in this study. Of these 985 patients, there were 72, 28, 84, and 801 cases of LC-CPFE, LC-PF, LC-Emp, and LC-Norm, respectively. Patients with LC-CPFE were all smokers, with a mean Brinkman index of 1158. Compared with the other groups, patients with LC-CPFE were predominantly men (n = 67, 93.0%) and were older (a mean age of 70.5); LC-CPFE was also associated with a larger tumor size (a mean tumor size of 29.5 mm), the presence of multiple tumors (n = 13, 18.0%), higher stage, squamous cell carcinoma-predominant histology (n = 46, 63.9%), and higher tumor grade (n = 45, 62.5%). Patients with LC-CPFE showed a significantly worse outcome than did patients with LC-Emp and LC-Norm, with a 5-year disease-free survival (DFS) rate of 63.5% and a 5-year overall survival (OS) rate of 53.5%. The OS rate of patients with LC-CPFE was worse than that of patients with LC-PF, although the statistical difference was not significant (p = 0.06), whereas the DFS rates between the LC-CPFE group and the LC-PF group were not significantly different (p = 0.664). In the LC-CPFE group, the tumors were mostly found in associated fibrotic areas (n = 56, 77.7%), followed by emphysematous areas (n = 9) and normal lung areas (n = 7). The pattern of the fibrotic area was as follows: 31 unclassified, 19 UIP, and 6 NSIP. In situ carcinomatous lesions were found in fibrotic areas of more than half of the LC-CPFE cases (n = 30, 53.5%).

Conclusion:
This study indicates that LCs in patients with CPFE syndrome developed in heterogeneous tumorigenic backgrounds. However, because patients with LC-CPFE showed significantly poorer outcomes compared with the other groups, CPFE should be considered an important background disease for patients after resection of lung cancer.

Background:
Activation of Epithelial-Mesenchymal Transition (EMT) mechanisms in tumor cells is known to be associated with its invasive and metastatic properties. However, this hypothesis has not been fully elucidated from the point of detailed clinicopathological view using surgically resected clinical samples. We, hereby, examined the expression levels of EMT marker and analyzed the integrated data of clinicopathlogical information including background genetic alterations.

Methods:
Clinical samples were obtained from the 256 cases of resected lung adenocarcinoma which were consecutively operated from January 2001 to December 2007 in Kyoto University Hospital. Pathological stage distribution of the cases by TNM classification (WHO, 7th edition) was below: 1A: 132, 1B: 56, 2A: 22, 2B: 4, 3A: 26, 3B: 2, 4:14. Mean survival time of all the cases was 62 months, and 5-year survival rate was 75.3%. The distribution of predominant histlogical subtype by IASLC/ATS/ERS classification was AIS (9, 3.5 %), MIA (13, 5.1 %), lepidic (18, 7.0 %), acinar （32, 12.5 %）, papillary (122, 47.6%), solid (44, 17.2 %), micropapillary (9, 3.5 %), mucinous (8, 3.1 %), others (1, 0.4 %). We performed immunohistochemical staining for the expression of E-cadherin and Vimentin on tissue microarrays of resected samples to assess the activation level of EMT. Then, we classified the cases with positive E-cadherin expression and negative for Vimentin as “null” activation of EMT mechanisms, called ‘Group N’, whereas loss of E-cadherin and positive for Vimentin as “full” activation, ‘Group F’, and, further, either loss of E-cadherin or positive Vimentin as “partial” activation, ‘Group P’. DNA samples were extracted from frozen surgical samples and the mutations for the hot-spot exons of EGFR, ALK, K-ras, and p53 were detected by SSCP or direct sequencing methods. Statistical analyses for survival were performed by Kaplan-Meirer curve and log-rank test. Categorical data were analyzed by Pearson’s test. P-values < 0.05 were considered to be statistically significant.

Results:
Histological subtypes were significantly associated with EMT activation level. Poorly differentiated tumors mainly comprising solid, micropapillary, and mucinous adenocarcinoma possessed highly activated EMT level, and vice versa. Group F showed the highest positivity both in local lymphatic/vascular invasion and lymph-node metastasis (35.7%/ 42.9%/ 46.3%, respectively), followed by group P and group N in order. Significant difference was found in 5-year disease-free/ overall survival rate among these 3 groups: group F, 46.9%/ 50.6%; group P, 64.9%/ 73.4%; group N, 74.9%/ 85.4%. Tumors harboring wild type EGFR or mutant p53 had tendency to acquire higher EMT activation level. Interestingly, p53 mutation rate significantly correlated with EMT activation level especially in mutant EGFR tumors, whereas no correlation in wild type EGFR ones. No significant correlation was shown between EMT activation level and the proportion of K-ras mutation or ALK fusion gene.

Conclusion:
Our results revealed that the activation of EMT mechanisms in human lung adenocarcinoma is significantly associated with histological subtypes and plays important roles in the tumor progression through its lymph-vascular local involvement leading to the node-metastasis. Among human lung adenocarcinomas harboring EGFR mutation, p53 alteration deeply correlates with EMT activation.

Background:
Pleomorphic carcinoma (PC) of the lung is rare, and it is classified as a subtype of sarcomatoid carcinoma of the lung in the WHO histologic classification of lung tumors. We here demonstrated the clinicopathologic characteristics of PC surgically resected in our hospital.

Methods:
In this study, 23cases (2.3%) of PC among 968 of non-small cell lung cancer surgically resected at the Kitasato University Hospital between January 2004 and January 2015 were reviewed. The registry data of the patients with PC were analyzed, and the clinicopathologic profiles and surgical outcomes of the patients were evaluated.

Results:
There were 19 men and 4 women, and their mean age was 64 years (range: 39 to 81 y). All but two patients were smoker, and their mean smoking index was 41 pack year. In this study, the mean diameter of the tumor was 48.8 mm; tumors over 50 mm in diameter comprised 45% of all cases. The TNM pathological stages of PC were classified as: 2 (9%) cases with stage IA, 5 (22%) with stage IB, 2 (9%) with stage IIA, 8 (35%) with stage IIB, 5 (22%) with stage IIIA, and 1 (4%) with stage IIIB carcinoma. Ten tumors contained identifiable epithelial components, and the other 12 consisted of spindle cells and giant cells alone: an adenocarcinoma component was found in 11 cases, and 4 of the 11 cases had a coexisting squamous cell carcinoma component. In all the 23 patients, lymphatic infiltration and/or venous infiltration were evident. Overall follow-up ranged from 60 to 2605 days, with a median (for patients still alive) of 896 days. The overall survival rate and disease-free survival rate were 65% and 57%, respectively. Furthermore, 58% of the clinical stage I patients with PC demonstrated advance in the pathological stage. Cancer recurrence was identified in 6 patients; local or local with distant recurrence in one each and distant in 4.

Conclusion:
We concluded that PC should be considered as an aggressive disease and vascular infiltration should be usually reported and used as a factor in clinical assessments.

Background:
Lung cancer remains the leading cause of cancer related death in USA and around the world. Multiple modalities are available for sampling lung neoplasms, mediastinal and hilar lymph nodes . Endobronchial ultrasound –guided transbronchial needle aspiration (EBUS-TBNA) has become an important diagnostic tool . Although the samples obtained by EBUS-TBNA are smaller than specimens collected by other surgical methods, the procedure has shown excellent specificity and sensitivity for the diagnosis of neoplastic diseases, is cost-effective compared to mediastinoscopy , and has become the procedure of choice for initial evaluation of patients with mediastinal and hilar lymphadenopathy . EBUS is currently performed by both interventional and general pulmonologists. Aim of the study: To assess the adequacy of tissue for diagnosis in relevance to PET scan, the diagnostic yield of the various lymph node (LN) stations and the level of experience of the operator.

Methods:
We reviewed the chart of 171 patients who underwent EBUS between the years of 2011-2013. We reviewed the pathological diagnosis, the LN stations, the PET scan results and the operator who performed the EBUS.

Results:
We included 171 patients where adequacy of tissue diagnosis was achieved by majority of patients in whom EBUS was performed (p<.0001). More tissue seemed to be positive in LN station 4 compared to the other LN stations but with no statistical significance. There was no correlation between the positivity of the PET scan and the tissue adequacy for diagnosis by EBUS (p=0.6410). PET scan showed a trend to increase in positive uptake in LN station 2 (p=0.0705). The adequacy of tissue diagnosis was achieved most significantly by Interventional Pulmonary (IP) trained operator, followed by an operator of more than 5 years’ experience followed by an operator of less than 5 years’ experience with 100% ‚ 93.33% ‚ 88.89% subsequently for tissue diagnosis accuracy (p=0.0019). The diagnostic tissue adequacy had a positive correlation with the PET scan when analyzed by operator, where the operator with more than five years’ experience had a closer correlation with the PET scan positive uptake. The percentage of tissue adequacy in relation to the PET scan positive uptake was of 54.64% ‚ 76.67% and 35.56% subsequently (p=0.0009).

Conclusion:
The adequacy of tissue diagnosis was achieved by majority of patients in whom EBUS was performed. There was no correlation between the positivity of the PET scan and the tissue adequacy for diagnosis by EBUS therefore PET scan and EBUS should be used complementary to each other for the appropriate diagnosis and staging of patients. The adequacy of tissue diagnosis was achieved most significantly by (IP) trained operator, followed by an operator of more than 5 years’ experience followed by an operator of less than 5 years’ experience.

Background:
Pulmonary lymphoepithelial-like carcinoma (LELC) is one of the rare histological non-small cell lung cancers. Only a few case reports have been published. The knowledge of its characteristics and prognosis is limited. Based on the data of the Surveillance, Epidemiology, and End Results Database (SEER), an analysis was performed to fill the gap of our knowledge

Methods:
Characteristics, treatment and outcomes of all pulmonary LELC patients was extracted both from the SEER database with 18 registered center from 1973-2011 using SEER*Stat 8.1.5 Statistical analysis was performed using SPSS 16.0 and GraphPad Prism 5.

Results:
A total of 62 patients with pulmonary LELC are identified and recorded. Among them, Caucasian patients account for the largest proportion (64.4%). The medium age at diagnosis is 65. The 1, 3 and 5 years survival rates of LELC are 85.6%, 74.5% and 55.2%. The median survival time of all LELC patients is 34 months. Comparing to other types of lung cancer, LELC has a better survival. 14 patients have received radiation, while most of the early stage LELC patients (30/34, 88.2%) have received surgical resection as the first treatment.Figure 1



Conclusion:
Pulmonary LELC is a rare pathological type of lung cancer . In this cohort, male and Caucasian patients account for a large proportion of LELC patients. The mean age of pulmonary LELC patients in this study is older than the patients in Asian studies. A large amount of patients are in the early stages (localized and regional) when they are diagnosed as LELC. LELC has a better prognosis than adenocarcinoma, most early stage patients have received surgical resection. However, no prognosis factor has been identified in our study. In order to understand pulmonary LELC more thoroughly, more cases are required.

Background:
The role of endobronchial ultrasound (EBUS) and transesophageal bronchoscopic ultrasound (EUS-B)-guided fine-needle aspiration (FNA) of intrathoracic lymphnodes has acquired paramount importance in obtaining definitive diagnosis in malignant diseases. Recently, the diagnostic value of cell block processing of EBUS-FNA samples to obtain accurate distinction between lung cancer subtypes has been studied, but the diagnostic yield have been marginally investigated in a larger cohort of patients.

Methods:
We review tertiary hospital experience with EBUS-FNA and EUS-B-FNA in obtaining tissue diagnosis for lung cancer and evaluate, if the cell block processing method increases diagnostic accuracy in pathological lung cancer subtyping. The pathological examination was based on smear cytology (SC) and cell block preparation (CBP) routinely obtained during EBUS (EUS-B)-FNA. After cell block embedding in paraffin and sectioning at 3 micrometer hematoxylin and eosin staining and if required immunostaining was available for microscopical histopathological examination.

Results:
From January 2011 to December 2013, 608 patients, including 208 lung cancer patients with mediastinal and hilar lymphnodes pathology, underwent EBUS-FNA or simultaneous EUS-B-FNA in North Estonia Medical Center. In lung cancer patients cytological assessment was performed in all 208 cases. Formalin fixed paraffin-embedded cell block for histopathological examination was available in 196 (94.2%) cases. The overall morphological verification rate in CBP group was 85.6% (n=178) and in SC group 79.3% (n=165). The pathological diagnosis of undifferentiated type of lung cancer was provided in 43.7% (n=91) of CBP cases and in 35.6% (n=74) of SC cases. Diagnostic yield in pathological subtyping of lung cancer was significantly higher in CPB compare to SC: 81.2 % (n=169) vs. 43.7% (n=91), respectively (p<0.005). Adding CBP to SC provided an accurate subtyping of lung cancer in 102 more patients and the diagnostic efficacy was increased by 49.0% (n=102/208) (p<0.005).

Conclusion:
Cell block processing method combined with smear cytology obtained from intrathoracic lymphnodes applying EBUS (EUS-B)-FNA significantly increases diagnostic yield in pathologic lung cancer subtyping. Both tissue processing techniques should be routinely applied simultaneously whenever possible with aim to facilitate clinical decision and make impact on lung cancer patients outcome.

Background:
We have recently demonstrated that presence of the micropapillary pattern increases the risk of local recurrence after limited resection for ≤2 cm lung adenocarcinoma (ADC). Currently, limited resection for small lung ADC has been done based on the definition of radiological non-invasive lung cancer, until histological subtype have not been examination. The purpose of this study is to investigate whether the presence of micropapillary pattern correlates with radiological non-invasive lung cancer in small lung ADC.

Methods:
All available tumor slides from patients with clinical stage IA, therapy-naive, surgically resected solitary lung ADC ≤2 cm in size (2001-2012) were reviewed. Comprehensive histologic subtyping was performed according to the IASLC/ATS/ERS classification. Tumor diameter and solid component diameter were measured at the maximum cut surface of the tumor using high-resolution CT (HRCT). HRCT findings were classified as three groups as pure ground glass nodule (GGN), part-solid, solid based on the IASLC/ATS/ERS classification. Recurrence-free probability (RFP) was estimated using the Kaplan-Meier method.

Results:
233 patients met inclusion criteria (50% women; median age: 67yrs; 48% never-smokers; median tumor size: 1.2cm; 68 pure GGN/ 76 part-solid/ 89 solid; 157 lobectomy; 43 AIS/ 77 MIA/113 IAD; 13 lymph node metastasis). Presence of the micropapillary pattern (≧5%) (MPP≧5) was identified in 21 cases (9%). MPP≧5 was significantly associated with tumor size, lymph node recurrence, lymphatic invasion, vascular invasion (P = .001, .003, .0017, .014, respectively) and was associated with increased risk of recurrence as compared to MMP<5% (5-year RFP: MIP≧5%:74.3%; MIP<5%:87.6%; P = .046). Twenty-one patients with MPP≧5 included 1 pure GGN / 5 part-solid / 15 solid in HRCT. The patient with pure GGN and MPP≧5 showed recurrence in lymph nodes in 10 months after surgery. In pure GGN group, MPP≧5 was associated with increased risk of recurrence as compared to MMP<5% (P=0.0001).

Conclusion:
The patient with radiological non-invasive lung cancer may be included in micropapillary pattern. It is necessary to consider lung adenocarcinoma histological subtypes for the patient with limited resection.

Background:
Individuals infected with HIV have a higher predisposition to develop malignancies. The spectrum of neoplastic diseases in HIV-infected patients has changed after the introduction of antiretroviral therapy which decreased the AIDS defining malignancies such as Kaposi's sarcoma (KS) and non-Hodgkin lymphoma (NHL), but augmented other tumor types contributing to increased mortality of patients on chronic treatment. We report a patient with HIV on more than 10 years of antiretroviral treatment in whom diagnosis of a metastatic adenocarcinoma of gastrointestinal origin and a concomitant primary lung squamous carcinoma was made.

Methods:
Clinical History Revission

Results:
A 68-year-old man with a history of HIV on antiretroviral treatment (ART) since 2004, ex-smoker with COPD, osteoporosis and chronic malnutrition, who presents with cough, dyspnea and hemoptysis. On the chest CT-scan a right paravertebral mass associated with atelectasis, a parahilar mass extending to the left upper lobe, and a mass in the pancreatic head is observed. A bronchoscopy with biopsies is performed. The morphological and inmunophenotypic expression patterns of the right lower lobe show metastatic adenocarcinoma of gastrointestinal origin while the left lower lobe biopsy shows primary squamous cell lung carcinoma and the presences of Aspergillus. The patient continued with hemoptysis, developed refractory respiratory failure and died.

Conclusion:
With the widespread use of potent ART there was a dramatic decrease in the incidence of KS and NHL and a significant increase in the incidence of several other malignancies. Although the biology of malignancy in HIV-infected people is often more aggressive than in those without HIV infection, standard treatment is generally indicated and can be associated with a favorable outcome, depending upon the tumor type, stage, and comorbidity. In this case two advanced stage tumor lesions associated with hemoptysis were documented, which finally led to the death of the patient. Figure 1 Figure 2

Background:
MAML2 (mastermind-like 2 (Drosophila)) gene, a transcriptional coactivator for NOTCH proteins, is known to be involved as a part of fusion gene (MECT1-MAML2) which is found in mucoepidermoid carcinoma (MEC) both of salivary gland and pulmonary origin. Ciliated muconodular papillary tumor (CMPT) is sharing morphologic features with mucoepidermoid carcinoma, at least in part, i.e. consist of mixture of mucinous and squamous epithelium. To determine whether these morphological mimics can share the molecular alterations, we evaluated MAML2 rearrangement of CMPT by FISH.

Methods:
Five cases of CMPT was recruited from pathologic diagnostic records between 2005 to 2014. Morphological assessment was done on routine HE stained slides of whole tumor specimen. Representative area was selected and submitted to FISH analysis. Fluorescence in situ hybridization (FISH) using break apart type MAML2 gene probes was performed on FFPE specimen. The break apart signal percentages on separated tumor nuclei was counted on the captured images of digital fluorescence microscope. 100 nuclei was counted in each cases. More than 30% of break apart signal is considered as positive result.

Results:
All five CMPTs were reviewed and confirmed the diagnosis on HE stained slides. These cases included 4 male, 1 female, were mean age of 71 years-old (range 60-83). There were three incidental cases which were patients with one primary lung adenosquamous carcinoma and two metastatic cancer (one colon cancer, one liposarcoma). All of five CMPT resulted negative for MAML2 break-apart FISH.

Conclusion:
These results indicated that CMPTs do not share the molecular alteration of MAML2, which is commonly detected in mucoepidermoid carcinoma of lung. In conclusion, CMPT is a distinct tumor or tumor-like lesion, does not related to MEC. Although, it is still uncertain whether CMPT is a true neoplastic lesion with multi-lineage differentiation potential or a reactive process with extensive epithelial proliferation.

Background:
According to the International Association for the Study of Lung Cancer (IASLC)/American Thoracic Society (ATS)/European Respiratory Society (ERS) classification, both solid- and micropapillary-predominant pulmonary adenocarcinoma have been reported to have a poor prognosis. Although pulmonary adenocarcinoma with some solid or micropapillary component have also been reported to have a poor prognosis, the ratio of these component to be chosen as the cutoff value for a prognostic factor remains controversial.

Methods:
A total of 115 patients with pulmonary invasive adenocarcinoma measuring ≦ 3 cm who underwent curative surgery at Tottori University Hospital between January 2005 and December 2008 were included. Patients with variants of invasive adenocarcinoma were excluded from this study. The median follow-up time was 78.0 months. A total of 84, 9, and 22 patients underwent lobectomy, segmentectomy, and wedge resection, respectively, and 100, 5, and 10 patients had stages I, II, and III, respectively. The tumors were divided into subtypes according to the IASLC/ATS/ERS classification. Cases with solid component occupying ≧ 5% of the entire tumor were defined as S-positive (S+), and cases with micropapillary component occupying ≧ 1% of the entire tumor were defined as MP-positive (MP+). Of the 115 adenocarcinoma, 30 and 85 were S+ and S-, and 27 and 88 were MP+ and MP-. The clinical characteristics and pathologic data of all 115 adenocarcinoma were retrospectively evaluated. The Kaplan-Meier method was used to estimate the recurrence-free survival (RFS) and overall survival (OS) rates, and the log-rank test was used to compare the RFS and OS among the subgroups.

Results:
The 5-year OS rate of cases that were S＋ and S- was 92.5% and 62.1%, respectively (log rank P < 0.001). The 5-year RFS rate of cases that were MP＋ and MP- was 77.3% and 51.9%, respectively (log rank P = 0.001). On multivariate survival analysis, the presence of solid component proved to be an independent prognostic factor, and the presence of micropapillary component proved to be an independent recurrence factor.

Conclusion:
The presence of solid component occupying ≧ 5% of the entire tumor was an independent predictor of a poor prognosis in pulmonary invasive adenocarcinoma measuring ≦ 3cm. The presence of any micropapillary component, even if only in 1% of the entire tumor, was a risk factor for post-operative recurrence and it affected the prognostic value.

Background:
Non-small cell lung cancer (NSCLC) comprises 85% of primary lung cancer, where adenocarcinoma, squamous cell and large cell carcinoma are the most common histological types. Recently a new classification of primary adenocarcinomas of the lung was published. The aim of this study was to review the histology of all primary lung adenocarcinomas operated on in Iceland during a 20 year period, 1991-2010, using the new criteria and assess the impact of histology on survival.

Methods:
This nationwide study included 301 patients with primary lung adenocarcinoma (mean age 65.5 yrs., 56% female) that underwent resection in Iceland between 1991-2010. Tumors were reclassified according to the current IASLC/ATS/ERS pulmonary adenocarcinoma classification system. Overall survival was estimated by the Kaplan-Meier method and multivariate Cox regression analysis used to evaluate prognostic factors of survival, including histological subtype

Results:
Acinar predominant adenocarcinoma was the most common histological subtype (45%). Solid predominant with mucin production comprised 24% of the cases, lepidic predominant 19% and papillary predominant 8%. There was one in situ adenocarcinoma, three minimally invasive adenocarcinomas and seven invasive mucinous adenocarcinomas. Overall survival at 1 year for all histological subtypes of adenocarcinoma was 81.1% and 42.6% at 5 years. A statistically significant difference in survival between the histological subtypes was not seen (log-rank test, p=0.43). Using multivariate analysis advanced stage and age predicted a worse outcome. Histologic subtyping did neither predict survival in uni- or multivariate analysis.

Conclusion:
Acinar and solid predominant adenocarcinoma are the most common histological subtypes for primary lung adenocarcinoma in Iceland. There was not a statistical difference in survival according to histological subtypes and the subtyping was not a prognostic factor of survival.

Background:
Pulmonary ossifications are heterotopic bone formations in the lung, previously thought to be a post-mortem finding only. Two types are described: nodular ossifications, smoothly edged, which are found in the alveoli themselves, and dendriform ossifications, branching through the alveolar septa. In this study the incidence of pulmonary ossifications was studied in a consecutive series of patients undergoing pulmonary surgery.

Methods:
From January 2008 to February 2015 19 patients with pulmonary ossifications were identified in patients undergoing thoracic surgery at Antwerp University Hospital. Diagnosis was made by the pathologist team. Neither PET nor CT was able to differentiate these ossifications from solid tumors.

Results:
15 patients (79%) were male. 8 received lung surgery for tumoral pathology. Most ossifications (58%) were found in the lower lung lobes without predilection for either chest side. 3 patients (16%) died during follow-up due to oncologic pathology unrelated to the ossifications. Most ossifications were nodular-type (12 or 63%), 6 or 32% were dendriform and one case contained both types.Figure 1



Conclusion:
Pulmonary ossifications are not as seldom as previously thought. These benign lesions are not simply a post-mortem finding and could be mistaken for a malignant space-occupying process. There appears to be a predilection for the lower lung lobes in male patients, without a clear association with other pathologies. Therefore, pulmonary ossifications deserve a place in the differential diagnosis of solitary pulmonary nodules.

Background:
The incidence trends of lung cancer (LC) by histology, gender, race/ethnicity and neighborhood socioeconomic status (nSES, derived from US Census data) have not been reported. To examine these trends, we conducted a population-based study, using data from the California Cancer Registry across three discrete time periods to correspond with Census data on nSES: 1988-1992; 1998-2002; 2008-2011.

Methods:
Incidence data for invasive LC were abstracted for the three time periods. In each time period, male and female age adjusted incidence rates of LC and incidence rate ratios were calculated by histologic cell type and stratified by nSES.

Results:
A total of 240,307 LC cases were identified across the three time periods. Histology incidence trends by race/ethnicity are shown in Figure 1 and by nSES in Table 1 for males and females. Larger declines in incidence were seen over the 3 time periods among males than females. Among males, incidence rate declines over time were seen in all race/ethnic and nSES groups, but were largest among Blacks and Hispanics. Across all races/ethnicities among males, there was a slight increase over time in the incidence of adenocarcinoma histology. Among females, incidence rate declines were seen among Hispanics regardless of nSES, mid- and high-nSES Whites, and low- and mid-nSES Blacks; incidence trends among Asian/Pacific Islander females did not change significantly over time, regardless of nSES. Among females, there were variations in incidence trends by histology with a slight increase in the adenocarcinoma histology. Figure 1Figure 2





Conclusion:
Our findings demonstrate differences in LC incidence over time by histology, gender, race/ethnicity and SES. While incidence rates consistently declined for males, there were greater declines in incidence for the high SES patient populations. For females, there were variations in trends by histology, race/ethnicity, and SES. These findings warrant further investigation.

Background:
In the evolving era of genetic sequencing of lung tumours and targeted agents, there is impetus to repeat biopsies of previously treated lung cancers when there is clinical and radiological evidence of relapse, justified on the basis that genetic status of the tumour may change over time. In our centre, even prior to genetic subtyping of lung cancers we have operated a policy of confirming relapse histologically and we present here the value of this strategy. This audit describes the outcome of repeat biopsies in the lung cancer population of Greater Glasgow over a five year period.

Methods:
The regional pathology database was interrogated for all patients with previous diagnosis of lung cancer who had repeat biopsy between February 2009 and March 2014. Inclusion criteria were those whose initial diagnostic biopsy was six months or more previously and included were CT guided biopsies, bronchoscopic brushings or washings, transbronchial or endobronchial guided biopsies, mediastinoscopies, surgical resections and cytology of pleural fluid. We excluded from our analysis those who were having lung biopsy due to metastasis from extra thoracic primary sites. We collated data on patient demographics, time between initial diagnosis and second biopsy, first and second pathology, stage and treatment at first presentation and again at second, whether second biopsy was a different site, and whether pathology was identical on second biopsy/similar (more or less well differentiated but same subtype of lung cancer)/ not malignant or different pathology.

Results:
103 patients fulfilled our inclusion criteria: 41 men, 62 women; age range 35-85y with initial stage of disease: 55 I, 18 II, 25 III, 4 IV and 1 small cell. 91 had primary treatment with curative intent: 66 surgery, 25 radical radiotherapy +/- chemo. 11 had chemo alone, 1 observation only as first treatment. Time to second biopsy ranged 6-52 months (median 17). 70 patients (70%) had identical or similar pathology at second presentation. 13 had different pathology: 2 patients with initial NSCLC developed a second tumour which was SCLC, 1 previously treated SCLC developed a second tumour which was NSCLC, 1 resected carcinoid developed subsequent adenocarcinoma and one adenocarcinoma developed subsequent carcinoid. 8 patients had change of NSCLC subtype at second presentation. The remaining 20 patients had no malignancy on second biopsy: these had had prior radiotherapy or surgery all with radiological/clinical suspicion of recurrence. Of the 103 patients 42 are still alive.

Conclusion:
Although lung cancer carries a high risk of relapse following primary therapy our results demonstrate that clinical or radiological suspicion of recurrence cannot justify treatment without confirmatory biopsy. One third of our cohort either had no malignancy or a second pathology.

Background:
To study the expression of fibroblast activation protein (FAP) in human lung cancer tissues and its clinical significance．

Methods:
Western-blot analysis was used to explore the expression of FAP in lung cancer tissues, paraneoplatic tissues (2cm beyond the cancer margin) and distal normal lung tissues(surgical resected specimens of lung tissue near the cut end) and also the lung cancer cell lines(A549, H1299, SPCA-1). Immunohistochemistry was performed to study the expression of FAP at protein level in tissues from 41 cases of lung cancer and 6 cases of benign pulmonary lesion．

Results:
Western blot analysis showed the expression of FAP was higher than in paraneoplatic tissues as well as normal lung tissues (P<0.05). It was also be positive in lung cancer cell lines (P<0.05). Immunohisto-chemistry showed that the positive rate of FAP staining was in fibroblast cells were 75.6%(31/41), the positive rate of FAP staining was in lung cancer cells were also 90.2%(37/41), whereas the expression in benign pulmonary lesion tissues was poor plus (1/6)．FAP expression was found to be significantly correlated with smoking status (x2=5.4085,P=0.02). However, there were no significant correlations between FAP and age, sex and TNM stage(P>0.05)．

Conclusion:
FAP could be over-expressed in lung cancer cells, which signifies that FAP also plays a role in the development of lung cancer and may serve as a potential biomarker in the treatment of lung cancer.

Background:
It is increasingly being recognized that adenocarcinomas (AC) of various organs with tumor cells arranged in a micropapillary pattern are more malignant than those without such a micropapillary component (MPC). However, the factors and mechanisms conferring the increased malignancy on MPC remain to be elucidated, so the exploration of such factors was the main purpose of the present study.

Methods:
We histologically reviewed the 629 radically resected lung adenocarcinomas at Kitasato University Hospital, Japan, from January 2002 to December 2012. MPC was defined as a small papillary tumor cell tuft without an obvious fibrovascular core. Tumors with ≥ 1% of their tumor cells arranged in a micropapillary pattern were diagnosed as AC with MPC (AC-MPC), while the remainder were diagnosed as conventional AC (CAC). The histological subtypes and differentiation grade of CAC as well as the background non-MPC of the AC-MPE were determined according to the 4th WHO classification. The clinicopathological features of AC-MPC and CAC were comparatively studied. The specific proteins expressed in AC-MPC were also analyzed using proteomic study based on 2-dimensional gel electrophoresis (2-DE), and the results were confirmed in vivo with imunohistochemistry.

Results:
One hundred and one (16.1%) of the 629 histologically reviewed lung adenocarcinomas met the criteria defined above and were thus diagnosed as AC-MPC. Compared with the CAC, the AC-MPC had worse statuses for tumor size, vascular invasion, pleural invasion, node metastasis, disease stage, postoperative up-staging, and overall survival (OS) and disease-free survival (DFS) (p<0.0001, respectively). On 2-DE, 19 proteins differentially expressed more than 1.5-fold in amount between lung CAC and AC-MPC; in particular, vimentin, one of the identified proteins, was most up-regulated (3.5-fold) in AC-MPC cases. Vimentin expression was detected in MPC of 95 (94.1%) AC-MPC, and when compared with the 119 control CAC, the expression scores in MPC were higher than those of well- and moderately differentiated CAC, as well as the background non-MPC of the AC-MPC (p<0.0001), but not significantly different from those of poorly differentiated CAC (p=0.561). Within the AC-MPC entity, higher vimentin expression was correlated with more frequent vascular invasion and more advanced node metastasis, and multivariate analysis showed that high vimentin expression was an independent indicator of worse prognosis (OS: p=0.012, DFS: p=0.047).

Conclusion:
Vimentin expression is prevalent and markedly up-regulated in MPC, which might reflect the biological essence of poorer differentiation or dedifferentiation of MPC, and this might have a role in the acquisition and increase of invasiveness and consequent more malignant nature of MPC.

Background:
The high incidence and mortality rates of lung cancer reflect the need for new diagnostic and prognostic markers capable to early detect the disease and also predict its recurrence. The ideal biomarker should be evaluated in biological samples obtained by minimally invasive procedures. In this context, sputum is an attractive biological sample for these tests since it may represent the field of injury. Because cell–extracellular matrix interactions participate in several steps required for tumor cell invasion and formation of metastases, cofilin-1, hyaluronic acid (HA) and CD44 have been targeted as potential useful tumor markers. To our knowledge, cofilin-1 has never been evaluated in sputum from lung cancer patients. Objective: To evaluate the diagnostic and prognostic role of sputum cofilin-1 and to the relationship between this biomarker with HA and its receptor (CD44) in patients with non small cell lung cancer (NSCLC).

Methods:
Cofilin-1 and CD44 were analyzed by Elisa immunoassay and HA by "Elisa-like" fluorometric assay in the sputum of 74 NSCLC patients, 13 cancer-free patients with obstructive lung disease and 8 healthy individuals. Statistical analyses included ANOVA, ROC curves, Spearman correlation, and logistic regression.

Results:
Sputum cofilin-1 levels were increased in patients with lung cancer (1475.83 pg/ml ±145.35) when compared to cancer-free patients (662.63 pg/ml ±5.74) and volunteers (415.25 ±3.68 pg/ml). A significant association was found between cancer patients with high levels of cofilin-1 and CD44 (R=0.21; P=0.04) as well as between HA and CD44 (R=0.46; P<0.01). Cofilin-1 did not correlate with HA (P>0.4). Univariate analysis demonstrated that high expression of sputum cofilin-1 significantly correlated to T4 (P=0.01) and M stage (P=0.03), tobacco history (P=0.01) and squamous cell carcinoma histologic type (P=0.04). Logistic regression analysis controlled for tobacco history, histologic types, stage, HA and CD44 expression showed that cancer cell–associated cofilin-1 was an independent predictor of metastases [OR=5.77 (0.78-42.74)]. Patients with sputum cofilin-1 >1475.83pg/ml had a high risk for metastasis. In relating to diagnosis, sputum Cofilin-1, at a cut off value of 248.9pg/mL presented sensitivity and specificity of 0.80 and 0.67 respectively, in distinguish healthy volunteers from NSCLC patients with AUC of 0.787. Cofilin-1 was also able to distinguish cancer-free patients from cancer patients at a cut off of 802.5pg/mL with AUC of 0.693 and respective sensitivity and specificity of 0.60 and 0.54.

Conclusion:
Cofilin-1 presented moderate sensitivity as diagnostic biomarker for lung cancer in sputum. Cofilin-1 was associated with metastases, but its prognostic value was dependent on the histological type and tobacco history. The association between sputum cofilin-1 and CD44 in cancer patients suggests that during tumor development, high levels of cofilin-1 facilitate tumors growth and the penetration of capillaries into the tissue milieu. Thus, increased tumor expression of cofilin-1 seems to be more associated to primary events, while increased angiogenesis seems to be related to a secondary event. Regardless of the involved mechanism, detection of sputum cofilin-1 provides important prognostic information on cancer patients. Larger series of NSCLC patients still needs to be studied to confirm the usefulness of sputum cofilin-1 as diagnostic and/or prognostic biomarker.

Background:
Pleural lavage cytology (PLC) is the microscopic study of cells obtained from saline instilled into and retrieved from the chest cavity (in patients without preoperative pleural effusion) during surgery for non–small-cell lung cancer. The solution is aspirated, and cytologic analysis is performed to screen for malignant cells. Results from this procedure have been published from Japan as early as 1989,1 and internationally, an increasing number of centers have adopted this practice.

Methods:
Between 1995 and 2013, 2616 patients underwent surgical pulmonary resection for primary lung cancer without disseminated disease at our institute. Cytology of pleural lavage immediately after thoracotomy before any manipulation of the lung was examined in 1563consecutive patients with lung cancer with no pleural effusion. The macroscopic status of the pleural cavity was evaluated before any manipulation, and when no malignant findings were noted, the pleural cavity was washed with 100 ml of physiologic saline solution.

Results:
The results of the cytologic examination were divided into two categories, positive and negative PLC group. Papanicolaou classes I to IIIa were regarded as negative, classes IIIb, IV and V as positive. Of the 83 patients (6.8%) whose specimens were positive for PLC. Of the 83 patients in the positive PLC group, 74 (4.7%) had adenocarcinoma, with a significantly higher ratio of adenocarcinoma compared with the negative PLC group. Survival in the positive PLC group was significantly worse than in the negative PLC group (p = 0.001), especially in pathologic stage II (p = 0.001). We assume that the PLC positive cases have a T4 status. All PLC positive cases are reassigned Stage III. The result showed almost similar curves was shown between PLC negative Stage III and the adjusted PLC positive Stage III. We propose that positive PLC positive disease should be classified to pathologic T4 and managed similarly to dissemination.

Conclusion:
A positive PLC result was a strong unfavorable prognostic factor, and almost all patients with positive PLC relapsed within 5 years. PLC should be considered in all patients with early stage lung cancer suitable for resection ,especially, done when assessing the final stage in patients with adenocarcinoma of the lung. A positive result is an independent predictor of adverse survival and carries a prognosis. That suggests it may be appropriate to upstage patients by 1 T category or consider as T4 disease.

Background:
Traditionally, squamous cell carcinoma of the lung (SqCC) is conceptualized as more of a central rather than a peripheral form of lung cancer. While there is some variability with respect to the definition in the literature of what constitutes the ‘peripheral’ portions of the lung, historically, rates of squamous cell carcinoma in the lung periphery are typically sited in the 15% to 30% range. More recently, and especially in light of the historical data, we increasingly observed that a significant portion of newly diagnosed peripheral lung lesions – perhaps even a majority, appeared to be squamous cell carcinomas. Therefore, a comprehensive review of the tumor data at our facility, a busy teaching hospital with a large cohort of cancer patients, was undertaken to assess whether there had been a substantive change in the traditional epidemiologic distributions of the lung cancer, specifically with respect to squamous cell carcinoma. Given the differences in cell biology and carcinogenesis of different types of cancer, a potential epidemiologic shift might suggest a change in tumor biology, etc.

Methods:
From May 12[th], 2012 through May 13[th] of 2013, all histopathologicallly confirmed diagnoses of squamous cell carcinoma of the lung at our facility were reviewed. Again, while there is some dissonance in the literature with respect to the definition of the ‘periphery’, a reasonable approach given the existing data is to define the lung periphery as the lateral or outer half of the lung with respect to the position of the lesion on the axial cuts of the computed tomography scans of the chest. Each patient’s lesion was then classified as peripheral or central using that definition. Furthermore, various demographic data points for each patient such as age, race, sex, smoking history, use of inhaled corticosteroids, and concomitant use of proton pump inhibitors were also collected and analyzed. In addition, a cohort of patients without a prior history of malignancy was also analyzed as a “de novo” subset.

Results:
During the evaluation period, a total of fifty-six patients were diagnosed with SqCC. Of these, 55% (31/56) had SqCC located in the lung periphery with the remaining 45% (25/56) being found in a central location. Of the 56 patients diagnosed with SqCC, 27 had a prior history of malignancy. These 27 patients were then removed from the analysis in an effort to assess whether this distribution would persist in the remaining 29 patients. This “de novo” SqCC subset was then analyzed. Of this subset of patients, 62% (18/29) had SqCC that were located in the lung periphery and 38% (11/29) had lesions that were located centrally. Other epidemiological features correlated with typical trends seen in patients with SqCC.

Conclusion:
Our findings appear to confirm our initial observation that, within our institution, there has been a substantive shift in the traditional distribution of Squamous Cell Carcinoma with the majority of these cancers now being diagnosed in the lung periphery as opposed to the more central locations. Further work will be needed to confirm these results.

Background:
Long-time survival is an important goal in NSCLC treatment. However, in patients (pts) with wild type EGFR and non translocated ALK (EGFRwt/ALKnt) advanced disease, median survival at diagnosis is around 9-14 months and long time survivors (LTS) constitute a very small proportion of these patients. The aim of our research was to explore the clinical-pathological characteristics of a population of EGFRwt/ALKnt LTS with an overall survival (OS) of at least 36 months, with the purpose to identify which clinical-pathological features could help to identify a better outcome in advanced NSCLC.

Methods:
We analyzed retrospectively data from patients diagnosed of EGFRwt/ALKnt advanced NSCLC with an OS of at least 36 months and treated in 8 institutions from Madrid (Spain). All these patients were selected in a period of 10 years (January 2002 to 2012). We analyzed clinical-pathological characteristics (age, sex, ECOG, stage IIIB vs IV, histology, smoking status, diabetes and vascular disease, weight loss >10%, symptoms at diagnosis and sites of metastasis), laboratory parameters (LDH and haemoglobin levels) and type of treatment administered (platinum based treatment, metasectomies, number of chemotherapy lines, maintenance, grade 4 toxicity as well as metformine intake). Finally, data from PFS and OS were also collected.

Results:
Among all patients diagnosed with EGFRwt/ALKnt NSCLC and treated in 8 institutions in 10 years, we identified 93 pts with an OS of at least 36 months. 55 pts (60%) were older 65 years, 67 pts (71%) male and 85 pts (91 %) were smokers/former smokers. Comorbidities (diabetes and vascular disease) were infrequent: 7pts (7%) and 13 pts (14%), respectively. Adenocarcinoma was most common pathological subgroup (60 pts, 65%) followed by squamous (21 pts, 22%), large cell carcinoma (7, 7%) and “other histologies” (6 pts, 6%). The majority of them, had a good PS; ECOG 0 32 pts (34%) and 1 57 pts (61%). A minority of patients had weight loss greater than 10% at presentation (12 pts, 14%). Most frequent symptoms were cough (41 pts, 44%), followed by pain (34 pts, 36%), dyspnea (25 pts, 27%) and haemoptysis (9 pts, 9%). LDH and haemoglobin leves were normal in the majority (65 pts, 70% and 72 pts, 77%, respectively). On the other hand, metformin intake was uncommon (16 pts, 17%). One or two metastatic sites at diagnosis were described in 44 pts, 47% and 29 pts, 31%, respectively and only 13 pts (14%) had brain metastasis (mts) and 5 (5%) adrenal mts. First-line chemotherapy based in platinum was administrated in 92 pts (98%), however, maintenance therapy only in 41 pts (44%). Local treatment (metasectomies +/-RT), was done in 35 pts (38 %). Grade 4 toxicity was detected in 7 pts (7%). Finally, we estimated a median PFS of 13.4 months and median OS of 40.5 months

Conclusion:
To our knowledge, this is the largest multicenter serie reported of very long-term survivors (OS >36 months) with with EGFRwt/ALKnt advanced NSCLC. This study includes an exhaustive clinical and pathological analysis of this specific population. In this moment, we are carrying out a comprehensive molecular analysis

Background:
Pulmonary papillary adenoma is a rare benign tumor of the lung, characterized by a histological appearance in which low-grade cuboid or single columnar cells proliferate, centering on the fibrovascular interstitium. Since diagnosis is made morphologically, genetic analysis is not used for its diagnosis in common practice.

Methods:
[Case presentation] A 60-year-old woman with no history of smoking or asbestos exposure was referred to our hospital with lung cancer suspected due to an abnormal shadow detected on a plain chest radiograph for screening. A mass lesion 3.5 cm in diameter was detected in the right middle lobe on chest computed tomography (CT). Although positron emission tomography (PET)/CT revealed fluorodeoxyglucose accumulation in the mass (SUV max 3.4), there was no other clear accumulation and there were no findings indicating lymph node metastasis or distant metastasis on enhanced CT and magnetic resonance imaging (MRI) scans. Therefore, the clinical stage was estimated as cT2aN0M0, stage IB. As a treatment strategy, we decided to use surgical biopsy because a definitive diagnosis could not be made from bronchoscopy. Thoracoscopy showed no apparent pleural dissemination or pleural effusion. Since the tumor in the middle lobe was difficult to resect, we resected the middle lobe and submitted it for intraoperative pathological examination. As a result, it was diagnosed as pulmonary papillary adenoma. Consequently, in surgery, systematic lymph node excision was not performed and samples were obtained from lymph nodes #11s and #11i. The permanent preparation showed the appearance of cuboid and low columnar-shaped cells proliferating on the fibrovascular interstitium, which was consistent with papillary adenoma. However, it also indicated a marginal region of lepidic pattern with focal low papillary projection. In immunohistological examination, both the marginal region and the body of tumor were stained with epidermal growth factor receptor (EGFR) mutation specific antibodies. Furthermore, genetic mutation was found in EGFR genetic analysis. Thus, this patient was diagnosed with invasive adenocarcinoma in papillary pattern predominant. The final pathological stage was estimated as T2aN0M0. However, adjuvant postoperative treatment was not performed. The patient was followed up in the outpatient department, and no recurrence has been observed eight months.

Results:
[Discussion] Pulmonary papillary adenoma is commonly diagnosed based on the histological forms and genetic examination is not usually used. Thus, we cannot deny the possibility that lesions with gene mutations have been present in the patients diagnosed with papillary adenoma in the past. In addition, in patients with a large primary tumor with a morphologically low grade, it is difficult to judge whether postoperative treatment should be provided and it is considered necessary to judge it by taking the clinical course into account.

Conclusion:
Pulmonary papillary adenoma is a rare tumor. To obtain the definite diagnosis with the exclusion of malignancy, it has been suggested that complete resection and genetic analysis is necessary .

Background:
Fetal lung interstitial tumor (FLIT) is a newly recognized and a rare lung lesion of neonates.　The entity was firstly proposed by Dishop MK, et al. in 2010. FLIT is histologically charized by unique mixture of immature interstitial mesenchyme with irregular airspace-like structures, which mimicks abnormal and incompletely developed lung.To date, only 13 cases of FLIT have been reported including 2 cases from Japan. We herein present another case of FLIT in a neonate with histopathological and immunohistochemical analyses to discuss its pathogenesis.

Methods:
not applicable

Results:
A 3 day-old boy was referred to our hospital with respiratory distress. He was born at 36 weeks of gestation, weighing 2070 g. Chest X-ray and computed tomography findings depicted a mediastinal tumor containing viscous fluid. However, the operation revealed that the tumor was developed in the left lung, and the tumor was resected. The postoperative course was uneventful and no recurrent disease was detected at his latest follow-up at 2 years after the resection. The tumor was 30x25x20mm-sized and its cut section showed a well-circumscribed solid and cystic mass. Histological examination revealed a well-circumscribed lesion with a fibrous capsule. The lesion consisted of immature airspace-like structures with widened septa containing immature mesenchymal cells, also, overlaying low cuboidal epithelium was observed. Immunohistochemically, the epithelial cells were positive for cytokeratin, EMA, and TTF-1. The interstitial cells were diffusely positive for vimentin and smooth muscle actin, and negative for desmin. Not nuclear but cytoplasmic staining of beta-catenin was observed in the epithelial cells. The tumor cells were immunohistochemically negative for ALK. The differential diagnoses of the tumor included pleulopulnmonary blastoma (PPB), and congenital cystic adenomatoid malformation/congenital pulmonary airway malformation (CCAM/CPAM) type 3. PPB is characterized by rhabdoid or blastemal cells and/or anaplastic features and CCAM/CPAM type 3 generally shows a diffuse lesion without a capsule.These histological features were not observed in the present tumor, and it was diagnosed as FLIT. Yoshida M, et al. noticed the nuclear stainig of beta-catenin in the epitheliaｌ cells of FLIT. Its nulcear staining is also observed in the epithelai cells in fetal lung at pseudo-glandular stage, and they discussed the resembrance between FLIT and fetal lung at pseudo-glandular stage. In the present case, beta-catenin expression was observed in the cytoplasm of epithelial cells, which rahter resembled to the lung in alveolar stage. In 2014, Onoda T, et al. reported a novel chromosomal rearrangement resulting in α-2-macroglobulin (A2M) and anaplastic lymphoma kinase (ALK) gene fusion in a case of FLIT. However, the present case was negative for ALK by immunohistochemistry. Immunohistochemical findings of beta-catenin and ALK of the present case suggested the pathogenic heterogeneity of FLIT.

Conclusion:
A case of FLIT in a neonate is presented with typical histopathological findings. Pathogenic heterogeneity of FLIT was suggested by beta-catenin and ALK immunohistochemistry of the case.

Background:
The Japan Lung Cancer Society, Japanese Association for Chest Surgery, and Japanese Respiratory Society jointly established the Japanese Joint Committee for Lung Cancer Registration. The Japanese Joint Committee reported that number of resected lung cancer patients under 40 years of age in Japan was 101 cases of 11663 registered patients in 2004. Apparently there are many people on their 50s to 70s who was resected for treatment of lung cancer. Lung cancer in patients under 40 years old is rare. Young lung cancer patients should have specific characteristics.

Methods:
We performed 2835 operations for lung cancer for 15years from 2000 through 2014 in our hospital. Among 2835 patients with lung cancer, 47 patients were younger than 40. Among 47 patients 26 patients were male and 21 patients were female. We examined characteristics of young lung cancer patients by clinicopathologic and molecular biologic characteristics.

Results:
Among patients with operation, pathological stage IA, IB, IIA, IIB, IIIA, IIIB were 24, 6, 3, 2, 6, 5 cases, respectively. 36 cases were diagnosed as adenocarcinoma. Squamous cell carcinoma was only one case. 3 cases were diagnosed as large cell carcinoma. Most of young lung cancer cases were diagnosed as adenocarcinoma. 5-year survival of resected lung cancer patients was 74%. 5-year survival of inoperable cases was 23.8%. We will show the biological characteristics of young age lung cancer patients. 9cases showed EGFR sensitive mutation. 4 cases showed the transforming EML4-ALK fusion gene.

Conclusion:
Young lung cancer patients showed specific clinicopathologic and molecular biologic characteristics compared with the older age patients.

Background:
Malignant melanoma is a refractory malignancy with a dismal prognosis. It generally arises from the skin in most cases, and cases of primary pulmonary malignant melanoma are rare and often behave aggressively. We have treated two cases of localized primary pulmonary malignant melanoma by surgical resection. Although cutaneous melanomas often carry activating mutations in the BRAF gene (V600E) and express programmed death ligand 1 (PD-L1), little is known about primary pulmonary malignant melanoma.

Methods:
We determined the BRAF mutational status (exons 11 and 15) by direct sequencing in the two tumors. Next, we performed a target sequencing analysis using the Human Lung Cancer Panel (Qiagen, Hilden, Germany), which targets 20 lung cancer-related genes including most of the exons in BRAF, using the same samples. We evaluated the expression of PD-L1 on the surface of the tumor cells by immunohistochemical testing in formalin-fixed, paraffin-embedded tumor specimens with the use of a rabbit monoclonal antihuman PD-L1 antibody.

Results:
No BRAF mutations and PD-L1 expression were detected in both of two cases. We detected a p53 mutation, which was thought to be a potential somatic mutation, in one of the two cases using a sequencing panel targeting 20 lung cancer-related genes.

Conclusion:
We encountered two cases of malignant melanoma of the lung that did not carry activating mutations in the BRAF gene. Further molecular analyses may uncover the characteristics of primary malignant melanoma.

Background:
(AS) are rare malignant soft tissue tumors of endothelial cell origin representing 2% of all sarcomas. Most AS develops in the absence of precursor lesions. To date, only 20 cases of (AS) have been described after renal transplantation, occurring mostly on the skin or in a dialysis fistula, their pulmonary location is very rare. We report the case of a patient with a renal transplant who presents a hypo pharyngeal and a right lower lobe (RLL) lesion where a Angiosarcoma was documented.

Methods:
Clinical History Revision

Results:
A 78 years-old patient, ex-smoker, with end-stage renal failure received a cadaveric donor renal transplant in 2000. Immunosuppressed with relatively low doses of tacrolimus and steroids, graft function remained stable with a serum creatinine in 1,2 until January 2015 when he consults with dysphonia, dysphagia, cough, and mild hemoptysis. The patient had bilateral neck lymphadenopathy, bilateral basal crackles and the rest of the physical examination within normal parameters. Pulmonology evaluated the patient finding a hypopharyngeal mass and another with round morphology in RLL that were metabolically active in PET-CT. A hypopharyngeal biopsy is taken and a CT-guided transthoracic puncture, finding a high grade undifferentiated tumor of mesenchymal origin, expressing Vimenin and CD10, with vascular marker CD31 and gene C-Myc. Gene p53 and Ki-67 in 90% of the tumor. No lymphoid or epithelial line markers are expressed. The patient is currently in chemotherapy and immunotherapy.

Conclusion:
The use of potent immunosuppressive agents has significantly reduced the rates of acute rejection after renal transplantation. However, in­creased cancer incidence after renal transplan­tation has become an important problem. Skin tumors, post-transplant lymphoproliferative dis­eases and organ cancers are the most common malignant tumors seen in these patients. Angiosarcoma is rarely seen in this group of patients, and location in lung and hypopharynx without evidence elsewhere of commitment affectation is very rare. Figure 1 Figure 2

Background:
Recently endobronchial ultrasound guided transbronchial needle aspiration system (EBUS-TBNA) was developed in Japan and probed to be an effective method to diagnose peribronchial tree lesions especially for lymphnode metastasis of lung cancer.　In this time, we present our experience of EBUS-TBNA for peritracheal mediastinal tumors and discuss current advances of EBUS-TBNA for lung peri-tracheobronchial lesions.

Methods:
Between 2008 and 2014, EBUS-TBNA was performed in nine patients with peritracheal mediastinal mass to diagnose them morphologically.

Results:
Final histological type of these nine cases were, 6 malignant tumors (two malignant lymphomas, one multiple myeloma, one thyroid carcinoma, one squamous cell carcinoma, one large cell carcinoma) and 3 benign tumors (one neurogenic schwanoma, one bronchogenic cyst and one thymic cyst). Of the detected 6 malignant tumors, cytology diagnoses were positive in three cases, suspicious in two cases and inadequate in one case. Of the detected three benign tumors, cytology diagnoses were benign in two cases and inadequate in one case. Among 7 solid mediastinal tumors, histological evaluation was capable in 3 cases and insufficient in 4 cases. Combined with cytological and histological examination, morphological diagnoses were useful in 6 cases (67%) with EBUS-TBNA for peritracheal mediastinal tumors.

Conclusion:
Although histological type of peri-trachal mediastinal tumor was various, morphological diagnosis with EBUS-TBNA was useful in 67% of the lesions in this series. Analysis of atypical cells in the cytology specimen was effective to estimate the histological figures of the lesions.

Background:
Surgical and pathological handling of lung physically affects lung tissue. This leads to artifacts that alter the morphological appearance of pulmonary parenchyma.

Methods:
In this study four mechanisms of ex-vivo artifacts and corresponding diagnostic pitfalls are described and illustrated.

Results:
The four patterns of artifacts are: 1) Surgical collapse, due to the removal of air and blood from pulmonary resections; 2) Ex-vivo contraction of bronchial and bronchiolar smooth muscle; 3) Clamping edema of open lung biopsies, and 4) Spreading of tissue fragments and individual cells through a knife surface. Morphologic pitfalls include diagnostic patterns of adenocarcinoma, asthma, constrictive bronchiolitis, and lymphedema.

Conclusion:
Four patterns of pulmonary ex-vivo artifacts are important to recognize, in order to avoid morphologic misinterpretations, possibly improving reproducibility in histopathological diagnosis of lung cancer

Background:
Lung cancer is the deadliest cancer worldwide and it is of particular concern in Brazil as the second cause of cancer death in both genders. The new classification proposed by International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society Classification has proven its prognostic value with also a better clinical understanding of lung adenocarcinoma. Prior to this classification all patients diagnosed with early stage adenocarcinoma were considered to have practically the same disease. In our country, however, medical literature is still incipient on this topic. We reviewed the histopathology of a consecutive series of patients diagnosed with stage I adenocarcinoma.

Methods:
Cross-sectional study including 50 patients diagnosed with stages IA or IB adenocarcinoma undergoing surgical resection of non-small cell lung cancer. The variables: histological subtype, sex, age and tumour size. We have divided patients in two groups based on the presence of lepidic features. Statistical analysis was performed by Anova and Bonferroni multiple comparison test, to correlate tumour size among histological subtype groups.

Results:
The mean age was 63.7 (11.5) years, 50% were men. The average size of resected tumours was 1.4 (0.7) mm; 45 cases (90%) were stage IA. The predominant subtypes were histologically lepidic (n=24, 48%); the acinar subtype was found in 20 (40%) cases. Patients and tumour characteristics according to histological subtype are showed in table 1. There was statistical difference in size (p<0.05) when comparing lepidic tumors with acinar tumors (1.3mm versus 2.3mm respectively). Table 1: Patients and tumour characteristics according to histological subtype.

Subtypes Variables	Lepidid	Acinar	Others	p
n (%)	24 (48)	20 (40)	6 (12)
Men n (%)	11 (45.8)	9 (45)	6 (100)
Age mean (SD)	63 (11.6)	66.3 (10.8)	62.7 (7.7)
Tumor size mean (SD)	1.3 (0.6)	2.3 (1.4)	1.6 (0.6)	0.02*

*Anova with Bonferroni multiple comparison test.

Conclusion:
In our brief series of lung adenocarcinoma patients, the most common subtypes were acinar and lepidic; the latter being larger in this sample. The long-term follow-up will give us important information about the prognosis of these patients treated exclusively with surgery.

Background:
Synchronous lung cancers are simultaneously diagnosed, physically distinct and separate lung cancers which have no common lymphatics with the primary tumor and may have same or different histology with the primary neoplasms. Although radiological imaging techniques guide in terms of initial diagnosis, histopathological evidence is required for definitive diagnosis of synchronous multiplee primary lung cancers. Early diagnosis represents the only chance to obtain a surgical cure in these patients.

Methods:
not applicable

Results:
Here, we present a case with synchronous multiplee primary lung cancers in whom both tumors are diagnosed simultaneously. A 69 year-old male patient with cough and left-sided chest pain complaints and 90 pack / year history of active smoking admitted to our clinic. Thoracic CT of the patient revealed a pleural-based mass in the right lower lobe and another mass on the left lung which is associated with the hilum and caused atelectasis in the distal airways. Diagnostic bronchoscopy was performed to the patient and separate biopsies were taken from the both lesions. Histological sections obtained from the bronchoscopic biopsy specimens revealed that there was an infiltrative tumor in both right and left lung. In right lung, the tumor composed of abortive glandular structures and single cell infiltrations within the desmoplastic stroma. The second tumor (left lung) was consist of solid islands composed of atypical squamous cells with eosinophilic cytoplasm and darkly basophilic nuclei. Histochemically, in the first tumor, neoplastic cells had intracytoplasmic vacuoles stained by mucicarmin indicating a feature of adenocarcinoma whereas there were no cells containing mucin vacuoles in the second tumor. Immunohistochemical study has supported the histological and histochemical findings. The tumor on the right side showed a diffuse immunoreactivity by CK7 which is a highly spesific marker for adenocarcinomas whereas the tumor on the left side was stained by the basal cell markers such as CK5/6 and p63 which are highly specific markers for squamous cell carcinoma. Briefly, histopathologic examination of the biopsies from left upper lobe and right lower lobe revealed squamous cell lung carcinoma and adenocarcinoma, respectively. Thereupon oncologic PET examination was performed for screening and evaluating if there is another primary tumor site for adenocarcinoma. In PET examination, FDG uptakes of extrapulmonary tissues were considered to be normal. Thus both lesions thought to be primary lung tumors.

Conclusion:
Our case is a good example of simultaneously detected synchronous primary tumors of the lung and we reported this case in order to emphasize the possibility of another primary tumor in the cases which are initially thought to be metastatic lesions and for sure the need of biopsies separately.

Background:
Malignant pleural mesothelioma is a difficult diagnosis, more severe cases may not perform by pleural biopsy. With these cases, the technique of immunocytochemistry was performed proposals.

Methods:
A prospective, cross-sectional descriptive statistics with clinical series cases.

Results:
§ Implementing 58 cases of malignant pleural mesothelioma by immunocytochemistry. § Technical Cytospin method has helped for immunocytochemistry have high results. § Should be done more staining with markers for the other diagnostic diseases: lung cancer and non-lung cancers that metastatic to pleura. § The results of the data : - Sensitivity: 84.06 % - Specificity : 100 % - Positive Prognostic Value : 100 % - Negative Prognostic Value : 92.25 % - Accuracy : 94.12 %

Conclusion:
The diagnosis of malignant pleural mesothelioma is always a difficult diagnosis. Needing the differential diagnosis with other types of diseases, such as lung cancer and non-lung cancers that to pleural pulmonary metastases. Staining with immunocytochemistry in cases of pleural effusion showed relatively high value. This suggests that the ability to diagnose initially screening properties of this technique is very good, especially valuable in the diagnosis of severe disease progression can not perform other diagnostic techniques.

Background:
The combined effect of improved cancer diagnosis and management has led to a marked increase in cancer survivors. Consequently, an appreciable proportion of cancer diagnoses are registered among patients who had already received a cancer diagnosis in the past, and more accurate diagnostic procedures lead to the identification of more than one cancer in a subset of patients. We present the case of a patient with breast cancer in whom a lepidic primary lung adenocarcinoma was discovered during a follow-up.

Methods:
Medical History Revission

Results:
A 68-years-old female with breast cancer EC EIII diagnosed in March / 2010, handled with lumpectomy and lymph node removal, solid mucinous ductal carcinoma Ki67 expression: 35% RH: E (+) 100%, P (-), HER 2 NEU negative, negative margins with angiolymphatic invasion 3/15. She received radiotherapy, tamoxifen and later anastrozole. In Dec / 2010 she presents tumor recurrence managed with radical mastectomy, received chemotherapy with Adriamycin and Cyclophosphamide. In April / 2013 she consults with two months of dry cough and dyspnea, normal physical examination, an unremarkable mammography, chest CT-scan with irregular right basal nodular lesion and mediastinal nodes. The patient underwent resection through thoracoscopy, pathology shows lepidic adenocarcinoma pattern with mutated EGFR exon 19 and exon 21 negative, with metastatic nodal involvement by the primary lung tumor and previous breast carcinoma.

Conclusion:
Patients that have been diagnosed with a cancer, have an increased lifetime risk for developing another de novo malignancy depending on various inherited, environmental and iatrogenic risk factors. Cancer patients could survive longer due to settling treatment modalities, and then would likely develop a new malignancy. The monitoring and evaluation procedures are especially useful for early detection of tumors associated when there’s a known tumor lesion. Figure 1Figure 2

Background:
IGNITE (a large, multicentre, interventional, non-comparative diagnostic study; NCT01788163) evaluated local diagnostic practices for patients with advanced non-small-cell lung cancer (aNSCLC) in Asia-Pacific/Russia.

Methods:
Eligible patients: local/metastatic aNSCLC; chemotherapy-naïve, newly diagnosed/recurrent disease after resection; ineligible for curative treatment. We report diagnostic assessments and epidermal growth factor receptor (EGFR) mutation test turnaround times (secondary endpoints) associated with tissue/cytology samples from patients in Asia-Pacific/Russia.

Results:
3382 patients enrolled (972 Russia). Immunohistochemistry (IHC) analysis was used to confirm diagnosis in 989/2093 (47%) and 165/949 (17%) patients in Asia-Pacific and Russia, respectively (where data were available). Where IHC was used, the markers assessed were: TTF-1 (Asia-Pacific 95% and Russia 90%); p65 (3% and 5%); and p40 (17% and 4%). EGFR mutation tests were not performed on samples from 262 patients and tested samples from 23 patients did not yield results. The most common reason for not testing was insufficient material provided to test (Asia-Pacific 93% [100/108 responses], Russia 67% [24/36]). The percentages of neoplastic cells in samples (data available: Asia-Pacific n=1042; Russia n=187) were: <20% tumour cells: Asia-Pacific 33% vs Russia 6%; 20–50% tumour cells: 28% vs 33%; and >50% tumour cells: 40% vs 61%. Considering sampling methodologies (data available: Asia-Pacific n=2410; Russia n=972), the most common sampling sites were the lungs (Asia-Pacific 68%; Russia 80%) or lymph nodes (Asia-Pacific 14%; Russia 10%); the most common sample collection method was bronchoscopy (Asia-Pacific 22%; Russia 45%; Table 1). Median EGFR mutation test turnaround time was within 2 weeks for all countries except Thailand (70 days; Table 2). Mutation test success rates were high for Asia-Pacific (99.5%) and Russia (98.7%).

Conclusion:
Diagnostic assessments, sampling methodologies and EGFR mutation testing practices vary between and within Asia-Pacific and Russia; further understanding of local practices will drive improvements and enable more patients to receive appropriate personalised treatment. Figure 1 Figure 2

Background:
Treatment and diagnosis of lung cancer is an interdisciplinary challenge. New treatment options can benefit from refined information on biological processes in the tumor. Two diagnostic disciplines, pathology and radiology, can provide oncology with valuable information allowing to select of the most appropriate treatment. Linking radiology and pathology on the imaging level requires sophisticated software. Therefore, we developed a computer tool combining quantitative functional MRI and CT with state-of-the-art whole-slide and 3D reconstructed histology.

Methods:
We selected a model lung cancer patient to investigate the requirements and possibilities for a combined software. The patient had a squamous cell carcinoma. Prior to excision, functional imaging using MRI as well as highly resolved CT and MRI volumes were acquired. These included a dynamic contrast-enhanced (DCE)- and a diffusion-weighted imaging (DWI)- MR scan. After imaging, the tumor was resected and a macroscopic slice of 5 mm thickness was resected from the center region of the tumor. This slice was further divided into 11 blocks, which were then cut into microscopic (~ 2 μm) slices. Staining was applied to these slices according to the requirements of the pathologist and then scanned by a whole-slide histological scanner (Hamamatsu, Japan). The obtained images of the microscopic slices were automatically reconstructed into histological 3D volumes by means of registration. Functional MRI and morphological CT data was imported into the software prototype. The tumor volume was determined in the CT image by a two-click automatic segmentation method. Quantitative parametric maps of the extended general kinetic model (eGKM) for vascular information and the apparent diffusion coefficient (ADC) for cell density were calculated. Afterwards, the histological blocks were aligned such that the blocks were located correctly on a photo of the macroscopic slice. They were then manually aligned to the morphologic contrast enhanced T1 image showing the best contrast for structures visible in the macroscopic slice. Finally, the pathological data were imported into the software for direct comparison of pathology and functional imaging in human lung tumors. Figure 1



Results:
We were able to combine histological and radiological information into a software solution which provids an integrated and improved research opportunity for pathologists, radiologists and biologists. It allows correlation of findings on molecular and cell level with findings from in-vivo functional imaging.

Conclusion:
We demonstrated that a combined evaluation of functional MRI and pathology can be facilitated. New studies will show the usage in more common lung cancers and larger numbers of patients.

Background:
Mitofusin-2 gene (MFN2) encodes a mitochondrial protein which is critical for mitochondrial fusion process. MFN2 was initially identified as a hypertension-associated gene and implicated in the pathogenesis of multiple cancer types. However, roles and underlying mechanisms of MFN2 in lung adenocarcinoma development remain to be determined.

Methods:
MFN2 expression at protein level was examined in 30 pair lung adenocarcinoma/adjacent normal lung samples with immunohistochemistry staining. Then MFN2 knockdown was performed in human lung adenocarcinoma cells A549 with lentiviral-mediated shRNA strategy. The effects of MFN2 knockdown on cell proliferation, cell cycle process, cell migration and invasion was investigated in A549 cells. Then MFN2-knockdown induced gene expression changes in A549 cells was analyzed by microarray assay and then functional pathway enrichment analysis was performed to identify critical pathways involved in MFN2-mediated lung adenocarcinoma development. The expression changes of downstream factors were further determined in A549 cells by western blot.

Results:
As compared to adjacent normal lung tissues, MFN2 expression was significantly higher in lung adenocarcinoma tissues with positive MFN2 signals in 90% (27/30) lung adenocarcinoma tissues and only in 26.7% (8/30) adjacent normal tissues (Fig. 1A, B). Furthermore, MFN2 knockdown inhibited cell proliferation (Fig. 1C), induced cell cycle arrest (Fig. 1E) and blocked invasion behavior (Fig. 1D) in A549 lung adenocarcinoma cells. Microarray analysis revealed that a lot of genes were deregulated in A549 cells with MFN2 knockdown (Fig. 1F). Then functional pathway enrichment revealed six pathways were enriched in deregulated genes including Cell cycle, DNA replication, ECM-receptor interaction, Focal adhesion, MAPK signaling pathway and Chemokine signaling pathway (Fig. 1G). Furthermore, the downregulation of RAP1A and upregulation of RALB and ITGA2 identified in MFN2-knockdown cells by microarray analysis were confirmed by western blot (Fig. 1H).Figure 1



Conclusion:
Our results confirmed the involvement of MFN2 in the pathogenesis of lung adenocarcinoma and revealed that MFN2 was critical for cell proliferation and invasion in lung adenocarcinoma cell line A549. Furthermore, microarray analysis identified multiple pathways deregulated in MFN2-knockdown cells, providing valuable insights about the mechanisms underlying MFN2-associated lung adenocarcinoma development.

Background:
Cyclin-dependent kinase 4 (CDK4)/RB and mouse double minute 2 (MDM2)/p53 are the two main regulators of the tumor-suppressor pathways that control cellular responses to potentially oncogenic stimuli. CDK4 inhibits RB by triggering its phosphorylation, leading to releasing the G1-S restriction point. MDM2 inhibits the transcription activity of p53 by blocking the transfer of p53 from cytoplasm to nucleus and by accelerating ubiquitination of p53. Our preliminary SNP microarray analysis using lung specimens from non-invasive tumor (adenocarcinoma in situ) and invasive tumor (lepidic predominant adenocarcinoma) showed the amplification of chromosome 12q13–15, including CDK4 and MDM2 gene regions. The aim of the present study was to determine the mechanistic implications of CDK4 and MDM2 in lung adenocarcinoma migration and invasion.

Methods:
Using siRNAs specific for CDK4 and MDM2, the expressions of CDK4 and MDM2 were knocked down in the human non-small cell lung cancer cell lines A549, H460, H1299, SK-Lu-1 and H23, which harbor wild-type RB yet contain other aberrations in p53 (wild-type in A549, H460, absent in H1299, and mutated in SK-Lu-1, H23). Cell proliferation (AlamarBlue staining), mobility (scratch assay), and invasion (transwell-matrigel chamber system) were investigated.

Results:
The knockdown of CDK4 (5.5, 18.5, 2.2, 22.8 and 8.3% compared to scrambled siRNA in A549, H460, H1299, SK-Lu-1 and H23, respectively) significantly inhibited cell proliferation in H23 and SK-Lu-1, and decreased cell migration in SK-Lu-1 and H460. It also repressed cell invasion in H460, SK-Lu-1 and A549. The decreased expression of MDM2 (43.4, 69.6, 6.4, 27.3, 8.7% compared to scrambled siRNA in A549, H460, H1299, SK-Lu-1 and H23, respectively) dramatically inhibited cell proliferation in H1299, SK-Lu-1 and H23, and diminished cell migration in H23, A549 and SK-Lu-1. It also hindered cell invasion in H460 and H23.

Conclusion:
These findings suggest CDK4 and/or MDM2 pathways may play critical roles in cell proliferation, mobility and invasion, and furthermore, the targeting CDK4 and/or MDM2 may provide therapeutic benefit to lung cancer patients.

Background:
Tumors exhibiting extensive hypoxia have been shown to be more aggressive than corresponding tumors that are better oxygenized, which suggests that hypoxic condition induces stem-like properties. The purpose of the present study was to investigate whether hypoxic stress induces acquisition of stem-like properties, and the mechanism is involved with DNA demethylation in lung cancer.

Methods:
Normal epithelial cell line (BEAS-2B) and human lung cancer cell lines (A549, H292, H226 and H460) were incubated either in normoxic or in hypoxic (below 1% O~2~) condition. The cell lines were treated with a DNA methyltransferase inhibitor (5-azacytidine, AZA) to determine whether the expression of stem cell markers (CD44, CD133, CXCR4, ABCG2, CD117, ALDH1A1, EpCAM, CD90, Oct4, Nanog, SOX2, SSEA4 and CD166) was reactivated. Methylation-specific PCR and bisulfite sequencing were used to analyze the methylation status, and real-time RT-PCR and western blotting were performed to analyze the expression of the stem cell markers. Cell migration and Matrigel invasion assay were performed for functional analysis

Results:
Among the 13 stem cell markers, CXCR4, Oct4 and Nanog were increased at least one lung cancer cell line in hypoxic condition compared with in normoxic condition. These three stem cell markers were reactivated by treatment with AZA. Methylation-specific PCR showed decreased promoter methylation of these three stem cell markers in hypoxic condition compared with in normoxic condition, which was further validated by bisulfite sequencing. Migration and invasion were increase in hypoxic condition compared with in normoxic condition

Conclusion:
These results suggest that under the hypoxic condition, reactivation of stem-like properties was related with promoter demethylation of stem cell markers. Further studies are needed to assess its value as a prognostic factor and potential therapeutic applications.

Background:
Our previous studies demonstrated that one third of lung cancers of chromate-exposed workers (chromate LC) showed high-degree microsatellite instability (MSI), most of which repressed DNA mismatch repair (MMR) hMLH1protein. MMR-deficient tumors are characterized by widespread changes in the number of microsatellites accumulating in both coding and non-coding sequences of many human genes. The MRE11–RAD50–NBS1 complex is essential for DNA double-strand break (DSB) repair performing by homologous recombination (HR) or non-homologous end joining (NHEJ). In gastric and colorectal cancers with MSI, the mono- or biallelic deletions in the poly(T)11 within MRE11 intron 4 and the frameshift mutations in the (A)9 repeat in RAD50 exon 13 were detected and the significant reduction of both proteins were identified.

Methods:
We used formalin-fixed paraffin-embedded materials from 36 chromate LC (28 cases; all male, mean age 56.8, squamous cell ca (SQ) 35, stage I 27, BI=488, mean chromate exposure 24 years) and 28 non-chromate LC (all male, mean age 61, SQ 28, stage I 8, BI=674). DNA was extracted and amplified using nested-PCR. The fragment analysis was performed using a capillary electrophoresis and GeneScan Analysis software (Applied Biosystem, USA).

Results:
In the poly(T)11 within MRE11 intron 4, 8 (29%) of 28 non-chromate LC showed 1bp deletion or insertion and 14 (52%) of 33 chromate LC showed 1bp deletion or insertion. In the (A)9 repeat in RAD50 exon 13, none of 27 non-chromate LC showed deletion or insertion and 4 (16%) of 25 chromate LC showed deletion. 67% of chromate LC with more than 3 MSI had abnormality of MRE11 gene, and 60% of chromate LC with 2 MSI had it. While, 27% of chromate LC with less than one MSI and 28% of non-chromate LC had it. 17% of chromate LC with more than 3 MSI had abnormality of MRE11 gene, and 10% of chromate LC with 2 MSI had it. While, 25% of chromate LC with less than one MSI had it and none of non-chromate LC had it.

Conclusion:
Half of chromate LC had the abnormality of the poly(T)11 within MRE11, which was associated with the degree of MSI. Sixteen percentage of chromate LC had the abnormality of the (A)9 repeat in RAD50. The carcinogenesis of chromate LC may be involved in the abnormality of DNA repair MRE11–RAD50–NBS1 complex.

Background:
The immune microenvironment of primary tumors has been reported to be a prognostic factor. We previously reported that the tumor-infiltrating regulatory T sell (Treg) count was positively correlted with the intratumoral cyclooxygenase-2 (COX-2) expession level and was associated with a poor survival among patients with non-small cell lung cancer (NSCLC). Recently, numerous single nucleotide polymorphisms (SNPs) in the COX-2 gene have been identified, and those SNPs may contribute to differential gene expression and enzyme activity levels. However, whether COX-2 genetic variants influence the functions of COX-2 in NSCLC remains unclear.

Methods:
Eighty NSCLC patients who underwent a complete recection at our institute ware enrolled. We extracted DNA from the peripheral blood and identified five different COX-2 SNPs. The correlations between the COX-2 SNPs and the expression levels of COX-2, Tregs and Ki-67 were studied. The prognostic significance of the COX-2 SNPs was also evaluated.

Results:
COX-2 SNPs were not correlated with the expression of COX-2. However, for the COX-2 -1195G/A polymorphism, the AA genotype group had a significantly higher Treg score. Furthermore, the AA group had a significantly higher Treg score regardless of the COX-2 expression level. The COX-2 -1195AA genotype group tended to have a shorter disease-free survival period than the GA/GG group.

Conclusion:
In conclusion, the COX-2 -1195G/A polymorphism influences the infiltration of Tregs into NSCLC, and the COX-2 SNP factor may be a prognostic factor reflecting Treg infiltration in NSCLC.

Background:
There are a number of genetic abnormalities that can occur in lung cancer. We, and others, have shown that receptor tyrosine kinases (RTKs), such as EGFR, c-MET, RON, and Eph, frequently harbor gain-of-function mutations in addition to being overexpressed in lung cancer. We also have shown that the soil nematode C. elegans overexpressing a c-Met mutant revealed an abnormal vulval phenotype with hyperplasia. Interestingly, exposure to nicotine significantly aggravated the phenotype suggesting that C. elegans can be used as an in vivo model for rapid screening of RTK mutants as well as carcinogens.

Methods:
C. elegans strains vab-1 (Eph receptor), RB2088 (MET receptor), SD551 (temperature sensitive strain expressing constitutively active form of KRAS), and three sli-1 mutants PS2728, PS1258, and MT13032 (inactive, c-CBL) were compared to wild-type N2 worms for survival, fertility, egg-laying capacity, locomotion, and phenotypic changes in the absence or presence of nicotine. Gene expression analysis was also performed on each of the strains in the absence or presence of nicotine.

Results:
Nicotine treatment reduced the lifespan of worms for all strains (p=.0034). Nicotine treatment adversely affected egg-laying capacity of all strains, including N2 control, reducing egg production by 13% at 50μM nicotine and 31% at 500 μM nicotine. Furthermore, the fertility (the number of eggs laid/worm) was significantly reduced in SD551 mutant worms compared to N2 worms (p=.003). Overall locomotion velocity did not change with increasing concentration of nicotine except in MT13032, a c-Cbl mutant. Heat map analysis of gene expression profiling data clearly revealed that the various kinases and phosphatases in C. elegans that are marginally expressed in N2 worms were significantly enhanced upon chronic nicotine exposure. The expression of these genes was already elevated in SD551 and that was further increased in response to nicotine.

Conclusion:
Taken together, chronic nicotine exposure adversely affects various biological functions of C. elegans and these effects are exaggerated in the mutants. Interestingly, nicotine treatment also upregulates the expression of various kinases and phosphatases thereby strengthening our contention that the initial screening studies for the oncogenic mutants detected in humans can be rapidly carried out in C. elegans.

Background:
TGF-β promotes tumor invasion and metastasis by inducing an epithelial-mesenchymal transition (EMT). EMT is often associated with acquisition of stem-like characteristics. In the present study, we investigated whether EMT induced by TGF-β could acquire stem-like characteristics in lung cancer.

Methods:
Normal epithelial cell line (BEAS-2B) and lung cancer cell lines (A549, H292, H226 and H460) were used in the study. These cell lines were incubated with 10 ng/ml of TGF-β for 3 days. Western blot was performed to analyze the expression of epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin, vimentin, fibronectin and alpha-smooth muscle actin). Real-time RT-PCR and western blot were performed to analyze the expression of stem cell markers (CD44, CD133, CXCR4, ABCG2, CD117, ALDH1A1, EpCAM, CD90, Oct4, Nanog, SOX2, SSEA4, and CD166). Wound-healing assay, Matrigel invasion assay and sphere formation assay were used to assess functional characteristics of EMT and stemness acquisition.

Results:
TGF-β induced EMT and stem cell markers with variable degrees according to lung cancer cell lines. Most of the stem cell markers were increased by treatment with TGF-β except H460 cell line. Increased expression of mesenchymal markers was associated with the acquisition of stem cell makers. Migration, invasion and sphere formation were increased according to the expression of stem cell markers.

Conclusion:
TGF-β induced EMT was associated with acquisition of stem-like characteristics which was different according to lung cancer cell lines. Further studies are needed to investigate the signal mechanism of EMT and stemness acquisition.

Background:
Lung cancer is a worldwide problem andthe leading cause of death among all malignancies. Despite tremendous progresses in diagnosis and treatment, the overall treatment outcomes for lung cancer patients remain poor, and metastatic lung cancer is responsible for more than ninety percent of lung cancer related deaths. However, the details for lung cancer invasion and thereafter metastasis remain unclear. In this study, we characterized the biological features of invasive human lung cancer cells, and investigated the potential therapeutic effects of Suberoylanilide Hydroxamic Acid (SAHA) on invasive cancer cell subpopulation.

Methods:
Boyden-type cell invasion chambers were used for isolation of cancer cell subpopulations with high invasiveness (H-INV) and low invasiveness (L-INV) from human lung cancer H460 cells. The potential enrichment of stem cell-like cancer cells in H-INV cells and the resistances of H-INV cells to chemotherapy and radiation treatment were investigated. We also tested the effects of SAHA on the differentiation of cancer stem cell and its consequences on cancer cell invasion and the sensitivities to radio/chemotherapies in H-INV cells. Furthermore, microarray for message RNA was performed for identification of gene expression profiling for invasive cancer cells.

Results:
Comparing to L-INV cells, H-INV cells are with enrichments of stem cell-like cancer cells, with increased positive staining of putative stem cell markers such as CD24[low]/CD44[+] and OCT3/4, and more tumorigenic. H-INV cells are also more resistant to treatments of chemotherapeutic agents and ionizing radiation. Treatment with SAHA can induce differentiation of stem cell-like cell in H-INV cells, causing reduced cancer cell invasion and increased sensitivity to chemo/radiotherapy in cells. With mRNA microarray assay, we identified 453 genes differentially expressed in H-INV versus L-INV, and five of these genes have been further tested for their significances in paired primary and metastatic lung tumors.

Conclusion:
Our study suggested putative roles of cancer stem cell in lung cancer invasion and migration. Study also showed that invasive lung cancer cells are resistant to most of first-line and second-line chemotherapeutic agents and radiotherapy, indicating novel therapeutic strategies are needed for the treatment of metastatic lung cancer. Of this setting, SAHA may serve as a chemotherapeutic agent for benefiting lung cancer patients. The candidate genes identified in this study may also have clinic impact as potential metastatic predictors for diagnosis and prognosis for human lung cancer.

Background:
Mitochondrial dysfunction is observed not only in lung cancer cel, but can develop in peripheral tissues. Peripheral blood mononuclear cells (PBMC) represent an available population of cells that can be used for the studies on remote effects of lung cancer. NADH dehydrogenase (ubiquinone) Fe-S protein 1 (Ndufs1) is located at the inner mitochondrial and transfers electrons from NADH to the respiratory system. Mitochondrially encoded cytochrome c oxidase I (MT-CO1) is involved in the coupling between O2 reduction and proton pumping. The changes in both key components of respiratory system reflect mitochodrial status. On the other hand the impairment of mitochondrial function may be crucial among pathomechanisms leading to neurological deficits. The aim of the study was evaluate mitochondrial status in PBMC in relation to the cognition in lung cancer patients.

Methods:
The study included 80 (24 females and 56 males, aged 61.5±6.7 years) consecutive lung cancer patients (5 small-cell lung cancer, 75 non-small cell lung cancer) hospitalized in Clinical Oncology with The Sub-department of Diurnal Chemotherapy Wielkopolska Center of Pulmonology and Thoracosurgery of Eugenia and Janusz Zeyland and Chair and Clinic of Oncology. PBMC were isolated by density gradient centrifugation. The expression Ndufs1 a marker of mitochondrial complex I and MTCO1 a marker of complex IV in PBMC was evaluated by means of ELISA and expressed in pg per mg of protein. Neurological examination, MiniMental State Examination (MMSE), Trail Making Test (TMT) A and B, and Hamilton scale were performed at baseline (time of lung cancer diagnosis) and after 6 months.. Patients serum was tested for the presence of onconeuronal antibodies with indirect immunofluorescence as a screening and Line blot as confirmation test.

Results:
Ndufs 1 expression in PBMC was lower in patient with peripheral nervous system involvement (0.00; 0.0-3.6218 ; median; minimum-maximum) than in subjects without neurological deficit (0.0; 0.0-8.61; median; minimum-maximum; P= 0,024). Up-regulated expression of Ndufs 1 in PBMC is associated with worse TMT- A (13.61±3.13s) than in patients with down-regulated complex I marker (8.60±4.51s; P=0.003). Similarly TMT- B results were worse in patients with higher Ndufs 1 expression (162.48±46.40s) than in cases with inhibited Ndufs1 (124.78±51.77s; P<0,05). Ndufs1 expression correlated negatively with MMSE 6 months after lung cancer diagnosis (Kendall tau =-0.310; P=0.0236), and positively with Hamilton scale score after 6 months (Kendall tau=0.288; P=0.0428), TMT-A (Kendall tau=0.301; P=0.0001) and TMT-B (Kendall tau=0.199; P=0.0120) at baseline. Up-regulated expression of MTCO1 was associated with worse TMT-A results (11.05±5.81s) compared to down-regulated marker of mitochondrial complex IV (8.52±4.14s; P=0.048). MTCO1 expression correlated positively with TMT-A results (Kendall tau=0.167; P=0.0344) at baseline. In 12 patients onconeuronal antibodies were identified (Ma/Ta, Yo, Ri). No differences in Ndufs1 and MTCO1 expression were found between patients with onconeronal antibodies and seronegative subjects.

Conclusion:
Up-regulation of mitochondrial complex I and IV in PBMC of lung cancer patients is associated with cognitive decline. Stimulation of mitochondria status in PBMC may indicate cytotoxic response leading to cognitive impairement.

Background:
Cancer-associated fibroblasts (CAFs) communicate with cancer cells and play important roles in cancer invasion. We previously reported that local invasion of cancer cells was frequently observed in lung adenocarcinoma patients with podoplanin(PDPN)-expressing CAFs. However, the underlying mechanisms of this phenomenon have remained unclear.

Methods:
We established a novel collagen invasion assay model in which cancer cells and CAFs were co-cultured; we analyzed the mechanisms governing how cancer cell invasion was promoted by PDPN(+)CAFs.

Results:
By observing the dynamic movement of both CAFs and cancer cells in the collagen matrix, we found that PDPN(+)CAFs invaded the matrix to a greater extent, with more cancer cells invading within the “tracks” created by the CAFs, compared with control CAFs. The knockdown of PDPN in CAFs decreased the invasion of both the CAFs and the cancer cells. PDPN(+)CAFs displayed a higher RhoA activity, and treatment with a ROCK inhibitor cancelled the increased invasion ability of PDPN(+)CAFs and subsequently decreased the number of invaded cancer cells. After intravenous injection in the mouse tail vein, PDPN(+)CAFs invaded and promoted cancer cell invasion into the lung parenchyma, compared with control CAFs. Among the patients with lung adenocarcinoma, we observed some cases with PDPN(+)CAFs at the invasive front of the tumor. These cases predominantly exhibited pleural invasion of cancer cells, known as pathological invasiveness.

Conclusion:
Our results indicated that PDPN(+)CAFs were tumor-promoting CAFs that lead and enhance the local invasion of cancer cells, suggesting that the invasion activity of CAFs themselves could be rate-determining for cancer cell invasion. For analysis of fibroblast-dependent cancer cell invasion, we established single-cell derived clones from primarily cultured CAFs and conduct further investigation.

Background:
Lung cancer remains the leading cause of cancer-related deaths worldwide. While significant knowledge has been gained regarding the characterization of mutational drivers in NSCLC, much less is known regarding interactions between tumor cells and the surrounding microenvironment that are critical for tumor progression. Additionally, a significant limitation in current understanding is the lack of knowledge regarding which tumor gene products are necessary for promoting cell survival in the context of the tumor microenvironment. We hypothesize that the use of pooled shRNA synthetic lethal screens within an in vivo murine model will allow for the elucidation of targetable microenvironment-dependent genes.

Methods:
We generated a custom murine shRNA lentiviral library targeting 250 genes implicated in the communication between cancer cells and the microenvironment, which was used to transduce two murine cell lines: Lewis Lung carcinoma (LLC) and CMT167 cells. Following puromycin selection of cells harboring incorporated shRNA’s of interest, populations were expanded and designated for in vitro versus in vivo replication and growth. Selected cells were allocated to either in vitro passage vs direct in vivo injection into the lungs of 18 week-old syngeneic C57BL6 mice. After 4 weeks, cells were harvested and gDNA was isolated. Sequencing and quantitation of shRNA was performed using an Illumina deep-sequencing platform. Both raw and normalized read counts were assessed and analyzed to determine the relative representation of a particular shRNA within an in vitro or in vivo sample. Following quality control assessments which demonstrated adequate read count numbers per sample, and appropriate correlation of sample similarity per groups, direct comparisons between in vitro and in vivo samples were performed.

Results:
Multiple gene candidates were identified and largely reproducible via either rank analysis, mean, or t-test analyses. Candidate genes included multiple chemokines, and their receptors, matrix proteases, complement factors, and growth factor receptors.

Conclusion:
These results suggest a list of genes that are both intriguing and diverse, pointing toward gene products that would not have been previously predicted to influence cancer cell survival and growth through a lung cancer cell-autonomous fashion. Furthermore, these genes appear to potentially interact with multiple compartments of the tumor microenvironment including the extracellular matrix, cytokine milieu, vascular structures (complement factors), and the adaptive immune system. Validation of specific gene targets are ongoing through assessment of tumor growth comparing murine cell lines transfected with individual shRNA’s of interest vs control tumor cells. Furthermore, parallel pooled shRNA synthetic lethal screens within selectively adaptive immune-deficient models are currently in progress.

Background:
Wnt signaling is invovled in driving cancer stem cells (CSCs) activity in a variety of cancers. The aim of this study was to explore the role of Wnt signaling in the lung cancer stem cells (LCSCs).

Methods:
Sphere culture was processed by treating human lung adenocarcinoma cell line SPC-A1 with IGF, EGF and FGF-10 to obtain the LCSCs. After confirming the stemness by immunoflurescence, functional genome screening and RT-PCR were employed to perform pathway analysis. The relationship between the identified signaling pathway and stemness gene expression was explored by agnoist/antagonist assay. Moreover, the effects on sphere formation, cell viability and colony formation by different signaling molecule inhibitors were also analyzed.

Results:
The results showed that LCSCs were successfully generated and the phenotype characterization was confirmed by expressing pluripotent stem cell markers Nanog and Oct 4, and lung distal epithelial markers CCSP and SP-C. The involvement of Wnt pathway in LCSCs was confirmed by functional genome screening and verified by RT-PCR. The expression of Wnt signaling components were related with the expression of the Nanog and Oct 4. Anti-cancer effects could be exerted by using different signaling molecule inhibitors targeting Wnt signaling pathway.

Conclusion:
In conclusion, Wnt signaling pathway is involved in the stemness regulation of LCSCs and could be considered as a potential therapeutic target in the lung adenocarcinoma.

Background:
Alterations of the EGFR/ERK and Hippo/YAP pathway have been found in non-small cell lung cancer (NSCLC).

Methods:
Luciferase reporter and downstream gene expression assays were used to test hippo pathway activity.

Results:
Herein, we show that ERK1 and ERK2 have an effect on the Hippo/YAP pathway in human NSCLC cells. Firstly, inhibition of ERK1/2 by siRNA or small-molecular inhibitors decreased the YAP protein level, the reporter activity of the Hippo pathway, and the mRNA levels of the Hippo downstream genes, CTGF, Gli2, and BIRC5. Secondly, degradation of YAP protein was accelerated after ERK1/2 depletion in NSCLC cell lines, in which YAP mRNA level was not decreased. Thirdly, forced over-expression of the ERK2 gene rescued the YAP protein level and Hippo reporter activity after siRNA knockdown targeting 3'UTR of the ERK2 gene in NSCLC cells. Fourthly, depletion of ERK1/2 reduced the migration and invasion of NSCLC cells. Combined depletion of ERK1/2 had a greater effect on cell migration than depletion of either one separately. Finally, the MEK1/2 inhibitor Trametinib decreased YAP protein level and transcriptional activity of the Hippo pathway in NSCLC cell lines.

Conclusion:
Our results suggest that ERK1/2 inhibition participates in reducing YAP protein level, which in turn down-regulates expression of the downstream genes of the Hippo pathway to suppress migration and invasion of NSCLC cells.

Background:
Proper epigenetic control of transcription is essential for stem cell homeostasis and is frequently disrupted in cancer, but this has not been well investigated in lung biology or lung disease. We have previously demonstrated that the stem cell marker Sca-1 enriches for mouse bronchioalveolar stem cells (BASCs) and lung adenocarcinoma cells with enhanced metastatic and tumor propagation abilities (TPCs).

Methods:
I performed a small chemical library screen using a Kras; p53-flox driven mouse lung adenocarcinoma cell line, CK1750 with high expression of the stem cell markers Sca-1 and CD24. Chemically treated cels were seperated and screened by FACS analysis for changes in the surface antigens Sca-1 and CD24. I treated Sca-1 low expressing mouse lung adenocarcinoma cell lines TM1 and TnM2 with either 1 uM UNC0638 or DMSO for 4 days. 100,000 cells per mouse were injected intravenously. After 3-4 Weeks mice were sacrificed and lungs and other organs were analyzed for tumor formation. Treated cells were also plated in 3D organoid culture and grown for 14 days. After this matrigel plugs were fixed and counted for organoid formation. I isolated BASCs and AT2 cells from 6 week old mice by FACS and plated cells in 3D organoid co-culture. Cells were grown with either 250 nM UNC0638 or DMSO for 21 days. At this point plugs were fixed, sectioned and organoids were stained for lung lineage markers SPC and CC10 and organoids expressing each were scored.

Results:
A FACS screen of adenocarcinoma cell lines revealed that UNC0638, an inhibitor of H3K9me1/2 methyltransferases G9a and Glp enriches for high Sca-1 expressing, TPC-like cells. Gene expression analysis of primary adenocarcinomas shows that G9a/Glp are down-regulated in Sca-1+ metastatic TPCs. Furthermore, analysis of 400+ early stage patient lung adenocarcinomas reveals that low G9a expression and high expression of KDM3A, an H3K9me1/2 demethylase, significantly correlate with worse survival (P=0.008, P=0.002). This implies that dysregulation of H3K9me1/2 is also a significant factor in human disease. Interestingly, G9a/Glp inhibition of adenocarcinoma cells prior to transplantation increases Sca-1+ cells but does not increase recipient lung tumor burden. Instead, significantly more tumors are found in non-lung locations (58% vs. 13%, P=0.02). Whilst inhibition does not affect cell proliferation or migration, colony forming efficiency in 3D organoid culture is significantly increased (1.1% vs 0.3%, P=0.04). This suggests that altered stem-like properties such as tumor initiation may underlie the more tumorigenic phenotypes of H3K9me1/2 low, Sca-1+ TPCs. H3K9me1/2 also regulates the behavior of lung stem cells. G9a/Glp inhibition of BASCs or alveolar type 2 cells in 3D co-culture assays increases both Sca-1+ cells and undifferentiated organoids, and significantly decreases alveolar-lineage organoids (P<0.0005). BASC cultures also show increases in bronchiolar-lineage organoids (P<0.0005), implying that cell fate decisions may regulated by H3K9me1/2.

Conclusion:
These findings suggest that common mechanisms of epigenetic regulation exist between mouse lung stem cells and lung TPCs. Determining the precise mechanisms of this regulation will be important for our understanding of lung biology and disease, with potential implications for the diagnosis and treatment of human adenocarcinoma.

Background:
Lung cancer is one of the biggest cancer killers in the world. Despite certain recent advances, mortality is still high. Targeted therapy has increased the time to death for metastastic lung cancer, but such therapy is not available for all lung cancer patients. Targeted therapy is more often available for never smokers, due to presence of druggable driver mutations. In order to search for new putative targets of therapy, we seek to identify pathways involved different subgroups of patients and in patients with early relapse.

Methods:
A total of 190 patients undergoing surgery for lung cancer were included in the study (154 EGFR positive, 23 EGFR negative, 170 smokers and 20 non-smokers). Lung cancer tissue and clinical information was available for all patients and normal lung tissue was available for 30 of the patients. Whole genome expression array analysis (Agilent) was performed using mRNA isolated from all samples and DNA-methylation was analysed for 168 tumours and 21 matched normal lung tissue samples. R was used for statistical analyses; annHeatmap (from Heatplus) for hierarchical clustering, limma to identify differentially expressed genes, SPIA for pathway analysis and canonical correlation of methylation and mRNA-expression was performed with the CCA function from the PMA package. Pathways with an FDR<0.1 were considered significant. DAVID was used for gene ontology analysis.

Results:
Based on correlation of mRNA and methylation, different pathways were identified as predominant in specific subgroups of lung adenocarcinomas. Preliminary results indicate that genes involved in the KEGG-pathways cell cycle are more highly expressed in EGFR positive than in EGFR negative tumours in smokers. In the EGFR-negative tumours, several pathways are up-regulated: Oocyte meiosis, progesterone-mediated oocyte maturation, HTLV-1 infection, p53 signalling pathway and small cell lung cancer. For non-smoking patients, four pathways were up-regulated in EGFR-positive tumours: ECM-receptor interaction, TGF-beta signalling pathway, bile secretion and cocaine addiction. There were no pathways up-regulated in EGFR-negative compared with EGFR-positive never-smokers. This may partly be due to small numbers. Similarly, pathways dominating the tumours of patients with early relapse will be identified. Genes whose expression and methylation status were correlated were identified within smokers and non-smokers separately.

Conclusion:
Based on correlation between mRNA and methylation, specific pathways were identified activated in subgroups of lung adenocarcinomas. There are significant differences between ever-smokers and never-smokers. Survival analyses are ongoing.

Background:
Hepatocyte Growth Factor and its corresponding receptor MET are potential therapeutic targets for NSCLC. In NSCLC MET is over expressed, activated and mutated and is involved in resistance to tyrosine kinase inhibitors. A high proportion of primary NSCLC cases show elevated MET expression on immunohistochemistry (IHC), however, it is not clear whether expression changes during the metastatic process. We investigated paired NSCLC tumour samples to determine whether MET receptor expression differs in the primary and its corresponding lymph node and distant metastases.

Methods:
Tissue Microarrays (TMAs) were constructed using 1mm cores of primary and metastatic matched NSCLC archival paraffin tissues in triplicate. For IHC, TMAs were stained with the SP44 clone (Ventana) and an H-score calculated based on the percentage of cells stained and their intensity with a minimum of 0 and maximum of 300. The mean of values from multiple cores was calculated. Two independent scorers assessed the tissues. Discordance was defined as an H-score difference of greater than 100 between the primary tumour and its metastasis.

Results:
61 patients with primary and matched distant metastasis were included in the main analysis with 38 (62%) male and brain the most common site of metastasis (27/61, 44%). The histology was adenocarcinoma in 26 patients, squamous cell in 21 and large cell, undifferentiated or mixed in 14. The median H-score was 100 in primary tissue and 120 in metastatic tissue. Brain secondaries showed the highest median H-score (140) compared with other metastatic sites (105). MET concordance was present in 50/61 cases (82%). MET discordance was found in 11/61 tumours including 6/26 (23%) of adenocarcinomas, 2/21 (10%) of squamous carcinomas and 3/14 (21%) of mixed or large cell tumours. Discordant elevation in metastases was present in 6/61 (10 %) and reduction in 5/61 (8%). MET FISH data was available for 54/61 of primary tissues and 60/61 of metastasis. MET was amplified only in 2/61(3%) cases, seen in both primary and secondary tissues, associated with strong positivity on IHC. An additional 75 patients had matched primary and lymph node metastasis MET IHC and FISH data available. High rate of concordance, 66/75 (88%) also was present in this nodal cohort. The median H-score was 100 in primary tissue and 117 in nodal metastatic, similar to the main primary and distant metastatic group. MET discordance between primary and nodal secondary was found only in 9/75 (12%) with discordant elevation in 4/75 (5%) and discordant reduction in 5/75 (7%). MET FISH true amplification was seen in 2 paired specimens and one primary with clonal amplification which was non-amplified in the lymph node metastasis.

Conclusion:
In this cohort of paired biopsies, increased MET expression in the primary was retained in a high proportion of distant and lymph node metastases. These data indicate that MET expression in metastases can be predicted by expression in the primary and further suggests that treatment decisions in the metastatic setting can be based on MET IHC results of archival primary or lymph node tumour.

Background:
Pulmonary adenocarcinoma is the commonest and the most histologically diverse form of lung cancer. ‘Cell-in-cell’ structures are frequently seen as incidental findings when making diagnoses of lung cancer. These structures arise when one viable tumour cell is enveloped within a vacuole within another viable tumour cell. They have been identified in a number of solid tumours including breast, lung, endometrial, pancreatic, melanoma and squamous cell carcinomas. The repeated occurrence of the phenomenon in such a range of tumour types suggests that it may represent a process that confers a selective advantage to the malignant clone (or subclone). Possible mechanisms for this include the acquisition of nutrition by the host cell, horizontal gene transfer, or simple clonal extinction of neighbouring subclones. The biological significance and mechanisms of action still all remain largely unknown. We set out to investigate these.

Methods:
100 recent consecutive cases of lung adenocarcinoma from our surgical centre were de-archived and examined. For each, high-resolution digital images of 10 high-power field (hpf) equivalents were examined. A scoring scheme for both cell-in-cell appearances and multinucleated cells was formulated and used to score the tumours. Independently from this they were classified by histological pattern and graded for numerous architectural and cytological characteristics. Results were collated and statistical tests carried out. In vitro experiments using cocultures of H1299 lung cancer cells transfected to express either GFP (Green Fluorescent Protein) or RFP (Red Fluorescent Protein) were also conducted. These cells were seeded on a selection of coated surfaces including gelatin, fibronectin and collagen to mimic the extracellular matrix proteins.

Results:
Cell-in-cell structures are frequently observed in primary lung adenocarcinomas. 15% of cases have frequent occurrences (≥0.8/hpf). Cell-in-cell structure frequently is strongly associated with multinucleation (P<0.0001). In addition, there is a strong association with cytological measures of tumour grade such as mitoses (P<0.0001), necrosis (P 0.015) and solid pattern (P=0.002). In cell co-culture experiments we are able to reproduce the same appearances, and to visualize cultured lung adenocarcinoma cells engulfing one another. The process is greatly enhanced by the presence of extracellular fibronectin or collagen, but gelatin has a negative effect. This suggests integrins may be involved in the process, as collagen and fibronectin are integrin activators, while gelatin is known for its lack of integrin activation.

Conclusion:
Lung adenocarcinomas frequently contain numerous cell-in-cell structures. This is related to the solid phenotype; this implies that engulfment requires full three-dimensional movement which is impossible in two-dimensional lepidic/acinar/papillary conformations. It is associated with high cytological grade, and may be a useful component of future grading systems. The association with multinuclearity implies that cell engulfment may be the mechanism that leads to the formation of multinucleated giant cells. The same appearances can be reproduced in cell culture systems, where they appear to be dependent upon the presence of signaling from the extracellular matrix.

Background:
Dysregulated inflammation is associated with the development and progression of lung cancer. Pulmonary diseases characterized by increased inflammation, including emphysema and pulmonary fibrosis, are strongly related to heightened risk of lung cancer. Moreover, lung cancer patients with increased levels of inflammatory mediators or inflammatory cells have poor outcomes. It has been shown that dysregulated inflammatory cytokines in the tumor microenvironment can promote cancer metastasis. However, the mechanisms of this effect in lung cancer have not been fully understood. Interleukin 1β (IL-1β), a key pro-inflammatory cytokine, is associated with tumor aggressiveness and poor patient outcomes in NSCLC. Herein, we report that treatment of IL-1β leads to epithelial-to-mesenchymal transition (EMT) in NSCLC cell lines. Delineation of the underlying molecular pathway(s) may potentiate novel therapeutic strategies.

Methods:
We treated NSCLC cell lines with IL-1β acutely (3 days) and chronically (21 days) in vitro and identified EMT mediators using RNA interference and chemical inhibitors. Histone modifications and DNA methylation were analyzed with chemical inhibitors, ChIPassays and methylation-specific PCR. We utilized transwell migration, cell proliferation and anchorage-independent cell growth assays to evaluate the functional phenotypes

Results:
We found that following acute IL-1β exposure (within 7 days), the activator protein 1 (AP-1) transcription factor components, including Fra-1 and c-jun, mediate EMT. AP-1 functions downstream of ERK1/2 and JNK signaling and resides upstream of the transcription factors Slug and Zeb2. Importantly, inhibition of slug, zeb2, fra-1 or ERK1/2 and JNK signaling by RNA interference or chemical inhibitor is sufficient to abolish IL-1β-induced E-cadherin repression. This occurs concomitantly with decreased cell migration and invasion. Surprisingly, following prolonged IL-1β exposure (21 days), cells do not revert back to the epithelial state despite inhibition of these acute EMT mediators. We also found that following withdrawal of IL-1β after twenty one-day exposure, the treated cells are able to maintain their mesenchymal phenotype for more than 30 days before reverting back to an epithelial phenotype. We refer to this prolonged but reversible EMT program that persists in the absence of the original inflammatory stimulus as EMT “memory.” Further studies showed that fra-1 is only required to establish but not to maintain EMT memory. Chemical inhibition of a variety of enzymes involved in histone modifications and DNA methylation indicates the repression of E-cadherin is mediated by different mechanisms depending on the duration of IL-1β exposure. H3K27Me3 and histone acetylation mediate E-cadherin repression during acute EMT but DNA methylation is responsible for the downregulation of E-cadherin in EMT memory. In fact, we have found increased CpG island methylation in the E-cadherin promoter region in EMT memory. In vitro functional studies further showed that EMT memory enables cancer cells to enhance their motility but gradually regain proliferative advantage.

Conclusion:
We conclude that lung cancer cells utilize distinct mechanisms for EMT in response to acute and chronic inflammation. We also demonstrate that dynamic alteration of histone modification and DNA methylation can lead to prolonged but reversible EMT, subsequently creating a time window for cancer cells to migrate to distant organs and eventually undergo mesenchymal-epithelial transition to form macro-metastases.

Background:
Epidemiologic studies suggest that citrus fruit consumption reduces cancer risk. We have previously shown that 2-hydroxyflavanone (2HF), a naturally occurring compound in citrus fruits, exerts antineoplastic effects in-vitro and in-vivo against renal cell carcinoma in a manner dependent on VHL function and expression of glutathione S-transferase p (GSTP). GSTP is a stress- and xenobiotic-defense protein that catalyzes the first step committed step of the mercapturic acid pathway (MPy).

Methods:
We performed mining of molecular databases, including the TCGA. Then, we conducted cytotoxicity assays, followed by signaling studies. Finally xenograft experiments, using nu/nu athymic nude mice were performed.

Results:
2HF inhibited non-small and small cell lung cancer cell line growth in-vitro. Reduced CDK4 and cyclin B1 levels were correlated with G~2~/M arrest. Apoptosis was accompanied by Bcl2 down-regulation and Bax upregulation. Inhibition of PI3K signaling was evident from reduction in AKT and p70S6K phosphorylation. Reduction of vimentin and fibronectin and increase in E-cadherin indicated inhibition of epithelial-mesenchymal transition. Remarkably, 2HF reduced the expression of RLIP76/RALBP1, a rate-limiting glutathione-conjugate/multidrug transporter of the MPy. 2HF also inhibited the transport activity of RLIP76. Dose-dependent 2HF cytotoxicity was enhanced by antibodies to RLIP76. Oral dosing of 2HF resulted in 3-5 mM serum concentrations and inhibited the growth of H1618 and H358 NSCLC cell line xenografts in nu/nu athymic nude mice. Residual tumors had reduced Ki67 and CD31 staining, indicating inhibited proliferation and angiogenesis. Higher MPy expression in lung cancer was evident from increased RLIP expression in human lung cancer tissues, and analyses of the TCGA database showed that RLIP76 expression was inversely correlated with survival in lung adenocarcinoma.

Conclusion:
Taken together, these studies demonstrate antineoplastic activity of 2HF against lung cancer cell lines in-vitro and in-vivo through a novel mechanism that simultaneously causes down-regulation of MaP, stress-signaling, EMT and angiogenesis. Excellent oral absorption of 2HF indicates its suitability for therapy and possibly prevention of lung cancer.

Background:
Cyclin dependent kinases (CDKs) are protein kinases that regulate cell growth and proliferation in cells. CDK11 belongs to transcriptional subfamilies of CDKs and has been reported to be crucial for survival of sarcoma and breast cancer cells. To examine its roles in lung cancer cells, we investigated the effect of CDK11 knockdown on non-small cell lung cancer (NSCLC) cell lines.

Methods:
17 NSCLC cell lines were used for expression analysis of CDK11. Among them, we used three lung cancer cell lines, H460, H1299 and H358 for functional analysis. Synthetic siRNAs were utilized to knockdown CDK11. mRNA and protein levels of CDK11 were evaluated by real-time PCR and western blotting, respectively. Changes in growth were examined by WST-1 proliferation assay and liquid and soft agar colony formation assays. Cell cycle and apoptosis were analyzed by FACS with propidium iodide staining.

Results:
Western blot analysis revealed that all of the 17 lung cancer cell lines expressed CDK11 mRNA and protein and that compared to a normal cell line, H460 expressed CDK11 at lower levels but H1299 and H358 expressed CDK11 at higher levels. CDK11 knockdown suppressed proliferation and anchorage-dependent and independent clonal growth in H460 and H1299 cell lines but not in H358. Induction of apoptosis was seen in H460 but not in the others.

Conclusion:
CDK11 knockdown suppressed proliferation and clonogenic growth in H460 and H1299 cells, and induced apoptosis in H460. These results suggest that inhibiting CDK11 may be an attractive target for the treatment of NSCLC.

Background:
High throughput sequencing methods such as exome sequencing, RNA sequencing, Chromatin–immunoprecipitation (ChIP) sequencing are essential tools for cancer research. However, these fine and delicate analyses contain several methodological problems. For example, although tumor mass may be suitable for mutation analysis, histological heterogeneity of the tumor tissue causes insufficient results especially for epigenetic or RNA analyses. Besides, the cancer-associated stromal cells and immune cells in the tumor will also affect the results. In this study, we tried ChIP for tiny but pure tumor samples which were selected by laser captured microdissection and verified its availability for ChIP sequence analysis.

Methods:
We used a lung adenocarcinoma frozen tissue harboring EGFR L858R mutation. After formalin fixation (1%, 10min), tumor cells, stroma cells and immune cells were microdissected separately by LMD4000 (Leica) and ChIP was performed to using H3K4me3 anti-body. Then, the quality was confirmed by real-time PCR for CCR7 which is one of the tumor specific markers and CD3 which is representative T lymphocyte marker. Sanger sequence for EGFR L858R mutation was also analyzed for confirmation that each sample was dissected and extracted correctly.

Results:
Only from the sample of tumor cells, we detected EGFR L858R mutation by Sanger sequence but from stromal cells and immune cells, we did not detect EGFR mutation. The result showed that we extracted samples correctly. And H3K4me3 mark at CCR7 gene was detected only from tumor cells and was not detected from the other samples. Moreover, H3K4me3 mark at CD3 gene was detected from stroma cells and immune cells but not tumor cells. These results indicated that microdissection method is useful and necessary method for ChIP analysis.

Conclusion:
Microdissection can be applied for epigenetic analysis like ChIP method. Our results indicated that microdissection method is useful for tumor-cell-specific epigenome profiling by ChIP sequencing.

Background:
One of the metabolic perturbations in cancer cells is the Warburg effect; glycolysis is preferred over oxidative phosphorylation (OXPHOS), even in the presence of oxygen. The precise mitochondrial alterations that underlie the increased dependence of cancer cells on aerobic glycolysis for energy generation may serve as an escape mechanism from apoptosis. Here, we aimed to profile the mitochondrial activity in different lung cancer cell lines in reference to their glycolytic activity and to their sensitivity to metabolic modifications.

Methods:
The metabolic profile of A549 and H358 cell lines were tested before and after glycolysis blockade (glucose starvation, 2DG) and mitochondrial induction (FCCP). Glycolysis inhibition and mitochondrial activity were assessed by western-blot quantification of key enzymes involved in the glycolysis pathway (e.g. Hexokinase I/II, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase 2) and of mitochondrial coded proteins (e.g. ND1, ATP6 synthase). The oxygen consumption rates (OCR) and extra cellular acidification rate (ECAR) were measured by XF[e]24 extracellular flux analyzer. Further, mitochondrial index was compared to the cells' sensitivity to glycolysis inhibition.

Results:
A549 cells were highly affected by glucose inhibition/starvation accompanied by ineffective mitochondrial compensation. On the other hand, H358 cells recovered completely from glucose starvation through mitochondrial hyper-activation (Fig 1); At the basal level (when no material was applied), A549 cells that were starved had a decrease of 68% in the ECAR, as compared to non-treated cells. Their recovery was limited after glucose injection (23 vs.41 mpH/min). In comparison, H358 cells had a 43% decrease in their glycolysis rate with a full recovery after glucose injection (44-46 mpH/min; pre & post respectively). Mitochondrial respiration was very low for A549 cells under starvation, while significantly increased in H358 cells (223 vs.143 pmol/min, *Pv<0.0001). Respectively, the expression level of mitochondrial coded proteins was higher in the cells that demonstrated higher mitochondrial capacity (Fig 2). Figure 1



Conclusion:
Cells with high mitochondrial capacity may tolerate glucose starvation/ blockade, while a limited mitochondrial reserve exposes the cells to higher sensitivity to glycolysis stress. This might suggest a potential therapeutic avenue with a companion predictive test.

Background:
Protein tyrosine kinase 7 (PTK7) is a catalytically inactive receptor tyrosine kinase that is also known as colon carcinoma kinase-4 (CCK-4). Recent reports have shown that PTK7 plays an important role in carcinogenesis, and it is known to be up-regulated in gastric cancer, colon cancer, esophageal cancer, and liposarcoma. However, we found that PTK7 expression was down-regulated at the mRNA as well as protein levels in human lung squamous cell carcinoma (SCC), unlike in other tumors. In this study, we attempted to explore the role of PTK7 in lung cancer

Methods:
We analyzed expression of PTK7 by RT-PCR and western blot analysis using tumor and normal lung tissue from 10 SCC patients. To explore the functional role of PTK7, the expression of PTK7 in SCC cells was examined using empty vector and PTK7 gene inserted vector.

Results:
We found that PTK7 expression was down-regulated at the mRNA as well as protein levels in human lung squamous cell carcinoma (SCC). Upon investigating the functional role of PTK7 in SCC, we found that overexpression of PTK7 in SCC cells resulted in inhibition of cell proliferation, invasion, and migration. Further, we confirmed that these phenotypic changes are associated with the activation of Akt and ERK.

Conclusion:
These observations may indicate a role for PTK7 in cell proliferation, wound healing and invasion via regulating Akt and Erk activation. Our findings suggest that PTK7 has different oncogenic roles in organs and target tumors.

Background:
LKB1 regulates both cell growth and energy metabolism. It remains unclear how LKB1 inactivation coordinates tumor progression with metabolic adaptation in non-small cell lung cancer (NSCLC).

Methods:
Mouse Colony, Mouse Treatment and Tumor Analyses Statistical Analysis Hematoxylin and Eosin (HE) Staining and Immunohistochemistry (IHC) Bioinformatics Analysis Cell Lines and In vitro Assays ShRNA, Plasmids, Lentivirus Production and Infection Analysis of Human Lung ADC and Ad-SCC Specimens Western Blotting Enzymatic Activity Assays and Liquid Chromatography-tandem Mass Spectrometry (LC-MS) Analysis Oil red O Staining Reverse Transcription and Quantitative PCR Analysis

Results:
Here in KRAS/LKB1 (KL) mouse model, we reveal differential reactive oxygen species (ROS) levels in lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC). ROS can modulate ADC-to-SCC transdifferentiation (AST). Further, pentose phosphate pathway deregulation and impaired fatty acid oxidation collectively contribute to the redox imbalance and functionally affect AST. Similar tumor and redox heterogeneity also exist in human NSCLC with LKB1 inactivation. In preclinical trials towards metabolic stress, certain KL ADC can develop drug resistance through squamous transdifferentiation.

Conclusion:
This study uncovers critical redox control of tumor plasticity that may affect therapeutic response in NSCLC.

Background:
Rac3 is a member of the Rac family of small guanosine triphosphatases (GTPases), regulates a variety of cell functions, including the organization of the cytoskeleton, cell migration, and invasion. The overexpression of Rac3 has been reported in several human cancers. However, the role of Rac3 in Lung cancer (LC) has not been determined yet. The purpose of this study is to investigate the effect of silence on Rac3 expression in human LC cells and the consequence of cell survival.

Methods:
Lentivirus small hairpin RNA (shRNA) interference techniques were utilized to knock down Rac3 gene. Gene and protein expression was quantified by quantitative Real-time polymerase chain reaction (qRT-PCR) and Western Blot. LC cell apoptosis was examined by Annexin V-APC /propidium iodide staining.

Results:
Efficient silence of of Rac3 strongly inhibited A549 cells proliferation and colony formation ability, and significantly decreased tumor growth. Moreover, flow cytometry analysis showed that knockdown of Rac3 led to G2/M phase cell cycle arrest as well as an excess accumulation of cells in the G1and S phase.

Conclusion:
Thus, functional analysis using shRNAs reveals a critical role for Rac3 in the tumor growth of LC cells. shRNAs silencing of Rac3 could provide an effective strategy to treat LC.

Background:
The epidermal growth factor receptor (EGFR) is one of the best-known targets of therapy for non-small cell lung cancer (NSCLC). Our purpose was to investigate whether ubiquitin-specific protease 8 (USP8) and stratifin (14-3-3σ or SFN) inhibit or stimulate lysosomal degradation of EGFR in lung adenocarcinoma.

Methods:
Using Western blotting and immunofluorescence analysis, we examined the effect of USP8 or SFN knockdown by siRNA and overexpression of USP8. Expressions of USP8 and SFN in normal and tumorous lung tissue were examined by Western blotting and immunohistochemistry.

Results:
USP8 or SFN knockdown led to downregulation of cellular proliferation, receptor tyrosine kinases such as EGFR and proto-oncogenes (c-Met), and downstream signaling pathways such as the AKT, ERK, and STAT3 pathways, whereas it upregulated the accumulation of EGFR at lysosomes for degradation. However, overexpression of USP8 led to an increase of EGFR and downstream signaling after EGF stimulation. Moreover, USP8 and SFN expressions were increased in the tumorous lung tissue in comparison with normal lung tissue from the same patient.

Conclusion:
USP8 and SFN inhibit ubiquitination of EGFR for lysosomal degradation in lung adenocarcinoma cells, suggesting that USP8 and SFN could be potential therapeutic targets for NSCLC.

Background:
Even among heavy smokers, the lifetime risk for developing lung cancer is between 15 and 20% which raises a question about whether there are protective factors that mitigate risk from toxic exposures in tobacco smoke. Previously we have shown significant gene-environment interaction effects between smoking and genetic loci on chromosome 15q25.1 near the nicotinic acetylcholine receptor locus contrasting minimal increases in risk for never smokers from SNPs in this region versus substantial impact on risk for smokers. Studies contrasting risk in heavy versus light smokers have not been conducted and little is known about protective factors that may reduce risk for some smokers.

Methods:
In order to identify genetic factors that might reduce lung cancer risk, we performed a genome-wide case only analysis of lung cancer risk, comparing heavy to light smokers. This approach to analysis is powerful for identifying gene-environment interactions provided there is no correlation between the genetic factor and the environment exposure. We performed a genome-wide association of heavy smokers who had a 30 packyear or more background of smoking versus 1824 lung cancer cases with less than 30 packyears of smoking exposure using data derived from studies conducted by 7 sites within the Transdisciplinary Research in Cancer of the Lung (TRICL) consortium. To improve our ability to identify genetic variants, we used imputation based on the March 2012 release of the 1000 Genomes to impute and analyze data from more than 9 million genetic variants.

Results:
This analysis identified one region showing many highly significant associations with the most significant being rs62180069 (p=5x10-8, OR = 0.76). This SNP lies between the SCHLAP1 gene encoding the SWI/SNF complex antagonist with prostate cancer 1, non protein coding, gene and the gene UBE2E3 on chromosome 2. At this locus there was no gene-smoking correlation in the controls and no evidence for heterogeneity in strength of association among the 7 contributing studies.

Conclusion:
UBE2E3 is the ubiquitin conjugating enzyme E3. This gene is overexpressed in a substantial number of lung cancers. Further studies to characterize the impact of this variant on lung cancer risk according to further variation in smoking behavior and also impacts on function are warranted.

Background:
Receiving a cancer diagnosis, particularly of lung cancer, has been shown to increase psychological and biological stress responses and the immediate risks of extreme adverse health outcomes, such as suicide and cardiovascular deaths. Data are scarce on the potential influence of this diagnosis on tumor progression. Prior studies lend suggestive evidence for an association of psychobiological stress-responses on lung cancer progression. The aim of this study is to improve understanding of determinants of the stress-response to a lung cancer diagnosis and explore potential role of this response in disease progression and survival.

Methods:
We have initiated a nationwide prospective cohort study of Icelandic lung cancer patients with a comprehensive questionnaire and biomarker measures of stress, as well as detailed documentation of clinical parameters and disease course. Eligible are all individuals diagnosed with lung cancer at Landspitali University Hospital in Iceland. The aim is to recruit 300 patients over a three year period between 2015 and 2017. Patients with clinical or radigraphic changes suggestive of lung cancer are referred to our hospital. They go through a diagnostic work-up, leading to a definite lung cancer diagnosis and staging during a 24 hour diagnostic course or within few days thereafter. Assessment of psychological stress and relevant biomarkers are integrated with clinical assessments at two time points, i.e. during the diagnostic work-up and at follow-up visit 1-3 weeks later (before treatment). The study participation involves questionnaire assessment of symptoms of anxiety, depression, posttraumatic stress, sleep disturbances and quality of life. Biomarker repositories include overnight urine collection, diurnal saliva and hair sampling for analysis of cortisol and catecholamines along with ECG to determine heart rate variability. Bronchoscopic and core needle biopsies as well as surgical tumor samples will be used for assessment of apoptosis, proliferation, microvascular density and adrenoreceptors expression. Radiographic progression will be assessed at baseline and every 6 months from diagnosis along with complete documentation of clinical parameters, disease course and survival.

Results:
In 4 weeks we have recruited 8 patients (80% acceptance rate). We will characterize determinants of a severe psychological-, neuro-endocrine- and cardiovascular stress-response to a cancer diagnosis, as well as the potential relevance of these responses on tumor characteristics, radiographic progression and disease-specific survival. We expect to present preliminary results from approximately 30 patients at the conference.

Conclusion:
Significance: This research program is the first comprehensive attempt to evaluate determinants of psychobiological-induced responses to a lung cancer diagnosis and their potential impact on cancer progression. The findings might guide intervention strategies to improve quality of life, reduce morbidity and prolong survival in lung cancer patients.

Background:
Lung cancer is the leading cause of cancer mortality and accounts for 1.6 million deaths annually in the world. Lung cancers may be classified into non-small cell (NSCLC) and small cell (SCLC) lung cancers, which individually account for approximately 85% and 15%, respectively, of lung cancer cases. Despite recent advances in cancer therapy, the overall 5-year survival rate of lung cancer remains low. There remains an urgent need for discovery of novel approaches for early diagnosis and therapy. Inositol-trisphosphate 3-kinase A (ITPKA) regulates inositol phosphate metabolism and calcium signaling by phosphorylation of the second messenger inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) to inositol-1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) (1). ITPKA has a very limited tissue expression, mainly in brain and testis. ITPKA, previously known as a neuron-specific F-actin bundling protein, has recently been shown to be overexpressed in lung adenocarcinoma and associated with increased metastatic potential (2). However, our understanding of the role and regulation of ITPKA in cancers is limited. Reference: 1. Shears SB. How versatile are inositol phosphate kinases? The Biochemical journal. 2004; 377:265-80. 2. Windhorst S, Kalinina T, Schmid K, Blechner C, Kriebitzsch N, Hinsch R, et al. Functional role of inositol-1,4,5-trisphosphate-3-kinase-A for motility of malignant transformed cells. International journal of cancer Journal international du cancer. 2011;129:1300-9.

Methods:
To identify potential oncogenes that are involved in the pathogenesis of lung cancer, cDNA microarray analysis was performed to search for up-regulated genes in primary lung adenocarcinomas. Inositol-trisphosphate 3-kinase A (ITPKA) was found to be overexpressed in lung ADC.

Results:
Using gain-of-function and loss-of-function approaches, we demonstrated that ITPKA contributes to cancer development. We also showed that methylation level in the ITPKA gene body is highly tumor-specific, and is positively correlated with its expression. Furthermore, DNMT3B-mediated methylation of the CpG island in ITPKA gene body regulates its expression via modulation of the binding of transcription activator SP1 to the ITPKA promoter. ITPKA gene body methylation first appeared at the in situ carcinoma stage and progressively increased during the multistage pathogenesis of lung carcinoma. Figure 1



Conclusion:
Altogether, deregulation of ITPKA may promote oncogenic transformation and function as a universal or near universal hallmark of malignancy. A novel regulatory mechanism of oncogene expression was demonstrated via gene body methylation which manipulates the binding of transcriptional factor(s) to its promoter and controls gene expression.

Background:
The kynurenine pathway is crucial for tryptophan metabolism, which has been shown to be active both in macrophages and microglial cells. L-Kynurenine (L-KYN) is transported across the blood-brain barrier and serves as a source for the synthesis of all the other metabolites of the kynurenine pathway. Glial cells have the enzymatic capability for the biosynthesis of brain kynurenines as kynurenine aminotransferase (KAT). KAT converts L-KYN to kynurenic acid, which is an inhibitor of glutamate neurotransmission. The lowered KAT activity was observed in the plasma and brains of patients with neurodegenerative disorders followed by a tendency to a decrease KYNA in plasma and brains. Peripheral blood mononuclear cells (PBMC) can be considered as representative for metabolic changes in peripheral tisues during the course of lung cancer. With this background in mind we have undertaken the evaluation of translocator protein 18 kDa (TSPO), which reflects microglia activation, G Protein-coupled Receptor (GPR35), a KYNA receptor and kynurenine aminotransferase II (KAT) in PBMC and serum L-KYN concentration in relations to cognitive functions in lung cancer patients.

Methods:
We included in the study 80 consecutive lung cancer patients (5 small-cell lung cancer, 75 non-small cel lung cancer, 24 females and 56 males, aged 61.5±6.7 years) hospitalized in Clinical Oncology with The Sub-department of Diurnal Chemotherapy Wielkopolska Center of Pulmonology and Thoracosurgery of Eugenia and Janusz Zeyland and Chair and Clinic of Oncology. PBMC were isolated by density gradient centrifugation. The expression of TSPO, GPR35 KAT in PBMC was evaluated with ELISA. Serum L-KYN concentration was measured by means of spectrofotometric method. Neurological examination, MiniMental State Examination (MMSE), Trail Making Test (TMT) A and B, and Hamilton scale were performed at baseline (time of lung cancer diagnosis) and after 6 months.

Results:
Decreased TSPO expression in PBMC was associated with better results of MMSE evaluation (29.00; 28.0–29.0; median, interquartile range) than in lung cancer patients with up-regulated TPSO (28.0; 26.0–28.7; P=0.016). Also, TMT-A results were better in lung cancer patients with down-regulated TPSO (8.41±3.68s) compared to the subject with stimulated TPSO (12.92±7.30s; P=0.002). TSPO expression in PBMC negatively correlated with MMSE score (Kendall’s tau = -0.182; P=0.0178) and positively with TMT-A (Kendall’s tau = 0.168; P=0.0309) evaluated at baseline. The up-regulation of KAT expression in PBMC was associated with better cognitive functions measured with MMSE 6 months after baseline (28.4±0.7) comparing to lung cancer patients with inhibited KAT (27.1±1.8). KAT correlated positively with MMSE scoring 6 months after baseline (Kendall’s tau= 0.308; P=0.0234).The expression of GPR35 in PBMC did not correlated with cognitive measures. Serum L-KYN concentration correlated negatively with TMT-A evaluated 6 months after baseline (Kendall’s tau= -0.586; P=0.0141). Moreover, TPSO expression correlated positively with KAT (Kendall’s tau= 0.253, P=0.0009) and negatively with GPR35 (Kendall’s tau= -0.173, P=0.0491), but no correlation with L-KYN was found.

Conclusion:
Stimulation of kynurenine pathway in PBMC seems to be protective against cognitive decline during the course of lung cancer. Microglial activation can be independent pathomechanism leading to cognitive impairement in lung cancer patients.

Background:
Progression through the mammalian cell cycle relies upon coordination of a complex network of proteins. Following genomic insult, checkpoints during each stage of the cell cycle are engaged to halt cell cycle progression to allow faithful DNA repair. Failure to arrest cell cycle may lead to genomic instability and cancer development. However, the molecular basis for the loss of genome integrity during cancer development remains to be determined. Cell division cycle associated 3 (CDCA3) is a key regulator of the normal cell cycle. CDCA3 modulates this process by enabling cell entry into mitosis through degradation of the mitosis-inhibitory factor WEE1. CDCA3 itself is also degraded in G1 yet re-expressed in G2/M phase, to allow successful progression through the cell cycle. Here we describe for the first time a novel function for CDCA3 in maintaining effective cell cycle progression in lung cancer.

Methods:
To examine the role of CDCA3 in modulating the cell cycle of lung cancer cells, CDCA3 was depleted using an siRNA approach in A549, SKMES and H460 cell lines. CDCA3 depletion was assessed using Western blot analysis. Cell proliferation assays were performed on control and CDCA3 knockdown cells over a period of 96 h using the Promega CellTitre-Glo cell viability assay. Cell cycle progression was assessed on propidium iodide stained cells using a Beckman Coulter Gallios flow cytometer. To determine if CDCA3 expression is associated with lung cancer progression, a tissue microarray (TMA) with cores from 600 patients was stained with an anti-CDCA3 antibody. Correlation of CDCA3 staining with clinical data and patient prognosis is ongoing.

Results:
As a cell cycle related protein, we tested if CDCA3 is required for effective proliferation of a range of lung cancer cell lines. CDCA3 depletion reduced the proliferation of U2OS (osteosarcoma), A549, MOR (lung adenomcarcenoma) and SKMES cancer cells (squamous lung cancer). Interestingly, depletion of CDCA3 did not affect proliferation of H460 cells (large neuroendocrine lung cancer). We next tested the cell cycle progression and noted that knockdown of CDCA3 induced an increase of all cell lines in G1 arrest with the exception of H460 cells. To observe if CDCA3 expression is linked with disease progression, TMA staining of lung cancer biopsies was performed. Accordingly, elevated expression of CDCA3 was identified specifically in tumour cells. These data highlight the potential prognostic value of CDCA3 expression.

Conclusion:
Our data point to a potential role for CDCA3 in the progression of lung cancer. While the precise mechanism for CDCA3-dependent cell cycle regulation remains unknown, it is possible that elevated CDCA3 levels modulate tumour cell proliferation. Identifying the molecular basis may yield novel therapeutic avenues worth targeting during aberrant cell cycle in cancer.

Background:
Non-small cell lung cancer has become the leading cause of cancer-related deaths worldwide. The subset of NSCLC can be further defined at molecular level by driver mutations that occur in multiple oncogenes, such as EGFR, KRAS and EML4/ALK alterations. The KIF5B-RET fusion gene has been established as a new oncogenic driver in NSCLC. Several studies have demonstrated that KIF5B-RET promote cell growth and tumorigenicity, however, few progress has been made further. Our study aims to investigate other characters of KIF5B-RET fusion gene and tries to explore the potential signaling pathways involved in the gene functions.

Methods:
Lentivirus（encoding KIF5B-RET）was used to transfect the lung epithelial cell line BEAS-2B and lung cancer cell line A549 to generate stable transfectant and the protein expression was analysed using western blot . To verify the oncogenic features of KIF5B-RET in vitro, we detected its expression genetically followed by CCK8 assay, colony formation assay, transwell and Annexin V-FITC/PI double staining to explore proliferation, invasion, migration and apoptosis. The mechanism by which KIF5B-RET kinase induced invasion and migration was investigated by western blot, and administration of RET and SRC inhibitions.

Results:
The stable transfected cell line expressed phosphorylation RET, examined by western blot, suggesting that KIF5B-RET could automatically activate RET protein in the absence of ligand. Firstly we detected the basic characters of KIF5B-RET, but found no significant difference in proliferation as it’s reported in previous studies. To further detect the function of KIF5B-RET fusion gene, we focused on characters of invasion, migration, and apoptosis. We demonstrated that KIF5B-RET showed a significantly increased ability of invasion and migration compared with control group, suggesting that KIF5B-RET fusion gene could promote cell invasion and migration. However, no change was observed after treating the transfected cells with Cisplatin, indicating the gene may have no influence on apoptosis. As we all know, RET tyrosine kinase can activate ERK which belongs to the downstream signaling system. Our restult showed that KIF5B-RET fusion kinase can also induced activation of ERK and even SRC kinase. Finally, we found that stable cells became sensitive to the RET tyrosine kinase inhibitors Sunitinib and Apatinib. The invasion and migration could be suppressed by RET or SRC inhibitors significantly.

Conclusion:
Out data showed that KIF5B-RET fusion gene can activate ERK and SRC kinase through activating RET tyrosine kinase, and promote migration and invasion in vitro ，but did not have an effort on proliferation and apoptosis. RET inhibitor Apatinib and Sunitinib and SRC inhibitor could suppress the phenomenon of invasion and migration, suggesting that KIF5B-RET promotes invasion and migration through activation of SRC kinase. Our preclinical data demonstrated the antitumor activities of Apatinib and Sunitinib against KIF5B-RET gene fusion-driven cells and indicated the therapeutic potential of tyrosine kinase inhibitors targeting RET, which may benefit this certain subpopulation of NSCLC patients.

Background:
About 10% of lung adenocarcinomas express neuroendocrine features, which are thought to denote a subset of adenocarcinomas with poor prognosis. Achaete-scute homologue 1 (ASCL1) is a transcription factor implicated in the neuroendocrine differentiation of lung tissue. Recently, ASCL1 was identified as a neuroendocrine marker in lung adenocarcinomas, and its expression was upregulated in lung adenocarcinomas of smokers when compared to adenocarcinomas of non-smokers and other types of lung cancers. ASCL1 expression in the peripheral airways of cancer-free smokers has not been studied. Moreover, the effect of cigarette smoke exposure on the neuroendocrine differentiation of lung cancer cells has not been examined in vitro.

Methods:
Distal airway brushings for epithelial cells were obtained in 8 subjects who participated in CT scan lung cancer screening at the NYU Lung Cancer Biomarker Center (part of the NCI Early Detection Research Network); never (n=1), former (n=4) and current (n=3) smokers. ASCL1 mRNA expression was measured using real time reverse transcription polymerase chain reaction (RT-PCR). A549 cell line was incubated with cigarette smoke condensate (CSC; extracted to DMSO) at 10 or 40 mcg/mL for 4, 24 or 48 hours. Following the incubation periods, ASCL1 expression levels were measured via western blot with lamin B1 as the nuclear protein loading control. Three individual experiments were performed. Statistical analyses were performed with Kruskal-Wallis test (RT-PCR) and Student's t-test (western blot). Figure 1



Results:
Real time RT-PCR data for ASCL1 in the distal airway brushing samples of the 8 subjects suggested a trend toward higher ASCL1 mRNA titers (p = 0.26) in current smokers (mean = 33 pack-years) compared to former smokers (mean = 51 pack-years), whose ASCL1 mRNA expression levels were higher than that of a never-smoker [Figure 1A]. A549 cells exposed to 10 mcg/mL of CSC for 4 hours had 4.1 fold (p = 0.023) and 2.0 fold (p = 0.017) increases in ASCL1 expression compared to those exposed to 1% DMSO and serum-free media (SF) only, respectively [Figure 1B]. No statistically significant change in ASCL1 expression was noted in the other CSC exposure groups.

Conclusion:
CSC induced ASCL1 expression in A549 cells, and the stimulatory effect of CSC was no longer observed at the higher concentration and the longer exposure times. This in vitro finding is in agreement with the RT-PCR data, which also suggest a trend toward increased ASCL1 expression with more recent smoking history in the distal airways of cancer-free human subjects.

Background:
Tobacco related death has become the first cause of death worldwide and it is estimated that approximately 1 millions patients each year died from tobacco related diseases in China ,the most common diseases from which are COPD and lung cancer.Recently,the effects of long-term exposure to PM2.5(particulate matter) on human health have drawn much attention from clinicians and researches.Cigarette smoke is one of the main sources of indoor PM2.5. At the same time, cigarette smoke also includes nearly six thousand kinds of chemical substances, most of which are harmful to the body, especially benzopyrene.It is proved that benzopyrene is a class of organic compounds with significant carcinogenic effect. However, the underlying mechanisms remain unclear. The aims of this study were to determine the involvement of RNA-binding motif protein 5 (RBM5) and Wnt/β-catenin signaling in cigarette smoke PM2.5 induced alveolar epithelial injury, as well as the interaction between both.

Methods:
A549 cells were treated with cigarette smoke extract (CSE). The MTT assay was used to assess the effects of CSE on cell viability. The levels of RBM5 and Wnt/β-catenin/GSK3β were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blots. A luciferase assay was used to assess the activity of β-catenin/TCF signaling

Results:
CSE inhibits the proliferation of A549 cells, with increasing CSE concentration and action time, the growth inhibition rate of A549 cells is more big, has the time and dose dependence; Cytosolic and nuclear β-catenin levels significantly increased following CSE treatment, compared with those in control cells (P < 0.05); The luciferase activity in CSE-exposed cells transfected with the TCF luciferase reporter wild-type plasmid (pGL3-OT) was significantly greater than that in cells without CSE exposure (33,167 ± 3085 vs. 19,978 ± 1916, respectively, P < 0.05);given CSE A549 cells, RBM5 mRNA increased with the increase of CSE concentration and action time prolonged expression gradually decreased, with time and dose dependence; with increasing concentrations of cigarette smoke extract, reduce the expression of RBM5 protein expression, with dose dependent(all P < 0.05);after pcDNA3.1-RBM5 transfection, Wnt/β-catenin signaling pathway inhibition; siRBM5 after transfection, Wnt/β-catenin signaling enhanced; give the Wnt signal pathway blocker ICG-001 blocked Wnt/β-catenin signaling pathway, the expression of RBM5 and the difference was not statistically significant.

Conclusion:
Down regulation of RBM5 and activation of Wnt/β-catenin signaling are involved in CSE PM 2.5 induced alveolar epithelial injury. RBM5 acts as an upstream molecule that negatively regulates the activity of Wnt/β-catenin signaling This study was supported by grants from the National Natural Science Foundation of China (No.81472169 and No.81241069)

Background:
Lung cancer is the leading cause of cancer mortality worldwide. Non-small cell lung cancer (NSCLC) is the most common subtype, accounting for about 80% of all lung cancers. miRNAs are small RNAs of 21-24 nucleotides in length, which play major role in cell proliferation and differentiation and their differential expression is known to be associated with various cancers including lung cancer. Role of miR-326 has been previously studied as a marker of bone metastasis in lung cancer. Moreover, we have previously shown that miR-326 plays a critical role in the epithelial to mesenchymal transition (EMT) by targeting transforming growth factor (TGF)-β1 and other members of TGF-β signaling pathway. The aim of present study is to check the expression and correlation of miR-326 and lung epithelial developmental gene nuclear factor IB (NFIB) in non-small cell lung cancer tissue samples as cancer metastasis is accompanied by EMT.

Methods:
We have examined eight pathologically confirmed non-small cell lung cancer cases. All patients were men and smokers with age ranged from 29 to 74 years (mean 54.6 years). Surgical resection was performed in all the cases which were either stage II or III. Histopathologically, 4 cases were squamous cell carcinomas, 3 were adenocarcinomas including one case of invasive mucinous carcinoma and one case was low grade mucoepidermoid carcinoma. RNA was isolated from fresh frozen tissue to check for miR-326 and NFIB levels by real time PCR. Protein expression was checked by immunohistochemistry (NFIB; 1:200; Abcam)) and in-situ hybridization (miR-326; Exiqon). Adjoining lung tissue served as normal control in each case.

Results:
Expression of both miR-326 and NFIB was found to be down regulated in non-small cell lung cancer tissue at both RNA and protein level (Fig 1A-C). Our in silico experiments identified a target site of miR-326 at the 3’UTR of NFIB gene; presumably it stabilizes the transcripts of NFIB (Fig 1D). Figure 1



Conclusion:
Our preliminary data suggests that miR-326 stabilizes the transcripts of NFIB in normal epithelial cells and maintain epithelial cell integrity. Dysregulation of miR-326 and NFIB in non-small cell lung cancer indicate that miR-326 and NFIB work synergistically and may contribute to the development of non-small cell lung cancer.

Background:
To explore the expression of PPAR-γ ligand in lung cancer and its effect on the apoptosis of lung cancer.

Methods:
The expression of PPAR-γ in 80 patients with lung cancer was detected using cell-culture method and its expression in the lung cancer cell lines was also determined by RT-PCR and Western blot. Additionally, TUNEL method was used to detect the apoptosis.

Results:
PPAR-γ was expressed in the lung cancer tissue and the tissue of lung benign lesions. Its optical density was the highest in the lung cancer tissue (0.1832±0.0407), then the tissue of lung benign lesions (0.1201±0.0308), and the lowest in the normal tissue (0.1185±0.0296). The total expression of PPAR-γ showed significant difference in the patients with different pathological types and differentiated degrees (P<0.05), and there was also statistical significance regarding the total expression of PPAR-γ in small cell lung cancer and non-small cell lung cancer (P<0.01). The expression of squamous cell carcinoma is the highest in non-small cell lung cancer. Significant difference was presented by comparison to the expression of PPAR-γ in poorly-differentiated and moderately, highly-differentiated lung cancer tissues (P<0.05).

Conclusion:
The expression of PPAR-γ is closely related to the pathological features of patients with lung cancer, and hence, to research PPAR-γ ligand can provide new evidences for the treatment of lung cancer.

Background:
Previous studies have shown that the levels of 15-lipoxygenase-1(15-LOX-1),15-lipoxygenase-2(15-LOX-2) and their metabolites 13(S)-HODE and 15(S)-HETE are significantly reduced in human non-small cell lung carcinoma (NSCLC). Furthermore, animal model experiments indicate that the reduction of these molecules occurs before the establishment of lung tumor, suggesting their roles in lung tumorigenesis. However, the functions of these molecules remain unknown in NSCLC.

Methods:
We treated NSCLC cells with exogenous 13(S)-HODE and 15(S)-HETE and then examined how they affected cell functions. We also over-expressed 15-LOX-1 and 15-LOX-2 in tumor cells to restore these two enzymes to generate endogenous 13(S)-HODE and 15(S)-HETE before the cell function was assessed.

Results:
The application of exogenous 13(S)-HODE and 15(S)-HETE significantly enhanced the activity of peroxisome proliferator-activated receptor-γ(PPARγ), inhibited cell proliferation, induced apoptosis, and activated caspase-9 and caspase-3. The overexpression of 15-LOX-1 and 15-LOX-2 could obviously promote the endogenous levels of 13(S)-HODE and 15(S)-HETE, which were demonstrated to be more effective in the inhibition of NSCLC.

Conclusion:
We have demonstrated that exogenous or endogenous 13(S)-HODE and 15(S)-HETE can functionally inhibit NSCLC likely by activating PPARγ. The restoration of 15-LOXs activities to increase the production of endogenous 15(S)-HETE and 13(S)-HODE may offer a novel research direction for the molecular targeting treatment of NSCLC and avoid potential side-effects associated with the application of synthetic PPARγ ligands.

Background:
Metastasis of non-small cell lung cancer shortens the survival time of patients and decreases their quality of life. This unfortunate situation could be improved if the functions of long noncoding RNAs (lncRNAs) were identified in depth.

Methods:
not applicable

Results:
In the present study, a large number of lncRNAs and mRNAs with different expression patterns were verified in 95D and 95C lung cancer cell lines. The lncRNA, LOC101448202, was highly expressed in 95D cells, and lentivirus-mediated RNA interference was able to silence its expression with a silencing efficiency of 92%. LOC101448202 gene silencing led to a decrease in cell proliferation, adhesion, migration, and invasion and the number of pseudopods and microvillion the cell surface was also reduced. At the mRNA level, her-2 expression was inhibited and the expression of nm23-H1 and E-cadherin was increased. At the protein level, β-catenin and ezrin levels were decreased both in vitro and in vivo. In clinical specimens and mouse models, LOC101448202 expression was positively related to tumor growth.

Conclusion:
These data indicate that LOC101448202 expression levels are associated with 95D cell metastasis, demonstrating the tumor-promoting function of this lncRNA.

Background:
Ubiquitin protein ligase E3C (UBE3C) has been recently proposed as a potential oncogene in hepatocellular carcinoma. However, its role and mechanism in non-small cell lung cancer remains unknown.

Methods:
Eight cases of NSCLC and matched nontumorous samples were used to analyze UBE3C expression at the level of protein. Then, we down-regulated the expression of UBE3C in NSCLC cells and assessed the biological role of UBE3C in NSCLC cell line. Finally, the prognostic role of UBE3C was examined by Immunohistochemistry (IHC) in tissue microarray (TMA) consisting of 208 cases of NSCLC.

Results:
The expression of UBE3C in NSCLC tissues was much higher than that in nontumorous samples. Downregulation of UBE3C expression suppressed the proliferation and invasion of lung cancer cells. Further analysis showed that downregulation of UBE3C expression mainly promoted cell apoptosis but without an effect on cell cycle. High levels of UBE3C expression were associated with higher tumor stage in NSCLC patients. The 5-year overall survival rate in the UBE3C[high] group was significantly lower than that in the UBE3C[low] group. In multivariate analysis, UBE3C was identified as an independent prognostic factor for overall survival.

Conclusion:
Our findings indicated that UBE3C may represent a candidate therapeutic target and a novel prognostic marker of NSCLC.

Background:
The invasion and metastasis of malignant cell is generally considered as a major reason why it is always failed in the therapy of lung cancer. The mechanism in metastasis is complicated and uncertain as well. The scientific research proves that Long non-coding RNA (LncRNA) is bound up with the occurrence, development and prognoses of lung cancer.

Methods:
Application of high throughput LncRNA chip to investigate the differentially expressed LncRNA between 95D and 95C cell lines. RNA interference (RNAi) approaches were used for the analysis of the biological functions and metastasis of TATDN1. Tumor growth was studied in nude mice.

Results:
TATDN1-1 was highly expressed in 95D cells lines and NSCLC tissues. In 95D cells knockdown of TATDN1 by using small interfering RNA (siRNA) resulted in significant reduction in proliferation, adhesion, migration and invasion of these cells in vitro. 95D cells xenografts with decreased TATDN1 expression were impaired in tumor formation and growth. On genetic level, MALAT-1displays the strongest association with genes involved in cancer like cellular growth, movement, proliferation, signaling.

Conclusion:
These data indicate that TATDN1expression levels are associated with 95D cells metastasis and identify tumor-promoting functions of TATDN1.

Background:
Lung cancer remains the leading cancer related cause of death in the United States and worldwide. Non-small cell lung cancer (NSCLC), the most common subtype (85%) of lung cancer, continues to be associated with a very poor 5-year survival rate of less than 15%. Despite the recent advances in systemic lung cancer treatment due to the introduction new therapies targeting angiogenesis, epidermal growth factor receptor (EGFR), and activin receptor-like kinase-1 (ALK1) in selected patient subgroups, the overall mortality of patients with advanced stage disease remains high. The development of new biomarkers and individualized therapies is needed to overcome these challenges and make significant strides towards improving the care of lung cancer patients. Dopamine (DA) has long been used in the treatment of Parkinson's disease and acute cardiac dysfunction. Given that DA is produced by the sympathetic nerves ending in blood vessels, we originally postulated and later revealed that DA and its dopamine D2 receptor (D~2~R) agonists inhibit VEGF-mediated angiogenesis and also completely block accumulation of tumor ascites and tumor growth in mice. Specifically, we demonstrated that DA stimulates endocytosis of VEGFR-2 via D~2~R thereby preventing angiogenesis by inhibiting VEGF binding, receptor phosphorylation and subsequent downstream signaling. These observations define a possible link between DA and vascular biology. Subsequent studies by numerous investigators clearly demonstrate that this strategy can be successfully applied to various diseases including cancer . Correspondingly, we observed significantly more angiogenesis, tumor growth, and VEGFR-2 phosphorylation in D~2~R knockout mice. We documented D~2~R colocalization with VEGFR-2 and described the molecular mechanism through which D~2~R/VEGFR-2 crosstalk can mediate the dephosphorylation of VEGFR-2. D~2~R agonists have been shown to increase the efficacy of anti-cancer drugs in preclinical models of breast and colon cancer. Here we show that D~2~R agonists inhibit tumor growth in orthotopic murine lung cancer models through inhibition of tumor angiogenesis and reduction of tumor infiltrating myeloid derived suppressor cells.

Methods:
We utilize syngeneic (LLC1) and human xenograft (A549) orthotopic murine lung cancer models as well as pathological examination of human lung cancer tissue to describe D~2~R agonist-mediated inhibition of lung tumor growth.

Results:
We sought to determine whether Dopamine D2 Receptor (D~2~R) agonists inhibit lung tumor progression and identify subpopulations of lung cancer patients that benefit most from D~2~R agonist therapy. We demonstrate D~2~R agonists abrogate lung tumor progression in syngeneic (LLC1) and human xenograft (A549) orthotopic murine models through inhibition of tumor angiogenesis and reduction of tumor infiltrating myeloid derived suppressor cells. Pathological examination of human lung cancer tissue revealed a positive correlation between endothelial D~2~R expression and tumor stage. Lung cancer patients with a smoking history exhibited greater levels of D~2~R in lung endothelium.

Conclusion:
Our results suggest D~2~R agonists may represent a promising individualized therapy for lung cancer patients with high levels of endothelial D~2~R expression and a smoking history.

Background:
Metastasis is a multistep process and the main cause of disease failure and mortality in lung cancer patients. Twist1 is a highly conserved developmental gene involved in embryogenesis that could be reactivated in cancers promoting both malignant conversion and cancer progression through epithelial-mesenchymal transition (EMT). The aim of this study was to investigate the role and mechanism of Twist1 in the pathogenesis of lung cancer.

Methods:
We examined a series of surgical lung cancer samples from Chinese patients (n=75) and showed that Twist1 expression was linked to lymph node status (P<0.05). To validate that Twist1 is a driver of EMT in non-small cell lung cancer (NSCLC), we used two human lung cancer cell lines (H1650 and H1975, EGFR mutation) and demonstrated that Twist1 was associated with cell growth and mobility.

Results:
Overexpression of Twist1 increased cell growth, mobility, and a decrease of Twist1 by shRNA technology reversed the phenomenon. Twist1 promoted the tumor growth in vivo and induced the expression changes of many genes by tumor gene RNA array. Twist1 significantly down-regulated p4EBP1 expression in H1650 cells and up-regulated p4EBP1 in H1975 cells by qRT-PCR and western blot assay.

Conclusion:
Collectively, both our in vivo and in vitro findings support that Twist1 in promoting lung cancer by upregulation p4EBP1, which are needed to further study the role of Twist1in NSCLC

Background:
Lung cancer is one of the most heterogeneous of all solid cancers. This may in part be due to hi-jacking and additional bystander affects that are exerted on the normal lung cell population by the cancer cells. A number of pathways may be stimulated through soluble factors or effector filled vesicles such as exosomes secreted by cancer cells. The aim of this project was to evaluate the effects of non-small cell lung cancer (NSCLC) cells on an immortalised normal bronchial epithelial cell line.

Methods:
A normal bronchial epithelial cell line (HBEC4) was exposed to adenocarcinoma, large cell and squamous NSCLC cell lines and a number of phenotypic and genotypic characterisations were undertaken. These included cellular proliferation (BrdU ELISA), gene (RT-PCR) and miRNA expression screening (Nanostring). The effect of cancer exosome fractions was also determined.

Results:
Exposure to various subtypes of NSCLC significantly increased the cellular proliferation rate of the immortalised cell line in a number of models. Expression of a number of miRNAs were altered in the normal cells pre- and post exposure to the cancer cells. Various stem cell factor markers (KLF4, Oct, c-myc) were also significantly changed at the mRNA level. In addition, exosome fractions altered the behaviour of the normal cell line, likewise stimulating cell proliferation.

Conclusion:
Lung cancer cells may influence normal cell behaviour in both a direct and indirect manner using multiple mechanisms. Normal bronchial epithelial cells with stem like features may be induced to proliferate and behave in a malignant manner. This, akin to Hodgkin’s lymphoma, may contribute significantly to the composition of the tumour. Furthermore this observation may contribute to the heterogeneity of lung cancer tumours and affect treatment response. Ongoing studies are evaluating these effects in novel 2D and 3D culture systems.

Background:
Lung adenocarcinoma gene expression is highly aberrant and heterogeneous and not due to mutations in all these protein-coding genes. Epigenetic regulation by microRNAs has been shown to explain some of this dysregulation. We aimed at identifying whether few microRNAs could explain multiple overexpressed genes in key pathways of lung adenocarcinoma.

Methods:
Publicly available gene expression profiling data from three different publications were included in this in silico analysis. In the lung adenocarcinomas (n=139/49/45) and normal lung tissue (n=17/9/65) among the common 8543 genes (based on the EntrezID), the differentially expressed and diagnostic genes were investigated. The genes with q-value under 0.01 and with concordant regulation among all three datasets were regarded as significantly differentially expressed. Diagnostic genes were identified through the performance of each differentially expressed gene as a classifier. Finally, the miRNAs with highly probable predicted (PCT>0.9 in TargetScan), miRNA targeting interactions (MTI) and/or with experimentally validated MTIs were assessed, the number of gene targets in the down- or up-regulated genes measured for each miRNA and an enrichment analysis was performed.

Results:
The common overexpressed and down-regulated genes among the three datasets were 534 and 638 respectively. Among the diagnostic genes with AUC over 0.8 were the known genes encoding recently discovered diagnostic proteins but also new unknown genes. Among the pathways in KEGG, genes of the Cell Cycle, Carbon-pool by Folate and Base Excision and Mismatch Repair were significantly overexpressed. Importantly, we identified few microRNAs (q‹ 0.01) that could target most of these genes and that are all previously shown to be down-regulated and validated in lung and/or other cancer. Among the top microRNA were the following; Mir-615-3p targets 86 highly over-expressed genes in all three datasets. This microRNA was recently shown to act as a tumor suppressor through inhibition of the AKT2 in pancreatic cancer cell lines. Let-7b targets 83 highly over-expressed genes in all three datasets. Let-7b is controlling genomic balance and is down-regulated in aggressive breast cancer and significantly reduced in serum is correlated to poor survival in resectable NSCLC. Mir-16 targets 75 of the up-regulated genes in adenocarcinoma and was verified down-regulated in NSCLC versus adjacent normal lung tissue. Mir-193b targets 65 genes and was identified as a tumor suppressor in NSCLC and hepatocellular carcinoma. Mir-320a targets 50 genes and was identified as a crucial miRNA regulating glycolysis and was verified down-regulated in NSCLC. Mir-34a targets 46 of the overexpressed genes and is a crucial miRNA down-regulated in lung cancer and already proposed as a treatment with cisplatin.

Conclusion:
By combined in-silico analysis of three large datasets on adenocarcinoma versus normal adjacent lung tissue we detected novel candidate diagnostic genes and important pathways that recapitulate the phenotype of this cancer. Importantly, we found microRNAs that could target and thus explain a large portion of the pathway dysregulation. One of these identified microRNAs, miR-34a, has already demonstrated a therapeutic potential in lung cancer.

Background:
MLF1IP was initially identified as a MLF1 interacting protein, which encodes a centromere protein essential for cell cycle and mitosis. It has been reported that MLF1IP depletion impaired the interaction between centromere and microtubules, finally inducing defects in cell mitosis. However, the involvement of MLF1IP in lung adenocarcinoma development and related mechanisms remain to be elucidated.

Methods:
MLF1IP expression at mRNA level in 15 pair lung adenocarcinoma/adjacent normal lung samples was examined with real-time PCR assay. Then the expression of MLF1IP in human lung adenocarcinoma cells A549 was inhibited with lentiviral-mediated shRNA strategy. Effects of MLF1IP knockdown on cell proliferation was analyzed by Cellomics cell counting method and MTT assay. Then the impact of MLF1IP knockdown on colony formation, cell cycle process and cell survival was determined in A549 cells by colonogenesis assay, PI staining and Annexin V-APC staining respectively.

Results:
MLF1IP expression was significantly increased in lung adenocarcinomas as compared to adjacent normal lung tissues (fold change=2.50, P<0.05), with higher MLF1IP expression observed in 66.7% (10/15) samples while lower expression observed in only 20% (3/15) samples (Fig. 1A). Furthermore, MLF1IP knockdown impaired cell proliferation (Fig. 1B, C), inhibited colony formation ability (Fig. 1D), induced cell cycle arrest (Fig. 1E) and promoted cell apoptosis (Fig. 1F) in A549 lung adenocarcinoma cells.Figure 1



Conclusion:
Our study showed that MLF1IP expression is correlated with lung adenocarcinoma development and MLF1IP expression is critical for cell proliferation and survival in lung adenocarcinoma cell line A549. MLF1IP represents a novel potential target for lung adenocarcinoma therapy.

Background:
CHK2 is a transducer protein that is involved in DNA damage response (DDR). CHK2 phosphorylates effector proteins that play roles in DNA repair, cell cycle regulation and apoptosis. Recently, CHK2 have been found to have a critical role in the mitosis, and disruption of CHK2-BRCA pathway caused chromosomal instability in colon cancer cell lines (Stolz et al. Nat Cell Biol 2010:12; 492). The purpose of this study was to investigate the biological role of CHK2 and related factors in lung adenocarcinoma.

Methods:
We investigated 60 surgically resected lung adenocarcinomas. CHK2 and BRCA1 mRNA expression levels were evaluated by qRT-PCR. Relative mRNA expression levels of each sample were standardized to those of β-actin. EGFR mutation (exon 19 deletion and 21 point mutation) was detected by PNA-LNA PCR clamp method. KRAS mutation (exon 2, codon 12, 13) and p53 mutation (exon 5-9) were examined by direct sequencing. p27 and p21 protein expression levels were assessed by immunohistochemistry. Chromosomal aberration (CA) was examined in 20 samples with single-nucleotide polymorphism–CGH (SNP–CGH).

Results:
CHK2 mRNA levels were significantly increased in the tumor tissues compared to the normal tissues (p=0.012). CHK2 mRNA level was not correlated with patients’ clinicopathological factors, EGFR mutation status or p53 mutation status. CHK2 mRNA levels were significantly correlated with BRCA1 mRNA levels (ρ = 0.569, p < 0.0001). High CHK2 mRNA expression and high p27 protein expression levels were associated with poor prognosis for recurrence free survival (P = 0.028, P = 0.048), although both expression levels were not correlated with each other. 7 samples were determined to be high CA, while 13 samples to be low CA according to SNP-CGH. CHK2 mRNA level was higher in high CA (7 samples) than in low CA samples (13 samples) (Ave. 0.326 vs. 0.185; p=0.0129).

Conclusion:
CHK2 mRNA expression level was increased in lung adenocarcinoma and was related to poor prognostic outcomes. CHK2 pathway may be important for the proliferation of lung adenocarcinoma, especially in tumors with chromosomal instability.

Background:
Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are considered as two distinct subtypes of lung cancer and derived from different types of lung epithelial cells and featured with different biomarker expression. Interestingly, there exist certain lung tumors so called adenosquamous cell carcinoma (Ad-SCC) containing mixed both adenomatous and squamous pathologies; more importantly, these two different pathologies within a single tumor are consistently shown to have identical gene mutations. In consideration the fact that most tumors are derived from a single epithelial cell, it’s reasonable to hypothesize that there must exist lineage transition between ADC and SCC subtypes. However, this fundamental question remains unanswered due to the difficulty of study of human clinical samples. Indeed, most studies of clinical samples can only provide indirect evidences to support this hypothesis. Taking advantage of mouse models mimicking human lung cancer, we have recently successfully shown that inactivation of a tumor suppressor LKB1 confers mouse lung ADC with strong plasticity and makes them transdifferentiate into SCC through mixed Ad-SCC as intermediates (Han XK, et al. Nat Commun, 2014). However, whether there exists a phenotypic transition from ADC to SCC in human lung cancer remains unknown.

Methods:
Immunohistochemical analyses Integrative genomic analyses Establishement of patient-derived tumor xenograft model Statistic Analyses

Results:
not applicable.

Conclusion:
We pathologically analyzed a large cohort of human NSCLC samples and carefully evaluate the prevalence of mixed pathologies in context with LKB1 genetic inactivation. Moreover, we took advantage of the established lung ADC PDX mouse models to perform serial transplantation w/o the interfere of essential signaling pathways identified from de novo animal model study and test if possible that human ADC with LKB1 inactivation can progress and transdifferentiate into SCC. Based on our current understanding of this type of phenotypic transition in mice as well as the resources and systems established in the lab, we here succeed in proving the transdifferentiation of human ADC to SCC.

Background:
Cyclin Y (CCNY) is a novel cyclin and is highly conserved in metazoan species. Cyclin Y mRNA has several transcripts and only ccny1 and ccny2 has been documented. A potential CDK partner of Cyclin Y is PFTAIRE kinase (PFTK1). In hepatocellular carcinoma, cell motility and invasion was enhanced by PFTK1 expression. But the function of CCNY1 and CCNY2 on cell migration and invasiveness has not been reported yet.

Methods:
Recombined plasmids carrying CCNY1 and CCNY2 were constructed and transfected to H1299 cells to obtain CCNY up-regulation cells. A lentivirus-based RNAi delivery system was used to inhibit CCNY mRNA expression. The role of CCNY in cell motility and invasion was investigated using wound healing and transwell assay. The protein levels in lung cancer cells were determined by western-blot, immunofluorescence technique and high-content cell analysis. Mouse xenograft experiments were carried out to study the metastasis ability of CCNY1 and CCNY2 in vivo. Immunohistochemistry was used to detect the CCNY protein level of lung cancer tissues.

Results:
cell motility and invasion activity were inhibited and MET (Mesenchymal - Epithelial Transition) was caused by down-regulating of CCNY in 95D and H1299 cells. CCNY2 could enhance cell migration and invasion activity in vivo and vitro. The F-actin level was regulated by CCNY2 expression. In non-small cell lung cancer tissues, CCNY2 was highly expressed and the CCNY2 expression was associated with histological grade.

Conclusion:
CCNY2 was firstly detected in lung cancer cells and non-small cell lung cancer tissues. Our findings demonstrated CCNY2 not CCNY1 promoted cell motility and invasion by regulating the expression of F-actin and modulating intracellular cytoskeletal components.

Background:
Background: CD73, is a glycosylphosphatidylinositol (GPI)-linked 70-kDa cell surface enzyme that catalyzes the dephosphorylation of extracellular AMP to adenosine, its dysregulation contributes to tumorigenesis and progression of a variety of malignancies, and it was suggested as a therapeutic target of cancers. But the functional relevance of CD73 and the mechanism underlying its dysregulation in lung tumorigenesis remained unclear. Here, we mainly focus on if CD73 has important functions in non-small-cell lung cancer (NSCLC) by: 1.evaluating the clinicopathologic significance of CD73 through analysing its expression in 38 human NSCLCs tissues using quantitative PCR, Western Blot; 2. determining its role in NSCLC using in vitro assays; 3. investigating the regulatory mechanism of CD73 dysregulation in NSCLS cell lines.

Methods:
Western blot analysis, real-time quantitative reverse transcriptase PCR, RNA interference microarray analysis and trans well were performed on human NSCLC tissues and cell lines. Thirty-one paired NSCLC tissues and adjacent noncancerous lung tissues were collected.

Results:
Figure 1Figure 2CD73 was upregulated in 52.38% of the lung tumor tissues and its expression was significantly related to histology and differentiation (P < 0.05). Reduced CD73 expression suppressed NSCLC cell growth in vitro. miR-30a-5p was significantly downregulated in 4 paired lung cancer tissues(with > 2.0-fold change and FDR < 0.05) and was validated in the 38 independent paired tissues (63.16%, P < 0.05), CD73 was a novel target of miR-30a-5p predicted by TargetScan, miRanda, PicTar, MirTarget2, PITA and detected by luciferase report assay, then ectopic miR-30a-5p expression in cancer cells reduced CD73 expression.





Conclusion:
CD73 play a very important role in NSCLC, and regulated by miR-30a-5p. CD73 may provide a potential target for diagnosis and treatment for lung cancer.

Background:
Integrin α11β1 is a stromal cell-specific receptor for fibrillar collagens and is over-expressed in carcinoma-associated fibroblasts (CAFs) in non- small cell lung cancer (NSCLC). We have studied the direct role of stromal integrin a11 on the growth and metastasis of NSCLC cells using novel immune-compromised a11 deficient mice.

Methods:
We developed α11 non-expressing immune-deficient mice by back-crossing for at least 10 times the α11-deficient heterozygous C57BL/6J mice (+/-) to obtain a homogenous C57BL/6 background. These were subsequently bred with the BALB/c SCID mice for 7 generations, producing α11-deficient heterozygous (+/-) in SCID background. In vivo studies were done using subcutaneous tumorigenicity assay and orthotopic model to evaluate metastatic potential of integrin α11. Immunostaining were carried out using integrin α11, α-SMA, and cytokeratin. PisroSirius red staining was used to visualize the collagen fibers. Images were taken by polarized-light microscopy using parallel and perpendicular polarizer orientations on an Olympus BX51 microscope. Second Harmonic Generation (SHG) was used to visualize fibrillar collagen and atomic force microscopy was applied to measure the stiffness in tumor stroma.

Results:
The tumor growth of both primary human lung cancer (PHLC) and established NSCLC cells in α11 knockout (α11[-/-]) mice was significantly impeded compared to wild type (α11[+/+]). Orthotopic implantation of a spontaneously metastatic NCI-H460SM cell line into the lungs of α11[-/-] and α11[+/+] mice showed significant reduction in the metastatic potential of these cells in the α11[-/-] mice. Using mouse WG-6v2 Illumina Bead Chips, we identified that alpha11 expression correlates with that of a fibrillar collagen cross-linking enzyme, LOXL1, in the xenograft stroma. Fibrillar collagen was highly disorganized and had a significantly lower elastic modulus in the alpha11 knockout xenografts compared to wildtype. The results suggest a role for α11 in promoting tumor growth and metastatic progression by affecting the collagen stiffness of the tumor stroma.

Conclusion:
The integrin a11β1 signaling pathway in CAFs promotes tumor growth and metastasis of NSCLC cells. This appears closely linked to collagen cross-linking, the organization, and stiffness of fibrillar collagen matrices.

Background:
Lung cancer (LC) is the first cause of cancer death in men and the third in women in Argentina. Multiple studies have shown that patients with LC treatment prolong their survival and thus increases the risk of second tumors. The prevalence of second tumors after lung cancer ranges from 2% to 15%, which further increases after ten years since the diagnosis was made. Reports from latinoamerica are scarce.

Methods:
We evaluated retrospectively the medical record of pts with LC from 2005 to 2014. We recorded the presence of second tumor in the follow-up.

Results:
Two hundred twelve patients were registered. Ten patients (4.56%) were recorded with second tumors. Median age was 68 years old (r. 59-80),most of them were men (80%) and smokers (90%). Regarding primary tumor, 90% (9 pts) were non small lung cancer (NSCLC) versus 10% SCLC. The most frequent histological type was squamous carcinoma 60% (6 pts) and adenocarcinoma 30% (3 pts). The most frequent second tumor site was: lung 40% (4 pts) followed by larynx , bladder, kidney, ovary, breast and NHL. Median survival was 22 months (r. 1-160) in pts without second tumors versus 65 months (r. 22-165) in pts with second tumors (p: 0,005 – Fisher Test). The median of diagnosis time of second tumor was 22.5 months.

Conclusion:
These results show the paramount importance of a correct follow-up in these patients. In our series, one each twenty patients with lung cáncer had a second cancer during their follow-up. The most frequent site were respiratory and genitourinary tracts. There was a significant difference in the survival in pts with second tumors.

Background:
DNA cytosine methylation profiles are important features of malignancy. This study was designed to identify 5-methyl cytosines on a genome-wide scale in non-small cell lung cancers (NSCLC) relative to paired non-tumor lung which, analyzed alone or coupled to transcriptome data, could suggest methylome-deregulated loci.

Methods:
Twenty-four NSCLC tumor (T) – non-tumor (NT) pairs were interrogated for 1.2 million CCGG-bounded fragments across all genomic compartments, using a methylation-sensitive restriction enzyme based HELP-microarray assay. Expression microarrays were also employed, from specimens from the same lung resections.

Results:
We found: (i) Good correlation (r[2] =0.52, p=0.0006) between HELP and the reference quantitative methylation assay MassArray ®; (ii) Wide distribution of differential methylation (DM) among 32,037 promoters (PR, 26% of array-represented loci), 248,721 gene bodies (GB, 39 %), and 171,996 intergenic (IG, 48%) loci; (iii) In PR CpG island (CGI) hypermethylation exceeded CGI hypomethylation; (iv) DM hypermethylation in adenocarcinoma specifically was observed in many unexpected PR [e.g., RASL12; SPTAN1, mir-26a,] and GB [e.g., AKAP13, ANK family, PRKCE, ROS1] regions; (v) Overlay of DMxDE (differential expression) for adenocarcinoma yielded loci with canonical DM:DE patterns (e.g. PR hyper/hypo-methylation:mRNA down/up-regulated n=80; GB hyper/hypo-methylated:mRNA up/down-regulated GB n=3,136). (vi) Examples in adenocarcinoma hypermethylated PR loci with reduced expression included: HBEGF, DPT, AGER, SPARCL1, PTPRM; GB hypermethylated loci with upregulated expression included FERMT1, SLC7A5, FAP, TFAP2a genes. (vii) IPA analyses showed adenocarcinoma-specific promoter DMxDE overlay identifying familiar lung cancer nodes [tP53, Akt] and less familiar nodes [HBEGF, NQO1, GRK5, VWF, HPGD, CDH5, CTNNAL1, PTPN13, DACH1, SMAD6, LAMA3, AR].Figure 1



Conclusion:
Methylome sampling, alone and combined with transcriptome data, yields new loci, as well as previously recognized ones, distributed throughout the genome that are deregulated in NSCLC.

Background:
There have been several studies demonstrating existence of cancer stem-like cells in lung cancers, and resistance of such cells to conventional chemotherapy or targeted agents. As such, targeting cancer stem-like cells is a potential strategy to prevent development of drug resistance and tumor recurrence. Previously our group has demonstrated that mithramycin, a specific inhibitor of transcription factor SP1, attenuates induction of side population (a phenotype of cancer stem-like cells) by cigarette smoke condensate, and modulates expression of multiple genes regulating stem-cell related pathways in lung cancer cells. The present study was performed to further examine the effects of mithramycin on stem cell signaling pathways, and ascertain if mithramycin can eliminate stem-like cells in lung cancer following exposure to conventional chemotherapeutic or targeted agents.

Methods:
Stem-like cell populations in cultured H358 and H2228 lung adenocarcinoma cells were identified based on expression of stem cell markers, ALDH1 and CD133 using ALDEFLUOR[TM] assay and flow cytometry, respectively. Sphere-formation assays were used to examine clonogenic growth of stem-like cells. qRT-PCR techniques were used to evaluate expression levels of stemness-related genes. Western blot techniques were utilized to assess activation of stemness-related (WNT/β-catenin and NOTCH) signaling pathways.

Results:
Small CD133[+] or ALDH1[+] fractions were detected in untreated H2228 and H358 cells, respectively. Consistent with notion that stem-like cells are present in these two lines, H2228 and H358 cells formed pulmospheroids when cultured in stem cell media in low attachment plates; these phenotypic changes were accompanied by increased expression of stemness-related genes including Oct4, Sox2 and Nanog. Cisplatin treatment enriched CD133[+] fraction in H2228 cells and ALDH[+] fraction in H358 cells. Mithramycin abolished this enrichment, and mediated dose-dependent decreases in Oct4, Sox2 and Nanog expression in a dose-dependent manner. Preliminary analysis demonstrated that mithramycin decreased total as well as active forms of β-catenin, but did not affect levels of cleaved NOTCH1, suggesting that mithramycin eliminates lung cancer stem-like cells partially through suppression of WNT/β-catenin signaling. The effects of mithramycin on lung cancer stem-like cells induced by targeted agents are currently under investigation.

Conclusion:
Mithramycin suppresses stemness-related signaling, and is a potential therapeutic agent for elimination of stem-like cells emerging in lung cancers after cisplatin therapy.

Background:
Previous study has confirmed that the occurrence of Wnt pathway activation is associated with risk of non-small-cell lung cancer recurrence. However, whether the pharmacologic blocking of the Wnt signaling pathway could provide therapeutic possibility remains unknown. The aim of the present study was to evaluate the therapeutic functions of the Wnt signaling pathway inhibitor pyrvinium pamoate (PP) on lung cancer stem cells (LCSCs) in vitro.

Methods:
Colony formation and sphere culture were performed to enrich LCSCs from three lung cancer cell lines: PC9, SPC-A1, and A549. After confirming stemness by immunofluo­rescence, PP was employed for cell viability assay by comparison with three other kinds of Wnt signaling inhibitor: salinomycin, ICG-001, and silibinin. The effect of PP on LCSCs was further verified by colony formation assay and gene expression analysis.

Results:
LCSCs were successfully generated by sphere culture from SPC-A1 and PC9 cells, but not A549 cells. Immunofluorescence assay showed that LCSCs could express pluripotent stem cell markers, including NANOG, Oct4, KLF5, and SOX2, and Wnt signaling pathway molecules β-catenin and MYC. Half-maximal inhibitory concentrations of PP on SPC-A1, PC9, and A549 were 10 nM, 0.44 nM, and 0.21 nM, respectively, which are much lower than those of salinomycin, ICG-001, and silibinin. Moreover, significantly decreased colony formation and downregulation of pluripotent stem cell signaling pathway were observed in lung cancer cells after treatment with PP.

Conclusion:
Wnt signaling inhibitor PP can inhibit proliferation of LCSCs, and the Wnt signaling pathway could be considered a promising therapeutic or interventional target in lung adenocarcinoma.

Background:
Primary resistance against epithelial growth factor receptor (EGFR) targetet therapy is often caused by K-Ras mutations or amplification of the MET-oncogene. HMG-CoA reductase inhibitors (statins) are well tolerated drug mainly prescribed for the the primary and secondary prophylaxis of coronary artery disease. However, the majority of statins are metabolized in the liver by Cyp3A4, which may lead to interactions with Erlotinib a tyrosin kinase inhibitor (TKI) which targets the EGFR. Therefore we tested the efficacy of Erlotinib in combination with Pitavastatin, which is metabolized by Cyp3A4, for the treatment of primary Erlotinib-resistant NSCLC celllines.

Methods:
Experiments were carried out with human NSCLC celllines A549 (K-RAS mutation), Calu-6 (K-RAS mutation, P53 mutation), HCC 827 (EGFR mutation, Erlotinib sensitive) and H1993 (MET amplification). Apoptosis was measured by the activity of caspase 3 by cleavage of specific fluorescent caspase substrates, by binding of AnnexinV by FACS analysis or by the cleavage of PARP by Western blot. Inhibition of growth was assessed by MTS assays. The effect of either drug alone or in combination on phosphorylation of AKT and ERK1/2 was evaluated by Western blot.

Results:
Inhibition of growth by erlotinib was seen in the sensitive cell line HCC 827 but not in A549, Calu6 and H1993. Pitavastatin led to growth inhibition in all 4 cell lines investigated in a dose dependant manner. However, the combination of Pitavastatin and Erlotinib was significantly more effective than either drug alone. Erlotinib and Pitavastatin did not induce apoptosis when used as single agents in A549, Calu6 or H1993. When a combination of TKI and Pitavastatin was used we observed significantly increased caspase 3 activity and a higher rate of annexin V positive cells. Moreover an increased cleavage of PARP was shown in the combination treatment. Taken together we could show increased rate of apoptosis when Erlotinib and Pitavastatin where used in combination. Importantly the activation of survival pathways mediated by phosphorylation of AKT was markedly decreased, by the combined treatment in A549, Calu6 and H1993 cells. ERK 1/2 phosphorylation was reduced in H1993 and Calu6 cells upon combined treatment.

Conclusion:
Our data indicate, that the treatment of primary EGFR-TKI resistant cells (A549, Calu6 and H1993) with Erlotinib in combination with Pitavastatin leads to growth arrest and an induced rate of apoptosis.

Background:
To evaluate the potential of conditional reprogrammed cells (CRCs) established from biopsy or effusion samples of advanced non-small cell lung cancer (NSCLC) for in vitro pharmacologic screen and identification of drug resistance mechanisms.

Methods:
A total of 48 tumor specimens obtained from 46 patients with NSCLC were cultured with irradiated fibroblast feeder cells and Rho kinase inhibitor (Y-27632) to induce tumor cells to proliferate indefinitely. The cell lines established from patients harboring EGFR mutation or other druggable oncogenes were subjected to genetic analyses and pharmacologic screen. Corresponding tumor cells were injected into nude mice to test for tumorigenicity and efficacy of targeted agents in vivo.

Results:
Twenty one male patients and twenty five female patients were assessed for establishment of CRC. Adenocarcinoma was the most frequent histologic type (84.7%). There were 21 patients (46%) who harbored an active EGFR mutation. There were four patients with ALK fusion and five with ROS1 fusion. Twenty-six patients experienced disease progressed while on treatment with EGFR (20), ALK (2) or ROS1 (4) tyrosine kinase inhibitors. Tumor cells came from primary or distant metastases in 48% and 52%, respectively. Thirty one (65%) samples were obtained by tumor biopsy and 17 from malignant pleural effusion. Nine CRC model were successfully established (18.7%, 9/48). The successful growth was not dependent on the clinicopathologic characteristics. Both cells from pleural effusion (4 of 17) and biopsy (5 of 31) and adenocarcinoma (8 of 41) and squamous cell carcinoma (1 of 3) were successfully cultured. For biopsy samples, the success rate of cells obtained from primary lung lesion was 21.7% (5 of 23) and cells from metastatic site outside lung was 0% (0 of 8) (P = 0.3). For effusion samples, volume of effusion required for CRC was not significant factors for establishment (success vs. failure cases: mean volume 500 ml vs. 267 ml). The genetic characteristics of patients with non-squamous cell carcinoma did not affect the success rate of CRC (EGFR mutation, 4 of 21; ALK translocation, 0 of 4; ROS1 translocation, 2 of 5; wild or unknown, 2 of 15). Two xenograft models with CRC were successfully established and passaged to maintain tumor in vivo.

Conclusion:
The CRC models derived from NSCLC patients provide useful in vitro platforms of preclinical studies evaluating novel targeted therapies and uncovering the drug resistance mechanisms.

Background:
Metastasis is the main cause of cancer-associated mortality. We recently developed an ex vivo 4D lung cancer model that mimics metastasis. One of the unique features of the model is its ability to isolate tumor cells in three different phases of cancer progression: primary tumors, circulating tumor cells (CTCs), and metastatic lesions. In this study, we want to further characterize the CTCs from the model and determine whether they enter a resting cell cycle phase, and conferring them to be resistant to chemotherapeutic drugs.

Methods:
We harvested rat lung and heart block and decellularized them using 0.1% sodium dodecyl sulfate and 1% Triton-X100. Acellular lung scaffolds were set up in a customized bioreactor and seeded with 50 million cells of human lung cancer cell line H1299 that were cultured on a petri dish (2D). Culture media was replenished and CTCs were collected daily. We measured and compared the cell cycle of 2D cells and CTCs using propidium iodide. Next, we tested the CTCs and 2D cells with 5 μM vinorelbine, 50 μM gemcitabine, 0.1 μM paclitaxel, or 10 μg/ml etoposide and measured total live cells after 2 days of culture. All analysis was performed using PRISM software and Student’s t-test was used to compare the significance of variance.

Results:
Cell cycle analysis of CTCs from a 4D model seeded with H1299 cells showed a significantly higher population of cells in G0/G1, resting cell cycle phase, than in respective 2D cells (65% vs 49%, p<0.01). Furthermore, our results showed a significant decrease in 2D cells upon treatment with all chemotherapeutic drugs. There was a significantly smaller number of 2D cells in the treatment group when treated with gemcitabine (p<0.0001), paclitaxel (p=0.01) and etoposide (p<0.0001) and vinorelbine (p=0.006) than in the control group. On the other hand, there was no significant effect of drugs on the total live CTCs from the 4D model with H1299 that were treated with all four drugs on a 96-well plate as compared to the untreated control group. For H1299 CTCs, there was no significant difference in the number of cells when treated with gemcitabine (p=0.38), paclitaxel (p=0.828), and etoposide (p=0.162), while there were significantly more CTCs with the vinorelbine treatment (p=0.04) compared to the control group.

Conclusion:
Overall, our results show that CTCs from the 4D model are different from parental 2D cells that were placed in the 4D model. These CTCs enter the G0/G1 phase, which may confer resistance to chemotherapeutic agents that are cell-cycle-dependent in efficacy. Further characterization of the CTCs from the model may provide the mechanism of the cell cycle arrest and chemotherapy resistance.

Background:
Lung cancer is the leading cause of cancer deaths in Japan and worldwide. Despite recent advances in targeted therapy, long-term survival of patients with lung cancer remains poor. Novel treatment approaches are needed to extend survival of these patients and improve control of this disease. Oncolytic virus therapy is a promising therapy for various tumor types. A third generation oncolytic herpes simplex virus type 1 (HSV-1), G47Δ, has been tested in clinical trials in Japan for glioma, prostate cancer, and olfactory neuroblastoma. In this study, we investigated the potential of G47∆ as a new therapeutic modality for human lung cancer.

Methods:
Human lung cancer cell lines A549 (adenocarcinoma), EBC-1 (squamous cell carcinoma), LU99 (large cell carcinoma) and SBC-3 (small cell carcinoma) were used. Infectivity and cytopathic effects of G47Δ on lung cancer cell lines were assayed in vitro. Viral replication was determined by standard viral plaque assay. For in vivo studies, athymic mice harboring established subcutaneous tumors and lung tumors generated with A549 or EBC-1 were used.

Results:
All cell lines were susceptible and sensitive to G47Δ irrespective of histological types. Viral replication assay resulted in approximately a 200-fold increase in virus titer by 48 h. In subcutaneous xenograft models, intraneoplastic inoculations with G47Δ significantly inhibited the tumor growth compared with those with mock. In orthotopic xenograft models, intrapleural inoculations with G47Δ prolonged the survival time.

Conclusion:
Oncolytic HSV-1 G47Δ was effective in human lung cancer cell lines. Direct intratumoral inoculation of G47Δ induced an obvious therapeutic effect on lung cancer, suggesting G47Δ may be a potent therapeutic modality for all histological types of lung cancer.

Background:
The discovery of “driver mutations” such as the Epidermal Growth Factor Receptor (EGFR) and the Anaplastic Lymphoma Kinase (ALK) has led to a remarkable improvement in the outcomes of lung adenocarcinoma, which accounts 50% of the non-small cell lung cancer (NSCLC) diagnoses. Up today, no druggable molecular targets have been identified for squamous carcinoma or small cell lung cancer, which are still treated with the “one-fits-all” therapeutic approach, as it is for a relevant percentage of adenocarcinomas too. The precise definition of molecular profile and, possibly, the description of predictive factors are research priority in the thoracic oncology field. The vast majority of preclinical data are based on in vitro studies, but cell lines models do not entirely reflect tumour characteristics and are hampered by genetic divergence from primary tumours. Patient derived tumour xenografts (PDTX) are a valuable alternative to closely reproduce tumour biology and to prospectively characterize in vivo mechanisms of cancer growth and therapeutic response. Through the generation of a cohort of lung cancer xenopatients, the project aims to confirm the reliability of such models in this disease and to prospectively characterize its biomolecular features.

Methods:
Metastatic and early stages lung cancer cases are considered for the enrolment. Written informed consent is requested from each patient. Fresh tumour tissue from lung biopsies or lung resections is collected and kept in serum free medium (4° C), embedded in 20% matrigel and subcutaneously engrafted into NSG and NOD SCID mice, within 24 hours from sample collection. The exponentially growing tumours are passaged subcutaneously to other mice for a second passage after which they are archived for subsequent analyses (formalin fixed, snap frozen and RNA later). Each sample from surgical resection is also stored to create a DNA lung cancer bank.

Results:
Fourteen samples from TC-guided lung biopsies and sixty-six from radically resected NSCLC were engrafted in NSG and NOD SCID mice lineage in a 1:1 ratio. Due to the low engraftment rate and high morbidity observed in NGS mice in the first 73 samples, subsequent engraftments and expansions were performed in NOD SCID mice only. The overall engraftment rate in biopsy samples was 0 % in NGS and 7.14 % in NOD SCID mice as opposed to 0 % in NGS and 27,27 % in NOD SCID for surgical samples (50% adenocarcinomas, 44,45% squamous carcinomas and 5,55% sarcomatoid carcinomas). Nineteen samples underwent the second passage: of those, 10 samples have been archived after the second successful passage and will be used for further analyses.

Conclusion:
The trial is still ongoing and a longer follow-up is needed. In biopsy-derived samples, engraftment is deeply limited by the paucity of tissue. The results of this study will possibly confirm the reliability of PDTX in lung cancer and provide prospective biomolecular characterization for different histological types.

Background:
The key mechanisms that underlie drug resistance in lung cancer have yet to be fully elucidated. A significant limiting factor is the lack of biologically relevant cellular models for basic laboratory research. To address these issues, many are now turning to three-dimensional (3D) based cellular assay systems that permit the formation of multicellular structures such as tumour spheroids. Depending on their size the internal microenvironment of these structures mimics closely that of those in vivo. In the majority of cases, spheroids with a diameter greater than 100µm exhibit an asymmetry in cellular proliferation and viability - proliferating tumour cells at the periphery; cell-cycle arrested cells at larger distances from the surface. Regions of necrosis associated with reduced oxygen tension and hypoxia have often been reported. This study compared drug resistant models of non-small cell lung cancer (NSCLC) in 3D culture with those in grown in two-dimensional (2D) culture. The behaviour of cells grown in these distinct geometric configurations was monitored and compared by measuring viability, proliferation and oxygen tensions.

Methods:
Happy Cell Advanced Suspension Medium[™] (ASM) was chosen to culture our 3D spheroids. This polymer-based formulation was selected for its ease of use, as well as its compliance with liquid handling, high content imaging and analysis (HCSA) and high throughput screening (HTS) systems. Isogenic NSCLC cell line models of cisplatin resistance were cultured in 2D and 3D cell culture systems. Cisplatin sensitive (Pt) and isogenic cisplatin resistant (CisR) NSCLC sub-types were studied. IC50 values were calculated and a positive control was selected. All cultures were grown in a range of cisplatin concentrations for 72 hours. Subsequently, viability and hypoxia assays were conducted in order to compare the response of Pt and CisR cells in both 2D and 3D culture systems. Morphological analysis was performed via high content analysis (HCA) and confocal microscopy.

Results:
At equivalent cisplatin concentrations 3D spheroids exhibit greater resistance compared with monolayers. Imaging experiments have shown that these 3D structures have a central necrotic core, a feature of the asymmetric growth patterns associated with 3D structures.

Conclusion:
Happy Cell ASM is a novel 3D culture medium for generating multicellular tumour spheroids and has potential for HTS and HCSA. When treated with cisplatin our spheroids exhibited resistance to therapy compared to 2D monolayer cultures. These results suggest that spheroids may provide a more accurate in vitro model to elucidate mechanisms of drug resistance and may aid the identification of novel targets to re-sensitise patient therapy.

Background:
G-CSF is a hematopoietic growth factor which enhances the proliferation and differentiation of neutrophil precursor cells. However the results of studies on G-CSF-induced tumor growth are controversial. Recently, some studies reported that G-CSF stimulates the growth of tumor cells such as colon cancer cells, small lung cancer cells , skin carcinoma cells and astrocytoma cells, In contrast, Brandstetter et al. reported that G-CSF does not exhibit any effect on the proliferation of ovarian carcinoma cell lines or tumor samples despite presence of the G-CSF receptor in the tested cell lines and biopsies.

Methods:
In vitro effects of G-CSF on tumor cell proliferation. Two mouse non-small lung cancer cell lines, Lewis lung cancer cell line (LL-2) and KLN-205 were grown in DMEM medium with FBS. In vivo evaluation of the effects of G-CSF on tumor growth. Seven week-old male C57BL/6 mice were purchased from CLEA Japan . LL-2 cancer cells were grown in culture, harvested and subcutaneously injected as a suspension into the C57BL/6 mice in the proximal dorsa midline. The mice were randomized into 4 groups, group 1) saline control, 2) G-CSF alone, 3) CDDP alone and 4) CDDP plus G-CSF group. The mice were injected 5 mg/kg CDDP intraperitoneally 2 hours after tumor cell transplantation and then, were given 5 mg/kg CDDP intraperitoneally each week. Two hours after CDDP or saline injection, the mice were given 30 mg/kg G-CSF or the same volume of saline intraperitoneally each day, and 21 days after tumor cell transplantation they were sacrificed and the tumors were removed.

Results:
We found that LL-2 and KLN-205 cell proliferation was unchanged significantly in the presence of various concentrations of G-CSF. To ensure that the results were due to the absence of the G-CSF receptor,we investigated the G-CSF receptor mRNA in these two cell lines by RT-PCR.Groups of mice were intraperiotoneally given 5mg/kg CDDP or saline per week starting 2 hours after tumor cell transplantation. Then, 2 hours after CDDP or saline injection the mice were intraperitoneally given 30m g/kg G-CSF or saline per day. Tumor growth was markedly inhibited in the CDDP and CDDP+G-CSF treatment group compared with the saline control group. Concurrent administration of G-CSF significantly enhanced the tumor suppressing effect of CDDP in early stage tumor growth. 7 days after tumor cells transplantation, the tumor volume were 6.84±9.07 for CDDP plus G-CSF treatment VS 16.34±10.29 mm3 for CDDP alone (p=0.047).

Conclusion:
In summary, our results provide evidence that G-CSF as a growth factor does not promote tumor cell proliferation. Concurrent (Combination) administration of G-CSF significantly enhances the tumor suppressing effect of CDDP in early stage tumor growth. Thus, concurrent (combination) administration of G-CSF with anticancer agents is a safe and effective method for reducing chemotherapeutic agent-induced myelosuppression. In spite of further studies are required to determine whether this effect of G-CSF is a common feature against lung cancer and the solid tumors of the other organs, in this time, our study suggested a novel importance of G-CSF treatment against cancer therapy.

Background:
The molecular target anticancer drugs such as EGFR-TKI and ALK inhibitor have dramatically changed the strategy of medical treatment for lung cancer. The investigation of each driver mutation is recommended to pick up the responder to the corresponding molecular targeting drugs. In-vitro anticancer drug sensitivity tests such as succinate dehydrogenase inhibition test (SDI) and the collagen gel-droplet embedded culture drug sensitivity test (CD-DST) are able to examine the sensitivities of the surgically resected fresh cancer tissue to multiple cytotoxic chemotherapeutic drugs at one time. We develop the method to predict the effect for multiple molecular targeting drugs for individual lung cancer patient by applying CD-DST or SDI.

Methods:
Firstly, we titrated the growth inhibitory effect of the molecular targeting drugs on cultured lung cancer cell lines (H460, A549, HCC827, H1975, H3122) using SDI and CD-DST. Secondly, we evaluated sensitivity of surgically resected cancer tissues obtained from 33 lung cancer patients to Erlotinib by using SDI or CD-DST. Finally, we compared the drug sensitivity and EGFR mutation profile.

Results:
Both Erlotinib and Crizotinib exhibited significantly stronger growth inhibitory effects on lung cancer cell lines with target gene alterations than the others without driver mutation or with T790M-mediated resistance to EGFR-TKI. In clinical study using SDI (n=21), 20μM of Erlotinib inhibited cell growth more in EGFR mutant cases (60.0 ± 9.8(%)), than in wild type EGFR cases (86.8 ± 13.9 (%)) (p = 0.0004). The area under the curve (AUC) of receiver operating characteristic (ROC) curve was 0.958 for cell viability. The ratio showed best combination of sensitivity and specificity for prediction of drug sensitivity at values >72.7 (93.3% sensitivity and 100% specificity). By using CD-DST method (n=12), the cell viabilities were 33.5 ± 21.2(%) in EGFR mutants, and 79.0 ± 18.6(%) in wild type EGFR cases (p = 0.026). The AUC of ROC was 0.963 for cell viability. The ratio showed best combination of sensitivity and specificity for prediction of drug sensitivity at values >55.9 (88.9% sensitivity and 100% specificity)

Conclusion:
The growth inhibitory effects of Erlotinib evaluated by both SDI and CD-DST were correlated with EGFR mutation profile. In-vitro drug sentivity tests may be able to predict the clinical effect of molecular targeted drugs.

Background:
Orthotopic models are likely to provide more relevant pharmacokinetic and pharmacodynamic information than subcutaneous models. We established an orthotopically implanted SCID mouse model of lung cancer without thoracotomy. This model is simple and reproducible and many transplanted mice can be produced at once. The main disadvantage of the orthotopic model is that tumor size and volume changes are difficult to continuously monitor reproducibly and can only be assessed at necropsy. In this study, we evaluated the usefulness of small-animal computed tomography (CT) to non-invasively and repeatedly monitored the inhibitory effect of cisplatin and erlotinib on lung cancer in an orthotopic SCID mouse model. Our goal was to establish a standard model to evaluate efficacy of novel treatment regimens in lung cancer.

Methods:
We created an orthotopic lung cancer transplantation model in mice. Suspensions of 2.0 × 10[4] cancer cells were injected into the left lung of SCID mice. We tested several non-small cell lung cancer cell lines, A549, FT821 and PC9 cells—only PC9 cells have an epidermal growth factor receptor (EGFR) mutation. We treated mice with cisplatin or erlotinib. When tumor volume had reached 1–3 mm[3], mice were divided into three groups: control, cisplatin and erlotinib. After treatment had begun, tumor volumes were evaluated by CT measurement every 3 days. All mice were sacrificed for histopathological analysis on day 18 after treatment began.

Results:
Mice implanted with A549, FT821 and PC9 cells were treated beginning on day 21, 50 and 35, respectively, after implantation. In mice transplanted with PC9 cells, tumor volume in the cisplatin group measured by CT was lower than in the control group, though not achieving statistical significance. In mice with A549 cells, tumor volume in the cisplatin group was similar to that in the control group. In mice with FT821 cells, tumor volume in the cisplatin group was significantly lower than in the control group. The mice in the cisplatin group showed temporarily decreased body weights. Histopathological analysis on day 18 after treatment showed necrotic lesions in lungs of mice transplanted with PC9 and FT821 cells but not in those with A549 cells. In mice with PC9 cells, which have a deletion of exon 19 in the EGFR gene, tumor volume in the erlotinib group was significantly lower than in the control group. In mice with A549 and FT821 cells, tumor volume in the erlotinib group was similar to that in the control group. There were no body weight changes in the erlotinib group. Histopathological analysis on day 18 after treatment showed necrotic lesions in lungs of mice implanted with PC9 cells but not in those with A549 and FT821 cells.

Conclusion:
This study supports using CT to monitor, non-invasively and repeatedly, tumor progression and therapeutic response of lung cancer in an orthotopic mouse model. This model is more analogous to the clinical condition than subcutaneously transplanted tumor models. Therefore, orthotopic tumor models have potential value as fundamental tools for the design and development of new therapies for cancer treatment.

Background:
To investigate the antitumor efficacy of histone deacetylase inhibitor (HDACi) or in combination with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) in non-small cell lung cancer (NSCLC) cell lines.

Methods:
Ten NSCLC cell lines with varying mutation status were treated with chidamide (HDACi) and icotinib (TKI) alone or in combination. MTS assay was performed to determine IC~50~ of each drug or in combination. Cell cycle was analyzed by flow cytometry. Markers of epithelial-to-mesenchymal transition (E-cadherin), apoptosis (caspase-3, PARP) were determined by western blot.

Results:
The results demonstrated that A549 (TKI-resistant, KRAS-mutated), HCC827 (TKI-sensitive, EGFR-mutated), HCC827IR (TKI-resistant, EGFR-mutated) was sensitive to chidamide, the IC~50~ of these three cell lines was less than 0.5nM and the IC~50~ of the other seven cell lines was more than 5μM. Chidamide increased the sensitivity of icotinib synergistically in EGFR and KRAS wild type cells (H292, Calu-3), KRAS mutant cells (A549, H460), and TKI resistant EGFR mutant cells (H1650, H1650GR, HCC827IR, H1975), but the synergistic effect was most meaningful in H1975 (EGFR L858R and T790M mutation). We also found that H460 and Calu-3 had no E-cadherin expression, H1975 had low level of E-cadherin expression, and the other seven cell lines had relatively high levels of E-cadherin expression. Moreover, with the increasing dosage of chidamide, E-cadherin expression was significantly increased in H1975 cell line, but was not changed in chidamide sensitive cell lines. In addition, chidamide alone or in combination with icotinib could induce H1975 cell cycle arrest at G1/S phase, and reduce the expression of casepase-3 and PARP.

Conclusion:
These results suggest that chidamide as a single agent exhibits antiproliferative effectives in NSCLC cells with EGFR and KRAS mutations. The combination of chidamide and icotinib may be a beneficial treatment strategy for NSCLC with EGFR-T790M mutation. But the role of chidamide in the antiproliferative or synergistic mechanisms should be further explored

Background:
Oncolytic viruses have been extensively studies in the past two decades and are promising for cancer treatment. We have shown previously that vesicular stomatitis virus expressing interferon β (VSV-IFNβ) has oncolytic activity in vitro and in vivo in an immune competent mouse model of NSCLC. However, for treatment of metastatic NSCLC, intravenous delivery of VSV-IFNβ still faces several challenges, such as rapid clearance from bloodstream due to serum complement as well as sequestration in lymphoid tissue. In order to overcome these problems, we are exploring the potential role of blood outgrowth endothelial cells (BOECs) as carrier cells to deliver VSV-IFNβ to lung tumor sites.

Methods:
Efficacy of VSV-IFNβ-infected BOECs in transferring VSV-IFNβ to co-cultured human lung cancer cell lines in presence or absence of VSV antiserum were tested in vitro. A/J mice intravenously injected with LM2 non-small cell lung cancer cells were treated with PBS, VSV-IFNβ or VSV-IFNβ-infected BOECs (3 sequential treatments 3 weeks after tumor cell injection). Tumor growth, intratumor viral titer and survival were tested.

Results:
We demonstrated that VSV-IFNβ-infected BOECs can effectively transfer VSV-IFNβ to co-cultured human lung cancer cells and result in viral oncolysis even in the presence of VSV antiserum. In mice bearing metastatic lung cancer, BOECs injected via tail vein preferentially accumulated in lung tumor tissues, and were absent in either normal lung or liver tissues. Moreover, treatment with VSV-IFNβ-BOECs had higher and more sustained intra-tumoral viral titers comparing with those treated with either PBS or naked VSV-IFNβ. Furthermore, there was a trend (p=0.09) towards reduced tumor burden in the VSV-IFNβ-BOEC treated mice (n=5). Currently, we are testing the survival benefit of VSV virus in metastatic lung cancer model.

Conclusion:
In summary, the pre-clinical data showed promise to support developing a clinical protocol in the near future to assess the safety, response and efficacy of VSV-IFNβ-infected BOECs in treatment of metastatic lung cancer.

Background:
To establish mouse models of radiation-induced esophagitis with fractionated irradiation using BALB/c or C57Bl/6 mice

Methods:
Thoracic irradiation at 0, 8, 12, or 15 Gy was given daily for 5 days by 320 kV X-ray irradiator to anesthetized, 6 week-old male BALB/c (n = 4~5 per group) or C57Bl/6 mice (n = 4 per group). Changes in the body weight and daily food intake were assessed for both strains of mice. At day 11, BALB/c esophagus was harvested and examined for the following assays: (i) histology by H&E staining; (ii) Cytokine array (R & D Systems); (iii) fluorescence-activated cell sorting (FACS) analysis by using Annexin V and propidium iodide (PI); (iv) quantitative real time-PCR (qRT-PCR) (Life Technologies) analysis.

Results:
We observed that fractionated irradiation produced a significant body weight reduction in Balb/c mice (20% by 12Gy X 5 and 30% by 15 Gy X 5). In contrast, C57Bl/6 mice seemed to be more resistant to fractionation irradiation as they exhibited little change in the body weight. As food intake in Balb/c mice was also significantly decreased at these doses compared to the control mice (p<0.05 for 12 Gy X 5 and P<0.01 for 15 Gy X 5), dose of 12Gy x 5 were selected for all assays. Histopathology of irradiated Balb/c mice showed erosive epithelium, mucosal detachment, and leukocyte infiltration. FACS analysis confirmed that irradiated esophagus had increased number of apoptotic cells, as evidenced by Annexin V and PI double positivity. We found that cytokines for C5/C5a, Timp-1 (tissue-inhibitor of metalloproteinases-1), Ccl2/Mcp-1 (monocyte chemoattractant protein-1), and Il-16 (interleukin-16) were increased in the irradiated esophagus compared to non-irradiated esophagus. qRT-PCR analyses revealed that Timp-1 as well as other genes involved in extracellular matrix remodeling including Pai-1 (plasminogen activator inhibitor-1), Gm-csf (granulocyte macrophage-colony stimulating factor), Vegf (vascular endothelial growth factor), and Sdf-1 (stromal-derived factor-1) were increased whereas Egf (epidermal growth factor), a potent mitogen for epithelial cells, was significantly decreased in the esophagus of irradiated mice.

Conclusion:
We established that BALB/c mice were more sensitive to fractionated irradiation than C57Bl/6 mice for developing symptoms reflecting radiation-induced esophagitis. In BALB/c mice, 12 Gy X 5 regimen seem to be the best schedule producing a significant reduction in the body weight and food intake, and histopathologic features similar to human esophagitis. Increased RNA transcripts for extracellular remodeling and cytokines indicate an active dynamics of tissue remodeling in the irradiated esophagus. Decreased Egf expression in the irradiated esophagus suggests that EGF may be a potential therapeutic strategy to treat radiation-induced esophagitis and we are currently investigating this strategy.

Background:
Mammalian target of rapamycin (mTOR) plays an important role in the physiological regulation of cell growth and development. Dysregulation of mTOR signaling frequently occurs in malignancies, including lung cancer. Inhibition of mTOR is a promising therapeutic strategy against lung cancer shown by clinical trials. But its mechanism remains unclear. Epithelial–mesenchymal transition (EMT) is critical in the pathogenesis of lung carcinoma. This study aimed to examine the effect of rapamycin on TGF-β1-induced EMT in lung carcinoma cells.

Methods:
Lung carcinoma cells (A549 cells) were pre-incubated with rapamycin and stimulated with TGF-β1. Morphological changes were observed under microscope. Cell phenotype markers were analyzed by western blotting and immunocytochemistry. F-actin cytoskeleton rearrangement was examined by phalloidin staining. Cell migration ability was measured by cell scratch test. The phosphorylated Smad2/3 and mTOR were measured by western blotting.

Results:
Firstly, TGF-β1 induced EMT in lung carcinoma cells confirmed by the morphological changes, as well as the down-regulation of epithelial marker (E-cadherin) and the up-regulation of mesenchymal marker (fibronectin) and the F-actin cytoskeleton rearrangement during which the mTOR pathway was activated. Secondly, rapamycin decreased the degree of TGF-β1 induced morphological changes, attenuated the down-regulation of E-cadherin and up-regulation of fibronectin, and inhibited the F-actin cytoskeleton rearrangement. Moreover, rapamycin inhibited the migration ability of lung carcinoma cells. Further research on mechanism showed that the attenuation TGF-β1-induced-EMT by rapamycin was associated with the down-regulation of the phosphorylation of Smad2/3.

Conclusion:
Rapamycin attenuated TGF-β1- induced EMT and migration in lung carcinoma cells which was mediated, at least in part, by decrease of Smad2/3 phosphorylation.

Background:
Bevacizumab is a recombinant, humanized, IgG1 monoclonal antibody that binds to and inhibits the activity of vascular endothelial growth factor (VEGF), and is approved to treat a variety of advanced solid tumors. PF‑06439535 is under development as a potential biosimilar to bevacizumab.

Methods:
Amino acid sequences of PF‑06439535 and EU‑ and US‑sourced bevacizumab (bevacizumab‑EU and bevacizumab‑US, respectively) were compared by peptide mapping; post-translational modifications and biochemical properties were analyzed by N-linked oligosaccharide profiling and imaged capillary electrophoresis. Functional analysis of PF‑06439535, bevacizumab‑EU, and bevacizumab‑US included an enzyme-linked immunosorbent assay to detect binding to the 4 major VEGF isoforms (VEGF~121~, VEGF~165~, VEGF~189~, VEGF~206~), and a cell growth inhibition assay in human umbilical vein endothelial cells (HUVEC). Toxicokinetics and potential toxicity of PF‑06439535 and bevacizumab‑EU were evaluated following intravenous (IV) administration (10 mg/kg twice weekly for 1 month, 9 doses total) in sexually- and skeletally-immature male cynomolgus monkeys; control animals received vehicle.

Results:
PF‑06439535, bevacizumab‑EU, and bevacizumab‑US had identical primary amino acid sequences and similar levels of N-linked oligosaccharides. Predominant charge isoforms were similar; charge heterogeneity was due to variations between PF‑06439535 and reference products in relative proportions of species with C-terminal lysines. Target binding to each VEGF isoform showed similar dose responses between PF‑06439535, bevacizumab‑EU, and bevacizumab‑US; comparable biological activity was observed by inhibition of VEGF-induced HUVEC proliferation. In cynomolgus monkeys, PF‑06439535 and bevacizumab‑EU (n=4 each) were well tolerated, with no PF‑06439535- or bevacizumab‑EU–related clinical, laboratory, or histopathology findings, except physeal dysplasia of the distal femur with similar incidence and severity for both molecules. Induction of anti‑drug antibodies was not observed in the PF‑06439535- or bevacizumab‑EU–dosed groups. Systemic exposure (mean area under the serum drug concentration–time curve from 0 to 72 hr ± standard deviation) was similar for PF‑06439535 and bevacizumab‑EU on Day 1 (12100 ± 876 vs 14700 ± 2260 μg·hr/mL) and Day 25 (45500 ± 5420 vs 45100 ± 3670 μg·hr/mL).

Conclusion:
Results from the analytical similarity assessments and nonclinical studies have supported the clinical development of PF‑06439535 as a potential biosimilar to bevacizumab. These data supported a randomized, double-blind phase I study (NCT02031991) in healthy human male volunteers in the United States, which assessed pharmacokinetics, safety, and immunogenicity of a single 5 mg/kg IV dose of PF‑06439535, bevacizumab‑EU, or bevacizumab‑US. A global, randomized phase III trial (NCT02364999) comparing PF‑06439535 and bevacizumab‑EU, plus paclitaxel/carboplatin, for first-line treatment of advanced non-squamous non‑small cell lung cancer is enrolling patients.

Background:
The study aimed to investigate the therapeutic effect of interleukin-2 (IL-2) treatment combined with magnetic fluid hyperthermia (MFH) on Lewis lung cancer-bearing mice.

Methods:
Magnetic fluids were prepared in vitro and directly injected into the tumors in the mice, which were subjected to an alternating magnetic field. The temperature in the tumor reached 43°C and was maintained by controlling the strength of magnetic field for 30 minutes. Twenty-four hours later, IL-2 was injected directly into the tumors. Mice were divided into four groups: group I (control), group II (MFH), group III (IL-2), and group IV (IL-2+MFH).

Results:
The tumor grew gradually in group II and group IV (both P<0.05) compared to the control group. Histological analysis showed that the tumor cells underwent apoptosis and necrosis. Immunohistochemistry results demonstrated that heat shock protein 70 (HSP70) and CD8-positive T cells were strongly expressed.

Conclusion:
The results have provided evidence that IL-2 treatment combined with MFH could improve the therapeutic effect on lung cancer-bearing mice

Background:
Clinical management of malignant pleural effusions (MPE) is a major problem in oncology with short survival. MPE is caused by various types of malignancies, especially lung, breast carcinomas, lymphomas besides ovarian carcinoma, malignant melanoma. Intrapleural chemotherapy might be a helpful treatment strategy in MPE cases. Intrapleural chemotherapy also enters systemic circulation and affects the primary tumor as well. The aim of this study is to evaluate ex vivo chemoterapy and targeted therapy sensitivity in MPE cases to project which drug might be effective.

Methods:
Effusion fluids from patients with MPE were fresh obtained. After centrifugation, the pellet was diluted in PBS and cell isolation was performed by Ficoll gradient to separate cells from erytrocytes. Cells were incubated in HITES supplemented complete RPMI medium at 37C with 5%CO2. After primary cell culture was obtained, cells were incubated to 96 well plates and agents ( Bevacuzimab, Cetuximab, Rituximab, Bortozemib, Gemsitabin, Vinblastin, Bleomycin, Docetaxel, 5 Florourasil, Cisplatin, Cyclophosphamide, Doxorubicine) in different dose ranges for 24 hours. WST-1 was performed to check cell viability.

Results:
The primary tumors of nine cases in this study with MPEs are breast carcinomas, lung adenocarcinoma, small cell carcinoma and mantle cell lymphoma. Resistance were observed in most drugs. Breast carcinoma cells and lung adenocarcinoma cells were sensitive to cisplatin and/or vinsblastin. Small cell carcinoma cells were sensitive to docetaxel and/ or bleomycin. Sensitivity was not observed to targeted therapy agents at single dose during 24 hours incubation.

Conclusion:
Our results indicate that ex vivo cancer cell culture and testing cell death results of various chemotherapoetic and new targeted drugs might help managing highly agressive disease of patients with MPE. Intrapleural chemotherapy application that is found sensitive by ex vivo tests might help patients.

Background:
The aim of the study is to estimate the role of JAK/STAT3 signaling pathway on apoptosis of lung adenocarcinoma induced by icotinib.

Methods:
EGFR mutation was detected in lung adenocarcinoma cell line PC-9 by ARMS assay; The inhibitory rates of cell proliferation, at different concentrations (0 ~ 100 umol/L) of icofinib and continued incubating for 24，48 and 72 h respectively, were evaluated by MTT assay; Apoptosis of PC-9 cells exposured to different concentrations of icotinib(0, 0.1, 1 and 10 umol/L) for 48 h were evaluated by TUNEL assay; JAK2, STAT3, Bcl-2, Bax mRNA expressions were evaluated by Real-time PCR assay; The protein levels of P-STAT3 and IL-6 were evaluated by Western-blot assay.

Results:
Human lung adenocarcinoma cell line PC-9 had an exon 19 deletion mutation in EGFR gene; Followed by treatment of icotinib，the proliferation of PC-9 cells were all inhibited significantly, especially in 48 and 72 h (P<0.01) in all concentrations; The inhibitory rates of cell proliferation in different treating time had statistical significance (P<0.01); Cell apoptosis at different concentrations were increased significantly (P<0.05); Along with the increasing concentrations, gen expression levels of JAK2, STAT3 and Bcl-2 decreased significantly (P<0.05), Bax increased significantly (P<0.05), JAK2/STAT3 ratios increased significantly (P<0.01), and Bcl-2/bax ratios decreased significantly (P<0.01); P-STAT3 and IL-6 protein levels were inhibited significantly at by higher concentration.

Conclusion:
JAK/STAT3 signaling pathway take a participate in apoptosis of PC-9 cells induced by icotinib. The most likely mechanism is icotinib inhibited the gen expression levels of JAK2, STAT3 and Bcl-2, so with the P-STAT3 and IL-6 protein levels, and mediated gene Bax overexpression.

Background:
In 2012, North Dakota enacted a comprehensive smoke-free law. In 2014, the 3rd phase of a stratified random sample longitudinal study of tobacco smoke pollution in restaurants and bars was conducted (n = 107). Phase 1 was conducted prior to passage of the law, Phases 2 and 3 were conducted 3 and 21 months post-implementation respectively.

Methods:
Tobacco smoke pollution levels were assessed by collection of particulate matter 2.5 microns aerodynamic in diameter or smaller using SidePak [TM] AM510 Personal Aerosol Monitors.

Results:
The geometric mean PM~2.5 ~was 6.9 microns/m[3]. Statistically significant reduction in mean PM~2.5~ occurred from Phases 1 to 3 but not from Phases 2 to 3 in all venues and for bars alone. A significant increase in indoor PM~2.5~ occurred when there was outdoor smoking or ashtrays within 20 feet of the venue entrance, exit, or windows and when smoking was observed within designated outdoor smoking shelters. Multi-level linear models found that the presence of a local ordinance and venue type were predictors of PM~2.5~ in Phase 1 but not in Phases 2 or 3. Significant decreases in mean PM~2.5~ by rurality occurred between Phases 1 and Phase 3. In contrast with the Phase 1 study, there were no significant differences in PM~2.5~ by rurality in only Phase 3.

Conclusion:
This longitudinal study is the largest rural pre and post-law rural study known globally. Passage of the comprehensive statewide smoke-free law effectively reduced PM~2.5~ levels in restaurants and bars statewide.

Background:
The Youth Smoking Prevention Foundation is taking the Kingdom of the Netherlands to court to end the structural and excessive influence exerted by the tobacco lobby on government anti-smoking policies. The Foundation is calling on the Dutch government to comply fully with the anti-smoking convention (WHO FCTC), which it signed and which is therefore legally binding. One of the most important articles in the convention states that every form of influence by the tobacco industry on policies to deter smoking must be avoided. In the court summons issued, the Foundation offers dozens of examples that show how the government has systematically violated this provision, and even invites the tobacco industry to clarify its position on matters of policy development. 19,000 tobacco-related deaths More than 19,000 Dutch people, half of them younger than 65, die each year as a result of smoking. In addition, an average of 120 children under the age of 18 start smoking every day. Some 60 of them will continue to smoke for the rest of their lives, and 30 of them will die prematurely from the effects of smoking. Smoking is by far the biggest cause of death that could be avoided through prevention. However, the marketing techniques deployed by the tobacco industry are so refined that many youths cannot resist the temptation to start smoking. Moreover, cigarettes are designed to be highly addictive. Children who start smoking end up addicted within weeks. For many of them, the question of ‘free will’ no longer applies: they are unable to stop smoking without help. Numerous national and international laws and conventions make it a duty of the Dutch government to protect the health of its citizens from a serious cause of illness like tobacco. With as many as 19,000 tobacco deaths every year, the government has an obligation to do all in its power to combat the massive scale of premature fatalities. And it should certainly prevent minors from starting to smoke, because almost nobody starts after they turn 18. Despite all this, the Dutch government has failed to implement measures that could be very effective in achieving results: imposing much higher taxes on tobacco and greatly reducing the current number of over 60,000 points of sale. Instead, the government listens to the tobacco industry, whose effective lobbying continues to successfully obstruct measures to discourage tobacco use.

Methods:
not applicable

Results:
ongoing lawsuit.

Conclusion:
The lawsuit is ongoing. Our foundation has had a lot publicity in all national media (television ,newspapers) As a chest physician working in an oncology center mainly treating patients with lungcancer it is very powerfull to start a lawsuit against the state to prevent lung cancer. We are making progress: we are at the table of several Ministries (finance, health department) to discuss the firewall protocol against the lobby of big tobacco. We use social media and patients advocates to make our message even stronger

Background:
Smoking prevention in schoolchildren with the aim to inform and prevent smoking initiation has been widely studied and has shown variable results. Interventions provided by physicians in a hospital setting have been rarely reported. Here we show the feasibility and gain of knowledge of our smoking prevention project in a hospital setting.

Methods:
Interventions performed from November 2009 - December 2014 were evaluated. Overall 790 children participated in our preventive intervention. A 7-item questionnaire was provided to the school classes (Grades 6 to 10) before and after a two-hour smoking prevention intervention consisting of anatomical models, oral presentations, videos, patient interviews and hands-on lung function tests. The goal was to show the anatomical and physiological basics as well as age-based information about the harms of smoking. During the intervention the children have been motivated to be actively involved. Class selection has been performed for groups of children in a highly vulnerable phase of age before smoking initiation.

Results:
The baseline questionnaire was completed by 768 children, the one after intervention by 719. The knowledge about which organs are affected by smoking increased from 7.1-99.3% to 64.5-99.5% (p<0.01). While only 58.9% knew that only a minority of people is able to quit smoking successfully, 96.3% answered the question correctly after intervention (p<0.001). Prior to the intervention only 75.6% believed that minor tobacco consumption is not damaging which increased to 87.8% after the teaching session (p<0.05). Smoking hookah was believed to be less harmful than cigarettes by 32.2% of children decreasing to 8.3% after the intervention (p<0.001).

Conclusion:
Information on health effects provided by lung specialists in the hospital leads to a statistically significant increase in knowledge as assessed by a short questionnaire. The intervention is feasible and well received. This kind of interventions might help to prevent schoolchildren from smoking in a highly vulnerable phase of age.

Background:
Smoking is a risk factor for several lung diseases. Quitting smoking provides positive outcomes and gives the best chance for the treatment in patients with pulmonary diseases, including lung cancer diagnoses. Currently few centers in Italy offer counseling for smoking cessation in cancer patients (and for patients with other lung diseases), despite the demonstrated efficacy of it.

Methods:
408 patients with pulmonary diseases (72% with lung cancer) were prospectively and sequentially evaluated from January 2013 to February 2015. An anonymous survey was developed with the aim to understand if current or former smoker patients received information by healthcare workers about smoking cessation before or after the diagnosis, their reaction and the actions adopted for quitting smoking. The survey included the Fagerström test for assessing the intensity of addiction to nicotine and it was conducted in several Italian Thoracic Oncology Units and Pulmonology Divisions.

Results:
After a pulmonary disease diagnosis, 72% of patients state to quit smoking, 20% to smoke less or not feel the same pleasure as before and only 8% confirms to continue to smoke or smoking even more. Among former smokers (298 people), 150 patients state how long they quitted smoking and in 45% of the cases was at the time of diagnosis or even later, about 35% 10 years before the diagnosis and 8% between 5 and 10 years earlier, while 12% more recently. Most of current smokers state that they continue because smoking helps them to control the stress, others because they like it or are not able to quit and very few because is a repetitive gesture. Data show that 39% of patients did not receive information about smoking cessation by health professionals, 26% received it before the diagnosis, 12% after it and 23% received it both before and after the diagnosis. Concerning the reaction to the counseling, 53% considers positively the health care provider action, even if 28% hoped they could have helped them more quit smoking and 19% reports a warning and paternalistic attitude of them. Only 23% of patients who attempted to quit smoking considers the gradual termination as the most effective measure, more than the sudden interruption. Regarding the smoking-cessation method or specific therapy adopted, 65% disclosed they simply quitted smoking overnight and 80% confirmed it as the most effective technique, while only 16% used electronic cigarettes, 8% a nicotine replacement treatment, 7% books and 4% attending a dedicated clinic. The Fagerström Test confirms that 50% has a low to moderate dependence to nicotine, while 50% has a high dependence.

Conclusion:
The survey was distributed to 293 lung cancer patients and 115 with pulmonary disease (mainly COPD patients). The result analysis underlines that the vast majority quitted smoking after having received their diagnosis. No main differences were seen evaluating the group with malignant and non-malignant diseases. Although many of them got advice by healthcare workers, the recourse to the use of techniques, drugs or access to specific clinic is still very low, especially considering that 50% of patients result highly dependent to nicotine.

Background:
Smoking is the largest cause of premature mortality. Smoking cessation is important, but is difficult to reach. A general underestimation of personal risk in smokers or a degree of misunderstanding around key risk factors for disease may be substantial. [1]The aim of this abstract is to calculate the reduction in average life expectancy per cigarette.

Methods:
Men born in 1900-1930 who smoked only cigarettes and continued smoking died on average about 10 years younger than lifelong non-smokers. Cessation at age 60, 50, 40, or 30 years gained, respectively, about 3, 6, 9, or 10 years of life expectancy.[2]Assuming that these men started at age 15 years and died at age of 72 this results on average in 57 years of smoking. Also assumed is that each day one pack of 20 cigarettes is smoked.

Results:
Smoking for 57 years 20 cigarettes per day results in a total of 416,100 cigarettes. The total number of minutes in 10 years is 5,256,000. The average decrease in life expectancy is 12.6 minutes/ cigarette or 4.2 hours /pack, equals more than a day/week. Discussion: If a smoker is aware of the reduced life expectancy then smoking of one cigarette may be looked-upon as a mini-suicide attempt. Taken also into account the passive smoking effect, the smoker may be seen as a mini-suicide-nano-terrorist.

Conclusion:
Conclusion The reduction in average life expectancy is 12.6 minutes per cigarette or 4 hours per pack. This knowledge may be of help to raise more awareness for the dangers of smoking. 1. Bethea J, Murtagh B, Wallace SE. “ I don ’ t mind damaging my own body ” A qualitative study of the factors that motivate smokers to quit. 2015;1–9. 2. Doll R, Peto R, Hall E, Wheatley K, Gray R. Mortality in relation to consumption of alcohol: 13 years’ observations on male British doctors. BMJ. 1994;309(6959):911–8.

Background:
It has been a normal practice for governments, not-for-profits and other platforms to provide Quitlines to help smokers quit. There has been positive results, however a recent study shows that a one-stop platform can offer more desirable outcomes.

Methods:
We conducted a 6-month study through an online survey involving 1,200 smokers who visited a revolutionary one-stop smoking cessation online platform, www.quitgate.com. Figure 1After visitors ordered a product or called the Quitline, a questionaire was emailed to them. Questions asked included year of smoking initiation, number of cigarettes smoked per day and number of quit attempts and through what means.



Results:
Interestingly, 37% of the participants reported that they were motivated to quit because when they called the Quitline and received counselling from the Tobacco Treatment Specialist, they were immediately provided without obligation, the option of getting a smoking cessation product on same platform with either some of the product free or highly discounted. Another group, 11% said they prefered the platform to quit because it was social, friendly, professional, non-judgemental and yet non-clinical. Overall, most of the participants said it was a great idea to have a one-stop platform which provided free professional counselling, smoking cessation products, tools/apps like smoking calculator, DNA (Dependence on Nicotine Assessment) low prices, free shipping and premium customer service to highly motivate smokers quit for good.

Conclusion:
It is great to note that while Quitlines are provided by several institutions to help in smoking cessation, an important area of also making smoking cessation products availble either for free or a little amount will go a long way to motivate smokers. The Centers for Disease Control and Prevention-CDC cites evidence-based counseling, behavioral cessation therapies, medication, and social support as treatments that increase the chances of tobacco cessation

Background:
When epidemiologic research first demonstrated an association between cigarette smoking and lung cancer in the early 1950s, adenocarcinoma comprised about 5% of lung cancers and appeared to be unrelated to smoking. In the 1960s and 1970s, adenocarcinoma increased sharply, and became strongly related to cigarette smoking. At the 2007 IASLC-sponsored 12th World Conference in Lung Cancer in Seoul, Korea, our group reported that by 2003, adenocarcinoma of the lung had risen to comprise 47% of all lung cancers in the US. The objective of this presentation is to update and expand upon our previous analysis.

Methods:
We analyzed time trends in lung cancer histology with changes in cigarette design and Tobacco Industry actions over six decades. We utilized Surveillance-Epidemiology and End Results (SEER) data on 419,941 lung cancers diagnosed between 1973 and 2011 to analyze time trends of age-standardized incidence rates of five histologic subtypes: adenocarcinoma, squamous cell, small cell, large cell, and adenosquamous carcinoma.

Results:
Over time, the percentage of lung cancers that were adenocarcinomas increased from 29% (in 1973-1974) to 55% (in 2010-2011). During this 38-year period, the percentage of lung cancers that were squamous cell carcinomas decreased from 41% to 26%. Among all patients, adenocarcinoma incidence surpassed squamous carcinoma by 1985-1989 to become the most common histologic subtype. Adenocarcinoma surpassed squamous cell in 1990-1994 in men, while it was already most common in women by 1973-1974. Adenocarcinoma rose 77% in men from 1973-1974 to 1990-1994, while it rose 197% in women between 1973-1974 and 2005-2006. Among whites, adenocarcinoma surpassed squamous carcinoma by 1985-1989, while this occurred among blacks by 1990-1994. It was already most common among other race individuals in 1973-1974. Adenocarcinoma was already most common among patients <50 years of age by 1973-1974, while adenocarcinoma rapidly increased and surpassed squamous carcinoma in all other age groups by 1990-1994.

Conclusion:
Incidence of adenocarcinoma of the lung has continued to increase to such an extent that it comprises a clear majority of all lung cancers in the US. Indeed, our analysis demonstrated that lung adenocarcinoma currently represents 55% of US lung cancers. It is the most common histology in men and women, in whites, blacks, and other-races, and in all age groups. The question of how the actions of Big Tobacco helped to create this epidemic will be addressed in a separate presentation at this meeting.

Background:
Lung cancer is one of the malignant tumors whose global incidence and mortality are very high. The chemoprevention has become an important prevention and control means of lung cancer except for giving up smoking and early detection. Research has showed the main component in green tea (-)-epigallocatechin-3-gallate (EGCG) is a potential chemopreventive agent for various tumors, especially lung cancer.

Methods:
The cells in each group were treated with different concentrations of EGCG for a certain time in the experiment. Two gene point mutation plasmid were constructed and transfected in A549 cells. Induction of apoptosis was examined using AnnexinV/Pl double staining flow cytometry. Western Blot detected the protein expressions of Bax, Bcl-xl and Caspase-3. Co-immunoprecipitation was used to detect the interaction of Ku70-Bax and acetylation status of Ku70. P<0.05 showed the difference had statistical significance.

Results:
Treatment of A549 cells with EGCG induced apoptosis with increasing expression of Bax and Caspase-3, but decreasing expression of Bcl-xl. EGCG could up-regulate K70 acetylation status of A549 cells，then down-regulate the interaction of Bax-Ku70 in the manner of concentration and time dependent. The apoptosis-promoting effect of EGCG on A549 cells was obviously weakened with the interaction of Bax-Ku70 strengthened and Caspase-3 (17KDa) expression declining after pCDNA3.1(+)-Ku70 plasmid and pCDNA3.1(+) -Ku70[539/542R] plasmid transfection.

Conclusion:
The authors induced apoptosis in human lung adenocarcinoma A549 cells after treatment with EGCG, and it was realized by interfering the interaction between Ku70 and Bax through regulating K70 acetylation. It verified that two loci K539 and K542 of Ku70 acetylation might play a crucial role in EGCG inducing apoptosis of A549 cells.

Background:
Indeterminate lung nodules are a common and increasing incidental finding on CT imaging and there are widely accepted surveillance protocols. However, even when using Low Dose (LD)-CT with a total effective dose of ~1mSv, concerns exist regarding the cumulative radiation exposure of subjects under surveillance, particularly in individuals not at high risk of lung cancer. By utilizing the Model Based Iterative Reconstruction (MBIR) technique, CT images can be obtained with a radiation dose comparable to chest x-ray (0.06-0.1 mSv). At this Ultra-Low Dose (ULD), MBIR images have generally less signal to noise ratio which may prevent small nodule detection. The aim of this prospective study was to assess the efficacy of ULD-CT in detecting clinically significant lung nodules (≥4mm) as compared to LD-CT.

Methods:
Following approval from the local Human Research Ethics Committee, adult subjects undergoing CT surveillance for incidental lung nodules were recruited from a tertiary hospital. Once informed consent was obtained, both standard LD- and a ULD-CT chest were performed. Scans were performed on the GE750HD Discovery scanner. Demographic information including lung cancer risk factor evaluation was obtained by questionnaire. Patients who withdrew consent or whose images were degraded by gross movement or metallic artefacts were excluded. Images from the ULD-CT were reconstructed with MBIR prior to reading. Each of LD/ULD-CT image sets was read blindly, randomly and independently by two experienced thoracic radiologists. The number, size and location of nodules was reported and subsequently compared.

Results:
100 subjects were recruited with a mean age of 65 years (range 32-87). Around 62% were ever smokers, with 30% smoking ≥30 pack years. Around 30% had risk factors other than smoking, but only ⅓ of these (9%) did not have a significant smoking history. Only a small proportion were high risk as evidenced by only 8 meeting Lung Cancer screening criteria (NLST criteria). A total of 200 nodules ≥4mm were detected, with all seen on both LD and ULD-CTs. In addition, there were 244 nodules <4mm seen on the LD-CT, with greater than 80% sensitivity for the ULD-CT, with minor variation between lobes. There were no false positive findings. There was a 10 fold reduction in effective radiation when comparing ULD-CT (0.09mSv) imaging with the standard LD-CT (1.11mSv). Lung nodules were subjectively better seen on the ULD-CT.

Conclusion:
ULD-CT with the advanced MBIR allows detection of all clinically significant lung nodules while achieving a radiation dose comparable to that of plain chest radiography. Particularly in low-risk populations, the use of ULD-CT for surveillance of lung nodules has the potential to significantly reduce cumulative radiation exposure.

Background:
In December 2013, the United States Preventive Services Task Force (USPSTF) provided a level B recommendation for the use of low-dose computed tomography (LDCT) to screen high-risk patients for lung cancer. Most recently, in February 2015, the Centers for Medicare and Medicaid Services (CMS) likewise approved coverage for at-risk patients, defined as those 55 years of age or older with a strong (30 pack-year) smoking history. The current USPSTF and CMS specified eligibility criteria for lung cancer screening are similar to those implemented by the National Lung Screening Trial and other studies which provided the evidence base that precipitated the decision to screen high risk patients, however these criteria may not adequately capture all sub-groups that comprise the complete population at risk for developing lung cancer. For example, younger patients (50+ years) who have a moderate (20 pack-year) smoking history and at least one other known lung cancer-related risk factor are considered to be at high risk by the National Comprehensive Cancer Network (NCCN). The purpose of this investigation is to investigate the prevalence of lung cancer among younger and older age groups of screening patients nationwide and to begin to provide important data that may assist with evaluating the adequacy of the eligibility criteria currently being used to define the population at-risk for developing lung cancer.

Methods:
The Center for Lung Cancer Screening and Prevention at the Stony Brook Cancer Center, recently conducted an electronic survey of all Lung Cancer Alliance Centers of Excellence for Lung Cancer Screening nationwide. The survey collected information regarding numbers and age groups of patients screened, numbers and stages of lung cancers detected, smoking history and other demographic variables. Lung cancer status (cancer detected vs. no cancer detected), stratified by age group (50-54 years vs 55-80 years) are presented here. A total of 24 Centers (among 240) provided data for the survey. Many Centers did not have available data for the younger subgroup of patients likely due to the implementation of the USPSTF criteria rather than the NCCN guidelines that recommend screening this younger, at-risk subgroup.

Results:
The survey data were cumulated over all 24 participating Centers of Excellence nationwide and included 7,252 patients. Of these, n= 697 patients were 50-54 years of age and n=6,555 were 55 years or older. Among the younger cohort, 16 patients (2.3%) were found to have lung cancer. In the older age category, lung cancer was detected in 130 patients or 2.0%.

Conclusion:
These findings suggest that this younger subgroup of at-risk patients warrant further consideration for lung cancer screening. Additionally the data suggest that this well-defined subgroup of 50-54 year old patients who have a moderate smoking history and at least one other known lung cancer-related risk factor may be at even higher risk for developing the disease than those 55+ years with a 30 pack-year smoking history. These nationwide data highlight the urgent need to re-evaluate the eligibility criteria currently being used to define the population at risk for developing lung cancer.

Background:
Following the publication of the National Lung Screening Trial (NLST) results in 2011, CT screening for lung cancer is now widely recommended in the US. However concerns remain with regards to patient selection according to risk level and overdiagnosis.Moreover adherence outside screening trials is typically about 50-60% and has been shown to be highly dependent on an individual's risk perception. This feasibility study explores the relevance of gene-based data on lung cancer risk assessment and adherence to screening, in a pilot screening program.

Methods:
This feasibility study was initiated in 2010 prior to NLST results being published. Following local media-based advertising, 157 current or former smokers (>50 years old with ≥20 pack year history), volunteered for lung cancer risk assessment and CT screening (using the IELCAP protocol). Participants were followed up for a mean of 2.4 years.At baseline CT screening, participants were assigned their lung cancer risk category according to a published and prospectively validated gene-based risk algorithm. This algorithm combines clinical risk variables with risk genotypes, derived from analysis of 20 risk single nucleotide polymorphisms (SNPS), to derive a composite lung cancer risk score categorised as moderate, high or very high.

Results:
SNP genotype results contributed to overall lung cancer risk in 88% of participants compared to the contribution from age = 68%, family history of lung cancer = 29% and self reported chronic obstructive pulmonary disease =15%. The SNP genotype results were the sole basis of risk in 18% of participants and contributed to risk in a further 70% of participants (total 88%). Adding SNP scores to the clinical risk score re-assigned screening participants into different risk categories in 28% (44/157) of participants (Figure 1). Importantly, timely adherence to the CT screening protocol was two-fold greater in those with a very high risk score compared to the high and moderate risk categories (71% vs 52% vs 52% respectively, OR =2.3, P<0.05). Figure 1



Conclusion:
In this feasibility study of a pilot community-based CT screening program we found gene-based risk assessment was of interest to all screening volunteers. As part of risk assessment, personalised SNP data made the greatest contribution to overall assignment of lung cancer risk in association with established clinical variables and significantly improved screening adherence. We conclude that gene-based risk stratification helps assign lung cancer risk and appears to improve adherence to screening.

Background:
The National Lung Cancer Screening Trial has demonstrated the efficacy of lung cancer screening based on annual low-dose computed tomography (CT) scanning in both former and current smokers. Nationwide lung cancer screening programs are therefore expected to be implemented. Adhesion to these programs will depend largely on public information regarding lung cancer screening. Here, we report on widespread beliefs regarding lung cancer screening in the general population prior to any information campaigns on lung cancer screening.

Methods:
The EDIFICE French nationwide observational surveys, conducted every 3 years since 2005, set out to characterize behaviors related to cancer screening. The 4th edition, EDIFICE 4, was conducted by phone interviews of a representative sample of 1602 subjects aged between 40 and 75 years, using the quota method, from June 12 to July 10, 2014. Attitudes and opinions regarding colorectal, prostate, breast, cervical and lung cancer screening were assessed.

Results:
For 43% of the French population, lung cancer screening is more reassuring than distressing. This figure is lower than those reported for perceptions of other screening programs, including colorectal cancer screening (51%) and breast cancer screening (63% vs. 46.7% for lung cancer screening in the female population). Eleven percent of the respondents (N=162) declared having already undergone a lung cancer screening test. For the vast majority (87%, N=140), this comprised a chest X-ray and for 63%, (N=101) the chest X-ray was not associated with another type of examination. Respondent-declared reasons for not undergoing screening included absence of risk factors (36%), absence of respiratory symptoms (34%), absence of physician recommendations for screening (29%) and futility (11%). Seven percent of current smokers and 32% of former smokers did not undergo screening because they did not consider themselves at risk for lung cancer. Fear of the results pushed 9% of current smokers to avoid lung cancer screening. However, 22% of all respondents and 38% of current smokers declared their intention to undergo a lung cancer screening test in the future.

Conclusion:
The general population has many misconceptions of lung cancer screening. Implementation of nationwide lung cancer screening programs should include information for the general public regarding selection criteria, techniques used and the benefits of lung cancer screening using low-dose CT scanning.

Background:
Visceral pleural invasion (VPI) of non-small cell lung cancer (NSCLC) has been recognized as a poor prognostic factor. Peripheral lung cancers often invade visceral pleura, and positive VPI upstages the T category of tumors from T1a to T2a. In addition, it is possible that peripheral lung cancers with positive VPI causes lymph nodes metastasis because of subpleural lymphovascular invasion. In this study, we statistically analyzed the correlation between VPI and lymph node metastasis.

Methods:
129 patients with NSCLC and a tumor diameter of ≤ 2cm underwent lobectomy or segmentectomy with systematic lymph node dissection in Tokushima University Hospital between January 2008 to December 2013. Excluding 11 patients who were not examined by FDG-PET before the surgery, we reviewed the medical records of 118 patients to obtain information on age, sex, CEA, SUVmax, CT findings, pathological VPI and lymph node metastasis.

Results:
Patient characteristics were as follows: median age of 66.5 (range: 41-86); male/female: 52/66; histologic type adenocarcinoma/squamous cell carcinoma/other: 103/12/3. 13(36.1%) of 36 patients who were suspected to be with visceral pleural invasion by preoperative CT findings were diagnosed with pathological visceral pleural invasion. The mean SUVmax on FDG-PET in patients with VPI was significantly higher than that of patients without VPI(p=0.01). Pathological visceral pleural invasion was identified in 19(16.1%) of 118 patients and associated with high incidence of lymph node metastasis significantly on multivariable analyses (p=0.00).

Conclusion:
VPI is important factors of lymph node involvement in small peripheral lung cancers. It is difficult to identify VPI of peripheral lung cancers by preoperative CT findings. FDG-PET may be useful for diagnose VPI.

Background:
Lung cancer (LC) is the most common cause of cancer death worldwide. At present, the diagnosis is primarily based on symptoms and detection occurs at late stages, thus resulting in a very poor prognosis. If the diagnosis could be shifted to early stages, then the overall morbidity for this disease could be dramatically altered. Metabolomics, an analytical platform used in combination with statistical techniques, has been shown to be a very powerful approach for the understanding of biological pathways involved in the onset and progression of diseases. The objective of this study was to identify, using metabolomics by NMR, a set of specific metabolites that could be used for LC screening in the clinical context.

Methods:
Metabolic profiles corresponding to a training set of serum samples from early-stage (n = 66) and advanced-stage (n = 69) NSCLC patients were obtained using [1]H-NMR spectroscopy. A matched control set of 71 serum samples from healthy subjects was also included. Furthermore, NMR experiments were also performed for an external validation set consisting of 20 early-stage and 20 advanced-stage NSCLC patients, 13 healthy individuals, and 27 benign pulmonary disease patients (BPD).

Results:
Multivariate statistical modeling of the data revealed that the serum of NSCLC patients, when compared with healthy individuals, exhibit a specific serum metabolic profile (R[2 ]= 0.931; Q[2 ]= 0.873) characterized by statistically significant differences in the concentrations of a number of lipids, organic acids and amino acids. The metabolic profiles obtained for NSCLC patients and healthy individuals were also different to that obtained for BPD patients. A similar analysis performed to compare the serum metabolomic profile of NSCLC patients at early and advanced stages of the disease (R[2 ]= 0.779; Q[2 ]= 0.592) showed that disease evolution has also a reflection in the metabolic profile of patients. Furthermore, a logistic regression analysis allowed the identification of a specific combination of five metabolites (threonine, glutamine, lactate, choline and methanol) that enables the discrimination between healthy individuals and NSCLC patients with a 77,5% sensitivity and a 76,9% specificity (70% for all non-cancer samples).

Conclusion:
Our results highlight the potential of metabolomics by [1]H-NMR for identifying biological pathways involved in the onset and progression of NSCLC, thus providing a sensitive, specific, minimally invasive and easily implementable method in clinical practice for the early diagnosis of NSCLC and for the optimization of risk profile models. Acknowledgements: Spanish Ministerio de Economía y Competitividad (MINECO, SAF2011-28350), Centro de Investigación Príncipe Felipe and Fundación Mutua Madrileña for their economic support and Red de Biobancos de Valencia and Bruker BioSpin for technical contributions. This study was also supported by the ISCIII (RTICC, RD12/0036/0025).

Background:
Altered expression of microRNAs is associated with development and invasion of cancers by regulating post-transcriptionally gene function. Possibility of detection of circulating miRNAs expression in patients’ plasma or serum make them valuable biomarkers of different neoplasms, such as lung cancer.

Methods:
We investigated potential role of miR-944 and miR-3662 expression analysis as a novel lung cancer biomarkers and their lung tumor specificity in plasma samples of 90 lung cancer patients (40 NSCLC patients in stage IA-IIIA and 20 NSCLC patients in stage IIIB-IV; 8 SCLC patients with limited and 22 SCLC patients with extensive disease) and 85 healthy individuals using qRT-PCR analysis.

Results:
Expression of miR-944 and miR-3662 was significantly upregulated in lung cancer patients in comparison to healthy individuals. Higher stage of lung cancer correlated with higher miRNAs expression (Figure 1). Receiver operating curves (ROC) analysis have presented diagnostic power of analysis of both miRNAs expression for detection of patients with I and II stage of NSCLC with area under curve (AUC) of 0.881. Moreover, miR-944 has shown diagnostic accuracy for operable squamous cell carcinoma detection (AUC=0.982) whereas miR-3662 - for operable adenocarcinoma (AUC=0.926) (Figure 2).Figure 1Figure 2





Conclusion:
Our research is a first study investigating the plasma expression of miR-944 and miR-3662 in patients with neoplasms and in healthy individuals. Moreover, this is a first study that described a miR-3662 expression. We have shown that examination of these two miRNAs may be considered as a tool for NSCLC early diagnosis as well as for non-invasive diagnosis of lung cancer late stages. Studied miRNAs have also shown high utility in detection of histological type-specific NSCLC subtypes, such as adenocarcinoma and squamous-cell carcinoma.

Background:
This study was conducted to investigate whether polymorphisms of genes involved in immune checkpoints can predict the prognosis of patients with early stage non-small cell lung cancer (NSCLC) after surgical resection.

Methods:
Twelve single nucleotide polymorphisms (SNPs) of PD-1, PD-L1, and CTLA-4 genes were selected and genotyped. A total of 354 patients with early stage NSCLC who underwent curative surgical resection were enrolled. The association of the SNPs with overall survival (OS) was analyzed.Twelve single nucleotide polymorphisms (SNPs) of PD-1, PD-L1, and CTLA-4 genes were selected and genotyped. A total of 354 patients with early stage NSCLC who underwent curative surgical resection were enrolled. The association of the SNPs with overall survival (OS) was analyzed.

Results:
Among the 12 SNPs investigated, PD-L1 SNP1C>G, SNP2G>C, and SNP3T>A were significantly associated with worse survival outcomes in multivariate analyses. When the three SNPs were combined, OS decreased in a dose-dependent manner as the number of bad genotypes increased (Ptrend = 0.0003). A higher expression of the reporter gene for the SNP2G- SNP3T haplotype was observed compared with the SNP2C- SNP3A haplotype by luciferase assay (P = 0.004). Patients with higher expression of PD-L1 mRNA had a better survival compared with lower expression (P = 0.03).

Conclusion:
PD-L1 SNP1C>G, SNP2G>C, and SNP3T>A polymorphisms may be useful for the prediction of prognosis in patients with surgically resected NSCLC. Further studies are needed to confirm our findings and to understand the role of PD-L1 in the antitumor immunity.

Background:
Targeted therapy is transforming the treatment of lung cancer. Such therapies are critically dependent on companion diagnostics that can predict the response to therapy. An ideal test is one that is quick, inexpensive, and non-invasive. In this regard, artificial intelligence nanosensor-based devices that profile volatolomic signatures (through volatile organic compounds (VOCs) analysis) have shown exciting potential. Numerous studies have shown cancer cells produce characteristic patterns of VOCs as a byproduct of their metabolism. These patterns can be used to diagnose patients with cancer using exhaled-breath samples. Here we asked whether the VOC patterns emanating from cancer cells could also be used to guide targeted therapy. In particular, we investigated whether lung cancer cell lines known to be sensitive to FGFR tyrosine kinase inhibitors (TKIs) can be distinguished from cell lines known to be resistant using an array of cross reactive, highly sensitive chemiresistors composed of gold nanoparticles (GNP) and carbon nanotubes (CNTs) coated with various recognition layers previously shown to be highly effective at profiling VOCs.

Methods:
Fourteen sensitive cell lines having an IC~50~ ≤ 50 nM for Ponatinib and AZD4547 (nonspecific and specific FGFR TKIs, respectively) and 21 resistant cell lines representing small cell and non-small cell lung cancers were cultured in complete media (RPMI 1640, 10% fetal bovine serum, and penicillin/streptomycin) under standard conditions to 50% to 75% confluency. SKC Tenax® TA Adsorbent resin was used to collect the VOCs from the head space of each cell line over a period of 60 to 72 hours. Triplicate measures were collected on each sample along with biological replicates. VOCs were also collected at the same time from control plates containing media only. After thermal desorption, the VOC pattern of each sample was characterized using a chemiresistor array of 36 sensors and 4 features per sensor. A statistical pattern recognition analysis was then conducted using a discriminant function analysis (DFA) algorithm to identify the most informative sensors and features.

Results:
We found that sensitive cell lines could be distinguished from resistant cell lines using only 4 sensors and one feature from each (GNP+dodecanethiol, CNT+PAH, GNP+thiol and CNT+β dextrin). Leave-one-out cross validation indicated a sensitivity of 88% for the FGFR TKI-sensitive cell lines with 100% specificity and 92% accuracy. The area under the receiver-operating characteristic curve was 70% and Wilcoxon p-value of 0.06.

Conclusion:
Profiling the VOCs emanating from lung cancer cells shows excellent diagnostic potential as a means of gauging initial sensitivity to FGFR1 TKIs. Consequently, this study suggests that the electronic nose devices currently being developed to profile exhaled breath for cancer detection could also play an important role in predicting responses to targeted therapies. Although cell lines are useful for identifying the VOC pattern that predicts the cancer cell response to therapy, they do not necessarily reflect the complexity that occurs in vivo due to interactions with the microenvironment. Therefore, future studies are needed to confirm if these results can be extended to project efficacy in patients assigned to FGFR TKI therapy.

Background:
More people die of lung cancer (LC) annually than of prostate, colon, and breast cancers combined, making it the leading cause of cancer-related mortality in the United States. LC can be divided into two main categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC is the predominant LC category accounting for roughly 85% to 90% of all diagnosed LCs. NSCLC can be further subdivided into three main histological subtypes including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Phenotypic characterization (i.e. histological features and LC subtypes) for NSCLC tissues remains a difficult task. Many studies have revealed certain genes that are associated with NSCLC; however, these genes cannot completely decipher between its varying phenotypes. CD36 is a biologically plausible candidate gene that is significantly under-expressed in NSCLC tissues compared to normal tissues. This differential expression is not observed in NSCLC tissue subtypes; however, significant differences in CD36 expression have been observed in NSCLC subtype-derived cell lines. Based on this previous expression data, we hypothesized that allelic heterogeneity within CD36 exons could disparately contribute to the development of NSCLC subtypes.

Methods:
To test this hypothesis, we obtained fresh-frozen LC tissues from the UAMS tissue bank and performed mutation screenings using Sanger sequencing methods and Mutation Surveyor software. Quantitative RT-PCR was performed on tissue mRNA and CD36 mRNA expression was normalized to HPRT1 (a housekeeping gene that is more stable in lung tissues) expression in the same samples. Genotype-specific CD36 expressions profiles were then identified and analyzed.

Results:
Several previously undiscovered variants were identified in Exon 4 of the CD36 gene. Two of these variants are associated with mRNA expression differences between the variant and wild-type genotypes that identify phenotypic heterogeneity. Adenocarcinoma samples with transcript harboring the first variant genotype overexpressed CD36 mRNA as compared to adenocarcinoma samples containing the wild-type genotype (p=0.013; N=37). In squamous cell carcinoma samples, there was no significant difference between samples with the first variant and wild-type (p=0.74; N=26). Squamous cell carcinoma samples with CD36 transcript harboring the second variant genotype was relatively under-expressed when compared to the squamous cell carcinoma samples with the wild-type genotype, though the comparison only approached significance at p=0.053 (N=37). A similar comparison in adenocarcinoma samples yielded non-significant results (p=0.59; N=25).

Conclusion:
Identification of NSCLC phenotypes is critical to treatment, but remains difficult with current histopathological methods. Our analysis of publicly available expression data has shown that probes used in global expression microarrays cannot completely and reliably distinguish between NSCLC phenotypes at the CD36 locus. We propose that allelic heterogeneity at the CD36 locus may alter array probe binding properties leading to inconsistent expression results. Our data has identified two previously undiscovered CD36 variants that may uniquely lead to altered CD36 mRNA expressions correlating to specific NSCLC subtypes. Hence, these results suggest that we may be able to accurately quantify transcripts associated with NSCLC subtypes using allele-specific probes.

Background:
Early detection and diagnostic clarification of indeterminate pulmonary nodules by less invasive methods could contribute to better intervention strategies and to the reduction of the high mortality in lung cancer patients. Endobronchial epithelial lining fluid (EELF) might contains molecular markers with diagnostic potential. With the bronchoscopic microsampling (BMS) technique, it is possible to collect EELF in close proximity to the suspected lesion without the risk of biopsy-associated complications. We investigated whether microRNA (miRNA) in EELF collected by BMS may be useful to facilitate preoperative diagnosis of indeterminate pulmonary nodules.

Methods:
The study included 24 non–small-cell lung cancer patients with 48 EELF samples. From each patient, EELF was collected from subsegmental bronchi close to the indeterminate pulmonary nodule, which was detected by computed tomography, and from the contralateral healthy lung. Diagnosis was confirmed by transbronchial biopsy or surgery. Global miRNA expression profile analysis (754 miRNAs) was performed using quantitative real-time polymerase chain reaction (qRT-PCR) with eight sample pairs. miRNAs potentially associated with a malignant phenotype were selected for further qRT-PCR analysis in an independent validation cohort (16 sample pairs).

Results:
All patients underwent BMS without complications. miRNA profiling by qRT-PCR could be reliably applied to EELF samples and resulted in potential miRNA markers for malignant pulmonary nodules. In particular, the miRNA pair miR-126/miR-126* significantly differentiated between EELF close to the indeterminate pulmonary nodules and the sample taken from the healthy contralateral lung (p<0.0001).

Conclusion:
Our study suggests that the analysis of miR-126/miR-126* in EELF collected by BMS could be a potentially useful adjunct to other diagnostic techniques aiming at the preoperative diagnosis of indeterminate pulmonary nodules.

Background:
Screening with low-dose computed tomography of high-risk group for lung cancer development allows for early detection of malignancy in a minor proportion of subjects and leads to improved outcomes. Implementation of complementary minimally-invasive molecular markers for more efficient pre-selection of candidates for imaging tests or help to further define detected changes is a rational way to further improve efficacy of such screening. Here we aimed to identify features of serum peptidome that could be used for differentiation of individuals with early lung cancer from other participants of lung cancer screening program.

Methods:
Blood samples were collected during lung cancer screening program performed in Pomerania district (Poland). MALDI-ToF mass spectrometry was used to characterize the low-molecular-weight fraction of serum proteome in the 800-14,000 Da range (i.e. endogenous serum peptidome). The analysis was performed in a group of 100 lung cancer patients (with early stage lung cancer diagnosed without clinical symptoms during the screening program or through routine diagnostic procedures) and a matched group of 300 controls (participants of the screening without malignancy).

Results:
Components of mass spectra were detected and specific features allowing differentiation of cancer cases were identified. The first group of 50 cancer cases and 150 matched controls was used to built and test multi-component peptide signature for cancer classification; obtained classifier showed about 70% specificity and sensitivity. The signature was validated in the second group of independently analyzed samples (50 cancer cases and 150 matched controls); the classifier performed well and the total number of misclassifications was below 25%.

Conclusion:
MALDI-based profiling of serum peptidome allowed identification of components differentiating patients with early stage lung cancer from healthy individuals. Hence, biomarker based on serum peptide signature has a potential applicability for early detection of lung cancer.

Background:
This study was undertaken to measure delays of diagnosis and treatment of lung cancer in a poor region of São Paulo, Brazil, where there are four million people. In addition, the relation of delay times and survival was analyzed

Methods:
We retrospectively reviewed 509 patients with lung cancer between July 2008 and December 2014. All patients admitted with lung cancer in our institution, which is the only reference for patients with cancer in this region, were considered eligible for this study once they had not undergone any previous oncology treatment. Dates for symptoms, visits to doctors, treatment and death were recorded. The delays in the diagnosis and treatment of lung cancer were arranged in the following time intervals: -Time (months) from the first symptoms experienced by the patient (history patients - HP) to the date on which the patient was diagnosed with cancer (DX); -Time (months) from initial presentation to the first appointment (first app) with a specialist in our institution to the date on which the patient was diagnosed with cancer (DX); -Time (months) from date on which the patient was diagnosed with cancer (DX) to the starting date of treatment (TTO). Descriptive analysis of data was carried out using measures of central tendency (median). Kaplan-Meier survival estimates were used to determine 5-year lung cancer specific survival for all patient and Log-rank (Mantel-cox) and Breslow (Generalized Wilcoxon) analyses were used to compare differences between factors. Survival was calculated from the date of patient admission at our institution to the date of last follow-up or until death from any cause. Statistical analyses were performed using SPSS v 17.0 for Windows.

Results:
Demographic characteristics of the 509 lung cancer patients were analyzed. The median age of these patients was 62 years (range 26 -96 years) and more than 75 percent of these patients were smokers. For all patients, median overall survival was 7 months (95% CI: 5.7 to 8.2) with 34.5% of these patients surviving one year and 8.1% surviving five years. Patients have spent a relevant time waiting in each interval period. For instance, the median time from the history patient (HP) to the diagnosis (DX) was 3 months. From the first appointment (first app) to diagnosis (DX) was 1 month, however, 79% of patients were diagnosed up to 2 months. Finally, the median time from the diagnosis (DX) to the starting date of treatment (TTO) was 1 month, but the majority of patients (82.5%) started the treatment up to 2 months. There was no statistical relationship between the delays and the mortality of patients. The time gap between the development of the first symptoms and the beginning of treatment was not relevant to the mortality rate of lung cancer, as shown in the survival data of the Kaplan-Meier graph.

Conclusion:
We have a relatively long time for confirmation of lung cancer and also to start treatment. Despite these data were not an independent significant factor for survival, this type of study is important to alert medical societies and government health agencies.

Background:
The literature is mixed regarding the impact of lung cancer surgery on physical and mental health quality of life (QoL)[1-4]. Some studies have found an improvement in QoL post surgery[1] while others have indicated a decrease in various aspects of QoL[2,3]. Further, the impact on QoL is often dependent on numerous factors such as type of surgery. The current study aims to assess the impact of surgery on both physical and mental health QoL in screening-diagnosed patients with early stage lung cancer, an under-studied population.

Methods:
SF-12 QoL indicators were collected from 86 participants (40 women, 46 men) at baseline CT screening and one-year follow up post-surgery for clinical stage IA non-small cell lung cancer. 69 had lobectomy and 17 had sublobar resection. Average time of follow up was 12 months since surgery (SD: 1.5 months; range: 9-15 months post surgery). Univariate and multivariate analyses were performed to examine the difference in physical (PHC) and mental (MHC) health component scores of the SF-12 before and after surgery using the Wilcoxon signed rank and Mann Whitney tests.

Results:

SF-12 Quality of Life Scores Pre and Post Surgery

		ALL M(SD)	MALE M(SD)	FEMALE M(SD)	LIMITED RESECTION M(SD)	LOBECTOMY M(SD)
PHC	Baseline (Pre-Surgery)	49.4(6.8)	49.8(5.8)	49.0(7.8)	47.8 (7.8)	49.8(6.5)
	Post-Surgery	48.7(7.1)	48.5(7.7)	49.0(6.4)	50.3(6.3)	48.3(7.2)
	Difference (Post-Pre)	-0.7(7.6)	-1.3(7.5)	0.0(7.6)	2.5*(6.0)	-1.5(7.7)
MHC	Baseline (Pre-Surgery)	53.7(8.6)	55.5(7.6)	51.7(9.3)	52.3(13.4)	54.0(7.1)
	Post-SurgerY	55.8(8.2)	57.3(8.1)	54.1(8.2)	55.7(6.3)	55.8(8.7)
	Difference (Post-Pre)	2.0*(9.6)	1.7*(8.5)	2.4*(10.9)	2.9(10.7)	1.8(9.4)
	*p<.05

There was no significant change in PHC post-surgery (Wilcoxon signed rank test, S=-216, p=0.32), but MHC significantly improved from baseline to post-surgery (S=527, P=0.01). Mean MHC was significantly higher among males as compared to females at both baseline (Chi-square=3.95, p=.047) and post-surgery (Chi-square=4.23, p=.039) and after controlling for age, ethnicity, and education, while no differences in PHC was observed. Further, there was an improvement in PCS score post-surgery among participants who underwent limited resection while a decrease in PCS score was observed among those who underwent lobectomy. The change in PCS score was significantly different between type of surgery (t=-2.01, p=0.048). After controlling for demographics, the difference was borderline significant (F=3.62, p=0.06).

Conclusion:
Surgery for early stage lung cancer was associated with an increase in mental health QoL one year after surgery, however, physical health QoL was not affected by surgery overall, but it did marginally improve among participants who underwent limited resection as compared to lobectomy. Further, although mental health QoL improved for both males and females, females had lower mental health QoL as compared to males at both time points. Current study findings have implications for lung cancer health professionals regarding how to most effectively present the possible impacts of surgery on the QoL of this subset of patients in which disease has not yet significantly progressed.

Background:
Pulmonary carcinoids are usually localized to the lungs but can also metastasize to mediastinal lymph nodes or to other organs. We studied the incidence and patient outcome in a well-defined population over a 60 year period.

Methods:
A nationwide study, including all pulmonary carcinoids diagnosed in Iceland from 1955 to 2014. Histologic specimens were re-evaluated and information retrieved from medical records. The tumors were staged according to the TNM staging system (6[th] edition). Survival was estimated using the Kaplan-Meier method, with end of follow-up on January 1[st] 2015. Mean follow-up was 186 months.

Results:
93 patients (62 females, average age of 52 years) were diagnosed during the 60 year period. Incidence increased from 0,2/100.000/year between 1955-1964 to 0,7 2005-2014. A total of 26 out of 85 patients (31%) were asymptomatic upon diagnosis and the rate of incidental detection increased from 17% in the first 30 years to 33% in the later 30 years. The most common symptoms were cough (56%), pneumonia (28%) and chest pain (11%). Mean tumor diameter was 2,7 cm (range: 0,3-6,3), 71 (84%) patients were diagnosed with typical carcinoid tumors and 14(16%) with atypical carcinoid tumors. Out of 77(91%) patients who had surgery, 65(84%) underwent a lobectomy. One patient died within 30 days of surgery. Most patients(n=67, 79%) were on stage I upon diagnosis and 4(5%) on stage II. Another 4 patients were on stage III with mediastinal lymph node metastases, all with typical histology. Out of six patients(7%) with distal metastases (stage IV), two had typical histology. Five patients(6%) had died from pulmonary carcinoids upon follow-up, but total 5-year survival was 92% for all patients and 87% for patients with typical carcinoids.

Conclusion:
The incidence of pulmonary carcinoids in Iceland has tripled over the last 6 decades, mostly due to steep increase in incidental detection on chest imaging. Most patients (>84%) are diagnosed with a localized disease, where long-term outcome is excellent.

Background:
We evaluated the associations between potentially functional variants in cancer-related genes and the risk of lung cancer to identify genetic factors responsible for lung cancer susceptibility in a Korean population.

Methods:
A total of 1,969 potentially functional single nucleotide polymorphisms (SNPs) of 1,151 genes involved in carcinogenesis were evaluated using the Affymetrix custom-made GeneChip in 610 NSCLC patients and 610 healthy controls. A replication study was performed on an independent set of 490 cases and 486 controls.

Results:
Eighty two SNPs with P < 0.05 for genotype distribution in the discovery set were tested in the replication study. Among the 82 SNPs, three SNPs (corepressor interacting with RBPJ 1 [CIR1] SNP1T>C, solute carrier family 38, member 4 [SLC38A4] SNP2C>T, ribonucleotide reductase M1 [RRM1] SNP3T>C) constantly showed significant associations with lung cancer (adjusted odds ratio [aOR] = 0.68, 95% CI = 0.59-0.84, P < 0.0001; aOR = 0.74, 95% CI = 0.63-0.88, P = 0.001; aOR = 0.72, 95% CI = 0.56-0.93, P = 0.01, respectively, under dominant model). Promoter assay demonstrated a decreased reporter gene expression for CIR1 SNP1 C allele was observed compared with T allele (P = 0.02).

Conclusion:
Our results suggest that the three SNPs, particularly CIR1 SNP1T>C, may contribute to lung cancer susceptibility in Koreans.

Background:
Only 30% of small cell lung cancers (SCLC) are diagnosed as limited stage (LS-SCLC), whereas the majority of cases show extensive stage disease (ES-SCLC). Specific frequency and outcome of SCLC within lung cancer screening trials have not been described. The purpose of this study was to describe the frequency and outcome of SCLC in lung cancer screening trials with annual or biennial LDCT controls.

Methods:
The population was selected from two lung cancer screening trials (one pilot study and one randomized controlled study) based on serial low-dose computed tomography (LDCT). Subjects with diagnosis of SCLC were selected and the stage of the disease was assessed at the time of diagnosis, as follows: a) TNM staging system; b) 2-stage staging system (e.g. LS-SCLC or ES-SCLC). Survival curves were estimated using Kaplan-Meier method and were compared by log-rank test.

Results:
5,134 subjects were recruited and, thereafter, followed up for a median time of 8.3 years, with 45,141 person-year of clinical follow up. Ten SCLC were reported with incidence of SCLC 22/100,000 person-year, notably, 8 in the LDCT arms with incidence of 24/100,000. SCLC was diagnosed in 3/1643 women and 7/3385 men, age at diagnosis 65 years (range 53-73), and cumulative tobacco consumption of 82 pack-years (range 30-113). The proportion of SCLC among all lung cancers diagnosed in the screening was 10/164. Six out of the 8 SCLC reported in LDCT arms were screen-detected, whereas 2 SCLC were non-screen-detected. Median standard uptake value (SUV) by [18]F-Fluorodeoxyglucose Positron Emission Tomography was 10 (range 5.5-14.4). According to TNM classification, all but 1 SCLC were advanced stage at the time of diagnosis, whereas according to the 2-stage system 5 LS-SCLC and 5 ES-SCLC were observed. The prevalence of LS-SCLC was 62.5% in LDCT arm, in particular, 66.7% among screen-detected and 50% non-screen-detected. The 2 SCLC reported in control group were both ES-SCLC. Six of the 10 subjects died from SCLC, with median overall survival of 21.2 months (95% CI 7.4 – nc months; Figure). Median overall survival was 12-month longer for LS-SCLC (p = 0.02). Survival at 5 years was 0%. Figure 1.



Conclusion:
SCLC was diagnosed with higher proportion of LS-SCLC in LDCT-based screening trials, as compared to data from the literature. Median overall survival of LS-SCLC was slightly longer than ES-SCLC, allegedly related to diagnosis anticipation. None of these patients was alive at 5 years.

Background:
Internationally, one of the most commonly reported quality outcome in surgery for lung cancer is 30 day mortality. However, is difficult to know what constitutes unacceptably high mortality or unacceptable variation between surgeons. In October 2014 national data was released from the Society for Cardiothoracic Surgery (SCTS) in the United Kingdom (UK) on hospital and individual surgeon volume performance for lung cancer resection in the UK. The implicit assumption is benchmarking of the performance. The aim of this study is to report on the impact of individual surgeon volume in relation to each death associated with the an average 30-day mortality rate of 2.2% using national data driven performance control limits (i.e. funnel plots), and determine the applicability on surgeon performance internationally.

Methods:
Data released by the SCTS were downloaded, complied and analysed. Each step change for individual mortality was calculated, and alert limits modelled using current UK national standard of the upper 99% binomial confidence limit.

Results:
Data from 29 units were published with the annual volume of 125 surgeons for 2012. Data from 6 surgeons were excluded for no lung resections performed. In the remaining 118 surgeons, the mean (SD) annual lung resection volume for cancer was 42 (27). A total of 25% of surgeons performed 18 resections (or less) per year. For 50% of surgeons undertaking 40 resections (or less) each death represents at least 2.5% (0 to 13%) of their annual work load. Using a 99% binomial confidence limit at 50 cases, the upper alert is 16%. Therefore for the majority of surgeons, a mortality rate of 15% which is 7.5 fold higher than average would not trigger the conventional national alert limits.

Conclusion:
Based on UK national data, lung cancer resection volumes for individual surgeons are low and for the majority even a single death (which could be due to chance), affects the overall mortality rate much more, carries a disproportionately high weighting and may encourage risk adverse behaviour whilst simultaneously failing to detect 7.5 fold increased mortality rates using conventional national limits. Such data driven limits would also not be applicable on an international level basis unless individual surgeon volume is higher than 100 resections per year, a level that was not achieved by most UK surgeons.

Background:
Lung Cancer is the leading cause of cancer death among New Zealand (NZ) Maori. Over the past twenty years lung cancer incidence has decreased in New Zealand for non-Maori but has increased for Maori, and is recognised to be the highest in the world of any ethnic group. Nationally, the incidence of lung cancer in Maori is 3.5 times higher than that in New Zealand Europeans, and lung cancer mortality in Maori males and females respectively, is 2.4 and 4.2 times higher than NZ Europeans. Maori have a higher incidence of lung cancer than countries with similar smoking rates. This suggests that there are additional factors other than smoking that predispose Maori to this disease. In the current study demographic and the well-established clinical risk variables for lung cancer were compared between New Zealand Maori and Europeans residing in the greater Auckland region and who were diagnosed between January 2004-January 2015.

Methods:
A retrospective review of patient clinical notes for those identified as being of NZ Maori ethnicity who were diagnosed with lung cancer (n=473) between January 2004 and January 2015 and treated within the greater Auckland region. Data extracted included histological type, smoking history, spirometry and basic demographics. This data was then compared with an established cohort of NZ European patients n= 417, with similar recruitment criteria over the period 2004-2008.

Results:
Despite comparable smoking exposure histories, NZ Maori patients were diagnosed on average 6 years younger than NZ European lung cancer patients (P<0.0001). At diagnosis, current smoking rate was 2 fold greater in NZ Maori compared to NZ Europeans (69% vs 36%, P<0.0001). Although NZ Maori patients had similar rates of COPD (≈64%), they had a trend towards less GOLD 1 (mild stage disease, P=0.08) and significantly greater airflow obstruction (worse COPD, FEV~1~%predicted 64% vs 73% in NZ Europeans, P<0.001). At lower smoking exposure (≤10 pk yrs), COPD rates in Maori with lung cancer were 2 fold greater than in NZ Europeans (64% vs 32% respectively, P<0.05). NZ Maori lung cancer patients had a lower prevalence of adenocarcinoma than in NZ Europeans (32% vs 43%, P=0.002) and a higher proportion of more aggressive lung cancer subtypes (squamous, non-small cell and small cell cancers) than NZ Europeans (61% vs 52%, P<0.0007).

Conclusion:
These results show that lung cancer in NZ Maori is associated with younger age at diagnosis, worse lung function and more aggressive histological subtypes compared to NZ Europeans. These results suggest that NZ Maori may have a greater inherent susceptibility to lung cancer compared to NZ Europeans. This greater susceptibility to lung cancer in Maori, along with socio-cultural factors, may contribute to their considerably greater mortality. These results suggest that for the future management of lung cancer, prevention measures (such as smoking cessation and tobacco control), risk assessment (such as lung function testing) and early diagnostic approaches (such as computed tomography screening) should be prioritised in high risk groups, particularly those with NZ Maori ancestry.

Background:
Insight Genetics Inc. and Biocept, Inc. have established a collaboration to develop a non-invasive work flow to enhance detection of the oncogenic Anaplastic Lymphoma Kinase (ALK) status in NSCLC patients. A barrier to detection of oncogenic transcripts in circulating tumor cells (CTCs) has been purification methods that are incompatible with downstream qPCR detection technologies. In contrast, Biocept's proprietary CTC capture technology has been shown to be benign for follow up qPCR detection using Insight Genetics proprietary qPCR-based oncogenic ALK detection assay.

Methods:
Initial studies were conducted to demonstrate cell capture on the Biocept platform with spiked ALK fusion positive H3122 cells. These studies show this to be a feasible option for non-invasive detection of ALK mRNA. A pre-amplification allele-specific approach including reference controls was incorporated. H3122 cells spiked into peripheral blood also demonstrated feasibility of the accurate detection of aberrant ALK expression using the Biocept CTC extraction methodology and Insight Genetics’ qPCR detection strategy.

Results:
Results from these studies and the detection of aberrant ALK expression from a cohort of ALK positive patients will be presented along with the potential to use CTCs to monitor ALK inhibitor resistant mutation profiles.

Conclusion:
Together, Biocept’s proprietary CTC capture technology coupled to Insight Genetics qPCR ALK detection assay appears to be a viable strategy to accurately monitor ALK status in NSCLC patient populations using a liquid biopsy.

Background:
Low dose computed tomography (LDCT) screening for lung cancer (LC) provides reduction in mortality rates among individuals at high risk. Pre-Test Probability of Malignancy (PTPM) is a common tool used during the decision process: when the probability of malignancy is moderate or high, patients should be referred for further testing or tissue sampling. However, in some cases, these statistic models may give an overestimated value, especially in countries with a high incidence of granulomatous diseases. We have calculated the PTPM in our LDCT screening program and this work explores its main results.

Methods:
Prospective cohort of current or former smokers, with a heavy smoking history. Data of the first LDCT were analyzed to calculate the PTPM. The inclusion criteria were similar to NLST. LDCT scans with indeterminate pulmonary nodules above 4mm in size were considered positive and were evaluated by a multidisciplinary team. The PTPM model used in this study was designed by Swensen et al and included patient’s age, smoking history, diameter of the nodule, spiculation and upper lobe location. A PTPM > 60% was considered high and between 6 and 60% was considered moderate.

Results:
From January 2013 to July 2014, 790 were included in the protocol. We found 310 positive LDCT at baseline (39%), 34 (11%) with high PTPM. Among them, 16 were followed with LDCT in 3 (56.2%), 6 (37.5%) or 12 (6.3%) months and the remaining were investigated with PET-CT and/or lung biopsy. From the patients followed by LDCT, one case showed an increase in nodule size and was investigated with lung biopsy; all others were stable in one-year follow up. LC was diagnosed in 7 patients and benign diseases in 5 patients with high PTPM, including 1 case of tuberculosis. Other 4 cases of NSCLC were found in the moderate PTPM group (n=272). Therefore, malignancy rate was 20.6% for high PTPM and 1.5% for moderate PTPM nodules.

Conclusion:
The Swensen’s PTPM model overestimates the prevalence of LC in both groups of moderate and high-calculated values of PTPM. The decision making process should include other variables discussed in a multidisciplinary board, been safe to follow patients with further image tests.

Background:
British Thoracic Society (BTS) Guidelines are aimed primarily at practitioners within the UK. They are National Health Service Evidence accredited which means they must adhere to robust guideline development methodology. The evidence base for this guideline comes mostly from countries outside the UK so the recommendations will have relevance to other countries healthcare systems.

Methods:
The recommendations are based on a comprehensive review of the literature on pulmonary nodules and expert opinion. A third of the 360 references cited were from 2012 onwards, reflecting the rapid expansion of the evidence base. The new evidence has resulted in important differences from guidelines previously published by the American College of Chest Physicians and the Fleischner Society.

Results:
There are four algorithms: initial approach to solid nodules; surveillance of solid nodules; management of sub-sold nodules; and pulmonary nodule treatment. Two malignancy prediction calculators are recommended to assess the risk of malignancy; one (the Brock University model) that performs best for smaller nodules and one that has the better accuracy for larger nodules following PET-CT (the Herder model). There are recommendations based on recent evidence from screening studies, for a higher nodule size threshold for follow up (≥5mm or ≥80mm[3]). This will reduce the number of follow up CTs which, in the UK at least, are not cost effective. Surveillance recommendations are also different from previous guidelines: people can be discharged after 1 year of stability if measured by semi-automated volumetry. Management is also dependent on the volume doubling time (VDT) with immediate further assessment for nodules that show a VDT of ≤400 days and either biopsy or further observation for nodules with VDTs of >400 to ≤600 days. People with nodules with a VDT >600 days have the option of discharge, if VDT is measured by volumetry. As in previous guidelines, a 3 month repaet CT is recommended for sub-solid nodules.After that, management is governed by risk assessment by the Brock tool (with the proviso that it may underestimate risk after the initial CT) and according to specific features that predict malignancy. Acknowledging the good prognosis of sub-solid nodules, there are recommendations for less aggressive options in their management. The guidelines provide more clarity in the use of further imaging, with ordinal scale reporting for PET-CT recommended to facilitate incorporation into the Herder risk model and more clarity about the place of biopsy and its influence on pre-test probability. Segmentectomy can be considered for primary diagnosis and treatment for nodules smaller than 2cm, and sub-lobar resection is recommended for pure ground glass nodules. Where fitness levels preclude surgery, non-surgical treatment with stereotactic ablative radiotherapy or radiofrequency ablation is recommended, even where biopsy is not possible, provided the probability of malignancy is high. Finally, there are evidence based recommendations about the information that people need that should be provided for them.

Conclusion:
The BTS guideline is intended to be used both as a summary in the day to day management of the person with a pulmonary nodule as well as a comprehensive reference text.

Background:
Lung cancer is the leading cause of cancer related deaths world-wide. While low dose computed tomography (LDCT) screening of the high risk patient population was recently shown to decrease deaths from lung cancer by 20%, LDCT also resulted in 18% over-diagnosis [c.f. Patz-E.-F.-JAMA-2003] with a positive predictive value of only 52.9% when a suspicious LDCT finding led to a biopsy [c.f. Church-T.-NEJM-368-2003]. We tested whether combining novel image-features (IF) with routinely collected baseline-features (BF) can improve the accuracy of diagnosing suspicious findings on baseline LDCT.

Methods:
This exploratory case-control study included N=123 (66-cancer, 57-no-cancer) high risk subjects with at least one suspicious finding (nodule >= 8mm [c.f. Lung-RADS-ACR-2014]) on baseline LDCT screening at Vanderbilt University on a VCT Discovery (GE-Healthcare, UK) or a Brilliance iCT 128 SP (Philips, Amsterdam) system. The cohort was randomly divided into a separate training-set (N=55, 32-cancer, 23-no-cancer) and a test-set (N=68, 34-cancer, 34-no-cancer). All model training and leave-one-out cross-validation were strictly restricted to the training-set. Performance was evaluated on the unseen test-set. Definitive lung cancer or no-cancer diagnosis, smoking history and at least 6 baseline-features (BF6) viz. age, family-history, pack-smoking-years, body-mass-index, nodule-location, nodule-size were recorded for all subjects. Baseline lung cancer predictions were generated by (a) using the Gould-model [c.f. Gould,M.-Chest-2007] and (b) fitting an Elastic-Net Regularized Generalized Linear Model (GLMnet [c.f. Zou-H.-Journ-Royal-Stats-Soc-B-2005]) to BF6. The final baseline model (“GLMnet:BF”) effectively utilized 4 baseline-features with the coefficients for age and body-mass-index shrunk to zero. New LDCT specific information was extracted by computing 589 intensity, shape, surface and texture features (IF589) [c.f. Aerts-H.-Nat-Comm-S2014, Way-T.-Med-Phys-2009] from a 3D volume-of-interest (VOI) encompassing a rough Graph-cuts [c.f. Li-K.-IEEE-PAMI-2006] segmentation for each suspicious nodule. A GLMnet was fit to all 595 features (BF6 and IF589) yielding a final enhanced model (“GLMnet:BF+IF”), which contained 12 features after GLMnet shrinkage : 10 IF related to VOI energy, nodule shape and surface statistics and image intensity variability and 2 BF (family-history, nodule-location).

Results:
Baseline AUC increase by 7.4% from 0.81 (Gould-model) and 0.80 (GLMnet:BF) to 0.87 (GLMnet:BF+IF). At 88% sensitivity, false positive rate reduced by 60% from 56% (Gould-model) and 44% (GLMnet:BF) to 18% (GLMnet:BF+IF); accuracy improved from 65% (Gould-model) and 71% (GLMnet:BF) to 84% (GLMnet:BF+IF). Fig.1 below shows more details: Figure 1



Conclusion:
This initial exploratory analysis showed that image-features extracted from suspicious LDCT findings may help reduce the number of unnecessary biopsies. Additional validation studies are warranted to determine the value of this structural imaging-based approach.