Virtual Library

Background
The status of lung cancer in rural and remote Aboriginal and Torres Strait Islander ( from this point referred to as Indigenous) communities in Queensland is unclear. It is not known how much of a problem lung cancer is in these communities nor how much awareness exists regarding lung cancer risk factors and early symptoms. Several factors contribute to the uncertainty of lung cancer status in rural and remote communities. Factors include the quality of reporting Indigenous status and cancer registration, cultural influences affecting treatment decisions, access to health services and availability of culturally appropriate lung cancer information resources . Research on lung cancer in rural and remote Indigenous communities in Queensland is needed to improve lung cancer diagnostic and referral pathways and develop culturally appropriate and effective lung cancer information resources. Study Aims: 1. Describe the local and regional health care facilities for Indigenous people who may be referred for suspected lung cancer across the state of Queensland. 2. Interview Indigenous people and health workers in 3 population sample groups from six rural and remote Indigenous communities in Queensland to identify if there are variations in patient flow relative to predicted utilisation of local and regional health care facilities.

Methods
1. Using publically available information, identify relevant health care facilities including those with diagnostic bronchoscopy (with or without endobronchial ultrasound (EBUS) services across Queensland to predict expected referral pathways for suspected lung cancer. 2. Using quantitative and qualitative approaches to learn preferred referral pathways from 3 target population groups including patients referred for medical treatment with symptoms suspicious of lung cancer or confirmed lung cancer, Indigenous health workers, Indigenous community members aged 18 years and older. Frequency distributions in terms of the following will be analysed: demographics, current health status, social situation, access to health services, social and financial impact of treatment and information resources. Frequency distributions will be cross tabulated with age, education attainment, socio-economic characteristics, cultural influences, lung cancer awareness and knowledge. The responses to narrative questions will be analysed to identify main themes. These themes will be categorised by issues relating to lung cancer knowledge, cultural influences and beliefs, the patient experience and access to lung cancer medical and support services.

Results
We identified a spectrum of health care services across Queensland where patients may be referred for lung cancer management, ranging from public to private facilities. There are seventeen discrete Indigenous communities in Queensland. Compared to the nearest health care facility which offer diagnostic bronchosopy, 5 discrete Indigenous communities are situated > 200km away, 9 > 500km away and 2> 1000km away. Only one is situated 50km away.

Conclusion
The research findings will provide a clear understanding of the affect of lung cancer in rural and remote Indigenous communities in Queensland. Knowledge gained from research will enable better health service planning and help reduce any health disparities experienced by Indigenous people; particularly those who live in less advantaged areas compared to other Australians when facing a diagnosis of possible or confirmed lung cancer.

Background
Adjuvant Cisplatin and Vinorelbine chemotherapy has been shown to improve overall survival for patients with resected non-small cell lung cancer. In the Auckland region, we administer 400mg/m2 of Cisplatin over 5 cycles in an attempt to reduce toxicity. The aim of this audit is to review the number of patients treated with this chemotherapy at our centre, assess the rates of toxicity, examine patient outcomes, and identify barriers to adjuvant chemotherapy administration.

Methods
Patients starting Cisplatin and Vinorelbine between 1 January 2008 and 31 December 2010 were identified by the Auckland Hospital oncology pharmacy department. Their clinical records were reviewed with respect to demographics, details of chemotherapy received, and toxicities encountered. Cancer outcomes were also analysed, including recurrence and mortality rates. Subsequently a review of our local thoracic multidisciplinary team meetings held over the same time period was undertaken to identify barriers to adjuvant chemotherapy access.

Results
A total of 29 patients received Cisplatin and Vinorelbine over the 3 years as adjuvant treatment for non-small cell lung cancer. 24 patients had stage 2 or early stage 3 cancer, four patients had stage 1B, and one patient had stage 4 cancer with an isolated brain metastasis which had been resected. 23 (79%) patients received all planned cycles of chemotherapy. Five patients did stop early due to toxicity and one because of travel plans. The average number of cycles given was 4.58 and the average dose of Cisplatin received was 349mg/m2. The average time from surgery to chemotherapy initiation was 87 days. There were no treatment related fatalities in our group. 25 (86.2%) patients had at least grade 3 toxicity requiring either dose reduction or deferral. The majority of side effects were haematological in origin. As of the 31/05/2013, 14 of 29 patients have had cancer recurrences (48.3%) and 14 patients have died (48.3%), the lung cancer specific mortality was 37.9%. One patient has been lost to follow-up. One, two, and three year survivals were 89.7%, 79.3%, and 56% respectively. Over the three years examined, 48 patients discussed at our thoracic multidisciplinary meeting had resection of a primary lung cancer between stages 1b (size >40mm) and 3a. 20 of these patients received adjuvant chemotherapy. Of the remaining 28 patients, four declined treatment, five were not medically fit enough for chemotherapy, and four developed cancer recurrence shortly after their operation. 15 (54%) patients were mistakenly not offered chemotherapy when it may have been of potential benefit.

Conclusion
This audit would suggest lower utilisation of adjuvant chemotherapy than predicted, and no improvement in toxicity rates by reducing the dose of Cisplatin used per cycle. A higher likelihood of completing all planned chemotherapy was found. It highlights the prolonged waiting times to access chemotherapy and identifies barriers that inhibit suitable patients from accessing this important treatment. Several areas for improvement are identified. Our main recommendation would be to present all patients and review their pathology at multidisciplinary meeting following surgery. Our data is limited by its retrospective nature and small study population.

Background
Lung cancer is the leading cause of cancer-related deaths globally, with a 15% 5-year survival rate. Platinum-based chemotherapy constitutes the main treatment modality, with a median overall survival (OS) of approximately 10-12 months. The current National Comprehensive Cancer Network (NCCN) guidelines recommend several options for first-line and second-line therapies but endorse only erlotinib/gefitinib as third-line therapy in unselected patients, as well as crizotinib in ALK-positive selected patients.The paucity of approved agents for third-line therapy and beyond for patients with non-small cell lung cancer (NSCLC) constitutes an important unmet medical need.

Methods
Retrospective analysis of 22 patients with advanced/metastatic NSCLC in progression after a minimum of 3 lines of therapy.

Results
Between January 2009 and October 2012, 22 patients were analysed. Median age at diagnosis was 62 years old. 15% of patients were never smokers and 72.7% had non squamous NSCLC histology. Stage at diagnosis resulted: 6 (27.3%) stage IIIA, 3 (13.6%) stage IIIB and 13 (59.1%) stage IV. 3 (13.6%) patients were EGFR mutation carriers and 1(1%) patient had ALK translocation. Third line therapy options resulted a clinical trial (27.3%), erlotinib (22.7%), paclitaxel/gemcitabine (13.6%), docetaxel (9.1%) and crizotinib (4.5%). Estimated median progression-free survival (PFS) between first and second line therapy was 5.3 months; PFS between second and third line resulted 4.4 months. Median PFS and overall survival (OS) beyond third line treatment has not been reached yet.

Conclusion
Currently, erlotinib/gefitinib and crizotinib, which target EGFR and ALK, are the only recommended agents for third-line therapy in patients with advanced/metastatic NSCLC. Real-world clinical practice reveals a variety of chemotherapeutic agents used in this setting. Additional systemic and/or targeted therapeutic under development, with complementary biomarker analysis, should be the key in identifying those patients most likely to benefit from newer agents.

Background
SABR is well established in medically inoperable patients with early lung cancer. These patients are usually elderly with multiple comorbidities. Chest wall toxicity is seen in 15% of cases, however in radiotherapy planning no chest wall constraints are used to minimise this risk. Some centres actively exclude the chestwall from PTV margin and some feel that the risk of sustaining a fracture is as high with 3 as with 5 fractions as rib is likely to behave similar to a serial structure.

Methods
Of the 85 patients treated with SABR from 2009 to 2013, cases with at least one year follow up were audited. Patients were treated with 18 Gy x 3# and 11 Gy x 5# on alternate days. Chest wall was defined as 3cm from the ipsilateral lung border excluding the skin. For the chest wall, the maximum dose and volume getting 30, 40 and 50 Gy was recorded. Patients were followed up three monthly for the first year with CT imaging and toxicity was scored using CTCAE v3.0. Dosimetric parameters were correlated against chest pain and rib fracture.

Results
50 patients with a median age of 70 (range 56-89) and follow up of 16.8 (range 1.2-44.9) months were audited. The majority (62%) of the patients had a PS of 2 and 66% had a T1 tumour. 6 patients developed a rib fracture and patients complained of dysaesthesia/chest pain. One patient sustained fractures in three adjacent ribs at two different time points. The rate of any grade rib fracture and chest pain was 12% and 16% respectively with median time to development of respective toxicities of 15.6 and 4.6 months. Grade 3 rib fracture was seen in one patient. All of the patients that sustained rib fracture/chest pain had SABR delivered over 5 fractions as the PTV overlapped the chest wall. Median survival is 34 months with a 1 year survival of 88%. No correlation was seen between rib fracture and chest wall dosimetric parameter. However chest pain correlated well to all dosimetric parameters.

Conclusion
Our series shows a similar rate of chest wall toxicity to that reported in the literature, which is largely less than grade 3 in severity. However it is important to minimize this side effect as much as possible as the patient population currently being treated with SABR is elderly with multiple comorbidities. As the incidence of this toxicity is low, large patient data is needed to identify useful dosimetric parameters to predict chest wall toxicity. Developing SABR in the UK within the consortium structure with national guidelines opens up the potential to obtain and analyse such data. However there are logistical challenges of pooling data across various hospitals.

Background
Therapeutic armamentarium for advanced NSCLC includes oral EGFR-TKIs. There is paucity of data from India on experience with oral EGFR-TKIs in newly diagnosed NSCLC. The current study sought to assess demographic profile and treatment outcomes for NSCLC patients receiving first line treatment with oral EGFR-TKIs at a tertiary care institute in North India.

Methods
Retrospective analysis of data on newly diagnosed NSCLC patients initiated on treatment with oral EGFR-TKIs over a 4 year period (January 2008 to December 2011). Demographic characteristics, histology, disease stage and smoking status were noted. Radiological response to treatment was assessed using RECIST. Toxicity was graded as per Common Toxicity Criteria (CTC v3.0). Numerical and categorical data were compared between groups using Mann Whitney U test and chi-square test respectively. Survival probabilities and median survivals were calculated by Kaplan-Meier method and group differences analyzed using the log-rank test.

Results
Of the 76 patients who comprised the study population, the percentage of males and current/ex-smokers was 67.1% (n=51) and 51.3% (n=39) respectively. Histological distribution was as follows: Adenocarcinoma 59.2% (n=45), Squamous cell 23.7% (n=18), NSCLC-NOS 11.8% (n=9) and Large cell carcinoma 5.3% (n=4). Majority of patients were in stages IV 64.5% (n=49) and IIIB 26.3% (n=20) while only 9.2% (n=7) were in stages I-IIIA. Malignant pleural effusion was present in 20 (26.3%) patients. Baseline Karnofsky performance status (KPS) was 80-100 in 25.5%, 60-70 in 42.6% and ≤50 in 31.9%. Gefitinib (n=70, 92.1%) was the most frequently used EGFR-TKI. Most common indications for use of oral EGFR-TKI were poor PS/physiological status in 65.8% (n=50) and unwillingness for chemotherapy in 27.6% (n=21). Overall, 47 patients had atleast one follow up visit after one month and were eligible for assessment for response and toxicity. There were no complete responses while partial response (PR), stable disease (SD) and progressive disease (PD) were documented in 36.2%, 29.8% and 34.0% respectively of assessable patients. The most common side effects were skin rash and diarrhoea that developed in 17.0% and 10.6% of patients respectively. These were mild (grade 1/2) in all except one patient each with grade 3. Objective PR was observed in all (100%) patients with skin rash as compared to 23.1% among those without skin rash (p<0.001). Among patients without skin rash, the median OS was 178 days (IQR 53-320 days) while among those with skin rash the median OS had not been reached (figure 1).Figure 1 Gender, histology and smoking status did not differ amongst patients with and without skin rash. However, skin rash occurred in 25.0% and 5.4% of patients with and without malignant pleural effusion (MPE) respectively (p=0.26). On multivariate logistic regression analysis, only MPE was associated with occurrence of rash [odds ratio=0.19 (95% CI=0.04-0.95); p=0.04].

Conclusion
Oral EGFR-TKIs are a useful treatment option for clinically selected patients with advanced NSCLC who have either poor PS or are unwilling for chemotherapy. Occurrence of skin rash has an independent association with objective treatment response and with better OS. Presence of MPE is associated with occurrence of skin rash.

Background
BALT Lymphoma accounts for only 1% of Non Hodgkin’s Lymphoma leading to the paucity of randomized clinical trials to outline the treatment options (1). There are a few recently published case reports in the literature claiming success with single agent rituximab therapy but none in the relapsed setting with repeat rituximab treatment.

Methods
A 67-year-old woman was initially diagnosed with BALT lymphoma involving the left lower lobe 10 years prior, for which she underwent wedge resection alone. Five years later, she developed recurrence with multiple bilateral lung nodules noted on her annual CT scan. She was then treated with chemotherapy using Rituximab, cyclophosphamide, vincristine and prednisone given for 12 cycles. She achieved completed remission. Her annual surveillance CT scan four years later showed a left lower lobe lesion. Staging PET/CT scan showed isolated uptake in the left lower lobe lesion with SUV of 4.7 with no evidence of disease elsewhere. The biopsy showed recurrent BALT lymphoma. Immunostains were positive for CD20 and CD79a. Bone marrow biopsy showed no evidence of lymphoma. She was then treated with four weekly doses of Rituximab at 375mg/m2. Follow up CT scan after completion of the treatment showed complete resolution of the left lower lobe lesion. She was started on maintenance treatment with Rituximab to be given every 3 months.

Results
There are no set guidelines for treatment of BALT lymphoma but general consensus has been local therapy with either surgery or radiation for early stage disease, and chemotherapy for late stages. Also resection might not possible for patients with poor lung function (1). A phase II study of monoclonal antibody rituximab showed overall response rate of 73% (2). Several case reports also suggested completed remission (CR) with 4 to 8 weekly doses of rituximab (3). However, high relapse rate of 36% , observed in a phase II study (2) suggests that perhaps the 4-weekly-doses regimen may not represent the best schedule. Recent meta-analysis showed improvement in the overall survival with maintenance rituximab treatment in the relapsed setting in patients with follicular lymphoma (4). Given that BALT lymphoma is a subtype of Non-Hodkins Lymphoma, we extrapolated the data to our patient and started her on maintenance treatment.

Conclusion
Although longer follow-up is needed in our case, the demonstration of minimal toxicity and considerable activity of this biologic agent in the relapsed setting is promising. 1. Ferraro P et al. Primary non-Hodgkin’s lymphoma of the lung. Ann Thorac Surg 2000;69: 993-7. 2. Conconi A et al. Clinical activity of rituximab in extranodal marginal zone B-cell lymphoma of MALT type. Blood 2003;102: 2741-5 3. Ahmet Bilici et al. Pulmonary BALT lymphoma successfully treated with eight cycles of weekly Rituximab: Report of first case and F-18 FDG PET/CT Images. J Korean Med Sci. 2011 April; 26(4): 574–576. 4. Vidal L et al. Rituximab maintenance for the treatment of patients with follicular lymphoma: an updated systematic review and meta-analysis of randomized trials. J Natl Cancer Inst. 2011 Dec 7;103(23):1799-806.

Background
Chemotherapy (CT) for advanced non-squamous non-small cell lung cancer (NS-NSCLC) without oncogenic drivers remains palliative with suggested similar efficacy and survival among different regimens. Histotype, maintenance therapy (m) and quality of life (QoL) have been explored to improve patients (pts) outcome. The ERACLE trial (NCT01303926), a QoL-oriented phase III trial, was designed to compare the QoL for two CT regimens.

Methods
Pts with stage IIIB/IV NS-NSCLC (ECOG 0/1) were randomized (1:1) to receive first-line CT. Arm A received 6 cycles of Cisplatin (C) (75 mg/m[2])/Pemetrexed (P) (500 mg/m[2]) q3w, followed by mP (500 mg/m[2]), while Arm B received Carboplatin (Cb) AUC 6/Paclitaxel (T) 200 mg/m[2] plus Bevacizumab (Be) 15 mg/kg q3w for 6 cycles, followed by mBe 15 mg/kg. Both treatments were administered until progression, unacceptable toxicity or death. Stratification was based on Study Centre and disease stage. Co-Primary endpoints were EQ5D Index (EQ5D-I) and EQ5D-VAS (Euro-QoL questionnaire). Quality of life data were collected at three time points during the induction phase and at 12 and 18 weeks during the maintenance phase. Secondary endpoints were QoL over time, safety and activity of CT arms. A sample size of 49 pts per arm (that have not progressed during initial CT and during maintenance therapy for at least 12 weeks) would have 91% chance to have 12-point Minimal Interesting Difference (MID) between arms for EQ5D-VAS, and 87% chance to find 0.137 MID between arms for EQ5D-I. It is assumed that about 20% of pts in both arms experience progressive disease before the evaluation of the primary endpoint. The study sample was then increased to 118.

Results
From 1/2011 to 3/2012, 118 pts were randomized to Arm A (n=60) or Arm B (n=58). Baseline demographics were well balanced across arms; Arm A/Arm B male: 70%/77.6%, PS 0: 78.3%/79.3%, stage IV 95%/93%, smokers: 63%/52% . Seventy four pts (62,7%) received maintenance chemotherapy. Treatment differences (mean change from baseline), EQ5D-VAS = 1.82 (95%CI -8.60 to 12.24; P=0.73), EQ5D-I = 0.15 (95%CI 0.01 to 0.29), favoured arm A. Safety was as expected without relevant haematological toxicity and with a significant impact of G3/4 alopecia (p=0.002) and G 1-3 neurotoxicity in ARM B (p=0.008) during induction. Response rates were (Arm A/Arm B) partial responses 40%/51%; stable disease 48.3%/27.6%. The Hazard Ratio (HR) for Progression Free Survival Arm A/Arm B [Cox's analysis] was 0.62 (95%CI 0.41 to 0.95) p=0.03 and HR for Overall Survival Arm A/Arm B [Cox's analysis] was 0.69 (95%CI 0.61 to 1.04) p=0.08.

Conclusion
Arm A showed better (over the MID) health profile (EQ5D-I) as compared to Arm B. EQ5D-VAS didn’t find any significant difference between treatment arms. By assuming equal activity, the choice of a treatment for advanced NSCLC should be mainly based on QoL.

Background
eviQ Cancer Treatments Online, launched in October 2009, is a Cancer Institute NSW flagship program. Its primary remit is to improve service delivery, standardise cancer treatment and improve patient outcomes. The primary audience is health professionals involved in delivering cancer care. Tailored information is also available for people with cancer and their carers, and other non-cancer health professionals. This open-access, web-based repository provides approximately 600 peer-reviewed cancer treatment protocols designed to support the way in which cancer care is implemented. eviQ treatment protocols and nursing resources provide detailed and extensive instructions on how to deliver evidence-based cancer treatments safely and appropriately. Provision of eviQ information promotes equity of access to the same information across rural, remote and metropolitan settings. eviQ provides reference standards that support the measurement and reporting of cancer treatment delivery to further determine strategies to reduce variation and improve patient outcomes.

Methods
Treatment protocols are developed according to the eviQ Governance Framework. Content specific reference committees, made up of clinical experts, discuss and review protocols at face to face meetings, and endorse them for publication on the site. Content is reviewed and updated in accordance with strict risk stratification criteria. This work is underpinned by the use of a tailor-made bibliometrics system, ensuring that the latest published evidence drives protocol development.

Results
eviQ includes a mechanism to record de-identified usage of the website. Usage has increased significantly; from 1932 in December 2009 to 27,853 (2,386 international) registered users in May 2013. Registered users by primary role: Australia (+ international) Medical 4,192 (+ 536) Nursing 11,377 (+653) Radiation therapy 1,031(+185) Allied Health 840 (+38) Other 4,889 (+486) There are currently 31 chemotherapy and 7 radiation respiratory cancer treatment protocols available on the site which were last reviewed and updated in 2012. The most frequently accessed respiratory treatment protocols (NSCLC carboplatin & gemcitabine and SCLC cisplatin & etoposide) are visited on average over 1000 times per month (based on figures from 2012-2013), this being comparable to similar breast treatment protocols (adjuvant adriamycin & paclitaxel and docetaxel & cyclophosphamide). Statistics confirm eviQ is accessed by a wide range of users in both public and private health care settings. eviQ is utilised in the tertiary setting as a teaching tool, further embedding the use of evidence based practice in the clinical setting. Independent evaluation undertaken in 2012 concluded eviQ is an easily usable, quality resource that supports evidence based practice in cancer care.

Conclusion
Every health professional engaged in cancer care has a responsibility to ensure work practice systems are optimised to ensure best patient outcomes. As a unique evidence- based online resource, eviQ contributes to reducing this variability. By harnessing the collective expertise of health care professionals in Australia, eviQ has developed an efficient, safe, quality national resource of evidence-based cancer treatment information for use at the point of care.

Background
PLCH is an orphan disease of unknown etiology, occurring almost exclusively in smokers, histopathologically characterized by focal Langerhans cell granulomas infiltrating and destroying distal bronchioles and lung parenchyma. Unlike other types of histiocytosis, PLCH was considered to be a polyclonal disease, but recently the monoclonal presence of the BRAF V600E mutation was demonstrated in some patients.

Methods
A 51-year old caucasian male smoker was referred due to lymphadenopathy and disseminated, progressive, apical accentuated pulmonary nodules (up to 12 mm) with beginning cavitation on CT-scans and elevated 18F-FDG uptake on PET/CT. The patient negated any B symptoms, while complaining of frequent coughs. Lung function tests showed a worsening restrictive pattern Hematological work up proved the diagnosis of a stage IV A SCL-NHL with marrow infiltration by a CLL-like lymphocyte population (Matutes score 4/5).No extrapulmonary involvement of PLCH was observed. Due to the CT findings suspicious of infectious disease, lymphoma infiltration or metastasis a CT-guided lung biopsy was performed. For BRAF analysis DNA was extracted from formalin-fixed paraffin-embedded lung biopsies and subsequent pyrosequencing.

Results
The biopsy revealed a granulomatous infiltration with CD1a positive Langerhans cells, molecular analysis confirmed a BRAF V600E mutation. Treatment with steroids and nicotine abstinence was initiated with impressive regression of pulmonary nodules after 1 month of therapy, as well as a decrease of F-18 FDG uptake on PET-CT. Lung function returned to normal.

Conclusion
PLCH should be considered in smokers with upper lobe predominant small nodules associated with cysts on high-resolution CT scans. These characteristic radiologic features are sufficient to establish a presumptive diagnosis. In case of doubt a lung biopsy should be performed. Our patient did not show typical PLCH HRCT aspects, presenting with large randomly distributed pulmonary nodules (up to 12 mm). The histological diagnosis of PLCH was clinically unexpected. The FDG uptake is compatible with this diagnosis, but unspecific. Predominant nodular patterns of PLCH have been described and may determine the early phase of the disease, whereas cystic patterns prevail later on. To the best of our knowledge, it has still to be determined how cystic lesions are formed, and how rapidly the dynamics of lung parenchymal abnormalities evolve. We demonstrate within a surprising short clinical course progressive nodular lesions, beginning with cavitation and partial regression after one month of therapy. Low incidence and spontaneous recovery of PLCH hamper the performance of clinical trials. Glucocorticoid therapy is successful only in a subgroup of patients and is advocated on empirical grounds in the treatment of nodular PLCH to accelerate the resolution of inflammation. Because of the predominantly macronodular pattern our patient received steroids in addition to nicotine abstinence and showed impressing regression within one month. The long-term impact of this strategy in BRAF V600E positive PLCH patients and the possible superiority of a targeted therapy with vemurafenib remain to be elucidated. But early therapeutic intervention, before conversion into cystic stages of PLCH may have the largest benefit.

Background
Patients with advanced non-small cell lung cancer are qualified to chemotherapy treatment with palliative intentions. Median survival time of those patients is 10 months and 24% live longer than 12 months. Current treatment modalities with targeted drugs and maintenance treatment extend survival time in limited group of patients beyond 24 months.The aim of the paper is to analyze selected patient-related factors, tumor factors and treatment regimens among patients who survived more than 24 months.

Methods
47 patients selected among 390 patients with advanced non-small cell lung cancer whose data were previously presented on WCLC 2011, not qualified to radical chemoradiotherapy treatment, who survived more than 24 months. Patients were treated with various platinum-based chemotherapy regimens – CDDP+Navelibine, CDDP+Gemcitabine, CDDP+Pemetrexed, Paclitaxel+Carboplatin and targeted drugs like erlotinib, gefitinib and bevacizumab. Usage of specific treatment regimen depended on clinical situation. Retrospective analysis of selected host factors, tumor factors and treatment regimen.

Results
Host-dependent factors: sex – 30 women, 17 men, age – range 39-67, median age 58 yrs, stage – IIIB – 15 patients, IV – 21 pts, reccurence after radical treatment – 9 pts; active smokers – 11 pts, former smokers – 32, non-smokers – 4; ECOG performance status – 0 – 32 pts, 1 – 15pts. Tumor-related factors: NSLC subtypes – not-otherwise specified – 28 pts, adenocarcinoma – 14, squamous cell carcinoma – 5 pts. Number of metastatic sites – 1- 30 pts, 2 – 17 pts; Metastatic locations – bone – 17 pts, lymph nodes – 20 pts, liver – 21 pts, brain – 0 pts. Treatment related factors – antyEGFR treatment – 6 patients, monoclonal anti body maintenance- 2 pts, premetreksed maintence-7 pts, taxenes maintenance- 8 pts. Patients recieved from 1 up to 4 lines of treatment. Median survival time was 34 months.

Conclusion
Survival time depends on various factors including usage of targeted drug therapies and maintenance treatment. Good performance status during treatment initialization still is one of the most important prognostic factors.

Background
Thoromboembolism has been reported as one of side effects of bevacizumab (BEV). An occurrence frequency of venous thoromboembolism caused by BEV is 0.2%. Specifically, it increases to approximately 4% within 1 to 3 months soon after chemotherapy. However, a ventricular thormbosis caused by BEV has almost never reported as far. We report a case of a ventricular thormbosis that occurred during chemotherapy using BEV for the lung cancer.

Methods
(case) 55 year-old, Japanese woman, who suffered myocardial infarction at 27 year-old and had a history of smoking (B.I.:350), complained of left.hemiplegia for brain tumor at first. After surgical treatment for brain tumor, she was diagnosed as lung adenocarcinaoma, cT1aN3M1b stage IV and was referred to our hospital. The first line pemetrexed(500 mg/m2) / carboplatin (AUC 6) /BEV (15 mg/kg) chemotherapy for two cycles every 3 weeks was performed. A partial response was found in the initial computed tomography (CT) evaluation, whereas the examination showed a thrombus of 26 × 25 mm in diameter in the left ventricle. D-dimer is 2.0 μg / mL at this point (D-dimer before treatment is 1.5 μg / mL). The anticoagulation therapy by using heparin continuous infusion and sequential oral agent therapy of warfarin and aspirin reduced the thrombus to 11 × 22 mm in diameter.

Results
(Discussion) Thromboembolism is clinically diagnosed by clinical findings, ultrasonography, contrast CT, and D-dimer. D-dimer is a simple indicator even for asymptomatic thrombus formation and has been reported to be valid for the evaluation of thrombus formation. In this case, D-dimer value was almost conserved through this event. We cannot be undeniable the relation the chemotherapy including BEV and left ventricular thrombosis. Also, since there is no evaluation of left ventricular function before chemotherapy treatment, it can not be denied the possibility of thrombus formation by reduced left ventricular function.

Conclusion
(conclusion) We have experienced a case that developed ventricular thrombosis during chemotherapy for lung cancer. It is believed to be evaluated, such as ultrasonography before using BEV in the case passing a long period of time after myocardial infarction.

Background
Lung cancer is the leading cause of cancer death yet public engagement with efforts against lung cancer is low. Public engagement with a cancer is critical to efforts to combat it yet the reasons for low support for efforts against lung cancer have not been systematically characterized.

Methods
We conducted a telephone survey of 1,071 people to determine levels of engagement and attitudes that might potentially drive engagement. These were then analyzed by univariate and multivariate analysis.

Results
8% of participants were involved with a lung cancer organization and 12% chose it among cancers to receive more support. Most participants felt that lung cancer was principally caused by external factors, that it could be cured if caught early, and that lung cancer patients were at least partly to blame for their illness. In multivariate analysis, participants who were supportive in some way of efforts against lung cancer were more likely to be employed, live in suburbia, and to be unsure of the cause of lung cancer. Potential supporters were more likely to be employed, female, younger, have higher income, to believe that genetics is the primary cause of lung cancer and to believe that lung cancer can be cured when caught early. Participants frequently noted that they supported a particular cancer because of knowing someone affected. Full demographic, univariate, and multivariate data will be presented.

Conclusion
As the lung cancer movement attempts to grow and increase its impact, the most successful recruitment efforts will be targeted to these groups.

Background
Background: Erlotinib is inhibitor of epidermal growth factor receptor tyrosine-kinase activity that has been shown to significantly increase survival, delayed symptom progression and improve quality of life in previously treated advanced non-small cell lung cancer (NSCLC). Here we report safety and efficacy data from open label, phase IV trial of erlotinib in Croatian population.

Methods
Methods: patients with advanced NSCLC who had failed prior chemotherapy were treated with oral erlotinib 150 mg daily until disease progression or unacceptable toxicity.

Results
Results: a total of 384 patients (276 men and 108 women, mean age 62) with advanced NSCLC were enrolled in the study from 2006 to 2012 in 10 centers in throughout Croatia. Patient characteristics: Non-squamous NSCLC had 57% of patients, squamous NSCLC 32% and NOS 11%; active smokers 41%, former and/or never smokers 57%; ECOG 0 36%, ECOG 1 54%, ECOG 2 7%, ECOG 3 1%. Most of the patients (83%) received erlotinib in third line of treatment. Progression-free survival (PFS) was 2,3 months showed trend to improved PFS in patients with squamous compared to non-squamous NSCLC (3,7 vs. 2,1 months). PFS in non-smokers was longer than in smokers (2,6 vs. 2,1 months). Disease control rate after two cycles of treatment was 40%; most patients had stable disease. Most common adverse event (AE) was rash which developed in 58% of patients and diarrhea in 28%. Most of AEs were grade I and II. Grade III rash developed only in 10% of patients.

Conclusion
Conclusion: These data confirmed favorable efficacy and safety profile even in non-selected NSCLC Croatian patients, including patients with squamous cell lung cancer.

Background
Various tumors metastasize to the lung and they are often detected as multiple nodules. Regardless of recent advances in computed tomography for detecting small pulmonary nodules and ground-glass opacity components which indicate possible primary lung cancer, preoperative differential diagnosis of either metastatic or primary lung cancer is usually difficult.

Methods
Four cases of such multiple lung metastases that coexisted with primary lung cancer were retrospectively examined: in three of the cases (case 1 is a myxoid liposarcoma in the right thigh, case 2 is a colon cancer, and case 3 is a renal cell carcinoma), a pulmonary metastasectomy revealed that one of the tumors was primary lung cancer. In case 4, the patient had a proven lung cancer that was combined with small nodules in the ipsilateral lung, one of which was pathologically diagnosed as a metastasis from rectal cancer.

Results
In case 1, the patient was diagnosed with clinical stage IA primary lung cancer (a well differentiated adenocarcinoma in the left lower lobe), and a left lower lobectomy was performed 17 days after the initial surgery. In case 2, a postoperative pathological examination revealed that one of the resected pulmonary tumors in the left upper lobe, measuring 5 mm in diameter, was Noguchi type B bronchioloalveolar carcinoma. In case 3, two nodules in the right lower lobe increased in size. An intra-operative pathological examination revealed that one of the pulmonary tumors in segment S9 measuring 7 mm in diameter was adenocarcinoma, and the other tumor in segment S8 located deeply near the pulmonary artery. Subsequently, a right lower lobectomy was performed. A postoperative pathological examination revealed that the tumor in segment S9 was Noguchi type A bronchioloalveolar carcinoma, and the other tumor measuring 8 mm in segment S8 of the resected lobe was metastatic clear cell carcinoma from renal cell carcinoma. In cases 2 and 3, the patients were diagnosed with clinical stage IA primary lung cancer and no additional treatment for lung cancer was required. In case 4, the patient, who had a history of rectal cancer, underwent left upper lobectomy with mediastinal lymph node dissection, combined with partial resection of the left lower lobe. A postoperative pathological examination using immunohistological staining revealed that the nodule in the left lower lobe and a hilar lymph node were metastasis from lung cancer (pT4N1M0, stage IIIA). The remaining nodule besides the tumor in the left upper lobe was diagnosed as metastasis from rectal cancer. The patient recovered uneventfully and was discharged with a treatment plan involving postoperative chemotherapy for lung cancer.

Conclusion
Possible coexistence of primary lung cancer should be considered in multiple metastases from other organs. On the other hand, the stage of the lung cancer depends on a definitive tissue diagnosis of the coexisting small nodules, and the importance of active tissue diagnosis including surgery should therefore be emphasized, especially in patients with previous malignancies.

Background
The diagnosis and treatment of cancer are traumatic events for patients with cancer and their families. It can be a time of a emotional distress for both and can evoke a wide range of emotions related to poor quality of life. The expression of these feelings is crucial in order to cope with a diagnosis of cancer and with treatment side effects and any action where people perceive that they’re moving those feelings outside may be beneficial to them emotionally and physically. Even patients who receive the best support from family and friends may need to connect with other patients, who are facing the same challenges. Patients may seek emotional and/or practical support from cancer patient organizations and find programs designed with the aim to help them and their families to alleviate the emotional concerns.

Methods
During the last five years, WALCE addressed their emotional care needs designing programs to ameliorate the cancer patient quality of life during the treatments. These projects are addressed to caregivers too and are observational studies. - The Look Good ... Feel Better® is active in 25 countries worldwide. WALCE started its collaboration with “La forza e il sorriso – L.G.F.B. Italia” in 2009. From March 2009 to June 2013, 68 make-up workshops were organised at the San Luigi Hospital (Orbassano, Italy) in collaboration with five local cancer centres. 487 ladies attended, guided by 7 voluntary beauticians, with the support of a psycho-oncologist. - Relaxation technique sessions may be helpful to face with stress. They are intended to promote physical, emotional and mental relaxation to get a better recovery of energies. From March 2012 to May 2013, 12 sessions were organised and 28 people attended. - Natural cooking classes are a good opportunity for patients and caregivers to learn that the health is closely related with nutrition and actively get involved during the preparation of food. The natural cuisine plays a key role in healthcare and these lessons provide useful tips for a balanced diet. Participating in these classes can empower people affected by cancer to learn vital skills that enable them to regain control, reduce isolation and restore hope. - Mindfulness Based Intervention (MBI) based on the assumption that a non-judgmental awareness and acceptance of one’s moment-to-moment experience have an effect on the distressing tendencies to escape from or to over-engage with one’s disturbing feelings, emotions and thoughts. MBI can positively impact on coping strategies and on the adaptation to the disease, by encouraging patients to relate differently to their physical and psychological symptoms.

Results
NOT APPLICABLE

Conclusion
Cancer patients tend to cope better with the illness and daily-life when self-confidence is regained. The sense of well-being shared in a relaxed atmosphere a acknowledging social, emotional and psychological needs, whilst being amongst other people with the same fears or anxieties, is an incentive to fight against cancer. It is important to know that there are ways to relieve the discomfort of most treatment-related side effects and to prevent them from becoming severe.

Background
We have previously characterized chemotherapy (CT) administration in the end of life (EOL) of solid tumor patients (pts) and found that close to 40% of them are treated with CT in the last month of life (LMOL). With this work we want to understand the reasons for CT administration in the EOL and chose lung cancer as a model.

Methods
We used a population of dead lung cancer pts that were treated by one oncologist in one hospital from 2004 to 2012. The oncologist was blinded to study design, data collection and analysis. We collected data retrospectively in the pt clinical charts and hospital electronic medical records. For group comparisons we used Chi square and Mann-Whitney tests and logistic regression for multivariate analysis.

Results
We identified 223 pts. The median age of the cohort was 65 years and 83% were males. The histology was NSCLC in 88% of the pts and the stage distribution was stage I or II in 6%, stage IIIA in 9%, stage IIIB in 23% and stage IV in 62%. The median survival of the cohort was 12 months. Of these, 190 pts were treated with CT, 74 (40%) in the LMOL and 50 (26%) in the last two weeks. Univariate analysis shows 11 variables significantly associated with CT administration in the LMOL: (1) dying in the hospital, (2) no asymptomatic interval after first therapeutic modality, (3) no opioid use, (4) less than 7 month survival, (5-6) progression to first and to last CT line, (7-8) worse performance status (PS) at first and at last CT line, (9) PS of 3 at last CT line, (10) having had less total time on CT and (11) having a low median duration of response to CT. In the logistic regression model, living less than 7 months, undergoing more than 3 lines of CT, dying in the hospital, no opioid use, progression to last CT line and PS of 3 at last CT line were considered predictors of CT administration in LMOL.

Conclusion
We show that CT administration in the EOL occurs in symptomatic pts that have short survival and chemoresistant disease. It is known that pts, families and society have unreasonable expectations on the efficacy of EOL CT and oncologists feel obliged to try to obtain disease control in poor PS pts with aggressive disease. Additionally, the subjective nature of the bond between oncologists, pts and families in the EOL is impossible to capture in this report.

Background
Recently, with the progress of treatment, the patients showing prolonged survival has increased in advanced lung cancer. Even though local treatments for the patients with vertebral metastasis, such as radiation therapy and/or surgery, are considered with effective, the indication of the treatment has not been determined. We report 5 cases who underwent posterior spinal fixation, they could maintain ADL(Activities of Daily Livings) well and could keep a long-term prognosis.

Methods
Between December 2012 and June 2004, we objectively evaluate postoperative state and prognosis of the 5 cases that underwent the posterior spinal fixation surgery for vertebral metastasis in advanced lung cancer.

Results
5 patients were underwent this surgery, 3 were male and 2 were female and the average age was 58.9 years. We added the surgery to remove the pressure of the spine in 3 cases, fortunately there was no spinal invasion in these 5 cases. We preformed chemoradiotherapy in 4 cases and chemotherapy alone in 1 case. EGFR mutation was positive in 2 cases. All patients were possible to ambulate in early postoperative day, and they showed improvement of neurological symptoms of paralysis. It was possible to maintain a relatively well ADL.

Conclusion
There may be an excessive burden to perform the surgery for the patients with vertebral metastasis in advanced lung cancer, the surgery for selecting patients may have been able to improve neurological symptoms such as paralysis. Therefore, we thought that the surgical procedure for the patients who are possible to survive long term period might have been one of the important treatment methods because of maintaining possibly their ADL well.

Background
Castleman’s disease, an uncommon disease that causes benign lymph node hyperplasia, must be distinguished from reactive lymph node hyperplasia and malignancies. Unicentric Castleman’s disease in patients with anemia is rarely described.

Methods
We describe the case of a 25-year-old woman with plasma cell type of unicentric Castleman’s disease presenting as iron deficiency anemia. We report the clinical course from the initial presentation to diagnosis and surgical cure and review the literature.

Results
Figure 1 A 25-year-old woman was referred with abnormal chest X-ray which was found during pre-employment screening. She had no other complaints, and her previous medical history was unremarkable, as was her family medical history. No fever, acute infectious symptoms and signs, or any other abnormalities were found in the physical examination. Normocytic anemia was found based on initial blood tests (hemoglobin 7.7 g/dl, mean corpuscular volume 88.9 fl, with normal white blood cells, red blood cell distribution width, liver renal functions, and electrolytes). Further laboratory examinations (ferritin 89.0 ng/ml; serum iron 23 mg/dl; and total iron binding capacity 206 mg/dl) demonstrated that she was iron deficient. Chest X-ray and chest CT scan confirmed a mass (6x4x4 cm) at right superior and mid-mediastinum (Fig. 1). PET/CT showed paratracheal mass shows inhomogeneously high FDG uptake. In suspicion of lymphoma, diagnostic thoracoscopic surgery was planned. However, intraoperative frozen section diagnosis of a specimen was benign. Thus, the mass was completely removed and diagnosis was consistent with unicentric plasma cell type Castleman’s disease. Three months after surgery, without any medical intervention, the laboratory data had recovered to normal range (hemoglobin 14.5 g/dl). She receives regular follow ups and no recurrence has been found for 4 years since surgery.

Conclusion
The surgical resection would appear to be the optimal treatment modality, and constitutional symptoms may be resolved. The prognosis following complete surgical resection appears to be good.

Background
Pleurodesis is an accepted therapy for patients with recurrent and massive pleural effusion in patients with advanced non small cell lung cancer.Bleomycin is safe and effective for chemical pleurodesis in several studies.The aim of this study was to study the effectiveness of bleomycin pleurodesis in advanced non small cell lung cancer.

Methods
Seventy five cases of proven non small cell lung cancer ; with recurrent and symptomatic pleural effusion admitted to our hospital between 2006 - 2012 were included in the study. All patients received chemical pleurodesis with 60 units of bleomycin after pleurocentesis using intercostals drainage.All patients received systemic chemotherapy following pleurodesis.

Results
The clinical control of pleural effusion by 4 weeks of procedure and the need for a repeat pleurodesis was analysed.The success rate was 76.7 per cent (95% CI,68.6 – 88.7 %). The only significant complication reported was chest pain of varying degrees. There were no deaths associated with bleomycin pleurodesis.

Conclusion
Bleomycin pleurodesis may be considered as safe and cost effective treatment in patients with advanced non small cell lung cancer with symptomatic pleural effusion in developing countries.

Background
A number of recent studies have reported poor cancer survival in England when compared to other developed countries worldwide. In particular, lung cancer has been highlighted as amongst the worst, with only Scotland and Malta showing poorer survival rates in Europe. The aim of this study was look for an explanation for these findings by assessing our post-operative survival in patients undergoing surgery for lung cancer. Through this we hope to establish whether the management of patients with curative disease is inferior or whether other key factors, such as stage and timing of diagnosis, are to blame.

Methods
501 patients were identified who had undergone lobectomy, bilobectomy or pneumonectomy for non-small cell lung cancer between January 2003 and January 2011. Information regarding patient demographics and histological stage of disease was collected. NHS number tracing was used to obtain patient status at follow-up. Survival data was plotted using the Kaplan-Meier method with comparison of stage 1 and 2 disease. In addition, information was collected from the NHS lung cancer database to identify the percentage of patients diagnosed with non-small cell lung cancer who underwent surgical management during the research period.

Results
Of the 501 patients within this cohort, 263 had stage 1 disease, 147 stage 2 disease and 91 stage 3 or 4 disease. Average age at the time of surgery was comparable between the groups (mean 67 years) and there were considerably more men within the study than women (M:F = 303:198). 5 year survival in patients with stage 1 or 2 disease was 75% or 40% respectively (Figure 1). On average, 12.9% of patients diagnosed with NSCLC underwent surgical resection. Figure 1: Comparison of survival following lung resection in patients with stage 1 and 2 NSCLC Figure 1

Conclusion
Cancer survival is an important measure of the effectiveness of both management strategies and healthcare systems. These findings demonstrate that in patients with early, operable lung cancers we are achieving survival rates comparable, and in some cases superior, to other developed countries. Only a very small number, 12.9% of patients, are diagnosed early enough to undergo surgery. This would suggest that it is not the treatment provided in England that is inferior, but our ability to diagnose lung cancer at an early stage.

Background
it is well known that combining chemotherapy and radiation therapy is beneficial to patients with locally advanced non small cell lung cancer compared to radiation alone or compared to a sequential approach using chemotherapy and radiation therapy. However, it is not obvious what is the best schedule. A few randomized trials assessed chemotherapy as induction before chemoradiotherapy (CT -> CTRT) versus chemotherapy as consolidation, after chemoradiotherapy (CTRT -> CT). Most of those trials are phase II trials with moderate sample sizes and were not designed to demonstrate treatment effect in terms of overall survival.

Methods
the study coordinators of those trials (T. Berghmans, H. Choy, P. Fournel, P. Garrido, J. Van Meerbeeck) agreed on a protocol for carrying out a meta-analysis of individual patients data and for sharing the individual patients data that were sent to the coordinating institution. Overall survival was the primary outcome, progression-free survival and toxic death occurrence were among the secondary outcomes. The treatment effect was assessed through the estimation of the hazard ratio of the survival distributions using CTRT -> CT as reference. Combined hazard ratio was obtained through Cox regression models (fixed effects) with a stratification by trial. Preplanned interactions between baseline covariates (age, sex, performance status, stage, histology) and treatment effect were assessed. Toxic death rates were analyzed per trial and odds ratios have been estimated to assess the treatment effect. Combined odds ratio was obtained by the Peto method.

Results
the data bases of the 5 eligible identified trials (3 with cisplatin based chemotherapy regimens, 2 with carboplatin based regimens) were shared for a total of 534 patients (CT -> CTRT 271, CTRT -> CT 263). Median ages were 60 and 61 years, stage IIIB represented 69%/70% of the patients and EOCG PS > 1 was rare (3%/2%). Median follow-up ranged from 12 months up to 66 months and rates of events from 44% to 88%. No significant difference was detected either for overall survival with an estimated HR of 0.96 (95% CI : 0.79-1.17) without heterogeneity between the 5 trials (I[2]=0) or for progression-free survival (analysis restricted to 4 out of the 5 trials), HR=0.91 (95% CI : 0.75-1.11) and absence of heterogeneity (I[2]=2%). For both outcomes, no interaction between the above specified covariates and treatment effect was found. Toxic deaths occurred overall in 3% of the patients, no detectable impact of treatment arm was found with a combined odds ratio of 0.40 and a 95 % CI overlapping 1 (0.15-1.06).

Conclusion
our results suggest that there is no argument in favour of one of the two therapeutic schedules when looking at overall survival or at progression free survival; however, in the absence of benefit in terms of prognosis, a more detailed evaluation of toxicity is warranted and is ongoing.

Background
Dyspnea is a distressing symptom that occurs in up to 75% of patients with lung cancer as measured by the Edmonton Symptom Assessment System (ESAS). Appropriate dyspnea management (DM) can improve the patient’s quality of life, performance status and emotional well-being. However DM is not uniform or standard across Regional Cancer Centers (RCC). A quality improvement intiative on DM was implemented through the Disease Pathway Management (DPM) of Cancer Care Ontario (CCO). DPM is a unifying approach to quality improvement that integrates program activity across the cancer continuum in order to advance system-wide improvements. This initiative provided advice on various delivery models and strategies for DM.

Methods
Seven RCCs received funding from CCO to undertake one year pilot projects in DM. These projects had to have potential for significant impact, be innovative and be cost effective. Each RCC project was required to address the physical and psychological aspects of dyspnea that affect the patient, their families and/or caregivers. The precise methodology was left to each RCC to develop and initiate within the specified criteria. Approaches included educational sessions for patients and family members, individual counseling and treatment plans, and symptom management clinics. Four measures were tracked: ESAS for patient-reported symptom severity, Palliative Performance Status (PPS) for evaluation of functional status, European Organization for Research and Treatment Quality of Life Questionnaire (EORTC-QOL) to measure quality of life and a Patient Survey to evaluate the patient’s knowledge of dyspnea, preparedness for self-management and overall satisfaction with the DM initiative.

Results
188 patients were evaluable. 45% of patients with an initial severe dyspnea score on ESAS reported a shift to either a moderate or mild score by the last visit. 32% of patients with an initial moderate dyspnea score on ESAS reported a shift to a mild score. Patient satisfaction was high, with feelings of empowerment to carry on daily activities as a result of the interventions offered; caregivers reported a better understanding of dyspnea and better ability to support their loved ones; clinicians noted a difference in patients attending the dyspnea care initiative and valued the helpful resource for their patients. Challenges encountered during the project were lower than expected recruitment due to lack of clinical engagement from busy clinicians, multiple additional visits that sometimes conflicted with other scheduled patient visits to the RCC, and declining performance status of the patients precluding in-person attendance for training in DM techniques.

Conclusion
DM can be effectively implemented and tailored to local needs of a RCC or program. Key factors for success included a clinical champion and a multidisciplinary team approach in order to build the necessary knowledge and expertise for DM. The lessons learned as a result of these pilot projects have led to a new initiative to improve the quality and consistency of DM across the province of Ontario. This new initiative will incorporate novel approaches for knowledge transference with the possibility of engaging healthcare providers beyond the RCC.

Background
At Cancer Care Manitoba (CCMB), Canada, lung cancer patients when considered incurable, are often enrolled into the palliative care program. One of the most important pre-requisites for patients to enroll into this program is to have a Do Not Resuscitate (DNR) status determined and signed by an authorized cancer care provider.Currently there are no guidelines on how and when to address advanced care planning and more specifically when to discuss DNR status. At this time the issue appears to be addressed on an individual basis depending on the patient’s unique circumstances and the personal practice of the physician involved. DNR discussions in the context of incurable lung cancer can be difficult for a number of reasons that include but are not limited to: social or family complexities such as dysfunctional dynamics or conflicted goals for medical attention, cultural differences, limited patient and/or family understanding of the patient’s prognosis, a recent diagnosis, limited time for discussion, physician preferences, and questionable patient cognitive competence secondary to pain, medications, brain metastases, whole brain radiotherapy, and emotional stress caused by the disclosure of incurable nature of the disease . Lung cancer team at CCMB has identified a need to explore the current practice and opinions around DNR discussion and future development of advance care planning guidelines. The findings from the current pilot study may serve the basis for a prospective randomized trial that tests an intervention that incorporates the timing of DNR discussions.

Methods
From January 2012 to November 2012 a total of 10 lung cancer patients who had previously discussed and determined their DNR status, 9 family members, and 10 health care providers were identified and agreed to participate. A mixed quantitative and qualitative methodology including a written questionnaire followed by a nurse directed audio recorded interview was employed to determine participant’s views. Interviews were transcribed and then content analysis and constant comparison techniques were used to identify, code, and categorize primary patterns in the collected data.

Results
Major themes identified from the patient and caregiver’s perspective include their trust in the health care system, the need for clear communication, the desire to be respected, and the benefit of having family present during discussion. Health care provider’s commonly expressed the importance of having adequate clinic time for discussions and the need for the process to be adaptable. Admission into a palliative care program is the most common trigger for initiating discussions but other points earlier in the disease course may also be appropriate. The presumed level of stress that patients are thought to undergo with DNR discussions is less than they actually report. Fnal data analysis is pending and subject to refinement before final presentation.

Conclusion
DNR status decision-making is a complex process, influenced by patient, family, and health care provider factors. The implementation of a standard approach to DNR discussions could potentially be restrictive. Potential areas of improvement include additional resources, and the specific guidelines. A need for a prospective trial addressing the timing of DNR discussion was identified but may not be feasible.

Background
Limited disease of small cell lung cancer (LD-SCLC) respond well to concurrent chemo-radiation (CCRT), but have high relapse rates and short relapse-free survival (RFS). We aimed to evaluate tumor metabolic activities measured by [18F]-fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET) as a prognostic factor and analyze its relationships with markers of tumor biologic behavior.

Methods
Forty-one LD-SCLC patients receiving 4 cycles of EP (etoposide 120 mg/m[2], days 1–3; cisplatin 60 mg/m[2], day 1), 2 cycles of EP (etoposide 130 mg/m[2], days 1–3; cisplatin 30 mg/m[2], day 1) with concurrent mediastinal irradiation were enrolled. SUVmax of primary tumor was revised with the SUV of liver (SUVlivermax). Differences between pre- and post-treatment average SUV uptake of the primary tumor and intrathoracic lymph nodes were presented as ∆SUVliveravg. Thirty-one tumor biopsy specimens were immunostained for glucose transporter-1 (GLUT-1), Bcl-2, and hypoxia inducible factor-1α (HIF-1α).

Results
Objective response rates (ORRs) after CCRT were 92.7% and 87.8% on chest CT and FDG-PET, respectively. Median overall survival (OS) and RFS were 13.7 (range 4.4–54.1) months and 10.4 (range 0.97–54.1) months, respectively. In multivariate analysis, pretreatment lactate dehydrogenase (LDH) and ∆SUVliveravg correlated significantly with RFS (hazard ratio [HR] 2.8, P = 0.043 and HR 0.3, P = 0.004, respectively). Gender, pretreatment LDH, objective tumor metabolic response, and SUVlivermax correlated significantly with OS (HR 12.1, P = 0.006; HR 3.7, P = 0.037; HR 10.1, P = 0.008 and HR 0.2, P = 0.014, respectively). High GLUT-1-positivity (>75%) and pretreatment LDH levels (>400 U/L) correlated significantly with better ORR (P = 0.012) and HIF-1α immunoreactivity score (IRS, P = 0.029), respectively.

Conclusion
∆SUVliveravg and GLUT-1, with respect to tumor glucose metabolism, might predict RFS and ORR, respectively, in definitive CCRT-treated LD-SCLC patients.

Background
Clinical data suggests low molecular weight heparin (LMWH) may produce a survival benefit when given to cancer patients. Several studies are being conducted in different cancer primaries to explore this hypothesis including FRAGMATIC, which will be presented at the World Lung Conference. The FRAGMATIC trial is an open label multi-centre Phase III randomised controlled trial in patients with lung cancer comparing anticancer treatment according to local practice plus dalteparin (Fragmin®), with anticancer treatment according to local practice alone. The primary outcome measure is overall survival and the secondary outcome measures include; toxicity, VTE-free survival, metastasis-free survival, quality of life, patient experience and cost utility. 2202 patients have been recruited in order to detect an advantage of 5% in overall survival at 1 year (to 30%). Fragmin is given as a daily subcutaneous injection for six months by the patients themselves or a willing partner/ carer. This may raise practical challenges regarding how applicable the results of the main trial will be to clinical practice especially with regard to quality of life and thus compliance. We conducted a longitudinal quality of life study to explore the experiences of patients participating in the FRAGMATIC trial to better inform the patient journey and practicalities of applying results to clinical practice.

Methods
Semi-structured interviews were held at up to three time points, with two groups of patients recruited from the intervention (n=4) and control (n=6) arms of the FRAGMATIC trial.A total of twenty interviews were analysed using Interpretative Phenomenological Analysis to identify emergent themes that reflect participants' lived experience. We report data focusing on participant views and experiences of self injection for six months.

Results
No discernable difference in quality of life was identified between patients in the control or intervention arm who reported similar experiences of the cancer journey and its treatments. Patients on the intervention arm found daily Fragmin injections to be acceptable and a small consequence of having additional treatment as they saw it. Patients developed processes in order to administer Fragmin at similar times and described adaptive techniques to reduce discomfort and bruising. Minimal training was required to be able to inject Fragmin and few side effects were reported. The most common side effect was stinging which was short lived and bruising around injection sites. Patients reported these events did not impact upon quality of life. Overall, Fragmin was found to be an acceptable intervention, which did not pose any compliance problems.

Conclusion
The FRAGMATIC trail will report the impact of daily subcutaneous LMWH on overall survival in lung cancer. In this trial 1100 patients self injected Fragmin or had it administered to them. Based on qualitative interview data, Fragmin injections were well tolerated and the experiences of patients suggest that it would be relatively simple to introduce self-injecting into standard lung cancer therapy should the results of the FRAGMATIC study demonstrate a survival benefit.

Background
A previous 2010 audit at the Royal Adelaide Hospital (RAH) found delays in treatment initiation when compared to targets set in the United Kingdom (UK) National Cancer Plan. The aim of this study was to review our performance in 2011 and to explore the impact of curative or palliative treatment intent and rural or urban based location, in order to guide future improvement.

Methods
Using a pathology database, we retrospectively reviewed 128 case notes of patients referred to the RAH in 2011 with new histologically confirmed lung cancer. We identified treatment intent, patient location and key dates in diagnosis and treatment by radiation oncology, medical oncology, cardiothoracic surgery or palliative care before calculating median intervals between these points.

Results
Figure 1 52% of patients were urban and 48% rural. 45% of patients were treated with curative intent and 55% with palliative intent. Pertinent median intervals include: referral to appointment (appt): 4 days (inpt: 0 days, outpt: 7 days); appt to diagnosis (a) rural outpt :16 days, urban outpt: 10 days (p=0.58); (b) surgical: 21 days, curative outpt CH-R: 7 days (P<0.001); (c) surgical rural 30.5 days, surgical urban 13 days (p<0.001); referral to treatment: 41 days (UK 2006: 41 days, RAH 2010: 48 days, (a) outpt: 54 days, inpt: 21 days (p<0.0001), (b) surgical: 62 days, outpt curative CH-R: 34 days (p=0.81), curative outpt CH-R: 34 days, palliative outpt CH-R: 46.5 days (p=0.79)). Inpts met both UK targets. There are a number of factors contributing to the delays in surgical cases including a higher proportion of cases needing multiple biopsy attempts and requiring CT-FNA, which takes twice as long to obtain as a bronchoscopy. Rural location did not impact on overall time to treatment but delays seen in appointment to diagnosis, in particular the surgical cases.

Conclusion
For 2011, the RAH achieved the first UK targets for first appointment to a specialist but did not meet the target set for referral to commencing treatment. Delays were seen in some rural subgroups, necessitating the need for a streamlined rural pathway to assist in managing investigations and appointments. In turn, this will also reduce the number of rural elective admissions. Initiatives to improve time to treatment with curative intent include pre-booking investigations, utilising EBUS for staging and improving access to CT-FNA. A follow up audit including analysis of mortality is being conducted.

Background
Locally advanced non-small cell lung cancer has poor prognosis with less than 20% probability of 5-year survival despite the use of concurrent chemoradiotherapy. Last generation chemotherapy regimen, high dose of radiotherapy, and surgery if feasible are options to achieve better outcomes in highly selected cases.

Methods
A case report of a 25-year woman with non-mutated adenocarcinoma of the right lung is presented in this work.

Results
The young woman, age 25 years, smoker of 1 pack/day since the age of 18 years, without family cancer history presented with right lung perihilar mass. CT scan showed tumor infiltration of the right upper lobe of 73x65x80 mm involving mediastinum as well as enlargement of right hilar and mediastinal lymph nodes. Suspicion of left aortic arc lymph nodes infiltration was described on FDG-PET/CT but no distant metastases were seen. Transbronchial biopsy confirmed adenocarcinoma. EGFR mutation and ALK translocation were not found. TNM stage (UICC7) was T4N2-3M0 – clinical stage IIIB. Performance status 1, cough, dyspnoea and mild weight loss at time of diagnosis were noted. Treatment consisted of 5 cycles of chemotherapy with cisplatin and pemetrexed in standard doses every 3 weeks and concurrent radiotherapy given in the dose of 72Gy/36 fraction/7.5 weeks during 3-5th cycle. Restaging after CRT showed substantial partial remission with negative mediastinal lymph nodes on PET/CT scan, therefore surgery was proposed, resulting in right upper lobectomy and mediastinal lymphadenectomy. The histology showed residual adenocarcinoma 35x20x45mm with regressive signs, stage ypT2 ypN0. Whole-body and brain FDG-PET/CT two months after surgery confirmed complete remission. No additional chemotherapy nor prophylactic cranial irradiation were indicated but close follow-up has been planned because of the high risk of recurrent distant metastases.

Conclusion
This case report showed successful multimodal therapy of advanced NSCLC in a very young woman whose age and good PS warranted aggressive treatment approach. This abstract was supported by a grant from the Ministry of Health of the Czech Republic – IGA MZ CR NT12331-5/2011 and by a grant from the Charles University Prague PRVOUK - P27.

Background
Photodynamic therapy (PDT) is a unique combination of photosensitizing drug and laser light for drug activation which results in tissue destruction by direct cellular damage and small vessel thrombosis. We have utilized PDT to manage airway malignancies since 1998. This large, single institution case series will describe technical considerations of airway PDT,incorporation of PDT into multi modality therapy, patient selection, patient management and clinical outcomes.

Methods
All patients receiving PDT were entered into an IRB approved outcomes registry. Data was abstracted from the medical record and the cancer registry. Photofrin was used as the photosensitizer at a dose of 2 mg/kg IV . Light dosimetry ranged from 100 - 200 J. Light was delivered at 630 nm. All patients were treated under general anesthesia for light application and airway debridement. Autofluorescent bronchoscopy was used to delineate extent of endobronchial disease.

Results
Between 1998 and 2013, we have performed PDT on 910 patients; 519 procedures (57%) have involved the tracheobronchial tree. The average age was 62 years with a range of 18 -92 years.The cell types managed by PDT included NSCLC, SCLC, carcinoid, and metastases to the airway. Indications for tracheobronchial PDT included symptom management/ palliation (63%), induction therapy (27%), and definitive therapy with curative intent (10%). Dyspnea and post obstructive pneumonia from airway obstruction were the most common symptoms. Photosensitivity rate was < 2%. There were no perforations of the tracheobronchial tree. There was a single stricture in this cohort; this occurred in a patient who had been previously resected.

Conclusion
PDT for primary or metastatic lesions to the tracheobronchial tree is safe. Early and advanced lesions can be treated. PDT can be used prior to surgical resection and may increase resectability by reducing endoluminal disease. PDT may be used after radiation therapy with good efficacy. PDT can be utilized during multiple courses of treatment to palliate obstructing or bleeding lesions. Photosensitivity is an infrequent complication. Our experience demonstates the utility of PDT across all stages of NSCLC when there is a visible endoluminal component. In order to more specifically define the utility of PDT in NSCLC, we have established a prospective multi center registry to delineate indications, contraindications and the role of PDT in treatment algorhythms for airway malignancies.

Background
A number of studies have demonstrated that early palliative care involvement in the care of metastatic non-small cell lung cancer patients leads to significant improvements in quality of life, symptom control, and survival. Most studies, however, were performed in large centres where palliative care service is readily available. The North West Cancer Centre is a comprehensive rural Australian cancer centre. In this study, we reviewed our patterns of palliative care involvement, seeking to examine the current practices in the light of our limitations.

Methods
Retrospective analysis of patients with metastatic adenocarcinoma of the lung in the North West Cancer Centre between January 2008 to December 2012.

Results
We identified 35 patients with stage IV adenocarcinoma of the lung. The median duration to first review by palliative care is 126 days (range 28 - 669 days). Median duration from first review by palliative care to time of death is 22 days (range 11 - 152 days). Both are calculated from time of first review in medical oncology clinic. Further analysis revealed only 10 patients reviewed by a palliative care physician, with a median duration to first review of 227 days (range from 76 to 670 days). 25 patients were reviewed by specialist palliative care nurses, with a median duration to first review of 382 days (range from 76 to 676.5 days). 30 patients required inpatient palliative care, however only 7 were admitted under a specialist palliative care unit. 8 patients are currently alive. 5 patients received no palliative care input.

Conclusion
Lung cancer patients in rural and remote communities are known to have poorer prognosis compared to their urban counterparts. Our study further highlights this disparity. Early palliative care is becoming a standard of care. However, there are unique challenges facing a rural cancer centre that limits feasibility of early palliative care involvement in rural and remote lung cancer patients. A limited number of specialist palliative care health care providers service a large area (map). Currently we have 1 part-time specialist palliative care physician and 2 specialist palliative care nurses, catering for inpatient, ambulatory, and community palliative care patients. Our study found significant involvement of non-palliative care specialist health care providers assisting in provision of palliative care services. It is our impression that increasing awareness and education of the supporting health care providers is imperative to improving delivery of palliative care in a rural setting.

Background
Docetaxel is an accepted second line drug for NSCLC not used frequently for its side effects. VT-122 (propranolol and etodolac) which has anti-angiogenic COX-2 inhibition properties. Modifying the tumor-microenvironment and sympathetic system has anticancer activity as well as ameliorates toxicity of anticancer drugs. Docetaxel efficiency can be improved with VT-122 which possesses the above property. To increase efficacy of docetaxel and safety profile using VT-122.

Methods
20 (10 in each arm) patients (55-65 years) were treated with docetaxel (75mg/m[2]) with or without VT-122. VT-122 was started 7 days before giving docetaxel. Propranolol doses were titrated to maintaining heart rate around 60. Etodolac was titrated on the basis of CRP to a maximum of 1200mg.

Results
Overall Response seen in 4 patients (VT-122 arm) and 2 patients (docetaxel arm). 6 patients in VT-122 arm completed 6 cycles of docetaxel while in non-VT-122 4 patients completed the 6 cycles. 1 year survival was 30% vs 10%; p=0.025. Grade III/IV hematologic toxicity was decreased by 50% ie 6 cases in docetaxel arm and 3 in VT-122. In non-hematological toxicity this trend was seen for asthenia, neuropathy, skin and nail changes and weight loss.

Conclusion
Addition of VT-122 (propranolol and etodolac) increases efficacy of docetaxel and is cost effective.

Background
Fatigue, depression, sleep disturbance, and dyspnea are symptoms frequently reported by patients with non-small cell lung cancer before anti-cancer treatment. The purposes of this study were to identify latent subgroups of patients with NSCLC using the above four symptoms assessed at pre-chemotherapy and to determine whether different patient subgroups based on their symptom profiles can predict their survival.

Methods
Patients with stage III or IV NSCLC were recruited from a large teaching hospital located in northern Taiwan. Fatigue subscale of the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) was used to measure fatigue. The Hospital Anxiety and Depression-Depression subscale was used to measure depressive symptom. The Pittsburg Sleep Quality Index (PSQI) was used to measure sleep disturbance. Four items from EORTC QLQ-C30 and QLQ-LC13 was used to measure dyspnea. All patients were followed up until the end of the study. The follow-up time ranged from 10 to 30 months. Latent class analysis was used to identify patient subgroups. Cox regression was used to examine the effect of patient subgroup on survival after adjusting the effect of covariates.

Results
The study sample consisted of 127 NSCLC patients. Two distinct patient subgroups were identified. The first group (30%) was characterized with high severity on all four symptoms (high symptom group), the second groups (70%) showed low severity on all four symptom (low symptom group). Before adjusting covariates, the “high symptom group” had significantly shorter survival time (median = 11.1 months) than the “low symptom group” (median = 21.1 months) (P = 0.016). After controlling for gender and disease stage, the group membership remain significantly associated with survival time (P=0.047). The risk of early death was 1.81 times higher in the “high symptom group” as compared to the “low symptom group” (HR=1.81, 95% CI=1.01 to 3.23).

Conclusion
The finding suggests that pre-treatment symptom profile has additional prognostic prediction in patients with advanced NSCLC. Early symptom assessment is recommended.

Background
The incidence of lung cancer in the Czech Republic has been unacceptably high for decades. It has reached 91/100 000 in men and 31/100 000 in women. Bronchoscopy plays an important role in the diagnostic process. To assess the quality and density of the bronchological net and to determine the current situation in the implementation of bronchoscopy in the Czech Republic we decided to carry out a national survey.

Methods
A bronchological questionnaire was sent by e-mail to every pneumologist performing bronchoscopy. With the help of repeated e–mails and additional telephone interviews we achieved a response rate of more than 95%.

Results
In 2012 there were 56 centers performing bronchoscopy in adults and 9 in children in the Czech Republic. The bronchological units of adult clinics employed 169 bronchologists using 231 fiberscopes, out of which 88 were video bronchoscopes and 79 rigid bronchoscopes. Altogether 30 354 bronchoscopies were performed in adults. General anesthesia was used in 2 146 cases, the rest was carried out under local anesthesia. The total number also includes 1767 bronchoscopies performed using rigid instrumentation. Cytological examination of the material obtained during bronchoscopy was carried out by a pathologist (Department of Clinical Pathology) at 34 centers, by a pneumologist trained in cytology at 13 centers and by both the pathologist and the pneumologist at 9 centers. Interventional bronchoscopic procedures (laser, electrocautery, stenting, brachytherapy, cryocautery and the introduction of endobronchial valves) were used in 17 departments. Altogether the above mentioned procedures were performed 654 times during one year. All 9 children's bronchological departments employed 12 bronchoscopists, using a total number of 29 fiberscopes, out of which 17 were video bronchoscopes. Seven pediatric bronchoscopy centers also used rigid instrumentation. The total number of pediatric bronchoscopies performed in 2012 was 682, the majority of them (621) under general anesthesia. Out of all bronchoscopies, the rigid bronchoscopy was performed in 32 cases. At five centers cytology examination was performed by a pathologist, at 3 centers by a pneumologist trained in cytology and at one center by both specialists together. Further analysis of the specialized bronchological procedures (endobronchial ultrasound, autofluorescence), including a comparison with previous surveys will be given in the lecture or on the poster.

Conclusion
The level of bronchological service offered in the Czech Republic is comparable with the most developed countries and serves a prompt and exact diagnostics of patients with respiratory disorders including lung cancer. "Supported by Projects (Ministry of Health) of conceptual development of research organization 00064203 (FN Motol, Prague, Czech Republic)".

Background
Dosing of many chemotherapy drugs is currently based on body surface area (BSA), which is partly derived from body weight. However, BSA has been shown to result in large inter-patient variability in drug exposure. Another measurement of body composition is fat free mass (FFM), which includes hepatic, renal and muscle metabolic tissue. Depletion of FFM is termed sarcopenia, which in breast and renal cancer populations is associated with increased chemotherapy toxicity and time to disease progression. Sarcopenia can occur regardless of weight or BSA. In order to explore the value of FFM assessment in lung cancer patients undergoing chemotherapy, we performed a systematic review to explore this relationship. We present an analysis of relationships between sarcopenia, body weight, BSA, chemotherapy toxicities and outcomes in lung cancer patients.

Methods
Two search strings (‘muscle mass loss’ AND ‘lung cancer’) were used as a basis for formulating specific literature search strategies in five databases. We included all publication types and the search was limited to articles written in English. From this larger review, we then narrowed our focus to chemotherapy-specific studies.

Results
Our systematic review of FFM loss in lung cancer consisted of 29 studies, from which 7 studies considered specifically the relationship between FFM and chemotherapy. One study of 250 obese patients found 15% were sarcopenic, FFM poorly correlated with BSA (r[2]=0.37), and variation in FFM per unit of BSA could account for up to three-times variation of chemotherapy volume of distribution, potentially indicating increased risk of chemotherapy toxicity. A further population-based study (N=441) found 46.8% were sarcopenic, despite only 7.5% being underweight. A further two smaller studies (N=40, 41) found the prevalence of sarcopenia to be 61% and 46% (despite approximately half being overweight or obese). Prospective evaluation of the effect of chemotherapy on FFM in 3 small studies (N<22) showed no significant change. Sarcopenia was also associated with poorer performance status and reduced median survival.

Conclusion
A more refined understanding of body composition is needed in lung cancer patients, in order to minimise toxicities of chemotherapy, whilst ensuring optimal chemotherapy dosage. The current literature suggests that a more detailed prospective study is required in order to explore the validity and advantage of measuring FFM rather than BSA in lung cancer patients, in the work-up to chemotherapy. It also suggests that targeting sarcopenia may offer a novel approach to improving chemotherapy tolerance and overall survival.

Background
The presence of EGFR activating mutations in NSCLC and sensitivity of these tumours to EGFR tyrosine kinase inhibitors (TKI) was first described in 2005. For NSCLC patients harbouring an activating EGFR mutation the treatment of choice is an EGFR TKI which is better tolerated and easier to administer than chemotherapy. However, questions remain about duration of therapy and optimal management on radiological progression.

Methods
A retrospective case note review was undertaken with the aim of establishing current practice in prescription and discontinuation of EGFR-TKIs, subsequent therapies and clinical outcomes. We identified consecutive patients from the database of the genetic testing laboratory (from Q4 2009 when routine testing commenced to a cut-off point of February 2013). 171 case- notes were reviewed for demographic and clinical data including survival.

Results
Of 171 cases 116 (69%) had received treatment with an EGFR TKI (Gefitinib 79%, Erlotinib 19%, both 2%). The reasons for not receiving treatment included: patient received radical therapy, patient died before oncology assessment and patient preference. 63% of patients were female, 26% never smokers, 44% ex-smokers, 6% current smokers and smoking history was not documented in 23%. 76% of patients had Stage IV disease and performance status was 0-1 in 47%, 2 in 22%, 3 in 7%, 4 in 2% and not documented in 23%. The average length of treatment on EGFR TKI was 10.5 months (range 0.5-40) and 36 (31%) patients were still on treatment at the time of analysis. Disease progression on the EGFR-TKI (PD) had occurred in 82 (71%) of patients and 28 (34%) of these continued EGFR TKI treatment beyond PD. The average length of time on treatment beyond PD was 5.6 months (range 1-16) and TKI treatment was ongoing in 9 of the 28 patients. 25 of the 73 patients (34%) with PD who had stopped EGFR TKI went onto receive a second line systemic treatment: pemetrexed and platinum 60%, gemcitabine and carboplatin 20%, single agent pemetrexed 8%, vinorelbine 8%, gemcitabine 4%. Third line therapy was received by 40% of those who had received 2[nd] line treatment.

Conclusion
Patients with EGFR activating mutations often derive a significant clinical benefit and marked reduction in tumour burden with oral EGFR TKI therapy, which results in a reluctance, from both patients and clinicians, to stop therapy at the time of radiological progression if the patient is still experiencing symptomatic improvement. Our results show that treatment beyond disease progression is common (34%) in ‘real –life’ clinical practice with some patients continuing to derive benefit for more than a year beyond the time of disease progression. .

Background
Patients with advanced lung cancer presenting with ECOG 2 at diagnosis trend to be no longer included in large, randomized, registration trials and then recommendations for their treatment are more difficult. Nonetheless, they account for a significant percentage of the cases attended in a Medical Oncology Department. We describe their characteristics and outcomes in our experience.

Methods
Medical records of lung cancer patients with ECOG 2 that were seen in our Department between april 2009 and april 2013 have been reviewed.

Results
124 patients (p) were found. They account for a 15.3% of the overall number of lung cancer patients attended. 106p were male (85.5%). Median age 66 years (44-83). By histology: adenocarcinoma 41p (33.1%), EGFR+ adenocarcinoma 4p (3.2%), squamous 36p (29.0%), small-cell 23p (18.5%), other 19p (15.3%), no histologic diagnosis 1p (0.8%). By stage: III 39p (31.5%), IV 85 (68.5%). By the time of analysis, 76p (61.3%) hade died, other 31p (25.0%) continued palliative care and 17 (13.7%) were still on active therapy. Initial intention of therapy was palliative in 94p (75.8%), radical/adjuvant in 9 /.3%) and 21p did not receive any active therapy beyond supportive care. Drugs administered at first line: carboplatin 43p (34.7%), cisplatin 12p (9.7%), EGFR-TKI 6p (4.8%), non-platinium chemotherapy 39p (31.5%), no Chemotherpy 24 (19.4%). No differences by gender existed in the drugs given, except for TKI which were more frequently given to women (5/6). Median overall survival was 34 weeks (IC: 26.0-41.9). No differences existed by gender (male 32 weeks, female 39 weeks) or stage (III 30 weeks, IV 36 weeks) but they differed by histology: adenocarcinoma 34 weeks, squamous 22 weeks, EGFR+ aenocarcinoma Not Reached, small cell 59 weeks.

Conclusion
A significant percentage of lung cancer patients are diagnosed with ECOG 2 performance status in every histological subtype. Minor differences existed with respect to clinical characteristicas and they benefit from receiving active therapy. This advantage seemed to be lesser in squanous carcinoma.

Background
Erlotinib (E) is a EGF-receptor tyrosine kinase inhibitor (TKI) approved for the treatment of NSCLC after progression to first-line chemotherapy irrespective of EGFR status. Currently EGFR mutation is usually performed upfront upon diagnosis and most mutated patients are treated with first-line TKI's. Nowadays, patients candidates for second line therapies are EGFR-wild-type.

Methods
Medical records of EGFR-wild-type patients treated with E in second or further linesbetween March/2008 and March/2013 were reviewed.

Results
85 patients (p) were found. Characteristics: Median age 61 years (38-83), 76.5% were male. Tobacco: 42.4% were smokers and 45.9% former smokers. PS 0, 16p; 1, 50p; 2, 19p; by stage: IIIb 27p, IV 58 p. Histology: adenocarcinoma 48p, squamous-cell 25p, undifferentiated or non-specified 11p, adenosquamous 1p. E was given: 40% as second, 44.7% as third and 15.3% as fourth or subsequent line. Effectivity: Partial response 9.4%, Stable disease 48.2%, Progressive disease 41.2%, Not assessable 1%. Overall Survival (OS): median 22 weeks (w) (95% CI, 17.4-26 .7), progression free survival (PFS) 12w (95% CI ,9.8-14 .2). In smokers PFS was 12w also. In squamous carcinomas 10w. In males 11w (these differences were non significant). Toxicity: 92.9%p presented some side-effect: 70.5% rash (49p, G1; 11p, G2); diarrhea 34.1%p (25p, G1; 3p, G2; 1p, G3); asthenia 29.4%p (12p, G1; 6p, G2; 7p, G3); ocular 2.4%p (2p, G1) and digestive 9.5% (2p, G1; 1p, G2; 1p, G3). Dose was reduced in 12.9%p (7p 100 and 4p 125 mg/day). Treatment was interrupted in 7.1%p (median 14 days (range 7-23)); most common causes were G3 skin rash and diarrhea.

Conclusion
We described the efficacy of Erlotinib in day-to-day clinical practice when was given beyond the first-line treatment in advanced and metastatic NSCLC without EGFR mutation. Our results are in accordance with has been reported from clinical trials and may reflect its efficacy in clinical practice.

Background
Creative art therapy (CAT) consists in the use of artistic activities to help patients manage physical and emotional problems in a therapeutic setting. In oncology, CAT has mostly been practiced in palliative-care units. CAT has not been used in lung cancer patients so far, especially when chemotherapy treatment is still delivered. Our objectives were to prospectively assess the feasibility of integrating CAT in the management of patients with metastatic lung cancer treated with chemotherapy.

Methods
From 2011/11 to 2013/05, CAT was offered to in- and out-patients who received chemotherapy for metastatic lung cancer in our department. Creative activities included the production of paintings, drawings, and/or sculptures. Patients were assessed by a trained art therapist for anxiety levels, self-awareness and satisfaction, before and after each art session.

Results
84 patients were included in the study, among whom 41 (55%) accepted CAT and received a mean number of 3 sessions. In this cohort of 13 men and 28 women, 31 (75%) and 17 (41%) patients reported improvements in anxiety levels and cancer-related symptoms after the art session, respectively. CAT gave satisfaction to 37 (90%) patients. These benefits were transient in all cases. Main reasons for refusing CAT in the remaining 33 patients included fatigue and lack of interest for arts. Painting and drawings from lung cancer patients along the disease management will be presented at the meeting.

Conclusion
Our study reports on the feasibility of CAT in lung cancer patients receiving chemotherapy. CAT may be considered part of the multimodal supportive care management of lung cancer patients. This study was supported by an unrestricted grant from Hoffmann-La Roche.

Background
The syndrome of combined pulmonary fibrosis and emphysema (CPFE) is characterized by imaging features consisting of the association of centrilobular and/or paraseptal emphysema and pulmonary fibrosis, associated with subnormal spirometry, contrasting with severe impairment of gas exchange with strong decrease in carbon monoxide diffusing capacity, and hypoxaemia at exercise. Virtually all patients presenting with CPFE are smokers and may be at high-risk to develop lung cancer; limited data have been made available on such association, mostly from Japanese cohorts.

Methods
This retrospective multicentre study was conducted by the Groupe d’Etudes et de Recherche sur les Maladies “Orphelines” Pulmonaires (GERM”O”P), a collaborative group of about 200 French physicians dedicated to the study of rare pulmonary diseases. Patients presenting with CPFE syndrome and lung cancer at the referring centers from 2003 to 2012, were included. The clinical, pathological, and therapeutic features, as well as the outcome of patients, were collected and analyzed.

Results
A total of 47 patients presenting with lung cancer and CPFE syndrome were identified. All patients but one were men, with a mean age of 68 years. All patients were smokers, with a mean of 47 pack-years. The CPFE syndrome was diagnosed synchronously with lung cancer in 27 (57%) patients. Detection of lung cancer was incidental in 22 (47%) patients. The tumour was diagnosed at an early-stage, i.e. stage I-II, in 55% of cases. A pathological diagnosis of lung cancer was obtained for only 38 (81%) patients. Histological type was squamous cell carcinoma in 17 (36%) patients, adenocarcinoma in 14 (30%) patients, non-small cell lung cancer not otherwise specified in 3 (6%) patients, small cell lung cancer in 3 (6%) patients, and sarcomatoid carcinoma in one (2%) patient. There was no significant relation between tumor histology and location in the lung parenchyma (p=0.32). Overall, 20 of the 47 patients could not receive standard-of-care treatment for lung cancer, as per international recommendations and guidelines; this limitation was considered to be directly related to the CPFE syndrome in 8 (40%) cases. After a mean follow-up of 17 months, 35 patients were dead, and 12 patients were alive. Causes of death included locoregional or systemic tumor progression in 5 (14%) and 17 (49%) patients, respectively, respiratory failure in 8 (23%) patients, treatment-induced toxicity in 4 (11%) patients, and post-operative exacerbation of pulmonary fibrosis in 1 (3%) patient.

Conclusion
Lung cancer in patients with CPFE syndrome represents a specific entity characterized by very strong association with tobacco-smoking and male gender, peculiar histological-radiological presentation, poor prognosis due to major limitations and risks to conduct standard-of-care diagnostic and therapeutic interventions in a significant proportion of patients, and possible interest of screening. Lung cancer in CPFE syndrome further represents the most characteristic and severe model of tobacco-related disease.

Background
Maintenance treatment, with either bevacizumab or pemetrexed, has been shown to increase PFS and overall survival. Two trials have compared double maintenance (DM) therapy (pemetrexed + bevacizumab), to single drug maintenance (bevacizumab in AVAPERL and POINTBREAK studies). Conflicting results were found. Before definitive conclusions can be driven from these studies and other ongoing study (ECOG 5508), the purpose of our retrospective study was to determine in real world setting the frequency of double maintenance discontinuation for adverse event, and to describe the main toxicities occurring during double maintenance.

Methods
All patients who received at least one cycle of pemetrexed and bevacizumab as maintenance treatment were identified from the Oncology Pharmacy database of participating centers since year 2011. All the charts were analyzed retrospectively to obtain clinical data. Lab results were noted for haemoglobin, creatinine and liver enzymes before starting and after receiving multiple doses of pemetrexed and bevacizumab.

Results
Included were 87 patients treated with two to six cycles of induction chemotherapy (median 4), combining platinum with pemetrexed and bevacizumab, followed by at least one cycle of bevacizumab and pemetrexed as maintenance treatment. All patients received supplementation of vitamin B12 and folic acid during chemotherapy. Baselines characteristics (%): male 54: stage IV 96,5; adenocarcinoma 96,5; median age 58 yr. 57,8% of patients had objective response after induction chemotherapy, and 42,2% had stable disease after induction chemotherapy. At cut off date: treatment was still ongoing for 17 patients (19,8%); 40,6% of patients stopped DM for progressive disease; 33,3% of patients stopped DM for toxicity (out of these 33% of patients, 42% went on single maintenance with Pemetrexed and 58% with Bevacizumab); 11,6% of patients stopped DM for patient/physician decision, and 14,4% for other reasons. The most common toxicity responsible for DM discontinuation was renal failure (52%).

Reason for discontinuation (%)	POINTBREAK	AVAPERL	This study
Progressive disease	61,0	54,7	40,6
Adverse event	13,7	21,6	33,3
Others reasons	19,8	23,5	25,8

Conclusion
This retrospective study suggests that in real world setting, double maintenance is frequently discontinued for adverse event (33,3% of patients). The most frequent adverse event was renal failure (half of the cases). Further analyses are ongoing in order to identify any predictive factors for renal failure occurrence and will be presented at meeting. These results suggest that particular caution should be taken in order to preserve renal function whenever double maintenance (pemetrexed + bevacizumab) is considered in a patient with stable or responding tumour after induction chemotherapy consisting in platinum, pemetrexed and bevacizumab.

Background
Multidisciplinary team (MDT) meetings are seen as vital to delivering a coordinated approach to the care of patients who have been diagnosed with cancer. Use of an MDT model of care has been shown to increase patient recruitment to clinical trials, ensure a shortened patient journey from diagnosis to treatment, and result in a higher likelihood that the patient receives evidence-based treatment, ensuring better survival outcomes. Additionally, MDT’s are essential in ensuring correct diagnosis and staging, appropriate treatment modalities, and timely referrals to different disciplines within the team. The aim of this study was to analyse the reasons why respiratory cancer patients diagnosed within the Illawarra Shoalhaven Local Health District (ISLHD) have repeat presentations at MDT meetings.

Methods
Patients diagnosed with lung cancer (diagnosis included Small Cell Lung Cancer (SCLC), Non Small Cell Lung Cancer (NSCLC) and Mesothelioma) and discussed in the Lung Cancer MDT meeting in the ISLHD between 1st January 2006 and 31st December 2011 were identified. These identified patients were then cross-referenced against MDT meeting data stored in patient records. Patients identified as having been discussed more than once at an MDT meeting were analysed in relation to the documented reason for re-discussion.

Results
There were 533 patients presented in an MDT meeting within the six year period analysed. 463 were discussed once; 57 twice; 12 three times; and 1 four times. Of those discussed more than once, 10 had pathological diagnosis confirmed after the initial MDT presentation, and were re-presented to discuss these results and formulate a treatment plan. Of the 10 patients, reasons for presentation without pathological diagnosis included: non-diagnostic tissue sample or origin not specified (4); issues around comorbidities and most appropriate avenue for obtaining diagnosis (3); and protracted inpatient stay prior to pneumonectomy (1). Two patients were discussed prior to pathological diagnosis for no apparent reason. 31 cases were re-presented, as initial discussion recommended further investigation/staging. Of these, 18 had initial diagnosis/presumed stage confirmed, 8 were up-staged. After initial treatment was completed, 13 patients were rediscussed with regard to future treatment/monitoring, 10 due to an increase in symptoms and 15 due to disease progression. 50 of the cases discussed more than once resulted in the patient being referred to additional Specialists.

Conclusion
A review of five years of Lung Cancer MDT data has shown that the MDT meeting is performing its role as a central point for discussion of treatment options for lung cancer patients. In a small number of cases (1.9%) pathological diagnosis had not been confirmed prior to the MDT. For the majority of these patients (8), the meeting provided expert guidance in regards to the most appropriate timing and procedure to obtain tissue diagnosis. The MDT may benefit from the development of a template/pathway to ensure a reduction in the number of patients presented without pathological diagnosis and avoid discussion of some of the patients recommended for further investigation/staging. A template/pathway may enable the MDT to have a more complete picture of the patient’s diagnosis and reduce re-presentation/delays in treatment.

Background
Treatment with inhibitors of the Epidermal Growth Factor Receptor (EGFR) commonly results in rash, which causes patients significant symptoms and embarrassment, and is not always easy to manage with existing treatments. BN83495 is a first in class inhibitor of steroid sulphatase (STS), which has been used in early phase studies as treatment for steroid-dependent cancers such as breast, prostate and endometrial cancer, with one side-effect noted to be dry skin. STS converts oestrone sulphate to oestrone, a precursor of oestradiol; and dehydroepiandrosterone sulphate (DHEA-S) to dehydroepiandrosterone (DHEA), a precursor of adrenal testosterone. Blocking these pathways can be helpful in the treatment of acne, and patients with X-linked ichthyosis, who have an inherited deficiency of STS, do not get acne. Hence we hypothesized that use of BN83495 could help to treat EGFR-associated rash.

Methods
Eligible patients had histologically proven, locally recurrent or metastatic NSCLC and were continuing to experience rash during treatment with an EGFR-TKI (Erlotinib or Gefitinib), despite standard management. Patients were treated with BN83495 at a dose of 40 mg/day orally for a period of 12 weeks and able to continue on standard treatment for rash such as topical steroids or antibiotics. For patients with no or minimal toxicity and minimal or no rash at 12 weeks, treatment with BN83495 could continue for an additional 3 months before being discontinued. If rash worsened on discontinuation of treatment at 6 months BN83495 could be resumed. The primary objective was to describe changes in the frequency and grade of EGFR-related rash and other cutaneous side-effects of the EGFR-TKI with the combination. A pragmatic sample size of 20 patients was proposed.

Results
Nine patients were registered but 2 withdrew prior to commencing BN83495. All patients had grade 2 rash at study entry. Of the 7 patients who commenced study treatment, 5 had an improvement in rash by one CTC grade. Patients received BN83495 for a median of 12 weeks (range 1 – 52) with 2 patients recommencing drug after rash worsened when BN83495 was stopped after 6 months of treatment as per protocol. BN83495 was generally well tolerated with no grade 3 or 4 toxicity. However, all participants experienced grade 2 dry skin related to BN83495 which was generally manageable with moisturizers, but 2 patients stopped study drug early for this reason. 3 patients discontinued study drug when the EGFR-TKI was stopped due to disease progression, and 2 other patients elected to cease the drug at 3 and 6 months respectively because rash was mild. The study was closed early due to poor accrual as many patients were not keen to enter a study which involved treating rash by taking an additional tablet.

Conclusion
Treatment of EGFR-TKI related rash with the steroid sulfatase inhibitor BN83495 improved the severity of rash in the majority of patients but commonly resulted in grade 2 dry skin. Accrual to a study which involved treating rash with an additional oral medication proved challenging. Alternative approaches such as topical use of a steroid sulfatase inhibitor could be considered for further investigation.

Background
Comorbidity, such as diseases of the cardiovascular, pulmonary, and other systems may influence prognosis in lung cancer as well as complicate its treatment. The performance status of patients, which is a known prognostic marker, may also be influenced by comorbidity. Due to the close link between tobacco smoking and lung cancer, and because lung cancer is often diagnosed in advanced ages (median age at diagnosis is 70 years), comorbidity iwill be present in a substancial proportion of lung cancer patients.

Methods
Patients with any stage lung cancer who did not have surgical treatment were identified in the Danish Lung Cancer Registry (DLCR). DLCR collects data from clinical departments, the Danish Cancer Registry, Danish National Patient Registry (DNPR) and the Central Population Register. A total of 22,999 patients with lung cancer were identified. Due to missing variables, 19,561 patients were available for analysis. Comorbidity was sought in the DNPR which is a register of all in and out patient visits to hospitals in Denmark. By record linkage, all lung cancer patients who had previously been diagnosed with any of a number of comorbid conditions was recorded using the Charlsson comorbidity score CCS. First treatment was categorized as chemotherapy, chemo-radiotherapy, radiotherapy or no therapy. Data on CCS, performance status, age, sex, stage, pulmonary function (Fev1), histology and type of first treatment (if any) were included in univariable and multivariable Cox proportional hazard analyses.

Results
For patients receiving chemotherapy as first treatment for lung cancer, survival was increasing worsened by increasing comorbidity (HR=1,00,1.10, 1.17, 1,15 for CCS scores 0, 1, 2, 3+ respectively). After adjustment for potential confounders, risk estimates was reduced somewhat (HR: 1.00, 1.05, 1.11, 1.11 for CCS scores 0, 1, 2, 3+ respectively). For patients receiving radiotherapy as first therapy, a different pattern was seen with better survival for patients with comorbidity (HR=1.00, 0.99, 0.94, 0.87 for CCS scores 0, 1, 2, 3+ respectively). After adjustment, this effect disappeared and survival was unaffected by CCS. For patients receiving combined radio/chemo therapy there was no significant association between CCS and survival.Throughout the analysis, performance score remained a strong and highly significant risk factor for survival, and was robust in multivariate analysis (HRunivariate, all patients= 1.0, 1.40, 1.95, 3.23, 5.91 for ECOG performance score 0,1,2,3 and 4 respectively).

Conclusion
Comorbidity has a limited effect on survival and only for patients treated with chemotherapy. It is rather the performance of the patient at diagnosis than the medical history that prognosticates survival in this patient group.

Background
In 2007, lung cancer was the leading cause of cancer-related mortality and morbidity in Australia for both men and women. Only 14% of those diagnosed with lung cancer survive five years beyond their diagnosis. People living in rural areas, those from lower socio-economic status or from certain cultural and linguistic backgrounds have poorer outcomes. Factors contributing to differences in survival and outcomes are varied. Cancer Australia is developing a best practice model of care for the management of lung cancer that has national relevance and can be implemented locally, with the aim of achieving consistency in approach and improving lung cancer outcomes. This abstract describes how the evidence base has been built to inform the model of care.

Methods
A systematic review of international and national literature on patterns and models of care for lung cancer informed themes explored through qualitative and quantitative research. A tiered approach to data collection included: (i) mapping of lung cancer services across Australia; (ii) health service consultation through interviews and site visits; and (iii) consumer consultation through a national survey and targeted interviews. Findings were presented and discussed at a national workshop with clinical leaders, consumers, researchers and service delivery experts to review and define principles and elements of best practice care.

Results
The patterns of care literature for lung cancer identified variations in time to diagnosis, access to active treatment, re-treatment and palliative care. Service delivery themes identified through the review and research included multidisciplinary care, specialist involvement in diagnosis and treatment, care coordination, early integration of palliative care, uptake of guidelines and quality measures, involvement of primary care and consideration of supportive care needs. Lung cancer service mapping identified 192 services across Australia providing some elements of lung cancer care. Approximately two-thirds were public and one third private. Multidisciplinary teams were identified in 58 services (30%), the majority in metropolitan locations (n=41, 71%). Consumer consultation identified variations in time to first specialist appointment (two weeks to two months), definitive diagnosis (two weeks to three months); and treatment (one week to two months). Consumers identified that improvements could be made in the way information about lung cancer is communicated across all stages of lung cancer. Health service consultation highlighted challenges and best practice approaches along with a range of systemic issues that influence how care is delivered. Challenges included streamlining a complex diagnostic pathway, managing multidisciplinary teams that include cancer and non-cancer specialists, early referral to palliative care, coordinating care, involvement of primary care, and a lack of standard guidelines for follow-up care. Best practice examples were identified across the diagnostic and treatment pathway. Results informed a set of national principles including: patient-centred care; timely diagnosis and staging; multidisciplinary care; appropriate treatment and supportive care; coordinated care; and collection and monitoring of data.

Conclusion
This research identified gaps and variations in the delivery of lung cancer care and has built the evidence base to inform the development of best practice approaches to support the consistent diagnosis and management of lung cancer in Australia.

Background
There are limited data on the relative effectiveness of biosimilar ESAs and other available ESAs for the treatment of CIA. In addition, it is unclear whether the most recent recommendations for more conservative use of ESAs to treat CIA are reflected in real-world clinical practice

Methods
We analysed 73 patients with NSCLC who were included in a retrospective audit of CIA treatment with ESAs in a large oncology centre in Spain, with patients treated by multiple physicians. The patients were treated for CIA with Binocrit[®] 40,000 IU QW (n=12), Binocrit[®] 30,000 IU QW (n=6), darbepoetin alfa 500 μg Q3W (n=36) or darbepoetin alfa 150 μg QW (n=19). In addition to overall haemoglobin (Hb) outcomes, comparisons were performed according to the different ESA treatments given

Results
The mean overall haemoglobin (Hb) at start of ESA treatment was 9.4 g/dL; the mean overall Hb level at the end of treatment was 10.6 g/dL. 36/73 patients (49%) achieved a Hb increase of at least 1 g/dL. There were no significant differences (p>0.05) between the groups in terms of Hb levels at the start of ESA treatment. At the end of treatment, however, the mean Hb level in the group treated with darbepoetin alfa 500 μg Q3W was significantly lower than that in the other three groups (Table). No drug-related adverse events were recorded

ESA	Mean treatment duration (weeks)	Mean Hb at start of treatment (g/dL)	Mean Hb at end of treatment (g/dL)
Darbepoetin 150 μg QW	4.16	9.2	11.3
Darbepoetin 500 μg Q3W	4.59	9.4	10.1
Binocrit 30,000 IU	3.50	9.4	11.1
Binocrit 40,000 IU	3.67	9.5	10.7

Conclusion
Our data indicate that Hb outcomes in a real-world clinical practice setting are similar for the ESA treatments used with the exception of Darbepoetin 500 μg Q3W, which achieved a significantly lower Hb at the end of treatment. We consider the use of ESAs in our centre to be conservative and safe, and to reflect the most recent change in ESA prescribing information and recommendations for more moderated use in patients with CIA (that is, use the lowest possible dose and duration of treatment necessary to avoid transfusions)

Background
Background: Lung cancer patients have a higher level of disease burden, higher unmet psychosocial needs and lower uptake of psychological services compared with other cancer groups. The Department of Clinical Psychology, in collaboration with the lung cancer service at PeterMac, revised the model of psychological care to optimise access and improve psychosocial well-being.

Methods
Method: This revised model of care involved realigning the Psychology Outpatient Clinic to run in parallel with the Lung Outpatient Clinic. The aims were to: 1) provide a timely and early intervention service; 2) increase patient access to psychology services; 3) reduce the burden of accessing psychology services; and 4) assess psychological needs of patients and provide appropriate psychological interventions. Patient demographics, uptake, referral information and session data were collected for a three month period and compared to data from the same period in the previous year.

Results
Results: Our sample included a total of 37 patients with lung cancer. The results indicated that the revised model of service delivery led to: a 21% increase in new lung patient referrals for psychology services; an almost 100% uptake of services; and a 186% increase in the number of scheduled sessions attended by lung patients. The main reasons that patients attended sessions were to address mood fluctuations, loss and grief issues, relationship and existential concerns.

Conclusion
Conclusion: The revised model made a significant impact on meeting the previously unmet needs of this patient group through providing timely assessments and interventions. This highlights the potential effectiveness of integrating psychology services within medical cancer streams. We received no funding and there was no duality or conflict of interest

Background
Pleurodesis is a palliative procedure which main purpose is to alleviate respiratory symptoms of patients with malignant pleural effusion. Nevertheless, quality-of-life is an outcome rarely explored in literature. The main purpose of this study was to evaluate the quality-of-life before and 30 days after pleurodesis in patients with malignant pleural effusion. The secondary objective was to identify predictors of quality-of-life improvement after pleurodesis.

Methods
Retrospective study including all patients with recurrent malignant pleural effusion who underwent pleurodesis at Hospital das Clinicas (University of Sao Paulo) and Hospital Aristides Maltez from 2008 until 2012 and who filled out quality-of-life questionnaires before and 30 days after the procedure. In both institutions the World Health Organization-bref general quality-of-life questionnaire has been regularly applied to all patients undergoing pleurodesis since 2007. Paired T-test was used to compare before and after scores and multivariable regression models were used to identify predictors.

Results
During the study period 132 patients were included (26 men, 106 women, mean age 58.1 +- 11.8). The primary tumors were: breast (84), lung (25), ovary (4), lymphoma (11) and other (8). The mean prepleurodesis quality-of-life scores were: physical domain 35.8+-17.7, psychological domain 58.8+-17.7, social domain 52.8+-14.8, and environmental domain 65.4+-18.8. Thirty days after pleurodesis, the physical aspect (8.1 pts, p=0.0001) and the environmental domain (3.6 pts, p=0.008) improved significantly while the other two domains remained unchanged. Predictors of improvement of quality-of-life in this sample were: ovary cancer (p=0.015), pleural fluid glucose (p=0.012), and low physical aspect score before pleurodesis (p=0.001).

Conclusion
All aspects of quality-of-life are deeply compromised in patients with recurrent malignant pleural effusion. Pleurodesis improves quality-of-life thirty days after the procedure particularly in patients with ovary cancer and with very low physical status scores.

Background
Patients with advanced lung cancer suffer from a high burden of tumor-related symptoms. Thus palliative care is an important treatment modality in these patients. Palliative care units have been established in many comprehensive cancer centers in order to achieve this. Here we report on the experience we have obtained at the Palliative Care Unit of the Medical University of Vienna in the management of patients with advanced lung cancer.

Methods
We retrospectively reviewed medical records of 86 patients with advanced lung cancer who were treated at our Palliative Care Unit between June 2010 and March 2013. We determined reasons for admission, duration of hospitalization, non-invasive as well as invasive medical interventions, and clinical outcome.

Results
We report on 86 patients with advanced lung cancer (74 % NSCLC, 26 % SCLC) who had been admitted to our Palliative Care Unit within a period of 34 months. Lung cancer patients comprised the largest group of cancer patients who are admitted to our unit. Reasons for admissions were deterioration of performance status (41 %), dyspnea (13 %), pain (38 %), psychosocial reasons (7 %), and other (1 %). Re-admissions occurred in 20 % of all patients. The patients had the following characteristics: median age 62 years (range 42-85 years), 38 % females and 62 % males, ECOG performance status 0-2 38 % and >2 62%, median body mass index (BMI) 24 (range 14-39). Median duration of hospitalization was 16 days (range 1-101 days). The following treatments were delivered during hospitalization: analgesic treatment according to the WHO I-III ladder (85 %), palliative radiotherapy (31 %), palliative chemotherapy (7 %), and invasive procedures (such as thoracocentesis, pleurX drainage system, pleurodesis, bronchial stenting, invasive neurolysis and other in 26%). Antibiotic therapy was delivered in 33 % and antipressants or antipsychotropic drugs in 38 % of all patients. Palliative sedation by means of a continuous intravenous or subcutaneous infusion with midazolam was administered in 25%. Dietary counseling and spiritual as well as psychosocial support was offered to all patients and accepted by most of them. 77 % of all patients died during their stay, mostly due to disease progression. The remaining 23 % of all patients were discharged with improvements in their tumor related symptoms or stable disease and were offered home care or hospice access.

Conclusion
Patients with advanced lung cancer did benefit from admission to the Palliative Care Unit. Medical as well as non-medical interventions resulted in improvements of cancer-related symptoms and better coping with the disease. Thus Palliative Care Units should be part of the multidisciplinary management of patients with advanced lung cancer. Schur S and Masel EK contributed equally to this work

Background
Non-small cell lung cancer is associated with a higher risk of thromboembolic events in comparison with SCLC. Adenocarcinoma represent roughly 75% of NSCLC patients. Lung adenocarcinomas harboring EGFR and KRAS mutations as well as EML4/ALK rearrangements represent distinct subsets of this disease. No data are available concerning the prevalence of pulmonary embolism in lung adenocarcinoma patients with these mutations. The aim of the study was to evaluate the prevalence of pulmonary embolism in patients with stage IIIB and IV lung adenocarcinomas harboring EGFR and KRAS mutations as well as EML4/ALK traslocations.

Methods
Patients with stage IIIB or IV NSCLC referred to Division of Medical Oncology at the Hospital of Perugia between 2008 and 2012 were included in the study. In these patients, contrast-enhanced CT scans of the chest were reviewed for the presence of pulmonary embolism by a panel composed by three radiologists. In the same patients, data regarding the molecular characteristics (EGFR exons 18-21 and KRAS exon 2 mutations as well as EML4/ALK traslocations) were collected.

Results
A total of 209 patients with stage IIIB or IV NSCLC were included in the study. A histologic diagnosis of lung adenocarcinoma was done in 173 patients (82.7%). In 127 of these patients sequence analysis for known EGFR (exon 18-21) and KRAS (exon 2) mutations was performed. In this population 31/173 patients were EGFR mutated (17.9%), 27/173 were K-RAS mutated (15.6 %) and 17/173 were EML4/ALK positive (9.8%). 41 patients with lung adenocarcinoma had a diagnosis of pulmonary embolism at CT scan (23.7%). Of these, 34.1% had no oncogene mutations in comparison with 28.8% of the patients without pulmonary embolism. Of the 41 patients with a diagnosis of pulmonary embolism 12.1% had an EGFR mutation and 12.1% a KRAS mutation, in comparison with 19.7% and 16.6% of patient without pulmonary embolism, respectively. In patients with lung adenocarcinoma, EML4/ALK rearrangements was observed in 19.5% among patients with pulmonary embolism and in 6.8% among patients without it. The risk of pulmonary embolism was 3.3-fold higher in presence of EML4/ALK rearrangements in comparison with no EML4/ALK rearrangements [OR: 3.3 (95%CI 1.2-9.2)].

Conclusion
In lung adenocarcinoma patients, the presence of EML4/ALK traslocation seems to be associated with a high risk of pulmonary embolism and could help in identifying patients at particular high risk who might benefit from an antithrombotic prophylaxis. These preliminary data need to be confirmed by further studies.

Background
Nitrogen-containing bisphosphonates(N-BPs) are usually administered to patients with advanced cancer harboring bone metastases to prevent skeletal related events(SREs). Preclinical studies have demonstrated the anticancer activity of N-BPs in vitro and in vivo. Zoledronate (ZOL) is a type of N-BP, and clinical trials of ZOL have demonstrated survival benefits in breast cancer and multiple myeloma. However, the benefit of ZOL to overall survival for lung cancer patients is still controversial. The first administration of intravenous N-BPs are occasionally induced fever, which is due to the activation of γδ T cells. γδ T cells are a small subset of T lymphocytes that play an active role in the immunosurveillance against infections and tumors as components of innate immunity. However, the significance of ZOL-induced fever is not clear in patients with advanced non-small cell lung cancer (NSCLC). The aim of this study was to estimate the prognostic value of fever after N-BP administration in advanced NSCLC patients.

Methods
We retrospectively reviewed 46 patients with advanced NSCLC who recerved ZOL treatment from March-2009 to March-2011 in the department of respiratory medicine in Tottori University hospital. ZOL-related fever was defined as body temprature more than 37.5℃ within 48 hours after the first administration of ZOL. Fever that persister for > 2 days, or treated with antibiotics were not defined as ZOL-related fever. Even if there were records of ZOL administration, we excluded the case with no record in terms of fever after treatment with ZOL. We analysed patients who had Eastern Cooperative Oncology Group (ECOG)-performance status (PS) ≦ 1. Clinicopathological factors such as age, gender, smoking history, disease stage, histological type, and epidermal growth factor receptor (EGFR) mutation status were analyzed using univariate and multivariate analyses, and these factors were compared between the fever group and non-fever group.P-value of < 0.05 were considered statistically significant.

Results
Fifteen out of 46 patients (32.6%) experienced fever after the first administration of ZOL. No significant differences in clinicopathological characteristics were seen between the two groups. The mean survival time (MST) observed in the fever group [MST 1003 vs. 471 day, hazard ratio (HR) 2.15, p=0.04] was significantly longer than the non-fever group. Univariate analyses for the probability of overall survival indicated that a positive EGFR mutation status (p=0.016), non-squamous type of cancer (p=0.02), no smoking history (p=0.001), and female gender (p=0.036) were predictors of significantly longer survival time. Comparatively, the presence of fever after ZOL administration [HR, 2.73; 95% confidence interval (CI) 1.17-6.34, p=0.020] and having no smoking history [HR, 0.13; 95%CI 0.20-0.85, p=0.033] were still significant predictors for longer survival time in multivariate analyses.

Conclusion
Fever after the first administration of ZOL was an independent prognostic factor in patients with advanced NSCLC.

Background
Lung cancer is the main cause of cancer death for both men and women in Australia. Non-small cell lung cancer (NSCLC) usually presents late in the disease when treatment options are limited. Surgery remains the main stay of curative treatment with definitive radiotherapy or chemoradiotherapy used in some patients unfit for surgery or with locally advanced disease. Increasing use of chemotherapy with introduction of new targeted therapies has broadened the therapeutic choices for patients. We wished to look at what happened to patients with NSCLC in our hospital (RHH)

Methods
An audit was performed of patients presenting at our MDT (at which all patients with lung cancer in southern Tasmania are discussed) in 2010 and 2011 with a diagnosis of NSCLC. We looked at the type of therapy assigned (or not) and where they had chemotherapy the number of lines and types of treatment given.

Results
177 patients presented with NSCLC. Histology showed 99 adenocarcinomas, 48 squamous cell, 20 NSCLC-NOS, 5 large cell neuroendocrine, 3 adenosquamous cell and 2 with synchronous primaries. 48 (27%) patients received no treatment beyond palliative care. Mostly this was due to comorbidities or patient wishes (8) but 13 (7%) patients died around the time of diagnosis. 38 (21%) patients underwent surgery (3 pneumonectomies, 31 lobectomies and 4 wedge resections). 3 had adjuvant radiotherapy and 5 had adjuvant chemotherapy. 79 (45%) patients under went radiotherapy: 59 had palliative radiotherapy, 12 chemoradiotherapy, 4 definitive radiotherapy, 1 brachytherapy and 3 had adjuvant radiotherapy to surgery. 84 (47%) were reviewed by medical oncologist of whom 56 (32% of total, 67% of those reviewed) received some form of 1[st] line chemotherapy. There were 13 different regimens of first line therapy, most common being carboplatin/gemcitabine (n=19) and carboplatin/vinorelbine (n=10). The reason for choice of therapy was not usually stated and varied between specialists but individual specialists also varied regimens used. 22 patients had 2nd line therapy, 12 patients had 3[rd] line and 5 patients had 4[th] line. 16 patients had pemetrexed maintenance therapy (1 in combination with carboplatin) of which 9 received 3 or less cycles, 1 patient received 26 cycles. 13 (7%) patients received erlotinib. The patients were reviewed by 7 different specialists (3 were seen elsewhere in the state) with one specialist seeing 36 patients but 4 seeing less than 10 over the 2 year period.

Conclusion
The majority of our patients received some form of therapy. Our radiotherapy usage is below expected numbers and chemotherapy usage is particularly low. This may reflect our patients’ demographics. The wide variety of first line regimens used makes over all conclusions about the effect of chemotherapy difficult. A number of medical oncologists treat less than 5 patients with lung cancer a year. Consolidation of care to a smaller number of specialists should be considered.

Background
Cicatricial fibromatosis arises in a surgical scar is a well-known clinical condition. However, its occurrence after thoracic surgery, especially at bronchial stump. is extremely rare.

Methods
42 years old male patient had undergone video assisted thoracoscopic right lower lobectomy 18 months earlier for primary lung cancer. There was no evidence of recurrence in last chest CT examination until 9 months ago. A 33mm sized mass was found beside right lower lobe stump margin in subcarinal space in chest CT examination. It had increased uptakes (SUVmax 6.5) in F-18 FDG PET/CT imaging. There were not any findings of distant metastasis in chest CT and F-18 FDG PET/CT imaging. Therefore we thought it would be a local recurrence of lung cancer at bronchial stump site. We performed biopsy of a mass using EBUS and EUS to confirm the diagnosis. The results were only fibrotic tissue without evidence of malignancy. So he had undergone a complete excision of subcarinal mass through thoracotomy. It was very hard and tightly adhesive to surrounding tissues, as bronchus and esophagus. Figure 1

Results
Pathologic report was that it was myofibroblast proliferation with infiltrative margin, which was diagnosed as cicatricial fibromatosis.

Conclusion
In this case, Cicatricial fibromatosis beside bronchial stump margin were suspected to a local recurrence. It has increased uptakes in PET-CT imaging, like as cancer recurrence. It should be completely excised as soon as possible because it is growing infiltrative to surrounding tissues and it would recur sometimes.

Background
Endothelial Growth Factor Receptor (EGFR) mutation testing is now recommended practice for lung adenocarcinoma. Those patients with EGFR mutations are eligible for targeted agents which offer survival and quality of life advantages with reduced toxicity in some. Several reports have suggested particular samples are more likely to give reliable EGFR mutation results than others. We were interested in investigating the types of samples locally that were viable for testing and to assess the population rate of EGFR mutation positivity in adenocarcinoma for which there is little published data in Australian (mainly Caucasian) populations.

Methods
Data was systematically collected for tissue samples sent for EGFR mutation testing over a three year period. Basic demographic data, sample type and test results were recorded. Testing was performed as part of the routine workup for patients with lung adenocarcinoma. Chart review was undertaken to clarify sampling methods and test results if required.

Results
214 sequential specimens from patients with proven adenocarcinoma were sent for analysis of the presence of EGFR mutation. 197 specimens were of adequate quality for analysis (>20% of specimen composed of tumour cells). There were 22 positive tests for EGFR mutations (16 female; 11 non- or never-smokers). Table 1 outlines the types and adequacy of specimens collected for testing. There was no association between specimen type and adequacy for analysis. 27 specimens were unable to be analysed. Of these 12 were labelled failed tests and 15 returned incomplete results for at least one of four exons. 17 specimens were labelled inadequate for testing; 9 of those were reported as negative for EGFR mutations despite being reported as having less than 20% tumour cells in the specimen. 6 of 12 failed tests were adequate for testing. 14 of 15 incomplete tests were adequate for testing.

Specimen Type	Number collected (% of total)	Number of specimens with >20% of tumour cells in specimen (% of number collected)	Number of failed or incomplete tests
Image guided FNA Bx	27 (12.5)	25 (93)	5
Image guided core Bx	16 (7.5)	15 (94)	3
Bronchial Bx/TBNA/EBUS/TBLB (bronchoscopy)	89 (42)	78 (88)	11
Lung/Pleura/Pericardium/ Mediastinal LN Bx (surgical)	49 (23)	48 (98)	5
Pleural fluid	9 (4)	8 (89)	0
Surgical LN Bx	4 (2)	4 (100)	0
Abdominal/oesophageal node	3 (1.5)	3 (100)	0
Brain/ cerebellar metastasis	11 (5)	10 (91)	1
Musculoskeletal metastasis (surgical)	6 (3)	6 (100)	2
	214 (100)	197 (92)	27 (13%)

Conclusion
In our population (majority Australian born Caucasians), the percentage and profile of EGFR positivity is similar to that reported in other studies. Representative samples can be obtained from many different types of tissue, in particular, fine needle aspirates and core biopsies of lung parenchyma and lymph nodes as well as from pleural fluid.

Background
Lung cancer is the most common cancer related death and the fourth most common cause of death in Germany. The five-year survival rate in European and North American countries is in between 5,5% and 15,7%. In the last couple of years though, significant progress has been made concerning personalized medicine in this disease entity. Novell targeted therapies with tyrosin kinase inhibitors (TKI) for patients with a mutation in the EGFR gene offer a new treatment option.

Methods
A 78-year old male patient was referred to the Thoraxklinik Heidelberg with atypical thoracic pain after cardiologic assessment. In 2001 he was given the diagnosis of pulmonary adenocarcinoma of the right upper lobe in the Asklepios Hospital Munich-Gauting. Because of his overall good clinical condition the patient had declined any treatment to this point. A new PET- CT was conducted and compared to the computer tomography of 2001 revealed a significantly grown lesion in the right upper lobe as well as new, small bipulmonar lesions suspicious of lung metastasis.

Results
A new histological assessment was performed on an endobronchial forceps biopsy of the now occluded right upper lobe. The diagnosis of a partial lepidic differentiated adeno-carcinoma was consistent with the diagnosis as well as the preserved specimens of 2001. We were able to perform an EGFR mutation analysis of the 2001,- as well as of the new tumor specimens through PCR-amplification and bidirectional sequencing of exons 18, 19 and 21. In each of the probes we detected a deletion combined with an insertion c.2127_29del (p.E709_T710delinsD) in exon 18.

Conclusion
The above mentioned mutation has already been described in the literature but there is no information concerning the responsiveness of tyrosine kinase inhibitors in this particular mutation. Now that the cancer had spread we decided to start treatment with erlotinib. Due to cutaneous side effects the patient decided to discontinue this therapy. Since then he has been in regular follow-up showing a stable disease and good general state of health.

Background
Lung cancer is one of the most appalling diagnoses, associated with significant patient distress, poured quality of life and often limited survival. Moreover, the current literature emphasizes the effect of distress level on survival rates. The purpose of this study was to explore the survival curve, considering the distress level reported by patients with advanced NSCLC cancer at the beginning of the treatment.

Methods
A sample of 24 patients with newly diagnosed stage IV NSCLC, answered the Distress Thermometer (DT) and the Problem List (PL) before start the first chemotherapy infusion. They treated at a private cancer center, located at Brasília, Brazil. Survival was calculated from time of the histopathological diagnosis. The data was analyzed with descriptive evaluation, followed by Kaplan-Meier models, to test associations with survival and distress.

Results
Figure 1Of those patients, 58.3% were male. They were between 54-81 years old (M = 66.2; SD = 6.6); 62.5% were married; 54.2% had at least college degree. The NSCLC were 87.5% adenocarcinoma and 12.5% epidermoid. The survival rate was 10.7 months for all patients (range from 1 to 50 months; SD = 10.7); being 6.4 months for moderate to severe distress and 14.2 for mild distress. Seven types of chemotherapy regimen were prescribed: Cisplatin+Gemcitabine (45.8%); Alimta+Carboplatin (16.7%); Carboplatin+Taxol+Avastin (8.3%); Erlotinib (8.3%); Taxol+Carboplatin (8.3%); Cisplatin+Alimta (8.3%); Cisplatin+Gemcitabine+Avastin (4.2%). Half of patients (54.2%) reported moderate to severe distress (DT ≥ 4). The most problems reported at PL were worry (70.8%), sadness (66.7%), fears (45.8%), sleep (70.8%), fatigue (66.7%), pain (58.3%), eating (50%) and breathing (50%). Considering the survival curves, patients with mild distress showed better survival rate than patients with moderate to severe distress (x[2] = 4.16; p = .04).

Conclusion
Despite the limited sample size, the data suggest that mild distress is a prognostic factor in survival rate. The high prevalence of patients with moderate to severe distress, as well as, the problems-related distress, highlighted the importance of the distress screening and also to create an advanced care planning for patients with lung cancer, from the time of diagnosis until the last phase of illness, helping them to cope with this reality. More studies are thus needed to investigate the implication of distress level at diagnosis as a prognostic factor, increasing and diversifying the sample in order to make the data more generalizable.

Background
Although lung resection still provides the best long-term outcome for lung cancer, it is also associated with a considerable decay of physical and psychosocial health status. Physical activity (PA) is considered beneficial for post-surgery recovery, and patients often are encouraged to increase PA in daily life. Surprisingly, only few studies objectively assessed daily PA in lung cancer patients, and none of these studies examined possible associations between daily PA and recovery of health status following lung resection. Therefore, our study aims to explore the relationship between daily PA and recovery of physical and psychosocial health status in lung cancer patients treated with lung resection.

Methods
Lung cancer patients undergoing curative lung resection at the Nederlands Canker Institute-Antoni van Leeuwenhoek Hospital in Amsterdam are enrolled in the study before surgery (T0). Follow-up measurements are scheduled at 1 (T1), 3 (T2) and 6 months (T3) post-surgery. PA levels are measured for three days on all occasions using a triaxial accelerometer and expressed as amount of activity in counts per minute. Secondary outcome measures include VO~2~max, pulmonary function, six-minute-walking-distance, times standing on the 30-seconds-chair-stand test, quality of life, mental distress, fatigue, and pain. The aim is to include 60 patients by October 2015.

Results
So far, fourteen lung cancer patients (6 male; mean age 65.4±10.4 yrs) are included in the study. Ten patients underwent lobectomy, four segment resection, while no patients underwent pneumectomy. Four patients dropped out due to comorbidity (n=1), disinterest (n=1), and inability to perform the measurements (n=2). First analyses show that both physical and psychosocial health are worse one month post-surgery, but improve during the following five months (table 1). Activity patterns from pre-surgery to 6 months post-surgery of the included patients are being analysed at the moment. Figure 1

Conclusion
As was expected from earlier research, physical and psychosocial outcome measures worsened following lung resection, but returned to baseline levels at 6 months post-surgery, except of VO2max. At this moment, we cannot calculate associations between daily PA and secondary outcome measures yet. However, patients are actively recruited and enrolled, and at the time of the conference we will be able to draw more firm conclusions about possible associations between daily PA and recovery of health status following lung resection.

Background
Use of tyrosine kinase inhibitors is a attractive option due to its predicatble profile of side effects & it been a oral tablet. Its efficacy and toxicities have been well described in both east asian and western population. However not much literature is available on its use from India. The aim of this paper was to audit the population which received TKI (Erlotinib or Gefinib) in a tertiary cancer center in India.

Methods
All patients who received TKI in lung cancer were identified from our OPD database. There case records, electronic medical records & our OPD databases were utilized. The demographic profile, the staging details , details of EGFR mutations, the RECIST response rates, the toxicities, progression free survival (PFS) & overall survival (OS). SPSS 16 was used for statistical analysis.

Results
Total of 105 patients were identified with a median age of 58 years (35-77 years). There were 56 females (52.8%). The 15.2 % (16) of patients were smokers & 22.3 % (18) were tobacco chewers. The median duration of smoking & tobacco chewing was 25 & 15 years respectively. All patients had stage IV disease. There were 2 cases of squamous cell carcinoma (1.9%)& rest all were adenocarcinomas. (98.1%) Extrathoracic disease was present in 42 patients (39.6 %) The EGFR mutation invoved exon 19 , 21 & 18 in 25, 72 & 8 patients respectively. The ECOF PS was 0 & 1 in 14 (13.2%) & 51 (48.1%) patients respectively.The incidence of any grade skin and mucosal toxicites were 53% & 38% respectively. The median PFS & OS overall were 11.1 & 21 months respectively. The PFS & OS were not affected by the performnce status, comorbidities & sites of metastasis

Conclusion
Use of TKI in Indian population had similar toxicity & efficacy as reported in literature.

Background
SBRT is an emerging technique, with numerous advantages over classical radiotherapy, namely in high surgical risk patients.

Methods
Critical review of a clinical case

Results
The authors report the case of a 37 year-old man, type 1 Neurofibromatosis patient, with dorsal paraplegia secondary to paravertebral tumours, severe kiphoscoliosis, and chronic hypercapnic respiratory failure requiring continuous non-invasive ventilation (NIV) since 2007. He was also medicated with Baclofen 125mg/day and botulinum toxin for severe spasticity, with poor response. In May 2010, he was diagnosed with testicular malignancy, which has undergone radical resection under local anaesthesia. In June 2010 he was submitted to a follow-up CT scan, which revealed a 9mm pulmonary nodule on the right upper lobe that remained under supervision, due to the risks of invasive diagnostic procedures. In January 2012, because of a significant size increase, a CT scan-guided biopsy of the lesion was performed, which revealed a lymphoepithelioma-like carcinoma. Subsequent CT-scans demonstrated progressive growth of the neoformation (4.9cm in July 2012) with invasion of the thoracic wall, and the PET/CT scan revealed FDG-F18 hyper fixation. Due to the comorbidities, and the need for continuous NIV, he had no surgical, chemotherapy or classical radiotherapy conditions. The remaining treatment option was SBRT. Therefore, a planning 4D-CT scan with a vacuum mattress for immobilization was performed, and a therapy plan was defined to deliver a total dose of 48Gy, in four fractions of 12Gy, in alternate days, with support of Image-Guided Radiation Therapy (IGRT) technology. There was a good clinical and haematological tolerance, no acute or late complications were referred, and the 6 months follow-up CT-scans showed partial response, according to RECIST criteria.

Conclusion
SBRT is nowadays a valid alternative for lung cancer treatment in patients with medical contraindications to surgical treatment, demonstrating a high local control of the lesion, associated with low toxicity effects. Figure 1Figure 2

Background
Discussion of patients at multidisciplinary meetings (MDMs) is now considered the standard of care for management of thoracic malignancies. However, there is limited evidence regarding concordance of decisions made at MDMs with subsequent actual treatment. This study aimed to assess the concordance between MDM outcomes and subsequent treatment received by patients with thoracic malignancy at our centre, and to identify factors that contributed towards any deviation from the MDM management plan.

Methods
In this retrospective audit, patients discussed at 12 Auckland City Hospital MDMs between 1[st] May and 31st July 2012 were identified, using the MDM lists for each session and chairperson summaries of discussion and decisions. The electronic cases records of these patients were then reviewed for demographical data, information on the MDM outcome and to ascertain the eventual treatment received by these patients. Any discrepancies between MDM recommendations and eventual patient management were noted, and the reasons stated for this.

Results
There were 121 cases identified from 138 documented patient presentations; 17 patients were re-presented within the study period, and the presentation at which anti-cancer treatment was determined was analysed (if achieved). Of the 121 cases, 93 included a pathologically confirmed diagnosis (80 primary lung or pleural tumours, 13 other tumours or pulmonary metastases), and 28 did not, including 4 with suspected malignant pleural effusions. A decision for radical therapy was made for 22 cases at the MDM. 18 of these cases received treatment as determined at MDM: 13 of 15 surgical resection, 1 of 3 radical radiotherapy, 3 of 3 radical chemo-radiotherapy, and 1 was deemed not to need adjuvant chemotherapy. A decision for one or more palliative treatments was made for 72 cases. Of these, the MDM determined treatment was actually received in 41 (57% of cases): 5 of 7 palliative surgery cases (1 died pre-op, 1 patient declined), 18 of 28 chemotherapy as initial treatment, and 13 of 23 radiotherapy as initial treatment. In 14 cases with an MDM decision recorded recommending both palliative chemotherapy and radiotherapy, 5 received both modalities, 7 radiotherapy alone and 2 neither. Reasons for discordance between MDM decision and treatment included patient deterioration or poor performance status (n=14), patient choice (n=8), patient and doctor agreed choice (n=5) and did not attend/lost to follow up (n=4) In the remaining 27 cases, best supportive care was recommended in 7 and no definitive treatment decision was able to be made in 20.

Conclusion
This study demonstrated excellent concordance between decisions made for radical therapy at the MDM and actual treatment. Concordance was less for palliative therapies, and this is likely to reflect on the poorer performance status of these patients at presentation and the perceived benefit versus adverse effects of palliative measures to the patient and the clinician. Since this study, a standardised electronic MDM template has been introduced to ensure accurate and complete information is available.

Background
Lung cancer is the second cause of cancer death in Vietnam. Most of patients with lung cancer are non small cell (90%) and present at advanced stage. Erlotinib was the first EGFR TKI permitted to be used for advanced stage non small cell lung cancer in Vietnam since 2007. Safety and efficacy of this treatment has never been reported in Vietnamese patients. We present the first case report of erlotinib in Vietnamese advanced-stage non small cell lung cancer patients to study the safety and the efficacy of this treatment.

Methods
From 12/2007 to 12/2011 advanced stage (wet IIIB and IV) non small cell lung cancer patients diagnosed at Cho Ray hospital were prescribed erlotinib as (1) second/third line treatment or as (2) first line treatment only if they could not use standard, state of the art chemotherapy after well informed about different treatment options.

Results
High overall response 35.5%, median progression free survival of 7 months, median overall survival of 11 months and 1 year survival rate 50% were seen in 31 patients using erlotinib. These results were similar to East Asia patients but better than ones of Caucasian patients. Common adverse events were rash and diarrhea, severe adverse events were rare.

Conclusion
Erlotinib had high efficacy and low toxicity in Vietnamese patients like East Asian patients.

Background
VATS lobectomy for Non-small cell lung cancer usually can be performed using one working window, and two or more ports for endoscopic instruments. The working window are made on the intercostal space space. However, these narrow Intercostal space could be the obstacle for the extraction of congested or huge resected lung lobe. So, we performed VATS lobectomy using 3 interocostal ports and the 3 cm subcostal incision.

Methods
Since March 2012, In Korea University Ansan Hospital, 20 VATS lobectomies was done for lung cancer. Of 20, we performed 4 cases of subcostal incision VATS lobectomy. At first, we performed entire procedure through the intercostal ports (10mm). After resection of lung, the subcostal incision (3cm ) was made anterior to the 10th rib. Because the 11th costal cartilage is abscent, there is no limitation during spreading the 10th intercostal space for the extraction of resected lung (Fig 1). And then, we dissect mediastinal lymph nodes systemically. During the subcostal incision, rectus muscle is resected partially. And the chest cavity was entered through the costophrenic junction. So,at the end the repair of diaphragm is needed. The chest tube can be placed properly through one of the intercostal ports.

Results
The characteristics of patients is summerized in Table 1.Of 4 cases of subcostal incision lobectomy, one case should be conversed to the anterior thoracotomy.That case was 6.5cm sized right middel lobe tumor.In the third case, we used two intercostal ports and one subcostal incision. In the question of post operative intercostal pain at operation day, patients did not complain severe pain (more than VAS score 5). However, we did not compare the pain score after this operation with conventional VATS lobectomy.

Talble 1.The patient's characteristics

Case No.	G/A	Lobe	Tumor size	LND	pTNM	3 ports	subcostal
1	M/67	LLL	3.5cm	30	T2aN0M0	5th/6th/7th	3cm
2	M/62	LUL	3.5cm	16	T2aN0M0	4th/6th/6th	3cm
3	F/69	RUL	1.8cm	47	T1aN1M0	5th/6th	3cm
4	F/43	RML	6.5cm	29	T2bN2M0	5th/6th/6th	1cm

Figure 1 Fig 1. Three intercostal ports (10mm) and one Subcostal incision (3cm)

Conclusion
The subcostal incision for extraction of lung specimen was useful in VATS lobectomy.

Background
Ranking hospitals, publishing reports of high and low performing facilities, and altering reimbursement for outlier sites is becoming common in many areas of healthcare. We explore challenges of profiling hospitals on appropriate use of advanced imaging in lung cancer care.

Methods
We identified 17,325 patients with newly diagnosed lung cancer over a 4 year period from the Veterans Health Administration’s Central Cancer Registry, of whom 5,424 (31%) were Stage IIB, IIIA, or IIIB and treated in one of 84 VA Medical Centers. Imaging was assessed 180 days pre and post diagnosis per National Comprehensive Cancer Network guidelines and American College of Radiology Appropriateness Criteria for Imaging. Risk-adjusted hospital rankings were estimated using a hierarchical logistic modelling approach.

Results
Recommended imaging was performed in 70% of patients overall for brain imaging and 51% of patients for positron emission tomography (PET). Overutilization, with combined bone scintigraphy and PET (BS/PET), occurred in 20% of patients. Widespread variation in non-adherence to guidelines was observed, with distinct geographic patterns. The New England region of the U.S. had imaging rates that were 27 percentage points higher than the Great Plains area, which had the lowest use of imaging. Receipt of recommended imaging ranged from a low of 61% of patients in one facility, to 94% in the best performing facility. Models calculating traditional observed-to-expected ratios identified 6 (7%) underperforming facilities, while hierarchical logistic models recommended by Centers for Medicare and Medicaid for hospital profiling, identified 14 (17%) underperforming facilities.Figure 1.

Conclusion
A significant proportion of patients do not receive recommended imaging, while other patients appear to undergo excessive imaging for lung cancer. These observations are hallmarks of poor quality. Hospital ranking and assessment of performance is sensitive to statistical approach. Lung cancer care providers should engage in developing uniform approaches to reporting and monitoring quality of care, and work to identify opportunities to improve efficiency and reduce waste.

Background
Five full-time doctors are sent to 5 affiliated hospitals from University Hospital of Kyoto Prefectural University of Medicine. To conduct a surgery in the affiliated hospitals, a doctor is sent from the university, for ensuring an efficient and secure medical care with limited members. Until now, each hospital performed surgeries in its own way. However, to perform safer and efficient surgery with a limited number of operating room nurses and thoracic surgeons, they need to work as one team. We report the standardization of thoracic surgery procedures.

Methods
First, to understand the current situation at each affiliated hospital, we survey all aspects of surgical procedures. Subsequently, we held 5 meetings among the group and standardized the surgical procedures. A thoracic surgeon and 3–4 operating room nurses from each hospital joined in the discussion. The topics of the discussion covered all aspects of surgery and methods from each hospital were analyzed and standardized. We have already standardized the thoracoscopy system and energy device in all hospitals. We also standardized the main surgical instruments and methods in these meetings. The content of the standardized main surgical procedure was thoroughly explained in a video distributed to each hospital in DVD format. We evaluated the frequency of use of the surgical instruments and excluded rarely used items. To increase the understanding on automatic suture instruments and energy device, the important usage points were shared in the meeting. Nurses were trained on the usage. In the 6[th] affiliated hospital meeting, a questionnaire was conducted to survey the awareness of this approach.

Results
The amount of time required from entering the operating room to starting the surgery was shortened from 62 to 55.5 minutes (average). The time required from the end of surgery to exiting the operating room was also shortened from 46.1 to 38.7 minutes (average). The difference among hospitals was successfully reduced. Because the main surgical instruments and methods were standardized, almost the same level of surgery could be performed in each hospital. Surgical instruments were reduced from 48.3 to 41.1 types (average). Total number of surgical instruments was successfully reduced from 91.8 to 73.5 items (average). In the questionnaire, all members referred to other hospitals devices, and they will attempt productions of their own device. All members confirmed improved understanding on thoracic surgery and 88% confirmed increased interest in thoracic surgery．

Conclusion
Standardization of the surgical procedures improved the workflow, enabled safe and efficient surgery among the affiliated hospitals, and increased awareness of the importance of workflow improvement. Change in awareness toward thoracic surgery was observed among participating members, suggesting that the present approach is highly useful.

Background
Background Radiation induced oesophagitis (RIO) is a significant toxicity of lung cancer treatment that has profound clinical, social and economic implications. The literature suggests there is minimal evidence to support current analgesic regimes with the exception of systemic analgesia. More information is required to better understand the patient experience of RIO and how it can be managed. Aim To identify the properties and characteristics of RIO experienced by patients having radiotherapy to the chest for lung cancer.

Methods
Methods A qualitative exploratory study conducted with patients with lung cancer receiving radiotherapy to the chest. Patients participated in semi-structured interviews exploring their experience of RIO. Interviews were recorded, transcribed and content analysed.

Results
Results Twenty six patients participated: six with grade 1; 14 with grade 2 and eight with grade 3 RIO. Patients were interviewed following recovery from grade 3 RIO. Four key domains were identified: 1.Pain descriptors such as “feels raw “, “burning”, “like reflux but worse” were reported 2. Swallowing difficulties varied over time and were described as “felt like there was a blockage, “afraid I would choke,” “unable to get anything through”. 3. Self care efforts employed by the patients to manage these difficulties ranged from diet modification, allowing food and drinks to go cold before eating and eating slowly. 4. An aversion to taking regular analgesia was also evident. The overall impact on participants’ lives was often understated, even in the context of hospital admissions, insertion of nasogastric tubes and poorly controlled pain.

Conclusion
Conclusions This study demonstrates the complexity of RIO and suggests clinicians may underestimate the effect and severity of RIO. Given patients appear to continue to experience problems, despite treatment, better prophylaxis and management regimes are required.

Background
Background: Thymidylate synthase (TYMS) is an important chemotherapeutic target in non-small cell lung cancer (NSCLC). Recently, arsenic trioxide (ATO) has been shown to downregulate TYMS in a colonic cancer model. Therefore, we examined the effect of TYMS suppression by ATO in NSCLC.

Methods
Methods: A panel of seven NSCLC cell lines (NCI-H23, NCI-H358, HCC827, NCI-H1650, NCI-H1975, HCC2935 and HCC4006, from ATCC) was used to determine the effects of ATO treatment on cell viability, TYMS protein and mRNA expression, E2F1 protein expression and TYMS activity. TYMS knockdown and overexpression were performed to confirm the importance of TYMS in cell survival and resistance to ATO respectively. Tumor growth inhibition in vivo was studied using a nude mice xenograft model.

Results
Results: ATO showed antiproliferative effects with clinically achievable concentrations (around 1.1-6.9 μM after 72 hours incubation) in NSCLC cell lines. Baseline TYMS protein expression was detected in H23, H358, H1650 and H1975 cells. Among them, downregulation of TYMS protein and mRNA expression, reduced total TYMS activity, and suppressed E2F1 expression were demonstrated in H23, H358, and H1650 cells with ATO. Cell viability was reduced by 15-50% with TYMS knockdown (to less than 30% protein expression) (Fig. 1). Overexpression of TYMS led to a 2.7-fold increase in IC~50~ value with ATO treatment for 72 hours in H358 cells, but not H23 cells. Using a xenograft model with H358 cell line, relative tumor volume was reduced to 44% that of control after 8 days of treatment with 7.5 mg/kg intraperitoneal ATO, and associated with significant downregulation of TYMS protein expression in tumor xenografts (Fig. 2). Figure 1 Figure 2

Conclusion
Conclusion: ATO has potent in vitro and in vivo activity in NSCLC, and is partially mediated by transcriptional downregulation of TYMS.

Background
Lung cancer is the most widespread cause of cancer death in the world. COX-2 derived prostaglandin E~2~ (PGE~2~) is well known to induce tumor growth and metastasis, and thus is associated with a poor prognosis. Lymph node metastasis is also one of the major determinant of the prognosis and is facilitated by lymphangiogenesis, however the precise of the mechanisms is not well understood. In the presenr atudy, we investigated the role of COX-2-derived PGE~2~ on formation of premetastatic niche facilitate the lymph node metastasis in Lung Cancer. Lung cancer is the most widespread cause of cancer death in the world. COX-2 derived prostaglandin E~2~ (PGE~2~) is well known to induce tumor growth and metastasis, and thus is associated with a poor prognosis. Lymph node metastasis is also one of the major determinant of the prognosis and is facilitated by lymphangiogenesis, however the precise of the mechanisms is not well understood. In the presenr atudy, we investigated the role of COX-2-derived PGE~2~ on formation of premetastatic niche facilitate the lymph node metastasis in Lung Cancer.

Methods
Male C57BL/6 (6-8 weeks-old, Wild type; WT) and EP3 knockout mice (EP3[-/-]), one of PGE~2~ receptor subtype, were used. Orthotopic intrapulmonary implantation model was made by injecting Lewis Lung Carcinoma (LLC 5.0X10[6] cells/ml) cells into the lung. We estimated the expressions of lymphangiogenic factors, SDF-1/CXCR4, subtypes of EP receptors by immunehistochemical staining, ELISA and real time PCR. The accumulation of immature dendritic cells (iDCs) and regulatory T cells (Tregs) were estimated by immunofluolecent analysis. Furthermore, we estimated the role of iDC on lymph node metastasis by injecting iDCs.

Results
The expression of COX-2 and SDF-1, and accumulation of iDCs and Tregs were increased before LLC cell accumulated in lymph node. Compared to vehicle mice, mediastinal lymph node metastasis formations were suppressed with a COX-2 inhibitor (celecoxib, 100mg/kg/day, p.o., P<0.05). Compared to other PGE~2~ subtype receptors, the expression of EP3 receptor was significantly suppressed in celecoxib treated mice (P<0.05). The expression of lymphangiogenesis markers and lymph node metastasis were suppressed in EP3[-/-]. The expression of SDF-1 and accumulations of CD11c[+]DCs and FOXP3[+]Tregs in lymph nodes were significantly suppressed in EP3[-/-] (P<0.05). In vitro study, under PGE~2~ stimulation, the SDF-1 conentration and expression of EP3 in culutured iDCs were significanltly enhanced compared to control. Furthrmore, WT transplanted with EP3[-/-]BM were significantly suppressed mediastinal lymph node metastasis formation compared to WT transplanted with WT mice.

Conclusion
These results suggested that premetastatic niche formation in mediastinal lymph node was induced by bone marrow derived immatured dendritic cells via PGE~2~-EP3 signaling by induction of SDF-1-expression. Thus, COX-2 inhibitors, CXCR4 antagonists and/or EP3 antagonists may become one of the options to suppress the lymph node metastasis.

Background
A hierarchical model in which a small population of stem cells or Cancer Initiating Cells (CIC) may be responsible of tumour growth has been proposed. CIC is defined as a cell able to regenerate detectable neoplastic populations in xenografted immunodeficient mice. By this experimental approach, convincing data have led to the identification of CIC in solid tumours including breast, brain, colorectal, melanoma and lung. However, the autonomous growth ability of CIC is allowed in specialized structures, called niches, in which a microenvironmental control is exerted by several stromal elements including mesenchymal cells. These attractive pathogenetic mechanisms encompass innovative therapeutic implications because the limited success of the actual treatment of lung cancer may be attributable to the lack of targeting on CICs and their niches. The aim of the present study was to identify mesenchymal stem cells (MSC) from Non Small Cell Lung Cancer (NSCLC) and to investigate their biological properties and in vivo tumorigenic potential.

Methods
Small fragments of NSCLC (53 Adeno- and 24 Squamous carcinoma) surgically removed from 77 patients (46 male) were subjected to enzymatic digestion whereas most of the original sample was processed for diagnostic purpose. To identify mesenchymal cells from human lung cancer (hLc-MSC) we used different conditioning media and immunomagnetic selection to separate neoplastic epithelial cells frequently growing together with adherent stromal cells at early passages. To determine their tumorigenic properties, female immunodeficient BALB/c nude mice were subcutaneously injected with either 10[6] hLc-MSCs (from male donors) alone or in combination with 10[6] Calu-3, a human adenocarcinoma cell line lacking Y chromosome and able to reproducibly induce xenografted tumours.

Results
We have been able to obtain stable primary cultures of hLc-MSC, expandable for at least 14 passages, from 62% of NSCLC samples. This cell population uniformly expressed the typical MSC markers CD90, CD105, CD73, CD13 and CD44 at FACS analysis. hLc-MSC were negative for EpCAM, CD45, CD14 and CD34 whereas CD117 and CD133 were expressed at low frequency. Clones and subclones were documented by limiting dilution analysis. The nuclear expression of transcription factors involved in stemness (OCT 3/4 and SOX2), and in bronchio-alveolar (TTF1) or squamous (p63) lineage commitment was consistently present in hLc-MSC isolated from Adeno- or Squamous cell carcinomas. In vivo xenotransplantation demonstrated that although hLc-MSC administered alone did not generate tumours, their co-injection with Calu-3 developed in all animals subcutaneous nodules that were 10-fold larger than those induced by equal doses of Calu-3 injected alone. To detect the fate of both injected cells on sections of xenografted tumours, FISH analysis of human specific sex chromosomes was combined with immunohistochemistry. We documented that cytokeratin positive adenocarcinoma structures showing X chromosomes only (Calu-3 derived) were surrounded by a thin layer of non neoplastic cells carrying XY chromosomes (hLc-MSC derived). The cross talk and phenotypic changes of E-Cadherin positive neoplastic cells co-cultured with hLc-MSC were also documented.

Conclusion
Our study demonstrates that the reciprocal inductive interactions between the mesenchyme and the neoplastic epithelium are essential for the fate induction of lung cancer.

Background
Activating mutations of K-ras are one of the most common alterations associated with tobacco exposure-related lung cancer(LC). There are two major types of LC - small-cell LC(SCLC) and non-small cell LC(NSCLC) - and the later accounts for 80% of the cases. NSCLC can be divided into three histological subtypes: Adenocarcinoma, large cells and squamous cell carcinoma. First-line treatment comprises surgical resection of tumor, followed or not by chemo/radiotherapy. In non-surgical cases, platinum compounds remain the cornerstone for both early and advanced NSCLC stages management, in spite of its toxicity, high rate of chemo-resistance and poor long term results. Several attempts to develop therapies based on molecular targets, such as K-ras, have been developed and thus far failed, clearly stating the need for new approaches to bring clinical benefits to patients. Among its several aberrations, NSCLC harbors alterations in the apoptotic pathways, leading to impaired pro-apoptotic signaling and positive modulation of anti-apoptotic pathways. Therapeutic strategies targeting such pathways can emerge as an alternative to the cytotoxic therapies selected to wild-type EGFR-patients – especially for K-ras mutated patients, comprising about 20% of this population.

Methods
Here we have analyzed a representative panel of NSCLC cell lines (A549, H460, Calu-1 and LC319), that display distinct sensitivities towards cisplatin, mutational profiles and histological subtypes, to test a pre-clinical therapeutic strategy engaging the TNF-related apoptosis-inducing ligand (TRAIL) receptor (Death receptor 4 and 5).

Results
TRAIL is a member of the TNF family of cytokines that induces apoptotic cell death in a variety of tumor cells by means of activation of its specific receptors, DR4 and DR5, while displaying low toxicity towards normal cells. We sought to investigate if DRs activation could subvert the relative resistance to cisplatin intrinsically presented by NSCLC cell lines. NSCLC cell lines treated with suboptimal concentrations of cisplatin (IC30) for 48h had their gene expression analyzed by qPCR and the results showed an increased expression of DR4 and DR5 in these cells. These results were confirmed at protein level by Western blot analysis. NSCLC cells are naturally regarded as resistant to TRAIL-induced cell death. Such resistance can rise, among other reasons, from low expression of DRs or increased expression of decoy receptors and/or anti-apoptotic proteins. LBY135 (Novartis) is a DR5 agonist monoclonal antibody that mimics TRAIL and induces cell death in DR5 expressing cells. As cisplatin modulated DR5 protein expression, we have combined it with LBY135 as a strategy to improve cell death induction upon NSCLC cells. LBY135 monotherapy did show some effects when analyzed by MTT assay, although it did not induce cell death accessed by Annexin/PI FACS analysis. However, when cisplatin IC30 and LBY135 were combined, we observed a significant decrease in MTT measurements and increased cell death incidence, suggesting a synergistic effect of these drugs. Such pro-apoptotic effect was blocked by zVAD-fmk, a pan-caspase inhibitor.

Conclusion
The synergistic effect observed was more pronounced in cell lines bearing the classical G12K-ras mutation, suggesting an alternative way to subvert chemo-resistance of K-ras mutated NSCLC and restore cisplatin-induced apoptotic signaling leading to cell death.

Background
Arsenic trioxide (As~2~O~3~), an ancient drug used in traditional Chinese medicine with the characteristics of high effectiveness and low toxicity, was used to treat acute promyelocytic leukemia at the beginning . Over the past decade, in our clinical practice, local injection of As~2~O~3~ into pleural cavity has been successfully used for more than 80 patients with malignant pleural effusions(MPE) caused by pleural metastasis of lung cancer. And interestingly, after local administration of As~2~O~3~, the composition of blood cells in pleural effusion was reduced and the color of MPE changed from red to light yellow in the majority of patients, indicating that As~2~O~3~ could be considered as an effective approach for the treatment of MPE, but the underlying mechanism remains unclear . The aim of this study was to investigate the mechanism of arsenic trioxide (As~2~O~3~) in the treatment of malignant pleural effusion (MPE) caused by pleural metastasis of lung cancer.

Methods
A mouse model of MPE caused by pleural metastasis of lung cancer was first established in our study, and then As~2~O~3~ was intraperitoneally injected to treat MPE. Those mice treated with bevacizumab and bleomycin were included as positive control animals, and placebo equivalents were also employed as negative control. The effects of As~2~O~3 ~on MPE volume, pleural vessel density, vascular permeability, expression of angiogenic function related factors including VEGF and TNF-α, as well as NF-κB activity in pleural carcinomatosis were observed.

Results
Intraperitoneal injection of As~2~O~3~ could reduce the volume of MPE and decrease vascular density and vascular permeability in pleural metastatic nodules, in a dose-dependent manner. Moreover, the dose-dependent decrease in VEGF and TNF-α expression in MPE and NF-κB activity in pleural carcinomatosis were also found after As~2~O~3 ~treatment.

Conclusion
Our work demonstrated that As~2~O~3~ could down-regulate the VEGF expression through NF-κB inhibition, and then decrease vascular density and permeability in pleural metastatic nodules, thereby playing its effects on MPE caused by pleural metastasis of lung cancer. Our results could provide a foundation for As~2~O~3~-based clinical treatment program.

Background
Small-cell lung cancer (SCLC) is the most aggressive subtype of lung cancers, with no early detection strategy or targeted therapy currently available. We hypothesized that difference gel electrophoresis (DIGE) may allow the identification of membrane-associated proteins specific to SCLC, the advancement of our understanding of SCLC biology, and the discovery of new candidate diagnostic or therapeutic biomarkers.

Methods
Membrane-associated protein lysates were prepared in quadruplicate from three SCLC, three non-small-cell lung cancer (NSCLC), and three immortalized normal bronchial epithelial cell lines. The 36 samples were co-analyzed by DIGE and subsequent protein identification was performed by mass spectrometry (MS). Identified proteins were submitted to Ingenuity pathway analysis (IPA). Candidate biomarkers were validated by Western blotting (WB) and immunohistochemistry (IHC), and tested against clinical outcomes.

Results
Principle component analysis on the global DIGE dataset demonstrated that the four replicates derived from each of the nine cell lines clustered very closely from one another, as did samples within the same histological group. A total of 135 distinct protein features were differentially expressed in SCLC as compared with NSCLC and bronchial airway epithelial cell lines (P<0.001). Those included 137 different proteins identified by tandem MS. IPA revealed that these proteins were overrepresented in the cellular assembly, organization, morphology, and tissue morphology networks. DPYL2, GNAQ, RSSA, RUVB1, and STMN1 were found to be the most discriminatory candidate biomarkers among the membrane-associated proteins overexpressed in SCLC as compared with NSCLC and normal bronchial airway cell lines. Overexpression of DPYL2, GNAQ, and STMN1 was verified in SCLC cell lines by WB. Intense protein expression of DPYL2, GNAQ, RUVB1, and STMN1 was also confirmed in SCLC by IHC on tissue microarrays (TMA). These proteins’ expression levels measured by IHC were significantly associated with the SCLC subtype and survival in a univariable analysis but could not be verified as independent in a multivariable analysis.

Conclusion
Proteomic analysis of membrane-associated proteins in lung cancer and bronchial airway epithelial cell lines revealed 137 proteins differentially expressed in SCLC. These proteins were enriched for cellular assembly, organization, morphology, and tissue morphology networks. Of the five proteins selected for clinical validation, DPYL2, GNAQ, RUVB1, and STMN1 overexpression in SCLC was verified by WB and IHC, suggesting that these results need to be tested for functional implication in SCLC progression. The association with survival requires further validation in a larger clinical dataset. Funding This work was supported by a VA Merit review award 1I01CX000242 to PPM. SO was supported by a Télévie Grant, a Fondation Mont-Godinne Grant and a Clinician-Researcher Mandate from Secteurs des Sciences de la Santé, Université Catholique de Louvain (UCL), Belgium.

Background
Cancer cells have defects in regulatory circuits governing proliferation and homeostasis. Consequently, cell metabolism is altered to meet the demand for accelerated, deregulated growth. Metabolic perturbations arising from malignant transformation have not been well characterized in human lung cancers in situ. The most well known metabolic derangement(s) in tumors is that of enhanced glycolysis and a decrease in mitochondrial oxidative phosphorylation. We wanted to characterize this phenomenon more accurately in human lung adenocarcinomas by metabolomic profiling.

Methods
We performed metabolomic analysis of matched pairs of solid, non-small cell lung adenocarcinomas and normal lung tissue from 25 surgically resected patients. Metabolites were extracted by a methanol-chloroform-water technique. The resulting extracts were divided into multiple fractions. Ultrahigh performance liquid chromatography/ mass spectrometry coupled with tandem mass spectrometry and gas chromatography/ mass spectrometry experiments were conducted. Agilent MassHunter Qualitative software was utilized. The Molecular Feature Extractor was utilized to find features in raw data files. Extracted peaks were retention time aligned using Mass Profiler Professional and unique features detected by least squares analysis. The Agilent version of the Metlin database was utilized to identify metabolites. Matched pairs t-test identified biochemicals significantly altered between tumor and normal specimens. The false discovery rate method assessed for significance; p-value ≤ 0.05 and q-value < 0.10.

Results
Based on known library standards to identify biochemicals, our global metabolomic profiling found 204 overexpressed and 42 underexpressed metabolites in tumors relative to normal lung (p< 0.05). We observed altered metabolite levels in lung tumors that mapped to not one, but two glucose utilization pathways. Glucose-6-P (2.7-fold), fructose-6-P (2.6-fold), fructose-1,6-bisP (6.9-fold), lactate (2.7-fold), and NAD[+] (1.4-fold) were significantly upregulated in tumors consistent with an aerobic glycolysis (i.e. Warburg) biosignature, the major source of ATP. Concurrently, pentose phosphate pathway (PPP) metabolites were upregulated in tumors: ribulose-5-P (2.6-fold), ribulose (3.6-fold), ribitol (4.6-fold), ribose (4-fold), and sedoheptulose-7-P (3-fold). Our data reveals evidence of multiple active pathways to explain glucose utilization in lung adenocarcinomas. The PPP is important to protect against oxidative stress as it serves to generate NADPH, and is a key anabolic pathway of nucleotide synthesis by generating the ribose-5-P backbone for proliferating cells. Observing both pathways simultaneously in lung adenocarcinomas suggests they are coupled to give tumors a growth advantage over normal tissue. Consistent with this, we observed an overall increasing nucleotide biosynthesis signature in tumors: multiple metabolites (range 2 to 17-fold) in purine and pyrimidine pathways were significantly elevated.

Conclusion
Metabolomic analysis identified a unique glucose energetic biosignature in lung tumors that is more complex that just a single process/ pathway. Our results suggest a specific strategy to target lung adenocarcinomas by exploiting their high glucose uptake.

Background
The etiology of malignant pleural mesothelioma (MPM) is closely linked with asbestos exposure. Asbestos is capable of inducing chronic inflammation which potentiates tumour suppressor gene silencing. Epigenetic silencing of the Wnt pathway, well characterized in the progression of colon cancer, is associated with chronic inflammation. As antagonists of Wnt pathways, the SFRPs are functional tumour suppressors of colon, gastric, breast, ovarian and lung cancers, with some members methylated in mesothelioma. In this study, we aimed to investigate the functional significance of the SFRP2 and 5 in MPM, and the effect of long-term asbestos exposure on epigenetic alteration in the immortalised mesothelial cell MeT-5A.

Methods
Gene expression and promoter DNA methylation of the SFRP family were analysed in MPM lines and MeT-5A with and without 5’Azacitidine treatment using RT-qPCR, MSP and COBRA. The effect of SFRP2 and SFRP5 re-expression on MPM cells was determined by cell growth and clonogenic assays in 2D and 3D culture. The expression and promoter DNA methylation of SFRP genes was also assessed in MPM patient samples using RT-qPCR and MSP. MeT-5A cells were cultured long-term (12 months) in the presence of asbestos, and SFRP mRNA expression and promoter DNA methylation was analysed.

Results
SFRP2 and SFRP5 were either absent or down-regulated MPM lines, and restored after 5’Azacitidine treatment. SFRP1 was highly expressed and unmethylated in MeT-5A line. Expression of the SFRP family was down-regulated in MPM patient samples and this correlated with methylation of promoter CpG islands. Ectopic expression of SFRP2 or SFRP5 inhibited MPM cell growth and colony formation in both 2D and 3D culture. SFRP1 was down-regulated and methylated following prolonged asbestos exposure in MeT-5A cells.

Conclusion
Our results indicate that both SFRP2 and SFRP5 function as tumour suppressor genes in MPM and long-term asbestos exposure induce gene silencing via DNA hypermethylation.

Background
Most patients die from lung cancer because of chemoresistant metastatic disease. We have previously identified the importance of fibroblast growth factor 2 (FGF2) /S6 kinase 2 (S6K2) signalling in mediating multidrug resistance in lung cancer through enhanced translation of antiapoptotic proteins, such as BCL-XL and XIAP. Here we investigate the downstream mediator(s) of this translational response and demonstrate its relevance in a lung cancer tissue array.

Methods
We used tandem affinity purification using S6K2 as bait as well as quantitative phospho-proteomics in the presence and absence of FGF2 stimulation in small cell lung cancer and HEK 293 cells to identify new S6K2 interactors and downstream mediators of the FGF2 pathway. Interactors were validated by co-immunopreciptation experiments and their functional significance examined by siRNA and expression of wild-type or phosphomutant forms. The clinical significance of our in vitro findings were examined in lung cancer tissue arrays.

Results
Here, we show that S6K2 interacts with and phosphorylates the heteronuclear ribonuclear protein hnRNPA1 on Ser 4 and 6. This increases the association of this protein with BCL-XL and XIAP mRNAs to promote their nuclear export while de-repressing their translation. A non-phosphorylatible S4/6A hnRNPA1 mutant prevented this process from occurring and impaired the pro-survival activity of FGF2/S6K2 signalling. Following phosphorylation and transfer to the cytoplasm in complex with mRNAs, phospho-hnRNPA1 associates with 14-3-3 to be sumoylated on K189 within a multi-protein complex involving UBC9. This targets hnRNPA1 for re-import into the nucleus in a caryopherin-dependent manner, a step that is essential for translational derepression of target mRNAs. Our in vitro findings predicted that in cancer cells where FGF2/S6K2 signalling is switched on, hnRNPA1 would be predominantly localised to the nucleus and cytoplasmic expression of anti-apoptotic proteins such as BCL-XL would be increased. To test this hypothesis, we stained a NSCLC tissue array for S6K2, hnRNPA1 and BCL-XL expression. Strikingly, this revealed that increased S6K2 expression tightly correlated with decreased cytoplasmic hnRNPA1 and increased BCL-XL levels.

Conclusion
FGF-2/S6K2 signalling promotes the nucleo-cytoplasmic cycling of hnRNPA1 to promote tumour cell survival through increased translation of the anti-apoptotic proteins BCL-XL and XIAP. Our immunohistochemical findings in NSCLC suggests that tumours which show absence of cytoplasmic hnRNPA1 in the presence of increased S6K2 and Bcl-XL expression might respond better to FGF receptor inhibitors.

Background
Tumor-derived microvesicles (TMV) contain bioactive molecules including proteins, RNA, microRNA, and DNA, and shed TMV propagate the horizontal transfer of their cargo in tumor microenvironment. TMV play important roles in cancer biology such as tumor progression, angiogenesis, escape from immune surveillance, metastasis and drug resistance. In this study, we investigated whether T790M EGFR mutation-related gefitinib resistance could be transferred via TMV using lung cancer cells harboring exon 19 del mutation (PC-9 cells) ± exon 20 T790M mutation (PC-9/GR cells).

Methods
TMV were isolated from the culture medium by serial centrifugations. Isolated TMV were checked by electron microscopy for size and intact structure, and characteristic TMV markers (CD81 and ezrin) were verified by western blot analysis. Cytotoxic assay was done using XTT assay and EGFR mutation testing was done using PNA-clamping method.

Results
Incubation of PC-9 cells with TMV (40 μg/ml) derived from PC-9/GR cells confer PC-9 cells gefitinib-resistant. EGFR genotyping of TMV in both cells revealed as the same with cellular DNA and this means that TMV carry cell-specific DNA. Passage study after PC-9/GR cell derived TMV transfer showed, however, regained gefitinib sensitivity suggesting that TMV-mediated gefitinib resistance is phenotypically transient. Genotyping after T790M-containing TMV revealed EGFR WT.

Conclusion
T790M-associated gefitinib resistance in lung cancer cells might be horizontally transferred via TMV but this type transfer of gefitinib resistance was phenotypically transient in cell-line system and there was no genotype transfer. Further investigation for TMV-mediated transfer of gefitinib resistance is necessary.

Background
Lung adenocarcinoma (AC) and squamous cell carcinoma (SCC) tumours have a large variance in tumour cell content. This heterogeneity is a concern for genomic studies, as it is difficult to distinguish mutational differences between tumour and non-tumour if low percentage tumour is used for analysis. In addition to this, tumour samples are affected by the amount of necrosis present, as the overall number of viable cells is decreased. We assessed tumour and necrotic content in lung tumour specimens from AC and SCC patients and aimed to identify possible implications for the suitability of these samples in molecular characterisation studies using next generation sequencing technology.

Methods
Lung tissue specimens were collected during the period of 1990 to 2013 from patients at The Prince Charles Hospital who consented to donate their surgically resected lung tissues for research. Tissues were macroscopically dissected, snap frozen in liquid nitrogen and stored at -80°C. A tissue section was taken and stained with haematoxylin and eosin (H&E) for two pathologists to independently assess tumour cell and necrotic content. Tumour cell content (TC) in each specimen was scored as percentage of viable cells as seen on the H&E slide, where necrotic content (NC) was recorded as a percentage of the whole slide section. Statistics were calculated using SPSS v21 software. Tumour specimens screened for eligibility to The Cancer Genome Atlas sequencing project are presented here.

Results
Tumours from 62 AC and 104 SCC subjects were scored (specimen characteristics in Table 1). Scoring between the two pathologists was highly correlated, with a high intraclass reliability (0.94 and 0.96 for TC and NC respectively).

Table 1: Clinical and Pathological Characteristics of Specimens

	AC	SCC
Number of Specimens	384	609
Number of Males/Females	36/26	84/20
Median Specimens per Subject	4	4
Range of Specimens per Subject	1-25	1-27
Median TC	35%	30%
Range of TC	0-88%	0-90%
Median NC	0%	6%
Range of NC	0-90%	0-100%
Median Age	62 yrs	68 yrs
Range of Age	45-85 yrs	46-91 yrs
Median Smoking Pack Years	40	56
Range of Smoking Pack Years	0-115	0-158

TC varied from 0-~90% for both subtypes. Comparing AC and SCC, the median TC was higher in AC than SCC (35% vs 30% respectively, p<0.05). NC varied from 0-~100%, but was generally low. The median NC was statistically significantly different between AC and SCC (0% and 6% respectively, p<0.001). TC was weakly correlated with NC (Spearman Rank r = 0.32, p<0.01). There were no clinically important correlations between smoking pack years, gender or age with TC and NC of specimens.

Conclusion
Lung AC and SCC specimens are heterogeneous in terms of TC and NC. Therefore, only a small proportion of resected lung cancer specimens meet the criteria required for massively parallel sequencing projects that require high quality tumour DNA and RNA (ie low NC) and relatively low stromal contamination (ie high TC).

Background
PI3K pathway activation in NSCLC has been shown by us and others to lead to a more aggressive disease correlating to poor prognosis for patients. Unfortunately, the success of PI3K targeted inhibition has been hampered by a high rate of innate and acquired resistance. Mutations in KRAS and B-RAF, ERK hyperactivation as well as extensive PI3K-MEK pathway cross-talk allow the MEK pathway to provide a bypass track. Preclinical studies demonstrate a rationale for a PI3K-MEK co-targeted treatment strategy which may provide a more effective response. A Phase I clinical trial is underway investigating the combination of GDC-0941, a pan-PI3K inhibitor, with GDC-0973, a MEK inhibitor. GDC-0980 is a dual PI3K-mTOR inhibitor which may offer improved pathway inhibition compared to GDC-0941. No data has been published to date on the combination of GDC-0980 and GDC-0973, which we believe may offer improved overall inhibition of survival signaling in NSCLC cells. We aim to elucidate the role of mutation status in response to this co-targeted inhibition approach in vitro, as well as investigating the frequency of PI3K and MEK pathway mutations in a well characterized Irish NSCLC patient cohort.

Methods
The effects of GDC-0941, GDC-0980 and GDC-0973 on proliferation and apoptosis in a panel of four NSCLC cell lines were analysed by BrdU Assay and HCA Apoptosis Assay, respectively. The four cell lines investigated were H460 (adenocarcinoma, PIK3CA mutant & KRAS mutant), A549 (adenocarcinoma, PIK3CA wild type & KRAS mutant), H1975 (adenocarcinoma, PIK3CA mutant, KRAS wild type & EGFR TKI resistant) and SKMES-1 (squamous cell carcinoma, PIK3CA wild type & KRAS mutant). Further investigation involved expression analysis of pAkt, pGSK-3β, pp70S6K, pS6RP, ERK and pERK in cell lines treated with each inhibitor alone or in combination using Mesoscale technology and Western blot. DNA was extracted from 120 NSCLC patient tissue samples, and screened for 547 mutations in 59 genes (including PI3K and MEK pathway members) using the Sequenom.

Results
GDC-0941 and GDC-0980 treatment induced dose-dependent anti-proliferative and pro-apoptotic responses across all four NSCLC cell lines, while GDC-0973 treatment induced only anti-proliferative responses. Protein expression analysis showed that GDC-0980 & GDC-0973 combination treatment induced significantly improved phosphoprotein inhibition compared to treatment with either inhibitor alone in cell lines harbouring PIK3CA mutations, while in one cell line bearing WT PIK3CA (SKMES-1), combination treatment actually increased pathway signalling. NSCLC patient mutational profiling data will be presented.

Conclusion
This research underpins the importance of mutation status in sensitivity to targeted therapies. While combination treatment approaches may be beneficial in certain molecular subtypes, in others they may be detrimental. In the era of personalised medicine, patient genotyping is crucial to improve patient survival and reduce toxicities.

Background
Malignant pleural mesothelioma (MPM) is a rare aggressive cancer of the pleura. Asbestos exposure (through inhalation) is the most well established risk factor for mesothelioma. The current standard of care for patients suffering from MPM is a combination of cisplatin and pemetrexed (or alternatively cisplatin and raltitrexed). Most patients however, die within 24 months of diagnosis. New therapies are therefore urgently required for this disease. Lysine acetyltransferases (KATs) including KAT5 have been linked with the development of cisplatin resistance. This gene may therefore be altered in MPM and could represent a novel candidate target for intervention

Methods
A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of KAT5 variants by RT-PCR. Levels were subsequently examined in a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies by RT-PCR. The effects of a small molecule inhibitor of KAT5 (MG-149) on cellular proliferation were examined.

Results
We show that the expression of KAT5A is dramatically increased in MPM. When separated according to histological subtype, KAT5A was significantly overexpressed in both the the sarcomatoid and biphasic subgroups for all transcript variants. Treatment of cells with the small molecule MG-149 caused significant inhibition of cellular proliferation (p<0.0001). We continue to asess this compound by other methodologies to confirm its potential utility in the treatment of MPM.

Conclusion
KAT5, a lysine acetyltransferase associated with cisplatin resistance in cancer is significantly altered in MPM. A small molecule inhibitor of this protein shows significant anti-proliferative effects in MPM cell lines. Targeting this protein may have important future implications for the management of MPM.

Background
Background: The PI3K/AKT signaling pathway is aberrantly active and has an important biologic impact in malignant pleural mesothelioma (MMe) cell cycle progression and chemo-resistance. Akt consists of three isoforms, Akt1, Akt2, and Akt3. Despite the growing amount of research demonstrating the existence of isoform-specific regulation, many papers still draw generalized conclusions about AKT without focusing on functional specificity of each isoforms. Recent data have clearly demonstrated a role of SIRT1 in the modulation of AKT1 activation and a role of PARP1 as a gatekeeper for SIRT1 activity by limiting NAD+ availability.

Methods
Methods: We explored the expression of AKT isoforms in MMe in vivo and in vitro and the balancing between their acetylation and phosphorylation status in human MMe derived cell lines in vitro.

Results
Results: We firstly described that MMe tumors in vivo and MMe derived cell lines express both AKT1 and AKT3 isoforms but not AKT2, and their expression results significantly increased in the biphasic histotype. Furthermore, we demonstrated an inverse correlation between AKTs acetylation and phosphorylation modulated by PARP1/SIRT1 activation status. By immunoprecipitation experiments, we evidenced that in basal conditions AKT1 is in part acetylated and in part phosphorylated and became highly phosphorylated and completely de-acetylated upon PARP1 inhibition. Interestingly, AKT1 activation, related to PARP1 inhibition, is unable to modulate pro-survival signals, because the downstream pathway is interrupted at the level of its effector mTOR. Conversely, SIRT1 inhibition or silencing result in a more evident AKT1 and its interactors acetylation.

Conclusion
Conclusions: In conclusion, our results clearly show how both PARP1 and SIRT1 affect critical cellular pathways involved in MMe progression and offer a model of a regulatory inter-relationship between these proteins. These data could be helpful for designing new effective therapeutic strategies.

Background
REV3, the catalytic subunit of the translesion synthesis (TLS) polymerase x, can continue replication past DNA adducts. Depletion of REV3 sensitizes A549 lung cancer cells to cisplatin. REV3 expression is part of a gene signature that predicted pemetrexed sensitivity in 17 NSCLC cell lines. BRCA1, TS, AEG1 and RAP80 are involved in DNA damage repair through homologous recombination. The homologous recombination and TLS pathways have non-redundant functions in response to cisplatin. We hypothesized that low mRNA expression of these genes – either alone or in combination – could confer improved outcome to cisplatin/pemetrexed in NSCLC patients.

Methods
REV3, BRCA1, RAP80, TS and AEG1 mRNA expression was examined by quantitative RT-PCR and categorized by terciles. Expression of each gene was correlated with outcome in 47 cisplatin/pemetrexed-treated NSCLC patients.

Results
63.8% male; 47% smokers; 80.9% ECOG PS 1; 80.8% adenocarcinoma. Overall response rate was 51%, with no differences according to expression levels of any of the genes. Progression-free survival (PFS) for patients with low, intermediate and high BRCA1 levels was 13.4, 5.5 and 3.9 months (m), respectively (P=0.005). Similar differences in PFS were observed according to TS (P=0.003) and AEG1 (P<0.001) expression. The hazard ratio (HR) for PFS for patients with high BRCA1 levels was 4 (P=0.002). Overall survival (OS) for patients with low, intermediate and high BRCA1 levels was 29.7, 7.4 and 6.3 m, respectively (P=0.05). Similar differences in OS were observed according to TS (P=0.005) and AEG1 (P=0.001) expression. HR for OS for patients with high BRCA1 levels was 3.6 (P=0.004). There were no differences in PFS or OS according to REV3 or RAP80 levels. However, the joint effect of BRCA1 and REV3 was significant for predictive modeling. PFS for patients with low, intermediate and high levels of both genes was 14.9, 7.2 and 2.8 m, respectively (P=0.001). OS for patients with low, intermediate and high levels of both genes was 29.7, 7.8 and 6.3 m, respectively (P=0.04).

Conclusion
Low BRCA1 expression predicts longer PFS and OS in pemetrexed/cisplatin-treated NSCLC p. Low TS and AEG1 levels have similar predictive value. The combination of low BRCA1 and REV3 expression confers longer PFS and OS. Analysis of these genes could be useful for customizing pemetrexed/platinum chemotherapy.

Background
Background: Small cell lung cancer (SCLC) is notoriously a highly fatal disease, with rapid emergence of resistance to first-line chemotherapy. Arsenic trioxide (ATO) has been used as a standard treatment for acute promyelocytic leukemia for the past decade. Our recent data have demonstrated in vitro activity of ATO, with clinically relevant concentrations, in SCLC cell line model. Although induced oxidative stress has been implicated as a possible mechanism of cytotoxicity, the exact mechanism of action of ATO in SCLC is not fully elucidated. In this study, we further explore the potential genetic changes and biological properties of acquired resistance to ATO in SCLC.

Methods
Methods: Using H841 SCLC cell line as a model, a daughter cell line resistant to ATO (H841AR) was established by culturing H841 cells in medium with progressively increasing concentrations of ATO for 12 months. Resistant clones were then selected and cultured in HITIS medium containing 20 μM ATO. Total RNA was extracted from both H841 and H841AR cells, followed by reverse transcription and hybridization with Affymetrix EXON 1.0 ST array. Gene chip signals were analyzed by Gene Spring 12 software. Cell proliferation (MTT) assay, wound healing (migration) experiment and invasion assay were performed to compare between H841 and H841AR cells.

Results
Results: Exponential growth of H841AR cells was shown in HITIS medium with 20 μM ATO. Comparing with H841 cells, 17 genes were upregulated by at least 5-fold in H841AR cells, consisting of stress response factors, immune system regulators and proliferation-associated or transcriptional factors. Similarly, 45 genes were downregulated by more than 5-fold in H841AR compared with H841 cells, mainly involving angiogenesis, signal transduction and neurodifferentiation. Apart from resistance to ATO (resistant index (RI) = 12.5 comparing H841AR cells with H841 cells), H841AR cells were also slightly resistant to cisplatin (RI = 2) compared with H841 cells. H841AR cells demonstrated faster proliferation, with greater migration and invasion properties.

Conclusion
Conclusion: There are distinct genetic alterations and biological properties in SCLC cell line (H841AR) with acquired resistance to ATO. Further analysis of the functional significance of various genetic alterations in ATO-resistant SCLC cell line is warranted. This study is partly funded by the CRCG Small Project Funding from the University of Hong Kong

Background
Recent studies in our laboratory show that tumor cells exposed to progressively higher concentrations of DETA-NONOate grow more aggressively than the parent cells. The currently accepted tumor stem cell model proposes that rare tumor stem cells produce both tumor cells (TCs) and rare tumor stem cells (TSCs). A number of TSC markers have been proposed, including CD antigens expression, Hoerscht 33342 exclusion, ALDH1/2 expression, and ALDH1/2 activity. In this study, we compare the TSC-like properties of parent and HNO cells. We hypothesized that human lung tumor cells adapted to high nitric oxide (HNO) levels will exhibit enhanced tumor stem cell-like properties relative to analogous cells not adapted to NO (“parent” cells).

Methods
The human lung adenocarcinoma cell line A549-Parent and the previously reported A549-HNO adapted cell lines were used in these studies. Immunohistochemistry was used to measure the expression of ALDH1/2 and six different CD antigens. Gene expression was measured using chip microarray analysis. FACS analysis was used to measure the population of H33342-positive and H33342-negative cells. ALDH activity was assessed using the ALDEfluor assay.

Results
The ALDEFLUOR assay demonstrated that ALDH activity of A549-HNO has increased compared to that of A549-parental. Relative to the A549-parental cells, A549-HNO cells showed markedly higher H33342-exclusion in the FACS analysis and greater ALDH1/2 expression in the immunostaining studies. Shocking these cells with 700 mM NO donor prior to treatment produced even more pronounced stem cell-like properties, suggesting that NO might be able to influence TCs into becoming more TSC-like. Gene chip results show that CD44, ALDH1A1, and ALDH2 are more highly expressed in A549-HNO cells than in A549-Parent cells. Cytospin results showed that ALDH1/2, CD44, and CD133 were more highly expressed in the A549-HNO cells, relative to A549-Parent cells.

Conclusion
The TSC markers tested in this study are consistent with the A549-HNO cells being more TSC-like than the A549-Parent cells. Having a large population of TSC-like cells would allow for improved drug screening of therapeutic candidates specifically designed to target TSCs.

Background
Hypothesis: Tumor stem cells (TSCs) are not rare cells and tumor cells TCs) have the plasticity to become TSCs. Objectives: Currently the rate limiting step in these efforts is the use of fluorescence-activated cell sorting (FACS) instrumentation to physically sort TSCs from TCs. We have shown that tumor cell lines could be adapted to comparatively high Nitric Oxide (NO) levels and that these cells had properties similar to TSCs. These cells were tested for various TSC properties using cellular, immunological, and molecular biology methods which included ALDH expression, ALDH activity, CD antigen expression, and H33342 dye exclusion. Proving that these are in fact TSCs would mean that we can produce large quantities of these cells to study. In a similar fashion, proving that TCs can become TSCs, would be very important.

Methods
We used the A549 and several Head & Neck SCC cell line in this study. They were FACS sorted five to seven times consecutively using H33342, to remove any chance of TSCs being present. The resulting TCs were cultured, and retested using H33342 at various times (several days to several weeks), for the presence of TSCs. The cells were cultured with or without NO.

Results
The FACS-sorted “pure” TCs, after being cultured, resulted in a TSC population re-immerging within days.

Conclusion
Collectively, these data support the idea that TSCs are not rare cells and that NO can drive TC populations to express properties of TSCs. This plasticity means that any TC can become a TSC.

Background
Malignant mesothelioma (MM), strongly associated with exposure to asbestos, is a growing worldwide problem (1). This aggressive tumour is largely resistant to oncological treatments and new approaches to therapy are urgently needed. Integrins are a class of adhesion molecules composed of an α and a β chain. Combinations of 18 α and 8 β subunits form the 24 members of the integrin family. The αv subunit can dimerize with β1, β3, β5, β6 and β8. Aberrant expression of αv integrins was reported in MM, and the integrins αv β3 and αv β5 have been implicated in tumour progression and metastasis. We have investigated the expression and function of αv integrins in MM cell lines and the effect of gene knockdown on cell invasion.

Methods
Expression of the integrin (ITG) genes was analysed by qPCR in 7 MM cell lines. Expression of the heterodimers was determined by Western blot, immunofluorescence and immunocytometry (monoclonal antibodies kindly provided by Simon Goodman, 2). In addition, we knocked down the genes potentially involved in tumour progression (ITGB3 and ITGB5) and analysed the in vitro 2D and 3D invasiveness with an agarose spot invasion assay (3) and MM spheroids.

Results
All 7 MM cell lines showed high ITGB1 expression, moderate ITGB5 expression, and a general low ITGB6 and ITGB8 expression. ITGB3 was expressed in one cell line, which accordingly had high αv β3 expression. ITGB3 knockdown of this cell line resulted in suppression of invasion both in 2D and 3D cultures.

Conclusion
We have found evidence that integrin αv β3 may play a role in MM invasion. Presently, we are testing cilengitide, a peptide antagonist of integrin αv, in MM cell lines. References: 1. Delgermaa F, Takahashi K, Park EK, Le GV, Hara T, Sorahan T. Global mesothelioma deaths reported to the World Health Organization between 1994 and 2008. Bull World Health Organ. 2011, 89:716-724. 2. Goodman SL, Grote HJ, Wilm C. Matched rabbit monoclonal antibodies against αv-series integrins reveal a novel αvβ3-LIBS epitope, and permit routine staining of archival paraffin samples of human tumors. Biol Open. 2012, 1:329-340. 3. Wiggins H and Rappoport J. An agarose spot assay for chemotactic invasion. Biotechniques. 2010, 48:121-124.

Background
Lung cancer remains the most common cause of cancer-related death in the world. The major advances in treatment of lung cancer have brought only minor improvements in survival; therefore novel strategic treatment approaches are urgently needed. Accumulating data allocate a central role for the cancer microenvironment including mesenchymal stem cells (MSCs) in acquisition of drug resistance and disease relapse. Therefore, we decided to study the effect of bone marrow (BM) MSCs on non small cell lung cancer (NSCLC) cell lines. Recent studies indicate that translation initiation factors are over expressed in multiple cancers and negatively impact NSCLC prognosis. Interestingly, translation initiation is highly modulated by microenvironmental cues. Hence, special emphasis will be attributed to the role of translation initiation in the crosstalk between the malignant lung cells and the BM-MSCs.

Methods
Specifically, we will determine the influence of MSCs (co-culture) and MSCs conditioned media on the proliferation, viability, migration of NSCLC cell lines (A549, H1299, and H460). We will also explore the effect on translation initiation factors implicated in cancer progression (eIF4E, eIF4GI, DHX29).

Results
Preliminary results demonstrate that 48h co-culture of NSCLC cell lines with MSCs conditioned media significantly decreased cell number (A549: 60%, H1299: 50%, H460: 10%, p<0.05) and viability (A549: 70%, H1299: 50%, H460: 60%, p<0.01) yet had no effect on cell cycle or death. Moreover, MSCs conditioned media decreased NSCLC cells motility (H1299: 60%, H460: 70%, p<0.05) and attenuated eIF4GI expression and activity as indicated by the levels of its targets (H1299: peIF4G 65%, total eIF4G 40%, SMAD5 30%, cMyc 80%; H460: peIF4G 90%, SMAD5 85%, cMyc 40%, p<0.05).

Conclusion
Ongoing work in our lab is aimed at dissecting whether the effects are dependent on direct cell-cell contact or mediated by soluble factors. Results that indicate translation initiation is modulated by MSCs derived components will mark new therapeutic targets for lung cancer treatment.

Background
A3 adenosine receptor mediates apoptosis in cancer cells via diverse signaling pathways. The present study examined A3 adenosine receptor-mediated apoptosis in Lu-65 cells, a human giant cell lung carcinoma cell line.

Methods
MTT assay, TUNEL staining, real-time RT-PCR, Western blotting, and assay of caspase-3, -8, and -9 activities were carried out in Lu-65 cells, and A3 adenosine receptor or p53 was knocked-down by transfecting each siRNA into cells.

Results
Extracellular adenosine induces Lu-65 cell apoptosis in a concentration (0.01-10 mM)-dependent manner, and the effect was inhibited by the A3 adenosine receptor inhibitor MRS1191 or by knocking-down A3 adenosine receptor or p53. Like adenosine, the A3 adenosine receptor agonist 2-Cl-IB-MECA also induced Lu-65 cell apoptosis. Adenosine upregulated expression of p53 and Noxa mRNAs and activated caspase-3 and -9, but not caspase-8. Those adenosine effects were still inhibited by knocking- down A3 adenosine receptor or p53.

Conclusion
The results of the present study show that adenosine upregulates p53 expression via A3 adenosine receptor, to promote p53-dependent Noxa gene transcription, causing activation of caspase-9 and the effector caspase-3 to induce Lu-65 cell apoptosis.

Background
Recently, a novel fusion gene resulting from linkage between the kinesin family member 5B gene (KIF5B; 10p11.22) and rearranged during transfection gene (RET; 10q11.21) was identified in non-small cell lung cancer (NSCLC). RET translocation was previously reported in thyroid cancer, as CCD6/RET translocation. However, the correlation between KIF5B/RET fusion gene status and clinicopathologic features of surgically-treated lung cancer has not been well characterized.

Methods
We have investigated KIF5B/RET fusion gene status in 371 surgically treated NSCLC (270 were adenocarcinoma and 101 were squamous cell carcinoma), 60 breast cancers, 11 metastatic lung cancers from colon cancers and thyroid papillary adenocarcinoma case at Nagoya City University Hospital. The fusion gene and CCD6/RET statuses were analyzed by RT-PCR based assay and direct sequencing. We have performed immunohistochemical (IHC) analysis using C-ternimal specific anti RET antibody (EPR2871, 1:250) (Epitomics Inc., Burlingame, CA, USA, n=86) with Dako linker kit using intercalated antibody-enhanced polymer (iAEP) method. Cytoplasm was stained either granular (G1) or diffuse (G2). G2 staining was defined as positive staining.

Results
We detected 3 of 270 cases of KIF5B/RET fusion genes in adenocarcinomas (1.5 %) from the present study; all were mixed subtype adenocarcinomas and three were female and never-smokers. The fusion genes were exclusive with the other mutations, such as EGFR, Kras, Braf, erbB2 mutations, and EML4-ALK fusion. KIF5B/RET fusion was not detected in the cases with squamous cell carcinoma or other types of cancers. Of the 3 cases, 2 cases were KIF5B (exon15): RET (exon12) fusions with papillary dominant and 1 cases were KIF5B (exon22): RET (exon12) fusion with solid dominant adenocarcinoma. Matched normal lung tissues did not show translocation. In the present study, we did not detect CCD6/RET fusion genes. as a driver somatic mutation of lung adenocarcinomas. Although all 3 had positive IHC staining, 35/86 had more than 10% staining and 15/86 had more than 50% staining.

Conclusion
In the present study, we reported KIF5B/RET fusion genes as a possible new driver somatic mutation of lung adenocarcinomas. Cinico-pathological backgrounds of the KIF5B/RET fusion positive patients were similar with that of the EML4/ALK fusion positive patients. The chimeric oncogene might be as a promising molecular target for the personalized diagnosis and treatment of NSCLC. However, the chimeric oncogene might not be determined using IHC analysis.

Background
Small cell lung cancer (SCLC) accounts for 13-15% of new lung cancer cases in worldwide and has the poor therapeutic outcomes with a median survival of just over one year. A CpG island methylate phenotype (CIMP) is well known as a methylator phenotype with characteristic promoter DNA methylation alterations, in colorectal cancers, glioblastoma and breast cancers, although there has been no report about any CIMP of SCLC. We investigated whether DNA methylation profiles can provide useful molecular subtyping of SCLC in terms of etiology and prognosis of SCLC.

Methods
We analyzed 28 fresh frozen samples from pure SCLC patients and 13 noncancerous lung tissues. All patients underwent surgical lung resection at the Cancer Institute Hospital, nine patients among them were treated with chemotherapy before surgery. After genomic DNA was treated with sodium bisulfite, bisulfite-converted genomic DNA was analyzed using Illumina’s Infinium HumanMethylation27 BeadChip. And, total RNA was extracted from twenty-five SCLC tumor samples and mRNA expression of these samples were analyzed by Agilent’s SurePrint G3 Human CGH Microarray. Next, we matched these two data sets by Gene Symbol, and identified fifty-five most differentially methylated CpG sites (corresponding to 46 genes) with a FDR p value cut off of 0.05. Gene ontology analysis was performed using DAVID Bioinformatics Resources.

Results
We selected a total of 1741 most differentially methylated CpG sites (s.d. > 0.20) across the 28 SCLC tumor tissues in each DNA methylation platform, after an elimination of the probes related with the X- and Y- chromosome. Unsupervised hierarchical clustering of methylation data from SCLC samples reveals two major subgroups with different prognosis: the 5 years disease-free interval (DFI) rate of patients in cluster 1 (11.1%) was lower than that of patients in cluster 2 (61.57%) (p = 0.001). By multivariate analysis for DFI, both postoperative chemotherapy and cluster 1 were a significant prognostic factor (p = 0.002 and 0.002; respectively). Next, among 1220 genes with methylation and expression data both available, the CpG sites were ranked on the basis of the spearman’s correlation coefficient between cluster 1 and cluster 2 into an ascending order. Finally, we identified that fifty-five CpG sites were nagetively correlated and found that apoptosis pathway was a most differentially expressed.

Conclusion
By comprehensive DNA methylation profiling, two distinct subgroups with different molecular and clinical phenotype were identified to evoke a CIMP of SCLC. We found some promoter markers in the apoptosis pathway could make a difference between the two groups, and we hope that our data can contribute to provide a useful resource for the construction of therapeutic strategy and the development of a new chemotherapeutic agent.

Background
Genetic variability can influence the pharmacokinetics and pharmacodynamics of paclitaxel and carboplatin in patients with non-small cell lung cancer (NSCLC). Additionally, the prevalence of genetic variations often differs between ethnic groups and may account for observed interethnic variability in drug efficacy. We aimed to undertake a pharmacogenomic investigation to account for patient variability in order to improve dosing and patient selection in NSCLC.

Methods
76 advanced NSCLC patients from Caucasian (n = 50) and Asian (n = 26) ethnicity were prospectively recruited into the study at Concord Repatriation General Hospital from 2007-2011. All patients received paclitaxel (175mg/m[2]) and carboplatin (target AUC 6 mg/mL•min) for an intended 6 cycles. A candidate gene approach was used to select single nucleotide polymorphisms (SNPs) for pharmacogenomic analysis. HPLC with population pharmacokinetic modelling (NONMEM) was used to obtain pharmacokinetic data (n = 61). Toxicities were assessed according to CTCAE v 4.0 and response was measured by CT scans according to RECIST criteria. Blood DNA was genotyped by the Australian Genetics Research Facility using a MassARRAY® iPLEX Gold system on a Sequenom mass spectrometer. SNPs were assessed for linkage disequilibrium, Hardy-Weinberg equilibrium and interethnic differences in allele frequency. Regression analysis was undertaken to associate SNPs with drug pharmacokinetics, toxicities and response.

Results
42 SNPs from 21 genes were selected and genotyped in patients. Regression analysis identified 12 SNPs significantly associated to paclitaxel pharmacokinetics, patient toxicities and response. SNPs rs2306283 and rs11615, in SLCO1B1 and ERCC1 respectively, associated with paclitaxel clearance. Previously published SNPs in CYP2C8, CYP1B1, ABCB1, ABCC10, SLCO2B1, GSTP1, ATP7A, ERCC1 and CCND1 were found to be associated with patient toxicities. Three SNPs, rs2306283, rs1056836 and rs2306168, encoded by SLCOB1, CYP1B1 and SLCO2B1 respectively, had interethnic difference in SNP prevalence and associated to patient outcomes. SNP rs2227291 in ATP7A associated with patient response and to nausea and anaemia.

Conclusion
The study associated various SNPs to drug pharmacokinetics, patient toxicities and response which could be integrated into future personalisation efforts of paclitaxel and carboplatin. Furthermore, SNPs with interethnic differences may provide clues to understand interethnic variability in drug efficacy. Finally, a novel SNP identified in ATP7A associated with patient response and toxicity. The ATP7A gene encodes a protein that has been linked to resistance to platinum based drugs, however, the SNP has unknown functional effects which require further elucidation.

Background
Malignant pleural mesothelioma (MPM) is a highly aggressive tumor caused by asbestos exposure after a long latency. The neurofibromatosis type 2 (NF2) tumor suppressor gene is mutated in around 50% of MPM cases, which encodes Merlin that regulates the Hippo tumor-suppressive signaling pathway. We previously reported occasional genetic alteration in the LATS2 and SAV1 genes in MMs, which encode components of the Hippo pathway. The LATS2 inactivation was shown to lead to constitutive activation of YAP, a prooncogenic protein and transcriptional coactivator, which enhances multiple cell cycle regulation genes including cyclin D1 (CCDN1). To further delineate the exact inactivation mechanism of this pathway in MMs, we analyzed MM cell lines for other multiple components that regulate activation or inactivation of this pathway.

Methods
We studied several new components that have been identified to be involved in the Hippo signaling pathway including a LIM protein Ajuba. Expression analyses such as conventional western blot and real time RT-PCR were performed. Using MPM cell lines and their transplanted mouse models, biological assays were conducted to study the effects of cell proliferation, motility and invasion after the induction of overexpression or knockdown of candidate genes. Immunohistochemical analysis with primary MPM tissues and xenografts was also carried out.

Results
We found that the expression of Ajuba was significantly down-regulated in 6 of 20 MM cell lines compared to an immortalized normal mesothelial cell line, MeT-5A. Interestingly, the 6 cell lines with low Ajuba expression showed decreased phosphorylation (activation) levels of YAP. We infected the MM cells with Ajuba-expressing lentivirus and found that exogenous Ajuba increased phosphorylation (inactivation) of YAP and inhibited cell proliferation. Dual-luciferase reporter assays demonstrated that Ajuba suppressed the promoter activities of YAP-transcriptional targets including CCND1. Knockdown of LATS2 effectively increased these promoter activities, suggesting the mediation of LATS2 for Ajuba to inhibit this cascade. Immunohistochemical analysis also showed frequent downregulation of Ajuba in primary MMs.

Conclusion
Inactivation of Hippo signaling is a key mechanism for MPM cell proliferation and progression. Our findings suggest that Ajuba is also one of the target components for Hippo signaling inactivation; Ajuba negatively regulates YAP in the presence of LATS2, and thus the downregulation of Ajuba serves to enhance constitutive activation of YAP in MM cells. Our study also indicates that a strategy to normalize this signaling cascade may be the rationale for developing a new target therapy against MPMs.

Background
Most of small cell lung cancer (SCLC) cases are diagnosed after metastatic spread of the diseases, and only 5% of SCLC patients survive beyond 5 years after diagnosis. Therefore, for the improvement of patients’ outcome in this disease, it is necessary to identify druggable targets that are activated by genetic alterations in SCLC cells.

Methods
To obtain a landscape of gross genetic alterations in SCLC, genome-wide copy number analysis and whole-transcriptome sequencing were performed in 58 and 42 SCLCs, respectively.

Results
Focal amplification of known oncogene loci, MYCL1 (1p34.2), MYCN (2p24.3), and MYC (8q24.21), was frequently and mutually exclusively detected. MYCL1 and MYC were co-amplified with other regions on either the same or the different chromosome in several cases. In addition, the 9p24.1 region was identified as being amplified in SCLCs without amplification of MYC family oncogenes. Notably, expression of the KIAA1432 gene in this region was significantly higher in KIAA1432 amplified cells than in non-amplified cells, and its mRNA expression showed strong correlations with the copy numbers. Thus, KIAA1432 is a novel gene activated by amplification in SCLCs. By whole-transcriptome sequencing, a total of 60 fusion transcripts, transcribed from 95 different genes, were identified as being expressed in SCLC cells. However, no in-frame fusion transcripts were recurrently detected in 2 SCLCs, and genes in the amplified regions, such as PVT1 neighboring MYC and RLF in MYCL1 amplicons, were recurrently fused with genes in the same amplicons or with those in different amplicons on either the same or different chromosome.

Conclusion
Amplification and fusion of several genes on chromosomes 1 and 8 were likely to occur simultaneously but not sequentially through chromothripsis in the development of SCLC. Amplification rather than fusion of genes was indicated to play an important role in its development.

Background
The epidermal growth factor receptor (EGFR) signaling pathway is involved in numerous biological processes including proliferation and apoptosis, migration/invasion, and angiogenesis, and has emerged as one of the most important and frequently deregulated pathways in NSCLC. The discovery of oncogenic, activating mutations in the tyrosine kinase domain of EGFR and DNA amplification of EGFR have led to the development of multiple targeted therapeutics against this pathway. While effective at prolonging survival, these targeted therapies are only applicable to a subset of patients (~15-20%) that harbour these alterations and resistance to treatment ultimately develops. As multiple genomic and epigenomic mechanisms can disrupt genes, a comprehensive understanding of the genetic alterations affecting genes within this pathway is required. An integrative, multi-dimensional genomics approach can detect genes disrupted by multiple mechanisms which may otherwise be overlooked if only a single genomic dimension were assessed, improving the ability to identify causal genetic events and decipher downstream consequences.

Methods
A multi-dimensional integrative analysis of copy number, DNA methylation and gene expression profiles on 77 adenocarcinomas and matched non-malignant tissue, was performed to investigate the complement of genetic alterations affecting the EGFR pathway. Novel candidate genes were validated in external datasets and immunohistochemical analysis of a tissue microarray was used to verify disruption at the protein level and to correlate expression with clinical features. The tumor suppressive effects of SIRPA were assessed by stable knockdown and in vitro assays on a panel of lung cancer cell lines. The effect of SIRPA downregulation on TKI sensitivity was assessed by dose response assays.

Results
Of the 35 genes examined, 11 were aberrantly expressed in over 50% of tumors, with 6 (RRAS, SIRPA, PIK3R1, TGFA, ERBB2 and EGFR) ranking in the 95th percentile of altered genes. Of these genes, all but SIRPA are known to be frequently disrupted in NSCLC and play a role in tumorigenesis. SIRPA is a transmembrane protein that negatively regulates receptor tyrosine kinsase activity and is frequently downregulated at both the mRNA and protein level in NSCLC tumors and cell lines. Underexpression of SIRPA is associated with EGFR mutations and is more prominent in adenocarcinoma than squamous cell carcinoma. Downregulation of SIRPA enhanced colony formation and wound healing but impaired viability and suppressed proliferation. Interestingly, SIRPA knockdown induced a senescent phenotype through the accumulation of p27 and Rb in its unphosphorylated state thereby blocking progression of the cell cycle. These results suggest senescence induced by SIRPA downregulation is a tumor suppressive mechanism that must be overcome to develop tumors.

Conclusion
Our integrative analysis of the EGFR pathway revealed SIRPA as one of the most frequently deregulated genes within the pathway. SIRPA functions as a tumor suppressor gene, controlling a number of biological functions through the inhibition of singaling pathways downstream of EGFR. To our knowledge, this is the first study to report a role for SIRPA in NSCLC tumorigenesis.

Background
MET is a tyrosine kinase receptor expressed on the cell surface of numerous cell types, including normal and malignant epithelial cells. MET is expressed on a proportion of non-small cell lung cancer (NSCLC) cases of adenocarcinoma and squamous-cell differentiation. In a phase II clinical trial evaluating the anti-MET monovalent antibody onartuzumab, patients with advanced MET-positive NSCLC – as determined by immunohistochemistry (IHC) – derived clinical benefit from onartuzumab and erlotinib. The prevalence of MET expression in two separate sample cohorts of NSCLC – vendor-procured and phase II clinical trial samples – is compared and correlated with MET gene copy number.

Methods
Formalin-fixed, paraffin-embedded NSCLC tissues were obtained from commercial sources or from the phase II clinical trial OAM4558g. Tumor histology was determined by morphology; IHC for TTF1 and p63, respectively; or both. MET IHC was performed with the SP44 rabbit monoclonal antibody (CONFIRM anti-Total c-MET) on a Ventana Benchmark XT platform. Patients were selected based on expression of MET by IHC as defined by moderate or strong staining in at least 50% of tumor cells (clinical score 2+/3+); MET gene copy number was determined by fluorescence in-situ hybridization (FISH) or by chromogenic in-situ hybridization (CISH) (INFORM® MET DNA probe, Ventana Medical Systems).

Results
Adenocarcinomas and squamous-cell carcinomas accounted for 46% (n=95) and 54% (n=110) of the vendor-procured samples and for 59% (n=75) and 28% (n=36) of clinical trial samples, respectively. In total, 16% (n=33) of vendor-procured and 52% of clinical trial samples were scored as MET-positive. The proportions of MET-positive cases among adenocarcinomas and squamous-cell carcinomas were 24% and 9% for vendor samples and 61% and 28% for clinical trial samples. FISH analysis of the OAM4558g clinical trial samples showed MET gene amplification in 8 of 96 cases (8%) with a high positive correlation to MET-positive IHC status; 6 of 8 gene-amplified cases were MET-positive by IHC (p<0.0001). Evaluation of MET copy number by CISH is ongoing and will be reported.

Sample Source	Clinical Trial OAM4558g		Vendor-Procured
Histology	Adenocarcinoma	Squamous-cell Carcinoma	Adenocarcinoma	Squamous-cell Carcinoma
Cases (n)	75	36	95	110
MET-positive (%)	61	28	24	9

Conclusion
These results demonstrate preferential MET expression for adenocarcinoma compared with the squamous-cell carcinoma subtype of NSCLC. The lower proportion of MET-positive cases in the vendor samples suggests that tissue quality is crucial for adequate MET assessment by IHC.

Background
Non-small-cell lung carcinoma (NSCLC) accounts for 85% of all malignant lung tumors. Our group previously identified Tripartite Motif-Containing 14 (TRIM14) as a component of a prognostic multigene expression signature for NSCLC patients. TRIM14 belongs to a conserved family of Tripartite Motif-encoding genes involved in a broad range of biological processes, such as transcriptional regulation, cell proliferation, and apoptosis. However, TRIM14 itself is poorly understood. Here we investigate the functional and prognostic role of TRIM14 in NSCLC.

Methods
Cox proportional hazards regression analysis was done on published mRNA expression datasets of primary NSCLCs to identify whether TRIM14 expression is associated with patient survival. The expression of TRIM14 was modified in human NSCLC cell lines (NCI-H1395, NCI-H1650, NCI-H520 and NCI-H157) by lentivirus-mediated overexpression and short-hairpin RNA (shRNA)-mediated silencing. Effects were assessed by examining in vitro cell proliferation and anchorage-independent growth, and in vivo tumorigenicity in mice.

Results
Univariate analyses demonstrated that TRIM14 expression is significantly associated with patient survival, with loss of expression correlated with poorer prognosis in resectable early stage NSCLC patients. In vitro studies showed that TRIM14 overexpression in H1395 cells suppressed proliferation and anchorage-independent growth, whereas shRNA knockdown of TRIM14 expression in H1650, H520, and H157 cells promoted cell proliferation and colony formation. In vivo studies demonstrated that suppressing TRIM14 expression in H1650 and H157 cells significantly promotes tumor growth in immunodeficient mice.

Conclusion
We show that TRIM14 may function as a tumor suppressor gene in NSCLC affecting cell proliferation and anchorage-independent growth in vitro and tumor growth in mice. We provide evidence that prognostic genes identified in microarray based gene expression analyses may have a strong biological rationale.

Background
Molecular cross-talk between EGFR and other signaling pathways creates alternative means of tumor cell proliferation and promotes resistance to single-agent erlotinib therapy in NSCLC driven by EGFR mutations. ROR1 knockdown inhibited the growth of NCI-H1975 cells (harboring EGFR L858R and T790M mutations). A pro-survival function for ROR1/MEK/ERK signaling in cooperation with AKT has been demonstrated. We have assessed ROR1 expression in 45 patients from the EURTAC trial (clinicaltrials.gov NCT00446225), 27 of whom harbored pretreatment concomitant EGFR T790M mutations, and correlated results with outcome.

Methods
ROR1 mRNA expression was examined by quantitative RT-PCR and categorized by terciles; patients were classified as having low/intermediate or high ROR1 expression. The T790M mutation was determined by Taqman with a PNA to inhibit amplification of the wild-type (wt) allele. Tumor samples were run in octuplicates; this method can detect 1 mutated allele among 10,000 wt alleles.

Results
Median age 65; 68.9% female; 57.8% never-smokers; 95.6% ECOG PS <2; 91.1% adenocarcinoma; 68.9% exon 19 deletion. No differences in baseline characteristics were observed according to ROR1 expression levels. 24 patients (53.3%) were treated with erlotinib and 21 (46.7%) with chemotherapy. 10 (41.7%) erlotinib-treated patients and 6 (28.6%) chemotherapy-treated patients had high ROR1 mRNA levels. Among erlotinib-treated patients, response rate (RR) was 40% for the 10 patients with high ROR1 levels vs 71.4% for the 14 with low/intermediate levels (P=0.058). Among chemotherapy-treated patients, RR for the 15 patients with low/intermediate ROR1 levels was 6.7%; the 6 patients with high ROR1 levels did not respond. Progression-free survival (PFS) was 11.8 months (m) for erlotinib-treated patients with low/intermediate ROR1 levels vs 5.8 m for those with high levels. PFS for chemotherapy-treated patients was 5.6 and 9 m, respectively (P=0.0165). 15 erlotinib-treated patients harbored concomitant T790M mutations; for these patients, PFS was10.8 m for those with low/intermediate ROR1 levels vs 2.7 m for those with high levels (P=0.0138).

Conclusion
ROR1 expression has a differential effect on outcome to erlotinib and chemotherapy in EGFR-mutant NSCLC patients. High ROR1 expression significantly limits PFS in erlotinib-treated patients with T790M mutations and ROR1-directed therapies can enhance the efficacy of treatment. In contrast, high ROR1 expression confers longer PFS to chemotherapy in the same group of patients. The role of chemotherapy and erlotinib in EGFR-mutant NSCLC patients with high ROR1 expression warrants further investigation.

Background
Cell transformation and tumour progression are associated with severe alterations of the epigenetic control of gene expression. Although the abnormal repression of tumour suppressor genes has been thoroughly investigated, the concept of tissue-specific gene aberrant reactivations in cancer is only starting to emerge. The extent of this process has not been reported yet, mainly because of the difficulties in detecting these expressions by the currently used transcriptomic analyses

Methods
We have developed a simple approach, exploiting genome wide expression data, which has enabled us to demonstrate that these "off-context" gene activations occur in any cancer, providing a universal source of cancer biomarkers. By applying this analysis to our series of lung cancer patients (n=297) with the corresponding precise clinical annotations and 5 to 10 years patient follow-up, we found that hundreds of germline-specific genes are ectopically expressed in the tumours and that a subset of 26 genes are specifically activated in a subgroup of highly aggressive metastatic-prone tumours, even at an early stage of the disease. This approach has enabled us to define and isolate a homogeneous group of very aggressive tumours called P3 with at least 3 genes ectopically expressed, independently of other prognosis parameters (histo-pathological or stage) (Rousseaux et al. Sci Transl Med 2013, 5 (186): 186ra66).

Results
Based on these data we setup a PCR based test to directly rank lung tumours by detection of the prognosis associated gene activations. This test was first validated on a series of 60 tumour samples from the same cohort (also analyzed by an affymetrix transcriptomic approach), which revealed the remarkable robustness of the PCR approach and showed a higher sensitivity of the PCR-based detections in tumour ranking. We then extended these PCR-based analyses to include additional patients with precise clinical and pathological annotations and 10 years follow-up but for whom transcriptomic data were not available. Here again we could show that our test successfully ranked the patients into groups with significantly different survival probabilities. we show here the intermediate analysis on a first group of 88 patients without lymph node metastasis treated by surgeryFigure 1

Conclusion
In conclusion we are ready to launch a prospective study based on this PCR test to evaluate its ability to predict tailored follow up of patients after surgery and also tumour sensitivity to design specific targeted therapies including immunotherapy since this ectopic activation may lead to very innovative treatment .

Background
Quantifying and comparing the degree of methylation in the promoter region of APC gene and coding region of FHIT gene in patients (p) with non small cell lung cancer (NSCLC) in different samples. Correlate these methylation patterns with clinical variables.

Methods
DNA was extracted from peripheral blood simples (B), sputum (S) and bronchial aspirate (BA), obtained from patients with NSCLC prior to the completion of surgery, as well as resected lung tumor tissue (TT) and normal lung tissue (TN). Methylation patterns were analyzed by bisulfito conversion and subsequent pyrosequencing (QIagen PyroMark System).We analyzed 5 CpG islands in the promoter region of the APC gene and 5 CpG islands in the coding region of the FHIT gene.

Results
We analyzed 20p, with a median age of 64 years (range 48-70), 16 men and 4 women. Smoking status: 2p never smokers, 11p former smokers, 7p current smokers. Histology: 10p adenocarcinoma, 9p squamous, 1p large cell. Stage: I 8p, II 8p, III 4p. No statistically significant differences were observed between the samples studied to any of the islands analyzed. The degree of methylation in TT CpG1 was higher in smokers and former smokers <5 years, compared to never smokers and ex-smokers >5 years, mean 4 (0-10) vs 0, p=0.022. There was no other difference when analyzing the degree of methylation as a function of the variables age, sex, smoking status, cumulative tobacco consumption, histological type and clinical stage. Respect FHIT gene, no statistically significant differences were found between the tissues studied respect to any of the CpG islands analyzed and like wise, no differences were observed when analyzed for degree of methylation depending on the clinical variables studied.

Conclusion
The area of ​​the APC gen promoter and FHIT coding region analyzed showed a low degree of methylation, with no significant difference observed between the samples studied. The degree of methylation in the TT CpG1 island of the APC gene was higher in current smokers and former smokers <5 years. However, these findings have to be confirmed in a larger sample. *This study was funded by the Carlos III Health Institute (PS09/00308), Spain.

Background
Lung cancer is one of the most common causes of cancer-related deaths worldwide. Effective early diagnosis and targeted therapies for lung cancer to reduce mortality and incidence would benefit from in-depth study on molecular mechanism of lung carcinogenesis, but these are largely unknown. In our previous study, we reported a novel hyper-methylated gene, CDO1, encoding cysteine dioxygenase 1 that involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In this study, we evaluated the functional characteristics of CDO1 in Lung squamous cell carcinoma (LSCC).

Methods
We analyzed expression levels of CDO1 mRNA and protein in tumor and normal tissue pairs from 12 LSCC patients were determined by RT-PCR and Western blot analysis. In order to determine whether high levels of CDO1 expression contributed to the LSCC cell proliferation, migration, invasion and colony formation, we employed a CDO1-expressing vector pEGFP_C3_CDO1 to transfect CDO1 into LSCC cell lines (HCC-95, HCC-1588). Furthermore, we performed gene expression profile analysis using human whole genome oligonucleotide chip (Agilent).

Results
Downregulation of CDO1 mRNA level was observed in LSCC cell lines and tumors derived from patient tissues. In cell proliferation assay, the number of HCC-95 and HCC-1588 cells transfected with pEGFP_C3_CDO1 decreased to 58-70% compared with pEGFP_C3 (p<0.05). Moreover, the forces expression of CDO1 in two different types of LSCC cell lines significantly decreased the cell migration, invasion, and colony formation ability (p<0.05). Furthermore, we showed that overexpression of CDO1 make change of several signal molecules in lung cancer cells using microarray analysis.

Conclusion
In this study, we found that CDO1 expression was regulated by DNA methylation, and this epigenetic regulated CDO1 might be a novel tumor suppressor gene and potentially valuable biomarker for lung squamous cell carcinoma.

Background
ATF2 is a member of the basic helix-loop-helix (b-ZIP) transcription factor family playing important roles in Stress and DNA damage. Several studies reported correlation between ATF2/JNK-mediated activation and resistance to damaging agents. Celastrol is an ATF2 inhibitor extracted from a Chinese plant known as “Tunder of God Vine”. Celastrol is used in traditional Chinese medicine for anti-inflammatory properties. In addition, Celastrol is a triterpene with promising anticancer activity in several cancer models both in vivo and in vitro. The purpose of the present study was to investigate whether ATF2 might play a role in inducing drug resistance in Non-Small Cell Lung Cancer (NSCLC).

Methods
In NSCLC cell lines ATF2 expression levels were evaluated by quantitative PCR (qPCR) and Western Blot (WB) and correlated to cisplatin (CDDP) resistance. Celastrol mediated ATF2/cJUN activity was assessed by Luminometry, qPCR and western blotting (WB). Furthermore, matched tumors and corresponding normal tissues of 88 surgically resected NSCLC specimens, were collected and both qPCR and immunohistochemical analyses for ATF2 were performed.

Results
NSCLC cell lines CDDP-resistant (H522 and H1395) expressed high levels of ATF2 protein. Moreover, CDDP treatment increased ATF2 phosphorylation levels leading to an enrichment within the nuclear cell compartment. In our study, Celastrol reduced ATF2 activity by decreasing the production of ATF2 mRNA and blocking the CDDP-mediated phosphorylation/mRNA expression of cJUN, a main ATF2 partner. Furthermore, ATF2/cJUN functional inhibition mediated by Celastrol restored the response to CDDP in resistant lung cell Lines. ATF2, at both protein and mRNA level, was significantly up-modulated in NSCLC tumor samples compared to the paired normal lung tissue (mRNA: p<<0.01, mean Log2(FC)=+4.7). Moreover, high expression of ATF2 mRNA was correlated with the smoking status of the patients. Relevantly, smoker or former smoker patients expressed significantly high ATF2 mRNA levels compared to non-smokers (p=0.02 and p=0.04, respectively).

Conclusion
This study shows that in NSCLC cell lines a correlation between ATF2 protein expression and CDDP resistance occurs. Furthermore, our results suggest a potential increase of CDDP sensitivity, following the Celastrol-mediated ATF2/cJUN inhibition. For the first time it has been shown in NSCLC an up-regulation of ATF2 mRNA/protein levels compared to normal tissues and consistent with that detected in CDDP resistant cell lines. This data suggest a possible involvement of ATF2 in NSCLC CDDP-resistance.

Background
Majority of investigation is focused on lung tumorigenesis in smokers, little in nonsmokers. Previously, Benzo[a]pyrene (B[a]P)-DNA adducts levels in nonsmoking female patients were higher than in nonsmoking male patients. However, p53 mutation rate in female patients did not differ from male patients. Moreover, HPV16/18 infection rate in female patients was much higher than in male patients. We therefore hypothesized that HPV infection could synergistically increased chromosomal instability (CIN) induced by B[a]P-DNA adducts and might contribute to lung tumorigenesis.

Methods
Herein, three HPV-positive and five HPV-negative lung cancer cells were enrolled to test the hypothesis. FISH analysis was used to determine the micronuclei formation when these cells were treated with various concentrations of B[a]P for different time-intervals.

Results
The efficacy of micronuclei and DNA adduct formation induced by B[a]P in HPV-positive cells were significantly higher than HPV-negative cells. Mechanistically, the micronuclei and DNA adduct formation are dependent on HPV E6 oncoprotein expression. We next questioned whether B[a]P-induced CIN enhanced by E6 could be through altering DNA repair gene expressions. The promoter hypermethylation and mRNA expression of six DNA repair genes including hMLH1, hMSH2, BRCA1, and BRCA2, and XRCC3, and XRCC5 were evaluated by MSP and real-time RT-PCR, and data indicated that XRCC3 and XRCC5 expressions in E6-positive cells were markedly lower than in E6-negative cells and the reduction of both genes was caused by promoter hypermethylation.

Conclusion
Collectively, the promoter hypermethylation of XRCC3 and XRCC5 induced by E6 may increase B[a]P-induced CIN and contribute to lung tumorigenesis in nonsmokers.

Background
Background: Antofagasta region in northern Chile shows the highest lung cancer (LC) mortality rate in the country. This population was exposed to arsenic (As) in drinking water of concentrations as high as 870 mg/L. Between 2003 and 2007, Antofagasta region showed a mortality rate of 30.8/100,000. It has been suggested that variation in As susceptibility among individuals might be partly due to differences in As biotransformation in addition to other genomic factors associated to the carcinogenic process. Objective: To determine the association of genomic variations (SNPs, and DNA copy-number alterations (CNAs) and susceptibility to LC, in blood and induced sputum samples collected from a LC risk sample population.

Methods
Materials and Methods: The 400 high-risk volunteers from Antofagasta and Metropolitan region was split in 2 groups, controls and cases, according to the result of early detection assays including automatic quantitative cytometry (AQC), DR70 and autofluorescence bronchoscopy (AFB). Copy number variants (CNVs) and single nucleotide polymorphisms (SNPs) were determined in genomic DNA of peripheral blood using TaqMan QPCR assays. Odd ratios (OR) were estimated by conditional likelihood and significance as exact mid-p values. Whole genome CGH profiles were generated with HEEBO 70-mer oligonucleotide microarrays.

Results
Results: Among 8 polymorphisms assayed (5 SNPs and 3 CNVs), CYP1A1 SNP rs1048943 was associated both to pre-neoplastic lesions (PNL) and cancer histopathology (OR 2.66, p=0.02). The CYP1B1 SNP rs1056836 was associated to LC, although no significantly. Some differences in the AS3MT SNP rs11191439 were observed between individuals from Antofagasta and Santiago. Additionally, the genomic profiles of sputum DNA and circulating cell-free DNA (cfDNA) from serum samples showed discrete differences among LC cases, pre-neoplastic lesions (PNL) and healthy controls. Chromosome 7p arm showed a significant gain in sputum DNA from cancer patients.

Conclusion
Conclusions: Conclusions: Our results suggest that SNPs associated to CYP450, like to CYP1A1 and 1B1, might be related to LC etiology. Additionally, genomic profiles from sputum DNA and cfDNA might be useful to detect PNL as complementary tools. Finally, this panel of genomic biomarkers in addition to DR70, AQC and AFB could be helpful to identify individuals susceptible to develop LC, and as complementary tools for LC early detection. These findings might be relevant in prevention and early detection of LC. Supported by INNOVA CORFO Chile: Grants 07CN13B48 and 11IDL2-10634.

Background
Plasminogen activator inhibitor type 1 (PAI-1) plays an important role in tumor growth and metastasis formation, directly via specific urokinase complexing or indirectly due to its affinity to vitronectin.

Methods
The aim of this study was to analyze the impact of the mutant forms of PAI-1: very long half-life (VLHL PAI-1) or devoid of affinity towards vitronectin (Vn neg PAI-1) and wild form (wPAI -1) on proliferation of lung cancer (A549 and H1299 ) and prostate cancer (LNCaP and DU145) cells characterized by different proteinase (urokinase) production.

Results
The dose- and time-dependent inhibition of cell proliferation in the presence of VLHL PAI-1 was evident in A549 and LNCaP cultures. In H1299 cells inhibitory effect was only dose-depended (p<0.001), while in DU145 only 100 mg/ml of VLHL PAI-1 in 72 hrs cultures suppresed prostate cancer cells proliferative activity (p <0.001). No significant effect of Vn neg PAI-1 on the proliferation of A549 and H1299 was observed while in prostate cancer lines (DU145, LNCaP) only the inhibitory effect of the highest Vn neg PAI-1 concentration (100μg/ml) was evident (p <0.001) but not time-dependent. wPAI-1 did’t affect A549 and LNCaP proliferation while in highest concentration it had the stimulating effect on H1299 and Du145 (24,48 hrs cultures).

Conclusion
PAI-1 is a negative regulator of cancer cells proliferation due to its anti-proteinase activity. Its biological effect on lung cancer cells is time and dose-dependent.

Background
Alpha-catulin is a cytoskeletal linker protein. Integrin-linked kinase (ILK) is a serine/threonine protein kinase implicated in cancer cell proliferation, anti-apoptosis, invasion and angiogenesis. Our previous study found that α-catulin is upregulated in lung cancer cells and directly interacted with ILK. Here, we further investigated the effect of α-catulin/ILK interaction in lung cancer metastasis and evaluated whether this interacting axis could be a potential therapeutic target.

Methods
Knockdown and overexpression of α-catulin and ILK genes were performed in non-small cell lung cancer (NSCLC) cell lines. Cell invasion and migration assays were done in 24-well Transwell polycarbonate filters coated with or without Matrigel. Spontaneous and experimental metastasis assays were performed in xenograft mice models to determine the effects of α-catulin on lung cancer cell metastasis in vivo. The public NSCLC cohort datasets were used for validation of the expression of α-catulin/ILK associated with patient survival.

Results
In the present study, we demonstrated that α-catulin promoted the migration and invasion of NSCLC cells through the ILK/NF-κB/integrin network. α-Catulin directly interacted with ILK, which in turn activated the ILK/Akt/NF-κB signaling pathway. This led to increased expression of the NF-κB downstream genes fibronectin and integrin α~v~β~3~, which sequentially activated NF-κB signaling and resulted in cancer cell migration, invasion and metastasis. The ILK plus CTNNAL1 two-gene signature was even more strongly associated with clinical outcomes of NSCLC patients.

Conclusion
Our data suggest that the alpha-catulin/ILK signaling axis is strongly associated with lung cancer cell migration and invasion and correlated with clinical outcome of NSCLC. Thus, this novel signaling axis could be a potential therapeutic target for treating NSCLC metastasis.

Background
Tyrosine Kinase (TK) domains are central regulators of signaling pathways that control differentiation, transcription, cell cycle progression, apoptosis, motility and invasion. EGFR TK mutations represent bona fide somatic mutations in NSCLC. Patients with specific mutations respond better to targeted therapy.Mutation analysis is known to be evaluated by several methods. Real time PCR using allele specific TaqMan primer probes is a simple, robust, highly sensitive, and selective method that is compatible with standard processes used for known gene expression analysis. The main objective of our study was to detect EGFR mutations in NSCLC patients registered in Tata Memorial Hospital (TMH) by using rapid and sensitive technique of RQ-PCR with In-house TaqMan primer probes. There is limited data regarding EGFR mutation in Indian population with variable mutation rate reported. This is an attempt to get actual mutation rate in our population, correlate the frequency of EGFR mutation and their subtypes and analyze across different variables of age, gender, habits and histology with different ethnicity groups.

Methods
1018 patients of NSCLC were referred for EFGR testing as a routine service over a 1.5-year period. Extracted DNA from FFPE blocks was amplified for the exons 18, 19, 20 and 21 using the specific TaqMan primer probes with the End point genotyping method on LC-480 II platform. Chi-square test was performed to reveal any significant correlation between the mutation status and age, gender and habits of the patient and tumor histology.

Results
Overall Mutation Rate (MR) was 25.0% with a higher mutation rate in females than males (34 vs.21) with a p value of 0.002. Within the age group, MR was high in > 60 yrs group as compared to <60 years (47.6 % vs. 31%). Total population of smoker vs. never-smoker was 37.5% vs. 58.6 %. (p value <0.001). MR was significantly higher in Never Smoker (NS) as compared to Smoker (S) (31.5 % vs.16 %), MR in Adenocarcinoma (ADC) was significantly higher than squamous (27.7 %vs. 5.6%) with a p value of <0.001. MR is higher in ADC NS female as compared to ADC NS male (37.2 %vs.29 %). MR of Exon 19 , 21, 18, & 20 was 53%, 38 %, 5.8 % & 2.4 % respectively. In the cohort of ADC NS gender, Exon 19 positivity was predominantly higher in male than female (61.8% vs.34 %) , whereas Exon 21 was marginally higher in ADC NS female than male (39 % vs 34 %). Exon 20 expressed 2.4%.

Conclusion
There is a heterogenous EGFR MR in diferent geographical population. We are in close association with East Asian countries than Western countries. In our data though the MR is high in NS, 16 % of EGFR MR in smokers is also startling reality. Another possibility of variation in EGFR mutation rate could be due to application of different technologies. Each technique differs in their sensitivity and specifictiy. EGFR tyrosine kinase domain define a new molecular marker for lung carcinoma.

Background
The spectrum and frequency of oncogenes in squamous cell lung cancers (SQCLCs) is actively being defined. Amplification of fibroblast growth factor receptor 1 (FGFR1) is the most common targetable oncogenic driver in SQCLCs, occurring in ~20%. Clinical trials of FGFR1 inhibitors for advanced SQCLCs are ongoing. The frequency, clinicopathologic features, and prognosis of FGFR1 amplification in early-stage SQCLCs have been reported but with discrepant results.

Methods
A cohort of histopathologically-defined and clinically-annotated resected SQCLCs was tested for FGFR1 amplification by FISH (Zytovision Dual Color Probe). Amplification was defined by FGFR1 copy number ≥2.2x CEP8 control copy number and was assessed by two evaluators (MW, LW) who were blinded to clinical results. Disease-free survival (DFS) defined as date of surgical resection until disease recurrent, relapse, or death, which ever occured first. DFS was estimated using Kaplan-Meier method. The association between FGFR1 status and clinical features (unpaired T-test, Fisher’s exact, Chi-square tests) and DFS (log-rank test for unadjusted analysis; Cox proportional hazards regression for multivariate analysis) were assessed.

Results
63 resected SQCLCs were evaluated. FGFR1 amplification was detected in 16 (24%). 56% were stage I, 24% were stage II, and 20% were stage IIIA. There was no association between FGFR1 amplification and age (p=0.86), sex (p=0.80), smoking status (p=0.37), or stage of disease (p=0.16). Median DFS was significantly longer in FGFR1-amplified cases compared to non-amplified cases: not reached vs 2.3 yrs (95% CI 1.1-3.4 yrs), p=0.02, with a corresponding unadjusted hazard ratio of 0.41 (95%CI: 0.19-0.88). Adjusted for sex and stage, multivariate analysis found FGFR1 amplification significantly associated with improved DFS (HR 0.31, 95%CI 0.1-0.89, p=0.03). Figure 1

Conclusion
FGFR1 amplification is associated with improved prognosis in this cohort of resected SQCLCs. The distinctive natural history substantiates FGFR1amplified SQCLCs as a unique, oncogene-defined subgroup. There was no association between FGFR1 status and sex, age, smoking status, or stage. FGFR1 amplification is common in SQCLCs.

Background
Molecular therapy targeted on tumour driving mutations should improve quality of life, PFS and overal survival in NSCLC patients. While EGFR mutations are widely accepted as targets for gefitinib and erlotinib in the first line treatment of advanced NSCLC, recently, translocation of EML4-ALK as well as amplification of ROS1, are used as guides for indication of crizotinib. Some other genetic changes, such as EGFR amplification, c-Met amplification, PIK3CA mutations and KRAS mutations are expected to serve as prognostic or predictive factors of targeted therapy currently or in near future.

Methods
We analyzed molecular predictors which were routinely tested at our department starting in 2004. We focus on EGFR mutations, EGFR gene amplifications, KRAS mutations, EML4-ALK translocations, c-Met amplifications and PIK3CA mutations. We analyzed the frequency of genetic changes and , where applicable, their impact on PFS and OS. We have also analysed comparability of PCR based detection (multiplex ligation-dependent probe amplification, MLPA) an FISH for EGFR gene amplifications.

Results
In the group of 890 NSCLC patients we found EGFR mutations on exon 19 in 62 cases, EGFR mutations on exon 21 in 24 patients. KRAS mutations were found in 141/819. Translocations of EML4-ALK were found in 7/203, c-Met amplifications in 5/104, EGFR amplifications in 11/34 and PIK3CA mutations in 8/220 patients. We confirmed a high correlation between FISH and MLPA-based testing of EGFR amplifications, as well as agreement of PFS and OS of both groups. However, EGFR gene amplifications were not prognostic, nor predictive marker of TKI therapeutic efficacy. Positive predictors of such treatment were in accordance with the literature and our expectiations, EGFR mutations for the treatment with gefitinib and erlotinib, and EML4-ALK translocations for crizotinib. We identified KRAS G12C mutation as the only negative predictor of EGFR inhibitor treatment. One hundred patients were defined as triple negative tumors (EGFR, EML4-ALK, KRAS negative). Some of our patiens had some combinations of genetic changes, such as concurrent EGFR and KRAS mutations, mutations and amplifications of EGFR gene and, in one patient, mutations and amplifications of EGFR, EML4-ALK translocation and c-Met amplification.

Conclusion
Precious morphologic and genetic investigations of NSCLC tumour tissue should serve as a guide for targeted therapy. However, in our population of non-squamous tumour lesions, we found predominantly a triple negative tumour type which should continue to be treated by first line chemotherapy.

Background
Lung cancer is the leading cause of cancer-related mortality in the world. These patients usually present at an advanced stage where treatment is mostly palliative. Hence, there is an urgent need to investigate the various pre-neoplastic pathways to identify suitable therapeutic targets to decrease lung cancer mortality. The expression patterns of mucins are drastically altered during lung cancer development, and these alterations facilitate lung cancer cell proliferation and metastasis. MUC16 mucin and Testis specific Y-like protein TSPYL5 are two proteins that are overexpressed in lung cancer and appear to promote growth of lung cancer cells. In addition overexpression of TSPYL5 facilitates chemoresistance and regulates aromatase (CYP19A1) enzyme expression. This study was conducted to assess the interplay between these two markers and evaluate their effect of cisplatin induced cytotoxicity in lung cancer cells.

Methods
Expression of MUC16 in normal lung as well as lung carcinoma tissues in a commercially available tissue array was assessed by immunohistochemical (IHC) analysis using MUC16 antibody (DAKO Company-M11 clone). MUC16 levels were semi-quantified using a composite score based on the intensity and extent of staining. Endogenously expressed MUC16 and TSPYL5 were stably knocked down using a MUC16 shRNA construct (pSUPER-Retro-MUC16-sh) and TSPYL5 ShRNA construct in H292 and H827 lung cancer cells by stable transfection method for investigating the oncogenic functions. Lung cancer cells were treated with cisplatin to examine the role of MUC16 and TSPYL5 on chemoresistance and survival in lung cancer cells.

Results
MUC16 is highly expressed in lung carcinoma and is not expressed in non-neoplastic lung tissues. MUC16 composite IHC scores increased progressively from stage I to stage III NSCLC. Knockdown of MUC16 led to decreased proliferation (due to G1 accumulation), invasion and motility in H292 lung cancer cells. TSPYL5 was significantly downregulated in MUC16 knockdown cells. TSYPL5 knockdown in H292 lung cancer cells resulted in decreased expression of MUC16 as well as aromatase. The knockdown studies suggested that silencing of MUC16 and TSPYL5 affected each other. These results indicate that there may be a feedback regulation between these two molecules during lung cancer cell proliferation. Furthermore, cisplatin treatment of these cells significantly abrogated MUC16 and TSPYL5 expression.

Conclusion
MUC16 is overexpressed in non small cell lung cancer. MUC16 plays an important role in lung cancer proliferation, through rapid G1/S transition in the cell cycle. MUC16 and TSPYL5 regulate each other during lung cancer cell proliferation, possibly through the aromatase pathway. One of the possible mechanisms through which cisplatin may decrease lung cancer proliferation is by downregulation of MUC16 and TSPYL5.

Background
Ataxia telangiectasia-mutated (ATM) is a DNA repair protein that is functionally absent in patients with A-T. Individuals with heterozygous or somatic homozygous mutations are predisposed to developing various cancers. Previous attempts to sequence ATM in cancer cell lines or in patient tissue samples have not identified any mutational hot-spots that are linked to the A-T phenotype or cancer predisposition, however much of the sequencing analysis performed to date has used traditional Sanger-based methodology, which is limited in its depth of coverage. Our lab has identified two non-small cell lung cancer (NSCLC) cell lines, NCI-H23 and NCI-H1395, which appear to be deficient for ATM. While H23 cells display sensitivity to ionizing radiation, typical of ATM deficiency, H1395 do not and moreover show downstream activation of several ATM targets. These previous results are important because the hallmark radiation sensitivity, and possible chemosensitivity of ATM-deficient patients may suggest that these patients would be more responsive to low-dose therapies. However, if cells show no detectable ATM but maintain an intact DNA repair mechanism, the usefulness of ATM as a biomarker is questionable. To assess if these observed differences were intrinsic to the ATM gene, we performed next-gen sequencing (NGS) to characterize the mutational spectra of H23 and H1395.

Methods
We used the semiconductor-based Ion Torrent PGM to sequence the coding region of ATM from genomic DNA isolated from H23, H1395, and H460 (ATM normal) cells. Variants were identified and mapped to the ATM gene. Additional western blots were performed to confirm mutation analysis.

Results
Several missense SNP mutations were identified in both H23 and H1395, however only one was shared by both, c.5948A>G, corresponding to amino acid substitution N1983S, which has previously been identified as a phosphovariant due to its proximity to the activation phosphorylation site, S1981. Interestingly, this SNP was not identified in H1395 in past sequencing attempts, demonstrating the power of NGS. Few of the other SNPs have previously been annotated in the COSMIC database, and it is as yet unknown whether they confer functional disruptions in the protein. Interestingly, the N1983S SNP is located in the same region of ATM used as the epitope for antibodies to both the phosporylated and unphosphorylated forms of ATM, raising the question of whether the antibodies are unable to bind to these variants and thus limiting detection. We performed western blots with antibodies to different epitopes to address this question.

Conclusion
We have yet to determine whether the mutations we identified in H23 and H1395 NSCLC cells are the cause of deficiency in these cells. However, use of deep-sequencing methodology has rapidly identified previously unknown mutations in these cell lines, and expansion of these studies to include a cohort of NSCLC patient samples may identify hot-spot mutations that were previously undetected by traditional sequencing methods. Moreover, these results imply that current methods for ATM detection may be insufficient; however supplementary deep-sequencing of these samples could be used to determine the nature of ATM deficiency, and to predict response to therapeutic treatments.

Background
Malignant pleural mesothelioma(MPM) is a locally aggressive disease characterized by a poor prognosis and increasing. MPM tumors are usually related to asbestos exposure, and the incidence is anticipated to peak between 2020 and 2030 because of the lag time between asbestos expousere and the development of the malignancy. MPM is difficult to detect at an early stage, and surgical and radiotherapeutic approaches are ineffective when used independently, because MPM spreads diffusely in the surrounding chest wall. No universally accepted treatment approach currently exists.

Methods
We examined whether combination treatment consisting of pemetrexed chemotherapy and photodynamic therapy (PDT) using the photosensitizer, NPe6, enhanced the antitumor effect in vitro and in vivo models. We also investigated preclinical treatment schedules. Four human malignant mesothelioma cell line, (MSTO-211H, H2052, H2452and H28) were assayed using the WST assy after treatment with pemetrexed and NPe6-PDT. The treatment schedule for the combination treatment was examined using nude mice.

Results
In nude mice injected with MSTO-211H cells and then treared using a combination of pemetrexed and NPe6-PDT (10 mg/kg NPe6, 10 J/cm2 laser irradiation), the tumor volume decreased by 50% but subsequently increased, reaching the pretreatment value after 14 days. Pemetrexed treatment followed by NPe6-PDT resulted in an 80% reduction in the tumor size and inhibited re-growth. NPe6-PDT followed by pemetrexed treatment resulted in a 60% reduction in tumor size but did not inhibit re-growth.

Conclusion
Pemetrexed reportedly inhibits multiple enzymes in the folate metabolic pathway, with TS being the main target. In non-small cell lung cancer cell line, high baseline TS expression levels confer resistance to pemetrexed, and the TS level is correlated with pemetrexed efficacy in a variety of solid tumors. These results suggest that the overexpression of TS protein induced by NPe6-PDT may be associated with the failure of pemetrexed to exert a tumoricidal action. Therefore, we concluded that NPe6-PDT followed by pemetrexed did not enhance tumor cell lethality in the in vivo model. Combination treatment, consisting of pemetrexed followed by NPe6-PDT, should be further investigated as a new treatment modality for malignant pleural mesothelioma. In the future, this combination treatment may contribute to a reduction in local recurrence and a prolonged survival period in patients with malignant pleural mesothelioma.

Background
Adenocarcinoma of the lung is a heterogenous group of disease. Even within the same patient, the tumor may show, various patterns of histopathology. In fact published data suggest that only 1/3rd of lung cancer are homogenous. Although known this factor is not taken into account in treatment planning and management. It is also known that treatment induces heterogenity and change in character of the tumor.

Methods
46 patients of Stage IV Adenocarcinoma of Lung were studied. All patients were confirmed as Adenocarcinoma by doing IHC TTF p63. All slides were viewed by two pathologists and all patients had EGFR mutation done.IHC marker for neuro-endocrine differentiation was done i.e. Chromogranin-A and Synaptophysin. Patients were treated with chemotherapy or EGFR-TKI. Patients were re-biopsied on progression. The same set of IHC studies was done.

Results

	IHC marker for adenocarcinoma	IHC marker for squamous cell	Neuro-endocrine marker
n=46
Pre-chemotherapy/TKI	41 (89%)	15 (32%)	17 (37%)
Post-chemotherapy/TKI	25 (54%)	25 (54%)	29 (63%)
n=18
Pre TKI	16 (89%)	5 (28%)	4 (22%)
Post TKI	8 (44%)	9 (50%)	14 (78%)
n=28
Pre-chemotherapy	25 (89%)	10 (36%)	13 (46%)
Post-chemotherapy	17 (61%)	16 (57%)	15 (54%)

Median duration of chemotherapy - 10 months Median duration of TKI - 16 months Change in character on IHC - seen in approximately 30% patients Infact change in post TKI occurring in upto 50% of patients with gain in Small Cell characters. Of the 50% patients who gained i.e. 10 patients - 7 of them also showed microscopic features of Small Cell Carcinoma of Lungs.

Conclusion
The study suggests that patients with advanced lung cancer have benefit from chemotherapy and TKI. On treatment and on progression there is a change in IHC and histopathology character in the patients. It occurs in close to 40% in such patients. These changes also call for changes in treatment. Hence re-biopy in such patients is essential.

Background
The CT-guided lung needle biopsy is a well-established and safety technique for diagnosis. A biopsy specimen often had loose connection and broke to tiny pieces before formalin fixation. Tumor invasion often involves the epithelial-mesenchymal transition (EMT) during which cells lose the lateral attachments to their neighbors and become more motile. The hypothesis is the fresh macroscopic appearance of specimens may relate pathological features and predict clinical features in patients with lung cancer.

Methods
The correlations between fresh macroscopic appearance of specimens and pathological findings or clinical outcomes were examined in patients who underwent CT-guided lung needle biopsy in our institution between May 2009 and May 2013. The intensity of fiber stained Azan staining (0, 1+, 2+, and 3+) and the percentage of positive cells (<1%, <25%, <50%, <75, and >75%) were assessed. The score of each case was multiplied to give a final score and the fibrosis was finally classified as low (<200) or high (>200). Comparisons of variables were performed by using Fisher exact tests.

Results
A total of 93 (86.1%) of 108 patients had adequate samples for diagnosis. The mean nodule diameter was 26 mm (range 4-75mm). CT findings revealed only three of 93 lesions showed ground-glass opacity, and all of them were in tight connection group. Macroscopically, 21.3% (n=23) specimens had loose connection, and 78.7% (n=85) specimens had tight connection. In loose connections, 73.9% (n=17) diagnosed as malignant and 26.1% (n=6) as benign, with sensitivity of 77.3%, specificity of 100%, and accuracy of 78.3%. In tight connections, 75.3% (n=64) as malignant and 24.7% (n=21) as benign, with sensitivity of 86.5%, specificity of 100%, and accuracy of 88.2%. There were 58 NSCLC samples, including 30 well or moderate (w/d or m/d), and 8 poorly differentiated (p/d) adenocarcinomas (Ad), 7 w/d or m/d, and 6 p/d squamous cell carcinomas (Sq), and 7 undifferentiated carcinoma. In 58 samples, 20.7% (n=12) specimens had loose connection and 79.3% (n=46) specimens had tight connection. In Azan staining, the tight connection group had 32 samples of high and 14 of low scores with the mean score of 213.6, and the loose connection group had 4 of high and 8 of low scores with the mean score of 118.6. The tight connection group had significantly higher scores than the loose connection group (p=0.042). The patients with loose connection had significantly higher rate of distant metastasis than those with tight connection (58.3% vs 21.7%, p=0.028). The median survival times are not reached in both groups.

Conclusion
Macroscopically loose connection specimens can afford to provide adequate amount of samples for diagnosis with sensitivity of 77.3%, and this appearance was negatively correlated with amount of fiber. Furthermore, the patients with loose connection tissue were associated with distal metastasis. The loose connection specimens may represent the status of EMT acquisition which is induced tumor initiation, growth, and metastasis. These findings suggest that the macroscopic appearance of tissue specimens obtained from CT guided biopsy can be an effective evaluation for prediction of metastasis in patients with NSCLC.

Background
Surgical adjuvant chemotherapy with platinum doublet is effective but not enough to suppress recurrence of NSCLC. We have applied combination chemoimmunotherapy with CBDCA+GEM and autologous tumor lysate transduced dendritic cell vaccination in patients with operable NSCLC in order to clarify that such a treatment can induce tumor specific immune response.

Methods
Eight patients with NSCLC whose pathological stages ranging from IB~IV were included in this study. The main purpose of the study is to obtain the principle of concept in which such vaccination can induce cellular immune response against autologous tumor cells detected by ELISPOT assay and establishment of CTL clones to recognize the tumor cell. Autologous tumor lysate was prepared from the surgical specimen aseptically by 5 times of freeze-thaw and sonication . DC cells were prepared from peripheral blood mononuclear cells obtained by apheresis, and in vitro culture with IL-4 and GM-CSF. Matured DC was induced by culture with TNF-α. Autologous tumor lysate-transduced DC was prepared by electroporation using MaxCyte cell loading system(MaxCyte[R]). More than 7x10[6] of the DC were injected intradermally at groin biweekly at 1, 3, 5, 7, 9 and 11week. Combination chemotherapy with CBDCA+GEM was administered at 0, 2, 4, 6, 8 and 10 week.

Results
Delayed type hypersensitivity skin reaction to the DC was observed in 5 of 8 patients after completion of the vaccination(6 times). ELISPOT assay revealed specific IFN-γ production in response to autologous tumor lysate-transduced DC following completion of the vaccination in 3 of the 5 patients. In one patient among them, CTL clones could be induced successfully in vitro, and identification of the Ag recognized by the CTL is now underway. No serious adverse effect was observed. Six patients have recurrent disease at 6, 6, 8, 10, 12 and 18months after operation. One patient(N0.6) died of the disease at 13 months after the operation due to systemic metastasis. However, the other patients are alive with(5 patients) or without(2 patients) evidence of the recurrent disease(ranging from 10~22 months as of February, 2013). Figure 1

Conclusion
The autologous tumor lysate-transduced DC vaccination is effective to induce tumor specific cellular immune response in some of such patients. Establishment of CTL clones recognizable autologous tumor cells can lead to identification of specific Ag coding genes which can be used as immunological targets.

Background
Malignant pleural mesothelioma (MPM) is an incurable and fatal malignancy of the pleural membranes. MPM has a poor prognosis and patients have a median survival of 9-13 months in clinical studies, with above 1,500 cases in the UK per year. No surgical approach has been shown to prolong survival and pemetrexed-cisplatin is now seen as the chemotherapy standard of care in the UK. However it is clear that new therapeutic strategies are urgently needed for MPM. Immunotherapy is potential new treatment approach to be considered in MPM, as the disease has been shown to respond to various immunotherapeutic strategies tested in animal models and early phase clinical trials. TroVax® consists of a highly attenuated vaccinia virus containing the human TAA 5T4 glycoprotein gene under regulatory control of a modified VV promoter, mH5. Velindre NHS Trust and the Wales Cancer Trials Unit (WCTU) have developed the SKOPOS trial to evaluate whether TroVax® is active in the treatment of MPM. SKOPOS is funded by the June Hancock Mesothelioma Research Fund, and the Velindre Cancer Centre Stepping Stones Appeal. Oxford BioMedica (UK) Ltd. is providing TroVax® vaccine free of charge, and also labeling and distribution costs. The trial is sponsored by Velindre NHS Trust, and coordinated by the WCTU.

Methods
A UK single centre, single arm, phase II trial. Eligible patients include locally advanced or metastatic, histologically or cytologically proven MPM, WHO performance status 0-1, life expectancy > 6months, at least four weeks since any previous therapy (surgery, radiotherapy). Enrolled patients will be treated with: TroVax® - intramuscular injection, dose 1 x 10[9] TCID ~50~/ml in 1ml, given on Day 1 of weeks 1, 3, 6, 9, 12, 15, 18, 21, 24. Folic Acid - 400μg oral daily from Day 2 of week 3 to Day 2 of week 16 B12 Vitamin – 1000μg intramuscular, Day 2 of weeks 3 and 12 Dexamethasone –4mg BD, Days 2-6 of weeks 4, 7, 10, 13 Pemetrexed - 500 mg/m[2] over 10 min, given on day 3 of weeks 4, 7, 10, 13 Cisplatin - 75mg/m[2] over 1h, given on day 3 of weeks 4, 7, 10, 13 The co-primary endpoints of the trial are i) cellular response to 5T4 and MVA antigens measured by intracellular cytokine staining (ICCS); and ii) antibody response to 5T4 and MVA antigens as measured by ELISA. Secondary outcome measures include safety, progression free survival, objective response rate and overall survival. The sample size was determined using Fleming’s single arm design. The outcome measure is the immune response to the 5T4 antigen. A success in this trial is defined as an increased response (at least a doubling in the 5T4 antibody or T-cell response) from that measured at baseline at any of the six follow-up time points. If an increased response is seen in at least 64% of patients then we will research the vaccine further in this group of patients. Using Fleming’s single-stage design, p1=0.40 and p2=0.64, setting alpha=0.05 (1-sided) and 80% power, 26 participants are required.

Results
Not Applicable

Conclusion
Not applicable

Background
The immune cells can prevent and inhibit tumour development but also may contribute to growth and progression of cancer. Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immature cells with immune suppressive properties, have been correlated with worse prognosis in several tumors. In addition, high levels of circulating CD4[+] T regulatory (Tregs) cells exhibit suppressive functional activity against anti-tumour T-cell responses and correlated with worse recurrence-free survival in NSCLC patients. In the present study, we investigated the expression and levels of MDSCs’ subpopulations and CD4[+]Tregs, their correlation to distinct immune cells, as well as to the clinical outcome of patients with advanced NSCLC.

Methods
Peripheral blood from 112 chemotherapy-naive patients with stage III/IV NSCLC and 27 healthy donors was analyzed with flow cytometry for the presence of monocytic and granulocytic subpopulations of MDSCs, CD4[+ ]Tregs, as well as the frequencies of distinct immune cells such as CD4[+ ]and CD8[+ ]cells, dendritic cells (DC) and B cells. The frequencies of these cells in the patients were compared to the healthy donors. The patients’ clinical outcome (PFS and OS) was compared according to the frequency of MDSCs and Tregs (high vs low expression as defined by their percentage above healthy donors).

Results
Two monocytic (CD14[+]CD15[-]CD11b[+]CD33[+]HLA-DR[-]Lin[-] and CD14[+]CD15[+]CD11b[+]CD33[+]HLA-DR[-]Lin[-]), and a granulocytic (CD14[-]CD15[+]CD11b[+]CD33[+]HLA-DR[-]Lin[-]) subpopulations as well as CD4[+] Tregs (CD3[+]CD4[+]CD25[+high]CD152[+]CD127[-]Foxp3[+]) were increased in patients compared to healthy donors (p<0.001 and p<0.007, respectively). Levels of MDSCs and CD4+ Tregs did not associated with tumor histology or stage of the disease. The levels of both subpopulations of monocytic but not of granulocytic MDSCs were reversely correlated with the levels of dendritic cells (DC) (r[2]=-0.3, p≤0.04) whereas the granulocytic subpopulation of MDSCs was reversely correlated with the levels of CD4[+] T cells (r[2]=-0.3, p=0.006). Increased baseline levels of both monocytic MDSCs’ subpopulations was associated with early relapse despite front-line platinum-based chemotherapy (p=0.05). The detection of baseline CD14[+]CD15[+]CD11b[+]CD33[+]HLA-DR[-]Lin[-] MDSCs within the normal levels was associated with longer OS compared to those with high levels (p=0.0035). Finally, patients with normal CD4[+ ]Tregs frequencies had a higher OS than those with high frequencies (p=0.05).

Conclusion
The data show that increased levels of monocytic MDSCs and CD4[+] Tregs in the peripheral blood of NSCLC patients are reversely correlated to the other normal immune cells. These cells could represent potential predictive/prognostic biomarkers since their increased levels were negatively correlated to the treatment outcome.

Background
Interleukin-15 (IL-15) is a potent pro-inflammatory cytokine, that plays a major role in stimulating immune effector cells following microbial and viral infection. IL-15 exhibits broad activity and induces the differentiation and proliferation of T, B and natural killer (NK) cells. IL-15 is primarily expressed by antigen presenting cells; however, in areas where there are high microbial loads, such as the lungs and colon, epithelial cells may also express IL-15 and its receptor (IL-15Rα). The expression of IL-15 and IL-15Rα by the lung epithelium facilitates immune responses by inducing local proliferation of NK and CD8+ T-cells in response to infection. Here we determine the status of IL-15 and IL-15Rα on lung cancers and assess their ability to stimulate the proliferation of these immune cells.

Methods
Western blotting, ELISA and qPCR were used to determine the expression of IL-15 and IL-15Rα in 6 human lung cancer cell lines and 1 normal human bronchial epithelial cell (HBEC) line. Further, mRNA from 146 primary lung cancers of all stages and tissues from 45 normal lungs were examined by multiplex qPCR for the expression of IL-15 and IL-15Rα. NK and T-cell proliferation assays were performed to determine the effects of IL-15 expression by the cell lines on these immune cells.

Results
IL-15 expression by lung cancer cell lines was significantly reduced compared to the HBEC cell line. Similar results were seen when we examined the expression of IL-15 in primary tumors, with lung cancer expressing less IL-15 than normal lung (P<0.001). When we compared IL-15 expression between stages, we found that increased stage correlated with a decrease in IL-15 expression with normal lung> stage I> stage II> stage III> stage IV. In contrast, IL-15Rα expression levels in the tumor samples were found to be largely unchanged across stage, P=0.417. Decreases in IL-15 with increasing stage may represent one mechanism in which lung cancers limit the immune response directed at the tumor and may aid in metastatic progression. In order to assess the immune effects of a reduction in IL-15 expression, we examined the ability of the lung cancer cell lines and HBEC to induce the proliferation of NK and T-cells following co-incubation. We found that the lung cancer cell lines significantly inhibited the proliferation of either NK or T-cells compared to HBEC.

Conclusion
Pro-inflammatory cytokines such as IL-15 are important for the induction of lung immunity. Decreases in IL-15 expression may reduce immune responses thereby aiding in the escape of lung cancer from immune detection and the dissemination of tumor. The differential expression of IL-15 across lung cancer stages and retention of IL-15Rα expression may make lung cancer a target for IL-15-based treatments and opens the potential for IL-15 to be used as a predictive biomarker in early stage patients.

Background
Tyrosine kinase inhibitors (TKIs) that target the epidermal growth factor receptor (EGFR) has become standard therapy in patients with EGFR activating mutations. Unfortunately, acquired resistance eventually limits the clinical effects and application of EGFR-TKIs. Although main mechanisms of acquired resistance have been detected with varying frequencies, including T790M mutation, amplicfication of other kinases and epithelial to mesenchymal transition(EMT), potential therapies for these tumors have not been modeled in vivo. Researches have shown that suppression of EMT and IL-6/STAT3 pathway may abrogate this acquired mechanism of drug resistance of TKI.

Methods
By using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), immunofluorescence, Western blot, cell tracking, invasion assay, ELISA assay and xenograft experiments, we analyzed the effect of metformin and TKIs on TKI resistance of tumor cells both in vitro and in vivo.

Results
Metformin, which is widely used as an antidiabetic agent, effectively increased sensitivy of TKI-resistant lung cancer cells to erlotinib or gefitinib. Metformin reversed EMT and decreased IL-6/STAT3 activation in TKI-resistant cells, while IL-6 addition in those cells bypassed the antitumor effect of metformin. Furthermore, overexpression or addition of IL-6 in TKI sensitive cells induced TKI resistance, which can be overcomed by metformin. Lastly, metformin-based combinatorial therapy effectively block tumor growth in xenografts involving TKI-resistant cancer cells, which is associated with EMT reversal and decreased IL-6/STAT3 activation.

Conclusion
Thus, the unexpected ability of metformin to reverse EMT and inactivate IL-6 axis further reveals that this biguanide, generally considered non-toxic and remarkably inexpensive, might be used in combination with TKIs in NSCLC patients harboring EGFR mutations to overcome TKI resistance. Our findings therefore offer preclinical proof of principle that combination of TKI and metformin may benefit patients with NSCLC.

Background
Lung cancer is the leading cause of all cancer deaths worldwide. Non-small cell lung cancer (NSCLC) is the most common type, accounting for 75–80% of all lung cancers. Bcl-2 is a central regulator of cell survival that is overexpressed in NSCLC and contributes to both malignant transformation and therapeutic resistance. We previously identified a small-molecule BH3 mimetic named S1 that exhibits nanomolar affinity towards Mcl-1, Bcl-2 and Bcl-XL. The purpose of this work was to study the key factors that determine the sensitivity of NSCLC cells to S1 and the mechanism underlying the resistance of this drug.

Methods
Acquired resistant cells were derived from initially sensitive NCI-H1975 cells. Western blot and co-immunoprecipitation were used to evaluate the contribution of Bcl-2 family members to the cellular response of several NSCLC cell lines to S1. Quantitative PCR and gene silencing were performed to investigate Bim down-regulation. PD98059 and MG-132 was used to inhibit the degradation of Bim.

Results
S1 can disrupt Bcl-2/Bim, Mcl-1/Bim and Bcl-XL/Bim complexes regardless of the levels of the anti-apoptotic proteins NSCLC cell lines. A progressive decrease in the relative levels of Bim characterized the increased de novo and acquired resistance of NSCLC cell lines. Furthermore, in resistant cells, acute treatment of S1 induced Bim phosphorylation on serine 69 and degradation via the proteasome pathway. ERK inhibitor PD98059 abrogated Bim phosphorylation and degradation and induced caspase activation and apoptosis. We showed that BH3 mimetics including S1 and ABT-737 induced ER stress and then activated MEK/ERK pathway in NSCLC cells. The function of MEK/ERK pathway in defining BH3 mimetics was illustrated: Bim was released from anti-apoptotic proteins by S1, ERK1/2 activation leaded to Bim sustained phosphorylation and then degraded by proteasome in naïve and acquired resistant NSCLC cells.Figure 1

Conclusion
We describe here a new mechanism for the regulation of Bim expression by phosphorylation protects NSCLC cells from BH3 mimetics induced apoptosis. These results provide significant novel insights into the molecular mechanisms for ERK1/2 mediated the crosstalk between the Bim regulation and ER stress to define BH3 mimetics in NSCLC cells.

Background
Lung cancer is the leading cause of cancer death worldwide. Non-Small Cell Lung Cancer (NSCLC) which is the predominant type is mostly advanced stage disease at diagnosis. Cytotoxic agents including chemotherapeutic drugs and radiation therapy, the mainstay of advanced NSCLC treatment are neither specific nor curative and are significant causes of morbidity in patients. This poor NSCLC outlook therefore requires a novel therapy as well as predictive markers of treatment response for an improvement. We propose Ataxia Telangiectasia Mutated (ATM), a critical player in the DNA double strand break repair pathway, as a potential predictor of treatment response in NSCLC. Radiation sensitivity is one of the hallmarks of ataxia telangiectasia (A-T), a condition due to ATM mutations. Our lab has previously demonstrated ATM deficiency in about 20% of resected NSCLC tumors and this is associated with poorer survival outcomes; however, no treatment guidelines currently take this into consideration.

Methods
We assessed in vitro the altered sensitivity of ATM deficient NSCLC cell lines to both targeted agents (e.g. Poly (ADP) ribose polymerase (PARP) inhibitors) and non-targeted agents (e.g. DNA damaging agents including ionizing radiation (IR) and the commonly used cytotoxic chemotherapies in NSCLC) using the clonogenic survival assay. A panel of NSCLC cell lines (NCI-H23, NCI-H226, NCI-H460, NC1-H522, NCI-H1793, NCI-H1395 and HCC 4006) were characterised for pre-existing ATM deficiency in terms of ATM protein expression levels and functionality using western blot. By examining the cells’ responses to IR, confirmation of ATM deficiency was achieved with clonogenic assay to assess cellular viability and western blot for the expression of IR inducible ATM-dependent phosphorylation sites on target proteins including phosphorylation of serine 1981 of ATM (p-S1981 ATM), serine 15 of p53 (p-S15 p53) and serine 824 of KAP1 (KRAB-associated protein 1).

Results
We identified NCI-H23 cells with pre-existing low ATM protein levels (11% relative to BT cells) and undetectable ATM protein in NCI-H1395 cells compare to our positive (BT cells) and negative (L3 cells) control lymphoblastoid cells derived from normal and A-T patients respectively. In addition, H23 and H1395 cells display altered ATM signaling as evidenced by no detectable level of p-S1981 ATM expression post-irradiation. There is increased sensitivity of H23 cells to DNA-damaging agents (such as IR, topotecan and cisplatin) and PARP inhibition compare to ATM proficient NSCLC cell lines.

Conclusion
Our results seem to delineate the therapeutic sensitivity of ATM deficient versus ATM proficient cell lines to both non-targeted agents (chemotherapy and radiation therapy) and to targeted agents (PARP inhibitors). ATM could serve as a potential marker in guiding the use of 1) targeted agents such as PARP inhibitors, and 2) conventional chemo-radio therapeutic agents in the treatment of NSCLC. We are in the process of establishing ATM knock-down cells from ATM proficient NSCLC cell lines. Results of the altered sensitivity of established ATM deficient NSCLC cells to our investigative agents will be discussed and presented.

Background
Background: Development of acquired resistance to cisplatin treatment is a major problem when treating patients with malignant pleural mesothelioma (MPM) and non-small cell lung cancer (NSCLC). Chemotherapy leads to tumor cell stress activation of glucosylceramide synthase (GCS) to eliminate ceramide by glycosylation and formation of glycosphingolipids (GSLs) such as globotriaosylceramide (Gb3), the functional receptor of verotoxin-1. Ceramide elimination leads to stimulated cell proliferation and blocked apoptosis, thus stimulating tumor progression. GSLs also transactivate multidrug resistance 1/P-Glycoprotein (MDR1) and possibly multidrug resistance protein 1 (MRP1) expression which confers tumour cell resistance by further preventing ceramide accumulation and stimulating drug efflux. We investigated if Gb3, MDR1 and MRP1 are co-expressed and co-localized in MPM and NSCLC cells with acquired cisplatin resistance and if GSC activity inhibitors DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) and cyclosporin A would reduce their expression and relieve cisplatin-resistance.

Methods
Methods: Cell surface as well as intracellular expression of Gb3, MDR1 and MRP1 was analysed by flow cytometry and confocal microscopy using specific protein antibodies on P31 MPM and H1299 NSCLC cell lines and corresponding sub-lines (P31res, H1299res) with acquired cisplatin resistance.

Results
Results: Gb3 and MDR1, but not MRP1 were co-expressed and partly co-localized on the cell surface, and Gb3 and MDR1 as well as MRP1 intracellular co-expressed but not co-localized in P31res and H1299res cells. P31 cells expressed minute cell surface Gb3 and the non-resistant cells had less cell surface and but similar intracellular expression of Gb3 and MDR1. Glycosphingolipid synthesis inhibitors PPMP and cyclosporin A radically decreased intracellular Gb3, MDR1 and MRP1-expression in all cell sub-lines whereas cell surface Gb and MDR1 expression was decreased only by PPMP but not by cyclosporin A.

Conclusion
conclusion: These results indicate that cell surface Gb3 that is co-expressed and co-localised with MDR1 is a likely tumour biomarker for acquired cisplatin resistance in MPM and NSCLC and that therapy with GCS activity inhibitors or Gb3 blockers affecting ceramide metabolism may overcome or substantially reduce acquired cisplatin drug resistance. Targeting the functional interplay between Gb3 and MDR1 might aid in the development of new drug therapies against acquired drug resistance in MPM and lung cancer.

Background
Background: Platinum-based drugs, such as cisplatin, are the standard treatment for aggressive malignant pleural mesothelioma (MPM) and non-small-cell lung cancer (NSCLC), but inherent as well as acquired resistance are major clinical problems leading to therapy failure and low median survival after diagnosis. Cisplatin exposure initiates the mitochondrial signaling pathway of apoptosis, by activation of BH3-only proteins i.e. pro-apoptotic members of the Bcl-2 family of proteins. Therapy failure may be the result of decreased apoptosis due to caspase-9-deactivation or over-expression of the anti-apoptotic proteins Bcl-X~L~ and Mcl-1. Affecting cisplatin resistance by targeting post- and off-target apoptosis signalling proteins with pro-apoptotic BH3-mimetics, would possible sensitize cancer cells to cisplatin treatment.

Methods
Methods: We investigated the expression of Bcl-2 family and other proteins involved in apoptosis signal transduction and the difference between the response to equiapoptotic cisplatin concentrations as well as the response to the pro-apoptotic BH3-mimetics ABT-737 and GX15-070, alone or in combination. To separate mitochondrial-dependent from –independent signalling we compared initiator-caspase-dependent parental P31 MPM cells with its acquired cisplatin-resistant (P31res) sub-line which has initiator-caspase-independent caspase-3-activated apoptosis,

Results
Results: On acquisition of cisplatin-resistance, the expression of the pro-apoptotic and anti-apoptotic Bcl-2-family proteins was either not changed or slightly decreased in P31res cell compared to parental P31 cells. A 6-h exposure to equiapoptotic concentrations of cisplatin, on the other hand, increased the expression of potent pro-apoptotic BH3-only proteins as well as the anti-apoptotic Bcl-x protein in P31 but not P31res cells whereas Bcl-x expression was almost annihilated in P31res cells. TUNEL results showed a synergic effect on apoptosis when cisplatin was combined with the BH3-mimetic GX15-070 in P31res, but only an additive effect in P31. The BH3-mimetic ABT-737 did not augment cytotoxicity or apoptosis either per se or when combined with cisplatin and/or GX15-070. GX15-070 efficiently inhibited anti-apoptotic Bcl-2-family-, inhibitors of apoptosis (IAP) - and heat shock protein (HSP) - family protein expression both with and without cisplatin in P31 cells, whereas preserved protein expression was noted in P31res cells after 6 h incubation with cisplatin. Sub-toxic concentrations of the IAP-inhibitor AT-406 and the HSP90-inhibitor 17-AAG with GX15-070 markedly potentiated cisplatin cytotoxicity in P31res cells.

Conclusion
Conclusion: P31 malignant mesothelioma cell acquisition of cisplatin-resistance led to deregulated Bcl-2 family protein expression and induced post-target apoptosis resistance to the BH3-mimetic GX15-070. GX15-070 had a synergistic effect on cisplatin-induced apoptosis in P31res cells. The synergy was due to efficient GX15-070 inhibition of expression of the anti-apoptotic Bcl-x protein despite apoptosis resistance by preserved IAP and HSP protein expression. Cisplatin therapy combined with GX15-070 in acquired cisplatin resistance was even more efficacious when combined also with inhibitors of IAP- and HSP- apoptosis signalling pathways.

Background
It has been found that HGF-dependent c-Met（HGFR） autocrine is activated in a wide variety of human primary and second malignancies. On the other hand, the metastatic growth potential of tumors can be activated through paracrine mechanism. c-Met dysregulation leads to lung cancer development through overexpression and mutation.

Methods
70 kinase enzymogram screening was proceeded by Z-lyte technique. MOA analysis was completed on the inhibited kinases. Cell vitality was determined at 24h, 48h, 72h after treatment through CCK8 method. Transwell system was used to observe the inhibition of cell migration. Difference of special gene expression was evaluated by Real-time PCR. Westernblot assay was used to compare the expression difference of c-MET and p-MET. HGF level in culture medium is determined by ELISA.

Results
SIM-89 can inhibit 3 kinases including c-Met（IC50=297nM）, AMPK, TRKA (IC50=150.2nM). SIM-89 has an ATP competive inhibition on c-Met. By Real-time PCR, SIM-89 has been found to inhibit STAT1, JAK1, c-Met gene expression in H460 cell. P-Met expression of A549, H441, H1299 and B16F10 cell can be inhibited by SIM-89. HGF level of supernatant in culture is significantly lower than control group. Vitality of NSCLC cell lines is inhibited dependent on time and concentration by SIM-89. Induced by HGF, migration of H460, H1299 cell is inhibited.

Conclusion
SIM-89 has significant inhibitive effect on c-Met, TRKA kinases. It also can inhibit proliferation, migration and HGF autocrine of NSCLC cell significantly. Further study in vivo should be carried to explore the pharmacokinetics of SIM-89.

Background
Lung cancer accounts for the greatest cancer related mortality world wide, with two thirds of all patients receiving radiation therapy as part of their cancer care. Despite advancements made, the overall 5-year survival in Canada remains less than 20%. Developments in nanotechnology have proven to possess exciting potential in the treatment of various malignancies. Nanoparticles with high atomic number, such as Gold (GNPs), facilitate surprising local enhancement secondary to gold’s strong photoelectric absorption coefficient. GNPs are capable of forming covalent bonds, enabling the creation of “targeted” radio-sensitizing agents. The goal of this study is to evaluate the in vitro and in vivo radiation potential and biodistribution profile of GNPs targeted against the epidermal growth factor receptor (EGFR) using the monoclonal antibody Cetuximab (GNP-cetuxumab) stabilized with thiolated polyethylene glycol (SH-PEG) for the treatment of non-small cell lung cancer (NSCLC). To date a comprehensive evaluation of such a platform has not been undertaken.

Methods
We examined the radiation enhancement of 50nm GNPs in vitro using SKMES-1 (High wild type EGFR expression) and h460 (low wild type EGFR expression) NSCLC cell lines, both possessing Kras mutations. MTS, clonogenic assays and flow cytometry were used to assess radiation effect using 4 groups (no GNPs, GNPs, GNPs bound to SH-PEG stabilizer, GNP-Cetuximab). In vivo biodistribution was conducted on balb-c nude mice bearing two flank subcutaneous SKMES-1 xenografts. One tumor received a single radiation exposure (200 kVp, 8 Gray) prior to tail vein injection of GNP-cetuximab or GNP-PEG in order to assess the effect radiation induced inflammation may have on GNP tumor uptake. Tumors and organs were harvested at various time points with GNP concentration determined using inductively coupled mass spectroscopy. In vivo radiation experiments were conducted on 3 flank tumor bearing groups (each group = 7): 1) radiation only, no nanoparticles 2) GNP-PEG, GNP stabilized by bound PEG and 3) GNP-Cetuximab. Four weekly radiation fractions (4Gy, 200 kVp) with weekly tail vein injection timing based on the biodistribution results were administered. Tumor growth kinetics was then evaluated.

Results
Significant in vitro radiation effect was observed in the GNP groups compared to the radiation only group. GNP-cetuximab and GNP-PEG demonstrated enhanced radiation effect as compared to unfunctionalized GNPs. In the biodistribution experiment, the peak intra-tumor concentration without pre-administered radiation in the GNP-PEG group was twice that of the GNP-Cetuximab group 5 days after tail vein injection. Tumor pre-irradiation resulted in a doubling of intra-tumor nanoparticle uptake in both groups. At the end of radiation therapy experiment, the GNP-PEG group demonstrated the greatest reduction in tumor growth as compared to the radiation alone group (52mm[3] vs 180mm[3 ]respectively, p<0.01).

Conclusion
Despite the superiority of GNPs bound to cetuximab in vitro as a radiation enhancer, the favorable tumor biodistribution of the GNP-PEG accounted for the most dramatically reduced tumor growth kinetics observed. The future utility of targeted nanoparticles requires further investigation in light of these findings. In this study we demonstrate the exciting in vitro and in vivo potential of GNPs in the radiation treatment of NSCLC.

Background
Constitutive activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway has been found in non–small cell lung cancer (NSCLC) and promotes cancer progression. Myristoylated alanine-rich C kinase substrates (MARCKS) is a substrate of protein kinase C (PKC), and acts as a key regulatory protein controlling cell motility and signaling. We previously reported that elevated MARCKS phosphorylation (pSer159/163) potentiates lung cancer cell malignancy by upregulation of AKT/Slug axis.

Methods
Tissue array and immunohistochemistry were performed to analyze MARCKS phosphorylation in 110 pairs of NSCLC tumor cells and corresponding normal tissues. The enforced and silenced expressions of MARCKS were performed in lung cancer cells to verify the role of MARCKS expression and its phosphorylation. In vitro and in vivo anticancer activities of a MARCKS ED domain peptide (MPS) were confirmed by MTS, colony formation, flow cytometry, invasion, migration assays and in vivo subcutaneous and orthotopic implantation. The molecules regulated by MPS peptide were determined by Western blotting, PIP3 pool assay and co-immunoprecipitation.

Results
We demonstrated that elevated MARCKS phosphorylation is correlated with advanced-stage and lymph node metastasis of lung cancer. siRNA knockdown of MARCKS expression confirmed the importance of MARCKS and its phosphorylation in modulating PIP3 pool and cancer cell survival. Furthermore, we have identified that a small peptide, MPS, which mimics the basic effector domain (ED) of MARCKS is very effective in suppressing phospho-MARCKS and PIP3 levels in lung cancer cells. MPS peptide treatment is able to inhibit cancer cell viability, colony formation, migration and invasion in vitro, as well as tumorigenesis and metastasis in vivo. Interestingly, MPS peptide is very cytotoxic to cancer cells with highly activating PI3K/AKT signaling and malignant phenotypes, while MPS has no cytotoxic effect on normal human bronchial epithelial cells. In addition, a co-treatment of MPS peptide with epidermal growth factor receptor inhibitor erlotinib could reverse drug sensitivity of these tyrosine kinase inhibitor (TKI) resistant cells, H1650 and H1975, toward erlotinib.

Conclusion
These results suggest a therapeutic potential in lung cancer treatment by MPS peptide through the sequestration of PIP2 pool and the suppression of MARCKS phosphorylation and PI3K/AKT pathway.

Background
Malignant mesothelioma (MM) is an aggressive, fatal, tumour of the pleura or peritoneum and is strongly related to asbestos exposure. Clinically, there is no curative therapy for MM and the profound chemoresistance of MM is well documented, reportedly being due to the ability of MM cells to escape cell death. Unravelling the molecular mechanisms employed by MM cells to evade cell death will therefore provide new insights into key cell death pathways that may be targeted for successful therapy. Currently, the Bcl-2 repertoire is an undervalued and attractive pharmacological target for mesothelioma therapy. The aims of this study were to investigate the sensitivity of MM to the BH3-mimetic, ABT-737, and identify mechanisms of overcoming resistance to support personalized therapy.

Methods
Sensitivity to the highly selective BCL-2/BCL-XL antagonist, ABT-737, was investigated using FACs-based analysis and immunoblotting techniques in a panel of MM cell lines and tumour cells isolated from freshly resected MM tumours. In addition, patient-derived tumour explants were similarly analysed, providing a clinically relevant 3D mesothelioma model. Furthermore, to explore the possibility that tumour cell metabolism may modulate the sensitivity of MM cells to ABT-737, we investigated the effect of inhibiting glycolysis with 2-deoxyglucose (2-DG) on ABT-737 -induced apoptosis in relevant pre-clinical models of MM.

Results
The majority of MM cell lines and freshly-derived cultured tumour cells were resistant to ABT-737, suggesting a deregulation of cell death signalling at the level of mitochondria. Aerobic glycolysis (Warburg effect) is a process by which tumour cells gain not only growth advantage (providing much needed “building blocks”) but also an increased survival potential. Importantly we report that, in MM, glycolysis can be inhibited by 2-deoxyglucose(2-DG), a competitive inhibitor of hexokinase. Although exposure to 2-DG did not induce cell death, 2-DG induced a time- and concentration-dependent potentiation of ABT-737 -induced apoptosis in either established mesothelioma cell lines or newly-derived tumour cells from patients. BCL-2-family member profiling revealed that, while the predominant pro-survival members expressed in MM were MCL-1 and BCL-XL, 2-DG selectively decreased MCL-1 protein levels, a leading cause of resistance to ABT-737 in other tumour models.

Conclusion
High constitutive levels of MCL-1 most likely explain the inherent resistance of MM cells to the highly selective BCL-2/BCL-XL antagonist, ABT-737. Importantly, 2-DG-dependent down-regulation of MCL-1 provides a potential mechanism for overcoming resistance to ABT-737 in the clinical setting.

Background
Met receptor phosphorylation is associated with poor prognosis in human SCLC. Several Met inhibitors are being tested for the treatment of different neoplasms. Met activation has been shown to be an inductor of epithelial to mesenchymal transition (EMT) in a number of tumor models. The aim of our work was to investigate the effects of hepatocyte growth factor (HGF)/Met induced EMT in SCLC, to evaluate the role of Met inhibition in mesenchymal/chemorefractory SCLC models and to investigate the significance of EMT in human SCLC.

Methods
Biological features (growth, invasiveness, tumorogenesis) and chemosensitivity of SCLC models (H69) of HGF-induced EMT (H69M) were evaluated in vitro and in vivo (subcutaneous xenografts in BALB/c nude mice). Mice with mesenchymal chemoresistant SCLC xenografts were treated with etoposide, the Met inhibitor PF-2341066 (Crizotinib) and the combination. Human SCLC samples at diagnosis and relapse were evaluated by immunohistochemistry and immunofluorescence for EMT markers and Met status and correlated these with patient outcome. Association between clinical-pathological characteristics were tested with Chi-Square and Fisher tests. Differences in survival according to biomarker status were evaluated by log-rank and Cox regression models, assuming a 2-sided statistical significance p< 0.05.

Results
We identified that the activation of the Met receptor through HGF induced expression of mesenchymal markers (Snail1, SPARC, vimentin) and downregulation of E-cadherin. This derived in increased tumorogenesis, local invasion and chemoresistance in xenograft models. The combination of etoposide and PF-2341066, but not the Met inhibitor alone significantly decreased tumor growth in this chemoresistant/mesenchymal models. Moreover, Snail1, SPARC and vimentin expression in human SCLC specimens (N:87) was significantly associated with Met activation (co-localization by immunofluorescence). Expression of mesenchymal markers predicted worse survival (all p-values <0.05) in the multivariate analysis. In 5 paired biopsies, we observed upregulation of mesenchymal markers and p-Met in chemorefractory disease.

Conclusion
These results provide novel evidence on an important role of Met-dependent EMT in the adverse clinical behavior of SCLC and support clinical trials of Met inhibitors and chemotherapy in this fatal disease.

Background
Gefitinib or Erlotinib (EGFR-TKI) is the first choice of treatment for advanced NSCLC patients harbouring activating EGFR mutations. In addition to primary resistance, acquired resistance to EGFR-TKI eventually occurred in patients after an initial response. New agents have been developed to specifically inhibit the signaling pathways involved in mediating resistance. Because of heterogeneous resistant mechanisms, single agent usually had limited efficacy. Thus drug combination therapy may offer more benefits by synergistic interactions and avoidance of resistance emergence. We studied the combination of afatinib (an irreversible ErbB family inhibitor) and dasatinib (an inhibition of Src, BCR-ABL, PDGFR, Eph) in 8 different genetically characterized NSCLC cell lines to evaluate resistance mechanisms and molecular predicting biomarkers.

Methods
Growth inhibition was assessed by MTS assay. The interaction between two different drugs was evaluated by the method of Chou and Talalay. EGFR and k-Ras mutations were tested by direct DNA sequencing. EGFR, HER2, Src and downstream proteins FAK, Akt, MAPK42/44, Stat3, Stat5 expressions were measured by western blot.

Results
The efficacy of dasatinib against NSCLC cells in vitro is significantly more potent than gefitinib (p<0.001) and anti-EGFR monoclonal antibody cetuximab (p<0.05). Afatinib demonstrated increased activity against gefitinib and cetuximab resistance cell lines. Synergistic interaction (combination index, CI< 1) between dasatinib and afatinib was found in 7 NSCLC cell lines except A549. The efficacy of dasatinib in combination with afatinib is significantly stronger than that of cetuximab in combination with afatinib (p<0.001). In gefitinib resistant cell lines, growth inhibition by dasatinib was correlated with the ratio of p-Akt/t-Akt (p<0.05); total FAK expression is correlated to the growth inhibition by afatinib (p<0.05); CI results between dasatinib and afatinib were correlated with the active ratio of p-FAK925/t-FAK (p<0.05). In H1650 cell line, which was resistant to both dasatinib and afatinib, the combination of both drugs significantly inhibited the activity of p-EGFR845, p-FAK925, p-Src416, p-Akt473 and p-MAPK42/44 when comparing with that treated by afatinib or dasatinib alone (p<0.05). No significant inhibition was found on p-Stat3 and p-Stat5 by dasatinib. Afatinib was able to reduce the activity of Stat3 but not Stat5. No significant effect was shown on p-Stat3 and p-Stat5 by the combination of afatinib and dasatinib.

Conclusion
Our study showed synergistic combination of afatinib and dasatinib is more potent than cetuximab plus afatinib against gefitinib resistant NSCLC cells; it inhibited cell proliferation in H1650 cell line via affecting SFK/FAK, PI3K/PTEN/Akt, and Ras/Raf/MEK/ERK, but not JAK/Stat pathways. The level of p-FAK925/t-FAK may be a useful biomarker predicating synergism between afatinib and dasatinib for the treatment of gefitinib resistant NSCLC cells. P-Akt/t-Akt and total FAK expression may be related to sensitivity to dasatinib and afatinib respectively.

Background
Psammplin A (PsA), a novel DNA methyltransferase (DNMT) inhibitor and histone deacetylase (HDAC) inhibitor, was reported to induce radiosensitivity in lung cancer cell line. Concerning in vivo tissue distribution characteristics, PsA was found to be highly distributed to lung tissues. However, it appeared to be unstable in biological matrices. Here we report the DNMT inhibitory effect of PsA derivatives and their potential for radiation sensitization in lung cancer cell line.

Methods
A549 lung cancer cell line was used in verification of DNMT inhibitory effect with cultured cell DNA extraction kit. A549 cell line was exposed to radiation with or without a total of 9 PsA derivatives for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays.

Results
These 9 PsA derivatives all showed DNMT inhibitory effect and inhibited cell proliferation at the ranges of IC50 30–120μM. Furthermore, radiation clonogenic assays revealed that these compound shows radiosensitizing properties in A549 lung cancer cell line.

Conclusion
This preliminary data support the further investigation of these derivatives for use as radiation sensitizing agents with potential for clinical application.

Background
Background: New therapeutic strategies are urgently needed for small cell lung cancer (SCLC) which accounts for approximately 29,000 cases annually in the U.S. SCLC tumors have rapid doubling times and a propensity for early development of widespread metastatic disease. There have been no therapeutic advances in recent decades. The microtubule-targeting agent eribulin is a mechanistically-unique inhibitor of microtubule dynamics. Eribulin binds with high affinity to a maximum of 15 distinct beta-tubulin binding sites inhibiting microtubule growth by depolymerization and sequestration of tubulin into non-productive aggregates. Eribulin is currently FDA approved for the treatment of metastatic breast cancer patients with a demonstrated significant increase in overall survival in patients that are refractory or resistant to multiple chemotherapy agents. We investigated the effects of eribulin in a panel of 15-human SCLC lines.

Methods
Methods: Growth inhibition by eribulin was assessed by tetrazolium based assays at 5-days post treatment with varying drug concentrations and the growth inhibitory (GI50) was calculated. Erbulin induced cell cycle arrest was monitored following propidium iodine staining and analysis by FACS. Apoptosis was determined by using the DNA binding dyes YOPRO and PI and analysis by FACS.

Results
Results: Eribulin inhibited the growth of 13 of the cell lines. Four SCLC lines had GI50s of < 1 nM and 9-lines had eribulin GI50 values of 1-6 nM. These GI50 are similar to those reported in breast cancer cell lines. The eribulin IC50 value in the remaining 2-lines was > 100 nM. We are currently exploring clinical and gene expression differences that may explain the high sensitivity and resistance of different cell lines. A two to three-fold increase in the % cells in the G2/M phase of the cell cycle were observed following an 18-hour exposure to eribulin at concentrations of ≤ 5 nM in all cell lines growth inhibited by eribulin. Apoptosis assays are ongoing and in vitro studies of eribulin in combination with radiation are planned.

Conclusion
Conclusions: Erbulin inhibited the growth of SCLC lines and induced a significant G2/M arrest. Confirmation of growth inhibition of SCLC cell lines in an in vivo nude mouse model would support human studies in SCLC patients.

Background
In vivo evaluation is essential for development of lung cancer treatment. However, the subcutaneous xenograft models are not closely reproducing microenvironment of lung cancer. Although orthotopic implantation SCID mouse model of lung cancer presents lymphatic metastasis to mediastinum or pleuritis carcinomatosa with progression of disease, it has been difficult to evaluate the efficacy of treatment without sacrifice of model mouse. Positron-emission tomography-computed tomography (PET-CT) with the glucose analogue [18F] fluorodeoxyglucose (FDG) has been recently applied for evaluating tumor response to anticancer therapy. We have evaluated the utility of FDG PET-CT in orthotopic implantation SCID mice model of lung cancer.

Methods
Animals: 6 weeks male SCID mice (n=12). Cell line: Ma44-3 cloned from Ma44 (human squamous cell lung cancer cell line). Under sufficient anesthesia, mice were placed in the left lateral decubitus position. A 1-cm transverse incision was made in the right lateral skin just below the inferior border of the scapula. After intercostal muscles were exposed, 2 x 10[6] tumor cells/ml with 400 μg/ml Matrigel® was injected into the right lung in a volume of 10 μl (2.0x10[4 ]cells) of medium. Four or 5 days after implantation (6 mice on day 4 and other 6 mice on day 5), the SCID mice were examined with FDG PET-CT and mice whose lung tumors were identified were randomized to treatment group and control group. Treatment group mice received intraperitoneal injection of cisplatin (7mg/kg) on day 6 after implantation. All mice were examined with FDG PET-CT on day 8 and 13 after implantation. Tumor volume and maximal standardized uptake value (SUV max) of the lung tumor were calculated for all mice. All SCID mice were sacrificed on day 13 after implantation for histopathologic analysis.

Results
Six mice whose lung tumors were identified at the first FDG PET-CT were randomized to treatment group (n=3) and control group (n=3). The average growth rates (day 13 versus day 5 or 6) of tumor volume and SUV max of the treatment group were 144% and 108%, respectively, whereas the average growth rates of tumor volume and SUV max of the control group were 1470% and 271%, respectively.

Conclusion
Tumor growth and inhibition were evaluated by FDG PET-CT in orthotopic implantation SCID mice model of lung cancer. This in vivo evaluation system is useful for development of lung cancer treatment.

Background
Targeted therapeutics to mutant-BRAF and MEK have demonstrated remarkable efficacy in BRAF[V600E] metastatic melanomas. Unexpectedly, similar responses to the BRAF inhibitor vemurafenib (PLX4032) were not observed in BRAF[V600E] colorectal cancers (CRCs). Approximately 3% of lung cancers carry BRAF mutations, with only half being BRAF[V600E]. Recent case studies have reported responses to vemurafenib in patients with BRAF[V600E] NSCLC, suggesting that inhibition of the MAPK pathway with targeted therapeutics may be a feasible treatment strategy for BRAF-mutant lung cancers.

Methods
A panel of NSCLC cell lines carrying BRAF[V600E], BRAF[G469A], BRAF[G466V] and BRAF[L597V] mutations were assessed for responses to the MEK inhibitors Selumetinib (AZD6244), Trametinib (GSK1120212) and PD-325901 using standard drug response assays. Cell lines were also analysed by Western Blot analysis at various time-points following treatment with MEK inhibitors to determine the effects on the signalling pathways within the cells.

Results
Although all the cell lines displayed some sensitivity to MEK inhibition, the level of cell death observed with MEK inhibitors as single agents was minimal. The responses to MEK inhibition varied in cell lines carrying the same BRAF mutation indicating that other genetic differences could influence responses to inhibition of the MAPK pathway. Inherent co-expression of specific Receptor Tyrosine Kinases such as EGFR was detected in some of the cell lines and an increase in P-EGFR was observed following MEK inhibition. Increase in P-EGFR has been reported as the mechanism of resistance to vemurafenib in BRAF-mutant CRCs. In cell lines carrying non-V600E BRAF mutations, MEK inhibitor treatment also resulted in an increase in P-MEK, revealing a release of the MAPK pathway negative feedback resulting in upstream activation of MEK. We are currently focusing on assessing various combination therapies with MEK inhibitors, such as RTK inhibitors or second generation RAF inhibitors to overcome the feedback effect and drug-induced enhanced RTK activity. We have observed synergy in some cases, such as with the BRAF inhibitor AZ628 and the MEK inhibitor PD-901 in the H1755 cell line carrying the BRAF[G469A] mutation. In other cases we have observed additive effects suggesting co-existing oncogenic signalling.

Conclusion
Our findings suggest that different combination therapies are effective in the various mutant-BRAF cell lines and efficacy may depend on co-existing oncogenic ‘drivers’ and compensatory signalling mechanisms. We predict that combination treatment strategies for effective responses in BRAF-mutant NSCLCs may need to be determined on a personalised basis following tumour characterisation for co-expression of additional activators of the MAPK signalling pathway.

Background
Inactivation of STK11/LKB1 is one of the most common genetic events in lung cancer, and understanding the cellular phenotypes and molecular pathways altered as a consequence will aid the development of therapeutic strategies targeting LKB1-deficient cancers.

Methods
We report the comprehensive analysis of gene and protein expression patterns associated with LKB1 loss in lung adenocarcinomas, through which we identify hallmarks of altered tumor metabolism and down-regulation of the PI3K/AKT pathway.

Results
Significant differences are observed between human tumors and those derived from a genetically engineered mouse model of LKB1 loss. A 16-gene signature is predictive of both mutational and non-mutational LKB1 loss in human tumors. Cell lines expressing this signature show increased sensitivity to MEK inhibition, independent of mutations in RAS and RAF family members. Restoration of LKB1 in lung cancer cell lines down-regulates the gene expression pattern, attenuates FOXO3, and induces resistance to MEK inhibition.

Conclusion
These findings identify characteristic phenotypic features of LKB1-deficient tumors and identify LKB1 loss as a novel determinant of MEK sensitivity.

Background
Respiratory gated radiotherapy (RGRT) is a modern radiotherapy technique, which is increasingly used technique to take account of respiratory motion. One potential risk of RGRT is the possibility of sublethal repair during treatment leading more tumor cell survival. This study assessd the influence of elongation of treatment time in respiratory gated radiotherapy by measuring cell survival in vitro.

Methods
Human lung cancer cell lines, H460 and H1299, were irradiated with 6MV photons using Varian 21EX linear accelerator in two different delivery models. Cells of conventional model irradiated continuously while the other cells irradiated with gated delivery. Doses of 2 Gy, 4 Gy and 8 Gy were studied. In conventional model, treatment times were 0.5 min for 2 Gy and 3min for 8 Gy including latency for multiple field. In gated delivery model, treatment times were 2.5 min for 2 Gy and 11 min for 8 Gy with 20% gating efficacy.

Results
H1299 cells were radioresistant than H460 cells (P<0.001). Elongation of treatment time caused significant increase in survival fraction in H1299 cell line (p=0.046). In H460 cell line survival fractions also increased but differences were not statistically significant (p=0.107). Relative differences between two delivery models in log scale survival fraction were not increased in higher dose.

Conclusion
The biologic effects of protracted delivery with gated technique were different between cell lines, suggesting influence of diversity of sublethal damage repair. More radioresistant cell lines were affected by elongation of treatment time. These results suggest that potential risk of sparing tumor in prolonged delivery time with gated radiotherapy should be considered in resistant tumor at the clinic.

Background
The pro-apoptosic Bcl-2 family member BIM is known to be a critical mediator of targeted therapy-induced apoptosis in lung cancer. Also, pretreatment level of BIM strongly predicted the capacity of EGFR inhibitors to induced apoptosis in EGFR-mutant cancers. But prevous studies were mostly done in two-dimensional (2D) cell cultures (monolayers).

Methods
So we used three-dimensional(3D) spheroid culture model that were proved to be a valuable platform to study acquired multicellular apoptotic resistance and effectively recapitulate some of the complexity of solid tumors such as mesothelioma. 3D spheroids with EGFR-mutant cell lines (H3255, HCC827, H1650, 11-18, H4006) were generated in polyHEMA-coated plates and TKI treatments were done both in 2D and 3D. The degree of apoptosis and expressions of BIM by immunoblotting before and after treatment were compared.

Results
Contrary to other solid tumors, spheroids showed more prominent apoptosis to TKI than monolayer. Spheroids expressed more BIM than did monolayers except H4006. BIM was not expressed in H4006 (2D, 3D), but BIM expression was found to be elevated after TKI treatment only in spheroids. Higher level of BIM in spheroids had the tendency of higher apoptotic response to TKI.

Conclusion
3D spheroids cultures are useful in studying apoptotic mechanism in EGFR-mutant lung cancer. BIM expression before and after treatment of TKI might be useful predictor in EGFR-mutant spheroids model. So therapies that upregulated BIM expression can improve the efficacy of TKI therapy.

Background
Current treatments for mesothelioma typically increase median survival by a matter of months. Progress in treatment has been hampered by lack of a suitable small animal model, which could guide clinical advances, given limited numbers of patients eligible for clinical trials. To this end we recently developed a transgenic mouse model of mesothelioma in which the viral oncogene, SV40TAg (TAg) is directed to mesothelial cells by use of the cell type specific mesothelin promoter. MexTAg mice develop mesothelioma rapidly and uniformly, but only following exposure to the natural carcinogen, asbestos. The model closely mimics the human disease and is thus ideal for both rapid analysis of novel therapeutic studies and for investigating factors that might act synergistically with asbestos to cause disease. Since all MexTAg mice develop mesothelioma following asbestos exposure the model is highly suitable for early intervention and cancer prevention studies. An effective cancer prevention strategy for the millions of people who have been exposed to asbestos could have enormous benefit worldwide. Epidemiological evidence indicates that supplementation with some dietary factors or use of common drugs such as statins and non-steroidal anti-inflammatory drugs is associated with a lower incidence of cancer.

Methods
not applicable

Results
We previously reported that dietary supplementation with a number of antioxidants did not alter the time to develop disease nor overall survival, despite the widely accepted hypothesis that asbestos catalyzed production of reactive oxygen and nitrogen species contribute to the development of this cancer. We have extended these studies to test whether vitamin D, non-steroidal anti-inflammatories, statins and some other candidate diets could alter the pattern of disease in the MexTAg model. Supplemented diets were provided at levels based on published data and began 2 weeks prior to asbestos exposure in order to maximize our chance of detecting a benefit. Preliminary data suggests a single agent or nutraceutical may not be enough to prevent the multiple pathways involved in tumorigenesis. This is somewhat supported by epidemiological data from the Wittenoom cohort of asbestos exposed workers and residents.

Conclusion
In conclusion, we think it is unlikely that antioxidants, anti-inflammatories or other nutrient-specific dietary supplements will moderate the rate of mesothelioma in asbestos exposed populations.

Background
Malignant pleural mesothelioma (MPM) is an aggressive cancer of the mesothelial cells and is becoming a worldwide health burden. Progress in the treatment of MPM remains difficult, underscored by the fact that no single treatment option has proven particularly effective and chemo-resistance is a common problem. However, epigenetic modifiers, such as histone deacetylase inhibitors (HDi) have emerged as a novel class of anti-cancer agent and are currently undergoing clinical trials in numerous cancers. There is also evidence to suggest that HDAC expression may play a role in the development of chemo-resistance. We investigated the levels of HDACs in MPM cell lines, patient samples and determined the effect of HDi on MPM cisplatin sensitive and resistant cell lines.

Methods
A panel of MPM cell lines (n=16) was screened for the expression of HDAC11, Class I (HDAC1/2/3/8) and Class II (HDAC4/5/6/7/9/10) HDACs at the mRNA level (RT-PCR) and at the protein level (Western Blot). The HDAC expression profile was determined in an isogenic cell line model consisting of a cisplatin sensitive (Parent) and resistant (CisR) MPM cell line, P31. In addition, HDAC expression was examined in panel of twenty patient samples (benign/biphasic/sarcomatoid/epithelial). Also, MPM TMAs were stained by imummo-histochemistry for HDAC5 expression. Furthermore proliferation assays (BrdU ELISA) were performed to determine the effect of two HDi on the p31 cell lines. The HDi used were Vorinostat (pan HDAC inhibitor) and 19i (selective HDAC5 inhibitor).

Results
HDAC11, Class I and II HDACs were detected to varying degrees at the mRNA and protein level within our cell line panel. In the P31 isogenic parent/cisplatin resistant, HDAC protein expression was decreased (HDAC2/3/4/5/7) in the CisR compared with the Parent. HDAC5 was significantly reduced at both the protein and the mRNA level (p<0.05). The expression of HDACs 1/2/3/5 were increased in the MPM patient samples (n=15) compared with benign (n=5) (HDAC2/3/5, p<0.05). HDAC5 staining in a cohort of MPM samples, demonstrated no significant association between survival and a low HDAC5 score. However females had a greater median survival than their male counterparts. Vorinostat and 19i significantly reduced the proliferative capacity of the p31 Parent and CisR. SAHA was more effective in the Parent cells compared with CisR. The effect of 19i was similar in both cell lines.

Conclusion
Altered HDAC expression was observed in an isogenic Parent and CisR MPM cell model. Work ongoing in non-small cell lung cancer (NSCLC) has also demonstrated significantly reduced HDAC5 levels in CisR compared with Parent. This may suggest that a reduction in HDAC expression is involved in cisplatin resistance. Reduced levels of HDAC5, in an MPM TMA, were not associated with survival. It should be noted, however that patient numbers were small and biphasic and sarcomatoid sub-types had the lowest expression levels of HDAC5. We are currently increasing our patient numbers for an additional IHC study. HDi significantly reduced the proliferative capacity of CisR and Parent MPM cells. HDAC levels may represent an important biomarker in stratifying patients for future epigenetic targeted therapies in MPM.

Background
Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer associated with exposure to asbestos. Untreated, MPM has a median survival time of 6 months, and most patients die within 24 months of diagnosis. There is an urgent unmet need to identify new therapies for this disease. Receptor tyrosine kinases (RTK) are an area of active research in cancer therapy. The identification of “addicted” signalling networks could rapidly lead to candidate inhibitors (RTKi) that could potentially be uused to target MPM. We have previously identified RON/MST1R as a candidate therapeutic target in MPM. Additional RTKs identified by other studies include Axl and c-MET. The agent BMS-777607 is an orally bioavailable small molecule with the capacity to inhibit c-MET, RON/MST1R, Axl and Tyro3 (at nanomolar concentrations) and has just completed a Phase I/II trial (ClinicalTrials.gov Identifier: NCT00605618). This drug may therefore have applicability in MPM.

Methods
A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of Tyro3, c-MET, RON/MST1R and Axl by RT-PCR. A cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies were subsequently examined for the expression of these RTKs. The effects of BMS-777607 on MPM cellular proliferation and viability are being assessed.

Results
Expression of various RON/MST1R isoforms, c-MET, Tyro3 and Axl were observed in all cell lines. Significantly higher expression of all genes were found in the malignant tumour material versus benign pleura. The effects of BMS-777607 on cellular proliferation/viability are ongoing and the results will be presented at the meeting.

Conclusion
BMS-777607 represents a unique compound capable of targeting four RTKs whose expression are significantly elevated in maligant MPM. Given the many indications that these RTKs are associated with “addicted” RTK networks, this compound may therefore have potential clinical utility in the clinical management of MPM.

Background
Activation of the mTOR pathway is a common down-stream mechanism of resistance to Epidermal Growth Factor receptor (EGFR) tyrosine kinase inhibitors. CC-223 (Celgene) is an mTOR kinase inhibitor under clinical development. We evaluated CC-223 in combination with erlotinib to overcome resistance to EGFR tyrosine kinase inhibition in non-small cell lung cancer (NSCLC) cell lines and xenografts in nude mice.

Methods
A panel of 18 NSCLC cell lines was used to evaluate the effect of erlotinib and CC-223 treatment on cell growth using an MTT assay. Intermediately (IC50 >1 and <10 mM) and highly (IC50 >10 mM) resistant cell lines to erlotinib were used in analyses of additive/synergistic effects for the combination treatment with CC-223.

Results
CC-223 demonstrated anti-proliferative activity in NSCLC cell lines with various degrees of sensitivity as reflected in different IC50 values, ranging from 0.15 to 12 mM. In combination with erlotinib, CC-223 showed synergistic anti-proliferative effects in NSCLC cells resistant to erlotinib with combination indices as low as 0.1-0.2. In vivo studies in mice xenografts demonstrated a strong synergistic effect of the combination treatment of erlotinib and CC-223 with a 90% decrease of tumor volume compared to untreated and 88% compared to erlotinib treated for A549 cells. IHC analyses of apoptosis and proliferation in the tumors are ongoing; mature data will be presented at the meeting.

Conclusion
The mTOR kinase inhibitor CC-223 demonstrated anti-proliferative activity in a panel of NSCLC cell lines. In NSCLC cells resistant to the EGFR tyrosine kinase inhibitor erlotinib, combining erlotinib with CC-223 demonstrated a synergistic anti-proliferative effect. In vivo studies of tumors in xenografted mice also demonstrated synergistic anti-tumor effects with the combination treatment even in erlotinib resistant tumors. The present data suggest that mTOR inhibition using the mTOR kinase inhibitor CC-223 may be a therapeutic strategy to overcome resistance to EGFR tyrosine kinase inhibitors in NSCLC.

Background
Nesfatin-1 is an anorexigenic neuropeptide. We have previously reported that serum nesfatin-1 level is decreased in lung cancer patients. In this study, we aimed to investigate possible effects of serum nesfatin-1 level in survival and chemotherapy response of non-small cell lung cancer patients.

Methods
Twenty-eight non-small cell lung cancer patients were included in this study. Data were obtained from clinical records and our previously study about nesfatin-1. The patients were divided into two groups according to high nesfatin-1 level or low. Survival data were compared between these groups. Moreover, serum nesfatin-1 levels were investigated in chemotherapy responder and non-responder patients.

Results
Three of patients received supportive care alone. Twenty-five patients were treated with platinum-based doublet chemotherapy. In chemotherapy non-responder patients, serum nesfatin-1 level was more lower than in the patients achieved clinical benefit (0.38 ng/ml ±0.12 ng/ml vs 0.55 ng/ml ±0.21 ng/ml; p:0.044). Patients with low nesfatin-1 level had shorter overall survival (OS), but it is not statistically significant (median OS 7.3 months vs 13.8 months, respectively; p: 0.829). Serum nesfatin-1 levels were not also different in between patients survived more than 12 months and not (0.62 ng/ml ±0.22 ng/ml vs 0.46 ng/ml ±0.19 ng/ml; p:0.077).

Conclusion
This preliminary study suggests that serum nesfatin-1 level is not prognostic factor in non-small cell lung cancer, but may be a predictive marker for chemotherapy response.

Background
Despite adjuvant chemotherapy improving overall survival of resected Stage II and III non-small cell lung cancer (NSCLC), acute and late toxicities of chemotherapy have highlighted the need to better predict which patients will benefit from treatment. RLIP76 is a ubiquitously expressed multi-functional transporter that is associated with cisplatin and vinorelbine resistance. Our aim was to analyse the prognostic and predictive value of RLIP76 expression in NSCLC.

Methods
We identified 367 NSCLC patients resected between 1996 and 2009. A tissue microarray was created and immunohistochemistry (IHC) performed with an anti-human RLIP76 rabbit polycloncal antibody (Millipore, Temecula, CA). The intensity (0-3) and proportion of tumour cells (0-100) with staining was scored. The product of RLIP76 intensity and proportion of tumour cells staining was calculated (range 0-300) and divided into high (>100) and no/low expression (≤100). RLIP76 expression was correlated with clinical features and patient outcome.

Results
IHC was available for 285 patients, 173(60.7%) of which were male. Number of patients according to stage I, II, III and IV was 150(52.6%), 83(29%), 44(15.4%) and 8(3%), respectively. RLIP76 was overexpressed in 117(41%) specimens. There was no relationship between RLIP76 expression and stage, histology, sex or age. High RLIP76 expression was associated with an improved prognosis on univariate (HR 0.62, CI 9.44-0.90, p=0.012,Figure 1) and multivariate analysis (HR 0.57, CI 0.39-0.83, p=0.003). Adjuvant chemotherapy was also associated with an improved survival on multivariate analysis (HR 0.52, CI 0.33-0.82, p=0.005). When stratifying by RLIP76 expression, the benefit of chemotherapy was limited to patients with no/low expression (HR =0.44, CI 0.24-0.8, p=0.008)(Figure 2). No benefit of chemotherapy was seen in patients with high RLIP76 expression (HR=0.75, CI 0.34-1.63, p=0.5). Figure 1 Figure 2

Conclusion
High RLIP76 expression is associated with an improved prognosis in resected NSCLC.Interestingly no/low RLIP76 expression may predict for benefit of adjuvant chemotherapy. Further studies are needed to confirm these results.

Background
Cabozantinib (C) is a potent ATP-competitive inhibitor of MET and vascular endothelial growth factor receptor 2 (VEGFR2) along with KIT, RET, AXL, TIE2, and FLT3. Hepatocyte growth factor (HGF), the ligand of MET, and VEGF act synergistically to promote angiogenesis. There are currently no widely accepted prognostic or predictive biomarkers for anti-angiogenic agents.

Methods
This is a retrospective correlative biomarker study from the phase 1b/2 trial of C+/-E in stage IIIB-IV NSCLC. All pts had to fail prior therapy with E. Both drugs are oral and dosed daily. C dosing is in the free-base equivalent weight. In phase I, there was a 2 week lead in with E and the cohorts included: 1A (60 mg C+150 mg E), 2A (60 mg C+100 mg E), 3A (100 mg C+100 mg E), 4A (100 mg C+50 mg E), and 2B (40 mg C+150 mg E). In phase II, both drugs started simultaneously: Arm A (100 mg C) and Arm B (100 mg C+50 mg E). Pts were included in the study if a pre-E+C and post-C > day 29 plasma sample was available. The Milliplex 13-plex and Luminex 51-plex assays were used. For this preliminary analysis, select markers previously implicated in angiogenesis (bFGF, VEGF, sVEGFR1-3, IL-6, IL-8, IL-12, IL-17, PDGF-BB, ICAM-1, VCAM-1) and those of interest (ligands of KIT and MET- SCF and HGF, respectively) were analyzed. Log transformed mean fluorescence intensity (MFI) values and Wilcoxon Rank paired sum tests were used to detect changes from day 1-29. A change from baseline was noted to be significant if at least 15% (median) with α<0.05 (2-sided) and a trend if 10-15% (median) with α<0.08 (2-sided).

Results
73 pts included: 52 phase I and 21 phase II; median age 60 years; 23M/50F; 56.2% nonsmoker; 91.8% adenocarcinomas. The pts with samples from both time points were divided into two groups due to limited sample size and included: Group R (complete/partial response and stable disease> 6 months; n=22) and Group NR (stable disease< 6 months and progressive disease; n=51). The only marker that changed in a single direction in all subjects within a group was sVEGFR2 in group R. Overall, significant decreases were noted in sVEGFR1-3, IL-6, PDGF-BB, and trended in IL12p70 and IL-17. By subgroups: Group R had significant decreases in bFGF, VEGF, sVEGFR1-3, IL-6, IL-8, IL-12(p40+p70), IL-17, PDGF-BB, SCF, and trended in HGF (median 10.1% ↓, p=0.0275); and Group NR had significant decreases in sVEGFR2-3, IL-6, PDGF-BB, and trended in sVEGFR-1, IL-6, and IL-17.

Conclusion
Both groups R+NR had a decrease in sVEGFR-2, suggesting that this is a marker of treatment with C rather than a marker of response. However, overall group R had larger dynamic decreases of immune markers than group NR. HGF, which is targeted downstream by C and plays a role in angiogenesis and E resistance, had a trend to decrease in group R but not group NR. This study is retrospective with a small sample size, imbalanced numbers per response subgroup, and is exploratory in nature.

Background
The T790M mutation is considered to be the major mechanism of acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). However, its clinical implication in patients with non-small cell lung cancer (NSCLC) is yet determined.

Methods
NSCLC patients with acquired resistance to initial EGFR TKIs such as gefitinib or erlotinib were identified, and post-progression tumor specimens were prospectively collected for T790M mutation analysis. Clinical features including the pattern of disease progression (intrathoracic only versus extrathoracic), treatment outcomes for initial or subsequent TKIs, post-progression survival, and overall survival were compared between patients with and without T790M.

Results
Out of 70 cases, 36 (51%) were identified to have the T790M mutation in the rebiopsy specimen. There was no difference in the pattern of disease progression, progression-free survival for initial TKIs (12.8 and 11.3 months), post-progression survival (14.7 and 14.1 months), or overall survival (43.5 and 36.8 months) in patients with and without T790M. In total, 34 patients received afatinib after post-progression biopsy as a subsequent treatment. Among them, six (18%) achieved objective response. The median progression-free survival for afatinib was 3.7 months for the entire group, and 3.2 and 4.6 months for the groups with (n = 21) and without (n = 13) T790M, respectively (p = 0.33). Figure 1Figure 2

Conclusion
Although T790M had no prognostic or predictive role in the present study, further research is necessary to identify patients with T790M-mutant tumors who might benefit from new treatment strategies.

Background
Molecular testing in lung cancer has dramatically evolved over the past few years with a large number of targeted therapies and associated biomarkers. This has created a demand for tissue preservation. A careful consensus for prioritization of the different assays is needed to identify the right therapy for the patient. It is necessary to have protocols available that give access to results rapidly and accurately without depleting the sample throughout the process. We have designed a prospective study to demonstrate an efficient biomarker testing workflow (EGFR, ALK and KRAS) in a real clinical setting that preserves limited, valuable clinical samples

Methods
This prospective study was conducted at two independent pathology laboratories (Laboratorio de Dianas Terapeuticas of Madrid, Queen’s University of Belfast). Each site performed tissue sectioning for diagnosis and molecular testing according to laboratory protocols or to the assay specific package insert. Digital pathology was used to annotate specimens. NSCLC specimens were obtained by each lab (surgical resections and small biopsies of primary and metastatic lesions), excluding cytology specimens, with the exception of cells blocks. EGFR and KRAS mutation testing was performed using the cobas[®] EGFR Mutation Test and the cobas[®] KRAS Mutation Test (both CE-IVD), using a single 5µm section per test. FISH analysis was performed using the Vysis LSI ALK probe. Figure 1

Results
A total of 103 cases were included. 100% of the specimens were successfully tested for EGFR mutation status. Failure rates were 1.9% for ALK and KRAS analysis. Failed DNA extraction/PCR amplification was 3.9%/2.9% for EGFR and 5.8%/12.6% for KRAS. The prevalence of molecular alterations identified in EGFR (4% and 13.2%), ALK (4% and 7.8%) and KRAS (26% and 29.4%) was somewhat similar to that described in earlier studies. ALK rearrangements were mutually exclusive with EGFR and KRAS mutations. The median turnaround time was almost identical between sites: 6-7 days for all biomarkers (EGFR range 1-12 days, ALK range 2-14 days and KRAS range 1-17 days).

Conclusion
We have demonstrated that it is feasible to incorporate this protocol into the routine analysis of thoracic samples. The pattern of retesting to achieve full analysis in all samples suggests that, while EGFR testing is easily achievable with a one-section approach, the analysis of KRAS and ALK may require another round of testing in a small percentage of cases. Our results thus provide a pre-analytical and analytical rationale for comprehensive genotyping in patients with NSCLC.

Background
Major obstacles limiting the clinical success of EGFR TKI therapy in EGFR mutant non-small cell lung cancer (NSCLC) patients are heterogeneity and variability in the initial anti-tumor response to treatment. The underlying molecular basis for this heterogeneity has not been explored in patients immediately after initiation of therapy.

Methods
We conducted CT-guided core needle biopsies immediately prior to erlotinib treatment initiation and at 6 days and 60 days post erlotinib initiation in a patient with histologically confirmed NSCLC harboring an established activating mutation in EGFR. DNA and RNA from each of the frozen biopsies were analyzed by whole exome sequencing and whole transcriptome sequencing, respectively. High-resolution CT images were also obtained at the time of each biopsy to assess the degree of molecular and radiographic responses observed.

Results
Two established activating somatic mutations were identified in EGFR (p.G719A and p. R776H). Gene expression analysis revealed that several proapoptotic genes including BID, CASP3 and several growth inhibitory genes including GADD45B, GADD45G were upregulated at 6 days post erlotinib treatment, while at 60 days their expression levels decreased to below pretreatment levels. Other proapoptotic genes such as BAD and BAX and were noted to be upregulated most significantly 60 days, as was growth inhibitory gene CDKN1A. In contrast, the growth-promoting genes CCNB1 and CCND3 exhibited a progressive decrease in expression across time points. Whole exome sequencing demonstrated the progressive evolution of global copy number abnormalities. High-resolution CT scans revealed no interval radiographic change in tumor size after 6 days of erlotinib treatment, and a decrease in tumor size 60 days into therapy. No clinical complications were encountered.

Conclusion
This study is the first reported longitudinal integrated genomic analysis of EGFR-mutant NSCLC in a patient treated with an EGFR TKI. We documented the feasibility, safety and utility of this strategy to establish initial drug efficacy at the molecular level prior to any radiographic evidence of response (6 days), as well as evidence that acquired resistance can emerge early in the course of TKI therapy. Serial integrated genomic analysis is ongoing in other TKI treated patients to enhance the management of NSCLC patients on targeted therapy.

Background
Circulating tumor cells (CTCs) are cells that originate from a primary solid tumor and are found transiting the circulatory system. CTCs are expected to provide useful clinical information on biology of their primary, as ”liquid biopsy.” We have developed a novel cell-sorting system equipped with a disposable microfluidic chip (On-chip Sort, On-Chip Biotechnologies, Tokyo, JAPAN). At American Association for Cancer Reseach meeting 2013, we previously reported about its CTCs enumeration capability when performed in a clinical setting. Currently, On-chip Sort enables recovery of more CTCs than conventional cell sorting systems for further characterization.

Methods
In a preclinical study, PC-9 and H1975 human lung cancer cells harboring EGFR mutations (E746_A750del and L858R, T790M, respectively) were spiked into the blood from healthy donors. After samples were negatively enriched using anti-CD45-coated magnetic beads, the spiked cancer cells in the samples were captured by On-chip Sort. The captured tumor cells were subjected to mutation detection by ARMS/Scorpion PCR assay. A clinical feasibility study was then conducted in lung cancer patients harboring EGFR mutations.

Results
On-chip Sort performed recovery of the spiked PC-9 and H1975 cancer cells (5 cells in 4 ml of blood) in a preclinical experiment. Successively, we were able to detect the EGFR mutations from the captured cells. In a clinical feasibility study, 4 blood samples from lung cancer patients harboring EGFR mutation were collected and were evaluated. All the samples were successful in capturing CTCs by On-chip Sort. ARMS/Scorpion PCR assay detected the EGFR deletion mutation from two of the samples (50%).

Conclusion
The preclinical study and the results of the clinical feasibility study suggested the possibility of the On-chip Sort assay to detect EGFR mutations from peripheral blood of patients with lung cancer. Further investigation is going to be conducted to evaluate the correlation of EGFR mutations in captured CTCs and primary lesions.

Background
Clinical implications of KRAS mutations in advanced non-small cell lung cancer remain unclear.

Methods
Kras mutation status was identified by direct sequencing test in 844 specimens that were diagnosed of NSCLC at Samsung Medical Center from 2006 to 2011. This study included stage IV NSCLC patients who were treated with systemic chemotherapy. We retrospectively evaluated the prognostic and predictive value of KRAS mutations in patients with advanced NSCLC.

Results
Among 484 patients with available results for both KRAS and EGFR mutations, 39 (8%) had KRAS and 182 (38%) EGFR mutations, with two cases having both mutations. The median overall survivals for patients with KRAS mutations, EGFR mutations, or both wild types were 7.7, 38.0, and 15.0 months, respectively (P < 0.001). The KRAS mutation was an independent poor prognostic factor in the multivariate analysis (hazard ratio=2.6, 95% CI: 1.8–3.7). Response rates and progression-free survival (PFS) for the pemetrexed-based regimen in the KRAS mutation group were 14% and 2.1 months, inferior to those (28% and 3.9 months) in the KRAS wild type group.

Conclusion
KRAS mutation tended to be associated with inferior treatment outcomes after gemcitabine-based chemotherapy, while there was no difference regarding taxane-based regimen. Although the clinical outcomes to EGFR tyrosine kinase inhibitors (TKIs) seemed to be better in patients with KRAS wild type than those with KRAS mutations, there was no statistical difference in response rates and PFS according to KRAS mutation status when EGFR mutation status was considered. Two patients with both KRAS and EGFR mutations showed partial response to EGFR TKIs. Although G12D mutation appeared more frequently in never smokers, there was no difference in clinical outcomes according to KRAS genotypes. These results suggested KRAS mutations have an independent prognostic value but a limited predictive role for EGFR TKIs or cytotoxic chemotherapy in advanced NSCLC.

Background
Neoadjuvant chemotherapy is used to help downstage cancer, and is widely used in locally-advanced breast cancer. Studies of Ki67 proliferation index in breast cancer have been fairly extensively evaluated. Comparison of core and surgical specimens in breast cancers without exposure to chemo- or radiotherapy revealed technical variation of up to 20% in the Ki67 index score. In non-small cell lung cancer (NSCLC), however, little is known about the association of rate of change of Ki67 after neoadjuvant chemotherapy with radiographic response and clinical outcomes. We surveyed NSCLC from patients treated with neoadjuvant chemotherapy.

Methods
NSCLC patients treated with neoadjuvant chemotherapy were identified from 3 Hungarian hospitals and 1 community hospital in the United States. Matched pre-chemotherapy and post-surgical resection formalin-fixed, paraffin-embedded (FFPE) tumor specimens were collected and the Ki67 index was scored under an IRB exemption. We set an absolute difference of 20% between pre-chemotherapy and post-resection Ki67 scores as “meaningful” to avoid possible technical variation reported in the literature. Radiographic response to neoadjuvant chemotherapy by RECIST 1.0 criteria was also measured. Fisher’s exact test was used to measure the relationship between gender, histology, and type of chemo with “meaningful” Ki67 index. Logistic regression was used to test the relationship between Ki67 index decline rate and outcome subgroup (response/no response). Decline rate was defined as a ratio of decrease of Ki67 to its level at baseline.

Results
63 matched cases were identified. 46 cases were analyzable for pre-chemotherapy and post-operative Ki67, and chemotherapy regimen; 40 cases also had response criteria by RECIST. Of the 46 cases, the median patient age was 59 years (range 40-77), 23 were men, and 30 of 34 had a smoking history. There were 24 adenocarcinomas and 22 squamous cell carcinomas. Stages I, II, III, and IV were 2, 9, 31, and 4; respectively. All but two patients received a platinum-doublet, with 24 containing gemcitabine. 5 patients also received neoadjuvant radiotherapy. The mean Ki67 index scores were 35% (range 1-100%) pre-chemotherapy and 32% (0-100%) post-resection. 9 patients (19.6%) had a paradoxical “meaningful” increase in Ki67 index after neoadjuvant chemotherapy. Of the 40 patients with RECIST response data, there was 1 complete response, 34 partial responses, 4 stable diseases, and 1 disease progression. There were no statistically significant differences between gender, histology subtype, or type of platinum doublet administered associated with this paradoxical increase. There was no statistically significant difference in Ki67 decline rate between responders and nonresponders.

Conclusion
In this cohort of patients, there was a paradoxical “meaningful” increase in Ki67 index after neoadjuvant chemotherapy in ~20% of patients, without a clear association between histology or platinum doublet administered. For patients receiving neoadjuvant chemotherapy, the Ki67 index decline rate was not associated with radiographic response. Approximately 1/5 of NSCLC may have selection of tumor cells for a higher proliferative index when undergoing neoadjuvant platinum-based chemotherapy. The effect of this on progression-free and overall survival is being evaluated.

Background
Pleomorphic carcinoma of the lung is a rare epithelial tumor, and its clinicopathological characteristics and prognostic factors are still controversial. Up-regulation of E-cadherin repressors is known to increase tumor cell invasion and motility in non-small cell lung cancer cells. The aim of this study was to clarify the clinicopathological characteristics and prognostic factors, such as E-cadherin repressors (e.g., Zinc finger E-box-binding homeobox [ZEB], Snail, and Twist), in pleomorphic carcinoma.

Methods
We reviewed 2,328 cases of resected lung cancers that occurred between 1988 and 2011 and identified 62 patients with pulmonary pleomorphic carcinoma. Immunohistochemical staining for ZEB, Snail, and Twist was performed, and nuclear expression was estimated by the percentage of positive cells. The patients were divided into two groups; a diffuse expression group (≥75%) or a focal expression group (<75%). Clinicopathological variables and the expression of E-cadherin repressors were investigated retrospectively to analyze prognostic factors for the disease-specific survival rate of 42 patients after lung resection.

Results
The TNM pathological stages of pleomorphic carcinoma were classified as follows: 7 (11.2%) cases with stage IA, 15 (24.1%) with stage IB, 3 (4.8%) with stage IIA, 20 (32.2%) with stage IIB, 10 (16.1%) with stage IIIA, 4 (6.4%) with stage IIIB, 3 (4.8%) with stage IV. The 5-year disease-specific survival rate after pulmonary resection was 68.3%. Forty-seven (75.8%) tumors contained at least 10% spindle and/or giant cells, and the other fifteen (24.1%) consisted entirely of spindle cells and giant cells. An adenocarcinoma component was found in 34 (54.8%) cases, a squamous cell carcinoma component in 7 (11.2%) cases, an adenosquamous cell carcinoma component in 4 (6.4%) cases, and a large cell carcinoma component in 2 (3.2%) cases. The pleomorphic carcinoma patients were divided into five groups according to the predominant epithelial component, and there were no significant differences in the disease-specific survival rate between the groups. Those with a predominance of spindle-cell or giant-cell components also showed no difference in disease-specific survival. Nuclear ZEB-1 expression was positive only in the pleomorphic component. Diffuse expression of ZEB1 was found in seven patients. Snail and Twist were positive in epithelial and pleomorphic components, but at various levels; however, expression tended to be higher in the pleomorphic component. Thirteen patients had diffuse expression of Snail, and eight had diffuse expression of Twist. Using multivariate analysis, lymph node metastasis, pleural invasion, and diffuse expression of ZEB1 in the pleomorphic component all predicted poorer disease-specific survival (p=0.007, 0.022, and 0.016, respectively). Nuclear ZEB1 expression was higher in cases with lymphatic permeation compared to those without lymphatic permeation, but this association was not statistically significant (p=0.107).

Conclusion
Several unfavorable prognostic factors for pulmonary plemorphic carcinoma have been reported, such as massive necrosis, TNM stages, lymph nodes metastasis, pleural invasion, and complete resection. Our current study showed that lymph node metastasis, pleural invasion, and diffuse expression of ZEB1 in the pleomorphic component suggested a worse prognosis for pulmonary pleomorphic carcinoma. ZEB1 may promote the aggressiveness and invasiveness of this malignant tumor.

Background
Pemetrexed (PEM) inhibits multiple enzymes in the folate (F) pathway. Several studies show that genetic polymorphisms in these enzymes influence the efficacy and toxicity of PEM. We aimed to investigate the relationship between genetic polymorphisms associated with the F pathway and clinical outcomes of Japanese patients with advanced non-squamous non-small cell lung cancer (NSQ-NSCLC) treated with PEM plus cisplatin (CIS).

Methods
We analyzed 34 polymorphisms in 14 genes associated with the F pathway in NSQ-NSCLC patients treated with PEM plus CIS: ABCC11, ADA, ATIC, DHFR, ERCC1, FPGS, GGH, MTHFD1, MTHFR, MTR, MTRR, SHMT1, SLC19A1, and TYMS. These polymorphisms were compared with clinical outcomes such as response, toxicity, and progression-free survival (PFS) using Pearson’s χ[2] test and the log-rank test.

Results
All 56 patients were Japanese, with a median age of 62 years; 57.1% were male, 96.4% had an Eastern Cooperative Oncology Group Performance Status of 0–1, 96.4% had stage IV disease, and 94.6% had adenocarcinoma. The response rate, disease control rate, and median PFS were 32.2%, 78.6%, and 4.7 months, respectively. Of the 38 polymorphisms tested, none were associated with response or toxicity, but 2 single nucleotide polymorphisms (SNPs) (in the gamma-glutamyl hydrolase [GGH 452C>T] and methionine synthase [MTR 2756A>G] genes) were significantly associated with PFS. Patients harboring the GGH-C452C variant had significantly longer PFS (5.6 vs 2.8 months; p < 0.0001) than those with the C452T or T452T variants. Further, patients harboring the MTR-A2756A variant had significantly longer PFS (5.3 vs 3.7 months; p = 0.036) than those with the A2756G variant. In addition, among patients with the GGH-C452C variant, those harboring the MTR-A2756A variant had significantly longer PFS (5.9 vs 4.3 months; p = 0.044) than those with the A2756G variant.

Conclusion
SNPs in GGH and MTR seem to predict differences in PFS in NSQ-NSCLC patients treated with PEM plus CIS, and a combination of these 2 SNPs may predict differences in PFS more accurately. These results should be validated in larger, adequately designed prospective studies.

Background
The ICE COLD-PCR (Improved and Complete Enrichment COamplification at Lower Denaturation Temperature PCR) technology is capable of high sensitivity mutation detection. The ICE COLD-PCR (ICP) reaction contains a reference sequence oligonucleotide (RS-oligo) that hybridizes to both alleles but will dissociate from the mutant strand at the critical temperature for all mutation types: point mutations, insertions, deletions and indels. Following ICP, samples with mutations present at 0.1% in the original sample can be enriched and determined using standard Sanger sequencing. Next-Generation Sequencing (NGS) allows high-throughput analysis of somatic mutations in cancer using targeted resequencing panels of genes. Thus a broad mutation signature of tumors can be determined. However, the level of detection is ~4% for mutations unless a higher depth of coverage is used; this comes at the price of reducing the number of samples that can be analyzed on a chip. ICP enrichment of mutations prior to NGS mimics an increased "depth of coverage" without reducing the throughput per chip.

Methods
Proof of concept experiments were performed using low level mutations: KRAS G12R (0.5, 0.1, 0.05%) PIK3CA E542K and E545K (1.0, 0.5, 0.1%) and EGFR Exon 19 E746_A750del (0.5, 0.1, 0.05%). In addition, DNA isolated from FFPE tissues and matched plasma were amplified by standard PCR or enriched by ICP and subsequently analyzed for PIK3CA, KRAS, BRAF and EGFR mutations using the Ion AmpliSeq Cancer Hotspot Panel on the Ion Torrent Personal Genome Machine (PGM) with the Variant Caller Plugin.

Results

ICE COLD-PCR Amplification of FFPE DNA with Sequence Confirmation by Sanger Sequencing or Next Generation Sequencing using the Ion Torrent PGM
Gene	Mutation in Sample	% Starting Mutant	% Variant Frequency (Sanger)	% Variant Frequency (NGS)	P-value	Coverage	Ref Cov	Var Cov
EGFR Exon 19	#1 E746_A750_Del	0.50%	30	9.1*	1.00E-10	2,000	1,812	182
#2 E746_A750_Del	0.10%	10	3*	7.82E-10	2,000	1,940	60
#3 E746_A750_Del	0.05%	Background	No Variant
KRAS	#1 G12R	0.50%	70	69.18	1.00E-10	19,174	5,819	13,264
#2 G12R	0.10%	10	23.58	1.00E-10	10,004	7,513	2,359
#3 G12R	0.05%	Background	2.86	5.01E-04	11,027	10,603	315
PIK3CA	#1 E542K	1.0%	15	16.48	1.00E-10	28,899	24,113	4,763
#1 E545K	1.0%	25	24.23	1.00E-10	30,027	22,700	7,275
#2 E542K	0.5%	10	9.19	1.00E-10	15,680	14,225	1,441
#2 E545K	0.5%	10	11.78	1.00E-10	15,267	13,405	1,798
#3 E542K	0.1%	Background	2.02	1.00E-10	15,358	15,045	310
#3 E545K	0.1%	Background	3.17	1.00E-10	15,154	14,657	481
* difficult to detect deletions by NGS

Conclusion
ICP is a flexible technology that results in a PCR product enriched for any mutation present in the sample analyzed. This upstream enrichment of low level somatic mutations combined with high throughput analysis by NGS brings NGS one step closer for use in high throughput screening of circulating mutations for patient treatment selection and monitoring of disease.

Background
As non-small cell lung cancer (NSCLC) is a leading cause of cancer death, new prognostic and predictive markers are highly warranted. miR-210 is an essential regulator of the hypoxia pathway. The biological and prognostic importance of miR-210 has not been thoroughly studied in NSCLC. We aimed to explore miR-210 expression in both cancer and tumor stromal cells in a large representative NSCLC patient cohort to assess its impact on disease-specific survival (DSS).

Methods
Tissue micro arrays (TMAs) were constructed, representing both cancer cells and tumor stromal cells from 335 unselected patients diagnosed with stage I-IIIA NSCLC. In situ hybridization was used to evaluate the expression of miR-210.

Results
In univariate analyses, high cancer cell (p=0.039) and high stromal cell expression (p=0.008) of miR-210 were both significantly associated with an improved DSS. Co-expression of miR-210 in cancer and stromal cells combined was also a positive prognostic factor for DSS (p=0.010). In multivariate analysis, miR-210 in stromal cells (p=0.011), and miR-210 co-expressed in cancer and stromal cells (p=0.011) were independent prognosticators for DSS.

Conclusion
High expression of miR-210 in tumor stromal cells, and high miR-210 co-expressed in cancer cells and tumor stromal cells mediates an independent favorable prognostic impact in NSCLC. miR-210 may be a candidate marker for prognostic stratification and therapeutic interventions in NSCLC.

Background
Treatment decisions in stage I and II resectable lung adenocarcinoma (ADC) are heterogeneous due to low efficacy of treatment and a high frequency of co-morbidities in the patient population. Currently, pathological stage is the main determinant of adjuvant treatment recommendations. The cell cycle progression (CCP) score is a proliferation based expression profile that has been shown to add significant prognostic stratification within stage I and II patients. We have developed an integrated prognostic model of pathological stage and the CCP expression score in order to maximize the prognostic utility of both markers.

Methods
Cox proportional hazards models with pathological stage and the CCP expression score were created from two data sets: 256 patients (190 stage I, 66 stage II) from the Director’s Consortium microarray cohort and 381 adenocarcinomas (337 stage I, 44 stage II) from a clinical study set analyzed by qPCR. Expression microarray data were scaled to adjust for differences in experimental platforms. The primary outcome measure was cancer-specific death, defined as death from lung cancer or death with recurrence within five years of surgery. Coefficients for modeling were derived from the hazard ratio in the Cox PH model.

Results
Both pathological stage and CCP score were independent predictors of lung cancer death in both cohorts. The coefficients for pathological stage and CCP score were consistent across both data sets and did not differ significantly between the analysis of all patients and a subset of untreated patients. A combined score (CS) of stage and CCP score (0.33 * CCP score + 0.52 * stage) was created from the subset of untreated patients. When applied to untreated patients in the clinical ADC cohort, pathological stage alone provided estimates of five-year risk of cancer-specific death of 12.6% (stage IA), 22.6% (stage IB), 38.4% (stage IIA) and 60% (stage IIB). In the same cohort, the combined score could separate stage IA patients with five-year risk estimates ranging from 6% to 24%. Similarly increased discrimination of risk estimates were observed for stage IB (10% to 42%), stage IIA (21% to 63%) and stage IIB patients (32% to 75%).

Conclusion
A combination of pathological stage and the CCP expression score is a more effective predictor of post-surgical risk of cancer-specific death than either marker alone. A more precise risk assessment provides better guidance in balancing treatment related risks with disease-specific survival.

Background
Prognostic biomarkers for patients with non-small cell lung cancer (NSCLC) who have been treated with neoadjuvant therapy have not been fully assessed. Identifying biological prognostic markers may help to distinguish patients who are likely to benefit from additional postoperative chemotherapy. The purpose of this study was to analyze prognostic biomarkers in surgically resected NSCLC after treatment with neoadjuvant therapy, with special reference to the immunophenotypes of both the cancer cells and the stromal cells.

Methods
A series of 66 patients with NSCLC who were treated with neoadjuvant chemotherapy, chemoradiotherapy, or radiotherapy followed by complete resection at our hospital between April 1992 and December 2009 were reviewed. Among the 66 surgically resected specimens, case with viable tumor cells remained in the specimens were included in this study (n =52). We examined the expressions of geminin and cleaved caspase 3 (proliferation and apoptosis markers), E-cadherin and vimentin (epithelial mesenchymal transition related molecules), ALDH1 and CD44v6 (Stem cells related molecules) in the cancer cells. Furthermore in the stromal cells, the expressions of podoplanin and CD90 in cancer associated fibroblasts (CAFs) and CD204 in tumor associated macrophages (TAMs) were also examined.

Results
The 5-year disease-free survival rate of patients with high ALDH1 expression levels in their cancer cells was significantly lower than those with a low ALDH1 level (47.3% vs. 21.5%, respectively; P =0.023). The expression statuses of geminin, cleaved caspase 3, E-cadherin, vimentin, and CD44v6 in the cancer cells had no prognostic impact. The 5-year disease-free survival rate of patients with low or high podoplanin and CD90 levels in CAFs and CD204 levels in TAMs expression levels were not any prognostic impact in the stromal cells (37.9% vs. 29.1%, 33.8% vs. 37.5%, 38.4% vs. 29.0%, P =0.90, P = 0.75, P =0.98). In NSCLC without neoadjuvant therapy matching for clinical stage and histopathology (case control, n =104), the 5-year DFS rate of the patients with a high ALDH1 expression level was 48.3%, while that of the cases with a low ALDH1 expression level was 59.8%. The expression of ALDH1 in cancer cells was not correlated with the prognosis in NSCLC without neoadjuvant therapy (P =0.507). A multivariate analysis identified ALDH1 expression in cancer cells as significantly independent prognostic factors for disease-free survival in patients who received neoadjuvant therapy (P =0.045).

Conclusion
The presence of ALDH1-positive cancer cells was an independent recurrence predictor in patients who received neoadjuvant therapy, while CAFs and TAMs did not provide any predictors. Although prospective studies with a larger number of patients are required to confirm the prognostic significance of ALDH1 expression in cancer cells in validation populations with neoadjuvant therapy, our results suggest that the immunophenotypes of ALDH1 expression can serve as a guide to additional treatment after surgical resection in patients who received neoadjuvant therapy.

Background
The insulin-like growth factor (IGF) pathway is involved in the development and progression of many tumours and there is growing preclinical evidence that blockade of this pathway has anti-tumour effects in NSCLC. IGF receptors (IGFR) are another potential target for targeted treatment of NSCLC and a number of agents are already undergoing clinical trial. Biomarkers are needed to select patients most likely to derive clinical benefit from these agents. The downstream pathway components of IGF1R and MET activation include PI3K and AKT, which are other potential biomarkers currently being investigated in this patient cohort. IGF1R has also been implicated in acquired resistance to EGFR-TKI treatments. Only a few small retrospective studies have investigated the prognostic role of IGF1R in NSCLC and the relationship with EGFR mutations is not known.

Methods
IGF1R status was evaluated by chromogenic silver in situ hybridization (ISH) and immunohistochemistry (IHC) in tissue microarray sections from a retrospective cohort of 264 surgically resected NSCLCs and results were compared to clinicopathological features and patient survival. Patients were classified as IGF1R gene amplification (either presence of tight gene clusters, IGF1R to CEN15 ratio ≥ 2, or ≥ 15 copies of IGF1R per cell in ≥ 10% of analysed cells); high polysomy (≥ 4 copies of IGF1R in ≥ 40% of tumour cells); low copy number (< 4 copies of IGF1R in < 40% of cells). Patients were also grouped as IGF1R-positve (amplification or high polysomy) or IGF1R-negative (low copy number).

Results
High IGF1R gene copy number was identified in 77 cases (29.2%) in which there were 32 amplified IGF1R cases (12.1%) and 45 high-polysomy IGF1R cases (17%). Increased copy number of IGF1R was more common in squamous cell carcinomas (SCC) compared to large cell carcinomas (LCC) or adenocarcinomas (ADC) (p<0.05). There was no correlation between IGF1R gene copy number status and other clinicopathological features including patient age, gender, smoking status, tumour size, vessel, perineural or lymphatic invasion, grade or stage. IGF1R copy number alteration in primary tumours was highly correlated with IGF1R copy number status in metastatic tumours (p<0.01). High IGF1R protein expression was observed in 61/259 (23.6%) primary tumours and 14/215 (6.5%) normal adjacent bronchial mucosae. High expression of IGF1R protein was significantly associated with SCC in comparison with non-SCC primary tumours, as well as with lymphatic and vessel invasion. There was a moderate correlation between IGF1R copy number status (positive versus negative) and IGF1R protein expression (high versus low) (Cramer’s V=0.3, p-value <0.001). Both IGF1R copy number status and protein expression were not associated with patient overall survival in univariate analyses (p>0.05).

Conclusion
High IGF1R gene copy number and its protein expression are frequent in NSCLC, particularly in SCC. However, alterations of IGF1R are not associated with patient prognosis. IGF1R gene copy number can readily be assessed in formalin fixed paraffin embedded tissue and warrants further investigation as a potential biomarker of targeted therapy in NSCLC.

Background
Background: In order to evaluate gene abnormality in lung adenocarcinoma at an early stage, genomic DNAs of six in situ adenocarcinomas (Ad) and nine small but invasive adenocarcinomas (INVAd) were examined by array-comparative genomic hybridization. Finally, 3q26 was detected as a significantly amplified region in INVAd relative to in situ Ad. Among the genes located at 3q26, we selected the epithelial cell transforming sequence 2 oncogene (ECT2) and examined it for abnormality in lung Ad, since ECT2 is related to Rho-specific exchange factors and cell cycle regulators, and is also thought to have an important role in the regulation of cytokinesis.

Methods
Methods: The clinical implications of ECT2 abnormality were examined by quantitative real-time genomic PCR (qPCR) and immunohistochemistry (IHC) using 158 cases of varies types of LAd, resected at Tsukuba University Hospital. The results were verified by cDNA microarray and 10k SNP array using another set of early-stage LAds, resected at the National Cancer Center Hospital.

Results
Results: qPCR and IHC analyses revealed overexpression of ECT2 in INVAd, and this was correlated with the MIB-1 index. ECT2 overexpression was significantly associated with lymph node metastasis, pathological stage, vascular invasion and the histological subtype of Ad. Patients with LAd overexpressing ECT2 showed an unfavorable outcome in terms of both disease-free and overall survival (Figure). These results were verified using another set of 144 early-stage Ads resected at the National Cancer Center Hospital. Figure 1

Conclusion
Conclusion: ECT2 is a new marker that can be used for prognostication of patients with early-stage LAd.

Background
The MDM2 protein plays an important role in regulating cell proliferation and apoptosis by interaction with multiple proteins including p53 and Rb. c.309 (rs2279744) polymorphism (T>G) in the MDM2 promoter has been shown to result in higher levels of MDM2 RNA and protein. In order to evaluate the association of the MDM2 309T>G polymorphism and lung cancer risk, we carried out a case-control study in a Japanese population.

Methods
The MDM2 genotypes were determined in 469 lung cancer patients and in 682 healthy control subjects using Smart Amplification Process (SmartAmp). Statistical adjustment was made for sex and age.

Results
The distribution of the MDM2 309T>G genotypes was not significantly different between control and overall lung cancer cases. Subgroup analysis of KRAS G to T transversion adenocarcinoma indicated that G/G genotype of SNP309 may be associated with lung cancer carcinogenesis (adjusted OR = 2.42 95% C.I. = 1.01-5.82 p = 0.05) compared to T/T + T/G genotypes. G/G genotype has lower-level exposure to cigarette smoke than T/T + T/G genotypes (p=0.03) among squamous cell lung cancer patients. There was however no effect of either polymorphism on age at diagnosis of lung cancer or on overall survival.

Conclusion
Our results indicate that the MDM2 309T>G polymorphism is not significantly associated with lung carcinogenesis but may be associated with smoking related cancer of the lung.

Background
Multimodal treatment provides the best outcome for some but not all malignant pleural mesothelioma (MPM) patients. Therefore the identification of prognostic markers helping to select patients remains a subject of key importance. Frequent loss of NF2 tumor suppressor gene, a regulator of Hippo pathway and mammalian target of rapamycin (mTOR) has been well documented in MPM. We recently observed loss of expression of phosphatase and tensin homologue (PTEN), a tumor suppressor gene of the phosphatidylinositol 3-kinase (PI3K)/mTOR pathway, in 62% of MPM patients. In this regard, increased activity of these pathways stemming from loss of abovementioned tumor suppressor genes may serve as potential prognosticator as well as therapeutic target. Here we aim to assess prognostic significance of PI3K/mTOR and Hippo pathways for MPM patients treated with a multimodal approach.

Methods
Large cohort of MPM patients uniformly treated with induction chemotherapy followed by extrapleural pneumonectomy was employed in this study. Tissue micro arrays were constructed composing of paired samples from patients (n = 153) collected pre- and post-induction chemotherapy. All tissues were evaluated for apoptotic index (cleaved caspase-3) and proliferation index (Ki-67). Expression levels of biomarkers of PI3K/mTOR (PTEN, p(phosphorylated)-mTOR and p-S6) and Hippo signaling pathway (nuclear-YAP and nuclear-Survivin) were evaluated by immunohistochemistry and correlated with overall survival (OAS) and progression free survival (PFS).

Results
Survival analysis showed that high p-S6 expression and high Ki-67 index in samples of treatment naïve patients was associated with shorter PFS (p = 0.02 and p = 0.04, respectively). High Ki-67 index after chemotherapy remained associated with shorter PFS (p = 0.03) and OAS (p = 0.02). Paired comparison of marker expression in samples prior and post induction chemotherapy revealed that decreased cytoplasmic PTEN as well as increased p-mTOR expression was associated with a worse OAS (p = 0.04 and 0.03, respectively). No correlation was observed between expression level of nuclear-YAP with PFS or OAS.

Conclusion
Ki-67 proliferation index has prognostic value for MPM patients treated with multimodality approach in when analyzed both pre- and post- induction chemotherapy specimens. Our results further reveal the prognostic significance of expression changes of PI3K/mTOR pathway components during induction chemotherapy. If confirmed, these data suggest PI3K/mTOR pathway to be a target for selected MPM patients.

Background
Antibodies targeting epidermal growth factor receptor (EGFR), such as cetuximab, may potentially improve outcome in non-small cell lung cancer (NSCLC) patients with high EGFR expression. The EGFR expression may be heterogeneously distributed within tumors and small biopsies may thus not accurately reveal the EGFR expression and EGFR expression may also change during chemotherapy.

Methods
EGFR expression in diagnostic biopsies and resection specimen was compared in 53 NSCLC patients stage T1-4N0-1M0 treated with surgery without preceding chemotherapy (OP-group) and from 65 NSCLC patients stage T1-3N0-2M0 (NAC-group) treated with preoperative carboplatin and paclitaxel were analyzed regarding EGFR expression to evaluate the concordance of EGFR expression between samples.

Results
No significant change in EGFR expression H-score was observed when comparing serial samples from either the OP-group (p=0.934) treated with surgery or the NAC-group (p=0.122) treated with preoperative chemotherapy. Discordance between tumors dichotomized according to EGFR expression (high: H-score≥200; low: H-score<200) in diagnostic biopsies and immediate resection specimens was 25% in the OP-group and 33% in the NAC-group (p=0.628).

Conclusion
EGFR expression in 25% of diagnostic biopsies may potentially not be accurate compared to the prevailing pattern in the whole tumor based on the larger resection specimens. This may potentially be an obstacle for proper use of antibodies targeting the EGFR in NSCLC. EGFR expression does however not change significantly during paclitaxel and carboplatin and rebiopsies in order to decide on anti-EGFR antibody therapy following chemotherapy does not seem warranted.

Background
Activating mutations in the EGFR gene identifies NSCLC patients sensitive to treatment with EGFR tyrosine kinase inhibitors. EGFR mutations are more prevalent in Asians compared to Caucasian. However, estimates of mutation frequencies are only available from selected patient cohorts and the incidence in an unselected Caucasian population is unknown. This study assess the prevalence of EGFR mutations in an unselected population based cohort, and the correlation between mutation and gender, age, ethnicity, smoking habits, as well pathological data.

Methods
Patients diagnosed with NSCLC June 1, 2010 - September 30, 2011 in a well-defined population of 1.7 million in the Capital Region, Denmark, were included in this single center study. The type and location of the diagnostic material was registered. All specimens were pretreatment, no residual tumors were included. Smoking data were also available. The mutation analyses were investigated by therascreen EGFR RGQ PCR Kit (detection of 29 somatic mutations in the EGFR oncogene, Qiagen) (n =1121) or direct sequencing (n=62). Twenty-two patients were analyzed with both methods.

Results
658 men and 598 women were included. All but 4 were Caucasians. 6.2% were never smokers, 38.9% were ex- smokers and 54.9% were current smokers. 1161 (92.4%) patients had material sufficient for mutation analysis. More than 50% malignant tumor cells were available for analyses in 68%, 76% and 56% of the histological, cytological clot, and cytological smear material respectively. Manual macro dissection to ensure this high concentration of tumor cells was done on 32%, 4%, and 10% of the histological, cytological clots, and cytological smears material, respectively. Cytological material was used for 38 % of the mutation analyses. 5.4 % of tested patients harbored mutation in the EGFR gene (4.3% men/ 6.7 % women). The majority of mutations (55 cases= 87%) were activating mutations (29 exon 19 deletions, 24 L858R mutations and 2 G719X mutations), 3 were rare mutations (two L861Q and one S768I) with unclear clinical response to TKI, five were insertions in exon 20 (resistance mutation). Three of the mutations (all exon 19 deletions) were detected in the three included Asian women. 8.0 % of adenocarcinomas, 1.9% of squamous cell carcinomas and one single case of adenosquamous cell carcinoma were mutated. No other tumor types were mutated. DNA yield was similar whether extracted from cytological or histological samples and these material types yielded similar mutation percentages (6.55% in cytology and 4.66% in histology of material analyzed). 29.4%, 4.4% and 2.9 % of never, ex- and current smokers, respectively, were mutated (p<0.001). No significantly difference between ex- and current-smokers was observed. Mutation status was not related to age.

Conclusion
EGFR mutations analysis on cytological as well as on histological material was possible in 92% of patients with NSCLC. 5.4% of the patients harbored EGFR mutation. Adenocarcinomas were mutated more often (8.0%) than squamous cell carcinomas (1.9%). Mutations were found in never smokers as well as in former and current smokers. No difference in gender and age regarding mutation status was observed.

Background
Pleural fluid samples are useful when tumor tissues are not available for mutation analysis. Moreover, the advantages of using pleural fluid are that it is easily accessible, can be sampled by relatively non-invasive methods, and can be repeatedly sampled. Approaches for examining molecular biomarkers in body fluids such as effusion may be clinically helpful in predicting the response to EGFR-TKI treatment.

Methods
Fifty-seven patients with malignant pleural effusion were enrolled in the study. EGFR mutations were assessed by direct sequencing and PNA clamping using tumor tissues, cell blocks, pleural effusions, and sera. The diagnostic performance of pleural effusion was investigated.

Results
Among the 57 patients with malignant effusion, 37 patients were NSCLC, 11 were SCLC and 9 were extrapulmonary cancer. Twenty patients including 11 SCLC and 9 extrapulmonary cancer showed negative for EGFR mutational status. Among the 37 NSCLC patients, the diagnostic performance of pleural effusion compared with the combination of tumor tissue and cell blocks showed 89% sensitivity, 100% specificity, positive predictive value of 100%, and negative predictive value of 95% by PNA clamping, and 67% sensitivity, 90% specificity, positive predictive value of 75%, and negative predictive value of 86% by directing sequencing. A patient in whom an EGFR mutation was identified in pleural effusion only by PNA clamping showed a significant response to EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment.

Conclusion
Pleural effusion had a diagnostic performance for the detection of EGFR mutations in NSCLC that was comparable to that of tumor tissues and cell blocks. The diagnostic performance of PNA clamping was good compared with that of direct sequencing. A more sensitive and accurate detection of EGFR mutations would benefit patients by allowing a better prediction of the response to EGFR-TKI treatment.

Background
Epidermal Growth Factor Receptor mutated (EGFR+) NSCLC patients have a median progression free survival (PFS) of approximately 12 months when treated with EGFR-tyrosine kinase inhibitors (TKI). One of the resistance mechanisms described is the T790M mutation. This mutation is reported in 49-65% of patients who are rebiopsied at disease progression. Here, we report on the incidence of T790M mutation in a cohort of patients who were sequentially rebiopsied.

Methods
EGFR+ patients or with TKI-response>24weeks and progressive disease on TKI’s were retrospectively analysed. Patients should have had at least 2 separate biopsies. All biopsies and treatments were collected from the medical record and pathological reports. Survival was calculated according to Kaplan-Meier. Overall survival (OS) was calculated from date of 1[st] diagnosis until death or June 2013, which ever was first

Results
68 patients with 2 biopsies or more were available for analysis. In the first biopsy at TKI-resistance; T790M mutation was detected in 34 patients (50,0%). 26/68 patients had later biopsies available; showing gain and loss of T790M in later biopsies (figure 1). Overall development of T790M was 57.4% (39/68). 7 patients had >3 biopsies available (figure 2). Patients developing T790M had numerically longer median OS of 3.8 years (range 2.8 – 4.9) as compared to median OS in T790M-patients (2.5 years, range 1.0 – 3.9) (P = 0.204).Figure 1Figure 2

Conclusion
57.4% of patients developed T790M. OS in patients developing T790M was longer than in patients not developing T790M, however the difference was not significant. Sequential data show that some patients ‘loose’ the T790M later on in the course of the disease. Data from this cohort suggests that T790M development is a dynamic process.

Background
The strategy for EGFR-TKI acquired resistance including continuous EGFR-TKI with chemotherapy or local therapy or chemotherapy alone. The aim of this study was to investigate the association of T790M mutation status with the efficacy of different modalities after acquired resistance to EGFR-TKI.

Methods
From Mar 2011 to Mar 2013, the advanced NSCLC patients who performed a rebiopsy after acquired resistance to EGFR-TKI in Shanghai Pulmonary Hospital were included into this study, Scorpion ARMS was used to detected the EGFR mutation status. SPSS 13.O was used to perform the statistical analysis.

Results
53 patients were enrolled in the study with a median age of 51.2 years old. 45.3% (25/53) were females and 54.7% (29/53) of patients had T790M mutation. Among them, 16 people with local disease progression received local therapy combined with TKI therapy, while 21 with a slow progress received chemotherapy plus TKI therapy. The median progression-free survival time (PFS1) of all patients according to RECIST criteria was 11.8 months. The median progression-free survival time (PFS2) of patients who received continuous EGFR-TKI treatment was 3.5 months (95% CI 2.689-4.311). Patients with T790M mutation had significantly longer PFS2 than those without T790M mutation (6.3 vs 3.0 months, p = 0.001), while there were no significant differences found in PFS1 between the two groups (13.0 vs 8.5 months, p = 0.999).

Conclusion
NSCLC patients who had T790M mutation after acquired resistance to EGFR-TKI benefited more from the continuous EGFR-TKI treatment and should be recommend to adopt this modality.

Background
In NSCLC, higher Thymidylate Synthase (TS) levels have been reported in both squamous and large cell carcinomas compared to adenocarcinoma. In clinical practice, Pemetrexed, a potent antifolate inhibitor of TS, showed a selective benefit in patients with "non-squamous" NSCLC. Two retrospective studies have shown that low TS protein levels are associated with better clinical outcome in NSCLC patients treated with pemetrexed. Aim of this study was to explore, in a series of advanced stage IV patients receiving pemetrexed-based regimens in first line of treatment, the association between TS mRNA and protein expression with overall survival (OS) and therapeutic response.

Methods
Two series of histologically confirmed non squamous-NSCLC, assessed in formalin-fixed and paraffin embedded specimens from patients treated with pemetrexed-based regimens were collected: the first series at San Luigi Hospital (n=64), the second series at Regina Elena National Cancer Institute (n=8). Due to the limited amounts of tissue specimens available, total RNA extraction was possible in 52 out of 72 cases. TS protein expression was performed using immunohistochemistry (mouse monoclonal TS106 antibody) and scored through H-SCORE method, considering both staining intensity (0 no staining; +1 weak; +2 moderate; or +3 strong) and percentage of tumor cells stained, resulting in semiquantitative H-scores ranging from 0 to 300. TS nuclear and cytoplasmic staining, respectively, were separately scored. Statistical analyses were performed using the STATISTICA10 software.

Results
The differential H-SCORE assessment showed a strong importance of TS localisation for clinical outcome prediction: in Cox regression analysis, a statistically significant association was observed between nuclear TS expression and OS (p < 0.009) indicating that lower nuclear TS expression levels were associated with longer OS. In addition, lower nuclear TS levels were significantly associated with a better response to therapy (p<0.001). On the contrary, TS cytoplasmic staining did not affect patients’ survival or clinical response (p>0.05). Four subgroups of patients, based on the dichotomized low/high TS expression in both nucleus and cytoplasm, were obtained: both high, both low, nucleus high/cytoplasm low and nucleus low/cytoplasm high. Significant differences in overall survival among these four groups were detected (p=0.017), confirming the strong and selective influence of nuclear TS, as compared to cytoplasmic TS, expression in clinical outcome. Moreover, Chi[2] test revealed a significant association between low nuclear TS and partial response to pemetrexed treatment, independently of cytoplasmic TS expression (p<0.001). No correlation between TS protein expression data and clinico-pathological data (age, gender) were identified. TS gene expression analyses are ongoing.

Conclusion
This retrospective study suggests that TS protein expression, selectively assessed at nuclear level, has a potential predictive role in advanced stage IV patients, receiving pemetrexed in first line of treatment. Patients with low nuclear TS expression showed prolonged overall survival and better response to therapy. Such preliminary results define TS assessment as a potential tool which may select the most appropriate group of patients to be treated with pemetrexed.

Background
Personalised treatments for lung cancer are being increasingly employed. Around 4% of pulmonary adenocarcinomas have rearrangements of the ALK gene resulting in oncogenic fusion proteins. Such cases are sensitive to the small molecule tyrosine kinase inhibitor Crizotinib, and detection of the ALK rearrangement is crucial to the treatment of these patients. Currently the only approved test for the detection of ALK-positive NSCLC is FISH. Various immunohistochemical assays have been proposed as alternatives or screening tools, which are cheaper, quicker and easier to perform and interpret than FISH. Validation of these immunohistochemical tests is therefore important.

Methods
15 FISH positive cases were obtained from local archives at the Royal Marsden and Royal Brompton Hospitals, with accompanying data on crizotinib therapy and clinical response. A further 14 FISH negative and FISH uncertain cases were also retrieved. 3 antibodies were optimised for immunohistochemical detection of ALK: D5F3, marketed by Ventana and using their proprietary automated system, ALK1 (Dako), and 5A4 (Abcam). All three antibodies were applied to sections from the archival cases. Antibodies were semiquantitatively scored on their intensity (0-3) and proportion of malignant cells stained (0-100%). Cutoffs for positivity/negativity were set by ROC analysis to optimise correct classification.

Results
Positivity according to the Vysis FISH assay (i.e. appropriately abnormal FISH signal in at least 15% of cells) was taken as the gold standard. The Ventana assay (D5F3) was markedly more intense but showed higher background than the other two antibodies. ROC analysis of staining intensity showed that the Ventana and Dako antibodies should be considered positive when staining is of 'moderate' intensity or above. The Abcam assay was discriminatory when any positivity was seen. Scoring of proportion did not improve specificity or sensitivity with any of the antibody tests. Subsequent analyses were of intensity scoring. Taking all cases together, all 3 antibodies were highly specific (100%) and sensitive (Ventana 86%, Abcam 79% and Dako 71%). In excision specimens, all 3 assays showed sensitivity of 83%. In cytology and small biopsy specimens, Ventana performed the best (specificity of 88% compared to 75% for Dako and 63% for Abcam) although the numbers are small. No ‘false positive’ (-ve FISH, +ve IHC) cases were seen; the only disparity between FISH and IHC were 'false negative' cases. Two cases were FISH-positive but universally IHC-negative (with all 3 assays). Of these, one was treated with crizotinib, and failed to respond.

Conclusion
IHC is a highly specific (100%) and sensitive (71%-86%) test for ALK rearrangement in archival FFPE tumour tissue. In this study of 15 ALK-rearranged tumours, the Ventana D5F3 assay performed the best, despite having relatively high background staining. Occasional cases are FISH-positive but IHC-negative. One such case was not responsive to Crizotinib, further supporting IHC as a test predictive of drug response. IHC has the potential to replace FISH for the detection of candidates for Crizotinib therapy. However, further work is required to direct the treatment of tumours with discordant FISH and immunohistochemical tests.

Background
Currently, biomarker testing for lung cancer is not uniformly integrated into the Canadian public healthcare system, despite its clear importance for patient outcomes. In order to better understand the current practice pattern for lung cancer biomarker testing, we assessed physician perspectives by specialty and region.

Methods
A national survey of Canadian specialists was conducted to understand their perspective on biomarker testing in lung cancer. An 11-item survey was developed to assess current practice and challenges related to biomarker testing. The survey was sent to 375 specialists (150 medical oncologists, 75 pathologists, 150 respirologists/thoracic surgeons) with a focus or interest in the management of lung cancer.

Results
The overall response rate for the survey was 36%, (38% of medical oncologists, 24% of pathologists, and 40% respirologists). It is understood that knowing tumour genotyping results at the time of the initial medical oncology consultation impacts patient outcome and influences the treatment decision (98%). To date, medical oncologists most commonly initiate molecular testing (67%), however, most respondents suggested a shared model for initiating the testing involving medical oncologists and pathologists is needed. The current perception is that less than two-thirds of patients have testing results available at the time of initial medical oncology consultation. Barriers identified to routine testing for all advanced lung cancer patients include cost, lack of funding for molecular testing, tissue availability and the quality of the tumour sample.

Conclusion
There was clear agreement that biomarker testing is important in determining the most appropriate initial treatment for patients with advanced NSCLC. There is a need for national consensus on who should initiate molecular testing. Moving forward, clear clinical guidance for pathologists needs to be established for molecular testing as part of the lung cancer diagnostic process. This includes defining the population to be tested, timing, and the tests to be performed. This may be facilitated by including more information on diagnostic sample requisitions, such as clinical suspicion of lung cancer primary (versus metastasis from another site), other cancer history, and other samples collected (and tested) previously or planned (e.g. planned resection). Pathologists need to incorporate routine EGFR, ALK testing into diagnostic lung cancer algorithm. This and greater clinical information on sample requisitions will minimize unnecessary tissue sections and allow the most efficient use of available tumour tissue for molecular testing. Incremental laboratory funding is required throughout the Canadian public healthcare system in order to provide the current standard of molecular testing required for NSCLC. Turnaround times need to be clearly established and monitored. Implementation of the College of American Pathologists (CAP) guidelines for transport from diagnostic lab to molecular testing lab (3 days), and turnaround for test results (10 days) will also improve the proportion of patients with test results available at initial consultation. Finally, feedback to clinicians, including sample quality, volume, whether or not testing was successful, and molecular results is necessary in a timely manner.

Background
Erlotinib is an epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitor. Clinical trials proved its efficacy in patients with advanced-stage NSCLC. Although, activating EGFR gene mutations are currently the strongest predictor of erlotinib efficacy, the majority of NSCLC patiens harbor wild-type EGFR gene and moreover there is still a large proportion of patients in whom it is not feasible to acquire an adequate tissue for EGFR mutation analysis. We conducted this retrospective study aiming to prove the predictive role of tumor markers in patients with advanced-stage NSCLC treated with erlotinib.

Methods
144 patients with advanced-stage (IIIB, IV) NSCLC were treated with erlotinib (150 mg daily). Pretreatment serum levels of CEA, CYFRA 21-1, NSE and TK were assessed. Comparison of patients’survival (PFS and OS) according to the level of assessed tumor markers was performed using the log-rank test.

Results
Median PFS and OS for patients with normal CEA levels was 2.9 and 16.1 vs. 1.9 and 8.6 months in patients with high CEA levels (p = 0.046 and p = 0.116). Median PFS and OS for patients with normal CYFRA 21-1 levels was 3.4 and 23.8 vs. 1.9 and 6.1 months in patients with high CYFRA 21-1 levels (p < 0.01 and p < 0.01). Median PFS and OS for patients with normal NSE levels was 2.1 and 9.8 vs. 1.1 and 3.8 months in patients with high NSE levels (p = 0.051 and p = 0.022). Median PFS and OS for patients with normal TK levels was 2.1 and 23.7 vs. 2.0 and 8.5 months in patients with high TK levels (p = 0.110 and p = 0.037).

Conclusion
The study proved that high pretreatment levels of CEA, CYFRA 21-1 and NSE predict low efficacy of erlotinib treatment in patients with advanced-stage NSCLC. Assessment of these tumor markers is a good predictive tool for erlotinib treatment efficacy especially for those patients with unknown EGFR mutation status.

Background
Circulating tumor cells (CTCs) are cells that have spread from the primary tumor into the bloodstream, and they play a crucial role in the development of distant metastases. CTCs have been detected in several cancers and associated with aggressive disease. The aim of this study was to evaluate the correlation between the numeric variation of CTCs in the blood of patients (pts) with advanced adenocarcinoma (ADK) of the lung during chemotherapy (CHT) and the radiological response to explore their potential role as an early predictive indicator of treatment response.

Methods
Peripheral blood samples and computed tomography (CT) scans were obtained before any treatment (baseline) from 25 pts with advanced ADK who were candidates for first-line CHT (platinum-based combination with pemetrexed). Blood samples were collected every two CHT cycles, and radiological responses were concomitantly assessed by CT according to the RECIST v. 1.1 criteria. CTCs were isolated from blood and diluted in a buffer containing formaldehyde to lyse red blood cells and fix CTCs using a filtration-based device (ScreenCell[®], France) to sort CTCs by size. CTCs were isolated by size using a microporous membrane filter composed of polycarbonate material containing circular pores that are calibrated (7.5±0.36 μm) and randomly distributed throughout the filter (1×10[5 ]pores/cm[2]). At the end of filtration, hematoxylin and eosin (HE) staining and immunofluorescence (IF) using CK7 were performed to enumerate and characterize the CTCs. Moreover, variations in the CTC count were compared with tumor size variations observed in CT scans.

Results
Baseline CTC counts and CT scans were obtained from 25 pts, including 18 males and 7 females, with a median age of 68 (range: 45-81) years. H&E staining revealed that the CTCs were morphologically compatible with tumor cells and were present in all 25 pts at baseline (range: 2-25 CTCs/ml). Furthermore, the epithelial origin of the CTCs was confirmed by CK7 positivity (demonstrated by IF). CTCs and CT images were assessed in 19 pts after at least two CHT cycles; the best response was a partial response (PR) in 2 pts, progressive disease (PD) in 3 pts and stable disease (SD) in 13 pts. The CTC number varied with the tumor size in 77.8% (14/18) of pts; decreased CTC counts were observed in 87.5% (7/8) of pts with reduced tumor size, whereas increased CTC counts were found in 70% (7/10) of pts with increased tumor size. Notably, variations in CTC count and tumor size were concordant in 100% (5/5) of pts achieving a PR or with PD as the best response.

Conclusion
This study demonstrates the feasibility of isolating CTCs in all advanced lung ADK pts using a low-cost, size-based technique. Interestingly, the concordance between the CTC analysis and CT suggests that CTCs may represent an early predictive marker of disease outcome. To our knowledge, this is the first study suggesting a relationship between CTC variation and treatment response in lung cancer.

Background
Molecular biomarkers in tissues and body fluids represent a promising source to improve cancer diagnostics. Bronchoscopic Microsampling (BMS) of endobronchial epithelial lining fluid (ELF) is a potent method to investigate potential biomarkers in lung diseases. Recent studies report that mRNA derived from ELF can be used to improve the differentiation of malignant and benign pulmonary nodules. Tenascin C (TNC) is an important component of the extracellular matrix involved in tissue remodeling and cell signaling. It has been reported that TNC is differentially expressed in malignant pulmonary nodules. We investigated whether specific splice variations of TNC are differentially expressed in ELF collected by the BMS method in proximity of pulmonary nodules.

Methods
ELF was collected by the BMS method from subsegmental bronchi close to the indeterminate pulmonary nodule and from the contralateral lung. Diagnosis was confirmed by transbronchial biopsy or surgery. In this study 176 ELF samples were included from a total of 88 patients (65 NSCLC and 23 benign cases) with indeterminate pulmonary nodules detected by computed tomography. Quantitative real-time PCR (RT-PCR) assays were used to detect different TNC variant patterns.

Results
All patients underwent BMS without complications. Quantitative RT-PCR was reliably applied to all ELF samples. A significant increase of TNC transcript variants close to malignant nodules was observed for most of the used qPCR assays. A better accuracy was observed for two protein-coding variants and an untranslated transcript indicating a distinct tumor-associated TNC variant pattern and a lower intraindividual variance.

Conclusion
Our results indicate that the analysis of individual TNC variants might improve the accuracy in detecting malignant nodules compared to the detection of whole TNC transcripts.

Background
Accurate assessment of ALK gene rearrangement in NSCLCs is critical to identify patients likely to respond to crizotinib. Currently, the gold standard for identifying ALK gene rearrangements is FISH and the Abbott Molecular ALK break apart probe is commonly used. We evaluated a new ALK/EML4 TriCheck FISH Probe for the detection of ALK rearrangements and confirmation of EML4 as the inversion partner. In addition, we evaluated its use as an ancillary FISH probe for use in cases with subtle, equivocal or atypical ALK FISH patterns.

Methods
ALK FISH was prospectively performed on 29 routine diagnostic cases using the ALK/EML4 TriCheck Probe (ZytoVision) and the Vysis ALK Break Apart FISH Probe (Abbott Molecular). ALK immunohistochemistry (IHC) was performed using the 5A4 clone antibody (Novocastra) and D5F3 clone antibody (Cell Signaling Technology).

Results
Both probes were concordant in all cases, except for one case which showed an atypical signal pattern using the Abbott Molecular ALK probe. This case was technically negative using standard scoring criteria for the Abbott probe, despite positive ALK IHC, but was confirmed as positive using the ZytoVision TriCheck probe. Two additional cases which were equivocal (10-16% ALK rearrangement), were confirmed to be positive for ALK rearrangement using the ALK/EML4 TriCheck probe. Of the 10 ALK rearranged cases, 4 showed evidence of EML4 translocation.

Conclusion
The ALK/EML4 TriCheck FISH Probe is useful for the detection of ALK gene rearrangements, including those involving EML4 as the translocation partner, especially for borderline cases or cases displaying atypical signal patterns, where an additional unique ALK FISH probe can provide further confirmation of rearrangement.

Background
Dysfunction of Nrf2-Keap1 interaction by Keap1 or Nrf2 mutations may promote tumor growth, drug resistance, and poor outcomes in non-small cell lung cancer (NSCLC). However, both gene mutations are uncommon in Asian patients, suggesting that a different mechanism might be involved in lung tumor progression via altering Nrf2-Keap1 pathway.

Methods
Two strategies—treatment of NO scavenger and a nuclear location sequence (NLS)-mutated Nrf2 expression vector transfected into Nrf2-knockdown stable clones—were used to explore whether IKKβ could be elevated by cytoplasmic Nrf2 and promotes cell invasion. The prognostic values of cytoplasmic Nrf2 and IKKβ mRNA levels in tumors from NSCLC patients were estimated by Kaplan Meier and Cox regression model.

Results
We showed that mutated p53 upregulated Nrf2 transcription by de-repression of Sp1 binding to the Nrf2 promoter. Interestingly, cytoplasmic Nrf2 expression was markedly increased in p53-mutated cells compared with p53 wildtype cells due to a decrease in iNOS-mediated NO production. The stability of IKKβ protein was also increased by the interaction of cytoplasmic Nrf2 with Keap1, which blocked IKKβ degradation by the Keap1-mediated proteasomal pathway. An increase in IKKβ expression due to cytoplasmic Nrf2 was responsible for soft agar growth and invasion capability. Positive cytoplasmic Nrf2 immunostaining or high IKKβ mRNA expression in tumors from NSCLC patients may predict poorer overall survival and relapse free survival. Moreover, patients with cytoplasmic Nrf2 positive tumors had unfavorable responses to cisplatin-based chemotherapy.

Conclusion
Cytoplasmic Nrf2 may promote tumor aggressiveness, poor outcome, and unfavorable response to chemotherapy in NSCLC.

Background
More than 70% of patients with lung cancer are diagnosed with advanced-stage, with short survival times. The discovery of epidermal growth factor receptor (EGFR) mutations is a major scientific breakthrough; tyrosine kinase inhibitor (TKI) treatment for patients with EGFR mutations reported a median overall survival of more than 2 years. Nevertheless, chemotherapy has steadfastly remained the most validated therapies in non-small-cell lung cancer (NSCLC) not only for vast majority of patients without EGFR mutations, but for those with EGFR-TKI resistance. There are many influence factors in platinum resistance, among them DNA repair genes have been extensively explored. Excision repair cross complementing 1 (ERCC1) and/or breast cancer susceptibility gene 1 (BRCA1) is generally associated with sensitivity to platinum, but the results were inconclusive and controversial. Apurinic/apyrimidinic endonuclease 1 (APE1), a bifunctional AP endonuclease/redox factor, is important in DNA repair and redox signaling, may be associated with chemoresistance.

Methods
In total, 172 patients with advanced NSCLC who received platinum-based doublet chemotherapy, ages 34 to 84 years at diagnosis from 2007 to 2012 were enrolled into this study. We evaluated the impact of APE1, BRCA1, ERCC1 expression levels on response to platinum-based chemotherapy and median progression-free survival (mPFS) and/or median overall survival (mOS) outcomes, and protein expression levels were determined by immunohistochemistry (IHC).

Results
The mPFS was 8.8 months, mOS 12.8 months. A partial treatment response was found in 55 patients (31.98%). No significant association was found between BRCA1 protein expression and response rate, mPFS or mOS. Interestingly, patients whose tumors did not express APE1 had 50.00% response rate compared to 25.00% whose tumors expressed the protein (OR 0.34; 95% CI 0.17-0.69; P=0.002). Patients negative for APE1 had a significantly longer mPFS (10.3 vs. 8.0 months, P=0.016), but not mOS (17.1 vs. 10.9 months, P=0.263), than those positive for APE1. Patients negative for ERCC1 expression experienced better response to chemotherapy (OR 0.36; 95% CI 0.18-0.72; P=0.003), longer mPFS (9.8 vs. 6.8 months, P=0.001), and longer mOS (14.4 vs. 10.3 months, P=0.011). In particular, the expression of ERCC1 had a positive trend of correlation with APE1 expression (r[2]=0.23, P<0.01), and patients negative for both APE1 and ERCC1 had significantly higher response rate (OR 0.18; 95% CI 0.07-0.45; P<0.001), longer mPFS (12.0 vs. 6.8 months, P<0.001) and mOS (18.8 vs. 10.1 months, P=0.010), than those positive for both APE1 and ERCC1. Multivariate analysis adjusting for possible confounding factors showed that negative APE1 and/or ERCC1 expression was a significantly favorable factors for mPFS (HR 1.86, P=0.002 and HR 1.83, P=0.001; respectively) and mOS (HR 2.02, P=0.002 and HR 1.78, P=0.007; respectively).

Conclusion
The results suggest that dual-function protein APE1 may be a novel prognostic and predictive factor, and the combined detection of APE1 and ERCC1 expression might better individualize the efficacy of chemotherapy and improve survival in advanced NSCLC.

Background
It has been demonstrated that the addition of bevacizumab to paclitaxel plus carboplatin (CPB) in the treatment of advanced NSCLC improves survival. Even though, there is a high variability in drug efficacy between patients, leading to different response rates. ANGIOMET is an exploratory study promoted by the SLCG in advanced NSCLC, non-squamous histologies (NS-NSCLC) treated in first line with a combination scheme based in CPB, designed to investigate the relationship between angiogenic mediators and the outcome and response to treatment. The primary end-point was progression-free survival (PFS), and the secondary end-points are the follows: OS, response-rates and toxicity profiles.

Methods
In this multicentric study, patients with stage IIIB/IV NS-NSCLC (ECOG status 0–2) were included and treated in first line with CPB. Peripheral blood samples were collected before treatment administration and DNA was purified from the leukocyte fraction. Ten SNPs of VEGF-pathway genes were genotyped in 186 samples by RT-PCR in duplicate. SNPs were related to PFS and OS (Kaplan-Meir method, log-rank test) and to response rate.

Results
10 SNPs were determined in 186 DNA samples. In this preliminary analysis there were data from 108 patients valid for PFS and OS analysis. Baseline characteristics of the patients were: median age, 63 years [37-80]; 74.5% male; 94.1% ECOG PS 0-1; 14% never-smokers, 100% caucasian; 89.7% adenocarcinomas, 2.8% large cell carcinomas; median number of CPB cycles was 4. There was no response assessment in 27 patients (25%), 30.6% PR, 31.5% SD and 13.0% PD. The SNP rs833061 (CC) in VEGFA correlated with lower response rates to CPB than the other genotypes (p=0.07). SNPs in KRAS and VEGFR2 were associated with PFS and/or OS in our cohort. The KRAS SNP rs10842513 (TT+CT) was associated with shorter PFS compared with the CC genotype (median: 5.39 vs 6.81 months; p=0.04, respectively). The VEGFR2 SNP rs2071559 (AA) was significantly associated with longer PFS and OS (Table 1). No significant differences in PFS or OS were observed according to other SNPs analyzed. Table 1: PFS and OS for VEGFR2 SNPrs2071559.

		PFS
	%	Median (months)	95%CI	p
VEGFR2 (rs2071559)
AA	25.6	9,408	5,084 - 13,732	0.01
GG+AG	74.0	5,724	4,902 - 6,546
		OS
	%	Median (months)	95%CI	p
VEGFR2 (rs2071559)
AA	25.6	NR	----	0.001
GG+AG	74.0	12,270	8,760 – 15.780

Conclusion
These preliminary data indicate that genetic variation in VEGFR2, SNP rs2071559 variant AA, is associated with prognosis in advanced NS-NSCLC patients treated with CPB and may have predictive implications as biomarkers in patients treated with chemotherapy with bevacizumab. On behalf of the Spanish Lung Cancer Group (SLCG)

Background
VeriStrat (VS) is a multivariate protein serum test that classifies Non Small Cell Lung Cancer (NSCLC) patients in 2 categories VS Good or VS Poor according to the overall survival (OS) of patients treated with EGFR-TKIs. Recently, the prospective Phase III PROSE study showed that the VS algorithm is predictive of differential OS benefit for erlotinib (E) vs second line standard chemotherapy (CT): VS Poor classified patients had worse OS on E compared to CT, while there was no significant difference between treatments outcome in the VSG group. Aim of the current study was to evaluate if baseline Standardized Uptake Value (SUV) of FDG-PET may help to optimize treatment choice between E or CT IN VS Good classified pts.

Methods
Plasma samples were collected before the beginning of E from metastatic NSCLC patients. Acquired spectra were classified according to the VS algorithm. The FDG-PET was performed tha day before the beginning of E. Survival curves were estimated using the Kaplan-Meier method.

Results
Thirty eight NSCLC patients on E therapy with the following characteristics were analyzed: median age 62 years old, 63% were males, 53% had adenocarcinoma histology, response rate was 26%, median OS 10 mos and TTP 3.4 mos. Twenty-six (68%) were classified as VS Good, 12 (32%) as Poor. TTP and OS for VS Good and Poor were 4.1 vs 2.1 mos (HR 0.86, log-rank p=0.6) and 11.1 vs 4.1 mos (HR 0.45,log-rank p=0.02), respectively. All VS Poor classified patients had SUV ≥ 7 and had the worst TTP and OS; VS Good classified patients with baseline SUV level ≥ 7 had worse OS (10 mos) and worse TTP (2.1 mos) compared to those who were VS Good and had SUV<7 (OS 16 mos) (TTP 13.8 mos).

Conclusion
The study confirmed that VS Poor classified patients had significantly shorter OS than those classified as VS Good. Patients with VS Good profile and with baseline FDG-PET SUVs levels <7 may benefit more from EGFR-TKIs than VS Good patients with higher FDG-PET SUV, suggesting that FDG-PET analysis may be a clinically useful tool for EGFR-TKIs therapy selection.

Background
Epithelial–mesenchymal transition (EMT) and expression of stemness features are key issues for tissue invasion and metastasis during cancer development. OCT4 and NANOG are homebox transcription factors essential to the self-renewal of stem cells and are expressed in several cancers. The role of OCT4/NANOG signaling in tumorigenesis and as biomarkers in non-small cell lung cancer (NSCLC) is still elusive.

Methods
mRNA was isolated from 177 frozen samples, corresponding to tumoral and normal parenchyma of NSCLC patients in resectable-stage. OCT4 and NANOG relative expression was determined by RTqPCR using hydrolysis probes. Expression levels were normalized using GUSB as endogenous housekeeping gene. Statistical significance was considered for p<0.05.

Results
Patient’s median age was 64 years [26-87], 87.6 % were males, 75.2 % presented performance status (PS=0) and 47.3% had squamous histology. We found a significant positive correlation between OCT4 and NANOG expression (r[2 ]=0.61 p<0.0001, Pearson test). Higher levels of expression of NANOG were related to poor differentiation grade (p= 0.04). Survival analysis revealed that there is a trend to a poorer progression free survival in the subgroup of patients with higher levels (> median) of expression of OCT4 levels

Conclusion
The transcription factor OCT4 may have a role as prognostic biomarker in resectable NSCLC. (Supported in part by ISCIII (PI12/02838), RTICC (RD12/0036/0025), Ministry of Economy and Competitiveness and SEOM Grants 2012)

Background
Neoplastic pericarditis is found in 60-80% of patients (pts) with large pericardial effusion (pe) (>2 cm on echocardiography). The most frequent causes of neoplastic pe are metastatic lung or breast cancer. Malignant pe is combined with high relapse rate after pericardiocentesis and poor prognosis. Early recognition of malignant pe and intensive local (+/-) systemic treatment is able to improve life expectancy. Nevertheless in 30-40% of pts the malignant cause of pe is not recognized during the first episode of disease. The aim of the study was to introduce the new scoring system assessing the probability of malignant pe in the pts requiring pericardial fluid drainage due to large pe/tamponade.

Methods
146 pts, median age 57 years (21-88), 80 with benign cause of pe, 66 with neoplastic cause of pe ( positive pe cytology or neoplastic infiltration in pericardial biopsy specimen) treated in the National Institute of Tuberculosis and Lung Diseases in 1982 - 2008 entered the study. Metastatic lung cancer was diagnosed in 67% of neoplastic pe.

Results
Based on previous results, the most important features distinguishing between neoplastic and benign pe were: cardiac tamponade on echocardiographic examination, HR>90 beats/min on ECG, enlarged mediastinal lymph nodes (>1cm) on chest CT scan, CEA> 5ng/ml in pe, Cyfra 21-1>95 ng/ml in pe, bloody pe. The sensitivity, specificity, PPV and NPV are listed in table 1.

Parameter Diag. value	HR>90/min	Cardiac tamponade	CEA>5 ng/ml (pe)	Cyfra 21-1>95 ng/ml (pe)	Bloody pe	Lymph nodes>1 cm (CT)
sensitivity	0.82	0.67	0.63	0.64	0.86	0.93
specificity	0.55	0.65	0.94	0.95	0.57	0.70
PPV	0.60	0.61	0.91	0.93	0.66	0.66
NPV	0.79	0.70	0.71	0.71	0.80	0.94

The original scoring system (-3 up to + 3 points) was developed based on PPV and NPV of the above mentioned parameters. The diagnostic value of the proposed scoring system was high, ( ROC: AUC 0.926 95%CI 0.85-0.96) and exceeding the value of single parameters (at a cut off of 0 points – sensitivity was 0.84 and specificity - 0.91).

Conclusion
Assessment of the probability of malignant pe according to the proposed original scoring system was able to improve the diagnostic algorithm based on single parameters, thus indicating pts in whom more invasive diagnostic methods should be applied to recognize malignant pe.

Background
Mucin 1 (MUC1), a glycoprotein highly expressed in many malignancies, is being explored as an antigen for immunotherapy. How best to measure MUC1 expression in non-small cell lung cancer (NSCLC) and its prognostic value in NSCLC are under discussion.

Methods
Tissue microarrays (TMAs) were constructed using triplicate 1mm cores of formalin-fixed paraffin-embedded tumour and stained with 214D4 (recognizes protein core) and MA695 (recognizes carbohydrate epitope) anti-MUC1 antibodies. TMAs were assessed for polarization, cytoplasmic and membranous staining intensity (scored 0–3) and proportion cells positive (0–100%; scored 0–5), averaged for multiple cores. A composite score (intensity x cells positive) was derived (range 0–15).

Results
TMAs from 518 patients were analyzed: 69% male, 95% Caucasian, 7% never-smoking; 49% adenocarcinoma, 35% squamous cell, 7% large cell; 43% stage I NSCLC, 33% stage II, 23% stage III. Immunohistochemistry staining intensity, proportion positive cells and depolarization were very similar between antibodies, with high concordance in composite score (R2=0.71, p<0.0001). Polarization was discordant in 8%. Similar scores were seen for N0, N1 and N2 when assessed by either antibody. For 77 cases with paired primary/N2 nodal tissue, mean 214D4 composite scores were 10.7 and 10.1 and MA695 scores 9.5 and 9.4, respectively. Discordant staining in primary but not node was seen in 5.2% and 10.4% with 214D4 and MA695, respectively. For 27 cases with neoadjuvant chemotherapy vs no chemotherapy, mean 214D4 scores were 10.2 vs 10.1 and MA695 9.3 vs 9.6, respectively. Higher scores with each antibody trended toward improved survival (non-significant). Polarization was associated with improved survival (whole cohort) with 214D4 (80.6 vs 47.8 months; HR 1.37 [95%CI 1.078–1.742], p=0.01 log rank test) and MA695 (95.8 vs 45.7 months; HR 1.48 [95%CI 1.159–1.878], p=0.002). Polarization with 214D4 was strongly associated with improved survival for adenocarcinoma (HR 1.92 [95%CI 1.385–2.668], p<0.0001) but not for non-adenocarcinoma. Similarly, polarization with MA695 conferred a survival advantage in adenocarcinoma (HR 1.68 [95%CI 1.225–2.311], p=0.001) but not non-adenocarcinoma cases. Data on MUC1 immunohistochemistry and circulating soluble MUC1 will be presented.

	214D4	MA695
No staining	3.5%	6.2%
Mean intensity
- Cytoplasmic	1.8	1.7
- Membranous	2.1	2.2
Mean cells positive	3.9	3.6
Mean composite score	10.1	9.6
- Adenocarcinoma	13.1	12.0
- Squamous cell	7.1	7.0
- Large cell	7.2	7.7
Depolarization	66.7%	62.4%

Conclusion
Over 93% were MUC1 immunohistochemistry positive, with higher scores in adenocarcinoma. Composite scores for the two antibodies were highly correlated and depolarization largely concordant. MUC1 expression was generally maintained in paired primary/nodal tumour and was similar across nodal stages and following neoadjuvant chemotherapy. Polarization was associated with improved survival in adenocarcinoma. Further investigation is needed to determine which antibodies best predict outcomes.