Virtual Library

Abstract:
Proton therapy, in particular, and ion therapy, just beginning, are becoming an increasing focus of attention in clinical radiation oncology. Proton therapy is a type of radiation treatment that uses protons rather than x-rays to treat cancer. Protons, however, can target the tumor with lower radiation doses to surrounding normal tissues. Proton therapy is particularly useful for treating cancer in children because it lessens the chance of harming healthy, developing tissue. In addition, proton therapy may be used to treat Lung cancer. Compared with standard radiation treatment, proton therapy has several benefits. It reduces the risk of radiation damage to healthy tissues; may allow a higher radiation dose to be directed at some types of tumors, which may keep the tumor from growing or spreading; and may result in fewer and less severe side effects (such as low blood counts, fatigue, and nausea) during and after treatment. However, there are some drawbacks such as higher cost and lack of convincing evidence from randomized trials proving their efficacy, justifying the higher costs involved in these therapies. Carbon ion therapy is another type of radiotherapies that can deliver high-dose radiation to a tumor while minimizing the dose delivered to the organs at risk; this profile differs from that of photon radiotherapy. Moreover, carbon ions are classified as high-linear energy transfer radiation and are expected to be effective for even photon-resistant tumors. There are several centers in Asia and Europe using carbon beam to treat lung cancer patients. In this session, we will provide an overview of the current status of clinical trials in proton therapy, including recent developments in ion therapy for lung cancer. We will also attempt to highlight some of the challenges that surround clinical trials in particle therapy.

Abstract not provided

Abstract:
Despite advances in treatment, lung cancer remains the leading cause of cancer mortality in most countries. About one third of patients with non-small cell lung cancer (NSCLC) presents with locally advanced non-metastatic disease (stages IIIA and B). Although 5-year overall survival (OS) ranges from 60 to 73% for completely resected pathologic stage I disease, OS decreases to less than 25% for pathological stage III disease. Patients with potentially resectable stage IIIA-N2 can be treated with induction chemotherapy followed by radical surgery. Prospective studies report 5-year OS rates from 20% to 30%, with about 30% of the patients reporting local recurrence (LR). When surgery is performed first, for patients presenting with node-positive disease at the time of resection, meta-analysis data demonstrate that adjuvant platinum-based chemotherapy has been shown to decrease distant metastases and locoregional recurrence (LRR), resulting in an approximately 5% OS advantage, and is now considered standard of care for patients with resected node-positive NSCLC. However, these patients have a 20% to 40% risk of LRR, and LRR correlates independently with worse OS. Thus, postoperative radiotherapy (PORT) holds great appeal as a means to reduce LRR and improve OS. Several phase III trials investigated the role of PORT after surgical resection in NSCLC. In 1998, the PORT Meta-analysis Trialists Group undertook an individual participant data (IPD) meta-analysis (of both published and unpublished trial data) of PORT versus surgery alone in NSCLC [1]. The original meta-analysis, based on 9 randomised controlled trials and 2128 patients, concluded that PORT was detrimental with a 7% absolute reduction in 2-year OS and a 4% reduction in recurrence-free survival. Subgroup analyses suggested that PORT was increasingly detrimental with decreasing stage (p = 0.0003) and lower nodal status (p = 0.016). The updated results for OS, and for local, distant and overall recurrence-free survival are unchanged, continuing to show a detrimental effect of PORT(2) : For the whole patient group, PORT decreased the OS at two years by 6% (52% vs. 58%). This deleterious effect was detected in patients with pN0–1 disease. No effect was detected in patients with pN2 disease. The PORT meta-analysis raised a lot of criticism for the following reasons: significant heterogeneity between trials, inclusion of trials with old radiotherapy techniques (notably, most of these trials, conducted principally in the 1960s to 1970s, included outmoded RT techniques and doses). The deleterious effect of PORT has been attributed to an excess of intercurrent deaths, with a high incidence of cardiac and respiratory complications due to poor radiotherapy techniques. In support of this hypothesis, several more recent trials with contemporary radiation techniques did not report an increase of death from intercurrent disease. Kepka et al. did not detect a difference in QoL scores, cardiopulmonary morbidity or non-cancer related deaths between patients receiving PORT and those treated with surgery alone (3). Two Surveillance, Epidemiology, and End Results (SEER) analyses and a secondary analysis of data from the Adjuvant Navelbine International Trialist Association (ANITA) trial suggest that PORT may be safely delivered in a modern cohort of patients with a potential OS benefit for stage IIIA (N2) disease (4, 5). In addition, being now established that the use of adjuvant chemotherapy in stage III disease prolongs OS, it is then hypothesised that with the reduction of distant metastases with chemotherapy, the survival benefit by improved local control after three-dimensional conformal (3D-CRT) PORT will occur. Then, recently, the National Cancer Data Base (NCDB), joint program of the Commission on Cancer of the American College of Surgeons and the American Cancer Society, has been queried to study the impact of modern PORT in the setting of standard-of-care adjuvant chemotherapy for pathologic stage IIIA (N2) NSCLC (6). Data of 1850 patients who received PORT between 1998 and 2010 were obtained. Use of PORT, compared with no PORT, was associated with a significant increase in median OS (45.2 v 40.7 months, respectively), 3-year OS (59.3% v 55.2% , respectively), and 5-year OS (39.3% v 34.8%, respectively; P=.014. This analysis of the NCDB for patients with pathologic N2 disease, receiving adjuvant chemotherapy, shows that PORT seems to confer an additional improvement in OS. In conclusion, modern radiotherapy techniques should be evaluated in stage III patients, as already stated in the initial individual-patient-data meta-analysis. This new evaluation is justified for several reasons: (a) the N2 population has changed because of a better selection with pre-treatment PET-CT scan and brain imaging; (b) adjuvant chemotherapy has become a standard of care in these patients; (c) technical advances of radiotherapy may enhance the ability of PORT to improve local relapse-free survival and possibly overall survival. Thus, based on the previous studies, the underlying hypotheses remain to be proven, with sufficiently powered new randomised trials. A prospective randomized phase III trial, LungART (Lung Adjuvant Radiotherapy Trial), designed with the primary aim of investigating the benefits of conformal radiotherapy in completely resected pN2 NSCLC, together with adjuvant chemotherapy, is currently ongoing in Europe, and should help answering definitely this question, investigators are strongly encouraged to enroll patients on this randomized trial. 1 : PORT Meta-analysis Trialists Group. Postoperative radiotherapy in non-small-cell lung cancer: systematic review and meta-analysis of individual patient data from nine randomised controlled trials. PORT Meta-analysis Trialists Group. Lancet 1998; 352:257-63. 2 : PORT Meta-analysis Trialists Group: Postoperative radiotherapy for non-small cell lung cancer. Cochrane Database Syst Rev 2:CD002142, 2005 3 : Kepka L, Bujko K, Orlowski TM, et al. Cardiopulmonary morbidity and quality of life in non-small cell lung cancer patients treated with or without postoperative radiotherapy. Radiother Oncol 2011;98:238–43. 4 : Lally BE, Zelterman D, Colasanto JM, et al. Postoperative radiotherapy for stage II or III non-small-cell lung cancer using the surveillance, epidemiology, and end results database. J Clin Oncol 2006; 24:2998-3006. 5 : Douillard JY, Rosell R, De Lena M, et al. Adjuvant vinorelbine plus cisplatin versus observation in patients with completely resected stage IB-IIIA non-small-cell lung cancer. Adjuvant Navelbine International Trialist Association ANITA: a randomised controlled trial. Lancet Oncol 2006;7: 719-27. 6 : Robinson CG, Patel AP, Bradley JD et al. Postoperative radiotherapy for pathologic N2 Non small Cell lung cancer treated with adjuvant chemotherapy : A review of the National Cancer Data Base. J Clin Oncol 2015 ; 33 :870-77

Abstract:
Evolving Role of Radiation for Oligometastases The key objectives of this presentation are to review the fundamental biology underlying metastatic disease, understand the definition and clinical evidence for oligo- (or limited) metastatic disease, analyze key clinical trials showing the potential role of stereotactic body radiation therapy (SBRT) for oligometastases and address challenges regarding this “high-tech” strategy. In the early 1900s, Halsted suggested that breast cancer spread via the local regional lymphatic vessels in a stepwise manner. Thus, once there are metastases, local therapy had no clear role. Later in the 20[th] century, an opposing theory (the “Fisher” theory) suggested that cancer is a systemic disease that, if it will ever metastasize, will already have done so early in the disease course. Local therapies are therefore less important than systemic therapies. A counterpoint to these approaches was proposed by Hellman and Weichselbaum, postulating that cancer is a spectrum from localized to wide-spread disease at the time of diagnosis, with many intermediate states (Hellman S, Weichselbaum RR. Oligometastases. J Clin Oncol. 1995 Jan;13(1):8-10). “Oligometastases” are essentially early metastases which are limited in number and location and based on a state of limited metastatic capacity. The hypothesis based on this approach is that there may be a subset of patients with oligometastatic disease for whom aggressive local treatment (such as surgery or SBRT) could change their outcome. Clinical evidence for oligometastasis includes the surgical experience for lung or liver metastases showing long-term survivals of approximately 20%. Studies are now emerging suggesting similar results utilizing SBRT. In an individual patient data meta-analysis of outcomes after surgery or SBRT, Ashworth et al. reported a 5-year survival rate of approximately 30% in patients with oligometastatic non-small cell lung cancer (Ashworth AB, et al. An individual patient data metaanalysis of outcomes and prognostic factors after treatment of oligometastatic non-small-cell lung cancer. Clin Lung Cancer. 2014 Sep;15(5):346-55). They developed a risk classification schema showing a better prognosis for metachronous vs. synchronous oligometastases (of which node-negative was favorable to node-positive). Other studies have shown that, among patients treated with SBRT for 1-5 oligometastases, those with ≤3 metastases had better outcomes compared to those with 4-5 metastases. The size of the metastases also appears to be important, as well as the biological equivalent dose (BED) of the SBRT. Studies have begun to explore the role of SBRT for oligometastases involving the lung, liver, adrenal, and other sites. It is likely that host-related factors (for example, immune mediated anti-cancer activity) and tumor related factors (such as genomics and proteomics) also affect the spectrum of disease aggressiveness. Challenges to this new “high-tech” approach will also be addressed, including issues related to patient selection, the level of evidence, and the cost effectiveness of this approach. Other approaches for improving the outcome for patients with metastatic disease will also be discussed, including the role of early palliative interventions. In summary, emerging (albeit non-randomized) data suggests that SBRT appears to be a promising strategy in selected patients with oligometastases. The patients most likely to benefit from SBRT have metachronous (vs. synchronous) metastases, N0 (vs. N+) disease, 1-3 metastases (vs. more), small metastases, and the ability to receive a higher radiation dose (BED >100Gy). Randomized trials are needed to establish whether SBRT improves progression free and/or over survival in this setting.

Background:
Despite their promise, therapies targeting driver receptor tyrosine kinases (RTKs) rarely produce complete responses and have shown modest clinical benefit in NSCLC. This suggests the presence of escape mechanisms that allow cells to survive and proliferate despite inhibition of an oncogenic driver.

Methods:
Using a genome-wide shRNA screen, we identified that the canonical Wnt/β-catenin pathway contributes to the survival of NSCLC cells during inhibition of the epidermal growth factor receptor (EGFR). In order to evaluate the effects of inhibiting the Wnt pathway on EGFR-inhibited cells, we categorized NSCLC cell lines as “Wnt-responsive” or “Wnt-non-responsive” based on their ability to upregulate β-catenin-dependent targets in response to treatment with exogenous Wnt3a. Using both shRNA knockdown and a novel tankyrase inhibitor, AZ1366, we evaluated the ability of tankyrase inhibition to synergize with EGFR-inhibition in multiple Wnt-responsive and Wnt-non-responsive cell lines. We then evaluated the effects of the combination of gefitinib and AZ1366 on the survival and tumor progression in an orthotopic mouse model. In order to comprehensively query transcriptional changes brought about by treatment, we performed RNA-seq on cells treated with gefitinib, AZ1366, or the combination of the two drugs.

Results:
We have demonstrated that inhibition of tankyrase, a key player in the canonical Wnt pathway, significantly increases the induction of senescense and/or apoptosis mediated by EGFR-inhibitors in cell lines with a Wnt-responsive phenotype, and that the ability of the tankyrase inhibitor to synergistically eliminate NSCLC cells is dependent on its actions within the canonical Wnt pathway. In Wnt-non-responsive cell lines, tankyrase inhibition did not synergize with inhibition of EGFR. We have further demonstrated that Wnt-responsive cell lines show evidence of EMT in response to Wnt ligand stimulation, and that this can be prevented with tankyrase-inhibitor treatment. Additionally, we have shown that mice orthotopically implanted with Wnt-responsive cell lines and treated with a combination of a tankyrase inhibitor and an EGFR inhibitor have a substantially reduced tumor burden and a significant improvement in survival when compared to treatment with an EGFR inhibitor alone. When Wnt-non-responsive cell lines were used, we noted no improvement in survival or reduction in tumor burden. RNA-seq analysis revealed that while most transcriptional changes present in the combination were driven by gefitinib, AZ1366 had the effect of significantly amplifying many of the changes thought to be instrumental in resistance to EGFR inhibition including increased expression of TP53 and apoptosis signaling machinery, increased expression of NF-kB signaling components, and a strong decrease in cell cycle drivers. Furthermore, treatment with AZ1366 alone resulted in decreased expression of Axl and its ligand, Gas6, a known mechanism of resistance to EGFR inhibition.

Conclusion:
Taken together, these results indicate that tankyrase inhibition impinges on multiple mechanisms of escape from EGFR-inhibition, and that its ability to synergize with EGFR-inhibition is dependent on its actions within the canonical Wnt pathway. As the goal of these studies is the development of combination therapies with EGFR inhibition, this suggest tankyrase as a promising target in the subset of NSCLC with known dependencies on signaling through the canonical Wnt pathway.

Background:
Precision therapy using EGFR small molecular inhibitors is the current standard-of-care in treatment of advanced non-small cell lung cancer (NSCLC) patients (pts) with EGFR mutations. Nonetheless, emergence of acquired resistance to therapy invariably occurs despite effective initial response. Classical rebiopsy studies of EGFR-mutant pts at clinical tumor progression based on RECIST criteria have identified diverse resistance mechanisms involving T790M-EGFR, MET amplification or activation and AXL upregulation. Tumor cells within minimal or microscopic residual disease during drug response may constitute founder cells for future disease relapse. The mechanisms of molecular changes intrinsic to these early therapeutic survivors are not yet well-understood. Our studies focus on tumor cells adaptation early during therapy to map the initial course of molecular drug resistance emergence and evolution.

Methods:
Drug-sensitive model studies of EGFR-mutant lung cancer were performed using HCC827 and PC-9 cells (exon 19 deletions EGFR) under erlotinib, and H1975 (T790M/L858R-EGFR) cells under CL-387,785 inhibition. Affymetrix microarray profiling was performed in triplicate at 0h, 8h, 9d and 9d tyrosine kinase inhibitor (TKI) followed by 7d drug-washout. Both in vitro and in vivo xenograft analyses, immunofluorescence, immunohistochemistry, time-lapse video microscopy analysis were conducted. Mass-spectrometry based global metabolomics profiling was also conducted under similar conditions as in gene expression profiling.

Results:
We identified an early adaptive and reversible drug-escape within EGFR-mutant cells that could emerge as early as 9 days during course of effective therapy with molecular drug resistance. Principal component analysis (PCA) of gene expression profiling data identified distinct transcriptome signatures in each cell state. Of note, the prosurvival cell state was independent of MET pathway activation, and had a TKI cytotoxicity escape at 100x higher IC50. The drug resistant cell state was associated with reversible cellular quiescence, suppressed Ki-67 expression, and profoundly inhibited cellular motility and migration. Transcriptome gene expression profiling revealed a remarkable adaptive genome-wide signature reprogramming, centered on the autocrine TGFβ2 cascade that involved pathways of cell adhesion, cell cycle regulation, cell division, glycolysis, and gluconeogenesis. Global metabolomic profiling of cellular metabolites in HCC827 cells under erlotinib inhibition also revealed a concurrent adaptive reprogramming of cellular metabolism during the early drug-resistant cell state, with suppression of glycolysis, TCA cycle, amino acids metabolism, and lipid bioenergetics. Our studies identified a direct link of TGFβ2 within the drug escaping cells, with the metabolic-bioenergetics quiescence, reverse Warburg metabolism and mitochondrial BCL-2/BCL-xL priming. Furthermore, this adaptive drug-resistant cell state also displayed an increased EMT and cancer stem cell signaling as adaptation to the drug treatment and that could be overcome by broad BCL-2/BCL-xL BH3 mimetic ABT-263, but not BCL-2 only mimetic ABT-199.

Conclusion:
We identified and characterized the emergence of early adaptive drug-escape within EGFR-mutant NSCLC cells amid an overall precision therapy excellent response, through a MET-independent mechanism. The profoundly drug-resisting prosurvival cell state undertook remarkable cellular transcriptome-metabolome adaptive reprogramming coorindated through autocrine TGFβ2 signaling augmentation. Our study results have important implications in lung cancer drug-resistant minimal/microscopic disease and future therapeutic remedies in precision therapy.

Background:
Non-small cell lung cancer patients harboring epidermal growth factor receptor (EGFR) mutation respond well to EGFR tyrosine kinase inhibitors (TKI). However, all patients develop resistance to EGFR TKI after long term use. EGFR T790M mutation can be found in about half of the resistant re-biopsy tumors. Afatinib is an irreversible EGFR TKI with in vitro activity against resistant T790M mutation. However, afatinib has little activity in EGFR TKI resistant patients whose tumors developed T790M mutation. A novel alinino-pyrimidine based WZ8040 has been developed to specifically inhibit phosphorylation of EGFR with T790M mutation and not on wild type EGFR. Similar compounds such as CO1686 or AZD9291 has demonstrated high activity against T790M mutations. We plan to develop afatinib or AZD9291, WZ8040 resistant PC9 cells to study afatinib or AZD9291 resistance.

Methods:
PC9 cells were grown in culture media containing escalating concentrations of afatinib, WZ8040 or AZD9291. When cells can grow in high concentrations of drugs, cells were cloned, expanded and grew in drug-free media for more than two weeks to obtain stable afatinib, WZ8040 or AZD9291 resistant clones. Gefitinib, afatinib, WZ8040, AZD9291. Cells viability were determined by surforodamine bromide method. PC9 parental and EGFR TKI resistant cells were treated with gefitnib, afatinib, WZ8040 or AZD9291 for one hour and EGFR, AKT, ERK phosphorylation were determined by Western blot. DNA repair capacity were compared between sensitive and resistant cells after exposure of cells to ultraviolet light and measured by pGL3-luciferase plasmid transfection methods. Epithelial mesenchyma transition of these cells were tested by snail, slug, vimentin and E-cadherin western blot. Autophagy was measured by LC3-II levels by Western blot. EGFR exon 18-21 sequence of each clones were determined by Sanger’s direct sequencing.

Results:
We developed afatinib resistant PC9 cells, PC9/AFAb2, PC9/AFAc3 and WZ8040 resistant PC9/WZd7, PC9/WZf6. PC9/AFA cells were more than 100-fold resistant to afatinib and PC9/WZ cells were more than 50-fold resistant to WZ8040. 10nM of afatinib treatment inhibits EGF-induced EGFR, AKT and EKR phosphorylation in PC9 cells, but phosphorylation of these kinases were only partially inhibited in PC9/AFA cells. Phosphorylation was completely blocked at 100nM afatinib. MEK inhibitor plus afatinib did not reverse resistance to afatinib in PC9/AFA cells. On the other hand, WZ8040 or AZD9291 alone completely reversed resistance in PC9/AFA cells. EGFR, AKT and ERK phosphorylation can be blocked by 100nM WZ8040 in PC9 and PC9/WZd7 cells. However, it is curious that phosphorylation of these proteins can be inhibited by 100nM gefitinib as well. EGFR T790M mutation was only detected in PC9/AFA cells and not in PC9/gef, PC9/WZ cells. None of the PC9/WZ cells have EGFR C797S mutation. We did not detect any other EGFR resistance mechanism in PC9/AFA cells. Other of comparing EMT, autophagy and DNA repair capacity of PC9 and their resistant cells are ongoing.

Conclusion:
We developed multiple gefitinib, afatinib, WZ8040, AZD9291 resistant PC9 cells. Only afatinib resistant cells develop EGFR T790M. We demonstrated that EGFR T790M was the predominant resistant mechanism in PC9/AFA cells. The characteristics of PC9/WZ and PC9/AZD9291 are still under investigation.

Background:
Rociletinib (CO-1686) is a novel, oral, irreversible tyrosine kinase inhibitor for the treatment of patients with mutant epidermal growth factor receptor (EGFR) non-small cell lung cancer (NSCLC) that has demonstrated efficacy against the activating mutations (L858R and del19) and the dominant acquired resistance mutation (T790M), while sparing wild-type (WT) EGFR. Although rociletinib has generated compelling objective responses in heavily-pretreated T790M positive and negative NSCLC patients, acquired resistance to rociletinib monotherapy has also been observed. We are currently exploring preclinical combinations to delay or prevent resistance to rociletinib.

Methods:
To study acquired resistance in an unbiased fashion, rociletinib resistant populations and clones were generated from the EGFR mutant NSCLC cell lines PC-9 (del19 EGFR), HCC827 (del19 EGFR), and NCI-H1975 (L858R/T790M EGFR) by chronic in vitro and/or in vivo exposure of rociletinib. Compound library screening was performed with rociletinib resistant cell lines to identify drug combinations that could restore rociletinib sensitivity. In vitro and in vivo validation, mechanism of action, and combination studies were performed to evaluate the potency of these combinations in rociletinib sensitive and resistant preclinical models. In addition, combination studies with therapies commonly used in NSCLC, including radiotherapy (RT), an anti-EGFR antibody, and anti-PD-1/L1 antibodies were also explored.

Results:
Multiple mechanisms of resistance were observed in rociletinib resistant cell lines, including MET amplification and an epithelial-mesenchymal transition (EMT). In a PC-9 resistant population (designated 2A10) generated by in vitro and in vivo selection, multiple agents including the aurora kinase inhibitor MLN8237, the MEK inhibitor trametinib, and an anti-EGFR antibody restored rociletinib sensitivity in cell viability assays. Western blot analysis demonstrated that the levels of p-ERK in the parental PC-9 cell line were comparable to p-ERK levels in 2A10 cells grown in the presence of 1 mM rociletinib. The combination of rociletinib and trametinib in the 2A10 cell line suppressed p-ERK signaling, concomitant with increased levels of apoptotic markers such as PARP cleavage. The combination of rociletinib and trametinib also demonstrated potent in vivo activity in the 2A10 xenograft model. In vitro and in vivo studies performed with additional cell lines and combinations are ongoing and will also be presented.

Conclusion:
Resistance to all 3[rd] generation EGFR inhibitors is likely to be observed, and identifying tolerable and effective combinations to delay or prevent resistance is critical in extending the clinical benefit of these therapies. In vitro and in vivo studies reported here highlight multiple combinations that restore the activity of rociletinib in rociletinib resistant models. In particular, the combination of trametinib and rociletinib restored MAPK pathway suppression and anti-tumor activity in the rociletinib resistant 2A10 model. These nonclinical data support the ongoing Phase 1/2 evaluation of the combination of trametinib and rociletinib.

Abstract not provided

Background:
The Lung Cancer Mutation Consortium (LCMC) 1.0 demonstrated multiplexed genomic platforms can assay 10 oncogenic drivers in tumor specimens from patients with lung adenocarcinomas. 28% of the patients with oncogenic drivers could be effectively targeted. The survival of these 275 patients treated with targeted agents was longer than the patients who were not treated with a targeted agent (Kris and Johnson JAMA 2014). The efficiency of Next-Generation Sequencing enables more comprehensive testing of additional aberrations with less tumor tissue. LCMC 2.0 was initiated to test tumor specimens for 12 oncogenic drivers and to provide the results to clinicians for treatment decisions and research purposes.

Methods:
The 16 site LCMC 2.0 is testing tumors from 1000 patients with lung adenocarcinomas in CLIA laboratories for mutations in KRAS, EGFR, HER2, BRAF, PIK3CA, AKT1, and NRAS, MET DNA amplification, and rearrangements in ALK as done in LCMC 1.0. The new genes that were added because of emerging information about potential therapeutic targets include MAP2K1 mutations, RET and ROS1 rearrangements, PTEN (MAb 138G4) loss and MET (MAb SP44) overexpression by immunohistochemistry (IHC). All patients were diagnosed with stage IIIB/IV lung adenocarcinoma after May 2012, had a performance status 0-2, and available tumor tissue.

Results:
Of 1073 patients registered, data is now reported for 759. The median age of the patients is 65 (23-90). The population includes 369 (55%) women; 164 (24%) never smokers, 399 (59%) former smokers, and 73 (11%) current smokers; 26 (4%) Asians, 58 (9%) African American, 548 (81%) Caucasian, and 43 (6%) of other races. As of April 2015 information on genomic and immunohistochemical changes for 675 eligible patients were recorded in our database. Alterations in oncogenic drivers were found in 45% of samples as follows: 159 KRAS (24%), 88 EGFR (13%), 25 ALK (4%), 19 BRAF (3%), 17 PIK3CA (3%), 9 HER2 (1%), 4 NRAS (1%) 0 AKT1, 28 had ≥ 2 findings (4%) and 25 MET DNA amplification (4%). The new genes studied in LCMC 2.0 revealed 1 MAP2K1 mutation (<1%), 19 RET (3%) and 9 ROS (1%) rearrangements, 94 had PTEN loss (14%), and 362 with MET overexpression (54%). As expected, PIK3CA mutations and PTEN loss by IHC were mutually exclusive in 109 of 111 (98%) patients’ tumors. Seventeen of the 23 (74%) with MET DNA amplification studied thus far with IHC had MET overexpression. Next-Generation platforms were used at 13 of 16 LCMC 2.0 sites.

Conclusion:
Next-Generation Sequencing is rapidly becoming routine practice at LCMC 2.0 centers with use going from 0 to 81% of sites since 2012. LCMC 2.0 identified additional targets (RET and ROS1 rearrangements and PTEN loss). PIK3CA and PTEN were largely mutually exclusive and an actionable oncogenic driver has been identified in the 45% of initial lung adenocarcinoma specimens. Supported by Free to Breathe

Background:
Fibroblast growth factor receptor (FGFR) tyrosine kinase plays a crucial role in cancer cell growth, survival, and resistance to chemotherapy. FGFR1 amplification occurs at a frequency of 10-20% and is a novel druggable target in squamous cell carcinoma of the lung (SCCL). A number of FGFR-targeted agents are currently being developed in SCCL harboring FGFR alterations. The aim of the study is to evaluate the activity of selective FGFR inhibitors (AZD4547, BAY116387) and the mechanisms of intrinsic and acquired resistance to these agents in SCCL.

Methods:
The antitumor activity of AZD4547 and BAY116387 was screened in a panel of 12 SCCL cell lines, among which 4 cell lines harbored FGFR1 amplification. To investigate mechanisms of acquired resistance, FGFR1-amplified H1581 cells which were exquisitely sensitive to FGFR inhibitors, were exposed to AZD4547 or BAY116387 to generate polyclonal resistant clones (H1581-AR, H1581-BR). Characterization of these resistant clones was performed using receptor tyrosine kinase (RTK) array, immunoblotting and microarray. Migration and invasion assays were also performed.

Results:
Among 12 SCCL cell lines, two FGFR1-amplified cells, H1581 and DMS114, were sensitive to FGFR inhibitors (IC~50~<250 nmol/L). Compared with resistant cells, sensitive cells showed increased phosphorylation of FRS2 and PLC-γ, but decreased phosphorylation of STAT3. There was no noticeable difference in FGFR1-3 protein expression level between sensitive and resistant cells. Importantly, phosphorylation of ERK1/2 was significantly suppressed upon treatment of FGFR inhibitors only in sensitive cells, suggesting phospho-ERK1/2 as a pharmacodynamic marker of downstream FGFR signaling. RTK array and immunoblots demonstrated strong overexpression and activation of MET in H1581-AR and H1581-BR, in comparison to almost nil expression in parental cells. Four different SCCL cells with intrinsic resistance to FGFR inhibitors also showed intermediate to high MET expression, suggesting that MET may be involved in both intrinsic and acquired resistance to FGFR inhibitors. Gene-set enrichment analysis against KEGG database showed that cytokine-cytokine receptor interaction pathway was significantly enriched, with MET contributing significantly to the core enrichment, in H1581-AR and H1581-BR, as compared with parental cells. Stimulation with HGF strongly activated downstream FGFR signaling or enhanced cell survival in the presence of FGFR inhibitors in both acquired and intrinsic resistant cells. Quantitative PCR on genomic DNA and fluorescent in situ hybridization revealed MET amplification in H1581-AR, but not in H1581-BR. MET amplification led to acquired resistance to AZD4547 in H1581-AR by activating ERBB3. The combination of FGFR inhibitors with ALK/MET inhibitor, crizotinib, or small interfering RNA targeting MET synergistically inhibited cell proliferation in both H1581-AR and H1581-BR, whereas it resulted in additive effects in SCCL cells with intrinsic resistance to FGFR inhibitors. Acquisition of resistance to FGFR inhibitors not only led to a morphologic change, but also promoted migration and invasion of resistant clones via inducing epithelial to mesenchymal transition phenotype, as documented by a decrease in E-cadherin and an increase in N-cadherin and vimentin.

Conclusion:
MET activation is sufficient to bypass dependency on FGFR signaling and concurrent inhibition of these two pathways may be desirable when targeting FGFR-dependent SCCL.

Background:
Activating mutations in the HER2 kinase domain are detected in 2-4% of non-small cell lung cancers (NSCLC), and are oncogenic in both in vitro and in vivo models. Current clinical strategies to target mutant HER2 include the use of covalent HER2 inhibitors afatinib, dacomitinib and neratinib; all of which have limited single agent activity. We evaluated how drug sensitive models of HER2 mutant lung cancer develop acquired resistance in vitro to gain biological insights and to predict how acquired resistance may develop in the clinic.

Methods:
Murin Ba/F3 cells expressing duplication/insertion of four amino acids (YVMA) between codon 775 and 776 in exon 20 of HER2 gene (A775_G776insYVMA (insYVMA)) were exposed to N-ethyl-N-nitrosourea mutagenesis and expanded in the presence of neratinib and dacomitinib. Total RNAs were extracted from resistant clones and sequencing of the HER2 tyrosine kinase domain was performed. Drug resistance was confirmed with cell growth assays and western blotting.

Results:
Total 5 clones for Neratinib and 7 clones for Dacomitinib were expanded from each 300 wells. Sequencing analysis revealed that all resistant clones retained original insertion mutation and acquired same substitution of Cysteine to Serine change in codon 805 (C805S) in exon 20 of HER2 gene. This mutation is analogous to the EGFR C797S mutation that mediates resistance to 3[rd] generation EGFR inhibitors. Next, we generated Ba/F3 cells co-expressing activating mutations; insYVMA and a dacomitinib hypersensitive insertion mutation (insertion of three amino acids (WLV) after codon 774 with deletion of M774 (M774del insWLV (insWLV))), in cis with the C805S mutation. Cell growth assay revealed these double mutants were resistant to all three second generation inhibitors for EGFR family; neratinib, dacomitinib, and afatinib, compared to parental cells which only have activating mutation. They were also resistant to 3[rd] generation EGFR inhibitors; WZ40002 and AZD9291. Phosphorylation of HER2 was not completely inhibited by these drugs. Resistant cells showed moderate sensitivity to mTOR inhibitor; rapamycin alone. Combination treatment with afatinib and rapamycin effectively inhibited growth of these cells.

Conclusion:
The C805S secondary HER2 mutation results in acquired resistance to covalent HER2 inhibitors in HER2 mutant NSCLC. Our results provide insights into drug resistance mechanisms and help predict likely clinical mechanisms of resistance to HER2 targeted therapies in HER2 mutant NSCLC.

Abstract not provided

Background:
LKB1 is a critical regulator of cell growth, metabolism and EMT, and it is mutated in 20-30% of non-small cell lung cancers (NSCLC). LKB1 mutations co-occur with KRAS-activating mutations in 7%-10% of all NSCLC and results in an aggressive phenotype and a worse response to chemotherapy compared to KRAS-mutated tumors. Because LKB1 activates AMPK (AMP-activated protein kinase) which functions as a cellular energy sensor, LKB1-deficient cells are unable to appropriately sense metabolic and energetic stress. LKB1 is also known to regulate angiogenesis, but the mechanism(s) by which this occurs remains unclear. Bevacizumab, the human anti-VEGF antibody approved for the treatment of NSCLC, improves the progression-free and overall survival of NSCLC patients combined with chemotherapy, but often the benefit is transient, and therapeutic resistance occurs. Our laboratory has previously identified phenotypical differences in vasculature patterns in A549 NSCLC tumors resistant to bevacizumab (LKB1 mutant), when compared to H1975 tumors, (LKB1 wild-type). In addition, LKB1 mutant NSCLC cell lines are highly vulnerable to agents acting on energetic pathways. These results may indicate that loss of LKB1 in NSCLC could alter the tumor vasculature and regulate sensitivity to anti-angiogenic therapies. Here, we investigate the hypothesis that combinations of energetic-depleting compounds along with blockade of tumor angiogenesis would be more effective in NSCLC LKB1 mutant tumors.

Methods:
mRNA and protein expression of 584 angiogenesis-related genes were analyzed in wild-type and LKB1 mutant NSCLC (TCGA, RPPA and PROSPECT databases). In vitro validation was performed using qPCR, immunohistochemistry and western blot analysis as well as pairs of isogenic LKB1 mutant cell lines with overexpressed or silenced LKB1. Endothelial cells were incubated with conditioned medium of wild-type and LKB1 mutant NSCLC cell lines, and tube formation matrigel, proliferation and migration (Boyden chamber) assays were performed.

Results:
We identify a group of new and classic angiogenesis-related molecules: VEGFA, VEGFR1, KDR, NRP1, PDGFB, PDGFRA-B, HIF-1A, C-KIT, VCAM1, hypoxia related molecules: HIF1AN, EGLN1, HIF3A, CA12, EPAS1 and immune related molecules: TNFSF11, NFKB1, CD47, PDL1 differentially expressed in LKB1-wild type and LKB1 mutant NSCLC (p<0.05 and fold-change ≥ or ≤1.5). LKB1 mutant cell lines showed higher protein expression of phospho-cKIT, a tyrosine-kinase receptor involve in cell proliferation and angiogenesis, and CA12 (Carbonic anhydrase 12), a known HIF-1α regulated molecule, involved in maintaining cellular pH homeostasis. Also, LKB1 mutant cells exhibit different quantitative vascular patterns in matrigel assays like number of nodes, junctions, length and branching of the endothelial matrix (p<0.05). Human endothelial cells exhibited an increase rate of proliferation and migration when incubated with conditioned medium from LKB1 mutant NSCLC cell lines compared with conditioned media from LKB1-wild type NSCLC cell lines (p<0.05).

Conclusion:
There are biological differences in vasculature patterns in LKB1 mutant NSCLC tumors and in LKB1 mutant cell lines comparing with wild-type LKB1. These differences are translated in biological alterations of human endothelial cells in vitro suggesting an important role of LKB1 in resistance to anti-angiogenic treatments in vivo.

Background:
RET gene fusions were identified as a novel oncogenic driver of ~1-2% of non-small cell lung cancer (NSCLC) patients and clinical trials investigating the use RET TKI therapy are underway. Like all NSCLC patients treated with TKI therapies, it is expected that drug resistance will emerge in this patient population. The mechanisms that drive acquired resistance to RET TKI therapy are still unknown. The objective of this study is to advance current understanding of RET signaling in NSCLC and to identify the cellular mechanisms of acquired RET TKI resistance that will eventually emerge in RET fusion positive NSCLC patients by using in vitro models of drug resistance.

Methods:
The LC-2/ad is a lung adenocarcinoma cell line that harbors the CCDC6-RET fusion. We created three distinct ponatinib resistant (PR) LC-2/ad cell lines (PR1, PR2, PR3) derived from three different dose-escalation strategies. RET break-apart fluorescence in situ hybridization (FISH) was performed on the parental LC-2/ad and PR-derivatives. Interactions between the RET kinase domain and known adaptor signaling molecules were assessed via proximity ligation assay (PLA) in parental LC-2/ad cells and resistant lines. Formation of RET-adaptor signaling complexes were confirmed via immunoprecipitation and western blot analysis. Next-generation RNA sequencing in conjunction with a high-throughput small molecule inhibitor screen were performed to elucidate the signaling pathways that drive resistance to RET-inhibition. Pathways and candidate molecules identified by these screens were validated using siRNA knockdown and pharmacologic inhibition in the context of a cell-proliferation MTS assay. Western blot analysis was utilized to identify the downstream signaling programs responsible for proliferation and survival in the RET-inhibition resistant cell lines.

Results:
MTS cell proliferation assay confirmed that all three ponatinib resistant cell lines are significantly less sensitive to ponatinib than parental LC-2/ad cells. RET FISH analysis demonstrated that the CCDC6-RET gene was retained in the PR1 and PR2 cell lines, but lost in the PR3 cell line. RT-PCR and western blot analysis confirmed the loss of the CCDC6-RET fusion in the PR3 cell line. DNA sequencing demonstrated no RET kinase domain mutations in either the PR1 or PR2 derivatives. Further, profound changes in the RET-signaling program have emerged in the PR1 and PR2 cell lines. Using a RET-GRB7 PLA, we have demonstrated that PR1 cells no longer form RET-GRB7 signaling complexes, while PR2 cells retain RET-GRB7 complexes even in the presence of ponatinib. Next-generation RNA sequencing of the PR1 cell line revealed an increase in expression of several known EMT markers including caveolin-1, vimentin, and ADAMTS1.

Conclusion:
Like many other targeted therapeutic strategies, resistance to small molecule Ret-inhibition in RET-fusion positive lung cancer cells can be driven by multiple mechanisms. Changes in the RET-adaptor programming appear to mitigate resistance in both the PR1 and PR2 cell lines, suggesting that RET-resistant cells may have successfully undergone an oncogenic switch to rely upon another known oncogenic driver in lieu of the CCDC6-RET fusion. Further, EMT reprogramming of the LC-2/ad cell may have contributed to the resistance phenotype in the PR1 cell line.

Background:
The BIM deletion polymorphism in intron 2 was found in a significant percent (~13%) of the Asian population, with 0.5% of individuals being homozygous for this deletion. Patients with EGFR mutant lung cancers harboring this BIM polymorphism have shorter progression free survival and overall response rates to 1[st] generation EGFR-TKIs, gefitinib and erlotinib. We recently reported that the histone deacetylase (HDAC) inhibitor vorinostat can epigenetically restore BIM function and death sensitivity of EGFR-TKI, in cases of EGFR mutant lung cancer where resistance to 1[st] generation EGFR-TKI is associated with a heterozygous BIM polymorphism. Here, we examined 1) whether BIM polymorphism associated with resistance to new generation EGFR-TKIs and 2) whether vorinostat could overcome EGFR-TKI resistance in EGFR mutant lung cancer cells with a homozygous BIM polymorphism.

Methods:
We used EGFR mutant lung cancer cells lines, PC-9, PC-9[i2BIM-/-] (a genetically engineered subclone that was homozygous for BIM deletion polymorphism), and PC-3 (heterozygous for BIM deletion polymorphism). These cell lines were treated with gefitinib, afatinib (2[nd] generation), and AZD9291 (3[rd] generation). Apoptosis was evaluated by FACS and expression of cleaved-caspase 3/7 and PARP by western blot.

Results:
While PC-9 cells were sensitive to all EGFR-TKIs in terms of apoptosis induction, both of PC-3 and PC-9[i2BIM-/- ] cells were resistant to 1[st] generation EGFR-TKIs and new generation EGFR-TKIs as well. Vorinostat combined with new generation EGFR-TKIs induced apoptosis of PC-3 and PC-9[i2BIM-/- ] cells in vitro. In the subcutaneous tumor model, AZD9291 regressed the tumors produced PC-9 cells but not PC-9[i2BIM-/- ] cells, indicating in vivo resistance of PC-9[i2BIM-/- ] cells to EGFR-TKIs. Combined use of vorinostat with AZD9291 successfully decreased the size of tumors produced by PC-9[i2BIM-/-] cells by inducing tumor cell apoptosis.

Conclusion:
These observations indicated that BIM deletion polymorphism is associated with apoptosis resistance caused not only by 1[st] generation EGFR-TKIs but also by new generation EGFR-TKIs. Moreover, combined use of HDAC inhibitor may overcome EGFR-TKI resistance associated not only with heterozygous deletion but also with homozygous deletion in the BIM gene.

Background:
Lung cancer accounts for one-fifth of cancer deaths worldwide with invasion, metastases and drug resistance representing major causes of mortality and barriers to cure. While lung cancers with activating mutations in the EGF receptor (EGFR) are susceptible to tyrosine kinase inhibitors (TKI), such as erlotinib and gefitinib, the efficacy of these agents is limited by the inevitable development of resistance. The epithelial-mesenchymal transition (EMT), by which epithelial cells acquire a mesenchymal and invasive phenotype, is one mechanism promoting EGFR-TKI resistance, including resistance to 3[rd] generation T790M-specific inhibitors. However, the molecular connections between EMT and resistance are not well understood. Here we report that upregulation of Neuropilin-2 (NRP2) is crucial for development of EGFR-TKI resistance associated with the EMT. NRP2 is a cell surface receptor for SEMA3F, a secreted semaphorin with tumor suppressor activity that is down-regulated during EMT. NRP1 and NRP2 are also co-receptors and signaling enhancers for several growth-promoting ligands such as VEGF, HGF and FGF. We previously reported that NRP2 was induced by TGFβ as part of an EMT response in lung cancers and that NRP2 knockdown suppressed the EMT phenotype, including local tumor invasion in a subcutaneous xenograft model.

Methods:
Immunohistochemistry (IHC) was performed for NRP2 on patient biopsies, before and after development of gefitinib resistance. EGFR mutant NSCLC cell lines, transfected with control or NRP2-specific shRNAs, were selected for gefitinib/erlotinib resistance in vitro, using progressively increasing concentrations or continuous exposure to IC~50~ levels of EGFR TKIs. Western blot analysis confirmed changes in NRP2 expression along with selected markers of EMT. MTS viability assays determined drug sensitivity while migration and invasion were assessed using Boyden chambers. Growth as spheroids was assessed in 1% methylcellulose medium in low-adherence plates.

Results:
Increased NRP2 was observed in lung tumor biopsies following acquisition of EMT-associated gefitinib-resistance, and in HCC4006-ER cells, which acquired a stable erlotinib-resistant EMT phenotype. In vitro, using multiple EGFR mutant cell lines, NRP2 knockdown blocked acquired gefitinib-resistance, arising both spontaneously following growth in IC~50~ concentrations or after exposure to TGFβ. Of interest, spontaneously-resistant cells exhibited increased migration similar to cells stimulated with TGFβ. NRP2 knockdown also blocked tumorsphere formation, which has been associated with stem-cell characteristics and drug resistance.

Conclusion:
Collectively, our results demonstrate that NRP2 is a mediator of acquired EGFR-TKI resistance. The results also suggest that NRP2 blocking antibodies could be useful for enhancing the duration of response to EGFR inhibitors, including those targeting the T790M mutation.

Abstract not provided

Background:
ROS1 fusion variants represent an important subset of oncogenic driver mutations in approximately 0.7 – 3.4% of non-small cell lung cancers. Since the frequency of ROS1 positive lung cancer patients is relatively low, it is unclear whether there are significant clinicopathologic associations for positive cases. Thus far, ROS1 positive patients tend to be younger and never-smokers with tumors displaying adenocarcinoma histology. This study describes a further cohort of ROS1 positive lung cancer patients in an effort to identify clinicopathologic associations.

Methods:
The data represent a retrospective analysis of the clinicopathological profiles of primary and metastatic lung cancer patients tested for ROS1 gene rearrangements by break-apart (BA) FISH at the University of Colorado School of Medicine.

Results:
The cohort consisted of 452 patients enriched for triple-negative (EGFR-, KRAS- and ALK-) non-squamous cell carcinomas screened for ROS1 rearrangements using the BA FISH assay. Nineteen cases (4.2%) were identified as positive for rearrangement, the majority (68%) of which were female, with a mean cohort age of 54.9 years (range 30-79); as compared to negative cases which included 56% female patients (P= 0.1083), and had a mean cohort age of 62.9 (range 21-90) (P= 0.0058). Seventeen out of the 19 ROS1 positive tumors were classified as adenocarcinomas, one was diagnosed as adenosquamous carcinoma, and the histology on one specimen was not otherwise specified (NOS). Among 12 individuals with information on pathologic stage at diagnosis, the majority (75%) were stage IV. The prevalent FISH pattern for rearrangement was a split 5’ and 3’ signal (68%) with the remaining specimens showing primarily single 3’ signals (21%) or a mix of split and single 3’ signals (11%).

Conclusion:
The ROS1 positive tumors in this cohort were primarily classified as adenocarcinomas, diagnosed at an advanced stage, in patients significantly younger and more likely to be women, although the sample set was biased for non-squamous lesions thereby limiting the application of this information to squamous cell lung carcinoma. The higher prevalence of ROS1 positive cases in this cohort compared to unselected cohorts is best explained by the inclusion of specimens with known negative status for EGFR and KRAS mutations and ALK fusions. As such, these data are in agreement with previous descriptions of ROS1 positive cohorts. Screening for ROS1 rearrangements in lung cancer patients displaying adenocarcinoma histology and negative for EGFR, KRAS and ALK activating events should identify a higher frequency of ROS1 rearranged tumors compared to unselected approaches and facilitate this subset of patients to be treated with targeted ROS1 inhibitors.

Background:
Primary pulmonary lymphoepithelioma-like carcinoma (LELC) is a rare histological type of large cell NSCLC. Histopathologically it is similar to nasopharyngeal carcinoma which is most commonly occurred in Southern China. It had close relationship with Epstein-Barr virus (EBV) infection. Over the past 28 years since it was first reported, less than 300 Primary pulmonary LELC cases have been reported in the literature. Due to its rarity, the treatment of advanced LELC is not only empirical, but controversial. Testing for EGFR mutation and ALK rearrangement are routine for NSCLC patients in clinical practice now. However, only few genotype studies have been done in pulmonary LELC, and till now no targeted therapy has been shown effective in the treatment of these patients.

Methods:
We investigated a cohort of 42 patients with primary pulmonary LELC and genotyped for ALK rearrangement and EGFR mutation. ALK rearrangement was detected by Fluorescence in situ Hybridization (FISH). EGFR mutational analysis of exons 18 through 21 was analyzed by TaqMan real-time polymerase chain reaction (PCR).

Results:
The clinicopathologic characteristics of 42 patients with pulmonary LELC are presented in Table 1. Twenty-seven of 42 patients were in stage I-IIIA (64.3%), and only 15/42 patients (35.7%) were in stage IIIB or IV. The female to male ratio was about 22:20, and the median age at diagnosis was 51 years (range, 29-67 years). Only 13 (31.0%) patients were smokers. In situ hybridization for Epstein-Barr virus-encoded RNA (EBERs) showed positive signals in all 42 patients. By immunohistiochemistry staining, all patients demonstrated positive expression of CK5/6 and P63, but almost all patients were negative for TTF-1(34/34, 100%) or CK7 (34/35, 97.1%). None of the 42 patients had ALK rearrangement. Of 42 patients tested EGFR mutation, only one patient (2.4%) harbored L858R mutation and gefitinib was applied to this case, however no objective response was observed and the progression free survival PFS time was only 1 month.Figure 1



Conclusion:
Primary pulmonary LELC is a unique histological subtype of non-small cell lung cancer. ALK rearrangement and EGFR mutation are lack and they may not be the oncogenic driver gene in pulmonary LELC. Conventional cytoxic chemotherapy is by far still a backbone treatment in advanced stage primary pulmonary LELC. Future efforts should be made to explore other oncogenic driver gene to guide targeted therapy in this rare disease to determine the optimal treatment.

Background:
Activation of the MET signaling pathway can propel the growth of cancer cells in non-small cell lung cancer (NSCLC). Increased MET gene by amplification and/or polysomy can cause MET protein overexpression; less common causes include mutations, translocations, and alternative RNA splicing. Clinical trials using MET as a biomarker for selection of lung cancer patients who might most benefit from targeted therapy have experienced variable outcomes. We aimed to characterize the relationship between MET protein overexpression and MET amplification or mean copy number alterations in patients with NSCLC.

Methods:
The Lung Cancer Mutation Consortium (LCMC) is performing an ongoing study of biomarkers with patients with NSCLC from 16 cancer center sites across the United States. For this analysis, 403 cases had complete data for MET protein expression by immunohistochemistry (IHC, monoclonal antibody SP44, Ventana) and MET gene amplification by fluorescence in-situ hybridization (FISH, MET/CEP7 ratio). Pathologists evaluated MET expression using the H-score, a semi-quantitative assessment of the percentage of tumor cells with no, faint, moderate, and/or strong staining, ranging from 0-300. Spearman's correlation was used to analyze the correlation between MET protein expression (H-scores) and FISH results (MET/CEP7 ratio (N=403) and MET copy number (N=341). Protein overexpression using 5 different cut-offs was compared with amplification defined as MET/CEP7 ≥ 2.2 and high mean copy number defined as ≥ 5 MET gene copies per cell using the Fisher’s exact test. Cox Proportional Hazards models were built to examine the associations of these different definitions of positivity with prognosis, adjusting for stage of disease.

Results:
MET protein expression was significantly correlated with MET copy numbers (r=0.17, p=0.0025), but not MET/CEP7 ratio (r=-0.013, p=0.80). No significant association was observed between protein overexpression using a commonly used definition for MET positivity (“at least moderate staining in ≥ 50% tumor cells”) and MET amplification (p=0.47) or high mean copy number (p=0.09). A definition for MET protein overexpression as “≥ 30% tumor cells with strong staining” was significantly associated with both MET amplification (p=0.03) and high mean copy number (p=0.007), but a definition of “≥ 10% tumor cells with strong staining” was not significantly associated with either. Definitions of protein overexpression based on high H-scores (≥200 or ≥250) were associated with high MET mean copy numbers (p=0.03 and 0.0008, respectively), but not amplification (p=0.46 and 0.12, respectively). All 5 definitions of MET protein overexpression demonstrated a significant association with worse prognosis by survival analyses (p-values ranged from 0.001 to 0.03). High MET copy number (p=0.045) was associated with worse prognosis, but MET amplification was not (p=0.07).

Conclusion:
Evaluation of NSCLC specimens from LCMC sites confirms that MET protein expression is correlated with high MET copy number and protein overexpression is associated with worse prognosis. Definitions of MET protein overexpression as “an H-score ≥250” and “≥30% tumor cells with strong staining” were significantly associated with high mean MET copy number. It may be worth reevaluating the performance of MET as a biomarker by different definitions of positivity to predict response to MET-targeted therapies.

Background:
Identification of driver mutations has led to the dramatic improvement in personalized therapy for lung adenocarcinoma. However, few targeted therapeutics are approved for treatment of squamous cell lung carcinoma. The identification of druggable molecular targets in SqCLC has been becoming a top research priority. Therefore, the aim of this study was to analyze the driver mutation profiles in a large cohort of Chinese squamous cell lung carcinomas to identify potential therapeutic targets.

Methods:
Approximately 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes were analyzed on 159 samples by using Ion Torrent semiconductor-based next-generation sequencing. The gene copy numbers of FGFR1, EGFR, HER2, PDGFRA, CCND1, SOX2, CDKN2A, and PTEN were assessed by FISH on 250 samples. In addition, the status of PTEN expression was examined by immunohistochemistry on 250 samples.

Results:
Somatic mutations were detected in 73.6% (117/159) of patients. The most commonly mutated gene detected in this study was TP53 (56.0%, 89/159), followed by CDKN2A (8.8%, 14/159), PI3KCA (8.8%, 14/159), KRAS (4.4%, 7/159), EGFR (3.1%, 5/159), FBXW7 (2.5%, 4/159), PTEN (2.5%, 4/159), FGFR3 (1.3%, 2/159), AKT1 (1.3%, 2/159) and KIT (0.6%, 1/159). Copy number alterations were present in 77.6% (191/246) of patients, including FGFR1 amplification (13.7%, 34/248), EGFR amplification (11.4%, 28/246), HER2 amplification (8.9%, 22/246), PDGFRA amplification (7.7%, 19/246), CCND1 amplification (11.0%, 27/246), SOX2 amplification (35.0%, 86/246), CDKN2A deletion (18.7%, 46/246), and PTEN deletion (29.3%, 72/246). The loss of PTEN expression was observed in 43.5% (108/248) of patients. TP53 mutations were significantly more common in men and smokers, while the frequency of EGFR mutation was significantly higher in women and never smokers. Amplification of FGFR1, CCND1 and SOX2 genes were significantly associated with smoking. The incidence of FGFR1 amplification in patients without lymph node metastasis was significantly higher than that in patients with lymph node metastasis (19.4% vs. 10.2%，P=0.043). The frequency of SOX2 amplification in tumors with moderate and poor differentiation was significantly higher than that in tumors with well differentiation (39.6% vs. 33.6% vs. 0%，P=0.036). The incidence of loss of PTEN protein expression in patients with pleural invasion was 51.2%, which was significantly higher than that in patients without pleural invasion (P=0.017). The loss of PTEN expression was significantly associated with PTEN gene deletion (P=0.001). No significant association was observed between the molecular abnormalities and disease-free survival and overall survival.

Conclusion:
Genetic alterations are common in squamous cell lung cancers. The findings of this study may facilitate the identification of molecular target candidates for precision medicine in patients with squamous cell lung cancers.

Abstract not provided

Background:
Identification of mutations which drive pulmonary adenocarcinomas (ADC) has rapidly moved from the research arena to clinical practice. The prevalence of these mutations has been suggested by a multitude of studies but here we describe the prevalence of mutations from a large study of patients with advanced ADC treated in the international phase III study INSPIRE (Lancet Oncology 2015) with all testing performed in one CLIA-certified laboratory under standardized conditions.

Methods:
Mutation testing was performed on 412 adenocarcinoma specimens using SNaPshot® methodology. Mutations were examined in the AKT, EGFR, KRAS, BRAF, NRAS, PIK3CA, TP53, PTEN, CTNNB1, and MEK1 genes. The relative frequencies of genetic alterations were calculated based on the total number of adequate specimens and specific consent for testing.

Results:
Of the 412 adenocarcinoma specimens tested, 372 (90.3%) had evaluable results from mutation testing. A single mutation was detected in 157 (42.2%) specimens, whereas mutations in two genes were detected in an additional 20 (5.4%). The overall prevalence of mutations for each specific gene was as follows: KRAS (34.2%), EGFR (12.2%), TP53 (4.9%), PTEN (2.8%), PIK3CA (2.2%), CTNNB1 (2.2%), NRAS (1.8%), BRAF (1.2%), MEK1 (0.3%), and AKT (0%). Figure 1 Evaluation of smoking status identified a substantially higher percentage of KRAS mutations in ex-light smokers and current smokers (38.2% and 40.5%) combined compared to never smokers (7.6%, p<0.0001) , and a lower proportion of EGFR mutations in ex-light and current smokers (10.9% and 4.9%) combined compared to never smokers (39.7%, p<0.0001). Patients ≥70 years old had a higher proportion of both NRAS (7.1% vs. 0.7%, p=0.009) and TP53 mutations (12.5% vs. 3.3%, p=0.010). In addition, males had a lower incidence of EGFR mutation (8.6% vs. 19.0%, p=0.007) as compared to females. Patients from North America, Europe, and Australia/New Zealand demonstrated lower rates of mutation in CTNNB1 (1.4% vs. 8.6%, p=0.030) and PIK3CA (1.4% vs. 8.3%, p=0.032) compared to patients from Central/South America, South Africa and India. Finally, among specimens with two mutations, combinations involving KRAS were the most prevalent (70%, 14/20) followed by TP53 (50%, 10/20).



Conclusion:
These results demonstrate the wide spectrum of mutations that can be detected in adenocarcinoma specimens, with high prevalence rates in the EGFR and KRAS genes. Most patients had only one identified driver mutation. The study revealed age and geographical associations in some mutations. The clinical relevance of the studied mutations in relation to chemotherapy and the human EGFR antibody, Necitumumab, will be studied.

Background:
Various protein kinases have been discovered as drivers in cancer and subsequently used as therapeutic targets.

Methods:
To discover potential target driver genes, we investigated characteristics of genome wide scans of genetic lesions in kinases from 103 Korean lung adenocarcinoma patients, whose driver mutations were unknown or negative after conventional screening of EGFR and KRAS mutations as well as EML4-ALK fusion. We employed targeted, pair-end deep sequencing and screened the coding sequences of 518 protein kinase and genes that are known to be mutated with considerable frequencies in lung adenocarcinoma such as TP53 and EGFR.

Results:
Pathway analysis revealed that recurrent alterations were enriched in p53 signaling (TP53, ATM, CHEK2, CDKN2A) and ErbB signaling (EGFR, BRAF, KRAS, ERBB4) pathways. Mutations in TP53 and an EGFR exon 21 hotspot regions were found in 28% (29/103) and 13% (13/103) of cases (Fig 1). TP53 mutation was significantly more common in older group (97%) than in younger group (3%). We identified novel somatic mutations of genes, including CHEK2, NEK2 (mitotic progression) and SMG1 (nonsense-mediated mRNA decay), that have not been highlighted in lung cancer previously. Figure 1 Fig 1. Discovery of recurrent mutations with identical substitutions at the same site



Conclusion:
As the inhibitors of these protein kinases can be therapeutic candidates to eradicate cancer cells, our results provide useful information for the development of effective therapeutic target agents, by which the activity of various kinases can be modulated, in adenocarcinoma of the lung.

Background:
The prognosis of lung SCC continues to be poor with no molecularly targeted agents specifically developed for its treatment. LCGI aims to identify potential targetable oncogenes in lung SCC.

Methods:
The LCGI study is being carried out in 500 patients with surgically resected lung SCC, treated at St James’s University Hospital and Beaumont University Hospital, Dublin. We used the platform of Sequenom’s MassArray to perform genotyping for accustomed panel of 258 somatic hotspot mutations in 49 genes including genes in the MAPK and PI3K pathways. We also evaluated FGFR1 amplification by fluorescence in situ hybridization (FISH) and MET protein expression by immunohistochemistry (IHC).

Results:
Lung SCCs from 258 patients have been tested by Sequenom MassArray to date. Lung SCCs from 150 patients have been evaluated for MET protein expression and 89 for FGFR1 amplification. 163 (63.2%) patients were male. The median age of the cohort was 68. The majority of patients were either current (39.5%) or former (58.1%) smokers at the time of diagnosis. 138 (53.5%) were stage I, 87 (33.7%) were stage II, and 33 (12.8%) were stage III SCCs. At least one aberrant, potentially targetable oncogene was identified in the SCC of 101 (39.1%) patients (see Table). The presence of PIK3CA or KRAS mutations, or FGFR1 amplification did not have a statistically significant impact on median overall survival or recurrence-free survival. However, the presence of two or more aberrations in driver oncogenes in a tumor (patients, n=19) was associated with a worse median overall survival compared to patients with either a single driver aberration (p=0.04) or no aberrations (p<.01). Table: Frequency of driver mutations in LCGI compared to The Cancer Genome Atlas (TCGA) study

Mutation	LCGI (n=258)	TCGA (n=178)
FGFR1 amp (n=89)	13 %	16.8 %
PIK3CA	15.1 %	10.1 %
KRAS	6.5 %	0.6 %
PTPN11	3.5 %	1.7 %
STK11	3.1 %	1.7 %
MYC	1.9 %	0.0 %
NRAS	1.6 %	0.0 %
BRAF	1.2 %	3.9 %
HRAS	1.6 %	1.7 %
CTNNB1	1.5 %	1.7 %
FBXW7	1.5 %	3.4 %
MET Overexpression (n=150)	1.3 %	NA
EGFR	0.9 %	2.8 %
AKT1	0.4 %	0.6 %
CDK4	0.4 %	0.0 %
GNA11	0.4 %	0.6 %
MAP2K1	0.4 %	0.6 %
DDR2	0 %	1.1 %

Conclusion:
39.1% of lung SCC patients have an aberrant, potentially targetable driver oncogene in their tumor. The presence of two or more aberrant oncogenes is a poor prognostic factor in lung SCC. These findings can be used to guide clinical trials in lung SCC.

Background:
Research into druggable driver gene mutations in non-small cell lung cancer has identified several molecule-targeting agents that are clinically prescribed to inhibit tumor growth in adenocarcinoma. However, lung cancers without targeted driver gene mutations show poorly differentiated histologies and unfavorable prognoses without medication. Pleomorphic carcinoma, a lung cancer that includes a mixed conventional histology and sarcomatoid components, is an extremely aggressive tumor and rarely responds well to anticancer treatments. However, it has recently been reported that lung cancer with a sarcomatoid component contains the ALK gene rearrangement. Therefore, we used a next-generation sequencing (NGS) analysis of the driver gene mutations in pleomorphic carcinoma (PC) of the lung to identify druggable target molecules.

Methods:
We selected PCs from primary lung carcinomas by reviewing the records of 944 patients who had undergone surgical resection between 2007 and 2014. The Ion PGM™ NGS sequencer (Life Technology Inc., US) was used to identify known gene rearrangements in lung cancers by analyzing RNA with IonAmpliSeq™ RNA Lung FusionPanel® (Life Technology). With our original panel for targeted gene amplifications, the Ion PGM was also used to analyze nucleotide sequence of whole exons of 41 driver genes reported to be involved in lung cancers. The cases examined were also analyzed with ALK immunohistochemistry (ALK-IHC) using the N-Histofine® ALK Detection Kit (Nichirei Bioscience Inc., Tokyo, Japan) on sliced paraffin-embedded tumor specimens.

Results:
Twenty-one lung cancer specimens diagnosed as PC and frozen material from nine patients were available for NGS. The EML4ex6/ALKex20 fusion gene was detected in one case, which was also positive on ALK–IHC. No other fusion genes were detected. Another case, which was negative for the fusion gene, was weakly positivity on ALK–IHC, so an unbalanced 5¢/3¢-end ALK expression test is planned. Six of nine cases but not the ALK-fusion-positive cases showed driver gene mutations in TP53. We also found several somatic mutations in druggable genes in this study, which should be considered carefully to determine whether they are driver or passenger genes.

Conclusion:
Although a small number of PCs were examined in this study, the ALK fusion gene was detected, which indicates the frequency of ALK fusion might be high in PC. Other possible druggable gene mutations were also identified in PCs. Further cases must be investigated to understand the mutation status of driver genes in PC.

Abstract not provided

Background:
Genotyping of non-small cell lung cancers (NSCLC) is important for directing patient care, particularly in adenocarcinomas (ADC) where targeted therapeutics are available. There is emerging evidence of efficacy of targeted therapy in the treatment and prevention of brain metastases. With recent adoption of next-generation sequencing technologies it is possible to test individual tumors simultaneously for somatic mutations in multiple genes. Here, we present the mutational spectrum of NSCLC brain metastases in our institution over a three year period.

Methods:
The department of pathology archives was searched to identify cases of NSCLC metastatic to brain that underwent surgical resections during 2012-2014. Clinicopathologic information was recorded from the pathology reports and the medical records. Molecular genotyping analysis was performed for EGFR/KRAS mutations using single gene analysis (prior to 2013) or by next generation sequencing (NGS) using the AmpliSeq Cancer Hotspot Panel v2. FISH was used to test for ALK rearrangements.

Results:
During 2012-2014, 31 NSCLC patients underwent surgical resection for brain metastases. Eighteen patients were female (58%) and 13 were male. The median age was 70 years (range 51-89). Tumor histology included 24 ADC and 7 squamous cell carcinomas (SCC). Twenty-three cases had molecular genotyping studies performed on the metastatic disease or the primary lung cancer. These included 3 SCC and 20 ADC. Of the ADC, 12 were tested with the NGS panel; 8 had been tested prior to 2013. The most frequently mutated genes were KRAS (8/20; 40%) and TP53 (7/12; 58%). Of the patients with KRAS mutations 7/8 were female (p = 0.085). The NGS assay detected clinically actionable mutations that would have not been detected with prior single gene assays including an EGFR exon 20 insertion, an ERBB2 exon 20 insertion, and two PIK3CA mutations. Additional mutations were detected in JAK3, FLT3, FBXW7, ATM, STK11, VHL and RB1.

Conclusion:
We found that 40% of the genotyped ADC metastases harbored KRAS mutations more frequently in females (although not significant), similar to prior reports. In addition, with NGS we were able to detect additional clinically significant targetable mutations. In summary, NSCLC genotyping can potentially help guide treatment and prevention of brain metastases.

Background:
Incresing data show advanced non-small cell lung cancer (NSCLC) patients with EGFR activating mutant have discrepant response to EGFR-TKI. The abundance of EGFR mutations may be a powerful explanation for the uneven clinical benefit. This study was designed to investigate the influence of EGFR mutant abundance on efficacy of EGFR-TKI by a quantitative method.

Methods:
201 NSCLC patients treated with EGFR-TKI with available tissue samples for EGFR mutation test were enrolled into the study. EGFR common mutations were detected by amplification refractory mutation system (ARMS) and percentage of mutant EGFR was tested with the method of an Allele Specific Quantitative PCR with Competitive Blocker (ASB-qPCR). In this assay, the copies of all mutations and EGFR locus were calculated by standard curve respectively. The cutoff values were obtained by the receiver operating characteristics (ROC) curve in training set. Further, the cutoff values were confirmed in validation set and the whole population. The relationship between the abundance of EGFR mutations and efficacy of EGFR-TKI was statistically analyzed.

Results:
Of the 201 samples, 72 harbored 19DEL mutation, 63 carried L858R mutant, and 66 with wild-type. The cohort was randomly divided into training and validation sets. The cutoff values of 19DEL and L858R mutation abundance were 4.84% and 9.47% determined by ROC curve in training set. 9.7% of patients with 19DEL positive were low abundance (<4.84%, LA group), while 33.3% of L858R-positive patients were LA (<9.47%).High abundance (HA) group, regardless of 19DEL or L858R positive had more longer median progression free survival (PFS) compared with LA and wild-type groups in either validation set or the whole population (15.0 vs 2.0 vs 1.9, 8.0 vs 1.9 vs 1.9; 15.0 vs 4.0 vs 2.0, 12.0 vs 2.0 vs 2.0; p<0.001). COX regression analysis showed that EGFR mutation abundance, together with smoking status, were independent factors of response to EGFR-TKI.

Conclusion:
The abundance of EGFR mutation could more precisely predict EGFR-TKI efficacy. NSCLC patients with LA mutation had inferior clinical benefit with EGFR-TKI. The heterogeneity in EGFR mutant abundance partly explain the efficacy discrepancy in patients with 19DEL or L858R positive.

Background:
EGFR-m in NSCLC and its responsiveness to TKI has proved beneficial in a subset of NSCLC patients (p) Breast cancer (BC) represents the most incident neoplasm among women. The co-existence of both EGFR-m-NSCLC and BC has been reported in p with Li-Fraumeni Syndrome. However, no systematic evaluation of the presence of both types of cancers has been performed in the general population.

Methods:
We sought to evaluate if EGFR-m-NSCLC associates higher rates of 2[nd] BC than the EGFR-wild type (WT)p. Clinical and molecular characteristics, as well as clinical outcomes of female p visited at our Institution from 2008 to 2014 and harboring both types of tumors were registered.

Results:
69/578 female p with EGFR-m were identified. Data on treatment and follow-up of 62p were available. 11/ 62p (17.7%) were diagnosed with both EGFR-m-NSCLC and BC. For EGFR-m-W-p, BC was diagnosed in 3 cases (0.52%) Regarding EGFR-m-NSCLC, age of diagnosis was 65y; 100% of p were Caucasian and never smokers. Del19, L858 and L861Q represented the 72.7%, 27.3% and 9% of EGFR-m. 36.3% had stage IV, 27.2% recurred after surgery and 36.3% initial stages never recurred. 63.6% received a TKI (42.8% erlotinib). 85.7% received TKI as 1[st] line. The overall response rate was 85.7%. 42.8% received subsequent therapies. The cause of death was related to lung cancer in all cases of death (54.5%). mOS was 29 months from the time of LC diagnosis. Regarding BC, the median age of diagnosis was 52y, BC was a prior diagnosis in 90.9%; the stage was 0, I, IIA and IIIA in 18.18%, 27.27%, 45.45% and 9.09%, respectively. After surgery (100%), 36.36% received ET, 36.36% both chemotherapy and ET and 54.54% radiotherapy (RT). 83.33% of p treated with RT developed the LC in the RT field. 90.90% of p never recurred.

Conclusion:
Diagnosis of BC appears to be higher in EGFR-m-NSCLC-p than in the general population. Evaluation of the molecular mechanism potentially related to this association is warranted.

Background:
Epidermal growth factor receptor (EGFR) mutations are relatively common oncogenic drivers in non-small cell lung cancer (NSCLC). Interestingly a number of patients have more than one mutation in EGFR. In order to understand whether these patients respond to EGFR inhibition, we reviewed our experience treating these patients. Herein we describe the Mayo Clinic experience with the S768I mutation of exon 20 of the EGFR gene.

Methods:
Relevant clinical and laboratory data were abstracted for selected cases, including evaluation of response after treatment with TKIs using the Response Evaluation Criteria In Solid Tumors (RECIST) version 1.1. Inclusion criteria were cases including EGFR S768I mutation performed at Mayo Clinic or elsewhere until December 2014. EGFR testing was performed following microscopic examination by a pathologist to identify and select areas of tumor for macrodissection and confirm sufficient tumor percent. The EGFR test is a PCR based assay employing allele specific amplification and is used to test for mutations within exons 18-21 of the EGFR gene, most recently using the FDA approved platform. When testing was performed elsewhere, bidirectional sequencing was used.

Results:
1,527 cases of NSCLC that underwent EGFR testing were reviewed and the S678I mutation was present in 9 patients (0.59%), 4 of which were female. Median age at diagnosis was 61 years (range 49-68 years), 5 patients were never smokers and no subjects were current smokers at the time of diagnosis. The stage at diagnosis was I in 2, III in 3 and IV in 4 patients, respectively. All specimens were adenocarcinomas with 5 of them being grade 3. Only 3 cases had an isolated S768I mutation, 4 cases had a concurrent G719S mutation and 2 cases had a concurrent L858R mutation. The tumor responses of patients with stage IV disease are shown in the table. One patient with a concurrent S768I and L858R mutation with stage IIIa disease received curative intent lobectomy after neoadjuvant treatment with erlotinib. Erlotinib was discontinued in one case due to fatigue. Table: Response of S768I mutations to erlotinib by RECIST 1.1

Mutation(s)	Best response on erlotinib	PFS (months)	Overall survival (months)
S768I alone	Progressive disease	3	5
S768I + G719	Partial response	6	23
S768I + G719S	Stable disease	12	33
S768I + L858R	Stable disease	30	51+

Conclusion:
S768I mutations in exon 20 of the EGFR are rare and are commonly seen in conjunction with common EGFR mutations. Due to its rarity and the variability of responses of treated cases, its exact prognostic and predictive role is not fully understood. Better understanding of its function and sensitivity to newer TKIs will allow for better management of patients with this mutation.

Abstract not provided

Background:
Gene fusions of anaplastic lymphoma kinase (ALK), ROS1, and RET are targetable oncogenes present in approximately 9% of advanced NSCLC. Current assays for detecting gene fusions are based on FISH (FDA-approved companion diagnostic test for ALK), immunohistochemistry (IHQ) and qRT-PCR. These tests, however, are complex and have disadvantages in terms of turnaround, sensitivity, cost and throughput. The nCounter platform allows joint detection, in a single tube, without any enzymatic reaction and in 72 hours, of multiple fusion genes by transcript-based method from formalin-fixed paraffin-embedded (FFPE) samples.

Methods:
A custom set consisting of 5´and 3´ probes and/or fusion-specific probes to detect ALK, ROS1 and RET fusion transcripts was evaluated. A panel of ALK-ROS-RET positive cell lines (H2228, H3122 [EML4-ALK], SU-DHL-1 [NPM-ALK], HCC78 [SLC34A2-ROS], BaF3 pBABE [CD74-ROS], LC2/ad [RET]) and control fusion negative cell lines (PC9, H1975 [EGFR mut], H460, H23 [KRAS mut]) were used for nCounter validation. To determine the minimum of tumor surface area for detection, ALK translocated cell line H2228 was tested in FFPE at increasing cell numbers (2500, 5000, 10.000, 25000, 50000) corresponding to a surface area of 0.27, 0.55, 1.1, 2.75 and 5.5 mm2, respectively, in the FFPE block. A total of 38 FFPE samples positive by FISH, IHC and/or qRT-PCR for ALK (n=30), ROS (n=7) and RET (n=1) were also analyzed. Total RNA was isolated from cell lines and FFPE and < 225 ng were used for hybridization. Raw counts were normalized using positive controls, negative controls and 4 house-keeping genes (GAPDH, GUSB, OAZ1 and POLR2A) as described in Lira et al. J Mol Diagn 2013. Positive and negative ALK fusion translocation was defined by a 3’/5’ ratio score of > 2.0 and ≤ 2.0 respectively. Response to crizotinib by RECIST criteria was retrospectively collected in patients with ALK-positive NSCLC.

Results:
nCounter sensitivity to predict fusion transcripts ALK, ROS and RET in cell lines by using both methods (3’/5’ and direct reporter probes) was 100%. Results indicate that samples containing as few as 10% positive tumor cells and a 2.75 mm2 tumor surface area were sufficient for adequate gene fusion detection. The accuracy of prediction (AUC) of ALK 3’-5’ ratio score in 45 independent samples was 82.6% (95% CI 69.3-95.6) with a kappa coefficient score of 0.637. Among 28 samples ALK-FISH-positive, ALK 3’-5’ scoring was positive in 27 samples (96%). One sample was non-evaluable by ALK 3’-5’ scoring. Among the 17 samples ALK-FISH-negative, ALK 3’-5’ score was negative and positive in 10 (59%) and 7 (41%) samples, respectively. All patients with ALK-FISH-negative samples but ALK 3’-5’ score positive (n=7) were positive for ALK IHC and 5 of them were treated with crizotinib. Response assessment was available in 3 of these patients and response rate was 100%. One patient non-evaluable by FISH but positive 3’-5’ scoring also responded to crizotinib.

Conclusion:
The ALK/ROS1/RET nCounter-based assay is a highly sensitive screening modality that might identify FISH-negative/non-evaluable NSCLC patients who could benefit from ALK inhibitors.

Background:
Mutation analysis plays a central role in the management of lung adenocarcinomas (LUAD). The use of multiple single gene or mutation specific assays, broadly adopted in many laboratories to detect clinically relevant genomic alterations, often leads to delays if sequentially performed, tissue exhaustion, incomplete assessment and additional biopsy procedures. Comprehensive assays using massively parallel “next-generation” sequencing (NGS) offer a distinct advantage when addressing the increased testing needs of genotype-based therapeutic approaches. Here we describe our experience with a 410 gene, clinically validated, hybrid-capture-based NGS assay applied to testing of LUAD.

Methods:
Consecutive LUAD cases submitted for routine mutation analysis within a 1 year period were reviewed. Unstained slides of formalin fixed, paraffin embedded tissue were received for each case (range 15-20 slides/case). Corresponding H&E stained slides were reviewed and cell counts were performed in a subset of cases with limited material to establish minimal tissue requirements. Testing was performed by a laboratory-developed custom hybridization-capture based assay (MSK-IMPACT) targeting all exons and selected introns of 410 key cancer genes (J Mol Diagn 17:251-264, 2015). Barcoded libraries from tumor / normal pairs were captured and sequenced on an Illumina HiSeq 2500 and analyzed with a custom analysis pipeline.

Results:
A total of 469 specimens were received for comprehensive testing (98 cytology samples, 239 needle biopsies, 132 large biopsies/resections) of which 93% (436/469) were successfully tested. Thirty four cases (7%, 34/469) failed due to very low tumor content or low DNA yield. Cell counts for failed samples averaged 239 cells / slide (range 10-270) while all successfully tested had over 1,000 cells / slide each. Failure rate was similar for cytologies and biopsies. An average of 10 genomic alterations were detected per patient (range 1-96). The most frequently mutated genes were TP53, EGFR, KRAS, KEAP1 and STK11. Copy number gains of NKX2-1 and EGFR genes and CDKN2A loss were most common. EGFR mutations and ALK fusions were detected in 28% and 4% of cases, respectively. Among the 299 EGFR / ALK WT cases, MSK-IMPACT uncovered targetable genomic alterations that would have remained undetected through focused EGFR/ALK testing alone. These included fusions in RET (10) and ROS1 (13), mutations in ERBB2 (11) and BRAF (19) and amplifications in MET (12, unrelated to EGFR), MDM2 (26) and CDK4 (20) among others. The higher than expected rates of RET and ROS1 fusions are related to enrichment of previously tested cases known to be negative for other driver alterations.

Conclusion:
Comprehensive hybrid-capture based NGS assays such as MSK-IMPACT are an efficient testing strategy for LUAD across sample types. This upfront broad approach enables more optimal patient stratification for treatment by targeted therapeutics.

Background:
Lung cancer is the most common cancer worldwide and has the highest mortality rate. Carcinomas comprise 95% of all lung cancers, the vast majority of which are non-small cell lung carcinomas (NSCC). It is critical to further distinguish adenocarcinomas from squamous carcinomas in order to optimize the efficiency of the precision medicine analysis for the detection of active molecular targets for therapies. Currently Thyroid Transcription Factor-1 (TTF-1) and Napsin-A are the most commonly used immunohistochemical (IHC) stains to identify primary lung adenocarcinoma, and p40 and cytokeratin 5/6 (CK5/6) are used for squamous cell carcinoma. IHC stains for these markers, are performed either individually (IHC brown staining) or in combination as dual immunostains (i.e. TTF-1 + Napsin-A and p40 + CK5/6, utilizing brown and red chromogens). Here we present a novel, truly multiplex immunohistochemical approach that combines staining with the above four antibodies on a single tissue section utilizing four different chromogens to accurately diagnose primary lung adenocarcinomas, squamous cell carcinomas, and combined adenosquamous carcinomas of the lung.

Methods:
Developmental reagents from Ventana Medical Systems, Inc. were leveraged for this study. Detection of CK 5/6 and p40 [BC28] was used to identify squamous cell carcinoma cells. Detection of Napsin A and TTF-1 was used to identify adenocarcinoma cells. Detection was accomplished using secondary antibody:enzyme conjugates and orthogonal chromogenic detection chemistries to simultaneously detect all 4 biomarkers. Fully automated multiplexed detection was performed on a Benchmark XT with 4 microns thick sections from formalin fixed paraffin embedded, non-small cell lung cancer specimens obtained from both the Ventana Medical Systems, Inc. tissue bank and from the Thomas Jefferson University’s Department of Pathology, Anatomy and Cell Biology laboratories. Detection of each marker in multiplex was compared to individual detections using diaminobenzidine deposition according to established Ventana Medical Systems, Inc. protocols. All detections were reviewed by a board certified pathologist.

Results:
Adenocarcinomas (7 of 7) and the adenocarcinoma components of the adenosquamous carcinomas (6 of 6) stained positive for TTF-1 (yellow nuclear stain) and Napsin-A (pink cytoplasmic granular stain). Squamous cell carcinomas (5 of 5) and the squamous cell carcinoma components of the adenosquamous carcinomas (6 of 6) stained positive for p40 (blue nuclear stain) and CK5/6 (brown cytoplasmic stain). The colors were clear, distinct, easily differentiated and recognizable. There was no discrepancy between the expression of the individual antibodies and the expression of the same antibodies in the multiplex setting.

Conclusion:
Increasingly, the diagnosis of lung cancer is established by examination of small tissue specimens obtained by minimally invasive techniques. It is critical to employ these tissues at maximum efficiency in order to render an accurate pathologic diagnosis and to perform theranostic studies, either genomic or IHC, to demonstrate genetic mutations that make patients eligible for molecularly targeted agents. This new quadriplex IHC offers the capability with a single 4 micron section to accurately diagnose primary lung adenocarcinoma, squamous cell carcinoma or adenosquamous carcinoma and while conserving tissue for additional molecular testing.

Abstract not provided

Background:
Next generation sequencing (NGS) has exceptional sensitivity, but at the expense of false positives. This can result in a less than optimal positive predictive value and eventually the futile treatment of patients. We have developed a unique set of quality control filters for both Ion Torrent and Illumina that minimize false positives, but have little negative impact on sensitivity. To address this paradoxical association of sensitivity and false positives, we developed a dual platform methodology of NGS using both the Ion Torrent and Illumina to solve this classical dilemma.

Methods:
A series of filters were developed to determine quality cutoffs for variant calls to minimize false positives that included the minimum quality score threshold (QUALT), minimum percent variant reads (MPVR), minimum variant reads (MVR), minimum variant reads threshold (MVRT), minimum variant allelic frequency threshold (MVAF), minimum variant reads positive predictive value (MVR-PPV), and systematic errors (SE). A parallel system of using the MiSeq and PGM to sequence all specimens within an IT systems control and a Classify Callsmatrix solution for mutational analysis was designed. Unique cohorts of patients with prior exome sequencing as part of TCGA were used as gold standard controls with matching fresh frozen and FFPE samples.

Results:
Table 1 provides the results of filters developed to maximize sensitivity versus PPV. Using our targeted sequencing panel the PGM consistently outperformed the MiSeq for the standard performance characteristics of sensitivity and PPV for both frozen and FFPE samples. Both platforms have systematic false positives that are unique and gene specific.

Table 1 Platform	Tissue	VAF setting	QUAL Cutoff	MVRT Cutoff	MVAF Cutoff	Mean Sensitivity	Range Sensitivity	Mean PPV	Range PPV
PGM	FF	0.2%	None	None	None	100%	93-100%	88%	70-96%
PGM	FF	0.2%	>99	>=20	>.035	99%	93-100%	95%	78-100%
PGM	FFPE	0.2%	None	None	None	99%	93-100%	58%	2-94%
PGM	FFPE	0.2%	>99	>=21	>.018	97%	63-100%	92%	40-100%
MiSeq	FF	1%	None	None	None	97%	79-100%	49%	31-66%
MiSeq	FF	1%	>99	>=5	>.017	95%	66-100%	82%	66-95%
MiSeq	FFPE	1%	None	None	None	94%	43-100%	10%	2-37%
MiSeq	FFPE	1%	>99	>=10	>.028	92%	39-100%	62%	6-100%

Table 2 provides the results for dual platform sequencing which show a marked reduction in false positives while maintaining sensitivity.

Table 2	FF	FF	FF	FF	FFPE	FFPE	FFPE	FFPE
	SNV(s)	SNV(s)	Indels	Indels	SNV(s)	SNV(s)	Indels	Indels
	Percent	VAF	Percent	VAF	Percent	VAF	Percent	VAF
Assay Sensitivity	99.8%	2.87%	100.0%	2.90%	98.3%	3.56%	100.0%	3.60%
Assay PPV	97.5%	2.87%	91.0%	2.90%	96.7%	3.56%	91.0%	3.60%

Conclusion:
Single platform NGS is plagued by false positives. Dual platform sequencing is a reliable method of diminishing false positives with minimal to no impact on sensitivity.

Background:
Molecular testing has become important in managing advanced non-small cell lung cancer (NSCLC), both in clinical practice, as well as in clinical trials. For the latter, tissue samples often have to be analysed in a central laboratory. We evaluated the turnaround time and possible delay in start of therapy in this process.

Methods:
We reviewed our prospective database on all molecular testing cases for clinical trial suitability in patients with advanced NSCLC between March 1, 2011 (start) and October 31, 2014. The following time points were considered: T1 (request for tissue sections from the pathology lab); T2 (receipt of sections and shipment); T3 (arrival of sections in central lab (CL)); T4 (receipt of biomarker result from CL).

Results:
251 patients were considered for biomarker-driven trials. Twenty-three cases did not have further analysis, as the request for central molecular testing was cancelled: insufficient tissue (n=11); exclusion criterion (n=10); patient refusal (n=2). Results for the remaining 228 patients were: failure of central biomarker analysis due to insufficient quantity of tissue (n=18), or quality of tissue (n=3, i.e. decalcification or poor fixation). Valuable results were obtained for 207 patients. In 91 of 228 (39.9%) samples sent, a biomarker of interest was documented. This led to 34 clinical trial inclusions. Other patients were no longer eligible due to loss of performance status (n=20), loss of contact (n=14), no trial slot available at the appropriate time (n=18), or exclusion criteria (n=5). The mean waiting time between signing informed consent (T1) and receiving results of the biomarker analysis (T4) was 25.1 calendar (SD 17.3) days (Table). The preparation of the unstained slides by the pathology lab took about 9.1 (SD 6.8) days, the time of the biomarker testing itself accounted for 12.8 (SD 7.3) days. For 18 of 228 (7.9%) patients, repeated sample shipments were needed because of insufficient tumor cells, their mean waiting time between informed consent and receiving the biomarker result was 62.2 (SD 38.4) days. Table: Waiting times (t) in molecular testing for 228 patients.

Time interval	Mean	StDev	Median	Range
Pathology lab (T2-T1)	9.1	6.8	7.0	1 - 70
Shipment (T3-T2)	1.8	1.6	1.0	0 - 17
Analysis (T4-T3)	12.8	7.3	12.0	2 - 58
Request to result (T4-T1)	25.1	17.3	22.0	7 - 184

Conclusion:
While molecular testing is important in many NSCLC trials, our results show that waiting times for central laboratory analysis can cause an important delay in treatment initiation, and even ineligibility for the trial(s) under consideration. Start of therapy based on properly validated local testing, with a posteriori central biomarker testing to guarantee the integrity of the trial, would be more rewarding for quite some patients.

Background:
Lung cancer is the leading cause of cancer mortality worldwide, and metastatic spread of cancer to distant organs is the main reason for lung cancer deaths. They spread to different distant organs, exhibit an outstandingly different situation of clinical characteristics and will be medically and surgically incurable. Thus, there is a clear need for a reliable and efficient in vitro culture model to enable transition to invasion and journey to distant organs of these critical steps in cancer metastatic progression.

Methods:
Here we report a biomimetic multi-organ microfluidic chip system more closely reconstituting the structural tissue arrangements, functional complexity and dynamic mechanical microenvironments and reproducing survival, growth, transition to invasion and journey to distant multi organs in lung cancer metastasis. To reconstitute the actual growth conditions of lung cancer in vivo, we created a thin, porous, flexible membrane, integrated microfluidic chip emulating the in vivo tissue structure and enabling heterotypic cell interactions, while maintaining cell compart-mentalization. The human bronchial epithelial cells and stromal cells were cultured on opposite sides of the membrane. Once the cells were grown to confluence, air was introduced into the epithelial compartment to create an air-liquid interface and more precisely mimic the lining of the lung air space. Then lung cancer cells were cultured on the human epithelial compartment to mimic lung cancer formation and the multi organ chambers were linked with side channels that supply lung cancer cells to the brain, bone or liver cells chamber to mimic lung cancer metastasis. In addition, the system provided analyzing cell physiology and visualizing complex cell behaviors in a more physiologically relevant context.

Results:
A biomimetic multi-organ microfluidic chip system was created. The quick formation of lung cancer cells that grow away from their natural margins and then attack adjacent components and spread to other organs were observed at all times and the cells characterizations were also detected accurately and effectively. In this multi-organ pathogenesis system, it might be possible to provide an ultrahigh level of reproducibility, authenticity and sensitivity.

Conclusion:
This microdevice provides a proof of principlefor this novel biomimetic strategy that is inspired by the integrated chemical, biological, and mechanical structures and functions of the living multi organs. This versatile system enables direct visualization and quantitative analysis of diverse biological processes of the intact lung cancer metastasis in ways that have not been possible in traditional cell culture or animal models.

Background:
Next-generation sequencing (NGS) can be used to interrogate multiple areas of the tumor genome. Several hot-spot panels have been developed to identify variants amenable to targeted therapies and enrollment into clinical trials. Variants of unknown significance (VUS) in the vicinity of hot-spots are routinely discovered. To better understand these obscure VUS, we built a Protein Viewer that displays the relationship of known actionable variant(s) to the VUS.

Methods:
We developed a web-based protein viewer that can be deployed across multiple browsers. The tool supports the visual representation of 23 genes which are interrogated by our NGS platform. We used the longest mRNA transcript (hg19) to define the protein domains. All actionable variants as reported by an knowledge database were included, with the selected VUS differentially highlighted. VUS is defined as a non-actionable variant that is not reported in dbSNP.

Results:
Approximately 50% of all stage III and IV lung cancer patients tested by our NGS platform have one or more VUS. After the variant information is loaded in the Protein Viewer, a two-dimensional image of the full length protein with actionable variants and VUS is displayed (Figure 1). The Viewer is utilized at RPCI to present cases at our molecular tumor board for quick visualization and discussion. Figure 1 Figure 1: Protein Viewer with a PIK3CA VUS harboring a Q546H (pink) in a lung adenocarcinoma. Top panel with PIK3CA exons 2-21 boundaries (vertical lines) with protein domains (blue rectangles along axis). Bottom panel with the zoom feature which allows more discreet visualization of the VUS, a neighboring Q546K actionable variant (green), and additional actionable variants for ovarian cancer (green rectangles).



Conclusion:
Understanding the relationship of VUS to protein domains and proximity to previously known actionable sites is a potentially powerful way to evaluate and determine whether a patient might be a candidate for targeted therapy. Because the exact effect of the VUS on the function of the protein is still impossible to discern (tyrosine kinase inhibitor sensitivity/ resistance/no effect), the next generation of protein viewers should incorporate 3D and protein folding/domain interaction prediction capabilities.

Abstract not provided

Background:
Genomic profiling is a powerful method for identifying mutations that drive tumors and matching patients to targeted therapies. However, this may only be a transient solution and resistance commonly emerges as the mechanism of targeted inhibition is overcome. Proteomic profiling of the tumor provides a dynamic tool to survey altered protein expression and deregulated pathways, which in turn may implicate specific treatments or identify novel therapeutic targets. Mass spectrometry offers highly multiplexed proteomic measurements, but extensive sample pre-processing and low sample throughput can lead to extended analysis times of weeks or months. Thus a need exists for a high throughput, sensitive and quantitative platform for proteomic analysis.

Methods:
We used the SOMAscan proteomic platform, which measures 1129 proteins with a median limit of detection of 40 fM and 5% CV, to analyze protein lysates from 63 lung tumor samples. The assay does not require sample pre-fractionation, and this study (which generated over 142,000 protein measurements) represents less than one day of SOMAscan throughput. The study consisted of matched tumor/non-tumor protein lysates prepared from 18 squamous cell carcinoma and 45 adenocarcinoma fresh-frozen resected specimens, 86% of which were Stage I/II. The paired log~2~ tumor/non-tumor ratio was calculated and hierarchical clustering heat maps and dendrograms were constructed to identify related protein regions and tumor phenotypes.

Results:
Common proteomic changes and unique tumor phenotypic groups were identified by unbiased clustering algorithms. Large, consistent tumor/non-tumor differences of at least 4-fold were observed for 35 proteins in at least 20 (32%) of the tumors. Some of these proteins were more than 100-fold higher in individual tumors. The two most commonly elevated proteins were thrombospondin 2 and MMP12, which were increased in 81% and 61% of the tumors, respectively. We have previously reported higher levels of MMP12 in the serum of lung cancer patients, and the current data supports a tumor-associated origin for circulated MMP12. A second analysis identified sub-phenotypes of tumors clustered by common protein alterations independent of histological classification or mutation status. Many of these tumor subsets had increased expression of known oncology drug targets.

Conclusion:
Broad, unbiased high-throughput proteomic profiling of tumor tissue may reveal individual phenotypes that hold the potential to respond to targeted therapies and to monitor therapeutic efficacy throughout treatment. Measuring proteins complements mutation analysis by enabling therapeutic selection beyond driver mutation targets, including immune modulator therapies, repurposing existing drugs and enriching clinical trials with likely responders. While genomics is a fixed snapshot, blood- and tissue-based serial proteomic measurements respond to change and can lead to the personalized adaptation of treatment and identification of novel therapeutic targets.

Background:
There is a clinical need to establish whether those pathways activated in vitro and in animal models, are also activated in human lung cancer. We established a window-of-opportunity clinical trial platform in lung cancer where novel agents are administered in the preoperative period. Intratumoral drug concentrations are correlated with molecular marker changes. Our four completed window-of-opportunity clinical trials established that optimal intratumoral drug concentrations are needed for the desired pharmacodynamic effects, providing direction for optimal dose and schedule. To further evaluate the value of this window-of-opportunity platform, we investigate the impact on standard postoperative outcome measures.

Methods:
39 consecutive patients enrolled under the window-of-opportunity platform were matched to 39 contemporary patients undergoing the same operation by the same surgeon. Co-morbidities and stage of lung cancer and postoperative complications were compared using univariate and multivariate analysis. Wilcoxon Scores (Rank Sums) for variable data elements and Fisher’s Exact Test was used for analysis.

Results:
When comparing window-of-opportunity patients to control patients, there was no difference in age, pack years of smoking, or incidence of comorbidities including diabetes, coronary artery disease, hypertension, chronic obstructive pulmonary disease, and previous cancer. There was no difference in the stage distribution, (stage I: 28 vs. 22, stage II: 5 vs. 3, stage III: 5 vs. 2 stage IV: 1 vs. 1, p=0.1642). There was also no difference in the incidence of postoperative pneumonia (4 vs. 9, p=0.2235), other infection (2 vs. 3, p=0.8208), atelectasis (2 vs. 4, p=0.6748), myocardial infarction (0 vs. 0, p=1.000), reoperation for bleeding (1 vs. 1, p=1.000), pulmonary embolism (1 vs. 2, p=1.000) or number patients experiencing any complication (14 vs. 8, p=0.131118). There was no difference in the distribution of survival at 2 years (27 vs. 30) or 5 years (10 vs. 15), p=0.2266.

Conclusion:
The window of opportunity platform does not increase the perioperative risk of complications in early stage NSCLC patients undergoing surgery. By evaluating drug effect and the potential toxicities, window-of-opportunity trials validate mechanisms established in the laboratory and facilitate bi-directional translation research.

Background:
Small Cell Lung Cancer (SCLC) is an aggressive, highly metastatic disease with dismal prognosis. Response rates to first line chemotherapy are generally high, but progression free survival is short due to development of chemotherapy resistance via mechanisms not well understood. Due to the difficulty in collecting tissue biopsies in SCLC, blood, which can be sampled simply and routinely, provides a means of inferring the current genetic status of a patients tumour via analysis of circulating tumour cells (CTCs) or circulating tumour DNA (ctDNA). These offer a minimally invasive opportunity to study drug resistance mechanisms, evaluate tumour heterogeneity and potentially reveal new drug targets in this disease. However, accurate assessment of both CTCs and ctDNA requires all blood cells be maintained intact until samples are processed, particularly when analytes present are at very low concentrations. Here we describe and validate a blood collection protocol that does not require on-site processing, and which is amenable for analysis of both CTCs and ctDNA following storage at ambient temperature in CellSave vacutainers for up to 96 hours after blood collection.

Methods:
To evaluate the suitability of using CellSave preserved samples for circulating free DNA (cfDNA) analysis, we undertook a 20 healthy normal volunteers (HNV) study and 45 patient sample study, with parallel EDTA and CellSave bloods collected. For each sample cfDNA was isolated between 4 hours and 96 hours post-draw and cfDNA yields determined. A potential issue with using CellSave blood was that the CellSave preservative could act as a DNA damaging agent and effectively increase background sequencing errors. To test this, the EDTA and CellSave cfDNA samples were subjected to next generation sequencing (NGS) to estimate the overall mutation burden. In addition, the utility of CellSave ctDNA for targeted NGS was also determined. Finally, SCLC-specific copy number aberrations (CNA) were analysed in ctDNA and CTCs isolated from the same CellSave blood sample from individual SCLC patients.

Results:
We demonstrate that yields of cfDNA obtained from 96-hour whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 hours of blood draw. Targeted and genome-wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples, thereby validating CellSave blood as a viable source of ctDNA. We also demonstrate that CTCs and ctDNA can be isolated from the same patient blood sample, and give the same patterns of CNA allowing direct comparison of the genetic status of patients’ tumours.

Conclusion:
In summary, we have demonstrated the suitability of whole blood CellSave samples for both CTC and ctDNA molecular analysis in SCLC. The ability to generate informative molecular profiles of both CTCs and ctDNA from a simple whole blood sample, up to 4 days post-draw represents a significant methodological improvement for clinical benefit. We posit that as minimally invasive, liquid biopsies become increasingly employed for cancer patient management, the ability to routinely and simply draw blood and ship samples to accredited biomarker assessment laboratories will greatly facilitate the delivery of personalised cancer medicines.

Abstract not provided

Background:
Plasma cell-free DNA (cfDNA) contains genetic information from primary and metastatic cancer foci. We utilized multiplex deep sequencing technology to investigate the clinical significance of cfDNA concentration and mutational burden in advanced non-small-cell lung cancer patients treated with EGFR tyrosine kinase inhibitors (TKIs).

Methods:
Between January 2012 and February 2014, seventy-one eligible patients from Sun Yat-sen University Cancer Center were enrolled. All the patients provided written informed consent and donated 2 ml plasma before taking EGFR-TKIs. Plasma DNA was isolated and purified using QIAamp Circulating Nucleic Acid Kit. CfDNA concentration was determined by Qubit Fluorometer. A set of 234 primer pairs were designed to amplify sequences covering hotspots of 35 genes. The amplicon libraries were prepared and entered into deep sequencing on Ion Torrent PGM chip. Variants were called by established bioinformatics methods. Circulating DNA mutational burden was defined as the number of somatic variants other than EGFR mutations. Objective response rate (ORR) and disease control rate (DCR) between different groups were compared using Fisher’s exact test while progression-free survival (PFS) and overall survival (OS) between different groups were compared by Kaplan-Meier curves and log-rank tests. Multivariate stepwise Cox regression analyses were performed to identify independent prognostic factors.

Results:
Forty-nine out of 71 patients were observed to harbor at least one variant and at most 7 variants, involving 10 genes (totally 124 variants). Higher cfDNA concentration was associated with impaired DCR (18.6% vs 81.4%; p=0.008), PFS (median PFS, 3.5 vs 15.2 months; HR=3.03; p=0.001) and OS (median OS, 27.3 vs not-reached; HR=2.38; p=0.042) compared with low cfDNA concentration group. Higher mutational burden was associated with unfavorable ORR (31.6% vs 73.7%; p=0.004) and PFS (median PFS; 8.6 vs 17.8 months; HR=1.61; p=0.050) compared with low mutational burden group. EGFR mutation conferred better ORR, DCR and PFS compared with EGFR wild-type (Figure 1). Multivariate analyses revealed that apart from EGFR mutation status, cfDNA concentration and mutational burden were also associated with the efficacy and/or the prognosis of EGFR-TKIs.Figure 1



Conclusion:
We for the first time showed that cfDNA concentration and mutational burden might influence the efficacy and prognosis of patients receiving EGFR-TKIs. These findings call for the need for the multiplex genetic analysis of patients’ cfDNA to tailor their treatment.

Background:
To detect the consistency of the c-MET gene amplification in peripheral blood and tumor tissue of patients with non small cell lung cancer and discuss the clinical application value of c-MET gene amplification in peripheral blood.

Methods:
Real-time fluorescent quantitative PCR was used to test the tissues in 257 patients of non small cell lung cancers and the peripheral blood samples in 318 patients of non small cell lung cancer, of which 185 cases of peripheral blood specimens could match the tissue samples, and detected the c-MET gene amplification in them by comparison of amplifications consistency in blood and tissue samples, and analysed the correlation between c-MET gene amplification and clinical characteristics of patients.

Results:
The c-MET gene amplification rate was 9.75% in peripheral blood of 31 patients with non small cell lung cancer，and was 8.95% in 23 cancer tissues, the amplification consistency was 81.25% in peripheral blood-tumor tissue matched samples. The difference was statistically significant (P<0.05).

Conclusion:
The consistency of the c-MET gene amplification in peripheral blood and tissue is high. c-MET gene amplification of peripheral blood could be used for clinical diagnosis and treatment in cases when tissue specimen is hard to get．

Abstract not provided

Background:
Ipilimumab (Ipi) is a humanized CTLA-4 antibody that blocks binding of CTLA-4 with its cognate ligands, permitting T cell activation through CD28 binding. There is evidence that phased in Ipi added to chemotherapy (C) may enhance efficacy in non-small cell lung cancer NSCLC. This trial was undertaken to gain a better understanding of the changes that occur in T cells, regulatory T cells (Tregs), and myeloid-derived suppressor (MDSC) in both the blood and tumor micro-environment with CTLA-4 blockade.

Methods:
Patients with stage T > 4 cm and/or N1, N2 NSCLC were offered neoadjuvant carboplatin AUC6 plus paclitaxel 200 mg/m[2] every 21 days 3 cycles with ipilimumab 10 mg/kg day 1 cycles 2 and 3. Blood for immune profiling of circulating T cells was collected prior to cycle 1, after cycle 1 chemotherapy alone, and after cycle 3 chemotherapy plus Ipi. If patients underwent tumor resection and excess tumor was available, viable tumor infiltrating lymphocytes (TILS) were disaggregated and stored for later analysis. Phenotypic and functional polychromatic flow cytometry (PFC) analyses were performed on peripheral blood mononuclear cells (PBMC).

Results:
Blood was successfully collected at all 3 time points for the first 17/18 patients who initiated trial therapy. Excess tumor (0.96-5 gms) was collected on 5 patients and ample viable CD45+ TIL cells (9.4-26x10[6]) were isolated and viably cryopreserved. Phenotypic analyses revealed that both CD4+ and CD8+ cells from all 17 patients were highly activated following two cycles of ipilimumab (cycle 3) as evidenced by greatly increased frequencies of CD28, HLA-DR, PD-1, and intracellular CTLA-4 expressing cells. The frequencies of Tregs, defined by CD4+CD25+FoxP3+ expression, were highly variable among the 17 participants, with 8 showing increased Tregs, 7 showing decreased frequencies, and 2 remaining unchanged over the course of therapy. Seven of the 17 participants had levels of MDSC cells at or above 5%, with two patients achieving MDSC levels of 13% and 26.7%. Tumor associated antigen (TAA)-specific CD4+ or CD8+ cells were detected at baseline on 4 patients (24%), but their relative frequencies were unaltered by Ipi therapy. The most commonly recognized TAA was Survivin, followed by MAGEA3 and PRAME. No patients developed detectable de novo TAA reactivities while on Ipi therapy. We will report the phenotypic and functional parameters of TILs isolated from 5 tumors of the patients enrolled on the current trial at the time of presentation.

Conclusion:
TAA-specific CD4+ or CD8+ cells were unexpectedly detected in the blood at baseline in a subset of patients. We were able to determine what common NSCLC antigens the circulating CD4+ or CD8+ T cells were activated against, and this has the potential to be a blood based biomarker for trials studying immunotherapy such as vaccines. Neoadjuvant ipilimumab therapy neither facilitated the development of anti-TAA reactivities nor enhanced the frequencies of existing TAA-reactive T cells in PBMC. Neoadjuvant ipilimumab therapy effectively enhanced the frequencies of highly activated T cells, but had no consistent effect of the frequencies of Tregs.

Background:
During the past decade, circulating tumor cells (CTCs) have been accepted as new prognostic and predictive factors for some type of cancers. In non small lung cancer cells (NSCLC) their detection and characterization is especially important to identify treatment resistances that some patients develop. Here, we report the value of characterization of CTCs and established cytological criteria to assess a good response for EGFR- tyrosine-Kinase inhibitors

Methods:
From Feb 2012 to October 2014, 39 patients (median age 63 years) with metastatic lung cancer were included in this study. NSCL (26 Adenocarcinomas and 13 Squamous cell carcinoma) EGFR wild-type patients were being treated with second or higher lines therapies with erlotinib. 10 ml of blood were collected from each patient into a CellSave TM Preservatives Tubes (Veridex, LLC, Johnson & Johnson Company) blood collection tube, maintained at room temperature and processed within a maximum of 72 hr after collection according to the protocol established by our group .For CTCs enrichment from PBMCs we used “Carcinoma Cell Enrichment and Detection kit: MACS technology (Miltenyi Biotechnology), using magnetic beads labeled with a multi-CK-specific. After isolation of CTCs, samples containing CTCs CK+ were stained for EGFR in double immunofluorescence (IF) experiments, following our standard protocol.The assessment of treatment was evaluated by histological criteria (CyCaR: Cytological Criteria of assessment Response).This way, as favorable response was defined when the number of CTCS was reduced more than 50% at 6 or 12 weeks of starting erlotinib treatment

Results:
Before treatment 18/39 patients (46%) were identified as positive for CTC[CK+ ]with an average number of 3.5 cell (range 1-11); 9 of which were presented persistence of CTCs after treatment. 7/ 18 patients positive for the presence of CTC[CK + ]were positive to presence EGFR marker (CTC[CK+ /EGFR +).]. More important was, that we found A positive correlation between CTCs and survival: patients with CTC(CK+ /EGFR-) presented a shorter OS (34 vs 53 weeks) and PFS (13 vs 17 weeks) compared with those patients with CTC(CK+ /EGFR +) which responded favorably to erlotinib. Patients without cytological response criteria had a worse survival OS (28 vs 53 weeks, p=0.057) and PFS (11,5 vs 21,3 weeks, p=0.06).

Conclusion:
Our study suggests that the characterization of CTC based in the EGFR expression might be useful as a marker for the therapeutic selection and monitoring of lung cancer patients sensitive to treatment with inhibitors of tyrosin-kinase.

Background:
Precision therapy with tyrosine kinase inhibitor (TKI) crizotinib and ceritinib against EML4-ALK (ALK+) non-small cell lung cancer (NSCLC) has advanced rapidly in recent years as a new paradigm in personalized cancer therapy. However, acquired drug resistance despite initial response still remains the rule, necessitating further investigations into mechanisms of resistance and novel therapies to overcome progressive resistant disease. Liquid-biopsy using peripheral blood circulating tumor cells (CTCs) as a minimally-invasive tool to determine patient’s disease status, tumor cells molecular-genomic make-up and evolution during therapies, is highly desirable. There is still an unmet need to develop affordable and robust technology platforms to empower such liquid-biopsy assay of CTC. There are relative advantages and pitfalls with various CTC platforms and a method to capture CTC in an unbiased fashion without pre-definition would be beneficial.

Methods:
We conducted pilot studies with adoption of two different CTC detection and enrichment platforms. First, we used the CellSearch[®] “positive-selection” platform through EpCAM immunomagnetic separation to profile 11 pts with ALK(+) NSCLC who were treated with crizotinib prospectively. Blood samples were collected (i) pretreatment, (ii) on TKI with CR/PR/SD, and (iii) at disease progression. Second, we evaluated a novel “negative-selection” CTCs capture-isolation platform, based on unbiased immunomagnetic removal of pan-leukocyte marker CD45+ cells coupled with RBC lysis, to enable CTC isolation without predefined CTC criteria. Pilot studies utilizing ALK+ H3122 cell line and ALK+ patients’ blood samples were performed for assay optimization and comparison. Whole genome sequencing using Illumina HiSeq x TEN was performed after whole genome amplification of the CTC tumor gDNA with paired-normal germline DNA for genomic interrogation.

Results:
Using ALK+ NSCLC patients’ peripheral blood samples, we demonstrated the presence of the EML4-ALK fusion (variant 1) in QPCR assay from the enriched CTC isolated using the CellSearch® platform. Also, CellSearch[®] enumeration in our pilot ALK+ cohort revealed a trend of correlation between the CTC numbers and disease status. The spike-in experiment in “negative selection” CTC platform enriched the spiked H3122 cells by 10,000 fold from the nucleated blood cells. Applying our “negative-selection” CTC assay to a patient with known EML4-ALK variant 1 (EML4-ex13/ALK-ex20) fusion lung cancer during disease progression on crizotinib, we detected the specific EML4-ALK variant 1 fusion in QPCR assay from the CTC enriched “eluate” fraction, but not in the “feed” fraction, whether the CellSearch® platform revealed any CTCs or not. In our index case of ALK-rearrangement NSCLC, we successfully performed whole genome sequencing analysis on the pretreatment negative-selection CTCs in comparison with the germline DNA and pretreatment lymph node tumor biopsied tissue tumor DNA. Our preliminary WGS results revealed similar genomic landscapes between the CTC and the biopsied tumor tissues.

Conclusion:
Taken together, our pilot CTC study results support the high sensitivity of the unbiased “negative selection” enrichment platform and its potential to empower molecular and genomic determinations in lung cancer. We also demonstrated the feasibility of the negative-selection CTC liquid-biopsy platform to achieve whole genome sequencing analysis of the captured CTCs.

Background:
The addition of bevacizumab, a monoclonal antibody against vascular endothelial growth factor, to a standard, first-line platin-based, two-agent chemotherapy regimen conferred a significant improvement in overall survival, progression-free survival, and response rate in patients with advanced non-squamous NSCLC (ns-NSCLC). The feasibility of the combination of bevacizumab and chemotherapy as the second-line or more therapy for ns-NSCLC is currently explored as well. However, no effective biomarker is validated to predict the response or clinical benefit of bevacizumab. This study aims to investigate the correlation between biomarkers and overall response to bevacizumab plus chemotherapy for patients with advanced ns-NSCLC.

Methods:
Patients with locally advanced or metastatic ns-NSCLC who were assigned to 7.5mg/kg 3-weekly doses of bevacizumab plus chemotherapy were eligible. Aaccording to investigators’ decision, chemotherapy regimens were pemetrexed (500mg/m[2]) with or without platinum (75mg/m[2]). Peripheral blood samples were collected at baseline and after 2[nd] cycle of therapy for the analysis of granulocyte colony-stimulating factor（G-CSF）, vascular endothelial growth factor A（VEGF-A） and VEGF receptor-2 (VEGFR-2). Plasma samples concomitantly with the radiological evaluation of disease progression were collected as possible. Correlation between biomarkers and overall response rate (ORR) were assessed by using a logistic regression model.

Results:
Baseline blood samples were available from 45 patients (100%, 19 patients were therapy naive, 26 patients failed of prior therapies), while samples after 2[nd] cycle were obtained from 37 patients (82.22%). For the primary analysis, there was no significant association between baseline plasma biomarkers and best overall response to the treatment. But, patients with low baseline plasma G-CSF level showed a trend toward improving ORR versus patients with high G-CSF level (odds ratio [OR], 3.846; 95%CI, 0.868 to 17.044，p＝0.076). Patients with high baseline plasma VEGF-A level showed a trend toward higher ORR versus patients with low VEGF-A levels (OR, 3.477; 95%CI, 0.857 to 14.113，p＝0.081). No significant correlations were observed between plasma biomarkers and progression-free survival (PFS). In comparison to baseline, plasma VEGF-A level increased significantly at 2[nd] cycle or radiological disease progression (p＜0.001). However, the magnitude of the difference did not correlate with ORR or PFS.

Conclusion:
Baseline or dynamic changes in plasma G-CSF, VEGF-A and VEGFR-2 did not correlate significantly with response of bevacizumab plus chemotherapy treatment for ns-NSCLC patients, while low baseline plasma G-CSF and high VEGF-A are possible candidate biomarkers for predicting response of bevacizumab plus chemotherapy for ns-NSCLC.

Background:
Circulating tumor cells (CTCs) and plasma circulating-free DNA (cfDNA) are promising candidates as non-invasive prognostic markers in malignant diseases. 18-fluorodeoxyglucose positron emission tomography integrated with computed tomography (18FDG-PET/TC) has a well-recognized diagnostic and prognostic value in non-small cell lung cancer (NSCLC). Very little is known about the mutual relationship between circulating biomarkers (CTCs and cfDNA) and 18FDG-PET/CT indicators in NSCLC.

Methods:
Peripheral blood samples from 28 patients affected by advanced/metastatic NSCLC were collected before starting first-line chemotherapy. CTCs were isolated by size using a filtration-based device (ScreenCell) and then identified and enumerated; cfDNA was isolated from plasma (QIAamp DNA Blood Mini Kit, Qiagen) and quantified by qPCR method using human telomerase reverse transcriptase (hTERT). All patients underwent 18FDG-PET/TC (Biograph 16 Siemens) at baseline. Maximum diameter (dmax) of the primary lesion (T), dmax of the greater lymph nodal (N), and metastatic (M) lesions were measured. Similarly, maximum and mean standardized uptake value (SUVmax, SUVmean) and size-incorporated SUVmax (SIMaxSUV) were computed for T, N and M, respectively; SIMaxSUV was calculated with the following formula for T, N, and M: SIMaxSUV= SUVMax*dmax. Presence (B+) and absence (B-) of metabolically active bone lesions (bone mets) were recorded. The association among CTCs, cfDNA and 18FDG-PET/CT-derived parameters was evaluated through multivariate analysis. T-test was performed to evaluate the difference in CTCs and cfDNA in B+ and B- groups, respectively.

Results:
Twenty-eight patients were evaluated; median age was 66 years (range: 51-80); male/female ratio was 18/10; 15 patients were current smokers, while 11 were former-smokers and 2 were never-smokers. Histo-types were grouped as it follows: adenocarcinoma= 22; squamous cell carcinoma= 5; not otherwise specified NSCLC= 1. Nine patients out of 28 had metabolically active bone lesions. Median CTC count was 7 CTCs/3ml (range: 0-47 CTCs/3ml), while median HTERT copy number was 109.0 (range: 16.7-1405-5).

	18FDG-PET/CT PARAMETERS	MEAN	STANDARD DEVIATION	P
T	Size	44.93	20.25	0.175
	SUV max	10.16	4.48	0.036
	SUV mean	10.6	3.4	0.994
	SIMaxSuv	487.7	333.5	0.472
N	Size	22.2	10.9	0.313
	SUV max	7.4	4.0	0.318
	SUV mean	5.8	3.0	0.294
	SIMaxSuv	172.8	158.1	0.231
M	Size	23.9	15.0	0.083
	SUV max	7.5	4.1	0.318
	SUV mean	7.4	1.2	0.307
	SIMaxSuv	216.4	206.5	0.463

At multivariate analysis, SUVmax of T was the only variable independently associated with cfDNA (p=0.036). No correlations were highlighted between CTCs and all PET-derived parameters. A trend towards significance between high HTERT and the presence of metabolically active bone lesions was observed (p=0.058).

Conclusion:
Our data demonstrate that the expression of cfDNA is correlated with the metabolic activity of the primary tumor lesion. Since SIMaxSUV was not correlated with HTERT, it appears that the expression of cfDNA depends from tumor metabolism rather than its burden. Further analyses on 18FDG-PET/TC-derived metabolic tumor volume are ongoing.

Abstract not provided

Background:
Detection of epidermal growth factor receptor (EGFR) mutation in non-small cell lung cancer (NSCLC) patients is mainly based on tissue biopsy, which is invasive and time consuming. Furthermore, there is still a need for serial monitoring of EGFR mutations and detection of EGFR tyrosine kinase inhibitors (TKIs) resistance. We hypothesized that plasma-based EGFR mutation analysis may be feasible for monitoring response to EGFR TKIs and could be used to predict the resistance.

Methods:
From January 2012 to October 2014, 200 EGFR mutant NSCLC patients were enrolled and treated with EGFR TKIs (141 patients for gefitinib, 46 patients for erlotinib, and 13 patients for afatinib). Plasma samples were prospectively obtained every 2 months from baseline until disease progression. The longitudinally collected plasma samples (n = 368) from 81 patients who progressed were analyzed using droplet digital PCR (ddPCR). We identified an association between serial EGFR mutant titers in plasma cell-free DNA (cfDNA) samples and the patient’s clinical response to EGFR TKIs.

Results:
Of a total 58 baseline cfDNA samples available for ddPCR, 43 (74%) samples demonstrated same mutation in the matched tumors (i.e. sensitivity: 70.8% (17/24) for L858R vs 76.5% (26/34) for exon 19 deletions). The concordance rate of plasma with tissue results of EGFR mutation was 88% for L858R and 86% for exon 19 deletion, respectively. Of the 54 patients with both before and after treatment plasma samples, 40 patients showed a dramatic decrease of mutant copies (greater than 50%) in blood in the first 2 months after treatment. We also found the secondary mutation (T790M) emerged in 28 patients around 3~13 months after treatment and in 4 patients before the treatment. Elevated circulating mutations (L858R/ex19/T790M) can be detected in 5 patients before disease progression as determined by CT scan.

Conclusion:
These results suggest that ddPCR is an appropriate method for determining plasma-based EGFR mutation status and may aid in monitoring response to EGFR TKIs and early detection of EGFR TKIs resistance.

Background:
Analysis of circulating tumor DNA (ctDNA) in plasma offers an opportunity to noninvasively monitor tumor burden and identify alternative drivers of disease progression in real-time. However, cancer progression during targeted therapy, such as EGFR-targeted therapies in non-small cell lung cancer (NSCLC), is driven by clonal evolution, and how this impacts the levels of targeted mutations in circulating tumor DNA (ctDNA) for monitoring disease burden is unclear.

Methods:
We collected serial plasma samples from 47 NSCLC patients receiving EGFR-targeted therapy (gefitinib) and hydroxychloroquine, and analysed mutations in EGFR, TP53, PTEN and PIK3CA in plasma by digital PCR and tagged-amplicon deep sequencing (TAm-Seq) of ctDNA.

Results:
We identified the same EGFR mutations in tumor and plasma samples in over 97% of patients, and found that patients with high pre-treatment levels of ctDNA are associated with worse progression-free survival and overall survival. Serial plasma analysis of 32 patients reveals clonal dynamics in ctDNA in response to treatment. In >72% of patients (23/32), EGFR mutations levels increased preceding clinical progression, with the resistant mutation T790M detected in around 50% of these patients (13/23) a median of 6 months before progression became clinically evident. In the remaining 9 of the 32 patients, EGFR-mutant ctDNA levels became uninformative during treatment, and in two patients we identified alternative driver mutations in ctDNA that correlated with progression. In one patient we also showed that the analysis of relative representations of resistant and sensitizing mutations may provide insight to the response to sequential treatment.

Conclusion:
Our results demonstrate the potential of ctDNA for noninvasive stratification and monitoring disease progression in NSCLC patients, and highlight that targeted therapy may drive the selection of alterative mutations. This may impact the representation of the targeted mutations in plasma for assessing disease burden. We therefore propose that effective ctDNA-based monitoring of targeted therapies in oncogene-addicted cancers requires tracking of multiple mutations beyond the targeted genes.

Background:
Plasma circulating tumor DNA (ctDNA) has been widely accepted as a form of liquid biopsy to detect EGFR mutations in NSCLC for its high concordance rate with tumor tissues. There are some retrospective studies about the ctDNA quantitative changes of EGFR mutations in EGFR-TKI treatment, but there is no report about serial ctDNA assessment of response and resistance to EGFR-TKI by detecting the dynamic changes of EGFR mutations during the whole course of EGFR-TKI treatment based on prospective clinical trial.

Methods:
Based on a randomized trial initiated to compare erlotinib with gefitinib in advanced NSCLC harboring EGFR exon 21 L858R mutation in tumor tissues (CTONG0901, NCT01024413), we prospectively collected serial plasma samples as preplanned schedule (baseline, one week after treatment, one month after treatment and then every 8 weeks until disease progression) and quantitatively detected EGFR L858R mutation in ctDNA by using fluorescence quantitative polymerase chain reaction. We made a serial ctDNA assessment of response and resistance to EGFR-TKI and its correlation with survival outcomes. Four patients’ serial plasma samples were selected to undergo next generation sequencing (NGS).

Results:
From 108 patients enrolled in the trial, serial plasma of 80 patients were collected as schedule and tested the quantity of L858R. As a whole, the quantity of L858R decreased to the lowest level when patients achieved best response to EGFR-TKI and increased to the highest level when disease progressed. Further analysis by Ward's Hierarchical Clustering Method showed that the dynamic changes of quantity of L858R could be categorized into two groups, Ascend Group and Stable Group (Figure 1A). Median progression-free survival (PFS) was 11.1 months (95%CI=6.6-15.6) and 7.5 months (95%CI=1.4-13.6) in two groups, respectively (HR=0.57, 95%CI=0.34-0.97, P=0.035) (Figure 1B). Median overall survival was 20.1 months (95%CI=15.7~24.5) vs. 16.4 months (95%CI=13.3~19.6) (HR=0.73, 95% CI =0.38~1.38, P=0.322). In multivariate Cox proportional hazards regression analysis, changing group was independent predictive factor for PFS. In plasma samples of 4 patients underwent NGS, similar dynamic changing characteristics were confirmed and more genetic mutations were found. Detailed data will be presented on site.Figure 1



Conclusion:
This is the first report about serial ctDNA assessment of response and resistance to EGFR-TKI by detecting the dynamic changes of EGFR mutation based on a prospective clinical trial. The quantity of plasma L858R has different changing patterns during EGFR-TKI treatment and higher L858R mutation abundance on EGFR-TKI resistance is correlated with longer PFS.

Background:
EGFR-T790M mutation, which is the valuable target for the next generation of EGFR-TKI, accounts for about half of the acquired resistance to current EGFR-TKI therapy in the EGFR sensitive mutation positive NSCLC patients. Due to clinical challenge in obtaining re-biopsy tumor tissues, noninvasive detection of EGFR-T790M in plasma circulating free DNA (cfDNA) has been proved to be feasible. Yet a highly sensitive assay needs to be developed to avoid false-negative detection. We here explored whether droplet digital PCR (ddPCR) of cfDNA can an alternative assay to identify the EGFR-TKI resistance mediated by EGFR-T790M in the clinical practice.

Methods:
The digital PCR method was recently developed for EGFR sensitive mutations, and its high sensitivity and specificity were validated in plasma cfDNA from EGFR-TKI-naïve NSCLC patients. In this study, we applied this method to detect EGFR-T790M in plasma cfDNA from metastatic NSCLC patients who initially responded but acquired resistance to current EGFR-TKI treatment. For the concordance analysis, the paired re-biopsy or pleural effusion cytology samples after failed EGFR-TKI were also collected for EGFR-T790M testing.

Results:
25 consecutive NSCLC patients were enrolled and analyzed in this study according to these criteria: 1. Metastatic NSCLC patients with acquired EGFR-TKI resistance. 2. The re-biopsy tissue or cytology samples and paired plasma samples were available after disease progression on EGFR-TKI. Among these 25 patients, 13 were positive and 9 were negative for EGFR-T790M mutation in both tumor tissue and plasma samples. 3 patients positive for EGFR-T790M mutation in tumor tissue were detected negative in their plasma. The overall concordance rate between plasma and tumor tissue testing was 88.00% (22/25) (Kappa=0.757, 95%CI: 0.4996-1.0). The sensitivity and specificity for plasma testing of EGFR-T790M mutation by ddPCR were 81.25% (13/16) (95%CI: 54.35%-96.00%) and 100.00% (9/9) (95%CI: 66.37%-100%), respectively. Figure 1



Conclusion:
Detection of EGFR-T790M in plasma cfDNA by ddPCR is highly sensitive and specific when compared to the pairedre-biopsy tissue or cytology samples. This noninvasive method could complement current invasive biopsy approach or provide an alternative method to identify specific mutation mediated resistance in clinic.

Abstract not provided

Background:
Primary pulmonary enteric adenocarcinoma (PEAC) is defined as a pulmonary adenocarcinoma with a predominant component (>50%) of intestinal differentiation and tumor cells positive for at least one intestinal marker. Due to its peculiarity and rarity, the optimal clinical management of PEAC patients remains unclear. A total of thirty cases have been described in literature to date, though familial aggregation has never been reported before. This is the first study describing the histological and molecular characterization of the PEAC from a patient with a family member affected by the same tumor.

Methods:
We evaluated the molecular characteristics of proband’s PEAC applying a previously validated 47-miRNA signature and applying the predictive method to estimate tissue-of-origin probabilities. Immunohistochemical (IHC) staining (TTF-1, Napsin A, CDX2, cytokeratonins, mucins) and mutational analyses (EGFR, K-RAS, ALK) were performed on formalin-fixed, paraffin-embedded tissue of a patient affected by PEAC.

Results:
The familial aggregation of PEAC was associated with similar clinicopathological features (age at diagnosis, smoking-habit, tumor localization, multiple colon polyps), histologic findings (IHC staining negative for TTF-1 and positive for CDX2) and genetic findings (K-RAS(Gly12Asp) mutation, but no EGFR/ALK aberrations). MiRNA profiling revealed main similarities with NSCLC (75.98%), and partial overlap with pancreatic cancer (PDAC, 23.34%), but not with colorectal cancer (less than 0.5%). Notably, this PEAC shares with PDAC key miRNAs associated with tumor aggressiveness (mir-31/-126/-506/-508-3p/-514). Figure 1



Conclusion:
In conclusion, we described, for the first time, PEACs in two members of the same family, associated with clinicopathological features. Moreover, miRNA profiling resembled mostly NSCLC, with a partial overlap with PDAC’s pattern that could explain the aggressive behavior compared to most NSCLC and guide future tailored-therapy approaches.

Background:
Apoptosis is a crucial pathway in tumor growth and metastatic development. Apoptotic proteins regulate the underlying molecular cascades and are thought to modulate the tumor response to chemotherapy and radiation. However, the prognostic value of the expression of apoptosis regulators in localized non-small-cell lung cancer (NSCLC) is still unclear.

Methods:
We investigated the protein expression of apoptosis regulators Bcl-2, Bcl-xl, Mcl-1, and pp32/PHAPI, and the expression of the lncRNA MALAT-1 in tumor samples from 383 NSCLC patients (median age: 65.6 years; 77.5% male; paraffin embedded tissue microarrays) For statistical analysis correlation tests, Log rank tests and Cox proportional hazard models were applied.

Results:
Tumor histology was significantly associated with the expression of Bcl-2, Bcl-xl and Mcl-1 (all p<0.001). Among the tested apoptotic markers only Bcl-2 demonstrated prognostic impact (HR=0.64, p=0.012). For NSCLC patients with non-adenocarcinoma histology, Bcl-2 expression was associated with increased overall survival (p=0.036). Besides tumor histology, prognostic impact of Bcl-2 was also found to depend on MALAT-1 lncRNA expression. Gene expression analysis of A549 adenocarcinoma cells with differential MALAT-1 lncRNA expression demonstrated an influence on the expression of Bcl-2 and its interacting proteins.

Conclusion:
Bcl-2 expression was specifically associated with superior prognosis in localized NSCLC. An interaction of Bcl-2 with MALAT-1 lncRNA expression was revealed, which merits further investigation for risk prediction in resectable NSCLC patients.

Background:
Malignant pleural mesothelioma (MPM) is an aggressive tumor mainly associated with asbestos exposure. MPM patients have a poor outcome (median overall survival (mOS) <1 year), therefore novel therapeutic approaches are needed. MiRNA have been demonstrated to have a role in tumorigenesis and progression in MPM. This study aimed to identify a miRNA signature associated with poor prognosis.

Methods:
We identified 26 un-resected MPM patients split as follows: 11 long survivors (LS) OS>3 years and 15 short survivors (SS) OS<1 year. MiRNA expression in 26 FFPE biopsy and 3 normal pleura (NP) was evaluated using Agilent Human miRNA Microarray platform including 2006 miRNA. Expression data were normalized by GeneSpring software (v.12.6). Class-comparison analysis between MPM/NP and SS/LS was performed using a t-test adjusted for multiple comparisons using Benjamini-Hochberg. OS curves were estimated using the Kaplan-Meier method and compared with the log-rank test. In silico validation was performed using miRseq data from TCGA portal based upon 16 patients (LS: 8; SS: 8). Candidate miRNA were assessed by univariate analysis using Kaplan-Meier method and median as cutoff.

Results:
Patients’ characteristics: median age 67 years (53-77); 81% males, 19% females; 73% epithelioid histotype, 12% sarcomatoid, 12% biphasic and 1 unspecified MPM. No differences in age, gender and histotype were observed between LS and SS. By class-comparison analysis, 30 miRNAs were significantly up-regulated and 11 down-regulated in MPM vs NP (adjusted p-value <0.05). Fourteen miRNAs were significantly associated with outcome, in the univariate survival analysis and differentially expressed in MPM. A miRNA signature, based on the top 6 prognostic miRNAs (unfavorable, miR-1224; favorable, miR-99a, miR-125b, let-7b, let-7c and let-7i) classified patients into low- or high-risk. High-risk patients showed a significantly shorter median OS (4.1 months, 95% CI 2.2-5.9) as compared with low-risk patients (median not reached, Log-rank p<0.001). In silico validation analysis confirmed that low expression of mir-99a, miR-125b and let-7c was associated with shorter OS. Relevant pathways, such as PI3K/AKT, WNT were associated with these top miRNAs by pathway analysis.

Conclusion:
A prognostic miRNA signature was identified by profiling a cohort of un-resected MPM, underlying the clinical potential of miRNA as predictors of survival. An additional validation in a larger independent cohort of MPM is ongoing.

Background:
Lung cancer is the leading cause of cancer mortality worldwide. About 30–40% of patients with lung cancer developed bone metastasis during the course of their disease, with the median survival time of 7 months approximately. Bone lesion is characterized by the imbalance of osteoblastic and osteoclastic activity induced by the tumor cells. Mesenchymal stem cells (MSCs) are the progenitor cells of osteoblast cells, and therefore it is interesting to investigate whether and how MSCs have biological alterations in the tumor microenvironment of lung cancer patients with bone lesion.

Methods:
We tested miR-139-5p and Notch1 expression during MSC osteogenic differentiation, and validated the effect of miR-139-5p in MSC osteogenesis by transfection of miR-139-5p mimic and inhibitor. We also collected blood samples of healthy donors and lung cancer with bone metastasis, and tested serum miR-139-5p expression.

Results:
We demonstrated that during MSC osteogenic differentiation in vitro, the expression of miR-139-5p increased significantly while Notch1 and its downstream factors decreased. By gain and loss of function studies we showed that miR-139-5p could positively regulate MSC osteogenic differentiation. And luciferase and western blot assays confirmed that miR-139-5p targeted Notch1 expression directly. Moreover, Notch1 knockdown by siRNA could no longer confer miR-139-5p induced MSC osteogenic differentiation. Lung cancer cell A549 and L9981 secreted factors upregulated miR-139-5p expression and meanwhile downregulated Notch1 expression in MSC, as well as impaired ALP activity, the early osteogenic marker of MSCs. More importantly, we observed that serum miR-139-5p was significantly lower in lung cancer patients with lytic bone lesion compared to healthy donors.

Conclusion:
We demonstrate for the first time, that miR-139-5p could regulate osteogenic differentiation of MSC by targeting Notch1 mediated signalling pathway. Lung cancer cells could decrease miR-139-5p expression and inhibit MSC osteogenic potential. Importantly, serum miR-139-5p is significantly lower in lung cancer patients with lytic bone lesion compared to healthy donors. Therefore, miR-139-5p might be a biomarker of bone metastasis in lung cancer patients and treatment with miR-139-5p mimic might be an interesting strategy to restore the impaired MSC osteogenic differentiation and to control bone disease in lung cancer patients with bone lesion.

Abstract not provided

Background:
EGFR Tyrosine Kinase Inhibitor (TKI) is now standard of care in lung cancer patients with EGFR mutation. Invariably, these patients develop resistance and would require treatment with another EGFR TKI or chemotherapy. The gate keeper mutation T790M is responsible for about 50% of resistance cases. There are other mechanisms that could confer resistance in these patients. High expression of microRNA (mir) 10 is associated with worse prognosis in resected lung cancer patients with EGFR mutation. The mir 27a and 27b is associated with increased expression of c-met which is a potential mechanism of resistance to EGFR. We explored the expression of mir 10b and 27a and 27b in lung cancer cell lines with EGFR mutation that were resistant to Erlotinib.

Methods:
Lung cancer cell lines with EGFR mutation CRL-2868 and CRL 2871 were treated with 5 nM of erlotinib for 24hours and 72 hours. The erlotinib resistant cells were then harvested and then microRNA profiling was done by real time PCR. HTB177 cell line without EGFR mutation was used as control.

Results:
We observed an increased expression of mir 10b, mir27a and mir27b in CRL-2871 compared to control HTB 177 cells. Mir 27b is upregulated in both cell lines. The increase expression of mir 10b and mir 27a were higher in CRL-2871 than the control cells 9 fold and 8 folds respectively. The mir 27b was increased 300 folds in the CRL 2868 compared with control.

Conclusion:
We observed upregulation of mir 10b, 27a and 27b in lung cancer cells lines with EGFR mutation that were resistant to Erlotinib treatment. This suggests an epigenetic mechanism of resistance other than the T790M mutation. Further research in patients with EGFR mutation resistance to EGFR TKI should be done to confirm this finding.

Background:
MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs that range in size from 19 to 25 nucleotides. Alteration in miRNA expression can cause them to act as either tumour suppressor or oncogenes. They have also been shown to regulate a number of processes involved in tumour biology such as metastasis, invasion and angiogenesis. More recently, miRNAs have been linked to chemo- and radio-resistance in many solid tumours, including lung cancer.

Methods:
An isogenic model of cisplatin resistance was established by chronically exposing a panel of NSCLC cell lines (MOR, H460, A549, SKMES-1, H1299) to cisplatin for 12 months, generating cisplatin resistant (CisR) sublines from their corresponding age-matched parental (PT) cells. MicroRNA expression profiling was carried out using 7th generation miRCURY LNA™ microRNA arrays consisting of 1,919 miRNAs (Exiqon). MicroRNAs that were significantly increased in CisR sublines were inhibited using antagomirs (Exiqon), while those that were significantly decreased were over-expressed using pre-miRs (Ambion). Functional studies examining clonogenic survival ability, proliferation (BrdU) and apoptosis (Annexin V/PI) were subsequently carried out in the presence or absence of cisplatin. To examine the translational relevance of these microRNAs, their expression was further examined in a cohort of pre-treatment matched normal and tumour lung tissues from NSCLC patients of different histological subtypes. Validation of this miRNA signature is currently being investigated in serum samples from this same cohort of patients and normal controls.

Results:
MicroRNA profiling analysis identified ten miRNAs which were significantly altered between parental and corresponding cisplatin resistant lung cancer cell lines. Validation of these miRNAs by real-time PCR (qPCR) identified a specific 5-miR signature that was significantly altered in CisR cells relative to their parental counterparts. Modification of these microRNAs altered the response of resistant cells to the cytotoxic effects of cisplatin and decreased the clonogenic survival of CisR cells when treated with increasing doses of cisplatin (0.1µM-10μM). Significant differential expression was found between normal and tumour tissues across each histological subtype, highlighting the potential use of these microRNAs as markers of response to cisplatin therapy in NSCLC patients. Three miRNAs (miR-A, B, C) belonging to the same family were significantly altered in tumour lung tissue of adenocarcinoma and squamous cell histology compared to matched normal lung tissue. MicroRNA-D expression was significantly altered in squamous cell carcinomas while miR-E was differentially expressed in adenocarcinomas only. Data relating to the expression of this novel signature in the circulation of our NSCLC patient cohort and normal controls will be presented at WCLC 2015.

Conclusion:
We have identified and validated a novel miRNA signature associated with cisplatin resistance in a panel of cisplatin resistant cell lines and in patient lung tumours. Genetic manipulation of these specific miRNAs in vitro altered the cisplatin resistant cell response to the cytotoxic effects of cisplatin chemotherapy. The data obtained from this study may provide a basis for the potential development of a companion diagnostic for lung cancer patients who are most likely to benefit, or not, from cisplatin chemotherapy.

Background:
Lung adenocarcinoma is one of the main pathological tissue types of lung small cell lung cancer (NSCLC). Tumor metastases are responsible for approximately 90% of lung adenocarcinoma-related death. The epithelial–mesenchymal transition (EMT) is regarded as a critical event during tumor metastasis. From our previous study, we found miR-33b might be involved in the EMT process of lung adenocarcinoma. However, the specific mechanism of miR-33b regulating EMT has not been established.

Methods:
Quantitative real time-PCR (qRT-PCR), in situ hybridization and immunohistochemistry were used to investigate expression of miR-33b and ZEB1 in paired lung adenocarcinoma tissues. MiR-33b target gene ZEB1 was investigated using luciferase reporter gene assays. Western blot, qRT-PCR, immunofluorescence were used to compare the expression levels of ZEB1, E-cad, Vim and β-catenin in A549 cells which were transfected miR-33b or miR-NC, anti-miR-33b or anti-miR-NC and in vivo. MTT was used to analysis growth curves of transfected cells with miR-33b or miR-NC, anti-miR-33b or anti-miR-NC, siRNA-NC or siRNA -ZEB1, siRNA-NC or siRNA-β-catenin. The transfected cells were subjected to Transwell migration and invasion assays.

Results:
Mean miR-33b levels were significantly lower in lung adenocarcinoma tissues compared with matched non-cancerous tissues (0.024± 0.028 vs 0.063 ± 0.074, P < 0.0001). The level of miR-33b in NSCLC was strongly correlated with lymph node metastasis (P < 0.0001). ZEB1 levels were significantly higher in lung adenocarcinoma tissues compared with matched non-cancerous tissues (0.447± 0.371vs0.084 ± 0.112, P <0.0001). The level of miR-33b in lung adenocarcinoma was negative correlated with ZEB1 (P < 0.0001,r=-0.6077). MiR-33b significantly inhibited EMT in lung adenocarcinoma metastasis in vitro and vivo (P < 0.05).MiR-33b directly targets the ZEB13[']UTR(P < 0.01).β-catenin was down regulation by overexpression of miR-33b (P < 0.01). MiR-33b overexpression and ZEB1 inhibition suppressed lung adenocarcinoma migration and invasion in vitro (P < 0.01).

Conclusion:
On the basis of our results, we propose that miR-33b suppress EMT in lung adenocarcinoma through direct targeting of ZEB1 in vitro and in vivo. Moreover, we found that Wnt/β-catenin/ZEB1 pathway regulated by miR-33b might involved in lung adenocarcinoma EMT. These findings provide new insights into the molecular functions of miR-33b as well asthe role of ZEB1 in lung adenocarcinoma metastasis.

Background:
Recent evidence indicates that long non-coding RNAs (lncRNAs) play a critical role in the regulation of cellular processes, such as differentiation, proliferation and metastasis. These lncRNAs are found to be dysregulated in a variety of cancers. In our previous study, we have demonstrated that lncRNA AK126698 regulated A549 cells cisplatin resistance partly through Wnt/β-catenin signaling. However, the clinical significance of lncRNA AK126698, and its’ molecular mechanisms of controlling cancer cell proliferation and migration are still unclear.

Methods:
Expression of lncRNA AK126698 was analyzed in 55 non-small cell lung cancer (NSCLC) tissues and 3 NSCLC cell lines using quantitative polymerase chain reaction (qPCR) assays. Gain and loss of function approaches were used to investigate the biological role of AK126698 in NSCLC cells. The effects of AK126698 on cell proliferation were evaluated by CCK-8 and colony formation assays. Apoptosis was evaluated by flow cytometry. Protein levels of AK126698 targets were determined by western blotting and fluorescent immunohistochemistry.

Results:
AK126698 expression was significantly decreased in NSCLC tumor tissues compared with normal lung tissues. Additionally, reduced AK126698 expression was associated with larger tumor size and advanced pathological staging of NSCLC patients. Ectopic expression of AK126698 impaired cell proliferation and migration and induced apoptosis in vitro. However, knockdown of AK126698 expression promoted cell migration and invasionin in vitro. In addition, the expression of FZD8 was inversely correlated with AK126698 in both NSCLC tissues and cell lines and that over-expression of AK126698 could significantly down-regulate the expression of gene downstream of FZD8, including β-catenin, CyclinD1 and c-Myc.

Conclusion:
The results of present study indicate that lncRNA AK126698 might serve as a suppressor that regulates the pathogenesis of NSCLC through Wnt/β-catenin signaling pathway. It may provide a new target for therapeutic intervention of NSCLC.

Abstract not provided

Background:
Deregulation of alternative splicing has become a hallmark of cancer. In non-small cell lung cancer (NSCLC), the biological importance of splicing is evidenced by the identification of aberrant RNA transcripts associated with somatic mutations in genes encoding splicing factors. We have previously developed ExonPointer, an algorithm optimized to detect differential splicing cassette events from data obtained in microarrays containing probes in exons and junctions. Our present objective was to apply this technology for the identification and characterization of cancer-associated splice variants and splicing factors in NSCLC.

Methods:
We applied ExonPointer for the identification of differential splice forms in lung cancer tissues (8 adenocarcinomas, 13 squamous cell carcinomas and 1 large cell carcinoma) and matched normal lung. We validated the events by RT-PCR and used bioinformatics tools, such as DAVID and Ingenuity Pathway Analysis, for cluster enrichment analyses. siRNA knockdown of specific splice isoforms was used for functional analyses. Prognostic studies were performed by immunohistochemistry in 127 primary tissues from patients with NSCLC.

Results:
The validation rate for the top 20 differentially expressed splice events identified by ExonPointer was 70%. Gene cluster analyses using the first 250 events showed a significant enrichment of cancer-related clusters such as Cellular Growth and Proliferation, or Cell Death and Survival. Among the validated genes, we identified Extended synaptotagmin-2 (ESYT2). ESYT2 is a membrane protein that mediates fibroblast growth factor receptor-1 (FGFR1) endocytosis and actin dynamics. A significantly different pattern of ESYT2 alternative splicing was found in primary lung tumors as compared to normal lung tissue (p<0.001). In particular, an isoform containing an extra exon was overexpressed in cancer tissues, while the expression of the canonical isoform was decreased. We found a significant correlation between the splicing pattern of ESYT2 and the expression of FGFR1 in a panel of 43 lung cancer cell lines (r=-0.724, p<0.001). Using siRNA downregulation, we analyzed the implication of the ESYT2 isoforms in tumor biology and demonstrated a distinct role of the splice isoforms in actin and tubulin cytoskeleton organization. We also searched for splicing factors responsible for the splicing of ESYT2. We found that the ratio of ESYT2 isoforms correlated with the expression of the splicing factor QKI in lung cancer cell lines (r=-0.793, p<0.001). Moreover, in vitro downregulation of QKI markedly affected ESYT2 splicing. Interestingly, we found a significant enrichment of QKI targets in the list of differentially spliced genes identified by ExonPointer (p<0.001), suggesting that this factor is a critical regulator of splicing in lung cancer. Finally, we observed a significant downregulation of QKI expression in primary NSCLC compared to adjacent normal lung cancer cells (p<0.001), and an association between the nuclear expression of this factor and disease-free survival (HR=0.61; 95%CI=0.35-1.05) or overall survival (HR=0.44; 95%CI=0.21-0.94).

Conclusion:
Using a novel analytical tool we have identified new splicing variants with functional relevance in lung cancer. Moreover, changes in splicing events in lung primary tumors were found to be largely regulated by the splicing factor QKI, a potential tumor suppressor gene downregulated in NSCLC and associated with the prognosis of the disease.

Background:
Fetal development shares many biological similarities with tumourigenesis such as high rates of cell proliferation and vasculature restructuring. Particularly in the lung, a number of genes and pathways involved in the development of this organ play important roles in the malignant transformation of adult lung cells. Despite these biological similarities, there is a paucity of information regarding how specific molecular regulators involved in fetal lung development become oncogenes in lung cancer. This is the case for micro RNAs (miRNAs), which have a pivotal role in regulating gene expression during organ development and tumourigenesis. Therefore, a better understanding of the human fetal lung miRNA-transcriptome has the potential to reveal key players in lung cancer development and progression.

Methods:
131 pairs of non-small cell lung cancer (NSCLC) tumour and adjacent non-malignant lung tissues and 15 human fetal lung tissue samples were profiled by miRNA-sequencing. Mann–Whitney U tests were performed with Benjamini-Hochberg multiple testing corrections to identify miRNAs abundantly expressed in fetal and tumour tissues but scarce in non-malignant adult lung (i.e. oncofetal miRNAs). To investigate protein-coding genes controlled by the oncofetal miRNAs identified, miRDIP was applied followed by gene reporter luciferase assay experiments. The assessment of associations between patient survival and mRNA expression of selected genes targeted by the oncofetal miRNAs was evaluated in multiple NSCLC cohorts (>1,400 tumour cases). To validate the protein expression of the most prominent gene regulated by the oncofetal miRNAs identified, immunohistochemical (IHC) analysis was performed on a lung adenocarcinoma (LUAD) tissue microarray (TMA).

Results:
We describe for the first time a comprehensive, unbiased characterization of miRNA expression in human fetal lung tissue by miRNA sequencing. Through comparison to a large cohort of NSCLC, we identified numerous miRNAs that recapitulate their fetal expression patterns in lung cancer. Strikingly, assessment of the genes potentially regulated by these oncofetal miRNAs led us to identify Nuclear Factor I/B (NFIB), a transcription factor essential for lung development, as being frequently targeted by oncofetal miRNAs. Concordantly, analysis of NFIB expression using RNA-sequencing data for multiple NSCLC cohorts revealed its frequent underexpression in tumours (>60%). Remarkably, low expression of NFIB was significantly associated with poorer survival in LUAD patients but not in squamous cell carcinoma patients, consistent with the functional role of NFIB in distal lung cell differentiation (i.e. cells that are the precursors of LUAD). Furthermore, an NFIB-related gene signature was identified in LUAD tumours and included several well-known lung differentiation markers (TTF-1, ABCA3, GPR116, SFTPB). Finally, the underexpression of NFIB protein was validated on a LUAD TMA, which also revealed that tumours presenting lower levels of this transcription factor are associated with higher grade, biologically more aggressive LUAD (invasive mucinous, micropapillary and solid subtypes).

Conclusion:
This work has revealed a prominent mechanism for the downregulation of a crucial gene for lung development, which we found to be associated with aggressive phenotypes of LUAD and consequently, poor patient survival. Elucidating the specific role of NFIB in lung cancer biology will likely lead to the identification of targetable tumour vulnerabilities.

Background:
Limited information exists regarding regulation of gene expression by long noncoding RNAs (lncRNAs) during initiation and progression of tobacco-associated lung cancers. In the present study, an in-vitro model system was used to examine the effects of cigarette smoke on lncRNA expression in human respiratory epithelia and lung cancer cells.

Methods:
Micro-array and qRT-PCR techniques were used to assess lncRNA and gene expression profiles in normal human small airway epithelial cells (SAEC) and immortalized human bronchial epithelial cells (HBEC) cultured in the presence or absence of cigarette smoke condensate (CSC). Quantitative chromatin immunoprecipitation (ChIP), methyl-DNA immunoprecipitation (MeDIP), and cross-link immunoprecipitation (CLIP) techniques were used to assess promoter occupancy, DNA methylation, and interaction of lncRNAs with target proteins. Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assays were used to identify a regulatory sequence for lncRNA BC070487.

Results:
Micro-array analysis demonstrated that under relevant exposure conditions, CSC consistently mediated a 2.5 fold increase in BC070487 expression, and 3.6 fold decrease in its immediate downstream target gene, ZNFX1, in SAEC. qRT-PCR experiments confirmed a 4-7 fold up-regulation of BC070487, with a 4-8 fold down-regulation of ZNFX1 in SAEC and HBEC, as well as Calu-6 and H841 lung cancer cells following CSC exposure. BC070487 expression correlated inversely with expression of ZNFX1 (BC070487: 1.38-10.31 fold up vs ZNFX1: 2.26-26.29 fold down) in primary lung cancers relative to adjacent normal lung parenchyma. Overexpression or depletion of BC070487 inhibited or enhanced expression of ZNFX1, respectively in normal respiratory epithelia and lung cancer cells. CSC as well as BC070487-mediated repression of ZNFX1 coincided with increased occupancy of EZH2, SUZ12, BMI1, and increased levels of H3K27Me3, with decreased H3K4Me3 within the ZNFX1 promoter region. CSC as well as BC070487 overexpression enhanced binding of BC070487 with EZH2, SUZ12 and BMI1 proteins in SAEC and Calu-6 cells. CSC exposure significantly increased DNA methylation in one of three CpG islands spanning the regulatory elements of ZNFX1. In addition, CSC enhanced binding of BC070487 with DNMT3A and DNMT3B with site-specific de novo DNA methylation of ZNFX1 in SAEC and Calu-6 cells. FAIRE assays identified a 800 bp regulatory sequence for BC070487; CSC-mediated activation of BC070487 coincided with increased levels of H3K4Me3, H3K27Ac, H3K36Me3, Sp1, and Ago proteins, with decreased levels of H3K27Me3 and H3K9Me3 within this regulatory region. miR-31, an oncomiR specifically induced by CSC, modulated the transcriptional activity of BC070487 via remodeling the distribution of histone modification marks (Up: H3K4Me3, H3K27Ac, H3K36Me3; Down: H3K27Me3 and H3K9Me3) in the regulatory region of BC070487. Overexpression or depletion of BC070487 increased or inhibited proliferation and invasion of lung cancer cells, and similarly modulated growth of SAEC and HBEC cells. In contrast, ZNFX1 overexpression or depletion decreased or enhanced growth of normal respiratory epithelial cells and lung cancer cells. BC070487 overexpression enhanced growth of lung cancer cells xenografts in athymic nude mice, while forced expression of ZNFX1 arrested growth of these xenografts.

Conclusion:
Collectively, these data suggest that CSC-mediated up-regulation of BC070487 suppresses ZNFX1 to promote pulmonary carcinogenesis.

Background:
The benefit from postoperative chemotherapy in non-small cell lung cancer (NSCLC) is modest and there are no reliable tools allowing for individual selection of high-risk patients for this management. We earlier demonstrated that overexpression of three miRNAs: miR-662, -192 and -192* may correlate with the risk of distant relapse in squamous cell lung cancer (SCC) patients undergoing pulmonary resection (Skrzypski et al. 2014, Br J Cancer). However, the role of these miRNAs in maintaining aggressive phenotype and/or chemoresistance of SCC is unknown. The aim of this study was to assess the biological role of three abovementioned miRNAs in SCC cells in vitro.

Methods:
In the search for appropriate in vitro model we screened by reverse transcription quantitative PCR 11 NSCLC cell lines (H1703, H520, H662, SK-MES-1, A549, H23, H1975, HCC 827, PC9, H460 and Calu-6) for miRNA expression profiles and analyzed their phenotypic features including migration, invasion and clonogenic potential. H520 and H1703 SCC cell lines were transfected with locked nucleic acid miRNA inhibitors. The sensitivity of transfected and wild type cells to cisplatin and etoposide was compared using cytotoxic MTT test. Soft agar colony formation assay was performed to assess the clonogenic potential of transfected vs. non-transfected cells.

Results:
Among the analyzed NSCLC cell lines, H520 cells showed natural overexpression of miR-662, -192 and -192*. These cells were resistant to both chemotherapeutics and exhibited an ability to form colonies in soft agar. The inhibition of miR-662 and miR-192 sensitized these cells to etoposide (p=0.004 and 0.016, respectively) but not to cisplatin. Similar results were obtained for H1703 cells (p=0.002 and 0.02, respectively). H520 cells treated with miR-192 and miR-662 inhibitors, compared to negative control, also demonstrated reduced number of colonies in soft agar (p<0.001).

Conclusion:
We developed and optimized a cellular model for in vitro studies of biological role of three prognostic miRNAs in SCC. Genes regulated by miR-662 and/or miR-192 may be involved in maintaining the chemoresistance and clonogenic potential of SCC, and these features may be inhibited in a miR-dependent specific manner. Further studies may elucidate predictive value of these genes and a possibility of their targeting with novel therapeutics.

Abstract not provided

Abstract not provided

Abstract:
Major advances in the understanding of molecular pathways that regulate tumor growth have led to development and FDA approval of new anticancer agents leading to improved survival in patients with certain cancer types. A common cancer like Non Small Cell Lung Cancer (NSCLC) is now subdivided in a number of molecularly defined subsets. Large randomized trials are not feasible anymore. This challenge to accrue to small subsets with availability of multiple drugs for each has led to novel trial design strategies like “Master Protocols” with multiple arms that are biomarker driven. Master Protocols are ideal if multiple molecular targets are known to drive tumor growth, if there is pre-clinical/clinical evidence that these targets can be pharmacologically inhibited and if the drugs to be tested are available. These protocols provide consistency of drug development approach regardless of intended target, utilize resources (including patient resources) in an efficient manner and have potential of bringing safe and effective drugs to patients faster. On the other hand these trials are time and resource intense, and some trials with public/private partnership have added need of a high level coordination. Part of NCI precision medicine initiative has led to “Master Protocols” like Lung-MAP, ALCHEMIST and NCI-MATCH. The Lung-MAP trial is evaluating patients with squamous cell lung cancer who have progressed beyond at least one line of therapy. The study divides patients into multiple treatment arms based on the molecular profiles of their cancers testing efficacy of targeted drugs. Promising results in any arm can lead to testing the drugs in that treatment arm in more patients, with the goal of more rapid drug approvals in these small subsets of squamous cell lung cancer patients. ALCHEMIST is testing the benefits of molecularly targeted adjuvant (post-surgical) treatment of patients with early-stage lung adenocarcinomas whose tumors have either an EGFR gene mutation or an anaplastic lymphoma kinase (ALK) gene rearrangement. Depending on the genetic abnormality in a tumor, the patient will be randomized to receive the EGFR protein kinase inhibitor erlotinib or the ALK protein kinase inhibitor, crizotinib against a placebo. The Food and Drug Administration (FDA) has approved these molecularly targeted therapies for advanced lung adenocarcinoma in patients with the relevant genetic changes. It is expected that most patients with early lung adenocarcinoma who are screened will not be eligible for the therapeutic portion of this trial because their tumors will not have the necessary mutations. However, the tumor samples from these patients will be saved, and, if they relapse while on standard treatment, their tumors will be biopsied again and analyzed for insight into the progression of their disease and for potential therapeutic approaches suggested by this analysis. The NCI-MATCH trial will test a large number of agents in virtually any tumor type in which appropriate abnormalities are identified. This umbrella protocol will examine between 20 and 25 drugs, including those that have been FDA-approved for the treatment of cancer at another tumor site or experimental agents that have shown activity against a known target at one or more tumor sites. If the response rate to a particular agent is high, the number of patients evaluated with that treatment would be expanded to further explore whether the targeted treatment represents a substantial advance over standard chemotherapy. If a tumor becomes resistant to the first test drug, it will be re-biopsied to see if another targeted therapy might be effective and to understand the basis for resistance to the initial treatment. By studying multiple agents at the same time, a higher proportion of patients will be eligible for the trial, and efficient progress can be made in the assessment of clinical benefit.

Abstract not provided

Abstract:
Over the past decade, there have been rapid advances in cancer drug discovery and development. Much of this progress has resulted from a better understanding of the genetic changes in cancer and the development of agents that target the underlying biology of disease. In addition, an improved understanding of the immune system and cancer has resulted in the development of immune checkpoint inhibitors that have profound clinical activity in many tumors. Finally, the role of the tumor microenvironment in the regulation of tumor growth, metastatic spread, and modulation of the immune system is an area of intense investigation[1]. This enhanced understanding of the complex biology of cancer, and novel drugs against these new targets, have ushered in an exciting era in drug development leading to important new drug approvals[2]. An increasingly important aspect in both clinical development and drug approval is the incorporation of biomarkers and companion diagnostics to select patients more likely to benefit from specific therapies[3]. In lung cancer, the utilization of companion diagnostics has been key in the clinical development of TKIs directed at a variety of EGFR mutations and ALK alterations[4,5,6]. Other biomarker tests have been used to identify genes such as BRAF and ROS1 in order to develop clinical trials to test approved targeted agents with activity in patients with these molecular alterations[7]. Ongoing clinical studies exemplified by the Lung Master Protocol and NCI Match Protocol are utilizing biomarker panel strategies, including next generation sequencing (NGS), to identify patients with specific mutations in lung and other cancers respectively[4]. The use of NGS to characterize molecular alterations is also becoming more common to characterize patients’ tumors in both the academic and community settings. Immunohistochemistry assays continue to be a mainstay for understanding tumor biology and are also being utilized to quantitate markers such as PDL-1 expression in tumor immune cells in order to identify patients who are more likely to respond to immune checkpoint inhibitors[8]. In addition, analysis of biomarkers in liquid biopsies (e.g. plasma, spinal fluid) are being analyzed to provide supplemental information and/or to obviate the need for ongoing tumor biopsies during therapy[9]. Clinical development based on patient subset will increasingly be the norm, rather than the exception, in oncology. Rather than exclusively utilizing histology to screen patients for clinical trials, the use of basket trials to identify and treat patients of various histologies with agents targeted to similar molecular alterations is an approach which is increasingly being utilized[10]. In addition, retrospective analyses are being conducted to understand underlying molecular abnormalities of patients with exceptional responses to existing therapies and to use the information to design future clinical trials. One of the major challenges in clinical development is tumor heterogeneity and drug resistance. To prevent or overcome the development of drug resistance, combination therapies to target specific pathways are being explored. One such example is the use of BRAF and MEK inhibitors in the treatment of metastatic melanoma[11]. Many clinical studies in lung cancer are now incorporating mandatory tumor biopsies during the course of EGFR and ALK inhibitor therapy to identify evolving genetic changes in tumors during therapy in order to incorporate second and third generation TKIs. New mechanisms to facilitate the drug review and approval processes are underway[12]. One such mechanism is the Breakthrough Therapy (BT) Program. Breakthrough Therapy is intended to expedite the development and review of drugs for serious or life threatening conditions. Designation of a drug as a BT is based on preliminary clinical evidence that demonstrates that a new drug may have substantial improvement over available therapy and facilitates ongoing communication between the sponsor and the FDA to streamline the drug development process. Accelerated approval has been developed by the FDA for speeding the development and approval of promising therapies to treat serious disease that provides a meaningful therapeutic benefit over available therapy. Accelerated approval is based on an improvement in patient benefit utilizing surrogates of survival that are reasonably able to predict clinical benefit. The accelerated approval mechanism has been essential in facilitating new drug approvals of promising therapies. In addition, fast track designation is an FDA program intended to facilitate the development and expedite the review of drugs to treat serious medical conditions. This program allows sponsors to facilitate the review process for drug approval by having ongoing FDA interactions and utilizing a rolling review of submissions. Given the importance of biomarker-directed therapy, an additional critical component of the regulatory landscape is the review and approval of companion diagnostics at the same time as specific drug approvals. The drug-diagnostic co-development model is becoming increasingly common in oncology as biomarker-driven patient selection is required for many of the new targeted and immune therapies. Thus, an evolution in both the clinical development paradigm and the regulatory landscape is occurring based on the discovery and development of more effective, biomarker-directed targeted agents and novel immune checkpoint inhibitors.REFERENCES 1. Hanahan, D. and Weinberg, RA. Cell 2011; 144: 646-674. 2. Wolford, JE. and Tewari, KS. Future Onc. 2015; 11: 1931-1945. 3. Shen, T., Hans Pajaro-Van de Stradt, S., Yeat, NC., et al. Front in Genet. 2015; 6:215 in press. 4. Morgensztern, D., Campo, MJ., Dahlberg, S., et al. J. Thor. Onc. 2015; 10: S1-S63. 5. Somasundarsm, A., Socinski, MA., and Burns, TF. Informa 2014; 15: 2693-2707. 6. Kwak EL, Bang Y-J, Camidge DR, et al. N Engl J Med. 2010; 18: 1693-1703. 7. Camidge, DR., Pao, W., and Sequist, LV. Nature Rev. Clin. Onc. 2014; 11: 473-481. 8. Carbognin, L., Pilotto, S., Milella, M., et al. PLOS ONE 2015; 10:1371 in press. 9. Francis, G. and Stein, S. Int. J. Mol. Sci. 2015; 16: 14122-14142. 10. Catenacci, DVT. Mol. Onc. 2015; 9: 967-996. 11. Long, GV., Stroyakovskiy, D., Gogas, H., et al. NEJM 2014; 371: 1877-1888. 12. Kesselheim, AS. and Darrow, JJ. Clin. Pharm. & Ther. 2015; 97: 29-36.

Abstract:
Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor issued from the mesothelial surface of the pleural space. A previous exposure to asbestos is the main risk factor of mesothelioma. Clinical signs are most of time late and unspecific. Chest CT-scan, a key imaging procedure, usually shows pleural effusion ±pleural thickening. PET-CT may help to differentiate MPM from pleural benign tumors, as well for distal tumor staging. But PET-CT is not recommended for the diagnosis of MPM, as well as soluble biomarkers, including mesothelin. A diagnosis of MPM based on pleural biopsies best obtained by thoracoscopy is recommended with compulsory immunohistochemistry (1, 2). The treatment of MPM is so far quite deceptive with median overall survival (OS) around 12 months, and relies mostly on chemotherapy and best supportive care (BSC). To date, only first line chemotherapy by cisplatin/carboplatin+pemetrexed is recommended by all guidelines for patients fitted for chemotherapy (3). The optimal duration of first line chemotherapy is unknown but a maximum of 6 cycles is recommended. There is no evidence supporting a maintenance treatment including by pemetrexed. Therapeutic options beyond first line treatment are presently highly limited despite nearly half of patients are clinically fitted for. According to guidelines, pemetrexed alone may be proposed again if patients did have tumor progression at least 3 to 6 months after stopping chemotherapy (1, 2). Other options exhibited deceptive response rates (4). Therefore, it is recommended in the other cases to propose patients to join clinical trials. Pathogenesis of MPM includes overexpression of growth factors (VEGF…), many genetic and epigenetic alterations and/or mutations of malignant cells (p16 INK4A/CDKN2A, BAP-1, NF-2…) responsible for cell proliferation and resistance to apoptosis, pleural inflammation and local immunosuppression induced by the tumor and favoring its growth. These elements provide the rationale for many targeted therapies and immunotherapy. But so far, very few drugs exhibited sufficient value to deserve further trials. Thus, first trials assessing anti-angiogenic drugs in MPM did not support their use in this cancer despite the key role of VEGF. A phase II trial of bevacizumab (anti-VEGF antibodies) combined with cisplatin+gemcitabine was negative (5). But other phase II trials evaluating bevacizumab with cisplatin+pemetrexed were promising with PFS of 6.9, 7.9 and 9.2 months, and DCR of 40, 57 and 88%, respectively. Therefore a phase III randomized (1:1) trial (« MAPS ») recruited 448 unresectable MPM patients to test cisplatin+pemetrexed with (arm B) or without (arm A) bevacizumab (6). Arm B non-progressive patients received bevacizumab maintenance until progression or toxicity. Median OS was significantly longer in the B arm: 18.8 [95%CI: 15.9-22.6] vs. 16.1 months [14.0-17.9] in the A arm, (adj.HR= 0.76, p=0.012). Thus bevacizumab addition to pemetrexed+cisplatin provided a significantly longer survival in MPM patients with acceptable toxicity, making this triplet a new treatment paradigm for this cancer. Pro-apoptotic agents such as proteasome inhibitors (bortezomib) or histones deacetylases inhibitors (HDACi) were also assessed with discordant results. In 2[nd]/3[rd] line treatment, vorinostat (HDACi) failed to show any significant OS gain versus placebo in a large phase III trial (7). Focal adhesion kinase (FAK) is a tyrosine kinase with multiple roles in tumor growth and resistance to chemotherapy. FAK is overexpressed with increasing activity in many human cancers, associated to a low tumor expression of the Merlin molecule, a potential predictive biomarker of FAK inhibitors. An inhibition of FAK may induce tumor cells apoptosis, reduce cancer stem cells, and modulate the activity of NF-2, frequently mutated in MPM. Thus, a trial is currently assessing FAK inhibitors (VS-6063/Defactinib) as maintenance treatment after 1[rst] line chemotherapy by platinum+pemetrexed. Phase I-II trials assessed antibodies targeting mesothelin, a mesothelial cell surface molecule overexpressed in (epithelioïd) MPM, alone or combined with Listeria toxin, showing promising results (8). Other innovative techniques including gene therapy, cellular therapy or oncovirotherapy, are also currently evaluated with first promising results. But, as in melanoma or in lung cancers, checkpoint inhibitors represent presently the most exciting tool. First results with anti-CTLA4 Ab (tremelimumab) were recently published: the main goal (RR) was not achieved but several prolonged response or stable disease were observed, justifying a larger phase II trial (n=564), assessing tremelimumab versus placebo in 2[nd]/3[rd] line treatment of MPM. Early data of a phase Ib basket trial with anti-PD-1 (Pembrolizumab) in the same setting found promising RR of 28% and DCR of 76% in PD-L1 positive MPM (2015 AACR meeting). Other trials with checkpoint inhibitors are underway. To date, the place of radiotherapy is limited in MPM, mostly with palliative intent (1). Prophylactic irradiation of chest scars and drains is highly discussed. A definitive answer on this controversial indication is hoped with a current randomized UK trial. Adjuvant radiotherapy is not validated yet as well. Limitations due to technical reasons and toxicities may be answered in the future by new modalities of radiotherapy such as IMRT. Multimodal treatment of MPM patients, whatever the surgery is (i.e. extrapleural pneumonectomy (EPP) or pleurectomy/decortication (P/D)) is not recommended outside clinical trials (1). Recent trials, even the highly controversial “MARS” trial, and meta-analysis undeniably plead for stopping EPP and to continue P/D only in clinical trials to find the best multimodal treatment for potentially resecable MPM patients, fitted for surgery (9). Additional intrapleural treatments (chemotherapy, photodynamic therapy (PDT) or immunotherapy) seem needed to improve significantly the post-surgery outcome, mostly as now targeted therapies such as bevacizumab may increase median OS close to 19 months in patients less selected than surgical patients! Thus, Friedberg and al found exciting OS over 31 months in patients treated by multimodal treatment including extensive P/D and intrapleural PDT (10). In conclusion, many research studies presently assess the value of targeted therapies and biomarkers, opening new perspectives in the management of MPM. Remaining questions are how to target the best patients for each drug or technique, and how to combine the different current and future therapeutic tools in MPM. But real hopes seem close now for our patients after a long dark age. References Scherpereel A, Astoul P, Baas P, Berghmans T, Clayson H, de Vuyst P, et al. Guidelines of the European Respiratory Society and the European Society of Thoracic Surgeons for the management of malignant pleural mesothelioma. Eur Respir J 2010 ;35:479-95. van Zandwijk N, Clarke C, Henderson D, Musk AW, Fong K, Nowak A, et al. Guidelines for the diagnosis and treatment of malignant pleural mesothelioma. J Thorac Dis. 2013; 5(6): E254-E307. Vogelzang NJ, Rusthoven JJ, Symanowski J, Denham C, Kaukel E, Ruffie P, et al. Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol 2003;21:2636-44. Zucali PA, Simonelli M, Michetti G, Tiseo M, Ceresoli GL, Collovà E, et al. Second-line chemotherapy in malignant pleural mesothelioma: results of a retrospective multicenter survey. Lung Cancer 2012;75:360-7. Kindler HL, Karrison TG, Gandara DR, Lu C, Krug LM, Stevenson JP, et al. Multicenter, Double-Blind, Placebo-Controlled, Randomized Phase II Trial of Gemcitabine/Cisplatin Plus Bevacizumab or Placebo in Patients With Malignant Mesothelioma. J Clin Oncol 2012;30:2509-15. Zalcman G, Mazières J, Margery J, Greillier L, Audigier-Valette C, Moro-Sibilot D, Molinier O, Corre R, Monnet I, Gounant V, Janicot H, Gervais R, Locher C, Milleron B, Tran Q, Lebitasy MP, Morin F, Creveuil C, Parienti JJ, and Scherpereel A. Bevacizumab 15mg/kg plus cisplatin-pemetrexed (CP) triplet versus CP doublet in Malignant Pleural Mesothelioma (MPM): Results of the IFCT-GFPC-0701 MAPS randomized phase 3 trial. ASCO 2015 annual meeting (Chicago, USA) Abstract #150191. Krug LM, Kindler HL, Calvert H, Manegold C, Tsao AS, Fennell D, Öhman R, Plummer R, Eberhardt WE, Fukuoka K, Gaafar RM, Lafitte JJ, Hillerdal G, Chu Q, Buikhuisen WA, Lubiniecki GM, Sun X, Smith M, Baas P. Vorinostat in patients with advanced malignant pleural mesothelioma who have progressed on previous chemotherapy (VANTAGE-014): a phase 3, double-blind, randomised, placebo-controlled trial. Lancet Oncol. 2015; 16(4): 447-56. Hassan R, Kindler HL, Jahan T, Bazhenova L, Reck M, Thomas A, Pastan I, Parno J, O'Shannessy DJ, Fatato P, Maltzman JD, Wallin BA. Phase II clinical trial of amatuximab, a chimeric antimesothelin antibody with pemetrexed and cisplatin in advanced unresectable pleural mesothelioma. Clin Cancer Res. 2014; 20(23): 5927-36. Cao C, Tian DH, Park J, Allan J, Pataky KA, Yan TD. A A systematic review and meta-analysis of surgical treatments for malignant pleural mesothelioma. Lung Cancer. 2014; 83(2): 240-5. Friedberg JS. Radical pleurectomy and photodynamic therapy for malignant pleural mesothelioma. Ann Cardiothorac Surg. 2012; 1(4): 472-80.

Background:
From our randomized controlled phase III trial of adjuvant chemo‑immunotherapy in lung cancer patients, the preliminary results indicated significant advantage in immunotherapy arm combined with chemotherapy. We report here the final analysis and long term results with 42.8 months of median follow up time.

Methods:
Between April 2007 and July 2012, 103 postsurgical non-small cell lung cancer patients were randomly assigned to receive either chemo-immunotherapy (group A) or chemotherapy (group B). The immunotherapy consisted of the adoptive transfer of autologous activated killer T cells and dendritic cells obtained from the lung cancer patients’ own regional lymph nodes.

Results:
The 2-year overall survival rates in groups A and B were 96.0 and 64.7 %, and the 5-year rates were 74.6 and 40.9 %, respectively, and the results confirmed the statistically significant difference analyzed 2 years previously. The hazard ratio (HR) was 0.321 (95% Confidence Interval 0.164~0.631). The 2- and 5-year recurrence-free survival rates were 68.0, 41.2 and 57.2, 29.2 % in groups A and B, respectively. Those differences were also statistically significant (log-rank test p = 0.0020). The HR was 0.435 (p = 0.0027) in favor of group A. Subgroup analysis between treatment groups using cox models indicated male (HR: 0.351, 95%CI: 0.171~0.721), Adenocarcinoma (HR: 0.279, 95%CI: 0.116~0.669), stage III (HR: 0.228, 95%CI: 0.092~0.564) and those who did not received preoperative chemotherapy had lower hazard ratio compared to other groups. Immunological analysis of cell surface markers in regional lymph-nodes of immunotherapy patients indicated the ratios of CD8 vs CD4 (CD8/4) are elevated in survivors.

Conclusion:
The final results of the statistical and immunological analysis of the study confirmed the efficacy of immunotherapy in adjuvant treatment of lung cancer patients. The study indicated the advantages and limitations of cell mediated immunotherapy and a large-scale multi-institutional RCT is inevitable for the clinical application of the study.

Background:
Postoperative adjuvant chemotherapy is extensively received due to its extension of the time to recurrence and enhancement of survival rate in NSCLC. However, it has reached the plateau presently, the beneficial cases are few, and drug-resistance and over-treatment phenomena are in most of patients, hence it is necessary to seek new postoperative adjuvant chemotherapy to improve the survival rate. Angiogenesis is one premise of occurrence, development and metastasize of malignant tumors, but VEGF is one of the most important tumors in the process of neovascularization. Under normal conditions, VEGF is hardly expressed in a lot of normal tissues in vivo, while highly expressed in the tumors. Endostatin can significantly intervene the angiogenesis-promoting effect to block the nutritional supply for tumors and inhibit tumor proliferation or metastasis. We compare the curative effect of endostatin plus adjuvant chemotherapy and adjuvant chemotherapy alone in the treatment of patients with completely resected NSCLC at stage IB-IIIA.

Methods:
This is an open, multicenter, randomized (1:1) study, stratified by gender, stage and histology. Completely resected pts (stage IB to IIIA) were randomized to receive adjuvant NP plus Endostatin (Vinorelbine 25mg/m2 on d1 and d8 plus Cisplatin 75 mg/m2 on d1, and iv plus endostatin 7.5mg/m2 per day iv for consecutive 14 days, every 21 days as one cycle, 4 cycles in total) or NP regimen alone. The primary endpoint was disease-free survival (DFS). Secondary endpoints included tumor response rate, overall survival and safety.

Results:
250 pts (1:1) were included from 07/2007 to 06/2009. Two arms were well-balanced with regard to age, gender, histology, staging, and resection type. The follow-up time was 60 months. The two groups had no significant difference in the incidence of toxicity reaction. Endostatin plus NP can prolong the DFS of patients with completely resectable NSCLC at stage IIIA with high security, but with no statistical difference (19.33±3.73 m vs 17.10±9.68 m). Cases with high expression of VEGF showed a better DFS than cases with low expression in endostatin plus NP group (48.45±3.52m vs 40.18±4.54m, P < 0.05). The level of peripheral circulating endothelial progenitor cells (EPCs) in NSCLC patients was significantly higher than that in healthy volunteers. EPCs level was associated with NSCLC stage. The EPCs levels after treatment significantly decreased than that before treatment (P=0.014) in beneficiaries of NP or NP plus with endostatin. The time to progression (TTP) was longer in patients with lower levels of EPCs (<0.35%) before chemotherapy or endostatin treatment (P<0.001). However, no statistically significant difference in OS was noticed between the two arms (P = 0.962). The survival rate of endostatin plus NP group was higher for patients in stage IIIA NSCLC, but the differences did not reach statistical significance (MST 41.267 months vs 39.533 months, P = 0.760).

Conclusion:
Vascular targeted therapy could prolong the DFS of patients with complete resectable NSCLC in stage IIIA, but did not show benefits in OS for stage IB−IIIA. We shall develop new strategies to identify the patient subgroups that will be benefited or harmed by vascular targeted therapy.

Background:
In resected early stage (II-IIIA) non-small cell lung cancer (NSCLC) adjuvant chemotherapy improves overall survival but the benefit is limited and pharmacogenomics tailored treatment is a potential way to further improve outcome. A phase III multicenter randomized trial comparing adjuvant pharmacogenomics-driven chemotherapy, based on thymidylate synthase (TS) and excision-repair cross-complementing-1 (ERCC1) gene expression versus standard adjuvant chemotherapy in completely resected Stage II-IIIA NSCLC recently completed patients’ (pts) enrolment (EudraCT #: 2008-001764-36).

Methods:
The mRNA ERCC1 and TS expression by qRT-PCR was centrally assessed on paraffin-embedded, post-surgical tumor specimens in all registered pts. Immunohistochemistry (IHC) straining for ERCC1 (using 2 monoclonal antibodies, 8F1 and 4F9) and TS protein expression was also performed. Randomization was stratified by stage and smoking status. Trial was emended on February 2011 to include the 7th staging system. The primary end point of the study is overall survival; secondary end points include recurrence-free survival, therapeutic compliance, toxicity profile and comparative evaluation of ERCC1 and TS mRNA versus protein expression. Study design was already reported [Novello S et al, JTO 2013; 8 (Suppl 2) P3.12-023].

Results:
Enrolment was concluded in August 2014 and at that time all gene expression data were available. Recruitment and gene expression results were completed in August 2014. 386 pts were included in the control arm, 375 in the tailored arm and 41 were excluded as screening failures (14) or are not yet fully evaluable (27). Statistical correlations to compare treatments received, toxicity profiles and pts’ survival data in the tailored and control groups are ongoing. Further data analyses will include the correlation between biomarker ERCC1/TS mRNA and protein expression levels, as well as compare ERCC1-IHC scores with the 2 ERCC1 antibodies. The distribution of some baseline characteristics depending on the molecular profile is shown in Table 1. Figure 1



Conclusion:
This trial will provide robust evidence if a tailored therapeutic strategy based on selected gene expression profile may contribute to improve efficacy and to ameliorate toxicity of adjuvant chemotherapy in completely resected early stage NSCLC.

Abstract not provided

Background:
Postop platinum-based CT improves outcomes in completely resected NSCLC with nodal involvement (St II-IIIA) but compliance and outcomes remain limited. Analysis of expression of genes involved in DNA repair could be used to individualize optimal CT. BRCA1 is primarily involved in the repair of double strand DNA breaks and functions as a differential regulator of response to cisplatin (Cis) and antimicrotubule agents. BRCA1 defficiency can enhance Cis resistance. Loss of BRCA1 function is associated to sensitivity to DNA-damaging CT and may also be associated with resistance to spindle poisons

Methods:
Randomized phase III multicenter trial. After surgery patients (p) with St II and III NCSLC were random 1:3 to control arm (3 cycles Cis-Docetaxel) or to experimental arm with treatment assigned according BRCA1 expression levels (low levels: Cis-Gemcitabine; intermediate levels: Cis-Doc; high levels: Docetaxel alone). Stratifification factors: N1 vs N2; age < or > 65 y; non-Squamous vs Squamous (Sq) histology; lobectomy vs pneumonectomy). Planned PORT in N2. Primary end-point OS. Secondary end-points DFS, toxicity profile (CTCAE v 3.0) /compliance, recurrence pattern. Statistical hypothesis: increase 20% 5y survival rate control group (45%)

Results:
From June/2007 to May/2013, a total of 591 p were screened and 500 of them were randomized in the study, 108 in control arm, 392 in experimental arm. In experimental arm 110 p received Cis-Gem, 127 Cis-Doc and 110 Doc alone. There were no significant differences between arm for known prognostic factors: Median age 64 y; 79% males, 21% females; 43% Sq, 49% Adenoca, 8% others; 57% former smokers, 32% current smokers, 11% never smokers; pneumonectomy 26%; N1 58%, N2 48%. Median tumor size 4.4 cm (0.8-15.5 cm). Median mRNA BRCA1 levels 15.78 (0.73-132). Mean BRCA1 levels 6.95 in Adenoca vs 20.29 in Sq (p<0.001). P with Sq histology showed a longer DFS (HR 0.73; p=0.05) but without differences in OR (HR 1) Median follow-up 28 months (0-79 m), with a cut-off of March 15[th] 2015, median survival has not reached both arms and no significant differences have been seen for OS with hazard ratio (HR) 0.866 (p=0.45) or DFS with HR 1. In experimental group HR for OS was 0.842 (NS) comparing low with high-BRCA1 levels. In p with high-BRCA1 levels control treatment (Cis-Doc) was superior to experimental (Doc) with HR 1.24 (NS).In non-Sq histology experimental treatment was superior to control with HR 0.75. For p receiving all planned treatment HR is 0.63 with p = 0.043 compared with p not able to complete treatment.

Conclusion:
Overall survival data are still immature because median survival is not reached with a median f-u 28 m for this N+ population. At this time analysis BRCA1 based adjuvant CT does not improve overall OS. In p with high BRCA1 levels Doc alone is inferior to Cis-Doc. BRCA-1 levels are higher in Sq and in non-Sq histology a trend to better survival in experimental arm was found. Full dose of planned treatment confers a survival advantage, however, longer follow-up is still warranted.

Background:
Non-small cell carcinoma is the most common type of lung cancer. A few recent prospective trials have shown that the addition of adjuvant chemotherapy after operable stage II non-small cell carcinoma patients is associated with an improved survival[1,2]. Adjuvant chemotherapy is effective but underutilized. Our aim was to analyze the practice patterns for adjuvant chemotherapy use in stage II non-small cell lung carcinoma using the National Cancer Database (NCDB).

Methods:
We selected a historical cohort of patients diagnosed with stage II non-small cell carcinoma between 2000 and 2012. This cohort is selected from the National Cancer Database (NCDB). NCDB is a national oncology outcomes database that includes 70% of new cancer diagnoses from more than 1,500 Commission on Cancer accredited programs in the United States and Puerto Rico. We studied this cohort to find out the difference in patterns of adjuvant chemotherapy use among stage II non-small cell carcinoma patients based on demographic and insurance characteristics. Two-tailed chi-square test was used as the test of significance with a p-value < 0.05 being considered significant. All values are given in percentages.

Results:
The total number of patients diagnosed with stage II lung cancer between the years 2000 to 2012 was analyzed (n=112430). We observed an increase in the percentage of patients receiving adjuvant chemotherapy from 9% to 18% from the year 2003 to 2004. The factors associated with increased adjuvant chemotherapy use were private insurance, ages 30 to 69, White/Hispanic race, higher education, higher income groups and female gender (p<0.0001)(Table 1). Table 1. Adjuvant Chemotherapy use in stage II Non-Small cell lung cancer.

VARIABLES INCLUDED	PERCENTAGE OF PATIENTS RECEIVING ADJUVANT CHEMOTHERAPY
Diagnosis year
2000-2003	6
2003-2012	23
Age
<30	7
30-49	22
50-69	25
69-89	9
Insurance status
Private/managed	26
Medicaid	19
Medicare	15
Uninsured	17
Sex
Male	17
Female	19
Annual Household Income (USD: 2012 census)
<36000	15
36000-43999	17
44000-52999	18
53000-68999	19
>69000	21
Educational Status (% Patients without HS degree)
>23%	15
15-22.9%	17
11- 14.9%	18
6-10.9%	19
<6%	21
Race
White	18
Black	17
Hispanic	19
Charlson comorbidity score
None	21
One	23
>= 2	18

Conclusion:
This cohort illustrates the increase in adjuvant chemotherapy use from 2000 to 2012 with significant increase during the year of 2004. Access to adjuvant chemotherapy is dependent on various demographic factors. The treatment and outcomes of non-small cell carcinoma is dependent on the type of treatment used, which itself is affected by the population demographics and health system factors. These variables should be studied in detail to find out the cause for the underutilization of adjuvant chemotherapy despite the evidence of survival benefit in patients with stage II non-small cell carcinoma of lung.

Background:
Adjuvant cisplatinum-based chemotherapy is recommended in patients with stages IB (≥ 4 cm), IIA, IIB, and IIIA of non small-cell lung cancer (NSCLC) after radical resection. Vinorelbine with cisplatin are preferable drugs in this indication, but the side effects of this treatment were not negligible in big adjuvant trials. Carboplatin with vinorelbine given intravenously switched to orally were applied in a multicentre prospective study SWITCH I to give better comfort, higher tolerability and comparable effectivenes as standard adjuvant chemotherapy. The recruitment period started in January 12[th], 2005 and lasted till September 5[th], 2008.

Methods:
Consecutive chemo-naive patients were recruited after complete resection of NSCLC stages IB, IIA, IIB and IIIA. Chemotherapy was applied from 2 to 6 weeks after complete resection. Four cycles of 21 days regimens were planned, Patients received carboplatin AUC 5 on the day 1,vinorelbine 25 mg/m[2] intravenously on the day 1 switched to 60 mg/m[2] orally on the day 8. Follow-up visits with physical evaluation, chest CT and laboratory tests have been realized every 3 months for 2 years and then every 6 months. Tolerability, side effects, relative dose intensity and survival were evaluated.

Results:
Seventy four patients (pts) were recruited to the SWITCH I study: 53 men and 21 women, 45 smokers, 23 ex- smokers and 6 non-smokers. Median age was 64 y (48-75 y). Tumor was squamous in 46, adenocarcinoma in 22, giant cell in 4 and NOS in 2 pts. Stage of the tumor was IB in19, IIA in 8, IIB in 22 and IIIA in 25. Mean number of applied cycles was 3.77 four planned cycles finished 82,4% patients. The most frequent hematological toxicities grade 3/4 were neutropenia (25.7 %), leukopenia (16.2 %), anemia (8.1 %) and trombocytopenia (2.7 %). Non-hematological toxicities were alopecia (12.2 %), nausea (4.1%), nefrotoxicity (1.4%) and diarrhoea (1,4%). Median of follow up was 4.73 y. Median of disease specific survival was 7.63 y (95% CI: 4.57 to NR), median of overall survival (MOS) was 5.9 y (95% CI, 3.7 to, NR) and median of disease free survival (DFS) 4.43 y . Three-year survival of 70.3% and five-year survival of 56,2% were reached.

Conclusion:
Adjuvant chemotherapy with carboplatinum and vinorelbine given intravenously on the day 1 and orally on the day 8 in 21 day regimen appears to be a comfortable and tolerable therapy in radically resected NSCLC. It provides higher dose intensity and more of acomplished treatments compared to big adjuvant trials and LACE meta-analysis, in which these parametres varied between 50 % to 76 % only. Survival results are comparable to LACE (3-year survival 70,3% vs 64.3%), 5-year survival 56,2% vs 55.1%) and MOS 5.9 vs 5.15 y). Supported by Grant Grant IGA MZ ČR NT/13569 of the Czech Ministry of Health.

Abstract not provided

Abstract:
With the dramatic clinical benefit that can be observed using tyrosine kinase inhibitors (TKIs) of the epidermal growth factor receptor (EGFR) in patients with non-small cell lung cancer (NSCLC) harbouring activating mutations in EGFR, there has understandably been a focus on the use of these agents in this subset of NSCLC. However, EGFR mutation positive NSCLC represents only approximately 10 – 15 % of all non-squamous NSCLC in non-East Asian patients, and a substantial proportion of East Asian patients do not harbour this mutation. Thus, world-wide, the vast majority of those with NSCLC are so-called “wild-type” for EGFR. For these patients, it is clear from randomized clinical trials that the treatment of choice in the first-line metastatic setting is platinum-doublet chemotherapy. Increasing data suggest that chemotherapy may be preferred in the second-line setting. Is there any role for the use of EGFR TKIs in the wild-type population? Randomized data in which an EGFR TKI is compared to placebo in both the maintenance and refractory settings suggest that there may be. NCIC Clinical Trials Group study BR21 [1] which randomized 731 unselected patients to either erlotinib or matching placebo, was designed and conducted prior to the discovery of activating mutations. Patients had received 1 (50 %) or > 2 (50 %) lines of prior therapy; > 90 % had received a platinum-doublet. An improvement in median survival (6.7 versus 4.7 months [HR 0.70, p < 0.001]) was also associated with a quality of life benefit. This benefit was consistent across subgroups, including in the 50 % of patients with non-adenocarcinoma histology. In a separate analysis of ever-smokers with squamous histology, patients highly unlikely to harbour an EGFR mutation, the magnitude of survival benefit was the same as in the overall study population (median 5.6 versus 3.5 months [HR 0.66, p=0.009])[2]. The SATURN trial [3] randomized 889 patients who had not progressed after 4 cycles of platinum-doublet chemotherapy to either erlotinib or placebo. While of debatable clinical relevance, there was a statistically significant one month prolongation of median survival with the use of erlotinib (HR 0.81, p=0.009). A similar effect was observed in the 44 % of patients with known EGFR wild-type status (HR 0.77, p=0.02). In a pre-planned subgroup analysis [4], a greater magnitude of benefit was observed in those patients whose best response to induction chemotherapy was stable disease (median overall survival 11.9 versus 9.6 months [HR 0.72, p=0.002]), with a similar effect noted in those patients with squamous histology (HR 0.67, p=0.01), and those known to be EGFR wild-type (HR 0.65, p=0.004). Maintenance erlotinib has been shown to not negatively impact quality of life [5], and when used in those with stable disease, to be cost effective [6]. Meta-analyses of placebo-controlled trials of EGFR TKIs in the maintenance setting have confirmed a modest progression-free survival benefit in squamous [7] and known wild-type [8] patients. Multiple trials have compared an EGFR TKI to either docetaxel or pemetrexed in the second-line setting. The TAILOR trial [9], the only trial to prospectively determine and enrol only wild-type patients, showed a clear PFS advantage to docetaxel, and a trend towards improved overall survival. However several other trials that enrolled patients who were unselected with regard to EGFR status had a substantial number of wild type patients, and none of these trials demonstrated a difference in overall survival in wild-type patients between an EGFR TKI and chemotherapy. While these were retrospective analyses on only a subset of enrolled patients with available tissue, wild-type patient numbers in many trials approached (and in one exceeded) the number of patients enrolled to TAILOR. Further, unlike other trials, TAILOR prohibited crossover, which may have impacted survival results, particularly for patients with squamous carcinoma in the erlotinib arm. Taken together these trials suggest that a treatment strategy that includes both chemotherapy and an EGFR TKI sequentially, irrespective of order, will lead to a similar length of survival provided patients receive both lines of therapy. In platinum-pretreated patients who are fit it is likely preferred to use chemotherapy and then at progression move on to an EGFR TKI, as the chance of patients receiving both treatments is higher. Additional data to suggest that EGFR TKIs may have activity in wild-type patients comes from several small, randomized phase II trials comparing second-line chemotherapy with the same chemotherapy with intercalated EGFR TKIs. These studies have shown prolonged PFS in patients treated with the combination. What these trials demonstrate is that EGFR TKIs appear to have a modest treatment effect in EGFR wild-type patients. In these days of targeted therapies leading to substantial treatment effects in a variety of tumours with oncogenic drivers, is this magnitude of benefit sufficient? In lung cancer, many other treatments have been adopted for a similar magnitude of benefit. Although objective response rates to EGFR TKIs are low in wild-type patients, they are also low to standard cytotoxic chemotherapies beyond first-line, and it seems possible that there is a larger proportion of patients with stabilization of disease and / or slowing of progression that is clinically relevant. Not all oncologists or patients will feel that a trial is warranted, but an EGFR TKI is a reasonable choice as last-line therapy when the option is no further treatment, or as maintenance treatment in patients with squamous histology following a best response of stable disease to induction platinum-based chemotherapy. EGFR “wild-type” is a heterogeneous, not homogeneous, population, and as with any therapy, only a subgroup of patients will benefit from treatment. However a consistent reproducible biomarker for benefit in the wild-type subgroup has not yet been discovered. EGFR protein expression, gene copy number, Kras status and serum proteomics have all been evaluated with at times conflicting results, due to limited samples and the retrospective nature of the analyses. The development of rash may be a pharmacodynamic predictor of greater efficacy [10]. Additional work is required to determine which wild-type patients may derive benefit from an EGFR TKI, to avoid needless toxicity and improve cost-effectiveness. References 1. Shepherd et al. N Engl J Med 353: 123-132, 2005 2. Clark et al. Clin Lung Cancer 7:389-394, 2006 3. Cappuzzo et al. Lancet Oncol 11:521-529, 2010 4. Coudert et al. Ann Oncol 23:388-394, 2012 5. Juhasz et al. Eur J Cancer 49:1205-1215, 2013 6. Walleser et al. Clinicoeconomics Outcomes Res 4:269-275, 2012 7. Ameratunga et al. Asia-Pacific J Clin Oncol. 10:273-278, 2014 8. Vale et al. Clin Lung Cancer 16:173-182, 2015 9. Garassino et al. Lancet Oncol 14:981-988, 2013 10. Ding et al. Contemp Clin Trials 29:527-536, 2008

Abstract not provided

Abstract not provided

Abstract:
Great advantages of next generation sequencing have been published so far, and many new genetic alterations were found with whole genome sequencing. Targeted sequencing using next generation sequencing technique can analyze FFPE small biopsy specimens, but may be equivalent or less than the current selected testing, such as EGFR and ALK testing. Although the targeted sequencing can actually analyze multiple genes, most diagnostic panels include the genes that are frequently altered in cancer generally, thus practically useful genes are limited in terms of lung cancer, such as EGFR, ALK, ROS1, and RET. In contrast, whole exome sequencing is potentially useful, as it can comprehensively examine mRNA expression on tumor cells. In general, mRNA in clinical samples well represents tumor genetic status even with significant dilution with the normal cells, which are less active in transcription. However, it is difficult to perserve high quality RNA with clinical samples, and it is unclear that the whole exome sequencing is constantly clinically applicable for small biopsy specimens. Furthremore, there are some cases that show discrepant results between DNA and RNA based assays. As EGFR transcript is suppressed in SCLC, EGFR mutation cannot be detected with the exome sequencing in SCLC transformed as a resistant mechanism to EGFR-TKI treatment. On the other hand, current selected testing for EGFR and ALK has been confirmed with clinical trials and are adjusted to clinical demands, e.g., short turnaround time and high sensitivity.