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Abstract not provided

Abstract:
Substantial progress has been made toward understanding the tumor biology of non-small cell lung cancer (NSCLC) during the last few years, and major discoveries in the molecular pathogenesis of lung cancer have led to successful applications of targeted therapeutic strategies. One representative of these successes is the subset of lung cancer patients with epidermal growth factor receptor (EGFR) mutations who have shown improved clinical response to tyrosine kinase inhibitors (TKIs).1 The prevalence of EGFR mutation is higher in the East Asian population than in Western populations. Asian NSCLC patients were reported to show a higher response rate and a longer survival to EGFR tyrosine kinase inhibitors (EGFR TKIs) as well as traditional chemotherapy. Therefore, genetic testing prior to treatment is becoming increasingly important and considered essential to select appropriate treatment strategies for NSCLC patients, especially in Asia. The exact mechanisms underlying these differences are not clear. However, approximately 45% of patients with NSCLC in the US are women whereas only 25% to 30% of patients with lung cancer are women in Eastern Asia. In addition, In the US, approximately 10% of patients with lung cancer are neversmokers (<100 lifetime cigarettes). In Asia, more than 30% of patients with lung cancer are neversmokers.2 Some international studies and global surveys have been conducted during the past few years to establish the current status of EGFR mutation testing and to establish the standard testing protocols.3-5EGFR mutation test is not yet available in many South East Asian medical institutions. In the medical facilities which do perform EGFR mutation test, the overall testing rate of newly diagnosed NSCLC is 31.8%, varying from 18.3% - 64.8%, the highest EGFR mutation testing rate was observed in Japan and the lowest in South East Asian Countries. Sex, smoking status and histological subtype are the main determining factors for EGFR mutation tests.5 However, the College of American Pathologists (CAP), International Association for the Study of Lung Cancer (IASLC), and Association for Molecular Pathology (AMP) guidelines recommended that patients with lung adenocarcinoma should not be excluded from testing on the basis of clinical characteristics that include ethnicity, smoking history, and sex. Factors associated with EGFR mutation status were country, sex, ethnicity, smoking status, smoking pack-years, disease stage and histology type. 4 In most countries, EGFR mutation rate and clinical pathologic factors are in line with previous reports in Asia, and the variations of EGFR mutation prevalence between countries are due to the selection of the tested populations. The majority of Asian institutions use PCR-based DNA direct sequencing methods to detect EGFR mutation. Other predictive biomarkers for EGFR TKI therapy are also available, including EGFR gene copy number, single-nucleotide polymorphisms of the EGFR gene, EGFR protein expression. 3 Next Generation Sequencing (NGS) and multiplex assays have made feasible the widespread adoption of molecular diagnostics for clinical use. According to our survey, the NGS is not available in clinics in most Asian medical institutions, and can be used for diagnosis of rare diseases and/or research. In China, sequencing based methods are the most commonly used, followed by the amplification mutation refractory system (ARMS).6 In Korea, the PNA clamp method is the most commonly used testing method, followed by direct sequencing methods.7 PCR-INVADER is the most commonly used test in Japan, followed by PNA-LNA PCR clamp.5 The materials for EGFR mutation analysis are usually from diagnostic samples, thus in most countries, small biopsy and cytology specimens are the most commonly used, followed by surgically resected tissues. Cytological specimens including smear slides and/or cell blocks have been shown to be suitable for EGFR mutation test.3, 5At present, the potential barriers to EGFR mutation testing in Asia include the cost, the number of laboratories capable of performing the test, communications between the clinicians and pathologists, and the predefined criteria for the type of patients who should be tested.3 The cost for EGFR mutation tests vary from U.S.$170 to 500. In most countries the costs are funded by the patients, but in a few countries are funded by pharmaceutical companies, while the insurance does not cover the cost of molecular testing in most Asian countries. Currently, National Comprehensive Cancer Network (NCCN)'s guidelines recommend EGFR-TKI as the first line treatment for patients with NSCLC harboring EGFR sensitizing mutations. Randomized first-line trials have demonstrated consistent improvement in tumor response rate and progression-free survival, but failed to prove overall survival benefit. These finding make TKI the second-line or third-line treatment options for NSCLC. Current data on treatment outcomes of second- and/or third-line EGFR therapy are still inconsistent.8 Patients treated with EGFR-TKI inevitably experience ac¬quired resistance by various molecular mechanisms. Many clinical trials are ongoing to explore the novel agents and strategies for better response and overcoming TKI resistance. In our questionnaire-based survey, four of seven Asia countries have ongoing clinical trials. The clinical trials include the first line and second line and maintance therapy using first generation and new generation EGFR TKIs. In 2013, more than 1000 studies with Chinese trials were registered on ClinicalTrials.gov and some trials used a local pharmaceutical company TKI, icotinib. Phase II studies of crizotinib in East Asian patients with ROS1-positive NSCLC are onging in China, Japan, South Korea and Taiwan. Anaplastic lymphoma kinase (ALK) rearrangement accounts for about 2–13% of unselected Asian patients with NSCLC, and occurs predominantly in younger individuals with adenocarcinoma who never smoked or light smokers (< 40 pack years). There is no strong evidence to suggest an ethnic difference of translocations among patients with NSCLC. The CAP/IASLC/AMP guideline recommended ALK FISH assay using FDA-approved specific companion test (Vysis ALK Break-Apart FISH Probe Kit, Abbott Molecular, Des Plaines, IL) for selecting patients for ALK tyrosine kinase inhibitor therapy. ALK immunohistochemistry (IHC) may be considered as a screening methodology to select specimens for ALK FISH testing. The ALK rearrangements have been shown to be 4.3% in men and 7.5% in women in Asian NSCLC by meta-analysis. Most Asia countries use FISH to detect ALK rearrangement, in Japan, reverse transcription–PCR (RT–PCR) is commonly used. In China, the Chinese Food and Drug Administration (CFDA) had approved VENTANA ALK IHC assay to aid the identification of patients for crizotinib treatment. The price of FISH detection range from US$415-800, and IHC range from US$14-220, most of which is funded by the patients.9 Recently, two additional oncogenes, RET and ROS1, were added to the list of driver oncogenes that are targetable with existing TKIs, and several clinical trials investigating the efficacy of such TKIs in Asia have been conducted. FISH and IHC are suitable for the diagnosis of ROS1 fusion, but some studies showed IHC is not suitable for the diagnosis of RET fusion.10 Over the past decades, we have witnessed rapid advances in molecular and cellular biology of lung cancer biology, and new data are upcoming which should facilitate personalized biomarker-based therapy in lung cancer, including characterization of driver mutations, genomic abnormalities and epigenetic changes. Individual centers should develop a multidisciplinary approach to integrate a molecular testing algorithm. Next generation sequencing should be able to resolve much of the complexity of molecular testing, especially in situations where there is only a small amount of tissue available. REFERENCES 1. Janku F, Stewart DJ, Kurzrock R. Targeted therapy in non-small-cell lung cancer--is it becoming a reality? Nat Rev Clin Oncol 2010;7:401-414. 2. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009;361:947-957. 3. Salto-Tellez M, Tsao MS, Shih JY, et al. Clinical and testing protocols for the analysis of epidermal growth factor receptor mutations in East Asian patients with non-small cell lung cancer: a combined clinical-molecular pathological approach. J Thorac Oncol 2011;6:1663-1669. 4. Shi Y, Au JS, Thongprasert S, et al. A prospective, molecular epidemiology study of EGFR mutations in Asian patients with advanced non-small-cell lung cancer of adenocarcinoma histology (PIONEER). J Thorac Oncol 2014;9:154-162. 5. Yatabe Y, Kerr KM, Utomo A, et al. EGFR mutation testing practices within the Asia Pacific region: results of a multicenter diagnostic survey. J Thorac Oncol 2015;10:438-445. 6. Wang S, Wang Z. EGFR mutations in patients with non-small cell lung cancer from mainland China and their relationships with clinicopathological features: a meta-analysis. Int J Clin Exp Med 2014;7:1967-1978. 7. Shim HS, Chung JH, Kim L, et al. Guideline Recommendations for EGFR Mutation Testing in Lung Cancer: Proposal of the Korean Cardiopulmonary Pathology Study Group. Korean J Pathol 2013;47:100-106. 8. Sculier JP, Berghmans T, Meert AP. Advances in target therapy in lung cancer. Eur Respir Rev 2015;24:23-29. 9. Fan L, Feng Y, Wan H, et al. Clinicopathological and demographical characteristics of non-small cell lung cancer patients with ALK rearrangements: a systematic review and meta-analysis. PLoS One 2014;9:e100866. 10. Kohno T, Nakaoku T, Tsuta K, et al. Beyond ALK-RET, ROS1 and other oncogene fusions in lung cancer. Transl Lung Cancer Res 2015;4:156-164.

Abstract:
Introduction Identification of molecular targets is now essential for the diagnosis, classification, selection and treatment monitoring of an increasing number of cancers. The analysis of these biomarkers must therefore be accessible to all patients, regardless of the healthcare facility where they are treated. Despite a decrease, lung cancer remains the leading cause of cancer-related deaths worldwide, often detected at an advanced stage with association with a poor prognosis. Recently, genetic alterations involved in adenocarcinomas have been discovered; some of these tumors harbor a driver mutation involving EGFR, KRAS, HER2, BRAF or PI3KCA gene or a rearrangement (ALK, ROS1, RET genes). Nearly 40% of NSCLC harbor one of those genetic abnormalities and several targeted therapies have been approved in USA and in Europe, such as erlotinib and gefitinib and crizotinib for NSCLC with EGFR mutation or ALK translocation, respectively. As many other targeted therapies are ongoing in lung cancer, molecular testing becomes a huge challenge worldwide, raising both financial and organizational issues.The French INCa molecular testing network: In 2006, the French National Cancer Institute (INCa) and the French Health Ministry have created a national network of 28 regional genetics platforms throughout the French territory, in order to offer molecular tests to all patients regardless of the institution where they are treated, i.e. university hospitals, cancer centers, hospital centers or private institutions, to enable that each innovative test could be rapidly implemented after new targeted therapies become available, and to guarantee the quality of the tests. The 28 regional platforms include a pathology laboratory in charge with the samples monitoring, and several laboratories with complementary expertise in molecular testing of hematological malignancies and solid tumors; as the tests are free of charge for the patients and the institutions, initial funds were given to all INCa genetics platforms to buy equipment (€4.7 million) and to hire non-medical personnel. The majority of these were technicians (58.32 FTE) and engineers (20.5 FTE); in addition, genetics platforms are financed on a basis of quarterly and annual activity reports to adjust budget and allocation. This initial funding was fol­lowed by the allocation of €4 million in annual funding for the centres and staff from the French Ministry of Health. The data sent in those reports are the number of tests performed each year, the number of patients undergoing tests, the percentage of patients found to have a molecular abnormality, the percentage of non-contributive results and the origin of requests. The INCa also set up a quality-assurance program with mandatory external quality evaluations, and publishes guidelines available on the Inca website. The markers studied are predictive markers that determine access to targeted therapy, markers guiding the diagnostic process, markers that contribute to establishing a diagnosis, prognostic markers guiding patient treatment strategy, and markers allowing the monitoring of residual diseases. Molecular genetics platforms do not have to perform all molecular tests: they must ensure that patients in their region have access to these tests via a referral platform. Tests concerning a large number of patients are carried out by all or almost all platforms (BCR-ABL quantification, KRAS and EGFR mutations, JAK2 mutations, MSI tests). For tests concerning a small number of patients, some platforms perform regional or national referral activity (ABL mutation screening in CML, cKIT and PDGFRA mutations in GIST, NMYC amplification in neuroblastomas, chromosomal abnormalities in sarcomas). Whereas the initial program included only EGFR and KRAS mutations’ detection in lung cancers and KRAS and BRAF mutations in colorectal cancers, a new program for detection of emerging biomarkers was set up in 2011; for lung non-squamous cell carcinoma patients at advanced stages those biomarkers were EGFR, K-RAS, HER2, BRAF and PI3KCA mutations and ALK translocations, and BRAF and KIT mutations in metastatic melanomas. In 2008, 1,269 EGFR activating mutation tests were performed, versus 2,667 in 2009, and 21,995 and 8,696 regarding EGFR mutation and ALK rearrangement, respectively in 2012. In 2013, 23,336 lung cancer samples were tested for EGFR mutations (10% were mutated), 18,861 for ALK rearrangement (3.5% rearranged), 22,9858 for KRAS mutations (27% mutated), 20,100 for BRAF mutation (2% mutated), 17,843 for HER2 mutation (0.7% mutated) and 17,375 for PI3KCA mutations (2.4% mutated).The German Network Genomic Medicine (NGM) Lung Cancer: The NGM is a health care provider network offering centralized high-quality next generation sequencing -based multiplex genotyping for lung cancer patients. Since NGS based genotyping is not reimbursed in Germany, the AOK Rheinland/Hamburg, one of the largest German public health insurances has contracted with the NGM for reimbursement of NGS-based multiplex genotyping of lung cancer in April 2014. In 2014, 4,500 lung cancer patients were gentotyped, representing nearly 10% of stage IV NSCLC patients in Germany.The Cancer Research UK (CRUK) Stratified medicine programme 1 and 2: There is no to date any national policy in UK for molecular testing for lung cancer patients, and a great variation exists regarding providers, funding and access; most of the time, the decision of referring for molecular testing depends on the clinician in reference to NICE clinical guidance. Nearly 7,300 UK patients were tested for EGFR mutations in 2010-2011 and depending on the units, ALK testing is performed either by IHC and /or FISH. In 2011, the Cancer Research UK (CRUK) Stratified Medicine Programme 1 and 2 has set-up a collaborative network of 26 hospitals and 8 CRUK Experimental Cancer Medicine Centres to provide genetic testing in lung, bowel, breast cancers and melanoma. The next plan for the NHS of England is to increase testing activity, to equitable access, to improve the quality assurance procedures and the cost values.References: 1. Collection Reports and summaries, collective volume edited by INCa, Boulogne-Billancourt, sept 2010 http://www.e-cancer.fr/Expertises-et-publications/Catalogue-des-publications/Molecular-genetic-testing-for-equal-access-to-targeted-therapies-in-France-in-2011 http://www.e-cancer.fr/Expertises-et-publications/Catalogue-des-publications/The-French-national-network-of-28-hospital-molecular-genetics-platforms-summary-of-the-activity-in-2009 http://www.e-cancer.fr/Expertises-et-publications/Catalogue-des-publications/Molecular-genetic-tests-for-access-to-targeted-therapies-in-France-in-2012 2. Nowak, F. et al. Tumour molecular profiling for deciding therapy—the French initiative. Nat. Rev. Clin. Oncol. 9, 479–486 (2012) 3. Nowak, F. et al. Europe Does It Better: Molecular Testing across a National Health Care System—The French Example; 2013 ASCO EDUCATIONAL BOOK | asco.org/edbook 4. Kostenko, A. et al. The network genomic medicine cost reimbursement model for implementation of comprehensive lung cancer genotyping in clinical routine. J Clin Oncol 33, 2015. ASCO Abstract e12556 5. CR UK Stratified Medicine Programme 1 and 2. http://www.cancerresearchuk.org/funding-for-researchers/how-we-deliver-research/our-research-partnerships/stratified-medicine-programme

Abstract:
This presentation comprises data from Central and South America, as well as Mexico since all of us, as Latin American countries share similar characteristics. The countries included in this group are considered “developing countries” and are characterized by their racial, geographic, cultural, political and economic heterogeneity. In order to obtain data about the current situation in the region, I have sent a survey to the institutions that perform molecular tests in these countries for which I could get contact information. The survey has been sent to 34 laboratories in 12 countries. I tried to be as inclusive as possible, although in some cases it was rather difficult to get adequate contact information. My apologies to those who did not received the survey and would have liked to participate. Nineteen laboratories answered the survey ¹. Although not updated, I also added information from 5 other laboratories presented at LALCA 2014². Despite these results do not represent every country nor all molecular laboratories in the region, many common factors can be identified which allow for a relatively accurate analysis. The results show: Molecular tests are run by a small group of laboratories concentrated in the main cities of these countries. Most frequently, molecular testing is financed by pharmaceutical companies or private health care programs; however, in some cases the government, through the public healthcare system, supports the cost of the tests, and occasionally the patient pays for it. The pharmaceutical companies centralize molecular testing in a few laboratories in each country. Although frequency is generally low, some specimens are analyzed abroad, mainly in the USA. Some regional laboratories perform the tests for those countries that do not have adequate technology for molecular testing. The general opinion was that sending specimens to molecular center did not pose major complications, except in big countries where the geographic distance tends to delay the transport. Pharmaceutical companies provide the logistic structure to aid in the transfer of specimens, thus, accelerating the process. Specimen rejection rate can be divided into two groups: insufficient tissue or inadequate specimen quality by poor tissue preservation. The average rejection rate was 5 to 22%, more frequently around 15%. As for quantity, some institutions improved the amount of tissue obtained and specimen handling over time. When consulted on the possibility to perform molecular tests for treatment and/or research, the answer were: 2 laboratories only make test for research, 10 only run them for treatment purposes and 9 perform tests both for treatment and research. In most of the countries research is more frequently economically supported by the government than by other sources. When oncologists participate in clinical trials the tests are usually run abroad, mainly in the USA. Table 1 shows the available test platforms in the laboratories that participated in the survey and their access to quality control (QC) programs. For sequencing, all the laboratories began with Sanger sequencing, but many of them have changed to PCR-allele specific real-time platform. Some countries are introducing NGS. Most countries do not have local regulations for quality control of molecular tests. A half of the laboratories included in the survey have a kind of international QC, represented by participation in CAP or European QC programs or sending material to reference laboratories for interobserver concordance of results. Table1

Country	Argentina	Brazil	Chile	Colombia	Costa Rica	Ecuador	Mexico	Peru	Uruguay
Laboratories	6	2/2*	2/1*	2/1*	1	1*	2	1	2
EGFR	6	3	3	3	1	1	2	1	2
ALK	6	4	3	3	1	1	2		1
Other tests	5	3	2	3		1	2	1	2
Sanger Seq.	5	2	1	2					2
PCR allele specific-real time	4	2	2	2	1	1	2	1	1
NGS	3	1							1
FISH	4	3	3	2	1	1	2		1
IHQ	5	2	1	2		1	1		1
External QC	3	1	1	1	1	1			1

* LALCA surveys information not updated Information on the total tests performed in the region is still incomplete and the number varies from country to country. In spite of this, the main reference laboratories are included in the survey and the data obtained reveals an insufficient number of tests related to the frequency of advanced lung cancer cases in the region. I did not include in this survey the test results but published Latin American data³¯⁴¯⁵ shown regional/country variability in the frequency of EGFR mutation and sometimes in ALK fusion test, probably related to genetic variability in the Latin American population. What are the challenges of molecular testing in Latin America? One of them is the quality and quantity of tissue available for molecular tests. In the region we still struggle against badly-fixed or inadequately processed specimens. In this field, probably, there is much education and interdisciplinary work to do yet. Reimbursement is another challenge. In most of the countries, pharmaceutical companies have financed so far the cost of molecular tests, but if this were not the case in the future when the need for many other tests arises, health care systems will have to bear testing and treatment costs. A careful evaluation will be required in each country to organize the most balanced use of the available resources. A particularly important challenge for the region is molecular testing quality certification. The access to international quality control programs is very expensive for the majority of the regional laboratories but quality control must be ensured. A group of us is trying to organize a stratified system that allows for a more affordable program to all laboratories. It is still a project in development. 1-Survey participants: Argentina: Esteban Mocetti: Hospital Italiano. Buenos Aires. Marina Gutierrez: Laboratorio Stamboulian. Buenos Aires. Erica Rojas Bilbao:Hospital Roffo. Buenos Aires. Guillermo Bramuglia: Argenomics. Fundacion Investigar. Buenos Aires. Valeria Denninghoff: Instituto CEMIC. Buenos Aires. Jorge Palazzi: IICT Labs. Rosario. Brasil: Fernando Soarez. Isabela Werneck da Cunha: AC Camargo Cancer Center. Sao Pablo. Fabio Tabora:Argos Lab / Messejana Hospital. Fortaleza. Chile: Cristina Fernández Ferradás:. Instituto Nacional del Tórax. Santiago de Chile. Antonio Piottante Becker: Clínica Las Condes. Santiago de Chile. Colombia: Andres Felipe Cardona. July Rodriguez: Foundation for Clinical and Applied Cancer Research Bogotá. Ruby E. Ríos Quintana-Roberto Jaramillo: Unidad de Diagnostico Hemto-Oncologico. Cali.Costa Rica: Luis Corrales Rodriguez: Centro de Investigación y Manejo del Cancer.(CIMCA y CCSS). San José. Mexico: Graciela Cruz Rico: Instituto Nacional de Cancerología. Distrito Federal. Erica Sagrario Peña Mirabal: Instituto de Enfermedades Respiratorias. Distrito Federal. Perú: Juan Carlos Gomez de La Torre Petrell: Laboratorios ROE. Lima. Uruguay: Alejandra Torres: Laboratorio Genia. Montevideo.Gonzalo Manrique, María Noel Zubillaga. Asociación Española. Montevideo. 2. LALCA 2014 surveys, not updated: Cintya Sternberg. INCA. Rio de Janeiro.Brasil, Vinicius Duval Da Silva Pontificia Universidade Catolica do Rio Grande do Sul. Brasil. Yumay Pires. Clinica Alemana. Santiago de Chile. Ana Margarita Baldión Elorza. Hospital Universitario Fundación Santafé de Bogota. Colombia. Nicolas Vivar Diaz. Hospital Carlos Andrade Marin. Quito Ecuador 3: Arrieta O, Cardona A, Martin C et al. Updated Frequency of EGFR and KRAS mutations in NSCLC in Latin America. The Latin-America Consortium for the Investigation of Lung Cancer (CLICaP) JTO 2015;10: 838-843 4: Bacchi C. et al. EGFR and KRAS mutations in Brazilian lung cancer patients. CLINICS 2012;67 (5):419-424 5: De Melo A et al. Mutational Profile and new IASLC/ATS/ERS classification provide additional prognostic information about lung ADC. A study of 125 patients from Brazil. Oncology 2015; Apr 1.(Epub ahead of print)

Abstract:
While genomic studies of lung adenocarcinomas over the past decade have enabled substantial improvements in treatment and patient outcomes, advances in squamous cell carcinomas of the lung have been far more modest. At present there are no targeted agents approved for the treatment of squamous cell lung cancers and there has been limited success in the use of targeted kinase inhibition in this disease. Here, I will review the results of genomic studies of squamous cell carcinomas and highlight the molecular features which drive these cancers and make them distinct from lung adenocarcinomas. I will discuss specific subtypes of squamous cancers, animal models of the disease and associations among genomic features and patient outcomes. I will discuss therapeutically relevant genetic alterations and efforts aimed at exploring these targets both pre-clinically and clinically with an emphasis on FGFR pathway genes. I will discuss heterogeneity and adaptive responses to therapy in squamous cell carcinomas as potential challenges to the treatment paradigms which have been successful in adenocarcinomas and these concepts in the context of the recent introduction of immunotherapeutic approaches.

Abstract:
Squamous cell carcinoma of the lung accounts for 20-30% of all non-small cell lung cancer (NSCLC). Until recently, treatment options for advanced squamous NSCLC (sqNSCLC) were limited. Compared to non-squamous NSCLC, standard care of sqNSCLC was restricted to first-line platinum-based doublet chemotherapy and second-line docetaxel or the epidermal-growth factor receptor (EGFR) inhibitor, erlotinib, and did not include pemetrexed because of inferior efficacy[1], bevacizumab because of increased risk of pulmonary hemorrhage[2] or agents active against known oncogenic driver mutations. Prompted by the high levels of EGFR overexpression in sqNSCLC and encouraging activity of EGFR-targeted therapies in patients with squamous histology [3,4] EGFR-inhibition trials limited to patients with sqNSCLC were initiated, the results of which are redefining the treatment of sqNSCLC. In the first-line setting, the addition of the second-generation recombinant human IgG1 EGFR monoclonal antibody (Mab), necitumumab, to gemcitabine and cisplatin has been shown to improve overall survival (OS) 11.5m vs 9.9 m (HR: 0.84, 95%CI: 0.74-0.96) in the phase III open-label SQUIRE trial, with comparable adverse events (AE) leading to treatment discontinuation in both treatment arms.[5] The better tolerability of necitumumab over the first-generation chimeric EGFR Mab, cetuximab, is supported by the similar OS efficacy in patients with good (PS: 0-1) (HR: 0.85, 95%CI: 0.74-0.98) and poor performance status (PS=2) (HR: 0.78, 95%CI: 0.51-1.21), in the absence of additional safety risk.[6] In fact, in SQUIRE, necitumumab was notably more effective at higher levels of baseline symptom severity[7] , which is contrary to the belief that patients with sqNSCLC deteriorate too quickly to benefit from combination approaches. In the second-line setting, the newer second-generation EGFR small molecule inhibitor, afatinib, has also been shown to improve OS. Most recently, the results of the phase III LUX-Lung 8 trial of afatinib vs erlotinib in patients with sqNSCLC progressing after four cycles of platinum-based chemotherapy have been published, demonstrating improved OS with afatinib 7.9m vs 6.8m (HR: 0.81, 95%CI: 0.69-0.95), with similar adverse events profiles noted between groups.[8 ]Based on these results, afatinib is clearly a treatment option for patients in the second-line management of sqNSCLC. Together, the recent results of these small molecule and MAb anti-EGFR studies support the continued relevance of EGFR as a target in the treatment of sqNSCLC and are shaping management strategies. Despite being a hallmark of cancer, the inhibition of angiogenesis has historically proven challenging in the treatment of patients with sqNSCLC due to the central location of these tumors and their close proximity to large blood vessels in the chest wall, and has been associated with an increased risk of bleeding. Findings from newer second-generation angiogenesis inhibitors, however, show comparable levels of gastrointestinal and respiratory tract bleeding events across all NSCLC histologies. [9 ] Compared to placebo, the anti-VEGFR-2 IgG MAb, ramucirumab, has recently been shown to improve progression-free survival (PFS) 4.5m vs 3.0 m (HR: 0.76, 95%CI: 0.68-0.86) and OS 10.5m vs 9.1m (HR: 0.86, 95%CI:0.75-0.98) in patients with advanced NSCLC progressing after first-line platinum-based chemotherapy, with significant improvements in patients with squamous histology in terms of overall objective response (ORR) (26.8% vs 10.5%, p=0.001), disease control rate (59.9% vs 45%, p=0.015) and PFS 4.2m vs 2.7m (HR 0.78, 95%CI0.61-0.96) and a numerically superior OS benefit 9.5m vs 8.2m (HR: 0.88, 95%CI: 0.69-1.13). [9 ] In Dec 2014, ramucirumab received FDA approval for use with docetaxel in the second-line management of advanced NSCLC, including patients with squamous histology. Finally, the inhibition of T-cell activation through programmed death (PD-1) receptor interaction with the tumor expressing PD-L1 ligand (immune checkpoint) is a noted mechanism of tumor immune surveillance escape in NSCLC. From early clinical trials immune checkpoint blockade is an attractive therapeutic strategy in NSCLC, given its ability to activate the immune system and produce long-term response. In the management of sqNSCLC, the fully human IgG4 anti-PD-1 monoclonal antibody, nivolumab, has recently replaced docetaxel as the preferred second-line therapy based on the results of CHECKMATE 017 [10], a phase III study of nivolumab versus docetaxel. Findings in CHECKMATE 017 demonstrated improved median OS 9.2m vs 6.0m (HR: 0.59, 95%CI: 0.44-0.79) and improved 1-year survival over docetaxel (42% vs 24%), with a more favorable safety profile and fewer treatment related grade 3/4 AE (7% vs 55%).[10] With the recent FDA approval of nivolumab in the second-line setting in March 2015, docetaxel will likely be relegated to third-line therapy in the management of sqNSCLC. However, additional studies are required to confirm the results of CHECKMATE 017 given the lower than expected median survival observed in the docetaxel arm, to identify biomarkers of response, and to better define the unique toxicities associated with these immune-modulating agents. The last year has seen an unprecedented evolution in the management of sqNSCLC, with survival gains noted in both the first and second-line setting in randomized clinical trials. Unfortunately, to date the identification of oncogenic driver mutations in sqNSCLC have yet to yield the significant improvements seen in non-squamous histology, however it is likely that the relevant biomarkers of efficacy will soon be identified. Regardless, with the current regulatory approvals and the numerous novel agents in development, improved outcomes in patients with squamous cell carcinoma of the lung are anticipated. The immediate task, with the expanded treatment options now available for sqNSCLC, is the interrogation of new combinations and the sequencing of available therapies to maximize the benefit for this historically underserved subgroup of patients with NSCLC. References 1. Scagliotti G, Brodowicz T, Shepherd FA et al. Treatment-by-histology interaction analyses in three phase III trials show superiority of pemetrexed in non-squamous non-small cell lung cancer. J Thorac Oncol 2011; 6: 64-70. 2. Johnson DH, Fehrenbacher L, Novotny WF et al. Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-small cell lung cancer. J Clin Oncol 2004; 22: 2184-91. 3. Pujol JL, Pirker R, Lynch TJ et al. Meta-analysis of individual patient data from randomized trials of chemotherapy plus cetuximab as first-line treatment for advanced non-small cell lung cancer. Lung Cancer 2014; 83: 211-218. 4. Kim JH, Grossi F, De Marinis F et al. Afatinib monotherapy in patients with metastatic squamous cell carcinoma of the lung progressing after erlotinib/gefitinib (E/G) and chemotherapy : interim subset analysis from a phase III trial. Proc Am Soc Clin Oncol 2012; 30 (suppl 15): abstr 7558. 5. Thatcher N, Hirsch F, Luft A et al. Necitumumab plus gemcitabine and cisplatin versus gemcitabine and cisplatin alone as first-line therapy in patients with stage IV squamous non-small-cell lung cancer (SQUIRE): an open-label, randomized, controlled phase 3 trial. Lancet Oncol 2015; 16(7): 763-774. 6. Socinski M, Luft A, Szczesna A et al. Subgroup analyses by performance status (PS) in the phase III SQUIRE study: First-line necitumumab (N) plus gemcitabine-cisplatin (GC) vs. GC in squamous non-small cell lung cancer (NSCLC). J Clin Oncol 2015; 33:suppl; abstr e19023. 7. Reck M, Gralla RJ, Bonomi P et al. Maximum severity score (MSS) of baseline patient-reported Lung Cancer Symptom Scale (LCSS) as a prognostic and predictive factor for overall survival (OS) in the Phase III SQUIRE study. ASCO Meeting 2015 abst; 33: 8099. 8. Soria J-C, Felip E, Cobo M et al. Afatinib versus erlotinib as second-line treatment of patients with advanced squamous cell carcinoma of the lung (LUX-Lung 8): an open-label randomised controlled phase 3 trial. Lancet Oncol 2015; dx.doi.org/10.1016/s1470-2045(15)00006-6. 9. Garon EB, Ciuleanu TE, Arrieta O et al. Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small cell lung cancer after disease progression on platinum-based therapy (REVEL): a multicentre, double-blind, randomized phase 3 trial. Lancet 2014; 384: 665-73. 10. Brahmer J, Reckamp KL, Baas P et al. Nivolumab versus Docetaxel in advanced squamous-cell non small cell lung cancer. NEJM 2015; doi: 10.1056/NEJMoa1504627.

Abstract:
In recent years, our understanding of non-small cell lung cancer (NSCLC) has evolved from thinking of this malignancy as a single disease, or a small number of histologic subtypes, to now a multitude of genomically-defined subsets, both in adenocarcinoma and squamous lung cancer. In development of new targeted therapies against these abnormalities, so-called Master Protocols offer a number of advantages over traditional single study designs for drug-biomarker approval, including a common infrastructure, homogeneous patient populations with consistent eligibility across multiple independent sub-studies, and the ability to screen large numbers of patients in rapid fashion. Thus, the Lung-MAP project was designed to facilitate approval of targeted therapy-predictive biomarker combinations in squamous lung cancer, a recognized area of unmet need. Lung-MAP is constructed as a unique public-private partnership engaging the National Cancer Institute (NCI) and its Thoracic Malignancies Steering Committee (TMSC), the Foundation of the NIH (FNIH), the pharmaceutical industry and advocacy groups such as Friends of Cancer Research (FOCR), along with an advisory role by the Federal Drug Administration (FDA). The design is multiple simultaneously running Phase II/III trials, each capable of independently opening and/or closing without affecting the other sub-studies, in which patients eligible for 2[nd] line therapy for lung SCC have their cancers genomically screened through a next generation sequencing (NGS) platform (Foundation Medicine). Patients are then randomized into one of several sub-studies, each comparing an experimental targeted therapy with standard of care therapy, based on identification of candidate predictive biomarkers associated with each sub-study. At launch, drug targets under study consisted of “match sub-studies” for PI3K, FGFR, CDK 4/6 and HGF, and a non-match sub-study testing PD-L1-directed therapy, as described below. Rapid turn-around time of NGS screening results, within 2 weeks, allows real time assignment into the appropriate sub-study. For those patients with cancers that do not “match” into a biomarker-driven sub-study, there is a ‘non-match” sub-study, in which a predictive biomarker is not yet of sufficient validation to utilize it in a drug-biomarker registration strategy. Due to changes in the therapeutic landscape since the launch of Lung-MAP, a number of amendments and modifications have been implemented, which will be discussed during this presentation.

Abstract:
Copy number alterations are common events in squamous cell lung cancers. A number of these play defined roles in tumorigenesis. Some are known to be oncogenic drivers in a subset of cases. Broad high-level amplification of the 3q26-28 cytoband occurs in about 30% of squamous cell lung cancers and was one of the first recurrent alterations characterized in this disease.(1) More focal amplification of 8p11 occurs between 10-20% of tumors.(2, 3) Specific genes in 3q26 that are recurrently amplified include SOX2, PIK3CA, and PRKCI. Other genes commonly amplified in 3q27-28 include BCL6, TP63, and EPHB3. Genes that are amplified at lower frequencies include EGFR, MYC, MCL1, RICTOR, CCND1, and CDK6. The table below summarizes these alterations.

Gene	Chromosome	Frequency
SOX2	3q26	35%
PIK3CA	3q26	30%
BCL6	3q27	20%
PRKCI	3q26	25%
TP63	3q28	21%
FGFR1	8p11	12%
MYC	8q24	8%
MCL1	1q21	6%
RICTOR	5p13	6%
EGFR	7p12	5%
CCND1	11q13	10%
CDK6	7q21-22	3%

Pharmacologic targeting of gene amplification events in squamous cell lung cancers has centered largely on 4 genes- FGFR1, PIK3CA, CCND1, and CDK6. The pre-clinical data and clinical trial work defining FGFR1 amplification as an oncogenic driver and drug target, respectively, are the most mature. Three abstracts summarizing the preliminary efficacy of the pan-FGFR inhibitors AZD4547, BGJ398, and JNJ42756493 were presented in 2014.(4-6) The overall response rates were low, ranging from 8-15%. Some of these have continued on as phase 2 trials (NCT02154490, AD4547). Other studies using less-specific FGFR inhibitors are also ongoing (NCT01935336, ponatinib; NCT02109016, lucitanib). Upstream PI3K pathway alterations have been the therapeutic targets for a number of trials of PI3K inhibitors, though only a subset have included PIK3CA amplification as a biomarker of interest. These include two phase 1 trials of PI3K-α or PI3K/mTOR inhibitors that have added expansion arms for PIK3CA amplified squamous cell lung cancers (NCT01296555, GDC0032; NCT01655225, LY3023414). Data for these studies have not yet been presented. Finally, G1/S checkpoint inhibitors, whose efficacy has been best defined in breast cancer, are now being tested for CCDN1 and CDK4/6 amplified squamous cell lung cancers. Drugs include palbociclib (S1400, NCT02154490) and abemaciclib (NCT02450539). It is worth noting, however, that the pre-clinical rationale for targeting the G1/S checkpoint alone is substantially weaker than for other pathways. The clinical experience derived from targeting FGFR1 amplification in squamous cell lung cancers can serve as a framework to understand, in general, which targeted therapy strategies are likely to fail both now and in the future. Comprehensive genomic analyses of squamous cell lung cancers have shown that these tumors are complex, with overlapping alterations in more than one oncogene and/or tumor suppressor occurring in most cases. This is particularly problematic for gene amplification targets, which are also plagued by questions of functional relevance apropos degree of amplification and association with protein expression. As borne out in the phase 1 trials of the pan-FGFR inhibitors, single-target inhibition is unlikely to generate the breadth and depth of responses seen with other drugs targeting other oncogenes. Issues surrounding pharmacodynamic efficacy and target inhibition may also play a role in limiting responses. Data from ongoing work will be presented identifying potential genomic and non-genomic modifiers of response to FGFR1 inhibition. References 1. Björkqvist A-M, Husgafvel-Pursiainen K, Anttila S, Karjalainen A, Tammilehto L, Mattson K, et al. DNA gains in 3q occur frequently in squamous cell carcinoma of the lung, but not in adenocarcinoma. Genes, Chromosomes and Cancer. 1998;22:79-82. 2. Paik PK, Shen R, Won H, Rekhtman N, Wang L, Sima CS, et al. Next generation sequencing of stage IV squamous cell lung cancers reveals an association of PI3K aberrations and evidence of clonal heterogeneity in patients with brain metastases. Cancer Discovery. 2015. 3. TCGA. Comprehensive genomic characterization of squamous cell lung cancers. Nature. 2012;489(7417):519-25. 4. Paik P, Shen R, Ferry D, Soria J-C, Mathewson A, Kilgour E, et al. A phase 1b open-label multicenter study of AZD4547 in patients with advanced squamous cell lung cancers: Preliminary antitumor activity and pharmacodynamic data. J Clin Oncol. 2014;32:suppl; abstr 8035. 5. Nogova L, Sequist L, Cassier P, Hidalgo M, Delord J-P, Schuler M, et al. Targeting FGFR1-amplified lung squamous cell carcinoma with the selective pan-FGFR inhibitor BGJ398. J Clin Oncol. 2014;32:suppl; abstr 8034. 6. Bahleda R, Dienstemann R, Adamo B, Gazzah A, Infante J, Zhong B. Phase 1 study of JNJ-42756493, a pan-fibroblast growth factor receptor (FGFR) inhibitor, in patients with advanced solid tumors. J Clin Oncol. 2014;32:abstr 2501.

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Background:
Programmed cell death-1 (PD-1) inhibitors have shown significant potential to induce durable responses in non-small cell lung cancer (NSCLC). Although responses have been seen in patients (pts) whose tumors harbor epidermal growth factor receptor (EGFR) mutations (EGFRm), data to date with inhibitors of PD-1, or its ligand PD-L1, suggest that responses are less frequent in EGFRm NSCLC. Studies in which EGFRm pts receive EGFR tyrosine kinase inhibitors (TKIs) and PD-1 inhibitors in sequence or concurrently are being conducted. However, based on the high response rate with EGFR TKIs in EGFRm pts, PD-1 inhibition does not precede the EGFR TKIs in these study designs.

Methods:
We evaluated data from our experience at UCLA as part of the KEYNOTE-001 clinical trial, in which pts received pembrolizumab 2 mg/kg every 3 weeks or 10 mg/kg every 2 or 3 weeks. Early in the trial, an amendment excluded EGFRm, EGFR TKI naïve pts, however a subsequent amendment allowed such pts if their mutation was non-sensitizing to approved EGFR TKIs. Although the trial employed central radiographic assessment by RECIST v1.1 (available to the sponsor but not the sites), clinical decisions and the assessment we describe were based on investigator-assessed immune-related response criteria. Groups were compared using Fisher’s exact test. Western blot was performed using standard techniques, exposing human non-small cell lung cancer cell lines HCC-827, H1975, Calu3 and H460 to erlotinib or afatinib at 1µM or control using the antibody PD-L1 mAb #1368 (Cell Signaling) and α-tubulin antibody #2144 (Cell Signaling).

Results:
We enrolled 29 EGFRm pts. 2 of 3 EGFR TKI naïve pts experienced a partial response (PR) compared to 1 of 26 enrolled after a prior EGFR TKI (p<0.001). 18 of these 29 pts had a 9 week scan. Of these, PR was seen in both EGFR TKI naïve pts (one L858R mutation and one exon 20 insertion) compared to 1 of 16 enrolled after a prior EGFR TKI (p<0.001). Of note, a similar trend of increased responses in EGFR TKI naïve pts was not seen in EGFR wild type pts. In vitro experiments using erlotinib and afatinib showed unchanged PD-L1 levels in cell lines not inhibited by the EGFR TKI used, but reduced PD-L1 in EGFRm cell lines inhibited by the TKI. Of note, the only responder among the EGFR TKI-treated EGFRm pts was one of only 4 of the 16 scanned post-TKI pts who had a non-sensitizing mutation. So, 0 of 22 EGFRm pts with a sensitizing mutation responded after an EGFR TKI.

Conclusion:
A retrospective analysis in EGFRm NSCLC showed a strong correlation between response and lack of prior EGFR TKI treatment. PD-L1 levels decrease in response to an EGFR TKI in cell lines sensitive to the TKI. Immunohistochemistry evaluating the presence and location of relevant proteins and immune effector cells are ongoing as is whole exome sequencing. These results have implications for the design of clinical trials of PD-1 inhibitors in EGFRm pts. Supported by: 1K23CA149079, One Ball Matt Memorial Golf Tournament, Kasdan Family

Background:
Although neoadjuvant chemotherapy (NAC) for advanced lung cancer can improve operability and local disease control, the duration of benefit is limited before resistance develops. PD-L1, which was a co-stimulatory molecule,interacting with PD-1, has a crucial role in T-cell regulation in immune response. Interest remains in combining chemotherapy and immune therapies to overcome resistance.

Methods:
In the study, we used immunohistochemistry, real-time PCR and flow cytometry techniques to investigatethe correlation between overall survival (OS) and disease free survival (DFS) of lung cancer patients and the expression of programmed cell death ligand1 (PD-L1) and the effect of NAC on the expression of PD-L1 in lung cancer cells.

Results:
Firstly, we identified PD-L1 was uprelugated in the SD lung cancer patient by the RNA-seq analysis. Therefore, we performed IHC evaluation in the total 194 patients of NSCLC. The patients with PD-L1 (−) had much better OS compared to those who were PD-L1 (+), and a high PD-L1 expression level in the cancer cells was significantly correlated with a shorter OS and DFS in patients with NAC from the 194 patient (n=78). Meanwhile,in patients who had stable disease (SD) to NAC, there was a rise in the expression of PD-L1, and patients with NAC (n=78) had significantly high rate of positive PD-L1 expression compared with those without NAC (n=116, p= 0.001). The chemotherapy of lung cancer can induce the expression of PD-L1, which may be one of the resistance mechanisms of NAC. Changes in PD-L1 expression were examined in vitro and vivo. Inhibition of the PI3K/AKT pathway reduced the up-regulation of PD-L1 induced by cisplatin, suggesting an involvement of PI3K/AKT pathway in up-regulation of PD-L1.Moreover, knock down of PD-L1 can lead to an increase in apoptosis, as well as cisplatin-induced apoptosis. And caspase7 might play an important role in the apoptosis of lung cancer cells after the knockdown of PD-L1.

Conclusion:
These findings support provide a relationship between PD-L1 expression and chemoresistance. All in all, these results suggest the use of PD-L1 inhibitor with chemotherapy after surgery, in lung cancer patients who received NAC.

Background:
In patients with previously treated NSCLC enrolled in KEYNOTE-001 (NCT01295827), the anti–PD-1 antibody pembrolizumab (MK-3475) has demonstrated promising efficacy and manageable safety when given at dosages of 10 mg/kg once every 2 weeks (Q2W) or once every 3 weeks (Q3W). In a prospectively defined validation set from KEYNOTE-001, the greatest efficacy was observed in patients whose tumors expressed PD-L1 in ≥50% of tumor cells. Here, we present data for patients with previously treated, PD-L1–positive advanced NSCLC enrolled in a KEYNOTE-001 expansion cohort added to evaluate pembrolizumab 2 mg/kg Q3W.

Methods:
Patients had measurable disease, ECOG performance status of 0 or 1, and adequate organ function. Prior therapy with ≥1 platinum-doublet chemotherapy regimen was required; an appropriate tyrosine kinase inhibitor was required for patients with sensitizing EGFR mutations or ALK translocations. All patients had PD-L1–positive tumors, defined as staining in ≥1% of tumor cells as determined by a prototype IHC assay using the 22C3 antibody. The percentage of PD-L1–stained tumor cells was also determined by a clinical trial IHC assay using the same antibody. Patients received pembrolizumab 2 mg/kg Q3W until investigator-determined progression according to immune-related response criteria, intolerable toxicity, patient withdrawal, or investigator decision. Response was assessed centrally every 9 weeks by RECIST v1.1.

Results:
Of the 55 patients enrolled, 41 (74.5%) received ≥2 prior therapies. Three (5.5%) patients experienced grade 3-5 drug-related AEs (grade 3 colitis and pneumonitis and grade 5 cardiorespiratory arrest). After a minimum of 27 weeks of follow-up by central radiology review of tumor imaging (median, 7.7 months; range, 6.4-9.7 months), confirmed overall response rate (ORR) in the 52 patients with centrally evaluable disease at baseline was 15.4% (95% CI, 6.9%-28.1%) and the disease control rate (DCR, complete response + partial response + stable disease) was 50.0% (95% CI, 35.8%-64.2%). At the time of analysis, all responses were ongoing, and the median response duration was not reached (range, 2.1+ to 6.2+ months). Median progression-free survival (PFS) was 3.3 months (95% CI, 2.0-6.0 months), with a 6-month PFS rate of 37.7%. Median overall survival (OS) was not reached, and the 6-month OS rate was 75.8%. In the 25 (45.5%) patients who had PD-L1 expression in ≥50% of tumor cells, confirmed ORR was 30.4% (95% CI, 13.2%-52.9%), DCR was 56.5% (34.5%-76.8%), median PFS was 4.2 months (95% CI, 1.9 months-NR), and 6-month PFS and OS rates were 49.0% and 81.8%, respectively.

Conclusion:
In this previously treated cohort of patients with PD-L1–positive advanced NSCLC, pembrolizumab 2 mg/kg Q3W demonstrated robust and durable antitumor activity, with improved efficacy in patients with PD-L1 staining in ≥50% of tumor cells.

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Background:
The humanized anti–PD-1 monoclonal antibody pembrolizumab (MK-3475) has demonstrated robust antitumor activity and a manageable safety profile in patients with advanced cancers, including NSCLC. In the first 495 patients with advanced NSCLC enrolled in multiple expansion cohorts of the phase 1b KEYNOTE-001 study (ClinicalTrials.gov, NCT01295827), pembrolizumab provided an overall response rate (ORR) of 19.4%. In a prospectively defined validation set, a relationship between tumor PD-L1 expression and pembrolizumab efficacy was demonstrated, such that patients with PD-L1 expression in ≥50% of cells had a 45.2% ORR compared with 16.5% and 10.7% in patients with PD-L1 expression in 1%-49% and <1% of cells, respectively. Using the total population of 550 patients with NSCLC treated with pembrolizumab in KEYNOTE-001, we assessed the relationship between antitumor activity and the level of PD-L1 expression in key patient subgroups.

Methods:
Patients with advanced NSCLC enrolled in the NSCLC-specific expansion cohorts of KEYNOTE-001 received pembrolizumab 2 or 10 mg/kg every 3 weeks (Q3W) or 10 mg/kg every 2 weeks (Q2W) until confirmed progression, intolerable toxicity, or investigator decision. Tumor PD-L1 expression was assessed by immunohistochemistry using a clinical-trial assay and scored as the proportion score (PS) (ie, percentage of tumor cells with membranous PD-L1 expression). Response was assessed every 9 weeks per RECIST v1.1 by central review. Patients evaluable for PD-L1 were those whose slides were prepared within 6 months of staining and for which a proportion score could be assigned.

Results:
ORR in the 550 patients who received ≥1 pembrolizumab dose was 18.9%. ORR was generally similar across subgroups (Table), although there may be a difference between ever and never smokers. Among the 409 patients evaluable for PD-L1 expression, ORR was highest in those with PS ≥50% as compared with PS 1%-49% or <1% (36.8%, 11.9%, and 10.0%, respectively). Within all subgroups, ORR was highest in patients with PS ≥50% (Table). Figure 1



Conclusion:
Pembrolizumab provides antitumor activity in a broad selection of subgroups of patients with advanced NSCLC. Improved response in patients whose tumors express PD-L1 in ≥50% of cells was observed for all subgroups. Ongoing analyses are investigating the interdependency between PD-L1 status, mutational status, and smoking.

Background:
Nivolumab (anti-PD-1, ONO-4538, BMS-936558), a fully human IgG4, PD-1 immune-checkpoint inhibitor antibody, has shown durable clinical activity in previous[MS誠1] phase I and II trials in several tumor types. In March 2015, U.S. Food and Drug Administration (FDA) has approved Nivolumab for the treatment of patients with metastatic squamous (SQ) non-small-cell lung cancer (NSCLC) with progression on or after platinum-based chemotherapy. Here, we report the results of two phase II studies to evaluate the efficacy and safety of nivolumab in previously treated advanced SQ (JapicCTI-No.132072) and NSQ (JapicCTI-No.132073) NSCLC pts.

Methods:
Both studies required pts aged ≥ 20 years with an ECOG performance status of 0 or 1, stage IIIB/IV, or recurrent NSCLC and at least one prior chemotherapy including platinum containing regimen. Pts received nivolumab 3 mg/kg IV Q2W until disease progression or unacceptable toxicity. The primary endpoint in both studies was the objective response rate (ORR) (RECIST v1.1). Planned sample size was 30 pts for SQ and 67 pts for NSQ, respectively (P~0~[MS誠1] =0.09 &[MS誠2] P~1~=0.26, P~0~=0.09 & P~1~=0.20 ; α=0.025 (one-side), 1-β=0.8).

Results:
From April 2013 to April 2014, a total of 111 NSCLC pts were enrolled in both studies (35 pts with SQ, 76 pts with NSQ, male/female: 81/30; PS 0/1: 46/55; aged 31 to 84 [median: 65.0] years; Stage IIIB/Stage IV/recurrence: 6/86/19). Objective response rates (ORRs) were 25.7% (9/35) [95% CI: 14.2, 42.1] in SQ and 19.7% (15/76) [95% CI: 12.3, 30.0] in NSQ, respectively. Complete Response was observed in 2.6% with NSQ. Median progression-free survival (mPFS) was 4.2 months (95% CI: 1.4, 7.1) for SQ and 2.8 months (95% CI: 1.4, 3.4) for NSQ, respectively. Median follow-up periods were 10.4 months and 8.4 months, respectively. Median duration of response was not reached in each study. Of 9 SQ pts and 15 NSQ pts who responded to nivolumab, durable and ongoing response was observed in 77.8% (7/9) and 80.0% (12/15), respectively. Median overall survival was not reached in either study. All Grade drug-related adverse events across both studies were 79.3% (88/111) and Grade 3-4 drug-related adverse events (G3-4 AEs) were observed in 16.2% (18/111) pts. Most common G3-4 AEs were lymphocyte count decreased 3.6% (4/111), hyponatremia 1.8% (2/111), interstitial lung disease 1.8% (2/111), pleural effusion 1.8% (2/111). Any grade of interstitial lung disease was observed in 4.5% (5/111) pts. No grade 5 AEs were observed.

Conclusion:
In these studies, nivolumab showed encouraging clinical efficacy in both SQ and NSQ NSCLC with a manageable safety profile.

Background:
The majority of lung cancer in the US is treated in the community. A prospective cohort study of stage IV non-small cell lung cancer (NSCLC) and extensive disease small cell lung cancer (ED SCLC) is being conducted in 70 US community oncology practices (Figure) to assess current standards of care (SOC) and outcomes in anticipation of immunotherapy as a new treatment modality. This study establishes a historical comparator cohort in a “pre-immunotherapy era” of lung cancer treatment. Figure 1



Methods:
Patients with stage IV NSCLC and ED SCLC, at any point in their care, with documented dates of diagnosis and prior treatment, are eligible for inclusion. Patients are followed prospectively for 36 months or until death, with data abstraction from medical records into electronic case report forms. Patient-reported outcomes are prospectively collected, as are archival tumor tissue and serial blood samples from consenting patients for molecular profiling studies.

Results:
This early analysis focused on patient clinical attributes and tumor sample characteristics of relevance to non-clinical trial patient populations and to biomarker testing (Table). Of 1,183 cases enrolled to date, at enrollment 17.6% were ECOG performance status (PS) 2 or 3, 18.8% of patients had brain metastases, 22.2% were on systemic steroids, 6.7% had history of a specific autoimmune condition, and 49.5% had had tissue samples from core needle or surgical specimens. Figure 1



Conclusion:
Many immunotherapy clinical trials exclude patients with brain metastases, certain steroid use, poor PS, and autoimmune disease, yet a substantial proportion of community-based lung cancer patients present with these attributes. Approximately half of advanced stage patients have tissue specimens amenable to current SOC biomarker testing. Efforts to develop additional biomarker tests for lung cancer patients need to consider the reality of limited tissue sample availability in the community setting.

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Background:
Epidermal growth factor receptor (EGFR) exon 20 T790M mutation may have a predictive role before EGFR-tyrosine kinase inhibitors (TKIs) treatment and it also might have a prognostic role after acquired resistance to EGFR-TKIs. However, its role in EGFR-TKI rechallenge after failure of initial EGFR-TKIs in EGFR-mutant advanced non-small cell lung cancer (NSCLC) remains unknown.

Methods:
We retrospectively evaluated the clinical course of 515 EGFR-mutant advanced NSCLC patients who received first generation EGFR-TKIs (gefitinib or erlotinib) from December 2009 to November 2014 at Guangdong General Hospital. Of these 515 patients, 65 patients recieved same EGFR-TKI rechallenge, including 51 patients who underwent rebiopsy and secondary EGFR mutation detection after failure of initial EGFR-TKIs. EGFR detection was performed by Sanger sequencing or Amplification Refractory Mutation System (ARMS) methods. Progression-free survival (PFS) and overall survival (OS) were both calculated from commencement of EGFR-TKI rechallenge. Survival data were analyzed using the Kaplan-Meier method and log-rank test.

Results:
EGFR activating mutations still existed in all the 51 patients who received rebiopsy and 18 patients were with T790M mutation while 33 patients were without T790M. The median PFS for the T790M+ and T790M- groups were 1.8 months (95%CI 1.180~2.420) and 2.0 months (95%CI 1.100~2.900), respectively (P=0.261). The median OS for the two groups were 7.7 months (95%CI 6.548~8.852) and 6.8 months (95%CI 4.730~8.870), respectively (P=0.565). No statistical difference was found in PFS or OS between two groups(Figure 1). Figure 1 Fig 1. Kaplan-Meier curves of patients in two groups. (A)Progression-free survival. (B) Overall survival.



Conclusion:
EGFR T790M mutation is neither a predictive nor a prognostic factor for first generation EGFR-TKI rechallenge in EGFR-mutant advanced NSCLC patients, indicating that whether T790M occurs or not, same EGFR-TKI rechallenge could not be recommended as a good strategy to overcome the resistance to first generation EGFR-TKIs.

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). Rociletinib has demonstrated efficacy against activating mutations (L858R and Del19) and the dominant acquired resistance mutation (T790M), while sparing wild-type EGFR. New insights into mutEGFR NSCLC suggest clonal heterogeneity – activating EGFR mutations are truncal (present in all tumor clones) and T790M is a dominant branch mutation with variable clonal frequency between patients and over time. The extent of this clonal heterogeneity may relate to rociletinib efficacy. Here we present preliminary findings to evaluate this hypothesis from an ongoing Phase 1/2 clinical trial.

Methods:
TIGER-X (NCT01526928) is a Phase I/II open-label, safety, pharmacokinetics and preliminary efficacy study of rociletinib in patients with metastatic or unresectable locally advanced EGFR mutation-positive NSCLC with progressive disease after ≥1 EGFR tyrosine kinase inhibitor (TKI). Screening included mandatory tumor biopsy and T790M testing. For Phase 1, patients could be T790M negative, positive or unknown. For Phase 2, T790M negative patients (by validated central testing) could have a contemporaneous local T790M+ result.

Results:
As of March 2015, 36 patients were enrolled in TIGER-X who were T790M central negative by cobas® or Qiagen therascreen® and evaluable for efficacy. Sensitivity analysis indicated that the 2 assay platforms were comparable for T790M detection. 69% (25/36) were T790M negative centrally but positive locally; 4/36 (11%) were negative by both central and local testing; and 7/36 (19%) were centrally negative with no local result. Median number of previous TKIs was 1 and median number of previous therapies was 2; 81% (29/36) were treated with a TKI as their most recent prior therapy. In central negative/local+ patients the ORR was 40% (10/25). In central negative/local negative patients the ORR was 25% (1/4). The most common treatment emergent adverse events in this subset (all grades) were fatigue, diarrhea, nausea and hyperglycemia.

Conclusion:
These preliminary findings suggest that patients who test negative for T790M using a sensitive tissue test may still benefit from treatment with rociletinib. In part, this clinical activity may be driven by T790M tumor heterogeneity, demonstrated by the discordant T790M results described. In addition, inhibition of IGF-1R/IR by the previously reported (Soria 2014) rociletinib metabolite M502 may also be driving some of the activity observed. This possible explanation is important, since the response rates reported herein are higher than described for other T790M inhibitors in T790M-negative patients. Furthermore, TKI re-treatment effect is unlikely to be a major driver of these results, since the majority of patients came on study directly after progression on another EGFR TKI. To further explore these findings, the open-label TIGER-2 (NCT02147990) and the randomized Phase 3 TIGER-3 (NCT02322281) studies include T790M negative patients.

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Abstract:
Introduction: Cancer cells express antigens that potentially differentiate them from normal cells. These are known to be numerous in lung cancer and characterized by a high mutational rate (7-11 mutations / MegaBase), especially in relation with smoking derived genetic instability, P53 mutations, and/or the presence of targetable mutations in adenocarcinoma. These tumor antigens should confer immunogenicity to lung cancer transformed cells. However, immune-editing occurs in most lung cancer along a three phases sequence: 1) Elimination, where transformed cells are destroyed by the immune system; 2) Equilibrium, equivalent to a functional state of dormancy in which tumor cells growth is controlled by adaptive immunity, a state characterized by typically dense lymphocytic infiltration rich in CD8 cytotoxic cells (E. Brambilla et al. JCO, under review); 3) Escape from immune surveillance. PD-L1 in NSCLC is expressed on the membrane of tumor cells, and/or on immune infiltrating cells dendritic cells (DC), other antigen presenting cells (APC) and T lymphocytes. PD-1, the PDL1 receptor, is expressed on tumor infiltrating lymphocytes (TILS), mainly CD4 T cells, T regulatory (T-reg) and B, NK, monocytes and DC. Upon PD-L1 binding, PD-1 inhibits kinases involved in T cell activation. There are two mechanisms of expression of immune checkpoints on tumor cells and their immune stromal counterparts: oncogenic signaling, and response to inflammatory signals, both of which occur potentially in lung cancer. Tumor cells express multiple ligands and receptors and antitumor immune response can be enhanced by multi-level blockade of immune checkpoints. PD-1/PD-L1 engagement leads to HSP-2 phosphatase activity which dephosphorylates Pi3K and thus downregulate AKT. The necessary patient selection for immunotherapy has stressed the search for predictive biomarker of PD-1/PD-L1 pathway inhibition. The cutoff for positivity on tumor cells[1–3]: The cutoff for positivity in and out of trials on tumor cells has never been assessed nor optimized or standardized. The percentage of PD-L1 membrane staining considered as the cutoff for positivity was from ≥1%, ≥5%, ≥10%, ≥50% and the intensity was or not defined and taking into account (any intensity, 1+, 2+, 3+, or a scale from 1 to 3+/H Score , or 2+3+only). At least, most if not all reports considered only membrane staining on tumor cells, although cytoplasmic staining was also considered with AQUA techniques. Stromal expression of PD-L1 on immune infiltrate (T cells, macrophages, DC) is also needed for scoring. Whereas DC and macrophages display a clear cytoplasmic membrane stain, this is not appreciated on lymphocytes. We have set up a study to assess a cutoff of positivity for prognosis analysis (1500 randomized early stage operable NSCLC patients with or without adjuvant cisplatin therapy after surgery) using E1L3N Cell Signaling antibody commercially available. We found that 20% of lung tumors cell expressed PD-L1 (≥20% intensity 2+3+), and 29% the immune stromal cells (T, macrophages, DC ) ≥10% intensity 2+3+. PD-L1 positivity in both tumor and immune cells were seen in only 9% of NSCLC, 20,7% were both negative . We double-check the scoring cells with Ming Tsao. The best concordance was for intensity 2+ /3+ (83%) although the intensity 1 was not reproducible ( 40%) . There was no prognostic relevance of PD-L1 (tumor cells or stroma) in the control arm and pooled analysis, whatever cutoff by 10% increment or linear scoring was used. There was no statistical correlation between PDL1 expression (Tumor or Immune cells ) with clinicopathological criteria or histology . Only immune PD-L1 expression was correlated with a highly intense immune infiltrations (TILs ) ( P = 002 ). Not surprisingly, previous published evaluations of prognostic value were discordant likely because immune checkpoints modulators play both positive and negative roles in the immune inhibitory pathways with some redundancy, and patients series and assays were not comparable .The two meta-analyses with their numerous biases ( different antibodies, cutoffs, patient series composition in early and advanced stage, ethnicities and contribution of oncogene driven cancers, time of use of the initial resection sample or contemporary biopsy…) rendered their interpretation extremely problematic . Global result was favoring a poor prognosis of “PD-L1 positivity” on tumor cells. PD-L1 expression as a predictive biomarker in cancer immunotherapy[1,4–7]: In the majority of phase I trials with four antibodies targeting the co-inhibitory receptor PD-1 or its primary ligand PD-L1 (Table 1), response rates appear higher in patients with increased tumor PD-L1 membrane expression by immunohistochemistry (IHC). However, different antibody assays, lack of standardization, different cutoff point to determine PD-L1 positivity, the usual various pharmaceutic companies to recommend their companion test, and the small number of specimens available for testing, in addition with the variability of the intervals between biopsy and test, has surely hampered the conclusion and prevent consensus to be reached[7,8]. The most pertinent threshold was provided by Garon et al, with ≥50% of tumor cells PD-L1 positive to allow the highest response rate of 45% in pembrolizumab treated patients in the validation group[1]. In most trial series, biopsies or resected specimen were used restropectively although considerable difference between these samples occurs due to tumor heterogeneity. The reliability of small biopsy samples is questionned[9]. Indeed lung tumor heterogeneity is exemplary , and PD-L1 is typically heterogeneous in its distribution in the tumor bulk as is PD-L1 positive immune cells . Multiple issues are yet addressed before PD-L1 is considered as a robust and definitive molecular predictor of efficacy. Various clones are currently being evaluated in and out of clinical trials (Ventana SP263, SP6242, Dako 28-8 and 22C3, Cell Signaling E1L3N). As for prognostic evaluations, thresholds of ≥1%, ≥5%, ≥10%, ≥50% or continuous H score have been used. In addition in a few trials, PD-L1 expression in TILs was predictive more than PD-L1 on tumor cells but the cutoff was not disclosed. IASLC pathology panel is leading a large multicentric reproducibility study ( Fred Hirsch )with lung pathologists of the IASLC Pathology Committee to address these questions. Alternative regulations of PD-1/PD-L1 pathway The ability of cancer cells to evade immunosurveillance results from the production of immunosuppressive chemokines by the tumor cells, loss of MHC antigen expression, a higher number of T-reg cells in the tumor microenvironment and inhibitory pathways referred to as immune checkpoints, which result in a link of inhibitory ligands to their receptors (CTLA~4~-PD-1, PD-L1/PD-L2-PD-1) are unfrequently upregulated in lung cancer. Moreover immune-editing was associated with the illegitimate expression of tumor germ cell (testis /placenta) antigens[10], normally absent in normal tissue but testis and placenta, inducing a state of immune escape when aberrantly expressed in lung cancer correlating with highly and metastatic aggressive behavior. While patients with PD-L1 overexpression based on different assays, cutoff, tumor material, have more robust response to PD-L1 (67-100% ORR), PD-L1 negative NSCLC ranges from 0 to 15%, suggesting that PD-L1 IHC is not a clear and exclusive predictive biomarker. This is not surprising due to multiple regulations at the two clinically relevant immunologic synapses: the tumor-T cell interface, and the APC-T cell interface, both playing role in tumor control. In all cohorts, PD-L1 in tumor cells was observed with or without immune infiltration. TILs intense infiltration occurred in 10% of NSCLC across histology and was a statistically significant good prognosis factor although the oncogene driven adenocarcinomas lack immune infiltrate. EGFR pathway upregulates PD-L1 as well as PTEN loss[11–14]. In addition the 2 synapses are functionally affected by HLA loss (>50% of NSCLC), EGFR signaling, PTEN loss, the density of CD8 in infiltrate available for cytotoxicity and even more CD8 +/PD1+ exhausted cytotoxic T cells among TILs . The best predictive biomarker might not be simply binary . Biopsies may underevaluate the pertinent tumor-stroma interface , PD-L1 biologically relevant ( more than 1-10% of tumor cell ! ) has already taken place and destroyed the potentially reactive CD8 T cells. Indeed secondary biomarkers may drive the tumor in association or independently of PD-1/PD-L1 pathway. Table 1: Prevalence of PD-L1 in NSCLC:

Percent tumor samples expressing PD-L1	Tumor surface expression cutoff for positivity	PD-L1 detection antibody	Reference
49%	5%	28-8	Grosso et al. JCO, 2013
52%	NR	R&D B7-H1	Gatalica et al. Cancer Epidemiology biomarkers prevention, 2014
95%	>10%	5H1	Dong et al. Nature Medicine, 2002
50%	11%	MIH1	Konishi et al. CCR, 2004
21% (squamous only)	>1% vs >5% vs H-score	5H1	Marti et al. JCO, 2014
60%	5%	DAKO IHC	Gettinger et al. JCO, 2014
50%	1%	NR	Sun et al. JCO, 2014
25%	≥50%	NR	Garon et al. NEJM, 2015

References: 1. Garon EB, et al. Pembrolizumab for the treatment of NSCLC. N Engl J Med. 2015;372(21):2018-2028. 2. Sorensen S, et al. PD-L1 expression and survival among advances NSCLC patients treated with chemotherapy. Ann Oncol. (25 (Supplement 4)). 3. Soria J-C, et al. Clinical activity, safety and biomarkers of PD-L1 blockade in NSCLC: Additional analyses from a clinical study of the engineered antibody MPDL3280A (anti-PDL1). 4. Patel SP, Kurzrock R. PD-L1 Expression as a Predictive Biomarker in Cancer Immunotherapy. Mol Cancer Ther. 2015;14(4):847-856. 5. Taube JM, et al. Association of PD-1, PD-1 ligands, and other features of the tumor immune microenvironment with response to anti-PD-1 therapy. CCR. 2014;20(19):5064-5074. 6. Herbst RS, et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature. 2014;515(7528):563-567. 7. Soria J-C, et al. Immune checkpoint modulation for non-small cell lung cancer. CCR. 2015;21(10):2256-2262. 8. Brahmer JR, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. NEJM. 2012;366(26):2455-2465. 9. Kitazono S, et al. Reliability of Small Biopsy Samples Compared With Resected Specimens for the Determination of PD-L1 Expression in NSCLC. Clin Lung Cancer. 2015. 10. Rousseaux S, et al. Ectopic activation of germline and placental genes identifies aggressive metastasis-prone lung cancers. Sci Transl Med. 2013;5(186):186ra66. 11. Akbay EA, et al. Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors. Cancer Discov. 2013;3(12):1355-1363. 12. D’Incecco A, Andreozzi M, Ludovini V, et al. PD-1 and PD-L1 expression in molecularly selected NSCLC patients. Br J Cancer. 2015;112(1):95-102. 13. Chen N, et al. Upregulation of PD-L1 by EGFR Activation Mediates the Immune Escape in EGFR-Driven NSCLC: Implication for Optional Immune Targeted Therapy for NSCLC Patients with EGFR Mutation. J Thorac Oncol. 2015 14. Lin C, et al. Programmed Death-Ligand 1 Expression Predicts TKI Response and Better Prognosis in a Cohort of Patients With EGFR Mutation-Positive Lung Adenocarcinoma. Clin Lung Cancer. 2015.

Abstract:
Multiple genetic and epigenetic changes in several cancer types cause resistance to immune attack of tumors by inducing specific T cells tolerance and by expressing ligands that engage inhibitory receptors and block T cells activation, all resulting on T-cells anergy or exhaustion within the tumor microenvironment (1). In this process, programmed death 1 (PD-1) protein, a T-cell co-inhibitory receptor, and one of its ligands, PD-L1 (B7-H1 or CD274), play a pivotal role in the ability of tumor cells to evade the host’s immune system. Antibody-mediated blockade PD-1/PD-L1 induced durable tumor regression and prolonged disease stabilization in non-small cell carcinoma (NSCLC) (2). Although these studies have reported correlations between PD-L1 immunohistochemical (IHC) expression levels on NSCLC tumor cells and clinical responses to PD-1 and PD-L1 inhibitors, there are patients with negative PD-L1 expression tumors who have showed similar responses than patients with positive expression. Recently, it has been shown that across multiple cancer types, including NSCLC, responses to anti-PD-L1 therapy were observed in patients with tumors expressing high levels of PD-L1, especially when PD-L1 was expressed by tumor-associated infiltrating cells (TAICs). Altogether, these findings suggest that there are other factors in the tumor microenvironment, including tumor infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) that may drive responses to anti-PD-1/PD-L1 therapies, and be involved in lung cancer pathogenesis and progression. A number of studies have characterized the PD-L1 protein expression by immunohistochemistry (IHC) or immunofluorescence (IF) in all NSCLC stages using formalin-fixed and paraffin-embedded (FFPE) tumor tissues, and correlated those findings with patient’s outcome, and in a limited number of cases with response to immunotherapy (3, 4). Those studies differ on the type of specimens (whole histology sections vs. tissue microarrays [TMAs]), the protein expression analysis (IHC vs. IF), and the quantification assessment (image analysis vs. microscope observation). Only few studies have attempted to correlate the expression of PD-L1 and TAICs, particularly TILs, using a limited number of IHC markers (e.g., CD8, CD45) (5). Up to date, there is no published study in which a comprehensive panel of immune markers, including PD-L1, has been performed attempting to develop a clinical relevant immuno-score system in surgically resected NSCLCs and explore their role as predictive markers of response to immunotherapy. We will present data on the characterization of TAICs in lung cancer tumor specimens using a large panel of markers (PD-L1, PD-1, CD3, CD4, CD8, CD45RO, CD57, Granzyme B, FOXP3, OX-40, and CD68) examined by both uniplex IHC and multiple immunofluorescence (IF) methodologies, and quantitated using image analysis systems (Aperio, Vectra and MultiOmyx). In surgically resected NSCLC tumor tissues the analysis was performed at both peri-tumoral and intra-tumoral compartments, and those data provided interesting data on the spatial distribution of TAICs and the expression of immune checkpoints in lung tumors. Our approach allowed us to devise an immuno-score system for lung cancer tissue specimens using both surgically resected and small diagnostic biopsies (core needle biopsies, CNBs) that correlated with clinical, pathological and molecular features of tumors. References: 1. Mellman I, Coukos G, Dranoff G: Cancer immunotherapy comes of age. Nature 2011, 480:480-9. 2. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, Powderly JD, Carvajal RD, Sosman JA, Atkins MB, Leming PD, Spigel DR, Antonia SJ, Horn L, Drake CG, Pardoll DM, Chen L, Sharfman WH, Anders RA, Taube JM, McMiller TL, Xu H, Korman AJ, Jure-Kunkel M, Agrawal S, McDonald D, Kollia GD, Gupta A, Wigginton JM, Sznol M: Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. The New England journal of medicine 2012, 366:2443-54. 3. Herbst RS, Soria JC, Kowanetz M, Fine GD, Hamid O, Gordon MS, Sosman JA, McDermott DF, Powderly JD, Gettinger SN, Kohrt HE, Horn L, Lawrence DP, Rost S, Leabman M, Xiao Y, Mokatrin A, Koeppen H, Hegde PS, Mellman I, Chen DS, Hodi FS: Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 2014, 515:563-7. 4. Taube JM, Klein A, Brahmer JR, Xu H, Pan X, Kim JH, Chen L, Pardoll DM, Topalian SL, Anders RA: Association of PD-1, PD-1 Ligands, and Other Features of the Tumor Immune Microenvironment with Response to Anti-PD-1 Therapy. Clinical cancer research : an official journal of the American Association for Cancer Research 2014, 20:5064-74. 5. Schalper KA, Brown J, Carvajal-Hausdorf D, McLaughlin J, Velcheti V, Syrigos KN, Herbst RS, Rimm DL. Objective measurement and clinical significance of TILs in non-small cell lung cancer. J Natl Cancer Inst. 2015 Feb 3;107(3).

Abstract not provided

Background:
Treatment options for patients with advanced SQ NSCLC who fail platinum-based doublet chemotherapy (PT-DC) are limited. NIVO, a fully human IgG4 programmed death-1 (PD-1) immune checkpoint inhibitor, demonstrates activity across NSCLC histologies and is approved in the US for treatment of metastatic SQ NSCLC with progression on or after platinum-based chemotherapy. We report results from a randomized, open-label, global phase 3 study (CheckMate 017; NCT01642004) comparing NIVO vs docetaxel in patients with previously treated SQ NSCLC and disease progression during/after one prior PT-DC regimen.

Methods:
Patients (N=272) were randomized 1:1 to receive either NIVO 3 mg/kg every 2 weeks (Q2W; n=135) or docetaxel 75 mg/m[2] Q3W (n=137) until disease progression or discontinuation due to toxicity or other reasons. For NIVO patients, treatment after initial progression was permitted at the investigator’s discretion, per protocol criteria. The primary objective was overall survival (OS). Secondary objectives included investigator-assessed objective response rate (ORR; per RECIST v1.1), progression-free survival (PFS), efficacy by PD-L1 expression (PD-L1 testing not required for enrollment), patient-reported outcomes (PRO), and safety. PRO analyses are presented in a separate abstract.

Results:
Treatment with NIVO led to 41% reduction in risk of death (hazard ratio [HR]=0.59; 95% CI: 0.44, 0.79; P=0.00025) and improved ORR (20% vs 9%; P=0.0083) and PFS (HR=0.62; 95% CI: 0.47, 0.81; P=0.0004) vs docetaxel (Table). Twenty-eight patients were treated with NIVO beyond initial progression, nine of whom demonstrated a non-conventional pattern of benefit (ie, reduction in target lesions with simultaneous appearance of new lesions, initial progression followed by tumor reduction, or no further progression for ≥2 tumor assessments). Across pre-specified cut-points (1%, 5%, and 10%), PD-L1 expression was neither prognostic nor predictive of benefit. OS HRs favored NIVO across most predefined patient subgroups. Grade 3–4 drug-related adverse events (AEs) were reported in 7% (9/131) of NIVO and 55% (71/129) of docetaxel patients. Grade 3–4 drug-related select AEs are shown below (Table). No deaths were related to NIVO vs 3 docetaxel-related deaths. Figure 1



Conclusion:
CheckMate 017 achieved its primary objective, demonstrating clinically superior and statistically significant OS with NIVO vs docetaxel in patients with advanced, previously treated SQ NSCLC. Benefit was seen regardless of PD-L1 status. The safety profile of NIVO 3 mg/kg Q2W is favorable vs docetaxel and consistent with prior studies. AEs were manageable with established guidelines. NIVO represents a new standard of care in this patient population. Updated OS and safety data will be presented.

Background:
Nivolumab (NIVO), a fully human IgG4 programmed death-1 (PD-1), immune checkpoint inhibitor antibody, has demonstrated durable responses and tolerability in heavily pretreated patients with advanced non-small cell lung cancer (NSCLC). NIVO was recently approved for the treatment of patients with metastatic squamous (SQ) NSCLC with progression on or after platinum-based chemotherapy. Conducted mostly in community-based oncology centers, this ongoing trial explores the safety of NIVO in patients with previously-treated PD-L1[+/-] metastatic SQ or non-squamous (NSQ) NSCLC.

Methods:
Eligible patients are enrolled in 4 subgroups: 1) SQ, performance status (PS) 0–1, ≥2 prior therapies; 2) SQ, PS 0–1, 1 prior therapy; 3) NSQ, PS 0–1, ≥1 prior therapy; and 4) SQ or NSQ, PS 2, ≥1 prior therapy. Patients with both PD-L1[+] and PD-L1[-] tumors are eligible. Patients receive NIVO 3 mg/kg IV (60 minutes) Q2W either until progressive disease (PD)/unacceptable toxicity (Cohort A) or for 1 year with the possibility of retreatment upon disease progression (Cohort B). Primary objective is to estimate incidence of high-grade (CTCAE v4.0 Grade 3–4 and 5), select treatment-related adverse events (STRAEs); exploratory efficacy assessments include ORR, PFS, and OS.

Results:
From 4/16/14 to 12/31/14, 824 patients were treated and have demographic and safety data available; 483 patients remained on study as of 12/31/2014. 395 patients had evaluable radiographic tumor assessments at first assessment (Week 9). Demographics, safety, and tumor response by PD-L1 status are reported. Figure 1



Conclusion:
Safety and tolerability are consistent with prior NIVO experience and no new safety signals have been identified in this trial of SQ/NSQ NSCLC patients. Immune-related toxicities are manageable in a community practice setting using previously-developed safety algorithms. The frequency of STRAEs of interest was similar between patients with PS 0–1 and those with PS 2. Early data from this large, multicenter trial suggests that patients with pretreated advanced NSCLC benefit from NIVO therapy regardless of tumor PD-L1 status, histology type, and PS status.

Background:
Patients with advanced, refractory squamous (SQ) non-small cell lung cancer (NSCLC) have historically poor outcomes and limited treatment options. Nivolumab (NIVO), a fully human IgG4 programmed death-1 (PD-1) immune checkpoint inhibitor antibody, has activity across NSCLC histologies and is FDA-approved for treatment of metastatic SQ NSCLC with progression on or after platinum-based chemotherapy. We report efficacy, safety, and biomarker analyses from a phase 2, single-arm study of NIVO in patients with SQ NSCLC who progressed during/after prior platinum-based doublet chemotherapy and ≥1 additional systemic regimen.

Methods:
Patients (N=117) received NIVO 3 mg/kg every 2 weeks until progressive disease (PD)/unacceptable toxicity; treatment beyond PD was permitted per protocol. The primary endpoint was independent radiology review committee (IRC)-assessed objective response rate (ORR), per RECIST v1.1. Additional objectives included investigator-assessed ORR, progression-free survival (PFS), overall survival (OS), safety, ORR by patient subgroups, efficacy by tumor PD-L1 expression (PD-L1[+]: ≥5% tumor cells expressing PD-L1), and blood-based biomarker analyses (measurement of circulating microRNA and cytokines).

Results:
IRC-assessed ORR was 15% (95% CI: 9, 22), with a minimum of 11 months follow-up. Median duration of response was not reached (range, 2+–12+ months); 76% (13/17) of patients had ongoing responses. Objective responses were observed across patient subgroups and regardless of PD-L1 expression (Table). Four of 22 patients treated beyond PD demonstrated a non-conventional pattern of benefit (ie, persistent reduction in target lesions in the presence of new lesions, regression following initial progression, or no further progression for ≥2 tumor assessments); OS for these patients was 6.6, 11.6+, 12.9+, and 13.5+ months. The 1-year OS rate was 41% (95% CI: 32, 50) and median OS was 8.2 months (95% CI: 6.1, 10.9). The 1-year PFS rate was 20% (95% CI: 13, 29); median PFS was 1.9 months (95% CI: 1.8, 3.2). Peripheral increases in serum IFN-γ-stimulated cytokines, including CXCL9 and CXCL10, were observed, and preliminary microRNA analyses identified altered gene expression following NIVO treatment. Grade 3–4 treatment-related adverse events occurred in 17% of patients, including fatigue (4%), diarrhea (3%), and pneumonitis (3%). Pneumonitis was manageable with corticosteroids; median time to resolution was 3.4 weeks (range, 0.7–13.4). Two treatment-related deaths (1 hypoxic pneumonia, 1 ischemic stroke) occurred in patients with multiple comorbidities and concurrent PD. Figure 1



Conclusion:
NIVO demonstrated clinically meaningful efficacy and an acceptable safety profile in patients with advanced, refractory SQ NSCLC. Updated 18-month OS, safety, and biomarker analyses will be presented.

Abstract not provided

Background:
Combined blockade of the PD‐1 and cytotoxic T‐lymphocyte‐associated antigen‐4 (CTLA‐4) immune checkpoint pathways has shown improved responses, encouraging survival rates, and a manageable safety profile in advanced melanoma. NIVO, a fully human IgG4 PD-1 immune checkpoint inhibitor antibody, has activity across NSCLC histologies and is approved in the US for treatment of metastatic squamous (SQ) NSCLC with progression on or after platinum-based chemotherapy. This phase 1 study evaluated the safety and efficacy of first‐line therapy with NIVO plus ipilimumab (IPI), an IgG1 CTLA‐4 checkpoint receptor blocking antibody, in chemotherapy‐naïve patients with advanced NSCLC.

Methods:
Patients (N=49) received NIVO plus IPI at the 1+3 mg/kg or 3+1 mg/kg combination dose, respectively (one SQ and one non‐SQ cohort per dose level), every 3 weeks for 4 cycles, followed by NIVO 3 mg/kg every 2 weeks until progression or unacceptable toxicity. Objective response rate (ORR; RECIST v1.1) was evaluated overall and by baseline tumor PD‐1 ligand 1 (PD‐L1) expression (PD‐L1[+]: ≥5% tumor cells expressing PD‐L1). Response was assessed at weeks 10, 17, and 23, and every 3 months thereafter until progression.

Results:
Median follow‐up for all patients was 50 weeks. Across histologies, confirmed ORR was 13% (3/24) for NIVO1+IPI3 and 20% (5/25) for NIVO3+IPI1. Two of 3 and 4/5 responders in the NIVO1+IPI3 and NIVO3+IPI1 arms, respectively, achieved a response by first scan. Median duration of response was not reached (NR) in either group, and responses were ongoing in 67% (2/3) and 60% (3/5) of patients treated with NIVO1+IPI3 and NIVO3+IPI1, respectively. Two patients in the NIVO3+IPI1 group exhibited an unconventional “immune-related” response with 56% and 64% maximum reductions in target lesions and simultaneous appearance of new lesions. The 24-week progression-free survival (PFS) rates and median PFS were 44% and 16.1 weeks, respectively, for NIVO1+IPI3 and 33% and 14.4 weeks, respectively, for NIVO3+IPI1. One-year overall survival (OS) rates and median OS were 65% and NR, respectively, for NIVO1+IPI3 and 44% and 47.9 weeks, respectively, for NIVO3+IPI1. Thirty-eight of 49 treated patients were evaluable for PD-L1 expression; objective responses were observed in PD‐L1[+] (19%, 3/16) and PD‐L1[-] (14%; 3/22) patients. Across arms, grade 3–4 treatment-related adverse events (AEs) were reported in 25 patients (51%); grade 3 pneumonitis was reported in 3 (6%) patients. Treatment‐related AEs led to discontinuation in 18 patients (37%); 15 (31%) patients discontinued treatment during induction. Treatment‐related deaths (n=3) were due to respiratory failure, bronchopulmonary hemorrhage, and toxic epidermal necrosis.

Conclusion:
Treatment with NIVO plus IPI was associated with durable responses and encouraging survival regardless of tumor PD-L1 expression. The safety profile was managed using established safety guidelines. Updated OS and results from additional doses and schedules will be presented.

Background:
PD-L1 expression on tumor-infiltrating immune cells (IC) and/or tumor cells (TC) can inhibit antitumor immunity. Atezolizumab (MPDL3280A) is an anti-PDL1 antibody that has shown efficacy across multiple tumor types. The efficacy and safety of atezolizumab in the Phase 2 FIR study has been reported previously (Spigel et al, ASCO 2015). Efficacy appeared to correlate with PD-L1 expression on IC and/or TC, with higher ORRs observed in patients with the highest expression of PD-L1, indicating that PD-L1 may be a predictive biomarker for response to atezolizumab. FIR was also designed to address questions of potential heterogeneity and changes in tumor PD-L1 expression in metachronous tissue samples, as well as the utility of using FDG-PET as a biomarker for response to atezolizumab in PD-L1–selected patients with NSCLC.

Methods:
FIR is a 3-cohort, single-arm, Phase 2 study of atezolizumab in PD-L1–selected patients with stage IIIB/IV NSCLC. Cohort 1 included chemo-naive patients, Cohort 2 included ≥ 2L patients without a history of brain metastases, and Cohort 3 included ≥ 2L patients with asymptomatic treated brain metastases. PD-L1 expression was centrally assessed by immunohistochemistry (IHC) using the SP142 antibody assay in archival and/or fresh tumor biopsies or resections and scored as IC0, 1, 2 or 3 and TC0, 1, 2 or 3. Patients with PD-L1 IC2/3 or TC2/3 tumors were enrolled and received 1200 mg atezolizumab IV every 3 weeks (last patient entered Jun 27, 2014). Responses were measured by RECIST v1.1, modified RECIST and FDG-PET using EORTC criteria. Exploratory objectives included the evaluation of potential predictive biomarkers, including the comparison of PD-L1 expression in matched archival and fresh tumor specimens, as well as the utility of FDG-PET in assessing response to immune checkpoint blockade.

Results:
From 1009 screened patients, 95 paired archival and fresh tumor samples were obtained. In these samples, the agreement of PD-L1 expression between fresh and archival tissue at the TC3 or IC3 cutoff was 88% when the same type of tissue procurement method was used (resection or biopsy), compared with 65% when different methods of procurement were used. To date, FDG-PET response has been centrally assessed in 71 of the 138 patients enrolled in FIR. Patients with metabolic response by EORTC criteria on 6-week scans had a higher ORR per RECIST v1.1 (72% [13/18]) than metabolic non-responders (ORR 4% [2/53]).

Conclusion:
There was a high agreement in TC3 or IC3 PD-L1 expression between archival and fresh tumor specimens. This work demonstrates that intra-patient heterogeneity in PD-L1 expression is low in metachronous tissues, indicating various types of tumor samples, including fresh or archival, can be reliably used to assess PD-L1 expression. In addition, FDG-PET has potential as an early on-treatment measure of response to atezolizumab. Further analyses will be presented. (NCT01846416)

Background:
Despite advances in treatment for NSCLC, the standard first-line treatment for metastatic disease remains platinum-based doublet chemotherapy with historical overall response rates (ORRs) of ≈30%. Preclinical data suggest that chemotherapy treatment can result in antigen release in the tumor microenvironment, potentially enhancing effects of cancer immunotherapy. Atezolizumab (MPDL3280A) is a human monoclonal antibody that targets the PD-L1/PD-1 immune checkpoint, while leaving the PD-L2/B7.1 interaction intact (which may reduce the risk of autoimmune lung toxicity). As atezolizumab has shown promising activity in advanced NSCLC, we investigated atezolizumab in combination with chemotherapy.

Methods:
A Phase Ib study was conducted to evaluate atezolizumab with chemotherapy in locally advanced or metastatic NSCLC patients who had not received chemotherapy for advanced disease. Pts received atezolizumab 15 mg/kg IV q3w with standard chemotherapy (carboplatin plus either paclitaxel [Arm C], pemetrexed [Arm D; nonsquamous] or weekly nab-paclitaxel [Arm E]) for 4-6 cycles followed by atezolizumab maintenance until progression. RECIST v1.1 was used to assess ORRs (unconfirmed) in pts dosed by Jun 29, 2014 (data cutoff: Sep 29, 2014). PD-L1 expression was centrally evaluated using the SP142 IHC antibody assay.

Results:
37 NSCLC pts were safety evaluable (8 in Arm C; 14 in Arm D; 15 in Arm E). Across these arms, 54% of pts were male, with a median age of 65 y (range, 40-82 y). 81% had non-squamous NSCLC, and 19% had squamous NSCLC. Median safety follow-up was 22.0 wks (range, 0.1-49.4 wks). Across arms, all-Grade AEs regardless of attribution included those commonly associated with chemotherapy, such as nausea (Arms C & D, 50%; Arm E, 73%), fatigue (Arm C, 38%; Arm D, 36%; Arm E, 73%) and constipation (Arm C, 25%; Arm D, 71%; Arm E, 27%). The most common Grade 3-4 atezolizumab-related AEs included anemia (Arms D & E, 7%), neutropenia (Arm C, 13%; Arm D, 7%) and thrombocytopenia (Arms D & E, 7%), with no pneumonitis or autoimmune renal toxicity observed. One potentially atezolizumab-related Grade 5 AE was observed in Arm D (candidemia after prolonged neutropenia). 30 pts were efficacy evaluable, and responses were observed in all arms regardless of PD-L1 expression (Table). Updated clinical data will be presented.

Table. RECIST v1.1 Responses in Patients with NSCLC

	Arm C: carboplatin + paclitaxel (n = 5)	Arm D: carboplatin + pemetrexed (n = 12)	Arm E: carboplatin + nab-paclitaxel (n = 13)	All Indicated Arms (n = 30)
ORR, %	60%	75%	62%	67%
95% CI, %	19%-92%	45%-93%	33%-83%	48%-82%
CR, n	0	0	2	2
PR, n	3	9	6	18

Conclusion:
Atezolizumab plus standard first-line chemotherapy was well tolerated in advanced NSCLC pts, with no unexpected toxicities. Clinical activity was promising and supportive of a potential synergy of atezolizumab with chemotherapy. Based on these results, several Phase III studies have been initiated.

Abstract not provided

Background:
TG4010 is an immunotherapy using an attenuated and modified poxvirus (MVA) coding for MUC1 and interleukin-2. Previous Phase 2 trials have demonstrated the efficacy and safety of TG4010 in combination with chemotherapy. In addition, Triple Positive Activated Lymphocytes (TrPAL; CD16+, CD56+, CD69+) was identified as a potential biomarker predictive of efficacy

Methods:
TIME is a double blind, placebo-controlled phase 2b/3 study. The Phase 2b part compared first line chemotherapy combined with TG4010 or placebo and further assessed the predictive value of baseline level of TrPAL. Eligibility criteria included stage IV NSCLC not previously treated, MUC1+ tumor by immunohistochemistry, PS ≤1. TG4010 10[8] pfu or placebo was given SC weekly for 6 weeks (w), then every 3w up to progression in immediate combination with chemotherapy. Patients were randomized using TrPAL cut-off value (normal vs high) that was previously pre-determined in healthy subjects. Primary efficacy endpoint was progression-free survival (PFS) using a Bayesian design to confirm that, with a 95% probability, the true hazard ratio (HR) is <1 in patients with normal TrPAL level. Secondary objectives were response rate (ORR), duration of response, survival, safety and subgroup analyses according to histology and level of TrPAL.

Results:
222 patients (pts) were randomized 1:1. In pts with normal TrPAL the study met the primary endpoint with a Bayesian probability of 98.4% that the PFS HR is <1 in favor of TG4010. In the whole study population, ORR was 39.6% vs 28.8% and duration of response was 30.1w versus 18.7w in the TG4010 and placebo arms respectively. Survival data will be presented at the time of the meeting. Preplanned subgroup analyses showed that PFS was significantly improved in the TG4010 arm in pts with low TrPAL (n=152; HR=0.66 [CI95% 0.46-0.95] p= 0.013) while it was not the case in pts with high TrPAL (n=70; HR=0.97 [CI 95% 0.55-1.73] p=0.463). In addition, PFS was also significantly improved in pts with non-squamous tumors (n=196; HR=0.69 [CI95% 0.51-0.94] p=0.009) as well as in pts with non-squamous tumors and low TrPAL (n=131; HR=0.61 [CI95% 0.42-0.89] p=0.005). In this last group, PFS at 9 months was 37% with TG4010 versus 18% with placebo. Frequency and severity of adverse events were similar in both treatment arms except injection site reactions which were more frequent in the TG4010 arm but all of mild or moderate intensity. Exploratory analysis of the impact of PDL1 expression in the tumor of patients treated with TG4010 in TIME study supports the activity of TG4010 whether the tumor is positive or negative for PDL1 expression.

Conclusion:
These results provide additional data supporting the efficacy of TG4010, particularly in patients with non-squamous tumors and/or a low level of TrPAL at baseline. The Phase 3 part of the TIME study is planned to continue in patients with non-squamous tumors with OS as primary endpoint.

Background:
MUC1, a tumor antigen, has been considered to a promising target antigen for cancer immunotherapy because it possesses a potent immunogenicity. It is processed and presented by antigen-presenting cells in a MHC-unrestricted pattern. Dendritic cell-based vaccine immunotherapy can elicit antigen-specific cytotoxic T lymphocytes in tumor-bearing hosts, and activated cytotoxic T lymphocytes are expected to attack cancer cells. In this study, we evaluated the efficacy of MUC1-targeted dendritic cell-based vaccine immunotherapy in patients with standard treatments-refractory advanced non-small-cell lung cancer (NSCLC).

Methods:
The eligibility criteria of this immunotherapy were as follows: histologic or cytologic evidence of NSCLC that had been proven to express MUC1 abundantly; an Eastern Cooperative Oncology Group performance status of 0-2; advanced stage of diseases refractory for other standard cancer treatments. The dendritic cells were prepared from peripheral blood mononuclear cells with cytokines interleukin-4 and granulocyte macrophage colony stimulating factor, were pulsed with MUC1 peptides, and subsequently administered to patients subcutaneously. The vaccinations were repeated bi-weekly, and assessable patients were received at least 6 vaccinations. Tumor response was assessed according to the Response Evaluation Criteria in Solid Tumors. Adverse events were graded according to National Cancer Institute Common Toxicity Criteria.

Results:
From June 2005 and March 2015, 42 patients were treated with dendritic cell-based vaccines, and 29 patients (69.0%) with median age of 61 years (range, 49-84 years) were assessable for tumor responses. The cohort consisted of 18 males and 11 females. As their histological types, 24 patients had adenocarcinomas; 4 patients with squamous cell carcinomas and 1 patient with pleomorphic carcinoma. Among these assessable patients, neither complete response nor partial response was obtained. Seventeen patients had progressive disease as the best response, and 11 patients had stable disease, yielding overall disease control rate of 39.2% (95%CI=20.3-55.6). Median survival time after the vaccines was 10.0 months, and 1-year survival rate was 39.6%. Adverse events related to the vaccines were less frequent. Immunological responses were able to be monitored in five patients, showing that MUC1-specific cytotoxic responses of effector immune cells were achieved in all of those patients, and the population of regulatory T lymphocytes in peripheral blood cells was decreased after the vaccines.

Conclusion:
MUC1-targeted dendritic cell-based vaccine immunotherapy is feasible, and has a potential to control the diseases in patients with refractory NSCLC.

Abstract not provided

Background:
There is a significant unmet medical need for effective and well-tolerated treatment options for advanced NSCLC patients. Angiogenesis is a fundamental step in tumor growth and progression; its inhibition has become an attractive target as anticancer therapy. We conducted this meta-analysis to evaluate the effectiveness of adding anti-angiogenic therapy (AT) to standard of care (chemotherapy, tyrosine kinase inhibitors (TKIs) or best supportive care) in advanced NSCLC

Methods:
The electronic databases Ovid PubMed, Cochrane Central Register of Controlled Trials, Embase, the websites of European Society of Clinical Oncology, the American Society of Clinical Oncology and the Lung Cancer Association were searched to identify all eligible phase 3, randomized, controlled trials with AT for the treatment of advanced NSCLC. Pooled hazard ratios (HRs) for overall survival (OS) and progression-free survival (PFS), and pooled odds ratio (OR) for overall response rates (RR) were calculated. We divided the population into 2 subgroups based on the dose of Bevacizumab administered: 7.5 mg/kg (group 1) and 15mg/kg (group 2)

Results:
Data of 19098 patients (9867 AT; 9231 controls) from 25 phase III trials were analyzed. Compared with the standard of care alone, the addition of AT did not prolong OS (HR 0.98; 95% [CI] 0.96-1.00; p=0.1 and HR 0.97; 95% [CI] 0.94-1.00; p=0.06 for group 1 and 2 respectively). In an exploratory analysis, the addition of AT did not prolong OS for the adenocarcinoma histology and when it was used in the 1[st] line setting. Furthermore, there was no OS benefit regardless of the type of AT therapy i.e. monoclonal antibodies (Mabs) versus oral TKIs. There was a significant improvement in PFS with the addition of AT (HR 0.85; 95% [CI] 0.79-0.91; p<0.00001 and HR 0.81; 95% [CI] 0.75-0.88; p<0.00001 for group 1 and 2 respectively) and overall RR (OR 1.61; 95% [CI] 1.30-2.01; p<0.0001 and OR 1.72; 95% [CI] 1.39-2.14; p<0.00001 for group1 and 2 respectively).

Conclusion:
This is the 1[st] meta-analysis to our knowledge including all phase 3 trials with AT in NSCLC and showing that the addition of AT to the standard of care in advanced NSCLC had no significant effect on OS and only improved PFS and overall RR. The role of AT in advanced lung cancer is still questionable; strong validated biomarkers are eagerly needed to predict which subgroup might benefit the most from such therapy.

Background:
The use of targeted drugs has implied the development of new imaging techniques able to assess in vivo processes as part of antitumor response. Functional imaging techniques may be more appropriate to study changes in vascularization parameters such as blood flow (BF), blood volume (BV) and permeability (PMB) after treatment with antiangiogenics. Perfusion-computed tomography (pCT) could be a useful technique to predict non-small cell lung cancer (NSCLC) (pts) that most benefit from antiangiogenic therapy by assessing early variations of perfusion parameters.

Methods:
IMPACT (NCT02316327) is an ongoing open-label, single arm phase II/IV study to evaluate the predictive value of early perfusion changes in pts diagnosed with advanced non-squamous (ns)-NSCLC treated with bevacizumab in combination with chemotherapy. Patients receive cisplatin (80 mg/m2 i.v. d1), gemcitabine (1250 mg/m2 i.v. d1 and 8) and bevacizumab (B, 7.5 mg/kg i.v. d1) up to 6 cycles each 21 days. Pts with non-progressive disease are allowed to continue with B maintenance until PD or unacceptable toxicity. pCT assessment is done basal (d-1), at d+7 and d+42. The primary endpoint is to evaluate whether early reductions (d-1 vs d+7) in pCT parameters in terms of BF (mL/100mL/min), BV (mL/100mL) and PMB (mL/100mL/min) may predict response to bevacizumab as compared to Objective Response Rate (ORR) in terms of RECIST after 2 cycles (d+42). All perfusion evaluation parameters during treatment are measured in the same single thoracic target lesion. Planned sample size is 20 pts.

Results:
A total of 12 pts with ns-NSCLC have been recruited and data is available for analysis in 8 pts. Mean age is 62 years, 7 males and 1 female. All pts were diagnosed of adenocarcinoma stage IV (63% stage IVb). All tumor samples were negative for EGFR/ALK and 50% positive for KRAS. Mean cycles of chemotherapy were 5 (range 2-6) and 3 (range 0-12) of B maintenance. Target lesions for perfusion were: lung 3 pts (38%), lymph nodes in 4 pts (50%) and pleura in 1 pt (12%). No differences were found in terms of basal BF, BV and PMB depending on perfusion-target chosen. Four pts (50%) achieved partial response (PR), 3 pts (38%) stable disease (SD) and 1 pt (12%) progressive disease (PD). Mean basal perfusion parameters were: BF 61,5 (34,4 - 109), BV 10,4 (3,7 - 22,2) and PMB 17 (5,5 - 27,9). Mean perfusion changes early assessed by pCT at d+7 were: BF 21,7%, BV -49% and PMB -34,4%, decreasing consistently at day +42 (BF -46,8%, BV -45,5% and PMB -53,9%). Mean early variation (d-1 vs d+7) of BF in pts with SD/PD was +1,7% as compared with -45,3% in pts with PR. Mean variation of BF compared with d+42 (d-1 vs d+42) was also greater in pts with PR (-50%). Similar trends were observed in BV and PMB.

Conclusion:
Early response to B as assessed with p-CT may help to select those pts with NSCLC who most benefit from antiangiogenic therapy. Early changes in perfusion parameters can be identified with B treatment. Recruitment is ongoing.

Background:
Bevacizumab is an anti-VEGF monoclonal antibody approved for the treatment of non-squamous NSCLC. It is widely accepted that immunosuppressive mechanisms dominate in patients (pts) with solid tumors, including NSCLC. MDSCs are a heterogeneous population of immature cells of myeloid origin, whose expression is induced by VEGF. We recently identified two monocytic and one granulocytic MDSC subpopulation which are significantly increased and functional in the peripheral blood of NSCLC pts.

Methods:
Peripheral blood immune cells from 46 pts with unresectable NSCLC were analyzed by flow cytometry before the initiation of chemotherapy and after 3 cycles. Changes in the frequencies of the three MDSCs subpopulations were correlated with clinical outcome. Isolated MDSCs were co-cultured with T cells in order to confirm their functionality through estimation of IFN-γ secretion.

Results:
At diagnosis, the CD15(-) monocytic MDSCs’ levels were significantly increased in male pts (p=0.03) and in smokers (p=0.01). Overall, chemotherapy had no effect on the frequency of the distinct MDSC subpopulations. However, after 3 cycles of therapy, levels of all three MDSC subpopulations numerically decreased in responders (n=11) compared to non-responders (n=4). In addition, bevacizumab-based chemotherapy regimens significantly reduced the frequency of the granulocytic MDSC subpopulation when compared to the effect of non-bevacizumab based therapy (p=0.02). Lastly, suppression of IFN-γ secretion in vitro, confirmed the inhibitory effect of isolated MDSCs on T-cell cytotoxic capacity.

Conclusion:
These data indicate that although chemotherapy has no effect on the levels of different immunosuppressive MDSC subpopulations, bevacizumab–based regimens seem to exert an effect on the granulocytic MDSC subpopulation. Additional studies are needed in a larger cohort of pts in order to document its impact in the clinical outcome of NSCLC pts.

Abstract not provided

Background:
Adoptive T-cell immunotherapy (ACT) has shown great promise in melanoma and hematologic malignancies; however one major limitation of engineered T cells targeting solid tumors is likely to be tumor microenvironment-induced hypofunction of the T cells. To study and limit this problem, we have developed a model in which human T cells engineered to target the antigen NYESO1 using a high-affinity engineered TCR (Ly95) are injected into mice bearing human A549 lung cancer cells. Using this model we demonstrate upregulation of PD1 and TIM3 on Ly95 TILs. We were able to augment T cell anti-tumor activity by combining Ly95 T cell therapy with anti-hPD1 and anti-hTIM3 antibodies.

Methods:
In vitro: Human T cells activated by anti-CD3/CD28 Dynabeads and transduced with lentivirus had 50% expression of Ly95 TCR as measured by flow cytometry. They were cocultured with marked tumor cells to measure IFNg release and antigen-specific killing. In vivo: Immunodeficient mice with 200mm[3] flank A549-A2-ESO (AAE) tumors received 10[7] T cells via tail vein. Three weeks later, tumors were harvested/digested, and human TILs were isolated/assesssed for tumor killing/IFNg secretion. This was repeated after the TILs were rested for 24hrs at 37[0]C/5%CO2. The number of TILs and PD1/TIM3 expression on the isolated TILs were assessed by flow cytometry at fresh harvest and post rest. The in vivo experiment was repeated comparing Ly95 T cells alone vs. Ly95 T cells plus either/both intraperitoneal (IP) anti-hPD1 or/and IP anti-hTIM3 at 10mg/kg every 5 days.

Results:
Ly95 TCR T cells were able to kill AAE tumor cells and secrete high amounts of IFNg in an antigen-specific/dose dependent fashion after 18hr coculture. 10[7 ]IV Ly95 T cells were able to slow AAE flank tumor growth as compared to control tumors (498mm[3 ]vs. 1009mm[3], p<0.05.) Flow cytometric analysis of harvested/digested tumors revealed that 5.2% of the tumor digest was human TILs. Freshly isolated TILs were hypofunctional in their ability to kill tumor cells and release IFNg when compared to cryopreserved Ly95 T cells (p<0.05.) After overnight rest away from tumor, TILs improved in function. Further analysis revealed that Ly95 TILs had upregulated their expression of PD1 and TIM3 (increase from 5 to 40% in PD1 and from 17 to 50% in TIM3.) Combining a single Ly95 T cell IV injection with multiple IP anti-hPD1 and anti-hTIM3 injections resulted in 43% reduction in flank tumor size compared to Ly95 T cell injection alone (189mm[3] vs. 332mm[3], p<0.05.)

Conclusion:
The PD1 and TIM3 pathways are involved in tumor-induced hypofunction of TCR engineered TILs. Combining anti-hPD1 and anti-hTIM3 antibodies with TCR T cells, and likely CAR T cells, will likely enhance the efficacy of these approaches in lung cancer and other solid tumors.

Background:
Adoptive T-cell therapy using chimeric antigen receptors (CAR) is an emerging strategy by redirecting T-cell effector functions against a cancer cell-surface antigen. To target lung adenocarcinoma (ADC) by CAR T-cell therapy, our laboratory has identified mesothelin (MSLN), a cell-surface antigen based on our published observation that MSLN is expressed in 60% of primary and metastatic lung ADC and is associated with tumor aggressiveness. Unlike hematological malignancies where CAR T-cell therapy has been successful targeting CD19, a cell-surface antigen that is uniformly expressed on B cells, MSLN expression intensity and distribution among lung ADC tumors is heterogeneous. The efficacy of CAR T-cell therapy in a heterogeneous antigen microenvironment is unknown. We hypothesized that the MSLN-targeted CAR T cells will be effective against high-antigen expressing lung ADC cells and the presence of even a small proportion of high MSLN expressing cells can enhance CAR T-cell cytotoxicity against low-antigen expressing lung ADC cells.

Methods:
Human peripheral blood T cells were retrovirally transduced with a 2[nd] generation of CAR targeting MSLN and bearing CD28 and CD3zeta activation domains. In vitro, we analyzed CAR T-cell cytotoxicity ([51]Cr release assay), effector cytokine secretion (Luminex assay), and proliferation (cell-counting assay) against lung ADC cell lines expressing variable levels of MSLN. In vivo, antitumor efficacy was evaluated by median survival and tumor bioluminescence (BLI) in mice bearing established homogeneous or heterogeneous lung ADC tumors.

Results:
In in vitro assays utilizing lung ADC cells with variable level of MSLN expression [low-antigen expression (EKVX or A549) or high-antigen expression (A549M and H1299M), control lung fibroblast (MRC5) or mesothelial cells (MET5A)], CAR T cells exhibit antigen-specific cytolytic activity, effector cytokine secretion and proliferation in proportion to the MSLN expression on cancer cells. In vivo, a single low dose of CAR T cells eradicates primary and metastatic established tumor expressing high-level of MSLN and prolongs tumor free survival (41 days vs not reached, p<0.0001). We next evaluated CAR T-cell efficacy in heterogeneous antigen microenvironment by mixing low and high antigen-expressing cells (A549 expressing firefly luciferase/A549M) and assessed the A549 tumor burden only by bioluminescence imaging. In the presence of A549M cells, CAR T cells are able to prolong progression-free survival of A549 tumor burden (22 days vs 0 days in absence of A549M cells). Further mechanistic studies demonstrated that CAR T cells lysed an additional 5%-15% A549 or EKVX cells in the presence of H1299M or A549M cells (p<0.05) without off-target cytotoxicity. Antigen-activated CAR T cells were effective against low-antigen expressing lung ADC cells without the need for high-antigen expressing cells in the coculture.

Conclusion:
Our results provide scientific rationale to translate MSLN-targeted CAR T-cell therapy for the treatment of the primary and metastatic lung ADC. A phase I clinical trial (NCT02414269) that includes lung ADC patients is initiated at our center.

Background:
This abstract is under embargo until September 8, 2015 and will be distributed onsite on September 8 in a Late Breaking Abstract Supplement.

Methods:


Results:


Conclusion:

Background:
Translating recent chimeric antigen receptor (CAR) T-cell therapy successes in hematologic malignancies to solid cancers requires overcoming barriers unique to solid tumors such as inadequate tumor infiltration, proliferation, and persistence. Our laboratory has published the rationale to target mesothelin (MSLN), a cell-surface antigen expressed in the majority of thoracic malignancies. We hypothesized that the immune modulating effects of low-dose radiation therapy (RT) would enhance the infiltration and proliferation of mesothelin-targeted CAR T-cell therapy for thoracic cancers, thereby achieving long-term tumor eradication.

Methods:
Using human T cells retrovirally transduced to express mesothelin-targeted CARs, we evaluated T-cell cytotoxicity by chromium release assay, proliferation by cell count assay, cytokine-release by multiplex ELISA, phenotype by flow cytometry, and chemokine receptor profiles by PCR against MSLN-expressing mesothelioma and lung cancer cell lines with and without localized RT. In clinically relevant mouse models (NOD/SCID gamma mice) with established MSLN-expressing tumors, we monitored therapy response, T-cell kinetics and anti-tumor efficacy by utilizing bioluminescent imaging (BLI), and conducted flow cytometric analysis of splenic/peripheral blood T cells for characterization of CAR T-cell effector phenotype.

Results:
RT did not enhance CAR T-cell cytotoxicity. In vitro, RT enhanced CAR T-cell migration in chemotactic assays, and correlatively induced the secretion of chemokines by tumor cells (Fig.1A). In vivo, RT resulted in dose dependent chemokine secretion with robust early intratumoral CAR T-cell accumulation (p<0.05, Fig.1B) as demonstrated by T-cell BLI. Ex vivo tumor analysis by flow cytometry on day 7 post T-cell administration confirmed that RT increased early infiltration and proliferation (p<0.05). Also, single low-dose RT potentiated the efficacy of systemically administered CAR T cells (median survival 30d vs. 79d, p= 0.02) with at least 50% tumor eradication up to 100 days even with a 30-fold decreased dose (Fig.1C&D). Furthermore, in mice with tumor eradication, harvested spleen T-cell analysis at day 56 demonstrated a greater number of persisting CAR T cells in mice treated with RT (p=0.02, Fig.1E).Figure 1



Conclusion:
Our data provides the rationale to use localized RT as a preconditioning regimen prior to CAR T-cell administration in a clinical trial for thoracic malignancies. Furthermore, our mechanistic observation of RT-induced, chemokine-mediated, enhanced T-cell infiltration may also assist the trafficking of endogenous anti-tumor T cells, thereby shifting the balance towards a cohesive anti-tumor immune microenvironment.

Background:
Chimeric antigen receptor (CAR) T-cell therapy has shown durable remissions in hematological malignancies targeting cancer-antigen CD19. Ideal cancer-antigen targets for CAR T-cell therapy are antigens overexpressed on cancer cell-surface with limited expression in normal tissues, associated with tumor aggressiveness and expressed in a large cohort of patients. In our search for such candidate antigens in lung adenocarcinoma (ADC), we investigated the overexpression of Mesothelin (MSLN), MUC16 (CA125), and the combination of MSLN-MUC16 as the interaction of both antigens has been shown to play a role in tumor metastasis.

Methods:
In patients with stage I lung ADC (n = 912, 1995 - 2009), a tissue microarray consisting of 4 cores from each tumor and normal lung tissue was used to examine the antigen-expression characteristics, and their association with cumulative incidence of recurrence (CIR). Autologous metastatic tumor tissue was available from 36 patients. Differences in CIR between groups were tested using the Gray method (for univariate nonparametric analyses) and Fine and Gray model (for multivariate analyses).

Results:
MSLN and MUC16 were not expressed in normal lung tissue. In primary and metastatic lung ADC tumors, MSLN was expressed in 69% and 64%, MUC16 was expressed in 46% and 69%, both antigens were present in 50% and 33%, and either antigen were present in 33% and 49% respectively. On univariate analysis, patients with high MSLN expression had high risk of recurrence than low expression [5-year CIR, High: 25.1% vs Low: 17.6%, P = 0.017]. Patients with high MUC16 expression had high risk of recurrence than low expression [5-year CIR, High: 24.2% vs Low: 14.0%, P < 0.001]. Patients with high MUC16 and high MSLN had higher risk of recurrence than low expression [5-year CIR, High risk (High MUC16 and High MSLN): 27.6%, Intermediate risk (High MUC16 and Low MSLN): 24.2%, Low risk (Low MUC16): 13.6%, P < 0.001]. On multivariate analysis, increased MUC16-MSLN expression was associated with recurrence [Hazard ratio, 2.57 95% Confidence interval 1.41 – 4.68 P = 0.002], even after adjustment for currently known markers of lung ADC aggressiveness (gender, surgical procedure, stage, architectural grade and lymphatic invasion). High expression of MUC16 in the primary tumor was associated with high expression at recurrence sites.

Conclusion:
MSLN, MUC16 or a combination of expression of both antigens in patients with primary lung ADC is associated with increased risk of recurrence, a retained overexpression at metastatic sites in advanced lung ADC indicating that MUC16-MSLN expression is a marker of tumor aggressiveness. Expression in the majority of lung ADC patients imparting aggressiveness with no expression in normal lung provides the rationale to target MSLN and MUC16 for lung ADC CAR T-cell therapy.

Abstract not provided

Background:
Clinical trials of immune checkpoints modulators, including both programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) inhibitors, have recently shown promising activity and tolerable toxicity in pre-treated NSCLC patients. However the predictive role of PD-L1 expression is still controversial. This pooled analysis aims to clarify the association of clinical objective responses to anti PD-1/PD-L1 monoclonal antibodies (MoAbs) and PD-L1 expression in pre-treated NSCLC patients.

Methods:
Data from all published studies, that evaluated efficacy and safety of PD-1/PD-L1 inhibitors in pre-treated NSCLC patients, stratified by tumor PD-L1 expression status (immunohistochemistry, cut-off point 1%), were collected by searching in PubMed, Cochrane Library, American Society of Clinical Oncology, and World Conference of Lung Cancer, meeting proceedings. Pooled Odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated for the Overall Response Rate (ORR) (as evaluated by Response Evaluation Criteria in Solid Tumors, version 1.1), according to PD-L1 expression status.

Results:
A total of six studies, with 776 patients, were eligible. Pooled analysis showed that patients with PD-L1 positive tumors (PD-L1 tumor cell staining ≥1%), had a significantly higher ORR, compared to patients with PD-L1 negative tumors (OR: 2.53; 95% CIs: 1.65-3.87). Figure 1Figure 2





Conclusion:
PD-L1 tumor expression seems to be associated with clinical activity of anti PD-1/PD-L1 MoAbs, in pre-treated, NSCLC patients, suggesting a potential role of PD-L1 expression, IHC cut-off point 1%, as predictive biomarker for the selection of patients who may benefit more from these therapies. Further analysis from ongoing phase II/III clinical trials will provide more information about this observation.

Background:
Pembrolizumab is a humanized monoclonal antibody against PD-1 that has demonstrated robust antitumor activity and a manageable safety profile in patients with advanced malignancies, including NSCLC. Similar to other immune checkpoint inhibitors, immune-mediated toxicities have been observed with pembrolizumab. We characterized the incidence of potentially immune-mediated adverse events (AEs) and the use of systemic corticosteroids for their management in patients with NSCLC treated with pembrolizumab in the phase 1 KEYNOTE-001 trial (ClinicalTrials.gov, NCT01295827).

Methods:
550 patients with advanced NSCLC received pembrolizumab 2 or 10 mg/kg every 3 weeks (Q3W) or 10 mg/kg every 2 weeks (Q2W). Potentially immune-mediated AEs were derived from a prespecified list and considered regardless of attribution to study treatment by the investigator. High-dose corticosteroid use was defined as an initial dose of ≥40 mg/day prednisone or equivalent. Low-dose corticosteroid use was defined as an initial dose of <40 mg/day prednisone or equivalent.

Results:
71 (12.9%) patients experienced ≥1 immune-mediated AE, including 17 (3.1%) who experienced grade 3-4 events, 1 (0.2) who died because of an immune-mediated AE (pneumonitis), and 14 (2.5%) who discontinued pembrolizumab because of immune-mediated AEs. The median time to onset of the first immune-mediated AE was 104 days (range, 2-393 days). Immune-related AE incidence was similar in patients treated with pembrolizumab 10 mg/kg Q2W and Q3W. The most common immune-mediated AEs were hypothyroidism, pneumonitis, and infusion-related reactions (Table). Pneumonitis was the most common grade 3-4 toxicity. Excluding hypothyroidism, 74.2% of immune-mediated AEs had resolved at the time of data cutoff. Of the 71 patients who experienced immune-mediated AEs, 30 (42.2%) received corticosteroids: 20 received high dose, 10 low dose. The highest incidence of corticosteroid use was for pneumonitis (84.2%) and colitis (80.0%) (Table). The duration of initial steroid use ranged from 1 to 129 days. Analyses related to the impact of steroid use on pembrolizumab efficacy are ongoing and will be available for presentation. Figure 1



Conclusion:
Potentially immune-mediated AEs, particularly those of grade 3-5 severity, are relatively infrequent in patients with advanced NSCLC treated with pembrolizumab. As evidenced by the low rate of pembrolizumab discontinuation, most immune-mediated events were managed by temporary pembrolizumab interruption and corticosteroid use.

Background:
The CheckMate 017 (NCT01642004) randomized, open-label, global phase 3 study evaluated efficacy and safety of second-line nivolumab vs docetaxel in patients with advanced squamous (SQ) non-small cell lung cancer (NSCLC). Overall survival was significantly superior and duration of treatment longer for nivolumab vs docetaxel. The study also evaluated disease-related symptoms using the Lung Cancer Symptom Scale (LCSS).

Methods:
The LCSS includes 100 mm visual analog scales for 6 major lung cancer symptoms plus three global items evaluating the impact of symptoms; 0 represents the least severity and 100 the greatest severity. Assessment was performed every 4 weeks for nivolumab and every 3 weeks for docetaxel for the first 6 months on treatment, followed by every 6 weeks for the remainder of the treatment period for both study arms. Following treatment discontinuation, the LCSS also was assessed at two follow-up visits. The LCSS average symptom burden index (ASBI) was computed from the 6 individual symptom scores. Mean baseline and mean change from baseline of the LCSS ASBI at each assessment were summarized by treatment group. A study secondary endpoint was to estimate the proportion of patients whose LCSS ASBI showed a clinically meaningful improvement by week 12 (10 point or greater decrease, the minimally important difference [MID]), which was based on all randomized patients.

Results:
Patient baseline characteristics were generally balanced across treatment groups. LCSS completion rates for baseline and at least one subsequent assessment were 68.9% and 62.8% for nivolumab and docetaxel, respectively. Completion rates remained relatively consistent throughout assessments and by treatment arm. Baseline LCSS ASBI values were similar for nivolumab (29.6; standard deviation [SD] 16.4) and docetaxel (29.6; SD 14.7). By week 12, 20.0% (27/135; 95% CI: 13.6, 27.7) of nivolumab patients demonstrated clinically meaningful symptom improvement compared to 21.9% (30/137; 95% CI: 15.3, 29.8) of docetaxel patients. Examining mean changes from baseline in patients’ LCSS ASBIs at each assessment, the nivolumab group demonstrated statistically significant improvements from baseline at each assessment from week 12 through week 54, after which sample sizes dropped to fewer than 10 patients; from week 40 through 54, the mean improvements exceeded the MID. In contrast, docetaxel patients remaining on treatment had no statistically significant changes in LCSS ASBI through week 18, after which the sample dropped to fewer than 10 patients. In the two follow-up visits after treatment discontinuation, the mean of the LCSS ASBI for both nivolumab and docetaxel patients indicated similar worsening of symptoms relative to baseline (range, 5.5–9.5); for docetaxel patients, the differences from baseline were statistically significant.

Conclusion:
By week 12, the proportion of patients showing meaningful symptom improvement was similar for both the nivolumab and docetaxel groups. However, the overall average symptom burden while on nivolumab improved from baseline over most of the year of available follow up, while average symptom burden for docetaxel patients remained stable relative to baseline during their shorter time on treatment. These results show statistically and clinically significant reductions from baseline in lung cancer symptoms for patients with squamous NSCLC treated with second-line nivolumab.

Abstract not provided

Background:
Responses to PD-1 blockade have been induced in approximately 20% of advanced non-small cell lung cancer (NSCLC) patients with progressive disease after standard therapy [Garon, NEJM 2015]. One challenge is to understand how the immune response was initiated in responding patients. Tumor mutational burden has been associated with response to PD-1 checkpoint inhibitors in NSCLC [Rizvi, Science, 2015]. In addition, studies in melanoma patient-derived tumor specimens revealed that responses to PD-1/L1 blockade rely on pre-therapy tumor infiltration of activated T effector cells [Tumeh, Nature, 2014]. We hypothesize that clonal T cell infiltration is correlated with tumor mutational load and clinical response with PD-1 blockade.

Methods:
We studied tumor specimens in NSCLC patients treated with pembrolizumab at UCLA on the KEYNOTE -001 clinical trial. All patients signed informed consent for the trial as well as separate specimen acquisition protocols. Responses were classified by the investigators according to irRC. DNA was extracted and whole exome sequencing was performed at the UCLA Immunogenetics Core. DNA from the same patient’s PBMC or other non-cancerous tissue was sequenced for baseline comparison. Immunohistochemistry (IHC) was done for CD8 (Clone C8/144B, Dako), CD4 (Clone SP35, Cell Marque) and PD-L1 (Clone SP142, Spring Bioscience).

Results:
We report results from 27 patients (14 responders, and 13 nonresponders). Significantly higher density of pre-dosing CD8+ cells (percentage of CD8+ nucleated cells) in the tumors of the responding patients was observed (mean of 17.7% in responders vs 5.6% in non-responders, p=0.02 by unpaired t test) suggestive of a pre-existing immune response. Mutational load in 5 patients (3 responders and 2 nonresponders) showed a trend towards correlation with response (mean of 19 nonsynonymous somatic mutations per MB in responders vs 6 in nonresponders, p=0.33). Interestingly, a strikingly significant correlation between mutational load and CD8 expression was observed (R[2]=0.96, p=0.003). In addition, pre-dosing tumor PD-L1 expression demonstrated a trend towards correlation with response (mean of 72.1% in responders vs 51.5% in nonresponders, p=0.07) but not with CD8 tumor infiltration (R[2]=0.05, p=0.28). No significant association of CD4+ T cell tumor infiltration with response (mean of 37.4% CD4 + cells in responders vs 27.0% in nonresponders, p=0.32) was observed.

Conclusion:
We observed strong correlation of pre-dosing intratumoral T cell infiltration with response and mutational load in NSCLC patients treated with pembrolizumab. Our results have direct implications for the design and interpretation of ongoing and planned immunotherapy studies for NSCLC and evaluation of potential predictive biomarkers to select patients most likely to benefit.

Background:
New therapeutic modalities in metastatic squamous non-small cell lung cancer (SQ NSCLC) focus on targeting pathways (programmed cell death 1 [PD-1]) involved in inhibiting anti-tumor T cell responses leading to tumor evasion. Nivolumab, an anti-PD-1 monoclonal antibody, blocks T cell inhibitory signal pathways by preventing engagement of PD-1. On March 4, 2015, FDA approved nivolumab for the treatment of patients with metastatic SQ NSCLC with progression on or after platinum-based doublet chemotherapy. The approval was based on a randomized study (CA209017) demonstrating a large magnitude of improvement in overall survival (OS) and was supported by single arm study (CA209063) demonstrating a 15% objective response rate (ORR), which appeared to be durable. We conducted a retrospective exploratory responder analysis to evaluate the association between response and OS in study CA209063. Figure 1



Methods:
CA209063 was a multicenter, multinational, single-arm, open-label study in patients with SQ NSCLC who previously received at least two lines of systemic therapies. Patients (n=117) received nivolumab 3 mg/kg as an intravenous (IV) infusion every 2 weeks until progressive disease (PD) or toxicity; treatment past PD was allowed if certain “clinical benefit” criteria were met. Response was defined as a partial response (PR) or complete response (CR) as determined by a blinded independent review committee (IRC) utilizing the Response Evaluation Criteria In Solid Tumors (RECIST) criteria (98 of 117 were evaluable). Responders were categorized into the following groups: A CR or PR, stable disease (SD), PD with continuation of treatment, and PD with discontinuation of treatment. A sensitivity landmark-based analysis was performed to exclude timing of response evaluation bias (Anderson et al, 1983).

Results:
The exploratory responder analysis showed that patients who achieved a best response of CR or PR had the longest survival with anti-PD1 therapy, followed by patients who either achieved a best response of SD or PD with continuation of treatment beyond RECIST progression. Patients whose best response was PD and no treatment beyond progression had poor survival (figure 1). The Landmark time-based sensitivity analysis at 3.5 months (median time to response) also suggested that responders had longer survival than non-responders.

Conclusion:
Our analysis suggests that patients with NSCLC who respond are likely to derive the most clinical benefit from anti-PD1 therapy. However, given the exploratory retrospective nature of this analysis, results should be interpreted cautiously. Further development of predictive biomarkers to identify patients most likely to respond is necessary.

Background:
Patients with advanced non-small cell lung cancer (NSCLC) often develop brain metastases (BrMs), and standard therapy such as surgery or radiation can cause toxicity and delay systemic treatment. Pembrolizumab is a PD-1 inhibitor with promising clinical activity and a favorable toxicity profile in patients with advanced NSCLC, however the efficacy of pembrolizumab in the central nervous system (CNS) is unknown. This trial aims to determine the safety and activity of pembrolizumab in patients with advanced NSCLC and untreated brain metastases.

Methods:
Eligibility for patients with NSCLC in this Phase II trial includes the presence of at least 1 BrM between 5 and 20 mm that is asymptomatic, untreated or progressing after prior local therapy, and not requiring urgent local therapy. PD-L1 expression in tumor obtained since the most recent systemic therapy is required. Patients are treated with pembrolizumab 10mg/kg every 2 weeks. Systemic response is determined by RECIST 1.1, and BrM response is determined by modified RECIST (mRECIST) in which brain lesions ≥ 5mm are considered measurable and up to 5 target lesions are allowed. The primary endpoint of this trial is BrM response rate.

Results:
Fifteen patients with NSCLC and untreated BrMs were treated with pembrolizumab, none of whom had a drug-related Grade ≥ 3 adverse event (AE) or any grade AE attributed to BrMs. Of the 10 patients evaluable for response, 5 (50% with 95% CI: 0.24-0.76) had a BrM response (4 partial and 1 complete) and 5 had a systemic response. Only one patient who responded in the body had progressive disease in the brain; all other patients who had a systemic response also had a CNS response. The duration of response in the brain was at least 12 weeks for 4 of the 5 responders, and all responses are ongoing at the time of data analysis.

Conclusion:
To our knowledge this is the first study to demonstrate that the PD-1 inhibitor pembrolizumab has activity in the CNS in patients with NSCLC and untreated brain metastases. To date there have been no drug-related neurologic or significant toxicity identified. Patient enrollment and biomarker analysis are ongoing.

Abstract not provided