Virtual Library

Abstract:
The majority of patients with lung cancer present in advanced stages, and small biopsy and cytology specimens most frequently provide the only tumor material for diagnosis. Furthermore, the same sample is also needed for molecular studies that guide treatment and management. Lung carcinomas often are diagnosed by minimally invasive techniques and specimens are obtained by bronchoscopic or transthoracic approaches. The choice of the procedure depends on the location, accessibility of the mass and other clinical parameters. Since most adenocarcinomas are peripherally located, the transthoracic approach is often chosen to provide diagnostic samples. Squamous cell carcinoma is most frequently centrally located and the bronchoscopic approach is more common. If cytology specimens, including pleural fluids, are obtained, cell blocks should be prepared. Despite the best efforts, the lesional tissue or the most representative area of the tumor may not be obtained in some cases due to sampling issues. Even when the tumor is sampled, poor tumor differentiation or insufficient characteristic morphological features in the tumor sample will cause the difficulty in rendering a specific diagnosis and would prompt pathologists to use immunohistochemistry. Use of immunohistochemistry greatly reduced the number of lung carcinoma cases classified as non-small cell carcinoma, NOS to only 3%. .In addition to immunohistochemistry, cytology samples should be interpreted in conjunction with histology of small biopsies whenever possible. Immunohistochemistry should be limited only to cases when classification is uncertain and every effort should be made to preserve as much material as possible for molecular studies. There are few strategies besides limited immunohistochemistry panels how this could be accomplished. One approach is to cut unstained slides from a paraffin block after initial hematoxylin-eosin stain sections were obtained. It is essential that histology technicians limit facing of the block and place only one tissue section per slide. Another approach is to have each core biopsy tissue fragment placed into separate blocks during specimen processing so only one block can be used for immunohistochemistry and all of the blocks can be used for molecular studies. Same laboratories in order to avoid tumor microdissection from the unstained slides prefer to core paraffin blocks by 1-mm needles after diagnostic work up. Formalin-fixed, paraffin-embedded tissue samples and cytology aspirates can be used for various testing platforms including next generation sequencing. Each molecular laboratory should establish criteria for specimen adequacy for molecular studies taking into account the specific testing platforms, while surgical pathologists should assess the specimen adequacy. Although PCR-based methods can detect mutations from a single cell, a low copy number DNA template can generate sequence artifacts leading to false results. Therefore, the assessment of adequacy is essential to avoid assays failures and false positive/negative results. Estimates of tumor content from H&E stained sections vary between pathologists and there is no true standard. Acceptable specimens should have a sufficient amount of tumor cells, but also a small proportion of admixed non-neoplastic cells, and no necrosis.[116] If the specimen is inadequate, a new specimen needs to be procured although in this situation decision regarding specimen type is often difficult and depends on many factors including the patient’s health. Alternate non-invasive highly sensitive methods so called “liquid biopsies” have been developed to detect the presence of cancer specific mutations in circulating DNA in blood samples. This approach may result in significant changes in the management of lung cancer patients and may replace invasive procedures. Until then, it is essential that each institution develops its own strategy that addresses the collection and processing of lung cancer samples. At the same time, pathology departments must implement the procedures that would precisely define how to spare the tumor tissue for molecular testing, and how to provide clinically acceptable turnaround time for molecular testing results. It is also essential to define how to integrate diagnostic interpretation and molecular results in a single pathology report. References: Travis WD et al. Diagnosis of lung cancer in small biopsies and cytology: implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. Arch Pathol Lab Med 2013; 137(5):668-84. Lindeman NI. et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from CAP/IASLC/AMP. J Thorac Oncol 2013:8(7):823-59. Francis G, Stein S.Circulating cell-free tumor DNA in the management of cancer. Int J Mol Sci. 2015;1 (6):14122-42

Abstract:
With 1.6 million new diagnoses and 1.4 million annual deaths worldwide, 230,000 annual cases and 160,000 deaths in the US, lung cancer is the oncologic scourge of the present age. It accounts for 23% of worldwide, and 28% of US, cancer deaths.1 Four decades of advances in diagnostic and treatment modalities, including the current ‘molecular’ decade of rapid-fire discovery of breakthrough therapeutics, have seen aggregate US 5-year survival improve from 12% to 17%.2 Although the deployment of effective population-based tobacco cessation and lung cancer screening programs can change these statistics, there remains the danger of blunted impact because of relatively little progress in the coordination of care, and major deficits in the use of curative-intent therapy. Lung cancer care is complicated. The disease is common and lethal; the primary at-risk population is ravaged by cumulative age- and tobacco-related comorbidities; the lungs and mediastinum are relatively inaccessible; multiple approaches and techniques for diagnosis, staging, and treatment exist, each requires different, highly-trained specialists (radiologists, pulmonologists, surgeons, medical oncologists, radiation oncologists, pathologists, palliative care specialists), using high-cost equipment, to perform high-risk procedures, any one of which may or may not be needed for specific patients. Determining which specific specialists and management approaches are needed for individual patients requires objective evaluation and careful coordination, in order to tailor management to patient needs. Prevailing nihilism about lung cancer care further complicates matters: ‘statistics suggest the patient will die anyway, so what’s the point?’, ‘he brought this on himself by smoking, whose problem is this?’ Care delivery must be better coordinated before we can achieve meaningful improvement in population-level survival statistics. All lung cancer care begins with an abnormal chest x-ray or CT scan. From then, it flows through certain ‘nodal points’: histologic confirmation, radiologic staging, histologic staging, selection of treatment, and (ultimately) outcomes. Each nodal point includes a myriad of options. Diagnosis is usually made by percutaneous (interventional radiologist), bronchoscopic (pulmonologist), or surgical (thoracic surgeon) biopsy ; radiologic staging often involves a PET/CT scan (nuclear radiologist), and brain MRI scan (neuro-radiologist); histologic staging requires an invasive biopsy procedure, which can be transbronchial needle biopsy during conventional bronchoscopy, endobronchial ultrasound-guided (EBUS) biopsy (pulmonologist), endoscopic ultrasound-guided (EUS) biopsy (gastroenterologist), percutaneous image-guided biopsy of a distant stage-defining lesion (interventional radiologist), mediastinoscopy, or other approaches to various parts of the mediastinum (surgeon). Treatment increasingly requires combinations of surgery, radiation therapy, chemotherapy and palliative care. Therefore lung cancer care demands a high degree of coordination. Major, well-described geographic, socio-economic, racial, and age-based disparities in diagnosis, staging, treatment, and outcomes suggest that healthcare systems fail to provide the required level of care coordination. The proportion of patients who make it to surgery, the most important curative treatment modality, varies from 9% in the UK, to 29% in the US.3 Use of invasive staging tests and surgical resection is significantly lower in African Americans than in Caucasians.4,5 Elderly patients are less likely to receive chemotherapy than younger patients.6 Although partly driven by patient choice, improvement in care coordination narrows or eliminates most disparities.7,8 Beyond disparities, access to high-quality care is generally low. ‘Trimodality’ staging (CT, PET/CT scan and invasive staging in combination) although associated with a 2-fold survival improvement was used in only 5% of US patients.9 In a high lung cancer mortality zone of the US, only 17% of curative-intent resections were preceded by invasive staging, including only one-third of patients with clinical N1, N2 or N3 disease (Osarogiagbon, unpublished data). Low rate of histologic confirmation of stage-defining lesions raises the danger of overuse and underuse of treatment modalities. At the extremes are primary surgical resection for patients with clinically evident mediastinal nodal disease, and palliative systemic chemotherapy for patients with false-positive radiologic staging tests or multiple primary cancers erroneously classified as stage IV. Avoiding misuse of diagnostic and staging modalities is equally difficult. The need to recognize and correctly act on non-diagnostic, false-negative and false-positive test results is great. Lung cancer care is often delayed when insufficient-quality diagnostic material or sampling error leads to erroneous reassurance that a high-risk radiologic lesion is benign. The problem is even greater in the use of invasive staging tests. More than half of US mediastinoscopy procedures fail to deliver lymph node tissue for pathology examination.10 This dismal statistic is probably worse with EBUS and EUS. The need for high-quality tissue rises as prognostic and response-predictive implications steadily increase our need for clear histologic categorization (and sub –categorization), and with the advent of molecular prognostication and treatment selection. The demand for high-quality tissue spans the stage spectrum. Therapeutic clinical trials now routinely demand tissue for molecular testing, surgical resection trials increasingly mandate a minimum quality of nodal staging. Patients’ eligibility for clinical trials and our ability to accelerate testing and deployment of novel treatments increasingly hinge on tissue procurement. Pathologists and interventionists who procure tissue must work collaboratively to increase tissue yield for the numerous purposes of treating clinicians. This must be achieved while maintaining patient safety and convenience. The countervailing forces of increasing adoption of minimally invasive diagnostic, staging and treatment modalities and ravenous hunger for high-quality tissue for prognostication and treatment selection collide within individual patients and healthcare systems. Only better coordination, involving all relevant clinicians in early strategic decision-making for each individual patient can prevent the delays, anxieties, exposure to harm, missed opportunity for better treatment outcomes, and looming medico-legal risk that the status quo in lung cancer care represents. Rising survival rates will only increase this conflict, as the need for re-characterization of disease rises, and lung cancer care evolves from a game of checkers to a chess match. REFERENCES 1. Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin 2011;61:69-90. 2. http://seer.cancer.gov/statfacts/html/lungb.html. Accessed May 28, 2015. 3. Moghissi K, Connolly CK. Resection rates in lung cancer patients. Eur Respir J 1996;9:5-6. 4. Bach PB, Cramer LD, Warren JL, Begg CB. Racial differences in the treatment of early-stage lung cancer. N Engl J Med 1999;341:1198-205. 5. Lathan CS, Neville BA, Earle CC. The effect of race on invasive staging and surgery in non-small-cell lung cancer. J Clin Oncol 2006;24:413-418. 6. Earle CC, Venditti LN, Neumann PJ, et al. Who gets chemotherapy for metastatic lung cancer? CHEST 2000;117;1239-1246. 7. Laroche C, Wells F, Coulden R, et al. Improving surgical resection rate in lung cancer. Thorax 1998;53:445-449. 8. Brawley OW. Lung cancer and race: equal treatment yields equal outcome among equal patients, but there is no equal treatment. J Clin Oncol 2006;24:332-333. 9. Farjah F, Flum DR, Ramsey SD, et al. Multi-modality mediastinal staging for lung cancer among Medicare beneficiaries. J Thorac Oncol 2009;4:355-363. 10. Little AG, Rusch VW, Bonner JA, et al. Patterns of surgical care of lung cancer patients. Ann Thorac Surg 2005;80:2051-6.

Abstract not provided

Abstract:
The personalized treatment of lung cancer begins with an accurate histologic diagnosis. Lung cancer is a heterogeneous disease and recent advances in understanding the genetic underpinnings of cancer coupled with the development of targeted therapeutics have added to the complexity of the diagnostic evaluation. The previous simple division of lung cancer into small cell and non-small cell histology is no longer adequate, and evaluation of tumors for specific genetic changes and their expression is essential for the precise and accurate diagnoses required for optimal treatment and patient management. In 2011, the International Association for the Study of Lung Cancer (IASLC), the American Thoracic Society (ATS), and the European Respiratory Society (ERS) published a new pathological classification of lung adenocarcinoma.[1] This new system established uniform terminology and diagnostic criteria for both resection specimens and small biopsies. Based on advances in the understanding of lung tumor biology, the correlation between specific tumor histology and outcomes with selected chemotherapy agents, as well as the successful development of novel targeted therapies, this new classification system emphasized the importance of a multidisciplinary approach to the diagnosis of lung cancer in order to best guide management decisions. In the IASLC/ATS/ERS system, several changes to pathological classification of adenocarcinoma were proposed.[1] The term “BAC” (bronchioloalveolar carcinoma) was discontinued and new pathological subtypes were added, including adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma. For patients with invasive adenocarcinoma, the new system proposed comprehensive histological subtyping with classification based on predominant histological pattern (lepidic, acinar, papillary, micropapillary and solid). Mucinous tumors were classified as mucinous AIS, mucinous MIA or invasive mucinous adenocarcinoma based on extent of invasion. In subsequent validation studies, pathological subtyping using the IASLC/ATS/ERS adenocarcinoma classification system has been demonstrated to have both prognostic and predictive significance.[2, 3] Patients with AIS and MIA have close to 100% disease-free survival. In contrast, invasive adenocarcinomas with solid and micropapillary predominant histological subtypes were associated with worse overall survival.[2, 4] Because most patients with lung cancer will be diagnosed with advanced-stage disease, one of the most important distinctions between the 2011 IASLC/ATS/ERS classification system and the previous 2004 World Health Association (WHO) classification is the establishment of diagnostic criteria for small biopsies and cytology. Although the focus was on adenocarcinoma, diagnostic criteria for other histologies were also included with the primary focus on distinguishing between squamous cell carcinoma and adenocarcinoma. A limited diagnostic work up was recommended to preserve as much tissue as possible for molecular testing. In cases where the diagnosis can be established based on light microscopy alone, the WHO classification criteria were maintained and no additional confirmatory testing was recommended. In tumors without definite squamous or adenocarcinoma morphology, limited immunohistochemistry may be used to refine the diagnosis. Most tumors can be classified using a single adenocarcinoma marker (i.e. TTF-1 or mucin) and a single squamous marker (i.e. p40 or p63.[5] The acknowledgement that one needs to “preserve as much tissue as possible” for biomarker testing reflects the changing uncertainty in which biomarkers need to be assessed and what techniques might be available for testing. With the identification and validation of actionable molecular targets that can guide therapy in patients with advanced adenocarcinoma, institutions are encouraged to develop consistent strategies for obtaining and managing tissue samples that are optimized for molecular testing. Because this approach has not been validated for squamous histology, tumors with equivocal morphology or IHC findings should be classified as NSCLC-NOS so as not to exclude patients from histology-specific chemotherapy or molecular testing. In 2013, the College of American Pathologists (CAP), IASLC and Association for Molecular Pathology (AMP) published a molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors that included 37 recommendations addressing 5 principal questions, including: (1) When should molecular testing be performed? (2) How should EGFR testing be performed? (3) How should ALK testing be performed? (4) Should other genes be routinely tested in lung adenocarcinoma? And, (5) How should molecular testing be implemented and operationalized?[6, 7] In addition to EGFR and ALK, a number of other molecular alterations have been described that are potentially treatable with targeted agents. Because the data were insufficient to support routine testing of other targets when the CAP/IASLC/AMP guidelines were finalized, the recommendation was to prioritize EGFR and ALK testing over other molecular markers in order to reserve tissue for these analyses.[6] The type and number of molecular alterations being evaluated ultimately determine how a limited specimen should be used. Immununohistochemical (IHC) and Fluorescent In Situ Hybridization (FISH) are the standard approaches for the detection of protein expression and chromosomal rearrangements and amplifications. Specific single nucleotide polymorphisms (SNPs) that are recognized mutations influencing response to specific therapies demand nucleic acid based tests utilizing a PCR based approach or, in the extreme, DNA sequencing. Most of these assays are typically developed as singleton tests for one specific SNP. As the number of necessary biomarkers increases, efficiency demands multiplexing these singleton assays or adopting a different technology, e.g. Next Gen Sequencing (NGS).[8-10] However, the timeliness for reporting results vary depending on the platform and the number of genes. It needs to be understood that none of these technologies can detect all of the types of genomic alterations of interest and that all of these methodologies must remain in the armamentarium available for proper specimen evaluation. With this in mind, efficient use of limited tissue demands clear communication between oncologists, surgeons, interventional radiologists and pathologists with regard what is needed for patient care and an understanding of available resources, either in house or through an appropriate reference laboratory. A further complexity is the timeliness in obtaining a result. While good practice suggests establishment of institutional protocols, individual patient needs ultimately dictate how best to proceed, emphasizing the importance of good interdisciplinary communication References: 1. Travis, W.D., et al., International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol, 2011. 6(2): p. 244-85. 2. Hung, J.J., et al., Prognostic value of the new International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society lung adenocarcinoma classification on death and recurrence in completely resected stage I lung adenocarcinoma. Ann Surg, 2013. 258(6): p. 1079-86. 3. Song, Z., et al., Prognostic value of the IASLC/ATS/ERS classification in stage I lung adenocarcinoma patients--based on a hospital study in China. Eur J Surg Oncol, 2013. 39(11): p. 1262-8. 4. Gu, J., et al., Prognostic significance of the IASLC/ATS/ERS classification in Chinese patients-A single institution retrospective study of 292 lung adenocarcinoma. J Surg Oncol, 2013. 107(5): p. 474-80. 5. Travis, W.D., et al., Diagnosis of lung cancer in small biopsies and cytology: implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. Arch Pathol Lab Med, 2013. 137(5): p. 668-84. 6. Lindeman, N.I., et al., Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. J Thorac Oncol, 2013. 8(7): p. 823-59. 7. Leighl, N.B., et al., Molecular testing for selection of patients with lung cancer for epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinase inhibitors: American Society of Clinical Oncology endorsement of the College of American Pathologists/International Association for the study of lung cancer/association for molecular pathology guideline. J Clin Oncol, 2014. 32(32): p. 3673-9. 8. Rekhtman, N., et al., Suitability of thoracic cytology for new therapeutic paradigms in non-small cell lung carcinoma: high accuracy of tumor subtyping and feasibility of EGFR and KRAS molecular testing. J Thorac Oncol, 2011. 6(3): p. 451-8. 9. Sholl, L.M., et al., Multi-institutional Oncogenic Driver Mutation Analysis in Lung Adenocarcinoma: The Lung Cancer Mutation Consortium Experience. J Thorac Oncol, 2015. 10(5): p. 768-77. 10. Gailey, M.P., et al., Multiplatform comparison of molecular oncology tests performed on cytology specimens and formalin-fixed, paraffin-embedded tissue. Cancer Cytopathol, 2015. 123(1): p. 30-9.

Abstract:
The ability to identify and therapeutically target specific mutations (typically exon 19 deletions and L858R) in the Epidermal Growth Factor Receptor (EGFR) gene marked the beginning of personalized medicine for NSCLC. Phase III clinical trials with the EGFR tyrosine kinase inhibitors gefitinib, erlotinib and afatinib demonstrated superiority of these agents over chemotherapy establishing these agents as standard therapy for EGFR mutation positive NSCLC. However, resistance to therapy invariably occurs through multiple, heterogeneous mechanisms of which a secondary gatekeeper mutation in EGFR, T790M, is the most frequent, being identified in 50-60% of patients at the time ofprogression after initial EGFR TKI. EGFR T790M is thought to result in resistance by increasing the affinity for ATP rather than simple stearic hinderance. Recently novel irreversible inhibitors, structurally distinct to earlier generation compounds, have been developed that inhibit T790M while having relatively less potency against wildtype EGFR including rocelitnib (C01686) and AZD9291. Phase I/II studies have demonstrated responses to rocilitinib (CO1686) and AZD9291 in about 60% of patients with T790M positive disease. Preliminary data indicates the degree of response may correlate with the allelic frequency of T790, with greater degrees of responses in patients with higher proportions of T790M. Resistance to these compounds has been described clinically and includes loss of T790M, small cell transformation as well asdevelopment of tertiary mutations C7957 (resistance to AZD9291).

Abstract:
Recent advances in targeted therapies and the molecular analysis of tumor samples have led to recommendations that EGFR mutation testing be implemented as standard of care in non-small cell lung cancer (NSCLC) of non-squamous type. This is in large part because of substantial benefits provided to patients treated with EGFR tyrosine kinase inhibitor (TKI) therapy, particularly those patients whose tumors are positive for activating, sensitizing mutations in EGFR. Despite these benefits, resistance to EGFR TKIs inevitably develops in all cases. The most common molecular mechanism of acquired resistance in this setting, occurring in approximately 50% of cases, is the evolution of a secondary ‘gatekeeper’ mutation which results in p.T790M (T790M). This acquired mutation results in a reduction in affinity of EGFR for the TKIs, while preserving catalytic function of the tyrosine kinase domain, The testing for T790M at the time of progression on TKI has emerged as an important clinical practice, as new-in-class EGFR TKIs demonstrate activity against this subset of resistant tumors. Because T790M initially emerges as a sub-clonal event, technical elements involved in its detection become paramount. Most importantly, achieving a high technical sensitivity to allow for detection of a sub-clonal (low alleleic frequency) phenomenon is critical for assays designed to detect this mutation. Sanger sequencing, for example, lacks the technical sensitivity to adequately identify low variant frequency events, and therefore alternate mechanisms of testing are required. Additional factors requiring major consideration in detection of T790M include potential sampling bias (particularly for the small biopsies or fine needle aspirates that are typically acquired), heterogeneity between multiple progressing lesions, cellular components of post-treatment biopsies, and technical ability to perform tumor enrichment to enhance detection. Genomic alterations resulting in T790M are uncommonly detected prior to TKI therapy, likely owing to the technical sensitivity of assays used to query for the presence of this alteration. Studies employing extraordinarily sensitive assays have demonstrated the presence of T790M as a subclonal event prior to TKI therapy in many tumors. Thus clonal selection under the pressure of TKI is a major mechanism allowing this alteration to be identified in the setting of progression on targeted therapy using less analytically sensitive assays. The secondary implication of this finding is that assays employed for detection of T790M must be adequately sensitive, but not over-sensitive to allow for the appropriate identification of what can best be considered the dominant mechanism of resistance. Others have postulated that highly sensitive detection of T790M prior to therapy could be used to determine a combination therapy approach which effectively prevents the evolutionary advantages of this sub-clone. In the uncommon instance that this alteration is identified at a high level pre-TKI therapy using standard assay approaches (<5% of cases), it can be associated with a germline alteration leading to a genetic predisposition for lung cancer. Recently, there has been great interest in the potential to monitor for the emergence of T790M alterations in the periphery, either via circulating tumor cells or circulating cell-free DNA. This approach is particularly attractive as it reduces requirements for invasive tissue sampling and can allow for a continuous monitoring approach. While the technical elements of liquid biopsy testing have been diversely applied, with very little in the way of consensus on methodology, numerous studies have demonstrated the promise of liquid biopsy approaches for both primary mutation detection as well as evaluation for T790M. In some cases, peripheral detection of T790M was demonstrated substantially before radiographic evidence of progression, a key proof-of-principle that such monitoring could be utilized as an effective approach for disease monitoring. Similar approaches for chimerism analysis or BCR-ABL1 transcript monitoring in the setting of bone marrow transplant or TKI therapy for chronic myelogenous leukemia, respectively, have been very successful. Major challenges still exist for both tissue-based and peripheral blood-based detection of T790M. Determination of the ideal level of assay technical sensitivity required for prediction of response to T790M-directed therapies will be a critical component to the implementation of these drugs in the clinic. In addition, for tissue biopsies, techniques to enhance the tumor cellularity of tested material and to avoid sample bias will need to be further refined. Liquid biopsy techniques, though demonstrating extraordinary promise, are widely divergent in terms of methodologies employed, and further study in this technological space is needed. 1. Arcila ME, Oxnard GR, Nafa K, et al. Rebiopsy of lung cancer patients with acquired resistance to EGFR inhibitors and enhanced detection of the T790M mutation using a locked nucleic acid-based assay. Clinical cancer research : an official journal of the American Association for Cancer Research 2011;17:1169-1180. 2. Inukai M, Toyooka S, Ito S, et al. Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer. Cancer Res 2006;66:7854-7858. 3. Kim Y, Ko J, Cui Z, et al. The EGFR T790M mutation in acquired resistance to an irreversible second-generation EGFR inhibitor. Mol Cancer Ther 2012;11:784-791. 4. Majem M, Remon J. Tumor heterogeneity: evolution through space and time in EGFR mutant non small cell lung cancer patients. Transl Lung Cancer Res 2013;2:226-237. 5. Newman AM, Bratman SV, To J, et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nature medicine 2014;20:548-554. 6. Oxnard GR, Paweletz CP, Kuang Y, et al. Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clinical cancer research : an official journal of the American Association for Cancer Research 2014;20:1698-1705. 7. Paweletz CP, Janne PA. Monitoring cancer through the blood. Cancer 2014;120:3859-3861. 8. Sorensen BS, Wu L, Wei W, et al. Monitoring of epidermal growth factor receptor tyrosine kinase inhibitor-sensitizing and resistance mutations in the plasma DNA of patients with advanced non-small cell lung cancer during treatment with erlotinib. Cancer 2014;120:3896-3901. 9. Tartarone A, Lerose R. Clinical approaches to treat patients with non-small cell lung cancer and epidermal growth factor receptor tyrosine kinase inhibitor acquired resistance. Ther Adv Respir Dis 2015. 10. Watanabe M, Kawaguchi T, Isa SI, et al. Ultra-Sensitive Detection of the Pretreatment EGFR T790M Mutation in Non-Small Cell Lung Cancer Patients with an EGFR-Activating Mutation Using Droplet Digital PCR. Clinical cancer research : an official journal of the American Association for Cancer Research 2015.

Abstract:
Acquired resistance to initial EGFR TKI invariably develops in patients with EGFR-mutant lung cancer after a median of 9-14 months. Though acquired EGFR resistance in NSCLC is a clinical condition that is treated and studied worldwide, there has until recently been a paucity of prospective data describing the best practices for managing these patients. In 2014, the first phase III trial studying EGFR-mutant lung cancer with acquired resistance was reported. Presented at ESMO 2014, the IMPRESS trial established platinum doublet chemotherapy as the standard second-line therapy for these patients, with no benefit to additionally continuing EGFR TKI at progression. And yet, even with standard second-line therapy established, there remain questions regarding how to manage patients with progressive disease (PD) on EGFR TKI, questions that will likely grow even more complex should newer agents reach the market. One way of framing this question is to consider the tools we have for managing acquired EGFR resistance, and then to consider how to best utilize them. The following outline provides a brief summary of therapeutic strategies that will be discussed further at WCLC 2015. · Continued TKI after PD - Feasible for a median of 3 months, especially in those with slow or asymptomatic PD (Lo et al, Cancer, 2015; Park et al, ESMO, 2014) · Retreatment with TKI after PD on chemo - 25% RR on a prospective phase II study of 20 patients, but responses were brief with a 3.4 month median PFS (Oh et al, Lung Cancer, 2012) · Afatinib – Limited activity in LUX-Lung 1 trial with 7% RR and 3 month median PFS (Miller et al, Lancet Oncol, 2010) · Afatinib / cetuximab – 29% RR and 4.7 month median PFS, with responses seen regardless of T790M status (Janjigian et al, Cancer Disc, 2014) · Cytotoxic chemotherapy – Cisplatin /pemetrexed has a 34% RR and 5.4 month median PFS after PD on gefitinib (Mok et al, ESMO, 2014) · Erlotinib & bevacizumab – May delay development of PD, but no prospective data for treatment of resistance (Seto et al, Lancet Oncol, 2014) · Erlotinib & crizotinib – Hypothetical option for MET-mediated resistance but requires dose reduction of both (Ou et al, ASCO, 2012) · Nivolumab – 82 patients with prior TKI and chemo treated on CheckMate 057, and there was no OS benefit seen compared to docetaxel (HR 1.18) (Paz-Arez, ASCO, 2015) · Erlotinib & nivolumab – 3 of 20 patients responded (15%) in the phase I study (Rizvi et al, ASCO, 2014) · Brain radiation – For CNS-only progression, radiation followed by restarting TKI can gain additional months of PFS (Weickhardt et al, JTO, 2012) · SBRT or surgical resection – An approach that is hypothesized to debulk a single resistant clone, thus delaying clinical resistance and prolonging the progression-free period (Weickhardt et al, JTO, 2012; Yu et al, JTO, 2013)

Abstract:
The introduction of EGFR TKIs has dramatically changed the natural history of advanced and/or metastatic NSCLC. The objective response rates of 50 to 70% are achieved and overall survival has improved from 4-5 months to over 30 months in EGFRm(+) NSCLC patients. However, unfortunately the patients are not cured of the disease and after a median PFS of 9 to 13 months, the disease comes back eventually with the emergence of acquired resistance(AR) to EGFR TKIs. Mechanisms of AR include target gene modification, activation of bypass tracks or histologic transformation, etc. In approximately 60% of patients, the mechanism of resistance is due to the acquisition of a gatekeeper T790M EGFR mutation. This T790M mutation leads to an enhanced affinity for ATP, thus reducing the ability of ATP-competitive reversible EGFR tyrosine kinase inhibitors, including gefitinib and erlotinib, to bind to the tyrosine kinase domain of EGFR. One strategy to overcome this mechanism of resistance mediated by target gene modification is through the use of more potent, novel, next-generation inhibitors. The ‘2[nd]-generation’ irreversible EGFR inhibitors such as afatinib and dacomitinib, covalent inhibitors of HER family kinases, showed preclinical activity against T790M in vitro. Both agents demonstrated excellent clinical activities in EGFR TKI-naïve patients with EGFR-mutant NSCLC in terms of response rate and progression-free survival as compared to cytotoxic chemotherapy. However, the results of treatment in patients with EGFR-mutant lung cancer who progress on an EGFR TKI are quite disappointing. Studies of afatinib monotherapy among patients with acquired resistance to erlotinib or gefitinib showed a response rate of only 7-8% and a progression-free survival of 3 to 4 months. This result may be due to the fact that physiologic doses of current generation irreversible EGFR TKIs do not fully inhibit EGFR T790M and dose escalation of 2[nd]-generation EGFR inhibitors is limited by on-target inhibition of wild-type EGFR, which leads to EGFR-mediated toxicity (skin rash and diarrhea). The so-called ‘3[rd]-generation’ EGFR TKIs are pyrimidine-based irreversible inhibitors and has mutant-specific activity including T790M mutation while sparing wild-type EGFR. There are several 3[rd]-generation EGFR TKIs under development, e.g., AZD 9291, CO-1686(Rociletinib), HM61713, ASP8273, EGF816, to name a few. The early clinical trials of the 3[rd]-generation EGFR TKIs have demonstrated a promising efficacy in patients with advanced EGFR-mutated NSCLC who have progressed on prior EGFR TKI therapy, including cohorts of patients with EGFR T790M-mutated NSCLC. For CO-1686, the reported overall response rate in the phase 1 study was 59%(27/46) in patients with centrally confirmed EGFR T790M-containing tumors. Median progression-free survival was 13.1 months. Likewise, initial results from the phase I trial of AZD9291 demonstrated a response rate of 61%(78/127) in patients with EGFR T790M positive tumors with median PFS of 9.6 months. Both AZD9291 and CO-1686 have recently been granted Breakthrough Therapy designation by the US FDA based upon results from early clinical studies. Early phase I/II results of HM61713 also showed encouraging anti-tumor activity with objective response rate of 55%(34/62) in T790M positive Korean NSCLC patients and global phase II trial is planned to launch. The early results of EGF816 and ASP8273, another irreversible 3[rd]-generation EGFR TKIs under clinical development, were recently reported and both agents demonstrated encouraging response rates of 50-60% in T790M(+) NSCLC patients after progression on a 1[st] or 2[nd] generation EGFR TKIs. Further studies are ongoing and mature results are awaited. In brief, many of the 3[rd] generation EGFR TKIs currently at various stages of development look so promising with encouraging clinical activities for T790M(+) NSCLC patients esp. in terms of response rate. In general these newer generation EGFR TKIs also have much better toxicity profiles as they spare the wild-type EGFR, e.g., less skin rash, diarrhea or paronychia compared with the 1[st]- or 2[nd]-generation EGFR TKIs though the toxicity profiles are slightly differerent one from another at some aspects. Since the follow-up is rather short we need longer follow up to confirm survival benefits. We certainly have made a significant progress in the management of advanced NSCLC with AR to EGFR TKIs, however, there are still several issues to be investigated to further improve the treatment outcomes, e.g., optimal timing and/or sequence of the 3[rd] generation EGFR TKIs, how to delay or prevent the emergence of resistance to 3[rd] generation agents, CNS progression, management of non-T790M-dependent AR to EGFR TKIs, etc. References Cong CR and Jänne PA. The quest to overcome esistance to EGFR-targeted therapies in cancer. Nat Med. 2013;19(11):1389-1400 Lovly CM and Shaw AT. Molecular Pathways: Resistance to Kinase Inhibitors and Implications for Therapeutic Strategies. Clin Cancer Res. 2014;20(9):2249–56. Jänne PA et al. AZD9291 in EGFR Inhibitor–Resistant Non–Small-Cell Lung Cancer, N Engl J Med. 2015 Apr 30;372(18):1689-99. Sequist LV et al. Rociletinib in EGFR-Mutated Non–Small-Cell Lung Cancer, N Engl J Med. 2015 Apr 30;372(18):1700-9. Park K et al. Updated safety and efficacy results from phase I/II study of HM61713 in patients (pts) with EGFR mutation positive non-small cell lung cancer (NSCLC) who failed previous EGFR-tyrosine kinase inhibitor (TKI). PASCO 2015 #8084 Tan D S-W et al. First-in-human phase I study of EGF816, a third generation, mutant-selective EGFR tyrosine kinase inhibitor, in advanced non-small cell lung cancer (NSCLC) harboring T790M. PASCO 2015 #8013 Goto Y et al. ASP8273, a mutant-selective irreversible EGFR inhibitor in patients (pts) with NSCLC harboring EGFR activating mutations: Preliminary results of first-in-human phase I study in Japan. PASCO 2015 #8014T

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Background:
Clinical studies with anti-EGFR agents demonstrate that EGFR TKIs play critical roles in the treatment of non-small cell lung cancer, especially in patients with positive EGFR mutation. Icotinib is an oral, selective EGFR TKIs. Phase 3 study showed that icotinib is non-inferior to gefitinib in treating unselected or EGFR-mutated advanced NSCLC patients as second-line therapy but better safety profile, which provide a rationale to examine icotinib in first-line setting. The objective of this study is to evaluate progression-free survival (PFS), overall survival (OS) and safety of icotinib in chemotherapy naïve NSCLC patients with EGFR mutation.

Methods:
In this phase 3, open-label, randomized study (CONVINCE, NCT01719536), 285 patients (pathologically confirmed NSCLC, positive 19/21 EGFR mutation, treatment naive) will be 1:1 randomized to receive oral icotinib (125 mg, three times daily) or cisplatine (intravenous [IV], 75 mg/m2, day 1) plus pemetrexed (IV, 500 mg/m2, day 1), patients achieving disease control after 4-cycle chemotherapy continue to receive single pemetrexed (IV, 500 mg/m2, day 1) as maintenance therapy until progression. Randomization will be stratified by performance status (0-1/2), smoking status (smoker/non-smoker), disease stage (IIIB/IV), and mutation type (19/21). A total of 228 events would provide 90% power to detect an HR for PFS of 1 at 2-sided significance level of 0.05. Response will be reviewed by both investigator and independent data monitoring committee. Patient enrollment was completed in June 2014, and the results are expected in June, 2015.

Results:
Not applicable

Conclusion:
Not applicable.

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). 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:
Targeted therapies have considerably improved the prognosis of patients with non-small cell lung cancer (NSCLC).Although not precision enough, RESIST criteria was still the most often used response assessment method to reflecting the clinical benefits. We propose a non-invasive, folate receptor (FR)–based circulating tumor cell (CTC) detection approach to interpret treatment response of targeted therapy between baseline and follow-up CTC values in EGFR mutation/ALK translocation advanced NSCLC.

Methods:
One hundred and thirty eight patients were enrolled in our study. Peripheral blood was analyzed for CTCs enumeration on negative enrichment by immunomagnetic beads. Changes of CTCs levels were correlated with radiological response. Sequential analyses were conducted to monitor CTC signals during therapy and correlate radiological effects with treatment outcome.

Results:
CTCs were detected (≥8.7CTC) in 84.8% of patients. Pretreatment and pro-treatment blood samples from all 118 EGFR-mutant (19deltion:56, L858R:57, G719x:3, L861Q:1, 19 deletion + L858R:1), 14 ALK translocation lung cancer patients and 6 EGFR wild type patients were collected. Of 89 eligible and evaluable patients, baseline CTC counts were not associated with response to treatment by RECIST (P=0.353). There is no difference between exon 19 deletion and L858R of baseline CTC values. (19deletion:19.4 CTCs, L858R:20.9 CTCs,P=0.222) The change of CTCs values increased correlation with radiological response (P=0.042) after treatment of targeted therapy. There is no significant difference between exon 19 deletion and L858R of CTCs values pre and pro EGFR-TKI treatment.(3.32 vs.12.1, P=0.783)

Conclusion:
This study confirms the predictive significance of CTCs in patients with EGFR mutation/ALK translocation NSCLC receiving targeted therapy. The change of CTCs value correlated significantly with radiological response. This strategy may enable non-invasive, specific biomarker assessment method for using CTC decreases as an early indication of response to targeted therapy and monitoring in patients undergoing targeted cancer therapies.

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Background:
Epidermal growth factor receptor (EGFR) and KRAS mutations, and echinoderm microtubule-associated protein-like 4 (EML4) anaplastic lymphoma kinase (ALK) translocation are generally considered to be mutually exclusive. However, some reports show that a number of patients may have concomitant mutations, and it is not yet clear what impact these double mutations could have on response to targeted therapy.

Methods:
We took into consideration 380 NSCLC patients who underwent non-sequential testing for EGFR and KRAS mutations and EML4-ALK translocation between January 2010 and December 2013. EGFR mutation and EML4-ALK translocation analysis were performed on the entire case series and KRAS mutation analysis was performed on 282 cases.

Results:
EGFR mutation and EML4-ALK translocation were present in 44 (11.6%) and 32 (8.4%) of patients, respectively. Ninety-two patients (32.6%) showed a KRAS mutation. Two concomitant mutations among EGFR, KRAS or EML4-ALK genes were observed in 16 patients. In particular, 6 of the 380 (1.6%) patients analyzed had concomitant EGFR mutation and EML4-ALK translocation. Of the 282 patients who also underwent KRAS mutation, 3 (1.1%) showed a concomitant EGFR and KRAS mutation and 7 (2.5%) a concomitant EML4-ALK and KRAS alteration. Of the 44 EGFR-mutated patients, 28 received a TKI-based treatment (24 with gefitinib and 4 with erlotinib) as first-line therapy, and 6 of these also had an EML4-ALK translocation. Among the 22 patients with EGFR mutation only, we observed 2 complete response (CR) (9%), 16 partial response (PR) (72.7%) and 4 progressive disease (PD) (18%). Of the 6 patients who also had an EML4-ALK translocation, one had CR (17%), 3 PR (50%) and 2 PD (33%). No differences were seen in terms of overall survival (OS). Of the 32 patients harboring the EML4-ALK translocation, 6 (those also carrying the EGFR mutation) were treated with a TKI as first-line therapy, while the others received chemotherapy. Twelve patients received crizotinib as second-line treatment and 7 progressed within 3 months of starting therapy. Of these, 2 showed a concomitant KRAS mutation (G12C) and one a concomitant EGFR mutation (exon 19 del). Two patients had stable disease, one of whom also showed a KRAS mutation (G12V). Two patients had PR and one had CR, all of whom showed a EML4-ALK translocation only. The median OS of the patients carrying an EML4-ALK translocation alone or a concomitant KRAS mutation was 57.1 (range 10.7-nr) and 10.7 (range 4.6-nr) months, respectively.

Conclusion:
The concomitant presence of EGFR, EML4-ALK or KRAS mutations is a possible event in NSCLC. KRAS mutation in patients with EML4-ALK translocation represents the most common double mutation and seems to confer a poor prognosis.

Background:
Genetic intratumoral heterogeneity has a profound influence on the selection of clinical treatment strategies and addressing resistance to targeted therapy. The purpose of our study is to explore the potential effect of intratumoral heterogeneity on both the genetic and pathologic characteristics of ALK-rearranged lung adenocarcinoma (LADC).

Methods:
We tested ALK fusions and EGFR mutations in 629 LADC patients by using laser capture microdissection (LCM) to capture spatially separated tumor cell subpopulations in various adenocarcinoma subtypes and test for ALK fusions and EGFR mutations in ALK-rearranged, EGFR-mutated, and ALK/EGFR co-altered LADCs in order to compare the oncogenic driver status between different tumor cell subpopulations in the same primary tumor.

Results:
Among the 629 patients, 30 (4.8%) had ALK fusions, 364 (57.9%) had EGFR mutations, and 2 had ALK fusions coexisting with EGFR mutations. Intratumoral heterogeneity of ALK fusions was identified in 9 patients by RT-PCR. In the 2 ALK/EGFR co-altered patients, intratumoral genetic heterogeneity was observed both between different growth patterns and within the same growth pattern. Genetic intratumoral heterogeneity of EGFR mutations was also identified in EGFR-mutated NSCLC. ALK fusions were positively associated with a micropapillary pattern (P=0.002) and negatively associated with a lepidic pattern (P=0.008) in a statistically-expanded analysis of 900 individual adenocarcinoma components, although they appeared to be more common in acinar-predominant LADCs in the analysis of 629 patients.

Conclusion:
Intratumoral genetic heterogeneity was demonstrated to co-exist with histologic heterogeneity in both single-driver and EGFR/ALK co-altered LADCs. As for the latter, one of the dual altered drivers may be the trunk-driver for the tumor.

Background:
Lung adenocarcinoma (LAC) patients (p) with EGFR mutations respond initially to EGFR tyrosine kinase inhibitors (TKIs) but invariably develop acquired EGFR TKI resistance. Prior studies identified the EGFR T790M mutation and activation of MET, NF-kB, PI3K, AXL, HER2 and the MAPK pathway as drivers of acquired EGFR TKI resistance. We hypothesized that tumor cell populations present pre-treatment harbor mechanisms of EGFR TKI resistance that are subsequently selected for by EGFR TKI therapy.

Methods:
We performed longitudinal comprehensive molecular tumor profiling on 10 p with metastatic EGFR-mutant LAC throughout the course of their disease. Exome sequencing to a mean depth of coverage of 100 X, was performed on FFPE or frozen patient tumor specimens as well as matched normal control specimens collected from patients prior to initiating standard erlotinib (erl) treatment, upon the development of erl resistance, and upon resistance to subsequent 2[nd] line therapy when available. One case of a patient with acquired resistance to the 3[rd] generation EGFR TKI Rociletinib was analyzed. We performed functional analysis of select mutations identified using established cellular models of EGFR-mutant LAC.

Results:
We constructed phylogenetic trees based on somatic mutations and copy number alterations identified by exome sequencing of longitudinally acquired patient specimens. Activating mutations (L858R or exon 19 deletion) were present in all tumor specimens analyzed, indicating that this is a ‘truncal’ event. We identified on-target mutation in EGFR (T790M) in ~ 50% of erl resistant specimens as expected. However, in three patients we identified concurrent low frequency oncogenic driver events pre-EGFR TKI treatment that subsequently increased in frequency upon erlotinib resistance. This included: 1) a BRAF V600E mutation that was detected pre-treatment at a low frequency that expanded in the erlotinib resistant tumor specimen; 2) a PIK3CA G106V mutation that was not present in a patient’s primary tumor, but developed in a lymph node metastasis at a low frequency and subsequently expanded in the erlotinib resistant tumor, and 3) a pre-treatment KRAS amplification that was found in a patient with de novo resistance to erlotinib. The functional significance of these mutations in driving tumor growth and EGFR TKI resistance will be discussed. We will also present exome sequencing analysis from multiple tumors (including a CNS and spinal metastasis) collected from the autopsy of a patient with initial response, but rapid development of acquired resistance to Rociletinib.

Conclusion:
These results indicate that EGFR-mutant LAC can harbor additional oncogenic driver mutations at low frequencies prior to therapy. EGFR TKI treatment can lead to expansion of these subclonal populations likely contributing to EGFR TKI resistance in patients with or without the EGFR T790M resistance mutation. These data demonstrate the utility of comprehensive molecular profiling of LAC p on targeted therapy beyond assessing EGFR T790M mutational status, and suggest that pre-treatment tumor analyses can in some cases predict mechanisms of EGFR TKI resistance before they become clinically significant.

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Background:
Identification of EML4-ALK fusion proteins has revolutionized the treatment of a subgroup of non-small cell lung cancer (NSCLC) patients. Although the gene inversion is regarded as the relevant event for therapy response, the relation between gene rearrangement, mRNA and protein levels has not been evaluated in detail. Thus, the objective of this study was to comprehensively define the molecular relations induced by ALK rearrangements in a large representative Swedish NSCLC cohort incorporating genomic, gene expression and protein data, as well as corresponding clinical correlates.

Methods:
ALK protein analysis was performed on 860 NSCLC patients (551 adenocarcinoma, 224 squamous cell carcinomas, 85 large cell carcinomas/NOS) using immunohistochemistry (IHC) on tissue microarrays (TMAs), applying an established monoclonal ALK antibody (clone D5F3, Cell signaling). In parallel, ALK rearrangement was determined by fluorescent in situ hybridization (FISH, Abbott, Vysis ALK Break Apart FISH Probe Kit) on the same TMAs. A subgroup of patients was additionally analyzed utilizing gene expression microarrays (Affymetrix, n=194) or RNA-sequencing (n=202). The RNA sequencing data was also used to identify ALK gene fusions.

Results:
ALK protein expression was observed in 12/860 (1.4%). ALK rearrangement was detected in 11/860 samples (1.3%) by FISH analysis. Of 194 patients evaluated by microarray, six (3.1%) showed high ALK gene expression and of 202 patients analyzed by RNA-seq, nine (4.5%) demonstrated high ALK transcript levels. Of the 11 FISH rearranged patients, eight (73%) showed positive protein expression. High ALK gene expression was observed in all four ALK-FISH rearranged samples with matching microarray or RNA-seq data. Of five patients with positive protein expression, only three (83%) showed high gene expression levels according to gene expression microarray and RNA-seq data. RNA-seq revealed that 2/202 samples were ALK rearranged, both of which were detected by FISH and IHC. One sample that was not rearranged according to RNA-seq-data did, however, demonstrate rearrangement with FISH.

Conclusion:
The overall frequency of ALK rearrangements in this NSCLC cohort was lower than previously reported, with a significant but variable correlation on different molecular levels. It is possible that technical issues with regard to the use of TMAs, where only a fraction of the whole tumor is represented, may have hampered the results. Therefore, the FISH and IHC analysis will be complemented with assessments on whole tissue sections. The discordant results also stress the need for careful validation of these methods before they can be implemented in the clinical practice.

Background:
EML4-ALK oncogene fusion in non- small cell lung cancer, identifies patients sensitive to ALK-targeted inhibitors. Estimates of the frequency of this fusion oncogene rearrangement are primarily available from selected patient cohorts. The true incidence in an unselected Caucasian population is unknown. This study assess the incidence of ALK rearrangement in a population based cohort, together with correlation to gender, age, smoking habits, as well as pathological and clinical.

Methods:
All patients in a well-defined catchment area of 1.7 million people in the capital region of Denmark diagnosed with pulmonary adenocarcinomas from April 1. 2013 to July 31. 2014 were prospectively included. The type and location of the diagnostic material, and data on smoking and clinical characteristics were registered. The rearrangement analyses were investigated by up-front analysis with immunohistochemistry (IHC) using clone 5A4 Novocastra and all IHC positive tests were also subsequently tested by FISH using Zytovision, spec. ALK Dual Color Break Apart.

Results:
Among 797 patients included in this study, 777 patients (97.5%) patients had sufficient material for mutation analysis. Fourteen patients (1.8%, 95% CI 1.1-3.0) were IHC positive, all with 3+ reaction. All but one of these were also FISH positive. Eight patients (57%) were women. Median age was 62.7 years. All tumors were strongly TTF1 positive with mucin present in the cytoplasm of the malignant cells without dominance of any subtype. Ten patients (71.4) were diagnosed in stage IIIa or higher. Nine patients (64.3%) were never smokers, 3 (21.4%) were light smokers (0.5-10 yrs), 2 (14.3 %) were heavy smokers (25-40 yrs). More than 1/3 (36%) of the analyses were done on cytological, cellblock material. Seven patients had localized or locally advanced disease and did not receive crizotinib. Among seven patients with advanced disease, six received crizotinib with one complete response in a light smoker (male) and three partial responses in two never and one light smoker (response rate 67%). One out of three females receiving crizotinib achieved a response while it was three out of three males. No heavy smokers received crizotinib despite an ALK translocation was identified. Median progression free survival for patients receiving crizotinib was 3.4 months (range 0-20 months).

Conclusion:
ALK rearrangement analysis was possible in 97.5% of all patients with pulmonary adenocarcinomas. 1.8% had a positive test. Rearrangements were primarily found in never/light smokers. No difference in gender regarding rearrangement status was observed. Response rate to crizotinib was 67% and was in this study more frequent in males than in females (not significant). Chance of response was equal in light and in never smokers.

Background:
The lung adenocarcinomas can be divided into terminal respiratory unit (TRU) and non-TRU types, as these tumors frequently show distinctive morphologic and gene expression characteristics. Tumors co-expressing thyroid transcription factor-1 (TTF-1) and mucins MUC5B and/or MUC5AC exhibit intermediate morphology between TRU-type and non-TRU-type adenocarcinomas. Few studies have focused on this type of adenocarcinoma.

Methods:
176 patients with lung adenocarcinoma were retrospectively reviewed. The tumors were divided into TRU type, non-TRU and the intermediate type by morphology and immunohistochemistry (TTF-1, MUC5B, MUC5AC). The expression of Napsin-A, CK20, epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) rearrangement were also evaluated.

Results:
TTF-1, MUC5B, MUC5AC, Napsin-A and CK20 were detected in 157 (89.2%), 52 (29.5%), 10 (5.7%), 143 (81.3%), and 10 (5.7%) patients, respectively. EGFR mutation and ALK rearrangement were present in 56 (31.8%) and 13 (7.4%) patients, respectively. 99, 44 and 33 patients, respectively, were defined as TRU-type, intermediate-type, and non-TRU-type by morphology and immunohistochemistry (Fig 1). A cribriform pattern and extracellular mucus were present in 44 (25.0%) and 38 (21.6%) patients, and the intermediate type was associated with a cribriform pattern and extracellular mucus morphologically, a transitional phenotype in Napsin-A and EGFR mutations (Fig 2). ALK rearrangement tumors were significantly associated with the expression of MUC5B (P = 0.026). The intermediate type present a higher incidence of ALK rearrangement compared with the other types (P = 0.005), which ALK rearrangement were detected in 8 of 44(18.2%) cases. There was no significant difference in prognosis between the morphological TRU and non-TRU types (P = 0.076). However, when the tumors were classfied into 3 groups, TRU type showed better prognosis than the other types (P = 0.038). Figure 1 Figure 2





Conclusion:
Tumors co-expressed TTF-1, MUC5B, and/or MUC5AC had a high incidence of ALK rearrangement and exhibited distinctive features in comparison with TRU-type and non-TRU-type adenocarcinomas.

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Background:
Non-small cell carcinoma (NSCLC) cases harboring deletions in exon 19 of EGFR typically respond to treatment with small molecule inhibitors of EGFR. Detection of EGFR deletions in routine clinical practice is performed using a large variety of assays and testing platforms, with varying performance characteristics that are often not readily available. Using a hybrid capture based comprehensive genomic profiling (CGP) assay we identified 250 consecutive NSCLC cases, obtained from a range of clinical institutions, harboring deletions in EGFR exon 19 and compared data from these cases with available prior EGFR testing results.

Methods:
DNA was extracted from 40 microns of FFPE sections and CGP was performed on hybridization-captured, adaptor ligation based libraries to a mean coverage depth of 678X for 3,769 exons of 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer. The results were evaluated for all classes of genomic alterations (GA). Clinically relevant genomic alterations (CRGA) were defined as GA linked to drugs on the market or under evaluation in mechanism driven clinical trials.

Results:
Of the 250 cases with exon 19 deletions excluding the C-Helix, consisting primarily of 746-750, 71 (28%) had previous EGFR testing results obtained through standard of care testing at multiple different institutions available for review. Of these 71 cases, 12 (17%) were negative for EGFR alterations, but were identified by CGP as harboring an exon 19 deletion. Of 14 cases with deletions affecting the C-Helix (753-761), 6 had previous EGFR testing results available for review, with 5 (83%) cases having a prior negative result. For select cases clinical histories were reviewed, and the clinical benefit from treatment with small molecule inhibitors of EGFR was observed, consistent with historic norms, including EGFR 746-750 deleted patients responding to erlotinib and afatinib, a patient with EGFR T751_I759>N responding to afatinib, and a patient with EGFR S752_I759del having an ongoing 18 month response to erlotinib.

Conclusion:
CGP in the course of clinical care can identify EGFR exon 19 deletions in NSCLC that may be missed by standard of care testing, including both the canonical 746-750 deletion as well as the less characterized C- Helix deletions. Tumors with either of these alterations that go undetected by standard testing but are identified by CGP can respond to anti-EGFR therapy. Given the proven improved extent and duration of tumor response and patient survival benefit conferred by anti-EGFR targeted therapy in patients whose NSCLC harbor EGFR exon 19 deletions, the 17% false negative rate in patients tested by standard hot spot assays is a concern. Further evaluation of the impact of the increased range and sensitivity of CGP to uncover EGFR alterations in NSCLC that have been missed by non-hybrid capture assays appears warranted.

Background:
Afatinib is an oral, irreversible ErbB family blocker and one of the key drugs for patients with EGFR mutation positive advanced non-small cell lung cancer (NSCLC). Although treatment with afatinib has a clinical benefit for these patients, such individuals inevitably develop drug resistance as with other TKIs. This is a multicenter prospective biomarker study to inform the usefulness of noninvasive liquid biopsy in the treatment of EGFR-tyrosine kinase inhibitors (EGFR-TKIs) and explore the molecular mechanism of acquired-resistance against afatinib.

Methods:
Eligible patients were EGFR-TKIs naïve and had histologically and cytologically confirmed stage IIIB/IV adenocarcinoma of the lung with activating EGFR mutations. Patients remained on afatinib treatment until disease progression or unacceptable toxicity. Tumor samples were collected upon before afatinib treatment and after disease progression. Plasma samples were collected upon before and during afatinib treatment (4 and 24 weeks after initiation) and after disease progression. DNA derived both from tumors and plasma was analyzed using Scorpion-ARMS (ARMS), digital PCR (dPCR) and next generation sequencing (NGS). We used a nanofluidic dPCR system (BioMark HD System; Fluidigm) with a digital chip to detect activating or resistance mutations of EGFR in a quantitative and highly sensitive manner. NGS on an Ion Torrent PGM device (Thermo Fisher Scientific) was applied to detect target molecules which contribute to the survival and growth of lung cancer cells. We compared the sensitivity of these methods in detection of EGFR activating mutations in plasma DNA.

Results:
A total of 35 EGFR mutation positive NSCLC patients were enrolled. Twenty one patients harbored a deletion in exon 19 and fourteen patients had an L858R missense mutation in exon 21. Twenty seven (77.1%) patients had an objective response. In plasma DNA obtained before afatinib treatment, dPCR and NGS detected EGFR activating mutations more sensitively compared with ARMS (83.9% v 58.1%; p <0.005, 74.2% v 58.1%; p =0.059, respectively). Concordance of EGFR activating mutations detected by dPCR and NGS was 26/31 (84%) (kappa value: 0.52). All of the mutation type detected by NGS on plasma DNA completely corresponded to that found in matching tumor tissue by NGS. As of March 2015, serial plasma DNA was analyzed in 9 patients. The copy number of activating mutation was markedly decreased in 5 of 9 patients.

Conclusion:
EGFR activating mutations in plasma DNA were frequently detected by dPCR or NGS. We will present the detailed data for monitoring mutation load in plasma DNA during the afatinib treatment.

Background:
Epidermal growth factor receptor (EGFR) gene mutations in tumors predict tumor response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). However, obtaining tumor tissue for mutation analysis is challenging. Recently, peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been widely used to detect diagnostic, prognostic, and predictive proteomic biomarkers. Here, we aimed to detect serum peptides/proteins associated with EGFR gene mutation status, and test whether a classification algorithm based on serum proteomic profiling could be developed to analyze EGFR gene mutation status to aid therapeutic decision-making.

Methods:
Serum collected from 223 stage IIIB or IV NSCLC patients with known EGFR gene mutation status in their tumors prior to therapy was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and ClinProTools software. Differences in serum peptides/proteins between patients with EGFR gene TKI-sensitive mutations and wild-type EGFR genes were detected in a training group of 100 patients; based on this analysis, a serum proteomic classification algorithm was developed to classify EGFR gene mutation status and tested in an independent validation group of 123 patients. The correlation between EGFR gene mutation status, as identified with the serum proteomic classifier and response to EGFR-TKIs was analyzed.

Results:
Nine peptide/protein peaks were significantly different between NSCLC patients with EGFR gene TKI-sensitive mutations and wild-type EGFR genes in the training group. A genetic algorithm model consisting of five peptides/proteins (m/z 4092.4, 4585.05, 1365.1, 4643.49 and 4438.43) was developed from the training group to separate patients with EGFR gene TKI-sensitive mutations and wild-type EGFR genes. The classifier exhibited a sensitivity of 84.6% and a specificity of 77.5% in the validation group. In the 81 patients from the validation group treated with EGFR-TKIs, 28 (59.6%) of 47 patients whose matched samples were labeled as “mutant” by the classifier and 3 (8.8%) of 34 patients whose matched samples were labeled as “wild” achieved an objective response (p<0.0001). Patients whose matched samples were labeled as “mutant” by the classifier had a significantly longer progression-free survival (PFS) than patients whose matched samples were labeled as “wild” (p=0.001).

Conclusion:
Peptides/proteins related to EGFR gene mutation status were found in the serum. Classification of EGFR gene mutation status using the serum proteomic classifier established in the present study in patients with stage IIIB or IV NSCLC is feasible and may predict tumor response to EGFR-TKIs.

Background:
AZD9291 is an oral, potent, irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) selective for the EGFR-TKI-sensitizing (EGFRm) and T790M resistance mutations. We examined how the level of AZD9291 brain penetration and activity compared to that of other EGFR-TKIs in preclinical models of EGFRm non-small cell lung cancer (NSCLC) brain metastases (BM).

Methods:
Brain exposure of AZD9291 and an active circulating metabolite of AZD9291 (AZ5104), CO-1686 (rociletinib), gefitinib, erlotinib, and afatinib were evaluated in mouse models. Brain distribution following intravenous administration of microdoses (<3 micrograms) of [[11]C]AZD9291 and [[11]C]CO-1686 were compared in healthy cynomolgus macaques using positron emission tomography (PET) imaging. In vivo efficacy of AZD9291 and CO-1686 were assessed in a mouse EGFRm (exon 19 deletion) BM xenograft (PC9) model. Human doses that could potentially deliver BM efficacy were predicted using a preclinical pharmacokinetic/pharmacodynamic (PK/PD) mathematical model, adapted to account for the differential exposure and binding of AZD9291 and AZ5104 in brain compared with plasma.

Results:
In preclinical studies, AZD9291 showed significant exposure in the brain with concentrations in mouse brain tissue approximately 2-fold higher than plasma, although the metabolite, AZ5104, did not show similar levels of exposure to parent. AZD9291 also showed higher brain exposure than other tested EGFR-TKIs. Furthermore, under microdosing conditions [[11]C]AZD9291 showed marked exposure in a cynomolgus macaques brain PET study in contrast to [[11]C]CO-1686. At clinically relevant doses, AZD9291 distribution to the mouse brain was approximately 10-fold higher than gefitinib. In the PC9 BM model, AZD9291 showed tumor growth inhibition at 5 mg/kg/day. Using an adapted preclinical PK/PD model, simulations with clinical AZD9291/AZ5104 PK data predicted that a human dose of 80 mg may be sufficient to target EGFRm BM.

Conclusion:
Preclinical studies indicate AZD9291 has significant exposure in the brain and activity against EGFRm BM, compared with the other EGFR-TKIs tested. In light of early clinical activity of AZD9291 observed in patients with EGFRm NSCLC BM, further investigation into the potential benefit of AZD9291 in patients with EGFRm NSCLC BM is warranted.

Background:
Increasing incidences of brain metastases (BM) and leptomeningeal metastases (LM) in lung cancer, especially in those patients with activating mutations of the epidermal growth factor receptor (EGFR) have been reported recently. However, there are currently no approved drugs available for the treatment of these diseases. AZD3759 is an oral EGFR TKI specifically designed to penetrate blood brain barrier (BBB) for the treatment of BM and LM.

Methods:
Efflux liability at BBB was assessed by using in vitro MDCKII-MDR1 and MDCKII-BCRP substrate assay, and central nervous system (CNS) penetration was evaluated in rats and mice and quantitatively measured by free brain to free blood ratio (K~p,uu,brain~) and CSF to free blood ratio (K~p,uu,CSF~). Brain uptake of [[11]C]-AZD3759 related radioactivity was also evaluated in two Cynomolgus monkeys by positron emission tomography (PET). Human clearance was predicted by different methods including In vitro In vivo extrapolation, liver blood flow method with fu correction and fu correction intercept method (FCIM). Human volume of distribution was projected from Oie-Tozer and PBPK methods. Human efficacious dose was predicted by achieving free brain concentration at steady state continuously covering the IC~50~ of pEGFR(Tyr1068) in PC-9(Exon19Del) cells.

Results:
AZD3759 has high passive permeability (29.5x10[-6] cm/sec) and is not a substrate of Pgp or BCRP. AZD3759 reached distribution equilibrium in rats and mice (K~puu,brain~ and K~puu,CSF~ > 0.5), suggesting good BBB penetration. The distribution of [[11]C]-AZD3759 related radioactivity to monkey brain was fast and homogenous. Estimated K~puu,brain~ in monkeys is greater than 0.5. In the animal model bearing EGFRm+ brain tumor, AZD3759 induced profound tumor regression and significantly improved animal survival. Predicted half-life in human PK is 13.3 hr and clinically observed t~1/2 ~ranges from 10 to 15 hr. Median K~puu,CSF ~value among six valuable BM patients is 1.2, suggesting good CNS penetration in human and consistent with the preclinical data. The predicted efficacious dose for AZD3759 in man is 100 mg bid. In an ongoing Phase I study, AZD3759 was well tolerated with no DLTs at 50 mg or 100 mg bid and some preliminary evidence of intracranial tumor shrinkage was observed.

Conclusion:
So far the preclinical predictions of CNS penetration of AZD3759 appear to be valid. Predicted and observed half-life of AZD3759 in human is consistent to date. Some preliminary evidence of intracranial tumor shrinkage was observed in clinical setting.

Background:
Prior studies have indicated higher epidermal growth factor receptor (EGFR) mutation rate in non-small cell lung cancer (NSCLC) patients (pts) with brain metastases; however, these studies did not adjust for the effects of potential confounding variables.

Methods:
This was a retrospective study of NSCLC pts diagnosed between 2007-2014 at the University of Nebraska Medical Center, USA and Tata Memorial Hospital, India. After excluding 87 pts due to missing data, a total of 1522 pts were included. Univariate analysis (Chi-square or Fisher’s exact tests) and multivariate logistic regression were used to determine any association between EGFR status and clinical factors.

Results:
EGFR mutations were more common in females than males (38% vs. 24%, p<.0001), Asians than Caucasians (31% vs. 13%, p<.0001), non-smokers than smokers (40% vs. 14%, p<.0001), alcohol non-consumers than consumers (32% vs. 15%, p<.0001), adenocarcinoma than other histologies (32% vs. 10%, p<.0001) and in pts with brain metastasis than extracranial metastases or no metastasis (39% vs. 29% vs. 15%, p<.0001). The type of EGFR mutation (exon 19 vs. 21) did not correlate with the presence of brain metastasis. Multivariate analysis demonstrated a higher likelihood of an EGFR mutation among Asians vs. Whites/other ethnic groups (odds ratio, OR 2.1, p=0.015), non-smokers vs. smokers (OR 2.8, p<0.0001), alcohol non-consumers vs. consumers (OR 1.6, p=0.022) and adenocarcinoma vs. other histologies (OR 3.1, p<0.0001). Pts with brain metastasis were 1.9 times more likely to have an EFGR mutation than pts with extracranial metastasis (p=0.0002). Pts with brain metastasis were 1.8 times more likely to have an EFGR mutation (p=0.0002) compared to those without. The distribution of EGFR mutations was similar between pts with brain metastasis vs. non-metastatic disease (p=0.86) and pts with extracranial metastasis vs. non-metastatic disease (p=0.44).

Conclusion:
Our study is the largest study to demonstrate almost two-fold higher likelihood of an EFGR mutation among newly diagnosed NSCLC pts with brain metastases vs. those without. Studies of prophylactic cranial irradiation in pts with earlier stages of EGFR mutation positive NSCLC may be warranted.

Abstract not provided

Background:
Resminostat, an oral hydroxamate-type inhibitor of class I and II histone deacetylases, has shown a broad spectrum of anti-tumor activity against human cancer cell lines, and synergetic or additive effects in combination with docetaxel in non-small cell lung cancer (NSCLC) cell lines. We initiated a phase I/II study to evaluate the safety and efficacy of combining resminostat and docetaxel in patients (pts) with NSCLC pretreated with platinum-based therapy. The purpose of the phase I portion was to evaluate dose-limiting toxicities (DLTs) in the first cycle, estimate the maximum tolerated dose (MTD) of resminostat when administered in combination with docetaxel, and determine the recommended dose (RD) for the phase II portion. Here, we report the results of the phase I portion.

Methods:
NSCLC pts with failure of a platinum-based therapy were eligible for the study. Patients were treated with docetaxel on day 1 and resminostat on days 1 to 5 every 21 days. Phase I was an open-label, 3+3 cohort, dose-escalation study. While the docetaxel dose was fixed at 75 mg/m[2], the resminostat dose was escalated from 400mg (Dose Level 1: DL1) to 600 mg (DL2). DLT was defined as follows: grade 4 thrombocytopenia, grade 4 neutropenia lasting >7 days, febrile neutropenia lasting >3 days, and any other clinically significant grade 3/4 non-hematological toxicity.

Results:
A total of 9 pts (DL1: 3 pts, DL2: 6 pts) were enrolled in the phase I portion: male/female, 6/3; median age, 60 yr (50-71 yr); histologically proven adenocarcinoma/squamous cell carcinoma, 7/2; performance status, 0/1 in 7/2 pts. No DLTs were observed at DL1 or DL2. The most frequent grade 3/4 adverse events in any cycle were neutropenia (8 pts, 88.9%), leukocytopenia (8 pts, 88.9%), and febrile neutropenia (4 pts, 44.4%). These events were transient and resolved prior to the next cycle. No pharmacokinetic (PK) interaction between resminostat and docetaxel was observed. A partial response was observed in 1 pts (DL1) and stable disease in 3 pts (DL2).

	DL1 N=3		DL2 N=6
PK parameters (Geometric Mean)	Resminostat	Docetaxel	Resminostat	Docetaxel
C~max ~(ng/mL)	3,010	2,840	5,610	3,140
T~max ~(h)	1.78	1.00	1.47	1.03
AUC~inf~(h∙ng/mL)	11,800	3,030	25,500	3,280
t~1/2~ (h)	2.98	8.21	3.02	8.73

Conclusion:
The combination of resminostat and docetaxel was tolerable up to DL2 (docetaxel 75 mg/m[2], resminostat 600 mg); the MTD was not reached. Dose Level 2 was determined as the RD for the phase II portion of this study, which is currently ongoing.

Background:
It is considered that there is a population of “fit-elderly” patients, but how to select this population is undetermined. Two-drug regimen consisted of carboplatin (CBDCA) + weekly paclitaxel (PTX) in elderly patients with non-small cell lung cancer (NSCLC) was reported to be active but to have 4.4% of toxic deaths. When considering to add bevacizumab (BEV) to the two-drug regimen, meta-analysis of BEV-related adverse events taught that congestive heart failure (CHF) and arterial thromboembolic events increased in elderly patients. In this phase II study, we employed exclusion criteria of having both congestive heart failure (CHF) and diabetes mellitus (DM), which relates to arterial thromboembolism.

Methods:
Elderly (≥70 years old) patients with chemotherapy-naive, stage IIIB/IV or recurrent non-squamous NSCLC, ECOG-PS 0-1, measurable target lesion, and adequate organ functions were eligible for this study. Pts with CHF (i.e. those with brain natriuretic peptide (BNP) ≥ 100 pg/ml and ejection fraction (EF) ≤ 50%) and with DM (i.e. those with HbA1c ≥ 7.0%) were excluded. Treatment included CBDCA at AUC 5 on day 1, PTX at 90 mg/m[2] on days 1 and 8, and BEV at 15 mg/kg on day 1 of each 21-day cycle for up to 4 cycles, followed by maintenance BEV.

Results:
Thirty-six eligible patients (14 male, 22 female; median age, 75 years) were enrolled between February 2012 and September 2014. Fifteen and 21 patients had ECOG-PS of 0 and 1, respectively. The median number of CBDCA + weekly-PTX + BEV treatment cycles received was 4, and that of BEV maintenance dosing was 5. Grade 3/4 non-hematological and hematological toxicities were observed in 13 (36.1%) and 20 pts (55.6%), respectively. The most common grade 3/4 AEs included neutropenia (52.8%), hypertension (11%), anemia (8.3%), and infection (8.3%). No fatal AE was observed. The response rate, the primary endpoint of this study, was 69.4% (95% CI = 51.9–83.7), and median progression free survival was 9 months.

Conclusion:
CBDCA + weekly PTX + BEV followed by BEV was a feasible and effective first-line regimen for selected elderly non-squamous NSCLC patients. BNP, EF, and HbA1c may aid in selecting “bevacizumab-fit” elderly patients.　 Clinical information: UMIN000006622.

Background:
The combination of epigenetic drug decitabine with genistein, a natural isoflavone, produces synergistic responses in preclinical studies with particular activity shown in lung cancer cell lines. Our phase I dose-escalation study of decitabine with a fixed dose of genistein to treat advanced solid tumor was followed by a phase II study in advanced lung cancer patients.

Methods:
In phase I, decitabine was administered over 10-hours at increasing doses (60, 120, 240 mg/m[2]) with continuous administration of genistein 300 mg/day orally. The MTD was 120 mg/m[2] with neutropenia as DLT. Decitabine at 120 mg/m[2 ]and genistein produced plasma levels of 0.62±0.06 µM and 8.5±5.6 µM, respectively.

Results:
The drug combination was well tolerated and produced stable disease for more than 6 months (7-14 months) in 5/10 patients. One gastric cancer patient had a 50% reduction in tumor burden after 6 months of therapy. Stable disease was also achieved in patients with desmoplastic small round cell tumor, oncocytic carcinoma, follicular thyroid carcinoma and bladder cancer. The phase II study was focused on nine patients with non-small cell lung cancer refractory to 3-4 lines of therapy. Eight progressed within the first radiologic evaluation at week 6. One NSCLC patient remains on therapy with SD after 3 months of treatment. A total of 60 adverse events were reported during the study with all patients experiencing at least one AE. Grade 3 & 4 treatment related toxicities were observed in 6/9 patients (66%) : neutropenia (4) anemia (2) febrile neutropenia (1) and hypertension (1).

Conclusion:
This combination of genistein with decitabine was well tolerated in advanced patients with solid tumors. The activity of the combination seen in some patients with tumors of more indolent biology was modest in the phase II cohort of heavily pretreated NSCLC patients. The efficacy profile observed in this trial suggests that tumors with slower tumor kinetics might benefit more from this type of epigenetic therapy.

Background:
Endostar [TM ](rh-endostatin, 7.5mg/m[2], 3-hour intravenous infusion (IV) daily for 14 days) was approved by Chinese FDA for treatment of advanced NSCLC in 2005. Considering continuous intravenous infusion (CIV) may be a more favorable way to deliver Endostar, we designed the phase I study to evaluate pharmacokinetics, tolerability and efficacy of Endostar CIV at different doses combined with pemetrexed and carboplatin in untreated advanced non-squamous NSCLC patients.

Methods:
In phase Ia, 19 patients were assigned to 4-6 cycles (21 days/cycle) of pemetrexed (500mg/m[2], day 1), carboplatin (AUC 5, day 1), and CIV Endostar from day 2 to day 21 at doses of 7.5mg (8 patients), 15mg (6 patients), 30mg (5 patients) /m[2]/d, respectively. Serum samples were obtained 0h、1h、2h、4h、8h、24h、48h、72h、96h、120h、122h、124h、128h、132h and 144h after the Endostar infusion. In phase Ib, another 21 patients received CIV Endostar at doses of 7.5mg (10 patients) or 15mg (11 patients) /m[2]/d with pemetrexed + carboplatin.

Results:
The AUC~0-120h~ of Endostar 7.5mg/m[2] CIV and 7.5mg/m[2] 3-hour IV daily were comparable (12.6±7.6 vs 13.3±8.8 ug/mL × hour). C~max~ (ng/ml) (7.5mg: 152.4±83.7; 15mg: 287.2±122.6; 30mg: 398.2±52.6) and AUC~0-120h~ (ug/mL × hour) (7.5mg: 12.6±7.6; 15mg: 21.2±10.8; 30mg: 33.4±8.5) were linear with dose. In phase Ia, the most common adverse events were anemia (78.9%, G3/4 10.5%), neutropenia (68.4%, G3/4 31.6%), thrombocytopenia (63.2%, G3/4 10.5%), LDH increase (47.4%), aminotransferase increase (42.1%), and supraventricular arrhythmia (26.3%). No grade 3 or 4 non- hematologic adverse event was observed. The incidence of supraventricular arrhythmia in 30mg cohort (40%) was higher than the other two cohorts. Thus 30mg cohort was excluded in Ib phase. Totally, 15 patients in 7.5mg cohort and 17 patients in 15mg cohort were evaluable for treatment response. The DCR and ORR were 80.0% and 60.0% in 7.5mg cohort, 94.1% and 76.5% in 15mg cohort, respectively.

Conclusion:
The pharmacokinetics of Endostar CIV and daily IV were comparable. At doses of 7.5mg and 15mg/m[2]/d, Endostar CIV was well tolerated with encouraging anti-tumor efficacy. Increasing dose of Endostar might lead to better response.

Abstract not provided

Background:
There is little evidence supporting the efficacy of third-line systemic therapy in non-small cell lung cancer (NSCLC) patients (p) with advanced (a) disease, except for erlotinib, and its role is unclear in unselected population. Nonetheless, further-line chemotherapy(CT) is frequently offered in daily clinical practice. We retrospectively analyzed the clinical, pathological characteristics and outcomes of p with aNSCLC who received >3 CT regimens to identify subsets of patients more likely to benefit. The presence of underlying molecular alterations has also been evaluated.

Methods:
The study included data from all consecutive p diagnosed with aNSCLC in our Institution from January 2008 to December 2013. Median overall survival (mOS) and progression free survival (PFS) were evaluated with Kaplan-Meier curves and groups were compared using the Log-rank test. Variables analyzed included p tumor and treatment characteristics. Overall response rate(ORR) was calculated according to the RECIST criteria.

Results:
A total of 486 p were included .175 p (36%) received >3 lines (group3+). Table 1 summarized p characteristics. Group 3+included more females (35.4%vs22.8%; p= 0.0041),younger p (58.9vs61.9;p =0.0016), more never-smoker p (26.9%vs18%;p=0.015), less lung (10.9%vs22.2%;p= 0.0020) and heart (4%vs11.6%;p=0.0020) comorbidities, a higher proportion of molecular alterations (EGFR/ALK) (25.7%vs12.9%; p= 0.0005), more adenocarcinoma (68.6%vs55%;p=0.0045) and less brain metastasis (14.3%vs23.5%;p=0.018). ORR to first line was higher in group 3+ (45.8%vs 29%;p=0.0009). 82.3% non-squamous histology were tested for at least one molecular alteration. There were no differences in PFS between both groups. The mOS of p in group 3+ was longer [24.3 m vs. 7.7 m, p<0.0001)], including p with EGFR/ALK/ROS1 wild-type or unknown [21.6 m vs. 7.4 m, p<0.0001) ]. OS was also longer in the group 3+ harboring a molecular alteration [32.2 m vs 12.7m;p=0.0002]. In the univariate analysis the presence of a molecular alteration were related to longer PFS. In univariate analysis having received >3, female gender, age<65 and the presence of molecular alterations were associated with longer OS. In the multivariate analysis >3 therapeutic lines and the presence of molecular alterations were related to longer OS.

Conclusion:
P treated with >3 systemic treatments were more likely to respond better, progress later and live longer. This better prognosis could be related to the presence of molecular alteration. However p without or unknown molecular alteration could benefit from receiving subsequent lines.

Background:
A recent study has shown pretreatment sodium levels to be a predictive and prognostic marker in NSCLC patients treated with erlotinib. The objective of this study was to evaluate the prognostic impact of pretreatment sodium levels on progression free survival (PFS) and overall survival (OS) in patients of NSCLC treated with pemetrexed-platinum doublet chemotherapy.

Methods:
Stage IIIB/ IV NSCLC patients aged ≥ 18 years treated between January 2011 to November 2014 at our centre were included in this retrospective study. Patients received pemetrexed 500 mg/m[2] with either cisplatin 75 mg/m[2 ]or car­boplatin (AUC 5) on day 1 of a 21 day cycle for 6 cycles followed by maintenance pemetrexed till progression. Electronic medical record (EMR) database of our hospital was used to retrieve demographic data, pretreatment sodium levels, PFS and OS data. LSS was defined as serum sodium < 136mEq/L. Survival analysis was performed using Kaplan-Meier curves and compared between LSS and normal serum sodium (NSS) groups using Log-Rank test and proportional hazard model.

Results:
Figure 1Data was available for 256 patients (M/F = 172/84) with median age of 53 (25-79) years. Majority had ECOG PS of 1 (0 = 34, 1 = 172, 2 = 44, 3 = 6). Stage IIIB = 24 (9%), stage IV = 232 (91%). Pretreatment LSS was observed in 75 (29%) patients while 181 (71%) had NSS. Median duration of follow-up was 17 months. Patients with NSS had significantly longer PFS (10.7 vs. 7.4 months; P < 0.05) and OS (17.6 vs. 13.4 months; P < 0.05) compared to LSS group. Cox-proportional hazard model has shown LSS was an independent prognostic biomarker for poor survival (P < 0.05).



Conclusion:
Pretreatment serum sodium level is an important prognostic marker in stage IIIB/ IV NSCLC patients. The simple possibility of testing coupled with low cost makes it an attractive marker to implement in clinical practice.

Background:
Non-small-cell lung cancer (NSCLC) accounts for >80% of all lung cancers, and the risk of lung cancer clearly increases with advancing age. Because of the progressive aging of population, the number of elderly patients with NSCLC is increasing and the desease is becoming an increasing public health problem worldwide. We previously reported a phase I study that recommended a dose of carboplatin (Cb, area under the curve = 5) plus pemetrexed (PEM, 500 mg/m[2]) for elderly (≥75-years-old) patients with non-squamous NSCLC. Furthermore, PEM maintenance therapy, following the combination therapy, was also found to be well tolerated. Therefore, we conducted a multicenter phase II trial to evaluated the efficacy and safety of Cb (area under the curve = 5) plus PEM (500 mg/m[2]) followed by maintenance PEM for elderly (≥75-years-old) patients with non-squamous NSCLC.

Methods:
Treated patients received 4 courses of Cb plus PEM, followed by maintenance PEM, without showing disease progression or severe toxicities. The primary endpoint was the 1-year overall survival (OS) rate, and the secondary endpoints were OS, progression free survival (PFS), response rate (RR), and safety.

Results:
Thirty four patients were enrolled between June 2012 and May 2013. All patients had an ECOG performance status 0 or 1, and adenocarcinoma. The median patient age was 77 years (75-84 years). Twenty four patients were male and ten patients were female. Three patients harbored activating epidermal growth factor recepter mutation (exon19 or 21). The median observation time was 22.7 months. In clinical outcome, the overall RR was 41.2%, and the disease control rate was 85.3%. No patient showed a complete response, 14 showed partial responses, 15 showed stable disease, 4 showed disease progression, and 1 was not evaluated. The maintenance therapy rate was 58.8%. The median PFS for all patients was 5.7 months (95% confidence interval, 3.3–8.5 months), whereas the median OS was 20.5 months (95% confidence interval, 7.8–25.4 months). The 1-year OS rate was 58.0%. In adverse events (total phase of this study), hematological adverse events ≥grade 3 were leucopenia (in 23.5% of patients), neutropenia (55.9%), anemia (35.3%), and thrombocytopenia (20.6%), and major non-hematological adverse events ≥grade 3 were febrile neutropenia (in 8.8% of patients), increased levels of aminotransferase (5.9%), infection (23.5%), and anorexia/fatigue (5.9%). There was 1 treatment-related death due to interstitial lung disease.

Conclusion:
The combination of Cb plus PEM followed by maintenance PEM was effective and reasonably well tolerated in chemotherapy-naïve elderly (≥75-years-old) patients with non-squamous NSCLC. This data was promising and valuable to conduct the phase III study compared with docetaxel (DOC) monotherapy in the first-line setting. Now, the phase III trial compared Cb plus PEM followed by maintenance PEM with DOC for chemotherapy-naïve elderly (≥75-years-old) patients with non-squamous NSCLC (JCOG1210/WJOG7813L: UMIN000011460) is ongoing and the result is warranted. Clinical trial information: UMIN000004810

Background:
Recent trials suggested that maintenance treatments improve outcomes for patients not progressing after first-line therapy for advanced NSCLC. However, physicians have little guidance on selecting which patients benefit the most and what drug or regimen is optimal. Here, we report a systematic review and network meta-analysis (NMA) of current evidence assessing relative efficacies of maintenance options in unselected populations, as well as in subgroups determined by EGFR mutation, histology, and response to induction.

Methods:
PubMed and conference proceedings were reviewed and individual study relative efficacy measures were meta-analyzed in a Bayesian hierarchical model. The primary and secondary outcomes, Overall Survival (OS) and Progression Free Survival (PFS), respectviely, were evaluated in terms of (i) posterior surface under cumulative ranking curve (SUCRA), (ii) probability of being best treatment, (iii) probability of outperforming no maintenance, and (iv) posterior median hazard ratios with 95% credible intervals, in an unselected population, as well as by EGFR mutation status, histology, and response to induction. Secondary outcomes were overall survival (OS) and adverse events.

Results:
Twelve trials evaluating eight maintenance treatments in 3,850 patients were included in NMA. Selected maintenance treatments showed substantial PFS and OS benefits with probabilities ≥99% and ≥92% respectively of outperforming no maintenance. Results suggest the following strategy for optimal OS and PFS: (i) switch to or continue pemetrexed or switch to anti-EGFR TKI for nonsquamous patients, (ii) continue gemcitabine for squamous patients, (iii) switch to docetaxel or continue gemcitabine for responders to previous induction, and (iv) switch to or continue pemetrexed or switch to anti-EGFR TKI for patients with stable disease post-induction.

Conclusion:
Maintenance treatments improve PFS and OS in good performance status patients with stage IIIb/IV NSCLC not progressing after first-line chemotherapy. Benefits are optimized by targeting specific maintenance treatments to selected patient groups guided by histology and response to previous induction.

Abstract not provided

Background:
Pemetrexed and cisplatin (P-C) has become the standard 1st line chemotherapy in NSCLC patients with wild-type EGFR. The recommended drugs in the 2nd line are docetaxel, docetaxel plus ramucirumab, gemcitabine or EGFR TKIs. Gemcitabine and vinorelbine have good clinical efficacies and low toxicity profiles, so this two drug combination therapy is challenged for their clinical efficacy as 2nd line treatment. The optimal 2nd line chemotherapy following failure of 1st line P-C treatment in advanced NSCLC patients with wild-type EGFR is not yet defined. Therefore, we evaluated the optimal 2nd line chemotherapy in P-C non-responders with advanced NSCLC.

Methods:
We conducted a retrospective analysis of patients with stage IIIB or IV NSCLC who had been treated with P-C as a first line treatment from February 2010 to May 2014. Patients who had EGFR mutation or were on pemetrexed maintenance therapy were excluded. We compared the progression free survival, overall response rate and adverse effects of each regimen.

Results:
Among 110 patients, 52 were eligible for the study. 28 received EGFR TKI (gefitinib or erlotinib); 13 received docetaxel monotherapy; 11 received gemcitabine-vinorelbine (G-V) combination therapy. Median age was 64.5, 61 and 63 years, respectively. All patients showed adenocarcinoma type histology except two in docetaxel and G-V group with large cell type histology. Best response rates were 15.4% in docetaxel group, 18.1% in G-V group and 11% in EGFR TKIs group. Median progression free survival time was 62 days(95% CI 54-70) in EGFR TKIs group, 63 days (95% CI 30-96) in docetaxel group, and 83 days (95% CI 55-111) in G-V group (P=0.17). In pairwise comparisons, p-value was 0.54 for EGFR TKI versus docetaxel group, 0.08 for TKI versus G-V group, and 0.23 for docetaxel versus G-V group. There were no difference in progression-free survival and response rate among the groups. There was a higher rate of grade 3/4 neutropenia in the Docetaxel group.

Conclusion:
Despite the absence of statistical significance, there was a trend that G-V combination therapy had longer progression-free survival outcome compared to EGFR TKI or Docetaxel groups. G-V as well showed better toxicity profiles compared to Docetaxel group. A larger study is required to confirm the efficacy of cytotoxic chemotherapy, especially G-V, as a second line treatment in EGFR mutation negative NSCLC patients.

Background:
In advanced non-small cell lung cancer (NSCLC) decisions regarding palliative treatment are based on tumor response, increasingly combined with patient reported outcomes, especially quality of life (QoL). However, considering treatment decisions in this manner ignores patients’ own opinion about (change in) QoL. A more patient-oriented view regarding therapy could offer valuable information in the process of shared decision-making about treatment initiation or continuation with chemotherapy. We assessed patients’ satisfaction with the received chemotherapy using the Cancer Therapy Satisfaction Questionnaire (CTSQ) in relation with QoL during treatment.

Methods:
In a prospective observational multi-center study, patients with stage IIIB or IV NSCLC receiving pemetrexed (PEM)-based chemotherapy as first or second line treatment were enrolled. Prior to and after four cycles of chemotherapy, patients completed the WHO Quality of Life-BREF (WHOQoL-BREF) and EORTC-Quality of Life Questionnaire-Core 30 (EORTC-QLQ-C30), which both contain one item measuring overall QoL on a 1-5 and 1-7 scale, respectively. After four cycles patients also completed the CTSQ, which consists of 16 items scored on a 1-5 scale and is divided in three domains, including the domain satisfaction with therapy (SWT). Linear transformation of the domain score results in a score range 0-100, with a higher score representing a better treatment satisfaction. Items of special interest were Question 7 (Q7) “Chemotherapy was worth taking even with side effects”, Question 16 (Q16) “If given the choice again, would you decide to take this chemotherapy treatment” and Question 2 (Q2) “Chemotherapy would cure the cancer”. From all patients tumor response measurements were obtained according to RECIST 1.1.

Results:
Of the 88 patients receiving four cycles of PEM-based chemotherapy, 65 patients completed the WHOQoL-BREF, EORTC-QLQ-30 and the CTSQ. The majority of these patients had stage IV NSCLC (87.7%) and received PEM-based therapy as first line treatment (92.3%). Treatment resulted in stable disease (53.8%), partial response (40.0%) and progressive disease (6.2%). Eighteen patients often (13.8%) or always (13.8%) expected chemotherapy would cure the cancer. During therapy, overall QoL measured by WHOQoL-BREF increased (1.3±0.6), remained stable (0±0) and decreased (-1.4±0.7) in respectively 15 (23.1%), 30 (46.2%) and 20 (30.8%) patients. The SWT domain score (77.5±12.3 vs. 83.8±13.1) and single item scores Q7 (4.1±0.9 vs. 4.4±0.8) and Q16 (4.4±0.7 vs. 4.5±0.6) in patients with decrease vs. increase of overall QoL did not differ significantly between the groups (p> 0.05). Change in overall QoL measured by the EORTC-QLQ-C30 related to SWT, Q7 and Q16 showed similar results.

Conclusion:
Despite a decrease of QoL during chemotherapy, patients still consider the treatment as worth taking and would decide to receive the chemotherapy again. Since the majority of patients understand that the treatment has no curative intentions, it is unlikely that the satisfaction with treatment only reflects false expectations of cancer cure. Our results represent a group of patients who mainly established disease stabilization, which could have influenced our findings. In shared decision-making on palliative treatment, patients’ QoL cannot be used as a single decision criterion because it does not reflect patients’ satisfaction with treatment. This study is funded by ZonMw, the Netherlands

Background:
Randomized clinical trials have demonstrated the benefits of chemotherapy in carefully selected NSCLC patients. How generalizable these results are to the general population of NSCLC patients, who often have multiple comorbidities that would have rendered them ineligible for licensing trials, is unresolved.

Methods:
The outcomes of unselected patients with stage IV NSCLC who did not participate in a clinical trial and who were treated with standard chemotherapy regimens (paclitaxel/carboplatin; gemcitabine/carboplatin; pemetrexed/carboplatin; paclitaxel/carboplatin/bevacizumab) as first line therapy between 2002 and 2012 at a tertiary teaching hospital were compared to the reported outcomes observed in the licensing trials supporting the use of these drug regimens.

Results:
Results are summarized in Table 1 Patients treated with three-weekly paclitaxel plus carboplatin at recommended dosages had a median progression free survival of 4.9 months for patients responding to this regimen and an overall survival of 13.1 months for responders vs 9.2 months for non-responders. In patients’ treated with gemcitabine plus carboplatin on a three or four week cycle the median progression free survival was 4.8 months for patients responding to the regimen and overall survival of 13 months for responders vs 8.9 months for non-responder group of patients. For patients receiving pemetrexed plus carboplatin the median progression free survival was 7.1 months for patients responding to the regimen with an overall survival of 15.5 months for responders vs 5.9 months for non-responders. Those patients’ treated with paclitaxel plus carboplatin plus bevacizumab the median progression free survival was 7.3 months for patients responding to treatment and overall survival was 16.7 months vs 14.6 months for those patients who did not respond to this regimen as initial treatment. Table I: Patient demographics and results for each regimen

	paclitaxel and carboplatin N=105	Gemcitabine and carboplatin N=35	pemetrexed and carboplatin N=26	paclitaxel and carboplatin and bevacizumab N=28
Age 70>	15.2%	31.4%	26.9%	25.0%
< 70	84.8%	68.6%	73.1%	75.0%
Gender Male Female	64.8% 35.2%	51.4% 48.6%	57.7% 42.3%	60.7% 39.3%
Smoker	89.2%	91.2%	84.6%	71.4%
ECOG 0	12.6%	12.1%	8.7%	23.1%
1	68.9%	63.6%	47.8%	76.9%
2	17.5%	24.2%	43.5%	0.0%
3	1.0%	0.0%	0.0%	0.0%
PFS (months)	4.9	4.8	7.1	7.3
OS (months) PR PD	13.1 9.2	13 8.9	15.5 5.9	16.7 14.6

Conclusion:
Progression free survival in an unselected population of NSCLC patients was similar to that reported in clinical trials supporting the approval of these drugs and regimens. The present analysis provides further support for the use of combination chemotherapy in patients with stage IV NSCLC, including those who would have been ineligible for many clinical trials due to comorbidities. Further analysis is ongoing.

Background:
The current available method to monitor response to treatment in lung cancer patient is by Computerized Tomography (CT) scans. However, time intervals between consecutive CT scans might be too long to allow early identification of treatment failure. The aim of this study is to examine the use of breath sampling as a tool for monitoring response to anti-cancerous treatment in patients with advanced lung cancer.

Methods:
In a prospective study, repeated exhaled breath samples were collected from patients with advanced lung cancer before and under systemic therapy. VOCs[1] profiles were determined by GC-MS[2] and nanomaterial-based array of sensors and correlated with response to therapy, assessed by CT scans as Complete Response (CR), Partial Response (PR), Stable Disease (SD), or Progressive Disease (PD). [1] Volatile Organic Compounds [2] gas-chromatography/mass-spectrometry

Results:
One hundred forty three breath samples were collected from 39 patients with stage III/IV lung cancer. GC-MS anaylsis identified 3 VOCs as significantly indicating PR/SD samples. One of them was also significantly discriminated between PR/SD and PD. Further, the NA-NOSE signals were able to alarm per a change in tumor response across therapy, i.e. indicating lack of further response to therapy, or developement of resistance to therapy. PR/SD was detected in a sensitivity of 93%, specificity of 85% and accuracy of 89% and ppositive/negative predictive values (PPV; NPV) of 86% and 92% respectively. PD was detected with 100% specificity and 92% accuracy, but the sensitivity was only 28%. The PPV and NPV were 100% and 91%, respectively. The achieved results indicate high reliability in predicting a progression of the disease and detecting patient's lack of response to treatment (i.e., PD).

Conclusion:
Breath analysis may serve as a serogate marker for response to systemic therapy in lung cancer. Such a monitoring tool can provide the oncologist with a quick and simple method to identify patient's response to anti-cancerous treatment in shorter intervals than currently available by CT scans.

Abstract not provided

Background:
ALK rearrangements in non-small cell lung cancer (NSCLC) have been associated with younger age of onset, East Asian ethnicity, non- or light-smoking history, and adenocarcinoma histology. The objective of this study was to evaluate data from two retrospective multicenter chart review studies conducted in 2013 and 2014 to assess characteristics of ALK+ NSCLC patients and further understand the epidemiology of ALK rearrangements in NSCLC patients.

Methods:
This analysis included data from two chart review studies of patients diagnosed with locally-advanced or metastatic ALK+ NSCLC conducted among two separate panels of US oncologists. In the first study conducted in September and October 2013, 27 oncologists contributed data on 273 patients; in the second study conducted between July and November 2014, 49 oncologists contributed data on 153 patients. In both studies, collected information included the age at diagnosis of NSCLC, sex, ethnicity, smoking history at primary NSCLC diagnosis, and tumor histology. Data from these studies were analyzed to assess ALK+ NSCLC patient characteristics.

Results:
Patients from the 2014 cohort tended to be younger than patients from the 2013 cohort at diagnosis of locally-advanced or metastatic NSCLC (Table). In both cohorts, a little over half of the patients were male. Racial composition was diverse in both patient groups. Patients had varied smoking histories in both studies, with approximately one third of patients reported as never-smokers, one third as light smokers, and one third as moderate/heavy smokers. Tumor histology was heterogeneous in both cohorts. However, a particularly large proportion of patients in the 2014 cohort had squamous cell histology (14%).

Table. Characteristics of ALK+ NSCLC Patients from the 2013 and 2014 Studies

	2013 Study[1] N=273	2014 Study N=153
Age (years), median (IQR)	67 (58-72)	59 (52-67)
Male (%)	52%	57%
Race/Ethnicity (%)
Caucasian	59%	63%
Black/African American	18%	14%
Asian	13%	14%
Hispanic/Latino	8%	2%
American Indian/Alaska Native	1%	6%
Unknown	0%	1%
Smoking History[2] (%)
Never	33%	27%
Light	33%	24%
Moderate/heavy	33%	37%
Unknown[3]	1%	13%
Cancer Histology[2] (%)
Adenocarcinoma	81%	65%
Squamous cell carcinoma	3%	14%
Large cell carcinoma	5%	8%
Mixed	11%	9%
Unknown	0%	4%

Notes: IQR = inter-quartile range [1] Wakelee HA, Sasane M, Zhang J, et al. Description of ALK+ NSCLC patient characteristics and ALK testing patterns. J Clin Oncol 32:5s, 2014 (suppl; abstr 8062). [2] Reported at primary NSCLC diagnosis. [3] Includes patients with unreported smoking history as well as 16 former smokers in the 2014 study with unknown smoking histories.

Conclusion:
Assessment of patient characteristics in the two chart reviews suggests that ALK+ NSCLC patients may have diverse characteristics with varied racial composition, smoking histories, and tumor histology to an extent not previously detected. These results suggest that physicians may be testing NSCLC patients more frequently, yielding more diverse histology than expected among ALK+ tumors. Molecular testing could be informative for all newly diagnosed NSCLC patients, including patients with squamous cell histology.

Background:
Clinical trials have shown superior efficacy of ALK inhibitors compared with chemotherapies for patients diagnosed with ALK+ non-small cell lung cancer (NSCLC). In Korea, crizotinib was approved for ALK+ NSCLC in 2011 but is not yet reimbursed. The objective of this study was to describe real-world patient characteristics, ALK testing and treatment patterns, and survival among Korean patients diagnosed with locally-advanced or metastatic ALK+ NSCLC.

Methods:
A retrospective patient chart review was conducted in two major cancer centers in Korea. Participating physicians (N=4) reviewed patient charts and reported patient characteristics, ALK testing and treatment patterns, and survival of patients diagnosed with ALK+ locally-advanced or metastatic NSCLC. ALK inhibitor treatment duration and overall survival (OS) were estimated using Kaplan-Meier analyses.

Results:
In late 2014, 55 ALK+ NSCLC patients were identified for this study. The median follow-up time among these patients was 24.8 months. The median age at locally advanced or metastatic NSCLC diagnosis was 60 years (interquartile range: 52 - 67); 53% of patients were female, 51% were never-smokers, 2% were former smokers, 33% were current smokers, 15% had unknown smoking status, and 98% were diagnosed with adenocarcinoma. At primary diagnosis, 67% of patients had metastatic disease. ALK rearrangement was confirmed by fluorescent in situ hybridization (78%) or immunohistochemistry (22%). 27% of patients had their ALK rearrangement detected more than three months after their locally-advanced or metastatic diagnosis. The majority of patients received initial systemic chemotherapy; only 13% received an ALK inhibitor in the first-line, and 62% received an ALK inhibitor by the end of follow-up. Out of 30 patients who received crizotinib, 83% discontinued (median duration of 6.3 months) and 13% died while still on crizotinib. Of those who discontinued, 32% switched to chemotherapy, 16% switched to a different ALK inhibitor, and 52% received no further antineoplastic therapy. After discontinuing crizotinib, the median OS was 4.3 months.

Conclusion:
In this study of locally-advanced or metastatic ALK+ NSCLC patients in Korea, OS was poor following discontinuation of crizotinib with a median survival of 4.3 months. Additionally, many patients had delays in receiving ALK testing. Among patients who failed crizotinib treatment, over half received no further antineoplastic therapy. These findings suggest the need to provide timely access to ALK testing and effective treatments following crizotinib discontinuation for ALK+ NSCLC patients in Korea.

Background:
Crizotinib is an oral small-molecule tyrosine kinase inhibitor, which was approved in the United States (US) in August of 2011 for the treatment of anaplastic lymphoma kinase (ALK)-positive advanced non-small cell lung cancer (NSCLC). There are currently limited data on crizotinib treatment and related outcomes in ALK-positive advanced NSCLC patients with brain metastases at diagnosis in real world settings. The main objective of the current analyses was to assess and report the response rates in the ALK-positive advanced lung cancer patients with brain metastases treated with crizotinib in regular clinical practice.

Methods:
Physicians in the US (N=107) and Canada (N=40) were recruited from cancer centers or teaching hospitals (48%) or free standing oncology clinics (47%) to abstract data retrospectively from medical records of adult (≥18 years) patients diagnosed with ALK-positive advanced NSCLC and treated with crizotinib as first- or later-line therapy from 8/1/2011-3/31/2013 (for the US) or 4/12012-3/31/2013 (for Canada) in non-clinical trial settings. IRB approval was obtained. A secure web-based form was used by physicians to abstract data and all patient data were de-identified and anonymous. Descriptive analyses were conducted to summarize objective response rate (ORR) in the subgroup with brain metastases. One-year survival rates from initiation of crizotinib were descriptively analyzed for the subgroup with brain metastases using the Kaplan-Meier method.

Results:
Data were extracted from 212 patient records in US (N=147) and Canada (N=65), which included 33 ALK-positive advanced NSCLC patients with brain metastases present prior to crizotinib initiation. The mean (SD) patient age at diagnosis for these 33 patients was 57.4 (12.0) years and a majority were male (58%), Caucasian (76%), current or former smokers (67%), ECOG status 0 or 1 (55%), adenocarcinoma histology (85%) and initially diagnosed at the locally advanced or metastatic stage (70%). The majority (71%) of these patients had been treated with either whole brain radiotherapy (16 patients) or stereotactic radiosurgery (8 patients) prior to initiation of crizotinib treatment. Of these 33 patients, 21 received crizotinib as 1[st] line therapy for advanced ALK-positive NSCLC. Approximately 61% were alive at time of medical record data abstraction. The ORR was estimated to be 61% and 60% for these patients with brain metastases who received crizotinib as 1[st] and later line treatment, respectively. The Kaplan-Meier estimates of 1-year survival from crizotinib initiation were 81% and 77% in patients with brain metastases, who received crizotinib as 1[st] line and later line treatment, respectively.

Conclusion:
In ALK-positive advanced NSCLC patients with brain metastases, complete or partial response was reported in majority of patients during treatment with crizotinib in real world settings.

Background:
The ongoing multicenter, randomized, open-label phase III study PROFILE 1014 recently demonstrated superior efficacy of crizotinib compared with chemotherapy in patients with previously untreated advanced ALK-positive NSCLC (Solomon et al, N Engl J Med 2014). Intracranial efficacy of crizotinib vs. chemotherapy was compared prospectively in this trial.

Methods:
Patients with previously untreated advanced non-squamous ALK-positive NSCLC (N=343) were randomized 1:1 to receive crizotinib 250 mg orally BID (n=172) or intravenous chemotherapy (pemetrexed 500 mg/m[2 ]+ cisplatin 75 mg/m[2] or carboplatin at AUC 5–6; all q3w for ≤6 cycles; n=171). Patients with treated brain metastases that were stable for ≥2 weeks with no ongoing requirement for corticosteroids were eligible. Treatment was continued until PD. Continuation of, or crossover to, crizotinib after PD (per independent radiology review [IRR]) was allowed for patients randomized to crizotinib or chemotherapy, respectively. Brain scanning was performed every 6 weeks in patients with baseline brain metastases and every 12 weeks in those without baseline brain metastases. Protocol-specified efficacy endpoints included PFS (primary endpoint), ORR, OS, and 12- and 18-month OS, as well as intracranial TTP. Intracranial DCR at 12 and 24 weeks was also evaluated. Efficacy was evaluated in the ITT population and in two subgroups of patients: those with and without baseline brain metastases.

Results:
Of 343 patients in the ITT population, 79 had brain metastases at baseline identified by IRR (23%) and 263 did not (77%; data not reported for one patient). Baseline characteristics of patients randomized to receive crizotinib or chemotherapy were generally well balanced within these two patient subgroups. Among the patients with baseline brain metastases, a significantly higher proportion achieved intracranial disease control with crizotinib than with chemotherapy at 12 weeks (33/39 [85%] vs. 18/40 [45%], respectively; P=0.0003) and at 24 weeks (22/39 [56%] vs. 10/40 [25%]; P=0.006). There was a numerical improvement in prospectively measured intracranial TTP with crizotinib in the ITT population (HR 0.60, P=0.069), as well as in patients either with baseline brain metastases (HR 0.45, P=0.063) or without baseline brain metastases (HR 0.69, P=0.323). The frequency of progression in the brain was low in the ITT population (15%) and in patients with and without baseline brain metastases (27% and 11%, respectively). Overall PFS was significantly longer with crizotinib than with chemotherapy in both subgroups (brain metastases present: HR 0.40, P=0.0007, median 9.0 vs. 4.0 months; brain metastases absent: HR 0.51, P≤0.0001, median 11.1 vs. 7.2 months), as it was in the ITT population (HR 0.45, P<0.0001, median 10.9 vs. 7.0 months). Twenty-five patients in the crizotinib arm of the study experienced intracranial PD; 22 of these patients received crizotinib for ≥3 weeks beyond PD and 19 also received intracranial radiotherapy.

Conclusion:
In this prospective assessment of intracranial efficacy, crizotinib demonstrated significantly greater intracranial disease control and overall efficacy compared with chemotherapy in patients with baseline brain metastases. These findings provide further confirmation of crizotinib as the standard of care for patients with previously untreated advanced ALK-positive NSCLC, including those patients with brain metastases at baseline.

Abstract not provided

Background:
Tyrosine kinase inhibitors (TKIs) have been associated with the development of a rare but serious and potentially fatal lung injury syndrome referred to as drug-induced interstitial lung disease (ILD). This has been best characterized for the epidermal growth factor receptor (EGFR) TKIs, with a typical presentation of delayed but rapidly progressive dyspnea leading to respiratory failure and death in up to one third of cases. While case reports of crizotinib-associated ILD have been published, little is known regarding the incidence, clinical characteristics, and mortality of crizotinib-associated ILD. In an effort to better understand this adverse event (AE), we performed a systematic review of respiratory-related events in the four largest clinical trials with crizotinib.

Methods:
An independent three-person panel composed of a pulmonologist, radiologist, and oncologist, none of whom were associated with any of the clinical studies, was convened to analyze respiratory AEs of grade ≥3 and any-grade AEs reported by the investigator as pneumonitis, ILD, or radiation pneumonitis in four crizotinib studies (PROFILE 1001, 1005, 1007, and 1014). After review by each panel member individually followed by consensus review, the events were classified either as disease progression or true respiratory AEs. Respiratory AEs were then further sub-classified as due to: 1) de novo ILD, 2) exacerbation or recurrence of pre-existing ILD, 3) concurrent illness (recurrence or progression of pre-existing condition), or 4) other toxicity not thought to be related to ILD (e.g. bacterial pneumonia).

Results:
There were a total of 1,669 patients in the four studies, among whom 446 events in 368 patients met the criteria for further analysis. Of these 446 events, 77 were attributed to progressive disease, 310 to other toxicity not thought related to ILD, 20 to de-novo ILD, 9 to concurrent illness, and 3 to exacerbation of underlying ILD. 27 cases (25 patients) were felt to have insufficient data to draw firm conclusions. Overall, the incidence of crizotinib-associated ILD was 1.2% (20/1,644) across the four studies, with no difference in incidence seen between Asian and Caucasian populations. The mean onset of ILD was 82 days. The mortality rate for patients identified with crizotinib-associated ILD was 50% (10/20). There was a trend towards improved survival if crizotinib was stopped rather than continued on presentation (64% [9/14] vs. 17% [1/6], P=0.065). Early administration of systemic corticosteroids did not seem to affect survival.

Conclusion:
This report represents the largest systematic review of TKI-associated ILD that has been performed by an independent multidisciplinary review panel. Crizotinib-associated ILD occurred in approximately 1.2% of patients who were administered crizotinib and displayed a characteristic pattern of delayed but rapidly progressive dyspnea and hypoxemia with a mortality of 50% (10/20). This pattern is similar to that seen with the EGFR TKIs, but appears to have a more delayed onset and a higher mortality rate. In contrast to EGFR TKI-associated ILD, which has a markedly higher incidence in the Asian population, there appears to be no such predilection for crizotinib-induced ILD. Immediate cessation of crizotinib may improve survival.

Background:
Crizotinib (XALKORI [TM], Pfizer) , a tyrosine kinase inhibitor targeting ALK, ROS1 and MET, is used for the therapy of advanced anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC). Cardiac toxicity is one of its adverse events which may interrupt the administration of crizotinib. Elevation of CK-MB has been reported but it remains to be determined whether the level of CK-MB can reflect cardiac toxicity of crizotinib therapy. We investigated the clinical manifestations and relevant frequency of heart-related side effects in 94 advanced ALK-rearranged NSCLC patients with treatment of crizotinib to share experiences of management of cardiac toxicity of crizotinib.

Methods:
A retrospective analysis was conducted to demonstrate the clinical manifestations as well as the corresponding frequency of cardiac toxicity in advanced ALK-rearranged NSCLC patients with treatment of crizotinib enrolled in our hospital in the past 4 years.

Results:
In the past 4 years, 95 advanced ALK-rearranged NSCLC patients were treated with crizotinib in our hospital, among which one patient dropped the treatment in 3 days due to grade 4 vomiting. In 94 eligible patients who continue the therapy more than one month, the heart-related side effects include QT interval prolongation (2/94), bradycardia (12/94), hypotension (3/94), aggravation of atrial fibrillation (1/94) and elevation of creatine kinase-MB(CK-MB) (59/94). Consequently, one of 2 patients with QT interval prolongation reduced dosage from 250 mg to 200mg twice daily for QT interval >500 ms on two electrocardiograms and then well tolerated. 12 patients with bradycardia presented asymptomatic and one patient with profound sinus bradycardia (heart rate [HR]≦45) continued crizotinib without dose reduction as she was asymptomatic and benefiting from continuous crizotinib treatment against the deadly disease. Patients with hypotension and aggravation of atrial fibrillation are tolerated and under close follow-up without dose reduction. Remarkably, we observed that majority of our patients (62.77%) experienced elevation of CK-MB and no correlation between age and CK-MB elevation (Pearson Correlation =-0.153，p=0.137).

Conclusion:
Cardiac toxicity is common during crizotinib treatment so that heart-related examinations, such as ECG as well as CK-MB, should be performed regularly especially for those with prior heart disease.

Background:
Crizotinib is an oral tyrosine kinase inhibitor that has been studied in patients with advanced ALK-positive non-small cell lung cancer and has demonstrated median therapy durations ranging from 23 weeks to 10.9 months. In the real-world setting, persistence and adherence to oral therapies may be suboptimal compared with physician-administered treatments. Little is known about crizotinib treatment experiences outside of clinical trials. The objective of this analysis was to evaluate persistence on and adherence to crizotinib using US pharmacy records.

Methods:
De-identified records from five closed-distribution US specialty pharmacies supplying crizotinib in the 50 states and US territories were analyzed. Patients with at least one record of a shipped crizotinib prescription between 8/30/2011 and 8/1/2014 were eligible. Persistence − the duration of time from initiation to discontinuation of therapy − was defined as the total number of days between the first and last prescription shipment dates plus the days' supply of the last prescription. Adherence − the extent to which a patient acts in accordance with the prescribed regimen − was evaluated using a medication possession ratio (MPR): the ratio of the total days' supply over the entire duration of therapy for patients with at least two crizotinib prescriptions. A grace period of 60 days between prescriptions was allowed. Sensitivity analyses of persistence and adherence were performed assuming grace periods between prescriptions of 30 and 90 days.

Results:
A total of 3845 crizotinib patients were identified and included in the analysis. Most were commercially insured (69%), <65 years of age (61%), and female (56%). The cohort was well balanced geographically. Most prescriptions (89%) were for doses of 250 mg BID. Average duration of therapy was 11.4 months (95% CI: 11.2−11.6), with a median of 7.5 months. 51% and 34% of patients remained on therapy at 6 and 12 months, respectively. 16% of patients remained on crizotinib >24 months (Figure 1). Figure 1 An average MPR of 95% was observed in the sub-cohort of 3072 patients with two or more prescriptions of crizotinib. Sensitivity analyses revealed that persistence (11.2−11.6 months) and adherence (93−96%) estimates were not impacted by the alternative grace-period definitions applied.



Conclusion:
In the real-world setting, the duration of crizotinib therapy was similar to that reported in clinical trials. Adherence to crizotinib was high and may be indicative of its acceptable tolerability. Further research is warranted to evaluate experiences associated with crizotinib using other real-world data sources.

Background:
Crizotinib, a standard care for advanced ALK-positive NSCLC, is a substrate for ABCB1-encoded P-glycoprotein, and is primarily metabolized by CYP3A4/5. The most common adverse events (AEs) are visual disorder, gastrointestinal disorders, and elevated transaminase levels. Serious AEs such as grade (Gr) ≥ 3 elevated transaminase levels and interstitial lung disease (ILD) occasionally develop.

Methods:
ALK-positive NSCLC patients were enrolled in cohort A (enrollment before starting crizotinib therapy) or cohort B (enrollment during crizotinib therapy). Trough concentrations of crizotinib at steady state were measured using LC/MS/MS and ABCB1 polymorphisms were analyzed. We evaluated clinically significant AEs, defined as Gr 4 hematological toxicity, Gr ≥ 3 non-hematological toxicity, or any ILD. AEs during 8 weeks were also evaluated prospectively on the patients enrolled in cohort A.

Results:
A total of 78 patients at 17 institutions were enrolled. In cohort A (n = 47), AEs which occurred in more than 40% of patients during 8 weeks were ALT increased (75.0%), visual disorder (47.2%), anorexia (45.5%), nausea (45.5%), and AST increased (43.2%). In both cohorts (n = 75), 26 clinically significant AEs (n = 25) were observed: Gr ≥ 3 elevated transaminase level (14.7%), ILD (4.0%), Gr 4 neutropenia (4.0%), Gr 3 thromboembolic event (4.0%), Gr 3 esophagitis (2.6%), and Gr 3 QTc prolongation (2.6%). There was one treatment-related death (1.3%) due to ILD. Clinically significant AEs tended to occur more frequently in females than males, albeit without significance (38.4% vs. 19.2%, respectively; p = 0.09). Blood samples for trough concentrations of crizotinib at steady state were collected from 63 patients. The geometric mean of trough concentrations were 396 (95% CI, 325-483) ng/ml in male and 395 (95% CI, 329-474) ng/ml in female, respectively (p=0.569, Mann-Whitney U test). No clinical factors including gender, weight, body surface area, and age which influenced trough concentrations or AEs of crizotinib were identified. Moreover, the trough concentration of crizotinib was not significantly different between patient with clinically significant and without (429 [95% CI, 361-509] ng/ml vs. 378 [95% CI, 313-456] ng/ml, respectively [p=0.365]).

Conclusion:
In this multicenter study, we observed crizotinib AEs as previously reported. Clinically significant AEs tended to occur more frequently in females than males, albeit without significance. Furthermore, we will present the association of clinically significant AEs and trough concentration with ABCB1 polymorphism.

Abstract not provided

Background:
The screening algorithm for detection of ALK-rearranged NSCLC is still under investigation. The Break-Apart ALK FISH is the standard diagnostic test for the treatment with crizotinib. However, immunohistochemistry (IHC) with different antibodies shows excellent results when compared to ALK FISH. The efficacy of crizotinib is studied in relation to ALK FISH. We compared IHC outcome and the number of ALK breaks estimated by FISH with clinical outcome and retested at disease progression on crizotinib treatment

Methods:
Patients treated with crizotinib who had biopsies with sufficient tumor tissue were selected. Tumor response was assessed by CT using RECIST v1.1. Fluorescence in situ hybridization (FISH) was performed with Vysis LSI ALK Break Apart FISH Probe KIT (Abbott Molecular Inc., Des Plaines, IL) and immunohistochemistry with Ventana (Ventana Medical Systems Inc, Tucson, AZ) ALK IHC Kit using D5F3 antibody. The same tests were performed on re-biopsy material after progression on crizotinib.

Results:
Twenty-nine patients with ALK positive advanced NSCLC were treated with crizotinib. Median (range) age was 54 yrs (21-75); 26 had an adenocarcinoma, 3 had large-cell carcinoma. We confirmed the presence of EML4-ALK fusions in all samples either by FISH or IHC. Median percentage of ALK breaks was 47% (2-76). Tumor responses occurred in 9 pts having a median of 51 (6-76) breaks per tumor sample. The 20 non-responders had a median of 27 (15-64) breaks per tumor sample with 64 being an outlyer. In IHC positive pts 9/11 had a response to crizotinib treatment. In those who were IHC negative no pts (0/14) had a response (12 PD; 2 SD). Median PFS for positive IHC is 7.9 mo (95% CI., 5.5 – 10.3) and for negative IHC 1.6 mo (1.3 – 1.8), n=17, p<0.0001. Overall survival is 6.5 mo (95% CI., 0 – 17.8) and 18.3 mo (95% CI., 11.1 – 25.6) respectively, n=29, p=0.05. In 8 pts with progressive disease re-biopsies were performed. Only in one pt FISH ALK became negative. In the other pts IHC and FISH remained both positive. In pts who progressed on crizotinib, 4 pts had extra red in the FISH, which may be treatment related.

Conclusion:
All IHC positive advanced NSCLC patients responded to crizotinib. Extra red in the FISH test may cause progression to treatment with crizotinib.

Background:
PROFILE 1014 compared the efficacy and safety of the ALK inhibitor crizotinib with platinum based chemotherapy in previously untreated advanced ALK-positive advanced NSCLC (Pfizer; NCT01154140). The primary endpoint was progression-free survival. The main objective of this post-hoc analysis was to compare patient-reported symptom and global quality of life (QOL) between crizotinib and chemotherapy in the subgroup of patients of Asian ethnicity in the ongoing study PROFILE 1014.

Methods:
Patients in the ongoing PROFILE 1014 study were randomized to crizotinib (250 mg PO bid; n= 172) or chemotherapy (pemetrexed 500 mg/m[2] + either cisplatin 75 mg/m[2] or carboplatin AUC 5–6; all IV q3w for ≤6 cycles; n= 171). Patient-reported outcomes were assessed at baseline, day 7 and day 15 of cycle 1, day 1 of subsequent 3-week cycles and end of treatment using the validated cancer specific questionnaire EORTC QLQ-C30 and its lung cancer module QLQ-LC13. Validated translations of the questionnaires in Asian languages (Japanese, Chinese, Korean etc) were made available. Higher scores (range 0−100) indicated higher symptom severity or better functioning/QOL. A positive change from baseline score indicates improvement for global QOL/functioning and deterioration in symptoms. Repeated measures mixed-effects analyses were performed to compare change from baseline scores between the treatment arms, with no adjustments made for multiple comparisons.

Results:
Of 343 patients randomized, 46% were of Asian ethnicity (crizotinib, n=77; chemotherapy, n=80). Completion rates at baseline were ≥95% in each group and scores were balanced. A statistically significantly greater overall improvement from baseline was observed with crizotinib compared with chemotherapy for global QOL (5.6 vs -7.7; p<0.001), emotional functioning (9.5 vs 2.7;p<0.05), physical functioning (5.0 vs - 2.7 p<0.001) and role functioning (3.7 vs. -7.2;p<0.001). A statistically significantly greater overall improvement was observed with crizotinib compared with chemotherapy for cough (-17.3 vs. -11.2; p<0.05), dyspnea (-9.5 vs.-1.1; p<0.001), pain in arm or shoulder (-11.4 vs.-2.2; p<0.001), pain in chest (-7.3 vs.3.3; p<0.001), pain in other parts (-11.2 vs. -0.4;p<0.001), fatigue (-9.9 vs. 3.9; p<0.001), insomnia (-10.3vs. -2.0; p<0.05), pain (-12.2 vs.-1.2; p<0.001) and appetite loss (-5.3 vs. 5.7; p<0.001). A statistically significantly greater overall deterioration was observed in the crizotinib arm for diarrhea (12.6 vs. 2.4; p<0.001) compared with chemotherapy. No statistically significant differences were observed for social functioning, sore mouth, dysphagia, nausea & vomiting, constipation and alopecia between crizotinib and chemotherapy.

Conclusion:
Consistent with previously reported results in the overall study population, treatment with crizotinib showed statistically significantly greater overall improvement in patient-reported lung cancer symptoms and global QOL compared with chemotherapy in the subgroup of patients of Asian ethnicity with previously untreated advanced ALK-positive NSCLC.

Background:
In the pivotal ASCEND-1 study, ceritinib, an anaplastic lymphoma kinase inhibitor (ALKi), showed clinical activity in patients with ALK-rearranged (ALK+) non-small cell lung cancer (NSCLC), including in patients with brain metastases (BrM). Here, patient-reported outcomes (PROs) from the recently reported ASCEND-2 study (NCT01685060) are described for chemotherapy- and ALKi-pretreated patients with ALK+ NSCLC with and without baseline BrM

Methods:
In ASCEND-2, adult patients with ALK+ NSCLC previously treated with chemotherapy and an ALKi (crizotinib) received oral ceritinib 750 mg daily. PROs were assessed at baseline and Day 1 of treatment cycles 2, 3, and every two cycles thereafter (1 cycle=28 days), using the Lung Cancer Symptom Scale (LCSS) and EORTC quality of life and lung cancer surveys (QLQ-C30 and QLQ-LC13, respectively). Data were analyzed by presence/absence of baseline BrM. Data beyond cycle 9 are not reported due to small sample sizes.

Results:
All 140 patients enrolled (median age [range] 51 [29–80] years; 50.0% male), had received ≥2 antineoplastic regimens and 100 (71.4%) had BrM at baseline. At data cutoff (13 August 2014), median follow-up was 11.3 months. PRO questionnaire compliance was at least 91.2% up to cycle 9. In the overall patient population, investigator-assessed disease control rate (DCR) was 77.1% and median duration of response (DOR) 9.7 months. Investigator-assessed whole-body DCR [95% confidence interval (CI)] in patients with and without baseline BrM was 74.0% [64.3, 82.3] and 85.0% [70.2, 94.3], respectively, while DOR [95% CI] was 9.2 [5.5, 11.1] and 10.3 [7.4, 16.6] months, respectively. Analysis of PROs data demonstrated that treatment with ceritinib improved lung cancer symptoms in patients with and without baseline BrM (Figure). QLQ-LC13 outcomes were broadly consistent with those of LCSS. In general, mean global quality of life (QLQ-C30) was maintained on treatment for both patient subgroups, with mean change from baseline in QLQ-C30 global health status ranging from -3.06 to +7.25 in patients without baseline BrM and -2.83 to +3.55 in those with baseline BrM. Figure 1



Conclusion:
In patients with ALKi-pretreated ALK+ NSCLC who received prior chemotherapy and ceritinib, clinical efficacy was demonstrated and cancer symptoms were mostly improved, with health-related quality of life generally maintained regardless of presence or absence of baseline BrM.

Background:
In the pivotal ASCEND-1 study, ceritinib, an anaplastic lymphoma kinase inhibitor (ALKi), demonstrated sustained clinical activity in ALKi-naive patients with ALK-rearranged (ALK+) non-small cell lung cancer (NSCLC), including in patients with brain metastases (BrM). ASCEND-3 (NCT01685138) evaluated patient-reported outcomes (PROs) as well as clinical outcomes with ceritinib, in ALKi-naive ALK+ NSCLC patients with and without baseline BrM.

Methods:
Adult patients with ALK+ NSCLC previously treated with up to 3 lines of cytotoxic therapy received oral ceritinib 750 mg daily. PROs were assessed using Lung Cancer Symptom Scale (LCSS) and EORTC (QLQ-C30, QLQ-LC13) quality of life and lung cancer surveys at baseline and Day 1 of treatment cycles 2, 3, and every two cycles thereafter (1 cycle=28 days). Data were analyzed by presence/absence of baseline BrM. Data beyond cycle 9 are not reported due to small sample sizes.

Results:
Of 124 enrolled patients (median age [range] 56 [27–82] years; 40.3% male), 50 (40.3%) had BrM at baseline. At data cutoff (27 June 2014), median follow-up was 8.3 months. Up to cycle 9, PRO questionnaire compliance was at least 97.0%. In the overall patient population, investigator-assessed disease control rate (DCR) was 89.5% and median duration of response (DOR) 9.3 months. Investigator-assessed whole-body DCR [95% confidence interval (CI)] in patients with and without baseline BrM was 86.0% [73.3, 94.2] and 91.9% [83.2, 97.0], respectively, while DOR [95% CI] was 9.1 [7.5, Not Estimable] and 10.8 [9.3, 10.8] months, respectively. Mean change from baseline in patients’ total LCSS score ranged from -3.4 to -11.4 while receiving ceritinib, with 82.1% of patients experiencing symptom improvement; symptoms improved in patients with and without baseline BrM (Figure). QLQ-LC13 outcomes were broadly consistent with those of LCSS in the full patient population and in the subgroups of patients with and without baseline BrM. In general, mean global quality of life (QLQ-C30) was maintained on treatment for all patients. Patients reported diarrhea and nausea and vomiting symptoms were worse than baseline, however, nausea and vomiting symptoms did reduce over time. Figure 1



Conclusion:
In ALKi-naive patients with ALK+ NSCLC, treatment with ceritinib demonstrated clinical efficacy and improved cancer symptoms, with health-related quality of life generally maintained regardless of baseline BrM status. Improvements were greatest for the lung-related symptoms, cough and pain.

Abstract not provided

Abstract:
Despite advances in genomic technologies, most advanced solid tumors remain incurable and drug resistance is almost inevitable with limited biomarkers available to personalize therapy. Two important lessons have emerged from the comprehensive genomic analyses of cancers, which may provide an explanation for difficulties that have been encountered in biomarker development. First, each tumor contains an individual assortment of multiple genomic aberrations, few of which are shared between patients with the same histopathological tumor subtype. Second, emerging evidence suggests that these anomalies appear to vary both spatially and temporally within the tumor, indicating substantial intratumor heterogeneity. Increasingly, molecular evidence suggests that intratumor heterogeneity may contribute to tumor growth through a branched (polytypic) rather than a linear pattern of tumor evolution. Branched evolutionary growth and intratumor heterogeneity results in coexisting cancer cell subclones with variegated genotypes and associated functional phenotypes that may be regionally separated within the same tumor or distinct within one biopsy and alter in dominance over time. Variegated phenotypes, resulting from intratumoral genetic heterogeneity and the emergence of new subclones at relapse, are likely to have important implications for developing novel targeted therapies and for preventing the emergence of drug resistance. Intratumor heterogeneity and tumour sampling bias, resulting from single biopsy-driven biomarker discovery and validation approaches, may also contribute to the recently reported failures in implementation of robust biomarkers in the clinical setting. Clinical trial efforts taking into account tumour heterogeneity and its relevance to lung cancer will be addressed.

Abstract:
There are many different manifestations of heterogeneity within lung tumours. The three main considerations are: Morphologically, all of the neoplastic cells in a tumour are not identical. These structural differences may be due to differential protein expression and cellular differentiation, in turn the result of either differential expression of wild type genes, post-transcriptional modification or expression of altered genes (mutation, fusion genes etc). Nuclear morphology varies at least in part due to alteration in chromosome structure and number. Genetically, tumour cell populations are heterogeneous, as mentioned above but also it can be demonstrated that there may be heterogeneity related to driver mutations and other functionally important changes which may not necessarily be deterministic of cell morphology. Compositely speaking, tumours are made up of more than just neoplastic cells; stromal cells, immune cells and vasculature for example may account for much of the tumour bulk. Potentially, all three of these may impact upon molecular testing practice in the initial diagnostic phase and at re-biopsy. Molecular testing may be executed in many different ways and may seek many different molecular changes, such that the potential for heterogeneity making an impact on testing is considerable. Some molecular tests involve the morphological examination of a histological or cytological slide for the presence or absence of a particular factor. Proteins are normally assessed at a morphological level using immunohistochemistry. In situ hybridisation can be used to visualise and assess the presence of specific mRNAs. The same technique is used to assess DNA; specific gene copy numbers (gene amplification, polysomy), the creation of new ‘fusion genes’ during rearrangement using break-apart probes etc. These techniques require the molecular signal to be visualised in the cells of interest (usually the tumour cells); morphological and compositional tumour heterogeneity greatly impact the ease with which these techniques are executed. In lung cancer molecular testing, most current interest is in mutation testing. Compositional heterogeneity is a significant practical issue and drives recommendations that samples are pathologically assessed before extraction and mandates steps be taken to maximize the proportion of the sample for extraction that is tumour (macro- or microdissection techniques are often used). The dilution of mutant alleles by wild type alleles from non-neoplastic tissue may lower the mutation allele frequency below the threshold for detection. Are therapeutically important mutations such as those in exons 18-21 of EGFR heterogeneously expressed in tumour cells? This remains a matter of some controversy. Some have argued that since these are addictive driver mutations, they are present from the start of tumourigenesis and therefore present in every tumour cell, as determined by clonal expansion of the neoplastic cell population. Studies which demonstrate mutations in some areas of extracted tumour but not in others, are criticized by failing to use sufficiently sensitive techniques to detect mutations which are over-diluted by non-neoplastic DNA. It is known that selective amplification of mutant alleles (MASI) is heterogeneous in tumours and this may lead to apparent heterogeneity of mutation (detectable in some areas and not in others) when the number of mutant alleles per tumour cell varies in different parts of the tumour, and those areas with fewer mutant alleles are not detected due to poor test sensitivity. This explanation for apparent mutational heterogeneity has been challenged by some studies, however, which have appeared to demonstrate heterogeneity, even when highly sensitive techniques are used. Heterogeneity appears to be associated with lower response rates to EGFR TKIs in EGFR mutant tumours. Discrepancy has been reported in mutational findings between synchronous primary tumour and metastatic deposits. These findings are not universal for EGFR mutations, but when present, tend to involve a mutated primary with wild type metastases more often than the reverse. Data are few but could influence biopsy strategies. More than one mutation may be present in a lung cancer. In the context of molecular aberrations commonly tested for (EGFR, KRAS mutation; ALK rearrangement), double mutations are described but are rare. It is rather more common, for example for double or even triple EGFR mutations to be found in the same tumour sample. For example, in the author’s laboratory, double EGFR mutations are found in 13.8% of EGFR-mutated cases; triple mutations in 0.6%. KRAS double mutations are exceptionally rare (0.8%). The presence of more than one mutation, often at different allelic frequencies (such as can be estimated in many studies), implies different clones of cells bearing different mutations, and from this comes the concept of minor clones of therapeutically resistant cells which are responsible for some, though probably not all disease recurrences on TKI therapy. The best known scenario fitting this ‘minor clone’ hypothesis is the emergence of tumour bearing the EGFR T790M resistance mutation, as well as the original sensitizing mutation, for which an EGFR TKI was given. Resistant minor clones of MET amplified cells may be an alternative source of recurrent, EGFR TKI resistant disease. Similarly with ALK mutated or KRAS mutated recurrences during ALK TKI therapy for ALK rearranged tumours. This increasingly recognised outcome in patients treated with EGFR or ALK TKIs is now driving re-biopsy of recurrent disease into standard of care. Testing approaches and strategy for recurrent disease are still evolving and are driven by this concept of minor clone heterogeneity. Another finding in the re-biopsy setting is histological subtype transformation. Whilst the initial EGFR or ALK altered tumour is almost always adenocarcinoma, recurrent disease may be small cell, sarcomatoid or even squamous cell carcinoma. Little is known about the mechanism of this transformation; emergent clones of different histology or differential stem cell differentiation? There are also emergent data demonstrating that where recurrence occurs at multiple sites, detectable resistance mechanisms may vary. In a broader sense, heterogeneity of sensitivity to particular therapies, amongst tumour cells, is a major driver of treatment resistance and/or relapse, and effectively why there are so very few instances of true cure of lung cancer as a consequence of systemic therapy. The development of effective treatment strategies to overcome recurrence will require a better understanding of how tumour heterogeneity influences this process.

Abstract:
Adenocarcinomas represent the most frequent subtype of lung cancer[1], and they are usually discovered late in the course of the disease even in the setting of vigilant radiographic and cytologic screening[2]. Despite improvements in molecular diagnosis and targeted therapies, the average 5 year-survival rate for lung adenocarcinoma remains only 15%[3]. Novel strategies based on the detection of genetic markers offer new hope for improved risk assessment, early cancer detection, therapeutic intervention and tumor surveillance, but the impact of these strategies has been limited by an incomplete understanding of the biology of lung cancer, particularly in its early developmental stages. Disappointingly, relatively few genetic alterations critical to the development of lung adenocarcinomas are currently recognized, and the timing and manner by which these alterations initiate and drive glandular neoplasia remains to be delineated. Recent refinements in the histologic classification of lung adenocarcinomas provide greater resolution of the sequential steps of glandular lung neoplasia[4]. Atypical adenomatous hyperplasia (AAH) is a microscopic discrete focus of cytologically atypical type II pneumocytes and/or Clara cells[5-6]. Once dismissed as a reactive change, AAH is now regarded as the first histologic step in a morphologic continuum culminating in the fully malignant adenocarcinoma. The link between AAH and invasive adenocarcinoma is strong and compelling: 5-20% of lungs resected for primary adenocarcinomas also harbor AAH, and AAH harbors some of the same genetic and epigenetic alterations found in adenocarcinomas including KRAS mutations, EGFR mutations, loss of heterozygosity at 9q and 16p, TP53 mutations, and epigenetic alterations in the WNT pathway. Like AAH, adenocarcinoma in situ (AIS) (formerly known as bronchioloalveolar carcinoma, BAC) is recognized as a non-invasive form of glandular neoplasia, but one that exhibits increased size, cellularity and morphologic atypia. In effect, it represents a next step in the continuum towards malignant adenocarcinoma. Minimally-invasive adenocarcinoma (MIA) is defined as a small adenocarcinoma (≤ 3cm) with a predominantly lepidic pattern and invasion of 5 mm or less in any one focus[4]. Invasive growth is present, albeit so limited that these carcinomas have been associated with 100% disease free survival[7,8]. This enhanced delineation of early glandular neoplasia provides a rational histologic framework for studying the timing of genetic alterations driving the early stages of lung tumorigenesis. “Branched evolutionary tumor growth” is the concept that cancers evolve by a repetitive process of clonal expansion, genetic diversification and clonal selection within the adaptive landscapes of tissue ecosystems[9]. In this study, to determine whether this phenomenon is operational during early stages of tumor progression, we evaluated lung glandular neoplasms spanning the full spectrum of early histologic progression using next generation sequencing (NGS) of coding regions from 125 well-characterized cancer-driving genes. We specifically targeted multifocal AAHs and advancing zones of histologic progression within individual AISs and MIAs. This multi-region sequencing revealed that clonal expansion is an early event that can be confirmed even in the earliest recognized step in glandular neoplasia. Moreover, the identification of significant genetic alterations such as KRAS mutations, loss of P53 activity and EGFR activation points to the presence of functionally relevant “drivers” that empower territorial expansion of subclones en route to malignancy. Importantly, these driver alterations are potentially measurable in clinical samples. Using ultra-sensitive droplet digital PCR (ddPCR), mutant DNA associated with early lesions was detected in a patient’s plasma and sputum providing proof of principle that even the earliest stages of glandular neoplasia can be detected via analysis of circulating DNA (circDNA). Our study provides the unique insight into the genetic alterations that initiate and drive the progression of lung glandular neoplasia and underlines the need for precise definition of these events to improve proper diagnosis and early detection of tumors. Identification of mutational features which characterize relevant lesions that actually progress to cancers will allow to better predict the fate of these early lesions and tailor the right therapy to prevent the progression. 1. Colby T. V., Koss M. N. & W., T. in Tumors of the Lower Respiratory Tract (eds Colby T. V., Koss M. N., & Travis W. D.) 91-106 (Armed Forces Institute of Pathology Washington DC, 1994). 2. Frost, J. K. et al. Early lung cancer detection: results of the initial (prevalence) radiologic and cytologic screening in the Johns Hopkins study. The American review of respiratory disease 130, 549-554 (1984). 3. Imielinski, M. et al. Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell 150, 1107-1120, doi:10.1016/j.cell.2012.08.029 (2012). 4. Travis, W. D. et al. Diagnosis of lung adenocarcinoma in resected specimens: implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. Archives of pathology & laboratory medicine 137, 685-705, doi:10.5858/arpa.2012-0264-RA (2013). 5. Weng, S. Y., Tsuchiya, E., Kasuga, T. & Sugano, H. Incidence of atypical bronchioloalveolar cell hyperplasia of the lung: relation to histological subtypes of lung cancer. Virchows Archiv. A, Pathological anatomy and histopathology 420, 463-471 (1992). 6. Chapman, A. D. & Kerr, K. M. The association between atypical adenomatous hyperplasia and primary lung cancer. British journal of cancer 83, 632-636, doi:10.1054/bjoc.2000.1317 (2000). 7. Borczuk, A. C. et al. Invasive size is an independent predictor of survival in pulmonary adenocarcinoma. The American journal of surgical pathology 33, 462-469, doi:10.1097/PAS.0b013e318190157c (2009). 8. Yim, J. et al. Histologic features are important prognostic indicators in early stages lung adenocarcinomas. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 20, 233-241, doi:10.1038/modpathol.3800734 (2007). 9. Greaves, M. & Maley, C. C. Clonal evolution in cancer. Nature 481, 306-313, doi:10.1038/nature10762 (2012).

Abstract not provided

Background:
Neuroendocrine lung tumours account for 25% of all lung cancer cases, and they range from low-aggressive pulmonary carcinoids (PCA) to highly malignant small-cell lung cancer (SCLC) and large-cell neuroendocrine lung carcinoma (LCNEC). The last two are strongly associated with heavy smoking and are typically detected at a clinically advanced stage, having a poor survival. Comprehensive genomic analyses in lung neuroendocrine tumours are difficult because of limited availability of tissue. While more effort has been done in the context of SCLC, the detailed molecular features of LCNEC remain largely unknown.

Methods:
We conducted 6.0 SNP array analyses of 60 LCNEC tumours, exome sequencing of 55 tumor-normal pairs, genome sequencing of 11 tumour-normal pairs, transcriptome sequencing of 69 tumours, and expression arrays on 60 tumors. Data analyses were performed using in house developed and published pipelines.

Results:
Analyses of chromosomal gene copy number revealed amplifications of MYCL1, FGFR1, MYC, IRS2 and TTF1. We also observed deletions of CDKN2A and PTPRD. TTF1 amplifications are characteristic of lung adenocarcinoma (AD); CDKN2A deletions are frequent alterations in both AD and squamous-cell lung carcinoma (SQ); FGFR1 amplifications are found in SQ and, less frequently, in SCLC; and MYCL1 and IRS2 amplifications are frequent events in SCLC. Similar to the copy number data, we found patterns of mutations characteristic of other lung cancer subtypes: TP53 was the most frequently mutated gene (75%) followed by RB1 (27%), and inactivation of both TP53 and RB1, which is the hallmark of SCLC, occurred in 20% of the cases. Mutations in STK11 and KEAP1-NFE2L2 (frequently seen in AD and SQ) were found in 23% and 22% of the specimens, respectively. Interestingly, mutations in RB1 and STK11/KEAP1 occurred in a mutually exclusive fashion (p-value=0.016). Despite the heterogeneity observed at the mutation level, analysis of the pattern of expression of LCNEC in comparison with the other lung cancer subtypes (AD, SQ, SCLC, and PCA) points to LCNEC as being an independent entity. An average mutation rate of 10.7 mutations per megabase was detected in LCNEC, which is in line with the rate observed in other lung tumours associated with smoking. We found that, similar to SCLC, the mutation signatures associated with APOBEC family of cytidine deaminases, smoking, and age (based on Alexandrov et al 2013) were the predominant ones in LCNEC. However, the contribution of the individual SCLC and LCNEC samples to these three signatures was quite different, and we are currently exploring it.

Conclusion:
Taking into account somatic copy number and mutation data, we distinguished two well-defined groups of LCNEC: an SCLC-like group, carrying alterations in MYCL1, ISR2, and in both RB1 and TP53; and a group resembling AD and SQ, with alterations in CDKN2A, TTF1, KEAP1-NFE2L2, and STK11. Although these results suggest that LCNEC might be a mix of different lung cancer subtypes, mutation clonality and expression analyses show that they are likely to be a separate entity, sharing molecular characteristics with the other lung cancer subtypes. Their heterogeneity suggests that LCNEC might represent an evolutionary trunk that can branch to SCLC or AD/SQ.

Background:
Identification of clinically relevant molecular drivers in patient tumors is essential in selecting appropriate targeted therapy. Using next-generation sequencing (NGS) -based clinical cancer gene test, we performed genomic profiling of lung adenocarcinoma tumors.

Methods:
We collected formalin-fixed paraffin-embedded tumors from 41 lung adenocarcinoma patients whose tumors previously tested negative for EGFR/KRAS/ALK by conventional methods in an ongoing trial (NCT01964157). We performed hybridization capture of 4,557 exons from 287 cancer-related genes and 47 introns from 19 genes frequently rearranged in cancer (FoundationOne). Illumina HiSeq2000 platform was used to sequence to high uniform depth.

Results:
Figure 1Tumors were sequenced to a median coverage of 529x. Overall, we identified a total of 170 known and 492 unknown individual genomic alterations. The number of known alterations per sample was average of 3.8 alterations (range 0-10). Cancer genomes are characterized by 45% (77/170) non-synonymous base substitutions, 17% (29/170) insertions or deletions, 2% (4/170) splice site mutations, 20% (34/170) gene amplifications, 5% (8/170) homozygous loss and 5% (8/170) gene fusions. TP53 was the most commonly mutated gene (13%, n=10/77) among non-synonymous base substitutions, followed by KRAS (10%, n=8/77) and PIK3CA (8%, 6/77). Insertions or deletions commonly occurred TP53 (17%, 5/29) and ERBB2 (14%, 4/29), and splice site mutations occurred in TP53, INPP4B, ATR, and MAP2K4 (n=1 each). Among gene amplification, MDM2 amplification was the most frequent (12%, 4/34), followed by ERBB2 (8%, 3/34) and CDK4 (8%, 3/34) amplification. All 8 cases of homozygous loss were observed with CDKN2A and CDKN2B. Fusion genes were most commonly observed with RET (50%, n=4/8). Based on NCCN guidelines, actionable genomic alterations with a targeted agent were identified in 16 patients (39%) (BRAF mutation [n=1], EGFR mutation [n=7], ERBB2 mutation [n=4], MET amplification [n=1], KIF5B-RET rearrangement [n=2], CCDC6-RET rearrangement [n=1], and CD74-ROS1 rearrangement [n=1]). Nine out of all patients (22%) showed discordance in targetable alterations when compared between NGS and conventional non-NGS methods.



Conclusion:
Thirty-nine percent of lung adenocarcinoma wild type for EGFR/KRAS/ALK may harbor a genomic alteration revealed by NGS approach. These results highlight the importance of profiling lung adenocarcinomas using NGS in the clinic.

Background:
Genome wide SNP array studies have identified systematic gene copy number aberrations (CNA) in non-small cell lung cancer (NSCLC), but their prognostic implication is unknown. This study aimed to investigate associations between CNAs and survival using the LACE-Bio bio-bank. The LACE-Bio consortium includes large clinical trials comparing adjuvant platinum-based chemotherapy to observation after complete resection of stage I-III NSCLC.

Methods:
DNA was extracted from FFPE tumor samples from 3 pivotal adjuvant chemotherapy trials (CALGB 9633, IALT, JBR.10); 1013 samples were profiled using Affymetrix OncoScan[TM] arrays with over 300,000 probes and normalized relative to a pool of normal tissues. Segmentation was performed using the CBS algorithm and minimally recurrent regions (MCR) across the series identified by CGHregions. All analyses were performed on the level of MCRs. CNAs were correlated with clinicopathological factors and adjusted for the False Discovery Rate (FDR). The primary endpoint, disease-free survival (DFS), was assessed via univariate Cox models stratified by trial and adjusted for treatment, age, sex, PS, histology, T, and N stage.

Results:
Among 976 successfully profiled samples, 414 (42%) were adenocarcinoma (ADC), 430 (44%) squamous cell carcinoma (SCC) and 132 (14%) other NSCLC; 710 (73%) were male. Across the 431 MCRs identified, patients had on average 94 (SD 69) CNAs: 51 gains and 43 losses. A gain or loss was observed in at least 10% of patients for 177 and 166 regions respectively. The most common gains (up to 48%) were on chromosomes 1p, 3q, 5p, 6p, and 22q. The most common losses (up to 40%) were on chromosomes 3p, 8p and 9p. The size of 253 of the 431 MCRs (59%) was smaller or equal to 3Mb (and 79% ≤10 Mb). Sensitivity analyses on the subset of samples with optimal quality (n=777, defined by MAPD<0.3) gave consistent results. The CNA frequency of 195 regions was significantly different with FDR≤0.05 between ADC and SCC (of which 49% regions of size ≤3Mb and 71% ≤10Mb); the most significant were more gains in 3q, 22q and 12 in SCC and more losses in 3p, 4, 5q in SCC. With a median follow-up of 5.3 years, 510 DFS events and 451 deaths were recorded. In univariate analyses for DFS, 13 regions in loci 19p11–13, 7p12, 9p21, 15q14 had a raw p-value <0.005 (FDR<0.13, the top 8 corresponded to FDR≤0.05); 9 of those 13 regions were of size ≤3Mb (12 regions ≤10Mb). In adjusted analyses, 10 of the 13 regions retained raw adjusted p-values ≤0.005 (FDR≤0.15). Losses of focal regions including CDKN2A/B and STK11 (≤3Mb) were associated with poorer DFS: the hazard ratio (HR) for a 2-fold copy number decrease in region 9p21.3 (including CDKN2A/B) was 1.50 (95% CI: 1.2–1.9, P<0.001, FDR=0.02), and the HR for a 2-fold copy number decrease in 19p13 (including STK11) was 2.4 (1.3–4.3, P=0.005, FDR=0.15). Similar results were obtained for overall survival and lung-cancer specific survival. Results of histology-specific analyses will be presented.

Conclusion:
These large-scale genome-wide analyses of gene CNA provide new candidate prognostic markers for stage I-III NSCLC.

Abstract not provided

Background:
Molecular biology has changed the treatment of advanced NSCLC, leading to many small subgroups of patients (pts) eligible for targeted therapies, many of them being not approved. Since 2010 we created a monthly MTB dedicated to NSCLC pts with potential driving molecular abnormalitie(s). MTB includes expert physicians from the lung tumor board and phase I unit, radiation therapists, researchers, geneticists, pathologists and biologists. A medical report summarizes the findings and treatment recommendations for each pts. We report 4 years of activity of MTB at Gustave-Roussy.

Methods:
All consecutive files discussed in MTB for a NSCLC were reviewed. MTB included pts with at least one molecular alteration based on a 75 gene panel (NGS analysis and FISH for ALK, HER2, MET, FGFR1, ROS1 and RET). Tumor and pts characteristics were collected as well as treatments. Pts outcome was calculated from the MTB date. Kaplan-Meier methods, and Cox proportional hazards models were used for survival analysis, adjusting for sex, histology, smoking status, metastasis at diagnosis, number of line(s) before MTB.

Results:
502 files were discussed between 02/2010 and 09/2014. Median age was 60 yrs (25–88 yrs), 53% were male, 86% Caucasian, 26% never-smokers, and 93% had PS ≤1. Initial clinical stage was III-IV in 417 pts (84%) and 79%/10%/11% were adenocarcinomas/squamous cell carcinomas/others NSCLC. Median number of treatment-lines before MTB was 1 (0-10), 86% were previously treated by a platinum-based chemotherapy regimen, 17% in a therapeutic trial, and median time from diagnosis to MTB was 5 months. Biopsy for Molecular Analysis (MoA) mostly came from CT guided biopsies (62%), surgery (21%) or endoscopy (16%). Biopsy was repeated in 19% of pts to get enough material for MoA. The MoA results were ALK rearrangement in 11%, exon 18/19/20/21 EGFR mutation (mut) in 2/14/4/7% respectively, KRAS mut in 32%, PI3KCA mut in 3%, BRAF mut in 5%, HER2 mut (Exon 20) in 2%, HER2 amplification in 2%, FGFR1 amplification in 3%, MET amplification in 3% and other rare mutations in 27%. MTB recommended a targeted therapy in 344 pts (68%) either within clinical trials (57%), EMA approved therapy (23%), an off label drug (9%), or an expanded access program (11%). 162pts (47%) actually received the recommended therapy, 141 (41%) did not and 41 (12%) might receive it at the time of progression. Median follow-up was 24 months (1-24; follow-up censored after 24 months). Median OS was 13.1 months [95%CI: 8.8; 18.2] for non-oriented pts, and 14.3 months [11.5; 16.7] for oriented pts (p=0.39). We observed a significant difference between EGFR/ALK/ROS1 mutated/rearranged pts (median 23.8 months) vs. pts with KRAS (8.6 months) or others mutations (11.1 months) or non-oriented pts (13.1 m; p=0.0008, HR=0.56, 1.15 and 0.97 respectively compared to non-oriented).

Conclusion:
MTB is feasible in daily practice with treatment recommendations in a majority of NSCLC pts (68%), enrichment in clinical trials or expanded access programs, and limitation of off-label drugs use. Benefit on survival for all oriented pts has to be clarified based on the type of molecular abnormality. Update results will be presented at the meeting.

Background:
Testing for biomarkers including EGFR mutations and ALK rearrangements is standard of care in the management of advanced non-small cell lung cancer (NSCLC), as it determines optimal systemic therapy (ST). Our centre began EGFR testing March 2010 and ALK April 2012. Initially, EGFR/ALK were requested by medical oncologists (MO) when patients were deemed eligible for EGFR or ALK targeted therapy. To expedite biomarker information to MO for rapid initiation of ST in patients with advanced stage or earlier stage disease that developed recurrence, June 2013 we implemented a multidisciplinary approach termed “reflex testing”. This was defined as our pathologists requesting EGFR/ALK at time of diagnosis of non-squamous NSCLC irrespective of a patient’s clinical stage. If tissue was at an outside centre, clerical staff requested EGFR/ALK at time of referral to MO. The objective of this study was to determine if reflex testing improved time-to-treatment (TTT) and the integration of personalized medicine in patients with advanced NSCLC.

Methods:
This was a retrospective chart review of patients with non-squamous NSCLC seen by MO at the Sunnybrook Odette Cancer Centre from March 18, 2010 to April 30, 2014. Patient and EGFR/ALK test characteristics were compared before and after reflex testing was implemented using Chi-square tests of association. Time outcomes were compared using Mann-Whitney U non-parametric tests. TTT was defined as the interval between first MO visit with advanced NSCLC to initiation of ST.

Results:
Of the 301 patients included, median age was 68, 43% female, 65% Caucasian, 75% smokers, 93% adenocarcinoma, 22% EGFR positive and 1% ALK positive. The majority presented with stage IV (65%) and 82% either presented with or developed advanced NSCLC. In advanced NSCLC patients (n=247), reflex testing significantly reduced median TTT compared to routine testing [(24 days (IQR: 7 to 42) vs. 36 days (IQR: 16 to 72), p=0.04)], reduced the rate of EGFR unknown (4% vs. 26%, p=0.002) and ALK unknown (10% vs. 50%, p<0.001). There was minimal impact on advanced NSCLC patients receiving any first-line ST (58% vs. 63%, p=0.48). However, among these patients, with reflex testing, fewer were initiated on first-line ST without biomarker results known by MO (EGFR 23% vs. 39%, p=0.12, ALK 17% vs. 42%, p=0.02), and at last follow up significantly fewer had EGFR unknown (0% vs. 13%, p=0.004) and ALK unknown (7% vs. 38%, p=0.003). Across all stages, rates of EGFR results available to MO at first consultation increased (34% vs. 4%, p<0.001). Reflex testing also impacted the quality of biomarker testing with a decrease in unsuccessful EGFR tests due to inconclusive results, insufficient or inappropriate tissue, or tissue not sent from holding lab to testing lab (4% vs. 15%, p=0.03).

Conclusion:
A multidisciplinary approach to earlier biomarker testing in NSCLC is feasible. Reflex testing for EGFR/ALK improved TTT and the integration of personalized medicine for patients with advanced NSCLC by improving biomarker testing rates, the quality of testing and fewer patients given ST without biomarkers known. These outcomes provide support for reflex EGFR/ALK testing by pathologists at time of diagnosis of non-squamous NSCLC.

Background:
The Network Genomic Medicine (NGM) Lung Cancer is an interdisciplinary and intersectoral network offering comprehensive and centralized next generation sequencing (NGS)-based multiplex genotyping for all inoperable lung cancer patients in Germany. In 2014 NGM and the AOK Rheinland/Hamburg, one of the largest German public health insurances, have successfully contracted and established the first "flat rate" cost reimbursement model for NGS-based comprehensive lung cancer genotyping in Europe. After a year the first joint health-economic evaluation of NGM patients was initiated.

Methods:
The AOK Rheinland/Hamburg cooperates with NGM within the integrated care contract (ICC) according to § 140 German Social Insurance Code. Besides the cost reimbursement model for the NGS-based diagnostics the ICC comprises optional second opinion consultation hours and a joint evaluation program. The NGS panel used for all patients currently consists of 14 genes and 102 amplicons to cover potentially targetable aberrations. Other German public and private health insurances are currently negotiating to join the ICC. In April 2015 we elaborated a model to analyze molecularly guided therapy cost and outcome of inoperable lung cancer patients integrating health insurance cost data (diagnostic, therapy and drug-related costs). This model includes NGS-based molecular diagnostic results, treatment strategies and cost-effectiveness. Additionally, time-points of molecular genotyping and their influence on patient-related outcome and quality of life will be examined.

Results:
In 2014 about 4500 lung cancer NGM patients were centrally genotyped on the central NGS platform in Cologne. Since April 2014 167 patients, insured by the AOK Rheinland/Hamburg, consented for ICC. 149 patients received NGS-based molecular diagnostic of their tumors. 18 samples were not suitable for testing. ICC patients were stratified according to their molecular diagnostic results and molecular guided therapy options (targeted drugs including off-label use, participating in clinical trials or standard chemotherapy). Clinical outcome data were collected within NGM (by over 200 clinical partners) and reimbursement data are provided by the AOK Rheinland/Hamburg. This model will be extended to all NGM patients independent of their insurance status. Final cost-effectiveness and outcome data will be presented.

Conclusion:
NGM stands for the implementation of personalized cancer therapy into clinical routine in Germany. Now we systematically evaluate NGS-based molecular results, clinical outcome and cost-effectiveness data besides of clinical trials. First-time in Europe data evaluation is provided in a close cooperation between health care providers and health insurance companies and even matching the patient’s data. Furthermore, in 2015 a joint database (NGM Cancer Information System) for retrospective evaluation of personalized cancer treatment in Germany will be launched. Our model of implementing personalized cancer care in broad clinical routine is currently transferred to other tumor entities.

Abstract not provided

Background:
It is estimated that 10-15% of lung cancer cases occur in never smokers. The cause of lung cancer in these patients includes many possible environmental factors but the precise cause in a given case is often uncertain. Additionally, there has been significant debate about whether the rate of lung cancer in these never smokers is increasing. Using our institutions’ cancer registry data, our objective was to determine if the proportion of never smokers with lung cancer is increasing.

Methods:
We conducted a retrospective study using lung cancer registry data from The University of Texas Southwestern Medical Center in Dallas, Parkland Hospital in Dallas, and Vanderbilt University in Nashville. These registries were queried between 1990 and 2013 for demographic information including gender, age at diagnosis, diagnosis [non small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)], and self-reported smoking history. A total of 10,568 NSCLC cases and 1504 SCLC cases were analyzed. Logistic regression analysis was performed to assess the incidence of never smokers with lung cancer.

Results:
The percentage of never smokers increased among NSCLC pts between 1990 and 2013 [Table 1]. Univariate logistic regression demonstrated an increasing proportion of never smokers among NSCLC cases (p < 0.0001 for year) and multivariate logistic regression also demonstrates this increase (p < 0.0001 for year) after controlling for age and gender. Never smokers with NSCLC were more likely to be female (65.3%, p < 0.0001) than males. The increase in the percentage of NSCLC never smokers was seen at both university hospitals and the Dallas county hospital. In contrast, the percentage of never smokers among SCLC cases did not significantly increase during this time period. Table 1: Percentage of never smokers Figure 1



Conclusion:
This multi-institution study demonstrates an increasing proportion of never smokers with NSCLC between 1990 and 2013 in a large, geographically and demographically diverse population. Because the biology and, thus, often the treatment options of lung cancer in never smokers differs from that of smokers, further investigation is warranted as to the etiology of the increasing incidence of never-smoker lung cancer.

Background:
EGFR T790M is most commonly seen as a somatic mutation in non-small cell lung cancer (NSCLC) following resistance to EGFR targeted therapies. Rarely EGFR T790M can be seen as a germline mutation where, in case reports, it has been associated with inherited lung cancer risk. However, the penetrance of the T790M germline mutation for NSCLC is not known, nor is it known whether germline carriers are also at risk for other cancers. The INHERIT study (INvestigating HEreditary RIsk from T790M, NCT01754025) is designed to prospectively identify and study individuals and family members with this rare germline mutation.

Methods:
Eligible subjects had EGFR T790M identified on routine cancer genotyping (excluding acquired T790M after therapy), or if they or a relative had already been found to carry a germline EGFR mutation. Confirmatory testing of saliva or blood was done to identify germline T790M carriers. Detailed 3-4 generation pedigrees of probands were constructed and analyzed for type of cancer, age at diagnosis, and relationship to proband with T790M mutation.

Results:
23 eligible kindreds were enrolled between 12/12 and 4/15, with 17 probands identified to have germline T790M and 6 probands shown to have acquired T790M. Average age at diagnosis for probands with germline T790M mutation was 55.8 (range 29 to 76) compared to 62 years (range 47 to 74) for non-germline probands. Pedigrees from confirmed T790M probands had an average kindred size of 28 members (range 3 to 40). Among the 325 1[st] and 2[nd] relatives, there were a total of 61 (18.7%) cancer diagnoses; 25 (39.7%) in lung, 4 (6.3 %) breast, 3 (4.8 %) colon, 4 (6.3) esophagus, 4 (6.3 %) leukemia/lymphoma, 3 (4.8 %) prostate, 3 (6.8%) bladder, 2 (3.2%) testes with about 1% or less with pancreatic, renal, brain, cervical cancer. Further, 7 of these 17 kindreds (41%) had multi-generational lung cancers consistent with autosomal dominant inheritance. In contrast, the cancer profile from the non-germline T790M kindreds showed high prevelance of breast cancer (61%; 13 of 21 relatives with cancer) and low prevalence of lung cancer (9%; 2 of 21). None of these 6 kindreds showed an autosomal dominant pattern of inheritance.

Conclusion:
A wide variety of tumor types were reported in this unique set of kindreds identified by tumor typing of probands for EGFR T790M mutations, with lung cancer as the most frequently reported cancer in close relatives. A high proportion of germline T790M kindreds also had a strong family history consistent with dominant inheritance. Future research will be needed to clarify the cancer risks in relatives of patients with EGFR T790M germline mutations and to develop guidelines and standards for prevention and early detection.

Background:
Lung cancer in never smokers may be enriched with oncogenic drivers. To explore patterns genomic changes among and within NS-LC, we performed multi-region whole genome sequencing (WGS) of primary pulmonary adenocarcinoma (LUAC).

Methods:
An observational study was performed on 8 cases of never-smoking LUAC resected with curative intent. Post-diagnostic residual fresh tumor was procured with informed consent, with constitutional samples from normal lung or blood. Selection criteria included: histologically confirmed LUAC; never-smoker [< 100 cigarettes in a lifetime]; no prior malignancy, cytotoxic therapy or thoracic radiotherapy. Tissue samples were procured by an anatomical pathologist (Table 1). Quality criteria were >40% tumor cellularity and <20% necrosis as assessed visually by 2 anatomical pathologists (Table 1). DNA was extracted using Qiagen AllPrep DNA/RNA Mini Kit and Blood Maxi Kit. WGS was performed on paired end libraries using Illumina's HiSeq 2000 platform (Table 1). Single nucleotide variants (SNVs) called by MuTect, Varscan, Strelka and SomaticSniper were considered ‘high priority’ if their predicted functional significance was ‘moderate’ or ‘high’ according to SNPEff. Genotyping was performed using Illumina’s HumanOmni2.5-8 array for copy number calling using the Genome Alteration Print tool.

Results:
14 tumour samples and 8 constitutional samples were sequenced (table 1). Figure 1 Common CNVs and SNVs were observed among and within cases (figure 1). Figure 2 In case 1, 3 of 6 (50%) genes harboring high priority variants were altered in all 4 regions. Similarly, for cases 2 and 3, 8/10 (80%) and 4/8 (50%) genes were altered by high priority variants in all regions.





Conclusion:
Patterns of SNVs and CNVs in LUAC demonstrate areas of common genomic changes and significant inter-, and intratumoral heterogeneity. These findings have significant implications for our understanding of lung cancer biology, also diagnostic testing of lung cancer and clinical trial design.

Abstract not provided

Background:
Primary lung cancer is increasingly understood as a heterogeneous disease made up of genomically defined subtypes requiring distinct treatment strategies. We hypothesize young age at diagnosis (< 40 years) is a clinical characteristic associated with an increased chance for a targetable genomic alteration. Our ALCMI study prospectively characterizes the somatic and germline genomics of young lung cancer (GYLC). Our goals are to identify a genomically enriched subtype of lung cancer, facilitate delivery of targeted therapy and lay groundwork for further studies of heritable and environmental lung cancer risk factors.

Methods:
Accrual opened July 2014. Patients are eligible if they were diagnosed with bronchogenic lung cancer less than age 40. A study website allows for virtual consenting so patients can participate remotely from anywhere in the world; and use social media to share our trial. We have an integrated data and bio repository that allows for seamless communication and completion of study activities like remote consenting and routing of blood and tumor specimens. We have defined 7 genomic alterations of interest based on the Lung Cancer Mutational Consortium (LCMC) (EGFR, KRAS, HER2, BRAF, ALK, ROS1, RET). We aim to demonstrate that the prevalence of targetable genomic alterations will be greater in our population compared to the LCMC and have powered our study to show an increase from 35% to 50%; and an improvement in use of targeted therapy from 22% to 40%. On study subjects without a known genotype will undergo comprehensive genomic profiling with the FoundationOne test to ensure that all of these genes have been tested. Subjects with advanced adenocarcinoma who are wild type for all 7 genes will receive additional genomic profiling using the FoundationOne Heme test; with the goal of identifying novel oncogenic drivers. Additional investigational genomics will include blood for germline analysis and plasma genomics. All on study genomic analysis is at no cost to the participant.

Results:
Preliminary results of the first 33 subjects show: Average age at diagnosis: 33 years; Range 22-39; Histology: adenocarcinoma n=29, squamous cell n=4; Stage at diagnosis: stage 4 n=26 (79%) stages 1-3 n=7 (21%). Of those with stage 4 adenocarcinoma (n=24); 18:24 (75%) have either an ALK re arrangement n=10 (42%), an EGFR activating mutation n=5 (21%) or a ROS1 fusion n=3 (13%).

Conclusion:
The trial is currently accruing (NCT02273336) https://www.openmednet.org/site/alcmi-goyl. We have accrued patients from the USA, Europe and Australia. Thus far in our prospective series those diagnosed with primary NSCLC < age 40 tend to have stage 4 adenocarcinoma. Preliminary results exceed our statistical expectation with 75% of our metastatic adenocarcinoma patients having an actionable mutation. We plan on presenting data for the first time at WCLC-2015 on the first 50 subjects. (Study, supported by grants from BJALCF, Beth Longwell Foundation, Peter Barker Foundation, Genentech, Schmidt Legacy Foundation, and Upstage Lung Cancer)

Background:
The incidence of NSCLC in pts 45 years of age or younger is ~2% of total cases, with annual newly diagnosed cases reaching 4,500 in the United States alone. Majority of these pts are diagnosed at an advanced stage with poor outcomes. Although several specific genomic alterations have been identified in young NSCLC pts particularly in light-/never smokers, e.g. EGFR mutations, the overarching underlying causative mechanisms in the pathogenesis and progression in these young pts remain largely unknown, and likely are different from older pts. The objective of this study is to examine the genomic landscapes of NSCLC in young pts through comprehensive whole exomic survey with the objective to arrive at novel predictive and prognostic genomic biomarkers and therapeutic opportunities in young NSCLC pts.

Methods:
We initially identified a cohort of 20 pts (40% male) diagnosed with NSCLC at an age of ≤45, who underwent surgical resection for the primary tumors or metastatic lesions at Cleveland Clinic from 2000-2012. Matching genomic DNA from FFPE lung tumor samples and paired-normal lung tissue/peripheral blood was subjected to whole exome sequencing using Illumina HiSeq 2000 platform. Exome variant calling was performed using GATK and/or SOAPsnp algorithms to identify somatic mutations in individual tumors. Pathway and protein-protein interaction (PPI) network analysis on mutant genes was performed using KEGG/NCI-PID databases and HotNet suite, respectively. Second cohort of young NSCLC tumor cases (n=50) were identified from the Sun Yat-sen University Cancer Center and genomic analysis is underway.

Results:
Majority of the tumors from the first cohort had adenocarcinoma (n=12) or squamous cell (n=4) histology. Six (6) pts were never-smokers, while the others had a median 30 pack-year cigarette smoking history. A significantly higher mutation burden was found in smokers (Median, 3.47/Mb) compared to never-smokers (Median, 0.76/Mb). We also found that the G:C→T:A transversions were more common in smokers, and C:G→T:A transitions more common among never-smokers. Key driver cancer genes such as TP53 (50%) and KRAS (17%) harbored mutations exclusively in smokers, whereas EGFR mutations (14%) were observed specifically in never-smokers. Interestingly, global pathway/PPI analysis of the mutant genes revealed distinct sub-networks associated with cell adhesion and epithelial mesenchymal transition (EMT) processes with a 7-fold enrichment in mutation frequency in these young pts when compared to their overall frequencies in the COSMIC/TCGA lung cancer dataset.

Conclusion:
Our study nominated novel candidate genes/pathways especially relating to cell adhesions and EMT processes, that potentially play a key role in early-onset NSCLC. Further analysis and validation of our findings could improve our understanding of lung cancer pathogenesis and eventually lead to precision therapies to benefit younger NSCLC pts.

Background:
EGFR, Kras mutations and EML4-ALK translocations were frequently positive in adenocarcinoma among lung cancer, and in fewer cases HER2, BRAF mutations or RET, ROS1 translocations were identified. Although adenocarcinomas emerged in the youth are estimatedly associated with some driver oncogenes including these mutations/translocations, the detail remains unknown.

Methods:
We retrospectively screened 55 consecutive patients who were diagnosed as stage I-IV adenocarcinoma at the age of 40 years or less in 2009-2014. We analyzed clinical and genetic characteristics among them.

Results:
Out of 55 patients, 21 (38%) were male, 24 (44%) were never-smoker, and 38 (69%) were stage IV, with the median age of 36 years (range; 26-40). Forty-five patients (82%) were identified some driver oncogene. 26 (47%) had EML4-ALK translocation, 13 (24%) had EGFR mutation, and 2 (4%) had Kras mutation. We examined rare oncogenes in 10 out of 14 triple-negative patients, which revealed three patients had HER2 mutation and two had RET translocation.

Conclusion:
82% of adenocarcinomas emerged in the youth were identified some targetable driver oncogenes. Not only EGFR mutation or EML4-ALK translocation, rare oncogene examination is necessary especially among these populations.

Abstract not provided

Background:
Phase III trials have demonstrated the superiority of the ALK tyrosine kinase inhibitor (ALK-TKI) crizotinib compared to standard chemotherapy in advanced ALK positive non-small cell lung cancers (ALK+ NSCLC) in first line and second line setting. Objective response rate (ORR) with crizotinib ranged from 65 to 75% and median progression free survival (PFS) from 7.7 to 10.9 months. However a resistance to crizotinib always occurs. The French Cooperative Thoracic Intergroup (IFCT)-1302 CLINALK study aimed to describe clinical outcome and post-progression management in a large cohort of French patients with ALK+ NSCLC treated with crizotinib.

Methods:
IFCT-1302 CLINALK is a multicentric observational retrospective study. Patients with ALK+ NSCLC from centers of the IFCT network were included according to the main following criteria: advanced stage III or stage IV NSCLC, ALK immunochemistry (IHC) and/or ALK FISH positivity, crizotinib treatment in the setting of the French expanded access cohort program or as approved drug. Epidemiological and clinical data, crizotinib efficacy (objective response based on RECIST, PFS, overall survival (OS)), duration of treatment with crizotinib after disease progression and post progression outcome were collected on a case report form. The study inclusion period was from November 18 2011 to December 31 2013. The data cut-off was December 31 2014.

Results:
318 patients were included (median age 58.3, female 49.4%, caucasian 98.6%, non-smoker 55.1%, performance status 0/1 78.7%, adenocarcinoma 91.7%, stage III 14.5%, stage IV 85.5%, brain metastasis 35.9%). IHC was positive in 151/173 patients and FISH in 279/283 patients. Before crizotinib treatment, patients received platinum-based chemotherapy in 89% of cases and pemetrexed-based chemotherapy in 76.1%. Crizotinib was prescribed as first-line treatment in 17 patients (5.3%), second-line in 168 patients (52.8%), third-line in 58 patients (18.2%) and more than third-line in 75 patients (23.7%). Objective response was complete response in 1 patient (0.3%), partial response in 126 patients (40.0%), stable disease in 62 patients (19.7%) and progression in 58 patients (18.4%). ORR was 40.3 % (95%CI, 34.9-45.7). 262/318 patients presented progressive disease (82.4%) at time of analysis. Median PFS was 6.9 months (95%CI, 5.7-8.6). Median OS with crizotinib was 18.7 months (95%CI, 15.2-22.5). Median duration of treatment with crizotinib after disease progression was 56 days (29-203). Among 143 patients with subsequent treatments, crizotinib was rechallenged in 32 patients (22.4%). 58/143 patients (40.6%) were treated after crizotinib failure with another ALK-TKI, either alectinib (19/58, 32.8%) or ceritinib (40/58, 69.0%). The ALK-TKI sequence was crizotinib-alectinib in 18 patients, crizotinib-ceritinib in 39 patients and crizotinib-alectinib-ceritinib in 1 patient.

Conclusion:
This retrospective study of 318 patients with ALK+ NSCLC showed a remarkable efficacy of crizotinib, with a 18.7 months median OS, a 40.3% ORR and a 6.9 months median PFS. However, ORR and mPFS were lower than those reported in phase III trials, which may be due to less stringent selection criteria. Analysis of predictive factors of response and survival including post-progression strategies will be presented.

Background:
There is strong interest in evaluating the outcomes of patients who have progressed after failing targeted agents. With different agents, the post-progression survival (PPS) may be either improved or shortened when a longer duration of time-to-progression (TTP) has been observed. This study evaluated the association between TTP and the duration of PPS among adult patients who received ceritinib for the treatment of advanced anaplastic lymphoma kinase-positive (ALK+) NSCLC.

Methods:
Patients experiencing disease progression during two single-arm, open-label, Phase I and II trials of ceritinib (ASCEND-1 [ClinicalTrials.gov Identifier: NCT01283516] and ASCEND-2 [ClinicalTrials.gov Identifier: NCT01685060]) were included in this analysis. For uniformity, all patients analyzed had received crizotinib prior to ceritinib. TTP after the initiation of ceritinib was studied as a predictor for the length of subsequent PPS using Cox proportional hazards models. Adjustments were made for patients’ baseline characteristics, including age, body mass index, gender, race, Eastern Cooperative Oncology Group (ECOG) performance score, number of prior regimens, tumor histology, and presence of brain metastases. A Kaplan-Meier analysis for PPS was performed stratified by shorter (< 6 months) versus longer TTP (≥ 6 months). As a secondary descriptive analysis, associations were quantified between the duration of TTP and the duration of survival (OS) measured as the sum of TTP and PPS.

Results:
Of 181 patients who experienced disease progression during study follow-up, 94% received at least one chemotherapy prior to baseline, 75% had an ECOG performance score greater than zero at screening, and 79 died during subsequent follow-up. In an unadjusted model, each 3 months of longer TTP was associated with a 24% lower hazard of death following progression (hazard ratio [HR]: 0.76, 95% confidence interval [CI]: 0.60-0.96). Results were similar after adjusting for baseline characteristics (HR: 0.77, 95% CI: 0.61-0.97). Patients with TTP ≥ 6 months experienced significantly longer PPS compared to those with TTP < 6 months (median: 9.8 vs. 6.5 months, log-rank p-value < 0.01). This positive relationship between TTP and PPS translated into the duration of OS: each 3 months of longer TTP was associated with a 58% lower hazard of death after adjusting for baseline characteristics (HR: 0.42, 95% CI: 0.32-0.54). Median OS was not reached for patients with TTP ≥ 6 months and was 10.3 months for patients with TTP < 6 months.

Conclusion:
A longer duration of TTP after treatment with ceritinib was significantly associated with both longer duration of PPS and longer OS.

Background:
ALK gene rearrangements occur in approximately 3–6% of patients with non-small-cell lung cancer (NSCLC). Crizotinib has demonstrated efficacy in ALK+ NSCLC, however many patients experience systemic and/or central nervous system (CNS) disease progression within one year of treatment. Alectinib, a CNS-penetrant and highly selective ALK inhibitor, has shown preclinical activity in the CNS (Ou, et al. JTO 2013) and clinical efficacy in crizotinib-naïve (Ohe, et al. ASCO 2015) and pre-treated (Ou, et al. ASCO 2015; Gandhi, et al. ASCO 2015) ALK+ NSCLC patients. We will present updated efficacy and safety outcomes from the phase II NP28761 study (NCT01871805).

Methods:
North American patients ≥18 years of age with ALK+ NSCLC (by FDA-approved FISH test), disease progression following first-line crizotinib, and ECOG PS ≤2 were enrolled. Patients received oral alectinib (600mg) twice daily until progression, death or withdrawal. The primary endpoint was overall response rate (ORR) by independent review committee (IRC) using RECIST v1.1. Secondary endpoints included investigator-assessed ORR; progression-free survival (PFS); quality of life (QoL); CNS response rate; disease control rate (DCR); and safety.

Results:
At data cut-off (24 October 2014), 87 patients were enrolled in the intent-to-treat population. Median age was 54 years; 74% had received prior chemotherapy; 60% of patients had baseline CNS metastases, of whom 65% (34/52) had prior brain radiation therapy. Median follow-up was 20.7 weeks. ORR by IRC was 48% (95% CI 36–60); median PFS was 6.3 months (Table 1). In patients with measurable CNS lesions at baseline (n=16), IRC CNS ORR was 69% (95% CI 41–89) and CNS DCR was 100% (complete response, 13%; partial response, 56%; stable disease, 31%). In patients with measurable or non-measurable CNS disease (n=52), IRC CNS ORR was 39% (95% CI 25–53) and 11 patients (21%) had complete CNS responses. The most common grade ≥3 AEs were elevated levels of blood creatine phosphokinase (8%), alanine aminotransferase (6%) and aspartate aminotransferase (5%); no GI toxicities leading to treatment withdrawal were reported. Clinically meaningful improvements were seen in EORTC QLQ-C30 items, including Global Health Status. Figure 1



Conclusion:
Alectinib (600mg twice daily) was well tolerated and demonstrated clinical efficacy in patients with ALK+ NSCLC disease who had progressed on prior crizotinib. A clinical benefit with alectinib was also observed in patients with CNS lesions at baseline. These data are preliminary; updated efficacy and safety data from a cut-off date of 27 April 2015 will be presented.

Abstract not provided

Background:
The central nervous system (CNS) is a frequent site of progression in ALK+ NSCLC patients treated with crizotinib, thus good CNS efficacy is of crucial importance for new ALK inhibitors. Two recent phase II studies examined the efficacy and safety of alectinib in patients with ALK+ NSCLC who progressed after crizotinib; data from both studies were pooled to further examine the efficacy of alectinib in the CNS.

Methods:
Both phase II, single-arm, multicenter studies enrolled ALK+ NSCLC patients previously treated with crizotinib. One study was conducted in North America only (NP28761; NCT01871805), the other was global (NP28673; NCT01801111). All patients received 600mg oral alectinib twice daily. A primary endpoint of both studies was objective response rate (ORR) by independent review committee (IRC) and key secondary endpoints included CNS ORR by IRC and CNS duration of response (DOR). Response was determined according to RECIST v1.1. All patients underwent imaging at baseline to assess CNS metastases.

Results:
The pooled analysis population comprised 225 patients (n=87 from NP28761 and n=138 from NP28673); baseline characteristics were similar to each study population, with most patients being non-smokers, <65 years old with ECOG performance status 0/1. Median follow-up was 27.7 weeks. Fifty patients had measurable CNS disease at baseline (MD) while a further 85 had non-measurable disease (NMD) at baseline; both groups together (M+NMD) comprised 135 patients, 60% of the overall study population. In the MD group, 34 patients (68%) had received prior radiotherapy, but 24 of them had completed that radiotherapy >6 months prior to starting alectinib. For the M+NMD group, 94 patients (70%) had received prior radiotherapy, with 55 completing this >6 months prior to starting alectinib. In the MD group, 30/50 patients had a CNS response (60.0%; 95% CI 45.2–73.6%), with 7 complete responses (CR; 14.0%) and a CNS DCR of 90.0% (78.2–96.7%). In the M+NMD group, 22 additional patients had a CR (29/135; 21.5%), giving a CNS ORR of 38.5% (30.3–47.3%), with a CNS DCR of 85.2% (78.1–90.7%). Complete responses were seen in patients with and without prior radiotherapy. Median CNS DOR after only 17% of events in both groups was 7.6 months (5.8–7.6) in the MD group (n=30) and 7.6 months (5.8–10.3) in the M+NMD group (n=52), which is similar to the systemic DOR reported in both studies (Ou et al, ASCO 2015; Gandhi et al, ASCO 2015). Tolerability was also similar to the overall study population.

Conclusion:
Alectinib showed promising efficacy in the CNS in ALK+ NSCLC patients previously treated with crizotinib, achieving a complete response rate of 22% and a DCR of 85%, irrespective of prior radiotherapy. The CNS response was sustained for an equivalent duration to the systemic response, suggesting that alectinib could provide an effective treatment for patients with ALK+ NSCLC while actively targeting CNS metastases. The ongoing phase III clinical studies will assess the systemic and CNS efficacy of alectinib versus crizotinib as front-line therapy for ALK+ NSCLC patients.

Background:
Brigatinib (AP26113), an investigational oral tyrosine kinase inhibitor with FDA breakthrough therapy designation for the treatment of patients with crizotinib-resistant advanced ALK+ NSCLC, has preclinical activity against both rearranged ALK and clinically identified crizotinib-resistant mutant ALK.

Methods:
This is an ongoing phase 1/2, single-arm, open-label, multicenter study in patients with advanced malignancies (N=137; NCT01449461). Patients received escalating total daily doses of brigatinib from 30–300 mg during phase 1. Daily regimens of 90 mg, 180 mg, or 90 mg for 7 days followed by 180 mg were evaluated in phase 2. Safety is reported for all treated patients; antitumor efficacy (ORR and PFS per RECIST v1.1) is reported for ALK+ NSCLC patients.

Results:
Seventy-nine (58%) patients had ALK+ NSCLC. Median age was 54 (29–83) years, 49% were female, 90% had prior crizotinib, and 47% had ≥2 prior chemotherapy regimens. As of February 17, 2015, 45/79 (57%) ALK+ NSCLC patients remained on study, with median time on treatment of 12.6 months (1 day to 35.5 months; n=79); ORR/PFS for evaluable ALK+ NSCLC patients was 74%/13.4 months (additional data shown in Table). In a post hoc independent radiological review of patients with brain metastases at baseline (as of January 19, 2015), 8/15 (53%) patients with measurable brain lesions ≥10 mm had an intracranial response (≥30% decrease in sum of longest diameters of target lesions) and 9/30 (30%) patients with only nonmeasurable lesions had disappearance of all lesions. Treatment-emergent AEs in ≥30% of total patients, generally grade 1/2, included nausea (52%), fatigue (42%), diarrhea (40%), headache (33%), and cough (32%). Early-onset pulmonary events, which occurred ≤7 days after treatment initiation and included dyspnea, hypoxia, and new pulmonary opacities on chest CT consistent with pneumonia or pneumonitis, were reported in 13/137 (9%) patients overall (6/44 [14%] at 180 mg qd; 2/50 [4%] at 90 mg qd [maintained or escalated to 180 mg qd after 7 days]).

Response and PFS With Brigatinib
	All Evaluable ALK+ NSCLC Patients n=78	Prior Crizotinib n=70	No Prior Crizotinib n=8
Response, n(%)
OR (CR+PR)	58(74)	50(71)	8(100)
[95% CI]	[63–84]	[59–82]	[63–100]
CR	7(9)	4(6)	3(38)
PR	51(65)	46(66)	5(63)
SD	11(14)[a]	11(16)[a]	0
PD	6(8)	6(9)	0
Termination before scan	3(4)	3(4)	0
Median duration of response,[b] mo	11.2[c]	9.9[d]	Not reached[e]
Median PFS,[b] mo	13.4	13.4	Not reached
[a]Includes non-CR/non-PD for 4 patients with no measurable disease at baseline [b]Kaplan-Meier estimate [c]n=55 evaluable [d]n=48 evaluable [e]n=7 evaluable

Conclusion:
Brigatinib has promising antitumor activity in ALK+ NSCLC patients with (71% ORR; PFS 13.4 months) or without (100% ORR) prior crizotinib, including patients with brain metastases (53% ORR in patients with measurable brain lesions). Early-onset pulmonary events were less frequent when starting at 90 vs 180 mg qd. A pivotal global phase 2 trial (ALTA) of brigatinib 90 mg qd vs 90 mg qd for 7 days followed by 180 mg qd in crizotinib-resistant ALK+ NSCLC is ongoing.

Background:
Overcoming acquired resistance in ALK+ and ROS1+ non-small-cell lung cancer (NSCLC) patients (pts) is key to optimizing therapy. PF-06463922 is a selective, brain-penetrant tyrosine kinase inhibitor (TKI) with demonstrated clinical activity against de novo fusions as well as resistance mutations, including ALK G1202R, that arise during treatment with other TKIs.

Methods:
In this ongoing phase I study, eligible pts had ALK+ or ROS1+ NSCLC, with or without brain metastases, and were treatment naïve or had disease progression after at least 1 prior TKIs. Pts with central nervous system (CNS) metastases, including untreated asymptomatic metastases, were eligible. Tumor tissue (archival sample or de novo biopsy) was required for enrollment. A modified continual reassessment method was used to estimate the maximum tolerated dose (MTD) and select a recommended phase II dose (RP2D). Once- (QD) or twice-daily (BID) dosing in 21-day cycles was explored. Secondary objectives included efficacy, safety, tolerability, pharmacokinetics (PK), effect on cognitive function, patient-reported outcomes, potential to induce/inhibit CYP3A4, biomarkers of drug response and resistance, and intracranial antitumor activity.

Results:
25 ALK+ pts (20 with CNS metastases, 23 previously treated with at least 1 ALK TKIs) and 5 ROS1+ pts (3 with CNS metastases, 3 previously treated with crizotinib) have been enrolled across 7 QD dose levels and 2 BID dose levels. Of 21 patients evaluable for intracranial response, 16 had stable disease or confirmed complete/partial response. The most common treatment-related adverse events (AEs) were hypercholesterolemia, peripheral neuropathy, and peripheral edema, occurring in 47%, 27% and 23% of pts, respectively. Peripheral neuropathy was reversible following treatment delay or dose reduction. The most common grade 3 and higher treatment-related AE was hypercholesterolemia, occurring in 10% of pts, which was well managed with statins. One DLT was reported for a pt at 200 mg QD, who received less than 16 of the 21 planned doses of study drug due to grade 2 CNS effects. To date, 20 pts remain on treatment. Preliminary PK analyses suggest that exposure increased linearly from 10 to 75 mg QD, with a terminal half-life of 20-28 hrs. At doses beyond 75 mg QD, the increase in exposure appeared to be non-proportional. PF-06463922 is considered a moderate inducer of CYP3A4.

Conclusion:
PF-06463922 is a potent ALK/ROS1 TKI that has demonstrated clinical activity in ALK+ and ROS1+ NSCLC pts, most of whom had CNS metastases and had received at least 1 prior TKI. The RP2D has not yet been established.

Abstract not provided