Virtual Library

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    MINI 10 - ALK and EGFR (ID 105)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 15
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      MINI10.01 - Frequency of Concomitant EGFR, EML4-ALK or KRAS Alterations in NSCLC Patients and Correlation with Response to Treatment (ID 942)

      P. Ulivi, E. Chiadini, C. Dazzi, A. Dubini, M. Costantini, L. Medri, M. Puccetti, L. Capelli, D. Calistri, A. Verlicchi, A. Gamboni, M. Papi, M. Mariotti, N. De Luigi, E. Scarpi, S. Bravaccini, G.M. Turolla, D. Amadori, L. Crinò, A. Delmonte

      • Abstract
      • Presentation
      • Slides

      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.

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      MINI10.02 - Intratumoral Heterogeneity of ALK-Rearranged and ALK/EGFR Co-Altered Lung Adenocarcinoma (ID 685)

      W. Cai, C. Zhou, D. Lin, C. Wu, X. Li, C. Zhao, L. Zheng, K. Fei, F.R. Hirsch

      • Abstract
      • Presentation
      • Slides

      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.

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      MINI10.03 - Evolution of Concurrent Driver Mutations in Lung Adenocarcinoma Patients on EGFR TKI Therapy Uncovered by Comprehensive Molecular Profiling (ID 2848)

      C.M. Blakely, S. Asthana, E. Pazarentzos, V. Olivas, L. Lin, J. Flanagan, A. Caulin, P. Giannikopoulos, T. Bivona

      • Abstract
      • Slides

      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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      MINI10.04 - Discussant for MINI10.01, MINI10.02, MINI10.03 (ID 3399)

      T. Mok

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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      MINI10.05 - ALK Rearrangements in Non-Small Cell Lung Cancer: Comprehensive Integration of Genomic, Gene Expression and Protein Analysis (ID 2731)

      J.S.M. Mattsson, M.A. Svensson, B. Hallström, H. Koyi, E. Brandén, H. Brunnström, K. Edlund, S. Ekman, L. La Fleur, M. Grinberg, J. Rahnenführer, K. Jirström, F. Pontén, M.G. Karlsson, C. Karlsson, G. Helenius, M. Uhlén, J. Botling, P. Micke

      • Abstract
      • Presentation
      • Slides

      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.

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      MINI10.06 - Incidence of ALK Gene Rearrangements in a Prospectively, Consecutively Collected Caucasian Population with Pulmonary Adenocarcinomas (ID 684)

      B.G. Skov, J.B. Soerensen, K.R. Larsen, P. Clementsen, A. Mellemgaard

      • Abstract
      • Presentation
      • Slides

      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.

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      MINI10.07 - Lung Adenocarcinomas Co-Expressing TTF-1, MUC5B and/or MUC5AC Show a High Incidence of ALK Rearrangement and a Poor Prognosis (ID 802)

      D. Lin, W. Sun

      • Abstract
      • Slides

      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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      MINI10.08 - Discussant for MINI10.05, MINI10.06, MINI10.07 (ID 3400)

      B. Besse

      • Abstract
      • Presentation

      Abstract not provided

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      MINI10.09 - Comprehensive Genomic Profiling Identifies EGFR Exon 19 Deletions in NSCLC Not Identified by Standard of Care Testing (ID 3042)

      A.B. Schrock, S.M. Ali, D. Herndon, G.M. Frampton, J. Greenbowe, K. Wang, D. Lipson, R. Yelensky, Z. Chalmers, J. Chmielecki, J.A. Elvin, M. Wolner, R. Bordoni, F. Braiteh, A. Dvir, R. Erlich, M. Mohamed, J.S. Ross, P.J. Stephens, V. Miller

      • Abstract
      • Presentation
      • Slides

      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.

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      MINI10.10 - A Multicenter Prospective Biomarker Study in Afatinib-Treated Patients with EGFR-Mutation Positive Non-Small Cell Lung Cancer (ID 472)

      K. Azuma, E. Iwama, K. Sakai, K. Nozaki, D. Harada, K. Hotta, F. Ohyanagi, T. Kurata, H. Akamatsu, K. Goto, T. Fukuhara, Y. Nakanishi, I. Okamoto, K. Nishio

      • Abstract
      • Presentation
      • Slides

      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.

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      MINI10.11 - Classification of EGFR Gene Mutation Status Using Serum Proteomic Profiling Predicts Tumor Response in Patients with Stage IIIB or IV NSCLC (ID 874)

      L. Yang, C. Tang, B. Xu, W. Wang, J. Li, X. Li, H. Qin, H. Gao, K. He, S. Song, X. Liu

      • Abstract
      • Slides

      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.

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      MINI10.12 - Preclinical Activity of AZD9291 in EGFR-Mutant NSCLC Brain Metastases (ID 410)

      P. Ballard, P. Yang, D. Cross, J. Yates, M.R.V. Finlay, M. Grist, M. Box, P. Johnström, K. Varnäs, J. Malmquist, C. Halldin, L. Farde, K. Thress

      • Abstract
      • Presentation
      • Slides

      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.

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      MINI10.13 - Prediction of Human Pharmacokinetics, Efficacious Dose and BBB Penetration of a Novel EGFR Inhibitor for Treating NSCLC with CNS Metastasis (ID 3282)

      K. Chen, P. Martin, S. Cohen-Rabbie, P. Johnstroem, Y. Wang, H. Jiang, Z. Yang, Z.(. Cheng

      • Abstract
      • Presentation
      • Slides

      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.

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      MINI10.14 - Brain Metastasis at Diagnosis and EGFR Mutational Status in Non-Small Cell Lung Cancer (ID 431)

      V.R. Bhatt, S.P. D`souza, L. Smith, A.M. Cushman-Vokoun, V. Noronha, V. Verma, A. Joshi, A. Chougule, N. Jambekar, A.K. Ganti, K. Prabhash

      • Abstract
      • Presentation
      • Slides

      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.

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      MINI10.15 - Discussant for MINI10.09, MINI10.10, MINI10.11, MINI10.12, MINI10.13, MINI10.14 (ID 3401)

      O. Arrieta Rodriguez

      • Abstract
      • Presentation

      Abstract not provided

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    PC 02 - Pro vs Con: Is There a Role for EGFR TKIs in EGFR Mutation Negative Disease? / Pro vs Con: Whole Exome Sequencing vs. Selected Testing (e.g., ALK and EGFR) (ID 48)

    • Event: WCLC 2015
    • Type: Pro Con
    • Track: Treatment of Advanced Diseases - NSCLC
    • Presentations: 5
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      Introduction (ID 2095)

      • Abstract

      Abstract not provided

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      PC02.01 - Is There a Role for EGFR TKIs in EGFR Mutation Negative Disease? - Pro (ID 2030)

      S.A. Laurie

      • Abstract
      • Presentation

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

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      PC02.02 - Is There a Role for EGFR TKIs in EGFR Mutation Negative Disease? - Con (ID 2031)

      L.V. Sequist

      • Abstract
      • Presentation

      Abstract not provided

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      PC02.03 - Whole Exome Sequencing vs. Selected Testing (e.g., ALK and EGFR) - Pro (ID 2032)

      I.I. Wistuba

      • Abstract
      • Presentation

      Abstract not provided

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      PC02.04 - Whole Exome Sequencing vs. Selected Testing (e.g., ALK and EGFR) - Con (ID 2033)

      Y. Yatabe

      • Abstract
      • Presentation
      • Slides

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

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