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R. Rosell

Moderator of

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    ED01 - Biology of Lung Cancer (ID 263)

    • Event: WCLC 2016
    • Type: Education Session
    • Track: Biology/Pathology
    • Presentations: 3
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      ED01.01 - Understanding Biology: The Road to Cure? (ID 6421)

      11:00 - 11:25  |  Author(s): D.P. Carbone

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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      ED01.02 - Tobacco Carcinogens and Lung Cancer Susceptibility (ID 6422)

      11:25 - 11:50  |  Author(s): S.S. Hecht, S..L. Park, S. Carmella, D.O. Stram, C.A. Haiman, L. Le Marchand, S.E. Murphy, J. Yuan

      • Abstract
      • Presentation
      • Slides

      Abstract:
      While cigarette smoking is clearly the major cause of lung cancer, only 11% of female and 24% of male lifetime smokers will get lung cancer by age 85 or greater, and this relatively small percentage is not due to competing causes of death from smoking (1) The major goal of the research approach discussed in this presentation is to identify individuals who are highly susceptible to the carcinogenic effects of cigarette smoke. These individuals would be candidates for intensive lung cancer surveillance and screening, increasing the probability of detection of a tumor at an early stage. We are not proposing methods for early detection of tumors such as the identification of metabolites or proteins characteristic of lung tumors, but rather early identification of susceptible individuals. While there are already algorithms relating various parameters to lung cancer susceptibility, they are mostly retrospective in nature, with pack-years of cigarette smoking being a major prognostic factor (2,3). Thus, these algorithms are typically applied to subjects who are older, when the process may be more advanced. Our ultimate goal is to develop a risk model that is prospective in nature. Overall, there would be a greater probability of success if one could identify high risk individuals early in the carcinogenic process. Even if this were effective in only 10% of tobacco users, the outcome could be prevention of more than 15,000 lung cancer deaths per year in the U.S. alone and massive financial savings. Among the more than 7,000 identified chemical compounds in cigarette smoke, there are 72 fully characterized carcinogens among which at least 20 are known to cause lung tumors in laboratory animals (4,5). Important among the lung carcinogens are polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene, tobacco-specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and volatiles such as 1,3-butadiene. Other related volatile compounds that may contribute to the carcinogenic process include acrolein, crotonaldehyde, and benzene. Perhaps the most important compound in tobacco smoke is nicotine – while not a carcinogen, it is the addictive constituent of smoke that causes people to continue to inhale this incredibly unhealthy mixture. In pursuit of our goal of identifying smokers susceptible to lung cancer, we have focused on several tobacco smoke toxicant and carcinogen parent substances and metabolites in urine (6). Thus, we and others have developed and applied analytically validated mass spectrometric methods for total nicotine equivalents (the sum of nicotine and six metabolites: nicotine glucuronide, cotinine, cotinine glucuronide, 3′-hydroxycotinine and its glucuronide, and nicotine-N-oxide); total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of NNK; phenanthrene tetraol (PheT) and 3-hydroxyphenanthrene (3-PheOH), metabolites of a representative PAH; S-phenylmercapturic acid (SPMA), a metabolite of the carcinogen benzene; 3-hydroxypropylmercapturic acid (HPMA), a metabolite of acrolein; and 3-hydroxy-1-methylpropylmercapturic acid (HMPMA), a metabolite of crotonaldehyde. We have collaborated with epidemiologists to evaluate the relationship of these urinary metabolites to cancer, as determined in prospective cohort studies. These studies collect and store bio-samples from large numbers of healthy subjects, then follow the subjects until sufficient numbers of cancer cases occur for statistical analysis. Samples from the cases and matched controls without cancer are retrieved from biorepositories and analyzed for specific biomarkers. The results of these studies have been reviewed (7,8). In summary, statistically significant relationships of urinary total cotinine (cotinine plus its glucuronide, the major metabolite of nicotine), total NNAL, and PheT with lung cancer risk were observed among male smokers in Shanghai. Urinary total cotinine and total NNAL were related to lung cancer risk in a study of male and female smokers in Singapore, and total NNAL in serum was related to lung cancer risk in a study of male and female smokers in the U.S. (7,8). Levels of urinary SPMA , HPMA, and HMPMA were not independently related to lung cancer in the Shanghai study. These results indicate that total cotinine, total NNAL, and PheT are possible biomarkers of lung cancer risk. We are also collaborating with scientists from the Multiethnic Cohort study, a prospective cohort study investigating the association of genetic and lifestyle factors with chronic diseases in a population with diverse ethnic backgrounds. They have reported that, for the same number of cigarettes smoked, and particularly at lower levels of smoking, African Americans and Native Hawaiians have a higher risk for lung cancer than Whites while Latinos and Japanese Americans have a lower risk (9). We are investigating the mechanistic basis for these remarkable differences. We analyzed urine samples from 300-700 subjects per group for total nicotine equivalents, total NNAL, PheT, 3-PheOH, SPMA, HPMA, and HMPMA. The results demonstrated that African Americans, although smoking fewer cigarettes per day than any of the other groups except Latinos, had significantly higher levels of total nicotine equivalents, total NNAL, PheT, 3-PheOH, and SPMA compared to Whites while Japanese Americans had significantly lower levels of most of these biomarkers than Whites. The relatively low level of urinary total nicotine equivalents in the Japanese American smokers was related to a high prevalence of CYP2A6 polymorphisms in this group (10). CYP2A6 is the primary catalyst of nicotine metabolism and the CYP2A6 alleles common in Japanese Americans code for low activity and non-functional enzyme. Therefore, Japanese Americans on the average have more unchanged nicotine circulating and will not need to obtain as much nicotine per cigarette. The biomarker profiles of Native Hawaiians and Latinos did not clearly relate to their relative lung cancer risks, but Native Hawaiians had high levels of the acrolein biomarker HPMA compared to other groups while those of Latinos were low. These results provide important new data pertinent to the relatively high risk of African Americans and the lower risk of Japanese Americans for lung cancer. Collectively, our results support the use of urinary nicotine metabolites, total NNAL, and PheT as biomarkers of lung cancer risk in cigarette smokers. Further studies are required to produce a reliably predictive algorithm for lung cancer susceptibility in cigarette smokers. These studies are likely to require the analysis of DNA adduct levels and to incorporate genetic and epigenetic information. Reference List 1. International Agency for Research on Cancer (2004) Tobacco Smoke and Involuntary Smoking. In IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 83 pp 174-176, IARC, Lyon, FR. 2. Tammemagi, C. M., Pinsky, P. F., Caporaso, N. E., Kvale, P. A., Hocking, W. G., Church, T. R., Riley, T. L., Commins, J., Oken, M. M., Berg, C. D., and Prorok, P. C. (2011) Lung cancer risk prediction: prostate, lung, colorectal and ovarian cancer screening trial models and validation. J. Natl. Cancer Inst. 103, 1058-1068. 3. Weissfeld, J. L., Lin, Y., Lin, H. M., Kurland, B. F., Wilson, D. O., Fuhrman, C. R., Pennathur, A., Romkes, M., Nukui, T., Yuan, J. M., Siegfried, J. M., and Diergaarde, B. (2015) Lung cancer risk prediction using common SNPs located in GWAS-identified susceptibility regions. J Thorac. Oncol. 4. Hecht, S. S. (1999) Tobacco smoke carcinogens and lung cancer. J. Natl. Cancer Inst. 91, 1194-1210. 5. Rodgman, A. and Perfetti, T. (2009) The Chemical Components of Tobacco and Tobacco Smoke. CRC Press, Boca Raton, FL. 6. Hecht, S. S., Yuan, J.-M., and Hatsukami, D. K. (2010) Applying tobacco carcinogen and toxicant biomarkers in product regulation and cancer prevention. Chem. Res. Toxicol. 23, 1001-1008. 7. Yuan, J. M., Butler, L. M., Stepanov, I., and Hecht, S. S. (2014) Urinary tobacco smoke-constituent biomarkers for assessing risk of lung cancer. Cancer Res. 74, 401-411. 8. Hecht, S. S., Murphy, S. E., Stepanov, I., Nelson, H. H., and Yuan, J.-M. (2012) Tobacco smoke biomarkers and cancer risk among male smokers in the Shanghai Cohort Study. Cancer Lett. 334, 34-38. 9. Haiman, C. A., Stram, D. O., Wilkens, L. R., Pike, M. C., Kolonel, L. N., Henderson, B. E., and Le Marchand, L. (2006) Ethnic and racial differences in the smoking-related risk of lung cancer. N. Engl. J. Med. 354, 333-342. 10. Park, S.-L., Tiirikainen, M., Patel, Y., Wilkens, L. R., Stram, D. O., Le Marchand, L., and Murphy, S. E. (2016) Genetic determinants of CYP2A6 activity across racial/ethnic groups with different risk of lung cancer and effect on their smoking behavior. Carcinogenesis in press.

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      ED01.03 - Insights from TCGA (ID 6423)

      11:50 - 12:15  |  Author(s): B. Ganesh, S. Devarakonda, R. Govindan

      • Abstract
      • Presentation
      • Slides

      Abstract:
      Advances in sequencing technologies have made it possible to characterize and catalogue genomic alterations in several cancers in an unbiased manner. Multiple individual groups and large-scale consortia such as The Cancer Genomic Atlas (TCGA), have sequenced close to a thousand lung cancer samples to date. [1-8]Apart from furthering our understanding of the frequently altered pathways in common histological subtypes of lung cancer, data from these studies have also highlighted the molecular heterogeneity underlying this disease. Investigators from TCGA initially reported genomic, transcriptomic, methylation and copy-number alterations in 230 adenocarcinoma (LUAD) and 178 squamous cell carcinoma (SQCC) samples.[1][,][2]An updated analysis, that included a total of 660 LUAD and 484 SQCC samples, was subsequently published in early 2016.[9] While the majority of lung cancer patients have a history of cigarette smoking, nearly 10% of patients are lifelong never-smokers.[3]Lung cancers that arise in smokers exhibit some of the highest mutational burdens across all human cancers (8-10 mutations/Mb). The vast majority of these mutations are C>A transversions. On the contrary, tumors from never smokers demonstrate a much lower mutational burden (0.8-1 mutations/Mb) and are enriched for C>T transitions. [1][,][2] Single nucleotide variations (SNVs) and copy number alterations (CNAs) While both LUAD and SQCC show frequent inactivation of the tumor suppressors TP53 and CDKN2A, these alterations are considerably more common in SQCCs. CDKN2A harbors the loci for two isoforms, p14ARF and p16INK4A, and is inactivated in SQCC through homozygous deletion (29%), methylation (21%), inactivating mutations (18%), or exon 1b skipping (4%). [1][,][2]These findings indicate a strong selective pressure for the loss of these tumor suppressors in NSCLC. The pattern of oncogenic alterations varies considerably between LUAD and SQCC. While LUADs typically showed activating RTK/RAS/RAF pathway mutations, these mutations are highly infrequent in SQCCs - which predominantly showed alterations in oxidative stress response (NFE2L2, KEAP1 and CUL3) and squamous differentiation pathways (SOX2, TP63, NOTCH1, etc.) in 44% of samples. [1,2]KRAS is the most commonly mutated oncogene in LUAD, followed by EGFR, BRAF, PIK3CA, and MET. The majority of EGFR mutations in LUAD are targetable (L858R or exon 19 deletion) with tyrosine kinase inhibitors (TKIs).[1]In contrast, such alterations are absent in SQCC. Two SQCC samples however demonstrated L861Q mutations in EGFR, which are potentially targetable with TKIs. [1][,][2]Although SQCC and LUAD shared several CNAs at the chromosomal arm level, amplification of 3q was frequent in SQCC. This region harbors important oncogenes such as SOX2, PIK3CA, and TP63. LUADs frequently showed amplifications in genes such as NKX2-1, TERT, MDM2, KRAS, and EGFR.[1][,][2]Oncogenic activation of kinases such as ALK, ROS1, and RET through rearrangement has been well described in LUAD, and these fusions are targetable with TKIs. These fusions were seen in 1-2% (ALK : 3/230, ROS1: 4/230, and RET: 2/230 samples) of LUADs. [1][,][2] Transcriptome analysis Deregulated splicing can be a consequence of mutations that alter splice-sites within a gene or splicing factors. Mutations in the proto-oncogene MET that lead to exon 14 skipping, and abnormal splicing of proto-oncogenes such as CTNNB1 as a result of U2AF1 mutation have been described in LUAD. [1] Transcriptome analyses have also enabled a reclassification of LUADs and SQCCs into three and four distinct subtypes, respectively. LUAD samples can be categorized as terminal respiratory unit (enriched for EGFR mutations and fusions; favorable prognosis), proximal-inflammatory (NF1 and TP53 co-mutation), or proximal-proliferative (KRAS and STK11 alterations) subtypes. Similarly, SQCCs can be classified as classical, basal, secretory, or primitive. Alterations in genes that participate in the oxidative stress response pathway, hypermethylation, and chromosomal instability are characteristic of the classical subtype (associated with heavy smoking and poor prognosis). [1][,][2] Key pathogenic alterations TCGA analysis revealed alterations in well known oncogenic drivers involving RAS signaling pathway in 62% of LUAD.. These samples with readily identifiable oncogenic driver alterations were collectively labeled ‘oncogene-positive’. Additional analyses of the ‘oncogene-negative’ sample cohort showed enrichment for RIT1, and NF1 mutations. Given the role of RIT1 and NF1 in RTK/RAS/RAF signaling, samples with these mutations were reclassified as oncogene positive, increasing the overall percentage of oncogene positive samples in LUAD to 76%. Nearly 69% of SQCC samples showed alterations in genes regulating PI3K/AKT, or RTK/RAS signaling. [1][,][2] The inability to readily identify an oncogenic driver in nearly a third of sequenced lung cancer samples highlights the need for greater powering of subsequent studies to identify novel low frequency genomic alterations. For instance, previously uncharacterized alterations in the RTK/RAS/RAF pathway were observed in RASA1, SOS1 in the updated TCGA analysis which analyzed a much larger cohort of samples.[9] Overall, despite showing a few similarities between LUAD and SQCC, investigators of TCGA reported prominent differences between the genomic landscapes of these subtypes. These subtypes have more of their alterations in common with other cancers than with one another. SQCCs more closely resembled head and neck squamous cell and bladder cancer, while LUAD resembled glioblastoma multiforme and colorectal cancer in this regard. [9] Immunotherapies The vast majority of lung cancers do not harbor alterations that are targetable by TKIs. [1][,][2 ]Immune checkpoint inhibitors are approved for use in patients with metastatic NSCLC. There is a clear need to develop optimal predictive biomarkers to identify those who are likely to respond to immune checkpoint inhibitors. Mutational burden has been correlated with better response to checkpoint inhibitors. Furthermore, using exome and transcriptome sequencing and sophisticated bioinformatics, it is now possible to identify mutated and expressed genes that could potentially serve as a trigger for immune response (so called neoantigens) once immune checkpoints like programmed death-1 or programmed death ligand-1 are inhibited.. Swanton and colleagues performed a neoantigen and clonality analysis on TCGA samples to examine characteristics such as neoantigen burden and intratumor heterogeneity (ITH), and their impact on survival. In LUAD, a higher neoantigen burden was significantly associated with longer survival. Although not statistically significant, there was a trend towards longer survival in molecularly homogeneous tumors (<1% ITH) as opposed to heterogeneous tumors. The updated TCGA analysis showed that 47% of LUAD and 53% of SQCC samples exhibited at least five predicted neoantigens. Efforts are ongoing to develop personalized vaccine therapy using predicted neoantigens in lung cancer and other malignancies. Outcomes for patients with advanced lung cancer are likely to improve in the near future with further advances in genome sequencing, molecularly targeted therapies and immunotherapies . [12] References 1. Network CGAR. Comprehensive molecular profiling of lung adenocarcinoma. Nature 2014;511:543-50. 2. Network CGAR. Comprehensive genomic characterization of squamous cell lung cancers. Nature 2012;489:519-25. 3. Govindan R, Ding L, Griffith M, et al. Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell 2012;150:1121-34. 4. Imielinski M, Berger AH, Hammerman PS, et al. Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell 2012;150:1107-20. 5. George J, Lim JS, Jang SJ, et al. Comprehensive genomic profiles of small cell lung cancer. Nature 2015;524:47-53. 6. Rudin CM, Durinck S, Stawiski EW, et al. Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer. Nat Genet 2012;44:1111-6. 7. Peifer M, Fernández-Cuesta L, Sos ML, et al. Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer. Nat Genet 2012;44:1104-10. 8. Seo JS, Ju YS, Lee WC, et al. The transcriptional landscape and mutational profile of lung adenocarcinoma. Genome Res 2012;22:2109-19. 9. Campbell JD, Alexandrov A, Kim J, et al. Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas. Nat Genet 2016;48:607-16. 10. Katayama R, Shaw AT, Khan TM, et al. Mechanisms of acquired crizotinib resistance in ALK-rearranged lung Cancers. Sci Transl Med 2012;4:120ra17. 11. Choi YL, Soda M, Yamashita Y, et al. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N Engl J Med 2010;363:1734-9. 12. McGranahan N, Furness AJ, Rosenthal R, et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 2016;351:1463-9.

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Author of

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    MA02 - RNA in Lung Cancer (ID 377)

    • Event: WCLC 2016
    • Type: Mini Oral Session
    • Track: Biology/Pathology
    • Presentations: 1
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      MA02.06 - Discussant for MA02.05 (ID 6957)

      14:56 - 15:08  |  Author(s): R. Rosell

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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    MA07 - ALK-ROS1 in Advanced NSCLC (ID 385)

    • Event: WCLC 2016
    • Type: Mini Oral Session
    • Track: Advanced NSCLC
    • Presentations: 1
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      MA07.05 - EUCROSS: A European Phase II Trial of Crizotinib in Advanced Adenocarcinoma of the Lung Harboring ROS1 Rearrangements - Preliminary Results (ID 4451)

      11:30 - 11:36  |  Author(s): R. Rosell

      • Abstract
      • Presentation
      • Slides

      Background:
      ROS1 rearrangements are present in the tumors of 1-2% of patients with lung adenocarcinoma (LAD). This patient subgroup is characterized by non-smoking history and younger than average age compared to the overall NSCLC population. In a phase I trial the ALK/ROS1/MET inhibitor crizotinib has shown to be highly effective in these patients (NCT00585195). EUCROSS is a prospective phase II trial of the Lung Cancer Group Cologne in collaboration with the Spanish Lung Cancer Group to evaluate crizotinib in ROS1-positive LAD. Here, we present preliminary data on efficacy and safety.

      Methods:
      Patients with advanced LAD harboring ROS1 rearrangements as confirmed by central FISH were eligible for the trial irrespectively of the number of prior treatment lines. Patients received treatment with crizotinib 250 mg BID - doses were adapted for management of AEs. Trial design: Fleming’s single stage phase II design. Primary endpoint: ORR (95% CI, H~0~: ORR≤20% vs. H~1~: ORR>20%). Secondary endpoints: a.o. PFS, OS and safety. All efficacy endpoints were assessed by investigator’s RECIST v1.1 and will be analyzed by IRB at a later stage. Baseline tumor tissue was analyzed by DNA-sequencing to identify the translocation Partners of ROS1, to validate FISH results and to identify additional biomarkers for prediction of response. Data-cut off for this report was March 2016.

      Results:
      In total, 34 patients were enrolled in EUCROSS at the time of data cut-off. Twenty-nine patients were eligible for efficacy assessment. Tumor tissue of 20 of these patients was suitable for further sequencing - 18 were sequenced positive for ROS1 fusion. The fusion partners involved were CD74 (N=9;50%), EZR (N=4;22%), SCL34A2 (N=3;17%), TPM3 and SDC4(N=1;6% each). The investigator assessed ORR was 69% (95% CI, 49.1-84.3) in the overall trial population and 83% (95% CI, 67.7-94.2) in the ROS1-positive by sequencing population (N=18;P=0.324 for difference of ORR). Three patients (10.3%;95% CI, 3.6-26.4) exhibited primary progression, two of them were sequenced ROS1-negative. All patients were included in the safety population (N=34). Most common AEs irrespectively of relatedness or grade were visual disorders (N=16;48%), edema (N=14;41%), diarrhea (N=13;38%) and bradycardia (N=11;32%).

      Conclusion:
      Crizotinib is a highly effective and safe treatment in the subset of ROS1 rearranged NSCLC patients as determined by FISH and DNA-sequencing. Although, the number of patients with tissue available for sequencing was low at the time of data cut-off, sensitivity and specificity support sequencing as the potential new gold-standard for the identification of clinically relevant ROS1 gene-rearrangements.

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    MA16 - Novel Strategies in Targeted Therapy (ID 407)

    • Event: WCLC 2016
    • Type: Mini Oral Session
    • Track: Chemotherapy/Targeted Therapy/Immunotherapy
    • Presentations: 1
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      MA16.03 - Global RET Registry (GLORY): Activity of RET-Directed Targeted Therapies in RET-Rearranged Lung Cancers (ID 4325)

      14:26 - 14:32  |  Author(s): R. Rosell

      • Abstract
      • Presentation
      • Slides

      Background:
      GLORY is a global registry of patients with RET-rearranged non-small cell lung cancer (NSCLC). In order to complement ongoing prospective studies, the registry’s goal is to provide data on the efficacy of RET-directed targeted therapies administered outside the context of a clinical trial. We previously reported results from our first interim analysis (Gautschi, ASCO 2016). Following additional accrual into the registry, updated results are presented here, with a focus on an expanded efficacy analysis of various RET inhibitors.

      Methods:
      A global, multicenter network of thoracic oncologists identified patients with pathologically-confirmed NSCLC harboring a RET rearrangement. Molecular profiling was performed locally via RT-PCR, FISH, or next-generation sequencing. Anonymized data including clinical, pathologic, and molecular features were collected centrally and analyzed by an independent statistician. Response to RET tyrosine kinase inhibition (TKI) administered off-protocol was determined by RECIST1.1 (data cutoff date: April 15, 2016). In the subgroup of patients who received RET TKI therapy, the objectives were to determine overall response rate (ORR, primary objective), progression-free survival (PFS), and overall survival (OS).

      Results:
      165 patients with RET-rearranged NSCLC from 29 centers in Europe, Asia, and the USA were accrued. The median age was 61 years (range 28-89 years). The majority of patients were female (52%), never smokers (63%), with lung adenocarcinomas (98%) and advanced disease (91%). The most frequent metastasic sites were lymph nodes (82%), bone (51%) and lung (32%). KIF5B-RET was the most commonly identified fusion (70%). 53 patients received at least one RET-TKI outside of a clinical protocol, including cabozantinib (21), vandetanib (11), sunitinib (10), sorafenib (2), alectinib (2), lenvatinib (2), nintedanib (2), ponatinib (2) and regorafenib (1). In patients who were evaluable for response (n=50), the ORR was 37% for cabozantinib, 18% for vandetanib, and 22% for sunitinib. Median PFS was 3.6, 2.9, and 2.2 months and median OS was 4.9, 10.2, and 6.8 months for cabozantinib, vandetanib, and sunitinib, respectively. Responses were also observed with nintedanib and lenvatinib. Among patients who received more than one TKI (n=10), 3 partial responses were achieved after prior treatment with a different TKI.

      Conclusion:
      RET inhibitors are active in individual patients with RET-rearranged NSCLC, however, novel therapeutic approaches are warranted with the hope of improving current clinical outcomes. GLORY remains the largest dataset of patients with RET-rearranged NSCLC, and continues to accrue patients.

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    MTE23 - Biomarker Characterization: Challenges and Perspectives (Ticketed Session) (ID 316)

    • Event: WCLC 2016
    • Type: Meet the Expert Session (Ticketed Session)
    • Track: Biology/Pathology
    • Presentations: 1
    • Moderators:
    • Coordinates: 12/07/2016, 07:30 - 08:30, Schubert 3
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      MTE23.02 - Biomarker Characterization: Challenges and Perspectives (ID 6581)

      08:00 - 08:30  |  Author(s): R. Rosell

      • Abstract
      • Presentation
      • Slides

      Abstract:
      Early adaptive resistance in EGFR mutant NSCLC Rafael Rosell Small molecule inhibitors are the current treatment for non-small-cell lung cancer (NSCLC), especially for tumours harbouring an active mutation of epidermal growth factor receptor (EGFR). Approximately 90% of EGFR mutations are exon 19 deletions or exon 21 single-point L858R substitutions and are associated with sensitivity to EGFR tyrosine kinase inhibitors (EGFR TKIs), like gefitinib or erlotinib. However, less than 5% of EGFR-mutant NSCLC patients achieve a complete response to EGFR TKI and the overall median progression-free survival is no longer than 9-11 months. Accumulated studies report several mechanisms of early adaptive resistance that can occur as early as two hours after starting EGFR TKI therapy. The activation of signal transducer and activator of transcription 3 (STAT3) signalling is among these mechanisms of resistance. Furthermore, EGFR blockage enriches lung cancer stem cells through Notch3-dependent signalling pathway. We have previously demonstrated that NF-κB contributes to gefitinib resistance in EGFR-mutant NSCLC. The efficacy of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) is jeopardized by the activation of signalling pathways. We examined the relevance of co-targeting EGFR, signal transducer and activator of transcription 3 (STAT3) and Src-YES-associated protein 1 (YAP1) signalling. We conducted clinical and preclinical studies of key components of signalling pathways limiting EGFR TKI efficacy in EGFR-mutant NSCLC. High levels of STAT3 or YAP1 mRNA expression were associated with worse outcome to EGFR TKI in two independent cohorts of EGFR-mutant NSCLC patients. In the initial cohort of 64 patients, median progression-free survival was shorter among the patients with high STAT3 than among those with low STAT3 (hazard ratio [HR] for disease progression, 3·02; 95% confidence interval [CI], 1·54-5·93; P=0·0013). Median progression-free survival was shorter among the patients with high YAP1 than among those with low YAP1 (HR for disease progression, 2·57; 95%CI, 1·30-5·09; P=0·0067). The results were similar in the validation cohort of 55 patients. We demonstrated that gefitinib augments STAT3 signalling in EGFR-mutant NSCLC cells. Gefitinib with TPCA-1 (STAT3 inhibitor) blocked STAT3, but not the YAP1 phosphorylation on tyrosine residue 357 by Src family kinases (SFKs) that occurs downstream of IL-6. The triple combination of gefitinib, TPCA-1 and AZD0530 (SFK inhibitor) ablated both STAT3 and YAP1 phosphorylation and markedly and safely suppressed tumour growth. Added value of our research: We found that EGFR TKI therapy activates STAT3 and that EGFR blockage enriches lung cancer stem cells with up-regulation of the YAP1 and Notch downstream effectors connective tissue growth factor (CTGF) and hairy-enhancer of split-1 (HES1), respectively. We sought to demonstrate that EGFR TKI treatment cannot abrogate STAT3 and Src-YAP1-Notch activation in EGFR-mutant NSCLC cell lines, leading us to examine whether the combination of gefitinib with compounds targeting STAT3 and Src, supresses the mechanisms of resistance. Nine days after gefitinib treatment, STAT3 mRNA level was significantly increased, as well as the fraction of ALDH positive cells. TPCA-1, a compound that targets STAT3, increases sensitivity to gefitinib in PC-9 and H1975 cells; however, neither gefitinib nor TPCA-1 inhibits Src or YAP1. The addition of the Src inhibitor, saracatinib, to the doublet of gefitinib and TPCA-1, was highly synergistic and abrogated STAT3, and Src-YAP1-Notch signalling. Implications: Treatment with single EGFR TKI can no longer be considered adequate for patients with EGFR mutant NSCLC. Our findings ultimately suggest that a clinical trial evaluating the co-targeted inhibition of STAT3 and Src is warranted. As a result, STAT3 and YAP1 mRNA levels could become important predictive biomarkers. References: We searched PubMed for English language reports published up to December, 2015 using the terms “non-small-cell lung cancer”, “STAT3”, “interleukin-6”, “NF-κB”, “aldehyde-dehydrogenase (ALDH)”, “integrin-linked kinase (ILK)”, “glycoprotein 130 (gp130)”, “Src-homology 2 domain-containing phosphatase 2 (SHP2)”, “the complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing protein-1 (CDCP1)”, “AXL”, “ephrin type-A receptor-2 (EphA2)”, “Src family kinases (SFK)”, “YES-associated protein 1 (YAP1)”, “Notch”, “cell migration, invasion and metastases” and “STAT3 inhibitors”.

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    OA10 - EGFR Mutations (ID 382)

    • Event: WCLC 2016
    • Type: Oral Session
    • Track: Biology/Pathology
    • Presentations: 1
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      OA10.03 - YAP-NOTCH and STAT3 Signaling Rebound as a Compensatory Response to Gefitinib or Osimertinib Treatment in EGFR Mutant Lung Cancer (ID 4144)

      11:20 - 11:30  |  Author(s): R. Rosell

      • Abstract
      • Presentation
      • Slides

      Background:
      Preclinical studies provide insights to therapy mechanisms of resistance that are not feasible with clinical studies. We investigated the signaling pathways that could be involved in adaptive resistance to gefitinib and/or osimertinib in EGFR mutant cells.

      Methods:
      We performed several laboratory methods to examine the signaling pathways involved in EGFR mutations. Signal transduction pathway analysis was designed using the Ingenuity Pathway Analysis (IPA) software (https://www.ingenuity.com/) Figure 1



      Results:
      Pathways mediating EGFR mutations are: i) ERK1/2 via Ras and MEK1/2 ii) AKT via PI3K and iii) STAT3 via JAK (Figure). By Western blot analysis, phosphorylation of Tyr705 on STAT3 was noted after 2 hours of gefitinib or osimertinib treatment in PC9 and H1975 EGFR mutant cells. Unexpectedly, YAP1 phosphorylation on Tyr357 and Notch activation was detected. Co-targeting STAT3 and Src with gefitinib or osimertinib ablates activation of STAT3 and YAP1-NOTCH3 signaling pathways (Figure). In vitro and in vivo, the combinatory therapy of gefitinib or osimertinib plus TPCA-1 (a dual inhibitor of IKKs and STAT3) plus saracatinib (a SFK inhibitor) leads to significant tumor shrinkage in PC9 and H1975 cells. In tumor samples of 64 EGFR mutant NSCLC patients treated with gefitinib, the median progression free survival (PFS) was significantly shorter in those with high levels of HES1, ALDH1A1, ALDH1A3, Bmi1, AXL, CDCP1, SHP2 and ILK (Figure). However, the mRNA levels of STAT3 and YAP1 stand out in the prediction of shorter PFS with a hazard ratio of 3.02 and 2.57, respectively (P<0.001)

      Conclusion:
      For the first time ever, we reported gefitinib induced activation of theYAP1-NOTCH signaling pathway, in addition to activation of STAT3, in EGFR mutant cells. Secondly, co-targeting STAT3 and Src, together with EGFR, causes significant tumor growth inhibition, in comparison with gefitinib or osimertinib single therapy.

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    P1.02 - Poster Session with Presenters Present (ID 454)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Biology/Pathology
    • Presentations: 4
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      P1.02-020 - The Effect of EGF-Pathway Targeted Immunization (EGF PTI) on STAT3 and Cancer Stem Cells in EGFR Mutant NSCLC Cells (ID 4698)

      14:30 - 14:30  |  Author(s): R. Rosell

      • Abstract
      • Slides

      Background:
      The vast majority of advanced non-small cell lung cancer (NSCLC) patients with EGFR mutant tumors will develop disease progression following successful treatment with an EGFR tyrosine kinase inhibitor (TKI). Resistance to EGFR-TKIs is due to various mechanisms, such as the secondary mutation (T790M) or the activation of alternative pathways (MET, AXL). What has not been fully appreciated is that EGFR blockade induces an imbalance in favor of survival, increases activity of STAT3 and enriches lung CSCs through Notch3-dependent signaling. EGF-PTI was designed to elicit an antibody response against EGF, in order to reduce EGF receptor signaling and limit tumor growth. We have explored whether EGF-PTI alone or in combination with EGFR TKIs may efficiently inhibit STAT3 and target CSCs.

      Methods:
      EGF PTI was provided by Bioven (Europe) Ltd. Gefitinib, erlotinib and the recently FDA-approved third-generation EGFR TKI, AZD9291 (osimertinib) were purchased from Selleck chemicals. Western blotting was used to assess the effect of the drugs on ERK, AKT and STAT3 phosphorylation and on Notch and PARP cleavage in EGFR (del19) mutant NSCLC PC9 cells and gefitinib-resistant PC9-GR4 cells. PC9-GR4 cells have been established in our lab and harbor the resistant T790M mutation (T790M+). The protein expression of AXL and CSCs markers such as HES1 (downstream effector of Notch) and Bmi1 was also examined.

      Results:
      Gefitinib, erlotinib or AZD9291 suppressed EGFR, ERK1/2 and AKT phosphorylation in PC9 cells but increased STAT3 phosphorylation on the tyrosine residue 705 in both PC9 and PC9-GR4 cells. EGF-PTI suppressed STAT3, EGFR and ERK1/2 and the combination of each of the three EGFR TKIs with EGF-PTI lead to more potent inhibition of STAT3, EGFR and ERK1/2. The EGF-PTI induced AKT phosphorylation was reversed when EGF PTI was combined with EGFR TKIs. Interestingly, EGF-PTI blocked Notch cleavage and decreased the expression of HES1. The expression of Bmi1 and AXL were also attenuated with EGF PTI and apoptosis was enhanced through the induction of PARP cleavage.

      Conclusion:
      EGF PTI may reverse mechanisms of resistance to single EGFR inhibition and the combination of EGF PTI with EGFR TKIs efficiently inhibits downstream signaling pathways in T790M+ cells. Based on these results, the design of a proof-of-concept trial with the combination of EGF PTI with gefitinib for the first line treatment of EGFR mutant NSCLC patients is in progress.

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      P1.02-050 - Acquired Resistance to EGFR Tyrosine Kinase Inhibitors (TKIs) in EGFR-Mutant Lung Adenocarcinoma among Hispanics (Rbiop-CLICaP) (ID 5955)

      14:30 - 14:30  |  Author(s): R. Rosell

      • Abstract

      Background:
      Patients with epidermal growth factor receptor (EGFR)-mutant lung carcinoma eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKI). In 50% of these cases, a secondary EGFR mutation, T790M, underlies the acquired resistance. Other alterations include amplification of MET, PI3K mutations, changes in MAPK1, Her2, AXL and even transformation to small cell lung carcinoma (SCLC). We assessed histological, clinical characteristics and survival outcomes in Hispanic patients with EGFR mutation after disease progression.

      Methods:
      34 EGFR-mutant lung cancer patients with acquired resistance to EGFR TKIs were identified as part of a prospective registry (active between January 2011 and January 2015) in which post-progression tumor specimens were collected for molecular analysis using SNaPshot tumor genotyping assay to detect mutations in EGFR, KRAS, BRAF, PI3KCA, TP53, MET and Her2, and FISH for MET, ALK and EGFR. Samples also underwent immunohistochemistry analysis for E-cadherin, synaptophysin, CD56 and PDL1. Post-progression interventions, response and survival were assessed and compared to those with and without T790M.

      Results:
      Mean age was 59.4±13.9 years; 62% were female, 65% were never-smokers and 53% had a performance status ≥80%; main metastatic sites were lung (16/47%), bone (20/58%), brain (18/52%) and liver (13/38%). All patients received erlotinib as first- line treatment and documented mutations were: 60% DelE19 (Del746–750) and 40% L858R. Overall response rate (ORR) with first line TKI was 61.8% and progression free survival (PFS) was 16.8 months (range, 13.7–19.9 m). After progressing to TKI, all patients were re-biopsied, of whom 16 had the T790M mutation (47.1%); 5 had PI3K mutations (14.7), 5 had EGFR amplification (14.7%), 2 had a KRAS mutation (5.9%), 3 had MET amplification (8.8%), 2 had Her2 alterations (5.8%, deletions/insertions in e20), and one had SCLC transformation (2.9%). 79.4% received treatment after progression. ORR for post-TKI treatments was 47.1% (CR 2/PR 14) and median PFS was 8.3 months (CI95% 2.2–36.6). There were no differences in PFS according to gender (p=0.10) or type of acquired alteration (p=0.63). Median survival was 32.9 months (CI95% 30.4–35.3), and only the use of post-progression therapy affected OS in multivariate analysis (p=0.05).

      Conclusion:
      Hispanic patients with acquired resistance to EGFR TKIs continued to be sensitive to other treatments after progression. Proportion of T790M+ patients appears to be similar to previously reported results in Caucasians.

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      P1.02-058 - EGFR Amplification and Sensitizing Mutations Correlates with Survival from Erlotinib in Lung Adenocarcinoma Patients (MutP-CLICAP) (ID 6335)

      14:30 - 14:30  |  Author(s): R. Rosell

      • Abstract

      Background:
      Tumor heterogeneity, which causes different EGFR mutation abundance, is believed to be responsible for varied progression-free survival (PFS) in lung adenocarcinoma (ADC) patients receiving EGFR-TKI treatment. Frequent EGFR amplification and its common affection inEGFR mutant allele promote the hypothesis that EGFR mutant abundance might be determined by EGFR copy number variation and therefore examination of EGFR amplification status in EGFR mutant patients could predict the efficacy of EGFR-TKI treatment.

      Methods:
      72 lung ADC patients who harbored EGFR activating mutations and received erlotinib as first line treatment, were examined for EGFR amplification by FISH. EGFR mutational and copy number status were compared with response, overall-survival (OS), and progression-free-survival (PFS).

      Results:
      Median age was 62-yo (r, 20-87 years), 53 patients were females (73%), and 89% have common mutations. Twenty-two (30.6%) samples with EGFR activating mutations were identified with EGFR amplification. EGFR amplification was more frequent in patients with exon 19 deletion (p=0.05) and in those with better performance status (p=0.01). Patients with EGFR gene amplification had a significantly longer PFS than those without [(25.2 months, 95%CI 22.0-38.5) vs. (12.4 months, 95%CI 5.3-19.5); p=0.002] as well as better OS [(EGFR amplified 37.8 months, 95%CI 30.9-44.7) vs. (EGFR non-amplified 27.1 months, 95%CI 12.8-41.3); p=0.009]. EGFR amplification significantly influenced the response to erlotinib (p=0.0001).

      Conclusion:
      EGFR amplification occurs in one third of patients with lung ADC harboring EGFR activating mutations, and could serve as an indicator for better response and survival from EGFR-TKI treatment.

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      P1.02-064 - MET-Dependent Activation of STAT3 as Mediator of Resistance to MEK Inhibitors in KRAS-Mutant Lung Cancer (ID 4240)

      14:30 - 14:30  |  Author(s): R. Rosell

      • Abstract

      Background:
      Targeting the MAPK pathway by MEK inhibition results in limited activity in patients with KRAS-mutant non-small cell lung cancer (NSCLC). The lack of effectiveness may be associated with activation of other effectors including STAT3, as well as MEK inhibition relief from negative feedback loops. Indeed, in KRAS-mutant colorectal cancer, MEK inhibition decreases the activity of the metalloprotease ADAM17, which normally inhibits MET signaling and STAT3 activation by promoting shedding of MET endogenous antagonist, soluble "decoy" MET. Herein, we explore the MET-dependent activation of STAT3 as a mediator of resistance to MEK inhibitors, and whether MET or STAT3 inhibitors can synergistically increase MEK-inhibitor-induced growth inhibition in KRAS-mutant NSCLC cells in vitro.

      Methods:
      Cell viability was assessed by MTT (thiazolyl blue) assay after treatment with the allosteric MEK inhibitor, selumetinib, the small-molecule dual inhibitor of the MET and ALK receptor tyrosine kinases, crizotinib, and evodiamine, an alkaloid isolated from the dried, unripe Evodia rutaecarpa (Juss.) Benth fruit, that exerts an anticancer effect by inhibiting STAT3. RNA was isolated from four KRAS cell lines and the STAT3 and MET mRNA expression analysis was performed by TaqMan based qRT-PCR. Western blotting was used to assess the effect of selumetinb on ERK, AKT and STAT3 phosphorylation.

      Results:
      We first evaluated the efficacy of the MEK inhibitor selumetinib in our KRAS-mutant NSCLC cell line panel using an MTT cell proliferation assay. H460 cells were relatively insensitive to selumetinib. Following 48-hour treatment with selumetinib, ERK1/2 and AKT phosphorylation were suppressed but a rebound activation of STAT3 occurred in H460 cells. We next investigated whether MET expression was related to the feedback activation of STAT3 signaling following MEK inhibitor treatment. We compared gene expression profiles of the H460 cell line before and after treatment with selumetinib. Interestingly, we found significant upregulation of MET and STAT3 mRNA expression after seven days of selumetinib treatment. To further interrogate the relationship between MEK inhibition and MET-mediated STAT3 reactivation, H460 cells were treated with the combination of selumetinib and crizotinib or selumetinib and evodiamine. A 72-hour exposure to both combinations resulted in a clear cell synergism, as measured by the combination index (CI) analysis, with a CI of 0.79 and 0.78 respectively.

      Conclusion:
      Collectively our results showed that the feedback STAT3 activation induced by MET, mitigates the effect of MEK inhibition, and provides rationale for further assessment of combined MEK and MET or STAT3 inhibition in KRAS-mutant NSCLC.

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    P2.01 - Poster Session with Presenters Present (ID 461)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Biology/Pathology
    • Presentations: 1
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      P2.01-008 - SiRe Next Generation Sequencing Panel: Effective Diagnostic Tool for Circulating Free DNA Analysis (ID 5624)

      14:30 - 14:30  |  Author(s): R. Rosell

      • Abstract

      Background:
      Tissue availability is a crucial point in NSCLC. The introduction of Liquid Biopsies allows to determine circulant biomarkers, specifically using free DNA. To simultaneously analyze multiple patients sample at high sensitivity, Next Generation Sequencing (NGS) can be narrowed to target a limited number of actionable genes. Here we prospectically applied a lab-developed narrowed gene panel (SiRe) to produce a DNA library covering 568 actionable mutations in six gene (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα).

      Methods:
      This daily clinical practice study was performed on cfDNA obtained from Non Small Cell Lung Cancer blood samples (serum and plasma) prospectically collected either prior to treatment administration in patients without tissue availability (n = 46) or after a progressive disease (n = 19) from a first line gefitinib (n = 14) or afatinib (n = 5) therapy.

      Results:
      SiRe detected an activating EGFR mutation in 4/46 (8.9%) cases and in T790M in 9/19 (47.4%) at the time of tumor progression. Using tissue data as gold standard, the SiRe panel showed a sensibility of 90.5% and specificity of 100%.

      Conclusion:
      The SiRe panel is an effective tool enabling the implementation of NGS for cfDNA mutational profiling in molecular pathology practice.

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    P2.03a - Poster Session with Presenters Present (ID 464)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Advanced NSCLC
    • Presentations: 1
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      P2.03a-007 - Pem/CBP/Bev Followed by Pem/Bev in Hispanic Patients with NSCLC: Outcomes According to Combined Score of TS, ERCC1 and VEGF Expression (ID 6293)

      14:30 - 14:30  |  Author(s): R. Rosell

      • Abstract

      Background:
      To evaluate the efficacy and safety of pemetrexed, carboplatin and bevacizumab (PCB) followed by maintenance pemetrexed and bevacizumab (PB) in chemotherapy-naïve patients with stage IV non-squamous non-small cell lung cancer (NSCLC) through the influence of thymidylate synthase (TS), ERCC1 and VEGF mRNA expression on several outcomes. The primary endpoints were the overall response rate (ORR), progression-free survival (PFS) and overall survival (OS).

      Methods:
      Patients were administered pemetrexed (500 mg/m2), carboplatin (AUC, 5.0 mg/ml/min) and bevacizumab (7.5 mg/kg) intravenously every three weeks for up to four cycles. Maintenance pemetrexed and bevacizumab was administered until disease progression or unacceptable toxicity.

      Results:
      One hundred forty-four Hispanic patients with a median follow-up of 13.8 months and a median number of maintenance cycles of 6 (range, 1- 32) were assessed. The ORR among the patients was 66% (95% CI, 47% to 79%). The median progression-free and overall survival (OS) rates were 7.9 months (95% CI, 5.9-10.0 months) and 21.4 months (95% CI, 18.3 to 24.4 months), respectively. Median TS, ERCC1 and VEGF mRNA levels were 1.45 (range, 0.17–2.52), 0.58 (range, 0.44-1.20), and 2.72 (range, 1.84-3.21), respectively. OS was significantly higher in patients with the lowest TS mRNA levels [29.6 months (95%CI 26.2-32.9) compared with those with higher levels 9.3 months (95%CI 6.6-12.0); p=0.0001]. ERCC1 mRNA levels also influenced the OS [median for ERCC1 mRNA˂0.58 28.7 months (95%CI 26.3-31.2) vs. ERCC1 mRNA˃0.58 11.1 months (95%CI 9.6-12.7); p=0.0001] as well as VEGF mRNA levels [median OS for VEGF mRNA˂2.72 26.4 months (95%CI 22.8-30.0) vs. VEGF mRNA˃2.72 18.2 months (95%CI 8.4-27.9); p=0.009]. TS mRNA did not influence treatment response, however the ORR was significantly higher in patients with low levels of ERCC1 (p = 0.003) and elevated VEGF (p = 0.005). Multivariate analysis found that TS mRNA levels (p=0.0001), VEGF mRNA levels (p=0.007) and PS (p=0.014) were independent prognostic factors.

      Conclusion:
      Overall, PCB followed by maintenance pemetrexed and bevacizumab was in Hispanic patients with non-squamous NSCLC. This regimen was associated with prolonged OS, particularly in patients with low TS, ERCC1 and VEGF mRNA expression. These biomarkers alone or in combination may be useful to assess the prognosis of patients with NSCLC treated with CBP/Pem/Bev.

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    P2.06 - Poster Session with Presenters Present (ID 467)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Scientific Co-Operation/Research Groups (Clinical Trials in Progress should be submitted in this category)
    • Presentations: 1
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      P2.06-010 - AZD9291 as 1st-Line Therapy for EGFR Mutant NSCLC Patients with Concomitant Pretreatment EGFR T790M Mutation. The AZENT Study (ID 4267)

      14:30 - 14:30  |  Author(s): R. Rosell

      • Abstract

      Background:
      Osimertinib (AZD9291) is a selective and irreversible pyrimidine-based inhibitor of the primary activating and the secondary EGFR mutation, T790M, which is the most common mechanism of acquired resistance to 1st and 2nd-generation EGFR tyrosine-kinase inhibitors (TKIs). Progression-free survival (PFS) with osimertinib was 9.6 and 2.8 months (m) for EGFR mutated (EGFR+) NSCLC patients progressing to prior EGFR TKI therapy with and without EGFR T790M mutation, respectively, indicating that the T790M is a predictive biomarker for osimertinib efficacy. Sixty patients from two expansion cohorts of the same study, received 1st-line osimertinib and obtained a PFS of 19.3m. T790M, arising in cis with the primary activating mutation, confers resistance to EGFR TKIs, even in the absence of drug selection. The coexistence of the pretreatment T790M mutation has been under appreciated, in spite of accumulative evidence that is present in a frequency of 35-60% using different detection methods. In our experience, pretreatment T790M mutation is frequently detected by three specific aspects of the method: tumor microdissection, examination of two separate tumor areas, and the use of a peptic nucleid acid clamp that inhibits wild-type allele amplification. Thus, we designed the first phase IIa study to evaluate the safety and efficacy of osimertinib as 1st-line therapy for patients with metastatic EGFR+ NSCLC and concomitant pretreatment T790M mutation.

      Methods:
      This is a multicenter, single-arm, open-label, non-controlled phase IIa clinical study in Spain. Eligible patients are aged ≥18 years with metastatic EGFR+ NSCLC and by central testing documented presence of pretreatment T790M mutation. Seventy-three patients will receive continuous treatment with osimertinib 80 mg daily until disease progression, intolerable adverse events, consent withdrawal or noncompliance with the study protocol. The primary endpoint is the objective response rate (ORR) assessed using Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. The trial is designed to detect a ≥70% ORR in this patient population. Secondary objectives include PFS, overall survival, time to treatment failure, duration of response and disease control rate. Additional pre-specified secondary objectives of the study are the longitudinal analysis of EGFR mutations (including the T790M and the C797S mutations) in plasma and serum and the expression analysis of a panel of biomarkers with possible predictive value for osimertinib treatment.

      Results:
      Not applicable

      Conclusion:
      Not applicable

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    P3.01 - Poster Session with Presenters Present (ID 469)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Biology/Pathology
    • Presentations: 1
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      P3.01-038 - STAT3 and Src-YAP1 Inhibition Results in Greater Necitumumab Sensitivity in Lung Squamous Cell Carcinoma (ID 4242)

      14:30 - 14:30  |  Author(s): R. Rosell

      • Abstract

      Background:
      The anti-EGFR monoclonal antibody (mAb), necitumumab, has been recently approved in combination with chemotherapy, as 1st-line treatment for advanced lung squamous cell carcinoma (LSCC) patients, but with minimal survival benefit. Evidence continues to accumulate that signal transducer and activator of transcription 3 (STAT3) is a promising molecular target for cancer therapies. STAT3 is activated by tyrosine phosphorylation in response to EGF and interleukin-6 (IL-6). In addition to STAT3, IL-6 activates the Src family kinases, and subsequently YES-associated protein 1 (YAP1). STAT3 and Src-YAP1 activation contributes to EGFR inhibitor resistance and concomitant targeting of EGFR and STAT3-Src may represent an effective treatment strategy for LSCC.

      Methods:
      RNA was isolated from six LSCC cell lines and the mRNA expression analysis of EGFR, STAT3, Src and YAP1 was performed by TaqMan based qRT-PCR. Cell viability was assessed by MTT (thiazolyl blue) assay after treatment with necitumumab and evodiamine, an alkaloid isolated from the dried, unripe Evodia rutaecarpa (Juss.) Benth fruit that exerts an anticancer effect by inhibiting STAT3 and Src. Western blotting was performed to assess the effect of necitumumab on EGFR downstream signaling pathways.

      Results:
      We first evaluated the expression of EGFR in our panel of LSCC cell lines. We found that almost all of them homogeneously express high levels of EGFR. We then assessed the effect of necitumumab on EGFR downstream signaling in the SK-MES1 cell line. Treatment of SK-MES1 cells with 25ug/ml of necitumumab for seven days was unable to ablate STAT3, Src or YAP1 mRNA expression. Consistent with this, we found that necitumumab suppressed EGFR, ERK1/2 and AKT phosphorylation but increased STAT3 phosphorylation on the critical tyrosine residue 705 in a time and dose-dependent manner. We examined the growth inhibitory effect of the necitumumab and evodiamine combination. We performed an MTT cell proliferation assay on SK-MES1 cells and we used a constant ratio drug combination method to determine synergy, additivity, or antagonism. The combination of necitumumab and evodiamine resulted in a clear synergism in SK-MES1 cells as measured by the combination index (CI) analysis, with a CI of 0.74. Experiments in the rest of our LSCC cell lines are ongoing.

      Conclusion:
      Herein we have examined the role of STAT3 and Src-YAP1 in the context of treatment with the FDA-approved EGFR mAb, necitumumab. Our data provide initial evidence that co-activation of STAT3 and Src-YAP1 may limit the cellular response to EGFR inhibition in LSCC.

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    P3.02b - Poster Session with Presenters Present (ID 494)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Advanced NSCLC
    • Presentations: 2
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      P3.02b-014 - Monitoring of T790M Mutation in Serum for Prediction of Response to Third Generation Inhibitors (ID 4097)

      14:30 - 14:30  |  Author(s): R. Rosell

      • Abstract
      • Slides

      Background:
      The emergence of T790M mutation (T790M) represents the main mechanism of acquired resistance (AR) to 1[st] and 2[nd] generation tyrosine kinase inhibitors (TKIs) in EGFR mutant patients (p). Recently, 3[rd] generation inhibitors (T790Mi) have demonstrated activity in EGFR mutant (mu) patients with AR to TKIs harboring T790M. Serum and plasma have been used as an alternative to tissue to detect both sensitizing EGFRmu and T790M. We evaluated if (1) T790M could be monitored along T790Mi therapy in p with baseline T790M in serum, (2) T790M loss could be correlated to clinical and radiographic response, and (3) T790M disappears soon in rapid responders.

      Methods:
      10 p out of a total of 15 T790M+p treated with T790Mi were selected according the baseline T790M+ in serum. Baseline characteristics, data on changes in T790M in serum; and radiographic and symptom changes along T790Mi therapy were collected. T790M in serum was detected using a PNA-locked nucleic PCR clamp-based technique. T790M was evaluated at baseline and at certain times after T790Mi initiation.

      Results:
      80% of the p were female and never smoker; 100% were adenocarcinoma, Caucasian, del19, and were treated with previous TKI, with a median (m) time to treatment failure of 11.25 months (mo) [range (r)1-19 mo]. P received 2 previous treatments (r1-6), 40% had a rebiopsy for T790M evaluation, had 3 metastatic sites (r1-6), and had a PS 1 in 70% of the cases. 5 p were evaluable for response with 2 SD and 3 PR as best response (BR) in the 1[st ]evaluation. 7 out of 9 p evaluable for clinical response, experienced an improvement in baseline symptoms as soon as 3 weeks (w) after starting T790Mi, only 1 p experienced an increase in pain, but not related to bone M1. T790M was lost in 80% of the p and it was not detected in serum at 3 or 6 w after the T790Mi initiation in 2 out of 4 and 4 out of 7 evaluable p, respectively.

      Conclusion:
      T790M detection can be lost early along T790Mi treatment. The decrease in symptom burden is seen in p with loss of T790M. PR and SD represent the BR in p with loss of T790M. The loss of T790M in the serum may be a marker of symptomatic and radiographic response to T790Mi. Future evaluation would demonstrate if the reappearance of T790M mutation in serum could be a marker of resistance to T790Mi.

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      P3.02b-047 - Co-Activation of STAT3 and YAP1 Signaling Pathways Limits EGFR Inhibitor Response in Lung Cancer (ID 4168)

      14:30 - 14:30  |  Author(s): R. Rosell

      • Abstract

      Background:
      EGFR tyrosine kinase inhibitors (TKIs) induce early activation of several signaling pathways. Interleukin-6 (IL-6) and signal transducer and activator of transcription 3 (STAT3) hyper-activation occur following EGFR TKI therapy in EGFR-mutant NSCLC cells. We explored the relevance of co-targeting EGFR, STAT3 and Src-YES-associated protein 1 (YAP1) signaling in EGFR-mutant NSCLC.

      Methods:
      We combined in vitro and in vivo approaches to explore whether concomitant activation of STAT3 and Src-YAP1 can limit the effectiveness of EGFR TKIs in EGFR-mutant NSCLC cells and xenograft models. In two cohorts of EGFR-mutant NSCLC patients, we examined messenger RNA (mRNA) gene expression within signaling pathways, leading to EGFR TKI resistance.

      Results:
      Gefitinib suppressed EGFR, ERK1/2 and AKT phosphorylation but increased STAT3 phosphorylation (pSTAT3-Tyr705). In EGFR mutant cells, gefitinib plus TPCA-1 (STAT3 inhibitor) abolished pSTAT3-Tyr705 but not the YAP1 phosphorylation on tyrosine 357 by Src family kinases (SFKs). The triple combination of gefitinib, TPCA-1 and AZD0530 (SFK inhibitor) ablated both STAT3 and YAP1 phosphorylation and was highly synergistic, according to the combination index. In two EGFR mutant xenograft mouse models, the triple combination of gefitinib, TPCA-1 and AZD0530 markedly and safely suppressed tumor growth. High levels of STAT3 or YAP1 mRNA expression were associated with worse outcome to EGFR TKI in 64 EGFR-mutant NSCLC patients. Median progression-free survival (PFS) was 9.6 (95%CI, 5.9-14.1) and 18.4 months (95%CI, 8.8-30.2) for patients with high and low STAT3 mRNA, respectively (p<0.001), (HR for disease progression, 3.02; 95% CI, 1.54-5.93; p=0.0013). Median PFS was 9.6 (95%CI, 7.7-15.2) and 23.4 months (95%CI, 13.0-28.1) for patients with high and low YAP1 mRNA, respectively (p=0.005), (HR for disease progression, 2.57; 95%CI, 1.30-5.09; p=0.0067). The results were similar in the validation cohort of 55 EGFR-mutant NSCLC patients treated with first-line EGFR TKI in the Department of Oncology of Shanghai Pulmonary Hospital.

      Conclusion:
      Our study reveals that STAT3 and Src-YAP1 signaling activation occurs following single EGFR TKI in EGFR-mutant NSCLC. STAT3 and YAP1 mRNA levels were significantly predictive of progression-free survival in the original as well as in the validation cohort of EGFR-mutant NSCLC patients. Co-targeting STAT3 and Src in combination with EGFR TKI could substantially improve survival.

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    P3.03 - Poster Session with Presenters Present (ID 473)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Mesothelioma/Thymic Malignancies/Esophageal Cancer/Other Thoracic Malignancies
    • Presentations: 4
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      P3.03-035 - Prognostic Role of hENT1 and RRM1 in Patients with Advanced Pleural Mesothelioma Treated with Second Line Gemcitabine Based Regimens (ID 6328)

      14:30 - 14:30  |  Author(s): R. Rosell

      • Abstract

      Background:
      Nucleoside transporter proteins mediates the transport of nucleosides and nucleoside analog drugs across the plasma membrane. The human equilibrative nucleoside transporter 1 (hENT1) is a nucleoside transporter protein that mediates cellular entry of gemcitabine, cytarabine, and fludarabine. The hENT1 expression has been demonstrated to be related with prognosis and activity of gemcitabine-based therapy in breast, ampullary, lung, and pancreatic cancer. In the same way, RRM1 encodes the regulatory subunit of ribonucleotide reductase and is a molecular target of gemcitabine. Previous studies showed increased RRM1 expression on continuous exposure of cell lines to gemcitabine and suggested improved survival for patients with low RRM1 expression.

      Methods:
      We measured hENT1 and RRM1 messenger RNA (mRNA) levels by quantitative real-time reverse transcription-polymerase chain reaction in tumor samples from 29 patients with advanced pleural mesothelioma (APM) treated in second line with gemcitabine based chemotherapy correlating these data with clinical parameters and disease outcomes (overall response rate-ORR, progression free survival-PFS and overall survival-OS).

      Results:
      The median age was 60-yo (r, 33-70 years), 15 patients were males (51%), 34% of cases undergone debulking surgery and the median follow-up was 13.4 months (95%CI 6.4-34). All patients were treated with Pem based chemotherapy in first line achieving a PFS of 8.1 months (95%CI 3.7-12.6), while PFS with second-line Gem based chemotherapy was 6.3 months (95%CI 3.6-8.8). 62% and 34.5% of patients had low levels of mRNA for hENT1 and RRM1, respectively. On univariate survival analysis, the hENT1 expression was associated with OS (p=0.001) and PFS (p=0.022). Specifically, those patients with overexpression of hENT1 showed a shorter OS p=0.021) and a shorter PFS (p=0.033). In contrast, mRNA expression of RRM1 did not influence the OS (p = 0.44) but it modifies positively the PFS (p=0.034). Multivariate analysis found that combined hENT1 (p=0.001) and RMM1 (p=0.012) predict survival in patients with APM treated with Gem based regimens.

      Conclusion:
      hENT1 mRNA expression carries prognostic information in patients with APM and combined with RRM1 holds promise as a predictive biomarkers in gemcitabine treated patients.

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      P3.03-045 - Treatment of Malignant Pleural Mesothelioma beyond First-Line among Hispanics (MeSO-CLICaP) (ID 6271)

      14:30 - 14:30  |  Author(s): R. Rosell

      • Abstract

      Background:
      Platinum/Pemetrexed chemotherapy is standard of care in first-line (FL) treatment of malignant pleural mesothelioma (MPM). Different second and third lines regimens are also considered, but the optimal treatment has not yet been defined.

      Methods:
      The aim of this study was to evaluate clinical outcomes of second (SL) and third line (TL) therapies in a series of MPMs included in a retrospective multinational database (MeSO-CLICaP). Clinical records of MPM-patients who received treatment beyond FL from 2008 to 2016 were reviewed. Study endpoints were response, overall-survival (OS), and progression-free-survival (PFS) for SL and TL, stratified for patient characteristics, FL-outcomes, and type of regimen. Out of 124 patients, 79 received SL/TL and had sufficient clinical data.

      Results:
      Of the 124 patients included in the MeSO-CLICaP registry, 79 (64%) received some treatment after first line. Median age was 59 years (range 33-81), 42 (53%) were men, 74% were current or former smokers and 77% had a baseline ECOG 0-1. After FL, 57 patients (76%) achieved disease-control (PR 24/32% and SD 33/44%) and 18 had a time-to-progression ≥8 months. Median PFS and OS to SL were 6.2 (95%CI 4.9-7.4) and 16.1 (95%CI 14.5-17.6) months, respectively. According to a multivariate analysis, disease control after SL-therapy was significantly related to pemetrexed-based treatment (OR 2.46; p=0.017) and FL-TTP≥12 months (OR 3.50; p=0.006). Improved PFS to SL was related to younger age (<65 years, HR: 0.60; p=0.045), ECOG 0-1 (HR 0.72; p=0.02), and FL-TTP≥12 months (HR 0.48; p<0.001). OS was significantly related to ECOG 0-1 (HR 0.43; p=0.011) and to FL-TTP≥12 months (HR 0.66; p=0.05). Fifty-two patients (42%) receive a TL achieving a disease control rate of 62% with a PFS of 5.9 months (95%CI 5.0-6.8).

      Conclusion:
      SL-chemotherapy appears to be active in Hispanic MPM-patients, particularly in younger patients with good PS and prolonged disease control with FL chemotherapy. Considering the important limitations of this study, due to retrospective nature and the possible selection bias, prospective clinical trials are warranted to clarify these issues.

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      P3.03-048 - Profiling Response to Chemotherapy in Malignant Pleural Mesothelioma among Hispanics (MeSO-CLICaP) (ID 6319)

      14:30 - 14:30  |  Author(s): R. Rosell

      • Abstract

      Background:
      Malignant pleural mesothelioma (MPM) is a rare malignant disease, and the understanding of molecular pathogenesis has lagged behind other malignancies.

      Methods:
      A series of 53 formalin-fixed, paraffin-embedded tissue samples with clinical annotations were retrospectively tested for BAP1 and PI3K mutations and for mRNA expression of TS and EGFR. Immunohistochemistry staining for CD26 (dipeptidyl-peptidase IV, DPP-IV) and Fibulin3 (Fib3) proteins were also performed. Outcomes like progression free survival (PFS), overall survival (OS) and response rate (ORR) were recorded and evaluated according to biomarkers. Cox model was applied to determine variables associated with survival.

      Results:
      Median age was 58 years (range 36-76), 27 (51%) were men, 89% were current or former smokers, and six patients had previous contact with asbestos. 77% had a baseline ECOG 0-1 and almost all patients (n=52/98%) received cisplatin or carboplatin plus pemetrexed (Pem) as first line; 58% of them were treated with Pem as maintenance for a mean of 4.7 +/-2.8 cycles. 53.5% and 41.5% of patients were positive for CD26 and fibulin-3, while 49% and 43.4% had low levels of EGFR and TS mRNA, respectively. The majority of epithelioid and biphasic types expressed CD26 (p=0.008), Fibulin3 (0.013) and had lower levels of TS mRNA (p=0.008). Mutations in PI3K (c.1173A> G, c.32G> C and c.32G> T) were found in 5 patients and only one patient had a mutation in BAP1 (c.241T> G). First line PFS were significantly longer in CD26+ (p=0.0001), in those with low EGFR mRNA expression (p=0.001), in patients with positive Fib3 (p=0.006) and lower TS mRNA expression (p=0.0001). OS were significantly higher in patients with CD26+ (p=0.0001), EGFR- (p=0.001), Fib3 + (p=0.0002) and low TS mRNA expression level (p=0.0001). Multivariate analysis found that CD26+ (p=0.012), Fib3 (p=0.020) and TS mRNA levels (p=0.05) were independent prognostic factors.

      Conclusion:
      CD26, Fib3 and TS were prognostic factors significantly associated with improved survival in patients with advanced MPM.

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      P3.03-060 - Characteristics and Long Term Outcomes of Advanced Pleural Mesothelioma in Latin America (MeSO-CLICaP) (ID 6265)

      14:30 - 14:30  |  Author(s): R. Rosell

      • Abstract

      Background:
      Malignant pleural mesothelioma (MPM) is an aggressive tumor, usually associated with a poor prognosis. MPM is a heterogeneous disease often associated with different clinical courses. Palliative platinum-based chemotherapy may help to improve symptoms and prolong life.

      Methods:
      The MeSO-CLICaP registry identified 124 patients with advanced MPM from 5 Latin American countries diagnosed and treated between January 2008 and March 2016. Data collected included age, gender, asbestos exposure, presenting signs/symptoms, performance status, histology, stage, treatment modalities including chemotherapy, and date of death or last follow-up. Outcomes like progression free survival (PFS), overall survival (OS) and response rate (ORR) were recorded. Cox model was applied to determine variables associated with survival.

      Results:
      median age was 59.5 years (range 33-84), 72 (58%) were men, 69% were current or former smokers and 37 patients (30%) had previous exposure to asbestos. Ninety-six patients (77%) had a baseline ECOG 0-1, 102 (82%) were epithelioid tumors, 47 (38%) and 77 (62%) cases had stage III or IV MPM. Only 20% (n=25) underwent pleurectomy, 28% (n=35) received radiotherapy and 123 patients received platinum-based chemotherapy in first line (plus Pem 68/54% and Gem 55/44%). ORR to first line chemotherapy was 48% (CR 3.2%/PR 43%), PFS was 10.5 months (95%CI 8.2-12.8) and 47 patients had Pem maintenance (mean number of cycles 4.4+/-3). Median OS was 25.3 months (95%CI 22.3-28.3) and according to a univariate analysis, stage (p=0.03), histology (p=0.005), and Pem manteinance (p=0.014) were associated with better OS. Multivariate analysis found that stage (p=0.002), histology (p=0.021), smoking history (p=0.001) and Pem manteinance (p=0.002) were independent prognostic factors.

      Conclusion:
      Our study identifies factors associated with a clinical benefit from chemotherapy among Hispanic patients with advanced MPM, and emphasizes the impact of histology and clinical benefit of chemotherapy on outcomes.