SCLC Transformation Research Articles

P2.14-62 Early, Subclinical SCLC Transformation in Patients with EGFR Mutant Lung Cancer Receiving Osimertinib, Detected Through Cell-Free DNA

P2.14-62 Early, Subclinical SCLC Transformation in Patients with EGFR Mutant Lung Cancer Receiving Osimertinib, Detected Through Cell-Free DNA

Concurrent RB1 and TP53 Alterations Define a Subset of EGFR-Mutant Lung Cancers at risk for Histologic Transformation and Inferior Clinical Outcomes

IntroductionEGFR-mutant lung cancers are clinically and genomically heterogeneous with concurrent RB transcriptional corepressor 1 (RB1)/tumor protein p53 (TP53) alterations identifying a subset at increased risk for small cell transformation. The genomic alterations that induce lineage plasticity are unknown. MethodsPatients with EGFR/RB1/TP53-mutant lung cancers, identified by next-generation sequencing from 2014 to 2018, were compared to patients with untreated, metastatic EGFR-mutant lung cancers without both RB1 and TP53 alterations. Time to EGFR–tyrosine kinase inhibitor discontinuation, overall survival, SCLC transformation rate, and genomic alterations were evaluated. ResultsPatients with EGFR/RB1/TP53-mutant lung cancers represented 5% (43 of 863) of EGFR-mutant lung cancers but were uniquely at risk for transformation (7 of 39, 18%), with no transformations in EGFR-mutant lung cancers without baseline TP53 and RB1 alterations. Irrespective of transformation, patients with EGFR/TP53/RB1-mutant lung cancers had a shorter time to discontinuation than EGFR/TP53- and EGFR-mutant –only cancers (9.5 versus 12.3 versus 36.6 months, respectively, p = 2 × 10-9). The triple-mutant population had a higher incidence of whole-genome doubling compared to NSCLC and SCLC at large (80% versus 34%, p < 5 × 10-9 versus 51%, p < 0.002, respectively) and further enrichment in triple-mutant cancers with eventual small cell histology (seven of seven pre-transformed plus four of four baseline SCLC versus 23 of 32 never transformed, respectively, p = 0.05). Activation-induced cytidine deaminase/apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like mutation signature was also enriched in triple-mutant lung cancers that transformed (false discovery rate = 0.03). ConclusionsEGFR/TP53/RB1-mutant lung cancers are at unique risk of histologic transformation, with 25% presenting with de novo SCLC or eventual small cell transformation. Triple-mutant lung cancers are enriched in whole-genome doubling and Activation-induced cytidine deaminase/apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like hypermutation which may represent early genomic determinants of lineage plasticity.

Diverse RB1 mutations and co-occurring molecular alterations in advanced NSCLC.

e20672 Background: Retinoblastoma transcriptional corepressor 1 (RB1) mutation is strikingly more frequent in non-small cell lung cancer (NSCLC) with SCLC transformation (SCLC-T) than that remain NSCLC. However, NSCLC patients harboring RB1 mutations do not necessarily transform to SCLC. Thus, exploring RB1 mutation frequency and co-occurring genomic alterations is potentially important to understand the SCLC-T mechanism. Methods: We investigated 47 NSCLC pts with RB1 aberrations at our institution. Submitted samples (tissue, plasma, cerebrospinal fluid and pleural effusion) were analyzed using next-generation sequencing across at least 139 genes (139-520). Demographics, molecular features and outcomes were analyzed. Results: Among 47 pts (27F/20M), median age was 58. Twenty (47%) had brain metastases and 7 (15%) underwent SCLC-T. A total of 39 types of RB1 mutations were identified, in which copy number deletion is the most common (40%). RB1 mutations were present from early NSCLC and were detected in 88%(29/33), 82%(9/11), 70%(9/13) and 42% (20/48) of tissue, pleural effusion, CSF and plasma samples, respectively. The most frequent co-occurring alterations were TP53 (100%, 95%) and EGFR (100%, 93%) in both SCLC-T group and non-transformed group. Besides, mutations involving in the PI3K pathway were often detected: PIK3CA (57%, 20%), AKT (43%, 10%), PTEN (14%, 15%), MET (14%, 15%), NTRK1 (14%, 13%). Notably, RB1 also co-occurred with ALK fusion (1/47). Moreover, SCLC-T group harbored significant higher frequency of RB1 mutation compared with non-transformed group. Conclusions: RB1 mutations in NSCLC are associated with a trend toward SCLC-T, and the mechanisms may be increased frequency of RB1 mutations and co-occurring similar molecular alterations to those in SCLC.

Clinical outcomes of EGFR+ NSCLC pts treated with immune checkpoint inhibitors (ICI).

9069 Background: ICIs have limited efficacy in EGFR+ NSCLC with ORR ~10% if PDL1 &gt; 25% in ATLANTIC, yet ICIs are often used in later lines of therapy as pts and providers feel there may be little risk. The impacts of ICI in this setting are poorly understood. We describe our institutional experience of ICI use in EGFR+ NSCLC. Methods: MGH pts with advanced EGFR+ NSCLC treated with ICI (any line) were retrospectively reviewed for demographics, PDL1, treatment duration and patient outcomes. Disease flare was defined as hospital/hospice admission due to progression or death (Chaft CCR 2011) within 30d of ICI. Results: 40 pts with EGFR+ NSCLC (22 del19, 11 L858R, 5 ins20, 2 other) received ICI between 7/2012-12/2018. 13 were on a clinical trial. 4 had SCLC transformation. Median # of prior therapies = 3 (range, 0-8). Of 16 with PDL1 quantified, 8 had PDL1 &gt; 25%. ICI regimens were: nivolumab (nivo; n = 16), pembrolizumab (pembro; 9), atezolizumab (atezo; 3), ipilimumab/nivo (7), carboplatin/pemetrexed (pem)/pembro (3), pem/pembro (1), paclitaxel/atezo (1). 18 pts stopped TKI ≤21d prior to ICI start. Median duration of treatment (DOT) was 25 days (range, 1-1482). DOT was &gt; 1 yr for 2 pts (5%), treated with 1st-line nivo/erlotinib (erlo) and 3rd-line nivo. All 8 pts with PDL1 &gt; 25% had DOT &lt; 2 mos. Disease flare within 30d of ICI occurred in 16/40 (40%) overall, 8/18 (44%) who stopped TKI ≤21d of ICI start, and 14/26 (54%) who received ICI in 4thline or later. 8 pts had concurrent TKIs (4 erlo/nivo, 2 erlo/pembro, 1 erlo/atezo, 1 osimertinib (osi)/nivo); 3/8 discontinued ICI for toxicity (all 3 treated first-line). 5 pts received osi immediately post-ICI. There was no pneumonitis on osi post-ICI; 1 pt developed gr3 LFTs and gr4 hypoNa. Conclusions: In this real world cohort of EGFR+ NSCLC, clinical benefit from ICI (assessed by DOT) was rare, including pts with high PDL1. 5% had durable benefit (both pts received ICI in earlier lines of therapy). A previously underappreciated negative outcome of ICI is that admission to hospital, hospice or death within 30d of ICI occur in up to 54% pts. This may be related to disease flare or hyperprogression and suggests that use of ICI in heavily pretreated EGFR+ NSCLC may negatively impact outcomes at end-of-life and should be used with caution.

From the beginning to resistance: Study of plasma monitoring and resistance mechanisms in a cohort of patients treated with osimertinib for advanced T790M-positive NSCLC

IntroductionAnalysis of circulating tumor DNA (ctDNA) for the identification of T790M mutation in advanced EGFR-mutated NSCLC patients can replace tissue re-biopsy for resistance characterization and, being non-invasive, may be applied for disease monitoring. We analysed ctDNA during osimertinib treatment to correlate mutational levels with clinical outcome and to predict pattern of resistance. Materials and MethodsForty patients with advanced NSCLC receiving osimertinib for T790M + disease after previous EGFR-TKI were enrolled in a pilot study to collect plasma at baseline and every 12 weeks until progression. Molecular analysis of ctDNA was performed by ddPCR and Therascreen®. When feasible at progression, tissue re-biopsy and NGS analysis were performed. ResultsThirty-eight patients had baseline plasma samples suitable for molecular analysis. Patients with low levels of the EGFR activating mutation in ctDNA [< 2200 copies/mL or allele frequency (AF) < 6.1%] showed better progression-free survival (17.8 or 17.8 months vs. 4.3 or 2.7, p = 0.022 or p = 0.018, respectively) and overall survival (23.6 or 23.6 vs. 7.7 or 7.3, p = 0.016 or p = 0.013, respectively) than patients with high levels (≥ 2200 copies/mL or AF ≥ 6.1%). Patients with detectable EGFR mutations in plasma (shedders) presented worse outcome than negative subjects (non-shedders). Low levels of T790M, higher T790M/activating mutation ratio and complete clearance after 2 months were associated with a trend towards better outcome. Tissue re-biopsy at resistance showed 3 patients with EGFR C797S, 1 with MET amplification, 1 with MYC amplification, 1 with PTEN loss, 3 with SCLC transformation. ConclusionsThe mutational analysis performed on plasma plays a significant role in prognostic stratification, especially for the EGFR activating mutation, since patients with absence or low levels of mutations presented a better outcome to osimertinib. At progression, tissue re-biopsy remains a crucial issue for the identification of resistance mechanisms.

EGFR-Mutant SCLC Exhibits Heterogeneous Phenotypes and Resistance to Common Antineoplastic Drugs

IntroductionApproximately 5% of patients with EGFR-activating mutations acquire EGFR tyrosine kinase inhibitor (TKI) resistance through SCLC transformation. However, the reason for the poor outcome and the molecular basis of EGFR-mutant SCLC that has transformed from adenocarcinoma remain unclear. MethodsIn this study, we established two EGFR-mutant SCLC cell lines from lung adenocarcinoma patients after failed EGFR-TKI treatment to investigate their molecular basis and potential therapeutic strategies in the hope of improving patient outcome. ResultsThese two EGFR-mutant SCLC cell lines displayed two different phenotypes: suspensive and adherent. Both phenotypes shared the same genomic alterations analyzed by array-based comparative genomic hybridization assay. Increased expression of EGFR and mesenchymal markers and decreased expression of neuroendocrine markers were observed in adherent cells. Principal component analysis and hierarchical clustering analysis of RNA microarray revealed that these two cell lines displayed a unique gene expression pattern that was distinctly different from that in NSCLC and classical SCLC cells. Combined treatment using an EGFR-TKI and an AKT inhibitor attenuated cell viabilities in our two cell lines. Moreover, the use of a histone deacetylase inhibitor significantly inhibited the cell viabilities of both cell lines in vitro and in vivo. ConclusionOur findings suggest that EGFR-mutant SCLC may be a distinct subclass of SCLC that exhibits epithelial-mesenchymal transition phenotypes, and adding an AKT or histone deacetylase inhibitor to pre-existing therapies may be one of the therapeutic choices for transformed EGFR-mutant SCLC.

A Brief Report of Transformation From NSCLC to SCLC: Molecular and Therapeutic Characteristics

IntroductionHistologic transformation from NSCLC to SCLC is a mechanism of resistance in EGFR-mutant tumors but is also occasionally observed in nonmutated NSCLC. MethodsWe performed a multicenter retrospective collection of cases presenting between 2005 and 2017. The objectives were to analyze survival data and to define epidemiologic, clinical, treatment and histomolecular characteristics at both the time of diagnosis of NSCLC and of SCLC. ResultsForty-eight EGFR-mutant NSCLC and 13 non–EGFR-mutant cases were registered. Most EGFR-mutant tumors retained the same EGFR mutation after transformation. The median time to SCLC transformation was shorter in the EGFR-mutant group than in non-EFGR mutants (16 months versus 26 months (p = 0.01)). Both tumors were responsive to platinum etoposide regimens (45% partial response for the EGFR-mutant group versus 40% for non-EFGR mutants). The median overall survival rates were 28 months in the EGFR-mutant group versus 37 months in the non–EFGR-mutant group, respectively. After transformation, the median overall survival was 9 months in the non–EGFR-mutant group versus 10 months in the EGFR-mutant group. ConclusionsTransformation into SCLC seems to occur more quickly in EGFR mutated tumors; however, once the tumor is transformed its survival and response to treatment seems comparable to that of classical SCLC.

Abstract 2495: RUNX1T1 is amplified in combined small cell lung cancer

Abstract Small cell lung cancer (SCLC) is a highly aggressive malignancy that expresses neuroendocrine (NE) genes and represents approximately 15% of all lung cancers; the remaining histological subtype is non-small cell lung cancer (NSCLC). Clinically, there are two types of SCLC, termed ‘pure' and ‘combined'. Combined SCLC presents as a mixture of SCLC and NSCLC components within the same tumor mass. We analyzed 88 SCLC patient tumors by targeted exome sequencing and found that four SCLC patients demonstrated RUNX1T1 amplification. Interestingly, among these 88 SCLC patients there were two with combined SCLC and both demonstrated RUNX1T1 amplification only in the small cell component. Neither MYC (8p24) nor FGFR1 (8p11), two genes commonly amplified in SCLC that are nearby the RUNX1T1 locus (8p22), were co-amplified. RUNX1T1, also called ETO or MTG8, was first identified in the oncogenic fusion transcript AML1/ETO in acute myelogenous leukemia (AML) and was previously reported as a transcriptional co-repressor. Based on our observation we hypothesized that: 1) RUNX1T1 may be a specific marker for SCLC and may play a role in determining the SCLC phenotype, including NE expression, and 2) RUNX1T1 may play a role in transforming NSCLC to SCLC in the early stages of combined SCLC tumor formation. First we examined RUNX1T1 mRNA expression among cancer cell lines using the CCLE database and found that RUNX1T1 is highly expressed in SCLC cells compared to other cancer cells, especially NSCLC cells. This was also true in tumors using RNAseq data. Consistent with these mRNA profiles, our western blot results showed higher RUNX1T1 protein expression in SCLC compared to NSCLC cell lines. These findings validate that RUNX1T1 expression is specifically upregulated in SCLC compared to NSCLC. To investigate a role for RUNX1T1 in determining the SCLC phenotype, we overexpressed RUNX1T1 in SCLC cell lines by lentiviral transduction and observed marked growth suppression in H82, H2171, and H841 cells. Conversely, transient knock down of RUNX1T1 expression in the SCLC cell line H446 using siRNA decreased the expression of two NE proteins (NEUROD1 and TTF1). To explore a potential role of RUNX1T1 in NSCLC to SCLC transformation, we overexpressed RUNX1T1 in three NSCLC cell lines (PC-9, H1650 and A549) but observed no obvious phenotypic changes and no increase in NE gene expression. In conclusion, we have demonstrated that RUNX1T1 expression is upregulated in SCLC compared to NSCLC. Moreover, the results from RUNX1T1 overexpression and knockdown experiments in SCLC cell lines indicates a potential role of RUNX1T1 in regulating the SCLC phenotype and NE expression. Citation Format: Tian He, Karen McColl, Alyssa Savadelis, Yanwen Chen, Gary Wildey, Afshin Dowlati. RUNX1T1 is amplified in combined small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2495.

Primary resistance to osimertinib due to SCLC transformation: Issue of T790M determination on liquid re-biopsy

ObjectivesEGFR T790M mutation is the most common mechanism of resistance to first-/second-generation EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) and could be overcome by third-generation EGFR-TKIs, such as osimertinib. Liquid biopsy, a non-invasive technique used to test the presence of the resistant mutation, may help avoiding tissue re-biopsy. However, analysing only circulating-free DNA, information about other less frequent and coexisting resistance mechanisms may remain unrevealed. Materials and methodsAll patients reported in this series participated in the ASTRIS trial, a real world treatment study testing the efficacy of osimertinib (80mg os die) in advanced T790M-positive NSCLC progressed to prior EGFR-TKI. Patients were considered eligible to osimertinib if T790M positive on tissue or plasma samples. In our patients, EGFR molecular testing on blood sample was conducted with digital droplet PCR (ddPCR). ResultsWe report our experience of five patients treated with osimertinib after T790M detection on liquid biopsy that presented a disease progression at first tumor assessment mediated by SCLC transformation, as evidenced at tissue re-biopsies. All patients showed low ratio T790M/activating mutation in the blood before osimertinib (lower than 0.03). For three patients, EGFR mutational analysis was T790M-negative when re-assessed by using a less sensitive method (therascreen®) on the same liquid biopsy sample analysed by ddPCR before osimertinib therapy. ConclusionAlthough liquid biopsy is a relevant tool to diagnose T790M presence in NSCLC patients resistant to EGFR-TKI, in case of a low ratio T790M/activating mutation, tissue biopsy should be considered to exclude the presence of SCLC transformation and/or other concomitant resistance mechanisms.

Acquired Resistance to Erlotinib in EGFR Mutation-Positive Lung Adenocarcinoma among Hispanics (CLICaP).

Lung cancer harboring epidermal growth factor receptor (EGFR) mutations and treated with EGFR tyrosine kinase inhibitors (TKIs) all eventually develop acquired resistance to the treatment, with half of the patients developing EGFR T790M resistance mutations. The purpose of this study was to assess histological and clinical characteristics and survival outcomes in Hispanic EGFR mutated lung cancer patients after disease progression. EGFR mutation-positive lung cancer patients (n=34) with acquired resistance to the EGFR-TKI erlotinib were identified from 2011 to 2015. Post-progression tumor specimens were collected for molecular analysis. Post-progression interventions, response to treatment, and survival were assessed and compared among all patients and those with and without T790M mutations. Mean age was 59.4±13.9years, 65% were never-smokers, and 53% had a performance status 0-1. All patients received erlotinib as first-line treatment. Identified mutations included: 60% DelE19 (Del746-750) and 40% L858R. First-line erlotinib overall response rate (ORR) was 61.8% and progression free survival (PFS) was 16.8months (95% CI: 13.7-19.9). Acquired resistance mutations identified were T790M mutation (47.1%); PI3K mutations (14.7%); EGFR amplification (14.7%); KRAS mutation (5.9%); MET amplification (8.8%); HER2 alterations (5.9%, deletions/insertions in e20); and SCLC transformation (2.9%). Of patients, 79.4% received treatment after progression. ORR for post-erlotinib treatment was 47.1% (CR 2/PR 14) and median PFS was 8.3months (95% CI: 2.2-36.6). Median overall survival (OS) from treatment initiation was 32.9months (95% CI: 30.4-35.3), and only the use of post-progression therapy affected OS in a multivariate analysis (p=0.05). Hispanic patients with acquired resistance to erlotinib continued to be sensitive to other treatments after progression. The proportion of T790M+ patients appears to be similar to that previously reported in Caucasians.

Resistance to epithelial growth factor receptor tyrosine kinase inhibitors in a patient with transformation from lung adenocarcinoma to small cell lung cancer: A case report.

First-generation epithelial growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have markedly improved the treatment of non-small cell lung cancer (non-SCLC) with EGFR-sensitive mutations. However, acquired resistance to these drugs was inevitable. The transformation of lung adenocarcinoma to SCLC following treatment with EGFR-TKIs is a rare phenomenon that contributes to resistance to EGFR-TKIs. The present case concerns a 74-year-old man previously diagnosed with and treated for pneumonia; however, this was later pathologically confirmed as lung adenocarcinoma by transbronchial lung biopsy. Deletion of exon 19 of EGFR was identified by next-generation sequencing technology. The patient improved markedly when treated with gefitinib, but relapsed after 1 year, with markedly increased serum levels of neuron-specific enolase (NSE). Transformation to SCLC was detected by endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) re-biopsy, which was negative for the deletion of exon 19 of EGFR. The patient was positive for vimentin expression and refractory to etoposide and cisplatin chemotherapy, and succumbed to the disease 18 months after diagnosis. Transformation of the disease from adenocarcinoma to SCLC may have been due to cancer heterogeneity. Re-biopsy is therefore important in EGFR-TKI-resistant patients for genetic and histological re-evaluation. NSE serum levels may also be useful for detecting early SCLC transformation.

MET amplification (amp) as a resistance mechanism to osimertinib.

9020 Background: Osimertinib (osi) is an EGFR T790M inhibitor. Mechanisms (mech) of acquired resistance (AR) are under study. We report a cohort of osi-AR pts with extensive pre/post-osi tissue and plasma. Methods: We analyzed 23 pts with AR to osi. Tumor biopsies (bx) underwent NGS (SNaPshot, MGH) and FISH for EGFR, MET amp (target:CEP7 &gt; 2.2.) Plasma underwent ctDNA NGS (Guardant360). Results: Of the 23 osi-treated EGFR-mutants (13 with EGFR del19, 10 L858R), 2 had de novo T790M, 21 acquired T790M. 13 had prior 3rd gen EGFR TKIs before osi - rociletinib(11), ASP8273(1), EGF816(1). Median time on osi was 12 mo (range 2-25), med total time on 3rd-gen EGFR TKIs was 18 mo. Bx types were tissue (16), plasma (18), and both (11, med interval 31d). All pts retained their founder EGFR mutation, but 15/23 (65%) “lost” T790M post-osi, suggesting AR arose from a T790wt subclone (Table). Common AR mech were MET amp (7/23; 30%) and EGFR C797S (5/23; 22%). 1 pt each had SCLC transformation, PIK3CA E545K/PIK3CA amp and FGFR1 D60N/FGFR1 amp. 2 pts had 2 distinct post-osi tissue bx showing heterogeneity. Pt 8 had plasma and a lung nodule with C797S/T790M while a mediastinal LN was wt at both loci. Pt 22 had MET-amp in pleural fluid but no MET amp on lung bx. Plasma identified AR mech not seen in tissue in 2/11 pts with both bx types. Among 7 pts with MET amp, 3 received combo EGFR/ MET TKI; all 3 had a RECIST PR. Conclusions: In this osi-resistant cohort, we commonly saw MET amp (30%) and C797S (22%). MET amp seems more common post-osi than after 1st-line TKIs, and 3/3 pts responded to EGFR+MET TKIs. AR heterogeneity was seen in 2 pts with matched tissue/plasma and 2 with &gt;1 tissue bx, underscoring the complementary roles of plasma and tissue. [Table: see text]

Heterogeneity in Immune Marker Expression after Acquisition of Resistance to EGFR Kinase Inhibitors: Analysis of a Case with Small Cell Lung Cancer Transformation

IntroductionExpression of immune markers is of scientific interest because of their potential roles as predictive biomarkers for immunotherapy. Although the microenvironment of metastatic tumors and/or therapy-inducible histological transformation may affect the expression of these immune markers, there are few data regarding this context. MethodsA 76-year-old never-smoking female with EGFR-mutated lung adenocarcinoma (AC) acquired resistance to gefitinib. After her death, an autopsy revealed SCLC transformation and EGFR T790M secondary mutation (T790M) as mutually exclusive resistance mechanisms occurring differently in different metastases; two liver metastases (SCLC versus AC with T790M) and two lymph node metastases (SCLC versus AC with T790M) were analyzed to compare the expression status of immune markers by immunohistochemistry and by an immune oncology gene expression panel. ResultsProgrammed death ligand 1 (PD-L1) protein was partially expressed in tumor cells with AC lesions (T790M) but not in tumor cells with SCLC transformation. The liver metastasis with SCLC transformation showed no stromal PD-L1 expression and scant tumor-infiltrating lymphocytes, whereas the other lesions demonstrated stromal PD-L1 staining and infiltration of CD8-positive T cells. Data generated using an immuno-oncology gene expression panel indicated a higher level of T-cell costimulatory molecules and lower expression of type I interferon–regulated genes in lesions with SCLC transformation. ConclusionThese data highlight the heterogeneity of expression of immune markers depending on the metastatic sites and histological transformation and indicate that the biopsy specimen from one lesion may not be representative of immune marker status for all lesions.

MTE21.01 Next Generation Sequencing

Non-small cell lung cancer (NSCLC) with sensitive epidermal growth factor receptor (EGFR) mutations invariably develop resistance to EGFR tyrosine kinase inhibitors (TKIs). 20%-30% of NSCLC patients harboring sensitive mutations have no good initial clinical response to EGFR-TKIs, which is defined as having intrinsic resistance to EGFR-TKIs; while the rest of patients with activating mutations who are initially responsive to EGFR-TKIs eventually develop acquired resistance after 10–12 months of consistent clinical benefit, followed by disease progression. The drug resistance is a really tough and urgent clinical problem. Part of resistant mechanisms have been reported, including BIM deletion polymorphism, combined with other bypass signal pathway activation, epithelial-mesenchymal transition (EMT) for primary resistance; T790M, cMET amplification, SCLC transformation for acquired resistance. However, partial resistant mechanisms still unknown. In contrast to acquired resistance to EGFR-TKIs, intrinsic resistance is more complicated. Next-generation sequencing (NGS) is a promising tool for analysis of tumor mutations. We aimed to investigate the intrinsic resistant mechanisms to EGFR-TKIs by NGS, further to optimize treatment strategies and improve clinical outcome in EGFR activating mutant patients having intrinsic resistance to EGFR-TKIs. At present, the study is underway, and the results will be presented at the 2016 WCLC. next-generation sequencing, NSCLC, EGFR-TKIs, drug resistance

Variation in Mechanisms of Acquired Resistance Among EGFR-Mutant NSCLC Patients With More Than 1 Postresistant Biopsy: Metastatic Non-Small Cell Lung Cancer

Acquired resistance (AR) typically develops after 9-12 mo of EGFR TKI therapy among EGFR-mutant patients (pts). Repeat biopsies after the development of AR have become a standard of care, and are increasingly obtained more than once during postresistance treatment. Here we report a unique series of 57 EGFR mutants who had >1 postresistant biopsy that illustrates the heterogeneity of AR within individual pts. We analyzed prospective data from 136 EGFR mutants with advanced NSCLC who had ≥1 biopsy after AR developed on EGFR TKI therapy. All biopsies underwent multiplexed CLIA-certified SNaPshot genotyping. When possible, preresistant biopsies were also studied. Some pts consented to provide tissue for patient-derived cell-line (PDCL) establishment. Fifty-seven pts had >1 post-AR biopsy. We collected data on EGFR TKI therapy, mechanisms of AR observed over multiple biopsies, biopsy safety, and correlations between clinical and PDCL data. Among 57 pts with multiple post-AR biopsies, median age= 58 (range 39-88), 72% were female, 58% had EGFR exon 19 deletion and 37% had L858R. The median number of post-AR biopsies was 2 (range 2-5). While the original EGFR mutation was uniformly maintained, we observed variation in AR mechanisms among 25/57 pts, with changes in T790M status over time in 14 (25%) pts. Both gain and loss of T790M were observed on serial biopsies, without a clear correlation to the timing of EGFR TKI therapy or biopsy site. Changes in histology (8), development of EMT (1) and EGFR amp (1) were also seen. Thirty-two of 57 pts had no variations in detected mechanisms of AR on serial biopsies, including T790M (n=19), MET amp (2), both MET and EGFR amp (1), SCLC transformation (1), BRAF mutation (1), and no AR mechanism identified (8). Repeat biopsies were safe, with 2.2% biopsy-related complications and no serious complications. We observed frequent and unexpected changes in the mechanism of AR, particularly T790M status, in pts undergoing serial biopsies, suggesting heterogeneity of resistant clones in these pts. Among pts with T790M observed at any time in their disease, 40% gained or lost the mutation over time. Relationship of AR mechanisms to therapy at the time of biopsy, biopsy location, and correlations with PDCL data to understand their biologic implications will be presented. Our data suggest serial biopsies in EGFR pts are informative and safe, and may become increasingly important as T790M-specific TKIs become available.

Abstract 955: Transformation from NSCLC to SCLC in EGFR mutant lung cancers with acquired resistance to EGFR inhibitors

Abstract Epidermal growth factor receptor (EGFR) mutant non-small cell lung cancers (NSCLCs) are sensitive to the EGFR inhibitors erlotinib and gefitinib. In the majority of patients with this type of cancer, EFGR inhibition leads to a dramatic reduction in tumor size coupled with an abatement of symptoms. These responses are temporary, and in most cases the cancers become resistant after an average of one year. Recently, we and others have found that a subset of these cancers transform from NSCLC to SCLC as they acquire resistance to EGFR TKI. Details underlying this novel resistance mechanism are largely undiscovered. To assess the genetic changes associated with NSCLC to SCLC transformation, we carried out next generation sequencing analysis on NSCLC and SCLC resistant tumors. The sequencing results revealed that Rb, along with p53 and PIK3CA were specifically altered in the SCLC transformed tumors. An IHC assay to detect Rb indicated that while 10 out of 10 SCLC transformed cases have loss of Rb, resistant cancers that maintained an NSCLC histology largely retained Rb. To address the mechanism underlying the resistance to EGFR inhibition in these tumors, we demonstrated that upon the transformation to SCLC there is a marked reduction in EGFR expression, suggesting that in transforming to SCLC these cells become a cell type that no longer requires EGFR for their survival. Finally, utilizing cell lines generated from biopsies taken from resistant cancers, we identify the Bcl-2/Bcl-XL inhibitor Abt-263 as a potential therapeutic approach to treat these resistant patients. Together, our results provide details underlying this novel mechanism of resistance to targeted therapy. Citation Format: Matthew J. Niederst, Lecia V. Sequist, Elizabeth L. Lockerman, Angel R. Garcia, Carlotta Costa, Farhiya Mohamoud, Darrell R. Borger, Toshi Shioda, Gad Getz, Anthony J. Iafrate, Mari Mino-Kenudson, Jeffrey A. Engelman. Transformation from NSCLC to SCLC in EGFR mutant lung cancers with acquired resistance to EGFR inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 955. doi:10.1158/1538-7445.AM2014-955

Abstract A87: Overcoming NSCLC to SCLC transformation as a resistance mechanism in EGFR mutant lung cancer.

Abstract The EGFR TKIs gefitinib and erlotinib produce dramatic responses in NSCLC patients with EGFR activating mutations, however, the efficacy of these drugs is severely limited by the development of acquired resistance. Recently, we and others have found that a subset of these cancers transform from NSCLC to SCLC as they acquire resistance to EGFR TKI. Here we report an initial characterization of the molecular mechanisms underlying the transformation from NSCLC to SCLC in samples and cell lines derived from resistant patients. Much like de novo SCLC, Rb and p53 are altered in the resistant tumors that transformed to SCLC, although loss of p53 and Rb function alone were insufficient to drive the transformation in NSCLC cell lines. In addition, we demonstrate that EGFR expression is lost in resistant samples, suggesting that in switching to SCLC these cancers become a cell type that is no longer dependent on EGFR activity and providing an explanation for lack of EGFR TKI sensitivity. Finally, we find that SCLC transformed cells are uniquely sensitive to the Bcl-2/Bcl-XL inhibitor Abt-263, offering a potential therapeutic option for these patients. Together, these results represent the first insights into the molecular and genetic details regarding this novel type of resistance to targeted therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A87. Citation Format: Matthew J. Niederst, Lecia V. Sequist, Elizabeth L. Lockerman, Carlotta Costa, Darrell R. Borger, Mari Mino-Kenudson, Anthony J. Iafrate, Jeffrey A. Engelman. Overcoming NSCLC to SCLC transformation as a resistance mechanism in EGFR mutant lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A87.

Occurrence of HER2 amplification in EGFR-mutant lung adenocarcinoma with acquired resistence to EGFR-TKIs.

8047 Background: Patients with EGFR-mutant lung adenocarcinoma develop progression of disease on TKIs therapy after a median of 12 months;this acquired resistance is mainly due to a secondary mutation in EGFR (T790 M) in about 50% of patients, amplification of MET in 15%, PIK3CA mutations in 5%, an unknown mechanism in almost 30% and a SCLC transformation in some pts. Recently, Takezawa and colleagues pointed out that HER2 amplification is a mechanism of acquired resistance to EGFR inhibition in EGFR-mutant lung cancers without EGFR T790M mutation. To aid in identification and treatment of these patients we examined a cohort of patients whose cancers were assessed with tumor biopsies at multiple times before and after their treatment with TKIs. Methods: 41 lung adenocarcinomas pts. (20 male, 21 female, median age 55 years) with EGFR mutations at 19 or 21 exons received TKIs as first line of treatment. 31 pts. (75%) showed a clinical response and relapsed after a mTTP of 12 months. At the time of relapse a new biopsy was performed, histologic samples were reviewed to re-confirm the diagnosis, EGFR, MET and HER-2 amplification were identified by FISH, while EGFR mutations have been tested by DNA sequencing. Results: At the time that drug resistence was acquired all 31 pts. retained their original activating EGFR mutations, 16 pts. developed EGFR T790M resistance mutation with pronunced EGFR amplification in 5, 4 pts. developed MET amplification, 3 pts. were found to have a diagnosis of small cell lung cancer. HER2 amplification was observed in four pts. (13%), with dramatic progression and a median OS of 5 months after treatment with CDDP + pemetrexed. Notably all 4 cases were EGFR T790M negative. Conclusions: Among pts. with acquired resistence to EGFR TKIs the presence of HER2 amplification defines a clinical subset with a more adverse prognosis and rapid progression. Interestingly, recent data suggest that afatinib combined with cetuximab could have promising activity in pts. with acquired resistance due to HER2 amplification.

A static diagnostic biopsy in patients with mutated EGFR lung adenocarcinom to guide therapeutic decisions.

e18039 Background: Approximately 70% of the pts. whose lung cancers harbor EGFR mutations acquire drug resistence after a response to EGFR tyrosine kinase inhibitors (TKIs) treatment; this acquired resistance is mainly due to a secondary mutation in EGFR (T790 M) in about 50% of patients, amplification of MET in 15%, PIK3CA mutations in 5%, an unknown mechanism in almost 30%, and SCLC transformation in some patients. Furthermore, clinical experience revealed that cancers with acquired resistance can respond again to TKIs, after a drug-free interval. To aid in identification and treatment of these patients we examined a cohort of patients whose cancers were assessed with tumor biopsies at multiple times before and after their treatment with TKIs. Methods: 21 lung adenocarcinomas pts. (10 male, 11 female, median age 53 years) with EGFR mutations at 19 or 21 exons received TKIs, as first line of treatment. All showed a clinical response and all relapsed ( mTTP 10 months). At the time of relapse a new biopsy was performed, histologic samples were reviewed to re-confirm the diagnosis, EGFR and MET amplification were identified by FISH, while EGFR mutations have been tested by DNA sequencing. Results: At the time that drug resistence was acquired all pts. retained their original activating EGFR mutations, 9 pts. developed EGFR T790M resistance mutation with pronunced EGFR amplification in 3, 2 pts. developed MET amplification, 8 biopsies did not reveal any new mutations, two pts. were found to have a diagnosis of small cell lung cancer in their drug resistant tumor biopsies and responded well to conventional chemotherapy regimen.15 of 19 confirmed lung adenocarcinoma patient underwent to a cisplatin - pemetrexed chemotherapy regimen and at the time of progression 10 of them accepted to undergo a new biopsy. Three pts. ( after 4, 5 and 6 months break from treatment with TKIs ) lost T790M mutation and their disease responded to a second-line course of erlotinib. Conclusions: In our cohort of pts. with acquired EGFR resistence some patients lost acquired T790M mutation and become sensitive to EGFR inhibitor, in addition, two pts. underwent the histologic transformation from NSCLC to SCLC at the time of TKI resistence.