Drivers In NSCLC Research Articles

Anti-HDGF antibody targets EGFR tyrosine kinase inhibitor-tolerant cells in NSCLC patient-derived xenografts.

Constitutively active mutant epidermal growth factor receptor (EGFR) is one of the major oncogenic drivers in NSCLC. Targeted therapy using EGFR tyrosine kinase inhibitor (TKI) is a firstline option in patients that have metastatic or recurring disease. However, despite the high response rate to TKI, most patients have a partial response, and the disease eventually progresses in 10 to 19 months. It is believed that drug-tolerant cells that survive TKI exposure during the progression-free period facilitate the emergence of acquired resistance. Thus, targeting the drug-tolerant cells could improve the treatment of NSCLC with EGFR mutations. We demonstrated here that EGFR mutant patient-derived xenograft (PDX) tumors responded partially to osimertinib despite near complete inhibition of EGFR activation. Signaling in AKT/mTOR and MAPK pathways could be reactivated shortly after initial inhibition. As a result, many tumor cells escaped drug killing and regained growth following about 35 days of continuous osimertinib dosing. However, when an antibody to hepatoma-derived growth factor (HDGF) was given concurrently with osimertinib, tumors showed complete or near-complete responses.There was significant prolongation of progression-free survival (PFS) of tumor-bearing mice as well. Immunohistochemistry and western blot analysis of tumors collected in the early stages of treatment suggest that increased suppression of the AKT/mTOR and MAPK pathways could be a mechanism that results in enhanced efficacy of osimertinib when it is combined with anti-HDGF antibody.

TWIST1 is a critical downstream target of the HGF/MET pathway and is required for MET driven acquired resistance in oncogene driven lung cancer.

MET amplification/mutations are important targetable oncogenic drivers in NSCLC, however, acquired resistance is inevitable and the majority of patients with targetable MET alterations fail to respond to MET tyrosine kinase inhibitors (TKIs). Furthermore, MET amplification is among the most common mediators of TKI resistance. As such, novel therapies to target MET pathway and overcome MET TKI resistance are clearly needed. Here we show that the epithelial-mesenchymal transition (EMT) transcription factor, TWIST1 is a key downstream mediator of HGF/MET induced resistance through suppression of p27 and targeting TWIST1 can overcome resistance. We found that TWIST1 is overexpressed at the time of TKI resistance in multiple MET-dependent TKI acquired resistance PDX models. We have shown for the first time that MET directly stabilized the TWIST protein leading to TKI resistance and that TWIST1 was required for MET-driven lung tumorigenesis as well as could induce MET TKI resistance when overexpressed. TWIST1 mediated MET TKI resistance through suppression of p27 expression and genetic or pharmacologic inhibition of TWIST1 overcame TKI resistance in vitro and in vivo. Our findings suggest that targeting TWIST1 may be an effective therapeutic strategy to overcome resistance in MET-driven NSCLC as well as in other oncogene driven subtypes in which MET amplification is the resistance mechanism.

Inflammatory parameters in NSCLC with driver mutation.

Aim: The tumor microenvironment of NSCLC with driver mutations, such as EGFR, ALK and ROS, is less inflammatory. Materials & methods: This retrospective study included 38 patients with NSCLC driver mutations. The relationship between clinical and inflammatory markers concerning progression-free survivaland overall survivalwas analyzed based on Kaplan-Meier curves. Results: The mean age of the patients was 59.8±11.9. Progression-free survivaland overall survivalwere significantly longer in patients under 65years of age and with low neutrophil-lymphocyte ratio, low systemic immune-inflammation index and high lymphocyte count (p<0.05). Conclusion: Unlike tumor biology, peripheral inflammatory parameters, such as neutrophil-lymphocyte ratio, systemic immune-inflammation indexand lymphocyte count may be associated with survival in NSCLC patients with driver mutations.

Targeting focal adhesion kinase boosts immune response in KRAS/LKB1 co-mutated lung adenocarcinoma via remodeling the tumor microenvironment

BackgroundKRAS mutation is one of the most common oncogenic drivers in NSCLC, however, the response to immunotherapy is heterogeneous owing to the distinct co-occurring genomic alterations. KRAS/LKB1 co-mutated lung adenocarcinoma displays poor response to PD-1 blockade whereas the mechanism remains undetermined.MethodsWe explored the specific characteristics of tumor microenvironment (TME) in KL tumors using syngeneic KRASG12DLKB1−/− (KL) and KRASG12DTP53−/− (KP) lung cancer mouse models. The impact of focal adhesion kinase (FAK) inhibitor on KL lung tumors was investigated in vitro and in vivo through evaluation of both KL cell lines and KL lung cancer mouse models.ResultsWe identified KL tumors as “immune-cold” tumors with excessive extracellular matrix (ECM) collagen deposition that formed a physical barrier to block the infiltration of CD8+T cells. Mechanistically, abundant activated cancer-associated fibroblasts (CAFs) resulted from FAK activation contributed to the formation of the unique TME of KL tumors. FAK inhibition with a small molecular inhibitor could remodel the TME by inhibiting CAFs activation, decreasing collagen deposition and further facilitating the infiltration of anti-tumor immune cells, including CD8+ T cells, DC cells and M1-like macrophages into tumors, hence, converting “immune-cold” KL tumors into “immune-hot” tumors. The combined FAK inhibitor and PD-1 blockade therapy synergistically retarded primary and metastatic tumor growth of KL tumors.ConclusionsOur study identified FAK as a promising intervention target for KL tumors and provided basis for the combination of FAK inhibitor with PD-1 blockade in the management of KL lung cancers.

Updated molecular analysis of MET exon 14skippingmutations (METex14) in non-small cell lung cancer (NSCLC).

9095 Background: METex14 is a heterogeneous family of mutations (mt) in NSCLC that can be effectively treated with approved targeted agents. Unlike some other drivers in NSCLC, METex14 occurs in both squamous and adenocarcinoma histology and in both smokers and non-smokers. Here, we present updated results from analysis of a large dataset to characterize the mutational landscape within METex14 NSCLC. Methods: NSCLC tissue samples were analyzed with DNA-based next-generation sequencing (NGS; 592 genes, NextSeq) or whole-exome sequencing (NovaSeq), RNA-based whole transcriptome sequencing (WTS, NovaSeq), and PD-L1 immunohistochemistry (Dako 22C3) at Caris Life Sciences (Phoenix, AZ). METex14 was detected by WTS. TMB-high was defined as ≥ 10 mt/Mb. Chi-square, Fisher’s exact or Mann-Whitney U tests were used to determine statistical significance and corrected for multiple hypothesis testing (q&lt;0.05). Immune cell estimates (quanTIseq) and pathway analysis (ssGSEA) were informed by WTS analysis. Results: A total of 711 METex14 cases were detected with 288 distinct METex14 mt. By histology, 79 (11.1%) were squamous (Sq), 478 (67.23%) were nonsquamous (nSq), and 24 (3.23%) were adenosquamous. The most common METex14 mt were D1028H (8.1%), D1028N (7.8%), c.3082+2T&gt;C (5.0%), D1028Y (4.6%), and c.3082+1G&gt;T (4.4%). Co-mutated TP53 was common (43.4%) but varied by specific METex14 mt, observed in 60.0% of MET c.3082+3A&gt;G vs 16.7% of MET G344R . Co-amplified CDK4 was found in 9.3%, with 42.9% in MET c.2924-1G&gt;A vs 6.7% in MET c.3802+1G&gt;T (p&lt;0.05). High TMB was seen in 9%; median TMB ranged from 2 mt/Mb in MET c.3082+2T&gt;A to 6.5 mt/Mb in MET c.3082+2T&gt;G (p&lt;0.05). PD-L1 ≥ 1% was seen in 80.8% compared to 56.2% in METex14-WT(p&lt;0.05), and median PD-L1 tumor proportion score (TPS) ranged from 0% in MET G344R to 75% in MET c.3082+2T&gt;A (p&lt;0.05). Co-mutations varied by histology: in Sq-NSCLC, 18.18% had TP53 mt (q&lt;0.05), 8.97% had POT1 mt (p&lt;0.05), 6.06% had TERT mt (p&lt;0.05), 5.13% CASP8 mt (q&lt;0.05), and 2.53% RNF43 mt (p&lt;0.05), while in nSq-NSCLC, 45.51% had TP53 mt, 3.38% had POT1 mt, 0.87% had TERT mt, and 0% in CASP8 and RNF43 mt. Smoking status was available for 120 cases: 88% were smokers and 12% were nonsmokers. Wnt, Hedgehog, and Notch signaling were enriched in nSq (q&lt;0.05) while upregulation of KRAS signaling, Epithelial-Mesenchymal Transition, and angiogenesis pathways were enriched in smokers with METext14 NSCLC (q&lt;0.2). Higher estimates of neutrophils and lower estimates of M2 macrophages, NK cells, and CD8+ T-cells were observed in Sq-NSCLC. PD-L1, PD-1, HAVCR-2, IDO-1 and IFN-γ expression were higher in nSq than Sq-NSCLC (q&lt;0.05). Conclusions: METex14 NSCLC is highly heterogenous, with variations in co-mutation, TMB, and PD-L1 expression. Although Sq- and nSq-NSCLC harbor METex14, the enrichment of oncogenic pathways and infiltrating immune cells differ between histology and smoking history.

Utility of ctDNA tumor fraction to inform negative liquid biopsy (LBx) results and need for tissue reflex in advanced non-small cell lung cancer (aNSCLC).

9076 Background: Negative LBx results can be challenging to interpret. Absence of a targetable alteration may accurately reflect the tumor genotype (true negative) or may represent a false negative due to insufficient ctDNA shed, concealing a targetable oncogenic driver. Reflex to tissue biopsy (TBx) profiling is thus advised, per FDA label, for negative LBx results. Here we investigate whether algorithmic quantification of ctDNA in a LBx sample can increase confidence in negative LBx results, reducing need for confirmatory TBx testing. Methods: This study used the nationwide (US-based) de-identified Flatiron Health-Foundation Medicine NSCLC clinico-genomic database (CGDB) from approximately 280 US cancer clinics (~800 sites of care), limited to patients (pts) with aNSCLC receiving any multi-gene LBx followed by TBx CGP. In parallel, we queried an institutional database of pts with both LBx (FoundationOneLiquid CDx, F1LCDx) and TBx (FoundationOneCDx, F1CDx) to calculate positive predictive agreement (PPA) and negative predictive value (NPV) of LBx compared to TBx for detection of 7 targetable NSCLC driver alterations: KRAS G12X, EGFR, MET exon 14, BRAF V600E mutations or ALK, ROS1, and RET rearrangements. Foundation Medicine’s ctDNA tumor fraction (TF) on F1LCDx is a composite algorithm prioritizing aneuploidy at higher levels to avoid germline signal and prioritizing variant allele frequency of canonical alterations at lower levels to maximize dynamic range. Results: Of 1,734 LBx sent prior to 1st-line therapy in pts with aNSCLC, 59% (1,024) were negative for an NCCN biomarker. For 465 pts with TBx after negative LBx (median 2.4 weeks from LBx to TBx result), 179 (38%) had a driver detected on reflex to TBx. In 45 receiving matched targeted therapy, median real-world PFS was 25 months. For pts starting therapy after reflex to TBx (n = 233), median time from LBx result to start of therapy was 4.7 weeks. To understand whether reflex to tissue may be avoided in some cases, LBx (F1LCDx) sensitivity was studied across 2,138 pairs in the institutional database. PPA for the 7 driver alterations was 59% overall (679/1,157) and improved to 96% (433/453) when limited to cases with TF ≥1%. Similarly, NPV was modest overall (67%) but improved when limited to LBx specimens with TF ≥1% (96%). Studying TF in the CGDB cohort, 33 of 85 pts (39%) with reflex TBx after negative LBx had a LBx TF ≥1% and, given high NPV for driver alterations, might have avoided reflex to confirmatory TBx. Conclusions: Elevated TF (≥1%) on F1LCDx informs assay sensitivity and identifies a subset of LBx results with high NPV where reflex to TBx, and the associated treatment delay, may be less valuable. Conversely, for pts with a negative LBx with low TF ( &lt; 1%) on F1LCDx, reflex to TBx is an important follow-on step to identify potentially missed driver alterations with potential for durable benefit on targeted therapy.

Clinical Utility Validation of an Automated Ultrarapid Gene Fusion Assay for NSCLC

IntroductionGene rearrangements are frequent oncologic drivers in NSCLC, and many are suitable for treatment with Food and Drug Administration–approved or experimental targeted therapies. We evaluated the accuracy, specimen acceptance profile, and limits of detection of a rapid fusion assay (Idylla GeneFusion Assay), a commercially available ultrarapid molecular assay, for its clinical utility. MethodsA collection of 97 specimens which had previously undergone next-generation sequencing testing were analyzed using the rapid fusion assay. Accuracy was evaluated by sensitivity and specificity compared with the next-generation sequencing results. The performance characteristics were tested by using a variety of different clinically relevant specimen types. Limits of detection were assessed by evaluating different input of tumor percentage and material amount. ResultsThe rapid fusion assay was found to have 100% sensitivity in detecting fusions of ALK, ROS1, RET, NTRK1, and MET exon 14 skipping and 83% sensitivity for NTRK2/3 fusions. There were 100% specificity in detecting fusions of ROS1, RET, NTRK2/3, and MET exon 14 skipping and 98% specificity for ALK. Testing was successful with formalin-fixed paraffin-embedded biopsy and surgical tissues, cell blocks from fine-needle aspiration and pleural fluid (down to 5% tumor content, 18 mm2 tissue scraped), cytology smears (≥300 cells), and previously extracted RNA (minimal 20 ng). ConclusionsThe rapid fusion assay is quick, accurate, and versatile, allowing reliable detection of ALK, ROS1, RET fusions, and MET exon 14 skipping in NSCLC, and NTRK fusions. Rapid molecular testing may expedite treatment with appropriate targeted therapies.

MA13.04 Entrectinib in Patients with ROS1 Fusion-Positive (ROS1-fp) NSCLC: Updated Efficacy and Safety Analysis

ROS1 gene rearrangements can lead to constitutively active fusion proteins that are targetable oncogenic drivers in NSCLC. Entrectinib, a potent ROS1 tyrosine kinase inhibitor (TKI), has demonstrated efficacy and CNS activity in patients with ROS1-fp NSCLC from the integrated analysis of the phase I/II studies STARTRK-1 (NCT02097810), STARTRK-2 (NCT02568267) and ALKA-372-001 (EudraCT 2012-000148-8): objective response rate (ORR) was 67% (n=108/161; data cutoff: May 1, 2019; median duration of survival follow-up: 15.8 months).

Radon and Lung Cancer: Current Trends and Future Perspectives.

Simple SummaryRadon represents the main risk factor of lung cancer in non-smokers and the second one in smoking patients. In Europe, there are several radon-prone areas, but regulatory policies may vary between countries. Radon causes DNA damage and high genomic tumor instability, but its exact carcinogenesis mechanism in lung cancer remains unknown. Molecular drivers in NSCLC are more often described in non-smoker patients and a potential association between radon exposure and oncogenic-driven NSCLC has been postulated. This is an updated review on indoor radon exposure and its role in lung cancer carcinogenesis, especially focusing on its potential relation with NSCLC with driver genomic alterations. We want to contribute to rising knowledge and awareness on this still silent but preventable lung cancer risk factor.Lung cancer is a public health problem and the first cause of cancer death worldwide. Radon is a radioactive gas that tends to accumulate inside homes, and it is the second lung cancer risk factor after smoking, and the first one in non-smokers. In Europe, there are several radon-prone areas, and although the 2013/59 EURATOM directive is aimed to regulate indoor radon exposition, regulating measures can vary between countries. Radon emits alpha-ionizing radiation that has been linked to a wide variety of cytotoxic and genotoxic effects; however, the link between lung cancer and radon from the genomic point of view remains poorly described. Driver molecular alterations have been recently identified in non-small lung cancer (NSCLC), such as somatic mutations (EGFR, BRAF, HER2, MET) or chromosomal rearrangements (ALK, ROS1, RET, NTRK), mainly in the non-smoking population, where no risk factor has been identified yet. An association between radon exposure and oncogenic NSCLC in non-smokers has been hypothesised. This paper provides a practical, concise and updated review on the implications of indoor radon in lung cancer carcinogenesis, and especially of its potential relation with NSCLC with driver genomic alterations.

Real-world (rw) analysis of quantitative MET copy number (CN) as a biomarker in NSCLC.

9123 Background: MET amplification (amp) can be a de novo driver or mechanism of resistance to targeted therapy. We explored the genomic landscape of MET amp NSCLC, including co-driver alterations and amplicon size, and its association with outcomes to MET tyrosine kinase inhibitors (TKIs) using rw data. Methods: Comprehensive genomic profiling (CGP) results from 64,521 tissue and 5,177 liquid NSCLC samples were queried for MET amp (focal amplicons ≥4 copies above specimen ploidy). Sub analysis comparing focal (≤20Mbp) and non-focal (&gt; 20Mbp) amp was performed. Using the nationwide (̃280 US cancer clinics) de-identified EHR-derived Flatiron Health-Foundation Medicine aNSCLC clinicogenomic database (CGDB) linked to tissue CGP (8/2014-9/2021), we assessed co-driver presence ( EGFR, ALK, RET, ROS1, NTRK, BRAF, KRAS) prior to therapy exposure and outcomes to MET TKIs. Results: MET amp was detected in 2,102 (3.3%) NSCLC tissue samples (median CN of 11, range 6-168). Evaluating full gene focal amplicons as well as non-focal MET amps (n = 398, median CN 7), smaller amplicon size significantly correlated with higher CN (p &lt; 0.001). In 5,177 liquid samples, MET amp was detected in 40 (0.77%) cases; frequency increased to 3.2% (33/1,033) in those with a tumor fraction above 10%. In the CGDB, 261 (3.2%) pts had NSCLC with MET amp (median CN 11). Among 241 pts with MET amp detected prior to receipt of any targeted therapy, MET amp co-occurred with another driver in 32 (13%) cases (81% KRAS, 16% EGFR, 3.1% BRAF) and with a MET exon 14 skipping alteration ( METex14) in 25 (10%). MET CN negatively correlated with the presence of a co-driver (median 11 with MET amp alone vs 9 with a non- METex14 co-driver, p &lt; 0.001); however, MET CN was similar in MET amp cases with and without METex14 (median 11 for both, p = 0.60). Co-occurring non- METex14 drivers were detected in 26% of MET amp cases with CN &lt; 8 and 19% with CN 8-9, but in only 9.1% with CN 10-20 and 2.6% with CN &gt; 20. Tumor mutational burden was not significantly correlated with MET CN. In 14 cases with an EGFR or ALK co-driver and MET amp detected post-treatment with an EGFR or ALK TKI, median MET CN was 13. Of 241 targeted therapy naïve MET amp pts, 39 received a MET TKI after CGP (30 crizotinib, 9 capmatinib) and 26 had rw response assessment available. Excluding 5 pts with co- METex14, 12/21 (57%) pts had a partial response and median rwPFS was 3.6 months. This rw cohort analysis was underpowered to assess the relationship between CN and outcome. Conclusions: MET amp was detected in 3.3% of NSCLC tissue samples and 3.2% of high tumor fraction blood samples. In TKI-naïve pts, MET CN negatively correlated with the presence of a concurrent NSCLC driver. MET amp was associated with response to MET TKIs. Further studies evaluating MET CN, amplicon size and presence of other potential drivers in both blood and tissue, as predictive biomarkers for MET TKIs and potential combination strategies for targeting MET amp, are warranted.

Distinct profile of cell-free DNA in malignant pleural effusion of non-small cell lung cancer and its impact on clinical genetic testing.

Cell-free DNA (cfDNA) in supernatant of pleural effusion from advanced NSCLC patients has been proved as surrogate sample detecting therapeutic targets as well as tumor mutation burden (TMB). As recently reported, cfDNA in pleural effusion supernatant is superior to plasma in TMB evaluation. It is reasonable to hypothesize that cfDNA profile in pleural effusion (PE) and plasma might be different. It remains to be elucidated why cfDNA in PE supernatant impacts on genetic analysis. Consequently, the approach dealing with cfDNA from PE supernatant might need to be different from that for plasma cfDNA in order to obtain accurate clinical genetic testing result.Methods: Pleural effusion samples from 32 patients with stage IV lung adenocarcinoma were collected. Supernatant and sediment were processed separately to extract Cell-free DNA as well as sediment DNA (PE-S). cfDNA from pleural effusion was analyzed by Agilent 2100 bioanalyzer. Libraries were prepared by 1) direct use of the total cfDNA without fragmentation step (PE-FL) or 2) use of full-length cfDNA fragmented to 150-250bp (PE-F), 3) use of cfDNA fragments enriched to ~167bp (PE-E167) as well as 4) use of cfDNA fragments larger than 500bp enriched (PE-E500). All samples were subjected to targeted next-generation sequencing (NGS) with a panel of 448 cancer-related genes as well as a panel of 10 NSCLC driver genes.Results: cfDNA were successfully extracted from 30 MPE samples. cfDNA displayed distinct profile in supernatant of malignant pleural effusion from that of plasma cfDNA. No statistical difference in detection of hotspot variations between PE-E167 and PE-F by 448-gene or 10-gene panel. While TMB from PE-F samples was significantly higher than that from PE-E167 and PE-FL. Higher TMB from PE-F was resulted from cancer-unspecific variants with low allele frequency (0.1%-1%) which were mainly introduced by long-fragment cfDNA. Similar genetic profile was observed between paired cfDNA of PE-FL and cfDNA of PE-E167.Conclusion: Long-fragment cfDNA in the PE supernatant will introduce low abundant cancer unrelated variants which leads overestimation of TMB. Paired PE-FL and PE-E167 gave comparable outcomes. Direct use of the total cfDNA without fragmentation step (PE-FL) is recommended for library preparation of NGS testing in clinical practice to exclude interference from long fragments of the cfDNA.

Duration of Targeted Therapy in Patients With Advanced Non–small-cell Lung Cancer Identified by Circulating Tumor DNA Analysis

BackgroundOutcomes of therapy targeting molecular driver alterations detected in advanced non–small-cell lung (NSCLC) using circulating tumor DNA (ctDNA) have not been widely reported in patients who are targeted therapy-naive. Patients and MethodsWe performed a multicenter retrospective review of patients with unresectable stage IIIB to IV NSCLC who received matched therapy after a targetable driver alteration was identified using a commercial ctDNA assay through usual clinical care. Eligible patients must not have received targeted therapy prior to ctDNA testing (prior chemotherapy or immunotherapy was permitted). Kaplan-Meier analysis was used to estimate the median duration of targeted therapy. Patients still on targeted therapy were censored at last follow-up. ResultsSeventy-six patients met inclusion criteria. The median age of diagnosis of NSCLC was 64.5 years (range, 31-87 years), 67% were female, 74% were never-smokers, and 97% had adenocarcinoma histology. Twenty-one (28%) patients received systemic treatment prior to targeted therapy, including chemotherapy (n = 17), immunotherapy (n = 5), and/or a biologic (n = 4). Thirty-three (43%) patients remain on targeted therapy at the time of data analysis. The median time on targeted therapy was similar to what has been reported for tissue-detected oncogenic driver mutations in the targeted therapy-naive setting. ConclusionsPatients with ctDNA-detected drivers had durable time on targeted therapy. These treatment outcomes data compliment previous studies that have shown enhanced targetable biomarker discovery rates and high tissue concordance of ctDNA testing when incorporated at initial diagnosis of NSCLC. Identification of NSCLC driver mutations using well-validated ctDNA assays can be used for clinical decision-making and targeted therapy assignment.

Characterization of 1,387 NSCLCs with MET exon 14 (METex14) skipping alterations (SA) and potential acquired resistance (AR) mechanisms.

9511 Background: METex14 SA are oncogenic drivers in NSCLC. Due to the numerous sites around ex14 that bind the spliceosome complex, many variations can result in deleterious alterations (alts). We present a comprehensive overview of these ex14 SA across 1,387 NSCLCs and characterized potential AR mechanisms. Methods: Hybrid-capture based comprehensive genomic profiling (CGP) was performed on samples from 60,495 NSCLC patients (pts). A scoring system was applied leveraging our large database of samples with METex14 SA to optimize accurate reporting of these variants. Paired samples were collected ≥ 60 days apart (median 462). Results: 1,393 METex14 SA were identified in samples (1,278 tissue, 109 circulating tumor DNA (ctDNA)) from 1,387 NSCLC pts (2.3%) spanning multiple functional sites: donor (42%), acceptor (4.7%), poly-pyrimidine tract (15%), acceptor and polypyrimidine tract (13%), D1010 (23%), Y1003 (2.1%), and whole exon deletions (0.3%). 6 samples (5 tissue, 1 ctDNA) harbored 2 METex14 SA, each including a mutation (mut) at the donor or acceptor site. MDM2 and CDK4 amplifications (amps) co-occurred in 32% and 19% of METex14 samples, respectively, but were more common with polypyrimidine tract (37% and 23%) vs donor site (32%, p = 0.07 and 18%, p = 0.07) alts. MET co-amp was present in 12% of cases and frequency did not significantly differ by functional site. 66 (4.8%) cases (57 tissue, 9 ctDNA) had known NSCLC co-drivers, including KRAS (68%) and EGFR (14%) mut, a subset of which may represent AR. Paired samples with a METex14 SA in the 1st sample were available for 36 pts. The METex14 SA was detected in the 2nd sample for 32 pts, excluding 3 with low ctDNA. 22/36 (61%) had reportable acquired alts detected including 9 with ≥1 acquired MET muts [D1228X (4), Y1230X (3), Y1003F (1), D1228A/E/H + L1195V (1)] and 3 with acquired MET amp. Other acquired alts included ERBB2 amp and mut (1 each), EGFR ex19ins (1), KRAS amp (1), PIK3CA mut (1), AKT2 amp (1) and others with unknown functional significance. Potential AR alts were present with primary METex14 SA spanning all functional sites. Conclusions: In a dataset of &gt; 60,000 advanced NSCLCs, METex14 SA were present in 2.3% of cases, and represented 6 major subtypes. Among paired cases, potential AR mechanisms included secondary MET alts (33%), and acquired alts in EGFR, ERBB2, KRAS, and PI3K pathways. Acquired alts were independent of the type of METex14 SA. Characterizing common co-occuring may be critical for predicting responses to MET inhibitors and informing rational combination strategies.

Real-world (RW) outcomes for advanced non-small cell lung cancer (aNSCLC) patients (pts) with EGFR exon 19 deletions (x19del) stratified by deletion size.

9591 Background: EGFR x19dels are well-established targetable drivers in NSCLC. Historically x19dels have been treated as a single group, but it’s unclear whether responses vary for distinct subtypes. We compared demographic, clinical and genomic characteristics as well as outcomes to EGFR tyrosine kinase inhibitors (TKIs) for aNSCLC pts with x19dels of varying lengths using a RW Clinico-Genomic Database (CGDB). Methods: Eligible pts had a diagnosis of aNSCLC, received care within the Flatiron Health network between 1/2011-9/2019, and had comprehensive genomic profiling (CGP) by Foundation Medicine. Clinical characteristics, treatment history and RW progression were obtained via technology-enabled abstraction as previously described (Singal G, JAMA 2019). x19del length was evaluated for association with overall survival (OS) and RW progression-free survival (rwPFS) with Kaplan-Meier analysis and unadjusted/adjusted (practice type, gender, age at TKI start, EGFR TKI type, race) hazard ratios (HR/aHR) from Cox proportional hazards models adjusted for survival bias. Results: Among 6,577 aNSCLC pts, EGFR x19dels were detected in 336 cases (5%). E746_A750del was the most frequent (214; 64%) and generally 5 amino acid (aa) deletions (x19del-5) were the most common (241; 72%). Other deletions (x19del-other) of 6 (61; 18%), 3 (20; 6%), 4 (11; 3%) or 8 aa (3; 1%) were also observed. Among pts treated with 1st-line EGFR TKI monotherapy after CGP, the x19del-5 (n = 70) cohort was more frequently female compared to x19del-other (n = 27) (90% vs 59%, p = 0.001). No statistically significant differences in the frequency of co-occurring alterations were observed, specifically for genes associated with response to EGFR TKI response such as CTNNB1 (14% vs 19%) and PIK3CA (10% vs 15%). 1st line EGFR TKIs used were similar for x19del-5 vs x19del-other (43% vs 41% osimertinib, 30% vs 37% erlotinib, 24% vs 22% afatinib, 3% vs 0% gefitinib). x19del-5 pts had similar median rwPFS (10.6 vs 10.6 months, HR: 0.73 [0.41-1.28], aHR:0.78 [0.38-1.59]) and median OS (29.2 vs 24.9 months, HR: 0.64 [0.32- 1.29], aHR: 0.75 [0.32-1.75]) compared to x19del-other. Conclusions: In a RW CGDB of 336 aNSCLC pts with EGFR x19dels, 5 aa x19dels were most common (71%) and 29% of cases had 3, 4, 6 or 8 aa x19dels . For pts included in the treatment cohort, no significant differences in rwPFS or OS were observed. These results suggest that x19del length does not significantly impact clinical outcomes to 1st-line EGFR TKIs.

Analysis of EGFR mutant urothelial carcinoma (UC) reveals distinct mutational landscape.

4545 Background: Genomic alterations (GA) of EGFR, are well recognized as druggable oncogenic drivers in NSCLC, but the druggable GA EGFR L858R and exon 19 deletion (ex19del), are rarely observed in genitourinary cancer. We reviewed the genomic landscape of advanced upper tract and bladder UC (UTUC and BUC) to assess the frequencies of druggable EGFR GA. Methods: Tissue specimens from patients with UTUC (407) and BUC (2,402) were assayed using hybrid capture-based comprehensive genomic profiling (CGP). Tumor mutational burden (TMB) was determined on up to 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 95 or 114 loci. Results: EGFR alterations ( EGFR alt) were present in 17 UTUC and 93 BUC (4.2% and 3.9%). Age distribution between the two subgroups was similar, but UTUC was more prevalent in female patients (47% v 29%). BUC had a higher median TMB (5.2mut/mb v 7.8 mut/mb; p = 0.046) and the prevalence of MSI-H cases was not significantly different. TERT (55% v 71%) and TP53 (59% v 74%) were the most frequently mutated genes in EGFR alt UTUC and BUC. Within EGFRalt, amplifications were the most common alterations in both UTUC and BUC (13/17, 76%; 57/93, 61%). Amplifications were mutually exclusive from cases with EGFR short variants (SV) in BUC (0/34, 0%), and co-occurred with EGFR SV in four UTUC cases (4/8 50%). The majority of EGFR SV were EGFR exon 20 insertions ( EGFRexon20), which made up a larger proportion of EGFR alt in UTUC than BUC (7/17, 41% v 13/93, 14%; p = 0.01). Compared to other EGFR alt , EGFRexon20 trended towards mutual exclusivity of GA in commonly altered UC genes: TP53 (UTUC EGFRexon20 v EGFR alt other: 0% v 100%, p = 5.1E-5; BUC EGFRexon20 v EGFR alt other: 0% v 86%, p = 2.2E-7) , PIK3CA (14% v 10%; 0% v 19%) , RAF1 (0% v 10%; 0% v 16%) , or FGFR3 (0% v 10%; 0% v 6.3%) alterations. Only 2.2% (2/93) of BUC EGFRalt were L858R mutations and none were ex19del (0/93), while neither mutation was detected in UTUC. Conclusions: EGFRexon20 defines a subset of UC cases, and is the most common non-amplification GA seen in EGFR in UC, with some enrichment in UTUC. Consideration should be given to developing a trial for EGFR exon20 UC patients given the recent investigational work on inhibitors with activity against EGFRexon20, such as poziotinib and TAK-788.

Genomic profiling of pulmonary lymphoepithelioma-like carcinoma (PLELC).

1572 Background: PLELC, a rare and distinct type of primary lung cancer, is characterized by Epstein-Barr virus (EBV) infection. Histologically, it resembles undifferentiated nasopharyngeal carcinomas (NPC). Only a few hundred cases have been reported since its discovery. Due to the extreme rareness, its genomic landscape remains elusive. Methods: Tissue samples of 27 PLELC patients (13 males and 14 females) with various stages (Ib to IV) were subjected to targeted sequencing using a panel consisting of 520 cancer-related genes, spanning 1.6Mb of human genome. Results: Collectively, we identified 184 somatic mutations spanning 109 genes, including 107 SNVs, 12 insertions or deletions (INDELs) and 65 copy-number amplifications (CNAs). Approximately, 50% of patients had CNAs. One patient had no mutation detected from this panel. Except for 2 patients, 1 with HER2 amplification and another with KRAS mutation, no other classic NSCLC driver genes were detected. The most frequently mutated genes were CCND1, TP53, DAXX and NF kBIA, occurring in 30%, 26%, 22% and 22% of patients, respectively. Interestingly, 78% (21/27) patients had mutations in epigenetic regulators. Of the 184 mutations identified, 51 occurred in epigenetics-related genes. Pathway analysis also revealed an enrichment of genes participating in chromatin remodeling and organization. Next, we compared the genomic profile of PLELC with lung adenocarcinoma and EBV positive NPC. The frequency of TP53 mutations was significantly higher in lung adenocarcinoma (68% vs 26%, p = 0.021). Comparing to NPC, PLELC had significantly more mutations in epigenetic regulators. TMB analysis revealed a median TMB of 1.6/Mb, significantly lowered than lung adenocarcinomas (p &lt; 0.01). We also assessed PD-L1expression and revealed that 67% had an overexpression of PD-L1. Interestingly, TP53-mutant patients were more likely to associated low PD-L1 expression (p &lt; 0.01). Conclusions: In this study, we elucidated a distinct genomic landscape associated with PLELC with no classic NSCLC driver mutation but an enrichment of mutations in epigenetic regulators. The observation of high expression of PD-L1 and lack of canonical druggable driver mutation raises the potential of immunocheckpoint blockade therapy for PLELC.

Analysis of EGFR mutant upper tract and bladder urothelial carcinoma (UC) reveals distinct mutational landscape.

416 Background: Genomic alterations (GA) of EGFR, are well recognized as druggable oncogenic drivers in NSCLC, but the druggable GA EGFR L858R and exon 19 deletion (ex19del), are rarely observed in genitourinary cancer. We reviewed the genomic landscape of advanced upper tract and bladder UC (UTUC and BUC) to assess the frequencies of druggable EGFR GA. Methods: Tissue specimens from patients with UTUC (407) and BUC (2,402) were assayed using hybrid capture-based comprehensive genomic profiling (CGP). Tumor mutational burden (TMB) was determined on up to 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 95 or 114 loci. Results: EGFR alterations ( EGFRalt) were present in 17 UTUC and 93 BUC (4.2% and 3.9%). Age distribution between the two subgroups was similar, but UTUC was more prevalent in female patients (47% v 29%). BUC had a higher median TMB (5.2mut/mb v 7.8 mut/mb; p = 0.046) and the prevalence of MSI-H cases was not significantly different. TERT (55% v 66%) and TP53 (58% v 74%) were the most frequently mutated genes in EGFRalt UTUC and BUC. Within EGFRalt, amplifications were the most common alterations in both UTUC and BUC (13/17, 76%; 57/93, 63%). Amplifications were mutually exclusive from cases with EGFR short variants (SV) in BUC (0/34, 0%), and co-occurred with EGFR SV in four UTUC cases (4/8 50%). The majority of EGFR SV were EGFR exon 20 insertions ( EGFRexon20), which made up a larger proportion of EGFRalt in UTUC than BUC (7/17, 38% v 13/93, 14%; p = 0.01). Compared to other EGFRalt, EGFRexon20 trended towards mutual exclusivity of GA in commonly altered UC genes: TP53 (0% v 100%, p = 5.1E-5; 0% v 86%, p = 2.2E-7) , PIK3CA (14% v 10%; 0% v 19%) , RAF1 (0% v 10%; 0% v 16%) , or FGFR3 (0% v 10%; 0% v 6.3%) alterations. Only 2.2% (2/93) of BUC EGFRalt were L858R mutations and none were ex19del (0/93), while neither mutation was detected in UTUC. Conclusions: EGFRexon20 defines a subset of UC cases, and is the most common non-amplification GA seen in EGFR in UC, with some enrichment in UTUC. Consideration should be given to developing a trial for EGFR exon20 UC patients given the recent investigational work on inhibitors with activity against EGFRexon20, such as poziotinib and TAK-788.

Abstract 4784: TAS0286/HM05, a novel highly selective RET inhibitor, prominently inhibits various RET defective tumor growth

Abstract The rearranged during transfection (RET) gene is a well-known proto-oncogene and encodes a single-pass transmembrane receptor tyrosine kinase. RET fusions and point mutations are oncogenic drivers in NSCLC, medullary thyroid cancer and other solid tumors, and therefore potential targets for cancer therapy. Multikinase inhibitors targeting the RET mutations have been tested in clinical trials with moderate efficacy in terms of tumor shrinkage and PFS.Moreover, multikinase inhibitors are characterized by poor tolerability due to off-target kinase inhibitory activities. To widen the therapeutic index, a selective RET kinase inhibitor is highly desirable. TAS0286/HM05 is a novel highly selective and potent RET kinase inhibitor.RET kinase assay was performed by homogeneous time-resolved fluorescence (HTRF) method. In-vitro proliferation studies and pharmacodynamics analyses were conducted in cancer cell lines with the RET fusions and RET activating mutations. The antitumor efficacy of TAS0286/HM05 was evaluated using mice xenograft models implanted with cancer cells with various RET gene abnormality. TAS0286/HM05 was orally administered for 14 or 28 consecutive days after grouping. In-vitro and in-vivo RET phosphorylation was detected using western blot analysis. The IC50 value for RET kinase of TAS0286/HM05 was below 1 nM. TAS0286/HM05 showed highly selective RET inhibitory activity among 283 kinases. In cellular assay, TAS0286/HM05 strongly suppressed phosphorylation of RET expressed in cells with various RET fusions and activating mutations, and inhibited cell proliferation at around 10 nM. At this concentration, apoptosis was also observed. The potency was higher than other multikinase inhibitors with RET inhibitory activity. Furthermore, in in-vivo efficacy studies, TAS0286/HM05 significantly inhibited the growth of tumors harboring various RET fusions and activating mutations at a range of 20 to 100 mg/kg/day without any body weight loss. The antitumor efficacy of TAS0286/HM05 was more potent than pre-existing multikinase inhibitors at their maximum tolerated dose. In particular, TAS0286/HM05 dramatically induced tumor regression of 40% within 15 days in animals implanted with LC-2/ad cells, a human lung adenocarcinoma cell line with CCDC6-RET fusion gene. TAS0286/HM05 is a novel and highly selective RET inhibitor with prominent tolerability. The potency of TAS0286/HM05 against tumors with RET abnormalities was stronger than currently marketed multikinase inhibitors being tested in NSCLC patients with RET fusions. TAS0286/HM05 is a promising agent for future clinical development in patients with RET gene abnormalities. Citation Format: Hidenori Fujita, Isao Miyazaki, Masanori Kato, Yukari Yamada, Keiji Ishida, Tomonori Haruma, Haruka Nagasaki, Kenjiro Ito, Akihiro Hashimoto, Yasuo Kodama, Kaoru Funabashi, Emanuela Lovati, Kazutaka Miyadera, Kenichi Matsuo, Yoshikazu Iwasawa. TAS0286/HM05, a novel highly selective RET inhibitor, prominently inhibits various RET defective tumor growth [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 4784.

Rare targetable drivers (RTD) in NSCLC: PD-L1 expression, tumor mutation burden (TMB), microsatellite instability (MSI) and outcomes with immune check-point inhibitors (ICPi).

9076Background: The efficacy of ICPi in NSCLC with RTD is unknown. Methods: 82 consecutive patients (pts) with RTD (non-EGFR/ALK) were selected from the Davidoff Cancer Center database. The correla...

Association between certain NSCLC driver mutations and sensitivity markers for chemotherapy or PD-(L)1 inhibition: A large-sample analysis.

8542Background: Driver gene alterations play a pivotal role in NSCLC and are predictive markers for specific targeted therapy. However, few evidence has correlated certain mutation types with the s...