Wild-type NSCLC Research Articles

Comparative Clinical Outcomes Between EGFR Ex20ins and Wildtype NSCLC Treated with Immune Checkpoint Inhibitors

IntroductionThe activity of immune checkpoint inhibitors (ICIs) in NSCLC harboring EGFR exon 20 insertion mutations (ex20ins) has not been closely examined due to the frequent exclusion of patients with EGFR mutations from large immunotherapy-based NSCLC trials. Patients and MethodsA real-world, retrospective study was conducted to compare outcomes of ICI-treated patients with EGFR ex20ins and wildtype NSCLC (wt-NSCLC; defined as EGFR and ALK test negative). Patients with advanced NSCLC from the Flatiron Health database (2015-2020) were included in the analysis. Real-world time to next therapy (rwTTNT) and overall survival (rwOS), stratified by ICI initiation line of therapy, were the prespecified primary and secondary endpoints, respectively. ResultsAmong 59 patients with EGFR ex20ins NSCLC and 5365 with wt-NSCLC, ICI treatment was received as first-line therapy in 25% and 39%, respectively. Patients with EGFR ex20ins had a 58% increased risk of shorter time to next-line therapy compared with wt-NSCLC (adjusted hazard ratio of 1.58 [95% confidence interval [CI], 1.2-2.1]; P = .0012). The median rwTTNT for first ICI line was 3.7 months (95% CI, 3.0-4.9) for EGFR ex20ins NSCLC compared with 5.8 months (95% CI, 5.6-6.0) for wt-NSCLC. No meaningful difference in rwOS between the groups was observed. ConclusionsICI therapy may be less effective for patients with EGFR ex20ins compared with wt-NSCLC. Consistent with prior data on exon 19 deletion and L858R substitution, tumors harboring ex20ins appear to be less responsive to immune checkpoint inhibition than wt-NSCLC.

Abstract 5113: Liquid biopsy-based multiomics profiling using low-pass whole genome sequencing and proteomics with computational modeling reveals molecular correlates of disease severity in EGFR/ALK wild type NSCLC patients

Abstract Background: Molecular features of disease severity provide a basis for patient stratification, and may hint at previously unrecognized drug targets. Liquid biopsy-based multiomics profiling of plasma coupled with computational modeling provides a promising approach to interrogate both tumor and non-tumor derived signals, to support patient-centric personalized precision oncology. Here, we utilized a blood-based multiomics platform to identify, refine, and combine signals to categorize late-stage, treatment-naive, EGFR/ALK wild type NSCLC. Methods: Cell-free DNA (cfDNA) was extracted from 34 late-stage, treatment-naive, EGFR/ALK wild type NSCLC patients and low pass whole-genome sequencing was performed to characterize cfDNA fragments, which reflect nucleosome protection and chromatin state. Gene activation for protein-coding genes (GAP scores) was modeled from fragment distribution around transcription start sites. The abundances of 644 plasma proteins including markers of the immune response, inflammation, cancer, and DNA repair were measured. Partial least-squares discriminant analysis (PLS-DA) was used for marker selection and sample separation by smoking status with either genomics or proteomics data. Results: By statistically modeling clinical data, we identified a significant association showing never-smokers being enriched in the highest N stage, (P = 7.95E-3). Using PLS-DA, we identified proteomic (top 3 proteins: ST1A1, AXIN1, STAMPB) and GAP score (top 3 genes: CHD3, FANCF, METTL3) markers that separated patients by smoking status, suggesting underlying molecular differences. By performing pathway analysis on GAP scores, we identified 108 significant pathways enriched in the never-smoker group (FDR < 5%), 18 of which were associated with immune cell signatures, specifically myeloid cells and macrophages. Based on our previous association of IL1RN GAP scores with disease progression in NSCLC, we examined whether these results are confirmed. In this cohort, patients with high IL1RN GAP scores were significantly associated with worse lymph-node staging (N) (P = 6E-3), which is indicative of worse prognosis. Pathway analysis comparing patients with high vs low IL1RN scores revealed 102 significant pathways (FDR < 5%), including leukocyte activity and regulation of inflammatory response. Conclusion: The liquid biopsy-based multiomics platform suggests an association between never-smoker status and pathways of inflammatory response, resulting in more severe lymph-node staging. Future applications of this multiomics platform provide promise for stratification of NSCLC patients for patient-centric precision oncology. Citation Format: Zheng Feng, Danyi Wang, Francesco Vallania, Neil Smith, Anupriya Tripathi, Juergen Scheuenpflug. Liquid biopsy-based multiomics profiling using low-pass whole genome sequencing and proteomics with computational modeling reveals molecular correlates of disease severity in EGFR/ALK wild type NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5113.

Treatment patterns and outcomes in resectable early stage NSCLC: Interim analysis of a global real-world study.

e18803 Background: Complete surgical resection is the preferred treatment for early stage NSCLC, with adjuvant chemotherapy as the standard of care in resected stage II/III and select stage IB NSCLC. Osimertinib, an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), is approved as adjuvant therapy in patients (pts) with resected stage IB–IIIA EGFR mutation-positive (EGFRm) NSCLC following results from the Phase III ADAURA trial. Understanding real-world clinical outcomes in early stage NSCLC, including EGFRm prevalence, will help inform unmet needs and further development of new treatment strategies in this population. We report interim results from a global non-interventional retrospective study of pts with resectable NSCLC using clinical data from medical records. Methods: Eligible pts (aged ≥18 yrs) had completely resected stage IA–IIIA NSCLC diagnosed between Jan 1, 2014 and Dec 31, 2017 with EGFR test results available and were followed to at least Dec 31, 2020. Primary endpoints included EGFRm prevalence, treatment patterns, and overall survival (OS); disease-free survival (DFS) was an exploratory endpoint estimated by Kaplan-Meier at predefined landmark timepoints. Results: Of 463 pts from 6 countries (31% from Taiwan, 21% Canada, 17% US, 13% Austria, 10% South Korea, 9% France), median age was 66 yrs (range: 33–86); 172 pts (37%) had stage IA NSCLC at initial diagnosis and 291 (63%) had stage IB–IIIA (22% IB, 13% IIA, 10% IIB, 18% IIIA). 213/463 pts (46%) were EGFRm (43% stage IA, 61% IB, 43% IIA, 26% IIB, 47% IIIA), of who 46% were from Taiwan, 21% South Korea, 14% Austria, 8% Canada, 7% US, and 4% France. In pts with EGFRm vs EGFR wild-type (wt) NSCLC, 84/213 (39%) and 83/250 (33%), respectively, received (neo)adjuvant therapy, of who 156/167 pts (93%) had stage IB–IIIA NSCLC. 106/156 pts (68%) had disease recurrence or death from time of surgery; recurrence rates were similar in pts with EGFRm vs EGFRwt NSCLC, though median DFS was longer in the EGFRm group (Table). Recurrence rates were high in both EGFRm and EGFTwt groups, with landmark DFS probability of 72% vs 77% at 12 mo and 29% vs 32% at 60 mo, respectively. Conclusions: In this real-world global study of surgically resected stage IA–IIIA NSCLC in pts who received an EGFR test, nearly half of the study cohort were EGFRm positive, of who 70% were treated in Taiwan/South Korea. The high rate of recurrence in pts with stage IB–IIIA NSCLC despite receiving (neo)adjuvant therapy reinforces the need for early diagnosis and EGFR testing to identify pts who might benefit from EGFR-targeted therapy, helping optimize clinical outcomes.[Table: see text]

61P IMpower110: Exploratory analyses of the impact of first-line (1L) atezolizumab on the efficacy of next-line of therapy in PD-L1–selected NSCLC

Progression-free survival 2 (PFS2), defined by the EMA as the time from randomisation to progression on next-line treatment (tx) or death from any cause, is emerging as a clinical trial endpoint to assess the impact of experimental therapy on next-line tx. The Phase III IMpower110 study of 1L atezolizumab (anti–PD-L1) vs chemotherapy (chemo) in PD-L1–selected NSCLC met its primary endpoint, showing improved OS in patients (pts) with PD-L1–high (TC3 or IC3) wild-type (WT; EGFR/ALK-negative) NSCLC (HR, 0.59; 95% CI: 0.40, 0.89).

P59.27 Complementary Utility of Combined ALK/ROS1 FISH with Immunohistochemistry for ALK/ROS1 Rearrangement Testing in Lung Cancer

Testing for EGFR mutations, ALK and ROS1 rearrangements and BRAF mutations are now recommended as the essential minimum predictive biomarkers to be tested in primary non-squamous NSCLC patients presenting with advanced disease. While the ALK D5F3 Ventana immunohistochemistry (IHC) Assay is approved for detection of ALK rearrangements and for targeted therapy, ROS1 IHC only serves as a screening tool for ROS1 rearrangements with demonstration of gene fusions by molecular methods required for confirmation and targeted treatment. Nevertheless, equivocal IHC results, although uncommon, still occur with the D5F3 ALK assay necessitating complementary methods for confirmation. The recent availability of combined ALK/ROS1 break-apart fluorescence in-situ hybridisation (FISH) probes offer the advantage of tissue conservation with ability to analyse both gene alterations in a single tissue section. The aim of the present study was to study the utility of the combined ALK/ROS1 FISH assay as a complement to ALK and ROS1 IHC in routine predictive biomarker testing algorithms. Over a 6-year study period, all EGFR wild type NSCLC samples were subject to automated ALK D5F3 Ventana IHC assay and ROS1 IHC using the D4D6 (Cell signalling technology) clone as a manual lab developed test. Diffuse strong granular staining in tumor cells for ALK and ROS1 protein was interpreted as positive. All cases of equivocal ALK staining and positive ROS1 staining was subject to combined ALK/ROS1 FISH using the FlexISH ALK/ROS1 DistinguISH Probe (Vysis) performed as per manufacturers’ instructions. Presence of ALK/ROS1 break apart signals and/or isolated ALK/ROS1 3’ signals in >15% of interpretable tumour nuclei was considered as positive for ALK or ROS1 rearrangement. The ALK Ventana Assay was performed in 711 formalin fixed paraffin embedded EGFR wild type NSCLC samples including biopsies, lymph node excisions and cell blocks. Of these, 12% (85/711) were positive, 2% (13/711) were equivocal, 75% (539/711) were negative, while in the remaining 11% (76/711), result could not be evaluated due to exhaustion of tumor tissue. FISH analysis in four of the ALK IHC equivocal cases show absence of ALK translocations while no tissue remained for FISH analysis in the remaining equivocal cases. ROS1 IHC was performed in 434 cases of the above cohort of which 7% (30/434) were positive. ROS1 immunopositivity was enriched among ALK IHC positive cases with 43% (19/44) of ALK IHC positive cases also showing protein expression of ROS1. FISH analysis revealed ROS1 translocation in two cases (both showing diffuse strong ROS1 protein expression and negative ALK IHC) while all dual ALK/ROS IHC positive cases tested by FISH showed presence of only ALK rearrangements with non-rearranged ROS1 signals. Tissue adequacy remains the major limiting factor for predictive biomarker testing in lung cancer. The use of combined ALK-ROS FISH allows for excellent demonstration of ALK and ROS1 rearrangements in the same section using the same criteria validated in single gene break apart FISH probes. ROS1 protein is commonly overexpressed in ALK rearranged NSCLC and may represent cross reactivity with ALK or a related protein.

SMARCA4 and Other SWItch/Sucrose NonFermentable Family Genomic Alterations in NSCLC: Clinicopathologic Characteristics and Outcomes to Immune Checkpoint Inhibition

IntroductionThe SWItch/Sucrose Nonfermentable (SWI/SNF) chromatin remodeling complex acts as a regulatory component of transcription, and inactivating mutations (muts) within the complex are implicated in genomic instability, higher tumor mutational burden, and an aggressive cancer phenotype. Whether SMARCA4 and other SWI/SNF alterations are independent prognostic factors or associated with clinical outcomes to immune checkpoint inhibitors (ICIs) in NSCLC remains unclear. MethodsWe collected clinicopathologic and genomic data from patients with NSCLC who underwent targeted next-generation sequencing at the Dana-Farber Cancer Institute. Tumors were characterized on the basis of the presence or absence of muts across a set of six SWI/SNF genes (ARID1A, ARID1B, ARID2, PBRM1, SMARCA4, and SMARCB1). ResultsOf 2689 patients with NSCLC, 20.6% (N = 555) had SWI/SNF genomic alterations. Compared with SWI/SNF wild-type (wt) NSCLC, patients with SWI/SNF-mutant NSCLCs had a lower prevalence of concurrent targetable driver muts (33.2% versus 22.2%; p < 0.001), a higher tumor mutational burden (median 8.5 versus 12.2 muts/megabase; p < 0.001), and a shorter median overall survival (mOS) from the time of advanced disease diagnosis (25.0 versus 19.3 mo, p = 0.01); the detrimental effect in OS seemed to be largely driven by SMARCA4 muts (mOS: 25.0 for SMARCA4 wt versus 15.6 mo for SMARCA4 mutant; p < 0.001). Among 532 patients who received ICIs, 25.5% (N = 136) harbored SWI/SNF muts. From the start of immunotherapy, there was no difference in objective response rate (ORR = 19.9% versus 25.0%, p = 0.2), median progression-free survival (mPFS = 3.0 versus 3.0 mo, hazard ratio [HR] = 0.96 [95% confidence interval [CI] = 0.77–1.18], p = 0.7), or mOS (13.1 versus 9.5 mo, HR = 0.81 [95% CI: 0.64–1.02], p = 0.07) in SWI/SNF-wt versus SWI/SNF-mutant NSCLC, respectively. Nevertheless, among KRAS-mutant NSCLCs treated with ICIs (N = 176), a concurrent SWI/SNF mut (N = 39) conferred a numerically lower ORR (21.9% versus 12.8%, p = 0.2), a significantly shorter mPFS (4.1 versus 1.8 mo, HR = 0.57 [95% CI: 0.38–0.84], p = 0.005), and a significantly shorter mOS (15.5 versus 8.2 mo, HR = 0.56 [95% CI: 0.36–0.86], p = 0.008). The deleterious effect on immunotherapy outcomes in KRAS-mutant NSCLC was most pronounced in the SMARCA4-mutant subset (N = 17), with a lower ORR (22% versus 0%, p = 0.03), a significantly shorter mPFS (4.1 versus 1.4 mo, HR = 0.25 [95% CI: 0.14–0.42], p < 0.001), and a significantly shorter mOS (15.1 versus 3.0 mo, HR = 0.29 [95% CI: 0.17–0.50], p < 0.001) compared with SMARCA4-wt KRAS-mutant NSCLCs. ConclusionsAlthough there were no associations between SWI/SNF mut status and immunotherapy efficacy in the overall NSCLC cohort, the presence of a SMARCA4 alteration may confer a worse outcome to immunotherapy among KRAS-mutant NSCLCs.

Investigation of immune microenvironment in EGFR mutant NSCLC.

e20517 Background: Immune checkpoint inhibitor therapy has made great achievements in NSCLC, but patients with EGFR mutations have poor efficacy with immunotherapy. Previous studies have explored the expression of PD-L1, neo-antigen, co-mutation and regulatory pathways in EGFR mutate NSCLC. This work compared the immune microenvironment of EGFR mutant and wild-type NSCLC. Methods: Patients: NSCLC. Using multi-color immunohistochemistry (multi-IHC) to evaluate the expression of 2 indicators in tumors and tumor stroma, namely CD8+ T cell and macrophage. Shapiro-Wilk was used for normality test, and t-test or Mann-Whitney U test was used according to the results. Two-sided P &lt; 0.05 was considered a significant difference. Results: The study included 119 NSCLC patients, including 59 women (49.6%) and 60 men (50.4%), with a median age of 57. There were 68 patients (57.1%) with EGFR mutations, 19 patients (16%) with KRAS mutations, and 58 patients (48.7%) with TP53 mutations. multi-IHC results showed that, EGFR mutation Vs EGFR wild type samples, (1) the number and proportion of CD8+ T cells in tumor were not statistically different. The median number of CD8+ T cells in tumor stroma was 231.5 vs 359, p = 0.05 and the proportion of CD8+ T cells was 3.92% vs 5.64%, p = 0.02; (2) The median number of macrophage in tumors was 1522 vs 110, p &lt; 0.01, and the proportion of macrophage cells was 24.93% Vs 1.38%, p &lt; 0.01. The median value of macrophages in tumor stroma was 617.5 Vs 208, p &lt; 0.01, and the proportion of macrophages cells was 10.88% vs 3.03%, p &lt; 0.01. Conclusions: Compared with EGFR wild-type patients, EGFR mutation patients have a lower proportion of CD8+ T cells and a higher proportion of macrophages in the immune microenvironment.

Exploring the applicability of immunotherapy in different EGFR mutation subgroups based on data analysis of 9,659 Chinese patients with NSCLC.

e21020 Background: Here we assessed TMB level, PD-L1 expression, and their correlation in 9649 Chinese NSCLC patients with or without EGFR mutation, and different subtypes of EGFR mutations, to find out the underlying mechanisms that different outcomes of ICI for EGFR wild type and EGFR mutation NSCLC patients, and the possibility of ICI therapy for NSCLC patients of different EGFR mutation subtypes. Methods: Tumor tissue samples of 9649 NSCLC patients were collected from Janary 2018, expression of PD-L1 were detected by using Dako PD-L1 IHC 22C3 pharmDx, Tumor Proportion Score (TPS) was used to determine expression of PD-L1. Gene mutation was detected by means of next generation sequencing (NGS). Performed Whole-Exome Sequencing (WES) on 70 tissue samples and corresponding White Blood Cells (WBCs) as matched control. Other samples were detected with panel covering whole exon regions of 733 cancer related genes. All these detections were performed in a College of American Pathologists (CAP)-certified and Clinical Laboratory Improvement Amendments (CLIA)-accredited lab for gene mutation analysis (3D Medicines Inc.,Shanghai, China). Statistical analysis was performed using GraphPad Prism (version 7.01) and SPSS version 21.0 (SPSS,Inc.). Results: Results showed that the proportion of EGFR mutation in Chinese patients with NSCLC was 51.3%, and the proportion of EGFR mutation subtypes were 42.6% L858R, 39.5% exon 19del, 2.3% exon 20in, 4.3% T790M. These samples were divided into different groups according to EGFR mutation, both WES based and panel-based results showed that EGFR wild type group displayed higher TMB level than EGFR mutation group (P &lt; 0.05). However, except for exon 19del, L858R, exon 20in, no significant differences were found between wild type and other EGFR mutation subtypes. Furthermore, results of IHC revealed that, higher proportion of strong positive PD-L1 expression (TPS≥50) were found in EGFR wild type than exon 19del, L858R and exon 20in, no significant correlation was found between TMB level and PD-L1 expression. Conclusions: EGFR mutations account for half of Chinese NSCLC patients. The biomarkers of immune checkpoint inhibitors (such as TMB and PD-L1) are different in various EGFR mutation subtypes, which may indicate that for some NSCLC patients with EGFR mutation subtypes, they may also respond to ICI treatment as wild-type.

Abstract PO-059: Investigating gene expression profiles associated with clinical radiation resistance in KEAP1/NFE2L2 wildtype lung cancer

Abstract Background: We previously reported that approximately half of local recurrences (LR) after radiotherapy for localized NSCLC harbor mutations in KEAP1 or NFE2L2. Here we sought to explore factors associated with LR after radiotherapy in KEAP1/NFE2L2 wildtype NSCLC. Methods: We identified consecutive patients with stage IA1-IIIC NSCLC treated at our institution with chemoradiotherapy (CRT) or stereotactic ablative radiotherapy (SABR) from 2009-2018 and who had genotyping performed on tumor tissue with full coverage of common recurrent lung cancer driver genes including TP53, KRAS, KEAP1, and NFE2L2. We defined LR as tumor regrowth within the prescription dose radiotherapy volume. Our primary objective was to identify factors associated with LR in KEAP1/NFE2L2WT tumors. We performed RNA-seq on a subset of cases with available tissue using the SMARTer Stranded total RNA-seq Kit v2 (Takara Bio USA, Inc.). Statistical analysis was performed using R (version 3.6) with differential gene expression performed using ‘DESeq2’. All P-values were two-sided and considered significant at P&amp;lt;0.05 with adjustment for multiple hypothesis testing when appropriate. Results: We identified 139 consecutive patients with localized NSCLC who received tumor genotyping and were treated with CRT for stage IIB-IIIC NSCLC (n=58) or SABR for stage I-IIB NSCLC (n=81). 26 (18.7%) of these patients harbored KEAP1/NFE2L2WT tumors. Clinical factors such as tumor volume (P=0.18), histology (P=0.87), and radiation dose (P=0.3) were not associated with LR in this subset. Similarly, somatic mutation analysis did not reveal association of any recurrent driver mutations with LR in these KEAP1/NFE2L2WT tumors, including in TP53 (n=19; P=0.73) or KRAS (n=9; P=0.98). Tissue was available for RNA-seq analysis of 38 KEAP1/NFE2L2WT tumors, of which the majority were adenocarcinomas (n=25, 65.8%) and approximately half each received CRT (n=20, 52.6%) and SABR (n=18, 47.4%). Gene set enrichment analysis revealed a trend for association of LR with expression of hypoxia genes (P=0.07, Q=0.28). Similarly, a previously reported 10-gene radiation sensitivity index (RSI) was not associated with LR (P=0.34). Individual gene analysis identified KRT14 as being significantly less expressed in cases with LR (adjusted P=2.2e-9). In a validation cohort of 24 stage I-IIA KEAP1/NFE2L2WT patients from the TCGA who were treated with radiotherapy, those who died had lower expression of KRT14 than those who did not (P=0.0003). Conclusions: In summary, we identify low expression of KRT14, a squamous cell carcinoma differentiation gene, as a potential biomarker for increased risk of LR after definitive radiotherapy of KEAP1/NFE2L2WT NSCLC. Validation in larger cohorts and biological characterization will be required to determine if this biomarker could be useful for guiding precision radiotherapy approaches. Citation Format: Michael S. Binkley, Young-Jun Jeon, Monica Nesselbush, Everett J. Moding, Barzin Nabet, Diego Almanza, Christopher Yoo, David M. Kurtz, Susie Grant Owen, Leah M. Backhus, Mark F. Berry, Joseph B. Shrager, Kavitha J. Ramchandran, Sukhmani K. Padda, Millie Das, Joel W. Neal, Heather A. Wakelee, Ash A. Alizadeh, Billy W. Loo, Maximilian Diehn. Investigating gene expression profiles associated with clinical radiation resistance in KEAP1/NFE2L2 wildtype lung cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-059.

Abstract 4384: Quantitative assessment of drug combination effects in NSCLC cell lines through network-based analysis of functional protein activity

Abstract Non-small cell lung cancer (NSCLC) is the most prevalent type of lung cancer, which is the leading cause of cancer death worldwide. The KRAS oncogene is one of the most common driver mutations in NSCLC patients but, despite that, there are no approved targeted therapies for tumors harboring this mutation. The MEK inhibitor Selumetinib (SE) turned out to be an ineffective drug against KRAS mutated lung cancer. Since combination therapy has shown to be beneficial in many tumors, we wanted to investigate the combination of MEK inhibition with other targeted therapies. Thus, we measured the proteomic response of KRAS mutant (MUT) and wild-type (WT) NSCLC cell lines (CLs) to the combination of SE with Everolimus (EV), an mTOR inhibitor, and Tamoxifen (TA), an Estrogen inhibitor. The expression level of 183 proteins was measured through Reverse Phase Protein Array (RPPA) in eight NSCLC CLs treated with SE, SE plus EV and SE plus TA at 6 time points: 5 minutes (min), 30 min, 1, 2, 6 and 24 hours (h). As control, measures were taken in baseline CLs and in CLs with only dimethyl sulfoxide. To study the drug combination effects, we applied a computational workflow centered on two algorithms initially developed for gene expression data. We analyzed the CLs with the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) to reconstruct a context specific signaling network of 2635 interactions between 73 kinases and 6 phosphatases and their substrates. Then, we analyzed the obtained network with the Virtual Inference of Protein activity by Enriched Regulon analysis (VIPER) algorithm to assess the mechanism of action of the drug compounds and how they dysregulate the activity of kinases and phosphatases over time. With respect to SE alone and apart from the drug targets, VIPER predicted the following proteins as highly inhibited over time across all CLs : 1) Aurora and LKB1 in SE+EV, 2) AMPK, c-RAF and ATR in SE+TA, 3) mTOR, p90, p70S6K, c-MET and GSK-3 in both combinations. On the other hand, Akt, ETK, Axl and FAK are all activated in SE+EV as well as PP2A, SGK1 and IGF1R in SE+TA, after 1h from the treatment. By running Gene Set Enrichment Analysis on the VIPER output against the primary drug target pathways, we found that mTOR signaling pathway exhibits a quite different evolution between KRAS MUT and WT CLs. On the other hand, other pathways revealed mechanisms of response that are CL specific. For instance, the main MEK pathways in H1734 CL (KRAS MUT) treated with SE+TA are all insensitive to the drug compounds. In conclusion, this computational approach successfully predicted protein-protein interactions and elucidated both proteomic mechanisms of drug combinations and CL specific dependencies. It allows to develop effective predictive and quantitative models reproducing each CL behavior, in order to realize the fullest potential of a targeted therapeutic approach. Citation Format: Chiara Antonini, George Rosenberger, Fortunato Bianconi, Lorenzo Tomassoni, Vienna Ludovini, Sara Baglivo, Sara Calandrini, Elisa Baldelli, Mariaelena Pierobon, Emanuel F. Petricoin, Andrea Califano. Quantitative assessment of drug combination effects in NSCLC cell lines through network-based analysis of functional protein activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4384.

CNS outcomes in EGFR/ALK wild-type NSCLC patients with brain metastases treated with immune checkpoint inhibitors (ICI).

e14505 Background: Anti-tumor lymphocytes activated in response to ICIs may cross the blood brain barrier with effect on brain metastases (BM). Preliminary evidence suggests that ICIs alone are effective for melanoma BMs. However, the ability of ICI to effectively control NSCLC BMs without radiotherapy is controversial. By comparing serial MRIs and patterns of progression in ICI and chemotherapy treated patients, we sought to investigate the role of ICI in BM control in NSCLC patients. Methods: Serial MRIs were reviewed in ICI treated NSCLC patients with known BMs. Eligible patients must have a minimum of one baseline MRI and a follow-up within 3 months of CNS directed treatment. Patients with EGFR/ ALK mutations were excluded. A control cohort of patients who had never received ICIs was selected using the same criteria. The primary outcome was cumulative CNS progression and intracranial progression free survival (iPFS) analyzed using an accelerated failure time model and competing risk analysis. Results: We identified 137 ICI treated NSCLC patients with known BMs. After excluding for patients without serial MRIs, we analyzed 40 ICI treated patients and 39 controls. All patients received upfront radiotherapy or surgery and the modality used was similar (p = 0.13). CNS failure was less common in the ICI group (p = 0.02). ICI treated patients had a trend to longer iPFS in the PD-L1 ≥50% subgroup (p = 0.058). Cumulative incidence of progressive BM at 12 and 24 months were significantly lower in the ICI treated PD-L1 ≥50% subgroup: 19%/19% vs 50%/56% (p = 0.02). Patients in the PD-L1 ≥50% subgroup who achieved a systemic partial response (n = 10) did not experience any CNS events despite extracranial progression in some. Conclusions: Durable CNS disease control is observed with ICIs in NSCLC with PD-L1 ≥50% and demonstrates a high level of concordance with systemic response. The need for radiotherapy in PD-L1 ≥50% disease should be investigated and may be reasonably delayed in asymptomatic patients with small BMs. Importantly, the same trend was not observed in PDL1 &lt; 50% patients and caution is needed in employing this strategy in this subset of patients.

LKB1 Deficiency Renders NSCLC Cells Sensitive to ERK Inhibitors.

Serine/threonine kinase 11 (LKB1/STK11) is one of the most mutated genes in NSCLC accounting for approximately one-third of cases and its activity is impaired in approximately half of KRAS-mutated NSCLC. At present, these patients cannot benefit from any specific therapy. Through CRISPR/Cas9 technology, we systematically deleted LKB1 in both wild-type (WT) and KRAS-mutated human NSCLC cells. By using these isogenic systems together with genetically engineered mouse models we investigated the cell response to ERK inhibitors both invitro and invivo. In all the systems used here, the loss of LKB1 creates vulnerability and renders these cells particularly sensitive to ERK inhibitors both invitro and invivo. The same cells expressing a WT LKB1 poorly respond to these drugs. At the molecular level, in the absence of LKB1, ERK inhibitors induced a marked inhibition of p90 ribosomal S6 kinase activation, which in turn abolished S6 protein activation, promoting the cytotoxic effect. This work shows that ERK inhibitors are effective in LKB1 and LKB1/KRAS-mutated tumors, thus offering a therapeutic strategy for this prognostically unfavorable subgroup of patients. Because ERK inhibitors are already in clinical development, our findings could be easily translatable to the clinic. Importantly, the lack of effect in cells expressing WT LKB1, predicts that treatment of LKB1-mutated tumors with ERK inhibitors should have a favorable toxicity profile.

Peri-operative adjuvant treatment for potential resectable NSCLC

Abstract Adjuvant chemotherapy is the standard treatment for resected stage 2-3 NSCLC since 2006. It increased OS by 5% at 5 years. There is a huge medical need to improve the long-term survival for these patients. NSCLC was divided to driver genes mutation and wild type NSCLC. The treatment strategy is different based these two type. For driver gene positive NSCLC small molecular target drugs play a critical role. For wild type patient check point blockade is an important treatment based The ADJUVANT trial showed that adjuvant gefitinib significant improved DFS. HR for recurrence was 0.60 with 28.7 months in gefitinib vs 18.0 months in chemotherapy. Subsequently the EVAN — another Chinese phase 2 trial and SELLECT trial from American once again confirmed that adjuvant EGFR TKI improved DFS. Recently Zhong et al reported a phase 2 trial on neo-adjuvant setting for EGFR mutant stage 3A resectable NSCLC. The ORR for neoadjuvant erlotinib versus GC chemotherapy was 54.1% vs 34.3% (p=0.092). Median DFS was significantly longer with erlotinib (21.5 months) vs GC chemotherapy (11.9 months; p=0.003). Definitely EGFR TKIs could be safely and effectively used in early resected NSCLC. The 3rd generation EGFR TKI – osimertinib in adjuvant setting also was tested. ALK is another important diver gene. ALINA trial–comparison alectinib with doublet chemotherapy after resection in NSCLC is ongoing. For wild type NSCLC some clinical trials on check point blockade in adjuvant setting are ongoing. BR.31 is an international intergroup trial. We expect the final result could answer whether duvarlumab could improve the OS for patients with early stage NSCLC. The characteristic of higher major pathological response, either check point inhibitor monotherapy or combination chemotherapy make neo-adjuvant immunotherapy with PD-l/L1 attractive treatment strategy for resectable even potential resectable NSCLC. In general peri-operative adjuvant treatment for NSCLC is becoming a hot research field.

Tepotinib in NSCLC patients with METex14 mutations: interim results from the phase II VISION study

Abstract Background Activating MET exon 14 skipping (METex14) mutations can be detected by liquid biopsy (LBx) or tumor biopsy (TBx) and are sensitive to MET inhibition. We report interim data from the phase II VISION study of tepotinib, a highly selective MET inhibitor, in NSCLC patients with METex14 mutations (NCT02864992). Methods Patients (pts) with advanced EGFR/ALK wild-type NSCLC harboring METex14 mutation identified by RNA-based testing with LBx or TBx (both ≥60 pts; overlap of LBx/TBx anticipated), enrolled at 113 sites in 8 countries (13 sites in Japan), receive tepotinib 500mg QD until progression, intolerable toxicity or withdrawal. Primary endpoint is ORR by independent review committee (IRC); secondary endpoints include investigator assessed ORR (INV) and safety. Results To date, 90 pts are enrolled, 15 are from Japan. At data cut-off (16/10/2018), in 35 evaluable LBx pts ORR (95% CI) was 51.4% (34.0, 68.6) by IRC with a median duration of response (mDoR) (range) of 9.8 months (1.1, 18.0) and 63.9% (46.2, 79.2) by INV with a mDOR of 17.1 months (1.3, 21.5). In 41 evaluable TBx pts, ORR was 41.5% (26.3, 57.9) by IRC and 58.5% (42.1, 73.7) by INV; mDoR was 12.4 months (1.1, 18.0; IRC) and 14.3 months (1.3, 21.5; INV). Of 11 evaluable Japanese pts, 6 were METex14 positive by LBx and 9 by TBx (overlap occurred). Confirmed responses were reported in 4/6 LBx pts and 3/9 TBx pts by IRC and in 4/6 LBx pts and 4/9 TBx pts by INV. Safety was evaluable in 69 pts. Main treatment-related adverse events (TRAEs; ≥15%) of any grade were peripheral edema (47.8%), diarrhea (18.8%), nausea (15.9%). The safety profile was similar in Japanese pts. TRAEs led to permanent discontinuation in 2 (2.9%) pts (1 ILD, 1 diarrhea & nausea). No TRAEs led to death. Conclusion In NSCLC pts with METex14 mutations, tepotinib had promising clinical efficacy. Similar activity was observed in Japanese pts. The safety profile was favorable. Recruitment, including a MET amplification cohort, is ongoing.

DYRK1A inhibition suppresses STAT3/EGFR/Met signalling and sensitizes EGFR wild-type NSCLC cells to AZD9291.

DYRK1A is considered a potential cancer therapeutic target, but the role of DYRK1A in NSCLC oncogenesis and treatment requires further investigation. In our study, high DYRK1A expression was observed in tumour samples from patients with lung cancer compared with normal lung tissues, and the high levels of DYRK1A were related to a reduced survival time in patients with lung cancer. Meanwhile, the DYRK1A inhibitor harmine could suppress the proliferation of NSCLC cells compared to that of the control. As DYRK1A suppression might be effective in treating NSCLC, we next explored the possible specific molecular mechanisms that were involved. We showed that DYRK1A suppression by siRNA could suppress the levels of EGFR and Met in NSCLC cells. Furthermore, DYRK1A siRNA could inhibit the expression and nuclear translocation of STAT3. Meanwhile, harmine could also regulate the STAT3/EGFR/Met signalling pathway in human NSCLC cells. AZD9291 is effective to treat NSCLC patients with EGFR‐sensitivity mutation and T790 M resistance mutation, but the clinical efficacy in patients with wild‐type EGFR remains modest. We showed that DYRK1A repression could enhance the anti‐cancer effect of AZD9291 by inducing apoptosis and suppressing cell proliferation in EGFR wild‐type NSCLC cells. In addition, harmine could enhance the anti‐NSCLC activity of AZD9291 by modulating STAT3 pathway. Finally, harmine could enhance the anti‐cancer activity of AZD9291 in primary NSCLC cells. Collectively, targeting DYRK1A might be an attractive target for AZD9291 sensitization in EGFR wild‐type NSCLC patients.

A plasma miRNA signature classifier identifies PD-L1 ≥ 50% NSCLC nonresponders to immune checkpoint inhibitors.

9096 Background: A sizable proportion of PD-L1≥50% NSCLC patients (pts) do not respond to single agent immune checkpoint inhibitors (ICI) and no biomarker is able to identify non responders. A three levels plasma microRNA signature classifier (MSC), reflecting an immunosuppressive profile of immune cell subsets, has already shown its ability to identify pts treated with ICI with a worse prognosis independently from PD-L1 expression. Aim of this trial is to prospectively test the ability of MSC to identify at diagnosis PD-L1≥50% non responders to ICI. Methods: we prospectively collected baseline plasma samples from 41 consecutive advanced EGFR/ALK/ROS1 wild-type NSCLC pts with PD-L1 TPS ≥50% treated with ICI as first (n = 28) or further line treatment to run the MSC molecular test. Pts were stratified in high (H) vs intermediate/low (I/L) risk levels. Overall response rate (ORR) and the relative risk of response (RR) were evaluated by 2x2 contingency table using Fisher exact test. Cox proportional hazard models were used to define crude and adjusted hazard ratio (HR). Results: According to RECIST 1.1 criteria, 14 (34%) pts respond to ICIs. With a global median follow-up of 9.8 months, median progression free survival (PFS) and overall survival (OS) were 7.9 months and not reached, respectively. Ten (24%) NSCLC pts were MSC H risk level. ORR was 0% in MSC H vs 45% in MSC I/L risk pts (RR = 0.10; 95%CI = 0.00-0.90; p = 0.0080). Median PFS was 11.4 months for MSC I/L pts vs 2.3 months for MSC H risk (HR = 0.26 95%CI = 0.11-0.62; p = 0.0021). Median OS was not reached for MSC I/L vs 2.7 months for MSC H risk pts (HR = 0.17 95%CI = 0.06-0.48; p = 0.0008). Data remained significant adjusting for age, sex, pack-years and ECOG performance status at the baseline: PFS HR = 0.24 (95%CI = 0.09-0.66; p = 0.0054) and OS HR = 0.15 (95%CI = 0.05-0.51; p = 0.0023). Conclusions: plasma MSC shows promising results for treatment selection with ICI. So far, MSC is the only molecular test able to identify a group of NSCLC pts with PD-L1≥50% who do not respond to single agent immunotherapy. Ongoing trials are validating these results and testing the possible predictive effect of MSC in chemotherapy plus immunotherapy combinations.

Soluble sPD-L1 and Serum Amyloid A1 as Potential Biomarkers for Lung Cancer.

SummaryBackgroundThe objective of this prospective study was to evaluate whether soluble programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) and serum amyloid A1 (SAA1) are potential diagnostic, predictive or prognostic biomarkers in lung cancer.MethodsLung cancer patients (n=115) with advanced metastatic disease, 101 with non-small cell lung cancer, NSCLC (77 EGFR wild-type NSCLC patients on chemotherapy, 15 EGFR mutation positive adenocarcinoma patients, 9 patients with mPD-L1 Expression ≥50% NSCLC – responders to immunotherapy), and 14 patients with small cell lung cancer (SCLC) were examined. ELISA method was used to determine sPD-L1 and SAA1 concentrations in patients’ plasma.ResultsSignificantly higher blood concentrations of sPD-L1 and SAA1 were noted in lung cancer patients compared with a healthy control group. In PD-L1+ NSCLC patients, a significantly higher sPD-L1 level was noticed compared to any other lung cancer subgroup, as well as the highest average SAA1 value compared to other subgroups.ConclusionsIt seems that sPD-1/PD-L1 might be a potential biomarker, prognostic and/ or predictive, particularly in patients treated with immunotherapy. Serum amyloid A1 has potential to act as a good predictor of patients’ survival, as well as a biomarker of a more advanced disease, with possibly good capability to predict the course of disease measured at different time points.

The Effect of LKB1 Activity on the Sensitivity to PI3K/mTOR Inhibition in Non–Small Cell Lung Cancer

IntroductionLiver kinase B1 (LKB1), also called serine/threonine kinase 11 (STK11), is a tumor suppressor that functions as master regulator of cell growth, metabolism, survival, and polarity. Approximately 30% to 35% of patients with NSCLC possess inactivated liver kinase B1 gene (LKB1), and these patients respond poorly to anti–programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) immunotherapy. Therefore, novel therapies targeting NSCLC with LKB1 loss are needed. MethodsWe used a new in silico signaling analysis method to identify the potential therapeutic targets and reposition drugs by integrating gene expression data with the Kyoto Encyclopedia of Genes and Genomes signaling pathways. LKB1 wild-type and LKB1-deficient NSCLC cell lines, including knockout clones generated by clustered regularly interspaced short pallindromic repeats–Cas9, were treated with inhibitors of mechanistic target of rapamycin kinase (mTOR) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and a dual inhibitor. ResultsIn silico experiments showed that inhibition of both mTOR and PI3K can be synergistically effective in LKB1-deficient NSCLC. In vitro and in vivo experiments showed the synergistic effect of mTOR inhibition and PI3K inhibition in LKB1-mutant NSCLC cell lines. The sensitivity to dual inhibition of mTOR and PI3K is higher in LKB1-mutant cell lines than in wild-type cell lines. A higher compensatory increase in Akt phosphorylation after rapamycin treatment of LKB1-deficient cells than after rapamycin treatment of LKB1 wild-type cells is responsible for the synergistic effect of mTOR and PI3K inhibition. Dual inhibition of mTOR and PI3K resulted in a greater decrease in protein expression of cell cycle–regulating proteins in LKB1 knockout cells than in LKB1 wild-type cells. ConclusionDual molecular targeted therapy for mTOR and PI3K may be a promising therapeutic strategy in the specific population of patients with lung cancer with LKB1 loss.

USP17 is required for trafficking and oncogenic signaling of mutant EGFR in NSCLC cells

BackgroundThe deubiquitinase USP17 is overexpressed in NSCLC and has been shown to be required for the growth and motility of EGFR wild-type (WT) NSCLC cells. USP17 is also required for clathrin-mediated endocytosis of EGFR. Here, we examine the impact of USP17 depletion on the growth, as well as EGFR endocytosis and signaling, of EGFR mutant (MT) NSCLC cells. In particular, we examine NSCLC cells harboring an EGFR activating exon 19 deletion (HCC827), or both the L858R activating mutation and the T790M resistance gatekeeper mutation (H1975) which renders them resistant to EGFR tyrosine kinase inhibitors (TKIs).MethodsMTT, trypan blue and clonogenic assays, confocal microscopy, Western blotting and cell cycle analysis were performed.ResultsUSP17 depletion blocks the growth of EGFRMT NSCLC cells carrying either the EGFR exon 19 deletion, or L858R/T790M double mutation. In contrast to EGFRWT cells, USP17 depletion also triggers apoptosis of EGFRMT NSCLC cells. USP17 is required for clathrin-mediated endocytosis in these EGFRMT NSCLC cells, but it is not required for the internalization of the mutated EGFR receptors. Instead, USP17 depletion alters the localization of these receptors within the cell, and although it does not decrease basal EGFR activation, it potently reduces activation of Src, a key kinase in mutant EGFR-dependent tumorigenicity. Finally, we demonstrate that USP17 depletion can trigger apoptosis in EGFRWT NSCLC cells, when combined with the EGFR tyrosine kinase inhibitor (TKI) gefitinib.ConclusionsOur data reveals that USP17 facilitates trafficking and oncogenic signaling of mutant EGFR and indicates targeting USP17 could represent a viable therapeutic strategy in NSCLC tumours carrying either an EGFR activating mutation, or a resistance gatekeeper mutation.

PC09.01 Debate 1: Early vs Delayed Treatment of Asymptomatic Brain Metastases in Wild-Type NSCLC - Early

PC09.01 Debate 1: Early vs Delayed Treatment of Asymptomatic Brain Metastases in Wild-Type NSCLC - Early