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Recent studies have linked elevated tumor aneuploidy to anti-tumor immune suppression and adverse survival following immunotherapy. Herein, we provide supportive evidence for tumor aneuploidy as a biomarker of response to immunotherapy in patients with non-small cell lung cancer (NSCLC). We identify a dose-response relationship between aneuploidy score and patient outcomes. In two independent NSCLC cohorts (n = 659 patients), we demonstrate a novel association between elevated aneuploidy and non-smoking-associated oncogenic driver mutations. Lastly, we report enrichment of TERT amplification and immune-suppressive phenotypes of highly aneuploid NSCLC. Taken together, our findings emphasize a potentially critical role for tumor aneuploidy in guiding immunotherapy treatment strategies., (© 2024. The Author(s).)

Purpose: Although immune checkpoint inhibitors (ICI) have extended survival in patients with non-small-cell lung cancer (NSCLC), acquired resistance (AR) to ICI frequently develops after an initial benefit. However, the mechanisms of AR to ICI in NSCLC are largely unknown., Methods: Comprehensive tumor genomic profiling, machine learning-based assessment of tumor-infiltrating lymphocytes, multiplexed immunofluorescence, and/or HLA-I immunohistochemistry (IHC) were performed on matched pre- and post-ICI tumor biopsies from patients with NSCLC treated with ICI at the Dana-Farber Cancer Institute who developed AR to ICI. Two additional cohorts of patients with intervening chemotherapy or targeted therapies between biopsies were included as controls., Results: We performed comprehensive genomic profiling and immunophenotypic characterization on samples from 82 patients with NSCLC and matched pre- and post-ICI biopsies and compared findings with a control cohort of patients with non-ICI intervening therapies between biopsies (chemotherapy, N = 32; targeted therapies, N = 89; both, N = 17). Putative resistance mutations were identified in 27.8% of immunotherapy-treated cases and included acquired loss-of-function mutations in STK11 , B2M , APC , MTOR , KEAP1 , and JAK1 / 2 ; these acquired alterations were not observed in the control groups. Immunophenotyping of matched pre- and post-ICI samples demonstrated significant decreases in intratumoral lymphocytes, CD3e + and CD8a + T cells, and PD-L1-PD1 engagement, as well as increased distance between tumor cells and CD8 + PD-1 + T cells. There was a significant decrease in HLA class I expression in the immunotherapy cohort at the time of AR compared with the chemotherapy ( P = .005) and the targeted therapy ( P = .01) cohorts., Conclusion: These findings highlight the genomic and immunophenotypic heterogeneity of ICI resistance in NSCLC, which will need to be considered when developing novel therapeutic strategies aimed at overcoming resistance.

Introduction: Although gene-level copy number alterations have been studied as a potential biomarker of immunotherapy efficacy in NSCLC, the impact of aneuploidy burden and chromosomal arm-level events on immune checkpoint inhibitor (ICI) efficacy in NSCLC is uncertain., Methods: Patients who received programmed cell death protein 1 or programmed death-ligand 1 (PD-L1) inhibitor at two academic centers were included. Across all 22 chromosomes analyzed, an arm was considered altered if at least 70% of its territory was either gained or deleted. Among nonsquamous NSCLCs which underwent targeted next-generation sequencing, we retrospectively quantified aneuploidy using the adjusted fraction of chromosomal arm alterations (FAA), defined as the number of altered chromosome arms divided by the number of chromosome arms assessed, adjusted for tumor purity., Results: Among 2293 nonsquamous NSCLCs identified, the median FAA increased with more advanced cancer stage and decreased with higher PD-L1 tumor proportion score (TPS) levels (median FAA in TPS < 1%: 0.09, TPS 1%-49%: 0.08, TPS ≥ 50%: 0.05, p < 0.0001). There was a very weak correlation between FAA and tumor mutational burden when taken as continuous variables (R: 0.07, p = 0.0005). A total of 765 advanced nonsquamous NSCLCs with available FAA values were treated with ICIs. With decreasing FAA tertiles, there was a progressive improvement in objective response rate (ORR 15.1% in upper tertile versus 23.2% in middle tertile versus 28.4% in lowest tertile, p = 0.001), median progression-free survival (mPFS 2.5 versus 3.3 versus 4.1 mo, p < 0.0001), and median overall survival (mOS 12.5 versus 13.9 versus 16.4 mo, p = 0.006), respectively. In the arm-level enrichment analysis, chromosome 9p loss (OR = 0.22, Q = 0.0002) and chromosome 1q gain (OR = 0.43, Q = 0.002) were significantly enriched in ICI nonresponders after false discovery rate adjustment. Compared with NSCLCs without chromosome 9p loss (n = 452), those with 9p loss (n = 154) had a lower ORR (28.1% versus 7.8%, p < 0.0001), a shorter mPFS (4.1 versus 2.3 mo, p < 0.0001), and a shorter mOS (18.0 versus 9.6 mo, p < 0.0001) to immunotherapy. In addition, among NSCLCs with high PD-L1 expression (TPS ≥ 50%), chromosome 9p loss was associated with lower ORR (43% versus 6%, p < 0.0001), shorter mPFS (6.4 versus 2.6 mo, p = 0.0006), and shorter mOS (30.2 versus 14.3 mo, p = 0.0008) to immunotherapy compared with NSCLCs without 9p loss. In multivariable analysis, adjusting for key variables including FAA, chromosome 9p loss, but not 1q gain, retained a significant impact on ORR (hazard ratio [HR] = 0.25, p < 0.001), mPFS (HR = 1.49, p = 0.001), and mOS (HR = 1.47, p = 0.003). Multiplexed immunofluorescence and computational deconvolution of RNA sequencing data revealed that tumors with either high FAA levels or chromosome 9p loss had significantly fewer tumor-associated cytotoxic immune cells., Conclusions: Nonsquamous NSCLCs with high aneuploidy and chromosome 9p loss have a distinct tumor immune microenvironment and less favorable outcomes to ICIs., (Copyright © 2023 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Purpose: Molecular factors predicting relapse in early-stage non-small-cell lung cancer (ES-NSCLC) are poorly understood, especially in inoperable patients receiving radiotherapy (RT). In this study, we compared the genomic profiles of inoperable and operable ES-NSCLC., Materials and Methods: This retrospective study included 53 patients with nonsquamous ES-NSCLC (stage I-II) treated at a single institution (University of Chicago) with surgery (ie, operable; n = 30) or RT (ie, inoperable; n = 23) who underwent tumor genomic profiling. A second cohort of ES-NSCLC treated with RT (Stanford, n = 39) was included to power clinical analyses. Prognostic gene alterations were identified and correlated with clinical variables. The primary clinical end point was the correlation of prognostic genes with the cumulative incidence of relapse, disease-free survival, and overall survival (OS) in a pooled RT cohort from the two institutions (N = 62)., Results: Although the surgery cohort exhibited lower rates of relapse, the RT cohort was highly enriched for somatic STK11 mutations (43% v 6.7%). Receiving supplemental oxygen (odds ratio [OR] = 5.5), 20+ pack-years of tobacco smoking (OR = 6.1), and Black race (OR = 4.3) were associated with increased frequency of STK11 mutations. In the pooled RT cohort (N = 62), STK11 mutation was strongly associated with inferior oncologic outcomes: 2-year incidence of relapse was 62% versus 20% and 2-year OS was 52% versus 85%, remaining independently prognostic on multivariable analyses (relapse: subdistribution hazard ratio = 4.0, P = .0041; disease-free survival: hazard ratio, 6.8, P = .0002; OS: hazard ratio, 6.0, P = .022). STK11 mutations were predominantly associated with distant failure, rather than local., Conclusion: In this cohort of ES-NSCLC, STK11 inactivation was associated with poor oncologic outcomes after RT and demonstrated a novel association with clinical hypoxia, which may underlie its correlation with medical inoperability. Further validation in larger cohorts and investigation of effective adjuvant systemic therapies may be warranted.

Over 500 clinical trials are investigating combination radiotherapy and immune checkpoint blockade (ICB) as cancer treatments; however, the majority of trials have found no positive interaction. Here we perform a comprehensive molecular analysis of a randomized phase I clinical trial of patients with non-small cell lung cancer (NSCLC) treated with concurrent or sequential ablative radiotherapy and ICB. We show that concurrent treatment is superior to sequential treatment in augmenting local and distant tumor responses and in improving overall survival in a subset of patients with immunologically cold, highly aneuploid tumors, but not in those with less aneuploid tumors. In addition, radiotherapy alone decreases intratumoral cytotoxic T cell and adaptive immune signatures, whereas radiotherapy and ICB upregulates key immune pathways. Our findings challenge the prevailing paradigm that local ablative radiotherapy beneficially stimulates the immune response. We propose the use of tumor aneuploidy as a biomarker and therapeutic target in personalizing treatment approaches for patients with NSCLC treated with radiotherapy and ICB., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Immune checkpoint blockade (ICB) improves outcomes in non-small cell lung cancer (NSCLC) though most patients progress. There are limited data regarding molecular predictors of progression. In particular, there is controversy regarding the role of CDKN2A loss-of-function (LOF) in ICB resistance. We analyzed 139 consecutive patients with advanced NSCLC who underwent NGS prior to ICB initiation to explore the association of CDKN2A LOF with clinical outcomes. 73% were PD-L1 positive (≥ 1%). 48% exhibited high TMB (≥ 10 mutations/megabase). CDKN2A LOF was present in 26% of patients and was associated with inferior PFS (multivariate hazard ratio [MVA-HR] 1.66, 95% CI 1.02-2.63, p = 0.041) and OS (MVA-HR 2.08, 95% CI 1.21-3.49, p = 0.0087) when compared to wild-type (WT) patients. These findings held in patients with high TMB (median OS, LOF vs. WT 10.5 vs. 22.3 months; p = 0.069) and PD-L1 ≥ 50% (median OS, LOF vs. WT 11.1 vs. 24.2 months; p = 0.020), as well as in an independent dataset. CDKN2A LOF vs. WT tumors were twice as likely to experience disease progression following ICB (46% vs. 21%; p = 0.021). CDKN2A LOF negatively impacts clinical outcomes in advanced NSCLC treated with ICB, even in high PD-L1 and high TMB tumors. This novel finding should be prospectively validated and presents a potential therapeutic target., (© 2021. The Author(s).)

Introduction: The 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., Methods: We 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)., Results: Of 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., Conclusions: Although 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., (Copyright © 2021 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Purpose: DNA damage response and repair (DDR) gene alterations are associated with increased tumor-infiltrating lymphocytes, higher genomic instability, and higher tumor mutational burden (TMB) in cancer. Whether DDR alterations are associated with clinical outcomes to programmed death ligand 1 [PD-(L)1] blockade in non-small cell lung cancer (NSCLC) is unknown., Experimental Design: Tumors from patients treated with PD-(L)1 inhibitors were analyzed using targeted next-generation sequencing (NGS). Cancers were categorized on the basis of the presence or absence of deleterious mutations across a panel of 53 DDR genes. Clinical outcomes to PD-(L)1 inhibitors were evaluated according to DDR mutation status., Results: Of 266 patients with successful NGS who received PD-(L)1 inhibitors, 132 (49.6%) were identified as having deleterious DDR mutations (DDR-positive). DDR-positive and DDR-negative groups were similar in terms of baseline clinicopathologic characteristics. The median TMB was significantly higher in the DDR-positive group compared with the DDR-negative group (12.1 vs. 7.6 mutations/megabase; P < 0.001). Compared with DDR-negative patients ( N = 134), DDR-positive patients had a significantly higher objective response rate (30.3% vs. 17.2%; P = 0.01), longer median progression-free survival [PFS; 5.4 vs. 2.2 months; HR, 0.58 (95% confidence interval (CI), 0.45-0.76); P < 0.001], and longer median overall survival [OS; 18.8 vs. 9.9 months; HR, 0.57 (95% CI, 0.42-0.77); P < 0.001] with PD-(L)1 therapy. After adjusting for PD-L1, TMB, performance status, tobacco use, and line of therapy, DDR-positive status was associated with a significantly longer PFS [HR, 0.68 (95% CI, 0.51-0.92); P = 0.01] and OS [HR, 0.60 (95% CI, 0.43-0.85); P = 0.004] in multivariate analysis., Conclusions: Deleterious DDR mutations are frequent in NSCLC and are associated with improved clinical outcomes in patients with NSCLC treated with PD-(L)1 blockade., (©2020 American Association for Cancer Research.)

Purpose: Molecular mechanisms of acquired resistance to MET tyrosine kinase inhibitors (TKI) are poorly understood. We aimed to characterize the genomic mechanisms of resistance to type I and type II MET TKIs and their impact on sequential MET TKI therapy outcomes in patients with metastatic MET exon 14-mutant NSCLC., Experimental Design: Genomic alterations occurring at the time of progression on MET TKIs were studied using plasma and tissue next-generation sequencing (NGS)., Results: A total of 20 patients had tissue or plasma available for analysis at the time of acquired resistance to a MET TKI. Genomic alterations known or suspected to be mechanisms of resistance were detected in 15 patients (75%). On-target acquired mechanisms of resistance, including single and polyclonal MET kinase domain mutations in codons H1094, G1163, L1195, D1228, Y1230, and high levels of amplification of the MET exon 14-mutant allele, were observed in 7 patients (35%). A number of off-target mechanisms of resistance were detected in 9 patients (45%), including KRAS mutations and amplifications in KRAS, EGFR, HER3 , and BRAF ; one case displayed both on- and off-target mechanisms of resistance. In 2 patients with on-target resistant mutations, switching between type I and type II MET TKIs resulted in second partial responses., Conclusions: On-target secondary mutations and activation of bypass signaling drive resistance to MET TKIs. A deeper understanding of these molecular mechanisms can support the development of sequential or combinatorial therapeutic strategies to overcome resistance., (©2020 American Association for Cancer Research.)