Supplementary Figure S1. Multi-institution cohort of patients with tumors harboring EGFR Exon 19 deletions. Primary analyses were performed on patients with tumors harboring E746_A750del and L747_A750>P, while investigation into additional uncommon variants was descriptive only. Patient numbers do not reflect the true prevalence of each variant since this cohort is selectively enriched for uncommon exon 19 deletions.

Supplementary Figure S2. Progression Free Survival for patients with tumors harboring select exon 19 deletions treated with first-line osimertinib. The point estimate for median progression free survival associated with each mutation is listed to the right of the figure legend.

Clinical and Molecular Characteristics of Cases of Acquired MET Amplification Identified by Next-Generation Sequencing after Selective RET Inhibitors

PURPOSE Anaplastic lymphoma kinase (ALK) is an established therapeutic target in non-small cell lung cancer (NSCLC), predominantly identified in adenocarcinomas. However, ALK rearrangements also occur in de novo squamous and adenosquamous NSCLCs and their clinicogenomic features remain poorly defined. METHODS This multi-institutional retrospective analysis included patients with advanced ALK+ NSCLC. Patients with de novo ALK+ squamous and adenosquamous NSCLCs were identified and compared with an ALK+ adenocarcinoma cohort treated with first-line (1L) alectinib. Overall survival (OS), time to progression (TTP), and time to treatment discontinuation (TTD) were analyzed using the Kaplan-Meier methodology. RESULTS Among 177 patients, 29 had ALK+ squamous (n = 17) and adenosquamous (n = 12) NSCLCs and 148 had ALK+ adenocarcinoma treated with 1L alectinib. Among patients receiving 1L alectinib, OS was significantly shorter for patients with adenosquamous (median, 31.0 months [95% CI, 17.0 to not reached {NR}]) and squamous (27.0 months [95% CI, 5.0 to 35.0]) tumors compared with that for patients with adenocarcinoma (median NR; median follow-up 51.2 months; P < .001). TTP was shorter for squamous (median, 8.0 months [95% CI, 2.0 to 12.0]) versus adenocarcinoma (median, 19.0 months [95% CI, 12.6 to 25.0]) cohorts (P < .001), although the adenosquamous cohort had comparable TTP (median, 20.0 months [95% CI, 9.4 to 30.6]) with the adenocarcinoma cohort (P = .70). TTD was significantly shorter for squamous (median, 9.5 months [95% CI, 1.5 to 13.0]) and adenosquamous (median, 20.0 months [95% CI, 3.0 to 24.0]) versus adenocarcinoma cohorts (52.3 months [95% CI, 40.5 to 57.5]; P < .001). Genomic profiling revealed more frequent TP53 (53% v 25%; P = .026), PDGFRA (16% v 0%; P = .009), KIT (11% v 0%; P = .045), PIK3CA (11% v 0%; P = .045), and MYC (11% v 0%; P = .045) coalterations in the squamous/adenosquamous cohort. CONCLUSION ALK+ squamous and adenosquamous NSCLCs are rare, but biologically distinct, with inferior outcomes on 1L ALK TKI, highlighting the need for further research to develop effective treatment strategies.

BACKGROUND:Although ALK-positive non-small cell lung cancer (NSCLC) derives limited benefit from immune checkpoint inhibitors, PD-L1 expression is frequently elevated at diagnosis. The clinical relevance of PD-L1 expression for the efficacy and durability of response to ALK tyrosine kinase inhibitors (TKIs) remains unclear. We investigated the association between tumor PD-L1 expression and outcomes with first-line ALK TKIs and used complementary preclinical models to explore potential biological mechanisms. METHODS:Clinical, pathologic, molecular, and treatment outcomes were evaluated among patients with ALK-rearranged NSCLC treated at the University of Colorado. PD-L1 expression was assessed by tumor proportion score (TPS). Murine EML4-ALK lung cancer cell lines were engineered to overexpress PD-L1, and effects on ALK TKI response were evaluated in vitro and in immune-competent mouse models. RESULTS:Among 130 TKI-naïve patients treated with first-line ALK TKIs, 57 (44%) had high PD-L1 expression (TPS ≥ 50%). After multivariable adjustment, high PD-L1 expression was associated with significantly shorter progression-free survival (PFS) compared with low PD-L1 expression (11 vs. 21 months; HR 2.29, 95% CI, 1.51-3.49; P ≤ .001), with no difference in overall survival (91 vs. 107 months; HR 1.16, 95% CI, 0.62-2.17). Objective response rates were similar between PD-L1-high and -low tumors (81.8% vs. 83.3%; Pearson r = -0.20, 95% CI, -0.40-0.07). In preclinical EML4-ALK models, tumor cell PD-L1 overexpression did not alter alectinib sensitivity, depth, or durability of response in vitro or in vivo CONCLUSIONS: High PD-L1 expression is associated with shorter PFS to first-line ALK TKIs without affecting the initial radiographic response. Preclinical findings suggest that PD-L1-intrinsic tumor cell signaling does not directly impair ALK TKI efficacy, implicating tumor microenvironment-mediated mechanisms in reduced response durability.

The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Small Cell Lung Cancer provide recommendations for diagnostic workup, staging, and treatment. These NCCN Guidelines Insights highlight recent updates, with a particular focus on changes to systemic therapies and radiation treatment in the NCCN Guidelines for Small Cell Lung Cancer.

Abstract Background: Immune checkpoint inhibition (ICI), alone and in combination with chemotherapy (ICI+C), has transformed the treatment landscape for non-small cell lung cancer (NSCLC). We have previously reported results from the myCare-040 study[1], where we validated an algorithm capable of distinguishing advanced NSCLC patients with favorable ICI+C benefit from those with no benefit. To understand whether the underlying molecular mechanisms used by the algorithm are conserved across disease stages, we have evaluated the algorithm in a cohort of patients with early stage NSCLC receiving adjuvant ICI or ICI + C. Design: The ΔTRI algorithm uses Cellworks’ computational model of a patient’s tumor genomics to predict biomarker changes related to disease progression and potential benefit from ICI+C therapy. The previously validated ΔTRI and clinical threshold (16) were evaluated in 51 non-squamous, early stage NSCLC patients (Stage I=20, Stage II=12, Stage IIIA=19) receiving adjuvant ICI or ICI+C ,with complete clinical and genomic information (Foundation One CDx) derived from the nationwide (US-based) de-identified ConcertAI Genomics360 database. Results: Patients in the ΔTRI High Benefit Group (ΔTRI ≥ 16, n = 11), had an incremental benefit in median OS of 19.4 months with the addition of chemotherapy to ICI (logrank p = 0.057, median OS ICI = 7 months vs ICI+C = 26.6 months). In contrast, patients in the ΔTRI No Benefit Group (ΔTRI < 16, n = 40) showed no improvement in OS when receiving ICI+C (logrank p = 0.84, median OS ICI = 13 months vs ICI+C = 9 months). A likelihood ratio test of interaction between the linear ΔTRI and treatment (ICI versus ICI+C) was significant (LR p = 0.038). Cut-point optimization for the early stage population (ΔTRI= 9) improved the logrank statistics in the High Benefit Group (ΔTRI ≥ 9; logrank p = 0.003). Conclusions: Although developed and validated in patients with advanced NSCLC, the ΔTRI also predicted incremental chemotherapy benefit in an real-world cohort of patients with early stage NSCLC receiving adjuvant ICI or ICI+C. Further work is needed to understand how these observations could be translated into clinical use. 1 Aggarawal et al, WCLC 2025 Citation Format: Prashant Nair, Kishor Promod, Ansu Kumar, Swati Khandelwal, Ambreen Ambreen, Susheel George, Mamatha Patil, Deepak Lala, Ashokraja Bala, Veena Balakrishnan, Shweta Kapoor, Drew Watson, James Wingrove, Tejas Patil. Computational modeling of comprehensive genomic profiling to predict chemo-immunotherapy benefit in early stage NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5252.

Abstract Background: Front-line genomic profiling in advanced NSCLC typically utilizes solid tissue, although assessment via liquid biopsy is becoming more frequent. In addition to detecting the presence of actionable mutations, genomic profiling can also be used to inform on the use of immune checkpoint inhibition (ICI), alone and in combination with chemotherapy (ICI+C). Using genomic profiling obtained from solid tissue (Foundation One CDx), we previously validated an algorithm capable of distinguishing advanced NSCLC patients with favorable ICI+C benefit from those with no benefit [1]. We have evaluated this algorithm in a cohort of advanced NSCLC patients receiving both liquid and solid tissue genomic profiling. Design: The ΔTRI algorithm uses Cellworks’ computational model of a patient’s tumor genomics to predict biomarker changes related to disease progression and potential benefit from ICI+C therapy. The previously validated ΔTRI and clinical threshold (ΔTRI score of 16, which equates to a 15% increase in 24 month OS when receiving ICI+C) were evaluated in 20 non-squamous, advanced NSCLC patients with complete clinical and genomic information (Foundation One Solid and Liquid), derived from the nationwide (US-based) de-identified ConcertAI Genomics360 database. Differences in solid and liquid-derived ΔTRI scores were assessed for association with clinical factors as well as genomic markers. Results: Mutational profiles were highly similar between both platforms (median Jaccard index = 0.84), with 20% (4/20) of the patients having a Jaccard index < 0.5. In the 16 patients with differences between solid and liquid aberrations, discordance was driven 81% of the time by novel mutations identified in the liquid samples. Despite these differences, the ΔTRI scores generated from liquid biopsy samples were significantly correlated with those generated from solid tissue (R2 = 0.61, p value < 0.001), with a median difference in ΔTRI score of 2.39, equating to roughly 2% difference in ICI+C benefit. Using the pre-defined clinical threshold of 16, 15% (n=3) of the samples switched from high ICI+C benefit category obtained from solid tissue (≥ 16 increase in 24 median OS with ICI+C) to a low/no benefit category ΔTRI score obtained with liquid samples, with an median difference in ΔTRI score of 7.4. Clinical factors such as the number of days between tissue and blood collection and number of metastatic sites were not associated with panel differences. Conclusions: Good similarity was observed in ΔTRI scores generated from liquid biopsy samples compared to scores generated from solid tissue, suggesting that with additional exploration liquid samples could be used for ΔTRI score generation. Discordant cases appeared to be driven by novel mutations identified via liquid biopsy. 1 Aggarawal et al, WCLC 2025 Citation Format: Charu Aggarwal, Prashant Nair, Anagha Jenu, Poornachandra G, Ansu Kumar, Swati Khandelwal, Jyoti Chauhan, Susheel George, Neelsh Lunkad, Vijayashree PS, Nagendra Prasad, Shweta Kapoor, Drew Watson, James Wingrove, Tejas Patil. Comparison of liquid versus solid tissue genomic profiling for the prediction of chemo-immunotherapy benefit in advanced NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2449.

Supplementary Figure S3. Progression Free Survival for patients with tumors harboring select exon 19 deletions treated with second or later line osimertinib (T790M+). The point estimate for median progression free survival associated with each mutation is listed to the right of the figure legend.

PURPOSE This observational study assessed the real-world characteristics, treatments, and outcomes of US patients with HER2-mutant advanced non-small cell lung cancer (NSCLC) overall and according to HER2 mutation type (tyrosine kinase domain [TKD] and non-TKD).METHODS Deidentified data were extracted for patients with advanced/metastatic NSCLC from the Flatiron Health-Foundation Medicine NSCLC Clinico-Genomic Database. Patients with oncogenic HER2 mutations were included. The primary objectives were to assess the prevalence of HER2 mutations and coaberrations, treatment patterns, and real-world overall survival (OS).RESULTS Overall, 559/14,768 (3.8%) patients had HER2 mutations; 262 (1.8%) were oncogenic. Patients with oncogenic TKD mutations (n = 197) were more frequently younger, female, and never-smokers than those with oncogenic non-TKD mutations (n = 65) and had fewer oncogenic coaberrations. Among patients with oncogenic HER2 mutations who underwent first-line treatment (n = 193), most received platinum-based chemoimmunotherapy (30.5%) or chemotherapy alone (27.9%); 119 patients (61.7%) received second-line treatment. Median OS after first and second lines of treatment was 13.5 months (95% CI, 11.6 to 16.9) and 11.1 months (95% CI, 9.2 to 13.6), respectively. Median OS with first-line platinum-based chemoimmunotherapy was 21.1 (95% CI, 12.2 to NA) and 11.7 months (95% CI, 8.3 to NA) in patients with TKD/non-TKD mutations, respectively, and median OS with platinum-based chemotherapy alone was 9.1 (95% CI, 5.7 to 16.0) and 17.3 (95% CI, 13.6 to NA) months, respectively.CONCLUSION NSCLC patients with oncogenic TKD HER2 mutations had different characteristics and genetic features than patients with non-TKD mutations. Real-world outcomes with first- and second-line standard-of-care treatment were suboptimal, highlighting the need for new treatment options for patients with advanced HER2-mutant NSCLC.

Clinical and Molecular Characteristics of Cases of RET Gain Identified by Next-Generation Sequencing after Selective RET Inhibitors

Purpose: Rearranged during transfection (RET) alterations are oncogenic drivers across solid tumors. Selective RET inhibitors (SRI) selpercatinib and pralsetinib have transformed outcomes for patients with RET-altered malignancies. Limited knowledge exists on genomic mechanisms of resistance to SRI.Experimental Design: We established "RETgistry," a global consortium of patients with advanced RET-altered solid tumors who received SRI and underwent postprogression tissue or plasma biopsies assessed by next-generation sequencing. Frequencies of secondary RET resistance mutations and acquired non-RET gene alterations were determined. Progression-free survival (PFS) and time to treatment discontinuation (TTD) on first SRI were estimated with the Kaplan-Meier method.Results: RETgistry included 109 patients with RET-altered advanced solid tumors (lung, n = 94; thyroid, n = 15) who underwent 143 post-SRI progression biopsies (tissue, 91; plasma, 52). The median PFS and TTD were 13.9 months [95% confidence interval (CI), 10.1-16.6] and 17.3 months (95% CI, 14-20.2), respectively. Secondary RET mutations were detected in 20 (14%) biopsies [lung cancer, 15 (12.4%) and thyroid carcinoma, 5 (22.7%)]. Common acquired off-target alterations involved MET (18.2%; amplification, 15%), TP53 (8.2%), APC (7.6%), KRAS (7.1%), KEAP1 (5.9%), and CDKN2A/B (5.3%). MET alterations were enriched in post-SRI versus pre-SRI specimens (full cohort, 17.6% vs. 2.0%, P = 0.022; lung cancer, 19.1% vs. 2.1%, P = 0.022).Conclusions: The prevalence of secondary RET mutations after SRI was low, underscoring a greater role for off-target resistance. Recurrent acquired alterations involving tumor suppressor genes or upstream regulators of MAPK and PI3K pathways were identified, most commonly MET amplification. Continued efforts to characterize SRI resistance biology are critical to guide the development of novel therapeutic strategies.

Oncogenic RET gene rearrangements drive a subset of lung adenocarcinomas (LUAD) and the tyrosine kinase inhibitors (TKIs) selpercatinib and pralsetinib are approved therapeutics. However, acquired resistance remains a hurdle to durable management. Two RET+ lung cancer cell lines (TR.1, TR.2) were established from a Trim24-Ret mouse model and lung tumors resulting from their orthotopic transplantation initially responded to selpercatinib followed by prompt progression within ∼3 weeks of initiating TKI treatment. Cell lines derived from the selpercatinib-resistant TR.1 and TR.2 tumors exhibited in vitro sensitivity to MET and ERBB-targeted TKIs, indicating acquired bypass signaling through these receptor tyrosine kinases (RTKs). The TKI-resistant cell lines showed no evidence for MET gene amplification, but exhibited transcriptional induction of genes that function within MET and ERBB2:ERBB4 interaction networks including ligands (HGF, NRG1), adaptors (GAB1) and co-receptors (NRP1). Exogenous HGF, but not NRG1 reversed in vitro growth inhibition by selpercatinib in TR.1 and TR.2 cells. Mice bearing orthotopic TR.1 or TR.2 lung tumors progressing on selpercatinib underwent significant re-shrinkage upon co-treatment with the MET inhibitor, crizotinib, although similar to the clinical experience, progression again occurred. By contrast, upfront treatment with selpercatinib and crizotinib in orthotopic tumors yielded complete elimination of 7 of 9 TR.1 tumors and both deepened and prolonged the duration of response in TR.2 tumors. The findings provide new insight into mechanisms of acquired resistance through bypass signaling and highlight the therapeutic benefit of simultaneous upfront blockade of driver oncogenes and dominant resistance mechanisms in LUAD.

The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Non-Small Cell Lung Cancer (NSCLC) provide recommendations for the treatment of patients with NSCLC, including diagnosis, primary disease management, surveillance, and subsequent treatment. The panel has updated the list of recommended targeted therapies based on recent FDA approvals and clinical data. This selection from the NCCN Guidelines for NSCLC focuses on treatment recommendations for advanced or metastatic NSCLC with actionable biomarkers.

Supplementary Table S1. Descriptive analysis of patient characteristics and outcomes for those with tumors harboring uncommon exon 19 deletions treated with osimertinib. Each variant represented ≤2.5% of the study cohort. Patients with + for “Smoking History” are those with either a prior or current smoking history. PFS=Progression free survival; OS= Overall survival; à denotes patients who have not yet experienced an event for a time to event endpoint.