Whole Transcriptome Sequencing (WTS) is a comprehensive alternative to targeted panels for detecting gene fusions and splice variants. To integrate WTS into clinical diagnostics, we compared its performance against established fusion assays (Archer FusionPlex and TSO500 RNA). WTS was evaluated in an initial cohort of 64 FFPE tumor samples, and quality control (QC) thresholds were defined based on missed fusions correlating with low tumor cell content (TCC < 40%). Key QC metrics included TCC ≥ 40%, RNA input ≥50 ng, ≥50 million reads, and median insert size >100 bp. WTS identified 92% of known fusions in the initial cohort. Validation in 357 samples showed 100% concordance with panel-based results when QC thresholds were met. Subsequent clinical deployment across 812 diverse tumor cases detected 121 fusions, though 423 (34%) required fallback to targeted assays due to low TCC. WTS provided added value by detecting novel fusions, pathogens, and enabling oncogenic pathway analysis. WTS is a reliable and informative method for fusion and splice variant detection in clinical diagnostics, provided rigorous pre-analytical and sequencing QC metrics are strictly applied.

e20751 Background: Tepotinib demonstrated clinical efficacy in NSCLC patients with MET ex14 skipping alterations in the single-arm VISION trial. Comparative effectiveness estimates against the contemporary therapeutic option of immunotherapy (IO) with or without chemotherapy (CT), were generated using external data. Methods: Eight international real-world datasets, which included patients who started treatment between 2010 and 2022 were pooled to form a real-world cohort. Inclusion and exclusion criteria based on VISION were applied to ensure generalizability and propensity scoring used to address differences in observed characteristics, stratifying by previous treatment status. Variables used were mean age, advanced vs. metastatic disease, sex, adenocarcinoma histology, and smoking history. Reweighted data were used to facilitate comparisons of real-world progression-free (PFS) and overall survival (OS) outcomes between therapy regimens in the first line (1L), second or later line (2L+), or line agnostic setting (LAs). Results: TOGETHER yielded information on 45 and 24 patients receiving IO or IO+CT in 1L, 83 and 6 patients in 2L+, respectively. In LAs 146 patients received IO±CT. TOGETHER patients were reweighted to match baseline characteristics of patients in VISION. As shown in the table (survival time in months), in 1L median (m) PFS was longest and the 24-month survival proportion was highest for tepotinib, compared to IO and IO+CT. In 2L+, due to the single digit sample size for those receiving IO+CT, only IO and IO±CT could be compared, with tepotinib showing longer mPFS and higher 24-month PFS. Due to the effects of prior or subsequent treatment lines which cannot be accounted for, 1L and 2L+ OS estimates are confounded; this notwithstanding, analyses of LAs showed the following: mOS for tepotinib was 19.2 months, compared to 19.0 for IO and 16.7 for IO±CT. Conclusions: Although MET ex14 skipping alterations are rare in NSCLC, pooling real-world datasets provided sufficient patient numbers to allow for comparative effectiveness estimates. In propensity score weighted analyses, tepotinib demonstrated longer PFS versus IO, as monotherapy or in combination with CT. The short PFS across comparators underlines the poor prognosis for patients with existing therapies, while also confirming tepotinib as an effective treatment option in this rare, biomarker-driven NSCLC subtype. Tepotinib IO IO+CT IO±CT 1L n 164 45 24 67 mPFS [95% CI] 8.7 [8.2, 12.6] 5.2 [2.7, 12.4] 8.0 [3.0, 17.5] 4.3 [2.7, 9.7] 24-month PFS 30% 13% Not reached 8% 2L+ n 149 83 6 88 mPFS [95% CI] 8.3 [7.0, 11.0] 4.8 [3.2, 7.7] - 4.8 [3.2, 7.7] 24-month PFS 19% 12% - 11% Line agnostic n 313 121 29 146 mOS [95% CI] 19.2 [16.3, 22.3] 19.0 [14.9, 22.4] 13.6 [12.3, 23.1] 16.7 [13.7, 21.7] 24-month OS 41% 33% 7% 32%

BACKGROUND:Molecular testing in NSCLC is essential for treatment selection, yet routine implementation remains inconsistent across institutions. Clinical evidence suggests that variability in testing may not be explained by patient or tumor characteristics but might be driven by institutional factors, potentially leading to adverse outcomes. We examined the extent to which variability in AGA testing is attributable to the treating institution. METHODS:We analyzed 6437 adults with stage IIIB/C or IV NSCLC enrolled in the prospective German real-world registry CRISP (2016-2022). Logistic mixed-effects models with AGA testing as the primary outcome were used to determine institutional variability across 171 institutions. Models included patient, tumor, and treatment-related fixed effects with institutions as random effects. Intraclass correlations (ICC) quantified institutional variability unexplained by other covariates. Institution type was tested in secondary analysis, and overall survival in exploratory analysis. FINDINGS:AGA testing was performed in 77.9 % of patients (n = 5016). Predicted probabilities for testing use ranged from 30.5 % to 93.2 % across institutions. Institutions significantly influenced testing use (p < 0.001), accounting for 21.4 % of the total variance. Variability significantly differed by institution type and was more pronounced in subgroups, e.g., squamous histology (ICC 29.5 %) and KRAS testing (ICC 34.4 %). Absence of AGA testing was independently associated with inferior survival (HRadj 1.11, 95 % CI 1.01-1.23, p = 0.029). INTERPRETATION:Substantial institutional variability exists in AGA testing for NSCLC, which was unexplained by patient or tumor characteristics. This objective evaluation of institution-based variability in testing may emphasize the importance of practice patterns on patient care and may therefore provide an avenue for change.

BACKGROUND:How frailty limits initial therapy in metastatic non-small-cell lung cancer (mNSCLC) remains poorly understood and was investigated in this study. METHODS:We retrospectively analyzed 2592 consecutive patients with mNSCLC treated between 2018-2023. RESULTS:Systemic therapy was initiated in 74% of patients with PD-L1 0-49% (n = 1306) vs. 79% with PD-L1 ≥50% (n = 507, p = 0.014), in whom availability of monoimmunotherapy reduced best supportive care (16.4%vs. 22.3%, p = 0.0002), while early death remained unchanged (ca. 4%). 70% of patients receiving mBSC were initially treatable but suffered early deterioration associated with comorbidities, metastatic burden, or protracted workup (p < 0.001). The atezolizumab Summary of Product Characteristics (SmPC) criteria, i.e. >80 years, or ECOG performance status (PS) ≥3, or comorbidities with PS ≥2 or age ≥70, were fulfilled by 38% (n = 501) and associated with 3-fold higher risk of death without therapy (230/501) compared to non-SmPC patients (p < 0.001). Under platinum, SmPC patients showed higher toxicity and shorter survival than non-SmPC patients, which for a platinum dose ratio ≤60% across 4 cycles (9% of 1306) resembled that with single-agent chemotherapy (median 5.1 months). SmPC criteria correlated with platinum use stronger than comorbidity scores, but predictability for individual patients remained modest (AUC 0.71, p < 0.001). CONCLUSIONS:The high pretherapeutic attrition of 26% in mNSCLC improved with availability of monoimmunotherapy, but requires more efficient, faster patient workflows for further mitigation. Adoption of the SmPC criteria could support identification of patients at higher risk for mBSC or potential platinum overtreatment to enhance utilization of novel platinum-free first-line options.

Background Small-cell lung cancer (SCLC) is an aggressive malignancy accounting for approximately 15% of all lung cancers, with over 70% of patients diagnosed at the extensive stage (ES). Phase III studies have shown significantly improved overall survival (OS) with the addition of PD-(L)1 inhibitors to platinum-based chemotherapy (CT), establishing chemoimmunotherapy (CIT) as the new first-line standard. This study investigates the real-world efficacy of CIT compared to alternative first-line regimens in patients with ES SCLC. Methods In this retrospective study, we analyzed 904 patients with newly diagnosed ES SCLC treated at a specialized thoracic cancer center between 2010 and 2022. The primary endpoints were OS and progression-free survival (PFS). Secondary endpoints included the impact of baseline brain metastases (BM), brain and thoracic radiotherapy (RT), and immune-related adverse events (irAEs). Results CIT (n = 203) resulted in longer OS than both platinum-based CT (n = 530) and nonplatinum CT (n = 171; median OS 10.2 vs. 9.4 vs. 3.6 months, P < .001). PFS under CIT and platinum-based CT was significantly longer compared to nonplatinum CT (median PFS 5.3 vs. 5.6 vs. 2.6 months, P < .001). Compared to platinum-based CT, CIT significantly reduced the risk of death or progression (OS HR 0.73, 95% CI, 0.62-0.87; P < .001; PFS HR 0.79, 95% CI, 0.67-0.93; P = .006), whereas nonplatinum CT was associated with a 2x higher risk (OS HR 2.19, 95% CI, 1.84-2.61; PFS HR 2.28, 95% CI, 1.91-2.72; both P < .001). Thoracic RT was an independent predictor of longer survival across the entire cohort (OS HR 0.57, 95% CI, 0.48-0.69; P < .001 in multivariable analysis [MVA] including sex, age, ECOG performance status, type of systemic treatment, and presence of baseline BM). While the presence of BM at diagnosis was prognostically neutral, brain RT among patients with baseline BM was also associated with longer survival (OS HR 0.47, 95% CI, 0.35-0.65; P < .001 in MVA). Immune-related adverse events (iAEs) were associated with longer OS and PFS (OS HR 0.67, 95% CI, 0.46-0.97; P = .032; PFS HR 0.68, 95% 0.47-0.97; P = .035 in MVA), while treatment discontinuation due to irAEs did not compromise survival. Conclusion Chemoimmunotherapy confers a clear OS advantage over conventional chemotherapy in real-world ES-SCLC. Local radiotherapy is associated with additional survival improvement, while the occurrence of irAEs may correlate with treatment efficacy.

BACKGROUND/OBJECTIVES:Following findings of the PACIFIC trial, consolidation therapy with durvalumab was established as standard of care for patients with unresectable, stage III non-small cell lung cancer (NSCLC) and no disease progression after chemoradiotherapy (CRT). This study evaluated real-world effectiveness of durvalumab consolidation in clinical routine in Germany. METHODS:Patients with unresectable, stage III NSCLC enrolled in the prospective, multicenter CRISP registry (August 2018-June 2023) were classified into two subgroups: treated with/without durvalumab consolidation therapy. Following stabilized inverse probability of treatment weighting, progression-free survival (PFS) and overall survival (OS) were assessed. RESULTS:This analysis included 231 patients, 124 with and 107 without durvalumab consolidation therapy. In the two subgroups (with/without durvalumab), median age was 67.2/64.6 years, 51.6%/47.7% were female, and 83.1%/86.0% had Eastern Cooperative Oncology Group performance status ≤1. Most patients (87%) received concurrent CRT. In the weighted analysis, median PFS was significantly longer with durvalumab consolidation (18.6 months, 95% CI [12.8, 24.5] vs. 7.5 months, 95% CI [5.5, 8.4]; HR 0.52, 95% CI [0.37, 0.73]); median OS was numerically longer but did not reach statistical significance (34.4 months, 95% CI [25.4, not available/not reached] vs. 27.5 months, 95% CI [17.4, 59.6]; HR 0.67, 95% CI [0.44, 1.02]). CONCLUSIONS:This real-world study confirmed significant PFS improvements with durvalumab consolidation in unresectable, stage III NSCLC, with effect sizes comparable to PACIFIC. Although OS differences were not statistically significant, these findings provide valuable real-world evidence supporting the effectiveness of durvalumab consolidation in routine clinical practice.

e20749 Background: MET ex14 skipping alterations in non-small cell lung cancer (NSCLC), recognized as important drivers of tumorigenesis, present in 3-4% of cases and are associated with poor prognosis. While case series suggest worse real-world outcomes in patients with MET ex14 skipping alterations compared to the broader NSCLC population, there is limited comparative effectiveness evidence on these outcomes for standard treatments prior to the first regulatory approvals of targeted therapy with selective MET receptor tyrosine kinase inhibitors (MET TKIs). Methods: Patient-level data from eight international real-world datasets of MET ex14 skipping NSCLC patients covering the period from 2010 to 2022 were pooled, applying inclusion/exclusion criteria consistent with the tepotinib MET TKI VISION trial. Propensity score (PS) weighting (based on clinical input) was applied to perform comparisons of real-world progression-free survival (PFS) and overall survival (OS) in four treatment classes: chemotherapy (CT), immunotherapy monotherapy (IO), IO plus chemotherapy (IO+CT), and an unapproved TKI (crizotinib). Results: Ultimately, 406 patients, treated across 661 lines of therapy, were incorporated into the analyses. Median PFS was, in months (95% CI): CT 4.8 (3.9–7.3), IO 5.7 (3.2, 10.7), IO+CT 4.9 (3.6, not evaluable), crizotinib 7.4 (4.7, 11.6). Median OS ranged from 11.9 months (8.4, 19.7) for crizotinib, to 18.4 months (11.0, 32.9) for IO. However, OS estimates are confounded by the effects of subsequent treatments and susceptible to immortal time bias due to the retrospective nature of data. Results were consistent across sensitivity analyses, including stratification by treatment line, disease subtype, and bootstrapping by sequential exclusion of datasets. Conclusions: Pooled real-world datasets of traditional therapies for MET ex14 skipping NSCLC demonstrated limited effectiveness. Median PFS was similar across treatment classes. IO (whether alone, or in combination with CT) did not demonstrate its clinical advantage in PFS or OS outcomes generally seen in the wider NSCLC population, while crizotinib did not outperform chemotherapy. These uniformly poor outcomes illustrate the need for further targeted treatment options in MET ex14 skipping alterations NSCLC. Confounding of OS, in part due to the use of subsequent therapies forms an important limitation of the analyses.

Abstract Introduction: Overcoming immunotherapy (IO) failure in advanced NSCLC requires distinguishing molecular features of acquired vs primary resistance. Here we used comprehensive multi-omic analyses to define resistance mechanisms. Methods: Analyses were performed on the largest-to-date IO resistance cohort: 1819 biospecimens from 892 patients with NSCLC (371 primary, 521 acquired) from the Phase 2 HUDSON study (NCT03334617) of combination regimens after progression on anti-PD-(L)1/chemotherapy. Targeted next-generation sequencing of unpaired tumor biopsies pre- (n=226) and post-IO (n=497) was used to profile mutation signatures, clonality, aneuploidy and genomic instability. Post-IO tumor burden was assessed by plasma ctDNA (n=445); peripheral T-cell repertoires were analyzed (n=335). Bulk (n=205) and single-cell (sc, n=75) RNA sequencing (RNAseq) of unpaired post-IO tumor biopsies enabled gene set enrichment analysis (GSEA) and high-resolution cell type annotation. Results: Comprehensive analyses revealed acquisition of sub-clonal genomic alterations at the time of acquired resistance. Despite lower systemic ctDNA burden at progression, activating FGF10 and RICTOR and inactivating RBM10 and MSH6 mutations were enriched in acquired vs primary resistance (false discovery rate [FDR] p<0.05). Post-IO tumors harbored more CDK4, CDK6, and CD22 activating mutations, and inactivating mutations in KDM6A, SMARCA4, and CDKN2A (FDR p<0.05). Genomic instability (increased homologous recombination deficiency signatures, elevated large-scale transitions, telomeric allelic imbalance) was noted in both primary and acquired resistant tumors. Bulk RNAseq GSEA detected upregulation of epithelial-to-mesenchymal transition (EMT), IFNγ response, and inflammatory pathways (FDR p<0.05) in acquired vs primary resistant tumors. Single-cell transcriptomics showed enrichment of tumor-reactive, tissue-resident memory CD8+ T-cell clusters in acquired resistant tumors. Notably, a naïve/stem-like CD8+ T-cell cluster was also enriched in acquired resistant tumors. GSEA in early, central, and tissue-resident memory CD4+ T-cell clusters revealed an upregulation of naïve/stem-like gene sets and a downregulation of antigen processing/presentation gene sets in acquired resistant tumors. scRNAseq and differential expression analysis of epithelial populations highlighted pronounced EMT activation, increased lineage plasticity, and neuroendocrine differentiation gene signatures, implicating cellular reprogramming and phenotypic plasticity as potential contributors to acquired IO resistance. Conclusion: Acquired IO resistance in NSCLC involves dynamic and unique genomic and transcriptomic remodeling, encompassing EMT, lineage plasticity, stem-like programs, and immune reprogramming—highlighting potential avenues for therapeutic intervention. Citation Format: Archana Balan, Sonia Iyer, James Conway, Christopher Cherry, Noushin Niknafs, Mohamed Reda Keddar, Avinash Reddy, Robert McEwen, James White, Grace Kim, Anissa Dallmann, Nima Boluriaan, Sreeharsha Gunda, Mark Awad, Glenwood Goss, Se-Hoon Lee, Keunchil Park, Martin Reck, Michael Thomas, Rachel Karchin, Jane Peters, John F. Kurland, Giuseppe Galletti, Simon T. Barry, Jan Cosaert, J. Carl Barrett, Benjamin Besse, John V. Heymach, Patrick M. Forde, Valsamo Anagnostou. Multi-modal multi-omic analyses reveal mechanisms of immunotherapy resistance in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr CT233.