ROS1 Mutations Research Articles

Overview of Systematic Reviews of ICIs in NSCLC With EGFR, ALK, ROS1, and RET Actionable Driver Mutations

The effectiveness and safety of immune checkpoint inhibitor (ICI) monotherapy in patients with previously treated advanced or metastatic non–small cell lung cancer with EGFR, ALK, ROS1, or RET actionable driver mutations or chromosomal rearrangements is currently uncertain. We assessed the efficacy and safety of ICIs in patients with this condition whose disease did not respond well to previous chemotherapy. We reviewed 13 systematic reviews of randomized controlled trials (RCTs). The quality assessment of these reviews revealed critical methodological flaws. All 13 systematic reviews focused on survival and progression-free survival (PFS) for patients with non–small cell lung cancer and EGFR gene mutations. The systematic reviews generally considered the same set of 4 clinical trials and did not report on other outcomes or patient groups, except for 1 review that looked at patients with different levels of anti-PD-L1 expression. We found no evidence on the efficacy and safety of ICIs in patients with ALK, ROS1, or RET mutations. Overall, the systematic reviews concluded that using ICIs alone, as a second-line therapy or beyond, does not significantly improve overall survival (OS) and PFS compared to chemotherapy in patients with non–small cell lung cancer with EGFR gene mutations. No conclusions can be made regarding the benefits of ICIs in patients with EGFR mutations based on histology or high antiprogrammed death-ligand 1 antibody expression levels. The safety of ICIs in patients with EGFR, ALK, ROS1, or RET actionable driver mutations could not be assessed because of the lack of evidence provided in the included systematic reviews.

CDX2-Suppressed Colorectal Cancers Possess Potentially Targetable Alterations in Receptor Tyrosine Kinases and Other Colorectal-Cancer-Associated Pathways.

Colorectal cancer, a prevalent gastrointestinal carcinoma, has a high risk for recurrence when locally advanced and remains lethal when in an advanced stage. Prognostic biomarkers may help in better delineating the aggressiveness of this disease in individual patients and help to tailor appropriate therapies. CDX2, a transcription factor of gastrointestinal differentiation, has been proposed as a biomarker for good outcomes and could also be a marker of specific sub-types amenable to targeted therapies. Colorectal cancers from The Cancer Genome Atlas (TCGA) colorectal cohort and colon cancers from the Sidra-LUMC AC-ICAM cohort were categorized according to their expressions of CDX2 mRNA. Groups with CDX2 suppression were compared with cancers showing no suppression regarding their clinical and genomic characteristics. CDX2-suppressed colorectal cancers showed a high prevalence of Microsatellite Instability (MSI) and a lower prevalence of chromosomal Instability (CIN) compared to non-CDX2-suppressed cancers. In addition, CDX2-suppressed cancers had a higher prevalence of mutations in several receptor tyrosine kinase genes, including EGFR, ERBB3, ERBB4, RET, and ROS1. In contrast, CDX2-suppressed cancers displayed lower mutation frequencies than non-CDX2-suppressed cancers in the genes encoding for the two most frequently mutated tumor suppressors, APC and TP53, and the most frequently mutated colorectal cancer oncogene, KRAS. However, CDX2-suppressed colorectal cancers had a higher prevalence of mutations in alternative genes of the WNT/APC/β-catenin and KRAS/BRAF/MEK pathways. In addition, they showed frequent mutations in DNA damage response (DDR) genes, such as BRCA2 and ATM. CDX2-suppressed colorectal cancers constitute a genomically distinct subset of colon and rectal cancers that have a lower prevalence of KRAS, APC, and TP53 mutations, but a high prevalence of mutations in less commonly mutated colorectal cancer genes. These alterations could serve as targets for personalized therapeutics in this subset.

Response to platinum-based therapies in second-line after immunotherapy in advanced or metastatic non-small-cell lung cancer PD-L1 ≥50.

Platinum-based therapies for patients with advanced non-small-cell lung cancer (NSCLC) have classically provided overall survival (OS) rates of six to nine months and objective response rates (ORRs) of 20-30%. Whether prior immunotherapy determines a different response to platinum is currently unknown. This study aimed to analyse the current response characteristics to platinum as a second-line treatment for advanced NSCLC (PD-L1 ≥50%) after first-line immunotherapy. This retrospective study was conducted at the University Hospital of Salamanca (CAUSA) between 2016 and 2023 with patients who had advanced NSCLC (PD-L1 ≥50%) treated with second-line platinum-based therapies after immunotherapy (without mutations in EGFR, ALK or ROS1 and with Eastern Cooperative Oncology Group (ECOG) ≤1 during the first- and second-line treatments). Survival and response correlation analyses (Kaplan-Meier and log rank tests in SPSS v. 25) were performed. Subsequently, the results were compared with historical cohorts (PubMed, COCHRANE, ScienceDirect, Embase, and the clinical trial registry) who had received platinum-based therapies for advanced NSCLC. Seventeen patients were analysed (11 male and 6 female). Their median age was 67 years (interquartile range, 50-77 years). Fifteen patients (88.2%) were smokers or former smokers. The patients' main histology was adenocarcinoma (9 patients, 52.9%). All first-line treatments applied pembrolizumab (median dose: 12 cycles). Second-line platinum-based therapy achieved OS of 25 months (95% CI: 7-45 months) and progression-free survival (PFS) of 6 months (95% CI: 2.5-95 months). The ORR was 47.1% [seven patients with a partial response (PR) and one patient with a complete response (CR)]. Of the patients with PRs or CRs, 75% were treated with platinum plus pemetrexed. The one-year survival rate was 58.8%. The historical OS for first-line platinum-based doublets is 7 to 12 months, with PFS of three to five months and an ORR of 17-30%. The current response to second-line platinum-based therapies for patients with advanced NSCLC after immunotherapy appears to achieve favourable response rates and be an optimal treatment after progression to immunotherapy. Prior immunotherapy appears to enhance these patients' platinum response, though future confirmatory studies are necessary.

A phase 2, open-label, single-arm study evaluating the combination of pembrolizumab (pembro), lenvatinib, and chemotherapy in patients (pts) with metastatic non-small cell lung cancer (NSCLC) harbouring a targetable mutation who experienced disease progression on standard tyrosine kinase inhibitors (TKIs).

198 Background: Pts with metastatic NSCLC harbouring a targetable mutation who progressed on standard TKIs often receive chemotherapy as the next line of treatment. We report efficacy and safety of a novel combination of pembro, lenvatinib and chemotherapy for these pts. Methods: This was a phase 2, open-label, single-centre, single-arm study enrolling pts with metastatic NSCLC with sensitizing EGFR, ALK or ROS1 mutation who progressed on standard TKIs. Eligible pts had measurable disease, no untreated brain metastasis and ECOG PS ≤1. Pts received pembro 200 mg, pemetrexed 500 mg/m2 and carboplatin AUC 5 every 3 weeks for 4 cycles plus lenvatinib 8 mg QD. The maintenance regimen without carboplatin was continued as tolerated for up to 2 years. The primary endpoint was objective response rate (ORR) by independent radiology review (IRR) as per RECIST 1.1. The secondary endpoints were PFS, overall survival (OS) and safety. Results: At the data cutoff date (2nd February 2024), 24 pts were screened and 19 were enrolled: median age 64 years (range 29–77); 10 (53%) female. Four pts were excluded at screening due to untreated brain metastasis. All enrolled pts were evaluated for safety while 15 pts were evaluable for response. All pts had adenocarcinoma with targetable mutation: EGFR mutation in 18 (95%) pts and ALK fusion in 1 (5%) pt. The median follow-up time was 11.3 months. Treatment was ongoing in 8 pts on the date of analysis, while 6 pts permanently discontinued due to disease progression and 1 pt permanently discontinued due to a treatment-related adverse event (TRAE). The ORR was 40% (6/15, 95% CI 19.8%–64.3%). All responses were partial responses. The median PFS was 11.9 months (range 3.5–20.3). The OS was not reached. TRAEs of any grade (Gr) occurred in 74% (14/19) of the pts. Most of the TRAEs were Gr 1–2, which occurred in 63% (12/19) of the pts. The most common TRAEs were hypothyroidism (31.6%), nausea (26.3%), neutropenia (26.3%), thrombocytopenia (26.3%), anorexia (21.1%), hypertension (15.8%), anaemia (10.5%), pruritus (10.5%) and rash (10.5%). G1 diarrhoea was reported in 1 pt (5.3%) and G1 adrenal insufficiency in 1 pt (5.3%). Gr 3 TRAEs were neutropenia in 2 pts, anaemia in 1 pt and thrombocytopenia in 1 pt. The only Gr 4 TRAE was thrombocytopenia. No treatment related death was observed. Conclusions: The combination of pembro, lenvatinib and chemotherapy demonstrated a favourable ORR and safety in treating metastatic NSCLC pts with a targetable mutation who progressed on standard TKIs. Clinical trial information: NCT04989322 .

Outcomes of EGFR, ALK, ROS1, BRAF, MET, and RET mutated non-small cell lung cancer with brain metastases (NSCLC BM).

201 Background: Non-small cell lung cancer (NSCLC) is a leading cause of brain metastases. Advances in gene-directed therapies have shifted the focus away from traditional platinum-based chemotherapies. Key targeted mutations in NSCLC include EGFR, ALK, BRAF, MET exon skipping, RET fusions, and ROS1 rearrangements. This retrospective study aims to explore overall survival (OS) and progression-free survival (PFS) in NSCLC BM patients with these gene mutations. Methods: In this retrospective study, conducted by the Department of Medical Oncology at Beijing Tiantan Hospital and the First Medical Center, PLA General Hospital, we reviewed 1526 patients with NSCLC brain metastasis from 2010 to 2022. Data collected included molecular marker status, systemic therapies, and dates of progression. The analysis focused on determining OS and PFS from the time of brain metastasis diagnosis to the last follow-up or death. Statistical analysis used the Cox proportional hazards model. Results: The study reviewed 1526 patients, identifying the following mutation distribution: EGFR mutations in 628 patients, ALK rearrangements in 207 patients, ROS1 rearrangements in 64 patients, BRAF mutations in 31 patients, MET exon skipping in 55 patients, and RET fusions in 42 patients. Significant variations in median OS (mOS) and median PFS (mPFS) were observed across these mutation groups. Patients with EGFR and ALK mutations demonstrated longer mOS and mPFS. Patients with RET and ROS1 mutations also showed better outcomes compared to those with BRAF and MET mutations. Conclusions: This study highlights the importance of molecular mutations in NSCLC BM as prognostic indicators and therapeutic targets. EGFR and ALK mutations were associated with more favorable outcomes, emphasizing the need for personalized medicine in managing NSCLC BM. The study suggests further research focused on specific mutation types is crucial to refine treatment strategies and improve patient care.

Integrating the Idylla™ System Alongside a Real-Time Polymerase Chain Reaction and Next-Generation Sequencing for Investigating Gene Fusions in Pleural Effusions from Non-Small-Cell Lung Cancer Patients: A Pilot Study.

Malignant pleural effusion (MPE) from patients with advanced non-small-cell lung cancer (NSCLC) has been proven valuable for molecular analysis; however, simultaneous detection of driver fusions in MPE is still challenging. In this study, we investigated the Idylla™ GeneFusion Panel, a stand-alone test in tissue samples, in the evaluation of ALK, ROS1, RET and MET ex14 skipping mutations in MPE and compared its performance with routine reference methods (Real-time-based and Next-generation Sequencing-NGS). The inclusion criteria for sample selection were as follows: advanced NSCLC harboring ALK, ROS1, RET fusions or MET exon-skipping alterations and the availability of MPE collected at diagnosis or disease progression. Molecular alterations have been investigated on tissue by fluorescence in situ hybridization (FISH) or Real-time PCR or NGS. For molecular profiling with the Idylla™ GeneFusion, 200 µL of MPE supernatants combined with 50 µL of RNA Later solution were loaded into the Idylla™ cartridge without cfRNA extraction. The Idylla™ GeneFusion Assay performed on MPEs was able to confirm molecular profile, previously diagnosed with conventional methods, in all cases. Our data confirm that MPE are suitable material for investigating fusion alterations. The Idylla™ GeneFusion, although indicated for investigation of tissue samples, offers the possibility of performing a molecular characterization of supernatants without undertaking the entire cfRNA extraction procedure providing a rapid and reliable strategy for the detection of actionable genetic alterations.

Correlation analysis between driver gene mutation and clinicopathological features in lung adenocarcinoma based on real-world cumulative clinical data.

Driver genes are essential predictors of targeted therapeutic efficacy. Detecting driver gene mutations in lung adenocarcinoma (LUAD) patients can help to screen for targeted drugs and improve patient survival benefits. This study aims to investigate the mutation characterization of driver genes and their correlation with clinicopathological features in LUAD. A total of 440 LUAD patients were selected from Sir Run Run Shaw Hospital between July 2019 and September 2022. Postoperative tissue specimens were analyzed for gene mutations using next-generation sequencing technology, focusing, including epidermal growth factor receptor EGFR, ALK, ROS1, RET, KRAS, MET, BRAF, HER2, PIK3CA and NRAS. At the same time, clinicopathological data were collected and organized for multidimensional correlation analysis. Of 440 LUAD patients, driver gene mutations were not detected in 48 patients. The proportion of patients with driver gene mutations was as high as 89.09%. The top three driver genetic mutations were EGFR, KRAS, and MET. Sixty-nine types of EGFR mutations were detected and distributed in the protein tyrosine kinase catalytic domain (56, 81.16%), Furin-like cysteine-rich region (9, 13.04%), receptor binding domain (3, 4.35%), and EGFR transmembrane domain (1, 1.45%). Single gene locus mutation occurred in 343 LUAD patients, but the mutation gene types covered all tested genes. Our findings showed that EGFR mutations were more commonly observed in non-smoking and female patients (P<0.01), KRAS mutations were more prevalent in male patients and smokers (P<0.01), ROS1 mutations had larger tumor diameters (P<0.01) and RET mutations were more prevalent in smokers (P<0.05). LUAD patients exhibit diverse genetic mutations, which may co-occur simultaneously. Integrated analysis of multiple mutations is essential for accurate diagnosis and effective treatment of the disease. The use of NGS can significantly expand our understanding of gene mutations and facilitate integrated analysis of multiple gene mutations, providing critical evidence for targeted treatment methods.

Association between PD-L1 expression with EGFR, ALK, and ROS1 driver oncogene mutations in non-small cell lung cancer.

Driver mutations and immunological expressions have gained importance in recent years for targeted therapies and immunotherapies of nonsmall cell lung cancer (NSCLC). This study examined the association between PD-L1 expression and ALK, ROS1, and EGFR driver oncogene mutations in patients with NSCLC. A total of 501 NSCLC patients were included for analysis. Immunohistochemistry was performed with a PD-L1 clone 22c3. EGFR mutations were detected by PCR. ALK and ROS1 rearrangement analysis was performed with FISH. Results: There was a highly statistically significant difference between PD-L1 expression and EGFR mutation. PD-L1 expression was higher in the EGFR wild-type than in mutated EGFR (P = 0.0002). There was no relationship between PD-L1 expression and ALK and ROS1 mutations (P = 0.8899, P = 0.2512, respectively). PD-L1 expression was higher in nonadenocarcinomas (non-AC) than in adenocarcinomas (AC) (P = 0.0438). The ALK rearrangement and EGFR mutations were higher in ACs (P = 0.0073, P = 0.0012, respectively). ALK, ROS1 rearrangements, and EGFR mutations were higher in nonsmokers (P < 0.05). EGFR mutations were detected more frequently in females than males (P = 0.001). There was no relationship between gender and ALK, ROS1, and PD-L1 (P > 0.05). The prevalence of EGFR, ALK, and ROS1 driver mutations in the Turkish population was 9.3%, 5.3%, and 2.4%, respectively. In conclusion, PD-L1 expression and mutated EGFR status have a highly negative association. PD-L1 expression was higher in EGFR wild-type patients. Therefore, it shows that the opportunity to receive PD-L1-related treatment may be higher in these patients. We think that PD-L1 immunohistochemical evaluation will increase the clinical predictive importance in EGFR wild-type cases.

Afatinib in patients (pts) with solid tumors with neuregulin 1 (NRG1) fusions: Results from the Targeted Agent and Profiling Utilization Registry (TAPUR) study.

3131 Background: TAPUR is a phase II basket study evaluating antitumor activity of commercially available targeted agents in pts with advanced cancers with genomic alterations. Results in a small cohort of pts with advanced solid tumors with NRG1 fusions treated with afatinib are reported. Methods: Eligible pts had measurable, advanced disease, NRG1fusion, ECOG performance status (PS) 0-2, adequate organ function, and no standard treatment (tx) options. Pts with non-small cell lung cancer (NSCLC) were eligible if they had no pathogenic mutations in ALK, BRAF, EGFR or ROS1. Genomic testing was performed in CLIA-certified, CAP-accredited site selected labs. Pts received 40 mg of afatinib daily until disease progression. Primary endpoint was disease control (DC) per investigator defined as complete (CR) or partial (PR) response per RECIST v. 1.1, or stable disease (SD) of at least 16 wks duration (SD16+). Secondary endpoints were progression-free survival (PFS), overall survival (OS), objective response (OR), duration of response, duration of SD, and safety. This cohort was closed after 2 years before completing stage 1 enrollment (n = 10) of the Simon’s 2-stage design used in TAPUR according to a prespecified cohort closure rule. Results: 4 pts with 3 tumor types with NRG1 fusion were enrolled between February 2020 and July 2021. 1 pt had NSCLC with a CD74- NRG1 fusion and achieved a PR that lasted 24 weeks (wks). This pt was a 58-year-old White, non-Hispanic female with ECOG PS 0, 2 prior systemic therapies and progressed after 32 wks on study tx. Co-alterations included CCNE1 amplification (amp), GATA amp, TP53 R65fs*58, and an ALK G1121D variant of unknown significance. 2 pts achieved SD16+: 1 was a 47-year-old White, Hispanic female with colorectal cancer (CRC) with a MATN2- NRG1 fusion, ECOG PS 1, 3 prior systemic therapies and co-alterations in APC, DDX11, FBXW7, FGFR1 and TP53; the other pt was a 49-year-old Black, non-Hispanic male with pancreatic cancer with a NRG1 exon 2 fusion, ECOG PS 1, 5 prior systemic therapies, a comutation in ARID1A. The durations of SD for these 2 pts were 136 and 64 wks, respectively. The pt with CRC underwent resection of a remaining target lesion and is still alive off study drug at 198 wks after the baseline visit as of Dec 2023. 1 pt was a 70-year-old White, non-Hispanic female with CRC with a CD74- NRG1 fusion, ECOG PS 0, 1 prior systemic therapy, and a best response of progressive disease. This pt had comutations in ARID1A, PBRM1 and TP53and progressed at 8 wks. Median OS was 81 wks. No grade 3-5 tx-related adverse events (AE) or serious AEs were reported. Conclusions: Though a small sample size, afatinib demonstrated promising activity in pts with advanced solid tumors with NRG1 fusion, including very durable DC. Additional study in a larger group of pts is warranted to confirm the efficacy of afatinib in pts with NRG1 fusion. Clinical trial information: NCT02693535 .

Transgenerational increases in DNA methylation in Arabidopsis plants defective in active DNA demethylation

Spontaneous gain or loss of DNA methylation occurs in plant and animal genomes, and DNA methylation changes can lead to meiotically stable epialleles that generate heritable phenotypic diversity. However, it is unclear whether transgenerational epigenetic stability may be regulated by any cellular factors. Here, we examined spontaneously occurring variations in DNA methylation in wild-type and ros1 mutant Arabidopsis plants that were propagated for ten generations from single-seed descent. We found that the ros1 mutant, which is defective in active DNA demethylation, showed an increased transgenerational epimutation rate. The ros1 mutation led to more spontaneously gained methylation than lost methylation at individual cytosines, compared to the wild type which had similar numbers of spontaneously gained and lost methylation cytosines. Consistently, transgenerational differentially methylated regions were also biased toward hypermethylation in the ros1 mutant. Our results reveal a genetic contribution of the ROS1 DNA demethylase to transgenerational epigenetic stability and suggest that ROS1 may have an unexpected surveillance function in preventing transgenerational DNA methylation increases.

Abstract LB182: Mutagenesis screens support potential best-in-class profile for selective, brain-penetrant, and TRK-sparing ROS1 inhibitor NVL-520

Abstract Introduction. ROS1 fusions are recurrent oncogenic drivers in non-small cell lung cancer (NSCLC). Tyrosine kinase inhibitors (TKIs) crizotinib, entrectinib, and repotrectinib have been approved for the treatment of ROS1-positive NSCLC. Response to these TKIs can be limited by emergence of drug resistance, with ~40% of patients acquiring on-target ROS1 mutations at progression on crizotinib. NVL-520 is an investigational, brain-penetrant, and TRK-sparing ROS1 TKI designed to maintain activity for ROS1 mutations that confer resistance to approved therapies. To investigate suppression of resistance by NVL-520, we performed two N-ethyl-N-nitrosourea (ENU) mutagenesis screens and determined the frequency and identity of resistance mutations following treatment with NVL-520 versus earlier-generation ROS1 TKIs. Methods. Cells were treated with ENU for 1 day followed by crizotinib, entrectinib, repotrectinib, or NVL-520 for 28 days. Frequency of resistance indicates the fraction of wells that proliferated on day 28 despite continuous TKI treatment. Resistance mutations in the ROS1 kinase domain were identified by Sanger sequencing. Resistant clones harboring ROS1 kinase-domain mutations were expanded and used to profile the inhibitory activity of ROS1 TKIs. Results. The first ENU screen was performed on Ba/F3 CD74-ROS1, simulating a first-line treatment setting. NVL-520 at an unbound drug concentration (Cunbound) of 8.2 nM yielded 1 resistant clone among 480 replicate wells (frequency of resistance = 0.2%). The second screen was performed on Ba/F3 CD74-ROS1 G2032R, which is the most common point mutation after progression on previous-generation therapies, simulating a later-line treatment setting. NVL-520 at Cunbound of 41 nM yielded 6 resistant clones among 480 replicate wells (frequency of resistance = 1.3%). These NVL-520 concentrations (8.2 - 41 nM) were well within its average Cunbound of 166 nM at the recommended phase 2 dose (RP2D) of 100 mg once daily. None of the 7 resistant clones harbored ROS1 kinase-domain mutations, suggesting an off-target resistance mechanism. Crizotinib, entrectinib, and repotrectinib at their respective average human Cunbound yielded frequencies of resistance ranging from &amp;gt;33% to 100% in at least one of the screens, indicating that they were less effective than NVL-520 at suppressing resistance in these screens. Conclusion. NVL-520 was highly effective at suppressing resistance in ENU mutagenesis screens simulating first-line (CD74-ROS1) and later-line (CD74-ROS1 G2032R) treatment. The screens did not reveal any novel ROS1 mutations that could confer resistance to NVL-520 at the RP2D. The broad preclinical activity against ROS1 and diverse ROS1 mutations, together with its brain penetrance and TRK-sparing design, suggests NVL-520 as a potential best-in-class ROS1 therapy. Citation Format: Anupong Tangpeerachaikul, Franklin Gu, Henry E. Pelish. Mutagenesis screens support potential best-in-class profile for selective, brain-penetrant, and TRK-sparing ROS1 inhibitor NVL-520 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB182.

Abstract 987: Novel ALK mutations in EML4::ALK+ NSCLC resistant to TKIs identified by liquid biopsy

Abstract The identification of Anaplastic Lymphoma Kinase (ALK) chromosomal translocations led to the development of effective targeted therapies in a subset of Non-Small Cell Lung Cancer (NSCLC) patients. Several drugs are currently in clinical practice for advanced ALK+ NSCLC. However, resistance remains a challenge in these patients. The possibility to analyze circulating tumor DNA (ctDNA) with non-invasive methods can greatly improve prognostic capabilities, by allowing early detection of relapse and of drug-resistant mutations from peripheral blood. We report a series of 15 consecutive relapsed NSCLC patients (12 ALK+, 3 ROS1+) treated at the Fondazione IRCCS-San Gerardo dei Tintori (Monza, Italy) that were investigated by liquid biopsy at tyrosine kinase inhibitor (TKI) failure, through amplicon deep sequencing of the ALK/ROS1 kinase domains. Median DNA yield was 4.8 ng per ml of plasma and mean fragment size was 176 bp. Mutational hotspot coding regions of ALK and ROS1 genes were amplified by high-fidelity PCR and sequenced. Mean coverage of called variants was 24,687x. The threshold for mutation calling was set at 0.6%, after correcting for mapping quality, minimum coverage, and strand bias. Analysis of background signal on healthy control DNA identified 0.0024 mismatches per mapped base. Overall, plasma ctDNA analysis detected an ALK mutation in 7/18 relapse samples from 7/15 patients. No ROS1 mutations were found in ROS1 fusion-positive patients. Among 12 patients carrying an EML4::ALK fusion, 10 variants potentially driving resistance were found. Along with mutations that were previously identified (L1196M/G1202R, E1154K, F1174L), novel ALK variants were detected such as L1198R, T1211I, C1235R, C1237Y, L1196P. Mutations were characterized for sensitivity to ALK inhibitors in a BaF3-EML4::ALK cell model, using cell proliferation assays and western blotting to evaluate the effects on ALK phosphorylation. C1237Y and L1196P mutants appeared to resist all TKIs; hence, we further tested their sensitivity to novel investigational drugs, zotizalkib and repotrectinib. Both mutants demonstrated resistance, suggesting that these two mutations confer broad resistance to TKIs. Molecular modelling of C1237Y and L1196P mutants was run to elucidate the mechanisms of resistance. The ADP-C1237Y complex showed a much smaller root-mean-square deviation than ADP-WT, indicating a higher affinity/activity of the mutant. L1196 interacts hydrophobically with a methyl group of lorlatinib, P1196 is predicted to lose such interaction, while ATP binding is not affected. The presence of C1237Y and L1196P variants, is a relevant finding due to their resistance, even after the exposure to the fourth-generation inhibitor zotizalkib. The clonal selection of these mutations, made by second/third generation ALK TKIs may lead to a complete refractory status, which would then render TKIs useless in a sequential therapeutic strategy. Citation Format: Federica Malighetti, Matteo Villa, Elisa Sala, Geeta Sharma, Giulia Arosio, Maria Gemelli, Chiara Manfroni, Diletta Fontana, Nicoletta Cordani, Raffaella Meneveri, Alfonso Zambon, Rocco Piazza, Fabio Pagni, Diego Cortinovis, Luca Mologni. Novel ALK mutations in EML4::ALK+ NSCLC resistant to TKIs identified by liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 987.

Abstract 6399: Real-world experience of an Asian pan-cancer combined DNA/RNA next generation sequencing (NGS) tissue biopsy assay

Abstract Introduction: Comprehensive molecular profiling with combined DNA-RNA next-generation sequencing (NGS) on tumor tissue is now increasingly being used as standard-of-care for sequence therapy selection to improve cancer survival. Combined DNA-RNA NGS is also considered standard-of-care for central nervous system (CNS) tumor classification. Despite the genetic heterogeneity and diversity of cancers, most commercial combined DNA/RNA NGS assays are validated in Western populations, resulting in knowledge gaps in patient outcomes. We report here for the first time a range of actionable outcomes from a comprehensive DNA/RNA tissue NGS assay designed specifically to probe Asian-prevalent cancers and biomarkers in an international multicenter real-world study in Asia. Methods: 1,002 formalin-fixed paraffin-embedded tissue samples from 995 Asian cancer patients across &amp;gt;70 centers in 7 countries and/or territories underwent real-world DNA/RNA NGS testing in a CAP-accredited CLIA-certified laboratory. Genomic alterations including SNVs, INDELS, gene fusions, CNVs, TMB, and MSI were analyzed using an Asian-centric pan-cancer hybrid-capture NGS panel that comprehensively profiles 572 cancer-related genes and 91 RNA fusion partners (UNITED). Biomarkers were considered actionable if the therapeutic indication in any solid tumor or hematological cancer was FDA-approved, guidelines-recommended, or supported by well-powered phase III clinical trials. Results: 27 cancer types were represented in 1,002 tissue samples, with lung, breast, colorectal, and prostate cancers comprising 45.5% of clinical volume. 79.5% of samples harbored ≥1 actionable biomarker, with the most frequently altered genes being TP53, KRAS, PIK3CA, EGFR, and NF1. Overall, 42.6% of samples had ≥1 biomarker with an indication in the patient’s cancer type, including 81.5% of lung, 59.0% of breast, 61.8% of colorectal, and 42.1% of prostate cancers. Across the pan-cancer cohort, 13.3% was TMB-high and 1.9% was MSI-high. 26.3% of samples were from cancer types outside the original assay design; the top 3 were pancreatic cancer (7.3%), sarcoma (3.3%), and CNS tumor (3.1%). Cancers of unknown primary (CUP) also constituted a significant proportion (3.8%). In CNS tumors, 38.7% harbored an actionable mutation in IDH1, BRAF, or H3-3A. In CUP, 78.8% harbored an actionable mutation; 50% of samples harbored biomarkers that additionally informed potential tumor origin, including mutations in EGFR, MET, ALK, ROS1, and BRCA1. Conclusion: The use of an Asian-focused pan-cancer DNA/RNA NGS panel resulted in detection of actionable biomarkers in 79.5% of clinical cases. These findings support the utility of DNA/RNA NGS assays in informing standard-of-care therapeutic decisions in Asian cancer patients for maximizing cancer survival. Citation Format: Jason Yongsheng Chan, Jing Yi Lee, Zi Yi Wan, Abner Herbert Lim, Tun Kiat Ko, Cedric Chuan-Young Ng, Donald Poon, Jens Samol, Tsz Him So, Su Pin Choo, Ravindran Kanesvaran, Cheng-Vai Hui, Joseph Siu-Kie Au, Aya El Helali, Timothy Yip, Vikneswari Rajasegaran, Sandy Lim, Ruifen Weng, Puay Hoon Tan, Bin Tean Teh, Min-Han Tan, Jonathan Poh. Real-world experience of an Asian pan-cancer combined DNA/RNA next generation sequencing (NGS) tissue biopsy assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6399.

Nationwide data from comprehensive genomic profiling assays for detecting driver oncogenes in non-small cell lung cancer.

Driver oncogenes are investigated upfront at diagnosis using multi-CDx systems with next-generation sequencing techniques or multiplex reverse-transcriptase polymerase chain reaction assays. Additionally, from 2019, comprehensive genomic profiling (CGP) assays have been available in Japan for patients with advanced solid tumors who had completed or were expected to complete standard chemotherapy. These assays are expected to comprehensively detect the driver oncogenes, especially for patients with non-small cell lung cancer (NSCLC). However, there are no reports of nationwide research on the detection of driver oncogenes in patients with advanced NSCLC who undergo CGP assays, especially in those with undetected driver oncogenes at diagnosis. In this study, we investigated the proportion of driver oncogenes detected in patients with advanced NSCLC with undetectable driver oncogenes at initial diagnosis and in all patients with advanced NSCLC who underwent CGP assays. We retrospectively analyzed data from 986 patients with advanced NSCLC who underwent CGP assays between August 2019 and March 2022, using the Center for Cancer Genomics and Advanced Therapeutics database. The proportion of driver oncogenes newly detected in patients with NSCLC who tested negative for driver oncogenes at diagnosis and in all patients with NSCLC were investigated. Driver oncogenes were detected in 451 patients (45.7%). EGFR was the most common (16.5%), followed by KRAS (14.5%). Among the 330 patients with undetected EGFR, ALK, ROS1, and BRAF V600E mutations at diagnosis, 81 patients (24.5%) had newly identified driver oncogenes. CGP assays could be useful to identify driver oncogenes in patients with advanced NSCLC, including those initially undetected, facilitating personalized treatment.

Mechanisms of Resistance to Tyrosine Kinase Inhibitors in ROS1 Fusion-Positive Nonsmall Cell Lung Cancer.

ROS1 fusion-positive (ROS1+) nonsmall cell lung cancer (NSCLC) patients are highly sensitive to tyrosine kinase inhibitor (TKI) treatments. However, acquired TKI resistance remains the major hurdle preventing patients from experiencing prolonged benefits. 107 advanced or metastatic ROS1+ NSCLC patients who progressed on crizotinib and lorlatinib were recruited. Tissue and plasma samples were collected at baseline (N = 50), postcrizotinib (N = 91), and postlorlatinib (N = 21), which were all subject to the 139-gene targeted next-generation DNA sequencing. Molecular dynamics modeling was performed to investigate the effects of ROS1 mutations on binding to different TKIs. In patients with postcrizotinib and postlorlatinib samples, an accumulation of on- and off-target resistance alterations after multiple TKI treatments was observed. ROS1 G2032R and MET amplification were the most common on-target and off-target alterations, respectively. Patients with CD74-ROS1 and SLC34A2-ROS1 had longer progression-free survival (PFS) (P < 0.001) and higher rates of resistance mutations (on-target, P = 0.001; off-target, P = 0.077) than other ROS1 fusion variants following crizotinib treatment. Ten distinct on-target resistance mutations were detected after TKI therapies, of which 4 were previously unreported (ROS1 L2010M, G1957A, D1988N, L1982V). Molecular dynamics simulations showed that all 4 mutations were refractory to crizotinib, while G1957A, D1988N, and L1982V were potentially sensitive to lorlatinib and entrectinib. This study provided a comprehensive portrait of TKI-resistance mechanisms in ROS1+ NSCLC patients. Using in silico simulations of TKI activity, novel secondary mutations that may confer TKI resistance were identified and may support clinical therapeutic decision-making.

Clinicopathological features of SMARCA4-deficient lung adenocarcinoma: a study of 42 cases

Objective: To investigate the clinicopathological characteristics and genetic mutations of SMARCA4-deficient lung adenocarcinoma. Methods: From January 2021 to April 2023 in the First Affiliated Hospital of Zhengzhou University, 42 cases of SMARCA4-deficienct lung adenocarcinoma were diagnosed and now analyzed. All cases were retrospectively studied using hematoxylin-eosin staining and immunohistochemistry. The clinicopathological features were reviewed. Next-generation sequencing (NGS) was performed to investigate the mutations of related genes. Results: Among the 42 cases, there were 35 biopsy and 7 surgical specimens. There were 38 males and 4 females. The male to female ratio was 9.5∶1.0, with an age range from 42 to 78 years. Thirty-three patients were smokers. Overall, 4 cases (9.5%), 2 cases (4.7%), 18 cases (42.9%) and 18 cases (42.9%) were at stages Ⅰ, Ⅱ, Ⅲ, and Ⅳ, respectively. Microscopically, all the cases were non-mucinous adenocarcinoma, without lepidic pattern. The morphology was diverse. Rhabdomyoid cells, tumor giant cells and tumor necrosis were present. Most of the tumor cells had eosinophilic cytoplasm and occasionally clear cytoplasm. Defined cell borders and variable cytoplasmic hyaline secretory globules could be found. Inflammatory cells infiltrated the tumor stroma. Immunohistochemistry showed 29 cases (69.0%, 29/42) expressed TTF1, 10 cases (40.0%, 10/25) expressed Napsin A, and 20 cases (100.0%, 20/20) expressed INI1. Forty cases (95.2%, 40/42) showed BRG1 loss in all tumor cells, while 2 cases (4.8%, 2/42) had partial BRG1 loss. PD-L1 (22C3) was positive in 59.2% of the cases (16/27). NGS revealed mutations in EGFR, ROS1, MET, RET and KRAS. Six cases (6/8) showed SMARCA4 mutation, while some cases were accompanied by mutations of TP53 (7/15), STK11 (4/8), and KEAP1 (1/8). Driver gene mutations were more common in women (P<0.05). Patients were followed up for 1-25 months. Four patients died and 20 patients' diseases progressed. Conclusions: SMARCA4-deficient lung adenocarcinoma lacks characteristic morphology. Most of them express TTF1 and harbor driver gene mutations. It is necessary to identify this subset of lung adenocarcinoma by carrying out BRG1 stain routinely on lung adenocarcinoma. These patients can then be identified and benefit from targeted therapies.

Genetic Profiling of Non-Small Cell Lung Cancer in Moroccan Patients by Targeted Next-Generation Sequencing.

We retrospectively analyzed the next-generation sequencing (NGS) results from diagnosed NSCLC patients to identify and compare genomic alterations of NSCLC between Moroccan patients and the Cancer Genome Atlas (TCGA). We also aimed to investigate the distribution and frequency of concurrent genomic alterations. From December 2022 to December 2023, a retrospective study of 76 formalin-fixed paraffin-embedded (FFPE) samples have been profiled using the Oncomine™ Precision Assay on the Ion Torrent™ Genexus™ Integrated Sequencer across the panel of 50 key genes that are applicable for the selection of targeted therapy. Seventy of the 76 FFPE sequenced samples carried at least one genetic alteration in the tested genes. The study identified 234 genetic alterations in 18 genes. Targetable genetic alterations in EGFR, KRAS, MET, BRAF, ALK, RET and ROS1 were identified in 84.3% of tumors. EGFR and KRAS mutations were frequently reported, occurring in 24.3% and 22.9% of cases, respectively. The untargetable genetic alterations were found in 74.3% of the specimens in FGFR3, TP53, ERBB2, PIK3CA, CDKN2A, PDL1, FGFR1, PTEN, CHEK2 and ERBB3. There were additional uncommon/rare mutations in EGFR, BRAF, RET and ROS1. Comparing the prevalence of selected mutated genes in the NSCLC patients from the TCGA database identified substantial differences in EGFR (24.3%, vs14.97%), KRAS (22.9%, vs 25.99%), and TP53 (34.3%, vs 50.94%). ALK, ROS1, and RET gene rearrangements were detected in 4.3% of the 70 tumors tested. The ALK/RET/MET/ROS1/EML4 fusions were detected in 11.4% of samples. Co-alterations occurred in 67.1% of specimens. Co-occurring driver gene mutations were observed in 44.3%. TP53 mutations co-occurred driver gene mutations in 30% of tumors. Three cases (4.3%) harbored concurrent FGFR3, TP53, and PIK3CA alterations. Our results regarding the proportion of samples with actionable mutations demonstrate the value of NGS testing for NSCLC patients in a real-world clinical diagnostic setting.

Non-invasive decision support for clinical treatment of non-small cell lung cancer using a multiscale radiomics approach

BackgroundSelecting therapeutic strategies for cancer patients is typically based on key target-molecule biomarkers that play an important role in cancer onset, progression, and prognosis. Thus, there is a pressing need for novel biomarkers that can be utilized longitudinally to guide treatment selection. MethodsUsing data from 508 non-small cell lung cancer (NSCLC) patients across three institutions, we developed and validated a comprehensive predictive biomarker that distinguishes six genotypes and infiltrative immune phenotypes. These features were analyzed to establish the association between radiological phenotypes and tumor genotypes/immune phenotypes and to create a radiological interpretation of molecular features. In addition, we assessed the sensitivity of the models by evaluating their performance at five different voxel intervals, resulting in improved generalizability of the proposed approach.Findings: The radiomics model we developed, which integrates clinical factors and multi-regional features, outperformed the conventional model that only uses clinical and intratumoral features. Our combined model showed significant performance for EGFR, KRAS, ALK, TP53, PIK3CA, and ROS1 mutation status with AUCs of 0.866, 0.874, 0.902, 0.850, 0.860, and 0.900, respectively. Additionally, the predictive performance for PD-1/PD-L1 was 0.852. Although the performance of all models decreased to different degrees at five different voxel space resolutions, the performance advantage of the combined model did not change. ConclusionsWe validated multiscale radiomic signatures across tumor genotypes and immunophenotypes in a multi-institutional cohort. This imaging-based biomarker offers a non-invasive approach to select patients with NSCLC who are sensitive to targeted therapies or immunotherapy, which is promising for developing personalized treatment strategies during therapy.

Association of mutation profiles with metastasis in patients with non-small cell lung cancer.

This study focused on the analysis of the correlation between common gene mutation types and metastatic sites in NSCLC patients. We retrospectively studied 1586 NSCLC patients and used fluorescence Polymerase chain reaction (PCR) to detect EGFR, ALK, ROS1, RET, MET, BRAF, HER2, KRAS, NRAS, and PIK3CA gene mutations, and also investigated sex, smoking status, age at diagnosis, histological type and TNM stage. In addition, we analyzed the site of metastasis in patients with stage IV NSCLC. The EGFR-mutation group more frequently metastasized to lung (18.9%, P = 0.004), brain (18.9%, P = 0.001) and bone (27.1%, P = 0.004) than wild-type patients. ALK-mutation group (71.0%, P < 0.001), BRAF-mutation group (82.4%, P = 0.005) and NRAS-mutation group (100%, P = 0.025) were more likely to metastasize than the wild-type group. In the ALK mutation, lung metastasis (24.2%, P = 0.013), brain (24.2%, P = 0.007), bone metastasis (32.3%, P = 0.024), liver metastasis (19.4%, P = 0.001), and pleural metastasis (29.0%, P = 0.021) were common. In the KRAS-mutation group, lung metastasis (21.7%, P = 0.012) and brain metastasis (23.3%, P = 0.001) were more common. Less metastasis occurred in the HER2-mutation group (28.3%, P = 0.014). There was no difference in the RET, MET and PIK3CA mutations. Patients with ALK mutant, BRAF mutant or NRAS mutant were more prone to metastasis, while the HER 2 mutation group was less metastatic. Patients with EGFR mutant NSCLC are more likely to develop bone, lung, or brain metastasis.

Comparison of Clinical and Genetic Characteristics Between Younger and Older Lung Cancer Patients

IntroductionThe aim of this study was to analyze the clinical and genetic characteristics of young lung cancer cases, and to compare them with those of older cases. MethodsWe used the Thoracic Tumors Registry (TTR) as a data source representative of lung cancer cases diagnosed in Spain, and included all cases registered until 9/01/2023 which had information on age at diagnosis or the data needed to calculate it. We performed a descriptive statistical analysis and fitted logistic regressions to analyze how different characteristics influenced being a younger lung cancer patient. ResultsA total of 26,336 subjects were included. Lung cancer cases <50 years old had a higher probability of being women (OR: 1.38; 95% CI: 1.21–1.57), being in stage III or IV (OR: 1.32; 95% CI: 1.08–1.62), not having comorbidities (OR: 5.21; 95% CI: 4.59–5.91), presenting with symptoms at diagnosis (OR: 1.53; 95% CI: 1.29–1.81), and having ALK translocation (OR: 7.61; 95% CI: 1.25–46.32) and HER2 mutation (OR: 5.71; 95% CI: 1.34–24.33), compared with subjects ≥50 years. Among subjects <35 years old (n=61), our study observed a higher proportion of women (59.0% vs. 26.6%; p<0.001), never smokers (45.8% vs. 10.3%; p<0.001), no comorbidities (21.3% vs. 74.0%; p<0.001); ALK translocation (33.3% vs. 4.4%; p<0.001) and ROS1 mutation (14.3% vs. 2.3%; p=0.01), compared with subjects ≥35 years. ConclusionsLung cancer displays differences by age at diagnosis which may have important implications for its clinical management.