ROS1 Gene Fusions Research Articles

Evolution of acquired resistance in a ROS1+ KRAS G12C+ NSCLC through the MAPK pathway

Patients with metastatic NSCLC bearing a ROS1 gene fusion usually experience prolonged disease control with ROS1-targeting tyrosine kinase inhibitors (TKI), but significant clinical heterogeneity exists in part due to the presence of co-occurring genomic alterations. Here, we report on a patient with metastatic NSCLC with a concurrent ROS1 fusion and KRAS p.G12C mutation at diagnosis who experienced a short duration of disease control on entrectinib, a ROS1 TKI. At progression, the patient continued entrectinib and started sotorasib, a small molecule inhibitor of KRAS p.G12C. A patient-derived cell line generated at progression on entrectinib demonstrated improved TKI responsiveness when treated with entrectinib and sotorasib. Cell-line growth dependence on both ROS1 and KRAS p.G12C was further reflected in the distinct downstream signaling pathways activated by each driver. Clinical benefit was not observed with combined therapy of entrectinib and sotorasib possibly related to an evolving KRAS p.G12C amplification identified on repeated molecular testing. This case supports the need for broad molecular profiling in patients with metastatic NSCLC for potential therapeutic and prognostic information.

Abstract C018: Disparities in the access to non-small cell lung cancer´s target therapies in Chile

Abstract Comprehensive next-generation sequencing (NGS) panels designed to identify the tumor mutational profile are becoming the standard care to prescribe target therapies in developed countries. In non-small cell lung cancer (NSCLC), this approach significantly impacts the patient´s clinical results, measured as progression-free survival and/or overall survival, compared to conventional chemotherapies. However, as Latin American patients tend to experience more significant health disparities because of structural, sociodemographic, and psychosocial factors, in this work, our purpose is to measure the disparities in the access to NSCLC´s target therapies, specifically in Chile. DNAs and RNAs from 1643 NSCLC samples from Chile, Brazil, and Peru were sequenced to assess the mutational status in fifty-two cancer genes. After an NGS quality control, variants were called and annotated using the Variant Effect Predictor, Annovar, COSMIC, and OncoKB, to categorize somatic mutations. The following analysis focused on today’s actionable genes in NSCLC, with FDA-approved target therapies (EGFR, KRAS, ALK, MET, ERBB2, BRAF, ROS1, and RET). In this analysis, 46.5% of tumors evidenced driver mutations (764/1643); interestingly, from this subset, 86.9% showed one driver variant, 11.2% two drivers, 1.4% three drivers, and 0.5% evidenced between 4-6 driver mutations. However, 19.4% (495/1643) evidenced actionable variants. The most mutated genes and the most common actionable variants were 15.3% EGFR (37% EGFR L858R), followed by 4.9% KRAS (100% KRAS G12C), 4.5% ALK (95.4% EML4-ALK fusion), 3% MET (100% MET exon 14 skipping), and 2.3% ERBB2. Finally, 1.5% BRAF, 1% ROS1 gene fusions and 0.9% RET gene fusions. Considering the target therapies approved by Chile´s Instituto de Salud Publica until October 2021, and if all these patients were diagnosed in Chile, only 64% would receive a targeted drug. EGFR is the gene with more target therapies validated in Chile, although drugs against exon twenty insertion have not been approved yet. Chile does not account for any targeted treatment for patients with alterations in KRAS, MET, RET and ERBB2; although the FDA approved a specific drug against KRAS G12C very recently (May 28, 2021), different is the case of MET because the first inhibitor, crizotinib, was FDA approved four years ago. Interestingly, in 2021, two inhibitors against the most common MET alteration were FDA approved, but none have been approved in Chile yet. In Chile, almost all target therapies have been validated against EGFR, ALK, and BRAF; however, patients with KRAS, MET, RET, and ERBB2 cannot access specific drugs, so in these cases, the recommended therapeutic option is chemotherapy. It is important to note that the target drugs approval only ensures the availability of the drug in Chile. Still, few of the target drugs are part of financed drugs by the Chilean health system, so the question is, how could we increase the national access to existing target therapies? Citation Format: Solange V. Rivas, Evelin González, Alejandro Blanco, Carolina Ibáñez, Alejandro Corvalán, Marcelo Garrido, Gareth Owen, Katherine Marcelain, Ricardo Armisén. Disparities in the access to non-small cell lung cancer´s target therapies in Chile [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C018.

Importance of ROS1 gene fusions in non-small cell lung cancer.

Targeted therapy has become one of the standards of care for advanced lung cancer. More than 10 genetic aberrations have been discovered that are actionable and several tyrosine kinase inhibitors (TKIs) have been approved to target each of them. Among several genetic aberrations that are actionable in non-small cell lung cancer (NSCLC), ROS1 translocations also known as gene fusion proteins, are found in only 1%-2% of the patient population. ROS1 mutations can usually be detected using a combination of techniques such as immunohistochemistry (IHC), Fluorescence in-situ testing (FISH), polymerase chain reaction (PCR), and next-generation sequencing (NGS). However, RNA NGS and ctDNA NGS (liquid biopsies) also contribute to the diagnosis. There are currently numerous FDA-approved agents for these tumors, including crizotinib and entrectinib; however, there is in-vitro sensitivity data and clinical data documenting responses to ceritinib and lorlatinib. Clinical responses and survival rates with these agents are frequently among the best compared to other TKIs with genetic aberrations; however, intrinsic or extrinsic mechanisms of resistance may develop, necessitating research for alternative treatment modalities. To combat the mechanisms of resistance, novel agents such as repotrectenib, cabozantinib, talotrectinib, and others are being developed. In this article, we examine the literature pertaining to patients with ROS1 tumors, including epidemiology, clinical outcomes, resistance mechanisms, and treatment options.

Detection of ROS1 gene fusions using next-generation sequencing for patients with malignancy in China.

Objective: This study aimed to identify ROS1 fusion partners in Chinese patients with solid tumors. Methods: Next-generation sequencing (NGS) analysis was used to detect ROS1 rearrangement in 45,438 Chinese patients with solid tumors between 2015 and 2020, and the clinical characteristics and genetic features of gene fusion were evaluated. H&E staining of the excised tumor tissues was conducted. Samples with a tumor cell content ≥ 20% were included for subsequent DNA extraction and sequencing analysis. Results: A total of 92 patients with ROS1 rearrangements were identified using next-generation sequencing, and the most common histological type lung cancer. From the 92 ROS1 fusion cases, 24 ROS1 fusion partners had been identified, including 14 novel partners and 10 reported partners. Of these, CD74, EZR, SDC4, and TPM3 were the four most frequently occurring partners. Fourteen novel ROS1 fusion partners were detected in 16 patients, including DCBLD1-ROS1, FRK-ROS1, and VGLL2-ROS1. In many patients, the ROS1 breakpoint was located between exons 32 and 34. Conclusion: This study describes 14 novel ROS1 fusion partners based on the largest ROS1 fusion cohort, and the ROS1 breakpoint was mostly located between exons 32 and 34. Additionally, next-generation sequencing is an optional method for identifying novel ROS1 fusions.

Clinicopathological and genetic analyses of pulmonary enteric adenocarcinoma

AimsPulmonary enteric adenocarcinoma (PEAC) is a rare variant of pulmonary adenocarcinoma. Due to its rarity, few pathological and molecular studies have been performed on PEAC. We herein conducted clinicopathological, immunohistochemical...

Evolution of lung adenocarcinoma from preneoplasia to invasive adenocarcinoma.

Mutations in driver genes contribute to the development and progression of lung adenocarcinoma (LUAD). However, in the dynamic evolutionary process from adenocarcinoma in situ (AIS) to minimally invasive adenocarcinoma (MIA) and eventually to invasive adenocarcinoma (IAC), the role of driver genes is currently unclear. This study aimed to analyse the role of driver gene status in the progression of LUAD from preneoplasia to IAC. Patients with LUAD who underwent surgery in our centre from March 2015 to December 2019 were retrospectively analysed, and LUAD patients with tumour sizes ≤3.0cm and pN0 were included in the final analysis. The mutation status of common driver genes, including EGFR, ALK and ROS1, was detected. According to the pathological characteristics, the patients were divided into three stages: AIS, MIA and IAC. We analysed the distribution of driver gene mutation frequencies across three stages of LUAD. In addition, we performed univariate and multivariate analyses of IAC patients to screen for relevant variables (driver genes and clinicopathological features) affecting their prognosis. Ultimately, 759 patients with LUAD were enrolled, including 135, 130, and 494 cases of AIS, MIA, and IAC, respectively. EGFR mutations were identified in 359 (61.8%) patients, and with the transition from AIS to MIA, the frequency of EGFR mutations increased from 33.3% to 50.8%, p=0.004, whereas the frequency of EGFR mutations was comparable for MIA and IAC (50.8% vs. 50.2%, p=0.922). Moreover, ALK and ROS1 gene fusions were identified in 17 cases (2.2%) and 2 cases (3.0‰) respectively. For AIS, neither ALK gene nor ROS1 gene fusions were observed. When the tumour progressed to MIA, the ALK fusion frequency was 2.3% (3/130), which was basically consistent with the ALK fusion frequency of 2.8% in IAC, p=0.143. For IAC, fusions of ROS1 fell into this category. In addition, we found that 40 patients (5.3%) developed metastasis/recurrence, and 14 patients (1.8%) died of cancer-specific related diseases. Notably, for AIS, there were no recurrences and no deaths, and for MIA, only 1 patient died with LUAD. Finally, survival analysis was performed in patients with stage IA invasive adenocarcinoma, and EGFR-mutant patients showed better DFS than EGFR-wild-type patients (p=0.036). Conversely, patients with ALK fusions showed worse DFS than those with ALK wild-type (p=0.004), and the same results were found in OS analysis. The accumulation of EGFR driver gene mutation frequencies mediates the progression of LUAD from AIS to MIA. When the tumour progresses to stage IA invasive adenocarcinoma, multivariate analysis based on driver gene status can be used as a pivotal prognostic factor.

21P Analytical performance of FusionPlex Dx

FusionPlex Dx (FP Dx) is a 2-part multimodal next-generation sequencing (NGS)-based in vitro diagnostic kitted device that detects structural alterations and oncogenic isoforms in RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples from solid tumors using proprietary Anchored Multiplex PCR (AMP™) chemistry. FP Dx is approved as a companion diagnostic for five indications: METex14 skipping events, ALK, RET, ROS1 gene fusions to guide therapy in non-small cell lung cancer and NTRK1/2/3 gene fusions for all solid tumors.

Cell block-based RNA next generation sequencing for detection of gene fusions in lung adenocarcinoma: An institutional experience.

Targeted therapy is an important part of the treatment of lung adenocarcinoma. Tests for EGFR mutation, ALK, ROS1, RET and NTRK gene fusions are needed to make a treatment decision. These gene fusions are traditionally detected by fluorescence in situ hybridisation (FISH) or immunohistochemistry. In this study, we investigated whether gene fusions in pulmonary adenocarcinoma could be accurately detected by RNA next-generation sequencing (RNA-NGS) and whether cytology cell blocks could be used effectively for this test. Archived cytological specimens of lung adenocarcinoma submitted for RNA sequencing between 2019 and 2022 at Fox Chase Cancer Center were retrospectively retrieved. Hybrid capture-based targeted RNA next generation sequencing was used, which covers 507 fusion genes, including ALK, ROS1, RET and NTRKs, irrespective of their partner genes. DNA NGS, FISH and chromosomal microarray analysis were used to confirm the results of the RNA-NGS. A total of 129 lung adenocarcinoma cytology specimens were submitted for molecular testing. Eight of 129 (6.2%) cases were excluded from RNA sequencing as their cell blocks contained inadequate numbers of tumour cells. One case (0.8%) failed to yield adequate RNA. The overall success rate was 93% (120/129). Ten of 120 (8.3%) cytology cases were positive for gene fusions, including 7 ALK, 2 ROS1 fusion genes, and 1 RET fusion gene. Twenty-two cell block cases were also tested for ALK fusion genes using FISH. However, 11 of 22 (50%) failed the testing due to inadequate material. Cytology cell blocks can be used as the main source of material for molecular testing for lung cancer. Detection of gene fusions by RNA-based NGS on cell blocks is convenient and reliable in daily practice.

EP08.02-041 NVL-520, a Highly Selective ROS1 Inhibitor, in Patients with Advanced ROS1-Positive Solid Tumors: The Phase 1/2 ARROS-1 Study

Oncogenic ROS1 gene fusions are implicated in the pathogenesis of various adult and pediatric cancers, including up to 3% of non-small cell lung cancers (NSCLC), where up to 40% of patients present with central nervous system (CNS) metastases. Tyrosine kinase inhibitors (TKIs) approved by the FDA and EMA for ROS1-positive NSCLC (crizotinib and entrectinib) are limited by acquired resistance, frequently mediated by secondary ROS1 kinase domain mutations. In addition, dual TRK/ROS1 kinase inhibitors such as entrectinib are associated with neurologic adverse events.

RNA-based next-generation sequencing in non-small-cell lung cancer in a routine setting: an experience from an Italian referral center.

Aim: ALK, ROS1, NTRKand RET gene fusions and MET exon 14 skipping alterations represent novel predictive biomarkers for advanced non-small-cell lung cancer (NSCLC). Therefore, testing patients for these genetic variants is crucial for choosing the best selective treatment. Over the last couple of decades, next-generation sequencing (NGS) platforms have emerged as an extremely useful tool for detecting these variants. Materials & methods: In the present study, we report our NGS molecular records produced during a year of diagnostic activity. Results: Overall, our in-house developed NGS workflow successfully analyzed n=116/131 (88.5%) NSCLC samples. Of these, eight (6.8%) and five (4.3%) out of 116 patients harbored ALK and RET gene rearrangements, respectively: one case harbored ROS1 gene fusion (0.7%). Conclusion: Our results highlight that an RNA-based NGS analysis can reliably detect gene fusion alterations, thereby playing a pivotal role in the management of NSCLC patients.

Treatment strategy, overall survival and associated risk factors among patients with unresectable stage IIIB/IV non-small cell lung cancer in China (2015–2017): A multicentre prospective study

SummaryBackgroundThere are limited studies on treatment and survival analysis among patients with unresectable Stage IIIB or IV non-small cell lung cancer (NSCLC) in routine practice in China. To address this gap, we conducted a prospective observational study in a cohort of patients treated at 11 hospitals in China.MethodsThis was a multicentre, prospective cohort study including patients with newly diagnosed unresectable Stage IIIB or IV NSCLC from June 26th, 2015 to April 28th, 2017. Patient baseline characteristics, disease characteristics, and anti-cancer treatments were obtained by medical chart review. The overall survival (OS) from the initiation of first-line treatment was analysed by the Kaplan-Meier method. Factors associated with survival were analysed by univariate and multivariate Cox regression models.FindingsAmong 1324 patients enrolled with median follow-up duration of 15·0 (range: 0·0–42·1) months, 83·5% (1105/1324) of them received first-line chemotherapy of which platinum-based compounds were the dominated agents. Overall, 30·9% (409/1324) of patients received targeted therapy as 1st-line treatment including 65·0% (266/409) EGFR-TKIs and 5·1% (21/409) ALK-TKIs. Of all eligible patients, gene testing rates were 44·0% (583/1324) for EGFR mutations, 17·0% (225/1324) for EML4-ALK gene fusions, and 8·3% (110/1324) for ROS1 gene fusions. The EGFR-TKIs were administered to 63·9% (179/280) of EGFR mutated patients as first-line treatment. The overall median OS was 23·2 (95%CI 19·5-25·5) months, and patients treated at tier 1 cities had better OS than that of tier 2 cities. Also, the OS in patients with EGFR mutation was longer than those with EGFR wild type. Multivariate Cox regression models suggested that male, education below high school, tier 2 cities, smoking history, and multiple metastases were associated with poor survival.InterpretationThe gene test coverage was relatively low among the studied population, and over half of EGFR mutated patients received EGFR-TKIs, suggesting that the result of genetic tests in real-world settings may not always indicate the selection of treatment. The OS benefit observed from patients treated in tier 1 cities and those with EGFR mutation may indicate a need for broader gene test coverage, providing NSCLC patients with personalized treatment according to the results of genetic tests.FundingRoche Holding AG.TRANSLATED ABSTRACT: This translation in Chinese was submitted by the authors and we reproduce it as supplied. It has not been peer reviewed. Our editorial processes have only been applied to the original abstract in English, which should serve as reference for this manuscript.摘要背景介绍:目前针对中国不可切除的IIIB期或IV期非小细胞肺癌(NSCLC)患者在临床实践中的治疗模式和生存分析的研究有限.为了填补这一空白, 我们开展了一项前瞻性,观察性研究, 纳入来自11家医院的患者.方法:这是一项多中心,前瞻性队列研究, 纳入了2015年6月26日至2017年4月28日新诊断为不可切除的IIIB期或IV期NSCLC患者.通过病历筛查获得基线特征,临床肿瘤特征和抗肿瘤治疗.采用Kaplan-Meier法分析从开始一线治疗后的总生存期(OS), 并通过单因素和多因素Cox回归模型分析与生存相关的影响因素.结果:在入组的1324例患者中, 中位随访时间为15.0个月(范围:0.0-42.1), 其中83.5%(1105/1324)的患者接受了一线化疗, 以铂类化合物为主.总体而言, 30.9%(409/1324)的患者接受靶向治疗作为一线治疗, 其中包括65.0%(266/409)的患者接受了EGFR-TKI治疗和5.1%(21/409)的患者接受了ALK-TKI治疗.在所有符合条件的患者中, EGFR突变,EML4-ALK基因融合和ROS1基因融合的基因检测率分别为44.0%(583/1324),17.0%(225/1324)和8.3%(110/1324).63.9%(179/280)的EGFR突变患者接受EGFR-TKI作为一线治疗.全组患者的中位总生存期为23.2 (95%置信区间 19·5-25·5) 个月, 在一线城市治疗的患者的总生存期优于二线城市的患者.此外, EGFR突变患者的总生存期长于EGFR野生型患者.多因素Cox回归模型分析表明, 男性,高中以下文化程度,二线城市,吸烟史及肿瘤转移与较低的生存率相关.解释:本研究人群中的基因检测覆盖率相对较低, 其中超过一半的EGFR突变患者接受了EGFR-TKI治疗, 这表明在真实世界诊疗中, 基因检测的结果可能并不完全指导治疗选择.总生存期在一线城市接受治疗的患者和EGFR突变的患者中更佳的现象, 可能提示需要更广泛的基因检测覆盖, 并根据基因检测结果为NSCLC患者提供个体化治疗.

Clinical characteristics of non-small cell lung cancer patients with EGFR mutations and ALK&ROS1 fusions.

ObjectiveTo study the relationship between clinical characteristics and anaplastic lymphoma kinase (ALK) fusions, c‐ros oncogene 1, receptor tyrosine kinase (ROS1) gene fusions, and epidermic growth factor receptor (EGFR) mutations in non‐small cell lung cancer (NSCLC) patients to distinguish these different types.MethodsBoth ALK, ROS1 gene rearrangements and EGFR mutations testing were performed. The clinical characteristics and associated pulmonary abnormalities were investigated.ResultsFour hundred fifty‐three NSCLC patients were included for analysis. One hundred seventy (37.5%), 32 (7.1%), and 9 cases (2.0%) with EGFR mutations, ALK gene fusions, and ROS1 gene fusions were identified, respectively. The EGFR‐positive and ALK&ROS1‐positive were more common in female (χ 2 = 61.934, P < 0.001 and χ 2 = 28.152, P < 0.001), non‐smoking (χ 2 = 59.315, P < 0.001 and χ 2 = 11.080, P = 0.001), and adenocarcinoma (χ 2 = 44.864, P < 0.001 and χ 2 = 12.318, P = 0.002) patients; proportion of patients with emphysema was lower (χ 2 = 35.494, P < 0.001 and χ 2 = 15.770, P < 0.001) than the wild‐type patients. The results of logistic regression analysis indicated that female (adjusted odds ratio [OR] 1.834, 95% confidence interval [CI] 1.069–3.144, P = 0.028), non‐smoking (adjusted OR 2.504, 95% CI 1.456–4.306, P = 0.001), lung adenocarcinoma (adjusted OR 4.512, 95% CI 2.465–8.260, P < 0.001), stage III–IV (adjusted OR 2.232, 95% CI 1.066–4.676, P = 0.033), and no symptoms of emphysema (adjusted OR 2.139, 95% CI 1.221–3.747, P = 0.008) were independent variables associated with EGFR mutations. Young (adjusted OR 3.947, 95% CI 1.873–8.314, P < 0.001) and lung adenocarcinoma (adjusted OR 2.950, 95% CI 0.998–8.719, P = 0.050) were associated with ALK/ROS1 fusions.Conclusions EGFR mutations were more likely to occur in non‐smoking, stage III–IV, and female patients with lung adenocarcinoma, whereas ALK&ROS1 gene fusions were more likely to occur in young patients with lung adenocarcinoma. Emphysema was less common in patients with EGFR mutations.

Fusion-positive non-small cell lung carcinoma: Biological principles, clinical practice, and diagnostic implications.

Based on superior efficacy and tolerability, targeted therapy is currently preferred over chemotherapy and/or immunotherapy for actionable gene fusions that occur in late-stage non-small cell lung carcinoma (NSCLC). Consequently, current clinical practice guidelines mandate testing for ALK, ROS1, NTRK, and RET gene fusions in all patients with newly diagnosed advanced non-squamous NSCLC (NS-NSCLC). Gene fusions can be detected using different approaches, but today RNA next-generation sequencing (NGS) or combined DNA/RNA NGS is the method of choice. The discovery of other gene fusions (involving, eg, NRG1, NUT, FGFR1, FGFR2, MET, BRAF, EGFR, SMARC fusions) and their partners has increased progressively in recent years, leading to the development of new and promising therapies and mandating the development and implementation of comprehensive detection methods. The purpose of this review is to focus on recent data concerning the main gene fusions identified in NSCLC, followed by the discussion of major challenges in this domain.

Abstract P224: Update from the Phase 2 registrational trial of repotrectinib in TKI-pretreated patients with ROS1+ advanced non-small cell lung cancer and with NTRK+ advanced solid tumors (TRIDENT-1)

Abstract Background: Repotrectinib is a next-generation ROS1/TRK tyrosine kinase inhibitor (TKI) that is currently in a registrational Phase 2 trial (TRIDENT-1). A previous update from the TRIDENT-1 trial showed a generally well tolerated safety profile in 185 treated patients. Interim efficacy data were previously presented. In ROS1+ NSCLC patients with 1 prior TKI and prior platinum-based chemotherapy (EXP-2), confirmed overall response rate (cORR) was 40% (2 of 5 patients); in ROS1+ NSCLC patients with 1 prior TKI without prior platinum-based chemotherapy (EXP-4), cORR was 67% (4 of 6 patients); in ROS1+ NSCLC patients with 2 prior TKIs without prior platinum-based chemotherapy (EXP-3), cORR was 40% (2 of 5 patients). In NTRK+ TKI-pretreated advanced solid tumor patients (EXP-6), cORR was 50% (3 of 6 patients). In addition, efficacy was demonstrated in patients who developed solvent front mutations (SFM) following prior ROS1/TRK TKI treatment. cORR was 67% (4 of 6 patients) and 50% (2 of 4 patients) in ROS1+ NSCLC and NTRK+ solid tumors patients with SFMs, respectively. One patient who developed a SFM after entrectinib treatment (EXP-2) had a cCR with 5.6+ months duration of response. Methods: TRIDENT-1 (NCT03093116) is an ongoing registrational Phase 2 trial enrolling patients whose cancers harbor a ROS1 or NTRK gene fusion. Patients are enrolled into 6 defined expansion cohorts (EXP 1-6), based on cancer type and prior therapy. The primary endpoint is cORR by Blinded Independent Central Review using RECIST v1.1. Results: An updated safety analysis across Phase 1 and Phase 2 patients (n=243) based on a data cut-off date of 4 May 2021 was conducted. Treatment-emergent adverse events (TEAEs) observed in ≥20% of patients were dizziness (62%), dysgeusia (43%), constipation (33%), dyspnea (30%), paresthesia (28%), anemia (26%), and fatigue (26%). Grade ≥3 treatment-related AEs (TRAEs) were observed in 17% of patients; no Grade 5 TRAEs were observed. The majority (77%) of dizziness TEAEs were Grade 1 and 4% were Grade 3; none of the dizziness events led to treatment discontinuation. Dose modifications remained infrequent including 24% of patients with a TEAE that led to a dose reduction and 10% of patients with a TEAE that led to drug discontinuation. Updated efficacy information for TKI-pretreated ROS1+ NSCLC and NTRK+ advanced solid tumor patients is being collected and will be available for presentation. Conclusions: Repotrectinib is a next-generation ROS1/TRK inhibitor. In an ongoing registrational Phase 2 trial, repotrectinib was generally well tolerated with low frequency of dose modifications. Updated Phase 2 efficacy data will be available for presentation. Citation Format: Jessica J. Lin, Byoung Chul Cho, Christoph Springfeld, D. Ross Camidge, Benjamin Solomon, Christina Baik, Vamsidhar Velcheti, Young-Chul Kim, Victor Moreno, Anthonie J. van der Wekken, Enriqueta Felip, Dipesh Uprety, Denise Trone, Shanna Stopatschinskaja, Alexander Drilon. Update from the Phase 2 registrational trial of repotrectinib in TKI-pretreated patients with ROS1+ advanced non-small cell lung cancer and with NTRK+ advanced solid tumors (TRIDENT-1) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P224.

Abstract P076: Novel mechanisms of acquired TKI resistance in ROS1+ NSCLC

Abstract ROS1 tyrosine kinase inhibitors (TKIs) entrectinib and crizotinib have significantly improved outcomes for patients with ROS1 gene fusion positive (ROS1+) lung adenocarcinoma. However, drug resistance inevitably develops, leading to disease progression. Approximately one-third of patients resistant to ROS1 TKIs demonstrate on-target ROS1 kinase domain (KD) mutations (Dziadziuszko et al. ESMO 2019), but the mechanism of resistance in the majority of patients remain unknown or poorly characterized. The purpose of this study was to model and characterize mechanisms of acquired ROS1 TKI resistance. To investigate resistance to ROS1 TKIs, we used patient-derived cell lines CUTO28 (TPM3-ROS1) and CUTO37 (CD74-ROS1) to generate drug-resistant derivatives to entrectinib (CUTO28-ER, CUTO37-ER) or crizotinib (CUTO28-CR,CUTO37-CR) in vitro. We utilized several techniques to probe the mechanisms driving resistance, including DNA and RNA sequencing, fluorescence in-situ hybridization (FISH), cell proliferation assays, and western blotting. DNA sequencing of the ROS1 KD in all resistant cell lines did not reveal any mutations. CUTO28-ER cells displayed sensitivity to MET-selective TKIs in cell proliferation assays. Consistent with MET dependency, MAPK and Akt pathways were inhibited by treatment with a MET-selective, but not a ROS1-selective, TKI. Western blot demonstrated MET overexpression and interphase FISH confirmed MET gene amplification (MET:CEP7 ratio 4.2) compared to parental cells (MET:CEP7 ratio 1.0). Notably, metaphase FISH revealed extrachromosomal DNA (ecDNA) amplification of MET. CUTO28-CR cells displayed sensitivity to Src family kinase (SFK) TKIs, and addition of pan-HER TKI afatinib resulted in synergistic inhibition of proliferation and downstream MAPK signaling. CUTO37-ER cells displayed sensitivity via proliferation and signaling to pan-HER inhibitor afatinib, and phosphorylation of EGFR Y845 consistent with Src-mediated activation of EGFR. Parental CUTO28 and CUTO37 cells were not sensitive to SFK or HER inhibition. Interphase FISH revealed modest EGFR gene amplification in CUTO28-CR cells—(EGFR:CEP7 ratio 2.1) compared to parental (EGFR:CEP7 ratio 1.0)—but not in CUTO37-ER or CUTO37-CR cells. HER2 gene amplification was not identified in any of the cell lines. Stimulation with epidermal growth factor (EGF) markedly enhanced MAPK signaling in CUTO28-CR, CUTO37-ER, and CUTO37-CR cells to a greater extent than in corresponding parental cells, consistent with EGFR or HER2 dependence. In conclusion, we demonstrated novel mechanisms of resistance to ROS1 TKIs entrectinib and crizotinib. These include ecDNA amplification of MET as an alternate oncogene driver and Src/EGFR axis as a driver of bypass signaling. Combination strategies with existing TKIs could be explored in patients with ROS1 TKI resistance. The discovery of ecDNA MET amplification is particularly intriguing; ecDNA is associated with more aggressive cancers and next generation sequencing analyses do not typically involve amplicon reconstruction to detect ecDNA. Citation Format: Logan C. Tyler, Anh T. Le, Hala Nijmeh, Liming Bao, Kristen Turner, Jason Christianson, Robert C. Doebele. Novel mechanisms of acquired TKI resistance in ROS1+ NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P076.

Reference standards for gene fusion molecular assays on cytological samples: an international validation study

AimsGene fusions assays are key for personalised treatments of advanced human cancers. Their implementation on cytological material requires a preliminary validation that may make use of cell line slides mimicking...

Abstract 1375: Activity of pemetrexed in patients with driver oncogene positive non-small cell lung cancer and squamous histology

Abstract Pemetrexed, a multi-targeted antifolate chemotherapy agent, has been used both as a single agent chemotherapy as well as in combination given its favorable side effect profile, and has been the only effective continuation maintenance chemotherapy regimen after initial doublet use associated with increased overall survival (OS) among non-squamous non-small cell lung cancers. The current labelled indications for pemetrexed in NSCLC are restricted to those with non-squamous histology due to previous studies that showed worse outcomes in patients with squamous histology when compared to non-squamous histology. More recently, however, in the era of increased molecular profiling of NSCLC, it has been observed that patients with ALK, ROS1, and RET gene fusions have demonstrated marked sensitivity to pemetrexed. This retrospective case series, therefore, evaluated the response to pemetrexed among a cohort of 9 oncogene-driven patients with squamous (or adenosquamous) histologies who did receive pemetrexed based chemotherapy. Response rates, progression free survival (PFS), and OS for these patients were compared to a historical group of patients published in previous studies that examined pemetrexed response. The results indicated a positive response to pemetrexed in patients with an actionable driver oncogene occurring in the context of squamous or adenosquamous histology. Patients in the oncogene-driven adenosquamous/squamous group had significantly higher response rates as compared to the historical squamous cell group and no significant difference in response rates as compared to the historical adenocarcinoma comparison group. In summary, contrary to prior studies, this case series does suggest that pemetrexed may be a viable treatment option in patients with squamous cell histology who also have a targetable mutation. Citation Format: Yunan Nie, Tejas Patil, David Ross Camidge. Activity of pemetrexed in patients with driver oncogene positive non-small cell lung cancer and squamous histology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1375.

Platform study of genotyping-guided precision medicine for rare solid tumours: a study protocol for a phase II, non-randomised, 18-month, open-label, multiarm, single-centre clinical trial testing the safety and efficacy of multiple Chinese-approved targeted drugs and PD-1 inhibitors in the treatment of metastatic rare tumours

IntroductionLimited clinical studies have been conducted on rare solid tumours, and there are few guidelines on the diagnosis and treatment, including experiences with targeted therapy and immunotherapy, of rare solid...

Actionable driver DNA variants and fusion genes can be detected in archived cytological specimens with the Oncomine Dx Target Test Multi-CDx system in lung cancer.

Molecular testing is critical for identifying actionable variants in lung cancer for precision medicine. When tumor tissue samples are unavailable, archived cytological specimens (ACSs) can be used. The authors examined whether oncogenic variants could be accurately detected in ACSs versus paired formalin-fixed, paraffin-embedded (FFPE) tumor tissues with in vitro diagnostic tests. The authors collected 18 ACSs and 15 FFPE tissues from 15 patients with lung cancer and investigated genomic profiles with the Oncomine Dx Target Test Multi-CDx system, which is an integrated next-generation sequencing platform that comprehensively examines 4 companion diagnostic target genes (epidermal growth factor receptor [EGFR]; B-Raf proto-oncogene, serine/threonine kinase [BRAF]; anaplastic lymphoma kinase [ALK]; and ROS proto-oncogene 1, receptor tyrosine kinase [ROS1]). They compared the quantity and quality of extracted nucleic acids, the sequencing quality control (QC), and the detected variants between ACSs and FFPE tissues. The total amount of DNA and RNA obtained from 1 slide was higher in FFPE tissues than ACSs. The RNA integrity number was higher in ACSs. There were no differences in sequencing QC between ACSs and FFPE tissues. A total of 21 variants, including EGFR mutations and ALK and ROS1 fusion genes, were detected in both ACSs and FFPE tissues with 100% concordance. ACSs can be a feasible alternative with which to identify actionable mutations and fusion genes via the Oncomine Dx Target Test Multi-CDx system.

P38.16 Expression of ROS1 Gene Rearrangement in Pulmonary Inflammatory Myofibroblastic Tumor With Cerebral Metastasis: Case Report

Primary pulmonary inflammatory myofibroblastic tumor (IMT) is a rare benign neoplasm; the tumor has been characterized by the application of immunohistochemical techniques, molecular biology and cytogenetics, which are very precious for the diagnosis. The purpose of this case was to determine whether immunohistochemistry for ROS1 could predict ROS1 rearrangement in inflammatory myofibroblastic tumor. A 50-year-old Moroccan man with history of mitral stenosis and mechanical valve replacement; presented to our hospital with a complaint of fatigue; headaches and lower back pain for one month. Contrast-enhanced computed tomography of his chest revealed a heterogenous well-defined mass lesion with central irregular calcification with no mediastinal lymphadenopathy or satellite nodules; this mass involving the left seventh rib and vertebral body and left atrial cavity. CT brain showed four solid enhancing irregular shaped mass lesions with hypodense areas consistent with metastasis CT-guided biopsy of the chest lesion showed fibrosis with dystrophic calcification; and non-specific chronic inflammation. He underwent radical excision of the right frontal lesion, and histopathology was distinctive fascicular growth of spindle cells with with a prominent inflammatory infiltrate, showed weak, diffuse and dot-like cytoplasmic staining with anaplastic lymphoma kinase (ALK)-negative immunohistochemistry and Fluorescence in situ hybridization (FISH) analysis was performed on interphase nuclei on 5 micron thin sections of formalin-fixed paraffin-embedded tissue that was found to be positive for TFG-ROS1 fusion. Because surgical excision was not possible crizotinib therapy (250 mg, bid) was chosen for the existence of ROS1 fusion gene with brain radiotherapy boost to metastasis. Treatment with glucocorticoids (methylprednisolone 1 mg/kg) and radiotherapy (5 days/week for 3 weeks at 3 Gy/fraction, 45 Gy/15 days) was started for cerebral metastasis . After taking crizotinib for 3 months, the strong changes in the size of the pulmonary mass was showed at chest chest computed tomography with decreased infiltration around the adjacent structures. Inflammatory pseudotumour of the lung with brain metastasis is very uncommon; we must continued therapy with crizotinib and long-term follow-up is necessary, for detection of recurrence, metastasis and to understand the natural course, especially in patients with myofibroblastic differentiation ROS1 positive Crizotinib is effective for pulmonary IMT, and should be taken into consideration for the treatment of inoperable pulmonary IMT patients who lack ALK rearrangement.