ROS1 Fusion-positive Patients Research Articles

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.

NVL-520 Is a Selective, TRK-Sparing, and Brain-Penetrant Inhibitor of ROS1 Fusions and Secondary Resistance Mutations.

The combined preclinical features of NVL-520 that include potent targeting of ROS1 and diverse ROS1 resistance mutations, high selectivity for ROS1 G2032R over TRK, and brain penetration mark the development of a distinct ROS1 TKI with the potential to surpass the limitations of earlier-generation TKIs for ROS1 fusion-positive patients. This article is highlighted in the In This Issue feature, p. 517.

Concurrent TP53 mutation adversely impact the efficacy of crizotinib in ROS1-rearranged lung cancer patients.

e20535 Background: ROS1 tyrosine kinase inhibitors (TKIs) are now standard of care for patients with advanced ROS1-rearranged NSCLC. But factors that may affect the efficacy of ROS1 TKIs remain to be explored. Methods: We conducted a retrospective multicenter study of lung cancer patients with ROS1 rearrangements. Treatment and survival follow-up was done and clinical records were reviewed. PFS distribution was analyzed by Kaplan-Meier method with log-rank test. Results: In total, we included 94 lung cancer patients with ROS1 fusion genes profiled by next-generation sequencing from May 2016 to September 2018. Fifty of them were female. The median diagnosis age was 54 (25-83). The most common histologic type was adenocarcinoma, which was confirmed in 75 of 78 patients with available pathological results. The most common fusion partners were CD74, EZR, SDC4 and SLC34A2 identified in 42, 19, 12 and 8 patients respectively. Concurrent actionable mutations were uncommon for ROS1 fusion-positive patients. The most frequent concomitant mutated gene was TP53, which was detected in 33% of all the patients. After excluding 29 patients who were lost to follow-up at the very start, the median follow-up time was 8.5 (0-28) months from the moment when mutation profiling was performed. Thirty-nine patients received treatment with crizotinib, among whom 27 were treatment-naïve patients. The median PFS of the 39 patients with crizotinib was not reached yet. Patients with baseline CNS metastasis tend to have shorter PFS compared to patients without (median, 12 vs NR, p = 0.0073). Besides, concurrent TP53 mutations were correlated with worse PFS (median, both NR, p = 0.0417). Mutation profiles of 10 patients were derived from ctDNA testing. No difference was found in PFS between these 10 patients with others whose genomic profiles were based on fresh tissue or FFPE specimens, suggesting that plasma ctDNA serves as good specimen source for mutation profiling to monitor clinical treatment. Conclusions: Concurrent TP53 mutation and presence of CNS metastasis are associated with decreased PFS of ROS1-positive patients treated with crizotinib.

Efficacy of crizotinib and pemetrexed-based chemotherapy in Chinese NSCLC patients with ROS1 rearrangement.

BackgroundROS1 rearrangement is a novel molecular subgroup of non-small-cell lung cancer (NSCLC). This study aimed to investigate the efficacy of crizotinib and pemetrexed-based chemotherapy in Chinese NSCLC patients with ROS1 rearrangement.ResultsA total of 2309 patients received ROS1 fusion detection and 51(2.2%) patients had ROS1 rearrangement. There was no significant difference between ROS1 fusion-positive and fusion-negative cohorts in demographic data. For the ROS1 fusion-positive patients, crizotinb-treated group had a higher overall response rate (ORR, 80.0%), disease control rate (DCR, 90.0%) and longer progression-free survival (PFS, 294 days) compared with the rates in pemetrexed-treated group (ORR, 40.8%; DCR, 71.4%; PFS, 179 days) and non-pemetrexed-treated group (ORR, 25.0%; DCR, 47.7%; PFS, 110 days). Besides, ORR, DCR and PFS were similar in three major ROS1 fusion partners. For the first-line treatment, patients received pemetrexed had a significant longer PFS than those received non-pemetrexed chemotherapy (209 vs. 146 days, P = 0.0107). In pemetrexed-treated cohorts, ROS1-positive patients with low TS expression had a statistically significant longer PFS than those with high TS expression (184 vs. 110 days, P = 0.0105).Materials and methodsWe retrospectively identified patients with NSCLC who were screened for ROS1 fusion using multiplex reverse transcription-polymerase chain reaction (RT-PCR) from October 2013 to February 2016. The thymidylate synthase (TS) mRNA levels were tested using quantitative real-time RT-PCR.ConclusionsCrizotinib was also highly active at treating Chinese NSCLC patients with ROS1 rearrangement. TS expression could predict the efficacy of pemetrexed-based therapy in ROS1 fusion-positive patients.

Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma

The success of crizotinib in ALK-positive patients has elicited efforts to find new oncogenic fusions in lung cancer. These efforts have led to the discovery of novel oncogenic fusion genes such as ROS1 and RET. However, the molecular and clinicopathologic characteristics associated with RET or ROS1 fusion, compared with ALK fusion-positive lung cancer, remain unclear. We accordingly analyzed the clinicopathologic characteristics of RET- and ROS1-fusion-positive lung adenocarcinomas. We further performed immunohistochemistry and fluorescence in situ hybridization analysis (FISH) in 15 cases of RET and 9 cases of ROS1 fusion tumors by identified NanoString's nCounter screening. RET fusion-positive patients were younger in age, never-smokers, and in early T stage; ROS1 fusion-positive patients had a higher number of never-smokers compared with patients with quintuple-negative (EGFR−/KRAS−/ALK−/ROS1−/RET−) lung adenocarcinoma. Histologically, RET and ROS1 fusion tumors share the solid signet-ring cell and mucinous cribriform pattern, as previously mentioned in the histology of ALK fusion tumors. Therefore, it can be presumed that fusion gene-associated lung adenocarcinomas share similar histologic features. In immunohistochemistry, the majority of 15 RET and 9 ROS1 fusion-positive cases showed positivity of more than moderate intensity and cytoplasmic staining for RET and ROS1 proteins, respectively. In FISH, the majority of RET and ROS1 rearrangement showed two signal patterns such as one fusion signal and two separated green and orange signals (1F1G1O) and an isolated 3′ green signal pattern (1F1G). Our study has provided not only characteristics of fusion gene-associated histologic features but also a proposal for a future screening strategy that will enable clinicians to select cases needed to be checked for ROS1 and RET rearrangements based on clinicohistologic features.

O1-9-2 - Tumor Genomic Profiling of Nsclc From the Phase III Trial of First-Line S-1/Cbdca Versus Paclitaxel/Cbdca (Wjog6611Ltr)

Background: Over the last decade, substantial advances have been made in developing genotype-based target therapies for advanced NSCLC. There is an increasing need to develop high-throughput genotyping tests to evaluate larger number genetic abnormalities.Method: Archived FFPE specimens obtained at the diagnosis were available for 304 (53.9%) of the 564 patients (Pts) who were enrolled in LETS study. With the use of Sequenom's MassARRAY system, we conducted high-throughput genotyping including somatic mutations and gene fusions (ALK, ROS1 and RET). We also evaluated MET amplification by FISH.Results: A somatic mutation in at least one was identified in 105 (48%) of the 217 pts with non-squamous cell carcinoma (SCC) and 26 (45%) of the 58 pts with SCC. Overall, we identified EGFR mutations in 46 patients (17%), TP53 mutations in 30 (11%), STK11 mutations in 27 (9.8%), MET mutations in 21 (7.6%), KRAS mutations in 17 (6.2%), PIK3CA mutations in 6 (2.2%), BRAF and NRAS mutations in each 3 (1.1%), and DDR2, ERBB2, NRF2 and PTEN mutations in each one patient (0.4%). The median overall survival (OS) of EGFR mutation-positive pts was significantly longer than that of pts without EGFR mutations (23.7 v 12.6 months; P = 0.004). Conversely, pts whose tumors had KRAS mutations had a significantly shorter survival than KRAS-wild type pts (9.9 v 15.3 months; P = 0.04). LungFusion panel identified ALK fusions in six cases (2.5%), ROS1 fusions in five cases (2.1%), and RET fusion in one case (0.4%), and these three rearrangements were mutually exclusive. Among five ROS1-fusion positive patients, two patients concomitantly had KRAS mutations, and one patient had EGFRL858R and PIK3CAE542K. Nine out of 229 advanced NSCLC pts (3.9%) were identified as having de novo MET amplification.Conclusions: MassARRAY-based multiplex genetic testing for somatic mutations and fusion genes performs well with nucleic acid derived from FFPE NSCLC tissues obtained from advanced NSCLC.

Clinical and the Prognostic Characteristics of Lung Adenocarcinoma Patients with ROS1 Fusion in Comparison with Other Driver Mutations in East Asian Populations

The prevalence, demographic features, and clinical outcomes of lung adenocarcinoma patients with novel ROS1 oncogenic rearrangement in East Asian populations are not clear. This study aimed to investigate the clinical and prognostic characteristics of lung adenocarcinoma in patients with ROS1 fusion compared with other driver mutations. Multiplex reverse transcription-polymerase chain reaction was used to detect the ROS1 fusion gene in lung adenocarcinoma cases. Immunohistochemistry was used to confirm the expression of ROS1. The demographic data and clinical outcomes of patients with the ROS1 fusion gene were compared with those of patients without the ROS1 fusion gene, including those with the EGFR mutation, EML4-ALK fusion, KRAS mutation, and quadruple-negative patients. Of 492 patients with lung adenocarcinoma, 12 (2.4%) had the ROS1 fusion gene. Their median age was 45.0 years, significantly younger than that of the ROS1 fusion-negative cohorts (p < 0.001). Acinar (including cribriform) and solid patterns were the two most common histologic subtypes in the ROS1 fusion tumors (7 of 12, 58.3%) and were predominantly seen in CD74-ROS1 fusion tumors (66.7%). There was no significant survival difference between the ROS1 fusion-positive and ROS1 fusion-negative cohorts in surgical group, but ROS1 fusion-positive patients might have worse outcomes than EGFR-mutant patients in the stage IV group. The ROS1 fusion gene can be successfully detected in East Asian patients with lung adenocarcinoma using multiplex reverse transcription-polymerase chain reaction. These patients tend to be younger and have characteristic histologic subtypes. Due to the small number of ROS1 fusion patients, the prognostic value of ROS1 fusion need further studies to confirm.

ROS1 fusions in Chinese patients with non-small-cell lung cancer

BackgroundTo determine the prevalence and clinicopathological features of ROS1 fusions in Chinese patients with non-small-cell lung cancer (NSCLC). MethodsFormalin-fixed and paraffin-embedded (FFPE) tissue sections from 392 patients with NSCLC were screened for ROS1 fusions by multiplex RT–PCR and all ROS1 fusions were validated by direct sequencing. The relationship between ROS1 fusions and clinicopathological features and the prognostic effect of the ROS1 fusion status on survival were analyzed. ResultsIn this study, 8 of 392 (2.0%) evaluable samples were found to harbor ROS1 fusions. Of the ROS1-positive patients, seven presented with adenocarcinoma, and one with adenosquamous carcinoma. The ratio of female to male and never smoker to smokers in a ROS1 fusion-positive group was 5:3. There was no statistically significant difference in age, sex, smoking history, histological type and pathological stage between ROS1 fusion-positive and ROS1 fusion-negative patients. ROS1 fusion-negative patients had a significantly longer survival when compared with ROS1 fusion-positive patients (P = 0.041). Lower pathological stage, younger age and ROS1 fusion-negative status were significantly associated with better prognosis on multivariate analysis. ConclusionsROS1 fusions occurred in ∼2.0% of Chinese patients with NSCLC and had no specific clinicopathological feature. ROS1 fusion-negative patients may have a better survival than ROS1 fusion-positive patients.

404 LY2228820 Dimesylate, a P38 MAPK Inhibitor, Demonstrates Anti-Neoplastic Activity in Mouse Models of Human Ovarian Cancer

The ROS1 tyrosine kinase receptor is activated in subsets of cancers including non-small-cell lung cancer (NSCLC), glioblastoma, and cholangiocarcinoma following chromosomal rearrangement. Resulting in-frame fusions of the ROS1 tyrosine kinase domain to multiple different 5′ fusion partners lead to constitutive kinase activity and have been shown to be transforming in vitro and in vivo. The ALK/cMET tyrosine kinase inhibitor crizotinib has been recently reported to inhibit ROS1 and cause tumor shrinkage in a ROS1 fusion containing NSCLC patient. However crizotinib has poor blood brain barrier penetration potentially hindering activity against metastatic brain tumors. In addition, crizotinib has reduced potency against the gatekeeper mutant form of ALK which has been reported as a mechanism of acquired resistance in ALK positive NSCLC patients. Given the structural homology between ALK and ROS1, it is feasible that a similar mechanism of resistance in ROS1-fusion positive patients will arise in the clinic. To confirm ROS1 fusions are oncogenic drivers in ROS1 fusion containing cancer cell lines, the effect of ROS1 knockdown on growth has been determined. ROS1 siRNA leads to preferential inhibition of growth of the ROS1 fusion containing cell lines U118MG (glioblastoma) and HCC78 (NSCLC) in soft agar compared to monolayer. We also identify that AZD1480, a JAK1/2 inhibitor currently in phase 1 clinical trials, inhibits ROS1 with equivalent potency to crizotinib in vitro. Both crizotinib and AZD1480 inhibit pROS1 and decrease phosphorylation levels of downstream signaling proteins including SHP and ERK in ROS1 fusion containing cell lines in vitro and in a U118MG xenograft in vivo. However, AZD1480 demonstrates more pronounced reduction in pSTAT3 levels compared to crizotinib, likely through the added inhibition of JAK1/2. Consistent with the siRNA findings, crizotinib and AZD1480 preferentially inhibit soft agar growth of the ROS1 fusion containing cell lines and lead to regression in the U118MG xenograft model. AZD1480 also penetrates the blood brain barrier in rats following oral administration as assessed using [C]AZD1480, with a brain:blood ratio of around 0.5 after 1 hour. Further, AZD1480 inhibits the L2026M gatekeeper mutation of ROS1 within two fold potency of its activity against wild type ROS1. Together, these data confirm ROS1 as a key genetic driver of tumors containing ROS1 fusions. They also suggest that AZD1480 is a novel ROS1 inhibitor with the potential to be efficacious in ROS1 containing cancer patients including those with brain metastases from NSCLC and treat or overcome gatekeeper mutation driven resistance.