ROS1 Alterations Research Articles

ROS1 Gene Fusion in Advanced Lung Cancer in Women: A Systematic Analysis, Review of the Literature, and Diagnostic Algorithm.

Crizotinib, a mesenchymal-epithelial transition/anaplastic lymphoma kinase/c-ros oncogene 1 (ROS1) inhibitor, has recently been approved by the US Food and Drug Administration for the treatment of patients with advanced ROS1-positive non-small-cell lung cancer (NSCLC). Therefore, interest in ROS1 testing is growing. ROS1 gene fusions affect approximately 0.5% to 2% of unselected NSCLCs. Limited data are available on the prevalence and distribution of ROS1 fusions in patients with advanced-stage NSCLC. A series of 727 lung adenocarcinomas from patients with stage IV disease, negative for epidermal growth factor receptor and anaplastic lymphoma kinase alterations, were tested for ROS1 fusions by fluorescent in situ hybridization analysis, with confirmation by immunohistochemistry. Results were correlated with clinicopathologic parameters and compared with data from the literature. ROS1 fusions were detected in 29 patients (4%), including 27 of 266 females (10.2%) and two of 461 males (0.4%; P = 1.2E-10). The mean age of patients with ROS1-positive disease was lower than that of patients with ROS1-negative disease (49.21 v 62.96 years, respectively; P = 1.1E-10). Eleven of 583 smokers (1.9%) and 18 of 144 nonsmokers (12.5%) showed ROS1 rearrangement (P = 4.05E-7). By logistic regression analysis, ROS1 fusions were independently associated with female sex, younger age at diagnosis, and absence of smoking history, (odds ratios, 12.4, 7.9, and 3.6, respectively). These data, integrated with those reported in the literature, indicate that the prevalence of ROS1 fusions in females and in nonsmokers was higher in patients with advanced disease than in patients with operable disease (11.2% v 3.1%, P < .001; 11.6% v 2.8%, P < .001, respectively). The mean age at diagnosis was significantly lower in patients with advanced disease (49.8 years) than in patients with operable disease (55.6 years; P < .001). Our data indicate that ROS1 fusions in patients with advanced-stage lung adenocarcinoma are more frequent in females, particularly if young and nonsmokers. A diagnostic algorithm for an accurate screening of ROS1 alterations was elaborated.

P3.03-029 ROS1 Alterations in Lung Adenocarcinoma: The Prognostic Role of Rearrangement and Copy Number Variation

ROS1 rearrangements define a distinct molecular subset of lung adenocarcinoma and patients (pts) experience significant improvements with oncogene-directed therapies. Break-apart/Split Fluorescence in situ hybridization (FISH) is a commonly used detection method for rearranged genes as well as the copy number variation (CNV). Based upon FISH, we aimed to thoroughly evaluate the prognostic role of ROS1 alterations in lung adenocarcinoma.

ALK and ROS1 testing on lung cancer cytologic samples: Perspectives.

Cytologic sampling is the mainstay of diagnosing advanced lung cancer. Moreover, to select patients for personalized first-line or second-line treatment, epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) rearrangements are tested on cytologic preparations. Commercially available fluorescence in situ hybridization (FISH) and immunocytochemistry (ICC) assays have primarily been used for the identification of cells harboring ALK or ROS1 gene fusions on histologic rather than cytologic preparations. However, it is now recognized that FISH and ICC also can be applied on cytologic samples provided the cytopathologist is aware that FISH and ICC results are not always concordant and that the performance of ICC largely depends on antibody clones, signal detection systems, and scoring systems. Notably, the routine clinical use of FISH and ICC may be replaced by emerging next-generation sequencing and digital, color-coded barcode technologies, which have the advantage of simultaneously evaluating ALK, ROS1, and EGFR alterations in a single analysis. Although their use in clinical cytologic practice remains to be fully established, it is conceivable that this technology will replace both FISH and ICC analyses in future diagnostic algorithms. Here, the authors review studies devoted to testing ALK and ROS1 on cytology specimens in an attempt to provide an update for the cytopathologist regarding current and evolving practice. Cancer Cytopathol 2017;125:817-30. © 2017 American Cancer Society.

Abstract CT030: STARTRK-NG: A phase 1/1b study of entrectinib in children and adolescents with advanced solid tumors and primary CNS tumors, with or without TRK, ROS1, or ALK fusions

Abstract Background: The STARTRK-NG (Studies of Tumor Alterations Responsive to Targeting Receptor Kinases - Next Generation) trial is a Phase 1/1b dose-escalation and expansion study of entrectinib in pediatric patients with cancer. Entrectinib is a potent oral, CNS-penetrant, inhibitor of the tyrosine kinases TRKA/B/C (encoded by the genes NTRK1/2/3, respectively), ROS1, and ALK with IC50s &amp;lt; 2 nM (biochemical kinase assay). Three adult studies (Phase 1 ALKA-372-001, Phase 1 STARTRK-1, and Phase 2 STARTRK-2) have enrolled hundreds of patients with advanced or metastatic solid tumors harboring TRKA/B/C, ROS1, or ALK molecular alterations, with or without CNS disease. Previously, we reported 400 mg/m2 daily as the adult body surface area (BSA)-based Recommended Phase 2 Dose (RP2D) (Patel et al, ASCO 2015). An objective response rate of 79% was seen in 24 tyrosine kinase inhibitor-naïve patients with TRK, ROS1, or ALK fusions who were treated at doses consistent with the RP2D (Drilon et al, AACR 2016). Gene fusions have been observed in a variety of adult solid tumor types, including non-small cell lung cancer, salivary gland cancers, soft tissue sarcomas and glioneuronal tumors. These fusions have also been found in cancers that affect the pediatric population, such as infantile fibrosarcoma, juvenile breast cancer, mesoblastic nephroma, intrinsic pontine gliomas, acute leukemias and anaplastic lymphoma. In addition, overexpression of TRKB and activating ALK point mutations have been observed in neuroblastoma. Thus, a pan-TRK, ROS1, and ALK inhibitor, like entrectinib, may potentially have broad therapeutic utility in pediatric patients. Methods: This is a multicenter, dose escalation study in pediatric patients (aged 2-21 years) with relapsed or refractory extracranial solid tumors (Phase 1), with additional expansion cohorts (Phase 1b) in patients with primary brain tumors harboring TRK, ROS1, or ALK molecular alterations inclusive of gene fusions, neuroblastoma, and other non-neuroblastoma, extracranial solid tumors harboring TRK, ROS1, or ALK gene fusions (NCT02650401). During dose escalation, a 3+3 schema will be used to determine the pediatric RP2D of entrectinib with a starting dose of 250 mg/m2 (approximately 60% of the adult BSA-based RP2D), administered orally once daily in repeated 4-week cycles, with concordant pharmacokinetics and pharmacodynamics studies. Up to four dose levels will be evaluated. Dose modifications, if necessary, will follow a protocol-specific dosing nomogram for each dose level. Once the pediatric RP2D is determined, the Phase 1b expansion cohorts will be opened simultaneously, and prospective molecular profiling will be performed to determine eligibility except for patients with neuroblastoma. Citation Format: Ami V. Desai, Garrett M. Brodeur, Jennifer Foster, Suzanne Shusterman, Amit J. Sabnis, Magaret Macy, Cynthia Wetmore, Ellen Basu, Zachary Hornby, Vanessa Esquibel, Edna Chow Maneval, Pratik S. Multani, Elizabeth Fox. STARTRK-NG: A phase 1/1b study of entrectinib in children and adolescents with advanced solid tumors and primary CNS tumors, with or without TRK, ROS1, or ALK fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT030. doi:10.1158/1538-7445.AM2017-CT030

Clinicopathologic features of non-small cell lung cancer (NSCLC) harboring an NTRK gene fusion.

11580 Background: Gene fusions involving NTRK1/2/3 can generate oncoproteins containing the kinase domains of TRKA/B/C, respectively. Inhibition of TRK signaling has led to dramatic responses across tumor types with NTRK fusions. An estimated 0.1 – 1% of NSCLCs harbor NTRK fusions. To date, clinical and radiographic responses to TRK inhibitors have been reported for 2 NTRK fusion-positive NSCLCs (Farago et al., 2015; Hong et al., 2016). Despite the potential benefit of identifying these fusions, the clinicopathologic features of NTRK fusion NSCLCs are not well characterized. Methods: Physicians across multiple institutions contributed deidentified cases to an NTRK fusion NSCLC database. A central pathologist (M.M.) reviewed tumor histology in cases with available tissue. Results: 10 NSCLC cases with NTRK gene fusions were identified. Of these, TRK kinase domain-containing potentially activating fusions were verified by next-generation sequencing (NGS) in 7, forming the study cohort. Fusions involved NTRK1 (6) and NTRK3 (1) with 6 different partners. Four (57%) patients were male. Median age at diagnosis was 47.6 years (range 27.9 – 86.0). The average smoking pack year history was 8.9 (range 0 to 30). Five (71%) presented with metastatic disease. No concurrent alterations in KRAS, EGFR, ALK, ROS1, or other known drivers were identified in the study cohort cases. On pathologic review of 4 cases, all were adenocarcinoma, including 2 invasive mucinous adenocarcinomas and 1 adenocarcinoma with neuroendocrine features. Of the 3 remaining non-study cohort cases, 1 was a non-kinase domain-containing NTRK1 fusion with a concurrent KRAS G12C mutation, 1 was an NTRK2 intragenic deletion disrupting the exon 18 3’ splice site, and 1 was an NTRK1 alteration detected by FISH but not verified by NGS and with a concurrent HER2 L755P mutation. Conclusions: NTRK fusions occur in both men and women across wide ranges in age and smoking history. We therefore suggest that all NSCLC adenocarcinomas without other oncogenic driver alterations be screened for NTRK fusions. Notably, not all NTRK alterations are activating, requiring validation of the specific position of the fusion.

Clinicopathologic features and genetic profile of the redefined large cell lung carcinoma

Objective: To investigate the clinicopathologic features and genetic profile of large cell lung carcinoma (LCC) redefined by new classification. Methods: Basing on 2015 WHO classification criteria in redefining large cell lung carcinoma, the expression of specific markers (TTF1, Napsin A, p40, CK5/6, CK, vimentin and ZEB1) was detected by immunohistochemistry and D-PAS staining in 303 surgically-removed lung specimens previously diagnosed as large cell lung carcinoma. The clinicopathologic and genetic characteristics (including EGFR, KRAS, BRAF, ALK and ROS1 gene mutation) were analyzed. Results: Based on the new definition of LCC, 116 cases (116/303, 38.3%) of LCC formerly diagnosed were reclassified as solid adenocarcinoma, 49 cases (49/303, 16.2%) as squamous cell carcinoma, 6 cases (6/303, 2.0%) as adenosquamous carcinoma, 22 cases (22/303, 7.3%) as spindle cell carcinoma and only 110 cases (110/303, 36.3%) as large cell carcinoma. Redefined LCCs were characterized as middle-age (range 40-80), male (102/110, 92.7%) and smoking patients (64/110, 58.2%) with intermediate-advanced stage. Among 110 cases, 9 cases with EGFR mutation and 10 cases with KRAS mutation and 1 case with ALK fusion were found. No BRAF and ROS1 alterations were identified. Conclusions: According to the new classification, LCCs formerly diagnosed are mostly reclassified as adenocarcinoma and non-keratinizing squamous cell carcinoma. The newly defined LCC may significantly benefit from clinical therapy.

Crizotinib chez les patients atteints de cancer bronchopulmonaire ROS1 positifs

ROS1 fusions are rare mutations that preferentially concern young and non-smoker women. The ROS1-rearranged protein conserves an intact tyrosine kinase domain, leading to the constitutive activation of the ROS1 tyrosine kinase function and of its downstream pathways, that are known to be involved in tumorigenesis. These molecular abnormalities have shown their oncogenic potential in animals' models and in human, with an early effect on carcinogenesis. Several partners have been identified. Patients with non-small cell lung cancers (NSCLC) harbouring ROS1 alterations can receive specific targeted therapies. Indeed, crizotinib has recently been approved in France in advanced ROS1-rearranged NSCLC. We propose a review of the oncogenic role of ROS1 rearrangements, the different methods for its diagnosis, and the available treatments.

P2.03b-068 The Druggable Mutation Landscape of Lung Adenocarcinoma: Topic: Biomarkers

Molecularly targeted therapies and immunotherapies have emerged as promising approaches in the fight against advanced-stage cancers, including lung adenocarcinoma. Identifying genomic alterations predictive of targeted therapy response, as well as biomarkers for immunotherapy response, such as tumor mutation burden (TMB), could minimize the utilization of ineffective therapies and help overcome tumor drug resistance, improving patient outcomes. We examined the largest previously described dataset, consisting of genomic alterations of ∼10,000 lung adenocarcinoma patients, to characterize the landscape of druggable alterations and identified previously undetected co-occurrence and exclusivity relationships between genomic alterations. Comprehensive genomic profiling (CGP) based on hybrid capture-based next-generation sequencing of the full coding regions of up to 315 cancer-related genes was performed on 10,472 lung adenocarcinomas. Base substitutions, indels, copy number alterations and gene fusion/rearrangements were assessed. TMB was calculated as the number of somatic, coding, base substitutions and indels per megabase of genome examined (high TMB: ≥20 mutations/Mb). Patient median age was 65 years (range: 13 to >90 years), and 56% were female. The alteration frequencies of the druggable NCCN lung adenocarcinoma guideline genes were: EGFR (20.5%), BRAF (5.8%), ERBB2 (5.7%), c-MET (5.2%), ALK (4.3%), RET (2.1%), and ROS1 (1.4%). 57.5% and 2.5% of samples had alterations in zero or multiple of these genes, respectively. Few cases with high TMB were found in samples with alterations in EGFR (3.6%) or ALK (2.6%), while a larger percentage with alterations in BRAF (12.9%) or zero NCCN genes (17.4%) had high TMB. 269 cancer-related genes were each altered in ≥0.1% of cases without alterations in NCCN genes or high TMB, including genes that are becoming clinically relevant, such as STK11 (24.8%), MYC (8.4%), NF1 (8.2%), PIK3CA (5.2%), RICTOR (3.7%), CDK4 (2.8%), CCND1 (2.8%), BRCA2 (1.7%), and NRAS (1.3%). Detailed co-occurrence and exclusivity relationships for all genomic alterations will be presented. EGFR, RET, and ROS1 alterations were most common in female cases, and ALK- and ROS1-altered tumors had the lowest patient age distributions (medians: 57 and 55 years, respectively). Using CGP, >50% of patients with lung adenocarcinoma had an alteration in at least one NCCN gene (42.5%), a high TMB status (12.3%), or both (2.3%). Amongst those with neither, 47.5% had an alteration in a gene with emerging evidence for clinical utility. Given the robustness of the dataset, this analysis suggests a significant expansion of the patient population eligible for personalized anti-lung cancer treatment through combination therapy and immunotherapy.

MET-inhibitors meet MET mutations in lung cancer

Mesenchymal-to-epithelial transition ( MET ) exon 14 mutation in non-small cell lung cancer (NSCLC) has been recognized. However, clinical, molecular, and pathologic features have not been well understood. Awad et al . described NSCLC patients with MET exon 14 mutations precisely in the ‘ Journal of Clinical Oncology 2016;34:721-30’. Among 933 non-squamous NSCLC patients, they found 28 (3.0%) patients who represented a unique clinical and molecular subtype of NSCLC. Median age of the 28 patients was 72.5 years, 68% were women, 36% were never-smokers, 64% had stage IV, 100% were white, non-Hispanic, and 64% had adenocarcinoma and 14% had pleomorphic carcinoma. Genomic deletions and point mutations occurred in 17 and 11, respectively, occurred of the 28 patients. Although none of the 28 patients with MET exon 14 mutations had KRAS , epidermal growth factor receptor ( EGFR ), ERBB2 , anaplastic lymphoma kinase ( ALK ), ROS1 , or RET alterations, mutations of TP53 , CDKN2A/B , BRAF600E , PIK3CA , PTEN , RB1 , ATM , BRCA2 , NF1 , or ARID2 were co-existed. Amplification of MDM2 was observed in 13 (46%), and 6 (21%) and 8 (29%) had high- and low-level MET copy gain, respectively. To date, two MET inhibitors, onartuzumab or tivantinib, combination with erlotinib in previously treated NSCLC were investigated in phase III trials. However, neither showed prolonged overall survival (OS) compared with erlotinib alone in molecularly unselected patients. Several publications including the report of Awad et al . revealed that patients with MET exon 14 mutation were successfully treated with MET-tyrosine kinase inhibitors (TKIs) such as crizotinib. Prospective trials using MET-TKIs in MET exon 14 mutated NSCLC are ongoing. Concerning translational research, significance of co-existed other mutations or amplifications and mechanism of acquired resistance to MET-TKIs remain to be clarified. Finally, the therapeutic strategies against the MET-TKI resistance and intracranial metastasis in NSCLC with MET exon 14 mutation should be elucidated.

Molecular analysis of peripheral non-squamous non-small cell lung cancer sampled by radial EBUS.

Treatment optimization of non-squamous non-small-cell lung cancers (nonSq-NSCLC) relies on the molecular analysis of the tumour. We aimed to assess the predictive factors of molecular analysis feasibility (MAF) from samples of peripheral nonSq-NSCLC obtained by radial endobronchial ultrasound bronchoscopy (r-EBUS) and 1.5 mm microbiopsy forceps. We reviewed data from consecutive peripheral lung nodules sampled with r-EBUS between January 2012 and July 2014 at a single French University Hospital. nonSq-NSCLC were systematically analysed for EGFR, KRAS, ALK, HER2, PI3K and BRAF throughout the study, and c-MET and ROS1 alterations for the last 10 months. Of 111 nonSq-NSCLC diagnosed by r-EBUS (113 procedures, mean nodule diameter 28 ± 15 mm), 88 were analysed for EGFR and ALK, 87 for KRAS, 86 for HER2, PI3K and BRAF and 14 for c-MET. Forty-one mutations were identified (23 KRAS, 10 EGFR, 2 BRAF, 1 HER2 and 5 ALK rearrangements). Four c-MET overexpressions were noted. MAF rose from 67% for the first 57 procedures to 89% for the last 56 procedures (P = 0.02) likely due to a higher number of biopsies performed (2 ± 1 vs 3 ± 2, P = 0.005). Upper or middle lobe location (OR 1.19, 95% CI: 1.02-1.38, P = 0.03), and at least three biopsies (OR 1.20, 95% CI: 1.04-1.40, P = 0.02) were predictive factors of MAF. Percentage of tumour cells, size of lesion and distance to the pleura did not correlate with MAF. Multi-gene molecular analysis could be performed in nearly 80% of paraffin-embedded biopsies or smear specimens sampled by r-EBUS assisted bronchoscopy of peripheral tumoral lung nodules.

Abstract LB-A08: ALK and ROS-1 status: A retrospective analysis in solid tumors

Abstract Background: Tyrosine kinase receptors are important regulators of cellular signal transduction pathways that play a crucial role in development of cancer. Among them, Anaplastic Lymphoma Kinase (ALK) and C-Ros Oncogene-1 (ROS-1) are activated by rearrangement in several tumors. Tailored therapies have been developed according to the status of ALK and ROS-1, successfully used in NSCLC and potentially useful in other solid tumors harboring ALK and ROS-1 aberrations. Material and methods: ALK and ROS-1 status was evaluated in 666 and 441 patients with solid tumors, respectively, since 2014 at the Pathology Department. ALK and ROS-1 rearrangement and gene copy number variation (CNV) were assayed by fluorescence in situ hybridization (FISH). Gene copy number gain was defined by 3 to 5 fusion signals on average in ≥10% of cells, with amplification being highlighted by the clusters or 10 to 15 fusion signals in 10% or more tumor cells. Results: ALK status was assessed in 650/666 (97%) cases, while ROS-1 status was evaluable in 426/441 (96%). We identified 15/650 (2.3%) cases with ALK rearrangment, including 13 lung adenocarcinomas (4.1%), 1 colon cancer (0.5%) and 1 myoepithelioma (0.5%). CNV for ALK was documented in 53% lung adenocarcinoma, 50% pancreatic, 41% biliary and 37% colon carcinoma patients. We found 7/426 (1.6%) tumors with ROS-1 rearrangement, corresponding to 5 lung (2.7%) and 2 colon (1.3%) adenocarcinomas. CNV for ROS-1 was more frequent in melanoma (77%), neuroendocrine tumors (39%) and NSCLC (32%, 3/4 cases of ROS-1 amplification were squamous carcinoma). Eight out of the 22 rearranged cases (5 ALK and 3 ROS-1) were tested for both genes: these aberrations were mutually exclusive. Three out of 5 (60%) ALK rearranged tumors showed ROS-1 deletion. No ALK and ROS-1 alterations were documented in 30 pancreatic, 25 H&amp;N, 22 biliary tract, 13 renal, and 13 breast carcinomas, as well as in 13 melanomas and 36 other solid tumors. Conclusions: ALK and ROS-1 rearrangement was found in 4.1% and 2.7% of NSCLC, respectively, in keeping with literature data. In other solid tumors, the percentage of rearrangements is exceedingly low, but melanoma, neuroendocrine tumors, pancreatic and colon cancer may show significant CNV. We speculate that such aberrations of ALK and ROS-1 could act as potential therapy targets, but additional investigation by immunohistochemistry and next generation sequencing is clinically warranted. Citation Format: Maria Silvia Cona, matteo Duca, Adele Testi, Sara Cresta, Katia Fiorella Dotti, Alice Indini, Diego Signorelli, Giuseppe Pelosi, Filippo Guglielmo de Braud, Silvia Damian. ALK and ROS-1 status: A retrospective analysis in solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A08.

Lung adenocarcinoma: Are skip N2 metastases different from non-skip?

ObjectiveThis study investigated the association between clinical pathologic features, especially adenocarcinoma subtypes and prognosis, and skip N2 metastasis in patients with lung adenocarcinoma. MethodsIn this study, 177 patients with lung adenocarcinoma and N2 metastasis were enrolled. Patients who had N2 lymph node metastases without N1 lymph node involvement were defined as skip N2 and otherwise as non-skip N2. We investigated the difference of clinicopathologic characteristics, recurrence-free survival, overall survival, and spectrum of well-identified molecular alterations in EGFR, KRAS, HER2, BRAF, ALK, ROS1, and RET genes in the 2 groups. ResultsSkip N2 metastasis was found in 45 patients, in whom a remarkably lower incidence of lymphovascular invasion was revealed (P = .01). Skip N2 metastasis was also associated with acinar subtype, good differentiation, and right lung cancer. The recurrence-free survival and overall survival were significantly better in the skip N2 group (5-year recurrence-free survival 37.4% vs 5.7%; log-rank P = .005; 5-year overall survival 60.7% vs 32.1%; log-rank P = .024). The predictive value of skip N2 was more significant in patients with lesions in the right lung (5-year recurrence-free survival 36.6% vs 0.0%; log-rank P = .002; 5-year overall survival 57.2% vs 27.9%; log-rank P = .016) and in patients whose tumor diameter was no more than 3 cm (5-year recurrence-free survival 43.1% vs 6.7%; log-rank P = .01; 5-year overall survival 74.6 vs 27.6%; log-rank P = .04). ConclusionsThere are distinct differences in clinicopathologic features and prognosis in patients with or without skip N2 metastasis. Considering the results of our study, subclassifications of mediastinal lymph node metastases could have clinical significance for patients with lung adenocarcinoma.

Alka-372-001: First-in-human, phase I study of entrectinib – an oral pan-trk, ROS1, and ALK inhibitor – in patients with advanced solid tumors with relevant molecular alterations.

2517 Background: Entrectinib (formerly RXDX-101) is a potent and selective oral small molecule inhibitor of the TrkA/B/C, ROS1, and ALK kinases. Schedule A (fasted, 4d on/3d off for 3wks, 1 wk rest) demonstrated significant antitumor activity (ASCO 2014). This abstract reports completion of Sch A and two other ongoing dosing schedules (B and C). Methods: Pts with advanced solid tumors with molecular alterations in TrkA, ROS1 or ALK were treated in Sch B (QD) or Sch C (4d on/3d off), both in fed state. Results: 31 pts were treated in Sch A (N=19), B (N=6), or C (N=6). In Sch A, doses > 800 mg/m2 did not increase exposure significantly. Thus, accrual in Sch A was closed. Various dose levels (mg/m2) were explored in Sch B [200 (n=3); 400 (n=3)] and Sch C [400 (n=3); 800 (n=3)]. 7 pts had objective responses: 6 PR, 1CR (see Table). We are the first to report clinical activity of a Trk inhibitor in a pt with NTRK1+ (encoding TrkA) CRC. Of 7 ROS1-rearranged evaluable/measurable NSCLC pts, 4 have had an objective response (ORR: 57%), with a median duration of 6+ months. Moreover, in ROS1 pts treated at ≥ 400 mg/m2, the ORR was 80% (4 of 5 pts). Entrectinib is well tolerated. The majority of pts reported G1/ G2 AEs. 13 pts reported ≥ G3 AEs. Asthenia and muscle weakness were possibly related ≥ grade 3 AEs (both reversible). No DLTs have been reported to date. In Sch A, entrectinib was readily absorbed and exposures increased dose-proportionally up to 800 mg/m2. In fed state, entrectinib exposures were approx. 2x compared to fasted state. Conclusions: In this trial with entrectinib administered in 3 different dosing schedules, significant antitumor response was observed in pts with relevant molecular alterations, notably ROS1-rearranged NSCLC at doses ≥ 400 mg/m2/day and the only NTRK1 rearranged pt treated to date. Accrual in Sch B and C continues until RP2D is achieved, followed by dedicated studies in selected tumor types. Clinical trial information: NCT02097810.Objective responses. Tumor type (alteration) Sch/Dose (mg/m2) Best Response Duration of Response (cycles) NSCLC (ALK) A/800 PR 10 NSCLC (ROS1) A/1200 PR 14+ C/400 PR 7+ C/400 CR 5+ B/400 PR 3+ CRC (NTRK1) A/1600 PR 3 Neuroblastoma (ALK) A/800 PR 9

Comprehensive Genomic Profiling of Carcinoma of Unknown Primary Site: New Routes to Targeted Therapies.

For carcinoma of unknown primary site (CUP), determining the primary tumor site may be uninformative and often does not improve outcome. To discover opportunities for targeted therapies in patients with CUP not currently searched for in routine practice. Comprehensive genomic profiling on 200 CUP formalin-fixed paraffin-embedded specimens (mean, 756× coverage) using the hybrid-capture-based FoundationOne assay at academic and community oncology clinics. Presence of targetable genomic alterations (GAs) in CUP and responses to targeted therapies. There were 125 adenocarcinomas of unknown primary site (ACUPs) and 75 carcinomas of unknown primary site without features of adenocarcinoma (non-ACUPs). At least 1 GA was found in 192 (96%) of CUP specimens, with a mean (SD) of 4.2 (2.8) GAs per tumor. The most frequent GAs were in TP53 (110 [55%]), KRAS (40 [20%]), CDKN2A (37 [19%]), MYC (23 [12%]), ARID1A (21 [11%]), MCL1 (19 [10%]), PIK3CA (17 [9%]), ERBB2 (16 [8%]), PTEN (14 [7%]), EGFR (12 [6%]), SMAD4 (13 [7%]), STK11 (13 [7%]), SMARCA4 (12 [6%]), RB1 (12 [6%]), RICTOR (12 [6%]), MLL2 (12 [6%]), BRAF (11 [6%]), and BRCA2 (11 [6%]). One or more potentially targetable GAs were identified in 169 of 200 (85%) CUP specimens. Mutations or amplifications of ERBB2 were more frequent in ACUPs (13 [10%]) than in non-ACUPs (3 [4%]). Alterations of EGFR (10 [8%] vs 2 [3%]) and BRAF (8 [6%] vs 3 [4%]) were more common in ACUPs than in non-ACUPs. Strikingly, clinically relevant alterations in the receptor tyrosine kinase (RTK)/Ras signaling pathway including alterations in ALK, ARAF, BRAF, EGFR, FGFR1, FGFR2, KIT, KRAS, MAP2K1, MET, NF1, NF2, NRAS, RAF1, RET, and ROS1 were found in 90 (72%) ACUPs but in only 29 (39%) non-ACUPs (P < .001). Almost all CUP samples harbored at least 1 clinically relevant GA with potential to influence and personalize therapy. The ACUP tumors were more frequently driven by GAs in the highly druggable RTK/Ras/mitogen-activated protein kinase (MAPK) signaling pathway than the non-ACUP tumors. Comprehensive genomic profiling can identify novel treatment paradigms to address the limited options and poor prognoses of patients with CUP.

O3.7 - Enriching phase I trials with molecular alterations: Interim analysis of 708 patients enrolled in the MOSCATO 01

ABSTRACT Background: Enrolling patients with ad hoc molecular alterations is one of the current challenges in early drug development. The MOSCATO 01 trial (NCT01566019) is one of the prospective trials ongoing worldwide using high throughput analysis for characterisation of the genomic alterations in cancer patients. Methods: An “on-purpose” tumor biopsy was collected from consenting patients with metastatic cancer referred for a phase I trial. DNA extracted from the tumor biopsy was analysed by comparative genomic hybridization (CGH) (if ≥ 30% tumor cells) and by hot spot mutations by Next Generation Sequencing (NGS) (if ≥ 10% tumor cells). Recently, WES and RNAseq were implemented in the MOSCATO trial. Results: Between December 2011 and November 2014, 708 patients were enrolled in the MOSCATO 01 trial, and 639 patients (90%) had undergone a tumor biopsy. Patient characteristics were as follows: median age at biopsy: 57 years (range, 18-78); main tumor types: lung (14%), Head and neck (13%), prostate and bladder (11%), breast (10%), median of 3 previous lines of treatment. Molecular profiling with CGH and NGS was performed in 510 (80%) and 565 patients (88%) respectively, with median analysis turnaround of 21 days. Clinical significant molecular alterations were found in 288 patients (45%), most frequently in PTEN/PI3K/AKT (n = 84), FGFR/FGF (n = 46), KRAS/NRAS/HRAS (n = 24), HER2/EGFR (n = 22/18) pathways. Patients with rare genomic alterations were also identified (eg MDM2, MET, AR, ALK, KIT, and ROS1 alterations among others). Therapy could be personalised in 141 patients (49%) with matched targeted therapy or dedicated phase I clinical trials. Preliminary results of WES and RNAseq as well as the response rate of patients with specific molecular alterations treated with matched targeted therapies will be updated and presented at the TAT meeting. Conclusions: Our updated results confirm in a larger cohort of patients the feasibility of molecular characterisation of cancer patients in routine clinical practice. Such approach allows the enrichment of early-phase clinical trials with specific and rare genomic alterations

Activation of RAS family members confers resistance to ROS1 targeting drugs.

The ROS1 tyrosine kinase is activated in lung cancer as a consequence of chromosomal rearrangement. Although high response rates and disease control have been observed in lung cancer patients bearing rearranged ROS1 tumors (ROS1+) treated with the kinase inhibitor crizotinib, many of these patients eventually relapse.To identify mechanisms of resistance to ROS1 inhibitors we generated resistant cells from HCC78 lung cancer cells bearing the SLC34A2-ROS1 rearrangement. We found that activation of the RAS pathway in the HCC78 cell model, due to either KRAS/NRAS mutations or to KRAS amplification, rendered the cells resistant to ROS1 inhibition. These cells were cross-resistant to different ROS1 inhibitors, but sensitive to inhibitors of the RAS signaling pathway. Interestingly, we identified focal KRAS amplification in a biopsy of a tumor from a patient that had become resistant to crizotinib treatment.Altogether our data suggest that the activation of members of the RAS family can confer resistance to ROS1 inhibitors. This has important clinical implications as: (i) RAS genetic alterations in ROS1+ primary tumors are likely negative predictors of efficacy for targeted drugs and (ii) this kind of resistance is unlikely to be overcome by the use of more specific or more potent ROS1 targeting drugs.

Parallel screening for ALK, MET and ROS1 alterations in non-small cell lung cancer with implications for daily routine testing.

ALK, MET and ROS1 are prognostic and predictive markers in NSCLC, which need to be implemented in daily routine. To evaluate different detection approaches and scoring systems for optimal stratification of patients eligible for mutation testing in the future, we screened a large and unselected cohort of NSCLCs for all three alterations. Using tissue microarrays, 473 surgically resected NSCLCs were tested for ALK and MET expression by IHC and genomic alterations in the ALK, MET and ROS1 gene by FISH. For MET IHC, two different criteria (MetMAb and H-score), for MET FISH, three different scoring systems (UCCC, Cappuzzo, PathVysion) were investigated. ALK and ROS1 positivity was seen in 2.6% and 1.3% of all ADCs, respectively, but not in pure SCCs. One ROS1 translocated tumor showed additional ROS1 amplification. MET IHC+/FISH+ cases were found in both histological subtypes (8.6% in all NSCLCs; 10.6% in ADCs; 5.0% in SCCs) and were associated with pleural invasion, lymphatic vessel invasion and lymph node metastasis. MET altered ADCs more frequently showed a papillary growth pattern. Whereas ALK testing revealed homogenous results in IHC and FISH, we saw discordant results for MET in about 10% of cases. Both METIHC scoring systems revealed almost identical results. We did not encounter any combined FISH positivity for ALK, MET or ROS1. However, three ALK positive cases harbored MET overexpression. In daily routine, IHC could support FISH in the identification of ALK altered NSCLCs. Further research is needed to assess the role of discordant MET results by means of IHC and FISH as well as the relevance of tumors with an increased ROS1 gene copy number.

Abstract 4656: The novel mRNA in situ hybridization method for the detection of ALK, RET, ROS1 and NTRK1 mRNA in non-small cell lung cancer

Abstract [Background]In recent years, tumor genotyping is critical for making decisions of non-small cell lung cancer (NSCLC) treatment. However genetic rearrangement of ALK, RET, ROS1 and NTRK1 were identified in the subset of NSCLC patients, these clinical characteristics were still remained to be clear. Immunohistochemistry (IHC) is widely used as a simple and quick method for screening for protein expression of these oncogenes. Fluorescence in situ hybridization (FISH) or RT-PCR are performed consecutively to confirm the genotyping. However it is often taking time and expensive and it is needed to consolidate novel substitute methods. [Purpose]We performed the in situ hybridization (ISH) analysis to detect cellular mRNA in formalin-fixed paraffin-embedded (FFPE) tissue samples of lung cancer. We also conducted IHC with the use of ALK, RET, ROS1 and NTRK1 specific antibodies and investigate a comparative study to assess the usefulness of mRNA-ISH to detect expression of these mRNA. [Materials and Methods]We made tissue micro array (TMA) slides using 40 FFPE samples of resected lung adenocarcinoma with known epidermal growth factor receptor (EGFR) mutation status by PCR (PNA-LNA PCR clamp method). The mRNA in situ hybridization was employed using RNA scope R 2.0 Regant Kit with targeting probes. IHC staining was conducted by antibodies using 5A4 and D5F3 for ALK, RET01 for RET, D4D6 for ROS1, and 14G6 for NTRK1. [Results]All of the IHC stained cases were clearly detected by mRNA-ISH respectively. There were some cases which were only visualized by ISH, each case was partially positive and not totally of the tumor. ALK: 5% of cases (2 out of 40) were detected in mRNA-ISH and were strongly stained with both 5A4 and D5F3. RET: 20% of cases (8/40) were detected in mRNA-ISH. Two cases (5% of total) had positive RET01 staining, while 6 cases had negative staining. (3 had no other genetic alterations, 2 had EGFR-L858R confirmed by PCR, and 1 had ALK by IHC and FISH.) ROS1: 20% (8/40) were detected in mRNA-ISH. Three cases (7.5% of total) presented positive D4D6 staining, while 5 had negative staining. (3 had no other alterations, 1 had EGFR-E746-A750 and 1 had EGFR-L858R by PCR.) NTRK1: 17.5% of cases (7/40) were detected in mRNA-ISH. 1 case (2.5% of total) presented positive 14G6 staining, while 6 cases had negative staining. (2 had no other alterations, 2 had EGFR-L858R, 1 had EGFR-E746-A750, and 1 had ALK.) [Conclusion] The novel mRNA in situ hybridization method combined with traditional IHC could improve detection rate of ALK, RET, ROS1 and NTRK1 gene alterations in NSCLC FFPE samples. Citation Format: Noriko Hirai, Takaaki Sasaki, Yoshinori Minami, Yoshinobu Ohsaki. The novel mRNA in situ hybridization method for the detection of ALK, RET, ROS1 and NTRK1 mRNA in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4656. doi:10.1158/1538-7445.AM2014-4656

Fibroblast growth factor receptor 2 translocations in intrahepatic cholangiocarcinoma.

Patients with cholangiocarcinoma often present with locally advanced or metastatic disease. There is a need for effective therapeutic strategies for advanced stage cholangiocarcinoma. Recently, FGFR2 translocations have been identified as a potential target for tyrosine kinase inhibitor therapies. This study evaluated 152 cholangiocarcinomas and 4 intraductal papillary biliary neoplasms of the bile duct for presence of FGFR2 translocations by fluorescence in situ hybridization and characterized the clinicopathologic features of cases with FGFR2 translocations. Thirteen (10 women, 3 men; 8%) of 156 biliary tumors harbored FGFR2 translocations, including 12 intrahepatic cholangiocarcinomas (12/96; 13%) and 1 intraductal papillary neoplasm of the bile duct. Histologically, cholangiocarcinomas with FGFR2 translocations displayed prominent intraductal growth (62%) or anastomosing tubular glands with desmoplasia (38%). Immunohistochemically, the tumors with FGFR2 translocations frequently showed weak and patchy expression of CK19 (77%). Markers of the stem cell phenotype in cholangiocarcinoma, HepPar1 and CK20, were negative in all cases. The median cancer-specific survival for patients whose tumors harbored FGFR2 translocations was 123 months compared to 37 months for cases without FGFR2 translocations (P = .039). This study also assessed 100 cholangiocarcinomas for ERBB2 amplification and ROS1 translocations. Of the cases tested, 3% and 1% were positive for ERBB2 amplification and ROS1 translocation, respectively. These results confirm that FGFR2, ERRB2, and ROS1 alterations are potential therapeutic targets for intrahepatic cholangiocarcinoma.

Novel Targets in Non-Small Cell Lung Cancer: ROS1 and RET Fusions

The discovery of chromosomal rearrangements involving the anaplastic lymphoma kinase (ALK) gene in non-small cell lung cancer (NSCLC) has stimulated renewed interest in oncogenic fusions as potential therapeutic targets. Recently, genetic alterations in ROS1 and RET were identified in patients with NSCLC. Like ALK, genetic alterations in ROS1 and RET involve chromosomal rearrangements that result in the formation of chimeric fusion kinases capable of oncogenic transformation. Notably, ROS1 and RET rearrangements are rarely found with other genetic alterations, such as EGFR, KRAS, or ALK. This finding suggests that both ROS1 and RET are independent oncogenic drivers that may be viable therapeutic targets. In initial screening studies, ROS1 and RET rearrangements were identified at similar frequencies (approximately 1%-2%), using a variety of genotyping techniques. Importantly, patients with either ROS1 or RET rearrangements appear to have unique clinical and pathologic features that may facilitate identification and enrichment strategies. These features may in turn expedite enrollment in clinical trials evaluating genotype-directed therapies in these rare patient populations. In this review, we summarize the molecular biology, clinical features, detection, and targeting of ROS1 and RET rearrangements in NSCLC.