ROS1 Gene Rearrangements Research Articles

Abstract A277: PF-06463922, a novel ROS1/ALK inhibitor, demonstrates sub-nanomolar potency against oncogenic ROS1 fusions and capable of blocking the resistant ROS1G2032R mutant in preclinical tumor models.

Abstract The oncogenic ROS1 gene fusion (Fig-ROS1) was first identified in glioblastoma cells over two decades ago. Recently, ROS1 gene rearrangements were further discovered in a variety of human cancers, including lung adenocarcinoma, cholangiocarcinoma, ovarian cancer, gastric adenocarcinoma, colorectal cancer, inflammatory myofibroblastic tumor, angiosarcoma, and epithelioid hemangioendothelioma, providing additional evidence for ROS1 as an attractive cancer target. The 1st generation Met/ALK/ROS1 inhibitor XALKORI ® (crizotinib) has demonstrated promising clinical response in ROS1 fusion positive NSCLC. But similar to what was seen with acquired ALK secondary resistant mutations in XALKORI refractory patients, a ROS1 kinase domain mutant–ROS1G2032R has been identified in a ROS1 positive NSCLC patient who developed resistance to XALKORI. Therefore, there is an urgent need to develop agents that can overcome this type of resistance. PF-06463922 is a novel, orally available, ATP-competitive small molecule inhibitor of ROS1/ALK with exquisite potency against ROS1 kinase. PF-06463922 inhibited the catalytic activity of recombinant ROS1 with a mean Ki of < 0.005 nM, and inhibited ROS1 autophosphorylation at IC50 values ranging from 0.1 nM to 1 nM cross a panel of cell lines harboring oncogenic ROS1 fusion variants including CD74-ROS1, SLC34A2-ROS1 and Fig-ROS1. PF-06463922 also inhibited cell proliferation and induced cell apoptosis at sub- to low-nanomolar concentrations in the HCC78 human NSCLC cells harboring SLC34A2-ROS1 fusions and the BaF3-CD74-ROS1 cells expressing human CD74-ROS1. In the BaF3 cells engineered to express the XALKORI resistant CD74-ROS1G2032R mutant, PF-06463922 demonstrated nanomolar potency against either ROS1G2032R cellular activity or cell proliferation. In vivo, PF-06463922 demonstrated marked cytoreductive antitumor efficacy at low nanomolar concentration in the NIH3T3 xenograft models expressing human CD74-ROS1 and Fig-ROS1. The antitumor efficacy of PF-06463922 was dose dependent and strongly correlated to inhibition in ROS1 phosphorylation and the downstream signaling molecules pSHP1, pSHP2 and pErk1/2, as well as inhibition of the cell cycle protein Cyclin D1 in tumors. To our knowledge, PF-06463922 is the first reported ROS1 inhibitor that is capable of blocking the resistant ROS1G2032R mutant at predicted pharmacologically relevant concentrations. Our data indicate that PF-06463922 has great potential for treating ROS1 fusion positive cancers including those from patients who relapsed from XALKORI therapy due to acquired ROS1G2032Rmutation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A277. Citation Format: Helen Y. Zou, Lars R. Engstrom, Qiuhua Li, Melissa West Lu, Ruth Wei Tang, Hui Wang, Konstantinos Tsaparikos, Sergei Timofeevski, Justine Lam, Shinji Yamazaki, Wenyue Hu, Hovhannes Gukasyan, Nathan Lee, Ted W. Johnson, Valeria Fantin, Tod Smeal. PF-06463922, a novel ROS1/ALK inhibitor, demonstrates sub-nanomolar potency against oncogenic ROS1 fusions and capable of blocking the resistant ROS1G2032R mutant in preclinical tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A277.

ROS1 Immunohistochemistry for Detection of ROS1-Rearranged Lung Adenocarcinomas

ROS1 gene rearrangements are reported in 1% to 2% of lung adenocarcinomas (ACAs) and are associated with response to the multitargeted tyrosine kinase inhibitor crizotinib. ROS1 rearrangements can be detected using fluorescence in situ hybridization (FISH); however, immunohistochemistry (IHC) for ROS1 protein is a promising alternate screening modality. In this study, we examine the correlation between ROS1 IHC and FISH and describe the clinicopathologic characteristics of ROS1-rearranged lung tumors. ROS1 IHC was performed using clone D4D6 on whole-tissue sections. In a validation cohort, IHC was compared with ROS1 break-apart FISH in 53 cases of lung ACA enriched for an absence of known genetic alterations and never-smoking status. In a screening cohort, we performed ROS1 IHC on 167 consecutive cases of lung ACA from a routine molecular diagnostic practice and confirmed positive results by FISH. In the validation cohort, 6 cases (11%) were both FISH and IHC positive. One FISH-negative case was strongly ROS1 IHC positive. All IHC-negative cases were FISH negative. In the screening cohort, 2 of 167 (1.2%) had strong, diffuse ROS1 protein expression; a rearrangement was confirmed by FISH in both. ROS1-translocated tumors were wild type for EGFR, KRAS, and ALK and commonly had solid growth with mucinous/cribriform features and psammomatous calcification. ROS1 protein expression in tumor cells is 100% sensitive and 92% specific for ROS1 rearrangements by FISH. ROS1 IHC is an effective screening tool for this rare but clinically important subset of lung ACAs.

A Case Series of Lengthy Progression-Free Survival With Pemetrexed-Containing Therapy in Metastatic Non–Small-Cell Lung Cancer Patients Harboring ROS1 Gene Rearrangements

c-ros oncogene 1 (ROS1), an orphan receptor tyrosine kinase, is a recently defined molecular subset of lung cancer identified in approximately 1% to 2% of screened patients.1,2 Like anaplastic lymphoma receptor tyrosine kinase (ALK) translocations, ROS1 rearrangements lead to constitutive kinase activation and commonly arise in young nonsmokers with adenocarcinoma tumor histology.3 Crizotinib (Pfizer), recently approved by the US Food and Drug Administration for treatment of metastatic non–small-cell lung cancer (NSCLC) patients with ALK rearranged tumors, has potent activity in cell lines with ROS1 translocations and recently demonstrated clinical activity in patients whose tumors harbor ROS1 rearrangements.3,4 Because of the recently discovered importance of ROS1 trans-locations in a small subset of NSCLC, we know little regarding the response to specific chemotherapeutics in patients whose tumors harbor these molecular alterations. Pemetrexed (Lilly) inhibits thymidylate synthase (TS) and other folate-dependent enzymes, and is a chemotherapeutic agent commonly used to treat nonsquamous NSCLC.5 Our institution conducted a retrospective case series of all meta-static lung cancer patients with ROS1 rearrangements. We recently began testing for ROS1 gene rearrangements using break-apart fluorescence in situ hybridization (FISH) in patients with lung adenocarcinoma and no other known mutations. A total of 129 ROS1 assays using FISH have been completed at Stanford with 5 ROS1 rearrangements identified; 1 additional patient treated at Stanford tested positive using FISH at an outside institution. Four of the 6 patients with detectable ROS1 rearrangements have metastatic cancer; and all 4 patients have a strikingly long progression-free survival (PFS) using pemetrexed—given as either single-agent second-line treatment or as part of front-line chemotherapy (Table 1). Table 1 Summary of ROS1 Cases Case 1 The patient is a 35-year-old never smoking woman who presented with dyspnea. Imaging revealed confluent pulmonary nodules in the left lung with a fluorine-18 fluorodeoxyglucose-avid pleural effusion. Mediastinoscopy showed N3 disease with right-sided mediastinal nodes positive for lung adenocarcinoma. The patient received 6 cycles of carboplatin, pemetrexed, and bevacizumab with a partial response followed by continuation maintenance with pemetrexed and bevacizumab for 21 cycles (Figure 1). During maintenance treatment, FISH was performed and revealed a ROS1 rearrangement. Bevacizumab was eventually discontinued because of nonspecific neurologic complaints. She continues on pemetrexed alone as maintenance therapy for > 14 cycles with minimal disease burden. Total time using pemetrexed is now > 36 months. Figure 1 Computed Tomography Scan of a NSCLC Patient With a ROS1 Gene Rearrangement Before Treatment (Left) and More Than 26 Months Later (Right) During Pemetrexed Continuation Maintenance Case 2 The patient is a 64-year-old man with a distant light smoking history who presented with a palpable left supraclavicular lymph node. Biopsy of this node revealed lung adenocarcinoma. The patient's NSCLC was initially stage IIIB and he was treated with concurrent chemoradiation with carboplatin and paclitaxel. He developed biopsy-proven recurrence of disease 1 year later with multiple bilateral pulmonary nodules. He then received 6 cycles of carboplatin, pemetrexed, and bevacizumab with a partial response, followed by continuation maintenance pemetrexed and bevacizumab for 37 cycles. During maintenance treatment, a ROS1 rearrangement was detected by FISH. Proteinuria eventually led to discontinuation of bevacizumab. He continues taking pemetrexed alone as maintenance therapy for additional > 19 cycles. Total time taking pemetrexed is now > 47 months.

Complete metabolic response in a patient with repeatedly relapsed non-small cell lung cancer harboring ROS1 gene rearrangement after treatment with crizotinib

A 55-year-old Caucasian woman with lung adenocarcinoma stage IV presented with repeated relapse after treatment with cytotoxic chemotherapy (carboplatin, gemcitabine, docetaxel, pemetrexed) and targeted agents (erlotinib, cetuximab, sunitinib). Comprehensive molecular diagnostics (EGFR-, ALK-, RAS-, BRAF-, PIK3CA-, HER2- and DDR2-aberrations) were performed and failed initially to detect any driver mutation. While the patient suffered from rapid deterioration of her general condition, in particular from progressive dyspnea due to lung metastases, we implemented screening for RET- and ROS1 translocations into our molecular diagnostic program based on recent reports of these new molecular subgroups in lung adenocarcinoma. On retesting the patient's tumor sample was found to harbor a ROS1-translocation. The patient was subsequently treated with crizotinib and experienced a pronounced clinical improvement corresponding to a complete metabolic response in 18F-FDG-PET and a good and confirmed partial response in CT (RECIST 1.1). Our case exemplifies the need for rapid implementation of newly discovered rare genetic lung cancer subtypes in routine molecular diagnostics.

IS7-16 - Clinical Activity of Crizotinib in Advanced Non-Small-Cell Lung Cancer (NSCLC) Harboring ROS1 Gene Rearrangement

ABSTRACT Background Chromosomal rearrangements of the ROS1 receptor tyrosine kinase gene define a new molecular subset of NSCLC. In cell lines, ROS1 rearrangements lead to expression of oncogenic ROS1 fusion kinases and sensitivity to ROS kinase inhibition. We examined the efficacy and safety of crizotinib, a small molecule tyrosine kinase inhibitor of MET, ALK and ROS, in patients with advanced, ROS1-rearranged NSCLC. Methods Patients with advanced NSCLC harboring ROS1 rearrangement, as determined using a break-apart FISH assay, were recruited into an expansion cohort of a phase 1 study of crizotinib. Patients were treated with crizotinib at the standard oral dose of 250 mg BID. The objective response rate (ORR) was determined based on RECIST 1.0. The disease control rate (DCR; stable disease [SD] + partial response [PR] + complete response [CR]) was evaluated at 8 weeks. Results Thirteen patients within the ROS expansion cohort received crizotinib and all were evaluable for response. The median age was 47 years (range 31–72), and all but one of the patients were never smokers. All patients had adenocarcinoma histology. Twelve of the 13 patients were tested for ALK rearrangement and all were negative. The median number of prior treatments was 1 (range 0–3). To date, the ORR is 54% (7 of 13), with 6 PRs and 1 CR, with 6 responses achieved by the first restaging scan at 7–8 weeks. There was one additional unconfirmed PR at the time of data cut-off. The DCR at 8 weeks was 85% (11 of 13). The median duration of treatment was 20 weeks (range 4+ to 59+). All responses are ongoing, and 12 patients continue on study. One patient had disease progression at first restaging and was discontinued from the study. The pharmacokinetics and safety profile of crizotinib in this group of patients were similar to that observed in patients with ALK-positive NSCLC. Conclusions Crizotinib demonstrates marked antitumor activity in patients with advanced NSCLC harboring ROS1 rearrangements. Like ALK, ROS defines a distinct subpopulation of NSCLC patients for whom crizotinib therapy may be highly effective. This study represents the first clinical validation of ROS as a therapeutic target in cancer.

1191PD - Clinical Activity of Crizotinib in Patients with Advanced Non-Small Cell Lung Cancer (NSCLC) Harboring Ros1 Gene Rearrangement

ABSTRACT Background Chromosomal rearrangements in the receptor tyrosine kinase (RTK) ROS1 define a new molecular subset of NSCLC. ALK inhibitors can inhibit ROS1 kinase activity in cell lines. We examined the efficacy and safety of crizotinib, a small molecule inhibitor of two other RTKs, MET and ALK, in patients with advanced, ROS1-rearranged NSCLC. Methods Patients with advanced NSCLC harboring ROS1 rearrangement, as determined using a break-apart FISH assay, were recruited into an expansion cohort of the original phase 1 study of crizotinib. Patients were treated with crizotinib at the standard oral dose of 250 mg BID. The objective response rate (ORR) was determined based on RECIST v1.0. The disease control rate (DCR; stable disease [SD] + partial response [PR] + complete response [CR]) was evaluated at weeks 8 and 16. Results At the time of data cut-off on April 19, 2012, 15 patients with ROS1 NSCLC had received crizotinib and 14 were evaluable for response. The median age was 54 years (range 31–72). Fourteen patients were never-smokers and all had adenocarcinoma histology. All patients were confirmed negative for ALK rearrangement. The median number of prior treatments was 1 (range 0–6). The first ROS1 patient received crizotinib on October 19, 2010. To date, the ORR is 57% (8/14; 95% CI 28.9–82.3), with 7 PR and 1 CR. There were 4 SD, one of which was unconfirmed as PR at the time of data cut-off. The DCR at 8 weeks was 79% (11/14). Median duration of treatment was 25.7 weeks (range 0.1 + to 65.3 + ), and 12 patients continue on study. The pharmacokinetics, antitumor activity and safety profile of crizotinib in this group of patients were similar to those observed in patients with ALK-positive NSCLC. Conclusions Crizotinib is associated with clinically significant antitumor activity in patients with ROS1 NSCLC. Similar to ALK, ROS1 rearrangement defines another unique molecular subset of NSCLC patients for whom crizotinib therapy may be highly effective. Importantly, this study represents the first clinical investigation of ROS1 as a driver mutation and therapeutic target in cancer. Disclosure S.I. Ou: Advisory role: Pfizer, Genentech. Compensated Honoraria: Pfizer, Genentech, Lilly. Myself Research funding: Pfizer. Myself (institution). D.R. Camidge: Advisory role: Pfizer. Myself. Honoraria: Pfizer. Myself. L. Tye: Employment: Pfizer. Clinician. Myself. Stock Ownership; Pfizer. Myself. K. Wilner: Emplyement: Pfizer. Senior Director. Myself. Compensated. Stock ownership: Pfizer. Myself. M. Varella-Garcia: Honoraria: Abbott Molecular. Myself. funding for the trial provided by NIH - yes. A.T. Shaw: Advisory role: Pfizer, Ariad, Chugai, Novartis, Daiichi-sankyo. Myself. Compensated Research funding: Astra Zeneca, Novartis. Myself. All other authors have declared no conflicts of interest.

Clinical activity of crizotinib in advanced non-small cell lung cancer (NSCLC) harboring ROS1 gene rearrangement.

7508 Background: Chromosomal rearrangements of the ROS1 receptor tyrosine kinase gene define a new molecular subset of NSCLC. In cell lines, ROS1 rearrangements lead to expression of oncogenic ROS1 fusion kinases and sensitivity to ROS kinase inhibition. We examined the efficacy and safety of crizotinib, a small molecule tyrosine kinase inhibitor of MET, ALK and ROS, in patients with advanced, ROS1-rearranged NSCLC. Methods: Patients with advanced NSCLC harboring ROS1 rearrangement, as determined using a break-apart FISH assay, were recruited into an expansion cohort of a phase 1 study of crizotinib. Patients were treated with crizotinib at the standard oral dose of 250 mg BID. The objective response rate (ORR) was determined based on RECIST 1.0. The disease control rate (DCR; stable disease [SD] + partial response [PR] + complete response [CR]) was evaluated at 8 weeks. Results: Thirteen patients within the ROS expansion cohort received crizotinib and all were evaluable for response. The median age was 47 yrs (range 31–72), and all but one of the patients were never-smokers. All patients had adenocarcinoma histology. 12/13 patients were tested for ALK rearrangement and all were negative. The median number of prior treatments was 1 (range 0–3). To date, the ORR is 54% (7/13), with 6 PRs and 1 CR, with 6 responses achieved by the first restaging scan at 7–8 wks. There was 1 additional unconfirmed PR at the time of data cut-off. The DCR at 8 wks was 85% (11/13). Median duration of treatment was 20 wks (range 4+–59+). All responses are ongoing, and 12 patients continue on study. One patient had disease progression at first restaging and was discontinued from the study. The pharmacokinetics and safety profile of crizotinib in this group of patients were similar to that observed in patients with ALK-positive NSCLC. Conclusions: Crizotinib demonstrates marked antitumor activity in patients with advanced NSCLC harboring ROS1 rearrangements. Like ALK, ROS defines a distinct subpopulation of NSCLC patients for whom crizotinib therapy may be highly effective. This study represents the first clinical validation of ROS as a therapeutic target in cancer.

Abstract 894: Targeting ROS1 receptor tyrosine kinase gene fusions in non-small cell lung cancer

Abstract Genomic rearrangements that fuse kinase domain-containing 3′ regions of tyrosine kinase genes with 5′ regions of unrelated genes are well characterized in cancer. The resulting fusion proteins often drive proliferation and invasion of tumor cells and thus represent attractive targets for therapeutic intervention. One such kinase that has been demonstrated to be involved in various genomic rearrangement events in non-small cell lung cancer (NSCLC), glioblastoma, and cholangiocarcinoma is the ROS1 receptor tyrosine kinase (also known as c-ROS and ROS). To date, three different 5′ fusion partners for ROS1 have been identified. In this study, we examined the frequency of ROS1 gene rearrangement in a panel of 428 patient-derived NSCLC tumor samples using a break-apart FISH assay. We found that approximately 1.2% of samples were positive. In positive samples, the expression of the fusion gene and the identity of the fusion partner were examined. HCC78 is a NSCLC cell line that has been demonstrated by us and others to express the SLC34A2-ROS1 gene fusion. In order to assess the cellular consequences of ROS1 inhibition, we treated HCC78 cells with two distinct small molecule kinase inhibitors that have activity against ROS1 (crizotinib and TAE684). We found that both drugs inhibited proliferation of HCC78 cells, down-regulated signaling pathways known to control cellular growth and survival, and disrupted normal cell-cycle progression. Finally, we report clinical data from a NSCLC patient with a ROS1 gene rearrangement, as detected by the FISH break-apart assay, who demonstrated tumor shrinkage when treated with crizotinib. This patient's tumor exhibited a novel ROS1 gene fusion partner. In conclusion, genomic rearrangements involving the ROS1 receptor tyrosine kinase are present in a small subset of NSCLC patients, and drugs that target ROS1 may be an effective treatment strategy for these patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 894. doi:1538-7445.AM2012-894