ROS1 rearrangements define a unique molecular class of lung cancers

Abstract

Purpose: Chromosomal rearrangements involving the ROS1 receptor tyrosine kinase gene have recently been described in a subset of non-small-cell lung cancers (NSCLCs). Because little is known about these tumors, we examined the clinical characteristics and treatment outcomes of patients with NSCLC with ROS1 rearrangement.

Patients and methods: Using a ROS1 fluorescent in situ hybridization (FISH) assay, we screened 1,073 patients with NSCLC and correlated ROS1 rearrangement status with clinical characteristics, overall survival, and when available, ALK rearrangement status. In vitro studies assessed the responsiveness of cells with ROS1 rearrangement to the tyrosine kinase inhibitor crizotinib. The clinical response of one patient with ROS1-rearranged NSCLC to crizotinib was investigated as part of an expanded phase I cohort.

Results: Of 1,073 tumors screened, 18 (1.7%) were ROS1 rearranged by FISH, and 31 (2.9%) were ALK rearranged. Compared with the ROS1-negative group, patients with ROS1 rearrangements were significantly younger and more likely to be never-smokers (each P < .001). All of the ROS1-positive tumors were adenocarcinomas, with a tendency toward higher grade. ROS1-positive and -negative groups showed no difference in overall survival. The HCC78 ROS1-rearranged NSCLC cell line and 293 cells transfected with CD74-ROS1 showed evidence of sensitivity to crizotinib. The patient treated with crizotinib showed tumor shrinkage, with a near complete response.

Conclusion: ROS1 rearrangement defines a molecular subset of NSCLC with distinct clinical characteristics that are similar to those observed in patients with ALK-rearranged NSCLC. Crizotinib shows in vitro activity and early evidence of clinical activity in ROS1-rearranged NSCLC.

Fig 1.

(A) A break-apart fluorescent in situ hybridization probe reveals separation of the 5′ ROS1 probe (green) from the 3′ ROS1 probe (red) in a non–small-cell lung cancer formalin-fixed paraffin-embedded specimen. Nuclei are stained with 4′,6-diamidino-2-phenylindole. Size bar = 10 μm. (B) Representative histologic appearance of a ROS1-rearranged tumor, stained with hematoxylin and eosin, showing a solid subtype of adenocarcinoma with highly atypical cytologic features. (C) Sanger sequencing of a reverse transcriptase polymerase chain reaction product from a tumor harboring a SLC34A2-ROS1 rearrangement.

Fig 2.

(A) Dose-response cell survival curves of ROS1-rearranged cell line (HCC78) and ROS1-negative cell lines in response to crizotinib (nM); ALK-positive lines H3122 and MGH006 are positive controls for crizotinib response, and the PC-9, HCC827, and crizotinib-resistant H3122 CR lines are negative controls. (B) Western blot reveals a three-fold reduction of phospho-ROS1 in HCC78 cells at the same concentration (300 nmol/L) of crizotinib that results in near-complete reduction of phospho-ALK in H3122 cells. Total ROS1 and ALK, as well as actin, are shown as controls. The concentration of crizotinib is indicated above each lane (in nM).

Fig 3.

Two hundred ninety-three cells were transfected with cDNAs encoding EML4-ALK E13;A20 or CD74-ROS1. At approximately 45 hours after transfection, cells were treated with increasing amounts of crizotinib for 2 hours. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins.

Fig 4.

Response of an ROS1-positive patient with advanced non–small-cell lung cancer to crizotinib. Computed tomography scans of the chest were obtained (A and C) at baseline and (B and D) after 12 weeks of crizotinib. Shown are (A and B) coronal reconstructions and (C and D) axial slices.

Fig A1.

Representative histologic appearances of ROS1-rearranged tumors, stained with hematoxylin and eosin, with (A and B) two tumors harboring adenocarcinoma with highly atypical cytologic features including malignant giant cells (A is a high-magnification image of patient in Fig 1), (C) a mucinous bronchioloalveolar carcinoma, and (D) a low-grade adenocarcinoma forming thin tortuous glandular structures.

Fig A2.

Kaplan-Meier overall survival curves of ROS1-positive and -negative populations.