ROS Immunohistochemistry Is a Sensitive and Specific Tool for Detection of ROS Rearrangements in Lung Adenocarcinoma
Lynette Sholl, Heather Sun, Mohit Butaney, Charles Lee, Pasi A Janne, Scott Rodig. Brigham and Women's Hospital, Boston, MA; Dana Farber Cancer Institute, Boston, MA
Background: ROS1 gene rearrangements occur in 1-2% of lung adenocarcinomas (ACA) and are associated with response to the multitargeted tyrosine kinase inhibitor, crizotinib. ROS1 gene rearrangements can be detected using fluorescence in situ hybridization (FISH), however a novel immunohistochemistry (IHC) antibody for ROS protein has shown promise as an alternate screening tool. In this study we examine the sensitivity and specificity of IHC for the presence of ROS1 rearrangements by FISH, and use ROS IHC to screen genetically characterized lung ACA for ROS1 rearrangements.
Design: ROS IHC was carried out using clone D4D6 (Cell Signaling Technology; 1:1000, 1 hour incubation) on 4 micron whole tissue sections; expression that was weak/multifocal or greater was considered positive. IHC was compared to breakapart FISH using probes (RP11-59K17 and RP1-92C8) flanking the ROS1 gene in 50 cases of lung ACA with known FISH translocation status. We then used ROS IHC to screen 167 cases of lung ACA with known EGFR and KRAS mutation and ALK rearrangement status. ROS1 FISH was performed on all IHC positive cases.
Results: In the IHC validation cohort, 6 cases were FISH positive. All 6 were IHC positive, 5 strongly. One additional FISH-negative case was strongly ROS IHC positive. 42 cases were negative by FISH and IHC. Two ROS-negative cases failed FISH. One case failed both FISH and IHC. In the screening study, 105 wild type cases were tested, and 2 had strong, diffuse ROS IHC; a rearrangement was confirmed by FISH in both. 18 EGFR mutated cases were tested and all were negative for ROS IHC. 39 KRAS mutated cases were tested, and one had weak, multifocal ROS staining; FISH was negative. 7 ALK rearranged cases were tested, one had weak, multifocal ROS expression; FISH was negative. ROS was expressed in reactive type II pneumocytes, occasionally strongly, in a subset of FISH-negative cases.
Conclusions: In this study, diffuse ROS protein expression in tumor cells was 100% sensitive and 98% specific for ROS1 rearrangements by FISH. The significance of strong ROS protein expression in FISH-negative tumor cells is unknown, but may be functionally significant and requires further study. In a screen of genetically characterized lung ACA, ROS protein expression was detected in 4 of 167 (2.4%) cases overall and FISH confirmed in 2 cases, for a detection rate of 1.2% overall and 1.9% in the pan-WT cohort. ROS IHC is an effective screening tool for this rare but clinically important subset of lung ACA.
Monday, March 4, 2013 2:00 PM
Proffered Papers: Section D, Monday Afternoon