Correlation of ROS1 (D4D6) Immunohistochemistry with ROS1 Fluorescence In Situ Hybridization Assay in a Contemporary Cohort of Pulmonary Adenocarcinomas

Abstract

Objective Repressor of Silencing (ROS1) gene rearrangement in the lung adenocarcinomas is one of the targetable mutually exclusive genomic alteration. Fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), next-generation sequencing, and reverse transcriptase polymerase chain reaction assays are generally used to detect ROS1 gene alterations. We evaluated the correlation between ROS1 IHC and FISH analysis considering FISH as the gold standard method to determine the utility of IHC as a screening method for lung adenocarcinoma. Materials and Methods A total of 374 advanced pulmonary adenocarcinoma patients were analyzed for ROS1 IHC on Ventana Benchmark XT platform using D4D6 rabbit monoclonal antibody. FISH assay was performed in parallel in all these cases using the Vysis ROS1 Break Apart FISH probe. Statistical Analysis The sensitivity, specificity, positive and negative likelihood ratios, positive and negative predictive values, and accuracy were evaluated. Results A total of 17 tumors were positive either by IHC or FISH analysis or both (true positive). Four tumors were positive by IHC (H-score range: 120–270), while negative on FISH analysis (false positive by IHC). One tumor was IHC negative, but positive by FISH analysis (false negative). The sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, positive predictive value, negative predictive value, and accuracy were 94.4% (confidence interval [CI]: 72.71–99.86%), 63.6% (CI: 30.79–89.07%), 2.6 (CI: 1.18–5.72), 0.09 (CI: 0.01–0.62), 80.95% (CI: 65.86–90.35%), 87.5% (CI: 49.74–98.02%), and 82.76%, respectively. Conclusion ROS1 IHC has high sensitivity at a cost of lower specificity for the detection of ROS1 gene rearrangement. All IHC positive cases should undergo a confirmatory FISH test as this testing algorithm stands as a reliable and economic tool to screen ROS1 rearrangement in lung adenocarcinomas.

The rapidly changing field of predictive biomarkers of non-small cell lung cancer

“…Membranous staining was seen in the presence of EZR-ROS1 fusion, presumably due to the ezrin protein binding to plasma membrane and actin cytoskeleton [ 153 ] A uniform scoring system for the ROS1 IHC reaction is still missing, most studies use the H-score calculated from the staining intensity and the proportion of positive tumor cells. With appropriate preanalytical and analytical standards, this antibody can achieve high sensitivity (95%–100%) but relatively poor specificity (63%–90%) [ 154 – 157 ]. The low specificity may potentially originate from a moderate ROS1 expression by macrophages, reactive alveolar epithelium, or even by tumors without ROS rearrangement.…”

Driver Mutations in Lung Cancer—Mapping the Way Forward

“…In the latest issue of South Asian Journal of Cancer ( SAJC ), two articles by Khaddar et al 1 and Sharma et al 2 address the clinical and pathological aspects of rare driver mutations in nonsmall cell lung cancer (NSCLC). The first article assessed the prognostic impact of baseline liver metastasis in anaplastic lymphoma kinase (ALK)-positive metastatic NSCLC.…”

“…The second article by Sharma et al 2 studied the utility of IHC as a screening tool for ROS1 translocation/ rearrangements. ROS1 prevalence in India is around 2.8 to 4.1%, 13 which is higher when compared to the western population.…”