Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers

Abstract

The recent discovery and characterization of an oncogenic ROS1 gene fusion in a subset of lung cancers has raised significant clinical interest because small molecule inhibitors may be effective to these tumors. As lung cancers with ROS1 rearrangements comprise only 1-3% of lung adenocarcinomas, patients with such tumors must be identified to gain optimal benefit from molecular therapy. Recently, immunohistochemical analyses using a novel anti-ROS1 rabbit monoclonal antibody (D4D6) have shown promise for accurate identification of ROS1-rearranged cancers. To validate this finding, we compared the immunostaining results of tissue microarrays (TMAs) containing 17 ROS1-rearranged and 253 ROS1-non-rearranged lung carcinomas. All 17 ROS1-rearranged cancers showed ROS1 immunoreactivity mostly in a diffuse and moderate-to-strong manner with an H-score range of 5-300 (median, 260). In contrast, 69% of ROS1-non-rearranged cancers lacked detectable immunoreactivity, whereas the remaining 31% showed reactivity mainly in a weak or focal manner. The H-score for the entire ROS1-non-rearranged group ranged from 0 to 240 (median, 0). The difference in H-score between the two cohorts was statistically significant, and the H-score cutoff (Z150) allowed optimal discrimination (94% sensitivity and 98% specificity). Similar but slightly less-specific performance was achieved using the extent of diffuse (Z75%) staining or Z2 þ staining intensity as cutoffs. CD74-ROS1 and EZR-ROS1 fusions were significantly associated with at least focal globular immunoreactivity and plasma membranous accentuation, respectively, and these patterns were specific to ROS1-rearranged cases. Although full-length ROS1 is expressed in some ROS1-non-rearranged cases, we showed that establishment of an optimal set of interpretative criteria makes ROS1 immunohistochemistry a valuable method to rapidly and accurately screen lung cancer patients for appropriate molecular therapy.

Analytical Validation of BRAF Mutation Testing from Circulating Free DNA Using the Amplification Refractory Mutation Testing System

“…Interpretation of ROS1 IHC is challenging, however, as expression can be seen in a patchy pattern, typically at weak intensity, in up to a third of tumors that do not have an underlying rearrangement. 44,45,48 Although some studies suggest that ROS1 expression in the absence of a rearrangement may have prognostic significance, 48 focal or patchy expression in tumor cells is rarely associated with a ROS1 rearrangement and therefore is unlikely to predict response to ROS1-targeted therapy. Moreover, the pattern of staining can vary among fusion types, including granular to globular staining in CD74-ROS1 fusions, weak membranous staining in EZR-ROS1 fusions, and vesicular localization staining in GOPC-ROS1 fusions.…”

“…Moreover, the pattern of staining can vary among fusion types, including granular to globular staining in CD74-ROS1 fusions, weak membranous staining in EZR-ROS1 fusions, and vesicular localization staining in GOPC-ROS1 fusions. 45 A single commercially available antibody clone (D4D6, Cell Signaling Technology, Danvers, Massachusetts) has been used in studies published to date. Most retrospective studies of ROS1 IHC using the D4D6 antibody demonstrate a sensitivity of 100% relative to FISH or RT-PCR.…”

“…Several cutoffs have been proposed using intensity alone or H score (intensity  percentage of tumor cells staining). In most studies, FISH-or molecularly confirmed ROS1-rearranged tumors have at least moderate-intensity ROS1 protein expression, but published evidence is insufficient to recommend one specific cutoff or scoring system, 42,45 and each laboratory must validate its own interpretive cutoff from known positive and negative samples.…”

ROS1 gene rearrangement and expression of splice isoforms in lung cancer, diagnosed by a novel quantitative RT-PCR assay

“…The sensitivity of ROS1 immunostaining for rearrangements was reported to be 100% [9][10][11][12][13][14] and, as such, IHC was suggested as an effective screening tool in NSCLC. However, ROS1 is more often expressed in NSCLC without concomitant translocation (4-30% NSCLC) [9,10,13,15].…”

Application of Immunohistochemistry in the Diagnosis of Pulmonary and Pleural Neoplasms

“…55 Similar to IHC for ALK, IHC stains for ROS1 have been shown to be an effective and cost-effective means to screen for ROS1 rearrangements. [56][57][58] A poorly differentiated adenocarcinoma of the lung shows acinar and micropapillary patterns of growth ( Figure 6, A) and expresses ROS1 ( Figure 6, B), detected by using a rabbit monoclonal antibody against ROS1 (clone D4D6; Cell Signaling Technology). Therefore, the judicious use of mutation-specific IHC stains may be used as a practical screening tool to detect certain actionable mutations (EGFR, ALK, and ROS1) amenable to targeted therapy.…”