Testing for ROS1 in non-small cell lung cancer: a review with recommendations

Abstract

Rearrangements of the ROS1 gene occur in 1-2 % of non-small cell lung cancers (NSCLCs). Crizotinib, a highly effective inhibitor of ROS1 kinase activity, is now FDA-approved for the treatment of patients with advanced ROS1-positive NSCLC. Consequently, focus on ROS1 testing is growing. Most laboratories currently rely on fluorescence in situ hybridisation (FISH) assays using a dual-colour break-apart probe to detect ROS1 rearrangements. Given the rarity of these rearrangements in NSCLC, detection of elevated ROS1 protein levels by immunohistochemistry may provide cost-effective screening prior to confirmatory FISH testing. Non-in situ testing approaches also hold potential as stand-alone methods or complementary tests, including multiplex real-time PCR assays and next-generation sequencing (NGS) platforms which include commercial test kits covering a range of fusion genes. In order to ensure high-quality biomarker testing, appropriate tissue handling, adequate control materials and participation in external quality assessment programmes are essential, irrespective of the testing technique employed. ROS1 testing is often only considered after negative tests for EGFR mutation and ALK gene rearrangement, based on the assumption that these oncogenic driver events tend to be exclusive. However, as the use of ROS1 inhibitors becomes routine, accurate and timely detection of ROS1 gene rearrangements will be critical for the optimal treatment of patients with NSCLC. As NGS techniques are introduced into routine diagnostic practice, ROS1 fusion gene testing will be provided as part of the initial testing package.

Keywords: Fluorescence in situ hybridisation; Immunohistochemistry; Non-small cell lung cancer; Predictive marker; ROS1; RT-PCR.

Fig. 1

a Schematic diagram of ROS1 fusions in NSCLC showing ROS1 tyrosine kinase domain (TKI, dark grey), ROS1 transmembrane domain (TM, mid-grey) and coiled-coil domains (CC, light grey) in ROS1 fusion proteins (KDELR2-ROS1 is not shown). Reproduced from Gainor and Shaw [35]. b Frequencies of different ROS1 fusion partners. Adapted from Gainor and Shaw [35], with additional data from more recent studies as reported in Table 1

Fig. 2

Examples of different FISH signal patterns using ROS1 break-apart assays. a–d Vysis LSI ROS1 (Cen) SpectrumGreen Probe and Vysis LSI ROS1 (Tel) SpectrumOrange Probe (Abbott Molecular, IL, USA) on histological specimens. a Normal (negative) ROS1 pattern: two fused signals. b Typical ROS1-positive pattern with fused and split signals. c Atypical ROS1-positive pattern with one fusion signal and isolated 3′ green signals. d Increased ROS1 copy number. This pattern should not be interpreted as positive; e–f ZytoLight SPEC ROS1 (Cen) Green Probe and (Tel) Orange Probe (ZytoVision, Bremerhaven, Germany) on cytological specimens. e Split signals. f Isolated 3′ green signals

Fig. 3

a–f Examples of ROS1 IHC in histological NSCLC specimens (D4D6 antibody, Ventana BenchMark XT; DAB chromogen). a HCC78 cell line (cellblock; ×400). b NSCLC with diffuse, strongly positive staining (×200). c NSCLC with diffuse, granular cytoplasmic staining (×400). d Adenocarcinoma with heterogeneous staining (×200). e Non-neoplastic type II pneumocytes with weak ROS1 staining (×630). f Bone metastasis of a ROS1-negative NSCLC showing strong granular staining of non-neoplastic osteoclastic giant cells (×400). g–h Aberrant immunostaining of ROS1 in a transbronchial biopsy with lung adenocarcinoma. g H&E stain, asterisks show tumour cells. h ROS1 IHC in adjacent hyperplastic type II pneumocytes (arrows) but not in tumour cells (asterisks)

Fig. 4

ROS1 IHC in ethanol-fixed and previously Papanicolaou-stained cytological specimens (D4D6 antibody, Leica BondMax; AEC chromogen, ×400). a HCC78 cell line (positive control; cytospin). b ROS1-positive adenocarcinoma. c Small group of ROS1-positive adenocarcinoma cells surrounded by numerous benign respiratory epithelial cells

Fig. 5

Algorithm for predictive genetic testing in advanced NSCLC: routine practice