Detection of ROS1 gene rearrangement in lung adenocarcinoma: comparison of IHC, FISH and real-time RT-PCR

Abstract

Aims: To compare fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and quantitative real-time reverse transcription-PCR (qRT-PCR) assays for detection of ROS1 fusion in a large number of ROS1-positive lung adenocatcinoma (ADC) patients.

Methods: Using IHC analysis, sixty lung ADCs including 16 cases with ROS1 protein expression and 44 cases without ROS1 expression were selected for this study. The ROS1 fusion status was examined by FISH and qRT-PCR assay.

Results: Among 60 cases, 16 (26.7%), 13 (21.7%) and 20 (33.3%) cases were ROS1 positive revealed by IHC, FISH and qRT-PCR, respectively. Using FISH as a standard method for ROS1 fusion detection, the sensitivity and specificity of IHC were 100% and 93.6%, respectively. Three IHC-positive cases, which showed FISH negative, were demonstrated with ROS1 fusion by qRT-PCR analysis. The sensitivity and specificity of qRT-PCR for detection for ROS1 fusion were 100% and 85.1%, respectively. The total concordance rate between IHC and qRT-PCR were 93.3%.

Conclusion: IHC is a reliable and rapid screening tool in routine pathologic laboratories for the identification of suitable candidates for ROS1-targeted therapy. Some ROS1 IHC-positive but FISH-negative cases did harbor the translocation events and may benefit from crizotinib.

Fig 1. Detection of ROS1 fusion in lung cancer patients by IHC, FISH and qRT-PCR assays.

(A–D) ROS1 IHC staining using D4D6 antibody. (A) Score 0/negative showing no staining; (B) Score 1+ showing faint cytoplasmic reactivity; (C) Score 2+ showing moderate cytoplasmic staining; and (D) Score 3+ showing intense granular cytoplasmic staining in ≥10% of tumor cells. Original magnification ×200. (E–H) FISH analysis using ROS1 dual color break-Apart FISH probes to detect ROS1 fusion as split red and green signals. (E) FISH-negative case showing intact two fused signals per nucleus. (F-G) FISH-positive cases representing split (red and green) signals. (H) FISH-positive case showing isolated green signals. Original magnification ×1000. (I–L) Graphs from qRT-PCR showing change in the normalized reporter signal (delta Rn) against PCR cycle number using AmoyDx ROS1 fusion gene detection kit.