Comparison of ROS1-rearrangement detection methods in a cohort of surgically resected non-small cell lung carcinomas
Viktoria Thurfjell1, Patrick Micke1, Hui Yu1
1Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
Background:
Patients with non-small cell lung cancer (NSCLC) harboring a ROS proto-oncogene 1 (ROS1)-rearrangement respond to treatment with ROS1 inhibitors. To distinguish these rare cases, screening with immunohistochemistry (IHC) for ROS1 protein expression has been suggested. However, the reliability of such an assay and the comparability of the antibody clones has been debated. Therefore we evaluated the diagnostic performance of current detection strategies for ROS1-rearrangement in two NSCLC-patient cohorts.
Methods:
Resected tissue samples, retrospectively collected from consecutive NSCLC-patients surgically treated at Uppsala University Hospital were incorporated into tissue microarrays [all n=676, adenocarcinomas (AC) n=401, squamous cell carcinomas (SCC) n=213, other NSCLC n=62]. ROS1-rearrangements were detected using fluorescence in situ hybridization (FISH) (Abbott Molecular; ZytoVision). In parallel, ROS1 protein expression was detected using IHC with three antibody clones (D4D6, SP384, EPMGHR2) and accuracy, sensitivity, and specificity were determined. Gene expression microarray data (Affymetrix) and RNA-sequencing data were available for a subset of patients. NanoString analyses were performed for samples with positive or ambiguous results (n=21).
Results:
Using FISH, 2/630 (0.3% all NSCLC; 0.5% non-squamous NSCLC) cases were positive for ROS1 fusion. Additionally, nine cases demonstrated ambiguous FISH results. Using IHC, ROS1 protein expression was detected in 24/665 (3.6% all NSCLC; 5.1% non-squamous NSCLC) cases with clone D4D6, in 18/639 (2.8% all NSCLC; 3.9% non-squamous NSCLC) cases with clone SP384, and in 1/593 (0.2% all NSCLC; 0.3% non-squamous NSCLC) case with clone EPMGHR2. Elevated RNA-levels were seen in 19/369 (5.1%) cases (Affymetrix and RNA-sequencing combined). The overlap of positive results between the assays was poor. Only one of the FISH-positive cases was positive with all antibodies and demonstrated high RNA-expression. This rearrangement was confirmed in the NanoString-assay and also in the RNA-sequencing data. Other cases with high protein/RNA-expression or ambiguous FISH were negative in the NanoString-assay.
Conclusions:
The occurrence of ROS1 fusions is low in our cohorts. The IHC assays detected the fusions, but the accuracy varied depending on the clone. The presumably false-positive and uncertain FISH results questions this method for detection of ROS1-rearrangements. Thus, when IHC is used for screening, transcript-based assays are preferable for validation in clinical diagnostics.
Insights
Immunohistochemistry (IHC) screening for ROS1 rearrangements in non-small cell lung cancer (NSCLC) shows variable accuracy. Transcript-based assays are preferred for validating ROS1 fusions in clinical diagnostics.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genetics
Background:
- Non-small cell lung cancer (NSCLC) patients with ROS1 rearrangements benefit from ROS1 inhibitors.
- Immunohistochemistry (IHC) is proposed for screening ROS1 rearrangements, but its reliability and antibody clone comparability are debated.
Purpose of the Study:
- To evaluate the diagnostic performance of current detection strategies for ROS1-rearrangement in NSCLC patient cohorts.
- To assess the reliability of IHC assays and compare different antibody clones for detecting ROS1 protein expression.
Main Methods:
- Retrospective analysis of resected NSCLC tissue samples (n=676) using fluorescence in situ hybridization (FISH) and IHC with three antibody clones (D4D6, SP384, EPMGHR2).
- Gene expression microarray and RNA-sequencing data were analyzed for a subset of patients.
- NanoString analyses were performed for samples with positive or ambiguous FISH results.
Main Results:
- ROS1 fusions were detected in 0.3% of NSCLC cases by FISH; nine cases had ambiguous FISH results.
- IHC detected ROS1 protein expression in 0.2%–3.6% of cases, varying by antibody clone.
- The overlap between FISH, IHC, and RNA expression results was poor; only one FISH-positive case showed high RNA expression and was confirmed by NanoString and RNA-sequencing.
Conclusions:
- The occurrence of ROS1 fusions is low in the studied NSCLC cohorts.
- IHC assays can detect ROS1 fusions, but accuracy varies significantly with the antibody clone used.
- Transcript-based assays are recommended for validating ROS1 rearrangements in clinical diagnostics when IHC is used for initial screening.
