Comparison of Different ROS1 Immunohistochemistry Clones and Consistency with Fluorescence In Situ Hybridization
Onur Dülger1,2, Büge Öz3
1Department of Molecular Medicine, Aziz Sancar Institute of Experimental Medicine, İstanbul University, İstanbul, Turkey
Background:
The study of ROS1 rearrangement in non-small cell lung carcinoma (NSCLC) has gained importance as it enables personalized treatment of NSCLC with tyrosine kinase inhibitors. Therefore, it is important that the ROS1 assessment tests become more standardized. In this study, we compared the two immunohistochemistry (IHC) antibodies (D4D6 and SP384 clones) and consistency with the fluorescence in situ hybridization (FISH) results in NSCLC.
Aims:
To investigate the effectiveness of the commonly used two IHC antibodies (SP384 and D4D6 clones) to detect ROS1 rearrangement in NSCLC.
Study Design:
A retrospective cohort study.
Methods:
The study included 103 samples diagnosed with NSCLC, confirmed using IHC and FISH ROS1 results (14 positives, four discordant, and 85 consecutive negatives), with sufficient tissue samples (≥ 50 tumor cells). All samples were initially tested with ROS1-IHC antibodies (D4D6 and SP384 clones); their ROS1 status was then analyzed using the FISH method. Finally, samples with discordant IHC and FISH results were confirmed using the reverse transcription polymerase chain reaction method.
Results:
The sensitivity of SP384 and D4D6 clones of ROS1 antibody was 100% with a ≥ 1 + cut-off. When the ≥ 2 + cut-off was used, the sensitivity rate for the SP384 clone was 100%, whereas the sensitivity for the D4D6 clone was 42.86%. ROS1 FISH rearranged samples were positive for both clones, but SP384 had generally higher intensity than D4D6. The mean IHC score was + 2 for SP384 and + 1.17 for D4D6. SP384 mostly tended to have a higher IHC score intensity, which made the evaluation easier than D4D6. SP384 has a higher sensitivity than D4D6. However, false positives were found in both clones. There was no significant correlation between ROS1 FISH-positivity percentage with SP384 (p = 0.713, p = 0.108) and D4D6 (p = 0.26, p = -0.323) IHC staining intensity. The staining patterns of both clones were similar (homogeneity/heterogeneity).
Conclusion:
Our findings show that the SP384 clone is more sensitive than D4D6. However, SP384 can also cause false positive results like D4D6. Knowing the variable diagnostic performance of different ROS1 antibodies before using them in clinical applications is necessary. IHC-positive results should be confirmed using FISH.
Insights
The SP384 antibody clone shows higher sensitivity for detecting ROS1 rearrangements in non-small cell lung cancer (NSCLC) compared to the D4D6 clone. However, both clones can produce false positives, necessitating confirmation with fluorescence in situ hybridization (FISH).
Area of Science:
- Oncology
- Molecular Diagnostics
- Immunohistochemistry
Background:
- ROS1 rearrangements are crucial biomarkers for targeted therapy in non-small cell lung cancer (NSCLC).
- Standardization of ROS1 detection methods is essential for accurate patient stratification.
- Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are key diagnostic tools for ROS1.
Purpose of the Study:
- To evaluate and compare the diagnostic performance of two common ROS1 IHC antibody clones (SP384 and D4D6).
- To assess the concordance of IHC results with FISH in NSCLC samples.
- To determine the sensitivity and specificity of each antibody clone for detecting ROS1 rearrangements.
Main Methods:
- Retrospective analysis of 103 NSCLC tissue samples.
- Initial testing using ROS1 IHC antibodies (SP384 and D4D6 clones).
- Confirmation of ROS1 status via FISH and reverse transcription polymerase chain reaction (RT-PCR) for discordant cases.
Main Results:
- SP384 clone demonstrated 100% sensitivity at a ≥1+ cut-off, outperforming D4D6 (42.86% sensitivity at ≥2+ cut-off).
- Both SP384 and D4D6 showed positive results for FISH-rearranged samples, with SP384 exhibiting higher staining intensity.
- False positive results were observed for both IHC clones; no significant correlation was found between IHC intensity and FISH positivity percentage.
Conclusions:
- The SP384 antibody clone offers superior sensitivity for ROS1 rearrangement detection in NSCLC compared to D4D6.
- Despite higher sensitivity, SP384 can yield false positives, underscoring the need for confirmatory FISH testing.
- Understanding the diagnostic variability of IHC antibodies is critical for reliable clinical application in NSCLC.
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