Comparison of in Situ and Extraction-Based Methods for the Detection of ROS1 Rearrangements in Solid Tumors

Area of Science:

• Oncology
• Molecular Diagnostics
• Genetics

Background:

• ROS1 rearrangements are key biomarkers in certain cancers, predicting sensitivity to targeted therapies.
• Accurate detection of ROS1 rearrangements is critical for effective patient treatment.
• Existing diagnostic methods have limitations, particularly with low sample numbers.

Purpose of the Study:

• To evaluate and compare the performance of various diagnostic techniques for detecting ROS1 rearrangements.
• To identify potential discrepancies and false-positive results among different methods.
• To provide recommendations for reliable ROS1 rearrangement detection in clinical practice.

Main Methods:

• Evaluated 35 samples (32 ROS1 FISH-positive, 3 ROS1 FISH-negative) using ROS1 chromogenic in situ hybridization (CISH), immunohistochemistry (IHC), and multiple next-generation sequencing (NGS) panels (Agilent, Archer, NanoString, Illumina).
• ROS1 FISH break-apart and isolated 3' green signal patterns were analyzed.
• Results were compared across methods to assess concordance and identify false positives.

Main Results:

• All 11 ROS1 FISH-positive samples with a break-apart signal pattern were confirmed by other methods.
• Of 21 ROS1 FISH-positive samples with isolated 3' green signals, 10 were confirmed by at least two other methods.
• 11 samples initially positive by ROS1 FISH with isolated 3' green signals were false positives, as indicated by negative IHC and other methods.

Conclusions:

• ROS1 fluorescence in situ hybridization (FISH) results showing isolated 3' green signals require confirmation by alternative diagnostic methods.
• Next-generation sequencing (NGS) approaches are valuable for verifying suspicious ROS1 FISH results, especially when sufficient sample material is available.
• Standardized and validated multi-method approaches are essential for accurate ROS1 rearrangement detection in clinical settings.