RNA Sequencing for Solid Tumor Fusion Gene Detection: Proficiency Testing Practice and Performance Comparison

Area of Science:

• Molecular Diagnostics
• Genomic Medicine
• Clinical Pathology

Background:

• RNA sequencing is vital for detecting genetic alterations like fusion genes in clinical labs.
• The College of American Pathologists (CAP) provides proficiency testing (PT) for RNA sequencing assays.

Purpose of the Study:

• To evaluate laboratory performance in RNA sequencing for detecting fusion genes, intragenic rearrangements, and exon-skipping events.
• Analyze CAP PT data from 2018-A to 2021-B to assess assay accuracy and laboratory practices.

Main Methods:

• Analysis of results from 153 laboratories participating in the CAP RNA sequencing PT program.
• Evaluation of 24 PT specimens with 22 distinct engineered fusion transcripts.
• Assessment of correct fusion event identification, performance variables, and laboratory practices.

Main Results:

• Overall sensitivity for fusion gene detection was 95.5%.
• False-negative rates were 3.6% for fusion genes and 18.3% for intragenic rearrangements/exon-skipping events.
• Few false positives were reported, likely due to pre/postanalytical errors; no practice characteristics correlated with detection results.

Conclusions:

• Clinical RNA sequencing demonstrates high sensitivity and specificity for fusion gene detection.
• Laboratory performance was consistent across different methodologies.
• Intragenic rearrangement and exon-skipping event detection showed higher false-negative rates compared to fusion genes.