RNA sequencing is an ideal strategy to confirm atypical RET arrangements detected by DNA-based next-generation sequencing (NGS) or fluorescence in situ hybridization (FISH), according to the results of a study published in Clinical Cancer Research.1
RET fusions are known drivers of several cancers, including cancers of the thyroid and lung. Several tyrosine kinase inhibitors are approved to treat these cancers, but require the presence of RET fusions as determined by diagnostic testing.
“Despite the growing number of diagnostic assays, the variability in their performance represents a significant challenge for harmonizing RET fusion testing,” the study authors wrote.
The aim of the study was to evaluate the accuracy of DNA- and RNA-based NGS, FISH, and immunohistochemistry (IHC) for detecting RET fusions. The study included a retrospective analysis of data from the MSK-IMPACT and MSK-Fusion panel from January 1, 2014 to May 9, 2020.
DNA-based NGS by MSK-IMPACT used a hybridization capture-based assay that evaluates the exons and select introns of 468 genes for mutations, copy-number variations, microsatellite instability, and select structural variants (SVs). DELLY was also used to evaluate SVs.
RNA sequencing was performed using MSK-Fusion, which is a custom RNA-based NGS assay that uses anchored multiplex polymerase chain reaction to evaluate select exons in 62 genes, including RET exons. A subset of cases were evaluated using break-apart FISH and RET IHC.
There were 171 RET SVs detected by DNA-based NGS from 46,897 tumors. Of the 171 SVs identified, 139 were predicted to form fusions with known partners and 32 were structural variants of unknown significance (SVUS).
Of the 139 RET SV cases thought to be oncogenic, 105 cases were not further analyzed “given the canonical nature of these fusions,” the study authors said. The remaining 34 cases were analyzed by RNA sequencing and confirmed to harbor a RET fusion. All 32 SVUS cases were also evaluated by RNA sequencing; 37.5% were found to harbor RET fusions.
Among the 16 patients who underwent circulating tumor DNA testing, 12.5% had no evidence of disease. In this cohort, RET SVs were identified in 31.3% of patients.
There were 66 samples cotested by MSK-IMPACT (DNA NGS) and MSK-Fusion (RNA sequencing). The sensitivity and specificity of MSK-IMPACT was 100% and 99.6%, respectively. There were 20 false-positives across the entire cohort, and 55% of these were related to non-lung and non-thyroid malignancies.
There were 48 samples evaluated by break-apart FISH, which demonstrated a sensitivity of 91.7% for detecting RET fusions overall, and 100% for RET fusions with KIF5B or CCDC6 partners. The sensitivity decreased to 66.7% for fusions with NCOA4.
Among 70 fusion-positive and 89 fusion-negative cases, the sensitivity of cytoplasmic staining (≥1% of tumor cells) was 89.5% and the specificity was 88.9%. A validation set demonstrated similar results.
“Our findings highlight the utility of RNA sequencing for confirming atypical RET rearrangements from DNA sequencing and FISH,” the study authors concluded.
Disclosures: Some of the study authors disclosed financial relationships with the pharmaceutical industry and/or the medical device industry. For a full list of disclosures, please refer to the original study.
Yang S-R, Aypar U, Rosen EY, et al. A performance comparison of commonly used assays to detect RET fusions. Clin Cancer Res. Published online December 3, 2020. doi:10.1158/1078-0432.CCR-20-3208