Enhancing variant interpretation in hereditary cancer testing with RNA analysis from residual blood samples.

Abstract

e22595 Background: Functional RNA studies can enable definitive classification for DNA variants of uncertain significance (VUS) identified via hereditary cancer testing and suspected of having an impact on mRNA splicing. However, when RNA analysis is performed as a reflex based on DNA results, the RNA analysis typically requires follow-up testing on an additional blood sample, placing extra burden on patients and clinics. In this study, we demonstrate that residual blood submitted for germline DNA testing is a suitable sample type for the isolation and analysis of RNA. Methods: RNA analysis was performed on selected residual EDTA blood samples submitted for hereditary cancer panel testing (March 2023-October 2023). These samples were known to carry a previously characterized or in silico -predicted splicing variant. RNA was isolated and subjected to reverse-transcriptase PCR (RT-PCR) and cDNA sequencing. Results were correlated with comparator data when available or with splice prediction models. Results: We analyzed 30 residual EDTA blood samples with 29 unique germline DNA variants. RNA and cDNA of sufficient quantity and quality were successfully isolated from 28/30 (93%) samples. Comparator data were available for 21 variants, consisting of previous RNA results generated in our laboratory from a secondary blood collection with RNA stabilization buffer, or from published evidence. Data for these 21 variants were concordant with comparator data showing complete or incomplete splice defects. For 7 variants with only in silico splice prediction for comparison, 5 were confirmed to result in a significant splicing defect. As a result of this study, RNA analysis is now a part of routine testing for blood samples meeting select criteria, with 26% of the variants tested to date successfully reclassified from VUS to likely pathogenic. Conclusions: Blood samples collected in EDTA tubes for germline DNA testing are a suitable sample type for the isolation and analysis of RNA by RT-PCR and cDNA sequencing. Under certain conditions residual samples may be used for RNA analysis, avoiding the need for secondary sample submission, and thus, significantly increasing the number of variants available for analysis. These functional studies may contribute to the reclassification of putative splice variants, providing definitive test results to more patients in a streamlined manner.