In a recent study published in the Journal of American Medical Association (JAMA) Oncology, researchers from California investigated the diagnostic outcomes of concurrent sequencing of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) to detect the predisposition to hereditary cancer in 43,524 individuals.
They found that adding RNA sequencing to germline genetic testing for cancer increased the positive yield and improved the detection of variants in the participants.
Study: Diagnostic Outcomes of Concurrent DNA and RNA Sequencing in Individuals Undergoing Hereditary Cancer Testing. Image Credit: Cryptographer/Shutterstock.com
The advent of precision medicine has revolutionized cancer management by predicting an individual’s predisposition to cancer via testing for tumor biomarkers and germline variants.
An increasing amount of evidence suggests that specific systemic therapy in individuals with germline pathogenic variants (PVs) may be effective in high-risk patients, including those with a familial history of cancer.
Early and accurate genetic testing in these patients could help improve prognosis using advanced technology, including next-generation sequencing and multigene panel testing. Although most precision medicine approaches are DNA-based, the more recent inclusion of RNA-based techniques has shown promise in identifying individuals with germline cancer susceptibility.
Therefore, researchers in the present study assessed the effectiveness of paired DNA and RNA testing in individuals undergoing hereditary cancer screening and measured the impact of the approach on the positive yield and rate of identifying variants of uncertain significance (VUS).
About the study
Clinical and molecular data were obtained from 43,524 individuals undergoing paired DNA and RNA testing at a testing laboratory between March 2019 and April 2020. The median age of the participants was 54 years, and 85.7% were female.
About 65.1% of these individuals had a history of cancer, with breast cancer being the most common type, followed by colorectal and ovarian cancer. The cohort included participants from the following races: Ashkenazi Jewish, Asian, Black, Hispanic, non-Hispanic White, and others.
Various genes (n=18) associated with breast, colorectal, ovarian, and pancreatic cancer were included in the study. DNA sequencing, RNA analysis, and deletion and duplication analysis were performed.
Sequence variations were classified as pathogenic (P), likely pathogenic (LP), VUS, benign (B), or likely benign (LB) based on guidelines from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP).
The approach was aligned with the ClinGen Sequence Variant Interpretation splicing subgroup to extract splicing-related evidence and standardize variant classification while interpreting RNA data.
The splicing impact of variants was predicted using in silico modeling via SpliceAI. The percent splicing index and number/type of splicing events were measured in patients compared to control data to identify abnormal transcripts.
The changes in variant classification were monitored over time. RNA-impacted variants were identified as those reclassified due to RNA evidence. The statistical analysis involved using the chi-square test and Fisher exact test.
Results and discussion
As per the study, while positive results were observed in 11.2% of participants, VUS was observed in 20.1%. RNA evidence impacted variant classification in 549 cases that underwent paired RNA-DNA testing.
This additional evidence improved the classification of LB and LP in 33.9% of cases, leading to reclassification to B or P. Furthermore, VUS was reclassified as LB or B in 45.5% of cases. Clinically significant reclassifications were made in 73.3% of cases.
RNA-impacted cases improved the positive rate and decreased the VUS rate. The relative increase in diagnostic yield was estimated to be 1.9%, suggesting that in the absence of RNA evidence, 1 in 54 patients with P/LP variants would have received incorrect or undetected results. The diagnostic yield was observed to vary across races.
Interestingly, about 75.7% of P/LP and 73.6% of B/LB RNA-impacted cases had recurrent variants observed in more than one individual. As per the results, PVs were common, emphasizing the need for RNA sequencing for improved accuracy.
Overall, RNA sequencing improved the sensitivity and accuracy of cancer detection in traditionally undetected cases and supported the previous clinically actionable classifications made with limited evidence.
Despite the improved detection rate and accuracy obtained via RNA sequencing tests, their widespread application is limited by their high cost to laboratories. The study findings call for further research and highlight the need for increased collaboration and data sharing among healthcare practitioners and researchers.
In conclusion, the present study provides evidence of the efficacy of RNA sequencing in improving the identification of high-risk individuals, especially missed by traditional DNA-based approaches.
In the future, studies including a greater number of cancer susceptibility genes could help improve our understanding of the effect of RNA splicing on cancer genetics, with the ultimate aim of improving patient outcomes via early detection.