Comparison of DNA and RNA-Based Mutation Testing for Non-Small Cell Lung Cancer

Molecular testing for oncogenic mutations is standard of care for patients with non-small cell lung cancer. While DNA sequencing effectively captures actionable substitution and indel mutations, the ability of RNA-based methods to detect these alterations remains unestablished. In this study, paired molecular analysis of 83 non-small cell lung cancers was performed via two clinical next generation sequencing tests, a DNA hybrid capture panel and an RNA anchored multiplex PCR panel. The two methods were concordant for 65 of 67 oncogenic substitution and indel mutations in EGFR, KRAS, PIK3CA, ERBB2, BRAF, CTNNB1, AKT1, ALK, NRAS, and ROS1. The two discordant mutations were identified near the assays' limit of detection. In concordant mutations, RNA sequencing showed a broader range of sequencing depth (27x to 55,916x) compared to DNA sequencing (424x to 4060x). The observed variant allele fraction was higher in RNA compared to DNA for 58 of 65 mutations (89%), with mean variant allele fraction of 45.2% in RNA compared to 26.9% in DNA (p < 0.0001). The analytical performance in the detection of somatic mutations in RNA is comparable to a reference DNA test. These results provide proof of concept for using RNA for mutation detection to complement DNA-based molecular testing for non-small cell lung cancer.