Confounding factors in assessing the enriched expression of somatic mutant alleles in bulk tumor samples

Allele-specific expression (ASE) of somatic mutations can be caused by <i>cis</i>-activation of the mutant allele or silencing of the wild-type allele and has been investigated by examining the enrichment of mutant allele in RNA relative to DNA. Here we show that this mutation-based approach can be confounded by gene expression differences in tumor and normal cells that coexist in most bulk tumor samples. We model mutant allele expression by incorporating tumor/normal expression difference, mutant allele dosage, tumor purity, and nonsense-mediated decay (NMD) efficiency, projecting that such enrichments can occur without ASE. This confounding effect is exacerbated with low tumor purity and is dependent on mutant allele dosage for NMD-triggering mutations. The model predictions are validated by a pancancer bulk tumor analysis with somatic insertions/deletions (indels) from 9101 The Cancer Genome Atlas (TCGA) samples. A single-cell analysis in five cutaneous squamous cell carcinomas demonstrates the robustness of this model to intratumor heterogeneity. As a byproduct of this confounding effect, we evaluate whether the inverse relationship between mutant allele enrichment in RNA and tumor purity could be leveraged to complement DNA-based somatic mutation detection in low purity samples. Indeed, our de novo somatic indel calling from TCGA RNA-seq increases the TCGA driver indel repertoire by ∼14%, especially in samples with purity less than 0.4, including actionable <i>EGFR</i> indels in lung adenocarcinoma and <i>FLT3</i> in acute myeloid leukemia. Our study not only reveals confounders in somatic mutant ASE analysis but also demonstrates their utility in RNA-based mutation calling.