Abstract 5403: Reproducible elevation of RNA versus DNA mutation signal in low purity breast tumors
Authors: Te Jerez , Viollet Coralie , Zhang Xijun , Singh Jatinder , Hooke Jeffrey A. , Pollard Harvey B. , Hu Hai , Shriver Craig D. , Dalgard Clifton L. , Wilkerson Matthew D. 
Published: July 2017

Abstract
Background: Accurate detection of somatic mutations is critical for informing targeted therapy options. Prevalent non-cancer cell admixture complicates this detection in breast cancer. Conventional mutation detection relies on DNA sequencing; however in prior work, we demonstrated that combining RNA and DNA sequencing increases mutation signal strength, or mutant allele fraction (MAF). The ratio of RNA MAF versus DNA MAF (RNA:DNA MAF) was greatest in low purity breast tumors. We hypothesized that this elevation is biologically driven and would be conserved in a second, distinct tissue specimen of the same tumors. Here, we compare mutation characteristics between two tissue blocks in a cohort of breast tumors (n = 8) to evaluate possible preservation of RNA versus DNA mutation signal throughout the tumor.
Methods: We selected four high purity and four low purity breast tumors (“Block1”) from The Cancer Genome Atlas (TCGA) cohort and associated ABSOLUTE purity analysis. For these tumors, we acquired a second tissue block (“Block2”) not analyzed by TCGA, cut sections, analyzed sections by H&E stains, and extracted nucleic acids. Whole genome DNA sequencing and mRNA sequencing was performed for Block2 specimens using Illumina X and NextSeq 500 sequencers, respectively. Somatic mutations in Block2 were detected using UNCeqR and compared to published UNCeqR somatic mutations from TCGA. We then evaluate MAF characteristics in the entire TCGA breast tumor cohort (n = 695).
Results: Tumor purity estimates, determined by histology and by sequencing, were reduced in Block2 of the low purity tumor set versus the high purity tumor set, consistent with Block1 analysis. Molecular properties of genome-wide gene expression and somatic DNA copy number were highly similar between block-mated specimens (p &lt; 0.01). We then identified expressed mutations present in Block1 and Block2 of the same tumor and compared the MAFs on these common mutations. DNA MAF and RNA MAF were each significantly correlated between Block1 and Block2 (p &lt; 1e-12 in both cases). The average RNA:DNA MAF was 2.5 for the cohort, indicating that RNA mutation signal is greater than DNA in general. In Block2 specimens, the RNA:DNA MAFs were significantly greater in the low purity tumor set than the high purity tumor set (mean 2.7 versus 2.1, p &lt; 6e-5), reflecting the same trend observed in Block1 specimens. Analyzing the entire TCGA cohort, RNA:DNA MAF was positively correlated with proliferation pathway gene expression (p &lt; 3e-16 ) and was greatest in the Basal subtype versus other subtypes (p &lt; 2e-9).
Conclusion: Mutant allele fraction both of DNA and of RNA was conserved across breast tumor subsections. Low purity and basal subtype breast tumors had elevated RNA:DNA MAF supporting a relationship to underlying biology and identifying classes of tumors with pronounced benefit for DNA and RNA integrated mutation analysis.
Citation Format: Jerez Te, Coralie Viollet, Xijun Zhang, Jatinder Singh, Jeffrey A. Hooke, Harvey B. Pollard, Hai Hu, Craig D. Shriver, Clifton L. Dalgard, Matthew D. Wilkerson. Reproducible elevation of RNA versus DNA mutation signal in low purity breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5403. doi:10.1158/1538-7445.AM2017-5403