Abstract 4267: Quantification of somatic hotspot mutations in KRAS, HRAS, BRAF, and PIK3CA: Comparing normal human breast and ductal carcinoma

Abstract

Abstract Data within the Catalogue of Somatic Mutations in Cancer indicates that a number of different hotspot oncogene mutations occur infrequently in breast tumors as compared to other tumor types (e.g., colon and lung). We hypothesized that greater sensitivity is required to detect somatic mutations in breast tumors, due to their highly-heterogeneous clonal structure. Therefore, the Allele-specific Competitive Blocker PCR method (with a sensitivity of 10-5), was used to quantify mutations in DNA isolated from normal human breast and ductal carcinoma (DC) samples (n = 10). The specific mutations quantified were: KRAS codon 12 GGT to GAT (G12D) and GGT to GTT (G12V), HRAS codon 12 GGC to GAC (G12D), BRAF codon 600 GTG to GAG (V600E), and PIK3CA codon 1047 CGT to CAT (H1047R). In addition, comparison of normal and malignant tissue was employed in order to define the lower bound on abnormal levels of mutation, as tumors that possess a mutant fraction (MF) greater than the upper 95% confidence interval of that present in normal breast. Interestingly, different patterns of mutation were observed for the different hotspot point mutations. We previously demonstrated that significantly higher levels of KRAS mutation are present in colon and lung tumors relative to the cognate normal tissue; however, the frequencies of KRAS codon 12 GGT to GAT and GGT to GTT mutation in normal breast and in DCs were quite similar (∼10-5 for the KRAS codon 12 GGT to GAT mutation in both tissues). Only one normal sample and two DCs had BRAF codon 600 GTG to GAG MFs >10-5. With respect to HRAS codon 12 GGC to GAC mutation, however, ∼80% of DCs had a MF greater than the upper 95% confidence interval of that present in normal breast tissue. Importantly, no breast tumor had a HRAS codon 12 GGC to GAC MF ≥10-1 (i.e., that detectable by DNA sequencing). Unexpectedly high levels of PIK3CA codon 1047 CGT to CAT mutation (>10-2) were observed in 4/10 normal breast samples and 4/10 DCs, suggesting that preexisting PIK3CA mutations in normal breast contribute to breast cancer susceptibility. In summary, all DC samples analyzed contained somatic mutations, none of which would be detectable by DNA sequencing. Consequently, these results indicate somatic mutations are more prevalent in breast cancer than is currently recognized. Ongoing analyses of additional tumors, corresponding to different breast cancer subtypes, will provide a better understanding of the role of somatic mutations in breast cancer. Citation Format: Malathi Banda, Meagan B. Myers, Karen L. McKim, Yiying Wang, Barbara L. Parsons. Quantification of somatic hotspot mutations in KRAS, HRAS, BRAF, and PIK3CA: Comparing normal human breast and ductal carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4267. doi:10.1158/1538-7445.AM2014-4267