Abstract
Information regarding the role of low-frequency hotspot cancer-driver mutations (CDMs) in breast carcinogenesis and therapeutic response is limited. Using the sensitive and quantitative Allele-specific Competitor Blocker PCR (ACB-PCR) approach, mutant fractions (MFs) of six CDMs (PIK3CA H1047R and E545K, KRAS G12D and G12V, HRAS G12D, and BRAF V600E) were quantified in invasive ductal carcinomas (IDCs; including ~20 samples per subtype). Measurable levels (i.e., ≥ 1 × 10−5, the lowest ACB-PCR standard employed) of the PIK3CA H1047R, PIK3CA E545K, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E mutations were observed in 34/81 (42%), 29/81 (36%), 51/81 (63%), 9/81 (11%), 70/81 (86%), and 48/81 (59%) of IDCs, respectively. Correlation analysis using available clinicopathological information revealed that PIK3CA H1047R and BRAF V600E MFs correlate positively with maximum tumor dimension. Analysis of IDC subtypes revealed minor mutant subpopulations of critical genes in the MAP kinase pathway (KRAS, HRAS, and BRAF) were prevalent across IDC subtypes. Few triple-negative breast cancers (TNBCs) had appreciable levels of PIK3CA mutation, suggesting that individuals with TNBC may be less responsive to inhibitors of the PI3K/AKT/mTOR pathway. These results suggest that low-frequency hotspot CDMs contribute significantly to the intertumoral and intratumoral genetic heterogeneity of IDCs, which has the potential to impact precision oncology approaches.
Highlights
Breast cancer is a heterogeneous disease, presenting with a spectrum of clinical features that impact prognosis and clinical response to treatment
A comparison of the distribution of measurements for PIK3CA H1047R, PIK3CA E545K, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E mutant fractions (MFs) in all invasive ductal carcinomas (IDCs) for the four subtypes is given in Figure 4, along with MF measurements previously reported in normal breast
This study has resulted in several critical findings, including the determination that: (1) all IDCs contain measurable levels of at least one cancer-driver mutations (CDMs), with the majority (59%) harboring three or more of the six CDMs, (2) CDMs are present in a subpopulation of cells, and would go undetected by standard DNA sequencing, (3) PIK3CA H1047R and BRAF V600E are correlated positively with maximum tumor dimension, whereas KRAS G12V is correlated negatively, and (4) subtype specific differences in MFs exist, most notably lower PIK3CA H1047R MFs in triple-negative breast cancers (TNBCs)
Summary
Breast cancer is a heterogeneous disease, presenting with a spectrum of clinical features that impact prognosis and clinical response to treatment. To examine the potential impact of low-frequency CDMs in breast carcinogenesis and targeted therapeutics, in previous work, we used ACB-PCR to quantify five hotspot CDMs (PIK3CA H1047R, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E) in human breast invasive ductal carcinomas (IDCs; [7]), and six hotspot CDMs (PIK3CA H1047R, PIK3CA E545K, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E) in the normal human breast [7,11] These targets were selected in part because they contain mutational hotspots (i.e., large percentages of the mutations within these genes occur within one or a few specific codons), making them ideal targets for the ACB-PCR methodology, which interrogates only a single base substitution in each assay.
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