Somatic mutations in benign breast disease tissues and association with breast cancer risk

Abstract

Background: Benign breast disease (BBD) is a risk factor for breast cancer (BC); however, little is known about the genetic alterations present at the time of BBD diagnosis and how these relate to risk of incident BC.

Methods: A subset of a long-term BBD cohort was selected to examine DNA variation across three BBD groups (42 future estrogen receptor-positive (ER+) BC, 36 future estrogen receptor-negative (ER-) BC, and 42 controls cancer-free for at least 16 years post-BBD). DNA extracted from archival formalin fixed, paraffin-embedded (FFPE) tissue blocks was analyzed for presence of DNA alterations using a targeted panel of 93 BC-associated genes. To address artifacts frequently observed in FFPE tissues (e.g., C>T changes), we applied three filtering strategies based on alternative allele frequencies and nucleotide substitution context. Gene-level associations were performed using two types of burden tests and adjusted for clinical and technical covariates.

Results: After filtering, the variant frequency of SNPs in our sample was highly consistent with population allele frequencies reported in 1 KG/ExAC (0.986, p < 1e-16). The top ten genes found to be nominally associated with later cancer status by four of 12 association methods(p < 0.05) were MED12, MSH2, BRIP1, PMS1, GATA3, MUC16, FAM175A, EXT2, MLH1 and TGFB1, although these were not statistically significant in permutation testing. However, all 10 gene-level associations had OR < 1 with lower mutation burden in controls compared to cases, which was marginally statistically significant in permutation testing (p = 0.04). Comparing between the three case groups, BBD ER+ cases were closer to controls in mutation profile, while BBD ER- cases were distinct. Notably, the variant burden was significantly higher in controls than in either ER+ or ER- cases. CD45 expression was associated with mutational burden (p < 0.001).

Conclusions: Somatic mutations were more frequent in benign breast tissue from women who did not develop cancer, opening questions of clonal diversity or immune-mediated restraint on future cancer development. CD45 expression was positively associated with mutational burden, most strongly in controls. Further studies in both normal and premalignant tissues are needed to better understand the role of somatic gene mutations and their contribution to future cancer development.

Keywords: Benign breast disease; Breast cancer risk; CD45 expression; Mutation burden; Somatic mutations.

Fig. 1

Gene-level association findings between cases (BBD with future cancer events) and controls (BBD without future cancer events up to 16 years): a analytical flows of 12 association and filtering methods. b Histogram with connected dot-plot summarizing consensus of significant genes detected by 12 methods

Fig. 2

Variant concordances with normal genetics finding and gene-level volcano plots: a population frequency’s variant-level (x-axis) concordances with observed allele frequencies in this BBD cohort (y-axis). b Volcano plots of weighted logistic regression-based odds-ratio (OR) and statistical significance, for all the cases versus controls, using the classic definition of AAF variant filtering

Fig. 3

De-novo mutational signatures of entire dataset: a four dinucleotide signatures found through NMF under the classic AAF filtering definition. b heatmap of found de-novo signatures’ coefficients across all samples. c Violin plots of signature-D’s coefficients with respect to block-year (after vs. before 1992). d Violin plots of signature-D’s coefficients with respect to sample groups (control, ER-negative, and ER-positive)

Fig. 4

Expression of CD45 by group and mutational burden. CD45 is presented as an H-score. a Example staining of low CD45 (18.62). Scale is 100 um. b Example staining of high CD45 (60.15). Scale is 100 um. c CD45 H score by group. d CD45 H score by mutational burden (classic AAF filtering)