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

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.

Resident microglia, and not peripheral macrophages, are the main source of brain tumor mononuclear cells.

Gliomas consist of multiple cell types, including an abundant number of microglia and macrophages, whereby their impact on tumor progression is controversially discussed. To understand their unique functions and consequently manipulate either microglia or macrophages in therapeutic approaches, it is essential to discriminate between both cell populations. Because of the lack of specific markers, generally total body irradiated chimeras with labeled bone marrow cells were used to identify infiltrated cells within the brain. However, total body irradiation (TBI) affects the blood-brain barrier integrity, which in turn potentially facilitates immune cell infiltration. In this study, changes on the blood-brain barrier were avoided using head-protected irradiation (HPI). Head protection and total body irradiated chimeras exhibited similar reconstitution levels of the myeloid cell lineage in the blood, enabling the comparable analyses of brain infiltrates. We demonstrate that the HPI model impeded a massive unspecific influx of donor-derived myeloid cells into naive as well as tumor-bearing brains. Moreover, experimental artifacts such as an enlarged distribution of infiltrated cells and fourfold increased tumor volumes are prevented in head-protected chimeras. In addition, our data evidenced for the first time that microglia are able to up-regulate CD45 and represent an inherent part of the CD45(high) population in the tumor context. All in all, HPI allowed for the unequivocal distinction between microglia and macrophages without alterations of tumor biology and consequently permits a detailed and realistic description of the myeloid cell composition in gliomas.