Total-genome analysis of BRCA1/2-related invasive carcinomas of the breast identifies tumor stroma as potential landscaper for neoplastic initiation

Abstract

We have shown that the tumor microenvironment of sporadic breast cancer is diverse in genetic alterations and contributes to the cancer phenotype. The dynamic morphology of the mammary gland might be of special interest in hereditary breast/ovarian cancer syndrome (HBOC). We hypothesized that hotspots of loss of heterozygosity or allelic imbalance (LOH/AI) within the tumor stroma of BRCA1/2-related breast cancers provide an impaired mammary stroma that could facilitate later malignant transformation of the breast epithelium. We conducted a total genome LOH/AI scan of DNA derived from the epithelium and stroma of 51 BRCA1/2-related breast cancers, using 372 microsatellite markers. We compared these data with those from a set of 134 sporadic breast cancers. HBOC-related breast cancers accumulated significantly more genetic alterations than did sporadic breast cancers. BRCA1/2-related breast cancer stroma showed LOH/AI at 59.7% of all loci analyzed, similar to the average frequency of LOH/AI observed in the epithelium (66.2%). This is remarkably different from sporadic breast cancers, for which the average epithelial LOH/AI frequency (36.7%) far exceeds the average stromal LOH/AI frequency (28.4%) (P=.03). We identified 11 hotspot loci of LOH/AI in the BRCA1/2 stroma, encompassing genes such as POLD1, which functions in DNA replication, and SDHB. In a subset of samples, enriched for BRCA1 cases, we found 45.0% overall LOH/AI in the stroma, which was significantly higher than the 41.8% LOH/AI observed in corresponding epithelium (P=.04). Together, our data indicate that, in HBOC-related breast cancers, the accumulation of genomic instability in the cancer stroma coincides with that in the neoplastic epithelium, and we postulate that such a genetically unstable stroma might facilitate a microenvironment that functions as a landscaper that promotes genomic instability in the epithelium and, subsequently, neoplastic transformation.

Figure 1

Pedigree of a family with HBOC segregating BRCA variants. The pedigree is shown across 4 generations, with affected members (with breast cancer) indicated by blackened circles. The proband (arrow) was diagnosed with breast cancer at age 49 years, and testing showed her as wild type for both BRCA genes. Other affected family members (stars) tested positive for the BRCA1 Ser1040Asn and BRCA2 Ser2483Gly variants.

Figure 2

Frequency of LOH/AI observed in the epithelium and stroma in HBOC-related breast cancer compared with sporadic breast cancers. Frequency of LOH/AI (Y-axes) is plotted on a markerwise level (X-axes). The average LOH/AI frequency of markers in the epithelium (top panel) and stroma (bottom panel) are shown for 51 cases of HBOC-related breast cancer (blue dots) and 134 cases of sporadic breast cancer (red dots). The bold horizontal lines indicate LOH/AI frequencies averaged over all markers.

Figure 3

Unsupervised hierarchical cluster analysis. Average linkage and the dissimilarity measure of proportion of discordant LOH/AI between samples are used. The analysis was based on the presence or absence of LOH at 371 informative loci for 51 HBOC-related breast carcinoma epithelial samples and the matching 51 stromal samples (for a total of 102 end branches). The numbers at each end branch indicate germline deleterious mutations in BRCA1 (1) or BRCA2 (2) or unclassified variants of these genes (uv1 and uv2, respectively). The stars indicate those samples for which the stroma and epithelium of one case cluster directly together. Note the clustering of BRCA1 samples near the left (black bar; see text for details). wt = Wild type.

Figure 4

Two-dimensional scaling of 51 HBOC-related breast cancer cases. Each case is represented by combining the stroma and malignant epithelium from the same patient. Samples were obtained from individuals with germline mutations in BRCA1 (black dots) or BRCA2 (red dots), with variants in BRCA1 (BRCA1uv [green dots]) or BRCA2 (BRCA2uv [blue dots]), or without mutations or variants in either gene (yellow dots). Note the clustering of samples with BRCA1 mutations to the left of the diagonal line.

Figure 5

Multidimensional scaling of LOH/AI patterns from 35 patients with deleterious BRCA1/2 mutations. The LOH/AI patterns from epithelium (EP; left panel) and stroma (ST; right panel) of individuals with germline mutations in BRCA1 (black dots) and BRCA2 (red dots) were analyzed separately in the context of the pattern of LOH/AI observed in sporadic breast cancers (green dots). Note that the stromal LOH/AI pattern differentiates BRCA1 and BRCA2 mutation-positive breast cancers from sporadic breast cancers.

Figure 6

Unsupervised cluster analysis by LOH/AI status in a combined stromal and epithelial data set for HBOC-related breast cancers and sporadic breast cancers. HBOC breast cancers are labeled at the end branches with “DM” (deleterious mutation) and “non-DM” (comprising those with variants of unknown significance and those without detectable mutations). The bar below the cluster plot visualizes the separation of HBOC breast cancers (black and gray bars) from sporadic breast cancers (white bars).