CpG island hypermethylation of BRCA1 and loss of pRb as co-occurring events in basal/triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) occurs in approximately 15% of all breast cancer patients, and the incidence of TNBC is greatly increased in BRCA1 mutation carriers. This study aimed to assess the impact of BRCA1 promoter methylation with respect to breast cancer subtypes in sporadic disease. Tissue microarrays (TMAs) were constructed representing tumors from 303 patients previously screened for BRCA1 germline mutations, of which a subset of 111 sporadic tumors had previously been analyzed with respect to BRCA1 methylation. Additionally, a set of eight tumors from BRCA1 mutation carriers were included on the TMAs. Expression analysis was performed on TMAs by immunohistochemistry (IHC) for BRCA1, pRb, p16, p53, PTEN, ER, PR, HER2, CK5/6, EGFR, MUC1 and Ki-67. Data on BRCA1 aberrations and IHC expression was examined with respect to breast cancer-specific survival. The results demonstrate that CpG island hypermethylation of BRCA1 significantly associates with the basal/triple-negative subtype. Low expression of pRb, and high/intense p16, were associated with BRCA1 promoter hypermethylation, and the same effects were seen in BRCA1 mutated tumors. The expression patterns of BRCA1, pRb, p16 and PTEN were highly correlated, and define a subgroup of TNBCs characterized by BRCA1 aberrations, high Ki-67 (≥ 40%) and favorable disease outcome. In conclusion, our findings demonstrate that epigenetic inactivation of the BRCA1 gene associates with RB/p16 dysfunction in promoting TNBCs. The clinical implications relate to the potential use of targeted treatment based on PARP inhibitors in sporadic TNBCs, wherein CpG island hypermethylation of BRCA1 represents a potential marker of therapeutic response.

Figure 1

BRCA1, pRb, p16 and PTEN define a sub-class within triple-negative (TN) breast cancers. (A) Hierarchical cluster analysis for the IHC expression profiles suggesting sub-structure within the TN subtype with at least two branches enriched for the basal/triple-negative phenotype. (B) The association matrix for IHC markers representing the log-transform of adjusted p-values (Bonferroni adjustment for multiple testing) derived from Fishers exact hypothesis testing of pair-wise comparisons revealing two groups of strong correlative relationships, i.e., the expression profiles of BRCA1, PTEN, pRb and p16 more strongly correlate with each other than to CK8, CK18, CK5/6 and EGFR. The Bonferroni adjusted p-values are shown and represented in a spectrum of blue colors ranging from <10⁻¹⁰ (deep blue) to <0.05 (light blue) and N.S = Not Significant (white). (C) Hierarchical cluster analysis performed separately on each of four main subtypes demonstrating that the TN subtype can be subdivided into two groups based on the co-ordinated expression of BRCA1, pRb, p16 and PTEN. Here, the familial tumors derived from BRCA1 mutation carriers along with sporadic tumors displaying CpG island hypermethylation predominantly cluster within the left branch (orange bar). The presence/absence of BRCA1 gene aberrations are indicated in cyan/black at the bottom of each dendrogram with white blanks representing unknown status.

Figure 2

Survival with respect to phenotype and RB/p16 dysfunction in TN breast cancers. (A) Breast cancer-specific survival with respect to phenotype without (left) and with (right) positivity for either EGFR or CK5/6 to define the basal-like phenotype. (B) The high/intense p16 expression pattern significantly associates with differential survival outcomes in TN breast cancer. (C) TN tumors lacking of Claudin-3 expression relate to reduced short-term survival. (D) Prognostic stratification of triple-negative breast cancer. Here, TN tumors lacking Claudin-3 are sub-categorized as TN/Claudin, with the remainder of TN tumors subdivided based on the prescence/abscence of high p16 levels (IHC 3⁺), referred to as TN/Claudin⁺/RB⁻ (loss), or TN/Claudin⁺/RB⁺, respectively.

Figure 3

Ki-67 in triple-negative (TN) breast tumors. (A) The proportions of tumor cells undergoing proliferation, estimated by looking at Ki-67 on TMAs, were found to be significantly higher in TN tumors displaying loss of RB (TN/Claudin⁺/RB⁻) compared with the other two TN subroups (TN/Claudin⁺/RB⁺ and TN/Claudin-Low). (B) The thresholds of optimal seperation for Ki-67 with respect to breast cancer-specific survival differ between luminal- and triple-negative subtypes. Here, Ki-67 does not identify prognostic subgroups within the HER2 subtype. (C) Kaplan-Meier plots, coupled with the log-rank test, demonstrating significantly more favorable disease outcome for patients with TN breast cancer displaying high levels of Ki-67 positive tumor cells (left part). In contrast, highly proliferating tumors of the luminal phenotype are associated with significantly less favorable disease outcome (right part).