Epigenetic silencing and deletion of the BRCA1 gene in sporadic breast cancer

Abstract

Introduction: BRCA1 or BRCA2 germline mutations increase the risk of developing breast cancer. Tumour cells from germline mutation carriers have frequently lost the wild-type allele. This is predicted to result in genomic instability where cell survival depends upon dysfunctional checkpoint mechanisms. Tumorigenic potential could then be acquired through further genomic alterations. Surprisingly, somatic BRCA mutations are not found in sporadic breast tumours. BRCA1 methylation has been shown to occur in sporadic breast tumours and to be associated with reduced gene expression. We examined the frequency of BRCA1 methylation in 143 primary sporadic breast tumours along with BRCA1 copy number alterations and tumour phenotype.

Methods: Primary sporadic breast tumours were analysed for BRCA1alpha promoter methylation by methylation specific PCR and for allelic imbalance (AI) at BRCA1 and BRCA2 loci by microsatellite analysis and TP53 (also known as p53) mutations by constant denaturing gel electrophoresis. The BRCA1 methylated tumours were analysed for BRCA1 copy alterations by fluorescence in situ hybridisation and BRCA1 expression by immunostaining.

Results: BRCA1 methylation was found in 13/143 (9.1%) sporadic breast tumours. The BRCA1 methylated tumours were significantly associated with estrogen receptor (ER) negativity (P = 0.0475) and displayed a trend for BRCA1 AI (P = 0.0731) as well as young-age at diagnosis (< or = 55; P = 0.0898). BRCA1 methylation was not associated with BRCA2 AI (P = 0.5420), although a significant association was found between BRCA1 AI and BRCA2 AI (P < 0.0001).Absent/markedly reduced BRCA1 expression was observed in 9/13 BRCA1 methylated tumours, most of which had BRCA1 deletion. An elevated TP53 mutation frequency was found among BRCA1 methylated tumours (38.5%) compared with non-methylated tumours (17.2%). The BRCA1 methylated tumours were mainly of tumour grade 3 (7/13) and infiltrating ductal type (12/13). Only one methylated tumour was of grade 1.

Conclusion: BRCA1 methylation is frequent in primary sporadic breast tumours. We found an indication for BRCA1 methylation to be associated with AI at the BRCA1 locus. Almost all BRCA1 methylated tumours with absent/markedly reduced BRCA1 expression (8/9) displayed BRCA1 deletion. Thus, epigenetic silencing and deletion of the BRCA1 gene might serve as Knudson's two 'hits' in sporadic breast tumorigenesis. We observed phenotypic similarities between BRCA1 methylated and familial BRCA1 tumours, based on BRCA1 deletion, TP53 mutations, ER status, young age at diagnosis and tumour grade.

Figure 1

Methylation-specific PCR analysis of the BRCA1 promoter region. Positive (+) and negative (-) controls are seen on the far right. Each lane contains products generated from separate PCR reactions using primers specific for (a) non-methylated and (b) methylated BRCA1 alleles. The tumour sample in lane 1 (L1) shows the presence of a band in both PCR reactions, indicating methylation of the BRCA1 promoter region.

Figure 2

Allelic imbalance analysis at the BRCA loci. The electropherograms represent quantification of PCR products from normal DNA (upper) and tumour DNA (lower) from the same individual. A signal reduction is evident in the tumour electropherograms, which suggest allelic imbalance (indicated by an arrow) at the (a) BRCA1 locus, using the D17S1323 marker, and (b) BRCA2 locus, using the D13S260 marker.

Figure 3

BRCA1 gene copy number and protein expression analysis on breast tumor sections. (a) Tumor cells in a BRCA1 methylated sample are seen to have reduced BRCA1 gene copy numbers (red signal) compared with centromere 17 (green signal) by FISH. Considerable heterogeneity in BRCA1 gene copy numbers was evident in all tumours analysed. (b) A BRCA1 methylated tumour section showing wild-type BRCA1 protein expression by immunostaining in normal breast ducts and (c) absent/markedly reduced BRCA1 protein expression in tumour cells. Bars = 7 μm.