Levels of DNA methylation vary at CpG sites across the BRCA1 promoter, and differ according to triple negative and "BRCA-like" status, in both blood and tumour DNA

Triple negative breast cancer is typically an aggressive and difficult to treat subtype. It is often associated with loss of function of the BRCA1 gene, either through mutation, loss of heterozygosity or methylation. This study aimed to measure methylation of the BRCA1 gene promoter at individual CpG sites in blood, tumour and normal breast tissue, to assess whether levels were correlated between different tissues, and with triple negative receptor status, histopathological scoring for BRCA-like features and BRCA1 protein expression. Blood DNA methylation levels were significantly correlated with tumour methylation at 9 of 11 CpG sites examined (p<0.0007). The levels of tumour DNA methylation were significantly higher in triple negative tumours, and in tumours with high BRCA-like histopathological scores (10 of 11 CpG sites; p<0.01 and p<0.007 respectively). Similar results were observed in blood DNA (6 of 11 CpG sites; p<0.03 and 7 of 11 CpG sites; p<0.02 respectively). This study provides insight into the pattern of CpG methylation across the BRCA1 promoter, and supports previous studies suggesting that tumours with BRCA1 promoter methylation have similar features to those with BRCA1 mutations, and therefore may be suitable for the same targeted therapies

Study population demographics according to blood and tumour tissue availability.

<p>Study population demographics according to blood and tumour tissue availability.</p

Morphological features and BRCA1 expression.

<p>H&Es demonstrating BRCA1 associated morphological features scores. A: Low score demonstrating good tubule formation, little nuclear pleomorphism, no lymphocytes and no mitoses. B: High score demonstrating syncytial islands, marked nuclear pleomorphism and heavy lymphocytic infiltrate. C, D&E: IHC performed using BRCA1 specific antibody Ab-1. C: Breast tumour demonstrating nuclear staining for BRCA1. D: Breast tumour demonstrating lack of nuclear or cytoplasmic staining for BRCA1. E: Normal breast tissue demonstrating normal nuclear staining for BRCA1. F: Histogram of the number of BRCA1-like morphological features from zero to 9 (n = 147).</p

Methylation plots comparing blood, tumour and normal breast tissue.

<p>Mean (+/-SD) methylation levels plotted against CpG site position along the chromosome in relation to the <i>BRCA1</i> transcription start site (position zero). (A) Blood DNA methylation level is shown in red and tumour DNA methylation level in blue for the matched samples (n = 170) and blood DNA methylation level for the whole sample set is shown in orange (n = 658). (B) Normal breast tissue DNA methylation level is shown in green (n = 26) and tumour DNA methylation level in blue (n = 170).</p

Morphological feature scoring.

<p>Morphological feature scoring.</p

Methylation plots comparing blood and tumour for BRCA1-like features, Triple negativity and BRCA1 protein expression.

<p>Mean (+/-SD) methylation levels plotted against CpG site position along the chromosome in relation to the <i>BRCA1</i> transcription start site (position zero). Blood methylation level is shown on the left and tumour methylation level on the right. A&B: Tumours with <5 BRCA1-like features are shown in orange and those with ≥5 BRCA1-like features are shown in green. C&D: Triple negative tumours are shown in maroon and non-triple negative are shown in teal. E&F: Tumours with high levels of BRCA1 protein expression are shown in brown and those with low levels are shown in cyan.</p

Details the primer sequences used for pyrosequencing.

<p>Details the primer sequences used for pyrosequencing.</p