Identification of BRCA1 Deficiency Using Multi-Analyte Estimation of BRCA1 and Its Repressors in FFPE Tumor Samples from Patients with Triple Negative Breast Cancer

<div><p>Purpose</p><p>Apart from germ-line BRCA1-mutated breast cancers, a significant proportion of women with sporadic triple negative breast cancer (TNBC) sub-type are known to harbour varying levels of BRCA1-dysfuction. There is currently no established diagnostic method to identify these patients.</p><p>Methods</p><p>The analysis was performed on 183 primary breast cancer tumor specimens from our longitudinal case-series archived as formalin-fixed-paraffin-embedded (FFPE) blocks comprising 71 TNBCs and 112 Hormone receptor positive HER2 negative (HR+HER2-) tumors. Transcript levels of BRCA1 and two of its repressors ID4 and microRNA182 were determined by TaqMan quantitative PCR. BRCA1 protein was detected immunohistochemically with the MS110 antibody.</p><p>Results</p><p>The representation of BRCA1 and its repressor ID4 as a ratio led to improved separation of TNBCs from HR+HER2- compared to either measure by itself. We then dichotomised the continuous distribution of each of the three measurements (Protein, MIRNA and transcript:repressor ratio) into categories of <b><i>deficient (0)</i></b> and <b><i>adequate (1)</i></b>. A composite BRCA1 Deficiency Score (BDS) was computed by the addition of the score for all three measures. Samples deficient on 2 or more measures were deemed to be BRCA1 deficient; and 40% of all TNBCs met this criterion.</p><p>Conclusion</p><p>We propose here a simple multi-level assay of BRCA1 deficiency using the BRCA1:ID4 ratio as a critical parameter that can be performed on FFPE samples in clinical laboratories by the estimation of only 3 bio-markers. The ease of testing will hopefully encourage adoption and clinical validation.</p></div

BRCA1 Deficiency Score (BDS).

<p>BRCA1 Deficiency Score (BDS): Amalgamated scores range from a low of 0 to a high of 3. Scores of 0 and 1 are almost exclusively seen in TNBCs. Scores of 2 and 3 were seen in both HR+HER2- as well as TNBCs. 23/27 HR+HER2- tumors scored adequate on all measures and hence had a score of 3. This method of selection classified 40% of TNBCs being BRCA1 deficient and only 3% of HR+HER2-ve to be BRCA1 deficient.</p

Relationship between BRCA1 and ID4.

<p>Relationship between BRCA1 and ID4.A four quadrant plot with “cut-off” lines for both transcripts that are at 1 SD above the mean value of all specimens. The upper right quadrant representing high ID4 and high BRCA1 is empty. The upper left quadrant of high ID4 and low BRCA1 is almost exclusively TNBCs while the lower right quadrant of high BRCA1 and low ID4 is exclusively luminal tumors.</p

Distribution of TNBCs and HR+HER2- tumors adequate and deficient in the three measurements.

<p>Distribution of TNBCs and HR+HER2- tumors adequate and deficient in the three measurements.</p

Assay part numbers (Gene expression and MicroRNA analysis).

<p>Assay part numbers (Gene expression and MicroRNA analysis).</p