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

Abstract

Purpose: 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.

Methods: 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.

Results: 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 deficient (0) and adequate (1). 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.

Conclusion: 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.

Fig 1. Identification of BRCA1 deficiency using multi-analyte estimation of BRCA1 and its repressors in FFPE tumor samples from Indian patients with Triple Negative Breast Cancer.

Flow chart depicting sequential elimination of tumors based on availability and QC criteria and the proportions of the sub-types used in this study. Panel A & B show the detailed break-up of numbers from each of the sub-types used in the different analytical tests. HR+: Hormone Receptor positive, TNBC: Triple Negative, QC: Quality Control.

Fig 2. Transnscript abundance of BRCA1 and ID4 in TNBC Vs HR+HER2-ve tumors.

Transcript abundance of BRCA1 and ID4 in HR+HER2-veand TNBCs. Median expression of BRCA1 and ID4 transcripts is significantly different in the two groups; *p = 0.008 for BRCA1, and **p = 0.001 for ID4, (Mann Whitney U Test). The high ranges of BRCA1 are exclusively HR+, and the high-ranges of ID4 are all TNBCs.

Fig 3. Relationship between BRCA1 and ID4.

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.

Fig 4. Distribution of BRCA1/ID4 ratio values in luminal tumors compared to TNBCs.

Distribution of BRCA1/ID4 ratio values in luminal tumors compared to TNBCs. An overlay of the histograms in panel A clearly shows the marked “Left-Skew” of TNBCs and a relatively “Right-shifted” distribution of luminal (HR+) tumors. The median value is significantly different in the two classes (*p<0.01- Mann Whitney U test).

Fig 5. Scatter plot of expression of MIR182 v BRCA1:MIR182.

Scatter plot of expression of MIR182 v BRCA1:MIR182 expression correlated to being highest in TNBC class (Pearson’s CC- −0.27, *p<0.05) There is also an inverse correlation between MIR182 and BRCA1transcripts.

Fig 6. Shared BRCA1 deficiency measures in TNBCs n = 55.

Shared BRCA1 deficiency measures in TNBCs n = 55.A Venn diagram, showing the overlap of TNBCspecimens deficient in BRCA1 by the three different measures.

Fig 7. BRCA1 Deficiency Score (BDS).

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.