BRCA1 protein expression and subcellular localization in primary breast cancer: Automated digital microscopy analysis of tissue microarrays

Abstract

Purpose: Mutations in BRCA1 are associated with familial as well as sporadic aggressive subtypes of breast cancer, but less is known about whether BRCA1 expression or subcellular localization contributes to progression in population-based settings.

Methods: We examined BRCA1 expression and subcellular localization in invasive breast cancer tissues from an ethnically diverse sample of 286 patients and 36 normal breast tissue controls. Two different methods were used to label breast cancer tissues for BRCA1: (1) Dual immunofluoresent staining with BRCA1 and cytokeratin 8/18 and (2) immunohistochemical staining using the previously validated MS110 mouse monoclonal antibody. Slides were visualized and quantified using the VECTRA Automated Multispectral Image Analysis System and InForm software.

Results: BRCA1 staining was more intense in normal than in invasive breast tissue for both cytoplasmic (p<0.0001) and nuclear (p<0.01) compartments. BRCA1 nuclear to cytoplasmic ratio was higher in breast cancer cells than in normal mammary epithelial cells. Reduced BRCA1 expression was associated with high tumor grade and negative hormone receptors (estrogen receptor, progesterone receptor and Her2). On the other hand, high BRCA1 expression correlated with basal-like tumors (high CK5/6 and EGFR), and high nuclear androgen receptor staining. Lower nuclear to cytoplasmic ratio of BRCA1 correlated significantly with high Ki67 labeling index (p< 0.05) and family history of breast cancer (p = 0.001).

Conclusion: Findings of this study indicate that alterations in BRCA1 protein expression and subcellular localization in breast cancer correlate with poor prognostic markers and aggressive tumor features. Further large-scale studies are required to assess the potential relevance of BRCA1 protein expression and localization in routine classification of breast cancer.

Fig 1. Dual IF staining for BRCA1 and CK8/18 and corresponding digital annotation for BRCA1 in a representative core.

A: Composite image where green fluorescent staining if for epithelial CK8/18, red is for BRCA1, and blue is for nuclear DAPI stain. B: Tissue segmentation. C: Nuclear score map. D: Cytoplasmic score map. The attached legend to the right indicates what each color stands for in the tissue segmentation and score maps.

Fig 2. IHC staining and corresponding digital annotation for BRCA1 in a representative core.

A: IHC staining utilizes MS110 mouse monoclonal antibody to assess the level of BRCA1 protein expression. B: Tissue segmentation where the epithelial compartment is pink and the stromal compartment is yellow. C: Nuclear segmentation. D: nuclear score map. Definition of the digital image annotation intensity score: blue = 0, yellow = 1, orange = 2 and red = 3. The attached legend to the right indicates what each color stands for in the tissue segmentation and score map.

Fig 3. Dual IF staining for BRCA1 and CK8/18 in representative cases of invasive breast cancer and normal breast tissues.

A and B: Normal breast ducts and glands with positive red IF for BRCA1 in myoepithelial cells (arrowheads) and luminal cells (arrows). C: BRCA1 positive ductal carcinoma. D: BRCA1 negative ductal carcinoma. E: BRCA1 positive lobular carcinoma. F: BRCA1 negative lobular carcinoma.

Fig 4. Summary of the nuclear and cytoplasmic BRCA1 staining.

A: A plot of pairwise nuclear versus cytoplasmic continuous digital H-score for BRCA1 showing a good correlation between the two locations. B: Percentage of cases that exhibit weak (H-score = <100), Intermediate (H-score > 100 and <200), and strong (H-score >200) cytoplasmic BRCA1 staining. C: Percentage of cases that exhibit weak, Intermediate, and strong nuclear BRCA1 staining.

Fig 5. Dual IF vs IHC staining for BRCA1 analysis.

Plot of pairwise dual IF versus IHC digital H-score for BRCA1 showing a good correlation between the two staining methods for cytoplasmic scores (A), nuclear scores (B), and nuclear/cytoplasmic ratios (C). Figures D to G demonstrate the correlation between the IHC staining (Figs. D and F) and the dual IF staining (Figs. E and G) in the corresponding cores.

Fig 6. Association between BRCA1 and other prognostic markers.

A: Positive BRCA1 staining in an invasive ductal carcinoma core. B, C, and D: Positive Her2, CK5/6 and AR staining in corresponding sections of the same core.