Validation of the BRCA1 antibody MS110 and the utility of BRCA1 as a patient selection biomarker in immunohistochemical analysis of breast and ovarian tumours.
BRCA1 protein measurement has previously been evaluated as a potential diagnostic marker without reaching a conclusive recommendation. In this study, we applied current best practice in antibody validation to further characterize MS110, a widely used antibody targeting BRCA1. Antibody specificity was investigated using different biochemical validation techniques. We found that BRCA1 could not be reliably detected using immunoprecipitation and Western blot in endogenously expressing cells. We used immunohistochemistry on formalin-fixed paraffin-embedded cell pellets to establish compatibility with formalin-fixed paraffin-embedded samples. We demonstrated that in transfected cells and cell lines with known genetic BRCA1 status, MS110 successfully detected BRCA1 giving the expected level of staining in immunohistochemistry. Following this, we investigated the use of BRCA1 protein measurement by immunohistochemistry in a cohort of triple negative breast and serous ovarian tumour samples to explore the use of BRCA1 protein measurement by immunohistochemistry for patient stratification. Using MS110 in repeated standardized experiments, on serial sections from a panel of patient samples, results demonstrated considerable run-to-run variability. We concluded that in formalin-fixed tissue samples, MS110 does detect BRCA1; however, using standard methodologies, BRCA1 expression levels in tissue samples is incompatible with the use of this protein as a statistically robust patient selection marker in immunohistochemistry. These results demonstrate the need for further development to deliver BRCA1 protein quantification by immunohistochemistry as a patient stratification marker.
AstraZeneca, Alderley Park, Macclesfield, Cheshire, UK.
Roy
R
Milner
Helen
H
Wombwell
Sonia
S
Eckersley
Donna
D
Barnes
Juli
J
Warwicker
Erica
E
Van Dorp
Rachel
R
Rowlinson
Simon
S
Dearden
Glen
G
Hughes
Chris
C
Harbron
Bob
B
Wellings
Darren
D
Hodgson
Chris
C
Womack
Neil
N
Gray
Alan
A
Lau
Mark J
MJ
O'Connor
Catherine
C
Marsden
Alexander J
AJ
Kvist
D000911:Antibodies, Monoclonal; D019313:BRCA1 Protein / Q000032:analysis* / Q000235:genetics; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000235:genetics; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism*; D059467:Transcriptome; D014162:Transfection
D000911:Antibodies, Monoclonal; D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D014408:Biomarkers, Tumor