Performance of Three-Biomarker Immunohistochemistry for Intrinsic Breast Cancer Subtyping in the AMBER Consortium

Abstract

Background Classification of breast cancer into intrinsic subtypes has clinical and epidemiologic importance. To examine accuracy of immunohistochemistry (IHC)-based methods for identifying intrinsic subtypes, a three-biomarker IHC panel was compared to the clinical record and RNA-based intrinsic (PAM50) subtypes. Methods Automated scoring of estrogen receptor (ER), progesterone receptor (PR) and HER2 was performed on IHC-stained tissue microarrays (TMAs) comprising 1,920 cases from the African American Breast Cancer Epidemiology and Risk (AMBER) consortium. Multiple cores (1–6/case) were collapsed to classify cases, and automated scoring was compared to the clinical record and to RNA-based subtyping. Results Automated analysis of the three-biomarker IHC panel produced high agreement with the clinical record (93% for ER and HER2, and 88% for PR). Cases with low tumor cellularity and smaller core size had reduced agreement with the clinical record. IHC-based definitions had high agreement with the clinical record regardless of hormone receptor positivity threshold (1% vs. 10%), but a 10% threshold produced highest agreement with RNA-based intrinsic subtypes. Using a 10% threshold, IHC-based definitions identified the basal-like intrinsic subtype with high sensitivity (86%), while sensitivity was lower for luminal A, luminal B and HER2-enriched subtypes (76%, 40% and 37%, respectively). Conclusion Three-biomarker IHC-based subtyping has reasonable accuracy for distinguishing basal-like from non-basal-like, while additional biomarkers are required for accurate classification of luminal A, luminal B and HER2-enriched cancers. Impact Epidemiologic studies relying on three-biomarker IHC status for subtype classification should use caution when distinguishing luminal A from luminal B and when interpreting findings for HER2-enriched cancers.

PAM50 and Risk of Recurrence Scores for Interval Breast Cancers

“…Positivity of EGFR was defined as any HER1 staining and positivity for CK 5/6 was defined as any cytoplasmic and/or membranous staining. Methods to distinguish intrinsic subtypes in CBCS Phase 3 are described in detail by Allott et al(26). Briefly, tissue microarrays (TMAs) were constructed and stained by TPL and were digitally imaged using the Aperio ScanScope XT (Aperio Technologies, Vista CA).…”

“…Automated digital image analysis was performed to quantify IHC staining using a Genie classifier and the Nuclear V9 algorithm (Aperio Technologies, Vista CA), for ER and PR and a Genie classifier and Membrane V9 algorithm for HER2. Three-marker intrinsic subtypes were defined using ER, PR, and HER2 status as described by Allott et al(26): Luminal A (ER+ and/or PR+, HER2−), Luminal B (ER+ and/or PR+, HER2+), HER2+ (ER−, PR−, HER2+) and triple negative (ER−, PR−, HER2−). Five-marker intrinsic subtypes were assigned to women who had complete data for ER, PR, HER2, CK5/6 and HER1 as described by Carey et al(24), where the three markers were used for Luminal and HER2 cancers, but basal-like cancer required positivity for either HER1 or Ck5/6 (ER−, PR−, HER2−, HER1+ or CK5/6+).…”

“…For samples from CBCS 1&2 (N=188), RNA was extracted from two unstained 10-μm FFPE slides per patient. For women in CBCS Phase 3, RNA was extracted from two 1-mm cores (N=377) or two 10 μm slides (N=79) as described previously(26). RNA was isolated using the RNeasy FFPE Kit (Qiagen) and Nanostring analyses were performed in the Rapid Adoption Molecular laboratory and the Translational Genomics laboratory at UNC.…”

FOXA1 hypermethylation: link between parity and ER-negative breast cancer in African American women?

“…Stained slides were digitally imaged at ×20 magnification using the Aperio ScanScope XT (Aperio Technologies, Vista, CA); automated image analysis of IHC staining was performed using a Genie classifier. Tumor cores on TMAs were collapsed into case-level data using a cellularity-weighted approach, as previously described [23]. With the weighted average of percent positivity values, an H-score was calculated, which reflects the extent of nuclear immunoreactivity, ranging from 0 to 300 [24].…”

Biology and Etiology of Young-Onset Breast Cancers among Premenopausal African American Women: Results from the AMBER Consortium

“…The collection of tumor characteristic and risk factor exposure data in the AMBER Consortium has been described previously(27,32). Briefly, each study contributed paraffin-embedded breast tumor tissue to two core research facilities (the Translational Pathology Lab (TPL) at the University of North Carolina at Chapel Hill (UNC) for the CBCS and the Roswell Park Cancer Institute for the WCHS and BWHS) where tissue microarrays (TMAs) were constructed for all available tumor specimens.…”