The Value of Internal Positive Controls and Synthetic Calibrators for Performance Assessment of Breast Carcinoma Estrogen Receptor Immunohistochemistry Assay

In 2010, the American Society of Clinical Oncology and the College of American Pathologists jointly issued guidelines for estrogen receptor (ER) and progesterone receptor testing and an update in 2020. Both guidelines recommended that ER and progesterone receptor assays be interpreted in the context of internal and external controls. Despite the considerable importance of internal controls for ER testing by immunohistochemistry (IHC), there are no studies that assess their performance and effectiveness. The Canadian Immunohistochemistry Quality Control performed an educational proficiency testing run for 70 laboratories using a tissue microarray with 80 samples of breast carcinoma enriched for tumors weakly positive for ER. The tissue microarray contained several internal positive controls and was run together with ER calibrators from Boston Cell Standards. The internal positive controls were scored as negative in 0/70 (0%), weak in 12/70 (17%), moderate in 29/70 (41%), and strong in 29/70 (41%) laboratories secondary to differences in analytical sensitivity of IHC protocols. For calibrators, only SP1 clone results (52 participants) are reported because of the small number of laboratories using other primary antibody clones. Thirty-nine laboratories (75%) had IHC protocols with (limit of detection) LOD < 25,000. Both internal positive controls and Boston Cell Standard ER calibrators showed excellent correlation with overall H-score mean, percent 3+ positive cells, and percent negative cells (P < .0001). The difference in diagnostic sensitivity was significant between weak and moderate and between weak and strong internal positive controls (t test, P = .015 and P = .009, respectively) and between IHC protocols with LOD < 25,000 and all others (t test, P = .001). The measured LOD had a significant association with diagnostic accuracy at 1% and 10% cutoffs, whereas the internal positive control score was significantly associated only with diagnostic accuracy at the 10% cutoff (1-way analysis of variance, P = .009). Negative results of the ER IHC assay should not be reported when internal positive controls are either negative or weak. Further studies with more advanced calibrators are needed in order to establish the recommended LOD range for optimal ER IHC assay performance with SP1 and other anti-ER primary antibody clones.