Background Despite the widespread use of immunohistochemistry (IHC) you will find no standardization recommendations that control for antibody probe variability. All EGFR antibodies but 2232 yielded specific results in western blotting; however only 31G7 and 2-18C9 were strongly connected (Pearson’s < 0.0001). HER3 staining was nonspecific and nonreproducible. KSR2 antibody High EGFR-expressing individuals experienced a worse prognosis when EGFR was measured with H11 or 31G7 (log rank = 0.015 and = 0.06). There was no statistically significant correlation between survival and EGFR detected by 2-18C9 15 or polyclonal 2232 antibodies. Conclusions Antibody validation is usually a critical analytic factor that regulates IHC readings in biomarker studies. Evaluation of IHC proficiency and quality control are key components toward IHC standardization. Impact This work highlights the importance of IHC standardization and could result in the improvement of clinically relevant IHC protocols. Introduction Immunohistochemistry (IHC) is frequently used in clinical diagnosis and the classification of neoplasms (1). Despite the widespread use of IHC in routine diagnostic practice no universally accepted standardization guidelines have YO-01027 been developed. IHC is subject to variable preanalytic analytic and postanalytic factors that decrease its reproducibility including tissue preparation and fixation diverse reagents different staining methods scoring systems and the definition of a “positive” result (2). Considerable variability has been attributed to the selection and insufficient validation of the primary antibodies poor quality controls and lack of rigorous assay optimization (1 3 Epidermal growth factor receptors (EGFR) are among the most analyzed cancer biomarkers because of their oncogenic activity in diverse tumor types (4). EGFR in particular was the first receptor to be proposed as a target for malignancy therapy and YO-01027 several EGFR-targeted therapies are now available in the medical center (5). EGFR IHC results highly depend on the type of antibody protocols scoring system and cutoffs used (6) rendering the prognostic role of EGFR protein expression as assessed by IHC highly controversial (7). This lack of reproducibility may be YO-01027 responsible for its failure as a companion diagnostic test and studies done to date largely nonstandardized have shown that detection of high EGFR expression by IHC does not reliably predict the clinical end result of EGFR-targeted treatment (8). Therefore IHC is not routinely used to determine EGFR status. In contrast IHC on formalin-fixed paraffin-embedded (FFPE) tissue is the current standard for evaluating estrogen receptor (ER) status in breast malignancy and IHC measurements directly affect the management of breast cancer patients (9). Standardized and validated IHC assays for ER include those that use monoclonal antibody (mAb) clones 1D5 and SP1 (10 11 and efforts to establishbest practice guidelines for ER IHC screening are ongoing (12). Nevertheless high false unfavorable rates of tissue samples evaluated by IHC have been reported when laboratory quality control was inadequate and proficiency screening requirements were not met (3); this highlights the critical need for the development of standardized IHC assays. Here we analyze validate and compare ER EGFR and human epidermal growth factor receptor (HER) 3 protein expression detected by commonly used ER EGFR and HER3 antibodies in breast and lung malignancy. Ultimately we show the effects of variable EGFR antibody selection and validation on end result in a breast malignancy cohort of 642 patients. Materials and Methods Cohorts FFPE main breast malignancy specimens from 642 patients that underwent surgery at Yale-New Haven Hospital (New Haven CT) between 1962 and 1983 were obtained from the archives of the Pathology Department of Yale University or college (New Haven CT). A smaller cohort was retrospectively collected from 42 non-small cell YO-01027 lung malignancy patients from Yale-New Haven Hospital between January 1995 and May 2003. The demographics tumor characteristics and routine IHC scoring for ER progesterone receptor and HER2 in the breast malignancy cohort are shown in Supplementary Table S1. The study was approved by the Institutional Review.