Original contribution Accurate detection of BRAF p.V600E mutations in challenging melanoma specimens requires stringent immunohistochemistry scoring criteria or sensitive molecular assays ☆ Kevin E. Fisher MD, PhD a, ⁎ , Cynthia Cohen MD a , Momin T. Siddiqui MD a , John F. Palma PhD b , Edward H. Lipford III MD c , John W. Longshore PhD c a Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322, USA b Roche Molecular Systems, Pleasanton, CA 94588, USA c Carolinas Pathology Group, Charlotte, NC 28203, USA Received 16 June 2014; revised 22 July 2014; accepted 23 July 2014 Keywords: BRAF; V600E; Melanoma; Immunohistochemistry; Molecular; Comparison; Criteria Summary Malignant melanoma patients require BRAF mutation testing prior to initiating BRAF inhibitor therapy. Molecular testing remains the diagnostic gold standard, but recent work suggests that BRAF immunohistochemistry (IHC) confers comparable results. Sample attributes and scoring criteria that may affect BRAF IHC interpretation, however, are poorly defined. We investigated formalin-fixed, paraffin-embedded samples with variable challenging interpretative attributes: metastases, core needle biopsies, sample tissues less than 60 mm 2 , samples with greater than 50% necrosis, and/or samples with greater than 10% melanin pigmentation. Three pathologists independently scored 122 BRAF V600E IHC-labeled melanoma samples for percentage (0%-100%) of staining intensity (0-3+). Interscorer BRAF IHC discrepancies were resolved by consensus review. Lenient (≥1+, N0%) and stringent (≥2+, ≥10%) IHC scoring criteria were compared to BRAF V600 mutation (cobas) results (n = 118). Specimens with greater than 10% melanin pigmentation and metastatic samples produced the majority of interobserver IHC and IHC/cobas scoring discrepancies. Consensus review using stringent scoring criteria decreased the number of discrepant results, yielded very good interobserver reproducibility, and improved specificity and positive predictive value for BRAF p.V600E detection. BRAF p.V600K mutations accounted for 57.1% of false-negative IHC results when stringent, consensus criteria scoring were used. The cobas test detected 75.0% (8/12) of BRAF IHC-negative BRAF p.V600K mutations confirmed by next-generation sequencing. Molecular BRAF testing is the preferred screening test for BRAF inhibitor therapy eligibility because of superior sensitivity in challenging interpretative melanoma specimens. However, BRAF V600E IHC has excellent specificity and positive predictive value when stringent, consensus scoring criteria are implemented. To decrease IHC scoring discrepancies, pathologists should interpret metastatic and pigmented samples with caution. © 2014 Elsevier Inc. All rights reserved. ☆ Conflict of interest statement: Roche Molecular Systems provided financial and technical resources for portions of the study. Dr Palma is employed by Roche Molecular Diagnostics (Pleasanton, CA, USA). ⁎ Corresponding author. Emory University Hospital, Department of Pathology and Laboratory Medicine, 1364 Clifton Rd NE, Atlanta, GA 30322. E-mail address: kfishe8@emory.edu (K. E. Fisher). www.elsevier.com/locate/humpath http://dx.doi.org/10.1016/j.humpath.2014.07.014 0046-8177/© 2014 Elsevier Inc. All rights reserved. Human Pathology (2014) 45, 2281–2293