Phospho-ERK THR202/Tyr214 Is Overexpressed in Hairy Cell Leukemia and Is a Useful Diagnostic Marker in Bone Marrow Trephine Sections Douglas W. Warden, MD, Sarah Ondrejka, DO, Jeffrey Lin, PhD, Lisa Durkin, BSc, Juraj Bodo, PhD, and Eric D. Hsi, MD Abstract: BRAF V600E mutations are present in virtually all cases of hairy cell leukemia (HCL). We hypothesized that de- tection of phospho-ERK (pERK) in tissue sections may be a useful marker for diagnosis of HCL. pERK/CD20 double im- munostaining was performed on 90 formalin-fixed bone marrow trephine samples affected with small B-cell lymphoproliferative disorders, including 28 cases of HCL. pERK staining was ob- served in all 28 cases of HCL and in 1 of 62 cases of non-HCL B-cell lymphoproliferative disorders. By allele-specific poly- merase chain reaction, all 11 cases of HCL with available DNA were positive for BRAF V600E, as was the 1 pERK + non-HCL case. The remaining 31 non-HCL cases tested were negative for BRAF V600E. The sensitivity and specificity of pERK for di- agnosis of HCL was 100% and 98%, respectively. We conclude that the presence of pERK as detected by immunohistochemical staining is a useful surrogate marker for BRAF V600E in the diagnosis of HCL. Key Words: hairy cell leukemia, chronic lymphocytic leukemia, BRAF mutation, phosphoprotein, phospho-ERK (Am J Surg Pathol 2013;37:305–308) B RAF V600E mutations have been identified in virtually all cases of hairy cell leukemia (HCL) but not in other small B-cell lymphoproliferative dis- orders (LPDs). 1 These findings have been corroborated by multiple studies 2–7 and show that detection of this mutation is a sensitive and specific test that will aid in the diagnosis of HCL. This mutation leads to constitutive activation/phosphorylation of the downstream kinases MEK and ERK. We hypothesized that immunostaining for phosphorylated ERK (pERK) in routinely processed bone marrow sections could be used as a surrogate marker for the BRAF V600E mutation and thus aid in the diagnosis of HCL. MATERIALS AND METHODS Case Selection This study was carried out with institutional review board approval. Bone marrow biopsies involved by HCL or one of the various small B-cell LPDs were retrieved from the Cleveland Clinic (Cleveland, OH) archives from 1999 through 2012. Components of each case including morphology, immunohistochemistry (IHC), flow cytometry, molecular and genetic testing, previous surgical pathology reports, and additional clinical and laboratory data were reviewed to confirm each diagnosis according to the 2008 World Health Organization classification system. 8 All cases of HCL displayed morphology consistent with hairy cells and had a classic immunophenotype diagnostic of HCL by flow cytometry (performed either on bone mar- row aspirate material or peripheral blood). Cases of “hairy cell variant” were identified by interstitial infiltrates of CD20-positive B cells that expressed CD11c, CD22, and CD103 with lack of CD25 by IHC analysis. Immunohistochemistry IHC was performed on HCL/ethylenediaminetetra- acetic acid–decalcified (Thermo Scientific decalcification solution 8340, Kalamazoo, MI), formalin-fixed, paraffin- embedded (FFPE) bone marrow trephine biopsies with an automated immunostainer (Discovery, Ventana Med- ical Systems, Tucson, AZ). We previously demonstrated the specificity of the pERK antibody for immunostaining in fixed tissues and that, in rapidly fixed tissues such as bone marrow biopsies, phosphoprotein levels reflect in vivo ex- pression. 9–11 Our sequential double-staining protocol used a rabbit monoclonal antibody to pERK1/2 THR202/Tyr214 (Cell Signaling Technology, Danvers, MA; clone D13.14.4E; 1:400) and mouse monoclonal antibody for CD20 (Dako, Carpinteria, CA; clone L26; 1:3200). Antigen retrieval was performed using a high pH tris-based buffer (Cell Conditioning 1; Ventana) for 30 minutes. Brown color (3,3 0 -diaminobenzidine, OmniMAP HRP; Ventana) in- dicated the presence of pERK, and red (UltraMAP AP; Ventana) indicated the presence of CD20. Staining was performed sequentially with pERK/3,3 0 -diaminobenzidine From the Department of Clinical Pathology, Cleveland Clinic, Cleveland, OH. Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Correspondence: Eric D. Hsi, MD, Department of Clinical Pathology, L-11, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195 (e-mail: hsie@ccf.org). Copyright r 2012 by Lippincott Williams & Wilkins ORIGINAL ARTICLE Am J Surg Pathol  Volume 37, Number 2, February 2013 www.ajsp.com | 305