Activating BRAF V600E Mutation in Aggressive Pediatric Langerhans Cell Histiocytosis Demonstration by Allele-specific PCR/Direct Sequencing and Immunohistochemistry Ga ´bor Me´hes, MD, PhD,* Ga ´bor Irsai, MD,* Judit Bedekovics, MD,* Lı´via Beke, MS,* Ferenc Fazakas, PhD,* Tı´mea Ro ´zsa, MD,w and Csongor Kiss, MD, PhDw Abstract: Langerhans cell histiocytosis (LCH) is a rare neo- plastic disease originating from cells characterized by antigen- presenting Langerhans cell phenotype. The clinical spectrum of LCH is highly variable including localized and disseminated forms mostly occurring in children. Recently, about 60% of LCHs were reported to carry the activating BRAF mutation V600E. In our retrospective study, we evaluated the occurrence and prognostic impact of the V600E mutation in formaldehyde- fixed, paraffin-embedded samples from 15 pediatric LCH cases treated at our institution. Allele-specific polymerase chain re- action (PCR) and direct sequencing were used to demonstrate the presence of V600E mutation, and immunohistochemistry (IHC) using the mutant protein–specific VE1 antibody clone was performed to confirm mutant BRAF protein expression. Eight of 15 (53.3%) cases proved to be BRAF mutants by any of the methods applied, with a single case showing a discrepancy (PCR negative/IHC positive). Four of the BRAF-mutant cases (50.0%) showed refractory disease and progressed to death within 43 months, whereas the remaining mutant cases were stable and responded well to therapy. Wild-type BRAF cases (7/ 15, 46.6%) with generally comparable initial presentation were all treated successfully. In conclusion, activating V600E BRAF mutation can be frequently demonstrated in pediatric LCH by both allele-specific PCR and IHC. Unfavorable risk cases po- tentially also responding to BRAF-inhibitory therapy can be identified by mutation testing using archival formaldehyde- fixed, paraffin-embedded tumor samples. Key Words: histiocytosis, molecular pathology, BRAF muta- tion, childhood cancer, prognosis, vemurafenib (Am J Surg Pathol 2014;38:1644–1648) L angerhans cell histiocytosis (LCH) is the neoplastic proliferation of histiocytes normally contributing to immune reactions of the skin and other mucus mem- branes. 1,2 The pathologic entity of LCH has been unified from 3 former clinical syndromes because of phenotypic similarities both at the histologic and at the clinical level. Eosinophilic granuloma is characterized by 1 or more lytic bone lesions, in which proliferating histiocytes are ac- companied by a prominent infiltrate of eosinophils. Hand- Schu ¨ller-Christian disease comprises the clinical triad of bone defects, exophthalmos, and polyuria because of the histiocytic infiltration of the pituitary stalk. Letterer-Siwe disease is a fulminant syndrome marked by hep- atosplenomegaly, lymphadenopathy, skin rashes, bone lesions, and hematological compromise. 3,4 Although these changes were regarded in part as inflammatory re- actions for decades, recent data provide evidence that pathologic Langerhans cells are clonal. 5,6 Despite uniform diagnostic criteria, the clinical course and the outcome of histiocytic proliferations are highly different. The incidence peaks in early childhood, but adolescents and adults are also affected in approx- imately one third of the cases. 1,7 Pediatric LCH is more frequently characterized by early dissemination and ag- gressive behavior. However, localized skin lesions, espe- cially in infants, can spontaneously regress. The diversity of clinical manifestations and the poor outcome of the recurrent disease call for new, clinically relevant biological features, which also enable the deci- sion of the most appropriate treatment. In addition to solid tumors, such as melanoma, 8,9 colorectal, 10,11 ovar- ian, 12 and thyroid cancer, 13,14 as well as hairy cell leuke- mia, 15,16 exon 15 V600 mutations of the serine/threonine kinase BRAF gene were recently found to be a frequent genetic aberration in parallel studies focusing on LCH. 17–19 According to these data primarily the BRAF V600E mutation may have a prognostic and predictive role in the treatment of LCH because of constant BRAF kinase activation. Previous studies focusing on BRAF From the *Department of Pathology; and wDepartment of Pediatric Hematology-Oncology, Institute of Pediatrics, University of Debrecen Clinical Centre, Debrecen, Hungary. Conflicts of Interest and Source of Funding: Supported by the project grants TA ´ MOP-4.2.2.A-11/1/KONV-2012-0045 (Vascular and car- diovascular research network) and TA ´ MOP-4.2.2.A-11/1/KONV- 2012-0025 of the European Union with cooperation with the Euro- pean Social Fund. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Correspondence: Ga´bor Me´hes, MD, PhD, Department of Pathology, University of Debrecen, Nagyerdei krt. 98., H-4032 Debrecen, Hungary (e-mail: gabor.mehes@med.unideb.hu). Copyright r 2014 by Lippincott Williams & Wilkins ORIGINAL ARTICLE 1644 | www.ajsp.com Am J Surg Pathol  Volume 38, Number 12, December 2014