RESEARCH ARTICLE MAP2K1 and MAP3K1 Mutations in Langerhans Cell Histiocytosis David S. Nelson, 1 Astrid van Halteren, 2 Willemijn T. Quispel, 2 Cor van den Bos, 3 Judith V.M.G. Bov ee, 4 Bhumi Patel, 1 Gayane Badalian-Very , 1,5 Paul van Hummelen, 6 Matthew Ducar , 6 Ling Lin, 6 Laura E. MacConaill, 6,7 R. Maarten Egeler , 8 and Barrett J. Rollins 1,5 * 1 Department of Medical Oncology, Dana-Farber Cancer Institute,Boston, MA 2 Immunology Laboratory,Willem Alexander Children’s Hospital, Leiden University Medical Center, Leiden, the Netherlands 3 Emma Children’s Hospital Academic Medical Center, Amsterdam, the Netherlands 4 Department of Pathology, Leiden University Medical Center, Leiden, the Netherlands 5 Department of Medicine,Brigham & Women’s Hospital and Harvard Medical School,Boston, MA 6 Center for Cancer Genome Discovery,Dana-Farber Cancer Institute,Boston, MA 7 Department of Pathology,Brigham & Women’s Hospital and Harvard Medical School,Boston, MA 8 Division of Hematology/Oncology, Hospital for Sick Children,Toronto,ON Langerhans cell histiocytosis (LCH) is now understood to be a neoplastic disease in which over 50% of cases have somatic acti- vating mutations of BRAF . However, the extracellular signal-related (ERK) pathway is activated in all cases including those with wild type BRAF alleles. Here, we applied a targeted massively parallel sequencing panel to 30 LCH samples to test for the pres- ence of additional genetic alterations that might cause ERK pathway activation. In 20 BRAF wild type samples, we found 3 somatic mutations in MAP2K1 (MEK1) including C121S and C121S/G128D in the kinase domain, and 56_61QKQKVG>R, an in-frame deletion in the N-terminal regulatory domain. All three variant proteins constitutively phosphorylated ERK in in vitro kinase assays. The C121S/G128D and 56_61QKQKVG>R variants were resistant to the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib in vitro. Within the entire sample set, we found 3 specimens with mutations in MAP3K1 (MEKK1), including two truncation mutants, T779fs and T1481fs; T1481fs encoded an unstable and nonfunctional protein when expressed in vitro. T779fs was present in a specimen carrying BRAF V600E. The third variant was a single nucleotide substitu- tion, E1286V, which was fully functional and is likely a germline polymorphism. These results indicate that LCH cells can harbor additional genetic alterations in the RAS-RAF-MEK pathway which, in the case of MAP2K1, may be responsible for ERK activa- tion in a wild type BRAF setting. The resistance of some of these variants to trametinib may also have clinical implications for the combined use of RAF and MEK inhibitors in LCH. V C 2015 Wiley Periodicals, Inc. INTRODUCTION Langerhans cell histiocytosis (LCH) is a rare disease characterized by the accumulation of cells that share phenotypic features with Langerhans cells (LCs), the primary antigen-presenting cells of skin and mucosal surfaces (Bechan et al., 2006; Egeler et al., 2010; Badalian-Very et al., 2013). It has a broad spectrum of clinical presentations but all include a prominent inflammatory component. The understanding of LCH pathobiology has evolved rapidly over the past 5 years. The discov- ery of activating somatic mutations of BRAF in over 50% of LCH cases has firmly classified this disease as a neoplastic disorder (Badalian-Very et al., 2010; Sahm et al., 2012; Satoh et al., 2012; Kansal et al., 2013; Berres et al., 2014), a categori- zation first implied by the clonality of LCH cells (Willman et al., 1994; Yu et al., 1994). Furthermore, analysis of gene expression patterns suggests that LCH cells are more closely related to myeloid dendritic cell precursors than to LCs (Allen et al., 2010). The demonstration of mutated BRAF alleles in CD341 bone marrow cells in some patients supports the characterization of LCH as a myeloid neoplasm albeit one with a strong inflammatory component (Berres et al., 2014). Additional Supporting Information may be found in the online version of this article. R. Maarten Egeler and Barrett J. Rollins contributed equally to this work. Supported by: Histiocytosis Association, Team Ippolittle/ Deloitte of the Boston Marathon Jimmy Fund Walk, Stichting 1000 Kaarsjes voor Juultje, the Say Yes to Education foundation, and Mr. George Weiss. *Correspondence to: Barrett J. Rollins, MD, PhD; Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215. E-mail: barrett_rollins@dfci.harvard.edu Received 29 August 2014; Accepted 22 January 2015 DOI 10.1002/gcc.22247 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). V V C 2015 Wiley Periodicals, Inc. GENES, CHROMOSOMES & CANCER 00:00–00 (2015)