BRAF--a new player in hematological neoplasms.

YBCMD-01791; No. of pages: 7; 4C: Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

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Review

BRAF — A new player in hematological neoplasms Marcin M. Machnicki, Tomasz Stoklosa ⁎ Department of Immunology, Medical University of Warsaw, Banacha 1A, 02-097 Warsaw, Poland

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Article history: Submitted 27 November 2013 Available online xxxx (Communicated by M. Lichtman, M.D., 31 December 2013) Keywords: BRAF kinase V600E mutation Leukemia Histiocytosis

a b s t r a c t BRAF oncogenic kinase has become a target for specific therapy in oncology. Genetic characterization of a predominant V600E mutation in melanoma, thyroid cancer, and other tumors became a focus for developing specific inhibitors, such as vemurafenib or dabrafenib. Our knowledge regarding the role of mutated BRAF in hematological malignancies has grown quickly as a result of new genetic techniques such as next-generation sequencing. This review summarizes current knowledge regarding the role of BRAF in lymphoid and myeloid neoplasms, with a focus on hairy-cell leukemia, Langerhans cell histiocytosis, and Erdheim–Chester disease. © 2014 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . BRAF — a protooncogenic kinase . . . . . . . . . . . . . . . . . . BRAF V600E — a predominant mutation and therapeutic target . . . . BRAF mutations in lymphoproliferative and myeloproliferative disorders BRAF mutations and multiple myeloma . . . . . . . . . . . . . . . BRAF mutations and hairy-cell leukemia . . . . . . . . . . . . . . BRAF mutations and histiocytosis . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction In 2002 Davies et al. [1] reported a new human oncogene BRAF, encoding a serine/threonine protein kinase involved in mitogenactivated protein kinase/extracellular signal-regulated kinase (MAPK/ ERK) pathway signaling. BRAF was found to be mutated in a variety of Abbreviations: MAPK/ERK, mitogen-activated protein kinase/extracellular signalregulated kinase; BRAFwt, wild-type BRAF; BRAFmut, mutated BRAF; PTC, papillary thyroid cancer; CRC, colorectal cancer; NSCLC, non-small-cell lung carcinoma; HCL, hairy-cell leukemia; CLL, chronic lymphocytic leukemia; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; DLBCL, diffuse large B-cell lymphoma; MM, multiple myeloma; PCL, plasma cell leukemia; IFN-α, interferon-α; PAs, purine nucleoside analogs; LCH, Langerhans-cell histiocytosis; ECD, Erdheim–Chester disease; CR, conserved region; KD, kinase domain. ⁎ Corresponding author at: Department of Immunology, Medical University of Warsaw, Banacha 1A, Bldg F, 02-097 Warsaw, Poland. E-mail address: [email protected] (T. Stoklosa).

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cancers and neoplasms and the newly discovered variants were oncogenic. Research that followed this initial discovery opened a new chapter in targeted cancer treatment with the introduction of BRAF kinase inhibitors into the clinic. However, despite a rapidly growing interest in BRAF over the last decade (Fig. 1), many questions are unanswered and, hence, the research in this field will continue to expand. This review summarizes the general findings on BRAF's role in cancer and focuses on hematological neoplasms that have been found to be associated with BRAF mutations. BRAF — a protooncogenic kinase BRAF is a 766 amino acid/95-kDa cytoplasmatic protein and belongs to the RAF-kinase family, together with its two paralogs, ARAF and CRAF [2]. The BRAF gene is located in the long arm of autosome 7 (7q34) and consists of 18 exons. Three conserved domains can be

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Please cite this article as: M.M. Machnicki, T. Stoklosa, BRAF — A new player in hematological neoplasms, Blood Cells Mol. Diseases (2014), http:// dx.doi.org/10.1016/j.bcmd.2014.01.001

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M.M. Machnicki, T. Stoklosa / Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx

1990 2002 cloning of BRAF detection of BRAF cDNA sequence mutations in human cancers

2011 BRAF mutations described in hairy-cell leukemia

2003 detection of BRAF mutations in papillary thyroid carcinoma

2010 BRAF mutations in langerhans cell histiocytosis

2011 FDA approval for vemurafenib

2004 functional characterization of frequent BRAF mutations

2010 results of phase I clinical trials of vemurafenib in treatment of melanoma

2012 detection of BRAF mutations in ErdheimChester disease

2008 BRAF fusions and mutations detected in brain tumors

2013 FDA approval for dabrafenib

Fig. 1. Short history of BRAF research.

found in the RAF family proteins, namely: conserved regions (CR) 1, 2 and 3. In BRAF, CR1 covers the cysteine-rich domain and most of the RAS binding domain, while CR3 is the kinase domain (KD). CR2 and CR3 contain several regulatory phosphorylation sites. Exons 11 and 15 which encode parts of KD are most frequently mutated in cancer [1,3]. In fact many members of the kinase superfamily share similar mutation spots in their KDs [4]. In normal cells, wild-type BRAF (BRAFwt) serves as a mitotic signal transponder in the ERK pathway. This conserved signaling pathway is activated after mitogen binding to its membrane ligand. The subsequent activation of a small GTPase RAS results in the RAS-dependent RAF activation. The signal is then passed through the phosphorylation cascade involving RAF, MEK and ERK kinases. Additionally, BRAF can signal to MEK through CRAF after formation of heterodimers and activation of CRAF (Fig. 2). Various cytoplasmic and nuclear substrates such as transcriptional factors are phosphorylated by ERK, subsequently influencing gene expression and making the ERK pathway an essential regulator of cell proliferation, differentiation, senescence or apoptosis [5,6]. It is thus not surprising, that the ERK pathway's hyperactivation occurs in

RTKs

HRAS

NRAS KRAS

ARAF

BRAF

CRAF

MEK1

MEK2

ERK1

ERK2

ERK targets in cytoplasm and nucleus Fig. 2. The mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. The receptor tyrosine kinase (RTK) binds a ligand and activates RAS, which then activates RAF–MEK–ERK phosphorylation cascade. ERK can phosphorylate proteins in cytosol and in nucleus.

substantial proportion of cancers, reflecting its important role in determination of cell fate [7]. Along with the initial discovery of BRAF mutations in cancer, Davies et al. observed elevated basal activity of various mutated BRAF (BRAFmut) proteins, as compared with BRAFwt, accompanied by the increase of ERK phosphorylation level. At the same time, BRAFmut showed high transforming potential in transfected NIH3T3 cells [1]. In general, BRAF mutations typically switch the kinase into constitutively active state which results in the RAS-independent, increased BRAFmut activity towards MEK. As a consequence, ERK pathway signaling is released from mitogen dependency and alters cell homeostasis. Among the BRAF mutants with impaired kinase activity, some still retain the ability to activate the ERK pathway in a CRAF-dependent manner [6]. Concurrently with mutated BRAF recognition as a new oncogene, it turned out that the constitutive BRAF activation is insufficient to drive oncogenesis without additional alterations. Pollock et al. found BRAF mutations to be present in more than half of melanomas, but they were even more prevalent in benign skin lesions such as nevi [8]. Moreover, experimental evidence for melanocytes and neural stem and progenitor cells proved that sole BRAFmut activity leads to the cancerprotective, oncogene-induced cell senescence [9,10] — a phenomenon that was previously observed in the context of the ERK pathway activation by oncogenic RAS [11]. Nevertheless, BRAF's oncogenic potential was confirmed by multiple diverse observations. BRAFmut cells are highly dependent on the effects of constitutive BRAF activation, as its direct suppression by RNA interference or inhibition of abnormal ERK pathway signaling with MEK inhibitors abrogates tumor growth [12–14]. BRAF mutation status can serve as a prognostic factor; according to recent meta-analyses BRAF mutation increases the risk of mortality in melanoma, colon cancer [15], and in papillary thyroid cancer (PTC), it correlates with high-risk clinicopathological factors (e.g. lymph node metastasis, advanced TNM stage) and poor outcome [16]. This is not a strict rule though, as in low-grade ovarian cancer presence of BRAF mutation correlates with improved outcome [17]. Furthermore, BRAF mutation status can predict drug response. Effectiveness of colorectal cancer (CRC) therapies based on anti-EGFR (an upstream receptor tyrosine kinase) antibodies depends on preserved wild-type status of BRAF, since in BRAFmut cancers the EGFR-related ERK pathway activation is bypassed [18]. Targeting the most common oncogenic BRAF variant (p.V600E) with its specific inhibitor vemurafenib improves the treatment of melanomas [19]. The majority of BRAF mutations identified by Davies et al. [1] were found in melanoma. However, this observation is no longer the case. BRAF is one of the most frequently mutated human kinases [4] (Table 1) and the occurrence of its variants is not limited to carcinomas — in fact, it is not limited to tumors [8].



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Table 1 BRAF mutations in human neoplasms. High frequency (over 33%) Hairy-cell leukemia Pilocytic astrocytoma Langerhans cell histiocytosis Erdheim–Chester disease Papillary thyroid carcinoma Malignant melanoma Low-grade ovarian cancer

Moderate frequency (10–33%) Up to 100% 72.7% 37.9–68.8% 51.4% 49.4% 47.8% 34.7%

Low frequency (under 10%)

Endometrial adenocarcinoma Colorectal cancer Hepatocellular carcinoma Cholangiocarcinoma

10.7% 9.6% 0–23.1% 2.1–21.7%

Adrenocortical carcinoma Diffuse large B-cell lymphoma Multiple myeloma Non-small-cell lung cancer Head and neck squamous cell carcinoma Chronic lymphocytic leukemia

5.7% 4.9% 2.8–4% 3.5% 3.4–7.8% 0.6–2.8%

Approximately half of cutaneous melanomas [15] and PTCs [16] harbor BRAF mutations. Other cancers with mutated BRAF include at least CRC [15], adrenocortical carcinoma [20], endometrial adenocarcinoma [21], low-grade ovarian cancer [17], head and neck squamous cell carcinoma [22,23] and non-small-cell lung carcinoma (NSCLC) [24]. The mutation prevalence in those diseases ranges from moderate to low. Few reports show high prevalence of BRAFmut in hepatocellular carcinomas and cholangiocarcinomas, yet the results remain contradictory [25,26]. BRAF does not seem to be involved in formation of leukemias and lymphomas with several exceptions, including hairy-cell leukemia (HCL) and its disease-defining BRAF p.V600E mutation [27,28]. This topic will be discussed later in this review. Not all of the diseases driven by mutated BRAF are progressive malignancies. For example, mutated BRAF plays a crucial role in pathogenesis of central nervous system tumors. Strikingly, mutations are found in up to three-fourths of cases, particularly in the benign, most common pediatric brain tumor, pilocytic astrocytoma [29], but frequencies also remain high for other tumor types [30]. Similarly, more than a half of the cases of two histiocytoses, Langerhans cell histiocytosis [31] and Erdheim–Chester disease [32,33] and more than two-thirds of nevi [8] harbor BRAF mutations. A rare but serious genetic disorder called cardio-facio-cutaneous syndrome arises on the basis of alterations of ERK pathway signaling and in most cases BRAF germline mutations were found to be the cause [34].

melanomas, both drugs showed remarkable activity in terms of response rate, but the survival benefits were limited by the rapid acquisition of resistance [19,41]. Various mechanisms can contribute to the reactivation of the ERK pathway (e.g. feedback activation of EGFR, aberrant splicing of BRAF or MEK1 mutations [42–44]), which is likely the major cause of resistance to BRAF inhibition, therefore new concepts are being tested, such as combined treatment with dabrafenib and MEK inhibitor trametinib, that delays the emergence of resistant clones and results in better outcomes [45]. While BRAF inhibitors are tested mainly in p.V600E melanoma treatment, many studies show that effective range of these drugs may be greater. For example, both vemurafenib and dabrafenib have been found to be effective against p.V600K melanomas [19,36,46] and their antitumor activity was also reported in patients with double p.V600E/ V600M mutation or p.V600R mutation [35,47], proving that even the infrequent BRAF variants can become therapeutic targets. Importantly, BRAF inhibitors may be effective in treatment of cancers other than melanoma. Responses were achieved in phase I trials of vemurafenib in treatment of metastatic CRC [48] and PTC [49] and some exceptional results were reported for hairy-cell leukemia and other hematological neoplasms, which will be discussed further.

BRAF V600E — a predominant mutation and therapeutic target

BRAF functions as an oncogene in lymphoid and myeloid neoplasms, but in most of them the occurrence of BRAF mutations is occasional and possibly restricted to some specific subtypes. These observations are now being confirmed by numerous studies, including those exploiting next-generation sequencing, which rules out the possibility of biased screening (mainly when searching for p.V600E only). In common chronic and acute leukemias, BRAF mutations are rare. Langabeer et al. [50] detected mutations in 1/151 chronic lymphocytic leukemias (CLL) and 1/4 B-cell prolymphocytic leukemias and Jebaraj et al. [51] 2.8% in CLL (4/138) and 0/32 B-cell prolymphocytic leukemias. In a large kinome study, Zhang et al. [52] analyzed BRAF coding sequences in 143 CLL cases and BRAF exons 11 and 15 in 130 CLL cases and found only 4 mutations. Finally, among 105 CLL exomes sequenced by Quesada et al. [53], 2 (1.9%) mutations were detected. BRAF mutations are also rare in acute myeloid (AML) or lymphoblastic leukemias

The mutational spectrum of BRAF appears to be very broad, yet taking the actual occurrence of identified mutations into consideration restricts this diversity dramatically. As stated above, mutations are generally found in exons 11 and 15 [1,3] but it is the valine codon in position 600 located in exon 15 that serves as a main hotspot. According to the study by Wan et al. [6] four out of seven most highly activating BRAF mutations involve substitution of p.V600 and only one mutation is located outside of exon 15. The c.T1799A substitution leading to p.V600E change is predominantly detected in most of the cases [1,18,27]. While at first this limited diversity seemed convenient for mutational screening and diagnostics, many studies showed that rare mutations other than c.T1799A remain relevant. In melanomas, activating [6] dinucleotide codon 600 mutations – including p.V600K, p.V600R and p.V600D – can be found in total in up to more than 25% of the cases [35,36] and in pilocytic astrocytoma a whole new group of mutations has been discovered, briefly various gene fusions between BRAF and KIA1499, arising from tandem duplications at 7q34 [29,37]. The most frequent, well-defined BRAF point mutations in cancers, gathered in the COSMIC database [38], are shown in Table 2. Establishment of BRAF mutation status as a biomarker in cancers like melanoma or PTC is an undoubted success; however, the real breakthrough came with the introduction of BRAF inhibitors into clinical practice. Two drugs thoroughly tested in various clinical trials and approved by the Food and Drug Administration are vemurafenib [39] and dabrafenib [40], both being highly specific for BRAF kinase and especially for its p.V600E variant. In phase III trials comparing vemurafenib/dabrafenib and dacarbazine in treatment of p.V600E

BRAF mutations in lymphoproliferative and myeloproliferative disorders

Table 2 Ten most frequent, well defined BRAF point mutations in the COSMIC database (COSMIC v66 Release) [38]. Residue

Coding sequence mutation

Amino acid mutation

Count

600 600 600 601 600 594 469 600 466 469

c.1799TNA c.1798_1799GTNAA c.1798_1799GTNAG c.1801ANG c.1799_1800TGNAA c.1781ANG c.1406GNC c.1798GNA c.1397GNT c.1406GNT

p.V600E p.V600K p.V600R p.K601E p.V600E p.D594G p.G469A p.V600M p.G466V p.G469V

19,076 432 68 63 51 43 30 25 19 18


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(ALL). While Lee et al. [54] found mutations in 9.1% (4/44) ALLs and AMLs and Gustafsson et al. [55] in 20.7% (6/29) ALLs, several screenings among large cohorts of patients with those diseases revealed no BRAF mutations at all [56–59]. There is also no evidence of any mutations in chronic myeloid leukemia and many other myeloid neoplasms [59]. Despite their rarity, those BRAFmut leukemias might still be considered as potential targets for ERK pathway inhibitors. For instance, in ALL the ERK pathway can be activated due to RAS mutations and such tumors are sensitive to MEK inhibition [58]. It is therefore possible that such inhibition would take place also in BRAFmut leukemic cells and the obvious effectiveness of vemurafenib in other hematological neoplasms with BRAF mutations (as discussed further) supports this idea. It must be noted though that in AML, ALL or CLL the frequency of p.V600E substitution among other alterations is generally lower than typically observed. While other mutations can still activate the ERK pathway [6], vemurafenib and dabrafenib are most active against p.V600E form. Only several BRAF mutations were detected in lymphomas, particularly in diffuse large B-cell lymphomas (DLBCL) [60,61]. In total, 6 mutations were found among 122 cases (4.9%) and these included highly activating [6] p.G468A substitutions but not p.V600E. However, Aggarwal et al. [62] screened 33 thyroid lymphomas and found 6 of them harboring BRAF mutations (including one p.V600E), all of which were DLBCL (6 of 25, 24%). NRAS mutations were also detected in another two cases of DLBCL. BRAF/RAS mutations are very common in papillary/follicular thyroid carcinomas, but in study by Aggarwal and colleagues none of the patients had coexistent carcinoma. The reason for the higher frequency of BRAF mutations in thyroid DLBCL is unknown and similarly to BRAFmut leukemias, the effectiveness of BRAF inhibition in DLBCL has to be further assessed. BRAF mutations and multiple myeloma After the discovery of BRAF mutations in melanoma, some attempts were made to determine BRAF mutation status in plasma cell neoplasms, including multiple myeloma (MM) and plasma cell leukemia (PCL), but three studies conducted at that time [63–65] failed to detect any BRAF alterations, except for the detection of p.V600E in the U266 myeloma cell line [64]. In contrary to those findings, Chapman et al. found one p.G469A, three p.K601N and four p.V600E mutations among 199 MMs [66], Mosca et al. identified a p.V600E mutation in 1/15 (6.7%) PCLs [67] and Boyd et al. [68] — a p.D594N mutation in 1/39 MMs (2.6%). Of clinical importance, Andrulis et al. [69] searched for p.V600E protein among a group of 379 patients with plasma cell disorders and found 2.8% of MMs expressing this variant (7/251), also reporting a patient with refractory, BRAF p.V600E MM, rapidly responding to vemurafenib. BRAF mutations and hairy-cell leukemia Mutations of the BRAF gene are commonly associated with a specific subtype of a particular cancer e.g. from all thyroid cancers only in PTCs BRAF is mutated [23,70]. Probably the most spectacular example of this selectivity is HCL, an uncommon B-cell neoplasm. While it was shown that BRAF mutations are virtually absent [27,28,68] or at most very rare in lymphoid neoplasms [50,57,58,60], in HCL the p.V600E substitution was found to be a disease-defining genetic abnormality, present in every case which was examined [27,28,68,71]. Moreover, the presence of phospho-ERK, the marker of ERK pathway activation, has been confirmed by immunohistochemistry [72]. HCL is characterized by hepatosplenomegaly, pancytopenia (including monocytopenia), specific immunophenotype and large B-cells that have “hairy” cytoplasmatic projections, notable in “wet preparations” under phase microscopy or using electron microscopy. Infiltration of marrow (accompanied by fibrosis) and spleen results in immunosuppression, frequently leading to death, despite the indolent disease course. However, HCL became easily manageable three decades ago, initially thanks to the

introduction of interferon-α (IFN-α) and purine nucleoside analogs (PAs), pentostatin and cladribine, afterwards. Treatment with those drugs induces complete and long-lasting remissions in majority of patients [73–75]. Discovery of p.V600E mutations in HCL had a major impact on diagnosis and treatment of this disease. So far, HCL has been differentially diagnosed from other B-cell malignancies by blood smear and marrow examinations, immunohistochemistry and flow cytometry [73]. In contrast to HCL, neoplasms that mimic HCL such as splenic B-cell marginal zone lymphoma or unclassifiable splenic B-cell lymphoma/leukemia (including HCL-variant) do not harbor BRAF mutations [27,68,76], do not express BRAF p.V600E protein [77] and do not present phosphoERK as assessed by immunohistochemistry [72]. Therefore, routine screenings using these markers could significantly improve HCL diagnostics. This discrimination is very important as “HCL-like” malignancies do not respond to IFN-α or PAs [74]. The actual p.V600E frequency in HCLs may be a bit lower than anticipated. Xi et al. found that classic HCLs expressing IgHV4-34 immunoglobulin rearrangement (which have a poor prognosis, similarly to HCL-variant), are BRAFwt, while Schnittger et al. identified additional BRAFwt cases which were negative for IGHV4-34 [76,78]. Such discrepancies emphasize the need for additional studies that would identify the subset of BRAFwt HCLs. HCL treatment with IFN-α and PAs may be very effective, but it is not curative and in many cases minimal residual disease remains. Patients who experience relapse can be treated with the same drugs, but after each line of treatment complete response rates drop, the duration of response shortens and some patients do not respond to the therapy from the beginning. Additionally, treatment with purine analogs may cause serious complications due to myelo- and immunosuppression [74,75,79]. While it was previously demonstrated that these problems can be overcome at least partially by using anti-CD20 monoclonal antibody rituximab [75], an obvious therapeutic possibility of using BRAF inhibitors emerged after p.V600E mutations were discovered in HCL. Indeed, first reports of poorly treatable HCL cases responding extremely well to vemurafenib [80–82] pave the way for the clinical trials. Overall, the identification of p.V600E mutation as a major genetic change in HCL may become a milestone in management of this disease, resembling to some degree the onset of BCR-ABL targeted treatment of chronic myeloid leukemia with imatinib. An interesting discovery was also made in the field of HCL thanks to the BRAF story. Given the uniform distribution of BRAF mutations, two groups checked whether cell lines that served as HCL models carry the p.V600E mutation [83,84]. Strikingly, none of them did, raising questions as to their usefulness for the study of HCL. BRAF mutations and histiocytosis Concurrently with the findings in HCL, two rare histiocytoses were found to be commonly driven by mutated BRAF: Langerhans-cell histiocytosis (LCH) and Erdheim–Chester disease (ECD). Similarly as in HCL, targeting BRAF may become an excellent alternative for the current therapies. LCH is a proliferation of histiocytes that have a phenotype similar to Langerhans cells and can be diagnosed primarily on the basis of CD1a expression. Other markers that can support identification are the presence of Birbeck granules and the expression of langerin (CD207) and S-100 protein [85–87]. Patients having a single-system LCH and lesions located in bones or skin, often have good outcomes, including spontaneously resolving cases. However, other sites including lungs or central nervous system may be affected, causing life-threatening, key organ failures. Likewise, if LCH progresses to a multi-system form, excellent outcomes are observed only when risk organs are uninvolved. Due to the rarity and heterogeneity of LCH, treatment strategies are diverse and their effectiveness is variable, with a notable exception of cladribine which seems to induce complete response in more than half of the refractory LCHs [85,87].


M.M. Machnicki, T. Stoklosa / Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx Table 3 Prevalence of BRAF mutations in Langerhans cell histiocytosis and Erdheim–Chester. BRAF mutation frequency

Paper

Langerhans cell histiocytosis 37.9% (11/29) 40% (2/5) 41.3% (19/46) 57.4% (35/61) 68.8% (11/16)

Haroche et al. [33] Yousem et al. [100] Sahm et al. [94] Badalian-Very et al. [31] Satoh et al. [96]

Erdheim–Chester disease 51.4% (19/37)

Haroche et al. [33] Update in: Emile et al. [32]

The second orphan disease, ECD, is characterized by the infiltration of single or multiple tissues and organs by foamy CD68+ CD1a− S100− histiocytes. Long bones are predominantly infiltrated and in such cases the disease course can be indolent. However, systemic forms with lesions localizing in other sites, including cardiovascular and central nervous systems, lungs or kidneys, are often fatal. The prognosis in ECD is worse than in LCH and more than half of the patients died of it [88,89] until the introduction of IFN-α, which was the first agent to improve survival in ECD [90]. Until recently, the etiology of both diseases was largely unknown, particularly it was not fully clear whether LCH and ECD are reactive inflammatory diseases or clonal neoplasias [88,91]. The current evidence strongly supports the latter possibility — in both diseases clonality was demonstrated long ago [92,93] and according to recent papers (Table 3) BRAF mutations are present in about half of the cases. Interestingly, the ERK pathway can be hyperactivated also in BRAFwt LCHs, thus one would expect that other gene alterations may exist in LCH or ECD. However, little additional genetic information has been reported so far [31]. The history of BRAF in melanoma seems to repeat itself in the case of LCH. The mutation rate is high and the p.V600E substitution is predominant. Regardless of the small number of LCH cases analyzed, several rare mutations were detected: p.V600K [94], p.V600D [95] and p.V600DLATV (insertion of 4 amino acids, functionally similar to p.V600D) [96] — all of which elevate BRAF kinase activity [6,96]. Additionally, as suggested by Badalian-Very et al. [86], self-healing LCH cases that carry BRAFmut may represent the same phenomenon of oncogene-induced senescence as observed in BRAFmut nevi [9] and the aggressive LCH forms may be driven by additional alterations. The finding of congenital, benign LCH harboring p.V600D mutation [95] further supports this concept. There is also growing evidence that the similar BRAF mutation rate in LCH and ECD is non-accidental. BRAF mutations are restricted to LCH and ECD among all histiocytoses [33] and there are some rare cases of those diseases coexisting in individual patients [97]. It was also shown that LCH cells of various maturation express BRAF p.V600E [94]. Finally, in a recent case report one of the patients was found to have coexisting ECD and LCH, both harboring p.V600E mutation [98]. The possibility that both types of histiocytes share a common progenitor cell which frequently acquires BRAF mutation is therefore likely. The clinical consequences of BRAF activation in LCH and ECD are yet unknown — previous studies failed to find any correlations with patient characteristics [31,33,94,96]. Nevertheless, excellent responses to vemurafenib have been observed in three patients with refractory, BRAF p.V600E ECD. Since one of the patients had p.V600E LCH skin lesions which rapidly disappeared, it is probable that vemurafenib will be also effective in LCH [98]. Conclusions As a result of encouraging clinical responses achieved with vemurafenib in HCL, MM or histiocytic disorders, it is important to

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identify those cases or subsets that carry BRAF mutations. As the prevalence of p.V600E substitution among other mutations is generally lower than in diseases with high BRAFmut frequency, it is necessary to widen the mutational screenings and investigate the functional consequences of other mutations. The clinically available BRAF inhibitors are primarily effective against BRAF p.V600E and in different cellular contexts they may even enhance proliferation [19,99], so their effectiveness against rare variants or the effectiveness of ERK pathway inhibition downstream of RAF should be closely examined. After the FDA approval for trametinib, MEK inhibitors emerge as a supplement or alternative for BRAF inhibitors. BRAF was considered as a therapeutic target primarily in melanoma. The latest experimental and clinical evidence suggests that inhibition of this kinase may become a new form of treatment of other cancers, at least for those cases which are otherwise refractory, which was clearly demonstrated for HCL and histiocytoses. These examples also prove that targeted therapies can be tailored even for rare cases and orphan diseases, which seems to be a requirement of tumor heterogeneity, and an expectation in the context of a high-throughput approach in genetics. Conflict of interest The authors declare no conflict of interest. Acknowledgments MMM was supported by the Postgraduate School of Molecular Medicine. TS was supported by EU program: FP7-REGPOT-2012CT2012-316254-BASTION. We also thank Eliza Glodkowska-Mrowka and Magdalena Machnicka for critical reading of the manuscript. References [1] H. Davies, G.R. Bignell, C. Cox, et al., Mutations of the BRAF gene in human cancer, Nature 417 (2002) 949–954. [2] R.M. Stephens, G. Sithanandam, T.D. Copeland, et al., 95-kilodalton B-Raf serine/threonine kinase: identification of the protein and its major autophosphorylation site, Mol. Cell. Biol. 12 (1992) 3733–3742. [3] E. Domingo, S.J. Schwartz, BRAF (v-raf murine sarcoma viral oncogene homolog B1), Atlas Genet Cytogenet Oncol Haematol2004. [4] C. Greenman, P. Stephens, R. Smith, et al., Patterns of somatic mutation in human cancer genomes, Nature 446 (2007) 153–158. [5] M.J. Garnett, R. Marais, Guilty as charged: B-RAF is a human oncogene, Cancer Cell 6 (2004) 313–319. [6] P.T. Wan, M.J. Garnett, S.M. Roe, et al., Mechanism of activation of the RAF–ERK signaling pathway by oncogenic mutations of B-RAF, Cell 116 (2004) 855–867. [7] R. Hoshino, Y. Chatani, T. Yamori, et al., Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signaling pathway in human tumors, Oncogene 18 (1999) 813–822. [8] P.M. Pollock, U.L. Harper, K.S. Hansen, et al., High frequency of BRAF mutations in nevi, Nat. Genet. 33 (2003) 19–20. [9] C. Michaloglou, L.C. Vredeveld, M.S. Soengas, et al., BRAFE600-associated senescence-like cell cycle arrest of human naevi, Nature 436 (2005) 720–724. [10] E.H. Raabe, K.S. Lim, J.M. Kim, et al., BRAF activation induces transformation and then senescence in human neural stem cells: a pilocytic astrocytoma model, Clin. Cancer Res. 17 (2011) 3590–3599. [11] M. Serrano, A.W. Lin, M.E. McCurrach, D. Beach, S.W. Lowe, Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a, Cell 88 (1997) 593–602. [12] S.R. Hingorani, M.A. Jacobetz, G.P. Robertson, M. Herlyn, D.A. Tuveson, Suppression of BRAF(V599E) in human melanoma abrogates transformation, Cancer Res. 63 (2003) 5198–5202. [13] D.B. Solit, L.A. Garraway, C.A. Pratilas, et al., BRAF mutation predicts sensitivity to MEK inhibition, Nature 439 (2006) 358–362. [14] N. Nakayama, K. Nakayama, S. Yeasmin, et al., KRAS or BRAF mutation status is a useful predictor of sensitivity to MEK inhibition in ovarian cancer, Br. J. Cancer 99 (2008) 2020–2028. [15] G. Safaee Ardekani, S.M. Jafarnejad, L. Tan, A. Saeedi, G. Li, The prognostic value of BRAF mutation in colorectal cancer and melanoma: a systematic review and meta-analysis, PLoS One 7 (2012) e47054. [16] T.H. Kim, Y.J. Park, J.A. Lim, et al., The association of the BRAF(V600E) mutation with prognostic factors and poor clinical outcome in papillary thyroid cancer: a meta-analysis, Cancer 118 (2012) 1764–1773.


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[17] R.N. Grisham, G. Iyer, K. Garg, et al., BRAF mutation is associated with early stage disease and improved outcome in patients with low-grade serous ovarian cancer, Cancer 119 (2013) 548–554. [18] F. Di Nicolantonio, M. Martini, F. Molinari, et al., Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer, J. Clin. Oncol. 26 (2008) 5705–5712. [19] P.B. Chapman, A. Hauschild, C. Robert, et al., Improved survival with vemurafenib in melanoma with BRAF V600E mutation, N. Engl. J. Med. 364 (2011) 2507–2516. [20] V. Kotoula, E. Sozopoulos, H. Litsiou, et al., Mutational analysis of the BRAF, RAS and EGFR genes in human adrenocortical carcinomas, Endocr. Relat. Cancer 16 (2009) 565–572. [21] M. He, V. Breese, S. Hang, et al., BRAF V600E mutations in endometrial adenocarcinoma, Diagn. Mol. Pathol. 22 (2013) 35–40. [22] A. Weber, L. Langhanki, F. Sommerer, et al., Mutations of the BRAF gene in squamous cell carcinoma of the head and neck, Oncogene 22 (2003) 4757–4759. [23] Y. Cohen, M. Xing, E. Mambo, et al., BRAF mutation in papillary thyroid carcinoma, J. Natl. Cancer Inst. 95 (2003) 625–627. [24] A. Marchetti, L. Felicioni, S. Malatesta, et al., Clinical features and outcome of patients with non-small-cell lung cancer harboring BRAF mutations, J. Clin. Oncol. 29 (2011) 3574–3579. [25] M. Colombino, P. Sperlongano, F. Izzo, et al., BRAF and PIK3CA genes are somatically mutated in hepatocellular carcinoma among patients from South Italy, Cell Death Dis. 3 (2012) e259. [26] A. Tannapfel, F. Sommerer, M. Benicke, et al., Mutations of the BRAF gene in cholangiocarcinoma but not in hepatocellular carcinoma, Gut 52 (2003) 706–712. [27] E. Tiacci, V. Trifonov, G. Schiavoni, et al., BRAF mutations in hairy-cell leukemia, N. Engl. J. Med. 364 (2011) 2305–2315. [28] L. Arcaini, S. Zibellini, E. Boveri, et al., The BRAF V600E mutation in hairy cell leukemia and other mature B-cell neoplasms, Blood 119 (2012) 188–191. [29] D.T. Jones, S. Kocialkowski, L. Liu, et al., Oncogenic RAF1 rearrangement and a novel BRAF mutation as alternatives to KIAA1549:BRAF fusion in activating the MAPK pathway in pilocytic astrocytoma, Oncogene 28 (2009) 2119–2123. [30] G. Schindler, D. Capper, J. Meyer, et al., Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma, ganglioglioma and extra-cerebellar pilocytic astrocytoma, Acta Neuropathol. 121 (2011) 397–405. [31] G. Badalian-Very, J.A. Vergilio, B.A. Degar, et al., Recurrent BRAF mutations in Langerhans cell histiocytosis, Blood 116 (2010) 1919–1923. [32] J.F. Emile, F. Charlotte, Z. Amoura, J. Haroche, BRAF mutations in Erdheim–Chester disease, J. Clin. Oncol. 31 (2013) 398. [33] J. Haroche, F. Charlotte, L. Arnaud, et al., High prevalence of BRAF V600E mutations in Erdheim–Chester disease but not in other non-Langerhans cell histiocytoses, Blood 120 (2012) 2700–2703. [34] P. Rodriguez-Viciana, O. Tetsu, W.E. Tidyman, et al., Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome, Science 311 (2006) 1287–1290. [35] O. Klein, A. Clements, A.M. Menzies, et al., BRAF inhibitor activity in V600R metastatic melanoma, Eur. J. Cancer 49 (2013) 1073–1079. [36] J.C. Rubinstein, M. Sznol, A.C. Pavlick, et al., Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032, J. Transl. Med. 8 (2010) 67. [37] S. Dahiya, J. Yu, A. Kaul, J.R. Leonard, D.H. Gutmann, Novel BRAF alteration in a sporadic pilocytic astrocytoma, Case Rep. Med. 2012 (2012) 418672. [38] S.A. Forbes, N. Bindal, S. Bamford, et al., COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer, Nucleic Acids Res. 39 (2011) D945–D950. [39] G. Bollag, P. Hirth, J. Tsai, et al., Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma, Nature 467 (2010) 596–599. [40] S. Laquerre, M. Arnone, K. Moss, et al., Abstract B88: a selective Raf kinase inhibitor induces cell death and tumor regression of human cancer cell lines encoding B-Raf V600E mutation, Mol. Cancer Ther. 8 (12 Suppl) (2009) B88. [41] A. Hauschild, J.J. Grob, L.V. Demidov, et al., Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial, Lancet 380 (2012) 358–365. [42] A. Prahallad, C. Sun, S. Huang, et al., Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR, Nature 483 (2012) 100–103. [43] P.I. Poulikakos, Y. Persaud, M. Janakiraman, et al., RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E), Nature 480 (2011) 387–390. [44] C.M. Emery, K.G. Vijayendran, M.C. Zipser, et al., MEK1 mutations confer resistance to MEK and B-RAF inhibition, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 20411–20416. [45] K.T. Flaherty, J.R. Infante, A. Daud, et al., Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations, N. Engl. J. Med. 367 (2012) 1694–1703. [46] G.V. Long, U. Trefzer, M.A. Davies, et al., Dabrafenib in patients with Val600Glu or Val600Lys BRAF-mutant melanoma metastatic to the brain (BREAK-MB): a multicentre, open-label, phase 2 trial, Lancet Oncol. 13 (2012) 1087–1095. [47] G. Ponti, G. Pellacani, A. Tomasi, et al., The somatic affairs of BRAF: tailored therapies for advanced malignant melanoma and orphan non-V600E (V600R-M) mutations, J. Clin. Pathol. 66 (2013) 441–445. [48] S. Kopetz, J. Desai, E. Chan, et al., PLX4032 in metastatic colorectal cancer patients with mutant BRAF tumors, J. Clin. Oncol. 28 (2010)(15s suppl; abstr 3534). [49] K. Kim, M. Cabanillas, A.J. Lazar, et al., Clinical responses to vemurafenib in patients with metastatic papillary thyroid cancer harboring V600EBRAF mutation, Thyroid 23 (2013) 1277–1283.

[50] S.E. Langabeer, F. Quinn, D. O'Brien, et al., Incidence of the BRAF V600E mutation in chronic lymphocytic leukaemia and prolymphocytic leukaemia, Leuk. Res. 36 (2012) 483–484. [51] B.M. Jebaraj, D. Kienle, A. Buhler, et al., BRAF mutations in chronic lymphocytic leukemia, Leuk. Lymphoma 54 (2013) 1177–1182. [52] X. Zhang, M. Reis, R. Khoriaty, et al., Sequence analysis of 515 kinase genes in chronic lymphocytic leukemia, Leukemia 25 (2011) 1908–1910. [53] V. Quesada, L. Conde, N. Villamor, et al., Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia, Nat. Genet. 44 (2012) 47–52. [54] J.W. Lee, Y.H. Soung, W.S. Park, et al., BRAF mutations in acute leukemias, Leukemia 18 (2004) 170–172. [55] B. Gustafsson, S. Angelini, B. Sander, et al., Mutations in the BRAF and N-ras genes in childhood acute lymphoblastic leukaemia, Leukemia 19 (2005) 310–312. [56] M.L. Smith, J. Snaddon, M. Neat, et al., Mutation of BRAF is uncommon in AML FAB type M1 and M2, Leukemia 17 (2003) 274–275. [57] J. Davidsson, H. Lilljebjorn, I. Panagopoulos, T. Fioretos, B. Johansson, BRAF mutations are very rare in B- and T-cell pediatric acute lymphoblastic leukemias, Leukemia 22 (2008) 1619–1621. [58] M. Case, E. Matheson, L. Minto, et al., Mutation of genes affecting the RAS pathway is common in childhood acute lymphoblastic leukemia, Cancer Res. 68 (2008) 6803–6809. [59] A.P. Trifa, R.A. Popp, A. Cucuianu, et al., Absence of BRAF V600E mutation in a cohort of 402 patients with various chronic and acute myeloid neoplasms, Leuk. Lymphoma 53 (2012) 2496–2497. [60] J.W. Lee, N.J. Yoo, Y.H. Soung, et al., BRAF mutations in non-Hodgkin's lymphoma, Br. J. Cancer 89 (2003) 1958–1960. [61] J.G. Lohr, P. Stojanov, M.S. Lawrence, et al., Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 3879–3884. [62] N. Aggarwal, S.H. Swerdlow, L.M. Kelly, et al., Thyroid carcinoma-associated genetic mutations also occur in thyroid lymphomas, Mod. Pathol. 25 (2012) 1203–1211. [63] L. Bonello, C. Voena, M. Ladetto, et al., BRAF gene is not mutated in plasma cell leukemia and multiple myeloma, Leukemia 17 (2003) 2238–2240. [64] M.H. Ng, K.M. Lau, W.S. Wong, et al., Alterations of RAS signalling in Chinese multiple myeloma patients: absent BRAF and rare RAS mutations, but frequent inactivation of RASSF1A by transcriptional silencing or expression of a non-functional variant transcript, Br. J. Haematol. 123 (2003) 637–645. [65] M. Velangi, E. Matheson, P. Taylor, et al., BRAF gene is not mutated in mismatch repair-proficient or -deficient plasma cell dyscrasias, Leukemia 18 (2004) 658–659. [66] M.A. Chapman, M.S. Lawrence, J.J. Keats, et al., Initial genome sequencing and analysis of multiple myeloma, Nature 471 (2011) 467–472. [67] L. Mosca, P. Musto, K. Todoerti, et al., Genome-wide analysis of primary plasma cell leukemia identifies recurrent imbalances associated with changes in transcriptional profiles, Am. J. Hematol. 88 (2013) 16–23. [68] E.M. Boyd, A.J. Bench, M.B. van 't Veer, et al., High resolution melting analysis for detection of BRAF exon 15 mutations in hairy cell leukaemia and other lymphoid malignancies, Br. J. Haematol. 155 (2011) 609–612. [69] M. Andrulis, N. Lehners, D. Capper, et al., Targeting the BRAF V600E mutation in multiple myeloma, Cancer Discov. 3 (2013) 862–869. [70] T. Fukushima, S. Suzuki, M. Mashiko, et al., BRAF mutations in papillary carcinomas of the thyroid, Oncogene 22 (2003) 6455–6457. [71] P.A. Blombery, S.Q. Wong, C.A. Hewitt, et al., Detection of BRAF mutations in patients with hairy cell leukemia and related lymphoproliferative disorders, Haematologica 97 (2012) 780–783. [72] E. Tiacci, G. Schiavoni, M.P. Martelli, et al., Constant activation of the RAF–MEK– ERK pathway as a diagnostic and therapeutic target in hairy cell leukemia, Haematologica 98 (2013) 635–639. [73] G. Jones, N. Parry-Jones, B. Wilkins, M. Else, D. Catovsky, Revised guidelines for the diagnosis and management of hairy cell leukaemia and hairy cell leukaemia variant*, Br. J. Haematol. 156 (2012) 186–195. [74] M.R. Grever, How I treat hairy cell leukemia, Blood 115 (2010) 21–28. [75] F. Forconi, Hairy cell leukaemia: biological and clinical overview from immunogenetic insights, Hematol. Oncol. 29 (2011) 55–66. [76] L. Xi, E. Arons, W. Navarro, et al., Both variant and IGHV4-34-expressing hairy cell leukemia lack the BRAF V600E mutation, Blood 119 (2012) 3330–3332. [77] M. Andrulis, R. Penzel, W. Weichert, A. von Deimling, D. Capper, Application of a BRAF V600E mutation-specific antibody for the diagnosis of hairy cell leukemia, Am. J. Surg. Pathol. 36 (2012) 1796–1800. [78] S. Schnittger, U. Bacher, T. Haferlach, et al., Development and validation of a real-time quantification assay to detect and monitor BRAFV600E mutations in hairy cell leukemia, Blood 119 (2012) 3151–3154. [79] M. Else, C.E. Dearden, E. Matutes, et al., Long-term follow-up of 233 patients with hairy cell leukaemia, treated initially with pentostatin or cladribine, at a median of 16 years from diagnosis, Br. J. Haematol. 145 (2009) 733–740. [80] S. Dietrich, H. Glimm, M. Andrulis, et al., BRAF inhibition in refractory hairy-cell leukemia, N. Engl. J. Med. 366 (2012) 2038–2040. [81] G.A. Follows, H. Sims, D.M. Bloxham, et al., Rapid response of biallelic BRAF V600E mutated hairy cell leukaemia to low dose vemurafenib, Br. J. Haematol. 161 (2013) 150–153. [82] F. Peyrade, D. Re, C. Ginet, et al., Low-dose vemurafenib induces complete remission in a case of hairy-cell leukemia with a V600E mutation, Haematologica 98 (2013) e20–e22.


M.M. Machnicki, T. Stoklosa / Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx [83] E. Tiacci, A. Pucciarini, B. Bigerna, et al., Absence of BRAF-V600E in the human cell lines BONNA-12, ESKOL, HAIR-M, and HC-1 questions their origin from hairy cell leukemia, Blood 119 (2012) 5332–5333. [84] N.J. Weston-Bell, D. Hendriks, G. Sugiyarto, et al., Hairy cell leukemia cell lines expressing annexin A1 and displaying B-cell receptor signals characteristic of primary tumor cells lack the signature BRAF mutation to reveal unrepresentative origins, Leukemia 27 (2013) 241–245. [85] O. Abla, R.M. Egeler, S. Weitzman, Langerhans cell histiocytosis: current concepts and treatments, Cancer Treat. Rev. 36 (2010) 354–359. [86] G. Badalian-Very, J.A. Vergilio, B.A. Degar, C. Rodriguez-Galindo, B.J. Rollins, Recent advances in the understanding of Langerhans cell histiocytosis, Br. J. Haematol. 156 (2012) 163–172. [87] B. Ng-Cheng-Hin, C. O'Hanlon-Brown, C. Alifrangis, J. Waxman, Langerhans cell histiocytosis: old disease new treatment, QJM 104 (2011) 89–96. [88] R.D. Mazor, A. Kesler, Y. Shoenfeld, Erdheim–Chester disease: an orphan condition seeking treatment, Isr. Med. Assoc. J. 14 (2012) 388–389. [89] C. Veyssier-Belot, P. Cacoub, D. Caparros-Lefebvre, et al., Erdheim–Chester disease. Clinical and radiologic characteristics of 59 cases, Medicine (Baltimore) 75 (1996) 157–169. [90] L. Arnaud, B. Hervier, A. Neel, et al., CNS involvement and treatment with interferon-alpha are independent prognostic factors in Erdheim–Chester disease: a multicenter survival analysis of 53 patients, Blood 117 (2011) 2778–2782. [91] G. Badalian-Very, J.A. Vergilio, M. Fleming, B.J. Rollins, Pathogenesis of Langerhans cell histiocytosis, Annu. Rev. Pathol. 8 (2013) 1–20.

7

[92] J. Chetritt, V. Paradis, D. Dargere, et al., Chester–Erdheim disease: a neoplastic disorder, Hum. Pathol. 30 (1999) 1093–1096. [93] C.L. Willman, L. Busque, B.B. Griffith, et al., Langerhans'-cell histiocytosis (histiocytosis X)—a clonal proliferative disease, N. Engl. J. Med. 331 (1994) 154–160. [94] F. Sahm, D. Capper, M. Preusser, et al., BRAFV600E mutant protein is expressed in cells of variable maturation in Langerhans cell histiocytosis, Blood 120 (2012) e28–e34. [95] R. Kansal, L. Quintanilla-Martinez, V. Datta, et al., Identification of the V600D mutation in Exon 15 of the BRAF oncogene in congenital, benign Langerhans cell histiocytosis, Genes Chromosomes Cancer 52 (2013) 99–106. [96] T. Satoh, A. Smith, A. Sarde, et al., B-RAF mutant alleles associated with Langerhans cell histiocytosis, a granulomatous pediatric disease, PLoS One 7 (2012) e33891. [97] S.L. Pineles, G.T. Liu, X. Acebes, et al., Presence of Erdheim–Chester disease and Langerhans cell histiocytosis in the same patient: a report of 2 cases, J. Neuroophthalmol. 31 (2011) 217–223. [98] J. Haroche, F. Cohen-Aubart, J.F. Emile, et al., Dramatic efficacy of vemurafenib in both multisystemic and refractory Erdheim–Chester disease and Langerhans cell histiocytosis harboring the BRAF V600E mutation, Blood 121 (2013) 1495–1500. [99] G. Hatzivassiliou, K. Song, I. Yen, et al., RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth, Nature 464 (2010) 431–435. [100] S.A. Yousem, S. Dacic, Y.E. Nikiforov, M. Nikiforova, Pulmonary Langerhans cell histiocytosis: profiling of multifocal tumors using next-generation sequencing identifies concordant occurrence of BRAF V600E mutations, Chest 143 (2013) 1679–1684.


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