BRAF

One of the first technologies capable of testing archived human samples for multiple alleles simultaneously was the Sequenom mass spectrometric genotyping platform. A modification specific for oncology applications known as OncoMap, which tests 983 specific mutations in 115 cancer-related genes, was applied to 61 LCH clinical samples. Overall, very few mutations were detected, a common finding in all subsequent studies (see later discussion) attesting to the stability of the LCH genome. However, a mutation in BRAF encoding the substitution of glutamate for valine at amino acid 600 (BRAF V600E) was observed in 57% of the samples. This mutation, which produces a constitutively active BRAF kinase, is the most commonly observed BRAF variant in cancer and is found in a variety of different cancer types in which it often plays a driver role in pathogenesis. The effect of mutationally activated BRAF is to stimulate signaling through the RAS/RAF/MEK/ERK pathway leading to constitutive transcription of genes involved in a variety of cellular responses including proliferation ( Fig. 1 ).

The ERK signal transduction cascade. The ERK signaling cascade transmits stimuli for a variety of cellular responses, such as proliferation, from the cell surface to the nucleus. In this example, engagement of a tyrosine kinase growth factor receptor by its cognate ligand leads to phosphorylation of amino acids in the cytoplasmic domain of the receptor, which, in turn, activates RAS through GRB2 and SOS. Activated RAS activates RAF through phosphorylation; activated RAF phosphorylates and activates MEK1/2; activated MEK1/2 phosphorylates ERK1/2, which then translocates to the nucleus to stimulate transcription of genes involved in cell proliferation. RAF, MEK1/2, and ERK1/2 are members of the mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK), MAP kinase kinase (MAPKK), and MAP kinase (MAPK) families, respectively (see Fig. 2 ). Constitutive activation of components of this cascade would produce constitutive transcription of target genes and unrestrained cell proliferation. This class of activating mutations in genes encoding RAF proteins (BRAF and ARAF) and MEK1 has been described in LCH (see text).

The presence in LCH of recurrent activating mutations in BRAF has been confirmed in several studies using a variety of different detection techniques ( Table 1 ). Among them are immunohistochemical studies with a V600E-specific antibody, which confirms that the variant is present specifically in LCH histiocytes, a fact that could previously be inferred only indirectly by molecular means. BRAF mutations occur in all clinical settings, including pediatric, adult, single system, and multisystem disease, and their overall prevalence in reported studies is 45% to 65%. Surprisingly, BRAF mutations appear at a nearly similar frequency in pulmonary LCH, a disease of adult smokers that has generally been thought to be polyclonal. However, about one-third of pulmonary LCH cases are clonal. It is also possible that cases that are polyclonal in the aggregate actually comprise several clones of BRAF mutant disease that arose independently.

In the original OncoMap analysis, the median age of patients whose histiocytes contain BRAF V600E was less than the median age of patients whose histiocytes did not, and younger age was associated with the presence of the mutation in an unadjusted exact logistic model but not in the adjusted model. The presence of mutated BRAF did not correlate with any other clinical features, although the clinical annotation of that sample set was limited. In contrast, in the largest sample set analyzed to date, clinical annotation was much more complete and the presence of BRAF V600E correlated with disease relapse. That study also identified BRAF V600E in bone marrow–derived hematopoietic precursors from patients with high-risk LCH. In patients with low-risk disease, the mutation was detected only in lesional cells. This finding suggests that the appearance of a driver mutation earlier in ontogeny might lead to a more widely disseminated and aggressive form of LCH.

Like other BRAF-driven diseases, LCH is also associated with activating mutations of BRAF other than V600E. For example, another acidic amino acid substitution for V600, BRAF V600D (a substitution of aspartate for valine), is an activating mutation that occurs rarely in melanoma and has been reported in a single case of LCH. A more commonly observed BRAF variant in melanoma, BRAF V600K (a substitution of lysine for valine), has not yet been reported in LCH. In addition, an insertional mutation substituting an additional 4 amino acids, DLAT (aspartate-leucine-alanine-threonine), for valine at amino acid position 600 has been found in a single LCH case. Although this variant has not been tested directly for constitutive kinase activity, structural inferences suggest that its effects on constitutive activation of BRAF kinase should be similar to that of V600E.

ARAF

Although activating BRAF mutations are present in 45% to 65% of LCH cases, the histiocytes in all LCH cases examined to date show high levels of phosphorylated ERK, implying ERK pathway activation (see Fig. 1 ). This finding has led several groups to perform even broader analyses such as whole-exome sequencing to find mechanisms for ERK activation other than activating BRAF mutations. A surprising outcome of one whole-exome analysis was the discovery of an activating mutation of ARAF . ARAF is another mitogen-activated protein (MAP) kinase kinase kinase in the same protein family as BRAF ( Fig. 2 ). The ARAF abnormality discovered in this study was actually a compound mutation comprising a single base substitution encoding leucine in place of phenylalanine at position 351 (F351L) and a 6-nucleotide deletion leading to loss of amino acids 347 and 348 (Q347_A348del). Biochemical analysis of this variant demonstrated that it is a constitutively active kinase capable of transforming mouse embryo fibroblasts, suggesting that it is likely to be a driver mutation in this clinical case. This ARAF variant was sensitive to inhibition by vemurafenib in vitro.

MAP kinase families. Signaling from the cell surface to the nucleus occurs through serial activation of members of the MAP kinase family of proteins. Generically, signaling proceeds from MAP kinase kinase kinase (MAPKKK) enzymes to MAP kinase kinase (MAPKK) enzymes and then to MAP kinase (MAPK) enzymes. Fig. 1 described the specific enzymes involved in growth and differentiation, portrayed here as the leftmost cascade. Other cascades result in activation of the MAP kinase JNK, which influences inflammation, apoptosis, differentiation, and survival, and the MAP kinase p38, which influences apoptosis and the release of cytokines. Note that there is cross talk between these cascades. In particular, the MAPKKKs MEKK1–3 stimulate the JNK pathway but can also stimulate the ERK1/2 pathway.

A distinct ARAF mutation, T70M (methionine substituted for threonine at position 70), has also been described in a single mixed case of LCH and Erdheim-Chester disease (ECD). Although this variant, which appears in the COSMIC (Catalog of Somatic Mutations in Cancer) database, has not been tested for RAF kinase activity, it is unlikely to be activated because it appeared in a case that also carried a BRAF V600E mutation. To date, no mutations in CRAF (also known as RAF1) have been described in LCH.

MAP2K1

Ongoing sequence analysis of LCH samples has uncovered additional somatic mutations that activate the ERK pathway. Most prominent are activating mutations in MAP2K1 , which encodes the MAP kinase kinase MEK1 (see Fig. 2 ). These mutations are found only in cases carrying wild-type BRAF alleles, supporting the notion that they are acting in the same signaling pathway as BRAF. The true prevalence of MAP2K1 mutations is uncertain at present because their frequency differs substantially among the samples tested in various reports: 28% in a 40-sample cohort, 19% in a different 36-sample cohort (of a 41-sample collection that included some mixed LCH/ECD and LCH/juvenile xanthogranuloma cases), and 10% in a 30 sample cohort. It is possible that this disparity arises from differences in the clinical characteristics of the patients who comprised these cohorts, but the presence of MAP2K1 mutations does not correlate with age, sex, sites of disease, or stage in these studies.

MAP2K1 mutations in LCH cluster vary specifically in 2 domains: an N-terminal negative regulatory domain and the N-terminal portion of the core kinase domain ( Table 2 ). Some MAP2K1 mutations found in solid tumors, leukemias, or lymphomas overlap these regions, but many more occur throughout the protein. Among the mutations in LCH are several that result in the same alterations of the MAP2K1 protein but involve different nucleotide substitutions (see Table 2 ). For example, the substitution of serine for cysteine at position 121 (C121S) is created both by a substitution of G for C at nucleotide position 362, an alteration also observed in melanoma, and by a substitution of A for T at position 361, creating a differently mutated codon that nonetheless still encodes serine at amino acid 121. Similarly, the deletion of amino acids 58 through 62 arises through an in-frame deletion of nucleotides 303 through 308 or 304 through 309. Finally, the frequently observed deletion of glutamate at position 102 (E102) and isoleucine at position 103 (I103) is produced through in-frame deletions starting at nucleotides 302, 303, or 304. This finding almost certainly reflects substantial selective pressure for these particular variant proteins, which can arise through a variety of alterations at the DNA level, which may be context- or patient specific.

Table 2

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