BACKGROUND: Hairy-cell leukemia (HCL) is a well-defined clinicopathological entity whose underlying genetic lesion is still obscure. METHODS: We searched for HCL-associated mutations by performing massively parallel sequencing of the whole exome of leukemic and matched normal cells purified from the peripheral blood of an index patient with HCL. Findings were validated by Sanger sequencing in 47 additional patients with HCL. RESULTS: Whole-exome sequencing identified five missense somatic clonal mutations that were confirmed on Sanger sequencing, including a heterozygous mutation in BRAF that results in the BRAF V600E variant protein. Since BRAF V600E is oncogenic in other tumors, further analyses were focused on this genetic lesion. The same BRAF mutation was noted in all the other 47 patients with HCL who were evaluated by means of Sanger sequencing. None of the 195 patients with other peripheral B-cell lymphomas or leukemias who were evaluated carried the BRAF V600E variant, including 38 patients with splenic marginal-zone lymphomas or unclassifiable splenic lymphomas or leukemias. In immunohistologic and Western blot studies, HCL cells expressed phosphorylated MEK and ERK (the downstream targets of the BRAF kinase), indicating a constitutive activation of the RAF-MEK-ERK mitogen-activated protein kinase pathway in HCL. In vitro incubation of BRAF-mutated primary leukemic hairy cells from 5 patients with PLX-4720, a specific inhibitor of active BRAF, led to a marked decrease in phosphorylated ERK and MEK. CONCLUSIONS; The BRAF V600E mutation was present in all patients with HCL who were evaluated. This finding may have implications for the pathogenesis, diagnosis, and targeted therapy of HCL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).
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机译：背景：毛细胞白血病（HCL）是一种定义明确的临床病理实体，其潜在的遗传病变仍不清楚。方法：我们通过对从HCL索引患者外周血中纯化的白血病和匹配正常细胞的整个外显子进行大规模平行测序，搜索与HCL相关的突变。通过Sanger测序对另外47名HCL患者进行了验证。结果：全外显子测序鉴定了Sanger测序证实的5个错义体细胞克隆突变，包括BRAF中的杂合突变，导致BRAF V600E变异蛋白。由于BRAF V600E在其他肿瘤中是致癌的，因此进一步的分析集中在这种遗传病变上。在所有其他47名HCL患者中，通过Sanger测序评估了相同的BRAF突变。被评估的195例其他外周B细胞淋巴瘤或白血病患者中，没有一个携带BRAF V600E变异体，包括38例脾边缘区淋巴瘤或无法分类的脾淋巴瘤或白血病患者。在免疫组织学和蛋白质印迹研究中，HCL细胞表达磷酸化的MEK和ERK（BRAF激酶的下游靶标），表明HCL中RAF-MEK-ERK丝裂原激活的蛋白激酶途径的组成性激活。来自5名PLX-4720患者的BRAF突变的原代白血病毛细胞的体外温育是活性BRAF的特异性抑制剂，导致磷酸化ERK和MEK显着降低。结论;在所有接受评估的HCL患者中均存在BRAF V600E突变。这一发现可能对HCL的发病机制，诊断和靶向治疗有影响。 （由Italianocial per la Ricerca sul Cancro和其他人资助。）。
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