Dissecting the pathogenesis of classical Hodgkin lymphoma (cHL), a common cancer in young adults, remains challenging because of the rarity of tumor cells in involved tissues (usually <5%). Here, we analyzed the coding genome of cHL by microdissecting tumor and normal cells from 34 patient biopsies for a total of ∼50 000 singly isolated lymphoma cells. We uncovered several recurrently mutated genes, namely, (32% of cases), (24%), (18%), and (16%), and document the functional role of mutant STAT6 in sustaining tumor cell viability. Mutations of genetically and functionally cooperated with disruption of, a JAK-STAT pathway inhibitor, to promote cHL growth. Overall, 87% of cases showed dysregulation of the JAK-STAT pathway by genetic alterations in multiple genes (also including ,, ,, and ), attesting to the pivotal role of this pathway in cHL pathogenesis and highlighting its potential as a new therapeutic target in this disease.

Hairy cell leukemia (HCL) is a distinct clinicopathologic entity that responds well to purine analogs but is sometimes difficult to differentiate from HCL-like disorders (eg, splenic marginal zone lymphoma and HCL variant). We recently identified the BRAF-V600E mutation as the disease-defining genetic event in HCL. In this study, we describe a new, simple, and inexpensive test for genetics-based diagnosis of HCL in whole-blood samples that detects BRAF-V600E through a sensitive allele-specific PCR qualitative assay followed by agarose-gel electrophoresis. IntroductionHairy cell leukemia (HCL) is a distinct entity usually characterized by splenomegaly (without lymphadenopathy), pancytopenia, and infiltration of BM, spleen, and liver by leukemic B cells with "hairy" appearance. In contrast to other chronic B-cell leukemias, HCL cells circulate at low percentages in the blood, 1 and exhibit distinct functional features and gene expression profile. 2,3 HCL diagnosis relies on morphologic and immunophenotypic criteria 1-4 that usually allow its distinction from HCL-like disorders of the 2008 World Health Organization classification, that is, splenic marginal zone lymphoma (SMZL) and splenic lymphoma/ leukemia unclassifiable (SLLU; including HCL variant [HCL-v]). 5 A correct diagnosis is critical because purine analogs (pentostatin and cladribine) are highly effective only in HCL. 4 The most problematic cases can be diagnosed using annexin-A1 immunostaining, 6-8 which we previously reported to be highly sensitive and specific for HCL among B-cell lymphomas. 6 However, because annexin-A1 is also expressed by myeloid and T cells, 6 this immunohistochemical staining may be difficult to interpret in BM biopsies with low percentages of HCL cells. Moreover, immunocytochemistry for annexin-A1 is not readily applicable to routine hematologic samples, such as peripheral blood or diluted BM aspirate (because of dry tap), that are also usually poor in HCL cells and rich in neutrophils and T cells.An ideal solution would be a sensitive and specific test for a genetics-based diagnosis of HCL. We recently identified BRAF-V600E as the HCL-defining genetic lesion (present in all HCL cases, absent in other B-cell neoplasms) 9 by Sanger sequencing of BRAF exon 15. However, this technique required Ն 30% leukemic cells for reliably detecting a clonal heterozygous mutation. Thus, the rare HCL cells typically present in the blood of most patients had to be purified through cell sorting, 9 a laborious procedure not amenable to a routine diagnostic setting.Aims of this study were: (1) to develop a sensitive, easy, and inexpensive test for the routine clinical diagnosis of HCL in blood samples, based on BRAF-V600E detection by allele-specific PCR (AS-PCR) followed by conventional agarose-gel electrophoresis; and (2) to assess the diagnostic accuracy of this test in a large cohort of HCL and HCL-like disorders. Methods Tumor samplesWe studied 117 HCL patients: 96 before therapy and 21 after therapy, all with detectable disease (at least 0.1% ...

Key Points• The V600E kinase-activating mutation of BRAF profoundly shapes the distinct identity of HCL among B-cell neoplasms.• Clinically available BRAF and MEK inhibitors exert potent antileukemic activity in patients' HCL cells in vitro and in vivo.Hairy cell leukemia (HCL) shows unique clinicopathological and biological features. HCL responds well to purine analogs but relapses are frequent and novel therapies are required. BRAF-V600E is the key driver mutation in HCL and distinguishes it from other B-cell lymphomas, including HCL-like leukemias/lymphomas (HCL-variant and splenic marginal zone lymphoma). The kinase-activating BRAF-V600E mutation also represents an ideal therapeutic target in HCL. Here, we investigated the biological and therapeutic importance of the activated BRAF-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway in HCL by exposing in vitro primary leukemic cells purified from 26 patients to clinically available BRAF (vemurafenib; dabrafenib) or MEK (trametinib) inhibitors. Results were validated in vivo in samples from vemurafenib-treated HCL patients within a phase 2 clinical trial. BRAF and MEK inhibitors caused, specifically in HCL (but not HCL-like) cells, marked MEK/ERK dephosphorylation, silencing of the BRAF-MEK-ERK pathway transcriptional output, loss of the HCL-specific gene expression signature, downregulation of the HCL markers CD25, tartrate-resistant acid phosphatase, and cyclin D1, smoothening of leukemic cells' hairy surface, and, eventually, apoptosis. Apoptosis was partially blunted by coculture with bone marrow stromal cells antagonizing MEK-ERK dephosphorylation. This protective effect could be counteracted by combined BRAF and MEK inhibition. Our results strongly support and inform the clinical use of BRAF and

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