Background Hairy cell leukemia (HCL) is a well defined clinico-pathological entity whose underlying genetic lesion is still obscure. Methods We searched for HCL-associated mutations by massively parallel sequencing of the whole exome of leukemic and matched normal mononuclear cells purified from the peripheral blood of one patient with HCL. Results Whole exome sequencing identified 5 missense somatic clonal mutations that were confirmed at Sanger sequencing, including a heterozygous V600E mutation involving the BRAF gene. Since the BRAF V600E mutation is oncogenic in other tumors, further analyses were focused on this genetic lesion. Sanger sequencing detected mutated BRAF in 46/46 additional HCL patients (47/47 including the index case; 100%). None of the 193 peripheral B-cell lymphomas/leukemias other than HCL that were investigated carried the BRAF V600E mutation, including 36 cases of splenic marginal zone lymphomas and unclassifiable splenic lymphomas/leukemias. Immunohistological and Western blot studies showed that HCL cells express phospho-MEK and phospho-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 cells from 5 HCL patients with PLX-4720, a specific inhibitor of active BRAF, led to marked decrease of phosphorylated ERK and MEK. Conclusions The BRAF V600E mutation was present in all HCL patients investigated. This finding may have relevant implications for the pathogenesis, diagnosis and targeted therapy of HCL (Funded by the Associazione Italiana Ricerca Cancro and others).

Summary Follicular lymphoma (FL) is an indolent disease, but 30-40% of cases undergo histologic transformation to an aggressive malignancy, typically represented by diffuse large B cell lymphoma (DLBCL). The pathogenesis of this process remains largely unknown. Using whole-exome sequencing and copy-number analysis, here we show that the dominant clone of FL and transformed FL (tFL) arise by divergent evolution from a common mutated precursor through the acquisition of distinct genetic events. Mutations in epigenetic modifiers and anti-apoptotic genes are introduced early in the common precursor, while tFL is specifically associated with alterations deregulating cell-cycle progression and DNA-damage responses (CDKN2A/B, MYC, TP53), as well as with aberrant somatic hypermutation. The genomic profile of tFL shares similarities with that of germinal center B-cell-type de novo DLBCL, but also displays unique combinations of altered genes, with diagnostic and therapeutic implications.

Sequencing studies from several model systems have suggested that diverse and abundant small RNAs may be derived from tRNA, but the function of these molecules remains undefined. Here, we demonstrate that one such tRNA-derived fragment, cloned from human mature B cells and designated CU1276, in fact possesses the functional characteristics of a microRNA, including a DICER1 -dependent biogenesis, physical association with Argonaute proteins, and the ability to repress mRNA transcripts in a sequence-specific manner. Expression of CU1276 is abundant in normal germinal center B cells but absent in germinal center-derived lymphomas, suggesting a role in the pathogenesis of this disease. Furthermore, CU1276 represses endogenous RPA1 , an essential gene involved in many aspects of DNA dynamics, and consequently, expression of this tRNA-derived microRNA in a lymphoma cell line suppresses proliferation and modulates the molecular response to DNA damage. These results establish that functionally active microRNAs can be derived from tRNA, thus defining a class of genetic entities with potentially important biological roles.

SUMMARY We report that diffuse large B-cell lymphoma (DLBCL) commonly fails to express cell-surface molecules necessary for the recognition of tumor cells by immune-effector cells. In 29% of cases, mutations and deletions inactivate the β2-microglobulin gene, thus preventing the cell-surface expression of the HLA class-I (HLA-I) complex that is necessary for recognition by CD8+ cytotoxic T-cells. In 21% of cases, analogous lesions involve the CD58 gene, which encodes a molecule involved in T and natural killer cell-mediated responses. In addition to gene inactivation, alternative mechanisms lead to aberrant expression of HLA-I and CD58 in >60% of DLBCL. These two events are significantly associated in this disease, suggesting that they are co-selected during lymphomagenesis for their combined role in escape from immune-surveillance.

Mutations in the gene encoding the KMT2D (also called MLL2) methyltransferase are highly recurrent and occur early during tumorigenesis in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). However, the functional consequences of KMT2D mutations and their role in lymphomagenesis are unknown. Here we show that FL/DLBCL-associated KMT2D mutations impair KMT2D enzymatic activity, leading to diminished global H3K4 methylation in germinal-center (GC) B-cells and DLBCL cells. Conditional deletion of Kmt2d early during B cell development, but not after initiation of the GC reaction, results in an increase in GC B-cells and enhances B cell proliferation in mice. In mice overexpressing BCL2, which develop GC-derived lymphomas resembling human tumors, genetic ablation of Kmt2d leads to a further increase in tumor incidence. These findings suggest that KMT2D acts as a tumor suppressor gene whose early loss facilitates lymphomagenesis by remodeling the epigenetic landscape of the cancer precursor cells. Eradication of KMT2D-deficient cells may represent a rational therapeutic approach for targeting early tumorigenic events.

Germinal centers (GCs) are sites of B-cell clonal expansion, hypermutation, and selection. GCs are polarized into dark (DZ) and light zones (LZ), a distinction that is of key importance to GC selection. However, the difference between the B cells in each of these zones in humans remains unclear. We show that, as in mice, CXCR4 and CD83 can be used to distinguish human LZ and DZ cells. Using these markers, we show that LZ and DZ cells in mice and humans differ only in the expression of characteristic "activation" and "proliferation" programs, suggesting that these populations represent alternating states of a single-cell type rather than distinct differentiation stages. In addition, LZ/DZ transcriptional profiling shows that, with the exception of "molecular" Burkitt lymphomas, nearly all human B-cell malignancies closely resemble LZ cells, which has important implications for our understanding of the molecular programs of lymphomagenesis. (Blood. 2012;120(11): 2240-2248)

Notch2 mutations represent the most frequent lesion in splenic marginal zone lymphoma.

The pathways regulating formation of the germinal center (GC) dark zone (DZ) and light zone (LZ) are unknown. In this study we show that FOXO1 transcription factor expression was restricted to the GC DZ and was required for DZ formation, since its absence in mice led to the loss of DZ gene programs and the formation of LZ-only GCs. FOXO1-negative GC B cells displayed normal somatic hypermutation but defective affinity maturation and class switch recombination. The function of FOXO1 in sustaining the DZ program involved the trans-activation of the chemokine receptor CXCR4, and cooperation with the BCL6 transcription factor in the trans-repression of genes involved in immune activation, DNA repair, and plasma cell differentiation. These results also have implications for the role of FOXO1 in lymphomagenesis because they suggest that constitutive FOXO1 activity might be required for the oncogenic activity of deregulated BCL6 expression.