Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

UGT2B17 modifies drug response in chronic lymphocytic leukaemia.

BACKGROUND: High UGT2B17 is associated with poor prognosis in untreated chronic lymphocytic leukaemia (CLL) patients and its expression is induced in non-responders to fludarabine-containing regimens. We examined whether UGT2B17, the predominant lymphoid glucuronosyltransferase, affects leukaemic drug response and is involved in the metabolic inactivation of anti-leukaemic agents. METHODS: Functional enzymatic assays and patients' plasma samples were analysed by mass-spectrometry to evaluate drug inactivation by UGT2B17. Cytotoxicity assays and RNA sequencing were used to assess drug response and transcriptome changes associated with high UGT2B17 levels. RESULTS: High UGT2B17 in B-cell models led to reduced sensitivity to fludarabine, ibrutinib and idelalisib. UGT2B17 expression in leukaemic cells involved a non-canonical promoter and was induced by short-term treatment with these anti-leukaemics. Glucuronides of both fludarabine and ibrutinib were detected in CLL patients on respective treatment, however UGT2B17 conjugated fludarabine but not ibrutinib. AMP-activated protein kinase emerges as a pathway associated with high UGT2B17 in fludarabine-treated patients and drug-treated cell models. The expression changes linked to UGT2B17 exposed nuclear factor kappa B as a key regulatory hub. CONCLUSIONS: Data imply that UGT2B17 represents a mechanism altering drug response in CLL through direct inactivation but would also involve additional mechanisms for drugs not inactivated by UGT2B17.

Sex-dependent association of circulating sex steroids and pituitary hormones with treatment-free survival in chronic lymphocytic leukemia patients.

Chronic lymphocytic leukemia (CLL) is not considered a hormone-regulated cancer although sex is a recognized risk factor with men more frequently diagnosed and developing progressive disease. We hypothesized that variable hormonal exposure may have a sexually dimorphic influence on treatment-free survival (TFS). In 156 CLL cases, we quantitatively profiled 29 circulating steroids (progesterone, adrenal precursors, androgens, estrogens, and catechol estrogens) as well as luteinizing hormone (LH) and follicle-stimulating hormone. Median TFS was shorter for men than that for women (80.7 vs. 135.0 months, P = 0.033). Circulating hormone profiles in CLL patients were significantly different from those of healthy donors. In male CLL cases, higher LH levels were associated with shorter TFS (adjusted hazard ratio (HRadj) 2.11; P = 0.004). In female CLL cases, high levels of the potent androgens testosterone and dihydrotestosterone and the sum of methoxy estrogens were associated with an improved TFS with HRadj values of 0.24 (P = 0.007), 0.54 (P = 0.023), and 0.31 (P = 0.034), respectively. Reduced TFS was observed for women with CLL exhibiting high expression of the steroid-inactivating UGT2B17 enzyme. This study is the first to establish a link between the outcome of CLL patients, sex steroids, and pituitary hormones, revealing a sex-specific hormonal imbalance associated with disease progression.

The Runx-PU.1 pathway preserves normal and AML/ETO9a leukemic stem cells.

Runx transcription factors contribute to hematopoiesis and are frequently implicated in hematologic malignancies. All three Runx isoforms are expressed at the earliest stages of hematopoiesis; however, their function in hematopoietic stem cells (HSCs) is not fully elucidated. Here, we show that Runx factors are essential in HSCs by driving the expression of the hematopoietic transcription factor PU.1. Mechanistically, by using a knockin mouse model in which all three Runx binding sites in the -14kb enhancer of PU.1 are disrupted, we observed failure to form chromosomal interactions between the PU.1 enhancer and its proximal promoter. Consequently, decreased PU.1 levels resulted in diminished long-term HSC function through HSC exhaustion, which could be rescued by reintroducing a PU.1 transgene. Similarly, in a mouse model of AML/ETO9a leukemia, disrupting the Runx binding sites resulted in decreased PU.1 levels. Leukemia onset was delayed, and limiting dilution transplantation experiments demonstrated functional loss of leukemia-initiating cells. This is surprising, because low PU.1 levels have been considered a hallmark of AML/ETO leukemia, as indicated in mouse models and as shown here in samples from leukemic patients. Our data demonstrate that Runx-dependent PU.1 chromatin interaction and transcription of PU.1 are essential for both normal and leukemia stem cells.

Overexpression of uridine diphospho glucuronosyltransferase 2B17 in high-risk chronic lymphocytic leukemia.

Uridine diphospho glucuronosyltransferase 2B17 (UGT2B17) glucuronidates androgens and xenobiotics including certain drugs. The UGT2B17 gene shows a remarkable copy number variation (CNV), which predisposes for solid tumors and influences drug response. Here, we identify a yet undescribed UGT2B17 mRNA overexpression in poor-risk chronic lymphocytic leukemia (CLL). In total, 320 CLL patients and 449 healthy donors were analyzed. High (above median) UGT2B17 expression was associated with established CLL poor prognostic factors and resulted in shorter treatment-free and overall survival (hazard ratio ([death] 2.18; 95% CI 1.18-4.01; P = .013). The prognostic impact of mRNA expression was more significant than that of UGT2B17 CNV. UGT2B17 mRNA levels in primary CLL samples directly correlated with functional glucuronidation activity toward androgens and the anticancer drug vorinostat (R > 0.9, P < .001). After treatment with fludarabine containing regimens UGT2B17 was up-regulated particularly in poor responders (P = .030). We observed an exclusive involvement of the 2B17 isoform within the UGT protein family. Gene expression profiling of a stable UGT2B17 knockdown in the CLL cell line MEC-1 demonstrated a significant involvement in key cellular processes. These findings establish a relevant role of UGT2B17 in CLL with functional consequences and potential therapeutic implications.

Recombinant antibodies encoded by IGHV1-69 react with pUL32, a phosphoprotein of cytomegalovirus and B-cell superantigen.

Leukemia cells from patients with chronic lymphocytic leukemia (CLL) express a highly restricted immunoglobulin heavy variable chain (IGHV) repertoire, suggesting that a limited set of antigens reacts with leukemic cells. Here, we evaluated the reactivity of a panel of different CLL recombinant antibodies (rAbs) encoded by the most commonly expressed IGHV genes with a panel of selected viral and bacterial pathogens. Six different CLL rAbs encoded by IGHV1-69 or IGHV3-21, but not a CLL rAb encoded by IGHV4-39 genes, reacted with a single protein of human cytomegalovirus (CMV). The CMV protein was identified as the large structural phosphoprotein pUL32. In contrast, none of the CLL rAbs bound to any other structure of CMV, adenovirus serotype 2, Salmonella enterica serovar Typhimurium, or of cells used for propagation of these microorganisms. Monoclonal antibodies or humanized rAbs of irrelevant specificity to pUL32 did not react with any of the proteins present in the different lysates. Still, rAbs encoded by a germ line IGHV1-69 51p1 allele from CMV-seropositive and -negative adults also reacted with pUL32. The observed reactivity of multiple different CLL rAbs and natural antibodies from CMV-seronegative adults with pUL32 is consistent with the properties of a superantigen.

Targeting sphingolipid metabolism in chronic lymphocytic leukemia.

Elevated levels of circulating C16:0 glucosylceramides (GluCer) and increased mRNA expression of UDP-glucose ceramide glycosyltransferase (UGCG), the enzyme responsible for converting ceramides (Cer) to GluCer, represent unfavorable prognostic markers in chronic lymphocytic leukemia (CLL) patients. To evaluate the therapeutic potential of inhibiting GluCer synthesis, we genetically repressed the UGCG pathway using in vitro models of leukemic B cells, in addition to UGCG pharmacological inhibition with approved drugs such as eliglustat and ibiglustat, both individually and in combination with ibrutinib, assessed in cell models and primary CLL patient cells. Cell viability, apoptosis, and proliferation were evaluated in vitro, and survival and apoptosis were examined ex vivo. UGCG inhibition efficacy was confirmed by quantifying intracellular sphingolipid levels through targeted lipidomics using mass spectrometry. Other inhibitors of sphingolipid biosynthesis pathways were similarly assessed. Blocking UGCG significantly decreased cell viability and proliferation, highlighting the oncogenic role of UGCG in CLL. The efficient inhibition of UGCG was confirmed by a significant reduction in GluCer intracellular levels. The combination of UGCG inhibitors with ibrutinib demonstrated synergistic effect. Inhibitors that target alternative pathways within sphingolipid metabolism, like sphingosine kinases inhibitor SKI-II, also demonstrated promising therapeutic effects both alone and when used in combination with ibrutinib, reinforcing the oncogenic impact of sphingolipids in CLL cells. Targeting sphingolipid metabolism, especially the UGCG pathway, represents a promising therapeutic strategy and as a combination therapy for potential treatment of CLL patients, warranting further investigation.

Distinct t(7;9)(q34;q32) breakpoints in healthy individuals and individuals with T-ALL.

After V(D)J-mediated translocations, signal joints are retained on one of the derivative chromosomes. We report here that such signal joints are highly reactive and constitute unstable genomic elements with potential oncogenic properties.

A Non-Canonical Role for the Glycosyltransferase Enzyme UGT2B17 as a Novel Constituent of the B Cell Receptor Signalosome.

In chronic lymphocytic leukemia (CLL), an elevated glycosyltransferase UGT2B17 expression (UGT2B17HI) identifies a subgroup of patients with shorter survival and poor drug response. We uncovered a mechanism, possibly independent of its enzymatic function, characterized by an enhanced expression and signaling of the proximal effectors of the pro-survival B cell receptor (BCR) pathway and elevated Bruton tyrosine kinase (BTK) phosphorylation in B-CLL cells from UGT2B17HI patients. A prominent feature of B-CLL cells is the strong correlation of UGT2B17 expression with the adverse marker ZAP70 encoding a tyrosine kinase that promotes B-CLL cell survival. Their combined high expression levels in the treatment of naïve patients further defined a prognostic group with the highest risk of poor survival. In leukemic cells, UGT2B17 knockout and repression of ZAP70 reduced proliferation, suggesting that the function of UGT2B17 might involve ZAP70. Mechanistically, UGT2B17 interacted with several kinases of the BCR pathway, including ZAP70, SYK, and BTK, revealing a potential therapeutic vulnerability. The dual SYK and JAK/STAT6 inhibitor cerdulatinib most effectively compromised the proliferative advantage conferred by UGT2B17 compared to the selective BTK inhibitor ibrutinib. Findings point to an oncogenic role for UGT2B17 as a novel constituent of BCR signalosome also connected with microenvironmental signaling.

Untargeted metabolomics identifies metabolic dysregulation of sphingolipids associated with aggressive chronic lymphocytic leukaemia and poor survival.

BACKGROUND: Metabolic dependencies of chronic lymphocytic leukaemia (CLL) cells may represent new personalized treatment approaches in patients harbouring unfavourable features. METHODS: Here, we used untargeted metabolomics and lipidomics analyses to isolate metabolomic features associated with aggressive CLL and poor survival outcomes. We initially focused on profiles associated with overexpression of the adverse metabolic marker glycosyltransferase (UGT2B17) associated with poor survival and drug resistance. RESULTS: Leukaemic B-cell metabolomes indicated a significant perturbation in lipids, predominantly bio-active sphingolipids. Expression of numerous enzyme-encoding genes of sphingolipid biosynthesis pathways was significantly associated with shorter patient survival. Targeted metabolomics further exposed higher circulating levels of glucosylceramides (C16:0 GluCer) in CLL patients relative to healthy donors and an aggressive cancer biology. In multivariate analyses, C16:0 GluCer and sphinganine were independent prognostic markers and were inversely linked to treatment-free survival. These two sphingolipid species function as antagonistic mediators, with sphinganine being pro-apoptotic and GluCer being pro-proliferative, tested in leukemic B-CLL cell models. Blocking GluCer synthesis using ceramide glucosyltransferase inhibitors induced cell death and reduced the proliferative phenotype, which further sensitized a leukaemic B-cell model to the anti-leukaemics fludarabine and ibrutinib in vitro. CONCLUSIONS: Specific sphingolipids may serve as prognostic markers in CLL, and inhibiting enzymatic pathways involved in their biosynthesis has potential as a therapaeutic approach.

Prolonged progression-free survival in patients with chronic lymphocytic leukemia receiving granulocyte colony-stimulating factor during treatment with fludarabine, cyclophosphamide, and rituximab.

The clinical benefit of the addition of granulocyte colony-stimulating factor (G-CSF) to standard immunochemotherapy of chronic lymphocytic leukemia (CLL) with fludarabine, cyclophosphamide, and rituximab (FCR) is still unclear. In this retrospective study we analyzed the outcome of 32 consecutive patients with CLL during treatment with FCR. Sixteen patients received G-CSF for treatment of CTC grade 3 or 4 neutropenia or febrile neutropenia at some point during therapy and 16 did not. Both groups were well balanced for clinical and biological risk factors. Overall response rates were not significantly different (94% vs. 75%; p=0.144). Interestingly, a significantly better progression-free survival (100% vs. 35.4% at 24 months; p<0.001) and even overall survival (100% vs. 77.8% at 24 months; p=0.022) was observed in patients receiving G-CSF. While the underlying cause remains to be elucidated, these data strongly suggest an association of the addition of G-CSF to FCR therapy with final patient outcome.

Prevalence and clinical impact of autoimmune diseases and chronic infections in malignant lymphomas at diagnosis.

There is increasing evidence for the role of chronic antigenic stimulation (CS) in the development of cancer. Clinical data, however, are rare as is the information on outcome. In this study, the occurrence of chronic infections (CI) and autoimmune diseases (AI) in patients with malignant lymphoma at diagnosis was assessed. Of 367 patients [non-Hodgkin's lymphoma (NHL) N = 297, Hodgkin's lymphoma N = 70], 9.8% (N = 36) had a history of chronic antigenic stimulation (4.4% AI, 5.4% CI) at diagnosis. After a median observation time of 74.7 months, 118 patients have died. There were more male patients in this cohort. However, sex ratio among patients with chronic antigenic stimulation was skewed in favor of women (p = 0.018), in particular among lymphoma patients with AI (p = 0.001). NHL patients with autoimmune diseases showed a tendency to develop diffuse large B cell lymphoma [8 of 12 AI + NHL patients (66.7%) vs. 100 of 266 non-CS NHL (37.6%); p = 0.066]. No significant difference in overall survival (OS) between CS and non-CS patients could be observed (median OS after 48 months was: CS 77.7% vs. non-CS 71.8%). In conclusion, chronic antigenic stimulation at diagnosis appears to be associated with a higher prevalence in women, in particular among patients with autoimmune disease. However, no difference in overall survival was observed. This suggests that the presence of chronic inflammatory conditions does not decisively influence the outcome of lymphoma patients.

NOTCH2 links protein kinase C delta to the expression of CD23 in chronic lymphocytic leukaemia (CLL) cells.

One characteristic of chronic lymphocytic leukaemia (CLL) lymphocytes is high expression of CD23, which has previously been identified as a downstream target for NOTCH2 signalling. The mechanisms regulating NOTCH2-dependent CD23 expression, however, are largely unknown. This study showed that peripheral CLL cells overexpressed transcriptionally active NOTCH2 (N2(IC)), irrespective of their prognostic marker profile. When placed in culture, NOTCH2 activity was spontaneously decreased in 25 out of 31 CLL cases (81%) within 24 h. DNA-bound N2(IC) complexes could be maintained by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or by gamma-interferon (IFN-gamma), two CLL characteristic inducers of CD23 expression. Inhibition of PKC-delta by RNA interference or by rottlerin antagonised PMA-induced NOTCH2 activation and also suppressed NOTCH2 activity in CLL cases with constitutively activated NOTCH2 signalling. In 23 out of 29 CLL cases tested (79%), DNA-bound N2(IC) complexes were found to be resistant to the gamma-secretase inhibitor (GSI) DAPT, suggesting that GSIs will be only effective in a subset of CLL cases. These data suggest that deregulation of NOTCH2 signalling is critically involved in maintaining the malignant phenotype of CLL lymphocytes and point to a link between PKC-delta and NOTCH2 signalling in the leukemic cells.

In vivo reinsertion of excised episomes by the V(D)J recombinase: a potential threat to genomic stability.

It has long been thought that signal joints, the byproducts of V(D)J recombination, are not involved in the dynamics of the rearrangement process. Evidence has now started to accumulate that this is not the case, and that signal joints play unsuspected roles in events that might compromise genomic integrity. Here we show both ex vivo and in vivo that the episomal circles excised during the normal process of receptor gene rearrangement may be reintegrated into the genome through trans-V(D)J recombination occurring between the episomal signal joint and an immunoglobulin/T-cell receptor target. We further demonstrate that cryptic recombination sites involved in T-cell acute lymphoblastic leukemia-associated chromosomal translocations constitute hotspots of insertion. Eventually, the identification of two in vivo cases associating episomal reintegration and chromosomal translocation suggests that reintegration events are linked to genomic instability. Altogether, our data suggest that V(D)J-mediated reintegration of episomal circles, an event likely eluding classical cytogenetic screenings, might represent an additional potent source of genomic instability and lymphoid cancer.

V(D)J targeting mistakes occur at low frequency in acute lymphoblastic leukemia.

Translocations of proto-oncogenes to the B-cell or T-cell antigen receptor loci in acute T- or B-cell leukemia and lymphoma have been, in most cases, accredited to V(D)J or switch recombination depending on the location of the breakpoint at the receptor locus. Only in rare instances, the reports take into account mechanistic characteristics of the translocation mechanism. To assess the functional ability of several sites implicated in supposedly V(D)J-mediated translocations, we tested five sites at four proto-oncogene loci in an ex vivo recombination substrate assay for their potential to act as direct target for V(D)J recombination. Our results show that the LMO2/RBTN2/TTG2 site and one LCK/P56 site readily engage in recombination with a genuine TCR element with the majority of breakpoint junctions showing the characteristics of V(D)J recombination, which strongly supports the involvement of this mechanism in the pathogenesis of the corresponding translocations in vivo. The site at the TLX1/HOX11 locus yielded 0.8% V(D)J-specific junctions. Sites at the LCK/P56 and TCF3/E2A proto-oncogenes resulted in exclusively unspecific breakpoints scattered over part of or the entire proto-oncogene region tested, marking them as unlikely V(D)J recombination targets. Our data suggest that, while being a potentially dangerous mechanism due to the introduction of DNA breaks, V(D)J recombination is a tightly controlled mechanism allowing for only few direct mistakes.

Inactivation of Prostaglandin E2 as a Mechanism for UGT2B17-Mediated Adverse Effects in Chronic Lymphocytic Leukemia.

High expression of the metabolic enzyme UDP-glucuronosyltransferase UGT2B17 in chronic lymphocytic leukemia (CLL) cells was associated with poor prognosis in two independent studies. However, the underlying mechanism remains unknown. We hypothesized that UGT2B17 impacts intracellular levels of hormone-like signaling molecules involved in the regulation of gene expression in leukemic cells. We initially confirmed in a third cohort of 291 CLL patients that those with high UGT2B17 displayed poor prognosis (hazard ratio of 2.31, P = 0.015). Consistent with the unfavorable prognostic significance of elevated UGT2B17 expression in CLL patients, high UGT2B17 expression was associated with enhanced proliferation of MEC1 and JVM2 malignant B-cell models. Transcriptomic analyses revealed that high UGT2B17 was linked to a significant alteration of genes related to prostaglandin E2 (PGE2) and to its precursor arachidonic acid, both in cell models and a cohort of 448 CLL patients. In functional assays, PGE2 emerged as a negative regulator of apoptosis in CLL patients and proliferation in cells models, whereas its effect was partially abrogated by high UGT2B17 expression in MEC1 and JVM2 cells. Enzymatic assays and mass-spectrometry analyses established that the UGT2B17 enzyme inactivates PGE2 by its conjugation to glucuronic acid (GlcA) leading to the formation of two glucuronide (G) derivatives. High UGT2B17 expression was further associated with a proficient inactivation of PGE2 to PGE2-G in CLL patient cells and cell models. We conclude that UGT2B17-dependent PGE2 glucuronidation impairs anti-oncogenic PGE2 effects in leukemic cells, thereby partially contributing to disease progression in high UGT2B17 CLL patients.

Autoimmune conditions and chronic infections in chronic lymphocytic leukemia patients at diagnosis are associated with unmutated IgVH genes.

Few data are available concerning the prevalence of autoimmune disease or chronic infections in chronic lymphocytic leukemia patients at diagnosis as well as their clinical outcome. We studied the frequency of such chronic conditions in relation to prognostic markers. A history of autoimmune disease or chronic infection was found in 21% of 186 chronic lymphocytic leukemia patients (12% in autoimmune diseases, 9% in chronic infections). Patients with a history of chronic stimulation were more likely to have unmutated IgV(H) genes (p<0.002), unfavorable or intermediate risk cytogenetics (11q, 17p deletions, trisomy 12) (p<0.001), and higher CD38 expression (p=0.004). Autoimmune conditions (n=22) were characterized by female predominance (55.0%) with a high frequency of unmutated IgV(H) (53,8%). Median time to first treatment was 83 months for the chronic stimulation group compared to 128 months for the non-chronic stimulation group (n.s.). Patients suffering from chronic conditions at chronic lymphocytic leukemia diagnosis are likely to have poor prognostic markers, particularly unmutated IgV(H) genes.

Signatures of CD8+ T cell dysfunction in AML patients and their reversibility with response to chemotherapy.

BACKGROUND: Our understanding of phenotypic and functional signatures of CD8+ T cell dysfunction in acute myeloid leukemia (AML) is limited. Deciphering these deranged T cell functional states and how they are impacted by induction chemotherapy is essential for incorporation of novel immune-based strategies to restore and maintain antileukemia immunity. METHODS: We utilized high-dimensional immunophenotyping, gene expression, and functional studies to characterize peripheral blood and bone marrow CD8+ T cells in 72 AML patients at diagnosis and after induction chemotherapy. RESULTS: Our data suggest that multiple aspects of deranged T cell function are operative in AML at diagnosis, with exhaustion and senescence being the dominant processes. Following treatment, the phenotypic and transcriptional profile of CD8+ T cells diverged between responders and nonresponders. Response to therapy correlated with upregulation of costimulatory, and downregulation of apoptotic and inhibitory, T cell signaling pathways, indicative of restoration of T cell function. In functional studies, AML blasts directly altered CD8+ T cell viability, expansion, co-signaling and senescence marker expression. This CD8+ T cell dysfunction was in part reversible upon PD-1 blockade or OX40 costimulation in vitro. CONCLUSION: Our findings highlight the uniqueness of AML in sculpting CD8+ T cell responses and the plasticity of their signatures upon chemotherapy response, providing a compelling rationale for integration of novel immunotherapies to augment antileukemia immunity. FUNDING: This work was supported by the Leukemia & Lymphoma Society grant no. 6449-13; NIH grants UM1-CA186691 and R01-HL110907-01; the American Society for Blood and Marrow Transplantation New Investigator Award/Gabrielle's Angel Foundation; the Vienna Fund for Innovative Cancer Research; and by fellowships from the Wenner-Gren Foundation and the Swedish Society for Medical Research.

Recombinase, chromosomal translocations and lymphoid neoplasia: targeting mistakes and repair failures.

A large number of lymphoid malignancies is characterized by specific chromosomal translocations, which are closely linked to the initial steps of pathogenesis. The hallmark of these translocations is the ectopic activation of a silent proto-oncogene through its relocation at the vicinity of an active regulatory element. Due to the unique feature of lymphoid cells to somatically rearrange and mutate receptor genes, and to the corresponding strong activity of the immune enhancers/promoters at that stage of cell development, B- and T-cell differentiation pathways represent propitious targets for chromosomal translocations and oncogene activation. Recent progress in the understanding of the V(D)J recombination process has allowed a more accurate definition of the translocation mechanisms involved, and has revealed that V(D)J-mediated translocations result both from targeting mistakes of the recombinase, and from illegitimate repair of the V(D)J recombination intermediates. Surprisingly, V(D)J-mediated translocations turn out to be restricted to two specific sub-types of lymphoid malignancies, T-cell acute lymphoblastic leukemias, and a restricted set of mature B-cell Non-Hodgkin's lymphomas.