An intragenic ERG deletion is a marker of an oncogenic subtype of B-cell precursor acute lymphoblastic leukemia with a favorable outcome despite frequent IKZF1 deletions.

Oncogenic subtypes in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are used for risk stratification. However, a significant number of BCP-ALL patients are still genetically unassigned. Using array-comparative genomic hybridization in a selected BCP-ALL cohort, we characterized a recurrent V(D)J-mediated intragenic deletion of the ERG gene (ERG(del)). A breakpoint-specific PCR assay was designed and used to screen an independent non-selected cohort of 897 children aged 1-17 years treated for BCP-ALL in the EORTC-CLG 58951 trial. ERG(del) was found in 29/897 patients (3.2%) and was mutually exclusive of known classifying genetic lesions, suggesting that it characterized a distinct leukemia entity. ERG(del) was associated with higher age (median 7.0 vs. 4.0 years, P=0.004), aberrant CD2 expression (43.5% vs. 3.7%, P<0.001) and frequent IKZF1 Δ4-7 deletions (37.9% vs. 5.3%, P<0.001). However, ERG(del) patients had a very good outcome, with an 8-year event-free survival (8-y EFS) and an 8-year overall survival of 86.4% and 95.6%, respectively, suggesting that the IKZF1 deletion had no impact on prognosis in this genetic subtype. Accordingly, within patients with an IKZF1 Δ4-7 deletion, those with ERG(del) had a better outcome (8-y EFS: 85.7% vs. 51.3%; hazard ratio: 0.16; 95% confidence interval: 0.02-1.20; P=0.04). These findings have implications for further stratification including IKZF1 status.

Clinical, cytogenetic and molecular characteristics of 14 T-ALL patients carrying the TCRbeta-HOXA rearrangement: a study of the Groupe Francophone de Cytogénétique Hématologique.

Recently, we and others described a new chromosomal rearrangement, that is, inv(7)(p15q34) and t(7;7)(p15;q34) involving the T-cell receptor beta (TCRbeta) (7q34) and the HOXA gene locus (7p15) in 5% of T-cell acute lymphoblastic leukemia (T-ALL) patients leading to transcriptional activation of especially HOXA10. To further address the clinical, immunophenotypical and molecular genetic findings of this chromosomal aberration, we studied 330 additional T-ALLs. This revealed TCRbeta-HOXA rearrangements in five additional patients, which brings the total to 14 cases in 424 patients (3.3%). Real-time quantitative PCR analysis for HOXA10 gene expression was performed in 170 T-ALL patients and detected HOXA10 overexpression in 25.2% of cases including all the cases with a TCRbeta-HOXA rearrangement (8.2%). In contrast, expression of the short HOXA10 transcript, HOXA10b, was almost exclusively found in the TCRbeta-HOXA rearranged cases, suggesting a specific role for the HOXA10b short transcript in TCRbeta-HOXA-mediated oncogenesis. Other molecular and/or cytogenetic aberrations frequently found in subtypes of T-ALL (SIL-TAL1, CALM-AF10, HOX11, HOX11L2) were not detected in the TCRbeta-HOXA rearranged cases except for deletion 9p21 and NOTCH1 activating mutations, which were present in 64 and 67%, respectively. In conclusion, this study defines TCRbeta-HOXA rearranged T-ALLs as a distinct cytogenetic subgroup by clinical, immunophenotypical and molecular genetic characteristics.

Cyclin D2 dysregulation by chromosomal translocations to TCR loci in T-cell acute lymphoblastic leukemias.

Strong expression of at least one of the three D-type cyclins is common in human cancers. While the cyclin D1 and D3 genes (CCND1 and CCND3) are recurrently involved in genomic rearrangements, especially in B-cell lymphoid neoplasias, no clear involvement of the cyclin D2 gene (CCND2) has been reported to date. Here, we identified chromosomal translocations targeting the CCND2 locus at 12p13, and the T-cell receptor beta (TCRB) or the TCRA/D loci in T-cell acute lymphoblastic leukemias (T-ALLs). Expression analysis demonstrated dramatic cyclin D2 overexpression in the translocated cases (n=3) compared to other T-ALLs (total, n=89). In order to evaluate dysregulation in T-ALL with respect to normal T-cell differentiation, we analyzed CCND2 expression in normal purified human thymic subpopulations. CCND2 levels were downregulated through progression from the early stages of human T-cell differentiation, further suggesting that the massive and sustained expression in the CCND2-rearranged T-ALL cases was oncogenic. Association with other oncogene expression (TAL1, HOXAs, or TLX3/HOX11L2), NOTCH1 activating mutations, and/or CDKN2A/p16/ARF deletion, showed that cyclin D2 dysregulation could contribute to multi-event oncogenesis in various T-ALL groups. This report is the first clear evidence of a direct involvement of cyclin D2 in human cancer due to recurrent somatic genetic alterations.

Disruption of T cell regulatory pathways is necessary for immunotherapeutic cure of T cell acute lymphoblastic leukemia in mice.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. In recent years, the outcome has been globally improved by current therapies, but it remains poor in patients with high, persistent residual disease following the first course of chemotherapy, prompting evaluation of the possible beneficial effects of immunotherapy protocols. In this study, we hypothesized that the disruption of two immunoregulatory pathways controlling the auto-reactive T cell response might synergize with dendritic cell-based immunotherapy of the disease, which is considered to be poorly immunogenic. In this study, we used TAL1xLMO1 leukemia cells adoptively transferred in mice, to generate murine leukemia with poorly immunogenic cells as a model for human T-ALL. Subsequently, these animals were treated with several different immunotherapeutic protocols. We compared the efficiency of a classical, dendritic cell-based immunotherapy (injection of dendritic cells loaded with tumor-derived antigenic products), to a combined treatment associating injection of antigen-loaded dendritic cells and disruption of the two immunoregulatory pathways: CD25+ suppressive T cells and cytotoxic T lymphocyte-associated antigens (CTLA-4). We show that this combined treatment resulted in cure, concomitantly with in vivo generation of immune memory, and TNF-alpha secretion. This study demonstrates that the disruption of these two immunoregulatory pathways synergized with immunostimulation by dendritic cells loaded with tumor-derived antigens, and paves the way for the testing of this combination in clinical trials.

[Development of a new strategy for minimal residual disease monitoring in children with B-precursor acute lymphoblastic leukemia]. / Mise en place d'une stratégie de suivi de la maladie résiduelle chez les enfants atteints de leucémies aiguës lymphoblastiques préB.

Despite modern regimen of chemotherapy, one-third of children with acute lymphoblastic leukaemia relapse. Recent studies have shown that a high level of minimal residual disease (MRD) at the end of induction is associated with an increased risk of relapse. We have developed and validated a real-time PCR method (RQ-PCR) to quantify MRD. We monitored 57 patients using IgH and TCR (Vdelta2Ddelta3) genes rearrangements as PCR targets. RQ-PCR was performed with a primer and a TaqMan probe designed to consensus sequences in VH segments in combination with one allele specific oligonucleotide primer complementary to the junctionnal region. A sensitivity of 10(-4) was reached for 72% of the IgH alleles (n = 50) and for 54,5% of the Vdelta2Ddelta3 alleles (n = 22). We compared the results with those obtained by competitive PCR in 53 patients: no discordance between the two methods was observed. Seventeen patients were found positive (32%) and 27 negative (51%) with both techniques and 9 children (17%) were positive only with TaqMan technology. RQ-PCR is more sensitive and more specific than competitive PCR. We thus propose that RQ-PCR might be used in first intention for MRD analysis.

Varicella-zoster viral meningitis mimicking lymphoma.

We report the case of a 30-year-old HIV-infected man admitted for a meningeal syndrome and a zoster rash. The CSF had cytological features suggesting a primary CNS lymphoma (PCNSL). The large lymphoid cells had a fine chromatin with nucleoli, a basophilic cytoplasm with azurophilic granules and high mitotic activity. Several arguments demonstrated the viral origin of the meningitis: the large lymphoid cells were of T origin with no evidence of clonal TCR gamma gene rearrangement. The PCR was positive for Varicella-Zoster Virus (VZV) and EBV DNA. Clinical evolution was favorable under acyclovir. We should be cautious in the differential diagnosis between viral meningitis and PCNSL.

LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.

We have previously shown correction of X-linked severe combined immunodeficiency [SCID-X1, also known as gamma chain (gamma(c)) deficiency] in 9 out of 10 patients by retrovirus-mediated gamma(c) gene transfer into autologous CD34 bone marrow cells. However, almost 3 years after gene therapy, uncontrolled exponential clonal proliferation of mature T cells (with gammadelta+ or alphabeta+ T cell receptors) has occurred in the two youngest patients. Both patients' clones showed retrovirus vector integration in proximity to the LMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2. Thus, retrovirus vector insertion can trigger deregulated premalignant cell proliferation with unexpected frequency, most likely driven by retrovirus enhancer activity on the LMO2 gene promoter.

Prognostic significance of FLT3 internal tandem repeat in patients with de novo acute myeloid leukemia treated with reinforced courses of chemotherapy.

FLT3 internal tandem duplications (FLT3-ITDs) are present in nearly 25% of patients with AML and have been associated with poor response to conventional therapy and poor outcome. We retrospectively evaluated the effect of reinforced courses of chemotherapy on the prognostic value of FLT3-ITDs in 159 AML patients prospectively enrolled in the ALFA-9000 trial, which randomly compared three reinforced induction regimens (standard 3+7 including high-dose daunorubicin, double induction, and timed-sequential therapy). FLT3-ITD was present in 40/159 (25%) blast samples and associated with high WBC (P = 0.002) and cytogenetics (P < 0.001) with a higher incidence (35%) in patients with a normal karyotype. There was no difference in CR rate between FLT3-wt and FLT3-ITD patients (80% vs 78%). Relapse-free survival (RFS) was similar in both groups (5-year RFS, 33% vs 32%; P = 0.41), even after adjustment for age, sex, WBC, cytogenetics, and treatment arm. A trend to a worse survival was observed in the FLT3-ITD group (estimated 5-year OS, 23% vs 37%; P = 0.09), mainly in patients with a normal karyotype. This was associated with a dramatic outcome in relapsing FLT3-ITD patients (estimated 3-year post-relapse survival, 0% vs 27%; P = 0.04). These results suggest that the bad prognosis associated with FLT3-ITDs in AML might be partly overcome using reinforced chemotherapy. Early detection of FLT3 mutations might thus be useful to intensify induction as well as post-remission therapy in FLT3-ITD patients.

Contribution of nitric oxide to the apoptotic process in human B cell chronic lymphocytic leukaemia.

B cell chronic lymphocytic leukaemia (B-CLL) is characterised by defective apoptosis that cannot be explained solely on the basis of the known chromosomal abnormalities. We and other have now reported that the leukemic cells spontaneously display the inducible isoform of nitric oxide synthase, iNOS. Inhibition of the iNOS pathway leads to increased apoptosis of the tumoral cells in vitro, indicating that the endogenous release of NO contributes to their resistance to the normal apoptotic process. The factors that induce the expression of iNOS in vivo in the leukemic cells are not yet identified. Yet, as interaction of B-CLL leukemic cells with bone marrow stromal cells promotes their survival, the involvement of adhesion molecules and integrins may be suspected. The engagement of CD23 stimulates iNOS activation in the tumoral cells, suggesting that in vivo interaction of CD23 with one of its recognised ligands may contribute to iNOS induction. A role for CD40-CD40 ligand interaction may also be hypothesised. The mechanisms involved in the anti-apoptotic role of NO are not fully understood, but may implicate the inhibition of caspase activity, hence the impairment of the Fas pathway. In addition, the mitochondrial membrane potential disruption appears to be a NO-sensitive step in the apoptosis cascade. The presence of a NOS displaying anti-apoptotic properties has now been recognised in different cell types, including various leukaemia. A better knowledge of the mechanisms governing the ultimate fate of NO, anti- versus pro-apoptotic would allow the development of new therapeutic approaches for the treatment of these diseases.

A complex pattern of recurrent chromosomal losses and gains in T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is a rare malignant proliferation of lymphoid cells with a postthymic phenotype. Previous cytogenetic and molecular studies reported complex karyotypes with recurrent chromosomal abnormalities, including translocations involving either TCL1 at 14q32.1 or MTCP1 at Xq28, inactivation of the ATM gene by deletion and/or mutation, and isochromosomes 8. For extensive study of chromosomal imbalances in T-PLL, we analyzed 22 tumoral DNAs using comparative genomic hybridization (CGH). Abnormal CGH profiles were detected in all cases, demonstrating highly recurrent gains and losses and largely extending the abnormalities previously established. Only a few nonrecurrent abnormalities were observed, in contrast to the genetic instability anticipated from ATM inactivation. Nine recurrent regions of loss were identified at 8p (frequency 86%), 11q (68%), 22q11 (45%), 13q (41%), 6q (36%), 9p (27%), 12p (23%), 11p11-p14 (23%), and 17p (23%), as well as four regions of gain at 8q (82%), 14q32 (50%), 22q21-qter (41%), and 6p (23%). Several recurrent chromosomal abnormalities were simultaneously present in each case (mean, 5.7; up to 10), none being mutually exclusive of another. Fluorescence in situ hybridization analysis confirmed and extended 22q11 and 13q losses, giving final frequencies of 55% and 45%, respectively. Analysis of one case over a 7-year period confirmed the overall genetic stability of T-PLL and showed that tumor progression was associated with the onset of a few chromosomal abnormalities. This study establishes a complex pattern of highly recurrent chromosomal abnormalities in T-PLL, including some, such as chromosome 13 deletion, commonly found in other lymphoid malignancies.

Diffuse large B-cell non-Hodgkin's lymphoma in a patient with autoimmune lymphoproliferative syndrome.

Mutations of Fas or Fas ligand genes result in the autoimmune lymphoproliferative syndrome (ALPS) in humans. We report here a diffuse large B-cell non-Hodgkin's lymphoma occurring in a man with ALPS. Fas-mediated lymphocyte apoptosis was defective in vitro, owing to a mutation within the death domain of the Fas molecule. High-dose methotrexate and doxorubicin-based chemotherapy led to complete remission of lymphoma.

A clinical, molecular and cytogenetic study of 12 cases of human herpesvirus 8 associated primary effusion lymphoma in HIV-infected patients.

INTRODUCTION: Primary effusion lymphoma is a rare type of B-cell lymphoproliferative disorder which is mainly observed in patients with HIV infection. Lymphomatous cells bridge features of immunoblastic and anaplastic cells with a non-B non-T phenotype and are characterized by the presence of the human herpesvirus 8 genome. We report on the retrospective analysis of 12 cases. PATIENTS AND METHODS: : Twelve HIV-infected patients with serous effusions containing large HHV8(+) lymphomatous cells were extensively evaluated to disclose associated visceral involvement. Clonality was assessed by IgH gene rearrangement PCR analysis (n = 11) or Southern blot (n = 1). EBV and HHV8 DNA sequences were detected by PCR analysis. Cytogenetics studies were performed in seven cases using RHG-banding. RESULTS: Extraserous localizations of lymphoma were present in six cases (50%): mediastinal (n = 2), mesenteric (n = 2), pancreatic (n = 1), and bone marrow involvement (n = 1). A monoclonal rearrangement of IgH genes was demonstrated in six cases, an oligoclonal pattern in one, whereas no clonality could be detected in five. High HHV8 copy numbers were demonstrated in all effusion fluids, with EBV-co-infection in all cases but one. Cytogenetic analysis displayed a complex karyotype in all cases without recurrent abnormalities. Eight patients have died. Three patients are in complete remission at 28, 53 and 55 months after high-dose chemotherapy (n = 1), cidofovir and alpha-interferon combination therapy (n = 1), and antiretroviral therapy alone (n = 1). CONCLUSION: The clinical and molecular pattern, as well as the response to therapy suggest that primary effusion lymphoma represents an heterogenous type of virus-induced B-cell lymphoproliferative disorder, sharing pathophysiological features with that induced by the Epstein-Barr virus and occurring in immunocompromised patients.

Expression and prognostic significance of survivin in de novo acute myeloid leukaemia.

Survivin is an inhibitor of apoptosis (programmed cell death) overexpressed in various human cancers, but undetectable in normal differentiated tissues. A potential distribution and prognostic significance of survivin in patients with de novo acute myeloid leukaemia (AML) was investigated. By immunofluorescence of bone marrow specimens and peripheral blood mononuclear cells, survivin was detected in 75 out of 125 interpretable AML cases (60%), with reactivity in 50-90% of AML cells. Survivin expression correlated with a lower white blood cell count (WBC) (P = 0.008 by the Mann-Whitney test) and was associated, in the 55 cases of FAB M0/M1/M2, with leukaemic granulocytic maturation (one out of five M/L0, 11 out of 22 M/L1 and 23 out of 28M/L2; P = 0.007 by the Fisher test). In 69 patients treated with the Acute Leukaemia French Association (ALFA) 9000 protocol, survivin expression was significantly associated with a lower WBC (P = 0.03 by the Mann-Whitney test) and favourable/intermediate cytogenetics (P= 0.03 by the Fisher test). There was no significant difference in complete remission rate or overall survival between survivin-positive and survivin-negative AML patients (P = 0.15 by the log-rank test). However, survivin expression became an independent negative prognostic factor for survival when adjusted with the Cox model for established prognostic factors in AML (cytogenetics, age and WBC) or for the ALFA 9000 treatment arm (RR = 2.8 and P = 0.026, by the likelihood-ratio test). These data suggest that survivin expression may be considered as a new unfavourable prognostic factor of de novo AML and suggest a role for apoptosis inhibition in influencing disease outcome.

Expression of a functional inducible nitric oxide synthase in hairy cell leukaemia and ESKOL cell line.

The expression of nitric oxide synthase (NOS) isoforms was investigated in the established ESKOL hairy cell line and in leukemic cells of patients with hairy cell leukemia (HCL). By reverse transcription-polymerase chain reaction (RT-PCR), these cells were found to spontaneously express inducible NOS (iNOS)-specific mRNA, but not endothelial constitutive NOS (ecNOS) mRNA. The iNOS protein was detected by immunofluorescence in the cytoplasm of permeabilized leukemic cells and ESKOL cells, using different anti-iNOS monoclonal antibodies. A protein of 135 kDa was identified by Western blotting in ESKOL and HCL lysates, confirming the presence of an iNOS in these cells. Cytosolic homogenates displayed NOS catalytic activity, as measured by the conversion of 14C-labelled L-arginine into 14C L-citrulline and by detection in situ using the DAF-2DA (diaminofluorescein diacetate) NO-sensitive fluorescent probe. Ligation of CD23 (low affinity IgE receptor) was found to increase iNOS expression in ESKOL and conversely to decrease the percentage of cells undergoing apoptosis, as measured by the percentage of cells expressing annexin V. These results indicate that, as in chronic B cell lymphocytic leukemia cells (B-CLL) a functional iNOS is expressed constitutively in hairy cells that contributes to protecting these tumoral cells from apoptosis.

Interferon-gamma-induced membrane PAF-receptor expression confers tumor cell susceptibility to NK perforin-dependent lysis.

Perforin is known to display a membranolytic activity on tumor cells. Nevertheless, perforin release during natural killer (NK)-cell activation is not sufficient to induce membrane target-cell damage. On the basis of the ability of perforin to interact with phospholipids containing a choline phosphate headgroup, we identify the platelet-activating factor (PAF) and its membrane receptor as crucial components in tumor cell killing activity of human resting NK cells. We demonstrate for the first time that upon activation, naive NK cells release the choline phosphate-containing lysolipid PAF, which binds to perforin and acts as an agonist on perforin-induced membrane damage. PAF is known to incorporate cell membranes using a specific receptor. Here we show that interferon-gamma (IFN-gamma) secreted from activated NK cells ends in PAF-receptor expression on perforin-sensitive K562 cells but not on perforin-resistant Daudi cells. In order to prove the capacity of PAF to interact simultaneously with its membrane PAF receptor and with perforin, we successfully co-purified the 3 components in the presence of bridging PAF molecules. The functional activity of this complex was further examined. The aim was to determine whether membrane PAF-receptor expression on tumor cells, driven to express this receptor, could render them sensitive to the perforin lytic pathway. The results confirmed that transfection of the PAF-receptor complementary DNA into major histocompatibility complex class I and Fas-receptor negative tumor cells restored susceptibility to naive NK cells and perforin attack. Failure of IFN-gamma to induce membrane PAF receptor constitutes the first described mechanism for tumor cells to resist the perforin lytic pathway.