Heterogeneous patterns of amplification of the NUP214-ABL1 fusion gene in T-cell acute lymphoblastic leukemia.

Episomes with the NUP214-ABL1 fusion gene have been observed in 6% of T-ALL. In this multicentric study we collected 27 cases of NUP214-ABL1-positive T-ALL. Median age was 15 years with male predominance. Outcome was poor in 12 patients. An associated abnormality involving TLX1 or TLX3 was found in all investigated cases. Fluorescent in situ hybridization revealed a heterogeneous pattern of NUP214-ABL1 amplification. Multiple episomes carrying the fusion were detected in 24 patients. Episomes were observed in a significant number of nuclei in 18 cases, but in only 1-5% of nuclei in 6. In addition, intrachromosomal amplification (small hsr) was identified either as the only change or in association with episomes in four cases and two T-ALL cell lines (PEER and ALL-SIL). One case showed insertion of apparently non-amplified NUP214-ABL1 sequences at 14q12. The amplified sequences were analyzed using array-based CGH.These findings confirm that the NUP214-ABL1 gene requires amplification for oncogenicity; it is part of a multistep process of leukemogenesis; and it can be a late event present only in subpopulations. Data also provide in vivo evidence for a model of episome formation, amplification and optional reintegration into the genome. Implications for the use of kinase inhibitors are discussed.

Immunopotentiation of the antibody response against influenza HA with apoptotic bodies generated by rabies virus G-ERA protein-driven apoptosis.

Apoptosis is considered to be a way of eliminating unwanted cells without causing major inflammation. Nevertheless, several lines of evidence show that apoptotic cell-derived antigens can be strong immunogens. The rabies virus glycoprotein G-ERA is an apoptotic molecule. We tested the ability of G-ERA to potentiate a B cell response against an exogenous antigen (influenza hemagglutinin, HA). We found that co-expression of G-ERA and HA in apoptotic bodies increased both the primary and memory HA-specific immune responses. The immunopotentiation of G-ERA is apoptosis-mediated but not necrosis-mediated. Our data indicate that G-ERA-mediated apoptosis might be useful to improve the immunogenicity of live vaccines.

Autologous stem cell transplantation for acute myelogenous leukemia in first complete remission: a 6-year follow-up study of 101 patients from a single institution.

The objective of the study was to assess the long-term outcome and impact of stem cell source in patients with acute myelogenous leukemia (AML) who received ASCT in first complete remission (CR). A total of 101 patients (median age 46 years) were included in the study. Cytogenetic categories distribution was: favorable: 18%, intermediate: 42%, and unfavorable: 7%. More than one induction course was needed for CR in 21% of patients. In all, 78% of patients had received at least one course of high-dose ara-C before autologous stem cell transplantation (ASCT). Bone marrow (n=58) or peripheral blood stem cells (PBSC) (n=43) transplantation was performed at a median of 3.5 months from CR. Hematologic recovery and hospitalization duration were significantly reduced in the PBSC group. No toxic death was recorded in this group. The median follow-up of survivors is 67 months (range: 15-183). The 6-year survival, disease-free survival (DFS), and relapse probabilities are 44%, 38%, and 54%, respectively. The presence of a favorable karyotype and the use of PBSC are independently associated to better survival, and DFS by multivariate analysis. Our results confirm that long-term DFS can be achieved with high-dose chemotherapy and ASCT in patients with AML. They show that use of PBSC is associated to very low mortality rate and acceptable morbidity and contributes to an improvement of autotransplant results.

New mechanisms of AML1 gene alteration in hematological malignancies.

The human AML1 gene (also named CBFA2 or RUNX1), located in the 21q22 chromosomal band, encodes for one of the two subunits forming a heterodimeric transcription factor, the human core binding factor (CBF). AML1 protein contains a highly evolutionary conserved domain of 128 amino acids called runt domain, responsible for both heterodimerization with the beta subunit of CBF and for DNA binding. AML1 is normally expressed in all hematopoietic lineages and acts to regulate the expression of various genes specific to hematopoiesis playing a pivotal role in myeloid differentiation. AML1 is one of the genes most frequently deregulated in leukemia through different mechanisms including translocation, mutation and amplification. Translocations lead to the formation of fusion genes encoding for chimerical proteins such as AML1-ETO which induces leukemogenesis. Recently, new mechanisms of AML1 deregulation by point mutations or amplification have been reported. To our knowledge, 51 patients (among 805 studied) with AML1 point mutations have been described. Forty of them have acute myeloid leukemia (AML) most often M0 AML. In this subtype of AML, the frequency of AML1 mutation is significantly higher; 21.5% of patients mutated (34/158). Mutations have also been found with lower frequency in other FAB subtype AML (6 cases), in myeloproliferative disorders (6 cases), in myelodysplastic syndrome (3 cases) and rarely in acute lymphoblastic leukemia (1 case). AML1 gene amplification has been found essentially in childhood ALL (12 cases) and more rarely in myeloid malignancies (4 cases). Here, we reviewed all these cases of AML1 point mutations and amplification and focused on the mechanisms of AML1 deregulation induced by these alterations.

Survey of early disapearance of BCR/ABL fusion transcript after allogeneic or autologous stem cell transplantation for chronic myelogenous leukemia.

The detection of BCR-ABL specific RNA by RT-PCR has been shown to predict relapse when positive 6 months after allogeneic stem cell transplantation (SCT) for chronic myelogenous leukemia (CML). In the present study, the focus was on evaluation of residual disease during the first weeks following SCT. In this study, 177 blood or marrow samples were obtained from 33 patients who received allogeneic (20 patients) or autologous (13 patients) SCT on day 0, day 30 and every 3 months for 1 year. T-cell depletion (TCD) was performed in 4 cases. On day 0 (day of graft infusion), 10/30 evaluable patients had negative RT-PCR (33%) regardless of pretransplant characteristics. On day 30, 14/18 patients (77%) from the allogeneic group had negative RT-PCR versus 0% in the autologous group. 2/4 patients who received TCD allogeneic grafts had day 30-positive PCR. Five patients in the allogeneic group had at least one positive RT-PCR sample between day 30 and day 90: 3 of them subsequently relapsed suggesting possible correlation between early positivity and relapse. Our results show that disappearance of MRD can be achieved within 3 months after transplantation in the majority of patients treated with allogeneic but not after autologous SCT. This suggests that the GVL effect might be operational early during the first weeks following transplantation.

Rabies virus ocular disease: T-cell-dependent protection is under the control of signaling by the p55 tumor necrosis factor alpha receptor, p55TNFR.

Following brain infection, the Challenge Virus Standard strain of rabies virus infects the retina. Rabies virus ocular infection induces the infiltration of neutrophils and predominantly T cells into the eye. The role of tumor necrosis factor alpha (TNF-alpha)-lymphotoxin signaling in the control of rabies virus ocular infection and inflammatory cell infiltration was assessed using mice lacking the p55 TNF-alpha receptor (p55TNFR(-/-) mice). The incidence of ocular disease and the intensity of retinal infection were greater in p55TNFR(-/-) mice than in C57BL/6 mice: the aggravation correlated with less neutrophil and T-cell infiltration. This indicates that cellular infiltration is under the control of the p55 TNF-alpha receptor and suggests that inflammatory cells may protect the eye against rabies virus ocular infection. The role of T cells following rabies virus ocular disease was assessed by comparison of rabies virus infection in nude mice with their normal counterparts. Indeed, the incidence and severity of the rabies virus ocular disease were higher in athymic nude mice than in BALB/c mice, indicating that T lymphocytes are protective during rabies virus ocular infection. Moreover, few T cells and neutrophils underwent apoptosis in rabies virus-infected retina. Altogether, these data suggest that T lymphocytes and neutrophils are able to enter the eye, escape the immune privilege status, and limit rabies virus ocular disease. In conclusion, rabies virus-mediated eye disease provides a new model for studying mechanisms regulating immune privilege during viral infection.

Selective role for the p55 Kd TNF-alpha receptor in immune unresponsiveness induced by an acute viral encephalitis.

Brain infection by the laboratory strain challenge virus standard (CVS), a highly neurotropic strain of rabies virus, causes splenocytes to become less responsive to in vitro stimulation with ConA. CVS-induced immune unresponsiveness is less severe in mice lacking the p55 Kd TNF-alpha receptor (p55TNFR(-/-)) than in C57BL/6 mice, despite a similar invasion of the brain. Comparison of CVS infection in these two strains of mice indicated that decreased immune responsiveness is associated with: (1) an in vivo reduction of the percentages of Th1 (IL-2, IFN-gamma and TNF-alpha) but not of Th2 (IL-4) cytokine-secreting T cells; and (2) an in vivo increase of the percentages of CD25 and CD69-expressing splenocytes. In contrast, CVS-induced immune unresponsiveness is not associated with abnormal percentage of T, B, NK cells or monocytes in vivo. The reductions of the CD4/CD8 ratio and of splenocyte expression of I-A(b) during CVS infection are similar in p55TNFR(-/-) and C57BL/6 mice indicating that these two parameters are not linked to the decreased responsiveness of splenocytes. These data suggest that CVS-induced immune unresponsiveness is under the control of the p55 Kd TNF-alpha receptor. We propose that T cell activation through this receptor, in an environment of poor antigen presentation, results in a state of T cells characterized by the reduced production of IL-2, TNF-alpha and IFN-gamma in vivo, the decreased responsiveness of splenocytes to ConA stimulation in vitro and the expression of the activation markers CD25 and CD69.

Absence of the p55 Kd TNF-alpha receptor promotes survival in rabies virus acute encephalitis.

We investigated the role played by inflammation in acute encephalitis following infection with a neurotropic virus by comparing the disease caused by the CVS strain of rabies virus in C57BL/6 and mice deficient for the p55 Kd TNF-alpha receptor (p55TNFR-/-). Morbidity (weight loss and paralysis) and mortality of infected mice were associated with viral propagation, cytokine (IL-6, IL-10, TNF-alpha and IFN-gamma) production, induction of apoptosis and infiltration of inflammatory cells. Mortality occurred later in p55TNFR-/- (than in C57BL/6 mice. In contrast, morbidity and the number of cells undergoing apoptosis were similar in C57BL/6 and p55TNFR-/- mice.) This suggests that morbidity and mortality are independently regulated and that the death of the animal was not due to CNS apoptosis. Delayed mortality correlated with: a reduction in viral load on day 9 p.i., an increase in IFN-gamma and IL-10 concentrations and a reduction in inflammatory cell infiltration in the CNS. Thus, these data indicate that CVS infection elicits an inflammatory response within the CNS and suggest that cytokines signaling via the p55 Kd TNF-alpha receptor is deleterious for the survival of the host. These results strongly suggest that, the modulation of TNF-alpha and upregulation of IFN-gamma would be a powerful anti-virus strategy in cases of viral encephalitis.

Treatment of chronic myelogenous leukemia with interleukin-2: a phase II study in 21 patients.

We designed a phase II study to assess the activity of recombinant interleukin-2 (rIL-2) in patients with chronic myelogenous leukemia (CML). Study population included 11 patients in the chronic phase of CML (6 in hematologic remission and 5 with active disease), 6 patients in the accelerated phase, and 4 in blastic phase of CML. Patients received three 5-day cycles administrated every other week. rIL-2 was given as intravenous bolus infusions of 8 x 10(6) IU/m2 three times a day during cycle 1 and twice a day during cycles 2 and 3. Response to rIL-2 was assessed on day 45. No hematologic response was achieved in the patients with evaluable disease. One patient in hematologic remission with rIL-2 achieved a major response (from 72% to 9% Ph+ metaphases), and two patients had some degree of reduction of Ph+ metaphases. Responses were short-lived (< 6 months), but two of these three patients achieved a new cytogenetic response with interferon given post-rIL-2. A significant immune activation was achieved with rIL-2 including a marked increase in CD3+/CD25+ cells, CD56+ cells, and in natural killer/lymphokine activated killer cell cytotoxic activity. These results confirm preclinical studies, which showed that IL-2 has antileukemic activity in CML. However, the responses observed were short lived and restricted to a subgroup of patients with low disease burden. This invites further studies testing its impact in situations of minimal disease or in combination with other cytokines.

Chromosome 8 tetrasomies and pentasomies--a clonal abnormality closely associated with acute monocytic leukaemia.

We report four cases of polysomy 8 (one tetrasomy and three pentasomies) observed in acute monocytic leukemia (FAB M4 and M5). Three of them showed a rearrangement of 11q23 identified by conventional cytogenetic analysis and/or chromosome painting. Our cases as well as a review of the literature, suggest that polysomy 8 is preferentially associated with monocytic differentiation (24/31). These polysomies have been observed in 21 de novo leukemias and in 10 secondary hematological disorders. A 11q23 rearrangement has been detected in 9 out of 32 patients, by conventional cytogenetic techniques in 7 and by FISH in 2. We suggest that these cases should be analysed by FISH and molecular studies in order to detect a rearrangement of MLL/11q23. Monocytic differentiation is often associated with a change of the MLL gene and the polysomy 8 might be a particular clonal evolution secondary to 11q23 abnormality.

Rabies virus infects mouse and human lymphocytes and induces apoptosis.

Attenuated and highly neurovirulent rabies virus strains have distinct cellular tropisms. Highly neurovirulent strains such as the challenge virus standard (CVS) are highly neurotropic, whereas the attenuated strain ERA also infects nonneuronal cells. We report that both rabies virus strains infect activated murine lymphocytes and the human lymphoblastoid Jurkat T-cell line in vitro. The lymphocytes are more permissive to the attenuated ERA rabies virus strain than to the CVS strain in both cases. We also report that in contrast to that of the CVS strain, ERA viral replication induces apoptosis of infected Jurkat T cells, and cell death is concomitant with viral glycoprotein expression, suggesting that this protein has a role in the induction of apoptosis. Our data indicate that (i) rabies virus infects lymphocytes, (ii) lymphocyte infection with the attenuated rabies virus strain causes apoptosis, and (iii) apoptosis does not hinder rabies virus production. In contrast to CVS, ERA rabies virus and other attenuated rabies virus vaccines stimulate a strong immune response and are efficient live vaccines. The paradoxical finding that a rabies virus triggers a strong immune response despite the fact that it infects lymphocytes and induces apoptosis is discussed in terms of the function of apoptosis in the immune response.

Early establishment of chimerism in the B and T lymphoid lineages after transplantation of allogeneic mobilized blood cells in leukemic patients.

BACKGROUND: The use of allogeneic recombinant human granulocyte colony-stimulating factor (rhG-CSF)-mobilized blood cells was recently evaluated in patients with malignancies. METHODS: Ten patients with leukemia were transplanted with allogeneic blood cells from HLA-identical sex-mismatched siblings; blood cells were mobilized with recombinant rhG-CSF. Up to 6 months after transplantation, blood and bone marrow samples were obtained from the recipient and analyzed for the presence of donor cells, using fluorescence in situ hybridization with specific probes hybridizing to sex chromosomes. RESULTS: Analysis of blood and bone marrow smears demonstrated a complete chimera, as early as day 15 after transplantation. Furthermore, marrow and blood CD4+, CD8+, CD19+, and CD34+ cells were sorted using direct immunofluorescence and flow cytometry: fluorescence in situ hybridization analysis on sorted cells demonstrated that most progenitors and most cells in the T- and B-cell lineages were of donor origin as early as day 15 after transplantation. CONCLUSIONS: Together with recently reported results, this study demonstrates that allogeneic rhG-CSF-mobilized blood cells contain primitive hematopoietic progenitors that can repopulate all lymphoid and myeloid lineages. Establishment of chimerism seems to be quick and stable, including the T- and B-cell lineages. Although establishment of chimerism in mitogen-responsive T cells is readily assayable with conventional cytogenetics, our study provides additional insight on the reconstitution of the B lineage and T-cell subsets after allogeneic transplantation in patients with leukemia.

A pilot study of busulfan and melphalan as preparatory regimen prior to allogeneic bone marrow transplantation in refractory or relapsed hematological malignancies.

In this pilot study; we assessed the immunosuppressive and the antileukemic potential of a combination of busulfan and melphalan prior to allogeneic BMT in 25 adult patients with refractory or relapsed hematological malignancies. Twelve patients were transplanted for acute myeloid leukemia (relapse: five patients; primary refractory: four patients; second remission: two patients), two patients for primary refractory acute lymphoblastic leukemia, nine patients for chronic myelogenous leukemia (accelerated phase: six patients; blastic phase: three patients) and two patients for primary refractory lymphoma. All received an unmanipulated marrow from HLA-identical siblings. All patients but one engrafted (median time to ANC > or = 0.5 x 10(9)/l = 17 days, to platelets > or = 50 x 10(9)/l = 29 days). Full chimerism was documented in the seven evaluable patients. The probability for developing acute GVHD was 58%. Complete remission was obtained in 17/18 measurable patients. With a 42 month median follow-up, eight patients are alive in unmaintained remission. The 4-year probabilities for relapse, survival, and DFS are respectively: 42%, 35%, and 31%. These results show that the combination of busulfan and melphalan ensures an effective immunosuppression allowing long-term engraftment. This regimen can provide long-term disease-free survival in patients with high-risk disease and thus represents an interesting alternative to the CY and/or TBI-containing regimens.

Atypical osteomalacia after 2 year etidronate intermittent cyclic administration in osteoporosis.

A 68-year-old postmenopausal woman with osteoporosis received intermittent cyclic etidronate therapy (400 mg/day). No concomitant diseases were noted. A baseline transiliac bone biopsy showed a high bone turnover without impairment due to mineralization. After 2 years of treatment, she had increased osteoid volume, and mineralization lag time corresponded to a variant form of osteomalacia called atypical osteomalacia. No clinical consequence was observed. This is the first case report of such a mineralization defect due to 2 year intermittent cyclic etidronate therapy.

Persistence of BCR/ABL mRNA-expressing bone-marrow cells in patients with chronic myelogenous leukemia in complete cytogenetic remission induced by interferon-alpha therapy.

Complete hematologic and cytogenetic responses can be obtained with interferon-alpha (IFN-alpha) in 15-25% of the patients with chronic myelogenous leukemia (CML). In these patients, reverse-transcription polymerase chain reaction (RT-PCR) can be used to evaluate minimal residual disease. We studied 12 patients who remained Philadelphia-negative for a median period of 21 months on IFN-alpha therapy. Using RT-PCR, the specific transcript was found in all bone marrow (BM) samples. Ten patients still exhibiting a persistent residual clone remained in cytogenetic remission for a median period of 14 months. As we observed a dissociation between bcr-abl expression in BM and peripheral blood (PB) cells, and given the known fluctuations of the bcr-abl PCR results, we suggest that PB negative results should be confirmed on BM specimens. Alternatively, it remains to be demonstrated whether longitudinal monitoring of residual disease would benefit from quantitative PCR or double fluorescence in situ hybridization.

Philadelphia chromosome-negative chronic myeloid leukaemia: a report of 14 new cases.

The Philadelphia chromosome (Ph) is the cytogenetic hallmark of chronic myeloid leukaemia (CML) and is used to confirm the diagnosis of CML based on clinical and morphological criteria. We investigated 14 patients with features of CML but without detectable Ph chromosome. In seven patients, referred to as BCR+, M-bcr/abl rearrangement was detected by polymerase chain reaction (PCR). The seven remaining patients did not have M-bcr/abl rearrangement and are described as BCR-. BCR- patients were younger, had lower white blood cell counts (WBC) and lower basophilia. Four BCR- and four BCR+ patients underwent blastic transformation (BT). Response to therapy was fairly similar in both populations. According to French-American-British (FAB) Cooperative Leukaemia Group guidelines, all BCR- patients were classified as having classic form CML or 'chronic granulocytic leukaemia' (CGL) when based only on morphological data. This study further confirms the existence of true CML cases without Ph chromosome or M-bcr/abl rearrangement and shows that this entity differs only slightly from classic form Ph+ CML. The Ph-BCR- subgroup raises two problems. First, the differential diagnosis with atypical CML or CMML, based on morphological data, and secondly, the therapeutic follow-up in the absence of a specific marker. In contrast, the residual disease of Ph-BCR- patients can be monitored by PCR. More advanced molecular and biochemical techniques will be required to understand which molecular mechanisms underlie Ph-BCR- CML, resulting in phenotypes sometimes indistinguishable from Ph+ CML.

Measurement of rabies virus N protein in rabies vaccines.

In this study, we designed a capture ELISA using a monoclonal antibody specific for the N protein, the major protein of the rabies virus nucleocapsid, to measure the N protein content in rabies vaccines. Free N protein content was compared in the two types of rabies vaccine currently used in humans: suckling mouse brain (SMB) vaccine prepared from rabies virus-infected brain tissue, and tissue culture (TC) vaccine prepared from supernatants of rabies virus-infected cells. It was found that SMB vaccines contained considerably higher amounts of N protein than most of the TC vaccines. Possible implications concerning the efficacy of rabies vaccines are discussed.