[Acute megakaryoblastic leukemia (AML-7) in a woman of 95 years]. / Leucémie aiguë mégacaryoblastique (LAM-7) chez une patiente de 95 ans.

Acute leukemia of megakaryocyte lineage (AML-7) is a rare entity defined by a blastic proliferation of which a part (>or= 50%) is represented by megakaryoblasts. We report the case of a 95 year old woman presenting a AML-7 secondary to a myelodysplastic syndrome (MDS), that represents an unusual form of MDS acutisation.

[Expression of proliferation marker Ki 67 in chronic lymphocytic leukemia]. / Expression du marqueur de proliferation Ki 67 dans les leucemies lymphoides chroniques.

Chronic lymphocytic leukemia (CLL) is characterized by a clonal expansion of low proliferating mature B and T lymphocytes in the bone marrow and peripheral blood. The nuclear antigen Ki 67 is a protein detected in G1, S, G2 and M phases of the cell cycle, but not in G0, and thus, is a widely accepted proliferation marker of Human tumors. The aim of this study was to evaluate Ki 67 monoclonal antibody in CLL. We studied 48 patients diagnosed as CLL on the presence of clinical signs, over 4.109/l circulating lymphoid cells and immunophenotyping by flow cytometry using CD19, CD5, CD22, CD23, FMC7 and immunoglobulin light chains monoclonal antibodies. Ki 67 immunostaining was determined by Avidin Biotin Complex method. Our results allows to characterize between CLL: one group which proliferation rate (percentage of Ki 67 positive cells) was equal or less than 2%, represented by 14 cases (29,2%) with morphological aspect of typical CLL, one group which proliferation rate was between 3% and 9% represented by 32 cases (66,6%) with morphological aspect of polymorph CLL or prolymphocytic leukemia, and a last group with proliferation rate equal or up to 10% and corresponding to two cases (4,2%) of transformation of CLL to high grade Non Hodgkin lymphoma. There were no correlation between Matutes immunological score and proliferation rate, as this rate was 2.9% in score < 3 and 2.7% in score > 3. This study confirm the Ki 67 usefulness in studying cellular proliferation, and underline that CLL with polymorphic cytology are more proliferate than typical CLL. These data reinforce the notion that CLL is a disease with heterogeneity in clinical behavior, immunophenotype, cytogenetic, molecular aspects, and thus, prognostic.

DEK-CAN molecular monitoring of myeloid malignancies could aid therapeutic stratification.

The t(6;9)(p23;q34) is a recurrent chromosomal abnormality observed in 1% of acute myelogenous leukemia (AML), which generates a fusion transcript between DEK and CAN/NUP214 genes. We used a DEK-CAN real-time quantitative (RQ)-PCR strategy to analyze 79 retrospective and prospective samples from 12 patients. Five patients reached DEK-CAN negativity (sensitivity 10(-5)); all underwent early allogeneic hematopoietic stem cell transplantation (median 5.5 months from diagnosis) with some demonstrating molecular positivity at the time of allograft. All four cases in CCR with adequate follow-up (median 18.5 months, range 13--95) demonstrate persistent molecular negativity, whereas all seven patients with persistent DEK-CAN positivity died at a median of 12 months from diagnosis (range 7--27). We conclude that DEK-CAN molecular monitoring by RQ-PCR in t(6;9) malignancies is a useful tool for individual patient management and that molecular negativity is indispensable for survival, but should not be a prerequisite for allografting in this rare, poor prognosis, subset of AML.

IgH DJ rearrangements within T-ALL correlate with cCD79a expression, an immature/TCRgammadelta phenotype and absence of IL7Ralpha/CD127 expression.

cCD79a and IgH VDJ/DJ rearrangements are considered to be relatively specific for B lymphoid precursors. We looked for both in cCD3+, CD7+, CD19- T-ALLs classified by TCR status into alphabeta or gammadelta/immature (IM) lineages, with individualization of HOX11L2+ T-ALLs since they represent an intermediate alphabeta/gammadelta category. cCD79a was expressed at low levels in 47% of T-ALL and was most frequent in IMgamma T-ALLs. IgH rearrangements were common in gammadelta/IM (45%) and HOX11L2+ (35%) T-ALLs compared to HOX11L2-negative cases (3%; P<0.001). CD127 (IL7Ralpha) expression was also more common in the gammadelta/IM lineage but its expression was virtually mutually exclusive of IgH rearrangement. Low-level cCD79a expression alone should therefore not be interpreted as evidence of B lineage affiliation in immature leukemias. gammadelta/IM lineage T-ALLs potentially include two distinct categories: predominantly IgH+, cCD79a+, CD127- cases which retain gammadelta and B lymphoid potential and IgH-, cCD79a-, CD127+ cases with restricted T lineage potential.

Human T-cell leukaemia/lymphoma virus type 1-associated infective dermatitis in Africa: a report of five cases from Senegal.

BACKGROUND: Infective dermatitis (ID) is a rare dermatological condition of childhood that has been linked to human T-cell leukaemia/lymphoma virus type 1 (HTLV-1). Most cases have been reported in the Caribbean. Although several million people are estimated to be infected by HTLV-1 in sub-Saharan Africa, no case of ID has been reported in this area. OBJECTIVES: To identify and to describe cases of HTLV-1-associated ID in Senegal, West Africa. METHODS: Over a 3-year period, a serological test for HTLV-1 was performed at a dermatological centre in Dakar, Senegal, in children who presented with a picture suggestive of ID. Complementary haematological, immunological and virological investigations were performed in infected children and in their mothers. RESULTS: Five patients with typical HTLV-1-associated ID were identified, of ages 17, 5, 4, 3 and 3 years; two patients belonged to the same family. They all presented with repeated flares of superinfected dermatitis involving typical sites of ID (mainly the scalp, external ears, nares and eyelids), associated with nasal discharge, and less commonly with a nonspecific papular rash on the face or trunk. Although oral antibiotic therapy always gave effective control of the symptoms, recurrences were constant. A persisting dry dermatitis of the retroauricular folds was common between flares. Infection in the oldest patient was associated with a chronic adult T-cell leukaemia/lymphoma. The mothers of three patients, and the grandmother of another, were all infected by HTLV-1 strains belonging to the Cosmopolitan molecular subtype, with a perfect nucleotide identity of long-terminal repeat and env gp21 genomic regions within each family. CONCLUSIONS: We present the clinical and virological features of the first reported African cases of HTLV-1-associated ID. When compared with data from the Caribbean, infectious features seemed particularly prominent. ID appears to be overlooked in sub-Saharan Africa, where it might be easily confused with common pyoderma. Breast feeding appears to be the origin of HTLV-1 contamination of the children.

Prognostic impact of p27KIP1 expression in cyclin D1 positive lymphoproliferative disorders.

Nodal mantle cell lymphoma (MCL) is a well-defined entity, but non-nodal leukemic cyclin D1 positive lymphoproliferative disorders have been reported and their relationship with MCL remains controversial and their prognosis heterogeneous. We prospectively studied the expression of cyclin D1 in CD5 positive leukemic B lymphoproliferative disorders at diagnosis and identified 65 cases overexpressing cyclin D1. We did not distinguish any clinical or biological criteria allowing one to identify a non-MCL group. Multivariate analysis identified age, anemia and p27kip1 expression as independent prognostic factors of survival. By univariate analysis, p27kip1 high expression proved to be the strongest predictor of prolonged survival. The median survival of p27 low expressors was 30 months, while it was not reached for p27 high expressors. A high level of p27 expression was often found associated with the absence of nodal involvement and the presence of somatic mutations, but neither of them was restricted to the p27 high expression group. In conclusion, we hypothesize that MCL and these cyclin D1 positive leukemic lymphoproliferative disorders represent a continuous spectrum of diseases. Determination of p27 expression level appears as a routine applicable test allowing identification of a subset of patients who could be considered for different therapeutic approaches.

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.

[Bone marrow aspiration for diagnostic purposes]. / La ponction aspiration médullaire à visée diagnostique.

Examination of bone marrow aspiration is an important tool in the diagnosis of haematological diseases. First attempts of bone marrow sampling took place at the beginning of the twentieth century. Thereafter, numerous methods were proposed and different materials were described. The commonly accepted sites for sampling are sternum and the iliac crest. We describe here a sampling procedure for each site. Bone marrow aspiration is a safely investigation, but not recommended for patients with impaired haemostasis. The physician must be aware of its side effects and complications which could occur. The consequence of the complications varies according to the type of iatrogenic injury. Prevention and rapid diagnosis are a crucial point in the management of bone marrow aspiration accidents. To avoid malpractice, the procedure should be taught by senior physicians including theoretical as well as practical learning. The purpose of the learning is a high quality of care to ensure patients the best comfort in subsequent bone marrow examinations, this point being particularly important in paediatrics.

Remission of transformed myelodysplastic syndrome with fibrosis after danazol therapy.

Danazol has been used with success in some hematological diseases, but there is no report of this treatment in acute leukemia. We report here a case of remission of myelodysplastic syndrome with myelofibrosis in transformation after danazol therapy in a 72-yr-old man. The role of danazol in remission induction is briefly discussed.

Sequential chemotherapy by CHOP and DHAP regimens followed by high-dose therapy with stem cell transplantation induces a high rate of complete response and improves event-free survival in mantle cell lymphoma: a prospective study.

Mantle cell lymphoma (MCL) is a distinct clinico-pathological entity with a poor prognosis. We have conducted a prospective study in patients with MCL to evaluate a therapeutic strategy in which CHOP polychemotherapy was followed by DHAP if CHOP failed to induce complete remission. Responding patients then proceeded to an intensification therapy with autologous peripheral blood stem cell transplantation (APBSCT). Twenty-eight consecutive patients with newly diagnosed aggressive MCL were included. After four cycles of CHOP regimen, two complete responses (CR) were obtained (7%) and 14 (50%), five (18%) and seven (25%) patients achieved partial (PR), minor (MR) and no response, respectively (one patient died from septic complications during CHOP induction). The two patients in CR after CHOP underwent intensification with TBI, high-dose cyclophosphamide-etoposide and APBSCT. The other twenty-five patients received DHAP and in this group a response rate of 92% (21 CR (84%), two PR (8%)) was observed. Two patients had progressive disease. The twenty-three responding patients received high-dose therapy (TAM8 regimen: TBI-cytarabine-melphalan) followed by APBSCT. One of the two partial responding patients achieved CR after TAM8. After a median follow-up of 47.6 months (range, 14-70), seven patients have relapsed. Our data confirm that: (1) CHOP regimen induces a low CR rate in MCL; (2) CHOP plus DHAP appears to be much more efficient and allows a large proportion of patients to proceed to high-dose therapy in CR; (3) consolidation therapy including TBI and high-dose Arac-C followed by APBSCT may improve event-free survival.

A multicentric study of 41 cases of B-prolymphocytic leukemia: two evolutive forms.

B-prolymphocytic leukemia (B-PLL) is an infrequent disease with a poor prognosis. We present the clinical and biological features of 41 patients. Median age was 67 years [42-89] and male-female sex ratio was 2.4. The immunophenotyping revealed B-cell phenotype, with a high level expression of surface IgM and/or IgD in all cases, FMC7+ in 76 % of cases and CD5+ in 67%. Marked spontaneous in-vitro apoptosis was observed in most cases tested (n = 12). The median overall survival time was 5 years and the event-free survival time was 37 months. As detected by univariate and multivariate analysis, the only variables associated with a poor prognosis were advanced age and anemia. No significant difference was observed between de novo PLL (n = 27) and prolymphocytoid transformation of chronic lymphocytic leukemia (n = 14). Two groups of patients were individualized according to their clinical course: patients who died within one year of diagnosis (n = 14) and patients who had a prolonged survival (n = 23) without any treatment in some cases. The comparison between the 2 groups showed that they differed in age (p = 0.01) and anemia (p = 0.02). We also observed that the patients with p53 mutations had a worse clinical outcome. Taken together these data confirm that B-PLL should be regarded as a distinct form of chronic lymphoproliferative disorder and suggest the existence of two patterns of clinical evolution.

Blastic variant of mantle cell lymphoma: a rare but highly aggressive subtype.

The blastic variant (BV) form of mantle cell lymphoma (MCL) is considered to be a very aggressive subtype of non-Hodgkin's lymphoma (NHL). In order to determine its clinico-biological features and response to therapy we studied 33 patients (17%) out of 187 suffering from MCL who were diagnosed with a BV of MCL. Blastic variant was diagnosed according to histopathological patterns, immunophenotyping, and bcl1 gene rearrangement and/or cyclin D1 overexpression. Three patients initially diagnosed with large cell NHL were classified as BV. Patients received front-line therapy including CHOP-like regimen or CVP (n = 29), or chlorambucil (n = 4) and CHOP or ESAP as second-line therapy. High-dose intensification with stem cell transplantation (SCT) was performed in 11 cases (autoSCT, n = 8; alloSCT, n = 3). All but two patients were in complete remission (CR) at the time of transplant (CR1, n = 5; CR2, n = 4). Clinical and biological characteristics did not differ from those of the common form of MCL. The median age was 62 years (29-80), with a sex ratio (M/F) of 2.6:1. Of the 33 patients, 66% had extranodal site involvement, 85% had an Ann Arbor stage IV, and 82% had peripheral lymphadenopathy. Circulating lymphomatous cells were seen in 48% of cases. Twelve patients (36%) entered a CR1 with a median duration of 11 months. Fifteen patients (46%) failed to respond and rapidly died of progressive disease. Second-line therapy led to a 26% (6/23) CR2 rate. Nine patients relapsed after high-dose therapy. Twenty-two of the 33 patients (66%) died of refractory or progressive disease. Median overall survival (OS) time was 14.5 months for the 33 BV patients as compared to 53 months for the 154 patients with a common form of MCL, P <0.0001. In the univariate analysis, OS was influenced by age, extranodal site involvement, circulating lymphomatous cells, and international prognosis index (IPI). In the multivariate analysis, only IPI affected OS: patients with IPI > or =2 had 8 months median OS as compared to 36 months median OS for patients with IPI <2, P = 0.003. Blastic variant is one of the worst forms of NHL. An improved recognition of BV of MCL is required, particularly in high-grade CD5+ NHL using immunophenotyping and bcl1 molecular study. Standard therapy using anthracycline or even high-dose intensification produce poor results and an alternative treatment should be proposed to such patients.

Caspase activation is required for terminal erythroid differentiation.

The cysteine proteases known as caspases play a central role in most apoptotic pathways. Here, we show that caspase inhibitors arrest the maturation of human erythroid progenitors at early stages of differentiation, before nucleus and chromatin condensation. Effector caspases such as caspase-3 are transiently activated through the mitochondrial pathway during erythroblast differentiation and cleave proteins involved in nucleus integrity (lamin B) and chromatin condensation (acinus)without inducing cell death and cleavage of GATA-1. These observations indicate a new function for caspases as key proteases in the process of erythroid differentiation.

Transforming growth factor inhibits erythropoiesis by blocking proliferation and accelerating differentiation of erythroid progenitors.

Erythropoiesis is positively regulated by stem cell factor, interleukin 3, and erythropoietin, which synergize to allow the production of hemoglobinized red blood cells from erythroid progenitors. In contrast, interferon gamma, tumor necrosis factor alpha, and transforming growth factor B(1), (TGF-beta(1)) are powerful inhibitors of erythropoiesis. Interferon gamma and alpha act principally by inducing apoptosis. The aim of this study was to elucidate the mechanisms by which TGF-beta(1) inhibits erythropoiesis. We used an in vitro serum-free system of human red blood cell production. From a virtually pure population of CD36(+) erythroid progenitors, stem cell factor, interleukin 3, and erythropoietin allowed massive proliferation (x300) and promoted terminal red blood cell differentiation. We show here that TGF-beta(1) (2 ng/mL) inhibited the growth of CD36(+) cells by 15-fold. TGF-beta(1) markedly accelerated and increased erythroid differentiation as assessed by hemoglobin and glycophorin expression. Furthermore, May-Grünwald-Giemsa staining and ultrastructural analysis revealed that TGF-beta(1) induced full differentiation toward normal enucleated red cells even in the absence of macrophages. This acceleration of erythroid differentiation did not modify the pattern of hemoglobin chains expression from adult or fetal erythroid progenitors. Analysis of apoptosis, cell cycle and Ki-67 expression showed that TGF-beta(1) inhibited cell proliferation by decreasing the cycle of immature erythroid cells and accelerating maturation toward orthochromatic normoblasts that are not in cycle. We showed that TGF-beta(1) is a paradoxical inhibitor of erythropoiesis that acts by blocking proliferation and accelerating differentiation of erythroid progenitors.

The incidence of clonal T-cell receptor rearrangements in B-cell precursor acute lymphoblastic leukemia varies with age and genotype.

B-cell precursor acute lymphoblastic leukemias (BCP-ALLs) are increasingly treated on risk-adapted protocols based on presenting clinical and biological features. Residual molecular positivity of clonal immunoglobulin (IG) and T-cell receptor (TCR) rearrangements allows detection of patients at an increased risk of relapse. If these rearrangements are to be used for universal follow-up, it is important to determine the extent to which they are informative in different BCP-ALL subsets. We show that IGH V-D-J rearrangements occur in 89% of 163 BCP-ALL, with no significant variation according to age or genotype (BCR-ABL, TEL-AML1, MLL-AF4, and E2A-PBX1). In contrast, TCRG rearrangements, which occur in 60% of patients overall, are frequent in BCR-ABL and TEL-AML1, are less so in MLL-AF4, and are virtually absent in infants aged predominantly from 1 to 2 years and in E2A-PBX1 ALLs. Incidence of the predominant TCRD Vdelta2-Ddelta3 rearrangement decreases with age but is independent of genotype. These differences are not due to differential recombination activating gene activity, nor can they be explained adequately by stage of maturation arrest. Analysis of MLL-AF4 BCP-ALL is in keeping with transformation of a precursor at an early stage of ontogenic development, despite the adult onset of the cases analyzed. We postulate that the complete absence of TCRG rearrangement in E2A-PBX1 cases may result from deregulated E2A function. These data also have practical consequences for the use of TCR clonality for the molecular follow-up of BCP-ALL.

Virological and molecular characterisation of a new B lymphoid cell line, established from an AIDS patient with primary effusion lymphoma, harbouring both KSHV/HHV8 and EBV viruses.

We report here a new case of primary effusion lymphoma (PEL), occurring in a French homosexual HIV-1 infected male with a pericardial, pleural and mesenteric tumour dissemination, and the establishment from his pleural effusion of a new cell line, Cra-BCBL, dually infected by EBV and KSHV/HHV8. Cra-BCBL cells are of B-cell origin as judged by their clonal immunoglobulin heavy chain (IgH) gene rearrangement, identical to that of the parental tumour. Both the cell line and the lymphoma cells expressed CD38 and CD45 antigens but no classical B-cell or T-cell lineage-restricted antigens. Cra-BCBL harbours a type I EBV virus, expressing a latency type II. Expression of KSHV/HHV8 ORF72 and ORF75 was detected by RT/PCR. In addition, KSHV lytic replication could be induced by treatment by n-butyrate. An equivalent and high copy number of KSHV genomes (20 to 200 copies by cell) was detected both in the primary tumour cells and in the cell line. Southern blot (SB) analysis of EBV terminal repeats (TR) displayed the same unique band in the cell line DNA and in the original tumour cells, consistent with a monoclonal infection of EBV. Furthermore, SB analysis of KSHV/HHV8 TR revealed the same hybridisation pattern between Cra-BCBL and the effusion cells, with a common band at around 30-40 kb corresponding to the fused termini of the viral episomes and a 5 Kb rearranged fragment. The new cell line characterised here could be a useful model to study interactions between two human herpes viruses and their contribution to lymphomagenesis.

Rapid, multifluorescent TCRG Vgamma and Jgamma typing: application to T cell acute lymphoblastic leukemia and to the detection of minor clonal populations.

Detection of clonal T cell receptor gamma (TCRG) gene rearrangements by PCR is widely used in both the diagnostic assessment of lymphoproliferative disorders and the follow-up of acute lymphoblastic leukaemia (ALL), when residual positivity in excess of 10(-3) at morphological complete remission is increasingly recognised to be an independent marker of poor prognosis. This is largely based on specific detection of V-J rearrangements from childhood cases. We describe rapid, multifluorescent Vgamma and Jgamma PCR typing of multiplex amplified diagnostic samples, as applied to 46 T-ALL. These strategies allow selected analysis of appropriate cases, immediate identification of Vgamma and Jgamma segments in over 95% of alleles, improved resolution and precision sizing and a sensitivity of detection at the 10(-2)-10(-3) level. We demonstrate preferential V-J combinations but no difference in V-J usage between children and adults, nor between SIL-TAL1-negative and -positive cases. A combination of fluorescent multiplex and Vgamma-Jgamma-specific monoplex follow-up, as described here, will allow detection of both significant clonal evolution and of the diagnostic clone at a level of prognostic significance, by techniques which can readily be applied to large-scale prospective studies for which real-time analysis is required.