[Characteristics of autoimmune hemolytic anemia in adults: retrospective analysis of 83 cases]. / Caractéristiques des anémies hémolytiques auto-immunes de l'adulte: analyse rétrospective d'une série de 83 patients.

PURPOSE: To describe the main characteristics and outcome of adult's acquired immune hemolytic anemias (AIHA). To analyse the relevance of the complementary tests performed for the search of an underlying disease. METHODS: Retrospective (1980-2000) monocentric study. INCLUSION CRITERIA: age above 16, AIHA defined by an hemoglobin level below 12 g/dl in men and 11 g/dl in women, with hemolysis and/or a positive direct Coombs test and/or the presence of cold agglutinins (threshold 1/500) and/or in the absence of any other cause. RESULTS: Eighty three patients included (56 women and 27 men), with a mean age of 56 years (+/- 22) at AIHA onset including: 72 patients (87%) with warm antibody AIHA and 11 (13%) with cold agglutinin disease. The mean follow-up was 48 months (median 22 months). Among the 72 patients with warm antibody AIHA, the specificity of autoantibodies was: IgG + complement (43%), IgG (32%) or complement alone (25%); cold agglutinins (titre from 1/60 to 1/512) were detected in 15 (20%) of the patients. Antinuclear antibodies were detected (threshold: 1/80) in 33% of the cases. Hypogammaglobulinemia on serum protein electrophoresis (SPE) was significantively associated with the presence of an underlying non-Hogkin lymphoma (NHL). The CT-scan of the the chest and abdomen which was performed in 50% of the patients, showed abnormalities other than a spleen enlargement in 25% of the cases. The medullar biopsy (MB) was abnormal in 7 of 26 cases (27%) but lead by itself to the diagnosis of NHL in a single case. Thrirty seven (51%) of warm antibody AIHA cases were finally considered to be "secondary" to an underlying disease namely: NHLs (n = 14), Hogkin's disease (n = 1) connective tissue disease (CTD) (n = 14), drug-induced AIHA (n = 3), miscellaneous (n = 5). In 6 out of 14 cases (43%) of NHL's associated AIHA, the onset of AIHA precedes the NHL from 22 to 66 months. The response rates to different therapeutic regimens did not significatively differ when "secondary" and "idiopathic" AIHA were compared. Overall, 13 patients (15.6%) died mainly from infectious complications (n = 5) or an underlying NHL (n = 5). CONCLUSIONS: In more than half of the cases AIHA are associated with an underlying disease and AIHA may precede the onset of a NHL for a long period. In the absence of a clinically apparent underlying disorder, testing for the presence of antinuclear antibodies, a SPE and a CT-scan must be systematic. Conversely, if no abnormalities are found, the relevance of a systematic MB at AIHA onset seems very low.

[Autoimmune hemolytic anemia]. / Anémies hémolytiques auto-immunes.

From the beginning of the century, autoimmune haemolytic anaemia (AIHA) was the first model of an auto-antibody mediated disease. Despite the variety of the clinical features, the diagnosis of AIHA provides few difficulties since the introduction of the popular Coombs' test. The clinical presentation of AIHA depends on the subclass type and on the thermal range activity of the causative auto-antibody, so that two main pictures occur usually: warm auto-antibody and cold auto-antibody types, the latter being less frequent than the former. In more than half the cases, AIHA is associated with another disease that must be considered more as the background of immune dysregulation than the cause of the disease. Systemic disorders, chronic lymphoid malignancy, primitive or acquired immunodeficiencies are the most common disorders associated with AIHA. Acute infections or drugs may give rise to acute transient AIHA. The clinical aspects and the links between AIHA and associated diseases are emphasised. No decisive advancement in therapy has arisen over the last decades. Some patients are still resistant to all therapeutic manoeuvres and may die. Much labour is to be done in order to discover more rational methods of therapy to restore a state of normal tolerance towards erythrocyte auto-antigens. Suppressing the production of pathogenic auto-antibodies by immunomodulation may be the first step of this task.

Quinine improves results of intensive chemotherapy (IC) in myelodysplastic syndromes (MDS) expressing P-glycoprotein (PGP). Updated results of a randomized study. Groupe Français des Myélodysplasies (GFM) and Groupe GOELAMS.

We designed a randomized trial of IC with or without quinine, an agent capable of reverting the multidrug resistance (mdr) phenotype, in patients aged < or = 65 years with high risk MDS. Patients were randomized to receive Mitoxantrone 12 mg/m2/d d2-5 + AraC 1 g/m2/12 h d1-5, with (Q+) or without (Q-) quinine (30 mg/kg/day). 131 patients were included. PGP expression analysis was successfully made in 91 patients and 42 patients (46%) had positive PGP expression. In PGP positive cases, 13 of the 25 (52%) patients who received quinine achieved CR, as compared to 3 of the 17 (18%) patients treated with chemotherapy alone (p = 0.02). In PGP negative cases, the CR rate was 35% and 49%, respectively in patients who received quinine or chemotherapy alone (difference not significant). In the 42 PGP positive patients, median Kaplan-Meier (KM) survival was 13 months in patients allocated to the quinine group, and 8 months in patients treated with chemotherapy alone (p = 0.01). In PGP negative patients, median KM survival was 14 months in patients allocated to the quinine group, and 14 months in patients treated with chemotherapy alone. Side effects of quinine mainly included vertigo and tinnitus that generally disappeared with dose reduction. Mucositis was significantly more frequently observed in the quinine group. No life threatening cardiac toxicity was observed. In conclusion, results of this randomized study show that quinine increases the CR rate and survival in PGP positive MDS cases treated with IC. The fact that quinine had no effect on the response rate and survival of PGP negative MDS suggests a specific effect on PGP mediated drug resistance rather than, for instance, a simple effect on the metabolism of Mitoxantrone and/or AraC.

A prospective study of autologous bone marrow or peripheral blood stem cell transplantation after intensive chemotherapy in myelodysplastic syndromes. Groupe Français des Myélodysplasies. Group Ouest-Est d'étude des Leucémies aiguës myéloïdes.

We prospectively assessed autologous stem cell transplantation for consolidation treatment in a trial of intensive chemotherapy in high risk myelodysplastic syndromes (MDS). In this trial, patients aged 55 years or less with no HLA-identical sibling and achieving CR were scheduled to receive unmanipulated autologous bone marrow transplantation (ABMT) preceded by a consolidation chemotherapy course. Forty-two of the 83 patients aged 55 years or less included in the trial (51%) achieved CR. Three were allografted in CR. Twenty-four of the remaining 39 patients who achieved CR (62%) received ABMT (16 patients) or autologous peripheral blood stem cell transplantation (APSCT) (eight patients). Indeed, as bone marrow harvest was often insufficient, APSCT was subsequently proposed after mobilization by consolidation chemotherapy followed by G-CSF. The conditioning regimen combined cyclophosphamide and busulfan. ABMT and APSCT were performed 1-7 months (median 3) after CR achievement. Hematological reconstitution occurred in all patients and tended to be faster after APSCT than ABMT although not significantly. Three patients died from the procedure, nine relapsed after 2-26 months and 12 (50%) were still in CR after 8-55 months. In autografted patients, median Kaplan-Meier disease-free survival and survival were 29 and 33 months from the autograft, respectively. Thus, ABMT or APSCT can be performed in almost two-thirds of MDS patients who achieve CR with intensive chemotherapy. PBSC collection may yield higher numbers of stem cells than marrow collection in some cases, and could improve the percentage of MDS patients autografted in CR. Longer follow-up is required to determine if autograft will prolong CR duration in at least some patients.

Quinine improves the results of intensive chemotherapy in myelodysplastic syndromes expressing P glycoprotein: results of a randomized study.

Intensive chemotherapy produces a lower complete remission (CR) rate in the myelodysplastic syndromes (MDS) than in de novo acute myeloid leukaemia (AML), possibly due in part to a higher incidence of P glycoprotein (PGP) expression in MDS blast cells. We designed a randomized trial of intensive chemotherapy with or without quinine, an agent capable of reverting the multidrug resistance (mdr) phenotype, in patients aged < or = 65 years with high-risk MDS. Patients were randomized to receive mitoxantrone 12 mg/m2/d days 2-5 + AraC 1 g/m2/12 h days 1-5, with (Q+) or without (Q-) quinine (30 mg/kg/d). 131 patients were included. PGP expression analysis was successful in 91 patients. In the 42 PGP-positive cases, 13/25 (52%) patients in the Q+ group achieved CR, compared to 3/17 (18%) patients in the Q- group (P = 0.02) and median Kaplan-Meier survival was 13 months in the Q+ group, and 8 months in the Q- group (P = 0.01). No life-threatening toxicity was observed with quinine. In conclusion, the results of this randomized study show that quinine increases the CR rate and survival in PGP-positive MDS cases treated with intensive chemotherapy.

[Myelodysplastic syndromes: unusual and mild forms]. / Syndromes myélodysplasiques: formes singulières et formes frontières.

Although the FAB classification of the myelodysplastic syndromes (MDS) allows to classify most patients, a few clinical patterns do not fit the FAB categories, including borderline forms with manifestations usually seen in other diseases and forms with atypical or unusual clinical or laboratory features that do not immediately suggest a MDS. Borderline forms are characterized by the presence of any of the following: high or very high platelet counts, myelofibrosis, bone marrow hypoplasia, eosinophilia, or systemic diseases such as relapsing polychondritis. Unusual or atypical forms include manifestations such as hemolysis, high reticulocyte counts, erythroblastopenia, or an abnormality of a single cell line such as isolated thrombocytopenia, isolated neutropenia, or isolated macrocytosis. The definitive diagnosis of these forms of MDS can require a number of investigations such as cytogenetic studies, bone marrow biopsy, and/or radionuclide evaluation, and may not be possible until the patient has been followed for some time.

Paroxysmal nocturnal haemoglobinuria: long-term follow-up and prognostic factors. French Society of Haematology.

BACKGROUND: Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired disorder of haematopoietic stem cells. Although knowledge about the pathophysiology of the disease is increasing, no multivariate analysis of factors influencing survival has been undertaken, mainly because the disease is rare. We undertook such an investigation. METHODS: Data were collected on 220 patients with PNH diagnosed over a 46-year period (1950-1995) from participating French centres. Diagnosis of the disease required, at least, an unequivocally positive Ham's test. FINDINGS: The Kaplan-Meier survival estimate was 65% (SE 4) at 10 years and 48% (6) at 15 years after diagnosis. 8-year cumulative incidence rates of the main complications (pancytopenia, thrombosis, and myelodysplastic syndrome) were 15% (3), 28% (4), and 5% (2), respectively. Demographic data, presenting features, initial treatment, complications, and causes of death were similar to those previously reported. In multivariate analysis, seven factors were significantly associated with survival in patients with PNH. Poor survival was associated with the occurrence of thrombosis as a complication (relative risk 10.2 [95% CI 6-17], p < 0.0001), evolution to pancytopenia (5.5 [2.8-11], p < 0.0001), myelodysplastic syndrome or acute leukaemia (19.1 [7.3-50], p < 0.001), age over 55 years at diagnosis (4 [2.4-6.9], p < 0.0001), need for additional treatment (2.1 [1.3-3.6], p < 0.003), and thrombocytopenia at diagnosis (2.2 [1.3-3.8, p < 0.003). Better survival was shown for patients in whom aplastic anaemia antedated PNH (0.32 [0.14-0.72], p < 0.02). Factors associated in multivariate analysis with a high risk of thrombosis during the disease course were thrombosis at diagnosis (5.1 [2.5-10.6], p = 0.0002), age over 54 years (2.6 [1.5-4.6, p = 0.0014), and infection at diagnosis (2.6 [1.3-5.2], p = 0.0099). The risk factors for progression to pancytopenia were absence at diagnosis of anaemia (4.03 [1.3-12.2], p = 0.03) and neutropenia (2.45 [1.1-5.7], p = 0.03). The risk factors for development of myelodysplastic syndrome or acute leukaemia were abdominal pain crisis at presentation (10.5 [2.5-44.0], p = 0.004) and year of diagnosis after 1983 (8.45 [1.8-40.7], p = 0.004). INTERPRETATION: This large number of cases permitted a detailed analysis of prognostic factors for the first time, in this rare disease. Estimates of PNH prognostic factors may serve as baseline data in the assessment of current and future treatments for this disease.

Impact of magnetic resonance imaging on the diagnosis of abdominal complications of paroxysmal nocturnal hemoglobinuria.

Magnetic resonance (MR) imaging is a method of choice for assessing vascular patency and parenchymal iron overload. During the course of paroxysmal nocturnal hemoglobinuria (PNH), it is clinically relevant to differentiate abdominal vein thrombosis from hemolytic attacks. Furthermore, the study of the parenchymal MR signal intensity adds informations about the iron storage in kidneys, liver, and spleen. Twelve PNH patients had 14 MR examinations of the abdomen with spin-echo T1- and T2-weighted images and flow-sensitive gradient echo images. Vessels patency and parenchymal signal abnormalities--either focal or diffuse--were assessed. MR imaging showed acute complications including hepatic vein obstruction in five patients, portal vein thrombosis in two patients, splenic infarct in one patient. In one patient treated with androgens, hepatocellular adenomas were shown. Parenchymal iron overload was present in the renal cortex of eleven patients with previous hemolytic attacks. On the first MR study of the remaining patient with an acute abdominal pain showing PNH, no iron overload was present in the renal cortex. Follow-up MR imaging showed the onset of renal cortex iron overload related to multiple hemolytic attacks. Despite the fact that all our patients were transfused, normal signal intensity of both liver and spleen was observed in three of them. MR imaging is particularly helpful for the diagnosis of abdominal complications of PNH.

Exhaustive analysis of the P53 gene coding sequence by denaturing gradient gel electrophoresis: application to the detection of point mutations in acute leukemias.

We report the set-up of a denaturant gradient gel electrophoresis (DGGE) assay to screen for mutations in the whole coding sequence of the p53 gene. These DGGE experimental conditions were applied to the analysis of the p53 gene in acute leukemias. Forty adults with acute myelogenous leukemia (AML) and 21 with acute lymphoid leukemia (ALL) were investigated. Eleven of the AML patients were investigated at the time of the initial diagnosis and at relapse. In contrast with most reports based on amplified fragments analyzed by single-strand conformation electrophoresis and focusing on exons 5 to 8, we analyzed the whole coding sequence of the gene. Two of the 40 AML patients displayed a point mutation in exon 7; it was either an A to G substitution that converted Tyr-234 to Cys, or a G to A change that converted Arg-248 to Gln. The screening procedure led to the discovery of several intronic and exonic polymorphisms. These results confirm the low incidence of p53 mutations in acute leukemias and suggest a limited role of the p53 protein in leukemogenesis. The computerized modeling and electrophoresis parameters presented here provide a powerful tool for the exhaustive characterization of p53 mutants in all kinds of malignancies.

A multicentric randomised study of alpha 2b interferon (IFN) and hydroxyurea (HU) with or without cytosine-arabinoside (Ara-c) in previously untreated patients with Ph+ chronic myelocytic leukemia (CML): preliminary cytogenetic results.

The CML 88 study was designed to evaluate the efficacy of maintenance therapy in a multicentric randomised protocol using IFN combined with low-dose Ara-C versus IFN alone, following an induction with IFN + HU. Between April 1988 and February 1991, 237 patients from 36 French Hematology Centres were entered in the study. Preliminary cytogenetic results show a slightly higher, although not statistically significant, proportion of major chromosomal responses, including complete cytogenetic remissions, in the IFN + Ara C arm.

Induction of cytolytic function in resting peripheral blood CD8+/Leu-7+ T cells through IL2/p 75 IL2-receptor interaction: a study in the allogeneic human bone marrow transplantation model.

CD8+/Leu-7+ T cells which circulate in increased proportions in the blood of long-term surviving BMT patients are for the most part high-density resting lymphocytes lacking IL2R-alpha (p55) expression. We show that they can be induced by IL2 to manifest cytolytic function after 24-48 hr stimulation by using rather high concentrations of IL2 (at least 50 U/ml). This function was much more readily induced in high-density CD8+/Leu-7+ T cells than in high-density CD8+/Leu-7+ T cells and occurred in the presence of minimal cell proliferation. Other cytokines involved in primary CTL differentiation (IFN-gamma, IL4 and IL6) were without effect suggesting that CD8+/Leu-7+ T cells are, in the BMT model, in vivo preactivated CTL ready to differentiate into cytolytic effectors under the sole IL2 stimulus. TU27 Mab directed at IL2R-beta (p75) subunit almost completely prevented IL2-induced cytolytic function of CD8+ T cells while 33B3.1 Mab directed at IL2R-alpha (p55) subunit was ineffective, suggesting that the signal for this function has its origin in IL2R-beta chains constitutively expressed by these cells.

Abnormally expanded CD8+/Leu-7+ lymphocytes persisting in long-term bone marrow-transplanted patients are resting pre-cytotoxic T-lymphocytes.

We analyzed the functional status of the small CD8+/Leu-7+ T-lymphocytes that circulate in increased proportions in the blood of many allogeneic bone marrow transplant (BMT) patients. Purified CD8+/Leu-7+ T cells were tested for their effect on T-cell proliferative responses. In contrast to CD8+/Leu-7-T-lymphocytes, such cells behaved as suppressor cells for lectin-induced mitogenic responses of the donor's peripheral blood lymphocytes. However, they did not interfere with the in vitro responsiveness to specific stimuli such as protein purified derivative (PPD) or alloantigens. We demonstrate that CD8+/Leu-7+ T cells are resting pre-cytotoxic T-lymphocytes (CTL) that can be induced by mitogenic lectins to express their cytolytic program in a non-specific, non-major histocompatibility complex-restricted manner against phytohemagglutinin-treated lymphoblasts or K562 target cells. The lectin-triggered cytotoxicity was achieved within a few days, together with limited cell division. Our results suggest that circulating CD8+/Leu-7+ T cells from BMT recipients are in vivo primed CTL awaiting cellular activation.

Frequent detection of minimal residual disease by use of the polymerase chain reaction in long-term survivors after bone marrow transplantation for chronic myeloid leukemia.

The polymerase chain reaction (PCR) allows the detection of minimal amounts of nucleic sequences and has been successfully used to test for the chronic myeloid leukemia-specific bcr/abl transcripts. We studied blood samples from 17 patients who had undergone allogeneic bone marrow transplantation for CML, using a modified polymerase chain reaction-based assay for the detection of leukemic mRNA. This nested PCR technique was found to be highly sensitive, detecting the chimeric bcr/abl transcript in 16 of 17 patients including several long-term survivors. Cytogenetic techniques failed to detect Ph mitoses. The clinical significance of the persisting bcr/abl transcript for long periods following BMT is poorly understood and remains to be elucidated by further studies.

Congenital sideroblastic anemia without clinical iron overload. A case report.

An 18 year old boy presented with microcytic hypochromic anemia. Erythrocytic abnormalities and family studies suggested congenital sideroblastic anemia (CSA), but atypical features included absence of clinical iron overload, scanty iron deposits in mitochondria of late erythroblasts and reticulocytes, and a high platelet count. An unusual adhesion between bone marrow macrophages and reticulocytes was observed by electron microscopy. Haematological response was seen following pyridoxine administration, thus fending support to the diagnosis of CSA.

Central nervous system relapses after bone marrow transplantation for acute lymphoblastic leukemia in remission.

This study defines the risk of central nervous system (CNS) relapse in patients undergoing bone marrow transplantation (BMT) for acute lymphoblastic leukemia (ALL) in remission, with no posttransplant prophylactic CNS therapy. Ninety-two consecutive patients in complete remission received BMT for ALL (n = 82) or high-grade non-Hodgkin's lymphoma with poor prognostic factors at diagnosis (n = 10). Sixty-six patients received allogeneic BMT (Allo-BMT) and 26 patients, without an identical sibling, underwent autologous BMT (Auto-BMT). Fifteen patients had CNS involvement at diagnosis and underwent BMT in first remission. Eight patients experienced CNS relapse after BMT, corresponding to a probability of 11% at 3 years. Apart from a history of prior CNS involvement, no patient characteristic evaluated statistically influenced CNS relapse after BMT. The probability of CNS relapse was 5.5% for the 70 patients without history of CNS involvement and 27.5% for the 22 patients with prior CNS involvement. However, subgroup analysis showed that the increased risk of CNS relapse is mainly observed in Auto-BMT patients with history of prior CNS involvement, particularly in patients undergoing BMT in first remission (three of five Auto-BMT versus one of ten Allo-BMT). Taking into account the multiple factors which influence the occurrence and the treatment of CNS leukemia, the results on this retrospective study suggests that (1) for patients without CNS involvement at diagnosis and for whom BMT is performed in first remission, cranial irradiation before BMT and posttransplant prophylactic CNS therapy can be omitted because of the low probability of CNS relapse after BMT (3.4%), when total-body irradiation (TBI) is included in the conditioning regimen; and (2) the difference observed between Allo-BMT and Auto-BMT patients with previous CNS involvement and undergoing BMT in first remission could indicate that graft-versus-host leukemia acts even in the CNS in Allo-BMT patients.

[Mixed lympho-epidermal cell cultures: value in bone marrow grafts]. / Cultures mixtes lympho-épidermiques: intérêt dans les greffes de moelle.

Graft-versus-host disease (GVHD) is the major complication of allogeneic HLA-identical bone marrow transplantation. GVHD is induced by the activation of mature T cells in the graft which react against minor antigens of the recipient. Mixed epidermal cell-lymphocyte cultures (MELC), which constitute an in vitro model of epidermal cell-lymphocyte interactions, make it possible to study the presentation of antigens to the lymphoid cells by epidermal Langerhans cells. We performed MELC in 66 patients who had received an HLA-identical bone marrow transplant for malignant blood disease. The bone marrow received by 29 recipients had been depleted of mature T cells, whereas 37 recipients had received a non-depleted bone marrow. A complete, uni- and multivariate statistical analysis was carried out on recipients of non-depleted bone marrow to evaluate the risk factors for acute and chronic GVHD. This study showed that MELC between donor and recipient was the most predictive parameter for the occurrence of GVHD. Other factors were a history of previous pregnancies in female donors and a diagnosis of chronic myelogenous leukaemia. These results may be of value in the selection of donors and for a better determination of the need for bone marrow depletion.

Adult patients presenting with pancytopenia: a reappraisal of underlying pathology and diagnostic procedures in 213 cases.

We reviewed 213 consecutive adult pancytopenic patients to determine the frequency of underlying pathology, to analyze our diagnostic procedure, and to determine the value of peripheral blood data for diagnosis. Pancytopenia was defined as the association of hemoglobin level below 12 g/dl in males and 11.5 g/dl in females, leukopenia below 4 x 10(9)/L, and thrombocytopenia below 150 x 10(9)/L. The bone marrow aspirates were normo- or hypercellular in 140 cases (66%). Bone marrow biopsies, performed in 93 cases, documented the presence of myelofibrosis in 67 cases. Aplastic anemia was diagnosed in 10% of the cases. Malignant myeloid disorders (acute myeloid leukemias, myelodysplastic syndromes, acute myeloid disorders with myelofibrosis) represented 42% of the cases and various malignant lymphoid disorders 18%. Vitamin deficiencies accounted for 7.5% and nonhematological pathology 10% of the cases. The bone marrow aspirate was sufficient for the diagnosis in 55% of the cases, and the trephine biopsy was necessary in 30%. In the remaining cases, other complementary tests were necessary to achieve final diagnosis. A discriminant analysis, focused on the hemogram data, showed that parameters obtained by analysis of blood smears were helpful for the diagnosis, especially the presence or absence of blast cells and/or of abnormal lymphoid cells.