Search

In this study we have identified frequent human leukocyte antigen (HLA)-A, -B, -C,-DRB1, and -DQB1 alleles, frequent HLA-B/C, HLA-DRB1/DQB1 two-allele associations, and the most common HLA-A/B/C/DRB1/DQB1 five-locus haplotypes in a population residing in the Paris, France, area. The study was carried out in 356 families of children awaiting hematopoietic stem-cell transplantation (HSCT), with the selection criterion that haplotypes could be assigned with certainty to both the patient and at least one parent. Parental haplotypes were HLA-A, -B serologically typed, and HLA-C, -DRB1, -DQB1 broadly typed by polymerase chain reaction-sequence-specific oligonucleotide probe. The alleles of the most frequent haplotypes were subsequently defined at a high-resolution level by polymerase chain reaction-sequence-specific primer. The results on the distribution of common alleles and common allele associations demonstrated similarities with the previously published data in Caucasian populations, as expected from the geographic origin of the studied population. More importantly, this study provides the largest listing of common B/C and DRB1/DQB1 associations and of common five-allele haplotypes defined with certainty in a Caucasian population to date. These results can be used to help estimate the likelihood of finding a suitable donor in unrelated HSCT and to delineate search strategies for potential donors.

Adenovirus infection remains an important cause of mortality after bone marrow transplantation (BMT). Currently no efficient antiviral treatment is known. Thus, testing new modalities of early diagnosis and treatment is a crucial objective. Adenovirus infection is defined by the combination of symptoms and the isolation of virus from the source of clinical symptoms. The involvement of two or more organs and the presence of virus in blood cultures define disseminated disease. Seven children with a median age of 7 years received bone marrow transplantation for leukemia. All received an unrelated graft without T cell depletion. Adenovirus was sought in blood, urine and biopsy specimens using PCR and culture. Analysis of biopsy specimens included systematic immunohistochemistry. Cidofovir treatment was initiated as soon as biopsy revealed the histopathological signs of adenovirus. Cidofovir was given at 5 mg/kg once weekly for 3 weeks then every 2 weeks. Six patients had diarrhoea and one patient had cystitis. Adenovirus infection and disseminated disease were diagnosed in four cases and three cases, respectively. In six cases, serotype A31 was isolated from gastrointestinal biopsy and in two cases serotypes B2 and C6 were detected in blood and urine. Cidofovir treatment was associated with clinical improvement of diarrhoea, cystitis and fever in five patients, in whom the virus became undetectable in cultures and PCR analyses despite the persistence of immunodeficiency. The median follow-up was 360 days after BMT (240-570). One child died of invasive aspergillosis and another of disseminated adenovirus after interruption of cidofovir therapy. Further studies in immunocompromised patients will be needed to extend these promising results concerning the role of cidofovir in adenovirus infection.

Improvements in HLA-typing by DNA-based methods now allow accurate genotyping of unrelated bone marrow (UBM) donors and recipients and also definition of haplotypes. In this regard, B*5002 has been predicted in linkage disequilibrium with Cw*0602, DRB1*0406 and DQB1*0402 based on the frequency of allele coexistence. Here, we confirm this assumption by HLA genotyping of four informative families and three unrelated individuals. In the four families, the extended haplotype HLA-B*5002, -Cw*0602, -DRB1*0406, DQB1*0402 can be definitely assigned by the mode of heritance. Furthermore, this association of alleles was also found in the three B*5002 unrelated individuals. Knowledge of the frequent linkage disequilibrium of these rare alleles can improve UBM donor search strategies.

Resume Ces dernieres annees, les medecins de l'hopital pediatrique Robert-Debre avaient la perception que l'activite liee a la drepanocytose augmentait. Objectifs Notre etude avait deux objectifs: decrire l'evolution de la prise en charge des patients drepanocytaires suivis, puis etre capable d'en determiner les causes si l'on mettait en evidence une prise en charge plus lourde pour l'hopital. Patients et methodes Une enquete descriptive retrospective a ete menee, couplant deux approches: d'une part, une comparaison de l'ensemble des enfants drepanocytaires suivis en 1992 avec l'ensemble de ceux suivis en 1996, du point de vue de leur profil phenotypique et de leurs hospitalisations (frequence, duree et type de complications) et, d'autre part, une description de revolution des complications et hospitalisations dans la cohorte suivie de 1992 a 1996. Les enfants ont ete reperes grâce aux listes etablies par le ≪ centre de la drepanocytose ≫, qui coordonne le suivi de tous les patients hospitalises dans l'hopital. Les donnees etudiees proviennent du systeme d'information de l'hopital, ainsi que des dossiers individuels medicaux, infirmiers, et du service social. Resultats Les resultats de cette enquete montrent une augmentation de l'activite liee a la drepanocytose pour l'hopital etudie; elle se traduit par une multiplication des hospitalisations et par une charge de travail plus importante pour les equipes soignantes. Cette augmentation d'activite est liee a la fois a un recrutement plus important, a un accroissement du taux d'hospitalisations non programmees et a une augmentation de l'incidence des complications aigues chez les enfants suivis. Ceci peut s'expliquer en partie par le vieillissement de la cohorte. Par ailleurs, la charge de travail du personnel soignant semble avoir augmente, a en juger notamment par la prescription plus frequente de morphiniques.

Gene transduction into immature hematopoietic cells collected at birth from the umbilical cord could be useful for the treatment of genetic or acquired disorders of the hematopoietic system diagnosed during pregnancy. The SCID-hu mouse is a convenient model to investigate T- cell lineage gene therapy, since it allows replication of human intrathymic T-cell development. CD34+ cells isolated from cord blood were cocultured with CRIP MFG-murine CD2 (mCD2) cells that produce recombinant retroviruses encoding the mCD2 antigen, a cell surface marker easily detectable by flow cytometry. After 3 and 4 days in coculture, a mean of 19% and 39% human hematopoietic cells, respectively, expressed the mCD2 antigen. CD34+ cells cocultured for 4 days were used to reconstitute human fetal thymus implanted in SCID mice. Five to 10 weeks later, the mCD2 antigen was detected on approximately 10% of human thymocytes repopulating the thymic grafts in four of nine SCID mouse chimeras. Vector genomes were detected in graft cell DNA by Southern blot. Analysis of vector integration indicated that positive cells were of polyclonal origin in three animals and predominantly monoclonal in the other one. Our data show that foreign genes can be transduced into CD34+ cord blood cells endowed with T-cell differentiation potential, and suggest strategies for T-cell lineage gene therapy in the neonate.

The spontaneous proliferation and the effects of 8 various growth factors (GF) were evaluated on leukemic cells from 27 patients with B-lineage ALL. Two groups of ALLs were distinguished. ALLs from group I (21 patients) exhibited a low spontaneous proliferative rate and were stimulated by IL-3 + IL-7 +/- SCF and/or LIF, while ALLs from group II (6 patients) had a high spontaneous proliferative rate and did no longer require this combination of GFs for proliferation. No effect of bFGF, IGF-I, IL-10 and IL-11 alone or in combination, was observed. Such differences in the behaviour of B-ALLs indicated that the GF requirement of ALL blasts was not related to the presence of serum in the culture nor to the pattern of reactivity of ALL blasts for B lymphoid markers or CD34 antigen. Furthermore, we showed in 1/9 cases that high proliferation might be due to an overexpression of the bcl-2 proto-oncogene and to the acquisition of an autocrine secretion.

1. Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the catabolism of thiopurine drugs, which are used to treat cancer patients and organ transplant recipients. Because TPMT activity is polymorphic and under genetic control, large interindividual variations in the immunosuppressive activity and toxicity of these drugs may, at least in part, be inherited. 2. We have developed a specific h.p.l.c. method for measuring 6-methyl mercaptopurine formed from 6-mercaptopurine (6-MP) in red blood cell lysates during the TPMT assay procedure. In blinded assays of 55 samples from adult blood donors, the results of the h.p.l.c. method correlated with those of the radiochemical reference method (r = 0.83, P < 0.001). 3. Using this h.p.l.c. assay, we tested the effect of known inhibitors of TPMT activity (syringic acid, p-anisic acid and tropolone) in vitro and showed that they were highly inhibitory. We also found that drugs often administered concomitantly with 6-MP (prednisone, prednisolone, 6-methylprednisolone, cyclophosphamide, methotrexate, and trimethoprim-sulphamethoxazole) had little or no effect on TPMT activity in vitro. 4. In a group of 300 French individuals, TMPT activity was highly variable, ranging from 4.7 to 35.3 nmol h-1 ml-1 of packed red blood cells (nmol h-1 ml-1 PRBC) with a mean value of 19.3 +/- 4.9. TMPT activity was not influenced by sex. 5. This sensitive and reproducible h.p.l.c. assay for TPMT activity in red blood cells may prove useful for prospective clinical studies designed to optimise dosage regimens of thiopurine drugs (detection limit for 6-methyl mercaptopurine is 5 ng ml-1, intra- and inter-assay variations are 6.8 and 8.2%, respectively).

The EORTC Children Leukemia Group (CLG) is part of the offspring of the EORTC Hemopathies Working Party which in 1978 split into a paediatric and to an 'adult' branch. At that time, the Berlin-Frankfurt-Munster (BFM) designed by H. Riehm for acute lymphoblastic leukaemia (ALL) appeared much more efficacious than all others and the CLG decided to adapt that treatment strategy for its own clinical trials. The main results of these may be summarised as follows:for standard risk patients, the deletion of cyclophosphamide from consolidation and reconsolidation courses does not jeopardise the patient's outcomefor medium- and high-risk patients receiving high-dose methotrexate (MTX), cranial radiotherapy is superfluouswith the dose scheduling of the BFM regimen, E-Coli L-Asparaginase is more efficacious than Erwinia L-Asparaginasethe addition of monthly intravenous (i.v.) 6-mercaptopurine to conventional maintenance chemotherapy is detrimentalthe assessment by quantitative polymerase chain reaction (PCR) of minimal residual disease at completion of induction is feasible in a cooperative setting and can be used as a powerful and independent prognostic factor. The CLG also conducted clinical studies of acute myeloblastic leukaemia. Since 1989, lymphoblastic non-Hodgkin's lymphomas have been treated within the ALL trials. The CLG collaborates with other Groups within the I-BFM Study Group and participants in the meta-analytic studies conducted by the Oxford team by the Oxford Children ALL Collaborative Group.

A case-control study was carried out to investigate the role of a family history of solid tumor or hematologic neoplasm in the etiology of childhood acute leukemia.Family cancer history in first- and second-degree relatives was compared in 279 incident cases (242 cases of acute lymphocytic leukemia and 37 of acute myeloid leukemia) and 285 controls. Recruitment was stratified by age, gender, hospital, area of residence, and ethnic origin. Odds ratios (OR) were estimated using an unconditional regression model taking into account the stratification variables, socioeconomic status, and familial structure.A significant association between childhood acute leukemia and a family history of hematologic neoplasm (OR = 2.7, confidence interval (CI) = 1.1-6.9) was found. This association was particularly clear-cut when the cases were restricted to acute myeloid leukemia (OR = 13.3, CI = 2.5-70.9). Childhood acute leukemia was associated with a family history of solid tumor (OR = 1.5, CI = 1.0-2.2), and elevated odds ratios were observed for family history of gastrointestinal cancer and melanoma. Those results are most unlikely to be explained by socioeconomic status and familial structure, which were very similar for the cases and controls. Differential misclassification is also unlikely for the first-degree relatives, even though it is difficult to rule it out for the second-degree relatives' history.The present study supports the hypothesis that a family history of cancer may be a risk factor for childhood acute leukemia.

Post-transplant lymphoproliferative disorder (PTLD) after haemopoietic stem cell transplantation is a serious complication that occurs in 8-22% of patients with high-risk factors. We retrospectively investigated tolerance and efficacy of humanized anti-CD20 monoclonal antibody (rituximab) as first-line treatment in 12 children with B-cell PTLD. At diagnosis, eight patients had tumoral involvement. The other four patients had fever, associated with raised Epstein-Barr virus (EBV) viral load and monoclonal gammopathy. Rituximab was given at the dose of 375 mg/m2 once a week by intravenous infusion (1-9 infusions). Only 1/48 infusions was associated with a grade 2 clinical adverse event. Eight out of 12 (66%) patients responded to the treatment and were in complete remission. All patients without tumoral involvement responded to the treatment. A rapid decrease in fever within 1 week was observed in all responders. Non-responders did not show any clinical response during the first week. Tumoral involvement and immunodepression seemed to be more marked in non-responders. Rituximab was an effective and well-tolerated treatment of B-cell PTLD. Early treatment before tumoral involvement seemed to be the most effective approach. Lack of rapid response should lead to intensification of PTLD treatment. Pre-emptive treatment should be considered and evaluated in further longitudinal multicentre studies.

Optimisation of busulfan dosage in patients undergoing bone marrow transplantation is recommended in order to reduce toxic effects associated with high drug exposure. A new method was developed coupling liquid chromatography with mass spectrometry (LC-MS) and was validated for the determination of busulfan concentrations in plasma. Recovery was 86.7%, the limit of detection was 2.5 ng/ml and linearity ranged from 5 to 2500 ng/ml. The correlation between the busulfan concentrations measured by our previously published HPLC-UV method and the new HPLC-MS method was highly significant (P0.0001). Sample volume was reduced and the method was rapid, sensitive and less expensive than the methods previously used in our laboratory. This method was used to determine the pharmacokinetic parameters of busulfan after the first administration of 1 mg/kg orally, in 13 children receiving the drug as part of the preparative regimen for bone marrow transplantation. Our results were similar to previously reported data. They showed that the apparent oral clearance of busulfan was 0.299+/-0.08 l/h/kg, and that it was significantly higher (P=0.02) in patients below the age of 5 years than in older children.

Strategies currently used for residual disease detection in acute lymphoblastic leukaemia (ALL) rely on polymerase chain reaction (PCR) detection of immunoglobulin and T-cell receptor rearrangements. The TEL-AML1 fusion transcript, which is associated with t(12;21) (p13;q22), is found in 25% of childhood B-cell precursor ALL, and represents an interesting alternative target. We compared two methods for quantitating TEL-AML1 fusion transcripts: competitive PCR and real-time PCR. These techniques showed similar sensitivity (5 x 10(-5)) and reproducibility. Giving highly correlated results, both techniques can be conveniently used for TEL-AML1 transcript quantification. The constancy of TEL-AML1 expression was evaluated by measuring TEL-AML1 transcripts at different steps of the cell cycle, and in 21 cases of ALL at diagnosis. No major variation in TEL-AML1 expression was observed during the cell cycle or in 20/21 of the ALL patients. Residual disease was then determined after completion of induction therapy in 20 patients with a TEL-AML1-positive ALL. Seven patients out of 20 (35%) were still positive, including two patients with high level of residual blasts (close to or beyond 10(-2)). When comparison was possible, results obtained using TEL-AML1 quantification were in accordance with those obtained using T-cell receptor rearrangements analysis.

We report a five-year experience of targeted neonatal screening for sickle cell disease in the northern part of the Paris area as well as the follow-up procedure of screened patients.This geographic area in France is characterized by a high frequency of populations at risk for sickle cell disease.Among 115,480 tested newborns, 250 patients were diagnosed (frequency: 1/462). The quality of the screening was attested by the high frequency (5.34%) of newborn carriers for a hemoglobin abnormality (n = 6168). We developed an optimized strategy which avoids the majority of pitfalls (false positive and false negative responses), except for S/HPFH. More than 95% of sickle cell disease was followed, the majority by medical sickle cell disease experts from hospitals. Only two deaths were recorded during this time period.We demonstrate the efficiency of targeting the proposed methodological strategy and the follow-up of affected newborns, a major argument demonstrating the importance of newborn screening.

Improvements in HLA-typing by DNA-based methods now allow accurate genotyping of unrelated bone marrow (UBM) donors and recipients and also definition of haplotypes. In this regard, B*5002 has been predicted in linkage disequilibrium with Cw*0602, DRB1*0406 and DQB1*0402 based on the frequency of allele coexistence. Here, we confirm this assumption by HLA genotyping of four informative families and three unrelated individuals. In the four families, the extended haplotype HLA-B*5002, -Cw*0602, -DRB1*0406, DQB1*0402 can be definitely assigned by the mode of heritance. Furthermore, this association of alleles was also found in the three B*5002 unrelated individuals. Knowledge of the frequent linkage disequilibrium of these rare alleles can improve UBM donor search strategies.

We present here the long-term results of three randomized clinical trials conducted on children with newly diagnosed acute lymphoblastic leukemia (ALL) between 1983 and 1998 by the Children Leukemia Cooperative Group (CLCG) from EORTC. In study 58831/32, the overall event-free survival (EFS) rates (+/- s.e.) at 6 and 10 years were 66% +/- 1.8% and 65% +/- 1.8%, respectively, and the risk of isolated central nervous system (CNS) relapse was 6% +/- 1% and 7% +/- 1%, respectively. In patients with a standard risk of relapse the omission of cyclophosphamide had no adverse effect on disease-free survival rates at 10 years (trial 58831). In medium- and high-risk patients the omission of radiotherapy did not increase the risk of CNS or systemic relapse (trial 58832). In study 58881 (1989-1998) the overall EFS rate at 8 years was 68.4% +/- 1.2% and the risk of isolated CNS relapse was 4.2%+/-0.5%. In this trial which adressed three randomized questions, the following results were obtained: the combination of cytarabine at high doses with methotrexate at high doses during interval therapy did not improve prognosis. The addition of 6-mercaptopurine iv during maintenance increased the risk of late relapse. E. coli asparaginase was more toxic and has a higher efficacy than Erwinia asparaginase. Leukocyte counts100 x 10(9)/l, specific genetic abnormalities, a poor initial response to steroids or a high level of minimal residual disease at early time points were consistently associated with an adverse prognosis in the 58881 trial.

In recent years, physicians at the Robert-Debré pediatric hospital in Paris perceived an increase in activity linked to sickle cell disease care. Our study had two objectives: first, to describe the evolution of care for children with sickle cell disease in the hospital, then, if a heavier intensity of care was shown, to try to investigate its causes.We conducted a retrospective study using two strategies. On one hand, we compared the group of children followed up in 1992 with the group of children followed up in 1996 for their phenotype and hospitalizations (frequency, length of stay and type of complications), on the other hand, we described the course of complications and hospitalizations in the cohort of children followed from 1992 to 1996. Children were spotted through lists established by the 'Center for sickle cell disease', which coordinates the follow-up of all sickle cell patients in the hospital. Data came from the hospital's information system, and from all (medical, nursing, and social) individual records.The major result of this study shows an increasing activity linked to sickle cell disease care in this hospital: multiplication of hospitalizations and increasing work load for the healthcare teams. This situation is due to a larger recruitment, a higher emergency hospitalization rate, and an increasing rate of complications among the sickle cell children. The children's ageing is part of the explanation. The work load for the healthcare team linked to each hospitalization has also grown, as shown by an increasing rate of morphine prescription.

Patient-controlled analgesia (PCA) has been shown to be superior to a continuous morphine infusion for the treatment of ongoing pain in children over five years of age. Nevertheless, prescription parameters such as the bolus dosage and the possible association of a continuous background infusion have not yet been standardized.Thirty-three children, aged four to 17, hospitalized in a pediatric hematology ward, benefited from PCA with a standardized prescription: a bolus dosage of at least 25 mg/kg, without a background infusion. Morphine consumption, side effects and efficacy on pain relief were followed.Median of mean morphine consumption was 0.32 mg.kg-1.d-1. Median of maximal consumption was 0.58 mg.kg-1.d-1. Mean duration was nine days. No important side effects were noted, except in two patients. They presented prolonged constipation and a poor quality of nocturnal sleep, but they also had a major depressive syndrome persisting after resolution of pain. Efficacy was comparable to a continuous intravenous infusion, and nocturnal sleep was of good quality for 31 children.This standardized technique of PCA can be used extensively in children over five years of age. It can be used as a reference for further studies.

Alloimmune neonatal neutropenia is a rare event. Usually asymptomatic, it may however in some cases result in a severe sepsis. Treatment with recombinant granulocyte colony-stimulating factor (G-CSF) has been recently proposed.We report two new cases in infected newborns of a successful treatment of alloimmune neonatal neutropenia with G-CSF, resulting in complete neutrophil recovery in less than 72 hours. Moreover, the treatment was well tolerated.The analysis of these two cases and of those previously reported indicates that G-CSF represents the first-choice treatment in this affection when infectious signs are present in the neonate.

Idiopathic thrombocytopenic purpurea (ITP) is an autoimmune disease that occurs frequently in Europe and the US, but has rarely been described in Africa. Case report. An 8-year-old girl was admitted for cutaneous and mucosal bleeding. She had a low platelet count (11 x 10(9)/l). ITP was suspected and the diagnosis was confirmed by bone marrow examination. Corticosteroid treatment was effective.This is the second case reported in Togo since 1982. The diagnosis of the disease is straightforward, so the lack of cases reported in central Africa suggests that the disease is rare in this region. The rarity of the disease may be due to genetic or environmental factors, or it may simply be that physicians overlook this disease when making their diagnosis. Corticosteroids are now the preferred treatment for ITP because of the risk of transmitting Creutzfeldt-Jacob's disease by intravenous administration of immunoglobulin. Splenectomy is the ultimate treatment for chronic forms.Unlike other diseases, the diagnosis and treatment of which require methods unavailable in parts of Africa, ITP treatment, as currently practiced in countries of the northern hemisphere, is within the reach of most African countries. Further studies are required to determine the true frequency of the disease in central Africa.

The implications of the detection of residual disease after treatment of acute lymphoblastic leukemia (ALL) are unclear. We conducted a prospective study at 11 centers to determine the predictive value of the presence or absence of detectable residual disease at several points in time during the first six months after complete remission of childhood ALL had been induced. Junctional sequences of T-cell-receptor or immunoglobulin gene rearrangements were used as clonal markers of leukemic cells. Residual disease was quantitated with a competitive polymerase-chain-reaction (PCR) assay. Of 246 patients enrolled at diagnosis and treated with a uniform chemotherapy protocol, 178 were monitored for residual disease with one clone-specific probe (in 74 percent) or more than one probe (in 26 percent). The median follow-up period was 38 months.The presence or absence and level of residual leukemia were significantly correlated with the risk of early relapse at each of the times studied (P0.001). PCR measurements identified patients at high risk for relapse after the completion of induction therapy (those withor =10(-2) residual blasts) or at later time points (those withor =10(-3) residual blasts). Multivariate analysis showed that as compared with immunophenotype, age, risk group (standard or very high risk), and white-cell count at diagnosis, the presence or absence and level of residual disease were the most powerful independent prognostic factors.Residual leukemia after induction of a remission is a powerful prognostic factor in childhood ALL. Detection of residual disease by PCR should be used to identify patients at risk for relapse and should be taken into account in considering alternative treatment.

Autoimmune neutropenia (AIN) is a frequent cause of chronic neutropenia especially in youngest children. Its diagnosis is established by immunological proof of the autoimmune mechanism. The aim of this study is to better describe this autoimmune process and to show the contribution of bone marrow smears to this diagnosis.Ten children, six girls and four boys, were examined between 1990 and 1995. Eight of them had typical AIN, confirmed by the presence of antibodies against neutrophils. Two other patients were included on the basis of bone marrow pictures. Five non-neutropenic children with normal bone marrow smears were chosen as controls. Bone marrow analysis was always performed by the same cytologist according to a reproducible technique.Six out of ten patients had important features of elective phagocytosis of neutrophils by marrow macrophages (unlike controls) without signs of dysgranulopoiesis or hemophagocytosis. Antibodies against neutrophils were detected in six patients with phagocytosis and in four patients without these cytological features. In two other children presenting the same bone marrow picture and clinical profile, an autoimmune process was probable, even in the absence of antibodies against neutrophils. Some patients had several infections and were given immunoglobulins and/or granulocyte colony-stimulating factor (G-CSF) therapy. The efficacy of Immunoglobulin was not constant, whereas G-CSF was effective at low doses and shortened the duration of infections.Prolonged neutropenia in childhood must lead to look for phagocytosis by marrow macrophages in bone marrow smears, as a possible sign of autoimmunity. Growth factors may temporarily be used associated with antibiotics therapy in severe and prolonged infections.

The pharmacokinetics of 6-mercaptopurine, including cerebrospinal-fluid (CSF) distribution, and the erythrocyte 6-thioguanine nucleotide concentrations were determined in children randomised to receive intravenous mercaptopurine for acute lymphoblastic leukaemia (ALL), according to the EORTC protocol ALL n.58881.After 1 month of oral treatment at a dose of 50 mg.m-2.day-1, the pharmacokinetic parameters were determined after the first i.v. administration of 1 g.m-2 (bolus dose of 0.2 g.m-2 followed by an 8-h infusion of 0.8 g.m-2) in 11 patients: systemic clearance was 23.02 1.h-1, volume of distribution was 0.75 l.kg-1, and elimination half-life was 1.64 h. The erythrocyte thioguanine concentrations were measured in the same 11 patients and increased significantly between the beginning and the end of infusion (10 pmol x 10(8) packed RBC) or within 24 h of infusion (223 pmol x 10(8) packed RBC). The CSF concentration was 3.78 mumol.1(-1), 1-6 h after the beginning of infusion (n = 28) and the CSF to plasma ratio was 0.15 (n = 16). In patients receiving the oral dose of 50-165 mg.m-2.day-1 of 6-mercaptopurine, CSF concentrations were below 0.18 mumol.1(-1), 1-24 h after drug intake (n = 67), and the CSF to plasma ratio was not calculated.Following the i.v. administration of 6-mercaptopurine, we observed high CSF concentrations of 6-mercaptopurine and an acute increase of erythrocyte thioguanine nucleotide concentrations. The clinical trial (EORTC protocol ALL n[symbol: see text]5881), comparing the oral and i.v. administrations of mercaptopurine, will demonstrate if the i.v. administration reduces the incidence of CNS relapses.

The gene encoding the CD2 mouse cell surface antigen was retrovirally transduced into cord blood CD34+ cells. On infection by culture at the contact of retrovirus-packaging cells, the mCD2 marker was expressed by 30-40% CD34+ cells, that included the most primitive stem cell-enriched Thy-1+ and CD38- subsets. Accordingly, sorted cord blood CD34+Thy-1+ cells could be directly infected in the same conditions. mCD2- transgenic cord blood CD34+ cells were then used to reconstitute human fetal thymus implanted in SCID mice. Five to 8 weeks later, the mCD2 antigen was detected on approximately 10% of the human thymocytes repopulating the thymus grafts and the transgene genome was detected in graft cell DNA by Southern blot. These results demonstrate efficient gene transfer into primitive cord blood hematopoietic cells endowed with lymphoid potential and suggest gene therapy schemes in neonates suffering inherited or acquired-such as HIV infection-disorders of the T-cell lineage.

Forty-six children with juvenile myelomonocytic leukemia (JMML) diagnosed between 1978 and 1993 in 12 centers were retrospectively studied. There is no evidence that any conventional treatment influences the long-term evolution of JMML. Among 28 patients treated without bone marrow transplantation (BMT), 26 died (median survival: 17 months), two are alive, one in complete remission (CR) after intensive chemotherapy. Allogenic BMT is the best treatment: 18 patients underwent BMT, 11 are in CR (at 9, 15, 22, 25, 41, 45, 49, 53, 66, 90 and 108 months). Conditioning regimens using chemotherapy alone may cure some patients (3/6) occasionally despite autologous reconstitution (1/3); if relapse occurs, a second BMT may be curative (2/3). Among the 12 patients conditioned immediately with TBI, six are in CR, one is in relapse, five died (one of them in durable autologus CR from Schwannoma). It is our opinion that splenectomy is of therapeutic value and seems not to have influenced the incidence of infections complications. We found no argument in favor of intensive chemotherapy before conditioning. Results with HLA-matched unrelated donors are satisfactory. One patient relapsed at 4 months after an unrelated BMT and entered a new CR after discontinuation of cyclosporine.

The marrows of patients with lysinuric protein intolerance (LPI) are generally considered as normal, even though autoerythrophagocytosis has been observed in some of them.Lysinuric protein intolerance was recognized in two 12 and 15-year-old brothers who had been diagnosed following an immuno-hematological investigation. Clinical history had been characterized by a neonatal macrophage activation syndrome (hepatosplenomegaly, pancytopenia, hypofibrinogenemia and hypertriglyceridemia). A putative diagnosis of familial lymphohistiocytosis had been ruled out because of unusual clinical and immunological course. Both brothers had displayed chronic aversion to high-protein foods, failure to thrive, osteoporosis and developmental delay. Metabolic investigations had revealed chronic hyperammonemia while cationic aminoaciduria (lysine, arginine and ornithine) was only present during L-citrulline supplementation. Bone marrow examinations had been performed during the neonatal period and during later metabolic investigations. They both displayed a peculiar red cell and granulocytes phagocytosis by histiocytes and granulocytes precursors.This aspect of bone marrow could be considered as a specific sign of LPI. This report suggests that appropriate metabolic investigations should be performed in any unexplained macrophage activation syndrome.

Gene transduction into immature hematopoietic cells collected at birth from the umbilical cord could be useful for the treatment of genetic or acquired disorders of the hematopoietic system diagnosed during pregnancy. The SCID-hu mouse is a convenient model to investigate T-cell lineage gene therapy, since it allows replication of human intrathymic T-cell development. CD34+ cells isolated from cord blood were cocultured with CRIP MFG-murine CD2 (mCD2) cells that produce recombinant retroviruses encoding the mCD2 antigen, a cell surface marker easily detectable by flow cytometry. After 3 and 4 days in coculture, a mean of 19% and 39% human hematopoietic cells, respectively, expressed the mCD2 antigen. CD34+ cells cocultured for 4 days were used to reconstitute human fetal thymus implanted in SCID mice. Five to 10 weeks later, the mCD2 antigen was detected on approximately 10% of human thymocytes repopulating the thymic grafts in four of nine SCID mouse chimeras. Vector genomes were detected in graft cell DNA by Southern blot. Analysis of vector integration indicated that positive cells were of polyclonal origin in three animals and predominantly monoclonal in the other one. Our data show that foreign genes can be transduced into CD34+ cord blood cells endowed with T-cell differentiation potential, and suggest strategies for T-cell lineage gene therapy in the neonate.

Bone marrow transplantation (BMT) from matched sibling donors has been useful for the treatment of acute lymphoblastic leukaemia in children with a poor prognosis but is not available to more than two-thirds of patients who do not have a matched allogeneic donor. This study was undertaken to assess one strategy of marrow graft rejection prevention when alternative marrow sources such as HLA-phenoidentical unrelated volunteers and HLA-partially incompatible relatives were used. Results have been compared with two matched groups of children with the same risks factors and disease status who underwent HLA-genoidentical or autologous BMT. The conditioning regimen was the same for the three groups of patients; in the study group anti-LFA-1 and anti-CD2 monoclonal antibodies combined with T-cell depletion of the marrow was added to prevent graft rejection and graft-versus-host disease. Nineteen patients were included and followed for a median of 25 months (14 months to 3 years). Bone marrow engraftment occurred in 83% of the evaluable patients. Post-transplantation infectious diseases were the most frequent causes of death in the study group, occurring in 31% of patients. No fatal infections occurred in the two control groups. Post-transplantation relapse of leukaemia occurred in 26% of study group's patients, in 58% of autologous BMT control group's patients and 5% of HLA-genoidentical allogeneic group's patients. The event-free survival was 83% in the HLA-genoidentical control group, and 30% and 24% in the study group and in the autologous control group, respectively. In conclusion, a high rate of engraftment was achieved by the use of anti-LFA-1 and anti CD2 antibodies. Occurrence of a long-lasting immunodeficiency, however, led to a high incidence of lethal infections and relapses. Combined approaches are therefore to be investigated accelerating immune reconstitution after transplantations of T-depleted HLA partially incompatible marrow.

The objective of this study was to evaluate the feasibility, the toxicity and the efficiency of a BFM-like treatment protocol for acute nonlymphoblastic leukemia (ANLL) of children in which mitoxantrone was substituted for conventional anthracycline. The chemotherapy called for induction (mitoxantrone, cytosine arabinoside, etoposide), consolidation (mitoxantrone, cytosine arabinoside, 6 thioguanine), followed by two intensification courses with cytosine arabinoside plus, respectively, mitoxantrone during the first and etoposide during the second courses. Maintenance therapy consisted of daily 6 thioguanine, four-weekly courses of cytosine arabinoside (s.c. daily during 4 days) and eight-weekly courses of mitoxantrone. The latter drug was pursued up to a total cumulative dose of 150 mg/sqm. Maintenance therapy was stopped at 2 years of diagnosis. Out of 108 patients, 84 (77%) achieved a complete remission, 10 died during induction of hemorrhage, sepsis or pulmonary infiltration by leukemic cells. A total of 32 relapses occurred. The median follow-up was 3.5 years. Actuarial event-free survival, disease-free survival and overall survival at 3 years as 41%, 52%, 56%, respectively. These results compare favorably with most reported data, and cytogenetic findings appear to be the most important prognostic factor.

A nationwide, longitudinal study of infants born to human immunodeficiency virus-seropositive mothers has been under way in France since 1986. After 7 years of follow-up observations, we will update our assessment of the transmission rate in France and analyze, on a larger number of mother-infant pairs, the influence of maternal factors. Among the 848 pairs included in this analysis, the transmission rate was 20.2 +/- 2.7%. The transmission rate has remained stable with time and was not influenced by the mode of delivery, the mode of maternal infection, or the mother's ethnic origin. It was twice as high among the breast-fed infants as among the bottle-fed infants (40 vs. 19%, p0.04). Two factors were identified in a multivariate analysis (that did not include lymphocyte subset counts and the mode of feeding) as being associated with an increased risk of maternofetal transmission: p24 antigenemia (odds ratio = 3.1, confidence interval, = 1.5-6.2; p0.003) and elevated maternal age (p0.05). In the subgroup of 277 women whose absolute CD4+ lymphocyte counts at the time of delivery were available, the risk of transmission increased gradually from 15% of counts of600 CD4+ cells to 43% at counts of200. The risk of transmission was also related to the percentage of CD8+ cells, but each of the two factors seemed to play an independent role: the risk was lowest (12%) when the CD4+ cell count was500 and the proportion of CD8+ cells wasor = 40%, and was highest (50%) for values200 and40%.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic relapsing microangiopathic hemolytic anemia is rare in children. This report describes a case associated with thrombocytopenia following idiopathic thrombocytopenic purpura.A 4 year-old girl was admitted for acute idiopathic thrombocytopenic purpura (platelet count: 12,000/mm3) without anemia or fragmented red cells. The patient was given intravenous gammaglobulins without success, followed by prednisone (2 mg/kg/day). The platelet count was normalized, but decreased when the treatment was discontinued. The patient developed acute intracranial hypertension at the age of 5 yr 8 mo, following two cerebral hematomas. The platelet count was 9,000/mm3. A second course of intravenous gammaglobulins and prednisone was unsuccessful, so a splenectomy was performed. One year later, the patient was admitted because of diffuse purpura, anemia and jaundice. Hematologic findings were: Hb 8.4 g/dl, reticulocytes 448,200/mm3, fragmented red cells 16%, platelets 15,000/mm3, WBC 22,400/mm3. Seroimmunologic investigation showed a high titer of antinuclear antibodies. Examination for viral etiology was negative. Intravenous gammaglobulins had a transient effect on platelets, reticulocytes and fragmented red cells. The patient was then given vincristine plus prednisone; they were only effective when high doses were used. A second intracerebral hemorrhage occurred when the patient was given low doses of drugs. After 3 other hematologic relapses, the vincristine was stopped without further complication.The criteria for systemic lupus erythematous were not satisfied, despite the presence of antinuclear antibodies. A congenital deficiency of an unidentified plasma factor that reverses microangiopathic hemolysis and thrombocytopenia was not demonstrated in this patient, who could not be given fresh frozen plasma.

Busulfan disposition is age-dependent with a higher clearance and a larger volume of distribution in children than in adults. The optimal dosage of busulfan needed to achieve bone marrow (BM) displacement in young children with malignant or nonmalignant disease remains to be defined. Using a gas chromatography-mass spectrometry assay, we evaluated plasma pharmacokinetics of busulfan in 33 children (median age, 9 months; range, 2 months to 2.75 years) with immune deficiencies, lysosomal storage diseases, acute leukemias, and malignant lymphohistiocytosis after an oral dose ranging from 0.9 to 2.6 mg/kg. The busulfan clearance (assuming a bioavailability of 1) ranged from 2.1 to 13.4 mL/min/kg with a mean of 6.8 mL/min/kg, which is higher than that reported in older children (4.5 mL/min/kg) and adults (2.9 mL/min/kg). Six children with lysosomal storage disease (5 with Hurler's disease, 1 with San Filippo's disease) had a prolonged elimination half-life (4.9 v 2.4 hours), a larger volume of distribution (3.4 v 1.2 L/kg) and a faster clearance (8.7 v 6.3 mL/min/kg) than the other 27 children. This suggests that a higher dose of busulfan will be required to achieve BM displacement in children with lysosomal storage disease. Over the dose range of 0.9 to 2.6 mg/kg, busulfan pharmacokinetics were linear. However, only 46% of the interpatient variation in systemic exposure could be ascribed to the dose. Given the wide interpatient variability in busulfan disposition, dose adjustment and drug monitoring will be needed to achieve the optimal dosage of busulfan in young children. The plasma busulfan levels required to achieve BM displacement need to be defined, especially in lysosomal storage diseases.

The diversity of human peripheral blood gamma/delta T cells is known to be limited by the preferential use of V genes coding for V gamma 9 (usually linked to JP) and V delta 2. We show that the diversity of these cells is further limited at the junctional region. First, an identical rearrangement is found in 10%-30% of all gamma/delta T cells which contain V gamma 9-JP rearrangements. Second, the vast majority of V gamma 9-JP rearrangements which are different from this predominant sequence have, nevertheless, the same length or code for variable regions whose length differs by only one amino acid (+/- 1). Overall, 30%-50% of V gamma 9-JP rearrangements have a junctional region which encodes for a peptide with the amino acid sequence E VX EL, in which EV is predominantly, but not exclusively, encoded by the germ-line V gamma 9 sequence and EL is encoded by JP. The X amino acid is variable, but a glutamine is over-represented. The diversity of the V gamma 9-JP repertoire is fairly constant in different individuals and at different ages, including before, during and after the post-natal expansion of peripheral blood gamma/delta T cells.

Since January 1988, 91 children with ANLL have been treated with a polychemotherapy regimen containing Mitoxantrone (MTZ), excluding other anthracyclines. Induction consisted of Ara-C, MTZ, and VP 16. Consolidation lasted 6 weeks with Vincristine, MTZ, Ara-C and 6-thioguanine (6TG), and was followed by 2 intensification courses combining High-dose Ara-C with respectively MTZ or VP 16. Maintenance therapy associated 6TG, Ara-C and MTZ up to a cumulative dose of 150 mg/m2. 91 patients are evaluable: 70 (76.9%) achieved complete remission, 59 (64.8%) after induction alone. There were 7 early deaths, 5 deaths in complete remission, and 17 relapses. Major toxic side effects were observed during the consolidation phase, mainly infectious complications, and the median duration of neutropenia was 82 days in this phase, leading to decrease the MTZ dose from 10 to 8 mg/m2. The event-free survival at three years is 38%. Cardiac toxicity is presently absent in children without previous cardiopathy.

The treatment of children with acute lymphoblastic leukaemias (ALL) according to EORTC 58951 protocol is divided into 3 phases (induction, consolidation, maintenance). During the induction phase, patients are randomly assigned to receive either prednisone or dexamethasone (associated with anticancer drugs). The response to corticotherapy is evaluated by the quantification of the residual disease (RD) at day 35 of treatment and has an major impact on prognosis and treatment: patients with an RD lower than 10−2/100 000 cells. The distribution of CYP3A genotypes was : *1/*1: 2,7%, *1/*3: 18,8% and *3/*3: 78,5%, in aggrement with already published data. CYP3A5*2 and *6 mutations were not found There was no difference in the response to corticosteroid treatment according to individual CYP3A5 genotypes or to the type of corticosteroid. Our results did not evidence an impact of CYP3A5 on the initial response to corticosteroids. Clinical Pharmacology & Therapeutics (2004) 75, P20–P20; doi: 10.1016/j.clpt.2003.11.075

Resume La decontamination digestive totale offre une alternative aux mesures prophylactiques de lutte contre les infections qui accompagnent les traitements immunosuppresseurs des enfants atteints de leucemie algue. Trente-deux enfants atteints de leucemie aigue, en periode d'aplasie therapeutique et beneficiant ou non d'une decontamination digestive ont fait l'objet d'une surveillance bacteriologique. La decontamination digestive ne semble pas diminuer la frequence des infections. Cependant, les germes responsables d'accidents infectieux chez les enfants decontamines appartiennent surtout aux bacteries de l'environnement alors qu'ils sont le plus souvent d'origine digestive, donc potentiellement plus dangereux, chez les enfants non decontamines. Ces resultats nous conduisent a n'appliquer une decontamination intestinale qu'aux formes graves de leucemie aigue.

Resume Dans notre serie historique de 60 patients greffes pour une aplasie medullaire grave ou une leucemie aigue, 30 % des malades presentent une infection fongique, lethale dans 12 cas. 67 patients ont recu un traitement prophylactique des infections fongiques par le ketoconazole. Ce traitement dont les posologies varient de 6 mg a 18 mg/kg est commence des le conditionnement et est poursuivi tant que persiste le deficit immunitaire. A ce traitement est associe un isolement en chambre sterile a flux laminaire et une decontamination digestive totale par des antibiotiques non absorbables. Aucun effet secondaire net peut etre attribue au ketoconazole dont la tolerance est excellente. La decontamination fongique de la gorge est obtenue chez 57 % des patients mais l'absence d'isolement fongique des selles n'est constatee que chez 12 % des greffes. Une infection systemique n'est observee que dans 4 cas (6 %). Si l'absorption du ketoconazole est diminuee apres greffe, les concentrations restent satisfaisantes sauf en cas de reaction severe du greffon contre l'hote.