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Ataxia-telangiectasia (A-T) is a recessive disorder caused by biallelic pathogenic variants of ataxia-telangiectasia mutated (ATM). This disease is characterized by progressive ataxia, telangiectasia, immune deficiency, predisposition to malignancies, and radiosensitivity. However, hypomorphic variants may be discovered associated with very atypical phenotypes, raising the importance of evaluating their pathogenic effects. In this study, multiple functional analyses were performed on lymphoblastoid cell lines from 36 patients, comprising 49 ATM variants, 24 being of uncertain significance. Thirteen patients with atypical phenotype and presumably hypomorphic variants were of particular interest to test strength of functional analyses and to highlight discrepancies with typical patients. Western-blot combined with transcript analyses allowed the identification of one missing variant, confirmed suspected splice defects and revealed unsuspected minor transcripts. Subcellular localization analyses confirmed the low level and abnormal cytoplasmic localization of ATM for most A-T cell lines. Interestingly, atypical patients had lower kinase defect and less altered cell-cycle distribution after genotoxic stress than typical patients. In conclusion, this study demonstrated the pathogenic effects of the 49 variants, highlighted the strength of KAP1 phosphorylation test for pathogenicity assessment and allowed the establishment of the Ataxia-TeLangiectasia Atypical Score to predict atypical phenotype. Altogether, we propose strategies for ATM variant detection and classification.

The rapid kinetics of hematopoietic stem cells induced by Plerixafor (Mozobil®, Genzyme) should be of particular interest in children. We therefore conducted a prospective trial to determine whether a one-day mobilization by plerixafor alone was efficient enough in children with cancer.Children with solid malignancies were consecutively recruited for this phase-IIA, Bayesian single-center prospective study. Mobilization consisted in one subcutaneous injection of 240 μg plerixafor/kg body weight at 8a.m. (h0). Collection by apheresis began at h5 provided that CD34+count exceeded 10 × 10(6)/L. Our main evaluation criterion was percent of children in which at least 5 × 10(6) CD34+/kg could be collected during the first apheresis.No patients fulfilled the success criterion, and so a stopping criterion was met after 5 patients. All patients reached the threshold value of 10 × 10(6) CD34+cells/L post-injection and so all were eligible for apheresis. Peak CD34+cell values were ranged from 11 to 44 × 10(6)/L and were reached in 4h to 6h. No side-effects were observed. Median number of CD34+cells collected per patient BW was 1.62 × 10(6)[0.47-3.5]. In 3 of the 5 patients, collection was1.5 × 10(6) CD34+/kg BW.In children, a 'one-day' mobilization regimen consisting of one injection of 240 μg/kg plerixafor alone in hematological steady state provides a faster and shorter mobilization than in adults. This strategy may be an attractive option for completing an insufficient graft. More studies are warranted to optimize the use of plerixafor in children.

Summary Corticosteroid and central nervous system (CNS) irradiation can induce cataract in childhood lymphoblastic leukaemia survivors. Few prospective studies with systematic ophthalmological evaluation have been published. Cataract was prospectively assessed by serial slip lamp tests in 517 patients. All had acute lymphoblastic leukaemia, all had been treated by chemotherapy with or without CNS irradiation, and none had received haematopoietic stem cell transplantation. Median ages at last evaluation and follow-up duration from leukaemia diagnosis were 16·8 and 10·9 years, respectively. Cataract was observed in 21/517 patients (4·1%). Cumulative incidence was 4·5 ± 1·2% at 15 years and reached 26 ± 8·1% at 25 years. CNS irradiation was the only risk factor: prevalence was 11·1% in patients who had received irradiation and 2·8% in those who did not. We did not detect any steroid dose effect: cumulative dose was 5133 and 5190 mg/m2 in patients with and without cataract, respectively. Cataract occurrence did not significantly impact quality of life. We conclude that, in the range of steroid dose reported here, the cataract risk proves very low 15 years after treatment without CNS irradiation but an even more prolonged follow-up is required because of potential very late occurrence.

Background Apheresis is a major challenge in peripheral stem cell collection from low-weight children with cancer. Comparisons between the new apheresis device Optia (TerumoBCT) and the earlier COBE Spectra (CaridianBCT) have been performed in adults but not in low-weight children. The objective was to compare the performance of these two devices in small children. Study Design and Methods In this retrospective study, all patients were reviewed weighing less than 15 kg undergoing stem cell collection using the Optia device between April 2011 and April 2012. They were paired on weight in a 3:1 ratio with patients whose cells had been collected with the COBE Spectra since 2006. Results Six patients were treated with the Optia and were matched with 18 patients treated with the Spectra. No side effects occurred. Collection efficiency (CE) was similar between the two groups (50% vs. 47%), but CD34 cell blood clearance was lower with the Optia (0.4 mL/min/kg vs. 0.6 mL/min/kg, p

The late effects and quality of life (QoL) in childhood acute leukemia survivors were compared between hematopoietic stem cell transplantation (HSCT) recipients and patients who underwent conventional therapy. The study included 943 patients, 256 of whom underwent HSCT (27.1%). Medical visits were conducted to detect the occurrence of physical late effects. Based on patient age, different questionnaires were used to assess QoL. To evaluate the association between HSCT and each type of late effect or QoL dimension, the appropriate multivariate regressions were performed. QoL mean scores were compared with those obtained for age- and sex-matched French control subjects. Of all the survivors, 674 (71.5%) had at least 1 late effect, with the risk being 5.0 CI95 (3.0-8.6) times higher for transplantation survivors. For child survivors, scoring of QoL showed no significant differences between the treatment groups. The adult HSCT survivors reported lower physical dimension QoL scores than chemotherapy survivors. Compared with French norms, the survivor group reported a significantly lower mental composite score; however, the physical composite score showed no significant difference. Thus, transplanted survivors have a high risk of developing late effects, resulting in a decreased physical well-being in adulthood. However, long after treatment completion, childhood leukemia survivors report that effects on psychological well-being are more important than they are in physical QoL dimensions.

As almost all patients with high-risk neuroblastomas have autograft, we aimed to determine if minimal residual disease (MRD) quantified by RT-PCR for tyrosine hydroxylase in PBSC is prognostic in neuroblastomas. PBSC harvests from 38 children were analyzed. Seven had harvests positive for TH-mRNA. Patients with a positive MRD had a lower 2-year-overall-survival compared to those with negative MRD (P = 0.04) regardless of whether or not PBSC were re-infused. Patients in CR/VGPR group with positive MRD have hazard ratio of death at 7.3 [1.3–40.5]. In conclusion, molecular MRD status in PBSC of good response group may be of interest as a survival prognostic factor in high-risk neuroblastomas. Pediatr Blood Cancer 2013;160:E109–E112. © 2013 Wiley Periodicals, Inc.

Resume La leucemie aigue megacaryoblastique, ou de type 7 (LAM7), represente 0,5 a 2 % des leucemies aigues (LA) de l’enfant et 3 a 10 % des leucemies aigues myeloides (LAM). La myelofibrose frequemment associee explique les difficultes de prelevement et de mise en evidence du clone leucemique. Nous rapportons le cas d’une enfant de 7 mois, sans antecedent, presentant une hepatomegalie et une pancytopenie qui avaient fait suspecter precocement une leucemie. Rapidement, le tableau clinique s’etait complete de douleurs osseuses et articulaires avec des arthrites aseptiques recidivantes. Les radiographies montraient des appositions periostees diffuses, des bandes claires metaphysaires, et des zones d’osteolyse egalement compatibles avec une LAM7. Malgre la repetition des examens (myelogrammes multisites, biopsies osteomedullaires), nous n’avons pu confirmer le diagnostic dans un premier temps du fait de la difficulte de prelevement et de l’absence de cellules anormales au plan morphologique, phenotypique ou cytogenetique. La clinique, les images radiologiques, l’aspect des myelogrammes et la myelofibrose etaient autant d’arguments en faveur d’une LAM7 mais nous n’avons pu mettre en evidence les blastes suspectes qu’apres plus de 2 mois d’investigations et 15 jours de corticotherapie.

New therapies for children with high risk neuroblastoma are needed, and haploidentical stem cell transplantation with NK post-graft injections is a potential option. To develop this strategy, we compared and correlated two methods of NK cytotoxicity assay. The aim of this work is to optimize in vitro NK cytotoxicity assays, investigate the effect of interleukin stimulation on NK cells and use of antiGD2 antibodies against tumor target cells and finally establish an in vitro model for haploidentical stem cell transplantation. Experimental design We evaluated NK cell cytotoxicity in vitro against NB cell lines (IMR-32 and SK-NSH) in different culture conditions using a Europium BATDA fluorescence test, and correlated the results with quantification of TH, Phox2B, and DCX transcripts evaluated by RT-PCR. Results Both IMR-32 and SK-N-SH neuroblastoma cell lines were sensitive to NK cells and particularly when NK cells were stimulated by interleukin IL-2 and IL-15 or when using anti-GD2 antibodies against tumor target cells. All these results were observed either with Europium fluorometry assay or with RT-PCR quantification. There is a clear correlation between the two methods, for the three transcripts at the ratio effector/target 50/1 (TH r = 0.75, Phox2B r = 0.79 and DCX r = 0.8), for all the values whatever the cell line. Besides for all three transcripts, the correlations were significantly independent of the cell line and the ratio E/T (all p values non-significant) even if the best correlation was observed for the ratio 50/1. After prolonged incubation times of effector and target cells (24 h), which could be evaluated only by RT-PCR, all the transcripts clearly decreased, confirming the haploidentical effect of NK against the two neuroblastoma cell lines in our two in vitro haploidentical models but no advantage of mismatch. Conclusions NK cytotoxicity against neuroblastoma cell lines can be evaluated by Europium assay and by RT-PCR with clear correlation for the three transcripts TH, Phox2B and DCX whatever the ratio E/T and cell line used. This new method of RT-PCR is simple and suitable for large-scale conditions like study of adherent tumor cells or prolonged incubations of target/effector cells which allowed us to observe haploidentical effect.

Background In France, as in other countries, there is a need for a population-based view of access to care and modalities of treatment for adolescents with cancer. Procedure Using a population-based registration, we report pathways of care for 15–19-year-old patients, diagnosed with cancer in 2006 and 2007, living in six French regions, accounting for 41% of the French population. Results The median times (inter-quartile range) for diagnosis and treatment of the 594 included adolescents were 8 weeks (3–17) and 3 days (0–16), respectively. First physicians met by the patients were mostly general practitioners (59%). Seventeen percent of patients were firstly seen on emergency wards. Most of the patients (82%) were treated in an adult environment. Management decisions were taken within the context of a multi-disciplinary team (MDT) in 54% of cases. Twenty-seven percent of patients were included in randomized or non-randomized clinical studies: percentage depended on the tumor type and on the number of on-going trials at the study period. Fifteen percent of patients were included in pediatric studies, 7% in adult studies, and 5% in studies including both adults and children. Conclusions The pathways of care for French adolescent patients with cancer are heterogeneous. Our results reveal differences in MDT meetings according to tumor types and a lack of effective collaboration between pediatric and adult wards. Efforts must be made to develop cancer networks to ensure that adolescents receive the optimal care in a suitable environment. Pediatr Blood Cancer 2012; 58: 924–929. © 2011 Wiley Periodicals, Inc.

Little is known on strategies to prevent or to treat relapses occurring after haploidentical stem cell transplantation (haplo-HSCT) performed for the high-risk neuroblastoma (NB). We describe a 6-year-old male with refractory NB who relapsed 22 months after haplo-HSCT. A complete remission was obtained with a combination of immuno-chemotherapy based on donor NK cells transplants, IL2 infusions and temozolomide/topotecan. This case is an incentive to explore both the immediate therapeutic effect of haplo-graft provided via haplo-NK cells and the immunogenic platform that haplo-HSCT offers for future treatment. Our post-relapse strategy shows that chemo- and bio-treatment should be viewed as complementary therapeutic options. Pediatr Blood Cancer 2012;59:739–742. © 2011 Wiley Periodicals, Inc.

Resume But de l’etude Six pour cent des produits sanguins labiles (PSL) transfuses en France sont delivres pour la pediatrie. Notre etude retrospective monocentrique realisee au centre hospitalier universitaire (CHU) de Clermont-Ferrand avait pour but d’evaluer les pratiques transfusionnelles en pediatrie. Patients et methodes Nous avons analyse les transfusions realisees dans l’ensemble du pole pediatrique pendant une annee : caracteristiques du receveur et du produit transfuse, indication de la transfusion, biologie pretransfusionnelle et effets indesirables. Nous avons ensuite cherche a determiner la conformite de la prescription aux recommandations de l’Agence francaise de securite sanitaire des produits de sante (Afssaps). Resultats Mille six cent sept PSL (806 concentres de globules rouges [CGR], 670 concentres plaquettaires [CP] et 131 unites de plasma therapeutique) avaient ete distribues a 233 enfants (5,3 % des transfusions realisees sur l’annee au CHU). Les services pediatriques les plus consommateurs etaient l’oncohematologie (68,2 %), la reanimation (15,4 %) et la chirurgie (10,2 %). Dix effets indesirables avaient ete rapportes. La conformite aux recommandations de l’Afssaps pour les concentres globulaires, les CP et les unites de plasma avait ete respectivement de 35,9 %, 41,6 % et 80,9 %. La non-conformite etait majoritairement due a la transformation et la qualification des produits (irradiation, « cytomegalovirus [CMV] negatif »). Conclusion L’oncologie, la reanimation neonatale et la chirurgie sont les principales disciplines pourvoyeuses de transfusion chez l’enfant. La discordance importante entre les prescriptions et les recommandations pour l’indication d’irradiation et la qualification CMV negatif illustre la complexite de l’appreciation du risque en transfusion pediatrique. Cela pourrait etre nettement simplifie par la generalisation des techniques photochimiques de securisation des PSL.

Background To improve outcome and overall survival (OS) in high-risk neuroblastoma, NB96 induction therapy was intensified using sequential high-dose chemotherapy and autologous stem cell rescue. Procedure Twenty children were included in this pilot study undertaken at seven reference centers in France, between May 1995 and October 1996. Induction began with one cycle of conventional chemotherapy followed by six sequential cycles of high-dose chemotherapy comprising two cycles of etoposide 800 mg/m2/day over 3 days, two cycles of cyclophosphamide 2,000 mg/m2/day over 3 days, and two cycles of carboplatin 400 mg/m2/day over 5 days, followed by stem cell rescue. Results Thirteen patients (13/20) received this induction with acceptable toxicity and adequate stem cell harvest. Of these, nine (9/13) underwent surgery according to the protocol, while one patient was given a consolidation regimen prior to surgery. No toxic death was recorded. At the end of induction, complete remission was achieved in 10 cases (50%), with six still alive in July 2009. The 5-year event-free survival and OS were 35 ± 11% and 40 ± 11%, respectively. Conclusion NB96 therapy is feasible and tolerated without lethal toxicity. Nevertheless, given the small sample size and absence of randomization in our study, the effectiveness of this strategy based on metastasis complete response rates and long-term outcome was not superior to other intensive chemotherapy regimens. Pediatr Blood Cancer 2011; 57: 965–971. © 2011 Wiley-Liss, Inc.

The literature contains a substantial amount of information about factors that adversely influence the linear growth in up to 85% of patients undergoing haematopoietic SCT (HSCT) with TBI and/or cranial irradiation (CI) for acute leukaemia (AL). By contrast, only a few studies have evaluated the impact of growth hormone (GH) therapy on growth rate and final height (FH) in these children. We evaluated growth rates during the pre- and post-transplant periods to FH in a group of 25 children treated with HSCT (n=22), TBI (n=21) or/and CI (n=8) for AL and receiving GH therapy. At the start of GH treatment, the median height Z-score was -2.19 (-3.95 to 0.02), significantly lower than at AL diagnosis (P

Better understanding of the antitumor effect of allogeneic transplant and the need to reduce the toxicity of the procedure, particularly in elderly patients have spurred the development of reduced-intensity conditioning regimens (RIC). These regimens allow fast engraftment with very low chemotherapy-induced toxicity. They are widely used in adults and there are numerous studies to demonstrate their feasibility and efficiency, but in pediatrics, the place of RIC remains to be determined. They can be proposed in two pediatric populations. First, solid tumors or hematological malignancies remaining unresponsive to the reference strategies according to best practices in pediatrics. Second, in children presenting malignancies for which allografting is the only recognized curative indication but is contraindicated with myeloablative conditioning regimens. More than 100 pediatrics cases have been reported in various pathologies, including blood diseases, acute leukemia, Hodgkin's lymphoma and solid tumors, and promising results published recently underline how RIC warrants further investigation in prospective comparative multicentric trials. The use of new post-graft treatment modalities is expected to pave the way to the development of RIC in pediatric patients.

Background A prospective clinical trial was performed in order to validate the pharmacokinetic (PK) and clinical benefits of a new dosing schedule of intravenous busulfan (IV Bu) in children. Procedure IV Bu was administered as a 2-hr infusion every 6 hr for 4 days. Five dose levels were given according to body-weight strata. Results The 67 children aged from 4 months to 17.2 years were followed up over 50 months after autologous or allogeneic stem-cell transplantation. Reduced PK variability was seen after IV Bu administration enabling efficient targeting with 78% of patients within the 900–1,500 µM · min therapeutic window and reproducible exposures across administrations. No neurological complications occurred. The low incidence of hepatic veno-occlusive disease (VOD) recorded was not correlated with high area under the curve (AUC). Only stomatitis was correlated with high AUC in the autologous group. The 4-year overall survival was 59% in the autologous group and 82% in the allogeneic group. Conclusion The new dosing schedule using IV Bu provides adequate therapeutic targeting from the first administration, with low toxicity and good disease control in high-risk children. The choice of this formulation of Bu should be considered because of its low morbidity and good outcome. Pediatr Blood Cancer 2012; 58: 90–97. © 2011 Wiley Periodicals, Inc.

BACKGROUND: Using autologous cryopreserved mononuclear cells (MNCs) for extracorporeal photochemotherapy (ECP) offers several advantages, such as treating patients from geographically distant care centers or maintaining ECP schedule while dramatically reducing number of apheresis sessions. We previously reported that cryopreserved cells retain their immunomodulatory properties when exposed to UVA and psoralen. To date, there are no clinical data on the use of cryopreserved MNCs for ECP (“cryo-ECP”). CASE REPORTS: Three patients were treated by cryo-ECP for refractory dermatomyositis, juvenile localized scleroderma, and acute graft-versus-host disease. For the first two patients, cryo-ECP aimed to reduce the number of apheresis sessions. Each cell product was split into three equal fractions: one was infused, and the other two were frozen for later infusion. The third patient was referred to our center from a hospital 700 km away. Fifteen apheresis procedures were performed during his stay: 12 were immediately treated and infused while three were cryopreserved. After discharge, the three cryopreserved bags were thawed, ECP-treated, and then sent back to the patient. CONCLUSION: In all three patients, cryo-ECP was safe and feasible. These cases illustrate promising clinical applications of the technique, opening perspectives for making ECP much more acceptable to patients while extending its indications.

Resume L’hypertension intracrânienne benigne (HICB) est caracterisee par une augmentation de la pression intracrânienne en l’absence de processus expansif intracrânien, de thrombose veineuse ou d’hydrocephalie. Contrairement a ce que pourrait laisser penser son appellation, elle met en jeu le pronostic visuel. Nous rapportons le cas d’une jeune fille de 15 ans qui a presente un tableau initial compatible avec une meningite virale, bien qu’il n’ait pas comporte de syndrome meninge franc. La pression du liquide cephalorachidien (LCR) etait de 70 cm d’eau. Il n’existait pas de dilatation ventriculaire. L’evolution etait marquee par une atteinte du nerf abducens (VI), une baisse d’acuite visuelle et un œdeme papillaire bilateral. Le diagnostic retenu etait celui d’HICB secondaire, de cause inconnue malgre un bilan exhaustif. Apres 8 semaines de traitement par acetazolamide per os et un traitement par ponctions lombaires iteratives, l’evolution etait favorable. Il n’y a pas eu de recidive apres 1 an de recul. Cette observation illustre les risques lies a une affection peu connue et improprement denommee benigne, dont la prise en charge symptomatique et etiologique est primordiale pour la preservation de la fonction visuelle.

Background Graft contamination has been blamed for causing relapse in children with high-risk neuroblastoma (HRNB) after autologous hematopoietic stem cell transplantation (HSCT). Procedure We report the long-term results of hematopoietic reconstitution, post-transplant complications, and clinical outcome of 44 children with HRNB treated with busulfan/melphalan high-dose chemotherapy followed by transplantation of purged CD34+ immunoselected autologous peripheral HSCT. Minimal residual disease (MRD) of grafts was evaluated by anti-GD2 immunofluorescence or tyrosine hydroxylase reverse transcriptase-polymerase chain reaction (RT-PCR). Results Contaminating neuroblasts were found in 19/38 grafts (50%) before CD34+ positive selection, and none after (technique sensitivity of one cell in 105). A median of 6.5 × 106 CD34+ cells/kg (range 0.8–23.7) were transplanted with only 2% of TRM. Neutrophils and platelet recovery occurred within a median of 12 days (range 9–47) and 44 days (range 12–259), respectively, without any secondary graft failure. Twenty-three percents of patients experienced a sepsis (10/44) and 14% a pyelonephritis (6/44). Recurrence of varicella zoster virus occurred in 21% of patients (9/44). Negative RT-PCR MRD within the leukapheresis product and cis-retinoic acid therapy were significantly and independently associated to a better survival (P

International audience; The management of immune diseases in children remains challenging , although significant advances have been made. In addition to pharmacological approaches, extracorporeal photochemotherapy (ECP) is distinctive in its ability to provide immunomodulation without immune suppression or toxicity. However, in practice, this therapy is not widely used because of logistical issues and the lack of robust clinical pediatric studies. Here, we discuss the potential clinical applications of ECP in children and emphasize the need for a rigorous and specifically pediatric clinical evaluation of ECP. ß 2010 Elsevier Masson SAS. All rights reserved. Résumé Malgré l'apport des biothérapies, le traitement des maladies dysim-munitaires sévères et des conflits allogéniques de l'enfant reste difficile et entaché de nombreuses complications. Dans ce contexte, la photo-chimiothérapie extracorporelle (PCE) (thérapie cellulaire qui repose sur l'effet immunomodulateur des cellules mononucléées du patient, prélevées par aphérèse et exposées ex vivo aux rayons ultraviolets A [UVA] en présence de psoralène) a l'avantage notable d'induire une tolérance immunitaire sans générer d'immunosuppression systémique ni de toxicité a ` court, moyen ou long terme. Cette immunomodulation fait intervenir notamment la génération de lymphocytes T régulateurs (T reg). Malgré cela, la PCE est peu utilisée en raison de ses contraintes logistiques et du manque de données cliniques. Nous proposons une revue des indications reconnues et potentielles de la PCE en pédiatrie. Nous insistons sur la nécessité d'une e ´valuation clinique spécifique a ` l'enfant qui ne peut se concevoir sans la participation active des cliniciens pédiatres en particulier dans le domaine de la transplanta-tion et des maladies auto-immunes et inflammatoires.

Quality control is essential to validate extracorporeal photopheresis (ECP) processes. There is just one protocol based on PHA-induced proliferation. Since it involves the use of radioactive thymidine, we developed another technique using CFSE labeling. We compared the two tests in a paired series including 18 procedures. The thymidine test was valid. Once proliferation was obtained (10 patients out of 13), the CFSE test was in close agreement with it. In particular, two cases of residual proliferation after ECP were simultaneously detected by both techniques. Only the CFSE test allows targeted analysis of lymphocytes, thus identifying a surviving lymphocytic sub-population.

Resume L’obesite est susceptible de s’accompagner d’anomalies metaboliques qui pourraient etre liees a la survenue d’une pathologie cardiovasculaire a l’âge adulte. En particulier, le « syndrome metabolique » (SM) est defini par la presence d’au moins 3 anomalies parmi 5 : obesite abdominale, hypertension arterielle (HTA), hypertriglyceridemie, hypoHDLcholesterolemie, intolerance au glucose. L’objectif de cette etude retrospective etait d’estimer la frequence des anomalies metaboliques et du SM dans une population semi-rurale d’enfants obeses. Materiel et methodes Les dossiers de 244 enfants obeses adresses en consultation d’obesite au CHU de Clermont-Ferrand entre janvier 2003 et janvier 2007 ont ete analyses selon la definition adaptee a l’enfant du National Cholesterol Education Program Adult Treatment Panel . L’insulinoresistance etait definie par un Homeostasis Model Assessment – Insulin Resistance (Homa-IR) superieur au 75 e percentile. Resultats Environ 95,9 % des enfants avaient une obesite abdominale, 38,1 % une HTA systolique, 19,3 % une HTA diastolique, 12,3 % une hypertriglyceridemie et 4,1 % une hypoHDLcholesterolemie. Le SM a ete trouve chez 11,5 % des enfants. La presence du SM etait correlee au Z-score de l’indice de masse corporelle (IMC). Les facteurs correles a la severite de l’obesite etaient le taux de triglycerides ( p p p p Conclusion Les anomalies metaboliques et l’insulinoresistance sont frequentes et precoces chez les enfants obeses ; dans la mesure ou elles sont etroitement correlees au degre d’obesite, leur prise en charge specifique apparait secondaire par rapport a celle de l’obesite.

We report 19 episodes of hypercalcemia in three children treated with 13-cis-retinoic acid (13-cis-RA) as a post-consolidation therapy for high-risk neuroblastoma. There was no concomitant overload in 13-cis-RA blood levels. Blood calcium fell after arrest of 13-cis-RA intake. Half dosage retinoid treatment resumption did not prevent the recurrence of hypercalcemia. Concomitant biological values showed massive bone resorption. Hence, hypercalcemia seemed not secondary to 13-cis-RA overload but rather to inter-individual variability in its interaction with bone metabolism. Current guidelines in case of hypercalcemia are to reduce 13-cis-RA dosage. Instead we propose to maintain the therapeutic dosage, but to shorten the duration of courses.

International audience; Our purpose was to assess success rates in children of achieving optimal hematopoietic progenitor cells (HPCs) harvest after mobilization with 300 microg/kg pegfilgrastim. Between January 2005 and January 2007, 26 children with solid malignancies who were referred for HPC collection were consecutively included. Hematopoietic progenitor cell mobilization consisted of one s.c. injection of 300 microg/kg body weight (BW) of pegfilgrastim. The success criterion was defined as at least 5 x 10(6) CD34+ cells/kg during the first standard apheresis (less than 3 blood volumes processed (BVP)). After 26 inclusions, the Bayesian analysis gave a mean estimated success rate of 60.7% (95% credibility interval: 42.0-78.0%). The first apheresis allowed the collection of 8.3 x 10(6) CD34+ cells/kg BW (range 0.6-37.8), with a median of 2.8 BVP (range 1.4-3.0). Overall, the median of CD34+ cells collected was 12.4 x 10(6)/kg (range 2.7-37.8). The cumulative dose of anthracyclin was the only variable associated with the total number of CD34+ collected cells (P or =3 occurred. We conclude that a single injection of 300 microg/kg pegfilgrastim in the hematological steady state is an efficient and well-tolerated method of HPC mobilization in children with solid malignancies.

New concepts of allogeneic hematopoietic SCT (allo-HSCT) for neuroblastoma and other solid tumors do not rely on escalation of chemotherapy intensity and tumor load reduction but rather on a graft-vs-tumor effect. At this point, this is still an investigational and unusual application of allogeneic transplant, with 78 neuroblastoma patients reported to the European Group for Blood and Marrow Transplantation activity survey from 2002 to 2007 and less than 100 published cases. Two trends can be observed in the reviewed data: some teams have used allo-HSCT in children with refractory or progressive disease and significant tumor burden and other teams in children with CR, PR or minimal residual disease earlier in their disease process. Early studies of allo-HSCT in children with high-risk neuroblastoma suggest that this is a feasible approach that may improve outcome in this deadly disease. However, the proper timing for allo-HSCT during the disease course remains to be determined.

The discovery of a liver nodule during postcancer follow-up suggests malignancy recurrence. However, patients previously treated for pediatric cancers are at greater risk of developing benign hepatic tumors, raising the problems of getting an accurate noninvasive diagnosis. Here we report 3 cases of focal nodular hyperplasia of the liver in patients previously treated for pediatric neoplastic diseases. Radiologic findings and needle-biopsies remained insufficient in 2 cases to rule out metastasis or a potentially threatening tumor. Only surgical resection led to positive diagnosis and prevented complications. However, a review of the literature shows that if the nodule presents with typical features of focal nodular hyperplasia a close imaging follow-up is all that is recommended.

Resume Introduction L’action immunomodulatrice de la photochimiotherapie extracorporelle ou photopherese et son efficacite dans d’autres dermatoses d’interface justifient son emploi dans le traitement du lichen muqueux. Nous rapportons deux cas de lichen erosif muqueux traites par photopherese. Observations Cas 1. Un homme âge de 61 ans avait un lichen erosif buccal evoluant depuis quatre ans et complique d’un carcinome lingual. Une pancreatite iatrogene rendant impossible une corticotherapie, des seances de photopherese etaient debutees. Des la neuvieme seance, les douleurs disparaissaient et les lesions s’amelioraient. Cependant, le malade n’etait pas en remission totale apres 32 seances. Cas 2. Une jeune femme âgee de 17 ans avait un lichen erosif oral et genital, corticodependant. La corticotherapie etait responsable de signes cliniques d’immunodepression. Apres sept seances de photopherese (une par semaine), les symptomes fonctionnels s’amendaient. A la quinzieme seance, alors que la corticotherapie avait pu etre arretee, les lesions cutanees avaient disparu et les lesions muqueuses etaient spectaculairement ameliorees. Une remission complete, sans corticotherapie, etait obtenue en une vingtaine de seances. Discussion Nos observations, associees a celles de 23 patients publiees dans la litterature, suggerent l’efficacite de la photopherese pour le traitement du lichen muqueux erosif. Les premiers signes de reponse therapeutique apparaissent au bout de quelques semaines. Les rechutes sont, en revanche, tres frequentes a distance de l’arret des seances, posant la question de la duree du traitement.

Resume Objectif Notre objectif etait d’ameliorer l’organisation et la gestion de la prise en charge des enfants atteints de cancer ou de leucemie et de repondre a la legislation en vigueur en France. Methodes Nous rapportons les etapes successives de la mise en place d’un systeme d’assurance qualite, les methodes utilisees, les motivations, le cout, les difficultes rencontrees ainsi que les avantages obtenus. Nous avons entrepris dans le centre regional de cancerologie pediatrique (CRCP) du CHU de Clermont-Ferrand une demarche qualite basee sur la norme ISO 9001/2000. La mise en place du systeme d’assurance qualite a ete conduite comme un projet de recherche et un projet medical d’etablissement avec le soutien de sa direction. La mission de notre CRCP est « la prise en charge de l’enfant et de sa famille ». Cette mission a ete eclatee en plusieurs « processus », approche qui consiste a considerer le service clinique en terme de flux et de successions de transformations (accueil, soins, soutien, accompagnement, etc.) qui produisent de la valeur ajoutee (des services et des produits adaptes aux besoins des « clients » : enfants, familles, correspondants, etc.). Resultats Nous avons individualise 10 processus « realisation » ou « metier » tels « diagnostic », « soins de support » ou « projet de l’enfant ». La cartographie qui est la representation systemique des processus et des interactions entre eux a permis d’avoir une vision globale de l’activite « soins » de notre CRCP. Conclusion La norme ISO 9001/2000 etant un outil d’aide a l’organisation et a la gestion, le benefice obtenu grâce a son implantation dans un service clinique a ete avant tout percu en terme organisationnel aboutissant a un veritable esprit d’equipe, une uniformisation des pratiques professionnelles et une valorisation du role de chacun. Les avantages apparaissent a 3 niveaux : l’enfant et sa famille, les equipes medicale et paramedicale et les tutelles.

Resume Au cours des dix dernieres annees, l’enteropathie auto-immune neonatale a pu etre individualisee au sein des diarrhees graves rebelles du nourrisson dans un cadre nosologique denomme syndrome d’immunoderegulation, polyendocrinopathies, enteropathie auto-immune, liees au chromosome X (Ipex). Par des etudes de liaison au sein de familles affectees, ce syndrome pediatrique rare et au pronostic defavorable, a pu etre rattache dans la majorite des cas a la mutation d’un gene, foxp3. Une perte de fonctionnalite homozygote de ce gene entraine la disparition d’une sous-population lymphocytaire de phenotype CD4+CD25+ ayant la capacite d’inhiber certaines reponses immunitaires. L’etude de ces lymphocytes T-regulateurs permet de mieux apprehender l’homeostasie du systeme immunitaire mais aussi la physiopathologie du syndrome Ipex, condition prealable indispensable avant tout traitement. La maitrise ex vivo de ces lymphocytes permettrait d’envisager de multiples et prometteuses applications de therapie cellulaire.

International audience; Relapse after transplantation is a major cause of treatment failure in paediatric acute lymphoblastic leukaemia (ALL). Here, we report the findings of a prospective national study designed to investigate the feasibility of immune intervention in children in first or subsequent remission following myeloablative conditioning. This study included 133 children who received a transplant for ALL between 2005 and 2008. Minimal Residual Disease (MRD) based on T cell receptor/immunoglobulin gene rearrangements was measured on days -30, 30, 90 and 150 post-transplantation. Ciclosporin treatment was rapidly discontinued and donor lymphocyte infusions (DLI) were programmed for patients with a pre- or post-transplant MRD status ≥10(-3) . Only nine patients received DLI. Pre- and post-transplant MRD status, and the duration of ciclosporin were independently associated with 5-year overall survival (OS), which was 62·07% for the whole cohort. OS was substantially higher in patients cleared of MRD than in those with persistent MRD (52·3% vs. 14·3%, respectively). Only pre-transplant MRD status (Hazard Ratio 2·57, P = 0·04) and duration of ciclosporin treatment (P < 0·001) were independently associated with relapse. The kinetics of chimerism were not useful for predicting relapse, whereas MRD monitoring up to 90 d post-transplantation was a valuable prognostic tool to guide therapeutic intervention.

International audience; The main aim of the Leucémies de l'Enfant et l'Adolescent (LEA) project (Childhood and Adolescent Leukaemia) is to study the determinants (medical, socioeconomic, behavioural and environmental) of medium- and long-term outcomes of patients treated for childhood acute leukaemia (AL). The LEA study began in 2004 and is based on a French multicentric prospective cohort. Included are children treated for AL since January 1980 (incident and prevalent cases), surviving at month 24 for myeloblastic AL and lymphoblastic AL grafted in first complete remission or at month 48 for lymphoblastic AL not grafted in first complete remission. Information is collected during specific medical visits and notably includes the following data: socioeconomic data, AL history, physical late effects (such as fertility, cardiac function and metabolic syndrome) and quality of life. Data are collected every 2 years until the patient is 20 years old and has had a 10-year follow-up duration from diagnosis or last relapse. Thereafter, assessments are planned every 4 years. In active centres in 2013, eligible patients number more than 3000. The cohort has already included 2385 survivors, with rate of exhaustiveness of almost 80%. Data access can be requested from principal coordinators and must be approved by the steering committee.

The cure rate for cancer in children is currently almost 75%. This rate has remained fairly constant over the past few years, which suggests that the limits of today's curative treatment potential have been reached. The development of cell therapy techniques opens up new therapeutic possibilities in paediatric oncology. Here, we deal both with a number of cell therapy techniques, which have already proved their efficacy in children, and other more innovative approaches, which require validation. Examples of the use of autologous and allogeneic cells are described. Clinical studies and their results, while often preliminary, are reported. The importance of well run clinical research, a clear and progressive legal framework and the necessary substantial economic support for the development of cell therapy are underlined.

Molecular detection of tumor cells is the most sensitive approach to study residual disease in bone marrow (BM), peripheral blood (PB), and peripheral blood stem cell (PBSC) autografts from children with metastatic neuroblastoma (NB). We have developed a real-time PCR assay that allows the quantification of tyrosine hydroxylase (TH) mRNA, a tissue-specific marker of neuroblasts. We investigated a total of 165 BM, PB, and PBSC samples from 30 children over 1 year of age with stage IV NB and correlated the findings with disease status and patient survival. The levels of TH mRNA agreed well with clinical status and were significantly different across the groups that included samples obtained from patients at diagnosis, after three cycles of chemotherapy, in complete or very good partial remission and at relapse. We found that overall survival was significantly worse for patients with1000 TH copies in BM after initial chemotherapy (p=0.0075). In 57% of cases, autologous PBSC harvests were found to be contaminated by neuroblasts, the level of TH500 copies being associated with a decreased survival (p=0.003). In addition, molecular quantification enabled an estimation of tumor depletion in contaminated autografts using CD34 selection (median, 3 logs). In conclusion, quantification of minimal residual disease in metastatic NB using real-time RT-PCR for TH mRNA appears to be of potential clinical value. Further studies are needed to ascertain prognostic implications of molecular analysis of residual disease.

A total of 254 extracorporeal photochemotherapy (ECP) procedures were performed in 8 children (median age 10 years; range 5-15) with extensive resistant chronic graft-versus-host disease (GVHD). ECP was carried out in the pediatric environment using a Cobe Spectra separator and UV-MATIC irradiator. A peripheral venous with a single-lumen permanent central catheter access (69% of ECP-apheresis) or a dual-lumen permanent central catheter access (26% of ECP-apheresis) were used preferentially. A median platelet decrease of 17% (0-71) (p = 0.0001) and median hemoglobin level decrease of 15 g/L (0-31)(p = 0.0001) were noted following each ECP-apheresis. However, none of the patients had profound thrombocytopenia or anemia. Two minor episodes of catheter related-bacteriemia (Staphylococcus aureus) were noted (2310 catheter-days). A negative correlation was found between lymphocyte collection efficacy (median = 38%) and pre ECP-apheresis lymphocyte count (r = 0.4, p = 0.00001). The median of 5 x 10(7) lymphocytes/kg (0.1-50.10(7)/kg) was irradiated in each procedure. All patients are alive and well, and 7/8 experienced a dramatic improvement in their cutaneous status. Liver and gut disease resolved completely in 4/6 and 5/5 patients, respectively. In all patients, a concomitant immunosuppressive therapy was stopped (5/8) or considerably reduced (3/8). Five patients with more than 2 years follow-up after discontinuation of ECP are in remission with no immunosuppression treatment. They have normal growth rates and normal school and sport activity. Our study shows that ECP is beneficial, well tolerated, and can be safely used for chronic GVHD treatment even in young children with low body weight and a poor performance status. We believe that having a dedicated pediatric environment together with an experienced, motivated, and specifically pediatric team is of crucial importance for improving patient's acceptance of this long-term therapeutic program.

International audience; BACKGROUND:Primary immunodeficiencies (PIDs) are inherited diseases associated with a considerable increase in susceptibility to infections. It is known that PIDs can also predispose to cancer and immune diseases, including allergy, autoimmunity, and inflammation.OBJECTIVE:We aimed at determining the incidence of autoimmunity and inflammation in patients with PIDs.METHODS:We have retrospectively screened 2183 consecutive cases of PID in the Centre de Référence Déficits Immunitaires Héréditaires registry (CEREDIH; the French national PID registry) for the occurrence of autoimmunity and inflammation.RESULTS:One or more autoimmune and inflammatory complications were noted in 26.2% of patients, with a risk of onset throughout the patient's lifetime. The risk of autoimmune cytopenia was at least 120 times higher than in the general population, the risk of inflammatory bowel disease in children was 80 times higher, and the risk of other autoimmune manifestations was approximately 10 times higher. Remarkably, all types of PIDs were associated with a risk of autoimmune and inflammatory complications, although the greatest risk was associated with T-cell PIDs and common variable immunodeficiency. The occurrence of autoimmune disease is a negative prognostic factor for survival.CONCLUSIONS:Our results provide the basis for a detailed prospective evaluation of autoimmunity and inflammation in the context of PIDs, with a view to accurately assessing these risks and describing the possible effect of medical intervention.

International audience; Minimal residual disease (MRD) is a major predictive factor of the cure rate of acute lymphoblastic leukaemia (ALL). Haematopoietic cell transplantation is a treatment option for patients at high risk of relapse. Between 2005 and 2008, we conducted a prospective study evaluating the feasibility and efficacy of the reduction of immunosuppressive medication shortly after a non-ex vivo T depleted myeloablative transplantation. Immunoglobulin (Ig)H/T-cell receptor MRD 30 d before transplant could be obtained in 122 of the 133 cases of high-risk paediatric ALL enrolled. There were no significant demographic differences except remission status (first or second complete remission) between the 95 children with MRD \textless10(-3) and the 27 with MRD ≥10(-3) . Multivariate analysis identified sex match and MRD as being significantly associated with 5-year survival. MRD ≥10(-3) compromised the 5-year cumulative incidence of relapse (43*6 vs. 16*7%). Complete remission status and stem cell source did not modify the relationship between MRD and prognosis. Thus, pre-transplant MRD is still a major predictor of outcome for ALL. The MRD-guided strategy resulted in survival for 72*3% of patients with MRD\textless10(-3) and 40*4% of those with MRD ≥10(-3).

Background Although controlled-rate freezing and storage in liquid nitrogen are the standard procedure for peripheral blood progenitor cell (PBPC) cryopreservation, uncontrolled-rate freezing and storage at -80 degrees C have been reported. Study design and methods The prospective evaluation of 109 autologous PBPC transplantations after uncontrolled-rate freezing and storage at -80 degrees C of apheresis products is reported. The cryoprotectant solution contained final concentrations of 1-percent human serum albumin, 2.5-percent hydroxyethyl starch, and 3.5-percent DMSO. Results With in vitro assays, the median recoveries of nucleated cells (NCs), CD34+ cells, CFU-GM, and BFU-E were 60.8 percent (range, 11.2-107.1%), 79.6 percent (6.3-158.1%), 35.6 percent (0.3-149.5%), and 32.6 percent (1.7-151.1%), respectively. The median length of storage was 7 weeks (range, 1-98). The median cell dose, per kg of body weight, given to patients after the preparative regimen was 6.34 x 10(8) NCs (range, 0.02-38.3), 3.77 x 10(6) CD34+ cells (0.23-58.5), and 66.04 x 10(4) CFU-GM (1.38-405.7). The median time to reach 0.5 x 10(9) granulocytes per L, 20 x 10(9) platelets per L, and 50 x 10(9) reticulocytes per L was 11 (range, 0-37), 11 (0-129), and 17 (0-200) days, respectively. Hematopoietic reconstitution did not differ in patients undergoing myeloablative or nonmyeloablative conditioning regimens before transplantation. Conclusion This simple and less expensive cryopreservation procedure can produce successful engraftment, comparable to that obtained with the standard storage procedure.

Aim: To evaluate the association between Down syndrome and retinoblastoma. Method: Presentation of a case report and review of the literature. Results: A retinoblastoma was observed in a 10-month-old boy with Down syndrome. A review of the literature yielded 14 other cases, suggesting a possible excess of retinoblastoma in Down syndrome, as previously proposed by two epidemiological studies. The possible roles of external physical agents and hyperplastic and dysplastic lesions of the retina in subjects with Down syndrome is discussed. Conclusion: A positive association between Down syndrome and retinoblastoma is possible. An epidemiological study on this subject is needed to better ascertain this potential link.

Mobilization of peripheral blood stem cells (PBSC) by granulocyte colony-stimulating factor (G-CSF), at 10 micrograms/kg/day vs. 20 micrograms/kg/day (in 42 and 29 patients, respectively), was compared in children with solid tumors or leukemias. During mobilization, differences were noted in the peak values of CD34+ cells in peripheral blood (PB) in these two groups (median 28 x 10(6)/L for 10 micrograms/kg/day vs. 61 x 10(6)/L for 20 micrograms/kg/day; p = 0.025). Similar numbers of progenitor cells were harvested for the two concentrations of G-CSF. However, similar CD34+ cell levels in the leukapheresis product were obtained after only the third dose of G-CSF at 20 micrograms/kg/day compared with the fourth dose of G-CSF at 10 micrograms/kg/day (1.7 and 1.2 x 10(6) CD34+ cells/kg/one patient's blood volume processed, respectively). Of note is the impact of diagnosis on PB CD34+ cell levels. We conclude that, in children, mobilization with G-CSF at 20 micrograms/kg/day could minimize the duration of priming but not reduce the number of leukaphereses. Thus, the impact on outcome, clinical practice, bed utilization, and health economics is uncertain.

Background This is the first report of the long-term results of CD34+ cell transplantation in children with neuroblastoma. We investigated the hematologic and immune recovery, posttransplant morbidity, and clinical outcome of these children. Procedure Twenty-three children with advanced neuroblastoma had PBPCs (20 patients) or BM (3 patients) collected, followed by CD34+ cell selection on Ceprate column. The purge of residual neuroblastoma cells was evaluated using an RT-PCR for tyrosine hydroxylase (TH) mRNA assay. Reinfusion of CD34+ cells followed busulfan + melphalan myeloablative chemotherapy. Results A median of 2.9 × 106 CD34+ cells/kg was reinjected. Median days to achieve ANC > 0.5 × 109/liter and platelets > 50 × 109/liter were 13 (range 9–33) and 59 (range 22–259), respectively. Circulating T cells were primarily CD4−/CD8+ with fewer than 0.2 109CD4+ cells/liter throughout the first 6 months. CD19+ cells and CD56+ cells were not detectable up to day +35 posttransplant. At 1 year posttransplant, 16 evaluable patients had stable hematopoiesis with 2.3 × 109 ANC/liter (range 0.8–4.1), 1.4 × 109 lymphocytes/liter (range 0.5– 2.0) and 251 × 109 PLT/liter (range 35–490). After the completion of hematopoietic reconstitution, six events of severe septicemia/septic shock were noted. Six children had severe VZV infections, and 2 had EBV-associated lymphoproliferation. Thirteen patients are alive with a median follow-up of 40 months (range 2–54). Ten patients have died; 8 relapsed or developed progressive disease, 1 died from nondocumented pneumopathy at day 56, and 1 developed AML-M4 at 3 years posttransplant. Conclusions In children, CD34+ cell transplantation can be accomplished with a reduction of neuroblastoma cell inoculum in the selected graft as assessed by RT-PCR analysis. CD34+ cell grafts provide successful neutrophil reconstitution. However, delayed platelet recovery, persistent decrease in CD4+ lymphocyte levels and a high incidence of serious and life-threatening late infections were observed in these children. There remains a critical need to evaluate any real clinical benefit of CD34+ cell autografts in neuroblastoma patients. Med. Pediatr. Oncol. 35:1–7, 2000. © 2000 Wiley-Liss, Inc.

To date, little is known about the fertility of women suffering from mucopolysaccharidosis type I (MPS I). We report on a female patient with MPS I treated by allogeneic bone marrow transplantation (BMT) at the age of 4 years (after a conditioning regimen containing busulfan 16 mg/kg and cyclophosphamide 100 mg/kg) who had four successful pregnancies without any reproductive assistance. Clinical and biological examinations of the children were normal. On the basis of this case, we discuss the fertility counselling of female MPS I patients at the time of BMT.

Background We update our experience on large-volume leukapheresis (LVL) in very small patients with malignancies. LVLs were performed with the aim of reducing the psychological impact of leukaphereses by reducing the number of procedures while collecting large numbers of cells. Procedure Seventeen LVLs were performed using a Cobe Spectra separator in 14 patients weighing ⩽15 kg. A median of 3.8 patient's blood volumes corresponding to 296 mL/kg (range, 202–565) of blood was processed per session of 190 minutes (120–279) duration. A femoral catheter was installed specially for collection for 88% LVL (vs. 35% for standard leukaphereses). A median volume of 16.9 mL/kg was collected with 5.4 × 108 MNC/kg (range, 0.6–16.3) and 8.2 × 106 CD34+ cells/kg (range, 1.3–31.7). Results No signs of complications due to citrate toxicity were encountered. No hypotensive or hypothermic episodes were observed. Platelet counts were significantly diminished after each procedure (median: −59%). When the extracorporal line was not primed with red blood cells (RBC), the difference between pre-LVL and post-LVL hemoglobin levels was significant with a median 32 g/L decrease. Conclusions The LVL approach for peripheral blood progenitor cells (PBPC) collection in very small children may expose them to the risk of anemia and thrombocytopenia and an excess of special central line installation. The application of this technique in these patients should be reserved for special cases when a very large number of cells must be collected and should be performed by an experienced team. Med. Pediatr. Oncol. 32:7–10, 1999. © 1999 Wiley-Liss, Inc.

No treatment has proved its efficiency in CLL. Autologous transplantation is now under consideration for the youngest patients. We assayed progenitor cells (CFU-GM, BFU-E, CD34) in the peripheral blood of 28 untreated CLL patients and found an increase of all these progenitors in CLL compared to controls. There was no statistical difference between stage A versus stages B and C for CFU-GM and BFU-E. In contrast, CD34 cells were higher in stages B and C as compared to stage A. This finding could be explained by a high number of circulating clonal cells in advanced stages of the disease.

Background Autologous peripheral blood stem cells (PBSCs) are frequently used to reconstitute hematopoiesis following administration of megatherapy in children with advanced stage IV neuroblastoma. Some centers prefer the use of autografts enriched for CD34+ progenitor cells because the positive selection procedure is believed to reduce indirectly tumor cell contamination. Procedure In this study, we monitored the efficiency of tumor cell purging following CD34 selection in PBSCs from seven patients with advanced neuroblastoma by using a highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Amplification of tissue-specific mRNA transcript of tyrosine hydroxylase gene with nested primers enabled the detection of residual neuroblastoma cells with a sensitivity of one malignantcell per 106 normals. Results Using this method, contaminating tumor cells were detected in seven of nine leukapheresis products of the patients. After positive immunoselection of CD34+ cells on Ceprate™ column, only one of nine enriched stem cell fraction still contained tumor cells detectable by RT-PCR. In six cases, PCR positive PBSCs became PCR negative after selection. Conclusions We conclude that tumor cell contamination may be frequently detected in PBSC harvests of stage IV neuroblastoma patients by sensitive molecular analysis. The load of contaminating malignant cells might be reduced following CD34 selection. Med. Pediatr. Oncol. 30:228–232, 1998. © 1998 Wiley-Liss,Inc.

To clarify the factors that may affect the peripheral blood stem cell (PBSC) collection in children weighingor = 15 kg, a consecutive registry of 109 leukapheresis procedures was analyzed. Collections were performed on a COBE Spectra separator. In 65.1% of the procedures, the peripheral vein, together with a central catheter inserted routinely at diagnosis, or 2 peripheral veins were used to access/return. For 84.4% of the procedures, the extracorporeal line was primed with red blood cells. The median granulocyte-macrophage colony forming unit (CFU-GM) number derived from 1 patient's blood volume processed was 13.8 x 10(4)/kg. Six times, a collection series failed, always in children treated foror = 26 weeks and 4 of those times in children weighingor = 11 kg. The patient's age, diagnosis, duration of preleukapheresis treatment, and mobilization regimens did not significantly affect the collection yield. Twenty-four transplantations were performed. The median times to neutrophils0.5 x 10(9)/L and platelets20 x 10(9)/L were 13 and 20 days, respectively. We conclude that even in very small children, leukapheresis can be performed safely, allowing adequate PBSC collection for transplantation and/or in vitro manipulations.

The kinetics of peripheral blood progenitor cell (PBPC) release induced by G-CSF-alone at 10 μg/kg/day were monitored daily in 42 children with solid tumors and leukemias. Median 16- and 27-fold enrichment of circulating CD34 + cells and granulocyte-macrophage colony-forming units (CFU-GM) was noted with peak values occurring after the 4th or the 5th G-CSF dose. Individual values of PBCD34 + cell levels in patients with solid tumors were not significantly different after the 4th and after the 5th dose. The day-of-collection PBCD34 + cell concentration was related to the harvested CD34+ cell (P = 0.0001) and CFU-GM numbers (P = 0.0001). No correlations were found between PBPC enrichment and either patient age, body weight, diagnosis or pre-mobilization treatment duration. The median numbers of 1.1 X 10 6 CD34 + cells/kg and 28.1 x 10 4 CFU-GM/kg were derived from one patient's blood volume processed. Nineteen patients received G-CSF-alone primed grafts and had successful engraftment. Our data indicate that in 88% of children a single standard leukapheresis is sufficient to obtain a minimum graft (2 x 10 6 CD34 + cell and/or 10 x 10 4 CGU-GM per kg) whether undertaken after the 4th dose of G-CSF or the 5th.