Abstract Background: The study of lineage markers by real-time quantitative polymerase chain reaction (RT-qPCR) at diagnosis enables differentiation between acute myeloblastic leukemia, B- or T-lineage acute lymphoblastic leukemia, without cell sorting. Our objective was to assess the relationship between protein expression and the amount of lineage marker mRNA in acute leukemia samples and to determine whether four lineage markers could be used to differentiate between normal and acute leukemia bone marrow (BM) without cell sorting. Methods: Quantification of the mRNA of CD19, CD79a, CD3e, and myeloperoxidase was performed by RT-qPCR on 130 acute leukemia BM samples at diagnosis and on 20 BM samples from healthy donors, without cell sorting. Immunophenotyping of leukemia samples was performed after manual gating around the blastic population. Results: Reference values for the four lineage markers were established by RT-qPCR for normal BM. The mRNA expression levels of these four lineage markers allowed the distinction between normal samples and 100% of acute leukemia samples. Conclusions: With 92% congruence for protein expression and amount of mRNA in acute leukemias, these four lineage markers, essential for diagnosis and subclassification of acute leukemias by flow cytometry, also represent excellent candidate genes when using RT-qPCR technology as a diagnostic tool for molecular cancer class prediction. Clin Chem Lab Med 2009;47:419–26.

Sickle celi anaemia is still responsible for severe crippling and death in young patients living in developing countries. Apart from prophylaxis and treatment of infections, no active treatment can be safely proposed in such areas of the world. Therefore a bone marrow transplantation was performed in 12 patients staying in Belgium and planning to return to Africa. Twelve patients, aged between 11 months and 23 years (median 4 years), underwent a HLA identical bone marrow transplantation. The conditioning regimen included oral busulphan for four consecutive days (4 mg/kg) followed by four days of intravenous cyclophosphamide (50 mg/kg). In 10 patients the engraftment was rapid and sustained. A further patient suffered transient red cell hypoplasia and another underwent a second bone marrow transplantation from the same donor at day 62 because of graft rejection. All patients are alive and well with a follow up ranging from 9-51 months (median 27 months). In all cases a complete cessation of vaso-occlusive episodes and haemolysis was observed as was a change in the haemoglobin pattern in accordance with the donor's electrophoretic pattern. Catholic University of Louvain, Brussels, Belgium, Department of Paediatrics Ch Vermylen G Cornu J Ninane A Borja Department of Internal Medicine A Ferrant

Before the Imatinib era, a prospective phase II trial was conducted in 2000 to assess the efficacy and the tolerance a combination of interferon alpha 2b (IFN) and cytarabine in children and adolescents with chronic myelogenous leukemia in first chronic phase without a suitable HLA-identical donor. Children received daily IFN (5 million units/m2) and subcutaneous cytarabine (20 mg/m2) for monthly course of 10 days. Between September 2000 and March 2003, 14 children (10 males, 4 females) median age 12 years (range 2–16.5) were recruited from 12 french centres. Patients (pts) were evaluated for time to, rate of hematologic and cytogenetic responses, toxicity and progression free survival. The median duration of follow-up is 13 months (range 2–32 mo). Eight pts achieved a complete hematologic response after a median time of treatment of 3 months (range 0–4 mo) including a pt enrolled in complete hematologic response after 1 month of therapy with hydroxyurea. Three pts were not evaluable for the cytogenetic response (early discontinuation for toxicity, no achievement of complete hematologic response within 3 months of treatment, progression). The best cytogenetic response by 12 months was: major response in 7 pts including complete cytogenetic response in 2 pts, minor response in 3 pts and failure in 1 pt. The median time to major cytogenetic response was 7 months (range 3–12 mo). Thirteen pts discontinued treatment protocol with a median time of 11 months for the following reasons: absence of complete hematologic response at 3 months (2 pts), loss of hematologic response (2 pts), absence of major cytogenetic response at 12 months (1 pt ), loss of major cytogenetic response (2pts), progression (3 pts), adverse event (1pt), other (2 pts). Probability of progression free survival at 18 months was 66% (95% CI, 34–98%). No treatment-related death occurred. The most frequently reported drug-related events were fever, mucositis, neutropenia and thrombocytopenia. Grade 3 and 4 non hematologic toxicity (fever and mucositis) was observed in 4 pts and grade 3 and 4 hematologic toxicity (anemia, neutropenia and thrombocytopenia) in 7 pts. The median total dose of IFN and cytarabine administered was 3.7 million units/m2 /day (range 2.3–5.1) and 20 mg/m2 /day (range 11–23). The median duration of IFN therapy was 11 months (range1–117 mo). The median number of courses of cytarabine was 7 (range 1–37). The combination of IFN and cytarabine is a well tolerated therapy providing hematologic and cytogenetic responses in children and adolescents with CML. Rates of hematologic and cytogenetic responses compare favourably with results observed in adults. Results should be compared to these of imatinib in children and adolescents.

The molecular basis of susceptibility to childhood malignant hemopathy remains largely unknown. An excess of skeletal congenital anomalies has been reported among children with hematological malignancy and points towards involvement of developmental genes, like those belonging to the HOX gene family. In addition to their role in embryogenesis, HOX transcription factors are known to be regulators of proliferation and differentiation of hematopoietic cells. We aimed to explore the possibility that germline alterations of HOX genes might be involved in childhood acute lymphoid malignancies. A cohort of 86 children diagnosed with acute lymphoid malignancy was studied, 20 of them concurrently presenting a congenital anomaly of the skeleton. First, We screened for nucleotide changes throughout the HOX genes of paralogous groups 4 to 13 in the 20 patients with skeletal defects, following a skeletal phenotype based strategy. Subsequently, we extended the HOX mutation screening to the other 66 children having a malignant lymphoproliferative disorder, but without skeletal defects. In total, 16 germline mutations were identified. While 13 changes were also observed in healthy controls, three variants were exclusively found in acute lymphoid malignancy cases. These comprised the germline c.242A > T (p.Glu81Val) missense mutation of HOXD4, detected in two children diagnosed with acute lymphoblastic leukemia (ALL). Furthermore, this mutation Was found in association with other specific HOX variants of cluster D (2q31-q37), defining a unique haplotype. Functional analysis of the murine Hoxd4 homolog revealed that mutant Hoxd4 protein had lower transcriptional activity than wild,type protein in vitro. The p.Glu81Val mutation of HOXD4 thus results in a partial loss-of-function, which might be involved in childhood ALL. (c) 2005 Wiley-Liss, Inc.

We report the history of a patient and his daughter, both affected with hypoplasia of the abdominal aorta and its branches, leading to early and dramatic complications. In the index patient, renal ischaemia as a result of severe hypoplasia of the abdominal aorta and the origin of renal arteries led to progressive renal failure and end-stage renal disease at the age of 32 years. Other vascular abnormalities included hypoplasia of the celiac trunk (CT) and superior mesenteric artery (SMA). After a successful kidney transplantation at the age of 40 years, he eventually deceased following an episode of possibly ischaemic acute pancreatitis at 47 years. The patient's daughter suffered from an haemorrhagic stroke at the age of 7 years, which led to the discovery of severe hypertension caused by bilateral narrowing of renal arteries, as well as hypoplasia of CT, SMA, subclavian and pulmonary arteries. Biopsy of the narrowed renal artery of the daughter showed a particular form of fibrodysplasia characterized by an unusual fibrosis of the inner part of the media, just beneath the internal elastic lamina. To our knowledge, this is the first report of familial hypoplasia of the abdominal aorta. It might be the cardinal manifestation of a familial form of fibromuscular dysplasia (FMD). Interestingly, the histological lesions described in the daughter's renal artery differ from the classical form of medial FMD.

BACKGROUND:Flow cytometry of lineage markers is useful in the classification of leukemias. Our aim was to assess whether the study of lineage genes at the RNA level would enable differentiation of acute myeloid leukemias (AMLs) from B-and T-lineage acute lymphoid leukemias (ALLs).METHODS:We measured mRNA of four lineage markers [CD19, CD79a, CD3e, and myeloperoxidase (MPO)] by reverse transcription followed by real-time quantitative (RTQ)-PCR. We investigated 72 acute leukemias (40 AMLs with 23-93% blast cells plus 27 B-lineage ALLs and 5 T-lineage ALLs) defined by morphologic criteria at diagnosis. RTQ-PCR analysis was performed on bone marrow without cell sorting. The expression of each gene was calculated as the difference in the threshold cycle [DeltaCT; CT value of target gene minus CT value of housekeeping gene (Abelson)].RESULTS:Three patterns of expression were detected. In the first, CD19, CD79a, and MPO mRNAs were less abundant than CD3e. In the second pattern, MPO mRNA was more abundant than the other three mRNAs. In the third, CD19 or CD79a was more highly expressed than CD3e and MPO. The three patterns corresponded to T-ALL, AML, and B-ALL, respectively. The use of cutoffs to establish qualitatively the pattern of coexpression of the four lineage markers provided the same information as the comparison among the four DeltaCT values. Prospective use of the scoring system correctly classified each of 13 additional cases (8 AML, 4 B-lineage ALL, and 1 T-lineage ALL).CONCLUSION:Study of lineage markers at diagnosis by RTQ-PCR allows differentiation of AML from B-ALL or T-ALL without cell sorting, even when the bone marrow contains few blast cells.

ABSTRACT A yellow-pigmented coryneform rod was isolated from the blood of a child with acute lymphoblastic leukemia who was perfused with a central venous catheter. The culture bottles were positive twice, at a 2-month interval. The isolate was identified as a Microbacterium sp. and studied along with five other similar strains. Phenotypic, chemotaxonomic, and genetic characteristics indicated that they are closely related to Microbacterium oxydans but that they belong to a distinct species, for which the name Microbacterium paraoxydans sp. nov. is proposed. The type strain of M . paraoxydans is CF36 T = DSM 15019 T . The G+C content of its DNA is 69.9 mol%.

BACKGROUNDTo assess the efficacy of chemotherapy (chemoreduction) plus local treatments as an alternative to external beam and enucleation for intraocular retinoblastoma.MATERIALS AND METHODSA prospective study was performed on 21 patients with retinoblastoma treated in our institution from September 1997 to December 2000 to study the ocular outcome of those 33 eyes.RESULTSThere were 9 unilateral and 12 bilateral retinoblastoma cases. There were 12 eyes with Reese-Ellsworth group I-IV and 21 eyes with group V. Among 33 eyes, nine eyes (27%) were initially managed by enucleation. The remaining 24 eyes (73%) were initially treated with chemoreduction (maximum of six cycles of carboplatin, vincristine, etoposide) or chemothermotherapy. Among those 24 eyes, 20 were successfully treated with local treatments (thermotherapy plus cryotherapy in 16 eyes and thermotherapy plus cryotherapy plus (125)I plaque radiotherapy in 4 eyes), enucleation eventually underwent in two eyes and was proposed but refused in one child with bilateral group V retinoblastoma. With a median follow-up of 21 months, conservative management without external beam radiation was successful in all 12 eyes with group I-IV and in a total of 20/33 eyes (60%). Among the nine cases of unilateral retinoblastoma, eight were enucleated but among the 24 eyes with bilateral retinoblastoma, 19 (79%) were successfully treated with conservative therapy.CONCLUSIONSIt may be possible to eradicate viable tumor in all eyes with Reese-Ellsworth group I-IV retinoblastoma by chemoreduction followed by local treatments. Although 8 out of 21 eyes (38%) with group V retinoblastoma may be salvaged after chemoreduction and local therapies, enucleation remained the treatment of choice in those eyes with total retinal detachment and diffuse vitreous seeding.

A 10-month-old girl with a history of recurrent candidiasis, developmental delay, and a fulminant varicella infection is described. The diagnosis of purine nucleoside phosphorylase (PNP) deficiency was suggested by a reduced level of serum uric acid and confirmed by measurement of PNP activity. A human leukocyte antigen-matched bone marrow transplantation resulted in immune reconstitution, but poor neurodevelopmental progression.

Le syndrome de thrombopenie et absence de radius (TAR) est une maladie congenitale rare associant une thrombopenie centrale et une absence bilaterale de radius. Il associe, dans une large variabilite phenotypique, des anomalies squelettiques, digestives, hematologiques et cardiaques. Nous decrivons et discutons le cas d'un nouveau-ne presentant une thrombopenie severe (29 000 PS/mm 3 ) associee a une phocomelie. Il presente les anomalies associees suivantes: une absence complete des os des bras et des avant-bras, un flessum des genoux, des clinodactylies multiples, une duplication oesophagienne, une intolerance vis-a-vis des proteines du lait de vache, une cryptorchidie droite et un hypospade balano-penien. Malgre un pronostic initial particulierement reserve, avec une evolution digestive et hematologique difficile a controler les dix-sept premiers mois, une prise en charge multidisciplinaire agressive, a permis de donner a cet enfant une qualite de vie normale. Nous soulignons qu'il s'agit a notre connaissance, de la premiere description d'une duplication oesophagienne dans ce syndrome.

Cord blood (CB) transplantations are associated with low graft-versus-host disease (GVHD). The pathophysiology of GVHD involves interaction and activation of different cell types, as lymphocytes and monocytes, and results in a cascade of cytokine production. After antigen or mitogen stimulation, CB monocytes release lower levels of cytokines than adult blood (AB) monocytes. In this study, the detection of intracellular IL-1 beta and TNF-alpha produced by monocytes was evaluated in response to tuberculin PPD to investigate whether the reduced capacity of CB monocytes to secrete cytokines could be related to an impaired functional activity and to a particular phenotypic profile. Results showed that the percentage of CD64(+)monocytes producing intracellular IL-1 beta and TNF-alpha was significantly lower in CB and that the phenotypic profile of CB monocytes producing these cytokine (CD64(+)CD14(+)) was different to that of AB monocytes (CD64(+)CD14(+), CD64(+)CD33(+) and CD64(+) CD45RO(+)). These results suggest that the lower capacity of CB monocyte populations to produce IL-1 beta and TNF-alpha might be due to a functional immaturity of CB monocytes at the cellular level as reflected by the different phenotypic profile of CB monocytes.

Fanconi's anemia is a rare autosomal recessive disease characterized by congenital abnormalities, a progressive pancytopenia and a predisposition to cancer. The diagnosis is based on an abnormal increase of spontaneous chromosome breakage, more specifically on a clear-cut increase of chromosome breakage in the presence of bifunctional alkylating agents. Eight complementation groups (A to H) have been defined, and the genes corresponding to four of these groups have been cloned (FANCA, FANCC, FANCF and FANCG). The function of the proteins encoded by the genes of Fanconi's anemia remains unknown. Numerous studies indicate that different cellular processes are probably involved, including DNA repair pathways, apoptosis, cell cycle regulation and oxygen metabolism. Nevertheless, the exact cellular and molecular mechanisms implicated in Fanconi's anemia remain a challenge for fundamental research. The treatment of Fanconi's anemia is also the subject of intense research, bearing principally upon bone marrow transplantation, which is successful in the case of HLA-identical sibling donors, and gene therapy, which is still at a preliminary stage on the clinical level.

Umbilical cord blood (CB) transplantations are associated with a lower risk of severe graft-versus-host disease (GVHD) compared to BMT. GVHD is an immune reaction that involves interaction between cell surface molecules resulting in cell activation and release of many cytokines. Monocytes are known to be an important source of cell adhesion (CAM) and co-stimulatory molecules which play a crucial role in the efficient activation of T and B cells. We analyzed the phenotype of CB monocytes in the presence or absence of an inflammatory signal (rIFN-gamma) and compared them to adult blood (AB); the expression of HLA-DR and 17 different markers (CD11a, CD11b, CD11c, CD18, CD29, CD40, CD44, CD49a, CD49d, CD49e, CD49f, CD54, CD58, CD62L, CD80, CD86 and CD102) was measured by flow cytometry. Statistical analysis showed that, compared to AB, CB monocytes did not express CD11b, CD11c, CD49d and after stimulation with rIFNgamma, they lost the expression of CD58 and CD102, whereas CD80 and CD86 expression was induced. The analysis of fluorescence intensity (MFI) revealed that CB monocytes expressed some CAM (CD29, CD54, CD102) with a lower intensity than AB monocytes except CD44. In conclusion, absence and reduced expression of some markers argue for a different phenotypic profile of CB monocytes compared to AB monocytes, which might partly contribute to their impaired immune response and to the low incidence of GVHD observed after CB transplantations. However, CB monocytes expressed CD80 and CD86 co-stimulatory molecules, but this expression did not prove a normal co-stimulatory function.

We report a 5.5-year-old boy who underwent autologous peripheral blood stem cell transplantation for high-risk acute lymphoblastic leukemia and who had two abdominal masses develop 6 months later. Macroscopically complete resection of the abdominal tumors was performed and revealed a well-differentiated leiomyosarcoma. Smooth muscle tumors, benign or malignant, are increasingly recognized in children with various immunodeficiencies; the association with acute lymphoblastic leukemia is rarely described.

Les douleurs abdominales sont un motif de consultation extremement frequent en Pediatrie. Souvent, l'etiologie est benigne. Cependant parfois, la mise au point revele un diagnostic grave. Et il arrive encore que, derriere ce diagnostic, s'en cache un autre … plus complexe. Le role du medecin est d'identifier les cas severes, afin de faire beneficier l'enfant d'un traitement adequat, a temps. Notre but est de presenter un cas de mise au point de douleurs abdominales liees a un lymphome de Burkitt sur un syndrome d'ataxie-telangiectasie meconnu jusque la. Les points essentiels de la litterature recente sur ce syndrome peu connu seront revus. Nos messages sont les suivants: des douleurs abdominales chez l'enfant demandent une anamnese et un examen clinique precis. Devant la survenue d'un lymphome chez l'enfant, il faut exclure une etiologie sous-jacente tel un deficit immunitaire. Enfin, nous rappelons les difficultes therapeutiques oncologiques dans le cas de l'ataxie-telangiectasie.

Lˈanémie de Fanconi est une maladie autosomique récessive rare caractérisée par des malformations congénitales, une pancytopénie progressive et une prédisposition au cancer. Le diagnostic est basé sur lˈaugmentation anormale du taux de cassures chromosomiques spontanées mais surtout, et de manière spécifique, sur une augmentation très nette de ces cassures chromosomiques en présence dˈagents alkylants bifonctionnels. Huit groupes de complémentation ont été définis (de A à H) et les gènes de quatre de ces groupes ont été clonés (FANCA, FANCC, FANCF et FANCG). La fonction des protéines pour lesquelles codent les gènes de lˈanémie de Fanconi nˈest toujours pas connue. Les nombreuses études réalisées semblent indiquer que différents processus cellulaires pourraient être impliqués dans lˈanémie de Fanconi. Il sˈagit essentiellement des mécanismes de réparation de lˈADN, de lˈapoptose, de la régulation du cycle cellulaire et du métabolisme de lˈoxygène. Les mécanismes cellulaires et moléculaires impliqués dans lˈanémie de Fanconi restent donc un défi pour la recherche fondamentale. Le traitement de lˈanémie de Fanconi fait lui aussi lˈobjet de recherches actives tant en ce qui concerne la greffe médullaire (pratiquée actuellement avec succès en cas de donneur apparenté HLA-identique) que la thérapie génique (encore à ses balbutiements sur le plan clinique).