Fabienne Gumy-Pause

ATM gene and lymphoid malignancies.

Inherited biallelic mutations of the ATM (ataxia-telangiectasia mutated) gene cause ataxia-telangiectasia, a rare autosomal recessive disorder associated with a high incidence of childhood leukaemias and lymphomas, suggesting that ATM gene alterations may be involved in lymphomagenesis. Loss of heterozygosity at 11q22–23 (location of the ATM gene) is a frequent event in sporadic lymphoid tumours, and several studies have reported a high prevalence of ATM gene alterations in diverse sporadic lymphoproliferative disorders, adding evidence to the postulated contribution of ATM in the pathogenesis of these tumours. This mini-review will summarize the recently published data concerning the ATM gene in sporadic lymphoid malignancies and will discuss the apparent paradox between the predominance of nonsense mutations observed in patient with ataxia-telangiectasia and the high proportion of missense alterations found in sporadic lymphoid tumours.

A simplified method for busulfan monitoring using dried blood spot in combination with liquid chromatography/tandem mass spectrometry

RATIONALE Busulfan (Bu) is an important component of the myeloablative conditioning regimen prior to hematopoietic stem cell transplantation (HSCT) especially in children. Intravenously administered Bu exhibits a therapeutic window phenomenon requiring therapeutic drug monitoring. Analytical methods developed for Bu routine monitoring were aimed at using low volumes of biological fluids and development of simple procedures to facilitate the dosage adjustment. In this report, we describe a simple, rapid method for Bu measurement using dried blood spots (DBS) from only 5 μL of whole blood. METHODS Bu extracted from DBS with methanol was measured by high-performance liquid chromatography with electrospray ionization and tandem mass spectrometry in multiple reaction monitoring mode using D8-Bu as an internal standard. The method was in-house validated evaluating trueness, repeatability, within-laboratory reproducibility, specificity and the lower limit of quantification (LLOQ). RESULTS The method was linear in the calibration range of 100-2000 ng mL(-1) (r(2)>0.99) encompassing the therapeutic concentrations of Bu. A good trueness (<14%), precision (<10%), and recovery (100%) were observed during validation of the method with quality controls of 300, 600 and 1400 ng mL(-1). The LLOQ was determined as 50 ng mL(-1) and no matrix or carryover effects were observed. The validated method was applied to measure Bu levels in four children receiving infusion of Bu prior to HSCT. A good correlation was observed between the Bu levels measured by DBS and dried plasma spot (DPS) (r(2) =0.96) and between DPS and the GC/MS method (r(2) =0.92). Bu was found to be stable in DBS up to 6 h at room temperature and for 24 h at 4 °C. CONCLUSIONS The new DBS method facilitates earlier dosage adjustment during Bu therapy by its specific and simple procedure using 5 μL of whole blood.

Adrenocortical oncocytoma in a child

Adrenocortical oncocytoma is a rare epithelial tumor only described in adults. We report the case of a 12‐year‐old female who presented a left adrenal mass with abdominal pain, fatigue, acne vulgaris, and elevation of the androstenedione and total testosterone. She had an adrenalectomy. A diagnosis of adrenocortical oncocytoma was made after detailed histological, immunohistochemical, and ultrastructural studies. Pediatr Blood Cancer 2008;50:718–721.

Report of a successful treatment of pulmonary Cunninghamella bertholletiae infection with liposomal amphotericin and posaconazole in a child with GvHD and review of the literature.

Zygomycosis is an emerging, opportunistic fungal infection particularly affecting immunocomprised patients. We report the case of a 10-year-old girl who developed pulmonary zygomycosis because of Cunninghamella bertholletiae 1 year after undergoing bone marrow transplantation complicated with severe cutaneous and digestive graft-versus-host disease. Treatment with surgery and liposomal amphotericin B followed by posaconazole successfully treated the infection.

Severe autoimmune hemolytic anemia in a liver transplanted child

Abstract:  AIHA can complicate solid organ and bone marrow transplantation early after transplant. We describe the first case report of a 16‐month‐old boy with mixed type warm‐acting IgM and warm IgG autoantibodies AIHA, occurring eight months after liver transplantation. This case describes the complexity of this very rare form of AIHA. It also illustrates the efficacy of rituximab in this indication, as well as the transfusion support with extremely rare blood, along with the importance of international collaboration to provide it. In this report, the etiologies of HA occurring in post‐transplant pediatric patients are reviewed and the different treatment strategies are discussed.

The Clinical Relevance of Pre-Formed Anti-HLA and Anti-MICA Antibodies after Cord Blood Transplantation in Children

Preformed anti-HLA antibodies (AHA) are known to be associated with delayed engraftment and reduced overall survival after adult hematopoietic stem cell transplantation. However, limited data is available in pediatric patients. In this study, we explored the role of AHA on clinical outcomes in 70 pediatric patients who received a single unit of HLA mismatch cord blood for hematologic malignancies, immunodeficiencies or metabolic diseases. The presence of AHA was detected in 44% (31/70) of the patients. Preformed class I AHA was associated with an increased occurrence of grade 1–4 acute graft-versus host disease (p<0.05). The presence of anti- major-histocompatibility-complex class I–related chain A antigens (MICA) antibodies was significantly associated with a reduced platelet recovery after transplantation (p<0.05). AHA of class II with the strength of antibody titer measured as the mean fluorescence intensity above 2000 was associated with reduced event-free survival (p<0.05). A reduction of high titer of AHA and anti-MICA antibodies might have to be considered before cord blood transplantation in pediatric patients for better outcomes.

STEPPING UP VERSUS STANDARD DOSES OF ERYTHROPOIETIN IN PRETERM INFANTS: A Randomized Controlled Trial

In this study, it is hypothesized that a planned increase in the dose of recombinant human erythropoietin (rh-EPO) can prevent transfusion in very low birth weight infants. Two different regimens of rh-EPO were administrated, one consisting in increasing dosage up to 5000 U/kg/wk, according to the individual reticulocytes response, and the second in a standard therapy of 1250 U/kg/wk. Fifty-one infants participated. Despite a significant higher reticulocytosis, the study was prematurely terminated due to the results of an interim analysis showing that transfusion was not avoided by increasing the rh-EPO. No significant differences were found between the two regimens concerning transfusion rate, volume transfused, gain in weight, and adverse effects. Progressive titration of rh-EPO to improve the biological response does not leave premature infants free of transfusion.

GSTP1 hypermethylation is associated with reduced protein expression, aggressive disease and prognosis in neuroblastoma.

Epigenetic modifications such as methylation of CpG islands in tumor‐suppressor gene promoter regions have been associated with tumor development in many human cancers. Using methylation specific multiplex ligation‐dependent probe amplification method, we analyzed the methylation status of 35 different genes in 16 neuroblastoma (NB) cell lines and 50 NB tumor samples (NBs), and investigated whether specific hypermethylation was associated with biological and/or clinical parameters. Among the genes found hypermethylated, the effect of GSTP1 hypermethylation on mRNA and protein expression was also explored. The median number of hypermethylated genes was higher in cell lines compared to NBs (5.5 vs. 2). For eight genes, aberrant methylation of CpG‐islands in NB was not (ESR1, PAX5, WT1, CADM1, MSH6, and CDKN2B) or very rarely (CDH13 and GSTP1) reported in literature. GSTP1 was found hypermethylated in 44% of the NB cell lines and in 33% of the stage 4‐11qLOH ‐non MYCN‐amplified high risk NBs. Hypermethylation was correlated with reduced mRNA and protein expression. In the whole NBs cohort, GSTP1 hypermethylation was less frequently detected (8%), but found to be associated with lower event‐free (EFS) and overall survival. Hypermethylation of GSTP1 showed also association with lower EFS in high risk subgroups as stage 4 and older patients (≥547 days). Our results suggest that, as in several adult cancers, aberrant methylation of GSTP1 may contribute to the carcinogenetic process in NB and could be potentially used as a new marker leading to define an ultra‐high risk subgroup.

Socioeconomic Status and Childhood Leukemia Incidence in Switzerland

Socioeconomic status (SES) discrepancies exist for child and adult cancer morbidity and are a major public health concern. In this Swiss population-based matched case–control study on the etiology of childhood leukemia, we selected the cases from the Swiss Childhood Cancer Registry diagnosed since 1991 and the controls randomly from census. We assigned eight controls per case from the 1990 and 2000 census and matched them by the year of birth and gender. SES information for both cases and controls was obtained from census records by probabilistic record linkage. We investigated the association of SES with childhood leukemia in Switzerland, and explored whether it varied with different definitions of socioeconomic status (parental education, living condition, area-based SES), time period, and age. In conditional logistic regression analyses of 565 leukemia cases and 4433 controls, we found no consistent evidence for an association between SES and childhood leukemia. The odds ratio comparing the highest with the lowest SES category ranged from 0.95 (95% CI: 0.71–1.26; Ptrend = 0.73) for paternal education to 1.37 (1.00–1.89; Ptrend = 0.064) for maternal education. No effect modification was found for time period and age at diagnosis. Based on this population-based study, which avoided participation and reporting bias, we assume the potential association of socioeconomic status and childhood leukemia if existing to be small. This study did not find evidence that socioeconomic status, of Switzerland or comparable countries, is a relevant risk factor or strong confounder in etiological investigations on childhood leukemia.

ATM variants and predisposition to childhood T-lineage acute lymphoblastic leukaemia

We read with a great interest the recent study published by Meier et al., on genetic variants of the ataxia telangiectasiamutated (ATM) gene, drug resistance, clinical outcome and predisposition to childhood T-lineage acute lymphoblastic leukaemia (T-ALL). They reported a high prevalence (60%) of nucleotide substitution in 16 selected ATM exons in 103 T-ALL, but this prevalence was similar to that of the controls (52%). Interestingly, five alterations located in the coding part of ATM were found to be 2.6-fold higher in T-ALL (13%) than in controls (5%, P1⁄4 0.06), associated with a higher white blood cell count at diagnosis (P1⁄4 0.05) and with an increased relapse-risk (P1⁄4 0.05). As mentioned by Meier et al., 21 different ATM alterations found in 7/31 (23%) childhood T-ALL samples were reported in our article published in 2003. In our study, 21 different rare variants and nine different well-known polymorphisms were found in all ATM exons, but only rare variants were reported in detail. Therefore, if we considered rare variants and polymorphisms located in the 16 exons analysed by Meier et al. (exons: 9, 12, 15, 17, 19, 20, 24, 31, 32, 39, 40, 41, 47, 53, 55 and 62), similar prevalence was found with 18/31 T-ALL (58%) harbouring ATM alteration(s). In contrast to the hypothesis proposed by Meier et al., this result suggests that we have the same sensitivity to detect ATM genetic abnormalities, even if the DHPLC conditions used in the two studies were different. In the last decade, a growing literature has been published concerning the association of genetic polymorphisms and the risk of childhood ALL. Enzymes involved in the folate homeostasis and in the metabolism of carcinogens were largely studied, but polymorphisms in genes involved in DNA repair could be also associated with a modified individual ability to maintain an intact genome in response to genotoxic stress. In the study of Meier et al., the intronic substitution IVS62þ 8A/C was found with a higher allelic frequency in T-ALL (12/206: 5.8%) as in controls (3/198: 1.5%) (w: P1⁄4 0.02). This high frequency was also observed in our 31 T-ALL samples analysed by direct sequencing: two samples were found homozygotes and two others heterozygotes for IVS62þ 8A/C (allelic frequency: 6/62: 9.7%). This frequency was not found statistically different from the healthy controls (6/114: 5.3%), but the number of samples analysed was small. As this alteration is located in the vicinity of the splicing site, we performed RT-PCR in order to determine a potential splicing defect. As shown in Figure 1, no defect was detected, suggesting that this substitution does not interfere with normal splicing. Altogether, these results suggest that IVS62þ 8A/C is a polymorphism unlikely to carry biological significance. However, the difference in allelic frequency found by Meier et al. between T-ALL and controls remains intriguing. Larger molecular epidemiology studies are needed to confirm this difference and to detect other ATM polymorphisms possibly involved in childhood ALL susceptibility. Acknowledgements