A Semi-Mechanistic Population Pharmacokinetic/Pharmacodynamic Model of Bortezomib in Pediatric Patients with Relapsed/Refractory Acute Lymphoblastic Leukemia.

INTRODUCTION: The pharmacokinetics (PK) of the 20S proteasome inhibitor bortezomib are characterized by a large volume of distribution and a rapid decline in plasma concentrations within the first hour after administration. An increase in exposure was observed in the second week of treatment, which has previously been explained by extensive binding of bortezomib to proteasome in erythrocytes and peripheral tissues. We characterized the nonlinear population PK and pharmacodynamics (PD) of bortezomib in children with acute lymphoblastic leukemia. METHODS: Overall, 323 samples from 28 patients were available from a pediatric clinical study investigating bortezomib at an intravenous dose of 1.3 mg/m2 twice weekly (Dutch Trial Registry number 1881/ITCC021). A semi-physiological PK model for bortezomib was first developed; the PK were linked to the decrease in 20S proteasome activity in the final PK/PD model. RESULTS: The plasma PK data were adequately described using a two-compartment model with linear elimination. Increased concentrations were observed in week 2 compared with week 1, which was described using a Langmuir binding model. The decrease in 20S proteasome activity was best described by a direct effect model with a sigmoidal maximal inhibitory effect, representing the relationship between plasma concentrations and effect. The maximal inhibitory effect was 0.696 pmol AMC/s/mg protein (95% confidence interval 0.664-0.728) after administration. CONCLUSION: The semi-physiological model adequately described the nonlinear PK and PD of bortezomib in plasma. This model can be used to further optimize dosing of bortezomib.

Is Acute Myeloblastic Leukemia in Children Under 2 Years of Age a Specific Entity? A Report from the FRENCH ELAM02 Study Group.

The clinical and biological characteristics of children under 2 years (infants) with acute myeloid leukemia (AML) are different from those of older children. We aimed to describe the specific characteristics of this population and the potential factors that influence the prognosis. We analyzed data concerning 438 children with newly-diagnosed AML treated in the ELAM02 protocol between March 2005 and December 2011, of which 103 were under 2 years old at diagnosis. The evaluation criteria were overall survival (OS) and event-free survival (EFS) of infants vs older children. The clinical and biological features were secondary criteria. Infants presented more frequent extra-medullary presentation than older children. They had a significantly higher proportion of skin lesions and central nervous system involvement (15% vs 3%, p < 0.0001 and 26% vs 12%, p = 0.0005, respectively). The global incidence of KMT2A rearrangements was nearly 55% for infants vs 11% for older children (p < 0.0001). Median 5-year OS was 70.4% for infants vs 71.4% for older children (p = 0.83). Five-year EFS was 67% for infants vs 58% for older children (p = 0.27). Infants with AML represent a cohort of patients with specific clinical and biological features. These remarkable differences had no significant impact on their outcome in the ELAM02 protocol.

Genetic polymorphisms of Th2 interleukins, history of asthma or eczema and childhood acute lymphoid leukaemia: Findings from the ESCALE study (SFCE).

BACKGROUND: Previous studies on the putative role of allergy in the aetiology of childhood leukaemia have reported contradictory results. The present study aimed to analyse the relation between a medical history of asthma or eczema and childhood acute lymphoid leukaemia (ALL) in light of potential candidate gene-environment interactions. METHODS: Analyses were based on a subset of 434 cases of ALL and 442 controls successfully genotyped and of European ancestry children enrolled in a French population-based case-control study conducted in 2003-2004. Information about medical history was obtained during a standardized interview with the mothers. Candidate polymorphisms in genes of the Th2 cytokines IL4, IL10, IL13 and IL4-receptor, were genotyped or imputed. RESULTS: None of the variant alleles were directly associated with childhood acute lymphoid leukaemia. A medical history of asthma or eczema was reported more often in the control group (OR = 0.7 [0.5-1.0]). This association was mostly seen in the group of children not carrying the IL13-rs20541 variant allele (Interaction Odds Ratio IOR 1.9, p-interaction = 0.07) and in those carrying the IL10 triple variant haplotype (IOR 0.5, p-interaction = 0.04). No interaction was observed with the candidate polymorphisms in IL4 and IL4R. CONCLUSION: This study provides a new insight into the relationship between allergic symptoms and childhood acute lymphoid leukaemia, by suggesting this inverse association could be limited to children carrying certain genetic polymorphisms. If confirmed, these results could help better understand the biological mechanisms involved in the development of childhood acute lymphoid leukaemia.

MN1 overexpression is driven by loss of DNMT3B methylation activity in inv(16) pediatric AML.

In acute myeloid leukemia (AML), specific genomic aberrations induce aberrant methylation, thus directly influencing the transcriptional programing of leukemic cells. Therefore, therapies targeting epigenetic processes are advocated as a promising therapeutic tool for AML treatment. However, to develop new therapies, a comprehensive understanding of the mechanism(s) driving the epigenetic changes as a result of acquired genetic abnormalities is necessary. This understanding is still lacking. In this study, we performed genome-wide CpG-island methylation profiling on pediatric AML samples. Six differentially methylated genomic regions within two genes, discriminating inv(16)(p13;q22) from non-inv(16) pediatric AML samples, were identified. All six regions had a hypomethylated phenotype in inv(16) AML samples, and this was most prominent at the regions encompassing the meningioma (disrupted in balanced translocation) 1 (MN1) oncogene. MN1 expression primarily correlated with the methylation level of the 3' end of the MN1 exon-1 locus. Decitabine treatment of different cell lines showed that induced loss of methylation at the MN1 locus can result in an increase of MN1 expression, indicating that MN1 expression is coregulated by DNA methylation. To investigate this methylation-associated mechanism, we determined the expression of DNA methyltransferases in inv(16) AML. We found that DNMT3B expression was significantly lower in inv(16) samples. Furthermore, DNMT3B expression correlated negatively with MN1 expression in pediatric AML samples. Importantly, depletion of DNMT3B impaired remethylation efficiency of the MN1 exon-1 locus in AML cells after decitabine exposure. These findings identify DNMT3B as an important coregulator of MN1 methylation. Taken together, this study shows that the methylation level of the MN1 exon-1 locus regulates MN1 expression levels in inv(16) pediatric AML. This methylation level is dependent on DNMT3B, thus suggesting a role for DNMT3B in leukemogenesis in inv(16) AML, through MN1 methylation regulation.

Age-related clinical and biological features of PTEN abnormalities in T-cell acute lymphoblastic leukaemia.

The tumour suppressor gene PTEN is commonly altered in T-cell acute lymphoblastic leukaemia but its prognostic impact is still debated. We screened a cohort of 573 fully characterised adult and paediatric T-cell acute lymphoblastic leukaemia (T-ALL) patients for genomic PTEN abnormalities. PTEN-inactivating mutations and/or deletions were identified in 91 cases (16%), including 18% of paediatric (49/277) and 14% of adult cases (42/296). Thirty-four patients harboured only mutations, 12 cases demonstrated only large deletions and 9 only microdeletions. About 36 patients had combined alterations. Different mechanisms of PTEN inactivation predicted differences in the clinical outcome for both adult and paediatric patients treated according to the GRAALL03/05 and FRALLE2000 protocols. Whereas large deletions predicted lower 5-year overall survival (P=0.0053 in adults, P=0.001 in children) and disease-free survival (P=0.0009 in adults, P=0.0002 in children), mutations were not associated with a worse prognosis. The prognostic impact of PTEN loss is therefore linked to the underlying type of genomic abnormality, both in adult and paediatric T-ALLs, demonstrating that detailed analysis of the type of abnormality type would be useful to refine risk stratification.

Cytogenetics and outcome of allogeneic transplantation in first remission of acute myeloid leukemia: the French pediatric experience.

We analyzed the impact of cytogenetics on 193 children enrolled in two successive French trials (LAME89/91 and ELAM02), who received hematopoietic stem cell transplantation during CR1. Detailed karyotype was available for 66/74 (89%) in LAME89/91 and 118/119 (99%) in ELAM02. Several karyotype and transplant characteristics differed according to therapeutic protocol: unfavorable karyotypes were more frequent in ELAM02 (36% vs 18%), pretransplant chemotherapy included high-dose cytarabine in ELAM02 and not in LAME89/91, IV replaced oral busulfan in the conditioning regimen, methotrexate was removed from post-transplant immunosuppression, and matched unrelated donor and cord blood transplantation were introduced. Five-year overall survival (OS) was 78.2% in LAME89 and 81.4% in ELAM02. OS was significantly lower for the unfavorable cytogenetic risk group in LAME89/91 when compared with intermediate and favorable groups (50% vs 90.6 and 86.4%, P=0.001). This difference was no longer apparent in ELAM02 (80.9% vs 71.3% and 5/5, respectively). Survival improvement for children with unfavorable karyotype was statistically significant (P=0.026) and was due to decrease in relapse risk. Five-year transplantation-related mortality was 6.75% in LAME89/91. In ELAM02, it was 3.2% for patients with a sibling donor and 10.9% with an unrelated donor or cord blood. We conclude that the outcome of children with unfavorable karyotype transplanted in CR1 has improved.

Prevalence and risk factors of iron overload after hematopoietic stem cell transplantation for childhood acute leukemia: a LEA study.

Data on post-transplant iron overload (IO) are scarce in pediatrics. We conducted a prospective multicenter cohort study (Leucémie de l'Enfant et de l'Adolescent cohort) to determine the prevalence and risk factors of IO in 384 acute leukemia survivors transplanted during childhood. Prevalence of IO (ferritin level ⩾350 ng/mL) was 42.2% (95%CI 37.2-47.2%). Factors significantly associated with IO were: 1) in univariate analysis: older age at transplant (P<0.001), allogeneic versus autologous transplantation (P<0.001), radiation-based preparative regimen (P=0.035) and recent period of transplantation (P<0.001); 2) in multivariate analysis: older age at transplant in quartiles (Odds Ratio (OR)=7.64, 95% CI: 3.73-15.64 for age >12.7 years and OR=5.36, 95% CI: 2.63-10.95 for age from 8.2 to 12.7 years compared to age < 4.7 years), acute myeloid leukemia (OR=3.23, 95% CI: 1.47-7.13), allogeneic graft (OR=4.34, 95% CI: 2.07-9.12 for alternative donors and OR=2.53, 95% CI: 1.2-5.33 for siblings, compared to autologous graft) and radiation-based conditioning regimen (OR=2.45, 95% CI: 1.09-5.53). Graft-versus-host disease was an additional risk factor for allogeneic graft recipients. In conclusion, IO is a frequent complication in pediatric long-term survivors after transplantation for acute leukemia, more frequently observed in older children, those transplanted from alternative donors or with graft-versus-host disease.

miR-139-5p controls translation in myeloid leukemia through EIF4G2.

MicroRNAs (miRNAs) are crucial components of homeostatic and developmental gene regulation. In turn, dysregulation of miRNA expression is a common feature of different types of cancer, which can be harnessed therapeutically. Here we identify miR-139-5p suppression across several cytogenetically defined acute myeloid leukemia (AML) subgroups. The promoter of mir-139 was transcriptionally silenced and could be reactivated by histone deacetylase inhibitors in a dose-dependent manner. Restoration of mir-139 expression in cell lines representing the major AML subgroups (t[8;21], inv[16], mixed lineage leukemia-rearranged and complex karyotype AML) caused cell cycle arrest and apoptosis in vitro and in xenograft mouse models in vivo. During normal hematopoiesis, mir-139 is exclusively expressed in terminally differentiated neutrophils and macrophages. Ectopic expression of mir-139 repressed proliferation of normal CD34(+)-hematopoietic stem and progenitor cells and perturbed myelomonocytic in vitro differentiation. Mechanistically, mir-139 exerts its effects by repressing the translation initiation factor EIF4G2, thereby reducing overall protein synthesis while specifically inducing the translation of cell cycle inhibitor p27(Kip1). Knockdown of EIF4G2 recapitulated the effects of mir-139, whereas restoring EIF4G2 expression rescued the mir-139 phenotype. Moreover, elevated miR-139-5p expression is associated with a favorable outcome in a cohort of 165 pediatric patients with AML. Thus, mir-139 acts as a global tumor suppressor-miR in AML by controlling protein translation. As AML cells are dependent on high protein synthesis rates controlling the expression of mir-139 constitutes a novel path for the treatment of AML.

[Multidisciplinarity, education, and training in pediatric oncology-hematology]. / Multidisciplinarité et formation des spécialistes à l'oncologie et à l'hématologie maligne pédiatrique.

INTRODUCTION: According to the European Society of Pediatric Oncology (SIOPE) standard of care guidelines, high-quality care of children and adolescents with cancer needs to be delivered by well-trained multidisciplinary teams in specialist centers working with designated shared-care local centers in a so-called hub-and-spoke model. The Diplôme Inter-Universitaire d'Oncologie Pédiatrique (DIUOP) is the only European training program in pediatric oncology in French for all physicians involved in care of patients with pediatric malignancies. In agreement with the SIOPE syllabus, the DIUOP is composed of training courses (120h), on-site practical training in a specialist center, and a research project to be defended before an examining board. METHOD: All graduates received a questionnaire to describe their current professional position. A comprehensive PubMed analysis retrieved all papers published form DIUOP research projects. RESULTS: From 2000 to 2011, 290 physicians were trained: 242 pediatricians, 21 surgeons, and 19 radiation therapists. Eight had another specialty including imaging, hematology, and pathology. Ninety-two were initially trained outside of France: 50% in Europe (mainly in Italy, Belgium, and Switzerland), 42% in Africa and the Middle East, and 8% in South America. Of the 266 graduates, 74% answered the questionnaire, and 90% of them take care of children and adolescents with cancer. Sixty-nine articles, i.e., one out of four research projects, were published in 34 journals with a median impact factor of 3.5 (0-22.6), 85% in English. CONCLUSION: DIUOP is the only French-speaking European education program providing a high-quality, professionalizing, and comprehensive multidisciplinary training program for French and international specialists taking care of children and adolescents with cancer.

Metabolic syndrome in adults who received hematopoietic stem cell transplantation for acute childhood leukemia: an LEA study.

We evaluated prospectively the incidence and risk factors of the metabolic syndrome (MS) and its components in 170 adult patients (mean age at evaluation: 24.8±5.4 years) who received an hematopoietic stem cell transplantation for childhood ALL, n=119, or AML, n=51. TBI was carried out in 124 cases; a busulfan-based conditioning was done in 30 patients. Twenty-nine patients developed a MS (17.1%, 95% confidence intervals: 11.7-23.6). The cumulative incidence was 13.4% at 25 years of age and 35.5% at 35 years of age. A higher body mass index (BMI) before transplantation and a growth hormone deficiency were associated with increased MS risk (P=0.002 and 0.01, respectively). MS risk was similar for patients who received TBI or busulfan-based conditioning. The TBI use increased the hyperglycemia risk (odds ratio (OR): 4.7, P=0.02). Women were at the risk of developing increased waist circumference (OR: 7.18, P=0.003) and low levels of high-density lipoprotein cholesterol (OR: 2.72, P=0.007). The steroid dose was not a risk factor. The MS occurs frequently among transplanted survivors of childhood leukemia. Its incidence increases with age. Both intrinsic (BMI, gender) and extrinsic factors (TBI, alkylating agents) contribute to its etiopathogenesis.

Parental smoking, maternal alcohol, coffee and tea consumption during pregnancy, and childhood acute leukemia: the ESTELLE study.

PURPOSE: To investigate the role of parental smoking during pre-conception and pregnancy, maternal beverage consumption (alcohol, coffee and tea) during pregnancy and their possible interactions, in the etiology of childhood acute leukemia (CL). METHODS: The ESTELLE study included 747 cases of CL [636 cases of acute lymphoblastic leukemia (ALL) and 100 cases of acute myeloblastic leukemia (AML)] diagnosed in France in 2010-2011 and 1,421 population controls frequency-matched with the cases on age and gender. Data were obtained from structured telephone questionnaires administered to the mothers. The odds ratios (OR) and their 95 % confidence intervals were estimated using unconditional logistic regression models adjusted for potential confounders. RESULTS: AML, but not ALL, was non-significantly associated with alcohol drinking during pregnancy [OR = 1.3 (0.8-2.0)] with a significant positive dose-response trend (p-trend = 0.02). Pre-conception paternal smoking was significantly associated with ALL [OR = 1.2 (1.1-1.5)] and AML [OR = 1.5 (1.0-2.3)]. CL was not associated with maternal smoking [OR = 1.0 (0.8-1.2)], or maternal coffee [OR = 0.9 (0.8-1.1)] or tea drinking [OR = 0.9 (0.8-1.1)] during pregnancy. However, a high consumption of coffee (>2 cups/day) was significantly associated with ALL [OR = 1.3 (1.0-1.8)]. CONCLUSIONS: The findings constitute additional evidence that maternal alcohol drinking during pregnancy may be involved in AML, and that paternal smoking before pregnancy may be a risk factor for CL. The role of maternal coffee drinking in CL remains unclear and should be investigated further in consortium analyses and in large birth cohort studies with exposure assessment more contemporaneous with the exposure, before the occurrence of the disease.

Adenovirus infection and disease in paediatric haematopoietic stem cell transplant patients: clues for antiviral pre-emptive treatment.

Human adenovirus (HAdV) infections constitute a major cause of morbidity in paediatric haematopoietic stem cell transplant (HSCT) patients. New antiviral treatments offer promising perspectives. However, it remains challenging to identify patients at risk for disseminated infection, and who should receive early antiviral intervention. We conducted a longitudinal study of allogeneic HSCT recipients, including weekly HAdV monitoring, to determine the risks factors associated with HAdV infection and dissemination, and to assess whether HAdV loads in stools may be used as surrogate markers for HAdV dissemination. Between September 2010 and December 2011, out of 72 patients, the cumulative incidence rates at day 100 of HAdV digestive infection, systemic infection and related disease were 35.9%, 24.0%, and 18.3%, respectively. In multivariate analysis, the risk factors for HAdV digestive and systemic infection were cord blood and in vitro T-cell depletion. Graft-versus-host disease (GVHD) grade >2 was also associated with systemic infection. In patients with HAdV digestive shedding, GVHD grade >2 and HAdV load in stools were the only risk factors for systemic infection. The median peak levels of HAdV in stool were 7.9 and 4.0 log10 copies/mL, respectively, in patients with HAdV systemic infection and in those without. HAdV monitoring in stools of paediatric HSCT recipients receiving cord blood or in vitro T-cell depleted transplants helps to predict patients at risk for HAdV systemic infection. Our results provide a rationale for randomized controlled trials to evaluate the benefit of anti-HAdV pre-emptive treatments based on HAdV DNA levels in stools.

Hematopoietic stem cell transplantation in relapsed ALK+ anaplastic large cell lymphoma in children and adolescents: a study on behalf of the SFCE and SFGM-TC.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is a treatment option for relapsed anaplastic large cell lymphoma (ALCL) in children, but reports on its efficacy in this disease are still limited. We analyzed data concerning 34 patients under 18 years of age prospectively registered in the French SFGM-TC database, who had undergone an allo-SCT for the treatment of ALK+ ALCL between 1993 and 2011. At transplant, 28 patients (82.4%) were in CR, whereas 6 exhibited detectable disease. Conditioning regimens were mostly myelo-ablative (n=31). With a median follow-up of 6 years, 5-year overall and event-free survival rates were 70% (SE=8%) and 58% (SE=9%), respectively. The 5-year cumulative incidence of relapse and treatment-related mortality was 18% (SE=7%) and 24% (SE=8%), respectively. Six patients had relapsed (median time, 141 days (35-235)). A durable CR had been obtained in 4/6 patients after injection of donor lymphocytes (n=1) or vinblastine-corticosteroid therapy (n=3). Ten patients had died, eight due to transplant toxicity and two due to progressive disease. Allo-SCT is an efficient treatment for pediatric patients with high-risk relapsed ALK+ ALCL. However, the overall morbidity of allo-SCT raises questions about its place, given the efficacy of targeted agents currently under development in this disease.

Childhood acute lymphoblastic leukaemia and indicators of early immune stimulation: the Estelle study (SFCE).

BACKGROUND: Factors related to early stimulation of the immune system (breastfeeding, proxies for exposure to infectious agents, normal delivery, and exposure to animals in early life) have been suggested to decrease the risk of childhood acute lymphoblastic leukaemia (ALL). METHODS: The national registry-based case-control study, ESTELLE, was carried out in France in 2010-2011. Population controls were frequency matched with cases on age and gender. The participation rates were 93% for cases and 86% for controls. Data were obtained from structured telephone questionnaires administered to mothers. Odds ratios (OR) were estimated using unconditional regression models adjusted for age, gender, and potential confounders. RESULTS: In all, 617 ALL and 1225 controls aged ⩾1 year were included. Inverse associations between ALL and early common infections (OR=0.8, 95% confidence interval (CI): 0.6, 1.0), non-first born (⩾3 vs 1; OR=0.7, 95% CI: 0.5, 1.0), attendance of a day-care centre before age 1 year (OR=0.7, 95% CI: 0.5, 1.0), breastfeeding (OR=0.8, 95% CI: 0.7, 1.0), and regular contact with pets (OR=0.8, 95% CI: 0.7, 1.0) in infancy were observed. CONCLUSIONS: The results support the hypothesis that conditions promoting the maturation of the immune system in infancy have a protective role with respect to ALL.

Methodological aspects of minimal residual disease assessment by flow cytometry in acute lymphoblastic leukemia: A French multicenter study.

Background: Minimal residual disease (MRD) assessment provides a powerful prognostic factor for therapeutic stratification in acute lymphoblastic leukemia (ALL). Multiparameter flow cytometry (MFC) has the potential for a rapid and sensitive identification of high risk patients. Our group has previously published that MRD levels analyzed by clone specific Ig/TcR-QPCR and MFC were concordant at a sensitivity of 10-4 . Here we report the MFC methodological aspects from this multi-center experience. Methods: MRD was assessed by MFC in 1030 follow-up samples from 265 pediatric and adult patients with de novo ALL treated in the FRALLE, EORTC or GRALL clinical trials. MRD assessment as applied by the eight participating MFC laboratories is described in detail regarding cell preparation, leukemia-associated immunophenotype (LAIP) markers and data analysis. Samples were obtained from bone marrow (BM) and peripheral blood (PB). Immunostaining was performed after erythrocyte lysis or Ficoll enrichment. Results: This study confirms the applicability of MFC-based MRD assessment in 97% of patients with ALL at the 10-4 cut-off. MRD values after Ficoll enrichment and erythrocyte lysis were found comparable. Higher MRD values were obtained in BM than in PB, especially for B-lineage ALL. Conclusions: Measurement of MRD by MFC at the 10-4 cut-off is applicable within a few hours for almost all patients and using a comparable analytical strategy allows for multicenter collaborative studies. The method can be introduced in a strategy aimed at defining the risk of failure of patients with childhood or adult ALL. © 2014 Clinical Cytometry Society.

miR-9 is a tumor suppressor in pediatric AML with t(8;21).

MicroRNAs (miRNAs) play a pivotal role in the regulation of hematopoiesis and development of leukemia. Great interest emerged in modulating miRNA expression for therapeutic purposes. In order to identify miRNAs, which specifically suppress leukemic growth of acute myeloid leukemia (AML) with t(8;21), inv(16) or mixed lineage leukemia (MLL) rearrangement by inducing differentiation, we conducted a miRNA expression profiling in a cohort of 90 cytogenetically characterized, de novo pediatric AML cases. Four miRNAs, specifically downregulated in MLL-rearranged, t(8;21) or inv(16) AMLs, were characterized by their tumor-suppressive properties in cell lines representing those respective cytogenetic groups. Among those, forced expression of miR-9 reduced leukemic growth and induced monocytic differentiation of t(8;21) AML cell lines in vitro and in vivo. The tumor-suppressive functions of miR-9 were specifically restricted to AML cell lines and primary leukemic blasts with t(8;21). On the other hand, these functions were not evident in AML blasts from patients with MLL rearrangements. We showed that miR-9 exerts its effects through the cooperation with let-7 to repress the oncogenic LIN28B/HMGA2 axis. Thus, miR-9 is a tumor suppressor-miR which acts in a stringent cell context-dependent manner.