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Clinical genome-wide next-generation sequencing (NGS) has brought new challenges to genetic laboratories. The identification of numerous patient-specific variants that may require to be screened for on multiple other samples poses an issue when striving for time and cost-effectiveness. Here, we propose d-multiSeq, a straightforward method utilizing the advantages of droplet PCR for multiplexing combined with amplicon-based NGS. By comparing d-multiSeq with a standard multiplex amplicon-based NGS, it was shown that partitioning prevents the amplification competition seen when multiplexing and leads to a homogeneous representation of each target in the total read count for up to a 40-target multiplex without the need for prior adjustment. Variant allele frequency was reliably evaluated with a sensitivity of 97.6% for variant allele frequency up to 1%. The applicability of d-multiSeq was also tested on cell-free DNA with the successful amplification of an eight-target multiplex panel. Preliminary application of the technique to assess the clonal evolution in a childhood leukemia harboring high interpatient variability in its somatic variants is shown. d-multiSeq represents a turnkey solution for analyzing large sets of patient-specific variants on low DNA amounts and cell-free DNA., (Copyright © 2023 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Aim: To evaluate the prognostic significance of initial central nervous system (CNS) involvement of children with acute lymphoblastic leukemia (ALL) enrolled in the EORTC 58951 trial., Patients and Methods: From 1998 to 2008, 1930 ALL patients were included in the randomized EORTC 58951 trial. Overall treatment intensity was adjusted according to known prognostic factors including the level of minimal residual disease after induction treatment. CNS-directed therapy comprised four to 11 courses of i.v. methotrexate (5g/m 2 ), and 10 to 19 intrathecal chemotherapy injections, depending on risk group and CNS status. Cranial irradiation was omitted for all patients., Results: The overall 8-year event-free survival (EFS) and overall survival (OS) rates were 81.3% and 88.1%, respectively. In the CNS-1, TPL+, CNS-2, and CNS-3 groups, the 8-year EFS rates were 82.1%, 77.1%, 78.3%, and 57.4%, respectively. Multivariable analysis indicated that initial CNS-3 status, but not CNS-2 or TLP+, was an independent adverse predictor of outcome. The 8-year incidence of isolated CNS relapse was 1.7% and of isolated or combined CNS relapse it was 3.7%. NCI high-risk group, male sex, CNS-2 and CNS-3 status were independent predictors for a higher incidence of any CNS relapse., Conclusions: CNS-3 status remains associated with poor prognosis and requires intensification of both systemic and CNS-directed therapy. This trial was registered at https://clinicaltrials.gov/under/NCT00003728., (Copyright © 2021. Published by Elsevier Masson SAS.)

Improved personalized adjustment of primary therapy to the perceived risk of relapse by using new prognostic markers for treatment stratification may be beneficial to patients with acute lymphoblastic leukemia (ALL). Here, we review the advances that have shed light on the role of IKZF1 aberration as prognostic factor in pediatric ALL and summarize emerging concepts in this field. Continued research on the interplay of disease biology with exposure and response to treatment will be key to further improve treatment strategies., (© 2020 by The American Society of Hematology.)

Mutations in LZTR1, already known to be causal in familial schwannomatosis type 2, have been recently involved in a small proportion of patients with autosomal dominant and autosomal recessive Noonan syndrome. LZTR1 is also a driver gene in non syndromal glioblastoma. We report a 26-year-old patient with typical Noonan syndrome, and the dominantly transmitted c.850C > T (p.(Arg284Cys)) variant in LZTR1. An oligoastrocytoma was diagnosed in the patient at the age of 22 years; recurrence of the tumor occurred at age 26, as a ganglioblastoma. The patient had been transiently treated with growth hormone between ages 15 and 17. Considering the implication of LZTR1 in sporadic tumors of the nervous system, we hypothesize that gliomas are a possible complication of LZTR1-related Noonan syndrome. This report also supports a possible link between occurrence of a cerebral tumor in Noonan syndrome and a previous treatment with growth hormone., (Copyright © 2019. Published by Elsevier Masson SAS.)

Purpose: Standardized and accurate variant assessment is essential for effective medical care. To that end, Clinical Genome (ClinGen) Resource clinical domain working groups (CDWGs) are systematically reviewing disease-associated genes for sufficient evidence to support disease causality and creating disease-specific specifications of American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines for consistent and accurate variant classification., Methods: The ClinGen RASopathy CDWG established an expert panel to curate gene information and generate gene- and disease-specific specifications to ACMG-AMP variant classification framework. These specifications were tested by classifying 37 exemplar pathogenic variants plus an additional 66 variants in ClinVar distributed across nine RASopathy genes., Results: RASopathy-related specifications were applied to 16 ACMG-AMP criteria, with 5 also having adjustable strength with availability of additional evidence. Another 5 criteria were deemed not applicable. Key adjustments to minor allele frequency thresholds, multiple de novo occurrence events and/or segregation, and strength adjustments impacted 60% of variant classifications. Unpublished case-level data from participating laboratories impacted 45% of classifications supporting the need for data sharing., Conclusion: RAS-specific ACMG-AMP specifications optimized the utility of available clinical evidence and Ras/MAPK pathway-specific characteristics to consistently classify RASopathy-associated variants. These specifications highlight how grouping genes by shared features promotes rapid multigenic variant assessment without sacrificing specificity and accuracy.

Noonan syndrome is a well-known genetic condition associating congenital heart defects, short stature, and distinctive facial features. Pulmonary valve stenosis and hypertrophic cardiomyopathy are the most frequent cardiac abnormalities, the latter being associated with a higher mortality. Here we report for the first time, a case of congenital left main coronary artery atresia in a Noonan syndrome associated with RIT1 variant, leading to unrescued sudden death. This case-report supports the already-suspected severity of the RIT1-related Noonan syndrome compared to average Noonan syndrome, and should encourage clinicians to be very cautious with these patients., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive stem cell disease of early childhood. RAS activation constitutes the core component of oncogenic signaling. In addition, leukemic blasts in one-fourth of JMML patients present with monosomy 7, and more than half of patients show elevated age-adjusted fetal hemoglobin (HbF) levels. Hematopoietic stem cell transplantation is the current standard of care and results in an event-free survival rate of 50% to 60%, indicating that novel molecular-driven therapeutic options are urgently needed. Using gene expression profiling in a series of 82 patient samples, we aimed at understanding the molecular biology behind JMML and identified a previously unrecognized molecular subgroup characterized by high LIN28B expression. LIN28B overexpression was significantly correlated with higher HbF levels, whereas patients with monosomy 7 seldom showed enhanced LIN28B expression. This finding gives a biological explanation of why patients with monosomy 7 are rarely diagnosed with high age-adjusted HbF levels. In addition, this new fetal-like JMML subgroup presented with reduced levels of most members of the let-7 microRNA family and showed characteristic overexpression of genes involved in fetal hematopoiesis and stem cell self-renewal. Lastly, high LIN28B expression was associated with poor clinical outcome in our JMML patient series but was not independent from other prognostic factors such as age and age-adjusted HbF levels. In conclusion, we identified elevated LIN28B expression as a hallmark of a novel fetal-like subgroup in JMML., (© 2016 by The American Society of Hematology.)

Noonan syndrome is associated with a range of malignancies including acute lymphoblastic leukemia (ALL). However, little information is available regarding the frequency, natural history, characteristics and prognosis of ALL in Noonan syndrome or RASopathies in general. Cross-referencing data from a large prospective cohort of 1176 patients having a molecularly confirmed RASopathy with data from the French childhood cancer registry allowed us to identify ALL in 6 (0.5%) patients including 4/778 (0.5%) with a germline PTPN11 mutation and 2/94 (2.1%) with a germline SOS1 mutation. None of the patients of our series with CFC syndrome (with germline BRAF or MAP2K1/MAP2K2 mutation - n = 121) or Costello syndrome (with HRAS mutation - n = 35) had an ALL. A total of 19 Noonan-ALL were gathered by adding our patients to those of the International Berlin-Munster-Frankfurt (I-BFM) study group and previously reported patients. Strikingly, all Noonan-associated ALL were B-cell precursor ALL, and high hyperdiploidy with more than 50 chromosomes was found in the leukemia cells of 13/17 (76%) patients with available genetics data. Our data suggest that children with Noonan syndrome are at higher risk to develop ALL. Like what is observed for somatic PTPN11 mutations, NS is preferentially associated with the development of hyperdiploid ALL that will usually respond well to chemotherapy. However, Noonan syndrome patients seem to have a propensity to develop post therapy myelodysplasia that can eventually be fatal. Hence, one should be particularly cautious when treating these patients., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Environmental causes of childhood acute lymphoblastic leukemia (ALL) remain largely undiscovered. In contrast, multiple germline ALL risk variants have been identified in the recent years. Apart from the low-risk common ALL risk alleles identified through genome wide association studies, rare germline mutations that cause cancer prone syndromes have been found to be associated with an increased risk of developing ALL. Here, we review the germline genetic changes known to be associated with ALL., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

If acute leukemia is the most frequent cancer in childhood (33%), it remains a very rare diagnosis in infants less than one year old, e.g. less than 5% of cases. At this age, the frequency of acute lymphoblastic leukemia (ALL) (almost all of B-lineage) is quite similar to the one of myeloblastic forms (AML). Infant leukemia frequently presents with high hyperleucocytosis, major tumoral burden and numerous extra-hematological features, especially in central nervous system and skin. Whatever the lineage, the leukemic cell is often very immature cytologically and immunologically. Rearrangements of the Mixed Lineage Leukemia (MLL) gene, located on band 11q23, are the hallmark of these immature leukemias and confer a particular resistance to conventional approaches, corticosteroids and chemotherapy. The immaturity of infants less than 1-year-old is associated to a decrease of the tolerable dose-intensity of some drugs (anthracyclines, alkylating agents) or asks questions about some procedures like radiotherapy or high dose conditioning regimen, responsible of inacceptable acute and late toxicities. The high level of severe infectious diseases and other high-grade side effects limits also the capacity to cure these infants. The survival of infants less than 1-year-old with AML is only 50% but similar to older children. On the other hand, survival of those with ALL is the same, then quite limited comparing the 80% survival in children over one year. Allogeneic stem cell transplantations are indicated in high-risk subgroups of infant ALL (age below 6 months, high hyperleucocytosis >300.10(9)/L, MLL-rearrangement, initial poor prednisone response). However, morbidity and mortality remain very important and these approaches cannot be extended to all cases. During the neonatal period, the dismal prognosis linked to the high number of primary failures or very early relapses and uncertainties about the late toxicities question physicians about ethics. It is an emergency to propose different strategies (targeted therapies) to these infants with acute leukemia as conventional trials failed to improve outcome., (Copyright © 2015 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

With intensified pediatric-like therapy and genetic disease dissection, the field of adult acute lymphoblastic leukemia (ALL) has evolved recently. In this new context, we aimed to reassess the value of conventional risk factors with regard to new genetic alterations and early response to therapy, as assessed by immunoglobulin/T-cell receptor minimal residual disease (MRD) levels. The study was performed in 423 younger adults with Philadelphia chromosome-negative ALL in first remission (265 B-cell precursor [BCP] and 158 T-cell ALL), with cumulative incidence of relapse (CIR) as the primary end point. In addition to conventional risk factors, the most frequent currently available genetic alterations were included in the analysis. A higher specific hazard of relapse was independently associated with postinduction MRD level ≥10(-4) and unfavorable genetic characteristics (ie, MLL gene rearrangement or focal IKZF1 gene deletion in BCP-ALL and no NOTCH1/FBXW7 mutation and/or N/K-RAS mutation and/or PTEN gene alteration in T-cell ALL). These 2 factors allowed definition of a new risk classification that is strongly associated with higher CIR and shorter relapse-free and overall survival. These results indicate that genetic abnormalities are important predictors of outcome in adult ALL not fully recapitulated by early response to therapy. Patients included in this study were treated in the multicenter GRAALL-2003 and GRAALL-2005 trials. Both trials were registered at http://www.clinicaltrials.gov as #NCT00222027 and #NCT00327678, respectively., (© 2014 by The American Society of Hematology.)

Childhood BCR-ABL1-positive B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has an unfavorable outcome and shows high frequency of IKZF1 deletions. The prognostic value of IKZF1 deletions was evaluated in 2 cohorts of BCR-ABL1-positive BCP-ALL patients, before tyrosine kinase inhibitors (pre-TKI) and after introduction of imatinib (in the European Study for Philadelphia-Acute Lymphoblastic Leukemia [EsPhALL]). In 126/191 (66%) cases an IKZF1 deletion was detected. In the pre-TKI cohort, IKZF1-deleted patients had an unfavorable outcome compared with wild-type patients (4-year disease-free survival [DFS] of 30.0 ± 6.8% vs 57.5 ± 9.4%; P = .01). In the EsPhALL cohort, the IKZF1 deletions were associated with an unfavorable prognosis in patients stratified in the good-risk arm based on early clinical response (4-year DFS of 51.9 ± 8.8% for IKZF1-deleted vs 78.6 ± 13.9% for IKZF1 wild-type; P = .03), even when treated with imatinib (4-year DFS of 55.5 ± 9.5% for IKZF1-deleted vs 75.0 ± 21.7% for IKZF1 wild-type; P = .05). In conclusion, the highly unfavorable outcome for childhood BCR-ABL1-positive BCP-ALL with IKZF1 deletions, irrespective of imatinib exposure, underscores the need for alternative therapies. In contrast, good-risk patients with IKZF1 wild-type responded remarkably well to imatinib-containing regimens, providing a rationale to potentially avoid hematopoietic stem-cell transplantation in this subset of patients.

Juvenile myelomonocytic leukemias (JMML) are rare but severe myelodysplastic and myeloproliferative neoplasms of infancy. They represent about 10 new cases per year in France and preferentially affect males. JMML are all stem cell diseases the common denominator of which is RAS pathway dysregulation, due to mutations in RAS (NRAS, KRAS) or RAS regulatory components (PTPN11, NF1 or CBL). This leads to an hypersensivity of myeloid progenitors to GM-CSF (granulo-macrophagic colony stimulating factor) which induces in turn excessive monocytic and macrophagic proliferation in blood and bone marrow. All organs can be infiltrated by this monocytic proliferation leading to multisystemic failure. Blast crisis with transformation into acute myeloid leukemia occurs in one third of patients. A salient feature of JMML is their frequent association with predisposition syndromes such as Noonan syndrome, neurofibromatosis and CBL syndrome, which are developmental diseases associated with a constitutional RAS pathway deregulation, now grouped under the name RASopathies. Clinical heterogeneity makes JMML diagnosis difficult. Splenomagaly is the most constant sign. Palor, adenopathy, respiratory or cutaneous symptoms can also be present. Blood smear shows monocytosis (>1×10(9)/L) presence of myeloid progenitors and abnormal basophils. The demonstration of an endogeneous in vitro growth of myeloid progenitors although not very specific can help JMML diagnosis. Nowadays, genetic typing has to be included in the workup of JMML diagnosis and allows to evidence a mutation in more than 90% of cases. JMML have a poor prognosis. The only curative treatment is bone marrow transplantation but approximately 35% of patients relapse. JMML clinical course is highly heterogeneous and unpredictable. Some rare patients have an indolent evolution or even spontaneous remission. Age over two years, thrombopenia below 33×10(9)/L and high foetal hemoglobin (HbF) level for age are poor prognosis criteria but hardly predict individual outcome. Several research directions are currently being explored to improve prognosis prediction and provide more effective targeted treatments.

Children with Down syndrome (DS) have an increased risk of B-cell precursor (BCP) acute lymphoblastic leukemia (ALL). The prognostic factors and outcome of DS-ALL patients treated in contemporary protocols are uncertain. We studied 653 DS-ALL patients enrolled in 16 international trials from 1995 to 2004. Non-DS BCP-ALL patients from the Dutch Child Oncology Group and Berlin-Frankfurt-Münster were reference cohorts. DS-ALL patients had a higher 8-year cumulative incidence of relapse (26% ± 2% vs 15% ± 1%, P < .001) and 2-year treatment-related mortality (TRM) (7% ± 1% vs 2.0% ± <1%, P < .0001) than non-DS patients, resulting in lower 8-year event-free survival (EFS) (64% ± 2% vs 81% ± 2%, P < .0001) and overall survival (74% ± 2% vs 89% ± 1%, P < .0001). Independent favorable prognostic factors include age <6 years (hazard ratio [HR] = 0.58, P = .002), white blood cell (WBC) count <10 × 10(9)/L (HR = 0.60, P = .005), and ETV6-RUNX1 (HR = 0.14, P = .006) for EFS and age (HR = 0.48, P < .001), ETV6-RUNX1 (HR = 0.1, P = .016) and high hyperdiploidy (HeH) (HR = 0.29, P = .04) for relapse-free survival. TRM was the major cause of death in ETV6-RUNX1 and HeH DS-ALLs. Thus, while relapse is the main contributor to poorer survival in DS-ALL, infection-associated TRM was increased in all protocol elements, unrelated to treatment phase or regimen. Future strategies to improve outcome in DS-ALL should include improved supportive care throughout therapy and reduction of therapy in newly identified good-prognosis subgroups.

Neonatal diabetes mellitus is a rare condition (1/90,000 to 1/260,000 live births) defined as mild-to-severe hyperglycemia within the first year of life. Permanent neonatal diabetes mellitus requires lifelong therapy, whereas transient form resolves early in life but may relapse later on. Two main physiopathological mechanisms may explain this disease: β cell functional impairment or absence (pancreas agenesis or β cells destruction). The main genetic causes of β cells impairment are 6q24 abnormalities and mutations in ABCC8 or KCNJ11 potassium channel (KATP channel) genes. Compared to the KATP subtype, the 6q24 subtype had specific features: developmental defects involving the heart, kidneys, or urinary tract, intrauterine growth restriction, and early diagnosis. Remission of neonatal diabetes mellitus occurred in 51% of probands at a median age of 17 weeks. Recurrence was common at pubertal age, with no difference between the 6q24 and KATP-channel groups (82% vs 86%, p=0.36, respectively). Patients with mutations in ABCC8 or KCNJ11 genes had developmental delay with or without epilepsy but also developmental coordination disorder (particularly visual-spatial dyspraxia) or attention deficits in all of those who underwent in-depth neuropsychomotor investigations., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

The aim of our study was to analyze the factors contributing to heterogeneity of prognosis in patients with hyperdiploidy>50 chromosomes (HD>50), a group of B-cell precursor acute lymphoblastic leukemia with favorable outcome. The 541 HD>50 patients registered prospectively in the 58951 European Organisation for Research and Treatment of Cancer (EORTC) Children's Leukemia Group (CLG) trial, identified by karyotype (446 patients) and by DNA index (DI) (490 patients), had a 6-year event-free survival (EFS) of 89.0% (standard error [SE] = 1.5%) and a 6-year overall survival (OS) of 95.9% (SE = 0.9%). The strongest prognostic factor was the modal number of chromosomes (MNC): the 6-year EFS of 51-53, 54-57, and 58-66 MNC groups were 80%, 89%, and 99%, respectively (P < .0001). Ploidy assessed by DI was also a favorable factor: the higher the DI, the better the outcome. The 6-year EFS of the 3 subgroups of DI < 1.16/≥1.16-<1.24/≥1.24 were 83%, 90%, and 95%, respectively (P = .009). All usual combinations of trisomies (chromosomes 4, 10, 17, 18) were significant favorable factors but had lower EFS when MNC was lower than 58. In multivariate analysis, MNC remained the strongest factor. Consequently, the best indicator for excellent outcome was ploidy assessed by karyotype because patients with 58-66 chromosomes stood every chance of being cured (OS of 100% at 6-year follow-up) with less-intensive therapy. This trial was registered at www.clinicaltrials.gov as #NCT00003728. Registered: http://www.eortc.org/, http://clinicaltrials.gov/show/NCT00003728.

Permanent neonatal diabetes mellitus is a rare disorder usually presenting within the first few weeks or months of life and defined by chronic hyperglycemia due to severe nonautoimmune insulin deficiency. Nonsyndromic neonatal diabetes is genetically heterogeneous and several genes have been linked to this disorder. Here, we report on a new homozygous recessive mutation in the INS gene in 2 siblings born to consanguineous parents and diagnosed with permanent neonatal diabetes without extrapancreatic features. Their clinically unaffected parents were heterozygous. Their phenotype was also characterized by severe intrauterine growth retardation, most likely reflecting severe insulin deficiency in prenatal life, hyperglycemia, and moderate dehydration in the first few days of life. Their clinical course was uneventful after introduction of insulin therapy with catch-up growth and acquisition of normal developmental milestones., (Copyright © 2012. Published by Elsevier SAS.)

Polyvalvular heart disease has been reported in a handful of "private" syndromes that have been recently suggested to represent a single dominantly inherited condition, the polyvalvular heart disease syndrome. We report five cases in two unrelated families (one sporadic case in the first family and three siblings and their father in the second family) with the same association of polyvalvular heart disease, distinctive facial appearance, and, except the father in family 2, major joint hypermobility. Interestingly, in three of our patients (2 siblings and the sporadic case), electron microscopy revealed characteristic ultrastructural skin abnormalities with abnormal amorphous or microfibrillar deposits under the capillary basal membrane in the papillary dermis, suggestive of a connective tissue disorder, but different from Marfan syndrome or Ehlers-Danlos syndrome. Moreover, in family 2, three others sibs died in early infancy of their heart defect. Our two families and the other published cases might illustrate intrafamilial and interfamilial variability within a single condition. However, our two families disclose major joint hypermobility, normal stature, and ultrastructural skin abnormalities that were not described in the previous reports. These discrepancies let us to consider them as affected by a distinct disorder of the connective tissue., (Copyright (c) 2009 Elsevier Masson SAS. All rights reserved.)

Transient and permanent neonatal diabetes mellitus (TNDM and PNDM) are rare conditions occurring in around 1 per 300,000 live births. In TNDM, growth-retarded infants develop diabetes in the first few weeks of life, only to go into remission after a few months with possible relapse to permanent diabetes usually around adolescence or in adulthood. In PNDM, insulin secretory failure occurs in the late fetal or early postnatal period. The very recently elucidated mutations in KCNJ11 and ABCC8 genes, encoding the Kir6.2 and SUR1 subunits of the pancreatic K(ATP) channel involved in regulation of insulin secretion, account for a third to a half of the PNDM cases. Molecular analysis of chromosome 6 anomalies and the KCNJ11 and ABCC8 genes encoding Kir6.2 and SUR1 provides a tool for distinguishing transient from permanent neonatal diabetes mellitus in the neonatal period. Some patients (those with mutations in KCNJ11 and ABCC8) may be transferred from insulin therapy to sulphonylureas.

This study investigates the extent of bone marrow (BM) involvement at diagnosis of apparent isolated extramedullary (AIEM) relapses of childhood acute lymphoblastic leukemia (ALL) and its relation to prognosis. Sixty-four children with first AIEM relapse treated in Germany, Czech Republic, or France were included. Real-time quantitative polymerase chain reaction using T-cell receptor and immunoglobulin gene rearrangements provided a sensitive measure of submicroscopic BM involvement, which was detectable at a level of 10(-4) or higher in 46 patients and less than 10(-4) in 11 patients, and was nondetectable (sensitivity: 10(-4)) in 7 patients. In the total cohort, the probability of event-free survival (pEFS) for children with BM involvement of 10(-4) or higher was 0.30 (0.09 +/- SE) versus 0.60 (+/- 0.12) for those with less than 10(-4) (P = .13). The cumulative incidence of subsequent relapse was 0.24 (+/- 0.01) for patients with BM involvement less than 10(-4) and 0.65 (+/- 0.01) for those with 10(-4) or higher (P = .012). Restricted to central nervous system (CNS) relapses, pEFS was 0.11 (+/- 0.09) for patients with BM involvement 10(-4) or higher and 0.63 (+/- 0.17) for those with less than 10(-4) (P = .053). CNS relapses were associated with a higher (> or = 10(-4): 80%) submicroscopic BM involvement than testicular relapses (> or = 10(-4): 57%, P = .08). In summary, we show marked heterogeneity of submicroscopic BM involvement at first AIEM relapse diagnosis in children with ALL, and demonstrate its possible prognostic relevance.

Transient (TNDM) and Permanent (PNDM) Neonatal Diabetes Mellitus are rare conditions occurring in about 1: 300,000 live births. In TNDM growth retarded infants develop diabetes in the first few weeks of life only to go into remission in a few months with possible relapse to a permanent diabetes state usually around adolescence or as adults. We believe that pancreatic dysfunction in this condition is maintained throughout life with relapse initiated at times of metabolic stress such as puberty or pregnancy. In PNDM, insulin secretory failure occurs in the late fetal or early postnatal period. A number of conditions are associated with PNDM, some of which have been elucidated at the molecular levels. Among those, the very recently elucidated mutations in KCNJ11 and ABCC8 gene, encoding the Kir6.2 and SUR1 subunit of the pancreatic K(ATP) channel involved in regulation of insulin secretion accounts for one third to a half of the PNDM cases. Patients with TNDM are more likely to have intrauterine growth retardation and less likely to develop ketoacidosis than patients with PNDM. In TNDM, patients are younger at the diagnosis of diabetes and have lower initial insulin requirements. Considerable overlap occurs between the two groups, so that TNDM cannot be distinguished from PNDM based on clinical features. Very early onset diabetes mellitus seems to be unrelated to autoimmunity in most instances. Recurrent diabetes is common in patients with "transient" neonatal diabetes mellitus and, consequently, prolonged follow-up is imperative. Molecular analysis of chromosome 6 anomalies, the KCNJ11 and ABCC8 genes encoding Kir6.2 and SUR1 provide a tool to identify transient from permanent neonatal diabetes mellitus in the neonatal period. This analysis also has potentially important therapeutic consequences leading to transfer some patients, those with mutations in KCNJ11 and ABCC8 from insulin therapy to sulfonylureas. Realizing how difficult it is to take care of a child of this age with diabetes mellitus should prompt clinicians to transfer these children to specialized centers. Insulin therapy and high caloric intake are the basis of the treatment. Insulin pump may offer an interesting therapeutic tool in this age group in experienced hands.

In a series of 153 children with T-cell malignancies enrolled in 2 consecutive European Organization for Research and Treatment of Cancer (EORTC) trials, we assessed the HOX11L2 expression and/or the presence of a t(5;14)(q35;q32). Additionally, in 138 of these patients, HOX11 expression and SIL-TAL rearrangement were also assessed. These alterations were mutually exclusive, and their frequency was 23% (n = 35), 7% (n = 10), and 12% (n = 17), respectively. HOX11L2/t(5;14) positivity was more frequent in acute lymphoblastic leukemia (ALL) with cortical T immunophenotype and in children aged between 6 and 9 years. In contrast with previously reported data, patients positive and negative for HOX11L2/t(5;14) were comparable with regard to clinical outcome as well as to the response to a 7-day prephase treatment or to residual disease at completion of induction therapy. The 3-year event-free survival (EFS) rate (+/- SE percentage) for patients positive and negative for HOX11L2/t(5;14) was 75.5% (+/- 8.1%) and 68.3% (+/- 5.0%), respectively; the hazard ratio was 0.84 (95% confidence interval, 0.40-1.80). Patients with HOX11-high expression and those with SIL-TAL fusion had low levels of residual disease at the end of induction and a favorable prognosis: the 3-year EFS rate was 83.3% (+/- 8.5%) and 75.3% (+/- 12.6%), respectively. The results obtained in HOX11L2/t(5;14) patients in this study do not confirm the unfavorable prognosis reported in previous studies.

Mutations in the DNA mismatch repair (MMR) system lead to an instability of simple repetitive DNA sequences involved in several cancer types. This instability is reflected in a high mutation rate of microsatellites, and recent studies in colon cancer indicate that defects in MMR result in frequent frameshift mutations in mononucleotide repeats located in the coding regions of BAX and transforming growth factor-beta (TGF-beta) receptor genes. Circumstantial evidence suggests that the MMR defect may be involved in some lymphoid malignancies, although several allelotype analyses have concluded on the low level of microsatellite instability in acute lymphoblastic leukemias. To further evaluate the implication of MMR defects in leukemogenesis, we have studied a series of 98 children with acute lymphoblastic leukemia and 14 leukemic cell lines using several indicators of MMR defects. Microsatellite markers were compared between blast and normal DNA from the same patients and mutations were sought in mononucleotide repeat sequences of BAX and TGF-beta receptor II (TGF-beta RII). The absence of microsatellite instability (MI) and the absence of mutations in the genes examined from patient's leukemic cells contrasted with the observation that half of the cell lines displayed a high degree of MI and that three of seven of these mutator cell lines harbored mutations in BAX and/or TGF-beta RII. From these results we conclude that MMR defects are very uncommon in freshly isolated blasts but are likely to be selected for during the establishment of cell lines.

Bone marrow (BM) trephine biopsies from 15 pediatric patients with acute lymphoid (ALL) or myeloid (AML) leukemia were engrafted subcutaneously into severe combined immunodeficiency (SCID) mice conditioned by 200 cGy total-body irradiation. Implants were harvested 5 to 19 weeks later for histologic, cytologic, and/or flow cytometric analysis of the residing marrow. Eighteen of 19 grafts contained viable human leukemic cells to various extents as assessed by one or more of these methods. Thirteen of 14 implants analyzed by flow cytometry included high numbers of tumor cells, accounting for 85% to 100% of the total nucleated cells in seven of them. Histologically, engrafted marrow samples exhibited areas of blastic infiltration, and tumor-specific gene rearrangements were retrieved in long-term engrafted biopsies. Importantly, engrafted mice remained perfectly healthy even 5 months posttransplantation, and no human tumor cell dissemination was detected in the hematolymphoid and nonhematopoietic tissues at the time of autopsy. These results demonstrate that human malignant hematopoiesis can be sustained long-term in its original, intact marrow stromal environment transplanted in appropriately conditioned immunodeficient mice.

OBJECTIVE: To reassess the occurrence of verocytotoxin-producing Escherichia coli (VTEC) in French hemolytic uremic syndrome (HUS) patients. METHODS: From March 1991 to January 1995, direct detection of verotoxin genes (VT) by the polymerase chain reaction (PCR) was performed on stool samples from 169 patients suffering from HUS. RESULTS: Fifty-one were PCR positive (30.1%); one was positive for the VT1 gene and the others for the VT2 gene. VTEC was isolated from only 32 of the 51 PCR-positive samples. E. coli O157:H7 was isolated from five patients. E. coli O111 was isolated from seven patients during an outbreak of HUS. Among the other VT2 E. coli strains, only four were serotypable. Of the 51 PCR-positive stools, 19 were culture negative for VTEC. CONCLUSIONS: This study provides evidence that in France E. coli O157 and other VTEC serotypes are involved in HUS.

Abnormalities of the short arm of chromosome 12 are relatively common in hematologic malignancies and deletions of the region. 12p12-13 are found in approximately 5% of the patients with acute lymphoblastic leukemia (ALL). As a potent inhibitor of cyclin-dependent kinases, p27KIP1 prevents the progression of the cell cycle and the gene encoding p27KIP1 represents a potential tumor-suppressor gene. Its recent assignment to the chromosomal region (12p12.3) prompted us to study the p27KIP1 gene in a series of 61 children with ALL. Microsatellite polymorphic markers flanking the p27KIP1 gene were analyzed to detect losses of heterozygosity (LOH). Eleven patients displayed LOH for at least one of the markers. The deleted are encompassed the p27KIP1 gene locus in 10 cases, but inactivation of the remaining allele by deletion, translocation, or mutation was never observed. In addition, in 1 patient, the p27KIP1 gene was situated outside of the region of LOH. Thus, p27KIP1 does not seem to be the target gene of 12p12-13 alterations. However, this study indicates that 12p12-13 alterations at the molecular level, which are present in about 27% of the children with B-lineage ALL, are much more common than had previously been reported by usual chromosome analysis. Moreover, LOH mapping allowed us to better define the location of a putative tumor-suppressor gene implicated in these malignancies and should therefore help in identifying this gene.

We have developed a strategy based on polymerase chain reaction (PCR) for detecting all possible gamma T-cell receptor (gamma TCR) rearrangements and the most common delta TCR rearrangements found in B-lineage and T-acute lymphoblastic leukemia (T-ALL). The segments amplified from blasts are then directly sequenced to derive clonospecific probes. From a series of 45 patients aged 1 to 15 years (42 B-lineage ALL, 3 T-ALL), 35 (83%) could be followed for minimal residual disease with at least one clonospecific probe. Detection of clonal markers using clonospecific probes routinely allowed the detection of 1 to 10 blasts out of 10(5) cells as determined by serial dilutions of the initial samples. Residual disease was quantitated by a competitive PCR assay based on the coamplification of an internal standard. Twenty children were prospectively followed for periods varying from 7 to 30 months. In most children, a progressive decrease of the tumor load was observed, and blasts became undetectable within 6 months after the initiation of treatment. A slower kinetics of decrease in tumor cells was found in three children. These three patients relapsed with blasts that continued to display the initial clonospecific markers. Three other children had a central nervous system relapse despite the absence of detectable medullary residual disease. The use of both delta and gamma TCR genes as clonal markers, as well as simplification in the methods to detect and quantify residual blasts reported here, will allow the study of the large number of patients required to determine the role of the detection of minimal residual disease by PCR in the follow-up of childhood ALL.

Fifty-nine Escherichia coli strains isolated from 54 unrelated patients over a six-year period, as well as the reference strain of the species, were studied by analysis of randomly amplified polymorphic DNA (RAPD) to assess the usefulness of this genotyping approach in molecular epidemiology. Using a 10-mer oligonucleotide primer, 28 different RAPD fingerprints were distinguished among the 60 strains previously delineated in 36 ribotypes by EcoRI and HindIII digests. The patterns were reproducible and stable after in vitro and in vivo studies. Some strains harbouring an identical ribotype exhibited distinct RAPD fingerprints. Thus, these data illustrate the usefulness of the association of two genetic markers in assessing the relationship between strains. Interestingly, among the RAPD patterns, several bands were present only in the highly virulent carboxylesterase type B2 strains, which are more homogeneous than the carboxylesterase type B1 strains. Because of its simplicity and rapidity, RAPD analysis appears to be a highly valuable tool for studying E. coli molecular epidemiology.