Whole-exome sequencing of pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL), the most common malignant disorder in childhood, is typically associated with numerical chromosomal aberrations, fusion genes or small focal deletions, thought to represent important pathogenetic events in the development of the leukemia. Mutations, such as single nucleotide changes, have also been reported in childhood ALL, but these have only been studied by sequencing a small number of candidate genes. Herein, we report the first unbiased sequencing of the whole exome of two cases of pediatric ALL carrying the ETV6/RUNX1 (TEL/AML1) fusion gene (the most common genetic subtype) and corresponding normal samples. A total of 14 somatic mutations were identified, including four and seven protein-altering nucleotide substitutions in each ALL. Twelve mutations (86%) occurred in genes previously described to be mutated in other types of cancer, but none was found to be recurrent in an extended series of 29 ETV6/RUNX1-positive ALLs. The number of single nucleotide mutations was similar to the number of copy number alterations as detected by single nucleotide polymorphism arrays. Although the true pathogenetic significance of the mutations must await future functional evaluations, this study provides a first estimate of the mutational burden at the genetic level of t(12;21)-positive childhood ALL.Leukemia advance online publication, 18 November 2011; doi:10.1038/leu.2011.333

Gemtuzumab ozogamicin as postconsolidation therapy does not prevent relapse in children with AML: results from NOPHO-AML 2004.

To access publisher full text version of this article. Please click on the hyperlink in Additional Links field.There are no data on the role of postconsolidation therapy with gemtuzumab ozogamicin (GO; Mylotarg) in children with acute myeloid leukemia (AML). The NOPHO-AML 2004 protocol studied postconsolidation randomization to GO or no further therapy. GO was administered at 5 mg/m(2) and repeated after 3 weeks. We randomized 120 patients; 59 to receive GO. Survival was analyzed on an intention-to-treat basis. The median follow-up for patients who were alive was 4.2 years. Children who received GO showed modest elevation of transaminase and bilirubin without signs of veno-occlusive disease. Severe neutropenia followed 95% and febrile neutropenia 40% of the GO courses. Only a moderate decline in platelet count and a minor decrease in hemoglobin occurred. Relapse occurred in 24 and 25 of those randomized to GO or no further therapy. The median time to relapse was 16 months versus 10 months (nonsignificant). The 5-year event-free survival and overall survival was 55% versus 51% and 74% versus 80% in those randomized to receive GO or no further therapy, respectively. Results were similar in all subgroups. In conclusion, GO therapy postconsolidation as given in this trial was well tolerated, showed a nonsignificant delay in time to relapse, but did not change the rate of relapse or survival (clinicaltrials.gov identifier NCT00476541).Swedish Childhood Cancer Foundation Danish Childhood Cancer Foundation Karen Elise Jensen Foundation Wyet

Fusion of ETV6 with an intronic sequence of the BAZ2A gene in a paediatric pre-B acute lymphoblastic leukaemia with a cryptic chromosome 12 rearrangement.

ETV6 at 12p13 is rearranged in a variety of haematological malignancies and solid tumours, with more than 20 different partners having been reported. These fusions result in either chimeric proteins or activation of the partner gene. However, there are a few examples of abnormalities resulting in truncated and, most likely, unproductive ETV6 proteins, suggesting that haploinsufficiency of ETV6 and/or the partner is leukaemogenic. We present a novel ETV6 rearrangement, identified in a paediatric pre-B acute lymphoblastic leukaemia. Fluorescence in situ hybridisation (FISH) and molecular genetic analyses revealed a fusion of ETV6 and BAZ2A (at 12q13), generated through a cryptic rearrangement between 12p13 and 12q13, consisting of exons 1 and 2 of ETV6 and a sequence from intron 1 of BAZ2A. This transcript is not expected to produce any chimeric protein, but may encode a truncated form of ETV6, containing the first 54 amino acids (aa), followed by 16 aa from the 3' fusion sequence, reminiscent of ETV6 fusions with MDS2, LOC115548, PER1, and STL. The rearrangement might also modify the regulation of BAZ2A by either activating a cryptic promoter or by coming under the control of the ETV6 promoter. The present case emphasises that 'unproductive'ETV6 rearrangements may play an important pathogenetic role in leukaemia

High dose methotrexate treatment in children with acute lymphoblastic leukaemia may be optimised by a weight-based dose calculation.

BACKGROUND: The inter-individual variation in exposure to methotrexate is considerable after intravenous high dose methotrexate (HDMTX) administration and both under- and over exposures may have dire consequences. Thus, optimal dose individualisation is of paramount importance. PROCEDURE: We studied how pharmacokinetic parameters were related to outcome in 340 patients with acute lymphoblastic leukaemia (ALL). A population pharmacokinetic model was developed with data from 1284 HDMTX courses in 304 children evaluating age, height, weight, body surface area (BSA), sex, serum creatinine and serum alanine aminotransferase as potential covariates. RESULT: Body weight improved the population pharmacokinetic model significantly more than any of the other patient characteristics, indicating that body weight may be the better way of dose normalisation. In a logistic regression analysis, higher values of clearance as well as volume of distribution were related to increased relapse risk in the standard (SR) and intermediate risk (IR) groups as well as in the entire cohort. A higher weight was strongly associated with worse outcome in the SR and IR groups, (P = 0.0186 and 0.0121, respectively). CONCLUSIONS: We conclude that dose normalisation of methotrexate according to body weigh may give more predictable pharmacokinetics of methotrexate and may also improve the outcome for children with ALL. Pediatr Blood Cancer © 2011 Wiley-Liss, Inc

B-cell precursor t(8;14)(q11;q32)-positive acute lymphoblastic leukemia in children is strongly associated with Down syndrome or with a concomitant Philadelphia chromosome.

We review the clinical and cytogenetic features of 44 acute lymphoblastic leukemias (ALLs) with t(8;14)(q11;q32), including three from our department and 41 ascertained in the literature, focusing on age and gender distribution, peripheral blood values, immunophenotypic data, survival and additional chromosomal changes. Most patients are children or young adults, with a median age of 10 yr for children and 28 for adults. There is a male preponderance, particularly in patients with Down syndrome (DS) or in children with concomitant t(9;22)(q34;q11). The median blood values are hemoglobin 72 g/L, platelets 17 x 10(9)/L and white blood cell count 9 x 10(9)/L, with hyperleukocytosis >50 x 10(9)/L having been reported in only approximately 10%. All reported cases have had a B-cell precursor immunophenotype, typically characterized by CD10+, CD19+, CD20+/-, CD22+, CD24+, CD34+, CD45dim/-, CD66c+/- and CD123+. At the time of reporting, 75% of the patients have been alive. The t(8;14) is the sole acquired change in 30%. The most common additional aberrations are t(9;22)(q34;q11), der(14)t(8;14), +21, +X and +14, the presence of which does not seem to confer a prognostic impact. A substantial proportion of the patients have DS (27%) or t(9;22) (16%). All patients with both t(8;14) and t(9;22) have been children without DS; the frequency of t(9;22) in that cohort is 30%. As t(9;22), or its molecular genetic correlate, may escape detection by conventional banding analysis we would strongly suggest that this aberration is actively looked for in pediatric ALL with t(8;14)

Platelet surface-bound IgG and platelet-specific IgG in plasma in childhood thrombocytopenia

Quantification of platelet-bound immunoglobulin is widely used in the evaluation of thrombocytopenia. Several methods have been devised among which labelled ligand-binding assays seem to be most appropriate. In series of adult patients such assays have been shown to be superior in separating immune-thrombocytopenia from thrombocytopenia of non-immune causes. We studied 62 children with thrombocytopenia of various causes, using radiolabelled protein A as a ligand to measure platelet-surface bound IgG. The test was highly sensitive (93%) in detecting immune-thrombocytopenia. The specificity, however, was only 57%, which is less than in published studies of adults. In a number of cases presumed to be non-immune-thrombocytopenia, notably a few patients with leukaemia and bone marrow aplasia, we found increased amounts of platelet surface-bound IgG. The significance of this finding is not clear. An indirect assay measuring platelet-specific IgG in plasma was less sensitive (46%) but highly specific for immune-thrombocytopenia (89%). The measurements of platelet-surface-bound IgG and platelet-specific IgG in plasma are of limited diagnostic value in childhood thrombocytopenia but are useful in following the treatment in chronic ITP

Simultaneous determination of folate and methotrexate metabolites in serum by LC-MS/MS during high-dose methotrexate therapy

High-dose methotrexate (HDMTX) is a central component in the treatment of acute lymphoblastic leukemia, osteosarcoma, and some lymphomas and brain tumors. MTX is given at lethal doses and then is followed by rescue treatment with folinic acid (FA). Despite FA rescue, many patients suffer severe toxicity. The pharmacokinetics of FA rescue have not been sufficiently studied. However, optimization of FA rescue could potentially increase anti-tumor effects, whilst decreasing organ toxicity. Here, we describe our efforts to establish and optimize a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the simultaneous determination of five essential components of the folate cycle, as well as MTX and its two metabolites. The method was applied to 6 individual patients receiving HDMTX, with 3 or 4 measurements for each patient. The method allows analysis of samples that were initially frozen. This notion, together with the test results in the 6 pilot patients, shows the feasibility of this method to study MTX and FA pharmacokinetics during HDMTX treatment. The method has the potential to optimize HDMTX and FA rescue treatment in individual patients