Britt-Marie Frost

Genome-wide signatures of differential DNA methylation in pediatric acute lymphoblastic leukemia

BackgroundAlthough aberrant DNA methylation has been observed previously in acute lymphoblastic leukemia (ALL), the patterns of differential methylation have not been comprehensively determined in all subtypes of ALL on a genome-wide scale. The relationship between DNA methylation, cytogenetic background, drug resistance and relapse in ALL is poorly understood.ResultsWe surveyed the DNA methylation levels of 435,941 CpG sites in samples from 764 children at diagnosis of ALL and from 27 children at relapse. This survey uncovered four characteristic methylation signatures. First, compared with control blood cells, the methylomes of ALL cells shared 9,406 predominantly hypermethylated CpG sites, independent of cytogenetic background. Second, each cytogenetic subtype of ALL displayed a unique set of hyper- and hypomethylated CpG sites. The CpG sites that constituted these two signatures differed in their functional genomic enrichment to regions with marks of active or repressed chromatin. Third, we identified subtype-specific differential methylation in promoter and enhancer regions that were strongly correlated with gene expression. Fourth, a set of 6,612 CpG sites was predominantly hypermethylated in ALL cells at relapse, compared with matched samples at diagnosis. Analysis of relapse-free survival identified CpG sites with subtype-specific differential methylation that divided the patients into different risk groups, depending on their methylation status.ConclusionsOur results suggest an important biological role for DNA methylation in the differences between ALL subtypes and in their clinical outcome after treatment.

Cellular cytotoxic drug sensitivity in children with acute leukemia and Down's syndrome: an explanation to differences in clinical outcome?

Cellular cytotoxic drug sensitivity in children with acute leukemia and Downs syndrome: an explanation to differences in clinical outcome?

Increased in vitro cellular drug resistance is related to poor outcome in high-risk childhood acute lymphoblastic leukaemia

Summary. We determined the in vitro cellular drug resistance in 370 children with newly diagnosed acute lymphoblastic leukaemia (ALL). The resistance to each of 10 drugs was measured by the fluorometric microculture cytotoxicity assay (FMCA) and was related to clinical outcome. The median follow‐up time was 41 months. Risk‐group stratified analyses indicated that in vitro resistance to dexamethasone, doxorubicin and amsacrine were each significantly related to the probability of disease‐free survival. In the high‐risk (HR) group, increased in vitro resistance to dexamethasone (P = 0·014), etoposide (P = 0·025) and doxorubicin (P = 0·05) was associated with a worse clinical outcome. Combining the results for these drugs provided a drug resistance score with an independent prognostic significance superior to that of any other factor studied, with a relative risk of relapse in the most resistant group 9·8 times that in the most sensitive group (P = 0·007). The results in the intermediate‐risk (IR) and standard‐risk (SR) groups were less clear cut. In conclusion, our data indicate that in vitro testing of cellular drug resistance can be used to predict the clinical outcome in HR ALL, while the final evaluation of the results in IR and SR patients must await longer follow‐up.

Vincristine in childhood leukaemia: no pharmacokinetic rationale for dose reduction in adolescents.

Aim: To investigate whether there is any pharmacokinetic rationale for the common practice of administering vincristine to adolescents at relatively lower doses than those to younger children. Methods: A total of 98 children, aged 1.3–17.3 y, with acute lymphoblastic leukaemia (ALL) were studied on day 1 of induction therapy. Plasma samples were drawn before and 10, 30, 360 and 1380 min after injection of vincristine 2.0 mg/m2 (maximum dose 2.0 mg) and analysed by high‐performance liquid chromatography. Results: The median value (and range) for distribution half‐life was 6.4 min (0.8–11.8), elimination half‐life 1014 min (258–2570), volume of distribution 445 L/m2 (137–1241) and total body clearance 362 ml/min/m2 (134–2553). No correlation was found between age and any of these pharmacokinetic parameters. The area under the concentration time curve (AUC) was significantly correlated to age (p= 0.002; ρ– 0.31), as expected from the dosage of vincristine. The lower AUC in children with a body surface area > 1 m2, which is reached at 8–9 y of age, indicates that they received a less intense treatment because of the capping of the vincristine dose at 2.0 mg.

Cellular drug sensitivity in MLL-rearranged childhood acute leukaemia is correlated to partner genes and cell lineage

Rearrangements in the 11q23 region, the site of the mixed lineage leukaemia (MLL) gene, are found in both childhood acute myeloid (AML) and lymphoblastic (ALL) leukaemia. We studied the in vitro drug resistance by the fluorometric microculture cytotoxicity assay (FMCA) in 132 children with AML and 178 children with ALL (aged 0–17 years). In AML, children with t(9;11) (n = 10) were significantly more sensitive to cytarabine (P < 0·001) and doxorubicin (P = 0·005) than non‐11q23 rearranged patients (n = 108). Children with other 11q23 rearrangements (n = 14) differed less from non‐rearranged children. The ‘AML‐profile’ common to all three groups included relative resistance to glucocorticoids and vincristine. In ALL, children with 11q23 rearrangement (n = 22) were significantly more sensitive to cytarabine (P = 0·026) than children without 11q23 rearrangement (n = 156), also after stratification for white blood cell count. In conclusion, the findings indicate that the cellular drug resistance is correlated to both the cell lineage and the type of 11q23 rearrangement. High cellular sensitivity to cytarabine and doxorubicin might explain the excellent treatment results in children with AML and t(9;11). The present study supports the strategy of contemporary protocols to include high‐dose cytarabine in the treatment of 11q23‐positive patients both in AML and ALL.

Vincristine pharmacokinetics is related to clinical outcome in children with standard risk acute lymphoblastic leukemia

Vincristine is a key drug in the treatment of childhood and adult acute lymphoblastic leukemia (ALL), and many other childhood malignancies. Despite decades of wide clinical use, no data on the correlation between vincristine pharmacokinetics and long‐term clinical outcome have been published. We here report clinical data (median follow‐up time 10·5 years, range 7·3–12 years) for 86 children with B‐cell precursor ALL, in whom vincristine kinetics were studied on treatment day 1. The median total plasma clearance was 429 and 331 ml/min per m2 and the area under the plasma concentration‐time curve (AUC) was 4·49 and 5·40 mg/l × min in relapse and non‐relapse patients, respectively (not significant). In standard risk patients, where treatment depends more heavily on vincristine than in other subgroups, the relative risk (RR) of relapse was significantly increased for patients with clearance values above median (RR 5·2; P = 0·036), or AUC values below median (RR 5·8; P = 0·025). Our data suggest a relationship between the antileukemic effect and the systemic exposure of the drug, which warrants further studies.

Translocation t(1;19) is related to low cellular drug resistance in childhood acute lymphoblastic leukaemia.

Translocation t(1;19) is related to low cellular drug resistance in childhood acute lymphoblastic leukaemia

Prognostic implications of mutations in NOTCH1 and FBXW7 in childhood T-all treated according to the NOPHO ALL-1992 and ALL-2000 protocols

In children, T‐cell acute lymphoblastic leukemia (T‐ALL) has inferior prognosis compared with B‐cell precursor ALL. In order to improve survival, individualized treatment strategies and thus risk stratification algorithms are warranted, ideally already at the time of diagnosis.

The Mutational Landscape in Pediatric Acute Lymphoblastic Leukemia Deciphered by Whole Genome Sequencing

Genomic characterization of pediatric acute lymphoblastic leukemia (ALL) has identified distinct patterns of genes and pathways altered in patients with well‐defined genetic aberrations. To extend the spectrum of known somatic variants in ALL, we performed whole genome and transcriptome sequencing of three B‐cell precursor patients, of which one carried the t(12;21)ETV6‐RUNX1 translocation and two lacked a known primary genetic aberration, and one T‐ALL patient. We found that each patient had a unique genome, with a combination of well‐known and previously undetected genomic aberrations. By targeted sequencing in 168 patients, we identified KMT2D and KIF1B as novel putative driver genes. We also identified a putative regulatory non‐coding variant that coincided with overexpression of the growth factor MDK. Our results contribute to an increased understanding of the biological mechanisms that lead to ALL and suggest that regulatory variants may be more important for cancer development than recognized to date. The heterogeneity of the genetic aberrations in ALL renders whole genome sequencing particularly well suited for analysis of somatic variants in both research and diagnostic applications.

Genome-wide analysis of cytogenetic aberrations in ETV6/RUNX1-positive childhood acute lymphoblastic leukaemia.

The chromosomal translocation t(12;21) resulting in the ETV6/RUNX1 fusion gene is the most frequent structural cytogenetic abnormality among patients with childhood acute lymphoblastic leukaemia (ALL). We investigated 62 ETV6/RUNX1‐positive childhood ALL patients by single nucleotide polymorphism array to explore acquired copy number alterations (CNAs) at diagnosis. The mean number of CNAs was 2·82 (range 0–14). Concordance with available G‐band karyotyping and comparative genomic hybridization was 93%. Based on three major protein‐protein complexes disrupted by these CNAs, patients could be categorized into four distinct subgroups, defined by different underlying biological mechanisms relevant to the aetiology of childhood ALL. When recurrent CNAs were evaluated by an oncogenetic tree analysis classifying their sequential order, the most common genetic aberrations (deletions of 6q, 9p, 13q and X, and gains of 10 and 21) seemed independent of each other. Finally, we identified the most common regions with recurrent gains and losses, which comprise microRNA clusters with known oncogenic or tumour‐suppressive roles. The present study sheds further light on the genetic diversity of ETV6/RUNX1‐positive childhood ALL, which may be important for understanding poor responses among this otherwise highly curable subset of ALL and lead to novel targeted treatment strategies.