DNA methylation and targeted sequencing of methyltransferases family genes in canine acute myeloid leukaemia, modelling human myeloid leukaemia.

Tumours shows aberrant DNA methylation patterns, being hypermethylated or hypomethylated compared with normal tissues. In human acute myeloid leukaemia (hAML) mutations in DNA methyltransferase (DNMT3A) are associated to a more aggressive tumour behaviour. As AML is lethal in dogs, we defined global DNA methylation content, and screened the C-terminal domain of DNMT3 family of genes for sequence variants in 39 canine acute myeloid leukaemia (cAML) cases. A heterogeneous pattern of DNA methylation was found among cAML samples, with subsets of cases being hypermethylated or hypomethylated compared with healthy controls; four recurrent single nucleotide variations (SNVs) were found in DNMT3L gene. Although SNVs were not directly correlated to whole genome DNA methylation levels, all hypomethylated cAML cases were homozygous for the deleterious mutation at p.Arg222Trp. This study contributes to understand genetic modifications of cAML, leading up to studies that will elucidate the role of methylome alterations in the pathogenesis of AML in dogs.

An immediate transcriptional signature associated with response to the histone deacetylase inhibitor Givinostat in T acute lymphoblastic leukemia xenografts.

Despite some success with certain hematological malignancies and in contrast with the strong pro-apoptotic effects measured in vitro, the overall response rate of acute lymphoblastic leukemia (ALL) to histone deacetylase inhibitors (HDACis) is low. With the aim to improve the understanding of how HDACis work in vivo, we investigated the therapeutic efficacy of the clinically approved HDACi Givinostat in a collection of nine pediatric human T-ALL engrafted systemically in NOD/SCID mice. We observed highly heterogeneous antileukemia responses to Givinostat, associated with reduction of the percentage of infiltrating blasts in target organs, induction of apoptosis and differentiation. These effects were not associated with the T-ALL cytogenetic subgroup. Transcriptome analysis disclosed an immediate transcriptional signature enriched in genes involved in cell-cycle regulation and DNA repair, which was validated by quantitative RT-PCR and was associated with in vivo response to this HDACi. Increased phospho-H2AX levels, a marker of DNA damage, were measured in T-ALL cells from Givinostat responders. These results indicate that the induction of the DNA damage response could be an early biomarker of the therapeutic effects of Givinostat in T-ALL models. This information should be considered in the design of future clinical trials with HDACis in acute leukemia.

Minimal residual disease is an important predictive factor of outcome in children with relapsed 'high-risk' acute lymphoblastic leukemia.

The aim of the study was to analyze the impact of minimal residual disease (MRD) after reinduction therapy on the outcome of children with relapsed 'high-risk' acute lymphoblastic leukemia (ALL). Sixty patients with isolated or combined marrow relapse were studied. All patients belonged to the S3 or S4 groups, as defined by the Berlin-Frankfurt-Münster stratification for relapsed ALL. MRD was studied by real-time quantitative PCR after the first, second and third chemotherapy course (time points 1 (TP1), 2 (TP2) and 3 (TP3), respectively). MRD results, not used for treatment refinement, were categorized as negative (NEG MRD), positive not-quantifiable (POS-NQ MRD) when MRD level was below quantitative range (a level <10(-4)) or positive within quantitative range (POS MRD) when MRD level was >or=10(-4). With a median observation time of 15 months, overall 3-year event-free survival (EFS) was 27%. The 3-year EFS was 73, 45 and 19% for patients with NEG-MRD, POS NQ-MRD and POS-MRD at TP1, respectively (P<0.05). The prognostic predictive value of MRD was statistically confirmed in multivariate analysis. MRD quantitation early and efficiently differentiates patients who benefit from conventional treatment, including allogeneic hematopoietic stem cell transplantation, from those needing innovative, experimental therapies.

Acute lymphoblastic leukemia with t(4;11) in children 1 year and older: The 'big sister' of the infant disease?

The t(4;11)-positive acute lymphoblastic leukemia (ALL) is a rare disease in children above the age of 1 year. We studied the clinical and biological characteristics in 32 consecutively diagnosed childhood cases (median age 10.0 years, range 1.0-17.1 years). Immunophenotyping revealed a pro-B and a pre-B stage in 24 and eight cases, respectively. IGH genes were rearranged in 84% of leukemias with a predominance of incomplete DJ(H) joints. Whereas IGK-Kde and TCRD rearrangements were rare, TCRG rearrangements were present in 50% of cases and involved mainly Vgamma11 or Vgamma9 together with a Jgamma1.3./2.3 gene segment, an unusual combination among t(4;11)-negative B-cell precursor ALL. Oligoclonality was found in about 30% as assessed by heterogeneous IGH and TCRG rearrangements. Our data are in line with transformation of a precursor cell at an early stage of B-cell development but retaining the potential to differentiate to the pre-B cell stage in vivo. Although a distinct difference between infant and older childhood cases with t(4;11) became evident, no age-related biological features were found within the childhood age group. In contrast to infants with t(4;11)-positive ALL, childhood cases had a relatively low cumulative incidence of relapse of 25% at 3.5 years with BFM-based high-risk protocols.