Molecular characterization of V(D)J rearrangements in immature acute leukemias.

Early T-cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL), T-Lymphoid/Myeloid Mixed Phenotype Acute Leukemia (T/M-MPAL), and Acute Myeloid Leukemia with minimal differentiation (AML-M0) are immature acute leukemias (AL) that present overlapping T-cell lymphoid and myeloid features at different degrees, with impact to disease classification. An interesting strategy to assess lymphoid lineage commitment and maturation is the analysis of V(D)J gene segment recombination, which can be applied to investigate leukemic cells in immature AL. Herein, we revisited 19 ETP-ALL, 8 T/M-MPAL, and 12 AML-M0 pediatric patients to characterize V(D)J rearrangement (V(D)J-r) profiles associated with other somatic alterations. V(D)J-r were identified in 74 %, 25 %, and 25 % of ETP-ALL, T/M-MPAL, and AML-M0, respectively. Forty-six percent of ETP-ALL harbored ≥ 3 V(D)J-r, while there was no more than one V(D)J-r per patient in AML-M0 and T/M-MPAL. TCRD was the most rearranged locus in ETPALL, but it was not rearranged in other AL. In ETP-ALL, N/KRAS mutations were associated with absence of V(D)J-r, while NF1 deletion was most frequent in patients with ≥ 3 V(D)J-r. Relapse and death occurred mainly in patients harboring one or no rearranged locus. Molecular characterization of V(D)J-r in our cohort indicates a distinct profile of ETP-ALL, compared to T/M-MPAL and AML-M0. Our findings also suggest that the clinical outcome of ETP-ALL patients may be affected by blast cell maturity, inferred from the number of rearranged TCR loci.

Association between Leukemic Evolution and Uncommon Chromosomal Alterations in Pediatric Myelodysplastic Syndrome.

Background: Pediatric myelodysplastic syndrome (pMDS) is a group of rare clonal neoplasms with a difficult diagnosis and risk of progression to acute myeloid leukemia (AML). The early stratification in risk groups is essential to choose the treatment and indication for allogeneic hematopoietic stem cell transplantation (HSCT). According to the Revised International Prognostic Scoring System, cytogenetic analysis has demonstrated an essential role in diagnosis and prognosis. In pMDS, abnormal karyotypes are present in 30-50% of the cases. Monosomy 7 is the most common chromosomal alteration associated with poor prognosis. However, the rarity of specific cytogenetic alterations makes its prognosis uncertain. Thus, this study aimed to describe uncommon cytogenetic alterations in a cohort of 200 pMDS patients and their association with evolution to AML. Methods: The cytogenetic analysis was performed in 200 pMDS patients by G-banding and fluorescence in situ hybridization between 2000 to 2022. Results: Rare chromosome alterations were observed in 7.5% (15/200) of the cases. These chromosome alterations were divided into four cytogenetic groups: hyperdiploidy, biclonal chromosomal alterations, translocations, and uncommon deletions representing 33.3%, 33.3%, 20%, and 13.3%, respectively. Most of these patients (10/15) were classified with advanced MDS (MDS-EB and MDS/AML) and the initial subtype was present in five patients (RCC). The leukemic evolution was observed in 66.66% (10/15) of the patients. Most patients had poor clinical outcomes and they were indicated for HSCT. Conclusion: The study of uncommon cytogenetic alterations in pMDS is important to improve the prognosis and guide early indication of HSCT.

Phenotypic profiling of CD34+ cells by advanced flow cytometry improves diagnosis of juvenile myelomonocytic leukemia.

Diagnostic criteria for juvenile myelomonocytic leukemia (JMML) are currently well defined, however in some patients diagnosis still remains a challenge. Flow cytometry is a well established tool for diagnosis and follow-up of hematological malignancies, nevertheless it is not routinely used for JMML diagnosis. Herewith, we characterized the CD34+ hematopoietic precursor cells collected from 31 children with JMML using a combination of standardized EuroFlow antibody panels to assess the ability to discriminate JMML cells from normal/reactive bone marrow cell as controls (n=29) or from cells of children with other hematological diseases mimicking JMML (n=9). CD34+ precursors in JMML showed markedly reduced B-cell and erythroid-committed precursors compared to controls, whereas monocytic and CD7+ lymphoid precursors were significantly expanded. Moreover, aberrant immunophenotypes were consistently present in CD34+ precursors in JMML, while they were virtually absent in controls. Multivariate logistic regression analysis showed that combined assessment of the number of CD34+CD7+ lymphoid precursors and CD34+ aberrant precursors or erythroid precursors had a great potential in discriminating JMMLs versus controls. Importantly our scoring model allowed highly efficient discrimination of truly JMML versus patients with JMML-like diseases. In conclusion, we show for the first time that CD34+ precursors from JMML patients display a unique immunophenotypic profile which might contribute to a fast and accurate diagnosis of JMML worldwide by applying an easy to standardize single eight-color antibody combination.

Prognostic Factors in Children and Adolescents with Lymphomas and Vertical Transmission of HIV in Rio de Janeiro, Brazil: A Multicentric Hospital-Based Survival Analysis Study.

Lymphomas related to HIV are generally aggressive and have a poor prognosis, despite the use of combined antiretroviral therapy (cART) and effective chemotherapy treatment. To determine survival and prognostic factors in children and adolescents living with HIV (CLWH) in Rio de Janeiro (RJ), Brazil, who developed lymphomas, we performed a retrospective and observational study of vertically infected CLWH aged from 0 to 20 incomplete years during1995 to 2018 at five reference centers for cancer and HIV/AIDS treatment. Of the 25 lymphomas, 19 were AIDS-defining malignancies (ADM) and 6 were non-AIDS-defining malignancies (NADM). The 5-year overall survival (OS) and 5-year event-free survival (EFS) probabilities were both 32.00% (95% CI = 13.72-50.23%), and the 5-year disease-free survival (DFS) probability was 53.30% (95% CI = 28.02-78.58%). In the multivariate Cox regression analysis, performance status 4 (PS 4) was considered a poor prognostic factor for OS (HR 4.85, 95% CI = 1.81-12.97, p = 0.002) and EFS (HR 4.95, 95% CI = 1.84-13.34, p = 0.002). For the DFS, higher CD4+ T-cell counts were considered a better prognostic factor (HR 0.86, 95% CI = 0.76-0.97, p = 0.017) in the multivariate Cox regression analysis. This study demonstrates, for the first time, survival and prognostic factors for CLWH who developed lymphomas in RJ, Brazil.

Immunophenotypic Analysis of Acute Megakaryoblastic Leukemia: A EuroFlow Study.

Acute megakaryoblastic leukemia (AMKL) is a rare and heterogeneous subtype of acute myeloid leukemia (AML). We evaluated the immunophenotypic profile of 72 AMKL and 114 non-AMKL AML patients using the EuroFlow AML panel. Univariate and multivariate/multidimensional analyses were performed to identify most relevant markers contributing to the diagnosis of AMKL. AMKL patients were subdivided into transient abnormal myelopoiesis (TAM), myeloid leukemia associated with Down syndrome (ML-DS), AML-not otherwise specified with megakaryocytic differentiation (NOS-AMKL), and AMKL-other patients (AML patients with other WHO classification but with flowcytometric features of megakaryocytic differentiation). Flowcytometric analysis showed good discrimination between AMKL and non-AMKL patients based on differential expression of, in particular, CD42a.CD61, CD41, CD42b, HLADR, CD15 and CD13. Combining CD42a.CD61 (positive) and CD13 (negative) resulted in a sensitivity of 71% and a specificity of 99%. Within AMKL patients, TAM and ML-DS patients showed higher frequencies of immature CD34+/CD117+ leukemic cells as compared to NOS-AMKL and AMKL-Other patients. In addition, ML-DS patients showed a significantly higher expression of CD33, CD11b, CD38 and CD7 as compared to the other three subgroups, allowing for good distinction of these patients. Overall, our data show that the EuroFlow AML panel allows for straightforward diagnosis of AMKL and that ML-DS is associated with a unique immunophenotypic profile.

KMT2A-MLLT1 and the Novel SEC16A-KMT2A in a Cryptic 3-Way Translocation t(9;11;19) Present in an Infant With Acute Lymphoblastic Leukemia.

About 25% of the patients with the translocation t(11;19)(q23;p13.3)/KMT2A-MLLT1 present three-way or more complex fusions, associated with a worse prognosis, suggesting that a particular mechanism creates functional KMT2A fusions for this condition. In this work, we show a cryptic three-way translocation t(9;11;19). Interestingly, long-distance inverse polymerase chain reaction sequencing revealed a KMT2A-MLLT1 and the yet unreported out-of-frame SEC16A-KMT2A fusion, associated with low SEC16A expression and KMT2A overexpression, in an infant with B-acute lymphoblastic leukemia presenting a poor prognosis. Our case illustrates the importance of molecular cytogenetic tests in selecting cases for further investigations, which could open perspectives regarding novel therapeutic approaches for poor prognosis childhood leukemias.

Immunophenotypic shifts during minimal residual evaluation in a case of leukemic form of anaplastic large cell lymphoma ALK.

BACKGROUND: This study aims to describe immunophenotypic explorations at diagnosis and follow up of a pediatric patient with leukemic phase of ALK+ anaplastic large cell lymphoma (ALCL) by multiparametric flow cytometry (MFC). CASE: An 8-color MFC combination of antibodies allowed to identify neoplastic cells in concentrations until 0.02% during minimal residual disease (MRD) monitoring. Immunophenotypic shifts occurred in key markers as CD30, CD7, CD2, and CD5, however neoplastic cells were clearly discriminated from normal populations. CONCLUSION: MFC can be a useful tool for ALCL diagnosis and MRD monitoring and may support therapeutic decisions.

IKZF1 deletions associate with CRLF2 overexpression leading to a poor prognosis in B-cell precursor acute lymphoblastic leukaemia.

Cytokine Receptor-Like Factor 2 (CRLF2) overexpression occurs in 5-15% of B-cell precursor acute lymphoblastic leukaemia (B-ALL). In ∼50% of these cases, the mechanisms underlying this dysregulation are unknown. IKAROS Family Zinc Finger 1 (IKZF1) is a possible candidate to play a role in this dysregulation since it binds to the CRLF2 promoter region and suppresses its expression. We hypothesised that IKZF1 loss of function, caused by deletions or its short isoforms expression, could be associated with CRLF2 overexpression in B-ALL. A total of 131 paediatric and adult patients and 7 B-ALL cell lines were analysed to investigate the presence of IKZF1 deletions and its splicing isoforms expression levels, the presence of CRLF2 rearrangements or mutations, CRLF2 expression and JAK2 mutations. Overall survival analyses were performed according to the CRLF2 and IKZF1 subgroups. Our analyses showed that 25.2% of patients exhibited CRLF2 overexpression (CRLF2-high). CRLF2-high was associated with the presence of IKZF1 deletions (IKZF1del, p = 0.001), particularly with those resulting in dominant-negative isoforms (p = 0.006). Moreover, CRLF2 expression was higher in paediatric samples with high loads of the short isoform IK4 (p = 0.011). It was also associated with the occurrence of the IKZF1 plus subgroup (p = 0.004). Furthermore, patients with CRLF2-high/IKZF1del had a poorer prognosis in the RELLA05 protocol (p = 0.067, 36.1 months, 95%CI 0.0-85.9) and adult cohort (p = 0.094, 29.7 months, 95%CI 11.8-47.5). In this study, we show that IKZF1 status is associated with CRLF2-high and dismal outcomes in B-ALL patients regardless of age.

Chronic Granulomatous Disease and Myelodysplastic Syndrome in a Patient with a Novel Mutation in CYBB.

Chronic Granulomatous Disease (CGD) is an inborn error of immunity characterized by impaired phagocyte function, recurrent fungal and bacterial infections and granuloma formation in multiple organs. Pediatric myelodysplastic Syndrome (MDS) is a rare hematological stem cell disease that leads to an ineffective hematopoiesis with variable risk of evolution to acute leukemias. Both disorders are rare and have distinct pathophysiologic mechanisms, with no known association. A 7-month-old boy presenting with recurrent infections and anemia at age 2 months underwent immunological, hematological and genetic investigation that culminated in the diagnosis of both CGD and MDS. Next generation sequencing was performed and identified a silent variant predicted as of Uncertain Significance, located in the splicing site at the end of exon 5 in CYBB. CYBB variants account for at least two thirds of CGD cases, but no previous descriptions of this variant were found in ClinVar or The Human Gene Mutation Database (HGMD) databases. We were able to demonstrate an exon 5 skipping on the proband's cDNA, which strongly suggests the disruption of the NADPH oxidase complex, abrogating the formation of reactive oxygen species from neutrophils. Moreover, erythroid cell lineage could be also affected by NADPH oxidase complex damages. Further investigation is needed to evaluate the potential effect of CYBB gene alterations in hematopoiesis, as well as in MDS and CGD association.

Updating recommendations of the Brazilian Group of Flow Cytometry (GBCFLUX) for diagnosis of acute leukemias using four-color flow cytometry panels.

INTRODUCTION: Flow cytometry has become an increasingly important tool in the clinical laboratory for the diagnosis and monitoring of many hematopoietic neoplasms. This method is ideal for immunophenotypic identification of cellular subpopulations in complex samples, such as bone marrow and peripheral blood. In general, 4-color panels appear to be adequate, depending on the assay. In acute leukemias (ALs), it is necessary identify and characterize the population of abnormal cells in order to recognize the compromised lineage and classify leukemia according to the WHO criteria. Although the use of eight- to ten-color immunophenotyping panels is wellestablished, many laboratories do not have access to this technology. OBJECTIVE AND METHOD: In 2015, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) proposed antibody panels designed to allow the precise diagnosis and characterization of AL within available resources. As many Brazilian flow cytometry laboratories use four-color immunophenotyping, the GBCFLUX has updated that document, according to current leukemia knowledge and after a forum of discussion and validation of antibody panels. RESULTS: Recommendations for morphological analysis of bone marrow smears and performing screening panel for lineage (s) identification of AL were maintained from the previous publication. The lineage-oriented proposed panels for B and T cell acute lymphoblastic leukemia (ALL) and for acute myeloid leukemia (AML) were constructed for an appropriate leukemia classification. CONCLUSION: Three levels of recommendations (i.e., mandatory, recommended, and optional) were established to enable an accurate diagnosis with some flexibility, considering local laboratory resources and patient-specific needs.

B-Cell Regeneration Profile and Minimal Residual Disease Status in Bone Marrow of Treated Multiple Myeloma Patients.

B-cell regeneration during therapy has been considered as a strong prognostic factor in multiple myeloma (MM). However, the effects of therapy and hemodilution in bone marrow (BM) B-cell recovery have not been systematically evaluated during follow-up. MM (n = 177) and adult (≥50y) healthy donor (HD; n = 14) BM samples were studied by next-generation flow (NGF) to simultaneously assess measurable residual disease (MRD) and residual normal B-cell populations. BM hemodilution was detected in 41 out of 177 (23%) patient samples, leading to lower total B-cell, B-cell precursor (BCP) and normal plasma cell (nPC) counts. Among MM BM, decreased percentages (vs. HD) of BCP, transitional/naïve B-cell (TBC/NBC) and nPC populations were observed at diagnosis. BM BCP increased after induction therapy, whereas TBC/NBC counts remained abnormally low. At day+100 postautologous stem cell transplantation, a greater increase in BCP with recovered TBC/NBC cell numbers but persistently low memory B-cell and nPC counts were found. At the end of therapy, complete response (CR) BM samples showed higher CD19- nPC counts vs. non-CR specimens. MRD positivity was associated with higher BCP and nPC percentages. Hemodilution showed a negative impact on BM B-cell distribution. Different BM B-cell regeneration profiles are present in MM at diagnosis and after therapy with no significant association with patient outcome.

Minimal residual disease assessment in acute lymphoblastic leukemia by 4-color flow cytometry: Recommendations from the MRD Working Group of the Brazilian Society of Bone Marrow Transplantation.

INTRODUCTION: The minimal residual disease (MRD) status plays a crucial role in the treatment of acute lymphoblastic leukemia (ALL) and is currently used in most therapeutic protocols to guide the appropriate therapeutic decision. Therefore, it is imperative that laboratories offer accurate and reliable results through well standardized technical processes by establishing rigorous operating procedures. METHOD: Our goal is to propose a monoclonal antibody (MoAb) panel for MRD detection in ALL and provide recommendations intended for flow cytometry laboratories that work on 4-color flow cytometry platforms. RESULTS AND CONCLUSION: The document includes pre-analytical and analytical procedures, quality control assurance, technical procedures, as well as the information that needs to be included in the reports for clinicians.

Bradyrhizobium campsiandrae sp. nov., a nitrogen-fixing bacterial strain isolated from a native leguminous tree from the Amazon adapted to flooded conditions.

The nitrogen-fixing bacterial strain UFLA 01-1174T was isolated from nodules of Campsiandra laurilifolia Benth. originating from the Amazon region, Brazil. Its taxonomic position was defined using a polyphasic approach. Analysis of the 16S rRNA gene placed the strain in the Bradyrhizobium genus, the closest species being B. guangdongense CCBAU 51649T and B. guangzhouense CCBAU 51670T, both with 99.8% similarity. Multilocus sequence analysis (MLSA) of recA, gyrB, glnII, rpoB, atpD, and dnaK indicated that UFLA 01-1174T is a new species, most closely related to B. stylosanthis BR 446T (94.4%) and B. manausense BR 3351T (93.7%). Average nucleotide identity (ANI) differentiated UFLA 01-1174T from the closest species with values lower than 90%. The G + C content in the DNA of UFLA 01-1174T is 63.6 mol%. Based on this data, we conclude that the strain represents a new species. The name proposed is Bradyrhizobium campsiandrae, with UFLA 01-1174T (= INPA 394BT = LMG 10099T) as type strain.

Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB, n = 41) and bone marrow (BM; n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r2 > 0.95 for all cell types in PB and r2 > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a "sample-in and result-out" approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal procedures.

Risk factors for the development of hospital-acquired pediatric venous thromboembolism-Dealing with potentially causal and confounding risk factors using a directed acyclic graph (DAG) analysis.

INTRODUCTION: Hospital-acquired venous thromboembolism (HA-VTE) in children comprises multiple risk factors that should not be evaluated separately due to collinearity and multiple cause and effect relationships. This is one of the first case-control study of pediatric HA-VTE risk factors using a Directed Acyclic Graph (DAG) analysis. MATERIAL AND METHODS: Retrospective, case-control study with 22 cases of objectively confirmed HA-VTE and 76 controls matched by age, sex, unit of admission, and period of hospitalization. Descriptive statistics were used to define distributions of continuous variables, frequencies, and proportions of categorical variables, comparing cases and controls. Due to many potential risk factors of HA-VTE, a directed acyclic graph (DAG) model was created to identify confounding, reduce bias, and increase precision on the analysis. The final model consisted of a DAG-informed conditional logistic regression. RESULTS: In the initial conventional univariable model, the following variables were selected as potential risk factors for HA-VTE: length of stay (LOS, days), immobility, ICU admission in the last 30 days, LOS in ICU, infection, central venous catheter (CVC), number of CVCs placed, L-asparaginase, heart failure, liver failure, and nephrotic syndrome. The final model using the set of variables selected by DAG analysis revealed LOS (OR = 1.106, 95%CI = 1.021-1.198, p = 0.013), L-asparaginase (OR = 26.463, 95%CI = 1.609-435.342, p = 0.022), and nephrotic syndrome (OR = 29.127, 95%CI = 1.044-812.508, p = 0.004) as independent risk factors for HA-VTE. CONCLUSION: The DAG-based approach was useful to clarify the influence of confounders and multiple causalities of HA-VTE. Interestingly, CVC placement-a known thrombotic risk factor highlighted in several studies-was considered a confounder, while LOS, L-asparaginase use and nephrotic syndrome were confirmed as risk factors to HA-VTE. Large confidence intervals are related to the sample size; however, the results were significant.

Efficient Nitrogen-Fixing Bacteria Isolated from Soybean Nodules in the Semi-arid Region of Northeast Brazil are Classified as Bradyrhizobium brasilense (Symbiovar Sojae).

Soybean (Glycine max L.) is an important legume that greatly benefits from inoculation with nitrogen-fixing bacteria. In a previous study, five efficient nitrogen-fixing bacterial strains, isolated from nodules of soybean inoculated with soil from semi-arid region, Northeast Brazil, were identified as a new group within the genus Bradyrhizobium. The taxonomic status of these strains was evaluated in this study. The phylogenetic analysis of the 16S rRNA gene showed the high similarity of the five strains to Bradyrhizobium brasilense UFLA03-321T (100%), B. pachyrhizi PAC48T (100%), B. ripae WR4T (100%), B. elkanii USDA 76T (99.91%), and B. macuxiense BR 10303T (99.91%). However, multilocus sequence analysis of the housekeeping genes atpD, dnaK, gyrB, recA, and rpoB, average nucleotide identity, and digital DNA-DNA hybridization analyses supported the classification of the group as B. brasilense. Some phenotypic characteristics allowed differentiating the five strains and the type strain of B. brasilense from the two neighboring species (B. pachyrhizi PAC48T and B. elkanii USDA 76T). The nodC and nifH genes' analyses showed that these strains belong to symbiovar sojae, together with B. elkanii (USDA 76T) and B. ferriligni (CCBAU 51502T). The present results support the classification of these five strains as Bradyrhizobium brasilense (symbiovar sojae).

Expression Profiles of DNA Methylation and Demethylation Machinery Components in Pediatric Myelodysplastic Syndrome: Clinical Implications.

PURPOSE: The aim of this study was to analyse the expression profiles of DNMT1, DNMT3A, DNMT3B (components of DNA methylation machinery), TET2 and APOBEC3B (components of DNA demethylation machinery) in pediatric MDS patients and investigate their associations with MDS subtypes, cytogenetics, evolution to acute myeloid leukemia (AML) and p15INK4B methylation level. PATIENTS AND METHODS: The expressions of DNMT1, DNMT3A, DNMT3B, TET2, and APOBEC3B were evaluated in 39 pediatric MDS patients by real-time quantitative PCR (qPCR). The quantification of p15INK4B methylation levels (MtL) was performed in 20 pediatric MDS patients by pyrosequencing. Mann-Whitney test was used to evaluate possible differences between the expression levels of selected in patients and donors, according to MDS subtypes, karyotypes, evolution to AML and p15INK4B MtL. The correlations between the expression levels of the different genes were assessed by Spearman rank correlation coefficient. RESULTS: We found that DNMTs expression levels were higher in pediatric MDS compared to donors [DNMT1 (p<0.03), DNMT3A (p<0.03), DNMT3B (p<0.02)]. TET2 and APOBEC3B expression levels did not show a statistically significant difference between pediatric patients and donors. Considering MDS subtypes, patients at initial stage presented DNMT1 overexpression (p<0.01), while DNMT3A (p<0.02) and DNMT3B (p<0.007) were overexpressed in advanced subtypes. TET2 and APOBEC3B expression did not differ in MDS subtypes. DNMT1 (p<0.03), DNMT3B (p<0.03), and APOBEC3B (p<0.04) expression was higher in patients with normal karyotypes, while patients with abnormal karyotypes showed higher DNMT3A expression (p<0.03). Karyotypes had no association with TET2 expression. DNMTs overexpression was observed in patients who showed disease evolution. A positive correlation was found between DNMTs expression and between APOBEC3B and DNMT3A/DNMT3B. However, TET2 expression was not correlated with DNMTs or APOBEC3B. p15INK4B MtL was higher in pediatric MDS patients compared with donors (p<0.03) and its hypermethylation was associated with increased DNMT1 expression (p<0.009). CONCLUSION: Our results suggest that the overexpression of DNMTs and an imbalance between the expressions of the DNA methylation/demethylation machinery components play an important role in MDS development and evolution to AML. These results have clinical implications indicating the importance of DNMTs inhibitors for preventing or delaying the progression to leukemia in pediatric MDS patients.