Characterisation of cells markers associated with IKZF1plus in BCP-ALL.

The presence of IKZF1 deletions has been associated with an increased relapse rate in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). There is a particular subset of IKZF1del cases called IKZF1plus (defined by the co-occurrence of IKZF1del and deletions in CDKN2A/B, PAX5, or the PAR1 region, in the absence of ERG deletions), which is also associated with worse prognosis, but some recent studies have not found major differences between the IKZF1del and IKZF1plus groups. Therefore, the IKZF1plus group still needs further comprehension and our study aims to characterise the molecular heterogeneity and identify molecular markers exclusively associated with IKZF1plus. Two independent series of cases (TARGET, n = 125 and GenLAb, n = 60) were evaluated by segregating patients into 3 groups: IKZF1plus, IKZF1del, and IKZF1wild. Differential expression analyses showed that the membrane protein-coding genes most associated with the IKZF1plus group were: KCNA5, GREB1, EPOR, SDK1, and PTPRB. Notably, KCNA5 and GREB1 differential expression levels were validated in the GenLAb validation series. Regarding copy number alterations, we observed a high frequency of VPREB1 deletions in the IKZF1plus group, as well as additional exclusive deletions in the CD200 and BTLA genes. Recent research suggests that the importance of the IKZF1plus profile varies depending on the genetic subgroup. In this scenario, we found associations between IKZF1plus and certain genes in BCP-ALL, being KCNA5 and GREB1 the most promising biomarkers for predicting IKZF1plus. A deeper understanding of these genetic profiles will allow a better risk assessment and offer precise rationale for therapeutic strategies in BCP-ALL.

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.

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.

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.

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.

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.

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.

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.

Aberrant Expression of EZH2 in Pediatric Patients with Myelodysplastic Syndrome: A Potential Biomarker of Leukemic Evolution.

Pediatric myelodysplastic syndrome (MDS) is an uncommon disease and little is known about the molecular alterations of its development and evolution to acute myeloid leukemia (AML). The Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 (PCR2). It is a histone methyltransferase, that targets lysine 27 of histone 3. This methylated H3-K27 is usually associated with the silencing of genes that are involved in fundamental cellular processes, such as cell proliferation and differentiation. There are only few studies showing the status of EZH2 expression in patients with MDS and they were performed in adult MDS patients. The aim of this study was to analyze the EZH2 expression in pediatric patients with MDS and its association with karyotypes and evolution to acute myeloid leukemia (AML). We conducted the first study of EZH2 expression in pediatric patients with MDS. Considering the EZH2 expression levels in 42 patients and 17 healthy pediatric donors, it was possible to define three groups of expression in patients: low, intermediate, and high. The intermediate level encompassed patients with normal karyotypes, low level included patients with monosomy 7 and del(7q) and high level included patients with trisomy 8 and del(11q) (p < 0.0001). Comparing the leukemic evolution, the low expression group presented disease evolution in 100% (8/8) of the cases, the intermediate expression group showed disease evolution in 4.34% (1/23) and in the high expression group, 63.63% (7/11) patients showed evolution from MDS to AML (p < 0.0001). It is important to note that low and high EZH2 expression are associated with leukemic evolution, however low expression showed a stronger association with evolution from MDS to AML than the high expression. Our results suggest a scale of measure for EZH2 expression in pediatric MDS, where aberrant EZH2 expression may be a potential biomarker of disease evolution.

Molecular cytogenetic studies characterizing a novel complex karyotype with an uncommon 5q22 deletion in childhood acute myeloid leukemia.

Deletions in the long arm of chromosome 5 or loss of the whole chromosome are rare in childhood Acute Myeloid Leukemia (AML) patients. It is also unknown if the wide variety of breakpoints have diverging implications in the patient's outcome. Despite -5/5q- abnormalities have usually been described as a poor prognostic feature, however, the low frequency of -5/5q- in pediatric AML patients limits a full knowledge about this cytogenetic and clinical category, which is an intriguing factor for further research and new findings. Here, we report an AML child showing an uncommon deletion in 5q associated with 2 new abnormalities involving chromosome 2 within a complex karyotype well-characterized by several molecular cytogenetic approaches. Our work stimulates upcoming studies with more detailed descriptions about 5q abnormalities to better define its role in the stratification risk of such cytogenetic subgroup in childhood AML.

Cytogenetic as an important tool for diagnosis and prognosis for patients with hypocellular primary myelodysplastic syndrome.

We analyzed cytogenetically 105 patients with hypocellular primary MDS and their clinical implications. The main chromosomal abnormalities found were del(5q)/-5, del(6q)/+6, del(7q)/-7, del(11q), and del(17p). Pediatric patients had a higher frequency of abnormal karyotypes compared with adult patients (P < 0,05). From our patients, 18% showed evolution of the disease. The chromosomal abnormalities presented in the diagnosis of patients who evolved to AML included numerical (-7, +8) and structural del(6q), del(7q), i(7q), t(7;9), i(9q), and del(11q) abnormalities and complex karyotypes. Although the frequency of evolution from hypocellular MDS to AML is low, our results suggest that some chromosomal alterations may play a critical role during this process. We applied the IPSS in our patients because this score system has been proved to be useful for predicting evolution of disease. When we considered the patients according to group 1 (intermediate-1) and group 2 (intermediate-2 and high risk), we showed that group 2 had a high association with respect to the frequency of abnormal karyotypes (P < 0,0001), evolution of disease (P < 0,0001), and mortality (P < 0,001). In fact, the cytogenetic analysis for patients with hypocellular primary MDS is an important tool for diagnosis, prognosis, in clinical decision-making and in follow-up.

Secondary abnormalities involving 1q or 13q and poor outcome in high stage Burkitt leukemia/lymphoma cases with 8q24 rearrangement at diagnosis.

Classical Burkitt lymphoma/leukemia (BL/L) presenting L3 morphology is found in 1% of childhood ALL. Recently, it has been described that secondary abnormalities could influence the prognosis of these patients. However, little information is available on these cytogenetic abnormalities and their prognostic importance in BL/L. Here, we report four new childhood BL/L cases associated with duplication within 1q or 13q, which exhibited a very unfavorable therapeutic response. We performed both classical and molecular cytogenetic analysis by multicolor chromosome banding of the secondary abnormalities involving the long arms of chromosome 1 or 13. These patients were previously treated with BFM-90 protocol. All of them died during or after the initial treatment. Here, for the first time, the exact breakpoints of the derivative chromosomes involved were determined at the cytogenetic level as 1q21 and 13q33 each.

Harmonization of light scatter and fluorescence flow cytometry profiles obtained after staining peripheral blood leucocytes for cell surface-only versus intracellular antigens with the Fix & Perm reagent.

Staining for intracellular markers with the Fix & Perm reagent is associated with variations in the scatter properties of leucocytes, limiting automated analysis of flow cytometry (FCM) data. Here, we investigated those variables significantly contributing to changes in the light scatter, autofluorescence, and bcl2 staining characteristics of peripheral blood (PB) leucocytes, after fixation with Fix & Perm. Our major aim was to evaluate a new mathematical approach for automated harmonization of FCM data from datafiles corresponding to aliquots of a sample treated with cell-surface-only versus Fix & Perm intracellular staining techniques. Overall, neither the anticoagulant used nor sample storage for <24 h showed significant impact on the light scatter and fluorescence properties of PB leucocytes; similarly, the duration of the fixation period (once >15 min were used) had a minimum impact on the FCM properties of PB leucocytes. Conversely, changes in cell/protein concentrations and the fixative/sample (vol/vol) ratio had a clear impact on the light scatter features of some populations of leucocytes. Accordingly, lower cell/protein concentrations were associated with lower scatter values, particularly for the neutrophils. Such changes could be partially corrected through the use of higher fixative to sample volume ratios. Despite the variable changes detected between aliquots of the same sample treated with cell surface-only versus intracellular staining procedures, the new mathematical approach here proposed and evaluated for automated harmonization of common parameters in both datafiles, could correct the FCM profiles of leucocytes derived from cells undergoing conventional fixation/permeabilization procedures, and made them indistinguishable from those corresponding to aliquots of the same sample treated with cell-surface-only staining techniques.

Sepsis-Related Mortality of Very Low Birth Weight Brazilian Infants: The Role of Pseudomonas aeruginosa.

The aim of this study is to identify risk factors for sepsis-related mortality in low birth weight (<1500 g) infants. We performed retrospective cohort study to investigate risk factors for sepsis-related mortality in all neonates birth weight <1500 g admitted to Level III neonatal intensive care unit, Brazil, April 2001/September 2004. Of the 203 cases, 71 (35%) had sepsis. Of those, gram-positive was identified in 52/87 blood cultures (59.8%), the most common Coagulase-negative Staphylococcus (31/87; 35.5%). Gram-negative was present in 29 of the 87 positive blood cultures (33.3%), with Pseudomonas aeruginosa (8/87; 9.1%), the most frequent agent. Overall 21 of 71 infants with sepsis (29.6%) died. Risk factors for sepsis-related mortality were gestational age 1000 g), five-minute Apgar

Racemic Etodolac is cytotoxic and cytostatic for B-cell precursor acute lymphoblastic leukemia cells.

Several epidemiological studies have provided evidence that administration of nonsteroidal anti-inflammatory drugs (NSAIDs) could have a prophylactic effect against some cancers such as sporadic colorectal cancer and leukemia. Indeed, various NSAIDs have been shown to induce apoptosis in malignant cells. We evaluated the effect of racemic Etodolac on proliferation and cell survival in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells. Etodolac decreased survival of Nalm-16 and Nalm-6 BCP-ALL cell lines and also decreased cell proliferation in Nalm-16 cell line. Ours findings indicate, for the first time to our knowledge, that Etodolac is cytotoxic and cytostatic for BCP-ALL cells.