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Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by clonal proliferation, with increased incidence with advancing age. AML with myelodysplasia-related changes (AML-MRC) represents an AML subtype with a poor prognosis and challenging treatment, particularly in elderly patients. We report the case of a 77-year-old patient diagnosed with high-risk AML-MRC, ineligible for intensive chemotherapy, with frequent need of transfusion of red cell concentrates. The authors present, to the best of their knowledge, the first patient in Portugal with AML-MRC treated with an hypomethylating agent, azacytidine, and a BCL2 inhibitor (venetoclax), and that association was essential in the treatment and overall survival, which was much higher than expected.

Oxidative stress and abnormal DNA methylation have been implicated in cancer, including myelodysplastic syndromes (MDSs). This fact leads us to investigate whether oxidative stress is correlated with localized and global DNA methylations in the peripheral blood of MDS patients. Sixty-six MDS patients and 26 healthy individuals were analyzed. Several oxidative stress and macromolecule damage parameters were analyzed. Localized (gene promotor) and global DNA methylations (5-mC and 5-hmC levels; LINE-1 methylation) were assessed. MDS patients had lower levels of reduced glutathione and total antioxidant status (TAS) and higher levels of peroxides, nitric oxide, peroxides/TAS, and 8-hydroxy-2-deoxyguanosine compared with controls. These patients had higher 5-mC levels and lower 5-hmC/5-mC ratio and LINE-1 methylation and increased methylation frequency of at least one methylated gene. Peroxide levels and peroxide/TAS ratio were higher in patients with methylated genes than those without methylation and negatively correlated with LINE-1 methylation and positively with 5-mC levels. The 5-hmC/5-mC ratio was significantly associated with progression to acute leukemia and peroxide/TAS ratio with overall survival. This study points to a relationship between oxidative stress and DNA methylation, two common pathogenic mechanisms involved in MDS, and suggests the relevance of 5-hmC/5-mC and peroxide/TAS ratios as complementary prognostic biomarkers.

Oxidative stress has been implicated in the development of several types of cancer, including myelodysplastic syndromes (MDS), as well as in the resistance to treatment. In this work, we assessed the potential of oxidative stress parameters to predict the response to erythropoiesis-stimulating agents (ESAs) in lower-risk MDS patients. To this end, we analyzed the systemic levels of reactive species (peroxides and NO), antioxidant defenses (uric acid, vitamin E, vitamin A, GSH, GSSG, TAS, as well as GPX and GR activities], and oxidative damage (8-OH-dG and MDA) in 66 MDS patients, from those 44 have been treated with ESA. We also calculated the peroxides/TAS and NO/TAS ratios and analyzed the gene expression of levels of the redox regulators, NFE2L2 and KEAP1. We found that patients that respond to ESA treatment showed lower levels of plasma peroxides ( p < 0.001), cellular GSH ( p < 0.001), and cellular GR activity ( p = 0.001) when compared to patients who did not respond to ESA treatment. ESA responders also showed lower levels of peroxides/TAS ratio ( p < 0.001) and higher levels of the expression of the NFE2L2 gene ( p = 0.001) than those that did not respond to ESA treatment. The levels of plasmatic peroxides shown to be the most accurate biomarker of ESA response, with good sensitivity (80%) and specificity (100%) and is an independent biomarker associated with therapy response. Overall, the present study demonstrated a correlation between oxidative stress levels and the response to ESA treatment in lower-risk MDS patients, with the plasmatic peroxides levels a good predictive biomarker of drug (ESA) response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gonçalves, Alves, Baldeiras, Jorge, Marques, Paiva, Oliveiros, Cortesão, Nascimento Costa and Sarmento-Ribeiro.)

Background: Despite bone marrow (BM) immunophenotyping by flow cytometry has progressively been recognized as an important tool for the diagnosis of myelodysplastic syndromes (MDS), the sparse knowledge about normal erythroid maturation and the lack of markers for erythroid characterization is a major shortcoming., Methods: Here, we analyzed the expression of CD43 and CD49d, two markers included in the diagnostic panel for B-cell chronic lymphoproliferative disorders (B-CLPD), in the CD34 + compartment of normal BM and along the normal and dysplastic erythroid maturation. For this, 13 normal BM aspirates and 18 BM aspirates from MDS patients were studied by flow cytometry., Results: Normal BM presented a higher expression of CD43 and CD49d among CD34 + erythroid precursors, compared to CD34 + cells committed to the remaining hematopoietic cell lineages. CD43 expression progressively decreased along the normal erythroid maturation, whereas CD49d levels increased from Stage I to Stage II, were maintained in Stages II and III, and then decreased until the last stage of maturation. In MDS, the expression of CD43 and CD49d followed a similar pattern, but with decreased expression levels for both markers, observed in all erythroid maturation stages (P < 0.05)., Conclusions: Our results point to the usefulness of CD43 and CD49d, two markers commonly present in B-CLPD diagnosis panels, in the identification of dysplastic phenotypic features in the erythroid lineage. This allows a feasible and inexpensive way to identify patients who would benefit from a more extensive study to evaluate the presence of MDS, during the processing of suspected B-CLPD samples. © 2019 International Clinical Cytometry Society., (© 2019 International Clinical Cytometry Society.)

Azacitidine (AZA) prolonged overall survival (OS) in the AZA-AML-001 trial. However, few subjects were randomized to AZA or intensive chemotherapy (IC). The Medical Research Council (MRC) and the Leukemia Research Foundation (LRF) developed a score for older AML patients receiving IC or non-intensive regimens, whereas the E-ALMA study validated a score for survival and response in elderly patients receiving AZA in daily practice. Both identified three groups with different risk estimates. This analysis evaluates the efficacy of frontline AZA in older AML patients (N = 710) unfit for IC from different national registries (E-ALMA + series) stratified by the MRC/LRF risk score. Median OS of patients categorized as good, standard and poor-risk groups by the MRC/LRF score was 13.4 (95% CI, 10.8-16), 12.4 (95% CI, 9.9-14.8), and 8.1 months (95% CI, 7-9.1), respectively (p = .0001). In conclusion, this is the largest retrospective cohort of older AML patients treated with AZA.

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) share common features: elevated oxidative stress, DNA repair deficiency, and aberrant DNA methylation. We performed a hospital-based case-control study to evaluate the association in variants of genes involved in oxidative stress, folate metabolism, DNA repair, and DNA methylation with susceptibility and prognosis of these malignancies. To that end, 16 SNPs (one per gene: CAT, CYBA, DNMT1, DNMT3A, DNMT3B, GPX1, KEAP1, MPO, MTRR, NEIL1, NFE2F2, OGG1, SLC19A1, SOD1, SOD2, and XRCC1) were genotyped in 191 patients (101 MDS and 90 AML) and 261 controls. We also measured oxidative stress (reactive oxygen species/total antioxidant status ratio), DNA damage (8-hydroxy-2'-deoxyguanosine), and DNA methylation (5-methylcytosine) in 50 subjects (40 MDS and 10 controls). Results showed that five genes (GPX1, NEIL1, NFE2L2, OGG1, and SOD2) were associated with MDS, two (DNMT3B and SLC19A1) with AML, and two (CYBA and DNMT1) with both diseases. We observed a correlation of CYBA TT, GPX1 TT, and SOD2 CC genotypes with increased oxidative stress levels, as well as NEIL1 TT and OGG1 GG genotypes with higher DNA damage. The 5-methylcytosine levels were negatively associated with DNMT1 CC, DNMT3A CC, and MTRR AA genotypes, and positively with DNMT3B CC genotype. Furthermore, DNMT3A, MTRR, NEIL1, and OGG1 variants modulated AML transformation in MDS patients. Additionally, DNMT3A, OGG1, GPX1, and KEAP1 variants influenced survival of MDS and AML patients. Altogether, data suggest that genetic variability influence predisposition and prognosis of MDS and AML patients, as well AML transformation rate in MDS patients. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Retrospective data was collected from 77 elderly acute myeloid leukemia (AML) patients treated with Azacitidine (AZA) and 50 elderly AML patients treated with intensive chemotherapy (IC) from 4 Portuguese Hospitals. Median OS was 10.6 months in those receiving AZA as 1st line. Response (overall response rate 44%) had a significant impact on overall survival (p=<0.0001). Median OS of the comparator IC cohort was significantly inferior to that observed in the cohort treated with AZA in first line (p=0.0104). These results support the efficacy of AZA in AML in the elderly in any line of treatment.

Background: Aberrant DNA methylation is considered a crucial mechanism in the pathogenesis of monoclonal gammopathies. We aimed to investigate the contribution of hypermethylation of 4 tumor suppressor genes to the multistep process of myelomagenesis., Methods: The methylation status of p15, p16, p53, and DAPK genes was evaluated in bone marrow samples from 94 patients at diagnosis: monoclonal gammopathy of uncertain significance (MGUS) (n = 48), smoldering multiple myeloma (SMM) (n = 8) and symptomatic multiple myeloma (MM) (n = 38), and from 8 healthy controls by methylation-specific polymerase chain reaction analysis., Results: Overall, 63% of patients with MM and 39% of patients with MGUS presented at least 1 hypermethylated gene (P < .05). No aberrant methylation was detected in normal bone marrow. The frequency of methylation for individual genes in patients with MGUS, SMM, and MM was p15, 15%, 50%, 21%; p16, 15%, 13%, 32%; p53, 2%, 12,5%, 5%, and DAPK, 19%, 25%, 39%, respectively (P < .05). No correlation was found between aberrant methylation and immunophenotypic markers, cytogenetic features, progression-free survival, and overall survival in patients with MM., Conclusions: The current study supports a relevant role for p15, p16, and DAPK hypermethylation in the genesis of the plasma cell neoplasm. DAPK hypermethylation also might be an important step in the progression from MGUS to MM., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Oxidative stress and abnormal DNA methylation have been implicated in some types of cancer, namely in myelodysplastic syndromes (MDS). Since both mechanisms are observed in MDS patients, we analyzed the correlation of intracellular levels of peroxides, superoxide anion, and glutathione (GSH), as well as ratios of peroxides/GSH and superoxide/GSH, with the methylation status of P15 and P16 gene promoters in bone marrow leukocytes from MDS patients. Compared to controls, these patients had lower GSH content, higher peroxide levels, peroxides/GSH and superoxide/GSH ratios, as well as higher methylation frequency of P15 and P16 gene promoters. Moreover, patients with methylated P15 gene had higher oxidative stress levels than patients without methylation (peroxides: 460 ± 42 MIF vs 229 ± 25 MIF, p = 0.001; superoxide: 383 ± 48 MIF vs 243 ± 17 MIF, p = 0.022; peroxides/GSH: 2.50 ± 0.08 vs 1.04 ± 0.34, p < 0.001; superoxide/GSH: 1.76 ± 0.21 vs 1.31 ± 0.10, p = 0.007). Patients with methylated P16 and at least one methylated gene had higher peroxide levels as well as peroxides/GSH ratio than patients without methylation. Interestingly, oxidative stress levels allow the discrimination of patients without methylation from ones with methylated P15, methylated P16, or at least one methylated (P15 or P16) promoter. Taken together, these findings support the hypothesis that oxidative stress is correlated with P15 and P16 hypermethylation.

Introduction: This myelodysplastic syndromes are a heterogeneous entity characterized by dysplasia, hypercellular bone marrow, cytopenias and risk of transformation to acute leukaemia. Prognostic factors, such as bone marrow fibrosis, lactate dehydrogenase and 2-microglobulin elevation have been described, but treatment is mainly based in the International Prognostic Scoring System., Material and Methods: Our aim was to analyze serum's erythropoietin at diagnosis in de novo myelodysplastic syndromes patients, through its impact in overall survival and possible implementation as prognostic marker. Clinical and laboratorial data from 102 patients with de novo myelodysplastic syndromes diagnosed between October/2009 and March/2014 were collected. Survival analysis was performed according to serum erythropoietin level stratification, using Kaplan-Meier methodology., Results: Our 102 patients had a median age of 74 years, with a male:female ratio of 0.8. Mean erythropoietin was significantly lower in refractory cytopenia with unilineage dysplasia patients in contrast with the higher values observed in 5q- syndrome (p < 0.05). Eleven patients progressed to acute leukaemia; these have higher mean erythropoietin values (p < 0.05). In addition, elevated serum erythropoietin was associated with lower survival rates (p = 0.0336). Predictive value of serum erythropoietin was maintained after Cox regression adjustment. In multivariate analysis, serum erythropoietin is an independent survival predictor (p < 0.001)., Discussion: Serum erythropoietin is a predictive factor for response to therapy with subcutaneous erythropoietin, and patients with myelodysplastic syndromes with higher values of erythropoietin have poorer response to administration of erythropoietin even at higher doses. Our sample shows that serum erythropoietin also has prognostic value, and in all myelodysplastic syndromes subtypes. Moreover, alone or in combination with other factors or prognostic indices, erythropoietin may enhance the prognostic indices such as the International Prognostic Scoring System, since high levels are associated with progression to acute leukemia and hence lower survival., Conclusion: This study suggests that increased erythropoietin levels at diagnosis can by itself be a poor prognosis factor inmyelodysplastic syndromes patients, with higher values in patients with progression to acute leukaemia and decreased overall survival.

An increasing body of evidence suggests the potential occurrence of antigen encounter by the cell of origin in chronic lymphocytic leukaemia (CLL) and CLL-like monoclonal B-cell lymphocytosis (MBL). However, the scenario in which this event might occur remains unknown. In order to gain insight into this scenario we investigated the molecular, cytogenetic and haematological features of 223 CLL-like (n = 84) and CLL (n = 139) clones with stereotyped (n = 32) versus non-stereotyped (n = 191) immunoglobulin heavy chain variable region (IGHV) amino acid sequences. Overall, stereotyped CLL-like MBL and CLL clones showed a unique IGHV profile, associated with higher IGHV1 and lower IGHV3 gene family usage (P = 0·03), longer IGHV complementary determining region 3 (HCDR3) sequences (P = 0·007) and unmutated IGHV (P < 0·001) versus non-stereotyped clones. Whilst the overall size of the stereotyped B-cell clones in peripheral blood did not appear to be associated with the CLL-related cytogenetic profile of B-cells (P > 0·05), it did show a significant association with the presence of myelodysplastic syndrome (MDS)-associated immunophenotypes on peripheral blood neutrophils and/or monocytes (P = 0·01). Altogether our results point to the potential involvement of different selection forces in the expansion of stereotyped vs. non-stereotyped CLL and CLL-like MBL clones, the former being potentially favoured by an underlying altered haematopoiesis., (© 2014 John Wiley & Sons Ltd.)

Long-term granulocyte-colony stimulating factor treatment has been shown to be safe and effective in severe chronic neutropenia patients. However, data on its use during pregnancy are limited. To address this issue, we analyzed all pregnancies reported to the European branch of the Severe Chronic Neutropenia International Registry since 1994. A total of 38 pregnancies in 21 women with chronic neutropenia (16 pregnancies in 10 women with congenital, 10 in 6 women with cyclic, 12 in 5 women with idiopathic neutropenia) were reported. Granulocyte-colony stimulating factor was administered throughout pregnancy in 16 women and for at least one trimester in a further 5 women. No major differences were seen between treated and untreated women with respect to pregnancy outcome, newborn complications and infections. In addition, we evaluated the genetic transmission of known or suspected genetic defects in 16 mothers having 22 newborns as well as in 8 men fathering 15 children. As a proof of inheritance, neutropenia was passed on to the newborn in 58% from female and in 62% from male patients with ELANE mutations, but also to some newborns from parents with unknown gene mutation. Based on our results, granulocyte-colony stimulating factor therapy has been shown to be safe for mothers throughout pregnancies and for newborns without any signs of teratogenicity. With an increasing number of adult patients, genetic counseling prior to conception and supportive care of mothers during pregnancy are crucial. The acceptance of having affected children may reflect the high quality of life obtained due to this treatment., (Copyright© Ferrata Storti Foundation.)

Chronic antigen-stimulation has been recurrently involved in the earlier stages of monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The expansion of two or more B-cell clones has frequently been reported in individuals with these conditions; potentially, such coexisting clones have a greater probability of interaction with common immunological determinants. Here, we analyzed the B-cell receptor repertoire and molecular profile, as well as the phenotypic, cytogenetic and hematologic features, of 228 chronic lymphocytic leukemia-like and non-chronic lymphocytic leukemia-like clones comparing multiclonal (n=85 clones from 41 cases) versus monoclonal (n=143 clones) monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The B-cell receptor of B-cell clones from multiclonal cases showed a slightly higher degree of HCDR3 homology than B-cell clones from mono clonal cases, in association with unique hematologic (e.g. lower B-lymphocyte counts) and cytogenetic (e.g. lower frequency of cytogenetically altered clones) features usually related to earlier stages of the disease. Moreover, a subgroup of coexisting B-cell clones from individual multiclonal cases which were found to be phylogenetically related showed unique molecular and cytogenetic features: they more frequently shared IGHV3 gene usage, shorter HCDR3 sequences with a greater proportion of IGHV mutations and del(13q14.3), than other unrelated B-cell clones. These results would support the antigen-driven nature of such multiclonal B-cell expansions, with potential involvement of multiple antigens/epitopes.

Central diabetes insipidus, is a syndrome characterized by the excretion of abnormally elevated volumes of diluted urine, due to the diminution of reabsorption of water in the collecting ducts, induced by the diminution of production of antidiuretic hormone. The involvement of the central nervous system in the leukaemia is frequent, but the association leukaemia/diabetes insipidus is rare. We describe a clinical case of a 40 years old female, whose first manifestation of leukaemia was diabetes insipidus; we discuss the difficulties of diagnosis, the particularities of the case and its evolution.