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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.)

Cancer cells alter their metabolism by switching from glycolysis to oxidative phosphorylation (OXPHOS), regardless of oxygen availability. Metabolism may be a molecular target in acute myeloid leukemia (AML), where mutations in metabolic genes have been described. This study evaluated glycolysis and OXPHOS as therapeutic targets. The sensitivity to 2-deoxy-D-glucose (2-DG; glycolysis inhibitor) and oligomycin (OXPHOS inhibitor) was tested in six AML cell lines (HEL, HL-60, K-562, KG-1, NB-4, THP-1). These cells were characterized for IDH1/2 exon 4 mutations, reactive oxygen species, and mitochondrial membrane potential. Metabolic activity was assessed by resazurin assay, whereas cell death and cell cycle were assessed by flow cytometry. Glucose uptake and metabolism-related gene expression were analyzed by 18 F-FDG and RT-PCR/qPCR, respectively. No IDH1/2 exon 4 mutations were detected. HEL cells had the highest 18 F-FDG uptake and peroxides/superoxide anion levels, whereas THP-1 showed the lowest. 2-DG reduced metabolic activity in all cell lines with HEL, KG-1, and NB-4 being the most sensitive cells. Oligomycin decreased metabolic activity in a cell line-dependent manner, the THP-1 resistant and HL-60 being the most sensitive. Both inhibitors induced apoptosis and cell cycle arrest in a cell line- and compound-dependent manner. 2-DG decreased 18 F-FDG uptake in HEL, HL-60, KG-1, and NB-4, while oligomycin increased the uptake in K-562. Metabolism gene expression had different responses to treatments. In conclusion, HEL and KG-1 show to be more glycolytic, whereas HL-60 was more OXPHOS dependent. Results suggest that AML cells reprogram their metabolism to overcome OXPHOS inhibition suggesting that glycolysis may be a better therapeutic target.

Splicing of pre-mRNA into functional mRNA, carried out by the spliceosome, represents a crucial step in eukaryotic gene expression. Mutations and other deregulation in some of the spliceosome components have been identified in multiple pathologies, including hematological malignancies. In this context, we evaluated the therapeutic potential of a splicing inhibitor, Pladienolide B (Pla-B), in two erythroleukemia cell lines. HEL and K562 cell lines were incubated with increasing doses of Pla-B in single and daily administration. Cell viability and density were evaluated using trypan blue assay. Flow cytometry was used to evaluate cell death, cell cycle, and caspase activity. NGS analysis was performed to assess the mutational status of 4 splicing-related genes (SF3B1, U2AF1, ZRSR2 and SRSF2). Expression levels of SF3B1 and unspliced DNAJB1 were evaluated by qPCR. Pla-B significantly decreased the viability and proliferation of both cell lines in time, dose, administration schedule, and cell line-dependent manner. HEL cells were more sensible to Pla-B (IC 50  = 1.5 nM) than K562 (IC 50  = 25 nM), with an IC 50 almost 17 times lower. Pla-B induced cell death, mainly by apoptosis, and cell cycle arrest in G 0 /G 1 phase. No mutations were found in any of the analyzed genes, suggesting that the observed cytotoxic effect is independent of the spliceosome mutations. Splicing modulator Pla-B showed high antitumor activity against HEL and K562 cell lines, inducing apoptosis and cell cycle arrest. These data suggest that Pla-B might represent a new therapeutic approach for erythroleukemia.

Although Imatinib and other tyrosine kinase inhibitors (TKIs) have excellent results, the appearance of resistance is a problem in chronic myeloid leukaemia (CML). PI3K/AKT/mTOR pathway is activated by BCR-ABL playing a crucial rule in CML. This study aimed to evaluate the therapeutic potential of Everolimus, in CML models sensitive and resistant to Imatinib. We used one CML cell line sensitive to Imatinib (K562) and two resistant (K562-RC and K56-RD). Cell lines were treated with Everolimus alone and in combination with Imatinib. Cell viability was analysed by resazurin assay. Cell death and cell cycle were analysed by flow cytometry. Additionally, we also studied peripheral blood samples obtained from 52 patients under TKI treatment. Everolimus reduced cell line viability in sensitive (IC 50  = 20 µM) and resistant models (K562-RC, IC 50  = 25 µM; K562-RD, IC 50  = 30 µM). This drug induced cell death by apoptosis and cell cycle arrest in G 0 /G 1 phase. Everolimus also reduced cell viability by increasing apoptosis of haematopoietic stem cells (CD34 + cells) with low cytotoxicity to lymphocytes. Everolimus at 25 µM increased apoptotic cells 18.7% in CD34 + cells and only 8% in lymphocytes. The response to Everolimus was influenced by TKI treatment, with a better response in samples from patients under 2nd and 3rd generation TKI and with less toxicity to lymphocytes. Our results reveal that Everolimus induce cell death in CML cells sensitive and resistant to Imatinib, with low cytotoxicity to normal cells, suggesting that Everolimus could be an alternative targeted therapeutic approach in CML patients, even in cases of Imatinib resistance.

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.)

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.)

Sorafenib is a multi-targeted tyrosine kinase inhibitor, with antiangiogenic and antiproliferative properties, approved for the treatment of advanced hepatocellular carcinoma. It induces a significant increase in the median overall survival, despite a complete response to treatment being rare. We report a clinical case of a 60-year-old male with hepatic cirrhosis, Child-Pugh class A and performance status 0, and advanced hepatocellular carcinoma. The primary tumor measured 17 x 8 cm and had diffuse intrahepatic metastization, extensive lung and left adrenal invasion, as well as thrombosis of inferior vena cava, with projection to the right atrium. This patient showed a rapid and complete response to sorafenib, evaluated by mRECIST (modified Response Evaluation Criteria in Solid Tumors), that remains after three years of treatment.

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.

The imbalance between reactive oxygen species (ROS) production and their elimination by antioxidants leads to oxidative stress. Depending on their concentration, ROS can trigger apoptosis or stimulate cell proliferation. We hypothesized that oxidative stress and mitochondrial dysfunction may participate not only in apoptosis detected in some myelodysplastic syndrome (MDS) patients, but also in increasing proliferation in other patients. We investigated the involvement of oxidative stress and mitochondrial dysfunction in MDS pathogenesis, as well as assessed their diagnostic and prognostic values. Intracellular peroxides, superoxide, superoxide/peroxides ratio, reduced glutathione (GSH), and mitochondrial membrane potential (Δψ(mit)) levels were analyzed in bone marrow cells from 27 MDS patients and 12 controls, by flow cytometry. We observed that all bone marrow cell types from MDS patients had increased intracellular peroxide levels and decreased GSH content, compared with control cells. Moreover, oxidative stress levels were MDS subtype- and risk group-dependent. Low-risk patients had the highest ROS levels, which can be related with their high apoptosis; and intermediate-2-risk patients had high Δψ(mit) that may be associated with their proliferative potential. GSH levels were negatively correlated with transfusion dependency, and peroxide levels were positively correlated with serum ferritin level. GSH content proved to be an accurate parameter to discriminate patients from controls. Finally, patients with high ROS or low GSH levels, as well as high superoxide/peroxides ratio had lower overall survival. Our results suggest that oxidative stress and mitochondrial dysfunction are involved in MDS development, and that oxidative stress parameters may constitute novel diagnosis and/or prognosis biomarkers for MDS.

Background: Farnesyltransferase inhibitors have the ability to interfere with various intracellular pathways, reducing cell survival and proliferation. They have become an attractive tool for cancer therapy, namely acute leukemias. In this work, we have studied the efficacy of α-hydroxyfarnesylphosphonic acid (α-HFPA) in CEM (acute T-cell lymphoblastic leukemia) in culture., Materials and Methods: CEM cells were incubated with α-HFPA at different concentrations; viability and proliferation studies were performed using the trypan blue exclusion assay and cell morphological analysis. Expression of lamin A/C, cyclin D1 and BAD were analyzed by flow cytometry., Results: Our results show that α-HFPA significantly decreases Farnesyltransferase activity, reduces cell proliferation and induces cell death through apoptosis in CEM cells, which is correlated with a reduction of cyclin D1 levels., Conclusion: This study suggests that α-HFPA blocks the cell cycle and induces cell death through apoptosis in CEM cells and may be a therapeutic approach in ALL.

Introduction: Hypovitaminosis D (hypoD) is a vitamin deficiency that has been rising in the developed countries, due not only to inappropriate eating habits, but also because of lower sun exposure and lack of exercise., Objective: To determine the prevalence of vitamin D deficiency and to analyze associated factors, in patients admitted to an Internal medicine Ward., Methods: Cross-sectional study which included 123 hospitalized patients admitted to an Internal Medicine Ward between April and May. Serum levels of vitamin D [25-hydroxyvitamin D, 25(OH)D] were determined and the sample divided into three groups based on 25(OH)D levels: severe hypoD (<10ng/mL), moderate deficiency of 25(OH)D (>10ng/mL and <20ng/mL) and third group with normal levels of 25(OH)D (>20ng/mL). Demographic variables were recorded as were factors potentially related to vitamin D deficiency., Results: In this sample (52.0% women) the average age was 71 ± 17 years, 67.5% of patients had severe hypoD, 25.2% moderate deficiency and 7.3% normal levels of vitamin D. The patients in the group with severe hypoD were older (p=0.027). In the same group there was a higher percentage of patients in the bedridden state (p=0.022), with higher impaired functional capacity (p=0.009) and with chronic renal insufficiency (p=0.011). In multivariate logistic regression analysis, factors associated with an increased likelihood of severe hypoD were: furosemide treatment [OR=3.673 (1.232-10.956) p=0.020] and albumin ≤3.25 [OR=5.617 (2.257-13.981) p<0.001]., Conclusion: The high prevalence of hypoD (67.5%) in this sample expresses the need for systematic evaluation of serum levels of 25(OH)D, in order to initiate early treatment in patients with inadequate levels. Furosemide treatment and hypoalbuminaemia present an increased likelihood of being associated with severe hypoD. Treatment of this hypovitaminosis is warranted not only because of the clinical consequences related to bone loss, but also because of its relationship with hypoalbuminemia which is associated with a poorer prognosis in hospitalized patients.

Churg-Strauss syndrome (CSS) is a systemic necrotizing vasculitis of the small and medium vessels, associated with extravascular eosinophilic granulomas, peripheral eosinophilia and asthma. This is a rare syndrome of unknown etiology, affecting both genders and all age groups. CSS patients usually respond well to steroid treatment, although relapses are common after it ends. Timely diagnosis and treatment generally lead to a good prognosis with a 90% survival rate at one year. A brief review of CSS is presented, with particular attention to diagnosis, therapy and recent developments in this area. The authors then report and discuss the clinical, laboratory and imaging characteristics of four patients admitted to an Internal Medicine Department with this diagnosis. The treatment, response and follow-up of the cases are also described., (Copyright © 2011 Sociedade Portuguesa de Pneumologia. Published by Elsevier España. All rights reserved.)

Introduction: Systemic sclerosis (SSc) is a systemic disease, characterized by fibrosis and vasculopathy, with variable internal organ involvement. Skin is very often involved, namely digital ulcers (DU), seldom treatment resistant, responsible for important functional limitation. The DU can evolve from sclerodactily with superficial ulcers, isquemic lesions, deep necrosis, gangrene, loss of tissue, and consequently, to finger amputation., Methods: The authors describe the case of a 36 year old female patient, with SSc diagnosed 6 years previously, with skin, lung and gut manifestations. The patient showed uncontrolled Raynaud's phenomenon (RF), despite the adequate treatment using nifedidpine and general local warming measures, with progressively worsening DU and isquemia, especially in cold seasons. Bosentan, 62.5 mg twice daily was started, and a significant improvement in the peripheral isquemic lesions was achieved. The ulcers' healing was fast, the patient totally recovered function and regained quality of life, and no further lesions developed., Conclusion: The authors review the RF and DU in SSc, as well as the use of bosentan, an endotheline receptor antagonist, and its indications. Although it is not formally approved, the use of bosentan in SS has shown benefits in reducing the incidence of DU, and despite no influence in the healing process, this drug prevents the development of new lesions.

Initially described 40 years ago (Rosai), the undifferentiated osteoclastic-type giant cell carcinoma (IOGCC) is a variant of ductal adenocarcinoma and accounts for less than 1% of exocrine pancreatic tumors. Its extreme rarity, with consequent existence of few reports and clinical experience, leads to the arousal of doubts with regard to its histogenesis, types of approach and therapeutical attitudes. It is important to note that in Portugal no similar case is registered in medical literature. A 61 year old patient admitted to the Internal Medicine Ward 3 at Coimbra University Hospital presents with a voluminous intra-abdominal mass in the left hypochondrium and microcytic anemia. During the investigation, a pancreatic neoplasm was identified, and the patient was submitted to surgical resection, the anatomo-pathological study of the tumor having revealed IOGCC. The particularities of the case, current available therapeutical options and its evolution are discussed, as well as a revision of the existing literature.