Subjects with chronic lymphocytic leukaemia-like B-cell clones with stereotyped B-cell receptors frequently show MDS-associated phenotypes on myeloid cells

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 0·05), 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.Funded by: Red Temática de Investigación Cooperativa en Cáncer (RTICC) of Instituto de Salud Carlos III – FONDOS FEDER. Grant Numbers: RD06/0020/0035, RD12/0036/0048, FIS PI06/0824-FEDER, PS09/02430-FEDER; FIS PI12/00905-FEDER Fondo de Investigación Sanitaria of Instituto de Salud Carlos III. Grant Numbers: GRS206/A/08, SAN/1778/2009; Gerencia Regional de Salud, Consejería de Educación and Consejería de Sanidad of Castilla y León. Grant Numbers: FS/1-2010, FS/19-2013; Fundación Memoria D. Samuel Solórzano, Universidad de Salamanca; Fundação para a Ciência e Tecnologia of Portugal. Grant Number: SFRH/BD/31609/2006; Fundación Científica de la Asociación Española contra el Cáncer. Grant Number: AECC-2008.Peer Reviewe

Molecular and cytogenetic characterization of expanded B-cell clones from multiclonal versus monoclonal B-cell chronic lymphoproliferative disorders

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

Travessias e travessuras nos estudos da criança: atas do III Simpósio Luso-Brasileiro em Estudos da Criança

Este livro de Atas contém as conferências e comunicações aceites para publicação do III Simpósio Luso-Brasileiro em Estudos da Crianças, que se realizou no Porto, em 2016. O Simpósio corresponde a uma iniciativa regular da comunidade científica luso-brasileira e compreende estudos neste campo inter/multidisciplinar oriundos de várias universidades portuguesas, brasileiras e ainda de países africanos de expressão portuguesa.Apoio financeiro do CIEC (Centro de Investigação em Estudos da Criança, IE, UMinho; UI 317 da FCT, Portugal) através do Projeto Estratégico UID/CED/00317/2013, financiado através dos Fundos Nacionais da FCT (Fundação para a Ciência e a Tecnologia), cofinanciado pelo Fundo Europeu de Desenvolvimento Regional (FEDER) através do COMPETE 2020 – Programa Operacional Competitividade e Internacionalização (POCI) com a referência POCI-01-0145-FEDER-00756

Caracterização Clínica e Molecular da Síndrome Mielodisplásica - Implicações no Prognóstico e Terapêutica

Tese de doutoramento em Ciências da Saúde, no ramo de Medicina, na especialidade de Medicina Interna, apresentada à Faculdade de Medicina da Universidade de CoimbraA Síndrome Mielodisplásica (SMD) é um grupo heterogéneo de doenças hematológicas caracterizado por citopenia(s) periférica(s) com medula hipercelular, hematopoiese ineficaz devido a aumento da apoptose e proliferação anormal de blastos. Esta patologia está associada a elevado risco de progressão para leucemia aguda com sobrevivência global baixa e resistência às terapêuticas convencionais. A etiologia e patogénese da SMD permanecem pouco esclarecidas. Para além da falência medular e das citopenias periféricas, comuns nas diversas formas de Síndrome Mielodisplásica, a proliferação clonal de progenitores hematopoiéticos associada a mutações genéticas e/ou epigenéticas hereditárias ou adquiridas pode também estar presente. Apesar de múltiplas tentativas para explicar os mecanismos moleculares desta entidade, pouco se sabe sobre a patogénese do passo promotor ou do estádio inicial. As alterações epigenéticas têm sido reconhecidas na última década como factores importantes no desenvolvimento do fenótipo maligno. Os padrões aberrantes de metilação são um mecanismo comum nas neoplasias humanas, especialmente do sistema hematopoiético. Podem estar envolvidos vários genes, dentro dos quais o p15, um gene frequentemente inactivado na SMD por metilação aberrante das ilhas 5’CpG. A inactivação deste gene tem vindo a ser associada ao risco de evolução da doença para leucemia mieloblástica aguda, conferindo mau prognóstico. Outros genes, como o p16, p53, DPAK, MGMT e receptores do TRAIL, têm sido menos estudados em séries de doentes com Síndrome Mielodisplásica. Os polimorfismos funcionais em genes chave, nomeadamente, da enzima metilenotetrahidrofolatoreductase (MTHFR), também podem influenciar a metilação do ADN. São conhecidos dois polimorfismos no gene da MTHFR, C677T e A1298C. Até à data, a Leucemia Mieloblástica Aguda (LMA) e a Síndrome Mielodisplásica constituem desafios terapêuticos porque o único tratamento curativo é o transplante de células pluripotenciais hematopoiéticas. No entanto, a maioria destes doentes apresenta uma média de idades superior a 70 anos, estando mais susceptível aos efeitos colaterais da quimioterapia. Estas dificuldades podem ser ultrapassadas por terapêuticas dirigidas a alvos moleculares que passam pela modulação dos mecanismos epigenéticos envolvidos na patogénese destas neoplasias. Os objectivos deste trabalho foram avaliar as alterações moleculares envolvidas na Síndrome Mielodisplásica, nomeadamente a relevância clinica da célula estaminal leucémica, das alterações epigenéticas e da apopotose na patogénese dos vários subtipos da Organização Mundial de Saúde (OMS), na evolução para Leucemia Aguda, sobrevivência e grupos de risco prognóstico. Além disso, pretendeu-se analisar o potencial terapêutico de fármacos dirigidos a alvos moleculares, como um inibidor da farnesiltransferase (α-HFPA), um inibidor do proteasoma (MG262), um inibidor tirosina cinase (Imatinib) e moduladores epigenéticos (Tricostatina A e Decitabina), identificando a associação com menor dose e maior eficácia terapêutica. Para o efeito estudámos o perfil de metilação de genes reguladores do ciclo celular, p15, p16 e p53, genes moduladores da apoptose, DAPK e receptores do TRAIL, e genes envolvidos na reparação do ADN, como o MGMT, em células da medula óssea de 102 doentes com SMD de novo, utilizando a Polymerase Chain Reaction (PCR) específica de metilação (MS-PCR). Os polimorfismos da MTHFR (C677T e A1298C) foram analisados por PCR Restriction Fragment Length Polymorphism (RFLP). A identificação e caracterização da célula estaminal hematopoiética (HSC) e estaminal leucémica (LSC) foi efectuada por citometria de fluxo recorrendo às marcações CD34/CD117/CD123/IL-6/TNF-α. A avaliação das proteínas reguladoras da apoptose (via membranar e mitocondial), em particular as proteínas da família BCL-2, BCL-2 e BAX, o TRAIL e seus receptores, o FAS-L, a IAP survivina, a caspase 3, o citocromo C e a p53, foi também realizada por citometria de fluxo utilizando anticorpos monoclonais marcados com sondas fluorescentes. O potencial terapêutico de fármacos em monoterapia e em associação foi avaliado em uma linha celular de SMD/LMA em cultura, as células F-36P, através de ensaios de proliferação celular recorrendo ao teste de azul de tripano. O tipo de morte celular foi analisado por citometria de fluxo, através da dupla marcação com anexina V e iodeto de propídeo, e por microscopia óptica, após coloração das células com May-Grümwald-Giemsa. Os doentes têm idade mediana de 74 anos (22-89), com uma razão Masculino/Feminino de 0,8. Os subtipos de SMD segundo a Organização Mundial de Saúde são: Citopenia Refractária com displasia multilinha (CRDM) (n=52), Citopenia Refractária com displasia unilinha (CRDU) (n=12), Anemia Refractária com Excesso de Blastos (AREB)-1 (n=8), AREB-2 (n=8), Anemia Refractária com sideroblastos em anel (n=6), Síndrome 5q- (n=4) e Leucemia Mielomonocítica Crónica (LMMC) (n=12) com o seguinte International Prognostic Scoring System (IPSS): baixo (n=37), intermédio-1 (n=39), intermédio-2 (n=10) e alto (n=1). Onze dos 102 doentes evoluíram para LMA. Evidencia-se um predomínio de subtipos menos agressivos, o que se reflecte em índices de prognóstico de risco baixo e intermédio. A análise de parâmetros bioquímicos permitiu identificar a eritropoietina como factor de mau prognóstico, quando elevada, estando associada aos doentes que evoluíram para LMA e a taxas de sobrevivência inferiores. Além disso, os níveis de plaquetas (média mais baixa), a maior percentagem de blastos (média mais elevada) e de células estaminais mielóides com expressão de IL-6 (CD34+/CD117+/IL-6+) e os índices de prognóstico (riscos intermédio e alto) são entre todos os parâmetros avaliados os que podem influenciar a evolução para LMA (p<0,05). Destas variáveis estudadas, somente a LDH e a eritropoietina relevaram impacto na sobrevivência, quando os seus níveis séricos estão elevados. Neste sentido, elaborámos uma proposta de um novo índice de progressão, que denominámos Índice de Progressão para Leucemia Aguda (IPLA). Este índice é caracterizado por uma pontuação que corresponde à soma dos parâmetros: hematológicos e bioquímicos, célula estaminal, metilação e apoptose, quando alterados. Com este tipo de análise verificámos que os doentes com os subtipos da OMS que apresentavam as pontuações mais elevadas foram os que evoluíram para LMA. Desta forma, este novo índice de prognóstico, poderá constituir uma nova “ferramenta” para avaliar o risco de progressão para LMA. A análise da célula estaminal hematopoiética permitiu a identificação de dois grupos de células, um com características fenotípicas “normais”, as HSC, e outro com características neoplásicas, as LSC, as quais expressam diferencialmente IL-6 e TNF-α. No entanto, os resultados são mais evidentes nos doentes do subtipo AREB-2 (a IL-6 predomina na HSC e o TNF-α na LSC). Estes resultados sugerem a relevância destes dois marcadores na distinção destes dois grupos de células estaminais. O estudo da apoptose mostrou um aumento tendencial de moléculas pró-apoptóticas nos subtipos sem excesso de blastos, embora apenas a caspase 3 tenha significado estatístico. Estes resultados corroboram o envolvimento da apoptose na SMD, em particular em doentes de baixo risco, podendo a caspase 3 vir a constituir um novo marcador de diagnóstico e prognóstico em doentes com SMD. Os resultados do perfil de metilação mostram que mais de 50% dos doentes apresentam dois ou mais genes metilados e que, somente 14,7% da população estudada não apresenta metilação de nenhum gene. Como a metilação é um evento mais frequente nos subtipos iniciais da SMD, nesta população, sugere a interferência da metilação numa fase inicial da doença. Os genes mais frequentemente encontrados metilados são os genes p15 e DAPK, em 56% e 54% dos doentes, respectivamente. O genótipo AC do polimorfismo A1298C da MTHFR mostrou associação com a SMD, mas sem relação estatística com os doseamentos séricos do folato e da vitamina B12 ou com o status de metilação dos genes estudados. Os estudos efectuados na linha celular de SMD permitiram a obtenção de dados experimentais relativamente ao potencial terapêutico de novos fármacos em SMD, nomeadamente das associações do α-HFPA com MG262 e/ou Imatinib e da TSA A com a DEC, mostrando que estas combinações induzem diminuição da proliferação das células F-36P de modo dependente da concentração, do tempo de exposição, do modo e esquema de administração dos fármacos, induzindo morte celular preferencialmente por apoptose. Em conclusão, este estudo sugere um papel da metilação na SMD, sobretudo nos grupos de baixo risco, o que poderá alterar a actual abordagem terapêutica. Revelou ainda uma associação entre o genótipo AC do polimorfismo A1298C da MTHFR com a SMD. Permitiu ainda identificar factores de mau prognóstico, como a eritropoietina e a LDH, e um novo índice de risco de progressão para LMA. Além disso, os novos fármacos testados na linha celular poderão constituir uma nova abordagem terapêutica na SMD, em monoterapia ou em esquemas terapêuticos combinados, o que permitirá a diminuição da toxicidade secundária, melhorando a qualidade de vida e a sobrevivência destes doentes.Myelodysplastic syndromes (MDS) are a heterogeneous group of hematologic disorders characterized by peripheral blood cytopenias with a hypercellular marrow, ineffective hematopoiesis due to increased apoptosis, and an abnormal proliferation of blast cells. These disorders associate with an increased risk of progression to secondary acute leukemia, with a markedly decreased overall survival and resistance to conventional antileukemic drugs. The etiopathogenesis of MDS remains unclear. In addition to the features of bone marrow failure and peripheral cytopenias that are common in MDS, a clonal proliferation of hematopoietic progenitor cells is characteristic of the disorder, resulting from genetic mutations and/or hereditary or acquired epigenetic changes. Despite ongoing attempts at clarifying the molecular mechanisms of this pathologic entity, data about the pathogenesis of the promoting step and of the initial stages of the disease remain scarce. Over the last decade, epigenetic changes have emerged as an important factor in the development of the malignant phenotype. Aberrant patterns of methylation are a common pathogenic mechanism in human neoplastic disorders, especially within the hematopoietic system. Several genes can be involved, including p15, a gene that is frequently inactivated in MDS through the aberrant methylation of 5’CpG islands. The inactivation of this gene associates with an increased risk of progression to acute myeloid leukemia (AML), and with a worse prognosis. Other genes, including p16, p53, DPAK, MGMT and TRAIL receptors, have been less extensively studied in vivo. Functional polymorphisms in key genes, such as the enzyme methylenetetrahydrofolate reductase (MTHFR), can affect DNA methylation. Two such polymorphisms have been described in the MTHFR gene: C677T and A1298C. AML and MDS are still major therapeutic challenges, with the only curative treatment being the transplantation of hematopoietic pluripotent cells. However, affected patients have an average age over 70 years old, which renders them more susceptible to the adverse effects of chemotherapy. These limitations can be overcome by therapies directed at molecular targets, including those responsible for the modulation of the epigenetic mechanisms involved in the pathogenesis of these neoplasms. This study aims to evaluate the molecular changes involved in MDS, especially the clinical relevance of the leukemic stem cell phenotype, of epigenetic changes, and of apoptosis, in the pathogenesis of the different World Health Organization (WHO) MDS subtypes, in the evolution to acute leukemia, and in survival rates and prognostic risk groups. Additionally, the therapeutic potential of drugs directed at specific molecular targets - such as a farnesyltransferase inhibitor (α-HFPA), a proteasome inhibitor (MG262), a tyrosine kinase inhibitor (imatinib) and two epigenetic modulators (trichostatin A and decitabine) – was studied, and the association using the lowest dose with the highest efficacy was identified. The methylation profile of the cell cycle regulator genes p15, p16 and p53, the apoptosis modulator genes DAPK and TRAIL receptors, and the DNA repair gene MGMT, was characterized in bone marrow cells from 102 patients with de novo MDS, through methylation-specific polymerase chain reaction (MS-PCR). The C677T and A1298C polymorphisms of the MTHFR gene were identified through PCR restriction fragment length polymorphism (RFLP). The identification and characterisation of the hematopoietic stem cell (HSC) and the leukemic stem cell (LSC) was achieved through the flow cytometric assessment of CD34, CD117, CD123, IL-6 and TNF-α. The expression of apoptosis regulator proteins from the mitochondrial pathway and the cell membrane pathway, such as the BCL-2 family proteins BCL-2 and BAX, TRAIL and its receptors, FAS-ligand, IAP survivin, caspase 3, citochrome C and p53 was also analysed through flow cytometry. The therapeutic potential of the cited drugs, both in monotherapy and in association, was determined in an MDS/AML cell line (F-36P), through cell proliferation assays and the trypan blue exclusion assay. The type of cell death was determined through flow cytometry, using annexin V and propidium iodide, and through optical microscopy, using the May-Grümwald-Giemsa stain. Patients had a median age of 74 years old (range: 22-89), with a male:female ratio of 0.8. Patients were diagnosed according to WHO criteria as refractory cytopenia with multilineage dysplasia (RCMD) (n=52), refractory cytopenia with unilineage dysplasia (RCUD) (n=12), refractory anemia with excess blasts (RAEB)-1 (n=8), RAEB-2 (n=8), refractory anemia with ring sideroblasts (RARS) (n=6), 5q- syndrome (n=4) and chronic myelomonocytic leukemia (CMML) (n=12), and were classified according to the International Prognostic Scoring System (IPSS) as low risk (n=37), intermidate-1 (n=39), intermediate-2 (n=10) and high risk (n=1). Eleven of the 102 patients progressed to AML. The higher rate of less aggressive WHO subtypes translated into a higher prevalence of low and intermediate risk prognostic groups. The analysis of biochemical parameters identified increased erythropoietin as a negative prognostic factor, associating with decreased overall survival and higher rates of progression to AML. Increased lactate dehydrogenase (LDH) levels were the only other biochemical predictor of decreased overall survival. On the other hand, decreased absolute platelet counts, increased percentage of blasts, increased percentage of IL-6-expressing (CD34+/CD117+/IL-6+) myeloid stem cells, and high IPSS risk groups were also predictors of the evolution to AML. As a result of our findings, we developed a proposal for a new prognostic index, the Acute Leukemia Progression Index (ALPI), calculated through the sum of altered hematologic, biochemical, stem cell, methylation and apoptosis parameters. We observed that patients with the highest scores were the most likely to progress to AML, suggesting that this new prognostic index could be a useful tool to evaluate the risk of progression to over leukemia. Our analyses of the hematopoietic stem cell identified two groups of cells, one with a canonical phenotype (HSC) and the other with neoplastic characteristics (LSC), which a different expression of IL-6 and TNF-α. Although our results suggest that these two markers are useful for the distinction of the two groups of stem cells, those differences were more marked in RAEB-2, with IL-6 prevailing in HSC, and TNF-α in LSC. Apoptosis studies showed a trend towards an increase in pro-apoptotic molecules in the WHO subtypes without excess blasts, although only caspase 3 had statistical significance, corroborating the previously described role of caspase 3 in MDS, especially in low-risk patients, and suggesting that it might be a new diagnostic and prognostic marker. Methylation profiles revealed that over half of patients had two or more methylated genes, while only 14.7% had none. Considering that, in our sample, aberrant methylation is more frequent in early-stage disease, this suggests that methylation is involved primarily in the initial stages of MDS. The genes that were most frequently methylated were p15 and DAPK, in 56% and 54% of patients, respectively. The AC genotype of the A1298C MTHFR polymorphism associated with MDS, but had no association with serum folate or vitamin B12 levels, or with the methylation status of the studied genes. Cell proliferation assays contributed new data on the therapeutic potential of novel drugs in MDS, especially of the associations of α-HFPA with MG262 and/or imatinib, and of trichostatin A and decitabine, which were shown to decrease the proliferation of F-36P cells and induce cell death mostly through apoptosis, in a concentration-dependent mode influenced by the duration of exposure, and the protocol of administration. In conclusion, this study suggests methylation plays a role in MDS, especially in low-risk groups, which could in the future affect the rationale behind current therapeutic approaches. In addition, it showed an association between the AC genotype of the MTHFR A1298C polymorphism and MDS. It also identified negative prognostic markers, such as increased erythropoietin and LDH levels, and proposed a new prognostic index for the progression to AML. The drugs tested in vitro could be a new therapeutic approach in MDS, in monotherapy or in combination, reducing the adverse effects of treatment, while improving the quality of life and overall survival of patients

Nutrição e alterações epigenéticas na síndrome mielodisplásica

Dissertação de mestrado em Medicina (Nutrição Clinica), apresentado à Faculdade de Medicina da Universidade de Coimbr

Oxidative Stress Parameters Can Predict the Response to Erythropoiesis-Stimulating Agents in Myelodysplastic Syndrome Patients

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

DNA Methylation Is Correlated with Oxidative Stress in Myelodysplastic Syndrome-Relevance as Complementary Prognostic Biomarkers

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.The present work was supported by CIMAGO (Center of Investigation on Environment Genetics and Oncobiology), Faculty of Medicine, University of Coimbra, Portugal; by a grant from Santander-Totta Bank/Gabinete de Apoio/Investigação (GAI) of the Faculty of Medicine, University of Coimbra, Portugal; and by the Foundation for Science and Technology (FCT), Portuga

Serum Erythropoietin as Prognostic Marker in Myelodysplastic Syndromes

Introdução: A síndrome mielodisplásica é uma doença heterogénea caracterizada por displasia, medula hipercelular, citopenias e risco de evolução para leucemia aguda. Outros factores de prognóstico, nomeadamente, fibrose medular, elevação da enzima desidrogenase do lactato e β2-microglobulina têm sido descritos, contudo, a decisão terapêutica baseia-se no score do International Prognostic Scoring System. Material e Métodos: Este trabalho teve como objectivo analisar a relevância da eritropoietina sérica ao diagnóstico, em doentes com síndrome mielodisplásica de novo, avaliando o seu impacto na sobrevivência global e a sua implementação como factor de prognóstico. Recolhemos dados clínicos e laboratoriais de 102 doentes com síndrome mielodisplásica de novo diagnosticada entre outubro/2009 e março/2014. A análise de sobrevivência foi efectuada recorrendo à metodologia de Kaplan-Meier, de acordo com os valores de eritropoietina. Resultados: A amostra, de 102 doentes, apresenta uma mediana de idades de 74 anos e relação masculino/feminino igual a 0,8. Os doentes com o subtipo citopenia refratária com displasia unilinha apresentam, em média, valores de eritropoietina significativamente mais baixos, em oposição aos doentes com o subtipo 5q- que apresentam a média de eritropoietina sérica mais elevada (p < 0,05). Onze doentes evoluíram para leucemia aguda; estes têm, em média, eritropoietina sérica superior (p < 0,05). Adicionalmente, a eritropoietina sérica acima do limite superior da normalidade associa-se a menor sobrevivência (p = 0,0336). Após ajuste do modelo de regressão de Cox, o valor preditivo da eritropoietina para a sobrevivência global manteve-se (p < 0,001). Em análise multivariada, a eritropoietina sérica demonstrou ser um factor de prognóstico independente (p < 0,001). Discussão: A eritropoietina sérica é um factor preditivo de resposta à terapêutica com eritropoietina subcutânea, sendo que os doentes com síndrome mielodisplásica com valores mais elevados de eritropoietina apresentam uma pior resposta à administração de eritropoietina, mesmo com doses mais elevadas. A nossa amostra demonstra que a eritropoietina sérica apresenta também valor prognóstico, e em todos os subtipos de síndrome mielodisplásica. Além disso, isoladamente ou em associação com outros factores ou índices de prognóstico, poderá melhorar o valor prognóstico de índices como o International Prognostic Scoring System, uma vez que valores elevados de eritropoietina estão associados a progressão para leucemia aguda e, consequentemente, a menor sobrevivência. Conclusão: Os resultados sugerem que o aumento dos níveis séricos de eritropoietina ao diagnóstico pode constituir um factor de mau prognóstico em doentes com síndrome mielodisplásica, associando-se a maior risco de evolução para leucemia aguda e menor sobrevivência global.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 β2-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 in myelodysplastic syndromes patients, with higher values in patients with progression to acute leukaemia and decreased overall survival