Extracellular matrix in hematopoiesis and hematologic malignancies

The extracellular matrix (ECM) is a complex structure composed of collagens, proteoglycans, glycosaminoglycans and adhesive glycoproteins. Interactions between the cells and the ECM are crucial to determine cell behavior, such as growth, death, differentiation and motility. Hematopoiesis is the system responsible for the production of blood cells. The control of proliferation and differentiation of these cells is attained through the interaction of the cells with the bone marrow microenvironment. The adhesion of hematopoietic progenitors to ECM molecules and the integrin activation are modulated by a variety of cytokines and growth factors, and this modulation seems to be the mechanism of regulation that influences proliferation of hematopoietic cells, transendothelial/transstromal migration and homing. Both in the migration and homing process, and in tumoral invasion the cells undergo the following steps: 1 - Degradation of the ECM by enzymes, including metalloproteinase, collagenase, plasmin, cathepsin, glycosidase and heparanase, secreted by the cells; 2 - Cell migration through the region previously degraded by enzymes; and 3 - Cell adhesion to specific receptors located on the cellular surface, that generally interact with ECM components. In onco-hematologic diseases, the interaction of neoplastic cells with the extracellular matrix also influences aggressiveness and prognosis of the disease.A matriz extracelular (MEC) é uma rede complexa composta por quatro grandes classes de macromoléculas: colágenos, proteoglicanos (PGs), glicosaminoglicanos (GAGs) e glicoproteínas adesivas. As interações entre as células e a MEC são cruciais para determinar os padrões de comportamento celular, tais como crescimento, morte, diferenciação e motilidade. A hematopoese é o sistema responsável pela produção das células sangüíneas. O controle da proliferação e diferenciação destas células é feito através da interação das células com o microambiente da medula óssea (matriz extracelular). A adesão de progenitores hematopoéticos a moléculas da MEC e a ativação das integrinas são modulados por uma variedade de citocinas e fatores de crescimento, e esta modulação parece ser o mecanismo de regulação que influencia a proliferação de células-tronco e progenitores hematopoéticos, migração transendotelial ou transestromal e homing. Tanto no processo de migração, homing e invasão tumoral, as células seguem os seguintes passos: 1 - Degradação da MEC por enzimas secretadas pelas células: metaloproteinases, colagenases, plasmina, catepsinas, glicosidases e heparanases; 2 - Locomoção das células na região da MEC previamente degradada pelas enzimas; 3 - Adesão das células via receptores específicos da superfície celular, que geralmente interagem com componentes da MEC. Nas doenças onco-hematológicas, a interação das células neoplásicas com a matriz extracelular também influencia na agressividade e prognóstico da doença.Universidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Late-onset noninfectious pulmonary complications after allogeneic bone marrow transplantation in Brazil: Incidence, clinical features and influence of tuberculosis

Universidade Federal de São Paulo, São Paulo, BrazilHosp Santa Marcelina, São Paulo, BrazilUniversidade Federal de São Paulo, São Paulo, BrazilWeb of Scienc

Hairy cell leukemia: a histo-cytochemical and ultra-structural study

We studied five patients with hairy cell leukemia (HCL) diagnosed within the last ten years at the Department of Hematology of Universidade Federal de São Paulo (UNIFESP) - Escola Paulista de Medicina. Our purpose was to analyze the value of transmission electron microscopy (TEM) by comparing this method with the conventional ones. At diagnosis, patients presented weight loss, spleen enlargement and hairy cells (HC) in peripheral blood and bone marrow slides. HC was characterized by morphology and tartrate test resistance in the acid phosphatase reaction (TRAP). At the evaluation time, the amount of HC ranged from 1% to 85% of WBC count. All patients, except two, had phenotype B. In these last two, TRAP as well as phenotype B could not be documented due to low HC numbers in their exams. Cytoplasmatic projections and the absence of lamellar ribosomic complex were the most frequent ultrastructural findings, even in those patients with the lowest HC numbers. Based on these features, TEM is an efficient method for searching for HC at HCL diagnosis and during the course of the disease.Estudamos cinco pacientes com leucemia de células pilosas (LCP), diagnosticada nos últimos dez anos na Disciplina de Hematologia da Escola Paulista de Medicina. O principal objetivo foi analisar o valor da microscopia eletrônica de transmissão (MET) pela comparação deste método com os convencionais. Pacientes apresentavam no diagnóstico perda de peso, esplenomegalia e células pilosas (CP) no sangue periférico e medula óssea. As CP foram caracterizadas pela morfologia e resistência ao tartarato na reação de fosfatase ácida (FATR). Na época em que foram avaliados, a quantidade de CP variou de 1 a 85% da leucometria. Todos os pacientes tinham fenótipo B, excetuando-se dois nos quais FATR e imunofenótipo não puderam ser documentados devido ao baixo número de CP no sangue periférico e medula óssea. Projeções citoplasmáticas e ausência de complexo ribosômico lamelar foram os mais freqüentes achados na MET, mesmo nos pacientes com baixa porcentagem de CP. Baseado nestes achados concluímos que MET é um método eficiente no diagnóstico e evolução da LCP.Universidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Fluorescent in-situ hybridization (FISH) for BCR/ABL in chronic myeloid leukemia after bone marrow transplantation

CONTEXT: Identification of Philadelphia chromosome or BCR/ABL gene rearrangement in chronic myeloid leukemia is important at diagnosis as well as after treatment. OBJECTIVE: To compare the results of karyotyping using fluorescent in-situ hybridization (FISH) upon diagnosis and 1 year after bone marrow transplantation in 12 patients. TYPE OF STUDY: Diagnostic test and residual disease detection. SETTING: Hematology and Hemotherapy Department, Federal University of São Paulo/Escola Paulista de Medicina, São Paulo, Brazil. SAMPLE: 12 patients with chronic myeloid leukemia at diagnosis and 1 year after bone marrow transplantation. DIAGNOSTIC TEST: Karyotyping was done in the usual way and the BCR/ABL gene-specific probe was used for FISH. MAIN MEASUREMENTS: Disease at diagnosis and residual. RESULTS: At diagnosis, 10 patients presented t(9;22)(q34.1;q11) as well as positive FISH. Two cases did not have metaphases but FISH was positive. After bone marrow transplantation, 8 patients presented normal karyotype, 1 had persistence of identifiable Philadelphia chromosome and 3 had no metaphases. Two cases showed complete chimera and 2 had donor and host cells simultaneously. FISH was possible in all cases after bone marrow transplantation and confirmed the persistence of identifiable Philadelphia chromosome clone in one patient, and identified another that did not present metaphases for analysis. Cases that showed mixed chimera in karyotype were negative for BCR/ABL by FISH. CONCLUSION: The applicability of FISH is clear, particularly for residual disease detection. Classical and molecular cytogenetics are complementary methods.CONTEXTO: Na leucemia mielóide crônica, a detecção do cromossomo Philadelphia ou o rearranjo gênico BCR/ABL é importante tanto ao diagnóstico como após o tratamento. OBJETIVO: Comparar os resultados do cariótipo com a hibridação in situ por fluorescência ao diagnóstico e, após um ano de transplante de medula óssea, em 12 pacientes com leucemia mielóide crônica. TIPO DE ESTUDO: Teste diagnóstico e detecção de doença residual. LOCAL: Disciplina de Hematologia e Hemoterapia da Universidade de São Paulo/Escola Paulista de Medicina. AMOSTRA: 12 pacientes com leucemia mielóide crônica ao diagnóstico e um ano após transplante de medula óssea. TESTE DIAGNÓSTICO: A análise do cariótipo (forma clássica) e a hibridação in situ por fluorescência com a sonda específica para os genes BCR/ABL. VARIÁVEIS ESTUDADAS: Doença ao diagnóstico e residual. RESULTADOS: Ao diagnóstico, 10 pacientes apresentaram t(9;22)(q34.1;q11) no cariótipo assim como pela hibridação in situ por fluorescência. Dois casos em que na citogenética não havia mitoses, apresentaram rearranjo pela hibridação in situ por fluorescência. Após o transplante de medula óssea, o cariótipo mostrou ausência do cromossomo Philadelphia em 8 casos, persistência em um e ausência de metáfases em três. Em dois casos havia quimera completa e em outros dois havia concomitância de células do doador e do receptor. A hibridação in situ por fluorescência foi possível em todos os casos após o transplante de medula óssea, confirmando a persistência do clone cromossomo Philadelphia num paciente em que o cariótipo também havia mostrado e identificando noutro em que não havia metáfases. Os casos de quimera mista foram negativos para BCR/ABL pela hibridação in situ por fluorescência. CONCLUSÃO: Ficou evidente a aplicabilidade da hibridação in situ por fluorescência, particularmente na detecção de doença residual. A citogenética clássica e a molecular são métodos complementares.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Discipline of Hematology and HemotherapyUNIFESP, EPM, Discipline of Hematology and HemotherapySciEL

Fludarabine induces apoptosis in chronic lymphocytic leukemia - the role of P53, Bcl-2, Bax, Mcl-1, and Bag-1 proteins

The expression of P53, Bcl-2, Bax, Bag-1, and Mcl-1 proteins in CD5/CD20-positive B-chronic lymphocytic leukemia (B-CLL) cells from 30 typical CLL patients was evaluated before and after 48 h of incubation with 10-6 M fludarabine using multiparametric flow cytometric analysis. Protein expression was correlated with annexin V expression, Rai modified clinical staging, lymphocyte doubling time, and previous treatment. Our main goal was to determine the predictive value of these proteins in CLL cells in terms of disease evolution. Bcl-2 expression decreased from a median fluorescence index (MFI) of 331.71 ± 42.2 to 245.81 ± 52.2 (P < 0.001) after fludarabine treatment, but there was no difference between viable cells (331.57 ± 44.6 MFI) and apoptotic cells (331.71 ± 42.2 MFI) before incubation (P = 0.859). Bax expression was higher in viable cells (156.24 ± 32.2 MFI) than in apoptotic cells (133.56 ± 35.7 MFI) before incubation, probably reflecting defective apoptosis in CLL (P = 0.001). Mcl-1 expression was increased in fludarabine-resistant cells and seemed to be a remarkable protein for the inhibition of the apoptotic process in CLL (from 233.59 ± 29.8 to 252.04 ± 35.5; P = 0.033). After fludarabine treatment, Bag-1 expression was increased in fludarabine-resistant cells (from 425.55 ± 39.3 to 447.49 ± 34.5 MFI, P = 0.012), and interestingly, this higher expression occurred in patients who had a short lymphocyte doubling time (P = 0.022). Therefore, we could assume that Bag-1 expression in such situation might identify CLL patients who will need treatment earlier.Universidade Federal de Minas Gerais Hospital das Clínicas Serviço de Hematologia e HemoterapiaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Disciplina de Hematologia e HemoterapiaUNIFESP, EPM, Disciplina de Hematologia e HemoterapiaSciEL

Proliferating cell nuclear antigen (PCNA), p53 and MDM2 expression in Hodgkin's disease

CONTEXT and OBJECTIVE: Tumor cells in Hodgkin's disease (HD) express cell proliferation markers that are evaluated according to the oncogenes involved or the expression of their proteins. Correlations between the protein expression grade and clinical data are now important for disease prognosis.DESIGN and SETTING: This was a retrospective analysis on proliferating cell nuclear antigen (PCNA), p53 and MDM2 (murine double minute-2) expression using immunohistochemistry, on formalin-fixed, paraffin-embedded tissues from diagnostic biopsies on 51 patients with HID. the study was conducted at the Division of Hematology and Transfusion Medicine, Hospital São Paulo, Universidade Federal de São Paulo.METHODS: Antigen expression was evaluated as the proportions of positive Hodgkin and Reed-Sternberg (HRS) cells and reactive lymphocytes (L), which were compared using Spearman correlation coefficients. the Friedman test was used for comparisons between the markers. the Pearson test was used to investigate associations between marker expression and clinical and laboratory parameters, marrow involvement, complete remission (CR) and overall survival (OS) rates.RESULTS: There was overexpression of antigen proteins in HRS, in relation to L (p < 0.001). in HRS, MDM2 was higher than p53 and PCNA (p < 0.003), while the latter two were equivalent. in L, p53 was lower than MDM2 and PCNA (p < 0.001), while the latter two were equivalent. There was no relationship between protein expression and clinical and laboratory variables or outcome.CONCLUSIONS: PCNA, p53 and MDM2 are tumor markers for HID, but showed no clinical or prognostic significance in our analysis.Universidade Federal de São Paulo, Hosp São Paulo, Escola Paulista Med, Div Hematol & Transfus Med, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Hosp São Paulo, Escola Paulista Med, Div Hematol & Transfus Med, BR-04023900 São Paulo, BrazilWeb of Scienc

Prognostic factors in non-Hodgkin lymphomas

CONTEXT: In Hodgkin's disease, each clinical or pathologic stage can be related to the extent of the area involved and predicts the next anatomical region at risk for tumor dissemination. OBJECTIVE: To determine the best prognostic factors that could predict survival in non-Hodgkin lymphoma cases. DESIGN: A retrospective study. LOCATION: Department of Hematology and Transfusion Medicine, Universidade Federal de São Paulo (UNIFESP) - Escola Paulista de Medicina. PARTICIPANTS: 142 patients with non-Hodgkin lymphoma diagnosed between February 1988 and March 1993. MAIN MEASUREMENTS: Histological subset, Sex, Age, Race, B symptoms, Performance status, Stage, Extranodal disease, Bulk disease, Mediastinal disease, CNS involvement, BM infiltration, Level of DHL, Immunophenotype. RESULTS: In the first study (113 patients), the following variables had a worse influence on survival: yellow race (P<0.1); ECOG II, III e IV (P<0.1) and extranodal disease (P<0.1) for high grade lymphomas; constitutional symptoms (P<0.1), ECOG II, III e IV (P<0.1) and involvement of CNS (P<0.1) for intermediate grade and the subtype lymphoplasmocytoid (P=0.0186) for low grade lymphomas. In the second survey (93 patients), when treatment was included, the variables related to NHL survival were: CNS involvement (P<0.1) for high grade lymphomas, constitutional symptoms (P<0.1), ECOG II, III, IV (P=0.0185) and also CNS involvement (P<0.1) for the intermediate group. There were no variables related to the survival for low-grade lymphomas. CONCLUSIONS: The intermediate grade lymphomas were more compatible with data found in the literature, probably because of the larger number of patients. In this specific case, the treatment did not have an influence on the survival.CONTEXTO: Na doença de Hodgkin, cada estágio clínico ou patológico pode ser relacionado com a extensão da área envolvida e predizer a próxima região anatômica de risco para disseminação. OBJETIVO: Estabelecer os fatores prognósticos que melhor predizem sobrevida em LNH. TIPO DE ESTUDO: Estudo retrospectivo LOCAL: Disciplina de Hematologia e Hemoterapia, Universidade Federal de São Paulo (UNIFESP) - Escola Paulista de Medicina. PARTICIPANTES: 142 pacientes com LNH diagnosticados entre fevereiro de 1988 e março de 1993. VARIÁVEIS ESTUDADAS: Tipo histológico, sexo, idade, raça, sintomas, sitação performance, estágio, doença extranodal, desenvolvimento de Bulk, comprometimento mediastinal, envolvimento do SNC, infiltração da medúla óssea, nível de desidrogenose láctica, fenótipo imune. RESULTADOS: Ao primeiro estudo (113 pacientes), as seguintes variáveis tiveram uma pior influência na sobrevida: raça amarela (P<0.1); ECOG II, III e IV (P<0.1) e doença extranodal (P<0.1) para os linfomas de alto grau; sintomas constitucionais (P<0.1), ECOG II, III e IV (P<0.1) e envolvimento de SNC (P<0.1) para os linfomas de grau intermediário e o subtipo linfoplasmocitóide (P=0.0186) para os linfomas de baixo grau. Ao segundo estudo (93 pacientes), quando inclui-se o tratamento, as variáveis relacionadas a sobrevida foram : envolvimento de SNC (P<0.1) para o linfomas de alto grau; sintomas constitucionais (P<0.1), ECOG II, III, IV (P=0.0185) e envolvimento de SNC (P<0.1) para o grupo intermediário. Nenhuma variável relacionou-se com a sobrevida para os linfomas de baixo grau. CONCLUSÕES: Os linfomas de grau intermediário, provavelmente devido ao maior número de pacientes, foram mais compatíveis com os dados encontrados na literatura. Neste caso específico, o tratamento não influenciou a sobrevida.Universidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Células CD34-positivas e suas subpopulações caracterizadas por análise de citometria de fluxo em doadores para transplante alogênico de medula óssea

CONTEXT AND OBJECTIVE: Counting and separating hematopoietic stem cells from different sources has importance for research and clinical assays. Our aims here were to characterize and quantify hematopoietic cell populations in marrow donors and to evaluate CD34 expression and relate this to engraftment. DESIGN AND SETTING: Cross-sectional study on hematopoietic stem cell assays, using flow cytometry on donor bone marrow samples, for allogenic transplantation patients at two hospitals in São Paulo. METHODS: Immunophenotyping of marrow cells was performed in accordance with positive findings of CD34FITC, CD117PE, CD38PE, CD7FITC, CD33PE, CD10FITC, CD19PE, CD14FITC, CD13PE, CD11cPE, CD15FITIC, CD22PE, CD61FITC and CD56PE monoclonal antibodies in CD45PerCP+ cells, searching for differentiation and maturation regions. CD34+ sorting cells were analyzed for CD38 and CD117. Rh-123 retention was done before and after sorting. Antigen expression and CD34+ cells were correlated with engraftment. RESULTS: In region R1, 0.1% to 2.8% of cells were CD34+/CD45+ and 1.1%, CD34+/CD45-. The main coexpressions of CD45+ cells were CD38, CD22, CD19 and CD56 in R2 and CD33, CD11c, CD14, CD15 and CD61 in R3 and R4. After sorting, 2.2x10(6) CD34+ cells were equivalent to 4.9% of total cells. Coexpression of CD34+/CD38+ and CD34+/CD117+ occurred in 94.9% and 82% of events, respectively. There was a positive relationship between CD34+ cells and engraftment. More than 80% of marrow cells expressed high Rh-123. CD34+ cell sorting showed that cells in regions of more differentiated lineages retained Rh-123 more intensively than in primitive lineage regions. CONCLUSION: We advocate that true stem cells are CD34+/CD45-/CD38-/low-Rh-123 accumulations.CONTEXTO E OBJETIVO: A contagem e separação de células-tronco hematopoéticas de diferentes fontes tem importância para ensaios clínicos e pesquisa basica. Nosso objetivo foi caracterizar e quantificar as populacões de células hematopoéticas, bem como avaliar a expressão do antígeno CD34 em populações mais primitivas e correlacioná-las com a enxertia nos doadores de medula óssea para transplante alogênico. TIPO DE ESTUDO E LOCAL: Estudo transversal no qual a diferenciação e a seleção de células-tronco hematopoéticas foram realizadas em amostras de medula óssea de doadores de pacientes submetidos a transplante alogênico nos Hospitais São Paulo e Santa Marcelina, São Paulo, Brasil. MÉTODOS: Imunofenotipagem de células mononucleares de medula óssea foi feita na população de células CD45PerCP+ com os seguintes anticorpos: CD34FITC, CD117PE, CD38PE, CD7FITC, CD33PE, CD10FITC, CD19PE, CD14FITC, CD13PE, CD11cPE, CD15FITC, CD22PE, CD61FITC e CD56PE. Após a definição de regiões de células positivas ao CD34, estas células foram selecionadas e analisadas para a co-expressão do CD38 e CD117. Células mononucleares totais de medula óssea e aquelas obtidas após a seleção foram testadas para a retenção de Rh-123. O teste de Friedman e o coeficiente de Sperman foram utilizados para comparar as expressões e correlacionar a contagem de células CD34+ com a enxertia. RESULTADOS: Na região R1, 0,1% a 2,8% das células foram CD34+/CD45+, porém apenas 1,1% das células foram CD34+/CD45-. As principais co-expressões de células CD45+ foram CD38, CD22, CD19 e CD56 na região R2 e CD33, CD11c, CD14, CD15 e CD61 nas regiões R3 e R4. Após a seleção, a mediana de 2,2x106 células CD34+ foi equivalente a 4,9% do total mediano de células da medula óssea. Co-expressões de células CD34+/CD38+ e CD34+/CD117+ ocorreram em 94,95 e 82%, respectivamente. Houve relação positiva entre o número de células CD34+ infundidas e o dia da enxertia. Observamos que mais de 80% das células mononucleares de medula óssea retêm intensamente a Rh-123. Após a seleção, células localizadas em regiões de maior diferenciação, regiões R3 e R4, acumulam mais fortemente a Rh-123 do que células mais primitivas da região R1. CONCLUSÃO: Postulamos que a célula tronco hematopoética mais primitiva expressa o seguinte fenótipo: CD34+/CD45-/CD38-/Rh-123 de baixa retenção

Nephrotic syndrome as a clinical manifestation of graft-versus-host disease (GVHD) in a marrow transplant recipient after cyclosporine withdrawal

GVHD is one of the most frequent complications of BMT and recently nephrotic syndrome (NS) has been described as a manifestation of chronic GVHD, Here, we present an AA patient who developed NS 1 year after BMT when cyclosporine was stopped. Renal biopsy showed focal sclerosis associated with membranous deposits. He also had other clinical manifestations of chronic GVHD: sicca-like syndrome and colestasis, After 15 days of CsA therapy, he experienced a remarkable improvement in the NS and GVHD as a whole. We comment on immunological mechanisms that could be involved in the pathogenesis of this manifestation.Universidade Federal de São Paulo, Escola Paulista Med, Disciplina Hematol, Dept Hematol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Pathol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Nephrol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Disciplina Hematol, Dept Hematol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Pathol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Nephrol, BR-04023900 São Paulo, BrazilWeb of Scienc

A retrospective comparison of cyclophosphamide plus antithymocyte globulin with cyclophosphamide plus busulfan as the conditioning regimen for severe aplastic anemia

Allogeneic hematopoietic stem cell transplantation (AHSCT) is the treatment of choice for young patients with severe aplastic anemia (SAA). The association of antithymocyte globulin (ATG) and cyclophosphamide (CY) is the most frequently used conditioning regimen for this disease. We performed this retrospective study in order to compare the outcomes of HLA-matched sibling donor AHSCT in 41 patients with SAA receiving cyclophosphamide plus ATG (ATG-CY, N = 17) or cyclophosphamide plus busulfan (BU-CY, N = 24). The substitution of BU for ATG was motivated by the high cost of ATG. There were no differences in the clinical features between the two groups, including age, gender, cytomegalovirus status, ABO match, interval between diagnosis and transplant, and number of total nucleated cells infused. No differences were observed in the time to neutrophil and platelet engraftment, or in the risk of veno-occlusive disease and hemorrhage. However, there was a higher risk of mucositis in the BU-CY group (71 vs 24%, P = 0.004). There were no differences in the incidence of neutrophil and platelet engraftment, acute and chronic graft-versus-host disease, and transplant-related mortality. There was a higher incidence of late rejection in the ATG-CY group (41 vs 4%, P = 0.009). Although the ATG-CY group had a longer follow-up (101 months) than the BU-CY group (67 months, P = 0.04), overall survival was similar between the groups (69 vs 58%, respectively, P = 0.32). We conclude that the association BU-CY is a feasible option to the conventional ATG-CY regimen in this population.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de Oncologia Clínica e ExperimentalHospital Santa Marcelina Serviço de Hematologia e HemoterapiaUNIFESP, EPM, Depto. de Oncologia Clínica e ExperimentalSciEL