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.

Karyotype of cryopreserved bone marrow cells

The analysis of chromosomal abnormalities is important for the study of hematological neoplastic disorders since it facilitates classification of the disease. The ability to perform chromosome analysis of cryopreserved malignant marrow or peripheral blast cells is important for retrospective studies. In the present study, we compared the karyotype of fresh bone marrow cells (20 metaphases) to that of cells stored with a simplified cryopreservation method, evaluated the effect of the use of granulocyte-macrophage colony-stimulating factor (GM-CSF) as an in vitro mitotic index stimulator, and compared the cell viability and chromosome morphology of fresh and cryopreserved cells whenever possible (sufficient metaphases for analysis). Twenty-five bone marrow samples from 24 patients with hematological disorders such as acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, chronic myeloid leukemia, megaloblastic anemia and lymphoma (8, 3, 3, 8, 1, and 1 patients, respectively) were selected at diagnosis, at relapse or during routine follow-up and one sample was obtained from a bone marrow donor after informed consent. Average cell viability before and after freezing was 98.8 and 78.5 percent, respectively (P < 0.05). Cytogenetic analysis was successful in 76 percent of fresh cell cultures, as opposed to 52 percent of cryopreserved samples (P < 0.05). GM-CSF had no proliferative effect before or after freezing. The morphological aspects of the chromosomes in fresh and cryopreserved cells were subjectively the same. The present study shows that cytogenetic analysis of cryopreserved bone marrow cells can be a reliable alternative when fresh cell analysis cannot be done, notwithstanding the reduced viability and lower percent of successful analysis that are associated with freezing

Fungal infections in marrow transplant recipients under antifungal prophylaxis with fluconazole

Fungal infection is one of the most important causes of morbidity and mortality in bone marrow transplant (BMT) recipients. The growing incidence of these infections is related to several factors including prolonged granulocytopenia, use of broad-spectrum antibiotics, conditioning regimens, and use of immunosuppression to avoid graft-versus-host disease (GvHD). In the present series, we report five cases of invasive mold infections documented among 64 BMT recipients undergoing fluconazole antifungal prophylaxis: 1) A strain of Scedosporium prolificans was isolated from a skin lesion that developed on day +72 after BMT in a chronic myeloid leukemic patient. 2) Invasive pulmonary aspergillosis (Aspergillus fumigatus) was diagnosed on day +29 in a patient with a long period of hospitalization before being transplanted for severe aplastic anemia. 3) A tumoral lung lesion due to Rhizopus arrhizus (zygomycosis) was observed in a transplanted patient who presented severe chronic GvHD. 4) A tumoral lesion due to Aspergillus spp involving the 7th, 8th and 9th right ribs and local soft tissue was diagnosed in a BMT patient on day +110. 5) A patient with a history of Ph1-positive acute lymphocytic leukemia exhibited a cerebral lesion on day +477 after receiving a BMT during an episode of severe chronic GvHD. At that time, blood and spinal fluid cultures yielded Fusarium sp. Opportunistic infections due to fungi other than Candida spp are becoming a major problem among BMT patients receiving systemic antifungal prophylaxis with fluconazole

Acute promyelocytic leukemia: the study of t(15; 17) translocation by fluorescent in situ hybridization, reverse transcriptase-polymerase chain reaction and cytogenetic techniques

Acute promyelocytic leukemia (AML M3) is a well-defined subtype of leukemia with specific and peculiar characteristics. Immediate identification of t(15;17) or the PML/RARA gene rearrangement is fundamental for treatment. The objective of the present study was to compare fluorescent in situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR) and karyotyping in 18 samples (12 at diagnosis and 6 after treatment) from 13 AML M3 patients. Bone marrow samples were submitted to karyotype G-banding, FISH and RT-PCR. At diagnosis, cytogenetics was successful in 10 of 12 samples, 8 with t(15;17) and 2 without. FISH was positive in 11/12 cases (one had no cells for analysis) and positivity varied from 25 to 93 per cent (mean: 56 per cent). RT-PCR was done in 6/12 cases and all were positive. Four of 8 patients with t(15;17) presented positive RT-PCR as well as 2 without metaphases. The lack of RT-PCR results in the other samples was due to poor quality RNA. When the three tests were compared at diagnosis, karyotyping presented the translocation in 80 per cent of the tested samples while FISH and RT-PCR showed the PML/RARA rearrangement in 100 per cent of them. Of 6 samples evaluated after treatment, 3 showed a normal karyotype, 1 persistence of an abnormal clone and 2 no metaphases. FISH was negative in 4 samples studied and 2 had no material for analysis. RT-PCR was positive in 4 (2 of which showed negative FISH, indicating residual disease) and negative in 2. When the three tests were compared after treatment, they showed concordance in 2 of 6 samples or, when there were not enough cells for all tests, concordance between karyotype and RT-PCR in one. At remission, RT-PCR was the most sensitive test in detecting residual disease, as expected (positive in 4/6 samples). An incidence of about 40 per cent of 5' breaks and 60 per cent of 3' breaks, i.e., bcr3 and bcr1/bcr2, respectively, was observed.

Granuloma letal da linha média: abordagem clínica e terapêutica de três casos / Lethal midline granuloma: clinical management of three cases

Objetivo. Relato de três casos do GLLM acompanhados pela Disciplina de Hematologia e Hemoterapia da Unifesp-EPM que tiveram boa resposta à terapêutica e evoluçao favorável. Métodos. Após confirmaçao histológica e histoquímica, os pacientes foram submetidos à tratamento quimio e radioterápico com boa resposta terapêutica. Resultados. Atualmente estes pacientes encontram-se em remissao total da doença, com sobrevida média de 45 meses. Conclusao. Levando-se em consideraçao nossa pequena experiência, acreditamos que o tratamento radioterápico e a abordagem quimioterápica inicial agressiva sao fundamentais para uma boa evoluçao deste tipo de linfoma.

Leucemia mielóide aguda Ph1-positivo de novo ou crise blástica de leucemia mielóide crônica? Análise molecular e evoluçäo clínica de um caso / Ph1-positive acute myeloid leukemia de novo or chronic myeloid leukemia? Molecular analysis and clinical evolution of a case

Os autores relatam um caso de leucemia mielóide aguda (LMA) que apresentava, ao diagnóstico, basofilia no sangue periférico e cariótipo com presença do cromossomo Filadélfia (Ph1). Após um ano de tratamento com quimioterapia intensiva e em fase de remissao clínica e hematológica, a análise molecular pela técnica da reaçao em cadeia da polimerase-trasncriptase reversa (RT-PCR) revelou presença de doença residual (rearranjo b2-a2). A seguir, o paciente apresentou primeira recidiva como LMA e, após a remissao, evoluiu com quadro hematológico sugestivo de leucemia mielóide crônica (LMC) em fase crônica. Após dez meses, apresentou nova recidiva da LMA. Os autores discutem a dificuldade do diagnóstico diferencial entre LMA Ph1-positivo de novo e crise blástica mielóide como primeira manifestaçao clínica da LMC, baseados nos aspectos clínicos e moleculares.

Necrólise epidérmica tóxica secundária ao uso da citosina-arabinosídeo em dose intermediária / Toxic epidermal necrolysis after the use of intermediate dose of cytosine arabinoside

A necrólise epidérmica tóxica é afecçao dermatológica secundária ao uso de drogas e corresponde à síndrome de Lyell, relacionada ao eritema multiforme e à síndrome de Stevens-Johnson. Objetivos. Relatar um caso de necrólise epidérmica fatal secundária à citosina-arabinosídeo (Ara-C) em dose intermediária. Relato de Caso. Paciente do sexo feminino, com 16 anos de idade, portadora de leucemia linfóide aguda - LLA-L1. Iniciou tratamento segundo o protocolo do Grupo Brasileiro de Tratamento da Leucemia Infantil/85, alto risco. Na fase II da induçäo, após o uso de Ara-C na dose de 1,5g/m2, intravenoso, 12/12h x três dias, desenvolveu múltiplas lesöes cutâneas bolhosas, que aumentaram rapidamente por progressäo das bordas. As bolhas continham secreçäo serosa, evoluíram para ulceraçäo superficial central, com infecçäo secundária múltipla. Faleceu por septicemia, no 13 dia após o início do quadro dermatológico. Conclusäo. O Ara-C tem sido relacionado a diversas manifestaçöes de toxicidade dermatológica; no entanto, até o momento, näo há relato de necrólise epidérmica tóxica, sendo este o primeiro caso da literatura.

Alteraçöes citogenética como fatores prognósticos em leucemias mielóides agudas / Cytogenetic abnormalities as prognostic factors in acute myeloide leukemia

O estudo das alteraçoes cromossômica nas leucemias mielóides agudas (LMA) vem-se tornando importante no diagnóstico e na caracterizaçao de subtipos, pois associam-se a características clínicas, morfológicas e imunológicas definidas à resposta a tratamento e à sobrevida. OBJETIVO. O presente trabalho objetiva avaliar a importância relativa das alteraçoes citogenética em portadores de LMA. MATERIAL. Foram estudados, ao diagnóstico, 13 pacientes com LMA e com idade mediana igual a 38 anos. O estudo citogenético foi realizado em material medular.RESULTADOS. Os subtipos FAB M1 e M2 foram o mais freqüentes (61,6 por cento). A análise citogenética mostrou cariótipo anormal em 61,5 por cento dos casos e, dentre estes, apenas 15,3 por cento tinham alteraçoes indicadoras de bom prognóstico [t(l5;17) e t(8;21)]. Na data de avaliaçao do estudo havia três pacientes vivos, dois em remissao completa contínua e um em segunda remissao. A sobrevida mediana global foi de 7 meses. Os pacientes foram divididos em dois grupos: um intitulado "bom prognóstico", que englobou cinco indivíduos com cariótipo normal e dois com as translocaçoes t(l5;17) e t(8;21), e outro, "mau prognóstico", com oito pacientes com alteraçoes cromossômicas desfavoráveis. O grupo "bom prognóstico" teve sobrevida mediana de nove meses, enquanto outro, de 6,2 meses, mas sem diferença estatisticamente significante (p= 0,180084), provavelmente devido ao pequeno número de casos em cada grupo. Entretanto, ao se analisar os casos em separado nota-se que os pacientes com translocaçoes (8;21) e (15;17), tidas como de bom prognóstico, tiveram sobrevidas mais longas. CONCLUSAO. Concluímos que o trabalho evidenciou sobrevida desigual entre os dois grupos, ressaltando a importância da análise citogenética que permite distinguir o paciente que terá evoluçao favorável.

Prognostic significance of BCR-ABL rearrangement in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the presence of a reciprocal translocation between chromosomes 9 and 22 in at least 95 per cent of cases. At the molecular level, this translocation results in the activation of the ABL oncogene of chromosome 9, which becomes contiguous with the 5'end of the BCR gene on chromosome 22. The breakpoint usually occurs between exons 2 and 3 (b2-a2 rearrangement), or 3 and 4 (b3-a2 rearrangement) of the major breakpoint cluster region (M-BCR) of the BCR gene. The aim of the present study was to characterize the type of BCR-ABL transcript in 32 patients with CML using the reverse transcriptase-polymerase chaim reaction (RT-PCR) and to determine if this type of rearrangement is related to the survival of the patients. Our results confirmed that RT-PCR is more sensitive than cytogenetic analysis for identifying the Philadelphia (Ph1) chromosome (96.9 per cent vs 79.3 per cent). The frequencies of b2-a2 and b3-a2 rearrangements were 28.1 per cent and 65.7 per cent, respectively. The survival of patients presenting the b2-a2 or the b3-a2 rearrangement was not significantly different (P = 0.27750). The data suggest that the type of transcript has no prognostic value for CML patients.

Fetal hemoglobin in patients with chronic renal disease

To investigate whether hemoglobin (Hb) synthesis is affected by different treatment protocols used for end-stage renal disease, we analyzed the electrophoretic pattern of hemoglobin in 136 adult patients with chronic renal failure. Forty-seven patients were not in a dialysis program (ND), 29 individuals were on continuous ambulatory peritoneal dialysis (CAPD), 33 patients were on hemodialysis(HD), and 27 subjects had received a kidney transplant (KT). We found 3.6 per cent hemoglobin C, 1.4 per cent hemoglobin S and 3.6 per cent beta-thalassemia minor as reported in other studies of Brazilian patients. In addition, we found increased fetal hemoglobin (Hb F) levels in 7.4 per cent of the patients which contrasts with the reported 0.01 per cent prevalence rate of hereditary persistence of Hb F in Brazil. Seven out of ten patients with elevated Hb F belonged to either the CAPD or the KT group. We postulate that stress erythropoiesis is probably the mechanism responsible for the Hb F increase in these patients. However, properly designed clinical studies are still necessary to clarify these questions.

Avaliaçäo do estado de ferro em indivíduos beta-talassêmicos heterozigotos / Evaluation of iron status in individuals with heterozygotic beta-thalassemia

A sobrecarga de ferro (Fe) é problema cosmopolita, que tem a hemocromatose idiopática e a talassemia como as doenças genéticas a que maisfreqüentemente está associada. Enquanto a sobrecarga de ferro em beta-talassêmicos em hipertransfusäo é situaçäo bem definida, existindo esquemas terapêuticos que visam minorá-la, o mesmo näo acontece com a talassemia menor, na qual estoques de ferro aumentados têm sido encontrados, de forma näo consensual. A ferritinasérica é considerada o melhor índice para avaliaçäo do estoque do ferro, uma vezque existe correlaçäo entre os seus valores e a concentraçäo de ferro hepático. OBJETIVO. O presente trabalho tem por objetivo verificar o risco de traços talassêmicos desenvolverem sobrecarga de Fe e a correlaçäo entre o grau de anemia e oestado do ferro. MÉTODOS. Foram avaliados o Fe sérico, TIBC, saturaçäo de transferrina, ferritina e homeglobina de 35 talassêmicos menores assintomáticos e 35 indivíduos normais, pareados por sexo e idade (20 mulheres e 15 homens, entre 20 e 54 anos de idade). RESULTADOS. Näo houve diferença estatisticamente significante para nenhuma variável estudada nos grupos do sexo feminimo, diferentemente dosexo masculino, em que a ferritina sérica nos traços talassêmicos apresentou média de 253,69ng/mL, e no controle normal de 107,79ng/mL (U calc

Padronizaçäo da técnica de imunofixaçäo na detecçäo e identificaçäo de paraproteínas séricas / Standartization of immunofixation technique for detection and identification of seric paraproteins

O diagnóstico e seguimento das paraproteinemias requer a identificaçäo e tipagem de paraproteínas (PP). A imunoeletroforese (IEF) é o método comumente usado embora demorado e pouco sensível. A técnica de imunofixaçäo (IF) é superior por ser mais sensível, rápida e de fácil interpretaçäo, particularmente no reconhecimento de PP presentes em baixa concentraçäo no soro e/ou urina. Consiste de fase eletroforética, seguida de fixaçäo, quando o anti-soro é colocado sobre o gel, precipitando a proteína. Objetivo. Este estudo objetiva padronizar a técnica de IF e compará-la à IEF. Métodos. Foram estudados os soros de 28 pacientes, sendo 25 portadores de mieloma múltiplo e 3 com hipergamaglobulinemia policlonal, comparados com 6 indivíduos normais. Todos foram submetidos à eletroforese (EF) em gel de agarose, à IEF e à IF. Resultados. O principal problema na padronizaçäo da IF foi a determinaçäo da diluiçäo que estabelecesse proporçäo ideal entre antígeno e anticorpo. A concentraçäo sérica ideal da PP, neste estudo, variou de 28 a 35 g/dL. A PP foi detectada e caracterizada por ambas as técnicas em 21 (84 por cento) dos indivíduos e näo detectada por nenhuma delas em 2 (8 por cento). Em outros 2, somente a IF conseguiu identificar a PP. Näo houve banda monoclonal à EF e à IEF que näo fosse identificada pela IF. Conclusäo. Nossos resultados permitem concluir que a IF é mais sensível que a IEF e deve ser incorporada à rotina de diagnóstico

Measurement of red blood cell antibodies in autoimmune hemolytic anemia

The enzyme-linked antiglobulin test (ELAT) was employed to measure the number of IgG molecules per red blood cell (IgG/RBC0 in 11 patients with autoimmune hemolytic anemia (AIHA). All patients with AIHA had high levels of red cell-associated IgG (110-3,650IgG/RBC). The control group consisted of normal volunteers (N=10) and patients with hereditary spherocytosis (N=1), ß--thalassemia (N=1), immunologic thrombocytopenic purpura (N=3) and IgG multiple myeloma (N=4). All control individuals presented low levels of red cell IgG (less than 38IgG/RBC) with the exception of one of four patients with myeloma who had a mildly elevated value (50 IgG/RBC). Since the multiple myeloma patients had > 2g/dl IgG, the possible nosnspecific uptake of IgG onto the RBCs of patients with elevated serum IgG values did not interfere with the results of ELAT. ELAT proved to be a useful method for accurate quantification of the amount of IgG specifically bound on the surface of RBC of patients with AIHA

Effects of whole-body x-irradiation on the peripheral blood of primate Cebus apella

The effects of ionizing radiation on the peripheral blood elements of primate Cebus apella were examined after a single 25.8mC/kg (100 R) dose of whole-body X-ray. We determined the number of white blood cells,neutrophils, osophils, basophils,lymphocytes,monocytes anderythrocytes up to 90 days after exposure of 5 animals. Hemoglobin content and microhematocrit were also determined. Under these experimental conditions, the total number of leukocytes was reduced from 9440/mm***3 to 5660/mm***3 on day6, mainly because of a decrease inthe lymphocyte population from 5843.2/mm***3 to 2349.0/mm***3 on day 6. No significant differences were observed in the values of erythrocytes, neutrophils,eosinophils,basophils,monocytes,hemoglobin content and microhematocrit. All hematological parameters studied returned tonormal values by day 90 after a single irradiation dose of 100R

Level of fetal hemoglobin as an indicator of clinical complications in sickle cell anemia

The fetal hemoglobin (HbF) level was used as an indicator of the development of severe clinical complications in 89 patients with sickle cell anemia (SCA). HbF was determined by the alkali denaturation technique. The mean HbF level was 6.7 + or - 4.2% (range, 0.7 to 19.2%) of total hemoglobin. Major organ failures were considered to be teerminal events of morbidity and included 8 cerebrovascular accidents, 13 aseptic necroses of the femoral head and 17 leg ulcer episodes. The characteristics of the test, including sensitivity, specificity and positive predictive value were analyzed for different levels of HbF. The overall specificities were 76,76, and 85% for HbF levels >=8,10 and 12%, respectively. The sensitivity of the test was low. The positive predictive value reached 71% for children with HbF >=8%. The data suggest that HbF level may be a useful indicator of the possibility of a patient developing serious clinical complications

Anemia falciforme e gravidez. / Sickle cell anemia and pregnancy

Os autores, analisando 5.876 partos ocorridos na Escola Paulista de Medicina, no periodo de 1978 a 1983, observaram 12 gestacoes em seis pacientes com anemia falciforme, sendo tres em homozigotas e tres em heterozigotas (AS). Todas eram oriundas da raca negra, e o diagnostico foi estabelecido antes da gravidez. A maioria das pacientes nao apresentou alteracoes da intensidade do quadro clinico, contudo, os tres casos homozigotos exibiam sintomatologia mais exuberante. Observam maior numero de intercorrencias clinico-cirurgicas e obstetricas. Apuram 25% de perdas conceptuais representadas por uma gravidez ectopica, um aborto espontaneo e um obito fetal. Nao registram mortalidade perinatal ou materna

Alguns aspectos anatomopatologicos das leucemias. / Anatomopathological aspects of leukemias

Quinze autopsias de pacientes leucemicos foram estudadas. Os principais achados macro e microscopicos sao analisados quanto a frequencia e intensidade de acometimento de diferentes orgaos. As principais causas de obito sao comentadas, bem como a relacao entre terapeutica e o grau de infiltracao leucemica