Rita de Cássia Tavares

Estimations of BCR-ABL/ABL transcripts by quantitative PCR in chronic myeloid leukaemia after allogeneic bone marrow transplantation and donor lymphocyte infusion

Serial assays of qualitative (multiplex and nested) and quantitative PCR were carried out for detecting and estimating the level of BCR-ABL transcripts in 39 CML patients following bone marrow transplantation. Seven of these patients, who received donor lymphocyte infusions (DLIs) following to relapse, were also monitored. Quantitative estimates of BCR-ABL transcripts were obtained by co-amplification with a competitor sequence. Estimates of ABL transcripts were used, an internal control and the ratio BCR-ABL/ABL was thus estimated for evaluating the kinetics of residual clones. Twenty four patients were followed shortly after BMT; two of these patients were in cytogenetic relapse coexisting with very high BCR-ABL levels while other 22 were in clinical, haematologic and cytogenetic remission 2-42 months after BMT. In this latter group, seven patients showed a favourable clinical-haematological progression in association with molecular remission while in 14 patients quantitative PCR assays indicated molecular relapse that was not associated with an early cytogenetic-haematologic relapse. BCR-ABL/ABL levels could not be correlated with presence of GVHD in 24 patients after BMT. In all seven patients treated with DLI, high levels of transcripts were detected at least 4 months before the appearance of clinical haematological relapse. Following DLI, five of these patients showed decreasing transcript levels from 2 to 5 logs between 4 and 12 months. In eight other patients studied long after BMT, five showed molecular relapse up to 117 months post-BMT and only one showed cytogenetic relapse. Our findings indicated that quantitative estimates of BCR-ABL transcripts were valuable for monitoring minimal residual disease in each patient.

Survival and graft-versus-host disease in patients receiving peripheral stem cell compared to bone marrow transplantation from HLA-matched related donor: retrospective analysis of 334 consecutive patients

The objective of this study was to compare the major transplant outcomes between patients receiving hematopoietic stem cell transplantation (HSCT) from bone marrow (BM) or peripheral blood stem cells (PBSC).

A Rare Case of Transfusion Transmission of Hepatitis A Virus to Two Patients with Haematological Disease

Background: This paper describes the transmission of hepatitis A virus (HAV) to two blood recipients from a healthy donor that later presented to the blood bank with jaundice. Methods: The RNA of HAV was detected by qualitative nested reverse transcription polymerase chain reaction (nested RT-PCR) and quantified by real-time RT-PCR. HAV RNA samples were genotyped by direct sequencing of PCR products. A sequence from a fragment of 168 bp from the VP1/2A HAV region was used to construct a phylogenetic tree. Case Report: A 31-year-old male donor accepted for donation of a whole blood unit returned to the blood bank with clinical jaundice 20 days after donation. His serological and NAT tests were negative for HBV and HCV. Serological tests for HAV IgM and IgG were negative on donation sample but positive on follow-up sample, confirming donors HAV acute infection. Both recipients of red blood cells (R1) and platelet concentrate (R2) from the same implicated donation were HAV IgM-negative and IgG-positive. Qualitative PCR was positive on samples from all three individuals and phylogenetic analysis of viruses proved HAV transmission to the two recipients of blood products. HAV viral load on donor follow-up sample and the platelet recipient was 1.3 and 1.5 × 103 IU/ml, respectively. The RBC recipient, also infected by HCV, was undergoing bone marrow transplantation and died from fulminant hepatitis, 26 days after the implicated HAV transfusion. Conclusion: The blood donor, a garbage collector, spontaneously returned to the blood bank when developing jaundice. This highlights the importance of donor education to immediately report to blood banks of any signs and symptoms related to infectious disease developed after blood donation. The fact that one immunocompromised patient with HCV infection died from fulminant hepatitis after receiving a HAV-contaminated platelet transfusion underpins the importance of a HAV vaccination program for these group of patients.

A multicenter feasibility study of chronic graft-versus-host disease according to the National Institute of Health criteria: efforts to establish a Brazil-Seattle consortium as a platform for future collaboration in clinical trials.

Background New criteria for the diagnosis and classification of chronic graft-versus-host disease were developed in 2005 for the purpose of clinical trials with a consensus sponsored by the National Institute of Health. Objectives The aim of this study is to present the results of a multicenter pilot study performed by the Brazil-Seattle chronic graft-versus-host disease consortium to determine the feasibility of using these criteria in five Brazilian centers. Methods The study was performed after translation of the consensus criteria into Portuguese and training. A total of 34 patients with National Institute of Health chronic graft-versus-host disease were enrolled in the pilot study between June 2006 and May 2009. Results Of the 34 patients, 26 (76%) met the criteria of overlap syndrome and eight (24%) the classic subcategory. The overall severity of disease was moderate in 21 (62%) and severe in 13 (38%) patients. The median time from transplant to onset of chronic graft-versus-host disease was 5.9 months (Range: 3 - 16 months); the median time for the overlap syndrome subcategory was 5.9 months (Range: 3 - 10 months) and for the classic subcategory, it was 7.3 months (Range: 3 - 16 months). At a median follow up of 16.5 months (Range: 4 - 39 months), overall survival was 75%. Conclusions It was feasible to use the National Institute of Health consensus criteria for the diagnosis and scoring of chronic graft-versus-host disease in a Brazilian prospective multicenter study. More importantly, a collaborative hematopoietic cell transplantation network was established in Brazil offering new opportunities for future clinical trials in chronic graft-versus-host disease and in other areas of research involving hematopoietic stem cell transplantation.

Diretrizes para o diagnóstico, classificação, profilaxia e tratamento da doença enxerto contra hospedeiro crônica

A falta de criterios diagnosticos padronizados, amplamente utilizados, pode comprometer tanto a avaliacao real da incidencia da doenca contra hospedeiro cronica bem como a correlacao de sua gravidade com a taxa de mortalidade pos-transplante. Na I Reuniao de Diretrizes da Sociedade Brasileira de Transplante de Medula Ossea, realizada em junho de 2009, o Grupo de Estudos de DECH Brasil - Seattle (GEDECH), baseado na realidade dos Centros brasileiros, apresentou as recomendacoes para diagnostico, classificacao, profilaxia e tratamento da doenca enxerto contra hospedeiro cronica propostas pelo National Institutes of Health. Estas propostas incluiram padronizacao das caracteristicas utilizadas no diagnostico e ferramentas para a pontuacao dos orgaos envolvidos e avaliacao global da gravidade a serem utilizados em estudos clinicos da doenca enxerto contra hospedeiro cronica. Estes criterios sao uteis para uma melhor analise da incidencia desta doenca, alem de poder avaliar a gravidade do comprometimento de um orgao ou sitio envolvido e a influencia na mortalidade tardia do transplante. A profilaxia e os tratamentos propostos para esta importante complicacao dos transplantes de celulas-tronco hematopoeticas foram discutidos e graduados de acordo com niveis de evidencia estabelecidos pelo National Institutes of Health.

Karyotype abnormalities and their clinical significance in a group of chronic myeloid leukemia patients treated with hematopoietic stem cell transplantation

CONTEXT AND OBJECTIVE Following hematopoietic stem cell transplantation (HSCT), karyotyping is a valuable tool for monitoring engraftment and disease status. Few studies have examined the prognostic significance of karyotypes in patients who underwent HSCT for chronic myeloid leukemia (CML). The objective of this study was to evaluate the significance of pretransplantation cytogenetic status in relation to outcomes following HSCT in CML patients. DESIGN AND SETTING Case series study at Instituto Nacional do Câncer (INCA), Rio de Janeiro, Brazil. METHODS Cytogenetic analysis was performed by G banding on 39 patients treated with HSCT. RESULTS Thirty-one patients were in the chronic phase and eight were in the accelerated phase. Prior to HSCT, additional chromosomal abnormalities on the Philadelphia (Ph) chromosome were found in 11 patients. The most frequent additional abnormality was a double Ph, which was observed in four cases. Following HSCT, full chimeras were observed in 31 patients (79.5%). Among these, 23 (82.3%) had presented Ph as the sole abnormality. Mixed chimeras were observed in seven patients, of which three had additional abnormalities. Only one case did not present any cytogenetic response. Five patients presented cytogenetic relapse associated with clinical relapse following HSCT. Twenty-seven patients are still alive and present complete hematological and cytogenetic remission. CONCLUSION In our study, the presence of additional abnormalities was not associated with worse outcome and relapse risk. Also, no differences in survival rates were observed. Our study supports the view that classical cytogenetic analysis remains an important tool regarding HSCT outcome.

Brazilian workshop model to train investigators in chronic graft-versus-host disease clinical trials according to the 2005-2006 National Institutes of Health recommendations

Background The lack of standardization of clinical diagnostic criteria, classification and severity scores of chronic graft-versus-host disease led the National Institutes of Health to propose consensus criteria for the purpose of clinical trials. Method Here we describe a one-day workshop model conducted by the Chronic Graft-versus-Host Disease Brazil-Seattle Consortium Study Group to train investigators interested in participating in multicenter clinical trials in Brazil. Workshop participants included eight transplant physicians, one dermatologist, two dentists, three physical therapists and one psychologist from five institutions. Workshop participants evaluated nine patients with varying degrees of severity of mucocutaneous lesions and other manifestations of the disease followed by a training session to review and discuss the issues encountered with the evaluation and scoring of patients and in the methods used to evaluate grip strength and the 2-minute walk test. Results Most participants had difficulties in rating the percentage of each type of mucocutaneous lesion and thought 20 minutes was insufficient to evaluate and record the scores of each patient using the National Institutes of Health criteria and other cutaneous assessments. Several specific areas of difficulties encountered by the evaluators were: 1) determining the percentage of erythema in movable and non-movable sclerosis, 2) whether to score all cutaneous findings in a particular area or just the dominant lesion; 3) clarification of the definition of poikiloderma in chronic graft-versus-host disease; 4) discrepant interpretation of the mouth score and 5) clarification on the methodology used for the evaluation of grip strength and the 2-minute walk tests. Conclusions Results of this workshop support the need to train investigators participating in clinical trials on chronic graft-versus-host disease.

Parvovirus B19 in the Context of Hematopoietic Stem Cell Transplantation: Evaluating Cell Donors and Recipients

Background Parvovirus B19 (B19V) is a common human pathogen, member of the family Parvoviridae. Typically, B19V has been found to infect erythroid progenitors and cause hematological disorders, such as anemia and aplastic crisis. However, the persistence of genomic deoxyribonucleic acid (DNA) has been demonstrated in tonsils, liver, skin, brain, synovial, and testicular tissues as well as bone marrow, for both symptomatic and asymptomatic subjects. Although the molecular and cellular mechanisms of persistence remain undefined, it raises questions about potential virus transmissibility and its effects in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Methods With this aim, we retrospectively screened allogeneic stem cell donors from 173 patients admitted for allo-HSCT from January 2008 to May 2013 using a seminested polymerase chain reaction approach. Results We found 8 positive donor samples, yielding a 4.6% of parvovirus prevalence (95% confidence interval, 2.36-8.85). Pre- and post-HSCT samples (n = 51) from the 8 recipients of the positive donors were also investigated, and 1 case exhibited B19V DNA in the post-HSCT follow-up (D + 60). Direct DNA sequencing was performed to determine the genotype of isolates and classification, performed by phylogenetic reconstruction, showed a predominance of genotype 1a, whereas the rare genotype 3b was detected in 2 additional patients. By molecular cloning, different B19V 1a substrains polymorphisms were evidenced in the single case in which donor and its recipient were B19V+. Conclusions Our results suggest that HSCT allografts are not a main source for B19V transmission, pointing to potential events of reinfection or endogenous viral reactivation.

Hyperdiploid karyotype in a child with hypocellular primary myelodysplastic syndrome.

To the Editor: Myelodysplastic syndrome (MDS) is unusual in childhood (1). Monosomy 7 is the most common acquired chromosomal abnormality in children with MDS (2). In this report, we describe a rare case of hyperdiploid karyotype in a child with hypocellular primary MDS, classified as refractory cytopenia (RC) (2) and submitted to bone marrow transplant (BMT). Cytogenetic and immunophenotyping studies were performed and their values in diagnosis and prognosis are discussed. A 16-year-old girl was referred in August 1999 to the Bone Marrow Unit, CEMO-INCA (Rio de Janeiro) with the suspicion of MDS-RC. She presented pancytopenia and hypocellularity of bone marrow showing megaloblastoid maturation. She was treated with vitamin B12 and folate with no response. In January 2000, a cytogenetic study revealed a normal karyotype. Granulocyte colonystimulating factor (G-CSF) was administered without success. In March 2001, the patient was indicated for human leucocyte antigen (HLA)identical sibling BMT. In order to choose the conditioning regimen, the diagnosis was discussed between hypoplastic MDS and aplastic anaemia (AA). Other laboratory tests were performed. Bone marrow analysis showed hypoplasia with the mielogram revealing some dysplastic features. Bone marrow biopsy revealed hypocellularity, with a decrease of erythroid cells with megaloblastoid changes, a decrease of granulocytic cells and the presence of dysmorphic megakaryocytes. The abnormal localisation of immature progenitor cells (ALIP) was not present. Cytogenetic analysis of bone marrow cells after GTG banding showed 41 normal cells (93%) and three (7%) with a hyperdiploid karyotype 51,XX,+4,+6,+8,+14,+20, according to the International System of Human Cytogenetic Nomenclature (3). Immunophenotyping was performed. A panel of the following directly conjugated antibodies was used: CD45, CD4, CD8, CD2, CD3, CD19, CD10, CD33, CD34, CD61, CD7, anti-HLA-Dr (Becton & Dickinson, San Jose, CA, USA). The immunophenotypic abnormalities observed in this patient were: hypogranular neutrophils demonstrated by CD45 vs. side light scatter, CD10 granulocytes and myeloid lineage expressing non-myeloid antigens such as CD2. The number of megakaryocytes detected by CD61 cells was 16.59%. This value is considered increased according to Stetler-Stevenson et al. (4) and are compatible with MDS patients. The percentage of CD34 cells was 1.72%. Based on the morphological, immunophenotypic and cytogenetic studies the final diagnosis was MDSRC. Her sister’s bone marrow was infused after conditioning with busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg). Graft-vs.-host disease (GVHD) prophylaxis consisted of cyclosporin A (CSA) (3 mg/kg from day 1) and methotrexate (15 mg/m on day +1 and 10 mg/m days +3 and +6) post-transplant. Neutrophil and platelet engraftment were achieved on days 17 and 20, respectively. She developed acute grade II GVHD (skin and liver) at day 42. Chronic progressive GVHD was diagnosed at her 11th month posttransplant along with liver dysfunction. Prednisone, CSA and mycophenolate mophetil were used. She had normalisation of hepatic enzymes. The patient is now 27 months post-transplant and remains in cytogenetic remission and complete donor chimaerism. The present case has proved to be peculiar in different aspects. In childhood MDS, Acar et al. described the first case of hyperdiploid karyotype in a patient (6-month-old boy) who had congenital anomalies, hypercellular bone marrow and classified as refractory anaemia with excess of blasts (RAEB) (5). Hyperdiploid karyotype in MDS was also described in a young woman (27-year old) with hypercellular bone marrow (6). According to literature, the present case represents the first case of paediatric MDS without congenital anomalies showing a hyperdiploid karyotype in a hypocellular Eur J Haematol 2003: 71: 399–401 Printed in UK. All rights reserved Copyright Blackwell Munksgaard 2003

Endocrine dysfunctions in adults post hematopoietic stem cell transplantation

Introduction: Hematopoietic stem cell transplantation (HSCT) is the standard treatment for some malignancies and hematological diseases. Many patients/year undergoes this procedure and were at high risk of development long-term complications, including endocrine dysfunctions. The aim of this study is to evaluate the frequency and nature of endocrine disorders in adult patients undergoing HSCT in Brazil. Materials and methods: Retrospective study from medical records of 494 post-HSCT patients, referred for follow-up in the Endocrinology Clinic of the National Cancer Institute (INCA). Results and discussion: Three hundred forty-two patients met criteria for inclusion, of these 52.3% had endocrine complications post-HSCT. The main endocrine dysfunction was hypogonadism, followed by hypothyroidism, hypocortisolism and hypopituitarism. Graft-versus-host disease was diagnosed in 42.4% of total population. Seventy-three (21.3%) patients of the cohort died during follow-up. Endocrine dysfunctions are common complications of HSCT. Early diagnosis and treatment can improve the quality of life and morbidity. Correspondence to: Carla Fernanda Nava, Praça da Cruz Vermelha, 23 Centro; CEP 20230-130 – Rio de Janeiro – Brazil, Tel: 55 021 3207-4625; E-mail: carlafernandanava@hotmail.com