Daniel Tabak

Granulocytic sarcoma of the small intestine with CBFβ/MYH11 fusion gene: report of an aleukaemic case and review of the literature

Granulocytic sarcomas (GS) are rare extramedullary tumours composed of immature myeloid cells. Inversion of chromosome 16 [inv(16)] is a cytogenetic marker for M4Eo subtype of acute myeloid leukaemia (AML). The possibility of an association between the development of granulocytic sarcoma of the small intestine (GSSI) and the M4Eo subtype of AML was suggested in nine previous case reports. Here we report an aleukaemic case of GSSI with inv(16) and its molecular equivalent, the CBFbeta/MYH11 fusion gene, detected by reverse transcriptase-polymerase chain reaction (RT-PCR), that after treatment with conventional AML chemotherapy followed by autologous bone marrow transplantation, achieved complete haematological and molecular remission on bone marrow examination. After chemotherapy, a thickened ileum wall positive for CBFbeta/MYH11 on tumour mass samples was still observed on computed tomography (CT) studies, raising the question of residual GS representing a reservoir of malignant cells. This case demonstrates the critical need of multidisciplinary diagnosis and follow-up of this entity combining immunopathologic, cytogenetic and molecular studies, reinforcing the potentiality of risk-adapted therapy strategies, as it is increasingly claimed for patients with overt AML.

A randomized, multicenter study of G-CSF starting on day +1 vs day +5 after autologous peripheral blood progenitor cell transplantation.

In order to assess the effect of delaying G-CSF administration after autologous peripheral blood progenitor cell (PBPC) transplantation on the duration of neutropenia, 87 patients were randomized to receive G-CSF 5 μg/kg/day starting on day +1 (n = 45) or +5 (n = 42) following PBPC transplantation, until recovery of the neutrophils. The duration of neutropenia (<0.5 × 109/l) was shorter in the day +1 group (7 vs 8 days; P = 0.02), especially in patients receiving melphalan 200 mg/m2 and CD34+ cell doses >3.0 × 106/kg. These patients had a later onset of neutropenia after transplant. There were no differences in time to neutrophil and platelet engraftment, or in the incidence of fever and documentation of infection. Although the duration of antibiotic therapy (7 vs 10.5 days; P = 0.01) and time to hospital discharge (13 vs 15 days; P = 0.02) were shorter in the day +1 group, these differences could not be predicted by the day of G-CSF initiation in multivariate analysis. Starting G-CSF on day +1 does not result in faster neutrophil engraftment but in later onset and consequently, slightly shorter duration of neutropenia in patients who receive melphalan 200 mg/m2 and CD34+ cell doses >3.0 × 106/kg.

Laboratory strategies for efficient handling of paraffin-embedded tissues for molecular detection of clonality in non-hodgkin lymphomas.

We herein present a technical strategy to optimize DNA isolation from paraffin-embedded tissue (PET). This includes the choice of adequate buffers for proteinase K digestion and multiplex PCR amplifications for assessing the appropriateness of DNA extracts for subsequent PCR assays for detecting clonality. We found that the association of proteinase K digestion in nonionic buffer and subsequent extract dilutions accounted for 79% of successful amplifications. A final efficiency of 88% was achieved by additional organic extractions and/or re-extractions. Comparisons were carried out with control DNA extracts from fresh samples to assess the efficiency of each clonality assay. Immunoglobulin CDRIII rearranged region amplification was more efficient for pregerminal center B-cell lymphomas in contrast to CDRII rearrangement detection, which was more effective for germinal and postgerminal lymphomas. T-cell clonality detection by TCR&ggr; PCR was less efficient in PET samples than in fresh tissues showing that DNA integrity is more critical for TCR than for IGH amplification. Two inconclusive cases without phenotypic markers and two other atypical lymphoproliferations masked by reactive T cells were diagnosed as plasmablastic lymphomas and as monoclonal B-proliferations, respectively, due to IGH rearrangements.

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.

A systematic approach to molecular quantitative determination of mixed chimaerism following allogeneic bone marrow transplantation: an analysis of its applicability in a group of patients with severe aplastic anaemia

Abstract:u2002 Mixed chimaerism (MC) following allogeneic bone marrow transplantation (allo‐BMT) is defined as the persistent cohabitation of haematopoietic cells from recipients and donors. Its kinetics, clinical implications and more efficient laboratory approaches for MC detection are the object of ongoing research in view of the possibility of developing useful markers. Here we describe a sequential analysis of chimaerism using variable number of tandem repeat (VNTR) polymerase chain reaction (PCR) followed by quantitative, fluorescent labelled, short tandem repeat (STR) PCR. A set of four, highly discriminative VNTR and four STR markers was used to assess chimaerism. Sensitivity and regression analysis indicated that this approach was reliable for routine application in a single BMT centre. We studied 12 patients with severe aplastic anaemia (SAA) who had received allo‐BMT, and had been conditioned with cyclosphosphamide (Cy) with or without anti‐thymocyte globulin (ATG). We found a 50% prevalence of MC in the whole group, with levels between 4% and 37% of recipient cells. A sustained stable MC pattern after BMT was characteristic of the Cy‐only conditioned patients but was also recorded in one patient treated with the Cyu2003+u2003ATG regime who showed a sustained MC pattern over a period of 24u2003months post‐BMT. In none of our patients, MC was associated with an increased risk of graft rejection in a median follow‐up of 39.5u2003months.

Quality of life of chronic myeloid leukemia patients in Brazil: ability to work as a key factor

PurposeThe purpose of this study was to evaluate the quality of life (QOL) of patients receiving treatment by the public health system in Brazil for chronic myeloid leukemia (CML), a disease requiring daily and strict compliance to oral medication and regular blood and bone marrow controls, which are invasive exams.MethodsBetween 2008 and 2010, patients with CML were surveyed by telephone. Quality of life was evaluated by the functional assessment of chronic illness therapy (FACIT) tool.ResultsThe mean QOL among CML patients was 92.53 (out of 124 total points) in the trial outcome index, 78.50 (out of 108) in the general total score, and 130.43 (out of 176) in the leukemia total score. Patients who had the prescriptions recently changed anyway had better QOL general score (pu2009=u20090.012) and leukemia-specific score (pu2009=u20090.043) than those who remained with the same treatment. Imatinib was not associated with this change in QOL (pu2009>u20090.797). The more the patient felt able to work, the higher the scores in all three FACIT scales (pu2009<u20090.001, Spearman’s correlation). The use of imatinib (pu2009=u20090.012) was associated with a better ability to work, while chemotherapy (pu2009=u20090.017) and the use of hydroxyurea (pu2009=u20090.001) were inversely associated with work capability.ConclusionsA recent change in medication can improve quality of life. The ability to work is an important component of quality of life of patients with CML. Ability to work should be specifically considered in CML treatment.

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

Patients' perceptions about diagnosis and treatment of chronic myeloid leukemia: a cross-sectional study among Brazilian patients

CONTEXT AND OBJECTIVESnChronic myeloid leukemia (CML) requires strict daily compliance with oral medication and regular blood and bone marrow control tests. The objective was to evaluate CML patients perceptions about the disease, their access to information regarding the diagnosis, monitoring and treatment, adverse effects and associations of these variables with patients demographics, region and healthcare access.nnnDESIGN AND SETTINGnProspective cross-sectional study among CML patients registered with the Brazilian Lymphoma and Leukemia Association (ABRALE).nnnMETHODSnCML patients receiving treatment through the public healthcare system were interviewed by telephone.nnnRESULTSnAmong 1,102 patients interviewed, the symptoms most frequently leading them to seek medical care were weakness or fatigue. One third were diagnosed by means of routine tests. The time that elapsed between first symptoms and seeking medical care was 42.28 ± 154.21 days. Most patients had been tested at least once for Philadelphia chromosome, but 43.2% did not know the results. 64.8% had had polymerase chain reaction testing for the BCR/ABL gene every three months. 47% believed that CML could be controlled, but 33.1% believed that there was no treatment. About 24% reported occasionally stopping their medication. Imatinib was associated with nausea, cramps and muscle pain. Self-reported treatment adherence was significantly associated with normalized blood count, and positively associated with imatinib.nnnCONCLUSIONSnThere is a lack of information or understanding about disease monitoring tools among Brazilian CML patients; they are diagnosed quickly and have good access to treatment. Correct comprehension of CML control tools is impaired in Brazilian patients.

Chromosome 17 abnormalities and mutation of the TP53 gene: correlation between cytogenetics, flow cytometry and molecular analysis in three cases of chronic myeloid leukemia

chronic myeloid leukemia (CML) have been described. This chromosomal region contains the tumor suppressor gene TP53 that may be an important factor in the evolution of this disease. In this study, we used flow cytometry and western blotting to assess p53 protein expression and single stranded conformational polymorphism to examine TP53 gene alterations in three patients with CML who showed alterations in 17p. Only the case with del(17)(p11) had p53 expression positive by flow cytometry and an abnormal migration pattern by SSCP analysis. The importance of the correlation between the results obtained with these techniques, as well as the clinical course of the patients, are discussed.

A Child with Philadelphia Positive (Ph+)-Acute Leukemia with Myeloid Morphology: One Case of Stem Cell Origin

Philadelphia positive (Ph+) acute myeloid leukemia (AML) is a rare and heterogeneous condition, mainly reported in adults, associated to poor prognosis and unfavorable response to therapy. Here we report clinical and laboratory findings in an 8-year-old patient diagnosed with Ph+ acute leukemia with myeloid (FAB M4) morphology. The patient consistently expressed variable levels of m-bcr, e1a2 transcripts during a 42-month follow-up after two different stem cell transplantation protocols. An immunophenotypic switch was documented, from a mixed, myeloid-lymphoid lineage to a full lymphoid phenotype following stem cell transplants, in association with an immature B-cell gene rearrangement profile and clonal instability during clinical progression. This report indicates a stem cell origin as previously suggested for Ph+ AML.