Hilda Rachel Diamond

Age-related changes in natural killer cell receptors from childhood through old age

Most studies on natural killer (NK) cells and aging have focused on overall cell numbers and global cytotoxic activity. NK cell functions are controlled by surface receptors belonging to three major families: killer cell immunoglobulin-like receptors (KIRs), natural cytotoxicity receptors (NCRs), and C-type lectins. The expression of these receptors was investigated from childhood through old age in T, NKT- and NK cells and also in the CD56(dim) (cytotoxic) and CD56(bright) (responsible for cytokine production) NK cell subsets. A decrease in the expression of activating receptors (NKp30 and NKp46) was observed in NK cells in elderly individuals. KIR expression was increased only in the CD56(bright) subset. Children presented similar results regarding expression of NKp30 and KIR, but not NKp46. NKG2D expression was decreased in T cells of elderly subjects. Analysis of KIR genotype revealed that KIR2DL5 and KIR2DS3 were significantly associated with old age. Cytotoxic activity was preserved from childhood through old age, suggesting that the increase of the absolute number of CD56(dim), observed in elderly, may represent a compensatory mechanism for the receptor expression alterations. This initial study provides the framework for more focused studies of this subject, which are necessary to determine whether the changing balance of NK receptor expression may influence susceptibility to infectious, inflammatory, and neoplastic diseases.

Cytogenetic and immunophenotypic evidence of independent clonal origins of concomitant chronic lymphocytic leukaemia and acute myeloid leukaemia

To the Editor: Although chronic B-cell leukaemia (B-CLL) is the most common leukaemia in the Western world, the association of CLL and acute leukaemia (AL) is a rare event. The presentation of acute myeloid leukaemia (AML) concomitantly with CLL is unusual and, so far, only 16 cases have been described (1). In the most of these cases the characterization of the diseases has been made by morphology and immunophenotyping studies (1–4), and only in two cases was a cytogenetic study carried out (5, 6). We report a case with the simultaneous occurrence of CLL and AML without previous exposure to a cytotoxic agent or irradiation. Immunophenotyping and cytogenetic studies were performed, and the clonal origins of the diseases are discussed. A 70-yr-old Portuguese woman was hospitalized with a one-month history of daily fever (38–39 uC), gum bleeding, an episode of epistaxis and weight loss. Physical examination was significant for cervical and inguinal lymphadenopathy, gum hypertrophy, and an enlarged liver and spleen (6 and 3 cm, respectively). The patient also had high arterial blood pressure and congestive heart failure. The leukocyte count was 61,600 cells/mm with 31% blast cells and 59% lymphocytes. Five months before this admission her haemogram had been normal. A bone marrow (BM) aspirate revealed approximately 27% blast cells and 49% apparently mature lymphocytes. Cytochemical analysis revealed the blastic cells to be positive Sudan black. The great majority of cells were negative to PAS, and few cells were weakly positive; a-naphtyl butyrate was also negative. LDH measured 5261 UI. Immunophenotyping studies showed the presence of two cell populations with different light-scatter in BM. Large cells which corresponded to blast cells had an immature myeloid immunophenotype, (HLA-Dr, CD13, CD33, CD34), and small cells, corresponding to a mature B cell population, displayed a typical B-cell phenotype (CD19, CD5, HLA-DR, weak SMIg and K). ANLL-M2 was diagnosed. Hydroxyurea 1.5 g/d was started. The number of blast cells was reduced in subsequent days, although the total leukocyte count had increased due to an increase in the absolute number of lymphocytes (93,000 cells/mm in 5 d with 93% lymphocytes and 5% blasts). The patient died on the tenth day of hospitalization due to complications of both metabolic functions and infection. A pathologic leg fracture was also report-ed. The karyotype of bone marrow cells was obtained after cultures in RPMI 1640 with 20% foetal calf serum (Gibco) and pokeweed mitogen at 37 uC for 72 h. Cell cultures were pulsed with colcemid (0.06 mg/ml) in the last hour of incubation. Cells were subsequently harvested by standard procedures (hypotonic shock with 0.075 M KCl) and fixed in methanol–acetic acid (3:1). GTG banding was per-formed as described by Seabright (7), and chromosomes were identified and arranged according to the International System for Cytogenetic Nomenclature (8). The cytogenetic analysis showed the presence of two abnormal clones: 47,XX,+12 [10]/46,XX,del(5)(q31),t(8;13) (q22;q21) [4]/46,XX [6]. Although other cases of CLL associated with AML have been reported (1–6), the present study showed some peculiarities. There were no data suggesting the presence of AML after the diagnosis of CLL, since the patient had received no prior chemotherapy or radiotherapy and the haemogram obtained 5 months before had been normal. The cytogenetic study showed two abnormal clones, and as yet only two cases reporting concomitant CLL and AML included cytogenetic studies (5, 6). The patient described by Lima et al. (5) had chromosome aberrations commonly associated with CLL and AML in the same metaphases, suggesting that both diseases might be derived from a single cell clone. The cytogenetic analysis for the case reported by Mateu Eur J Haematol 2001: 66: 281–283 Printed in UK. All rights reserved Copyright # Munksgaard 2001

Acute myeloid leukemia of donor origin after allogeneic stem cell transplantation from a sibling who harbors germline XPD and XRCC3 homozygous polymorphisms

A 54-year-old woman was diagnosed with infiltrative ductal breast carcinoma. Two years after treatment, the patient developed an acute myeloid leukemia (AML) which harbored del(11q23) in 8% of the blast cells. The patient was submitted for allogeneic stem cell transplantation (aSCT) from her HLA-compatible sister. Ten months after transplantation, she relapsed with an AML with basophilic maturation characterized by CD45low CD33high, CD117+, CD13-/+, HLA Drhigh, CD123high, and CD203c+ blast cells lacking expression of CD7, CD10, CD34, CD15, CD14, CD56, CD36, CD64, and cytoplasmic tryptase. Karyotype analysis showed the emergence of a new clone with t(2;14) and FISH analysis indicated the presence of MLL gene rearrangement consistent with del(11q23). Interestingly, AML blast cell DNA tested with microsatellite markers showed the same pattern as the donors, suggesting that this AML emerged from donor cells. Additionally, polymorphisms of the XPA, XPD, XRCC1, XRCC3 and RAD51 DNA repair genes revealed three unfavorable alleles with low DNA repair capacity.In summary, we report the first case of AML involving XPD and XRCC3 polymorphisms from donor origin following allogeneic stem cell transplantation and highlight the potential need for careful analysis of DNA repair gene polymorphisms in selecting candidate donors prior to allogeneic stem cell transplantation.

Immunological recovery after bone marrow transplantation for severe aplastic anaemia: a Brazilian experience.

Abstract: Twenty‐nine patients with severe aplastic anaemia (SAA) were submitted to bone marrow transplantation (BMT) and their immunological recovery analysed. Total lymphocyte counts, estimation of B lymphocytes, T lymphocytes and their subsets, natural‐killer (NK) activity were performed. Cells with the CD8 + phenotype and NK activity were the first signs of immunological recovery, whereas the CD4 + subset recovered later in patients who suffered from acute graft versus host disease (GvHD) and infections. Acute and chronic GvHD, cirrhosis, rejection and HIV viral infection contributed to the persistence of the profound immunodeficiency status observed after BMT. Our results did not differ greatly from the others and confirmed that BMT may be performed in underdeveloped countries despite the difficulties it might pose.

Study of Natural Cytotoxicity Receptors in Patients with HIV/AIDS and Cancer: A Cross-Sectional Study

The NCR receptors play a fundamental role in the cytotoxicity mediated by NK cells against tumor cells. In the current study, we investigated possible HIV/AIDS-related changes in the expression of the NCR receptors comparing healthy donors, HIV/AIDS patients, and HIV/AIDS patients with cancer (HIV/AIDSWC). The NCRs were quantified in NK cells (NKdim and NKbright) and T lymphocytes from peripheral blood samples by flow cytometry. We found a significant decrease in the frequency of NK cells expressing NKp46 in HIV/AIDS group (p = 0.0012). There was a decrease in the frequency of NK cells expressing NKp46 in the HIV/AIDSWC group; however, this was not statistically significant. We found a significant decrease in the frequency of NK cells expressing NKp30 in the HIV/AIDS group (p = 0.0144). There was a decrease in the frequency of NK cells expressing NKp30 and in the HIV/AIDSWC group, but this was not statistically significant. There were no changes in the distribution of NK cells and their subtypes in both groups.

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

Flow cytometry as a diagnostic support tool in juvenile myelomonocytic leukemia.

Th e diagnosis of juvenile myelomonocytic leukemia (JMML) follows a diffi cult question in pediatric hematology [1]. No consistently recurring cytogenetic abnormalities are reported in JMML, and normal karyotypes are found in 40 – 70% of patients. Monosomy 7, del(7q) or other abnormalities of chromosome 7 have been reported in ∼ 25% of cases [2,3]. Mutations in NRAS, NF1 and PTPN11 genes are found in ∼ 20 – 35% of JMML patients and are mutually exclusive; thus, overall, nearly 75% of patients with JMML have one of these abnormalities [3,4]. Although these molecular abnormalities are of help in the diagnostic workup of JMML, they are not pathognomonic, since these abnormalities are also found in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and others myelodysplastic syndrome/myeloproliferative disorders (MDS/ MPL) [3]. Moreover, these genetic tests are not widely available for clinical use. In addition, no specifi c phenotypic abnormalities have been reported in JMML to date [3]. Based on this, new diagnostic tools are urgently required to improve patient care. For several decades now, fl ow cytometry (FCM) immunophenotyping has been shown to be essential for rapid diagnosis, classifi cation and monitoring of most hematological malignancies [5,6] and also holds promise for the diagnosis of pediatric solid tumors [7]. However, the role of FCM in the diagnosis of JMML has been restricted to blast cell compartment analysis. Our aim was to identify abnormalities in the relative distribution and phenotypic characteristics of the diff erent compartments of hematopoietic cells that could contribute to the diagnosis of JMML. Here, we describe recurrent immunophenotypic abnormalities in three children ( � 22 months of age) with JMML that were referred to our hospital to investigate intermittent fever due to recurrent episodes of severe infections. Th e study was approved by the Ethical Committee from IPPMG/UFRJ and is in accordance with the Helsinki Declaration. Clinical and laboratory fi ndings are summarized in Table I. In all cases, fi nal diagnosis of JMML was established based on World Health Organization (WHO) 2008 criteria. Th e identifi cation, quantifi cation and characterization of hematopoietic cells were done by FCM on bone marrows (BM, all cases) and also on peripheral blood (PB) from case#1 at diagnosis and evolution. Briefl y, all samples were stained with the following three-color combinations of antibodies (all from BD Biosciences) conjugated with fl uorescein isothiocyanate (FITC)/phycoerythrin (PE)/perid inclorophyll proteincyanine 5.5 (PerCP-Cy5.5): CD19/CD10/CD45; CD34/ HLA-DR/CD45; CD15/CD34/CD45; CD7/CD34/CD45; CD7/CD117/CD45; CD13/CD11b/CD45; Cy MPO/CD13/ CD45; CD36/CD64/CD45; CD14/CD33/CD45; CD14/ HLA-DR/CD45; CD4/CD33/CD45, CD4/CD13/CD45. Th e samples (50 μ l per tube) were incubated for 15 min at room temperature in the dark, in the presence of saturating amounts of each of the above-mentioned monoclonal antibodies (MAb). Afterward, 2 ml of FACS lysing solution (BD) diluted 1:10 (v/v) in distilled water was added and the samples were incubated for another 10 min under the same conditions as those mentioned above. Th en, cells were centrifuged (5 min at 540 g) and the cell pellet was washed with 2 ml of PBS BSA 0.5%. Finally, cells were resuspended in 0.4 ml of PBS BSA 0.5%. Stained cells were acquired at low speed in a FACSCalibur fl ow cytometer (BD) using the

Transient myelodysplasia in an infant with Down syndrome preceding acute megakaryoblastic leukemia: cytogenetic and immunophenotypic findings

The incidence of leukemia in children with Down syndrome is 10to 20-fold higher than in other children. The Down syndrome group exhibit a 46-fold excess incidence of acute myeloid leukemia (AML), with acute megakaryoblastic leukemia (AML-M7) accounting for at least 50% of these cases. A myelodysplastic syndrome (MDS) generally precedes this malignancy [1,2]. The clinical course of MDS can vary from stable disease to rapid progression into acute leukemia. Spontaneous remission of MDS has rarely been observed. This phenomenon is extremely heterogeneous, perhaps related to differing pathogenesis of disease [3,4]. Here, we describe an uncommon case of evolution from primary MDS to AML-M7 in an infant with Down syndrome who showed spontaneous remission during MDS phase. An 8-month-old girl with Down syndrome, interventricular cleft and patent arteriosus duct was referred to our center because of thrombocytopenia in a preoperative blood count (platelet count, 46 10/L). The white blood cell count was 4 10/L, hemoglobin level was 11.7 g/dL. Analysis of bone marrow (BM) smear and histopathologic examination of BM fragment revealed a hypocellular morphology with dysplastic features. Cytogenetic analysis of bone marrow cells with GTG banding revealed the karyotype as 47,XX,þ21c[20]. Analysis of a peripheral blood cell culture stimulated by phytohemagglutinin confirmed the constitutional abnormality, with trisomy 21 observed in 25 cells. Chromosomes were identified and analyzed in accordance with ISCN 2005 [5]. Thrombocytopenia was maintained (platelet count, 49 10/L), and white blood cell count and hemoglobin levels had decreased to 2 10/L and 9 g/dL, respectively. Analysis of a new BM aspirate revealed an increased cellularity, with 12% myeloblasts, compatible with refractory anemia with excess of blasts (RAEB). Cytogenetic analysis of bone marrow cells with GTG banding showed trisomy of chromosome 21 in 25 metaphases as the sole anomaly. Flow cytometry showed 8% blasts positive for CD34, CD7, CD117, and CD33, with CD10 negative granulocytes. After a 10-month history of MDS, the patient presented spontaneous remission and the hemogram normalized, with 300 10/L platelets. She was submitted to cardiac surgery under extracorporeal circulation with success and without

Immunophenotypic analysis and evaluation of nutritional status in childhood acute lymphoblastic leukemia

Abstract The cure rate for childhood acute lymphoblastic leukemia (ALL)differs between developed and developing countries. In developingcountries there is a high prevalence of malnutrition thus it isimportant to evaluate the association between factors of nutritionand ALL prognosis, as well as to identify the prevalence ofimmunophenotypes and their association with nutritional status.Eighty-six children with acute lymphoblastic leukemia diagnosedin two universities in Rio de Janeiro were studied. The frequenciesof each immunological subtype were: common ALL 57%, pre-B9.3%; pro-B 8.1%; T-ALL 18% and biphenotypic ALL 7.0%. Itwas noticed that the typical incidence peak of common ALL isbetween 1 and 6 years old. The small number of malnourishedchildren did not allow statistical analysis to compare data betweenthe immunophenotype and nutritional status. For the same reason,a statistical approach comparing malnutrition status with com-plete remission and relapse rates was impaired. The relativeincidence of each immunological subtype was similar to thosefound in developed countriess. Rev. Bras. Hematol. Hemoter.2008;