Mihoko Yamamoto

SAGE analysis highlights the importance of p53csv, ddx5, mapkapk2 and ranbp2 to multiple myeloma tumorigenesis

Serial analysis of gene expression (SAGE) allows a comprehensive profiling of gene expression within a given tissue and also an assessment of transcript abundance. We generated SAGE libraries from normal and neoplastic plasma cells to identify genes differentially expressed in multiple myeloma (MM). Normal plasma cells were obtained from palatine tonsils and MM SAGE library was generated from bone marrow plasma cells of MM patients. We obtained 29,918 SAGE tags from normal and 10,340 tags from tumor libraries. Computer-generated genomic analysis identified 46 upregulated genes in the MM library. Ten upregulated genes were selected for further investigation. Differential expression was validated by quantitative real-time PCR in purified plasma cells of 31 patients and three controls. P53CSV, DDX5, MAPKAPK2 and RANBP2 were found to be upregulated in at least 50% of the MM cases tested. All of them were also found upregulated in MM when compared to normal plasma cells in a meta-analysis using ONCOMINE microarray database. Antibodies specific to DDX5, RANBP2 and MAPKAPK2 were used in a TMA containing 57 MM cases and confirmed the expression of these proteins in 74%, 96%, and 21% of the MM samples, respectively. Analysis of differential expression using SAGE could identify genes important for myeloma tumorigenesis (P53CSV, DDX5, MAPKPK2 and RANBP2) and that could potentially be useful as therapeutic targets.

Human neutrophil alloantigens systems

Neutrophil alloantigens are involved in a variety of clinical conditions including immune neutropenias, transfusion-related acute lung injury (TRALI), refractoriness to granulocyte transfusions and febrile transfusion reactions. In the last decade, considerable progress has been made in the characterization of the implicated antigens. Currently, seven antigens are assigned to five human neutrophil antigen (HNA) systems. The HNA-1a, HNA-1b and HNA-1c antigens have been identified as polymorphic forms of the neutrophil Fcgamma receptor IIIb (CD16b), encoded by three alleles. Recently, the primary structure of the HNA-2a antigen was elucidated and the HNA-2a-bearing glycoprotein was identified as a member of the Ly-6/uPAR superfamily, which has been clustered as CD177. The HNA-3a antigen is located on a 70-95 kDa glycoprotein; however, its molecular basis is still unknown. Finally, the HNA-4a and HNA-5a antigens were found to be caused by single nucleotide mutations in the alphaM (CD11b) and alphaL (CD11a) subunits of the leucocyte adhesion molecules (beta2 integrins). Molecular and biochemical characterization of neutrophil antigenshave expanded our diagnostic tools by the introduction of genotyping techniques and immunoassays for antibody identification. Further studies in the field of neutrophil immunology will facilitate the prevention and management of transfusion reactions and immune diseases caused by neutrophil antibodies.

Expression levels of CD47, CD35, CD55, and CD59 on red blood cells and signal-regulatory protein-α,β on monocytes from patients with warm autoimmune hemolytic anemia

BACKGROUND: Animal models have shown that CD47‐deficient mice develop severe autoimmune hemolytic anemia (AIHA) because the binding of red blood cell (RBC) CD47 to signal‐regulatory protein (SIRP‐α) on macrophages contributes to the inhibition of phagocytosis. In contrast, complement‐inhibitory proteins such as CD35, CD55, and CD59 may protect RBCs against the lysis by complement.

Combined flow cytometric assessment of CD45, HLA-DR, CD34, and CD117 expression is a useful approach for reliable quantification of blast cells in myelodysplastic syndromes†

Quantification of bone marrow (BM) blasts by cytomorphology is essential for the diagnosis of myelodysplastic syndromes (MDS). Owing to its subjectivity and the potential impact of dysplastic features on accurate identification of blast cells, more objective approaches are required, multiparameter flow cytometry (MFC) being a particularly promising approach in this regard. However, no consensus exists about the optimal combination of markers and strategy to be used.

Molecular studies reveal that A134T, G156A and G1333A SNPs in the CD177 gene are associated with atypical expression of human neutrophil antigen-2.

Background and Objectives  The human neutrophil antigen‐2 (HNA‐2) is expressed on a subpopulation of neutrophils as most subjects present a negative plus a positive HNA‐2 population of neutrophils. The number of neutrophils expressing HNA‐2 is variable and may increase in pregnancy, infections, myeloproliferative disorders and after G‐CSF. This study investigated the presence of polymorphisms in the gene encoding HNA‐2 (CD177) in individuals presenting different patterns of antigen expression and determined the association of single nucleotide polymorphisms (SNPs) with the heterogeneous HNA‐2 expression.

Altered immunophenotypic features of peripheral blood platelets in myelodysplastic syndromes

Background Multiparameter flow cytometric analysis of bone marrow and peripheral blood cells has proven to be of help in the diagnostic workup of myelodysplastic syndromes. However, the usefulness of flow cytometry for the detection of megakaryocytic and platelet dysplasia has not yet been investigated. The aim of this pilot study was to evaluate by flow cytometry the diagnostic and prognostic value of platelet dysplasia in myelodysplastic syndromes. Design and Methods We investigated the pattern of expression of distinct surface glycoproteins on peripheral blood platelets from a series of 44 myelodysplastic syndrome patients, 20 healthy subjects and 19 patients with platelet alterations associated to disease conditions other than myelodysplastic syndromes. Quantitative expression of CD31, CD34, CD36, CD41a, CD41b, CD42a, CD42b and CD61 glycoproteins together with the PAC-1, CD62-P, fibrinogen and CD63 platelet activation-associated markers and platelet light scatter properties were systematically evaluated. Results Overall, flow cytometry identified multiple immunophenotypic abnormalities on platelets of myelodysplastic syndrome patients, including altered light scatter characteristics, over-and under expression of specific platelet glycoproteins and asynchronous expression of CD34; decreased expression of CD36 (n=5), CD42a (n=1) and CD61 (n=2), together with reactivity for CD34 (n=1) were only observed among myelodysplastic syndrome cases, while other alterations were also found in other platelet disorders. Based on the overall platelet alterations detected for each patient, an immunophenotypic score was built which identified a subgroup of myelodysplastic syndrome patients with a high rate of moderate to severe alterations (score>1.5; n=16) who more frequently showed thrombocytopenia, megakaryocytic dysplasia and high-risk disease, together with a shorter overall survival. Conclusions Our results show the presence of altered phenotypes by flow cytometry on platelets from around half of the myelodysplastic syndrome patients studied. If confirmed in larger series of patients, these findings may help refine the diagnostic and prognostic assessment of this group of disorders.

Acute myeloid leukemia in elderly patients: experience of a single center

Acute myeloid leukemia (AML) is a disease predominantly of older adults. Treatment of AML in the elderly is complicated not only by comorbidities but also by the high prevalence of poor prognosis markers. Thirty-one consecutive unselected patients with AML older than 60 years (representing 33% of all AML cases diagnosed at our institution during the same period) were followed over a period of 5 years (1997-2002). A high incidence of AML with multilineage dysplasia (45%) and no favorable cytogenetic abnormalities but 62% intermediate and 38% unfavorable karyotypes were found. Sixteen patients (52%) were selected for induction of intensive cytotoxic treatment and complete remission was achieved only by some of these intensively treated patients (7 of 16). Of these, 3 remained alive without disease (median: 11 months), 1 patient died shortly after complete remission, and 3 patients relapsed and died from refractory disease. Only 1 patient that was refractory to intensive cytotoxic treatment remained alive with disease under supportive care. Fifteen patients (48%) were managed with palliative/supportive care: 7 received palliative treatment and supportive care, 8 received supportive care only, and 4 patients remained alive with disease under supportive care (median: 9 months). Mortality rate was 74% and overall survival at two years was 12%. To the best of our knowledge, there is no previous report regarding elderly patients with AML in Brazilian subsets. The present data are similar to previously reported studies showing that elderly AML patients are not only older but also biologically distinct from younger AML patients, particularly in terms of the high incidence of poor prognostic karyotypes and resistance to therapy.

Can thalidomide be effective to treat plasma cell leptomeningeal infiltration

To the Editor: Dear Sir, In May 2000, a previously healthy, 52-yr-old woman was diagnosed with Salmon-Durie stage IIIA, IgG j multiple myeloma (MM). She was initially treated with six cycles of intravenous (i.v.) vincristine 0.4 mg days 1–4; i.v. doxorubicin 9 mg/m days + 198 1–4; P. O dexamethasone days 1–4, 9–12, 17–20 (VAD) every 4 weeks), the last interrupted because of Staphylococcus aureus endocarditis. At this time, during the infection treatment, she developed radicular symptoms associated with a paravertebral mass identified on computed tomography scan. The biopsy ruled out an abscess and showed a plasmacytoma. The patient underwent lumbar spinal irradiation (4000 cGy) followed by six cycles of i.v. cyclophosphamide 750 mg/m day 1: i.v. doxorubicin 50 mg/m day 1; vincristine 1, 4 mg/m day 1; P. O prednisone 60 mg/m days 1–5 (CHOP), achieving complete clinical response. In December 2000, few days before the harvest of peripheral stem-cell for autologous transplantation, she was confused and hemiparetic on physical examination. A cranial magnetic resonance imaging showed a left parietal mass. Cerebrospinal fluid (CSF) showed 250 cells/ mm, with 100% of plasma cells (Fig. 1). This finding was confirmed by flow cytometry analysis of CSF (CD45–, CD38+, j+). There were no other signs of systemic activity of the disease (no serum or urinary M protein, <5% plasma cells in the bone marrow). She was treated on oral dexamethasone (40 mg on days 1–4, 9–12, 17–20 with interval doses of 16 mg/d) and three doses of weekly intrathecal chemotherapy (methotrexate 12 mg and dexamethasone 2 mg). Despite a brief period of symptomatic relief, there was no CSF cleansing (CSF on 11/30/2001: 640 cells/mm, 100% plasma cell; CSF on 02/14/2002: 80 cells/ mm, 100% plasma cells) and the neurologic symptoms ended up progressing (see Table 1). The patient was then treated with thalidomide (Thal) 800 mg/d for 30 d with simultaneous cranial radiation, achieving total dose of 1000 cGy. Radiotherapy was interrupted because the patient’s focal deficits worsened and her mental status deteriorated, resulting in progression to death 3 months after detection of the central nervous system involvement. In this study, we describe a very unusual complication in MM with 53 cases previously related in the literature. Meningeal myelomatosis, defined as meningeal involvement by plasma cells in the CSF, although might be a presenting feature, has usually been described in the setting of pre-existing MM (1). It tends to occur in stage III disease and is associated with plasma cell leukemia in 20% of patients. Few patients were reported to relapse with meningeal compromising and limited disease outside the central nervous system (CNS) (2). We questioned the role of the paraspinal plasmacytoma as the seeding source of plasma cells into CSF (3). As the meningeal myelomatosis represents a dismal event, with 1– 2 months median survival despite aggressive local treatment, associated or not with systemic therapy (1), we tried to treat this patient with Thal and radiotherapy following intrathecal chemotherapy and high dose glucocorticoid. We were expecting a better outcome than that previously reported, assuming the usefulness of Thal in relapsed/ refractory MM patients (4), the high angiogenesis Fig. 1. Cytospin of CSF sample showing 100% of dysplasic plasmocytes, confirming the diagnosis of leptomenigeal infiltration in our patient, ·1000. Eur J Haematol 2003: 70: 198–199 Printed in UK. All rights reserved Copyright Blackwell Munksgaard 2003

Septic arthritis as the first sign of Candida tropicalis fungaemia in an acute lymphoid leukemia patient

Fungal infections caused by Candida species have increased in incidence during the past two decades in England, North America and Europe. Candidal arthritis is rare in patients who are not intravenous drug users or are who not using a prostheses. We report the case of a 24-year-old man with acute lymphoid leukemia, who developed Candida tropicalis arthritis during an aplastic period after chemotherapy. This is the eighth case described in the literature of C. tropicalis causing arthritis without intra-articular inoculation. We call attention to an unusual first sign of fungal infection: septic arthritis without intra-articular inoculation. However, this case differs from the other seven, since despite therapy a fast and lethal evolution was observed. We reviewed reported cases, incidence, risk factors, mortality and treatment of neutropenic patients with fungal infections.

Megakaryocytic blast crisis as a first presentation of chronic myeloid leukemia

Abstract: Acute megakaryocytic leukemia (AmegL) corresponds to 5.0–10.0% of all acute myeloid leukemias (AML). Blast crisis as the first presentation of chronic myeloid leukemia (CML) accounts for 10.0% of all cases. Objective: We report a case of megakaryocytic blast crisis as the first presentation of CML. Case report: A 25‐yr‐old‐female with a 2‐month history of dry cough and a large, non‐tender splenomegaly was found to have a hemoglobin concentration of 10.5 g/dL, a hematocritof 33.0%, a white blood cell count (WBC) of 11.4 × 106 L with 38% small blasts, eosinophilia of 5%, basophilia of 8%, and a platelet count of 580 × 109 L. Bone marrow aspiration revealed 24% of blast cells with cytoplasmatic blebs and hyperplastic megakaryocytic lineage with dysplasia. Cytochemical stains were all negative, immunophenotyping studies showed CD41 and CD61 positivity in blast cells. Bone marrow biopsy showed grade II fibrosis. Karyotype revealed 46, XX, t(9,22) (q34.1;q11.2)[20] and the reverse‐transcriptase‐PCR (RT‐PCR) gave rise a product with a size corresponding to the 210 kDa protein (p210). No matched donor was found. After induction therapy 5.9% of blast cells persisted. The patient received Imatinib Mesylate and is doing well after a 12‐month follow‐up. Discussion: AmegL as the first presentation of CML is a rare and often fatal event. Some characteristics point towards the diagnosis of a blast crisis instead of AmegL de novo with t(9,22).