Nobuyoshi Hanaoka

NKG2D-mediated immunity underlying paroxysmal nocturnal haemoglobinuria and related bone marrow failure syndromes.

It is considered that a similar immune mechanism acts in the pathogenesis of bone marrow (BM) failure in paroxysmal nocturnal haemoglobinuria (PNH) and its related disorders, such as aplastic anaemia (AA) and myelodysplastic syndromes (MDS). However, the molecular events in immune‐mediated marrow injury have not been elucidated. We recently reported an abnormal expression of stress‐inducible NKG2D (natural‐killer group 2, member D) ligands, such as ULBP (UL16‐binding protein) and MICA/B (major histocompatibility complex class I chain‐related molecules A/B), on granulocytes in some PNH patients and the granulocyte killing by autologous lymphocytes in vitro. The present study found that the expression of NKG2D ligands was common to both granulocytes and BM cells of patients with PNH, AA, and MDS, indicating their exposure to some incitement to induce the ligands. The haematopoietic colony formation in vitro by the patients’ marrow cells significantly improved when their BM cells were pretreated with antibodies against NKG2D receptor, suggesting that the antibodies rescued haematopoietic cells expressing NKG2D ligands from damage by autologous lymphocytes with NKG2D. Clinical courses of patients with PNH and AA showed a close association of the expression of NKG2D ligands with BM failure and a favourable response to immunosuppressive therapy. We therefore propose that NKG2D‐mediated immunity may underlie the BM failure in PNH and its‐related marrow diseases.

Establishment of a novel CD20 negative mature B-cell line, WILL2, from a CD20 positive diffuse large B-cell lymphoma patient treated with rituximab.

Rituximab is a chimeric monoclonal antibody that binds to human CD20 antigen expressed on cell surface of mature B-cells, and depletes CD20 positive cells through direct signaling of apoptosis, complement activation and cellmediated cytotoxicity [1]. Use of this antibody accompanied with conventional chemotherapies resulted in better clinical outcome than chemotherapies only in various types of mature B-cell malignancies [2]. However, proliferation of CD20 negative B-cell clone after rituximab treatment is an emerging issue [3–6]. Here, we report the establishment of a novel CD20 negative mature B-cell line, WILL2, which was derived from a CD20 positive diffuse large B-cell lymphoma patient treated with rituximab repeatedly. A 62-year-old Japanese woman was admitted to our hospital because of malaise and liver dysfunction. Pathological examination of her lymph node biopsy specimen showed diffuse proliferation of CD20 positive B-cells (Fig. 1a); thus she was diagnosed as diffuse large B-cell lymphoma with CD10 and CD20 expression. Abnormal lymphocytes appeared in her peripheral blood (PB), and they were positive for CD20 as detected by flow cytometry (Fig. 1b). After eight courses of chemotherapy including rituximab, cyclophosphamide, doxorubicine, vincristine, and prednisolone (R-CHOP regimen), she received autoperipheral hematopoietic stem cell transplantation after high dose chemotherapy including melphalan, etoposide, cyclophosphamide dexamethazone, and rituximab (R-LEED regimen). She achieved complete remission for 3 months; however, she was died of relapsed disease sixteen months after initial diagnosis. A postmortem study revealed that the tumor cells infiltrated into multiple organs, pleura, and peritoneum. Pathological examination of a pleural tumor revealed diffuse proliferation of lymphoma cells lacking CD20, though CD10 remained to be positive (Fig. 1a). The WILL2 cell line was established from ascites obtained at the autopsy. The WILL2 cells were positive for CD10 and CD19 but negative for CD20 expression (Fig. 1b). Reverse transcription PCR (RT-PCR) analysis suggested that this cell line expresses less amount of CD20 mRNA than other CD20 positive B-cell lines (Fig. 1c). Karyotypic analysis of the WILL2 cells showed multiple structural abnormalities; 46,X,add(X)(q13),dup(1)(q32 q21),ins(1;?)(q21;?),der(4)t(4;6)(p16;p21),t(5;9)(q13;p22), t(8;22)(q24;q11),t(14;18)(q32;q21) (Fig. 1d). Fluorescent in situ hybridization (FISH) demonstrated a fusion of immunoglobulin lambda and C-MYC loci; indicating that t(8;22)(q24;q11) occurred (Fig. 1d). The t(14;18)(q32;q21) and 8q24 translocation were also found in the tumor cells in ascites at the autopsy as detected by FISH. (data not shown). Thus, the patient suffered from a diffuse large B-cell lymphoma with t(14;18)(q32;q21) and t(8;22) (q24;q11). A PCR method for amplification of T. Sonoki (&) N. Hanaoka H. Matsuoka H. Nakakuma Department of Hematology/Oncology, Wakayama Medical University, 811-1 Kimi-idera, Wakayama 633-1611, Japan e-mail: sonoki@wakayama-med.ac.jp

Acute Leukemia Showing t(8;22)(p11;q11), Myelodysplasia, CD13/CD33/CD19 Expression and Immunoglobulin Heavy Chain Gene Rearrangement

t(8;22)(p11;q11) is a rare but recurrent chromosome translocation that has been reported in 11 cases of myeloproliferative neoplasm or B-acute lymphoblastic leukemia. This translocation results in an in-frame fusion of FGFR1 on 8p11 and BCR on 22q11, and causes constitutive activation of the tyrosine kinase of the BCR/FGFR1 chimera protein. Here, we report the twelfth case of hematological tumor bearing t(8;22)(p11;q11). The bone marrow showed hypoplastic and tri-lineage dysplasia with 24.4% abnormal cells. The abnormal cells were not defined as myeloid or lymphoid morphologically, lacking a myeloperoxidase reaction. Flow cytometric analysis of the bone marrow cells revealed that the abnormal cells expressed CD13, CD33, CD34, and CD19, and that a fraction of the abnormal cells was positive for CD10. Southern blot analysis of the bone marrow cells showed rearrangement of the immunoglobulin heavy chain gene, a genetic hallmark of B-cell differentiation. Previously reported cases with t(8;22)(p11;q11) suggested an association between myeloid and B-lymphoid tumors, whereas other chromosome translocations involving FGFR1 on 8p11 showed a link between myeloid and T-lymphoid tumors. Our observation supports that t(8;22)(p11;q11) might define a dual myeloid and B-lymphoid disorder.

Molecular cloning of IGλ rearrangements using long‐distance inverse PCR (LDI‐PCR)

Malignant cells of mature B‐cell origin show tumor‐specific clonal immunoglobulin gene (IG) rearrangements, including V(D)J recombinations, nucleotide mutations, or translocations. Rapid molecular cloning of the breakpoint sequence by long‐distance inverse PCR (LDI‐PCR) has so far been applied to rearrangements targeted to IGH joining, IGH switch, and IGκ regions. We tended to apply LDI‐PCR method for cloning of IGλ rearrangements.

A nationwide survey of hypoplastic myelodysplastic syndrome (a multicenter retrospective study)

Hypoplastic myelodysplastic syndrome (hMDS) is a distinct entity with bone marrow (BM) hypocellularity and the risk of death from BM failure (BMF). To elucidate the characteristics of hMDS, the data of 129 patients diagnosed between April 2003 and March 2012 were collected from 20 institutions and the central review team of the National Research Group on Idiopathic Bone Marrow Failure Syndromes, and compared with 115 non‐hMDS patients. More RA and fewer CMMoL and RAEB‐t in French‐American‐British (FAB) and more RCUD and MDS‐U and fewer RCMD in World Health Organization (WHO) classifications were found in hMDS than non‐hMDS with significant differences. The overall survival (OS) and AML progression‐free survival (AML‐PFS) of hMDS were higher than those of non‐hMDS, especially in patients at age ≥50 and of lower risk in Revised International Prognostic Scoring System (IPSS‐R). In competing risks analysis, hMDS exhibited decreased risk of AML‐progression in lower IPSS or IPSS‐R risk patients, and higher risk of death from BMF in patients at age ≥50. Poor performance status (PS ≥2) and high karyotype risks in IPSS‐R (high and very high) were significant risk factors of death and AML‐progression in Cox proportional hazards analysis.

Real-time monitoring of antimicrobial use density to reduce antimicrobial resistance through the promotion of antimicrobial heterogeneity in a haematology/oncology unit

BACKGROUND In haematology/oncology units, the frequent and heavy use of broad-spectrum antimicrobials can lead to outbreaks of antimicrobial resistance. Increasing antimicrobial heterogeneity might be a useful strategy for preventing such resistance. METHODS A real-time antimicrobial use density (AUD) monitoring system (RAMS) was developed to precisely assess antimicrobial heterogeneity. This study was prospectively conducted over a 39 month period and involved 970 patients. Patient-specific antimicrobial therapy with five carbapenems (meropenem, biapenem, panipenem/betamipron, imipenem/cilastatin and doripenem) and four non-carbapenems (piperacillin/tazobactam, ceftazidime, cefozopran and cefepime) was prescribed in the first 12 months. A first-line antimicrobial was selected from among nine antimicrobials according to a predetermined schedule for the next 15 months. AUD-based antimicrobial selection was implemented using the RAMS during the last 12 months. We compared our findings for the RAMS period with those for the other periods to determine the effects of RAMS-based AUD monitoring on antimicrobial resistance. RESULTS The mean absolute difference between the AUD values of carbapenems and non-carbapenems (AUD deviation) was 6.0% in the RAMS period (range 0.5%-15.8%) and antimicrobial heterogeneity (AUD deviation <10%) was achieved in 10 out of 12 months (83.3%). Furthermore, during the RAMS period, AUD deviation was significantly smaller and the frequency of outbreaks of antimicrobial-resistant strains other than Stenotrophomonas maltophilia was significantly decreased (from 7.9% to 3.5%; P < 0.01) compared with the other periods. CONCLUSIONS The longer period of stable antimicrobial heterogeneity achieved by the RAMS strengthened its preventive effects against antimicrobial resistance. Optimal antimicrobial heterogeneity based on real-time AUD monitoring could reduce the frequency of outbreaks of antimicrobial resistance.

Occupancy of whole blood cells by a single PIGA-mutant clone with HMGA2 amplification in a paroxysmal nocturnal haemoglobinuria patient having blood cells with NKG2D ligands.

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Aggressive CD5-positive B-cell lymphoma after remission of CD5-negative follicular lymphoma with distinct immunoglobulin heavy chain rearrangement and translocation

We report a unique, aggressive B-cell lymphoma that developed after the long-term remission of follicular lymphoma (FL). FL cells were negative for CD5, whereas aggressive lymphoma cells were positive for CD5. In FL, one immunoglobulin heavy chain gene (IGH) allele underwent V/D/J recombination and another t(14;18)(q32;q21). In aggressive lymphoma, one IGH allele underwent D/J recombination and another translocation, but not t(14;18)(q32;q21). An aggressive lymphoma-specific D/J sequence was detected in FL tissue. Our results indicated that the two tumors arose from distinct B cells and that they existed concurrently in the same lymph node.

Mobile Cecum in a Young Woman with Ehlers-Danlos Syndrome Hypermobility type: A Case Report and Review of the Literature

Ehlers-Danlos syndrome, hypermobility type (EDS-HT) is unexpectedly common and is associated with a high rate of gastrointestinal manifestations. We herein report the first documented case of mobile cecum associated with EDS-HT. A 21-year-old woman with repeated right lower abdominal pain was initially diagnosed with EDS-HT. Abdominal examinations performed in the supine position, such as CT and ultrasonography, showed no gross abnormalities. In contrast, oral barium gastrointestinal transit X-ray images obtained with changes in the patients body position revealed position-dependent cecal volvulus with mobile cecum. She was finally discharged with a dramatic resolution of her symptoms after laparoscopic cecopexy for mobile cecum.

Refractory Ascites with Liver Fibrosis Developed in Late Phase Allogeneic Hematopoietic Stem Cell Transplantation: Report of Three Patients.

We report cases of three patients of refractory ascites without other fluid retention that occurred around five months after allogeneic hematopoietic stem cell transplantation (allo-HSCT). All three patients expired and postmortem examinations revealed unexpected liver fibrosis lacking histological evidences of graft-versus-host-disease (GVHD). The three patients showed normal hepatic function and size before transplantation. During their clinical courses, serum biochemistry test showed no elevation of hepatic enzymes and bilirubin; however, imaging studies demonstrated hepatic atrophy at the onset of ascites. One of the liver specimens showed bile obstruction, which could be seen in hepatic damage by GVHD. Although ascites resulting from venoocclusive disease in early phase allo-HSCT is well documented, ascites associated with hepatic fibrosis in late phase allo-HCST has not been reported. Further clinico-pathological studies on similar patients should be required to ascertain refractory ascites associated with liver fibrosis after allo-HSCT.