Comparison of two apheresis systems for granulocyte collection without hydroxyethyl starch.

BACKGROUND AND OBJECTIVES: Granulocyte transfusion (GTX) is a treatment option for severe infections in patients with neutropenia. In previous studies, hydroxyethyl starch (HES) was used to enhance red blood cell sedimentation for granulocyte collection (GC). However, there are safety concerns about HES, and HES is not readily available in some countries. Therefore, we compared the granulocyte counts and GC efficiency achieved by two apheresis systems without HES. MATERIALS AND METHODS: All consecutive GC procedures performed between July 2011 and March 2018 at our hospital were analysed. COBE Spectra was used until 5 February 2016, and Spectra Optia was used afterwards. HES was not used. RESULTS: Twenty-six GC procedures were performed, including 18 performed using COBE Spectra and 8 using Spectra Optia. When Spectra Optia was used, >1 × 1010 neutrophils were collected from seven of the eight (88%) procedures. Although there was no significant difference in the granulocyte yield between COBE Spectra-based and Spectra Optia-based GC procedures, the collection efficiency of Spectra Optia was significantly higher than that of COBE Spectra (p = 0.021). Furthermore, the granulocyte yields of Spectra Optia-based GC tended to be more strongly correlated with the peripheral blood neutrophil count on the day of apheresis than those of COBE Spectra-based GC. CONCLUSION: Our results suggest that Spectra Optia achieves greater GC efficiency than COBE Spectra, even without HES. GTX may be a therapeutic option for severe neutropenia, even in places where HES is not available.

Detection of the JAK2 V617F Mutation in Urinary Cell-free DNA in Patients with Myeloproliferative Neoplasms.

Objective Testing for the Janus activating kinase 2 (JAK2) V617F mutation is important for diagnosing and treating myeloproliferative neoplasms (MPNs). Recently, urine cell-free DNA (ucfDNA) was reported to be useful for detecting tumor-specific gene mutations in several solid tumors. However, its utility in detecting such mutations in hematological malignancies has not yet been assessed. In this study, we assessed whether or not the JAK2 V617F mutation could be detected in ucfDNA and whether or not its positivity rate in ucfDNA was associated with the JAK2 V617F allele ratio of peripheral blood cells in patients with MPN. Methods The JAK2 V617F allele ratio of genomic DNA from peripheral blood cells was determined using quantitative polymerase chain reaction (qPCR) or droplet digital PCR (ddPCR). ucfDNA was subjected to ddPCR. The correlation between the JAK2 V617F mutation positivity rates of blood-derived DNA and those of ucfDNA was assessed. Materials Twelve patients with polycythemia vera and 12 patients with essential thrombocythemia were enrolled. Ethylenediaminetetraacetic acid-treated peripheral blood (100 mL) and 15-30 mL of fresh urine were used. Results The JAK2 V617F mutation was detected in the ucfDNA from all 20 JAK2 V617F mutation-positive patients. In addition, the JAK2 V617F mutation positivity rate of ucfDNA was correlated with the JAK2 V617Fｓ allele ratio of blood-derived DNA, including in both estimated glomerular filtration rate (eGFR) groups (patients with an eGFR ≥50 or <50 mL/min/1.73 m2). Conclusion Our results indicate that ucfDNA is a valuable tool for diagnosing and monitoring MPN. Given these findings, other disease-specific gene mutations in hematological malignancies may also be detectable in ucfDNA.

A cluster of BK polyomavirus-associated hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: BK polyomavirus (BKV) can cause hemorrhagic cystitis (HC) in immunocompromised patients after hematopoietic stem cell transplantation (HSCT). It remains unclear whether nosocomial BKV infections occur. During a 9-month period, an increase in BKV-associated HC (BKV-HC) cases was observed at our institution. AIM: The BKV-HC cluster population was compared with populations of HSCT patients from before and after the BKV-HC cluster to evaluate whether nosocomial BKV transmission had occurred. METHODS: A retrospective analysis was carried out to assess the risk of patients developing BKV-HC after HSCT. The background data of the cluster patients were compared with those of the patients who underwent HSCT before or after the cluster, and the collected BKV isolates were serotyped. RESULTS: BKV-HC involving grade ≥2 hematuria occurred in six of 15 HSCT recipients during a 9-month period. The incidence of BKV-HC was significantly higher in this period than in the other periods (p = 0.0014). There were no significant differences in the patients' background data between the cluster and non-cluster periods, including in terms of risk factors for BKV-HC. Serotype analyses of BKV revealed that the BKV detected in the urine samples from four of the six BKV-HC patients belonged to subtype Ic. The gene sequences of these four BKV exhibited >99.5% homology. CONCLUSION: Our study suggests that nosocomial BKV infections may occur after HSCT. Although many cases of BKV-HC are caused by the reactivation of a latent virus, it is necessary to employ appropriate hygiene measures when cases of BKV-HC occur.

[Paroxysmal nocturnal hemoglobinuria treated with eculizumab in Wakayama, Japan: a retrospective analysis].

Currently, the humanized anti-C5 monoclonal antibody, eculizumab, is widely used for treating paroxysmal nocturnal hemoglobinuria (PNH) due to its effects on suppression of intravascular hemolysis and resulting improvement in quality of life. However, in some cases, this treatment is refractory or is associated with meningococcal meningitis. No region-specific analyses have been published, and currently, information on region specificity and genetic factors is limited. We present here the results of a retrospective study involving eight patients with PNH who were treated with eculizumab in our hospital in Wakayama, Japan. The median age of these patients was 77 (range 23-88) years. Six patients had a complication of aplastic anemia, four patients had a history of thrombosis, and two experienced hemolytic episodes. Before initiating eculizumab treatment, the median serum LDH level was 1,192 IU/l (range 755-1,525 IU/l). Serum LDH levels normalized in five patients within a month of initiating therapy and PNH-related symptoms disappeared. C5 gene mutations were identified in the three patients who did not respond to eculizumab.

Soluble NKG2D Ligands Are Potential Biomarkers and Sentinels of Immune-Mediated Bone Marrow Injury in Bone Marrow Failure Syndromes.

Immune-mediated processes are considered important in the pathogenesis of bone marrow failure syndromes (BFS). We previously reported that natural killer group 2D (NKG2D) ligands were expressed on pathological blood cells of patients with BFS and that NKG2D immunity may be involved in bone marrow failure. In addition to membranous NKG2D ligands on the cell surface, soluble NKG2D ligands can exist in plasma. We therefore examined the relationship between soluble NKG2D ligands and blood cell counts in 86 patients with BFS, including aplastic anemia, myelodysplastic syndrome with single lineage dysplasia, and paroxysmal nocturnal hemoglobinuria. Approximately half of the BFS patients were positive for soluble NKG2D ligands in the plasma by enzyme-linked immunosorbent assay, and soluble NKG2D ligand-positive BFS patients exhibited severe cytopenia regardless of membranous NKG2D ligand expression. In vitroanalyses demonstrated that soluble ULBP1, an NKG2D ligand, down-regulated NKG2D receptors on CD2-positive cells in peripheral blood. Moreover, soluble ULBP1 attenuated the cytotoxic effects of peripheral blood mononuclear cells on K562, which express membranous ULBP1. Our results suggest that soluble NKG2D ligands can be easy-to-measure biomarkers for the prediction of activity of immune-meditated bone marrow injury in BFS and that soluble NKG2D ligands suppress redundant immune-mediated bone marrow injury.

Long-term complete remission of early hematological relapse after discontinuation of immunosuppressants following allogeneic transplantation for Sezary syndrome.

Sezary syndrome (SS) is a leukemic form of cutaneous T-cell lymphoma and is chemo-resistant. Allogeneic hematopoietic stem cell transplantation is a promising therapy for SS; however, relapse is common. Therapeutic options after relapse have not been established. We managed an SS patient with hematological relapse within one month after transplantation. After discontinuation of immunosuppressants, she achieved complete remission and remained relapse-free. The chimeric analyses of Tcells showed that the full recipient type became complete donor chimera after immunological symptoms. This clinical course suggested that discontinuation of immunosuppressants may result in a graftversus- tumor effect, leading to the eradication of lymphoma cells.

Enforced expression of MIR142, a target of chromosome translocation in human B-cell tumors, results in B-cell depletion.

MicroRNA142 (MIR142) is a target of chromosome translocations and mutations in human B-cell lymphomas. We analyzed an aggressive B-cell lymphoma carrying t(8;17)(q24;q22) and t(6;14)(p21;q32), and sought to explore the role(s) of MIR142 in lymphomagenesis. t(8;17)(q24;q22) involved MYC on 8q24 and pri-MIR142 on 17q22. MYC was activated by a promoter substitution by t(8;17)(q24;q22). t(8;17)(q24;q22) was an additional event after t(6;14) (p21;q32), which caused the over-expression of CCND3. Southern blot analyses revealed that the MIR142 locus was deleted from the affected allele, whereas Northern analyses showed over-expression of MIR142 in tumor cells. Although previous studies reported an over-expression of mutations in MIR142 in B-cell lymphomas, limited information is available on the functions of MIR142 in lymphomagenesis. Therefore, we generated bone marrow transplantation (BMT) and transgenic (Eµ/mir142) mice, which showed enforced expression in hematopoietic progenitor cells and B cells, respectively. BMT mice showed decreased numbers of all lineage-positive cells, particularly B cells, in peripheral blood. Eµ/mir142 mice showed decreased numbers of IgM-positive splenocytes, and exhibited altered B-cell phenotypic changes induced by lipopolysaccharide. Our results suggest that over-expression of MIR142 alters B-cell differentiation, implying multi-step lymphomagenesis together with MYC activation and CCND3 over-expression.

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 patient's 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.

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.

Successful Intrathecal Chemotherapy Combined with Radiotherapy Followed by Pomalidomide and Low-Dose Dexamethasone Maintenance Therapy for a Primary Plasma Cell Leukemia Patient.

Primary plasma cell leukemia (PPCL) is a rare aggressive variant of plasma cell disorder and frequently presents with extramedullary disease. Central nervous system (CNS) involvement with PPCL has an extremely poor prognosis. We describe a 46-year-old man with PPCL treated with a combination of lenalidomide, bortezomib, and dexamethasone as induction therapy following upfront allogeneic stem cell transplantation (allo-SCT). Despite achieving a very good partial response, the patient suffered from an isolated CNS relapse 12 months after allo-SCT. He was immediately started on concurrent intrathecal chemotherapy (IT) and cranial irradiation (RT). Subsequently, pomalidomide and low-dose dexamethasone (Pd) were given as maintenance therapy. He has been without CNS recurrence for more than 18 months. Our case suggests that concurrent IT and RT followed by Pd maintenance therapy may be an effective option to control CNS relapse of PPCL after allo-SCT.

An Epstein-Barr virus susceptible immature T-cell line, WILL4, established from a patient with T-lymphoblastic lymphoma bearing CD21 and a clonal EBV genome.

We managed a patient with an Epstein-Barr virus-associated T-cell lymphoblastic lymphoma. Mediastinal tumor cells at initial admission were positive for CD4, CD8, and TdT. Interestingly, a lymph node at necropsy was compatible for a CD4-positive peripheral T-cell lymphoma without CD8 and TdT expression, suggesting a different phenotype from the mediastinal tumor. Tumor cells in pleural effusion continued to proliferate in in vitro and were designated as WILL4. WILL4 cells were positive for CD3, CD4, CD8, CD21, T-cell receptor (TcR) αß, and TdT, indicating a similar phenotype to thymocytes. Southern blot analyses showed that the pleural tumor and WILL4 cells shared a TcR gene rearrangement, and that both contained a clonal EBV genome in an episomal form. RT-PCR showed that EBNA1 and LMP1 were expressed in the fresh tumor and WILL4 cells. Southern blot analyses revealed that WILL4 cells were susceptible to EBV infection in vitro using B95-8 supernatant. Anti-CD21 antibody inhibited in vitro infection of EBV, suggesting that CD21 plays a role in EBV infection into WILL4 cells. In vitro infection of EBV did not affect latent gene expression in WILL4 cells. WILL4 is a useful tool for analyzing the roles of EBV in onocogenesis in immature T-lymphoid malignancies.

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.

B-Cell-Rich T-Cell Lymphoma Associated with Epstein-Barr Virus-Reactivation and T-Cell Suppression Following Antithymocyte Globulin Therapy in a Patient with Severe Aplastic Anemia.

B-cell lymphoproliferative disorder (B-LPD) is generally characterized by the proliferation of Epstein-Barr virus (EBV)-infected B lymphocytes. We here report the development of EBV-negative B-LPD associated with EBV-reactivation following antithymocyte globulin (ATG) therapy in a patient with aplastic anemia. The molecular autopsy study showed the sparse EBV-infected clonal T cells could be critically involved in the pathogenesis of EBV-negative oligoclonal B-LPD through cytokine amplification and escape from T-cell surveillances attributable to ATG-based immunosuppressive therapy, leading to an extremely rare B-cell-rich T-cell lymphoma. This report helps in elucidating the complex pathophysiology of intractable B-LPD refractory to rituximab.

Successful Immunosuppressive Therapy for Severe Infectious Mononucleosis in a Patient with Clonal Proliferation of EBV-infected CD8-positive Cells.

A 30-year-old woman was diagnosed with severe infectious mononucleosis (IM). The Epstein-Barr virus (EBV) had infected both CD19- and CD8-positive cells, and clonal proliferation of EBV-infected cells and T-cells was detected. Although we suspected malignant lymphoma, her condition improved following immunosuppressive therapy. A similar case was recently reported; therefore, this case is the second case of IM with EBV-infected CD8-positive cells and clonal proliferation of EBV-infected cells. Our results demonstrate that the clonal proliferation of EBV-infected cells is not always an indication for chemotherapy in the primary infection phase and that monitoring the EBV viral load is useful for therapeutic decision-making.

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.

Severe enteropathy caused by α-heavy chain disease lacking detectable M-proteins.

A 57-year-old Japanese man was admitted to our hospital with diarrhea, weight loss and malabsorption. Due to a high serum IgA level, we suspected α-heavy chain disease (α-HCD). However, no monoclonal IgA was detected on protein electrophoresis or immunofixation. Immunohistochemical staining of intestinal biopsy specimens showed proliferation of CD138(+)IgA(+) cells, compatible with a diagnosis of α-HCD. Most α-HCD patients exhibit M-proteins in the serum on electrophoresis or immunoelectrophoresis; however, some patients lack detectable M-proteins, similar to our patient. Therefore, when α-HCD is suspected based on the clinical features, immunohistochemistry is required to make a diagnosis.

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).

OBJECTIVES: 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. METHODS: To identify which IGλ isotype segment was rearranged, we performed Southern blot analysis using isotype-specific probes. We set inverse primers on the telomeric side of each joining region and amplified rearranged bands detected by Southern blot analysis as corresponding PCR products. RESULTS: All germline IGλ segments were successfully amplified as expected PCR products. We determined breakpoint sequences of five chromosome translocations involving IGλ locus: three novel t(8;22)(q24;q11), one known t(3;22)(q27;q11), and one partially known t(11;22)(q13;q11). Two of the three t(8;22)(q24;q11) were involved in Jλ with a recombination signal sequence and one of three in the first exon of IGLL5, which lies upstream of Jλ1. Three 8q24 breakpoints were widespread at 132, 260 and 366 kb downstream of MYC locus. The t(3;22)(q27;q11) showed a juxtaposition of Jλ2 and the first intron of BCL6, as previously reported. In t(11;22)(q13;q11), 3'UTR of cyclin D1 fused to the constant region of λ7 with nucleotide mutations. We also amplified four Vλ/Jλ recombination sequences. CONCLUSION: Our method is a useful tool for molecular analysis of genetic events in IGλ.