Rie Yamazaki

Dose-adjusted preemptive therapy for cytomegalovirus disease based on real-time polymerase chain reaction after allogeneic hematopoietic stem cell transplantation

We have prospectively evaluated the efficacy of real-time PCR-guided preemptive therapy for CMV diseases in allogeneic hematopoietic stem cell transplant recipients with grades II–IV acute GVHD. The dose of ganciclovir was adjusted according to the viral load determined by real-time polymerase chain reaction (PCR). On detecting CMV reactivation in the plasma, ganciclovir was initiated at a dose of 5 mg/kg body weight once daily, and the dose was increased to twice daily if viral load continued to increase after initiating ganciclovir. In 39 evaluable patients, CMV reactivation assessed by real-time PCR became positive in 30 (77%). One developed CMV gastroenteritis before PCR became positive. Thus the remaining 29 patients were treated preemptively with ganciclovir. The dose of ganciclovir was increased in 12 patients (41%) of preemptively treated patients for increasing viral load. CMV diseases were diagnosed in two patients (one gastroenteritis and one retinitis), and late CMV disease was diagnosed in one patient (gastritis). The treatment was generally well-tolerated, but three patients (10%) developed neutropenia (neutrophil count less than 1.0 × 109/l). In conclusion, real-time PCR-guided preemptive therapy with decreased dose of ganciclovir is feasible and does not increase the frequency of CMV diseases if the dose is adjusted according to the viral load.

Evaluation of cytomegalovirus-specific T-cell reconstitution in patients after various allogeneic haematopoietic stem cell transplantation using interferon-γ-enzyme-linked immunospot and human leucocyte antigen tetramer assays with an immunodominant T-cell epitope

Cytomegalovirus (CMV) infection is a major complication for patients who received allogeneic haematopoietic stem cell transplantation (HSCT). Accurate monitoring of CMV‐specific T‐cell reconstitution is required for appropriate decision on treatment, such as anti‐viral drugs, which have adverse effects. Although human leucocyte antigen (HLA) tetramer and interferon‐γ‐enzyme‐linked immunospot (IFN‐γ‐ELISPOT) assays have been used to measure CMV‐specific T cells, detailed comparison of these assays and kinetics of anti‐CMV T‐cell reconstitution between reduced‐intensity transplantation (RIST) and conventional HSCT has not yet been performed. In this study, we performed prospective comparative monitoring of CMV‐specific T cells using HLA tetramer and IFN‐γ‐ELISPOT assays with a single immunodominant CMV495 peptide in 28 HLA‐A*0201 and 9 HLA‐A*0206 patients after various allogeneic HSCTs. The IFN‐γ‐ELISPOT assay was more sensitive for evaluation of functional T cells than the HLA tetramer assay, and CMV‐specific T cells were reconstituted earlier in patients who received RIST without anti‐thymocyte globulin (ATG) than those receiving RIST with ATG or conventional HSCT. The threshold level for protection from CMV reactivation was estimated as over 1u2003×u2003106 cells/l peripheral blood with the IFN‐γ‐ELISPOT assay. These results demonstrate that the IFN‐γ‐ELISPOT assay with CMV495 provides more accurate evaluation on CMV immunity in HLA‐A*0201 and ‐A*0206 patients, and may be useful for determining timing of various treatments.

Prolonged thrombocytopenia after allogeneic hematopoietic stem cell transplantation: associations with impaired platelet production and increased platelet turnover

To evaluate the mechanisms underlying prolonged thrombocytopenia after allogeneic hematopoietic stem cell transplantation (SCT), an index for plasma glycocalicin normalized for the individual platelet count (GCI), plasma thrombopoietin (TPO), and circulating B cells producing anti-GPIIb-IIIa antibodies were measured in 50 SCT recipients with or without prolonged thrombocytopenia, 42 patients with idiopathic thrombocytopenic purpura, nine patients with aplastic anemia, and 22 healthy individuals. All three indices were significantly higher in the SCT recipients with thrombocytopenia than in those without (P<0.01 for all comparisons), and were significantly correlated with the platelet count in SCT recipients. Stepwise multiple regression analysis of the samples from the SCT recipients revealed that GCI and TPO independently pointed to specific mechanisms of thrombocytopenia. The GCI and TPO status in SCT recipients with thrombocytopenia had a pattern similar to that seen in aplastic anemia, suggesting a major role for impaired thrombopoiesis. An antiplatelet antibody response was frequently detected in SCT recipients, but the development of thrombocytopenia is likely to depend on additional factors, such as reticuloendothelial function. In summary, post transplant prolonged thrombocytopenia is associated with complex mechanisms, including impaired thrombopoiesis and increased platelet turnover.

Preferentially expressed antigen of melanoma (PRAME) in the development of diagnostic and therapeutic methods for hematological malignancies.

PRAME (Preferentially expressed antigen of melanoma), highly expressed in various solid tumor cells and normal testis, was first isolated as a human melanoma antigen recognized by cytotoxic T cells (CTL). This gene was also expressed in some of the hematological malignancies, including acute myelogenous leukemia (AML) and multiple myeloma. We and others have extensively evaluated the PRAME expression in various hematological malignancies and demonstrated high expression of the PRAME gene in subsets of AML, chronic myelogenous leukemia, acute lymphocytic leukemia, lymphoma and multiple myeloma. In addition, we have demonstrated that PRAME was a useful marker for detection of minimal residual disease (MRD) in patients with leukemia, particularly those leukemias in which tumor specific markers are currently unavailable. Since PRAME was first identified as a tumor antigen recognized by T cells, the possibility that PRAME is a leukemia antigen recognized by T cells was evaluated, and it was found that PRAME-positive leukemia cell lines and fresh leukemia cells were susceptible to lysis by the PRAME-specific CTL. Five CTL epitopes associated with either HLA-A * 0201 or HLA-A * 2402 have recently been identified. It is, therefore, an attractive strategy to apply PRAME specific immunotherapy on patients with PRAME positive leukemia in MRD condition.

Transmission of chromosomally integrated human herpesvirsus 6 (HHV-6) variant A from a parent to children leading to misdiagnosis of active HHV-6 infection

Abstract: Only a handful of cases of chromosomally integrated human herpesvirus 6 (CI‐HHV‐6) have been reported, suggesting that this phenomenon is rare. We here present a familial case of HHV‐6 variant A (HHV‐6A) transmission through a generation, which was identified in the setting of allogeneic hematopoietic stem cell transplantation (HSCT). A 31‐year‐old man with myelodysplastic syndrome underwent allogeneic HSCT from a human leukocyte antigen‐identical sibling, and was found to be continuously yielding high copy numbers of HHV‐6A DNA in plasma evaluated by real‐time polymerase chain reaction (PCR). Antiviral therapy with ganciclovir or foscarnet failed to decrease the copy numbers. HHV‐6A DNA was detected in the patients buccal mucosa and hair follicles, and was also detected in the plasma, whole blood, and buccal mucosa of the patients father and 2 siblings, but not in his mother. The sequences of HHV‐6A DNA isolated from all family members were identical. Since monitoring of HHV‐6 by PCR has been widely introduced to the field of HSCT, transplant physicians should be aware of such an alternative form of HHV‐6 transmission, particularly when HHV‐6A is detected.

Greater impact of oral fluconazole on drug interaction with intravenous calcineurin inhibitors as compared with intravenous fluconazole

ObjectiveAlthough the drug interaction between fluconazole and calcineurin inhibitors has been established, the influence of the route of fluconazole administration on its drug interaction with calcineurin inhibitors has yet to be fully examined. The aim of the present study is to examine whether different routes of fluconazole administration alter its drug interactions with intravenous calcineurin inhibitors.MethodsIn 53 recipients of allogeneic hematopoietic cell transplantation receiving calcineurin inhibitors intravenously, steady-state whole-blood levels of cyclosporine A (CsA) or tacrolimus were measured after the route of fluconazole (200xa0mg/day) administration was switched from intravenous to oral.ResultsThe mean steady-state whole-blood level of CsA or tacrolimus significantly increased after the route of fluconazole administration was switched from intravenous to oral (CsA: to 394u2009±u200928.4 from 362u2009±u200917.8xa0ng/mL; tacrolimus: to 18.8u2009±u20090.64 from 17.4u2009±u20090.39xa0ng/mL, Pu2009<u20090.05).ConclusionThis finding strongly suggests that oral fluconazole has a greater impact on its drug interactions with intravenous calcineurin inhibitors than intravenous fluconazole. Monitoring of blood levels of intravenous calcineurin inhibitors is recommended when the route of fluconazole administration is switched from intravenous to oral.

Effects of immunosuppressive agents on magnesium metabolism early after allogeneic hematopoietic stem cell transplantation

Background. The calcineurin inhibitors, cyclosporine A (CSA) and tacrolimus, cause hypomagnesemia by suppressing reabsorption of magnesium (Mg) from renal tubules. To assess whether the effect on Mg metabolism after allogeneic hematopoietic stem cell transplantation (HSCT) differs among calcineurin inhibitors, we prospectively evaluated the Mg metabolism in recipients of allogeneic HSCT who received CSA or tacrolimus Methods. Patients who underwent allogeneic HSCT were enrolled. CSA and tacrolimus were given by continuous infusion starting from day –1. Serum Mg and the total amount of urinary Mg excretion were measured once before starting of CSA or tacrolimus, and once weekly after HSCT for 4 weeks. Mg was supplemented with magnesium l-aspartate by continuous infusion to maintain the serum Mg level >1.4 mEq/L. Results. Thirty-six patients were evaluated (12 in the CSA group, 24 in the tacrolimus group). The serum Mg level began to decrease in both groups at the first week after HSCT, and the mean serum Mg levels were significantly lower in the tacrolimus group than in the CSA group from the first to the third week. The total amount of urinary Mg excretion and Mg supplementation began to increase in both groups at the second week after HSCT, and the amounts in the tacrolimus group were significantly higher than those in the CSA group. Conclusions. Although both calcineurin inhibitors increased urinary Mg excretion and caused hypomagnesemia shortly after HSCT, the effect was more significant with tacrolimus than with CSA. This observation may explain the higher incidence of renal impairment and encephalopathy in patients receiving tacrolimus.

Tacrolimus–azithromycin interaction in a recipient of allogeneic bone marrow transplantation

Tacrolimus is a potent macrolide immunosuppressive agent, which is used for the prophylaxis and treatment of organ rejection and graft-versus-host disease (GVHD) after allogeneic organ or stem cell transplantation. Tacrolimus undergoes extensive metabolism in the liver, which is mediated by cytochrome P450 (CYP) 3A4 isoenzymes [1]. Therefore, the metabolism of tacrolimus is affected by the drug interaction with several drugs metabolized by CYP 3A4. There is nearly a complete overlap between its drug interactions and those of cyclosporine A, another calcineurin inhibitor. However, fewer drug interaction studies for tacrolimus have been carried out. Macrolide antibiotics including erythromycin and clarithromycin have been known as the inhibitors of CYP, and their concomitant use with tacrolimus has been reported to increase the whole blood tacrolimus concentration [2–5]. Azithromycin, possessing a unique azalide structure, shows a pharamacokinetics and metabolism different from other macrolides. Indeed, unlike erythromycin and clarithromycin, azithromycin does not affect CYP [6,7]. Thus, its drug interaction with tacrolimus has not been reported, although the interaction between azithromycin and CSA has been described in a renal transplant recipient [8]. A 27-year-old woman with acute myelogenous leukemia underwent allogeneic bone marrow transplantation (BMT) from an human leukocyte antigen matched unrelated donor after the conditioning with total body irradiation and high-dose cytarabine. For the prophylaxis of GVHD, she received continuous intravenous tacrolimus at the initial dose of 0.03 mg/kg starting 1 day before BMT, and short-term methotrexate. The concentration of tacrolimus in the whole blood was measured by an automated microparticle enzyme immunoassay at least two or three times a week during the first 2 months after BMT. Engraftment was achieved on day 17. On day 37, she developed folliculitis on her face and head, and azithromycin (500 mg/day) was given orally on days 38–40. During the 1 week before initiating azithromycin, the dose of intravenous tacrolimus was not changed (0.02 mg/kg/day), and the concentration of tacrolimus had ranged between 15.8 and 17.5 ng/ml. On day 40, 3 days after starting azithromycin, the concentration of tacrolimus exceeded the measurable level without dilution (>30.0 ng/ml), although the dose of tacrolimus was not changed. Intravenous tacrolimus was discontinued for 24 h, and the blood level of tacrolimus decreased to 16.0 ng/ml. Tacrolimus was then restarted at a reduced dose (0.016 mg/kg). On day 44, the dose was increased to 0.02 mg/kg, which kept the blood level between 16.0 and 19.2 ng/ml. During this period, any additional drugs known to affect CYP isoenzyme activity were not given. Although azithromycin has been reported to have minimal effects on CYP, the marked increase in the tacrolimus blood level after only two doses of azithromycin in this case strongly suggested the possibility of its interaction with tacrolimus. Thus, transplant physicians should be aware of this drug interaction, because the chance of receiving both drugs is not infrequent among organ or stem cell transplant recipients. Further cases should be accumulated in order to evaluate and confirm this drug interaction.

Blood eosinophilia as a marker of favorable outcome after allogeneic stem cell transplantation.

Eosinophilia is observed in a variety of disorders including acute and chronic graft‐versus‐host disease (GVHD). The clinical records of 237 patients who underwent allogeneic stem cell transplantation (allo‐SCT) were retrospectively reviewed. Eosinophilia, defined as a relative eosinophil count >4% within the first 100u2003days, was observed in 135 patients (57%). The incidence of grades II–IV acute GVHD was significantly higher in patients without eosinophilia than in those with eosinophilia (68% vs. 43%; Pu2003<u20030.001). The incidence of chronic GVHD was significantly higher in patients without eosinophilia than in those with eosinophilia (73% vs. 56%; Pu2003=u20030.011). Relapse rate was similar between patients with and without eosinophilia (33% vs. 27%; Pu2003=u20030.438). The probability of nonrelapse mortality was 10% in patients with eosinophilia, which was significantly lower than that in patients without eosinophilia (31%; Pu2003<u20030.001), and the overall survival (OS) at 3u2003years was 67% in patients with eosinophilia, which was significantly higher than that in patients without eosinophilia (51%; Pu2003=u20030.003). Multivariate analysis identified older age, high‐risk disease, acute GVHD, sex disparity between patient and donor, and the absence of eosinophilia as significant factors for reduced OS. These data lead us to conclude that eosinophilia after allo‐SCT may serve as a favorable prognostic marker.

Long-Term Follow-up of Allogeneic Hematopoietic Stem Cell Transplantation for De Novo Acute Myelogenous Leukemia with a Conditioning Regimen of Total Body Irradiation and Granulocyte Colony-Stimulating Factor-Combined High-Dose Cytarabine

We retrospectively evaluated the efficacy and safety of total body irradiation (TBI) and granulocyte colony-stimulating factor (G-CSF)-combined high-dose cytarabine as a conditioning regimen for allogeneic hematopoietic stem cell transplantation (HSCT) in patients with de novo acute myelogenous leukemia (AML). The conditioning regimen consisted of 12 Gy of TBI followed by high-dose cytarabine (3 g/m(2)) every 12 hours for 4 days in combination with the continuous administration of G-CSF. Stem cell sources included bone marrow or peripheral blood stem cells (PBSC) from human leukocyte antigen (HLA)-identical siblings (n = 24), or bone marrow from HLA serologically matched unrelated donors (n = 26). Fifty patients (median age, 38 years) were evaluated. At HSCT, 35 patients were in the first or second complete remission (CR1/2), and 15 patients were not in remission (n = 14) or in the third CR (n = 1). Thirty-six of 50 patients are currently alive, with a median follow-up period of 5.6 years (range: 1.1-12.1 years). The 5-year estimated overall survival (OS) and disease-free survival (DFS) rates were 85.5% (95% confidence interval [CI], 73.7%-97.3%) and 82.1% (95% CI, 69.0%-95.2%) in patients with AML in the first or second CR, 46.7% (95% CI, 21.4%-72.0%), and 40.0% (95% CI, 15.3%-64.7%) in patients with AML in other stages. The 2-year cumulative incidence of treatment-related mortality (TRM) of all patients was 10.4% (95% CI, 1.8%-18.6%). The only factors affecting the OS and DFS were disease status at transplant and cytogenetics by multivariate analysis. These results suggest that G-CSF-combined high-dose cytarabine could be a promising component of the conditioning regimen for allogeneic HSCT for AML, providing a high DFS and low TRM.