ALBERT

Description: Varicella-zoster virus (VZV) infection remains a common complication after hematopoietic stem cell transplantation (HSCT). The introduction of long-term prophylaxis with low-dose acyclovir against VZV reactivation has been investigated, because VZV-related complications including post-herpetic neuralgia and secondary infection significantly affect the patient’s quality of life. We started long-term oral acyclovir at 200 mg/day in July 2001. Acyclovir was continued until the end of immunosuppressive therapy and at least one year after transplantation. To evaluate the efficacy of this long-term prophylaxis with ultra low-dose acyclovir against VZV reactivation, we analyzed the records of 242 Japanese adult patients who underwent allogeneic HSCT for the first time from June, 1995 to November, 2006 at University of Tokyo Hospital. Sixty-six patients developed VZV reactivation at a median of 248 days after HSCT, with a cumulative incidence of 34.7%. There was no VZV-related death. Only one breakthrough reactivation occurred during long-term acyclovir, responding well to the therapeutic dose of valacyclovir. The use of long-term acyclovir was the only independent determinant that significantly decreased the overall incidence of VZV reactivation (20.4% vs 50.5%, P

Description: Cardiac toxicity due to conditioning regimens is a critical problem in the hematopoietic stem cell transplantation (HSCT). High-dose cyclophosphamide, which is a major component of conventional myeloablative regimens, is considered to be a main cause of cardiac toxicity. Although reduced intensity conditioning regimens have been developed in order to diminish regimen related toxicities for patients with pre-transplant co-morbidity, their anti-neoplastic effects have been revealed to be insufficient especially for patients with acute lymphoblastic leukemia. Therefore, it is imperative to establish an alternative preparative regimen with myeloablative intensity for patients with impaired pre-transplant cardiac function. From June 1995 at the University of Tokyo, we have adopted a heart protective regimen (VP/rCY/TBI) which is composed of continuous infusion of VP-16 20 mg/kg for 2 days, CY 40 mg/kg for 1 day, and 12 Gy of fractionated total body irradiation (TBI) for 17 patients because they had impaired cardiac function defined by ejection fraction (EF) less than 0.55 (n=15) or a history of congestive heart failure (n=1) or pulmonary hypertension (n=1). The clinical relevance of VP/rCY/TBI regimen was evaluated by retrospectively comparing the outcome of VP/rCY/TBI recipients with that of conventional CY/TBI (CY 60 mg/kg for 2 days + fractionated TBI 12 Gy, which was administered to 140 patients during the same period) recipients. The characteristics analysis revealed that VP/rCY/TBI recipients had higher cumulative doses of anthracyclines (354 mg/m2 vs 150 mg/m2 calculated as the equivalence of native doxorubicin, p

Description: Late occurrence of viral infections beyond day 100 after hematopoietic stem cell transplantation (HSCT) was widely recognized to depend on the profound immune suppression due to severe chronic GVHD and its treatment. However, there have been few reports clarifying the direct relationships between the development of late viral infections and immune reconstitution after HSCT. To evaluate the correlation of the immune recovery with the occurrence of late cytomegalovirus (CMV) or varicella-zoster virus (VZV) infections, we retrospectively analyzed the records of 60 Japanese adult patients who underwent allogeneic HSCT for the first time from April, 2002 to February, 2007 at the University of Tokyo Hospital, and survived longer than 180 days after HSCT. Absolute lymphocyte subset counts (CD3+ T cells, CD3−CD19+ B cells, CD3+CD4+ helper T cells, CD4+CD45RO+ memory T cells, CD4+CD45RA+ naïve T cells, CD3+CD8+ cytotoxic T cells, CD3−CD56+ natural killer cells), absolute monocyte counts, serum IgG, IgA, and IgM levels were measured at 3 and 6 months after HSCT. As a prophylaxis against late CMV disease, risk-adopted preemptive therapy with ganciclovir was performed by monitoring CMV antigenemia beyond day 100 after HSCT. For late VZV disease, oral administration of acyclovir at 200 mg/day was principally continued until the end of immunosuppressive therapy and at least one year after HSCT in 52 patients, whereas valacyclovir at 500 mg/day three times a week was administered until one year after HSCT in eight patients. Two out of 60 patients have already developed CMV disease within 100 days after HSCT. Thirteen of the remaining 58 patients developed late CMV infection defined as 10 or more CMV-Ag positive cells per two slides at a median of 125 days (101 to 546 days) after HSCT. CD3+ T cells less than 400x106/L (P=0.003), CD3+CD4+ T cells less than 200 x106/L (P=0.013), CD4+CD45RO+ T cells less than 100x106/L (P

Description: Background: Identification of functional T-cell receptors (TCRs) to their cognate antigens is the key to the development of effective anti-viral or anti-tumor T-cell therapy. Deep sequencing of rearranged complementarity-determining region 3 (CDR3) regions of TCRA and TCRB gene segments is an emerging technology that facilitates high-throughput and semi-quantitative analysis of TCR repertoire with high resolution and accuracy. However, this method alone does not yield data for correct pairs of TCRA and TCRB sequences required for the structural determination of TCRαβ heterodimers expressed in a single T cell. "Human TCR efficient cloning within 10 days" (hTEC10) is a powerful novel technology that enables concurrent sequencing of paired TCRA and TCRB gene segments at a single cell level (Nat Med. 2013;19:1542-6). In this study, we attempted to elucidate the comprehensive TCR repertoire of cytomegalovirus (CMV)-reactive cytotoxic T-cells (CTLs) by combining quantitative deep sequencing and hTEC10. Methods: 20 ml peripheral blood samples were collected from healthy adult volunteers who gave written informed consent for the study. All donors were screened for CMV serostatus and typed for HLA-A. CMV-specific CD8+ T-cells were isolated and flow-sorted from peripheral blood (PB) mononuclear cells using an HLA-tetramer, NLV/A2, specific for HLA-A2-restricted CMV pp65-derived epitope (NLV peptide: NLVPMVATV). In some experiments, sorted NLV/A2-positive cells underwent one or two rounds of expansion with autologous PB mononuclear cells depleted of CD8+ and CD4+ T cells in the presence of NLV peptide. Using these samples, massive parallel sequencing of TCRA and TCRB V-D-J segments was performed after unbiased amplification of the target sequences by adaptor-ligation PCR. Concurrent TCRA and TCRB sequencing of TCRs expressed in a single sorted cell was performed by the hTEC10 protocol. Results: Of 20 donors who participated in the study, 8 were found to be CMV-seropositive and HLA-A2-positive (CMV+A2+). The samples obtained from 2 of CMV+A2+ donors, V001 and V004, were subjected to comprehensive TCR analysis by quantitative deep sequencing and single cell cloning: the respective frequencies of NLV/A2 tetramer-positive cells in PB of these donors were 0.36% and 0.25% of a CD8+ T-cell fraction. After two rounds of NLV-peptide stimulation, NLV/A2-tetramer-positive cells obtained from V001 and V004 were enriched to 84.3% and 90.5% of CD8+ T-cells, respectively. By deep sequencing analysis, the total number of unique TCRA/TCRB reads in PB samples from these donors was 1472/5787 in V001 and 2054/9179 in V004, while that of the enriched NLV/A2 tetramer-positive fractions was 178/62 in V001 and 100/104 in V004. However, in both donors, TCR repertoire of the expanded tetramer-positive cells was extremely skewed and the number of the most abundant top 3 reads comprised more than 90-95% of total reads for both TCRA and TCRB. To confirm the correct pairing of these TCRA and TCRB clonotypes at a single cell level, we also examined TCR sequences of the enriched tetramer-positive cells by hTEC10. A total of 180 cells each obtained from V001 and V004 were subjected for analysis. We identified 3 TCRA and TCRB clonotypes in samples from V001 and 6 clonotypes in those from V004. Importantly, 7 of 9 clones identified by hTEC10 were not listed in the clonotype determined by deep sequencing. To evaluate the effector functions of the cloned TCRs, paired TCRA and TCRB gene segments obtained by hTEC10 were transduced into PHA blasts established from T cells derived from CMV seronegative HLA-A2-negative donor. We confirmed that these PHA blasts transduced with HLA-A2-restricted CMV-specific TCRA and TCRB genes were NLV/A2-tetramer positive. Furthermore, these cells secreted INF-γ in response to NLV peptide as measured by ELISA. Conclusions: Ex vivo expanded anti-CMV CTLs reactive with NLV/A2 tetramers were extremely oligoclonal and consisted of only a few dominant clones. Determination of CDR3 sequences of these clones was feasible by quantitative deep sequencing combined with single cell cloning of TCRA and TCRB gene segments, although the results were complementary and not identical. This method could be useful for the efficient screening of the highly functional TCRs for adoptive T-cell immunotherapy. Disclosures No relevant conflicts of interest to declare.

Description: Background: Although the onset of invasive pulmonary aspergillosis (IPA) after allogeneic hematopoietic stem cell transplantation (HSCT) was bimodal, IPA early after HSCT has become less frequent, partly due to the shortening of neutropenic periods by the use of peripheral blood stem cells or colony-stimulating factors. Therefore, the validity of empiric anti-Aspergillus treatments based on sustained febrile neutropenia according to the IDSA guideline should be re-evaluated. Patients and Methods: We retrospectively reviewed the clinical records of 114 adult patients who underwent allogeneic HSCT between September 2002 and December 2005 in HEPA-filtered clean rooms at the University of Tokyo Hospital. Fluconazole at 200 mg/day was given as anti-Candida prophylaxis. In general, anti-Aspergillus agents were not started empirically, but presumptively started after the detection of positive Aspergillus antigen, positive beta-D-glucan, halo-sign on chest X-ray or CT-scan, and so on, associated with sustained febrile neutropenia. For the definition of early IPA, we employed proven or probable IPA according to the EORTC/MSG criteria that developed between the day of HSCT and seven days after engraftment. Results: All but two who experienced early death showed neutrophil engraftment at a median of 16.5 days after HSCT. Although 15 patients developed IPA after HSCT, early IPA was observed only in 2 (1.8 %) patients. Among 73 patients who experienced sustained febrile neutropenia for more than 7 days before engraftment, we empirically started anti-Aspergillus agents in 13 patients a median of 9 days after the development of febrile neutropenia, whereas fluconazole was continued in 60 patients who had febrile neutropenia for 15 days in median. Four of the 60 patients received presumptive anti-Aspergillus therapy, one of whom developed probable IPA after the initiation of treatment. There was another patient who received anti-Aspergillus treatment only after the development of probable IPA because he showed no prior signs for presumptive therapy. There was no difference in the incidence of early IPA between patients who received empiric anti-Aspergillus therapy and those who did not (0% vs. 3.3%, P〉0.99). The two patients who developed early probable IPA were successfully treated with anti-Aspergillus agents. Conclusions: These findings throw doubt on the validity of empiric anti-Aspergillus treatments for allogeneic HSCT recipients with sustained febrile neutropenia, provided that they are treated in clean units with anti-Candida prophylaxis. A randomized controlled trial is warranted to compare empiric and presumptive anti-Aspergillus treatments for allogeneic HSCT recipients with sustained febrile neutropenia.

Description: Background Cytomegalovirus (CMV) infection is still a major infectious complication following allogeneic hematopoietic cell transplantation (allo-HCT). Recently, it is reported that CMV reactivation is associated with a decreased risk of relapse in patients with acute myeloid leukemia (AML). The aim of this study is to evaluate the impact of early CMV reactivation on the incidence of disease relapse after allo-HSC in a large cohort of patients. Patients and Methods The Transplantation-related Complication Working Group of JSHCT retrospectively surveyed the database of the Transplant Registry Unified Management Program (TRUMP) at the JSHCT. Patients with AML (n=1836), acute lymphoblastic leukemia (ALL) (n=911), chronic myeloid leukemia (CML) (n=223) and myelodysplastic syndrome (MDS) (n=569) who underwent their first allo-HCT from HLA matched related or unrelated donors between 2000 and 2009, and who survived without disease relapse until day 100 after transplantation were analyzed. Patient who received umbilical cord blood transplantation were not included. Patients underwent surveillance by pp65 antigenemia from the time of engraftment and the start of preemptive therapy was defined as CMV reactivation. Cox proportional hazards models were used to evaluate the risk factors of relapse, non-relapse and overall mortality. CMV reactivation and acute/chronic GVHD were evaluated as time-dependent covariates. Results CMV reactivation was associated with a decreased cumulative incidence of relapse among patients with AML (20.3% versus 26.4%, p=0.027), but not in patients with ALL, CML or MDS by Gray’s test. Among 1836 AML patients, CMV reactivation occurred in 795 patients (43.3%) at median 42 days (-8 - 407 days), and 436 patients (23.7%) relapsed at median 221 days (101-2057 days) after allo-HCT. Grades II to IV acute GVHD developed in 630 patients (34.3%). On multivariate analysis taking competing risk factors into account, three factors significantly associated with a decreased risk of AML relapse: CMV reactivation (HR 0.765, 95%CI 0.59-0.99), unrelated donor compared to related donor (HR 0.593, 95%CI 0.42-0.84) and development of chronic GVHD (HR 0.773, 95% CI 0.60-0.99). However, no benefit in relapse-free survival associated with CMV reactivation was observed (HR 1.025, 95%CI 0.85-1.24). CMV reactivation was associated with increased non-relapse mortality (HR 1.160, 95%CI 1.02-1.32) and overall mortality (HR 1.370, 95%CI 1.11-1.69). Pre-transplant CMV serostatus was not extracted as a risk factor for relapse, non-relapse and overall mortality. Conclusions A beneficial effect of CMV reactivation on the subsequent relapse risk was observed in patients with AML but no other hematological malignancies in our large cohort study. However, this benefit was canceled out by the increased non-relapse mortality. The underlying mechanism is unclear, but the immunological activation against CMV reactivation plays an essential role in this association. Thus, the immune augmentation treatment options including vaccination and adoptive T-cell transfer might be useful to take advantage of the efficacy of CMV reactivation with minimal increase of non-relapse mortality. Disclosures: Miyamura: Novartis: Speakers Bureau; JRC Nagoya 1st Hospital: Employment. Nakamae:Kyowa Hakko Kirin Pharma, Inc.: Research Funding, Speakers Bureau, Travel/accommodations/meeting expenses Other; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Travel/accommodations/meeting expenses, Travel/accommodations/meeting expenses Other. Fukuda:the Japanese Ministry of Health, Labour, and Welfare: Research Funding.

Description: Alemtuzumab, a humanized monoclonal antibody directed against human CD52, has a strong lympholytic effect. We performed a study to evaluate the safety of unmanipulated HSCT from 2 or 3 loci-mismatched related donors using in vivo alemtuzumab. A total body irradiation-based regimen was used in young patients, while those 50 years old or older received fludarabine-based but myeloablative conditioning. Alemtuzumab was added to these regimens by intravenous infusion at 0.2 mg/kg/day for 6 days (days −8 to −3). We have treated 14 patients thus far. All achieved neutrophil engraftment with complete donor-type chimerism. Only 1 developed grade III-IV acute GVHD. There was no infection-related death. However, 8 patients (57.1%) developed grade II-III cardiac complications, mainly with congestion, according to Bearman’s criteria. We thus retrospectively analyzed the records of 143 adult patients who underwent allogeneic HSCT from 1995 to 2004 to evaluate whether the use of alemtuzumab was an independent risk factor for cardiac complications. On univariate analyses, the cumulative dose of anthracyclines (P=0.001), smoking history (P=0.036), serum ferritin level (P=0.033), left ventricular ejection fraction (P=0.070), HLA-mismatch (P=0.054), and the use of in vivo alemtuzumab (P=0.0002) affected the incidence of grade II to IV cardiac complications with at least borderline significance. Of these, multivariate analysis with backward stepwise selection revealed that the cumulative dose of anthracyclines and the use of in vivo alemtuzumab were independent significant risk factors for cardiac complications (P=0.0016 and P=0.0001, respectively). The duration from transplantation to the onset of cardiac complications was significantly longer in the alemtuzumab group (P=0.02). Fortunately, all the cardiac complications in the alemtuzumab group were successfully treated by the use of diuretics and/or catecholamines. In conclusion, in vivo alemtuzumab has an excellent efficacy to prevent severe GVHD even in HLA-mismatched HSCT. However, the use of in vivo alemtuzumab along with myeloablative conditioning for HLA-mismatched transplantation may affect the incidence of cardiac complications after transplantation. Considering the low incidence of cardiac complications in HLA-matched HSCT with reduced-intensity conditioning, frequent cardiac complications in our series might have resulted not only from alemtuzumab but also from intensive conditioning and/or cytokines excretion associated with the engraftment of HLA-mismatched donor cells. Close monitoring of the cardiac function may be important in such transplantation, especially in patients who had received a high cumulative dose of anthracyclines, because cardiac dysfunction could be manageable by an early detection and early treatment.

Description: Background It is of great importance to know the impact in quality of life (QOL) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) according to types and severity of chronic graft-versus-host disease (GVHD), to care for physical, mental and social aspects of transplant survivors. We conducted a cross-sectional questionnaire study to examine the relationship between patient-reported QOL and chronic GVHD defined by National Institute of Health (NIH) criteria. We also investigated the degree of agreement between visual analogue scales provided by patients (VASpt) and physicians (VASdoc). Methods The protocol was approved by the Institutional Review Board at each of the participating centers, and all subjects provided informed consent in accordance with the Declaration of Helsinki. Patients enrolled in the cohort were identified in the national transplant registry database registered from 47 centers which participated this study. Eligibility criteria included recipients of allo-HSCT for hematological diseases between 1995 to 2009, who were aged 16 years or above at transplant and 20 years or above at survey, and were relapse-free at survey. A total of 3301 eligible patients were registered in the database. SF-36, FACT-BMT, and VAS were administered to assess patient-reported QOL. Physicians evaluated the types and overall and organ-specific severity grades of chronic GVHD defined by NIH criteria (skin, mouth, eyes, gastrointestinal tract, liver, lung symptom, pulmonary function tests [PFT], joint and fascia, and genital tract), oral administration of immunosuppressant (IS), and VAS of respective patients at survey. Multivariable models were constructed to examine the relationship between QOL scores and types and severity of chronic GVHD after controlling for background covariates. Adjusted mean scores for the SF-36 summary scores were obtained as norm-based scores (mean=50, SD=10 based on general population). Interclass correlation coefficient (ICC) was obtained to evaluate the degree of agreement between VASpt and VASdoc. Data were analyzed with SPSS statistical software. Results In 1250 patients who were informed of the study, consent was obtained from 1216, and 1149 patients returned the questionnaires. Nine patients were excluded because of a time lag (〉4 months) that patient/physician completed reports, consequently, 1140 pairs of patient's and physician's questionnaires were included in the analysis. Males accounted for 52%, the median age at survey was 51 (range, 20-77), and the median time after allo-HSCT was 7.1 years (3.3-18.9). By the global severity score, 34% of patients had no GVHD, 29% mild, 25% moderate, and 9% had severe chronic GVHD. Frequently affected organs were eye (31%), skin (25%) and oral cavity (21%). The median scores were 49.3 for physical component summary (PCS), 52.4 for mental component summary (MCS), and 52.3 for role/social component summary (RCS). Overall, VASdoc were higher than VASpt (median, 91.0 vs 75.6). By the NIH global severity, PCS, FACT-BMT total score, VASpt and VASdoc were significantly (p

Description: Recently, a growing body of evidence has suggested that adiponectin, which is secreted by adipose tissues, plays a critical role in obesity-related and autoimmune diseases. We compared the concentrations of adiponectin among 26 normal subjects and 34 allogeneic stem cell transplantation recipients. The concentrations of adiponectin were significantly higher in recipients with chronic graft-versus-host disease (cGVHD) than those in subjects without cGVHD (21.7 ± 11.0 vs 9.1 ± 6.1 μg/mL in females, P 〈 .001; and 10.1 ± 6.8 vs 4.3 ± 2.9 μg/mL in males, P = .003). Multivariate analysis revealed that a higher concentration of adiponectin was associated with female sex (β-coefficient 8.2, P 〈 .0001) and the severity of cGVHD (β-coefficient 6.6, 12.7, and 15.6, P 〈 .01, each for mild, moderate, and severe cGVHD, respectively). In addition, adiponectin levels increased as cGVHD progressed, decreased as cGVHD improved, and did not change with stable cGVHD. In conclusion, adiponectin was associated with the severity of cGVHD and might play a role in the pathophysiology of cGVHD.