Search

[Introduction] Cytomegalovirus (CMV) infection is a common viral infection in recipients of allogeneic hematopoietic stem cell transplantation (allo-SCT). Early CMV reactivation after allo-SCT is associated with worse non-relapse mortality (NRM) and overall survival (OS). Recently, T-cell replete HLA-haploidentical SCT using post-transplant cyclophosphamide (PTCy-haplo SCT) has been developed and spread rapidly worldwide. Rationale of this strategy is assumed to be selective and cytotoxic depletion of alloreactive T cells which are responsible for graft-versus-host disease (GVHD), while preserving non-alloreactive T cells which can contribute to fight infections. However, recent studies showed that PTCy-haplo SCT was associated with the increased incidence of CMV infection. Letermovir (LET), a novel anti-CMV agent, which inhibits the CMV DNA terminase complex, was approved for the prevention of CMV reactivation in allo-SCT recipients in 2018 in some countries including Japan based on the result of a phase 3 trial. Our facility performs LET prophylaxis in allo-SCT recipient if either donor or recipient is seropositive CMV. Although LET is effective for the prevention of CMV reactivation in allo-SCT recipients, the clinical effectiveness of LET prophylaxis in PTCy-haplo SCT is not well elucidated. Based on these things, we retrospectively evaluated the efficacy of LET prophylaxis in PTCy-haplo SCT. [Methods] We retrospectively analyzed consecutive 99 recipients who received PTCy-haplo SCT at Hokkaido University Hospital from March 2013 to March 2021. We compared the cumulative incidence of CMV reactivation between the LET prophylaxis group (LET group, 33 patients) and LET non-prophylaxis group (non-LET group, 66 patients). LET was initiated on the day 0 at a dosage of 480mg daily. All patients were monitored for CMV reactivation by using the anti-CMV pp65 monoclonal antibody HRP-C7 assay at least once a week from the time of engraftment. CMV reactivation was defined as the detection of CMV antigen positive cells per 50000 white blood cells, whereas CMV disease was defined by organ dysfunction attributable to CMV. [Results] As baseline patient's characteristics were summarized in Table1, there were no difference between LET and non-LET group in terms of age, sex, underlying disease, disease risk at transplantation, prior transplantation, conditioning intensity, and CMV serostatus. All patients received peripheral blood stem cell transplantation. GVHD prophylaxis consisted of Cy (40-50 mg/kg on day 3 and 4), tacrolimus (from day 5), and mycophenolate mofetil (from day 5). The cumulative incidence of CMV reactivation at 150 days after transplantation in LET group was significantly lower than that in non-LET group (30.3% versus 69.7%; P [Conclusion] To our best knowledge, this is the largest retrospective study about the efficacy of LET in PTCy-Haplo SCT. LET is effective for prevention of CMV reactivation in PTCy-haplo SCT. Further studies focused on the long term effect of LET prophylaxis in PTCy-haplo SCT, such as the incidence of relapse and chronic GVHD, is warranted. Figure 1 Figure 1. Disclosures Nakagawa: AbbVie GK: Research Funding; Takeda Pharmaceutical Company: Research Funding. Teshima: Gentium/Jazz Pharmaceuticals: Consultancy; Merck Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; Pfizer Inc.: Honoraria; Nippon Shinyaku Co., Ltd.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Fuji pharma CO.,Ltd: Research Funding; Takeda Pharmaceutical Company: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis International AG: Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; TEIJIN PHARMA Limited: Research Funding; Astellas Pharma Inc.: Research Funding; Bristol Myers Squibb: Honoraria; Janssen Pharmaceutical K.K.: Other; Kyowa Kirin Co.,Ltd.: Honoraria, Research Funding; Sanofi S.A.: Research Funding.

Graft-versus-host disease (GVHD) is the major complication after allogeneic stem cell transplantation (SCT). Emerging evidence indicates that GVHD leads to injury of intestinal stem cells. However, it remains to be investigated whether skin stem cells could be targeted in skin GVHD. Lgr5+ hair follicle stem cells (HFSCs) contribute to folliculogenesis and have a multipotent capacity to regenerate all epithelial cells in repair. We studied the fate of Lgr5+ HFSCs after SCT and explored the novel treatment to protect Lgr5+ HFSCs against GVHD using murine models of SCT. We found that GVHD reduced Lgr5+ HFSCs in association with impaired hair regeneration and wound healing in the skin after SCT. Topical corticosteroids, a standard of care for a wide range of skin disorders including GVHD, damaged HFSCs and failed to improve skin homeostasis, despite of their anti-inflammatory effects. In contrast, JAK1/2 inhibitor ruxolitinib significantly ameliorated skin GVHD, protected Lgr5+ HFSCs, and restored hair regeneration and wound healing after SCT. We, for the first time, found that GVHD targets Lgr5+ HFSCs and that topical ruxolitinib represents a novel strategy to protect skin stem cells and maintain skin homeostasis in GVHD.

Background: In the absence of an HLA matched related donor, unrelated donor hematopoietic cell transplantation (HCT) is an alternative. Recently, HLA-haploidentical HCT using posttransplant cyclophosphamide (PTCY-haplo) has been increasingly performed. Unrelated donor peripheral blood stem cell transplantation (PBSCT) program was initiated in 2011 in Japan with rather slow increase, therefore, bone marrow is the main stem cell source for HCT from unrelated donors, while PTCY-haplo is mainly PBSCT. We compared outcomes of unrelated donor bone marrow transplant (UBMT) and PTCY-haplo. Methods: This is a retrospective analysis of a registry data of the Japan Society for HCT and the Japanese Data Center for HCT using the Transplant Registry Unified Management Program (TRUMP). Patients with acute leukemia and myelodysplastic syndromes, aged between 16 and 69 years, who undergone their first HCT between 2012 and 2015 were included in the study. HLA-A, -B, -C, and -DRB1 allele-level 8/8 matched (n=1,470), 7/8 matched (n=859), and 6/8 matched (n=186) T-cell replete UBMT recipients, and 140 recipients of PTCY-haplo (PBSCT, n=133; BMT, n=6; PBSCT+BMT, n=1) were identified as subjects for analyses. Adjusted comparison of the groups on overall mortality was performed with the use of the Cox proportional-hazards regression model. For other outcomes with competing risks, Fine and Gray's proportional-hazards model for subdistribution of a competing risk was used. The models were used to estimate adjusted probabilities, with consideration of other significant clinical variables in the final multivariate models. Results: The median age for 8/8 matched UBMT, PTCY-haplo, 7/8 matched, and 6/8 matched UBMT were 52(16-69), 46.5(17-68), 50(16-69), 50(16-68). According to refined disease risk index (rDRI), patients with high or very high rDRI were 32%, 54%, 34%, 34% for 8/8 matched UBMT, PTCY-haplo, 7/8 matched, and 6/8 matched UBMT. Myeloablative conditioning was used in 74%, 51%, 73%, 68% of patients for 8/8 matched UBMT, PTCY-haplo, 7/8 matched, and 6/8 matched UBMT. Median follow-up period of survivors for 8/8 matched UBMT, PTCY-haplo, 7/8 matched, and 6/8 matched UBMT were 2.79 (0.03-5.603),1.82 (015-3.89), 3.00 (0.09-5.57), 3.17 (0.27-5.58). In adjusted comparison by multivariate analyses setting 8/8 matched UBMT as the reference, PTCY-haplo showed similar overall mortality (relative risk [RR]=0.97, 95% confidence interval [CI], 0.75-1.26, p=0.823), decreased risk of non-relapse mortality (RR=0.43, 95% CI, 0.25-0.74, p=0.003), increased risk of relapse (RR=1.57, 95% CI, 1.21-2.03, p=0.001), and decreased risk of grade II to IV acute GVHD (RR=0.68, 95% CI, 0.48-0.95, p=0.023). Relative risks for 7/8 matched and 6/8 matched UBMT were 1.08 (p=0.223) and 1.27 (p=0.032) for overall mortality, 1.29 (p=0.004) and 1.73 (p Conclusions: PTCY-haplo showed notably low non-relapse mortality which contributed to comparable short-term survival outcomes with 8/8 matched UBMT. Disclosures Ishiyama: Alexion Pharmaceuticals, Inc.: Honoraria. Ichinohe:Otsuka Pharmaceutical Co.: Research Funding; Nippon Shinyaku Co.: Research Funding; Alexion Pharmaceuticals: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; JCR Pharmaceuticals: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Takeda Pharmaceutical Co.: Research Funding; Zenyaku Kogyo Co.: Research Funding; Mundipharma: Honoraria; Novartis.: Honoraria; Taiho Pharmaceutical Co.: Research Funding; Sumitomo Dainippon Pharma Co.: Research Funding; Repertoire Genesis Inc.: Research Funding; MSD: Research Funding; Chugai Pharmaceutical Co.: Research Funding; CSL Behring: Research Funding; Eisai Co.: Research Funding; Kyowa Hakko Kirin Co.: Research Funding; Ono Pharmaceutical Co.: Research Funding; Pfizer: Research Funding; Astellas Pharma: Research Funding.

To the editor: Dasatinib can inhibit T-cell activation through inhibition of the Scr family of tyrosine kinases such as p56 (Lck).[1][1] It has been reported that some chronic myeloid leukemia (CML) patients who were treated with dasatinib developed chronic large-granular lymphocytosis (LGL) with

Abstract 4544 Background: Current prognostic models, including the International Prognostic Index (IPI), incorporate both patient and tumor characteristics. In contrast, recent studies show that variables related to the host adaptive immunity and the tumor microenvironment are significant prognostic variables in cases of diffuse large B-cell lymphoma (DLBCL). Recently, Wilcox and co-workers reported that lymphopenia, defined as an absolute lymphocyte count (ALC) 630/μL, at diagnosis was associated with inferior survival in patients with DLBCL treated with CHOP/R-CHOP (Leukemia, 2011). The same group also reported that the ALC/AMC ratio at diagnosis can be a biomarker to predict the clinical outcome in DLBCL patients treated with R-CHOP (Porrate et al. ASH 2011). However, it remains to be determined if these parameters can predict the outcome of autologous peripheral blood stem cell transplantation (APBSCT) in patients with DLBCL in the first remission. Methods: We retrospectively examined the predictive value of the AMC and ALC in a cohort of 55 consecutive DLBCL patients who uniformly underwent APBSCT in their first remission at Hokkaido University Hospital and Sapporo City General Hospital. At presentation, all patients were at high risk (Coiffier et al. J Clin Oncol, 1991) (1997 to 2000), or high (H)/high-intermediate (HI) risk, in the age-adjusted IPI (aaIPI) (2001 to 2012). After six cycles of CHOP (before 2000, N=16) or R-CHOP (after 2001, N=39), all patients were treated with APBSCT, followed by the MCVC regimen, consisting of ranimustine, carboplatin, etoposide and cyclophosphamide. We performed a receiver operating characteristics (ROC) analysis to determine the optimal cut-off point for both the AMC and ALC in our patients, and values of 551/uL and 1,000/uL were set for the subsequent analyses. The disease free survival (DFS) and overall survival (OS) were estimated using the Kaplan–Meier method and two-tailed log-rank test. Results: Twenty-five patients were male and thirty were female. According to the aaIPI, 15, 31, and nine patients were classified as H, HI, and low (L)/low intermediate (LI), respectively. The median duration of follow-up after APBSCT was 85 months (range 1 to 179 months). At diagnosis, the median ALC was 1,095/μL (range 286–3,396/μL) and the median AMC was 551/μL (range 63–1,870/μL). The estimated 5-year OS and DFS for the entire cohort were 78% (95% confidence interval (CI) 64–88%) and 73% (95% CI 59–83%), respectively. In contrast to the previous study, the ALC did not predict an inferior OS or DFS in a univariate analysis of dichotomized variables. The estimated 5-year OS and DFS for those who had lymphopenia (ALC551/μL) had no significant impact on the 5-year OS, with rates of 88% for those who had an elevated AMC and 73% for those who did not. However, an elevated AMC was associated with a superior 5-year DFS of 88% compared to that of 59% in the cohort which did not have an elevated AMC (hazard ratio 3.18, 95% CI 1.10–11.41, p=0.03). Conclusions: Lymphopenia or an elevated monocyte number at diagnosis did not predict a poor outcome for high-risk patients with DLBCL when APBSCT was given in the first remission. Our results suggest that the dismal outcome obtained with CHOP/R-CHOP in high risk DLBCL patients who concomitantly had lymphopenia and elevated AMC could be overcome by an APBSCT in the first remission, although these results should be confirmed in a prospective study. Disclosures: No relevant conflicts of interest to declare.

Abstract 4154 Background: Monosomal karyotype (MK) has been defined as the presence of two or more autosomal monosomies or of a single monosomy associated with at least one structural abnormality (Breems et al, JCO 2008). The presence of MK has been associated with extremely poor prognosis in patients with not only acute myeloid leukemia (AML) but myelodysplastic syndrome (MDS) (Patnaik et al, Leukemia 2011). Our goal was to investigate the efficacy of allogeneic hematopoietic stem cell transplantation (HSCT) for myeloid malignancies with MK. Patients and methods: We combined data from two transplant centers, Sapporo Hokuyu Hospital and Hokkaido University Hospital, and analyzed consecutive patients who underwent allogeneic transplantation for AML and MDS between January 2003 and July 2010. Patients were divided according to MK classification scheme into four groups (Oran et al, BBMT 2011), CN; cytogenetically normal, MK; monosomal karyotype, CBF; core binding factor abnormalities, Oth (Others); non-CBF and non-MK abnormalities. Patients with acute promyelocytic leukemia were excluded. Resuts: One-hundred eighty three out of 229 patients were analyzed with a median age of 48 years(15–68). Sixty one (33%) were from HLA-matched related donors, 86 (47%) from unrelated and 36 (20%) were cord blood.Conditioning regimens were myeloablative (MAC, n=102, 56%) or reduced intensity(RIC, n=81, 44%). Seventy patients (38%) were cytogenetically normal, 27 (15%)had CBF abnormalities, 70(38%) had non-CBF and non-MK abnormalities and 16(9%) had monosomal karyotype. There was no statistically difference between four groups in age, donor source and conditioning regimen. In the MK group, the proportion of MDS and non-remission state at stem cell transplantation were significantly higher than other groups (p=0.002, p Seven of 16 patients with MK died within the first 50 days after transplantation, and 9 patients died within 120 days. Five patients died of infection and 2 died of complicated organ failure and 2 died of progression disease. Three patients who underwent transplantation at non-remission setting, survived more than 1-year experienced chronic graft-versus-host disease, suggesting the existence of GVL effect to myeloid malignancies with MK. Conclusion: This retrospective analysis revealed the dismal prognosis of myeloid malignancies with MK, even after allogeneic HSCT. Novel therapies and strategies are urgently needed for this very poor prognostic group. Disclosures: No relevant conflicts of interest to declare.

Innate immune cells such as natural killer (NK) cells play a crucial rule in antitumor immune responses. NK cells have functionally opposite receptors for activating and inhibiting signals to exert cytotoxicity. NKG2D is a C-type lectin-like activating receptor and is expressed in various immune cells, including NK cells, NKT cells, CD8+α/β+T cells, and γ/δ+T cells. NKG2D recognizes its ligands, MHC class I-related chain A and B (MICA, MICB). NKG2D ligands are not present on normal cells but are induced by various stresses such as viral infection. Moreover NKG2D ligands are expressed in many malignant cells including hematological malignancies. It has been suggested that involvement of NKG2D in NK or CD8+Tcell-mediated cytotoxicity correlates with the expression levels of ligands on target cells. Therefore induction of NKG2D ligands may lead to enhance the sensitivity to NKG2D-mediated cytotoxicity. In this study, we could enhance expression levels of MICA and MICB by treatment with the histone deacetylase inhibitor trichostatin A (TsA) in lymphoid leukemic cell line BALL1 and some primary patients’ lymphoid leukemic cells. Treatment of BALL1 and patients’ leukemic cells with TsA for 12 hr increased MICA and MICB mRNA expression at least by more than 2 fold by Real-time PCR. Treatment of BALL1 with TsA for 12 hr increased MICA and MICB protein expression on the cell surface by more than 2 fold by flow cytometry analysis. These results suggested that expression of MICA and MICB is partly regulated by histone acetylation. Chromatin immunoprecipitation assay revealed that treatment with TsA resulted in increased acetylation of histone H3 and decreased association with the counteracting enzymes of histone acetyltransferases HDAC1 at the MICA and MICB promoter in BALL1 cell and patients’ leukemic cells. To examine the impact of the cytolytic activity of NKG2D-expressing cells on leukemic cells in which expression of NKG2D ligands was induced by TsA treatment, we performed standard 4 hr 51Cr release assays using BALL1 cells and patients’ leukemic cells. Up-regulation of NKG2D ligands by TsA treatment led to enhance the susceptibility of BALL1 and patients’ leukemic cells by 2 or 3 times to the cytolytic activity of NKG2D-expressing cells. Blocking experiment using specific antibodies for MICA and MICB inhibited the NKG2D-expressing cell-mediated cytolytic activity against BALL1 cells. Our results suggest that regulation of NKG2D ligands expression by treatment with chromatin-remodeling drugs may be an effective strategy to enhance the susceptibility of leukemic cells to the cytolytic activity of NKG2D-expressing cells for immunotherapy.

It has recently been shown that inhibitory natural killer cell receptors (NKRs) on not only NK cells but also on T cells negatively regulate NK cell and T cell functions through their binding to MHC class I molecules. The C-type lectin superfamily inhibitory NKR CD94/NKG2A heterodimer recognizes an HLA-E that preferably bound to a peptide derived from the signal sequences of most HLA class I. Therefore, CD94-expressing cells can monitor the global status of HLA class I on the tumor and leukemic cells and induce cytolytic attack without inhibitory signal against HLA class I decreased target cells resulting induction of graft-versus-leukemia (GVL) effect but does not attack normal cells with HLA class I expression resulting no enhancement of graft-versus-host disease (GVHD). On the other hand, CD4+ CD25+ regulatory T cells (Treg) contribute to suppress allogeneic immune responses and prevent transplant rejection and GVHD. In this study, we tried to expand CD94-expressing T cells and Treg cells from the same cord blood cells and then investigated their cytolytic characteristics and immunoregulatory function in order to develop a potential strategy of cell therapy for hematological malignancy. After CD4 enrichment by negative selection using magnetic cell sorting (MACS) (Miltenyi Biotec)(CD4-enriched fraction) from cord blood, CD4+ CD25+ cells were isolated by positive selection with anti-CD25 magnetic microbeads. We could get more than 1,000 fold expansion of CD94-expressing CD8 T cells from CD4-depleted fraction after 8 days culture with immobilized anti-CD3 monoclonal antibody (mAb) (1 μg/mL) and IL-15 (5 ng/mL). Isolated CD4+ CD25+ cells were cultured with anti-CD3/CD28 mAb-coated dynabeads and IL-15 (5 ng/mL) and we could get about 50 fold expansion of Treg cells for 8 days. These expanded Treg cells could suppress allogeneic mixed lymphocyte culture more than 80% (effector cells: Treg cells= 2:1) and expressed FoxP3 mRNA about 100 fold compared with isolated CD25-negative cells. Cytolytic activities of purified CD94-expressing cells (CD94 > 90%) detected by 4 hours 51Cr release assay against K562 were 68.8 ± 16.8 % (n=5). Coculture of CD94-expressing cells with expanded Treg cells (CD94-expressing cell: Treg cells= 1:1, preincubation 4 hours) did not have any effect on cytolytic activities of purified CD94-expressing cells against K562 cells (66.1 ± 19.8 %, n=5). CD94-expressing CD8 T cells with cytolytic activity could be expanded from CD4-deplted fractions and Treg cells with immunosuppressive activity and increased expression level of FoxP3 mRNA could be expanded from CD4-enriched fractions of the same cord blood. Expanded these cytolytic CD94-expressing CD8 cells might be able to induce GVL effect without enhancing GVHD and Treg cells might be able to suppress allogeneic response including GVHD and graft rejection. Therefore, this strategy may be useful to differentiate lymphocytes in cord blood to two different kinds of effector cells exhibiting cytolytic or immunoreguratoly characters.

Buckgrand. Graft-versus host disease (GVHD) is a major complication after hematopoietic stem cell transplantation (HSCT). Macrophage migration inhibitory factor (MIF) plays a pivotal role in systemic as well as local inflammatory and immune responses. Recent reports that MIF expression is up-regulated in the allo-immune reaction during renal transplantation. Otherwise, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein belong to the TNF family. The level of TRAIL expression in T cells as well as NK cells can be markedly up-regulated after cell activations. In this study we report that kinetics of serum level of MIF and TRAIL in GVHD patients before and after HSCT. Patients and Methods. Date randomly obtained from 16 patients (10 males and 6 females) who underwent allo-SCT for treatment of hematological malignancies at Hokkaido University Hospital during the period May 2001 to January 2005. All patients were informed consent of peripheral blood smpling. Eight patients were received conventional transplantation and the others were reduced intensity stem cell transplantation (RIST). Seven patients have HLA identical sibling donor, but the others were received unrelated donor. Twelve of the 16 patients was achieved acute GVHD (aGVHD), gradeIto IIin 8 patients. Twelve patients survived day 100 after allo-SCT, 9 of those 12 patients developed chronic GVHD(cGVHD). Serum MIF and TRAIL concentration were measured at various time points using enzyme-linked immunosorbent assays (ELISAs). Results. Serum MIF concentration analysis by ELISA showed that only patients who developed aGVHD significantly increased (two folds) before and after allo-SCT (avelage, from 7.34 ng/ml before allo-SCT to 14.7 ng/ml after allo-SCT, p=0.018). However, we could not detect any correlation of MIF levels and aGVHD severity, donor sources. On the other hand, serum TRAIL concentration analysis by ELISA showed that patients who developed aGVHD were not associated (avelage, from 458.6 pg/ml before allo-SCT to 484.12 pg/ml after allo-SCT, p=0.632). We could not detect association aGVHD severity, donor sources. However, peak titer in aGVHD patients tends to decrease in unrelated transplantation (related 580.86pg/ml, unrelated 415.59pg/ml, p=0.22). Interestingly, we showed that average serum TRAIL concentration before allo-SCT associated with aGVHD and cGVHD. Serum TRAIL concentration with aGVHD patients (n=12, 458.85pg/ml) was tended to increase than without aGVHD (n=4, 330.45pg/ml, P=0.063) and with cGVHD patients (n=9, 535.21pg/ml) was significantly increase without cGVHD patients (n=3, 282.0 ng/ml, P=0.007). Discussion. The present study demonstrated the kinetics of MIF and TRAIL. Systemic up-regulation of MIF expression is associated with the occurrence of aGVHD. This data suggested that MIF after allo-SCT might play a pathogenetic role in aGVHD. On the other hand, we suggested that high level of TRAIL before allo-SCT associated acute and cGVHD. Maybe we might be to estimate acute and cGVHD in examining TRAIL level before allo-SCT. In conclusion our data are the first to establish an association TRAIL and GVHD in allogeneic stem cell transplantation.