A prospective dose-finding trial using a modified continual reassessment method for optimization of fludarabine plus melphalan conditioning for marrow transplantation from unrelated donors in patients with hematopoietic malignancies.

BACKGROUND: Because of the less graft-facilitating effect by bone marrow (BM), we need to assess a dosage of conditioning more accurately particularly in combination with reduced-intensity conditioning. Thus we examined that modified continual reassessment method (mCRM) is applicable for deciding appropriate conditioning of allogeneic BM transplantation. PATIENTS AND METHODS: The conditioning regimen consisted of i.v. fludarabine (125 mg/m2) plus an examination dose of i.v. melphalan. The primary endpoint was a donor-type T-cell chimerism at day 28 with successful engraftment defined as >90% donor cells. Five patients per dose level were planned to be accrued and chimerism data were used to determine the next dose. RESULTS: Seventeen patients were enrolled at doses between 130 and 160 mg/m2. The dose was changed from 160 to 130 mg/m(2) (second level) after five full-donor chimerisms. With one patient of 0% chimera in the second level, the dose was increased to 135 mg/m2 (third level). Following five full-donor chimerisms in the third level, the study was complete as projected. CONCLUSIONS: mCRM was shown to be a relevant method for dose-finding of conditioning regimen. The melphalan dose of 135 mg/m2 was determined as the recommended phase II dose to induce initial full-donor chimerism.

Clinical characteristics of chronic graft-versus-host disease following umbilical cord blood transplantation for adults.

Chronic GVHD is a significant complication following allogeneic hematopoietic stem cell transplantation; however, the clinical characteristics of chronic GVHD following cord blood transplantation (CBT) in adults have not been well described. Between March 2001 and November 2005, a total of 77 patients underwent CBT at eight transplantation centers of the Nagoya Blood and Marrow Transplantation Group. Of 77 patients, 29 survived without graft failure or progression of underlying diseases for at least 100 days after transplantation. The median age of the 29 patients was 42 years (range, 18-67 years). Seven patients developed chronic GVHD (extensive, n=4; limited, n=3) disease. The cumulative incidence of chronic GVHD 1 year after day 100 was 24% (95% confidence interval (CI), 11-41%), and the organs involved were the skin (n=6), oral cavity (n=4), liver (n=1) and gastrointestinal tract (n=1). In three patients, chronic GVHD was resolved with supportive care. The remaining four were successfully treated with additional immunosuppressive therapy. Event-free survival rates of the 29 patients with and without chronic GVHD 3 years after day 100 were 83 (95% CI, 27-97%) and 36% (95% CI, 17-56%), respectively (P=0.047). These results suggest that chronic GVHD following CBT is mild and has a graft-versus-malignancy effect.

The HLA-A*0201-restricted minor histocompatibility antigen HA-1H peptide can also be presented by another HLA-A2 subtype, A*0206.

HA-1(H) is one of the most attractive minor histocompatibility antigens (mHA) as a target for immunotherapy of hematopoietic malignancies, but HLA-A*0201 and HLA-B60 molecules capable of presenting HA-1(H)-derived peptides are less common in eastern Asian populations when compared with Caucasian populations. Therefore, an attempt was made to search for novel epitopes presented by HLA alleles other than those previously reported by generating CTL lines from patients undergoing HLA-identical, HA-1 disparate hematopoietic stem cell transplantation (hematopoietic SCT) by stimulation with a 29-mer HA-1(H) peptide spanning a central polymorphic histidine (His). Two CTL clones established were found to be restricted by HLA-A*0206, which is the second or third most common HLA-A2 subtype worldwide. Epitope mapping revealed that the clones recognized the same nonameric peptide as A*0201-restricted HA-1(H), VLHDDLLEA. This epitope was unexpected, since it does not contain any preferred anchor motifs for HLA-A*0206. However, an HLA peptide binding assay revealed stronger binding of this peptide to A*0206 than to A*0201. Interestingly, HLA-A*0206-restricted CTL clones could lyse both HLA-A*0206(+) and HLA-A*0201(+) targets (including leukemic blasts) that express HA-1(H) peptide endogenously, whereas an HLA-A*0201-restricted, HA-1(H)-specific CTL clone failed to lyse HLA-A*0206(+) targets. This finding will expand the patient population who can benefit from HA-1(H)-based immunotherapy.

Short-term methotrexate could reduce early immune reactions and improve outcomes in umbilical cord blood transplantation for adults.

Post transplant immune disorders are problematic in cord blood transplantation (CBT) for adult patients, and optimal prophylaxis has not been established. We investigated whether intensive graft-versus-host disease (GVHD) prophylaxis using short-term methotrexate (MTX) has a prognostic impact on CBT. Post-CBT immune reactions were classified according to time course as pre-engraftment immune reaction (PIR), engraftment syndrome (ES) or acute GVHD. Between March 2001 and November 2005, a total of 77 patients underwent CBT at eight transplantation centers. Median age was 48 years (range, 18-69 years). Preparative regimens comprised myeloablative (n=31) or reduced-intensity (n=46). Acute GVHD prophylaxis included cyclosporine alone (n=23), tacrolimus alone (n=12), cyclosporine plus MTX (n=17), tacrolimus plus short-term MTX (n=23) or cyclosporine plus methylprednisolone (n=2). Cumulative incidences of PIR, ES and grade II-IV GVHD were 36, 12 and 23%, respectively. Short-term MTX exerted significant favorable effects on post-CBT immune reactions (hazard ratio, 0.55; 95% confidence interval (95% CI), 0.31-0.98; P=0.04) in multivariate analysis. Overall survival rates for patients with and without short-term MTX at day 180 were 59% (95% CI, 42-73%) and 16% (95% CI, 6.6-30%) (P=0.0001), respectively. Short-term MTX could offer one optimal regimen to reduce immune reactions and improve outcomes in CBT.

Increase of bone marrow macrophages and CD8+ T lymphocytes predict graft failure after allogeneic bone marrow or cord blood transplantation.

Graft failure (GF) remains an obstacle to survival after allogeneic hematopoietic stem cell transplantation. However, differentiating GF from delayed engraftment (DE) can be difficult. Host CD8+ lymphocytes have been reported to mediate graft rejection, but the impact of macrophages on DE or GF is yet to be clarified. Peri-engraftment bone marrow (BM) specimens of 32 adult patients with normal engraftment, DE or GF were retrospectively evaluated to identify the potential associations of CD163+ macrophage and CD8+ lymphocyte infiltration into BM. The macrophage or CD8+ lymphocyte number/total nucleated cell number was defined as the Mac ratio and CD8 ratio, respectively. Both DE and GF groups had significantly higher Mac ratios at day 14 than the normal group (P<0.0001), but no significant difference was observed between the DE and GF groups (P=1.000). The CD8 ratio at day 14 was significantly higher in the GF than in the normal group (P=0.005), whereas the CD8 ratios of the DE and normal groups were similar (P=0.07). A high Mac ratio at day 14 was associated with a risk of DE or subsequent GF. Patients with increased CD8 ratio at day 14 had a further risk of GF. The Mac ratio and the CD8 ratio appear to be well suited for predicting engraftment status.

Exploratory research for optimal GvHD prophylaxis after single unit CBT in adults: short-term methotrexate reduced the incidence of severe GvHD more than mycophenolate mofetil.

In order to examine GvHD prophylaxis in umbilical cord blood transplantation (UCBT) in more detail, we compared transplant outcomes after UCBT for acute leukemia among GvHD prophylaxes using registry data. We selected patients transplanted with a calcineurin inhibitor and methotrexate (MTX)/mycophenolate mofetil (MMF) combination. A total of 1516 first myeloablative UCBT between 2000 and 2012 (Cyclosporine A (CyA) plus MTX, 824, Tacrolimus (Tac) plus MTX, 554, Tac plus MMF, 138) were included. With adjusted analyses, Tac plus MMF showed a significantly higher risk for grade II-IV and III-IV acute GvHD than CyA or Tac plus MTX. Although NRM was similar, Tac plus MMF showed a significantly lower risk of relapse than CyA or Tac plus MTX. A significant difference was observed in the risk of overall mortality (OM) between the MTX-containing group and MMF-containing group. In patients with standard-risk disease, there was no significant difference in the risk of OM in any GvHD prophylaxis. However, in patients with advanced-risk disease, Tac plus MMF showed a significantly lower risk of OM. Therefore, MTX-containing prophylaxis is preferred in UCBT for standard-risk disease, whereas MMF-containing prophylaxis is preferred for advanced-risk disease. A prospective study to identify optimal GvHD prophylaxis for UCBT is warranted.

GvHD prophylaxis after single-unit reduced intensity conditioning cord blood transplantation in adults with acute leukemia.

To investigate better GVHD prophylaxis in reduced intensity conditioning umbilical cord blood transplantation (RIC-UCBT), we compared transplant outcomes after UCBT among GvHD prophylaxes using the registry data. We selected patients transplanted for AML or ALL with a calcineurin inhibitor and methotrexate (MTX)/mycophenolate mofetil (MMF) combination. A total of 748 first RIC-UCBT between 2000 and 2012 (MTX+ group, 446, MMF+ group, 302) were included. The cumulative incidence of neutrophil and platelet counts higher than 50 000/µL was significantly better in the MMF+ group (relative risk (RR), 1.55; P<0.001: RR, 1.34; P=0.003, respectively). In multivariate analyses, the risk of grade II-IV and III-IV acute GvHD was significantly higher in the MMF+ group than in the MTX+ group (RR, 1.75; P<0.001: RR, 1.97; P=0.004, respectively). In disease-specific analyses of AML, the risk of relapse of high-risk disease was significantly lower in the MMF+ group (RR, 0.69; P=0.009), whereas no significant difference was observed in the risk of relapse-free and overall survival in high-risk disease. In patients with standard-risk disease, no significant differences were noted in the risk of relapse or survival between the MTX+ and MMF+ groups. Collectively, these results suggest that MMF-containing prophylaxis may be preferable in RIC-UCBT, particularly for high-risk disease.

Impact of graft-versus-host disease on outcomes after unrelated cord blood transplantation.

The effect of graft-versus-host disease (GVHD) on transplant outcomes after unrelated cord blood transplantation (UCBT) has not been fully elucidated. We analyzed the impact of acute and chronic GVHD on outcomes in adult patients with acute leukemia or myelodysplastic syndrome who underwent their first UCBT (n=2558). The effect of GVHD on outcomes was analyzed after adjusting for other significant variables. The occurrence of GVHD was treated as a time-dependent covariate. The occurrence of grade 1-2 or 3-4 acute GVHD was significantly associated with a lower relapse rate. Grade 3-4 acute GVHD was associated with a higher risk of non-relapse and overall mortality than no acute GVHD, whereas grade 1-2 acute GVHD was associated with a lower risk of non-relapse and overall mortality than no acute GVHD. Limited or extensive chronic GVHD was significantly associated with a lower relapse rate. Limited chronic GVHD was associated with a lower overall and non-relapse mortality than no chronic GVHD. In conclusion, mild acute or chronic GVHD was associated not only with a low risk of relapse but also with a low risk of non-relapse mortality, and provides a survival benefit in UCBT.

Increased risk for treatment-related mortality after bone marrow transplantation in GSTM1-positive recipients.

Treatment-related mortality (TRM) is a major obstacle to successful allogeneic hematopoietic stem cell transplantation (HSCT). A variety of drugs are used in allogeneic HSCT, and a genetic polymorphism in metabolic enzymes could affect the metabolism of drugs and potentially influence TRM. Here, we focused attention on GSTM1 and GSTT1 enzymes, which metabolize chemotherapeutic agents, chemical carcinogens and by-products of oxidative stress and are absent from more than 50% of some populations. To assess the significance of homozygous GSTM1 and GSTT1 gene deletion in HSCT, we analyzed DNA from 373 patients with hematological disease and their HLA-identical unrelated bone marrow donors using PCR. Homozygous GSTM1 and GSTT1 gene deletions were observed in 56 and 45% of patients, respectively, and 57 and 46% of donors, respectively. There was no significant association between GSTT1 polymorphism and any outcome. However, a GSTM1-positive genotype in recipients was significantly associated with higher TRM and lower survival. These results suggest that a GSTM1-null genotype in recipients protects against TRM after allogeneic HSCT. Further studies are needed to elucidate a mechanism of increased risk for TRM in GSTM1-positive recipients.

Risk and prognostic factors for Japanese patients with chronic graft-versus-host disease after bone marrow transplantation.

The incidence and prognostic factors for chronic graft-versus-host disease (cGVHD) were evaluated for 255 Japanese patients who survived more than 100 days after bone marrow transplantation, and of whom 119 (47%) developed cGVHD. Prior acute GVHD (grade 2-4) and use of an unrelated donor were significantly associated with the onset of cGVHD. Presence of cGVHD did not have an impact on mortality (hazard ratio (HR) = 0.89; 95% confidence interval (CI), 0.59-1.3). Three factors at diagnosis were associated with cGVHD-specific survival: presence of infection (HR = 4.1; 95% CI, 1.6-10.3), continuing use of corticosteroids at the onset of cGVHD (HR = 3.9; 95% CI, 1.7-9.1), and a Karnofsky performance score <80 (HR = 4.7; 95% CI, 2.0-11.3). The probability of cGVHD-specific survival at 4 years was 79% (95% CI, 70-86%). The severity and death rate of Japanese patients with cGVHD was lower than those for populations in Western countries, which might be the result of greater genetic homogeneity of Japanese ethnics. Our patients could not be accurately classified when the proposed prognostic models from Western countries were used, thus indicating the need for a different model to identify high-risk patients.

Increased frequency of the angiotensin-converting enzyme gene D-allele is associated with noninfectious pulmonary dysfunction following allogeneic stem cell transplant.

Noninfectious pulmonary dysfunction (NIPD) is a common and often fatal complication associated with allogeneic hematopoietic stem cell transplantation (HSCT). An insertion/deletion polymorphism in the angiotensin-converting enzyme (ACE) gene has been extensively studied in relation to cardiovascular and renal disease, and lung fibrosis. In pulmonary fibrosis, D-allele frequency is significantly higher than in the control population. We hypothesized that a similar mechanism exists between post-HSCT NIPD and pulmonary fibrosis in the absence of HSCT. We retrospectively analyzed the incidence of NIPD and the ACE genotype polymorphism in 118 Japanese patients who underwent HSCT from HLA-identical sibling donors. NIPD occurred in 17 cases. Deletion/deletion genotype carriers were more common in the NIPD group than in the other 101 patients (41.2 vs 11.9%; hazard ratio, 5.19; 95% confidence interval, 1.67-16.21). There were no significant relationships between the clinical characteristics of patients and the development of NIPD. These findings suggest that the ACE genotype is associated with the development of NIPD following HSCT. This study is the first to report the relationship between genetic background and NIPD.

Integration of humoral and cellular HLA-specific immune responses in cord blood allograft rejection.

In allo-stem cell transplantation (SCT), it is unclear whether donor-specific anti-HLA Abs (DSAs) can actually mediate graft rejection or if they are simply surrogate markers for the cellular immunity that causes graft rejection. Here, we first analyzed a case of cord blood allograft rejection in which DSA and cytotoxic T lymphocyte (CTL) specific for donor HLA-B*54:01 were detected at the time of graft rejection. Both the DSA and CTL inhibited colony formation by unrelated bone marrow mononuclear cells sharing HLA-B*54:01, suggesting that the humoral and cellular immune responses were involved in the graft rejection. Interestingly, the DSA and CTL were also detected in cryopreserved pre-transplant patient blood, raising a hypothesis that the presence of anti-HLA Abs could be an indicator for corresponding HLA-specific T cells. We then evaluated the existence of HLA-specific CD8(+) T cells in other patient blood specimens having anti-HLA class I Abs. Interferon-γ enzyme-linked immunospot assays clearly confirmed the existence of corresponding HLA-specific T-cell precursors in three of seven patients with anti-HLA Abs. In conclusion, our data demonstrate that integrated humoral and cellular immunity recognizing the same alloantigen of the donor can mediate graft rejection in DSA-positive patients undergoing HLA-mismatched allo-SCT. Further studies generalizing our observation are warranted.

Lymphoid blast crisis of chronic myelogenous leukemia occurring more than 11 years after receiving an allogeneic bone marrow transplant for chronic myelogenous leukemia in myeloid blast crisis at onset.

A 25-year-old male developed lymphoid blast crisis (BC) of chronic myelogenous leukemia (CML) more than 11 years after receiving an allogeneic bone marrow transplant (alloBMT) for CML with myeloid BC at presentation from his HLA-identical brother. The lymphoid BC of CML probably occurred without a preceding chronic phase of CML. This case illustrates the difficulties involved in determining the appropriate length of follow-up after alloBMT.

Intestinal thrombotic microangiopathy after allogeneic bone marrow transplantation: a clinical imitator of acute enteric graft-versus-host disease.

Thrombotic microangiopathy after bone marrow transplantation (post-BMT TMA) is a serious transplant-related complication. We identified 16 patients with TMA after allogeneic BMT who showed histopathological evidence of intestinal TMA in their gut specimens (six autopsies, 10 biopsies). In all, 14 patients had grade II-IV acute graft-versus-host disease (GVHD). The first seven patients were retrospectively diagnosed with TMA. Since six of them were diagnosed with progressive GVHD at that time because there was no awareness of the existence of intestinal TMA, they received more intensive treatment for GVHD, but all died between days +49 and +253. In contrast, the remaining nine patients were recently diagnosed with intestinal TMA on the basis of colonoscopic biopsies. For eight of these patients, the immunosuppressants were reduced, and the patients' intestinal symptoms improved gradually. Six of the nine patients were still alive 12 months after the diagnosis of TMA. Our findings suggest that the gut may be a site involved in post-BMT TMA, presenting as ischemic enterocolitis. Differentiating intestinal TMA from acute GVHD is important in patients suffering from severe and refractory diarrhea after BMT.

Unrelated cord blood transplantation vs related transplantation with HLA 1-antigen mismatch in the graft-versus-host direction.

Little information is available regarding whether an unrelated cord blood (UCB) unit or a related donor with a 1-antigen mismatch at the HLA-A, HLA-B or HLA-DR locus in the graft-versus-host direction (RD/1AG-MM-GVH) should be selected as an alternative donor for patients without an HLA-matched related/unrelated donor. Therefore, we conducted a retrospective study using national registry data on patients with leukemia or myelodysplastic syndrome who received transplantation using a single UCB (n=2288) unit or an RD/1AG-MM-GVH (n=525). We found that the survival rate in the UCB group was comparable to that in the RD/1AG-MM-GVH group, although the RD/1AG-MM-GVH group with an HLA-B mismatch showed significantly higher overall and non-relapse mortality. Neutrophil and platelet engraftment were significantly faster, whereas the incidence of acute or chronic graft-versus-host disease (GVHD) was significantly higher in the RD/1AG-MM-GVH group. The incidence of acute or chronic GVHD in the RD/1AG-MM-GVH group with in vivo T-cell depletion was comparable to that in the UCB group, which translated into a trend toward better overall survival, regardless of the presence of an HLA-B mismatch. In conclusion, UCB and RD/1AG-MM-GVH are comparable for use as an alternative donor, except for RD/1AG-MM-GVH involving an HLA-B mismatch.

Eosinophilia predicts better overall survival after acute graft-versus-host-disease.

The prognostic significance of eosinophilia after allogeneic hematopoietic SCT (HSCT) and the relationship between eosinophilia and acute GVHD are not well studied. We retrospectively analyzed 201 adult patients who underwent their first allogeneic HSCT. Seventy-three (36%) patients developed eosinophilia within the first 100 days after HSCT. Eosinophilia was observed more frequently among those patients with acute GVHD than those without it (48 vs 25%, P=0.009). However, it was associated with milder acute GVHD and lower incidence of gut and liver acute GVHD. Among patients with acute GVHD, the 3-year OS for patients with and without eosinophilia was 63.4 and 47.2% (P=0.02), respectively, and 3-year nonrelapse mortality (NRM) was 20.2 and 37.5% (P=0.01), respectively. Multivariate analysis confirmed that eosinophilia was associated with a better OS (P=0.03) and lower NRM (P=0.046) in patients with acute GVHD, whereas it was not associated with a higher relapse rate (P=0.45). In contrast, eosinophilia was not associated with outcomes in those patients without acute GVHD. In conclusion, eosinophilia was associated with milder acute GVHD and better prognosis among patients with acute GVHD. The pathophysiology behind eosinophilia after allogeneic HSCT remains to be investigated.

Donor single nucleotide polymorphism in the CCR9 gene affects the incidence of skin GVHD.

The interactions between chemokines and their receptors may have an important role in initiating GVHD after allogeneic hematopoietic SCT (allo-HSCT). CCL25 and CCR9 are unique because they are exclusively expressed in epithelial cells and in Peyer's patches of the small intestine. We focused on rs12721497 (G926A), one of the non-synonymous single nucleotide polymorphisms (SNPs) in the CCR9 gene, and analyzed the SNP of donors in 167 consecutive patients who received allo-HSCT from an HLA-identical sibling donor. Genotypes were tested for associations with acute and chronic GVHD in each organ and transplant outcome. Multivariate analyses showed that the genotype 926AG was significantly associated with the incidence of acute stage > or =2 skin GVHD (hazard ratio: 3.2; 95% confidence interval (95% CI): 1.1-9.1; P=0.032) and chronic skin GVHD (hazard ratio: 4.1; 95% CI: 1.1-15; P=0.036), but not with GVHD in other organs or with relapse, non-relapse mortality or OS. To clarify the functional differences between genotypes, each SNP in retroviral vectors was transfected into Jurkat cells. In chemotaxis assays, the 926G transfectant showed greater response to CCL25 than the 926A transfectant. In conclusion, more active homing of CCR9-926AG T cells to Peyer's patches may produce changes in Ag presentation and result in increased incidence of skin GVHD.