Donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene: detailed immunological function following add-back after haplo-identical transplantation.

BACKGROUND AIMS: Haplo-identical hematopoietic stem cell transplantation (HSCT) with add-back of donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene (TK cells) is one of the most widely applied promising new gene therapy approaches. However, the immunological status of added-back TK cells after HSCT has yet to be well characterized. METHODS: We investigated TK cells through the use of flow cytometry, T-cell receptor (TCR) Vß repertoire spectratyping and linear amplification-mediated polymerase chain reaction followed by insertion site analysis in a patient enrolled in our clinical trial. RESULTS: A comparison of onset with remission of acute graft-versus-host disease confirmed that TK cells were predominantly eliminated and that proliferative CD8(+) non-TK cells were also depleted in response to ganciclovir administration. The TCR Vß-chain repertoire of both TK cells and non-TK cells markedly changed after administration of ganciclovir, and, whereas the TCR repertoire of non-TK cells returned to a normal spectratype long after transplantation, that of TK cells remained skewed. With the long-term prophylactic administration of acyclovir, TK cells oligoclonally expanded and the frequency of spliced variants of TK cells increased. Known cancer-associated genes were not evident near the oligoclonally expanded herpes simplex virus (HSV)-TK insertion sites. CONCLUSIONS: We demonstrate obvious differences in immunological status between TK cells and non-TK cells. In addition, we speculate that long-term prophylactic administration of acyclovir increases the risk of oligoclonal expansion of spliced forms of TK cells.

Infusion of donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene for recurrent hematologic malignancies after allogeneic hematopoietic stem cell transplantation.

The infusion of donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene (TK-cells) is a promising strategy for the treatment of hematologic malignancies relapsing after allogeneic hematopoietic stem cell transplantation. Here we report the results of a phase I clinical trial designed to examine the feasibility, safety, and efficacy of donor lymphocyte infusion (DLI) of TK-cells. Three patients (two with malignant lymphomas, one with acute myeloid leukemia) were enrolled in the trial and received a single DLI of 1 × 10(7) or 5 × 10(7) TK-cells/kg. No local or systemic toxicity related to the gene-transfer procedure was observed. Two patients achieved stable disease. No patient had severe graft-versus-host disease requiring systemic steroid and/or ganciclovir administration. TK-cells were detected in the peripheral blood of all three patients by PCR, but did not persist longer than 28 days. Analysis of cytotoxic T lymphocyte activity detected no immune response against TK-cells by the recipient's own T cells. Flow cytometric analysis showed low proliferative activity and cytotoxic function of TK-cells. In conclusion, DLI of TK-cells was safely performed in all three patients. Our analysis suggests the probable cause of rapid disappearance of TK-cells to be insufficient in vivo expansion of TK-cells in these patients.

In vivo T-cell depletion with alemtuzumab in allogeneic hematopoietic stem cell transplantation: Combined results of two studies on aplastic anemia and HLA-mismatched haploidentical transplantation.

We evaluated the efficacy of in vivo T-cell depletion with alemtuzumab in two prospective studies according to the International Conference on Harmonisation (ICH)-Good Clinical Practice (ICH-GCP) guidelines; one was for patients with aplastic anemia (AA study) and the other was for patients who were undergoing hematopoietic stem cell transplantation (HSCT) from a 2- or 3-antigen-mismatched haploidentical donor (MM study). The final dose of alemtuzumab in these studies was 0.16 mg/kg/day for 6 days. At this dose, all of the 12 and 11 patients in the AA and MM studies, respectively, achieved initial engraftment and the incidences of Grade II-IV acute graft-versus-host disease (GVHD) were 0% and 18%. While cytomegalovirus (CMV) frequently reactivated, none of the patients developed fatal CMV disease. Transplantation-related mortality within 1 year after HSCT was observed in only two and one patients, respectively. The numbers of CD4+ and CD8+ T-cells and T-cell receptor rearrangement excision circles remained low within 1 year after HSCT. These findings suggest that the use of alemtuzumab at this dose in a conditioning regimen enables safe allogeneic HSCT even from a 2- or 3-antigen-mismatched donor. However, the use of a lower dose of alemtuzumab should be explored in future studies to accelerate immune recovery after HSCT.

Diagnosis and evaluation of intestinal graft-versus-host disease after allogeneic hematopoietic stem cell transplantation following reduced-intensity and myeloablative conditioning regimens.

Colonoscopic evaluation of mucosal tissues after allogeneic hematopoietic stem cell transplantation (HSCT) is very useful in evaluating pathogenesis and diagnosis of intestinal graft-versus-host disease (GVHD). However, information on the timing and sites of biopsies and the immunohistological evaluation of mucosal tissues for diagnosing intestinal GVHD, especially following reduced-intensity (RIC) regimens, remains very limited. A total of 33 patients with histologically proven GVHD after allogeneic HSCT with RIC (n = 23) and myeloablative conditioning (MAC, n = 10) regimens were enrolled in the present study. Colonoscopy was performed due to gastrointestinal symptoms, especially diarrhea and anorexia. Sites of biopsies with the worst histopathological grading were the terminal ileum in 67 % of patients. In the RIC group, the onset of diarrhea prior to colonoscopy examination was later (median: RIC, 57 vs. MAC, 27 days) and the number of patients who developed abdominal pain tended to be higher (RIC, 70 % vs. MAC, 30 %). A lower number of CD4+ cells and a higher ratio of Foxp3+ cells to CD4+ cells were detected in the involved lesions of intestinal GVHD following RIC. These differences in the RIC and MAC groups suggest that regimen-specific therapeutic strategies are required for diagnosing intestinal GVHD.

Comparison of outcomes after allogeneic hematopoietic stem cell transplantation in patients with follicular lymphoma, diffuse large B-cell lymphoma associated with follicular lymphoma, or de novo diffuse large B-cell lymphoma.

The outcome after allogeneic hematopoietic stem cell transplantation (allo-HCT) for diffuse large B-cell lymphoma (DLBCL) associated with follicular lymphoma (FL), which includes DLBCL with pre- or co-existing FL, remains controversial, and few previous reports have compared the outcomes after allo-HCT for FL, DLBCL associated with FL, and de novo DLBCL. We retrospectively analyzed 97 consecutive patients with FL (n = 46), DLBCL associated with FL (n = 22), or de novo DLBCL (n = 29) who received allo-HCT at our institute between 2000 and 2010. With a median follow-up of 53 months, the 5-year overall survival (OS) and progression-free survival (PFS) were, respectively, 77% and 70% for FL, 62% and 57% for DLBCL associated with FL, and 26% and 23% for de novo DLBCL. The 5-year cumulative incidences of non-relapse mortality and disease progression/relapse were, respectively, 16% and 15% for FL, 19% and 24% for DLBCL associated with FL, and 36% and 41% for de novo DLBCL. By a multivariate analysis, the OS and PFS for DLBCL associated with FL were significantly better than those for de novo DLBCL, whereas they were not significantly different from those for FL. These results suggest that allo-HCT may be a promising option for patients with not only advanced FL but also DLBCL associated with FL.

Real-time in vivo cellular imaging of graft-versus-host disease and its reaction to immunomodulatory reagents.

Visualizing the in vivo dynamics of individual donor cells after allogeneic hematopoietic stem cell transplantation (HSCT) will enable deeper understanding of the process of graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL). In this study, using non-invasive in vivo fluorescence imaging of the ear pinna, we successfully visualized green fluorescent protein (GFP) donor cells at the single cell level in the skin. This imaging model enabled visualization of the movement of GFP cells into blood vessels in real time after allogeneic HSCT. At day 1, a few donor cells were detected, and the movement of donor cells in blood vessels was readily observed at day 4. Early donor cell infiltration into non-lymphoid tissue was increased by treatment with croton oil, as an inflammatory reagent. Treatment with dexamethasone, as an anti-inflammatory reagent, suppressed donor cell infiltration. The in vivo cellular fluorescence imaging model described here is a very useful tool for monitoring individual donor cells in real-time and for exploring immunomodulatory reagents for allogeneic HSCT, as well as for understanding the mechanism of GVHD.

Feasibility of reduced-intensity cord blood transplantation as salvage therapy for graft failure: results of a nationwide survey of adult patients.

To evaluate whether rescue with cord blood transplantation (CBT) could improve the poor survival after graft failure (GF), we surveyed the data of 80 adult patients (median age, 51 years) who received CBT within 3 months of GF (primary 64, secondary 16), with fludarabine-based reduced-intensity regimens with or without melphalan, busulfan, cyclophosphamide, and/or 2-4 Gy total-body irradiation (TBI). A median number of 2.4 × 10(7)/kg total nucleated cells (TNC) were infused, and among the 61 evaluable patients who survived for more than 28 days, 45 (74%) engrafted. The median follow-up of surviving patients was 325 days, and the 1-year overall survival rate was 33% despite poor performance status (2-4, 60%), carryover organ toxicities (grade 3/4, 14%), and infections (82%) prior to CBT. Day 100 transplantation-related mortality was 45%, with 60% related to infectious complications. Multivariate analysis showed that the infusion of TNC ≥2.5 × 10(7)/kg and an alkylating agent-containing regimen were associated with a higher probability of engraftment, and that high risk-status at the preceding transplantation and grade 3/4 organ toxicities before CBT were associated with an increased risk of mortality. In conclusion, in an older population of patients, our data support the feasibility of CBT with a reduced-intensity conditioning regimen for GF.

Phase 1 trial of Wilms tumor 1 (WT1) peptide vaccine and gemcitabine combination therapy in patients with advanced pancreatic or biliary tract cancer.

An open-labeled, dose-escalation phase 1 trial of Wilms tumor 1 (WT1) vaccine and gemcitabine (GEM) combination therapy for patients with advanced pancreatic cancer or biliary tract cancer was performed. The primary end point was evaluation of toxicity, safety, and optimal immunologic dose of vaccine. Human leukocyte antigen (HLA)-A 0201, HLA-A 0206, and/or HLA-A 2402-positive patients with inoperable advanced pancreatic or biliary tract cancer who had not previously been treated with GEM were eligible for this study. Six doses of GEM and 4 doses of WT1 peptide (1 or 3 mg) emulsified in Montanide adjuvant were administered over 2 months. Twenty-five patients (13 male and 12 female) were enrolled. Nine patients had inoperable advanced pancreatic cancer, 8 had gallbladder cancer, 4 had intrahepatic, and 4 had extrahepatic bile duct cancer. The adverse events were comparable to those with GEM alone. Delayed-type hypersensitivity test was positive after vaccination in 2 patients, and WT1-specific T cells in peptide-stimulated culture were detected by tetramer assay in 59% (13 of 22) of patients. The disease control rate at 2 months was 89% for pancreatic cancer and 50% for biliary tract cancer. With a median follow-up time of 259 days, the median survival time for biliary tract cancer was 288 days, and that for pancreatic cancer was 259 days. Although objective clinical efficacy was not apparent, the safety of WT1 vaccine and GEM combination therapy was confirmed in this study.

A Markov decision analysis of allogeneic hematopoietic cell transplantation versus chemotherapy in patients with acute myeloid leukemia in first remission.

Various prospective trials have been performed to assess the roles of allogeneic hematopoietic cell transplantation (allo-HCT) and chemotherapy in patients with acute myeloid leukemia (AML) in first complete remission (CR1). However, the results have not always been consistent, and there has been a limited evaluation of quality of life (QOL) in these postremission strategies. We performed a Markov decision analysis that enabled us to compare survival outcomes with a QOL evaluation using a database of 2029 adult AML patients who achieved CR1. The Markov decision model compared 2 strategies: allo-HCT or chemotherapy in CR1. Patients who had intermediate- or unfavorable-risk AML had a longer life expectancy when they received allo-HCT in CR1 than patients treated with chemotherapy alone. Likewise, patients who had a suitable related donor who received allo-HCT in CR1 had a longer life expectancy. The life expectancy was shortened to a greater degree by adjustment for QOL in the allo-HCT group. Nevertheless, QOL-adjusted life expectancies in most of the subgroups remained longer in the allo-HCT group than in the chemotherapy group. Our results showed that older patients with a related donor and younger patients with unfavorable cytogenetics benefited the most from allo-HCT in CR1.

Comparison of allogeneic hematopoietic cell transplantation and chemotherapy in elderly patients with non-M3 acute myelogenous leukemia in first complete remission.

The benefits of allogeneic hematopoietic cell transplantation (allo-HCT) for patients with acute myelogenous leukemia (AML) in first complete remission (CR1) have mostly been evaluated in younger patients. Although favorable outcomes of allo-HCT over chemotherapy have been reported with the use of reduced-intensity conditioning (RIC) regimens in elderly patients with AML in CR1, information is still limited, especially on the effects of cytogenetic risks and donor sources. We collected data from AML patients aged 50 to 70 years who achieved CR1, and compared the outcome in 152 patients who underwent allo-HCT in CR1 (HCT group) to that in 884 patients who were treated with chemotherapy (CTx group). The cumulative incidence of relapse in the HCT group was significantly lower than that in the CTx group (22% versus 62%). Both overall survival (OS) and relapse-free survival (RFS) were significantly improved in the HCT group (OS: 62% versus 51%, P = .012), not only in the whole population, but also in the intermediate-risk group. Among patients who had a suitable related donor, the outcomes in the HCT group were significantly better than those in the CTx group. The introduction of appropriate treatment strategies that include allo-HCT may improve the outcome in elderly patients with AML in CR1.

Cost and effectiveness of reduced-intensity and conventional allogeneic hematopoietic stem cell transplantation for acute myelogenous leukemia and myelodysplastic syndrome.

GOALS OF WORK: Allogeneic stem cell transplantation with a reduced-intensity regimen (RIST) has been evaluated mostly in terms of its clinical benefit, and the pharmacoeconomic aspects of this procedure remain unclear. We compared the cost and effectiveness of RIST with those of stem cell transplantation using a conventional myeloablative regimen (CST). PATIENTS AND METHODS: Fifty consecutive patients who underwent transplantation for myeloid malignancy were included. Life years and medical costs during the entire treatment course for up to 2 years after transplantation were evaluated, and cost-effectiveness was assessed from the payer's perspective. MAIN RESULTS: Of these 50 cases, 35 were treated with CST and 15 were treated with RIST. The mean survival time was 1.5 years in CST and 1.2 years in RIST, while the mean total cost per patient within the first 2 years was $29,630 for CST and $29,466 for RIST, with no significant difference. The duration of total hospitalization was shorter in RIST than in CST; then, the cost for hospitalization represented a lower proportion of the total cost in RIST (49% of total cost) than in CST (63%). In contrast, the cost related to the conditioning regimen was significantly higher in RIST than in CST. CONCLUSIONS: This result suggests that the increased cost of the conditioning regimen offsets the reduced cost of hospitalization in RIST. Although some differences were observed in the details of the cost, the total cost and mean survival were comparable between CST and RIST, and this result was confirmed by a probabilistic sensitivity analysis.

Hematopoietic stem cell transplantation for therapy-related myelodysplastic syndrome and acute leukemia: a single-center analysis of 47 patients.

The prognosis of therapy-related myelodysplastic syndrome and acute leukemia (t-MDS/AL) remains poor. We retrospectively analyzed the data of 47 patients (31 AL and 16 MDS) who were treated at our institute. Thirty-three patients received disease-adapted chemotherapy, with a response rate of 73%, while 14 received no interventions due to an indolent course, such as MDS. The median follow-up of surviving patients was 1.9 years (range 0.1-10.5) after the diagnosis of t-MDS/AL, and the estimated 3-year overall survival (OS) for all patients was 55%. Twenty-seven patients underwent allogeneic hematopoietic stem cell transplantation (HCT), and the 3-year non-relapse mortality was 17%. Twenty patients did not undergo HCT due to various reasons including advanced age or comorbidities. The 3-year OS was better in patients who received HCT than in those who did not (71 vs. 31%; p = 0.018). A multivariate analysis revealed that HCT was associated with a better OS. Although this study has several limitations, including a potential selection bias due to the retrospective nature of the analysis and a small number of patients, the results show that modern HCT may be useful for inducing long-term survival in a fraction of patients suffering from t-MDS/AL. The present findings warrant future prospective studies.

Prognostic factors and outcomes of adult patients with acute myeloid leukemia after first relapse.

BACKGROUND: Patients with acute myeloid leukemia who are treated with conventional chemotherapy still have a substantial risk of relapse; the prognostic factors and optimal treatments after relapse have not been fully established. We, therefore, retrospectively analyzed data from patients with acute myeloid leukemia who had achieved first complete remission to assess their prognosis after first relapse. DESIGN AND METHODS: Clinical data were collected from 70 institutions across the country on adult patients who were diagnosed with acute myeloid leukemia and who had achieved a first complete remission after one or two courses of induction chemotherapy. RESULTS: Among the 1,535 patients who were treated with chemotherapy alone, 1,015 relapsed. Half of them subsequently achieved a second complete remission. The overall survival was 30% at 3 years after relapse. Multivariate analysis showed that achievement of second complete remission, salvage allogeneic hematopoietic cell transplantation, and a relapse-free interval of 1 year or longer were independent prognostic factors. The outcome after allogeneic transplantation in second complete remission was comparable to that after transplantation in first complete remission. Patients with acute myeloid leukemia and cytogenetic risk factors other than inv(16) or t(8;21) had a significantly worse outcome when they did not undergo salvage transplantation even when they achieved second complete remission. CONCLUSIONS: We found that both the achievement of second complete remission and the application of salvage transplantation were crucial for improving the prognosis of patients with acute myeloid leukemia in first relapse. Our results indicate that the optimal treatment strategy after first relapse may differ according to the cytogenetic risk.

A potential activity of valproic acid in the stimulation of interleukin-3-mediated megakaryopoiesis and erythropoiesis.

OBJECTIVE: Although the anticancer activities of histone deacetylase (HDAC) inhibitors have been studied, a role for HDAC in normal hematopoiesis has not been clearly defined. Previous studies have shown that the potent HDAC inhibitor FK228 stimulates interleukin (IL)-3-mediated erythropoiesis. Here, we examined whether the widely used valproic acid (VPA) affects megakaryopoiesis as well as erythropoiesis. MATERIALS AND METHODS: CD34(+) cells were incubated in serum-free or serum-containing cultures with cytokines, with or without VPA. RESULTS: In the serum-free cultures containing IL-3+stem cell factor (SCF), VPA significantly increased generation of CD61(+)GPA(-) megakaryocytic and a CD61(+)GPA(+) mixture of megakaryocytic and erythroid precursors from CD34(+) hematopoietic precursors at a pharmacological concentration (100 microg/mL). The increase in generation of megakaryocytic and erythroid precursors by VPA was confirmed by replating cultured cells with thrombopoietin+SCF and erythropoietin+SCF, respectively. VPA was as potent as FK228. In cultures with granulocyte-macrophage colony-stimulating factor+SCF, where CD61(-)GPA(+) erythroid precursors were mostly developed, VPA mainly enhanced the generation of CD61(-)GPA(+) erythroid precursors. In serum-containing cultures, only low numbers of CD61(+) or GPA(+) cells were developed with IL-3+SCF. Nevertheless, a substantial number of these cells were generated with VPA. Furthermore, these stimulating effects of VPA were observed by incubating CD34(+) cells from patients with myelodysplastic syndrome. Quantitative reverse transcription polymerase chain reaction showed that VPA enhanced GATA-2, but not GATA-1, messenger RNA expression with IL-3+SCF. CONCLUSIONS: These results indicate a novel role for VPA in enhancing the potential of IL-3 to stimulate megakaryopoiesis as well as erythropoiesis and suggest a new therapeutic approach of epigenetic therapy for hematological disease.

Outcome of 93 patients with relapse or progression following allogeneic hematopoietic cell transplantation.

Relapse/progression after allogeneic hematopoietic cell transplantation (allo-HCT) remains the major cause of treatment failure. In this study, the subsequent clinical outcome was overviewed in 292 patients with leukemia/myelodysplastic syndrome who received allo-HCT. Among them, 93 (32%) showed relapse/progression. Cohort 1 was chosen to receive no interventions with curative intent (n = 25). Cohort 2 received reinduction chemotherapy and/or donor lymphocyte infusion (n = 48), and Cohort 3 underwent a second allo-HCT (n = 20). Sixty-three patients received reinduction chemotherapy, and 27 (43%) achieved subsequent complete remission (CR). The incidence of nonrelapse mortality (NRM) was similar among the three cohorts (4, 15, and 5%). The 1-year overall survival (OS) after relapse was significantly better in patients with a second HCT (58%) than in others (14%, Cohorts 1 and 2; P <.001). However, the 2-year OS did not differ between the two groups, which suggests that it is difficult to maintain CR after the second HCT. Multivariate analysis showed that reinduction chemotherapy, CR after intervention, second HCT, and longer time to post-transplant relapse were associated with improved survival. In conclusion, for patients with relapse after allo-HCT, successful reinduction chemotherapy and a second HCT may be effective for prolonging survival without excessive NRM. However, effective measures to prevent disease progression after a second HCT clearly need to be developed.

Regular dose of gemcitabine induces an increase in CD14+ monocytes and CD11c+ dendritic cells in patients with advanced pancreatic cancer.

OBJECTIVE: Chemotherapy and immunotherapy often seem to contradict each other. However, recent reports suggested that the anticancer effects in some chemotherapeutic agents were concerned with immune response. This study was designed to evaluate the immunological reaction by gemcitabine for future clinical trial of combination therapy with gemcitabine and cancer vaccines. METHODS: We evaluated several immunological parameters in patients with advanced pancreatic cancer who received a conventional dose of gemcitabine for 2 months. Twenty-eight patients with metastasis or locally advanced tumor, including 18 gemcitabine-naïve and 10 with a history of preceding gemcitabine treatment, were enrolled in this study. The patients received gemcitabine 1000 mg/m(2) for 3 weeks, followed by 1 week of rest. We monitored the kinetics of lymphocytes, natural killer cells, monocytes, dendritic cells (DC), human leukocyte antigen (HLA)-multimer conjugated with CMV or WT1 peptide, and intracellular cytokine production of interferon-gamma and interleukin-4 by flow cytometry. The T cell receptor (TCR) repertoire was also analyzed. RESULTS: The absolute number and percentage of CD14(+) monocytes and CD11c(+) (myeloid) DC increased with gemcitabine treatment (P = 0.033 and P = 0.021). The percentage of CD123(+) (plasmacytoid) DC also increased (P = 0.034), whereas no significant change was observed in other immune parameters, including multimer, intracellular cytokine production and TCR repertoire. CONCLUSIONS: Our finding that gemcitabine treatment induced the proliferation of CD14(+) monocytes and CD11c(+) DC could support combination therapy with gemcitabine and specific immunotherapy such as peptide vaccination against pancreatic cancers.

Reduced-intensity unrelated donor bone marrow transplantation for hematologic malignancies.

To review a current experience of unrelated bone marrow transplantation (BMT) with reduced-intensity conditioning (RIC) regimens, we conducted a nationwide survey with 77 patients (age, 25-68 years). The backbone RIC regimen was a combination of fludarabine or cladribine, busulfan or melphalan and total body irradiation at 2-4 Gy. Five patients died early, but 71 (92%) achieved initial neutrophil recovery. Thereafter, 36 patients (47%) died of therapy-related complications, 23 (30%) of whom died within day 100. Grades II-IV acute graft-versus-host disease (GVHD) occurred in 34 of the 68 evaluable patients (50%). In a multivariate analysis, a regimen containing antithymocyte globulin (ATG) was significantly associated with a decreased risk of acute GVHD (P = 0.041). Thirty-three patients are currently alive with a median follow-up of 439 days (28-2002 days), with an OS of 50% at 1 year. In conclusion, unrelated BMT with RIC regimens can be a curative treatment in a subset of patients.

Impact of T cell chimerism on clinical outcome in 117 patients who underwent allogeneic stem cell transplantation with a busulfan-containing reduced-intensity conditioning regimen.

Within the concept of reduced-intensity stem cell transplantation (RIST) there is a wide range of different regimens used, and little information is available on the clinical impact of chimerism status in patients conditioned with a busulfan-containing regimen. Therefore, we retrospectively reviewed lineage-specific chimerism and the subsequent clinical outcome in 117 patients (median age, 55 years; range: 29-68) who underwent busulfan-containing RIST. The conditioning regimen consisted of busulfan (oral 8 mg/kg or i.v. 6.4 mg/kg) and fludarabine (180 mg/m(2), n = 64) or cladribine (0.66 mg/kg, n = 53), with or without 2-4 Gy total-body irridiation (TBI) (n = 26) or antihuman T-lymphocyte immunoglobulin (ATG; 5-10 mg/kg; n = 31). Chimerism was evaluated with peripheral blood samples taken on days 30, 60, and 90 after transplantation by polymerase chain reaction (PCR)-based amplification of polymorphic short tandem repeat regions. The median follow-up of surviving patients was 1039 days (153-2535). The percent donor-chimerism was significantly higher in granulocyte than T cell fraction throughout the entire course, and the median (mean) values were, respectively, 100% (96%) versus 95% (83%), 100% (98%) versus 100% (89%), and 100% (98%) versus 100% (91%) at days 30, 60, and 90 after RIST. In a multivariate analysis, having received <2 types of chemotherapy regimens before RIST was the only factor that was significantly associated with low donor T cell chimerism (<60%) at day 30 (hazard ratio [HR]: 6.1; 95% confidence interval [CI], 2.1-18.4; P < .01). The median percentage of donor T cell chimerism at day 30 was 9% (0%-63%) in 5 patients who experienced graft failure, which was significantly lower than that (97%; 15%-100%) in the rest of the patients (P < .01). No correlation was found between the kinetics of T cell chimerism and the occurrence of acute or chronic GVHD (aGVHD, cGVHD). The stem cell source and the addition of TBI or ATG were not associated with the degree of T cell chimerism, overall survival (OS) or event-free survival (EFS). In a Cox proportional hazard model, low donor T cell chimerism of <60% at day 30 was associated with both poor OS (HR: 2.2; 95% CI, 1.1-4.5; P = .02) and EFS (HR: 2.0; 95% CI, 1.1-3.8; P = .02). In conclusion, we found that 43% of the patients retained mixed donor T cell chimerism (<90% donor) at day 30, whereas 92% achieved complete chimerism in granulocyte fraction. Low donor T cell chimerism of <60% at day 30 may predict a poor outcome, and a prospective study to examine the value of early intervention based on chimerism data is warranted.