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A nationwide survey of hematopoietic cell transplantation (HCT) was started in Japan in 1991, and the analyzed survey data have been presented as the annual report of the Japan Society for Hematopoietic Cell Transplantation. The 10-year overall survival (OS) rates after HCT for each disease are as follows: acute myelogenous leukemia, 44.2%; acute lymphocytic leukemia, 33.7%; adult T-cell leukemia, 24.6%; chronic myelogenous leukemia, 53.3%; myelodysplastic syndrome, 37.3%; non-Hodgkin's lymphoma, 41.5%; Hodgkin's lymphoma, 50.8%; aplastic anemia, 72.5%; breast cancer, 37.1%; germ cell tumor, 52.6%; and ovarian cancer, 44.2%. The 5-year OS rates for multiple myeloma and lung cancer were 40.6% and 23.6%, respectively. Except in cord blood transplantation, engraftment was accomplished in more than 90% of patients. The respective frequencies of acute graft-versus-host disease (GVHD) and chronic GVHD were 41.1% and 34.9% for related bone marrow transplantation (BMT), 66.8% and 34.5% for unrelated BMT, 52.9% and 36.0% for allogeneic peripheral blood stem cell transplantation, and 53.3% and 32.1% for allogeneic cord blood transplantation. OS for each disease was analyzed by patient age, stem cell source, donor type, disease status, and disease type. These data provide objective and valuable information for hematologists as well as for patients who need HCT.

Post-transplantation lymphoproliferative disorder (PTLD) is usually an aberrant proliferation of EBV-infected B cells. We report the case of a 31-year-old man with severe aplastic anemia who suffered PTLD 42 days post-BMT from an unrelated donor. At the onset of PTLD, peripheral blood lymphocytes were comprised of 40% CD20(+) cells, 3% CD4(+) cells, and 56% CD8(+) cells. A highly sensitive in situ hybridization (ISH) method was used to detect EBV-encoded small non-polyadenylated RNA 1 (EBER-1) in 33.9% of sorted CD20(+) cells, 4.4% of CD4(+) cells, and 1.4% of CD8(+) cells. Each T-cell fraction contained less than 0.034% of contaminated EBV-infected B cells. Clonal proliferation of both B and T cells was demonstrated by Southern blotting. The patient did not respond to donor leukocyte infusion and died due to deterioration of PTLD. At autopsy, examination of multiple organs revealed B-cell (rather than T-cell) infiltration. This case clearly indicates that EBV can simultaneously infect B and T cells and can induce clonal proliferation of both lymphocyte subsets in severely immunocompromised patients.

Mismatches of minor histocompatibility antigens (mHas) between HLA-identical stem cell donors and recipients are known as a major risk factor for graft-versus-host disease (GVHD). We determined alleles of 9mHas for 40 patients who had undergone stem cell transplantation from HLA-identical donors and investigated the association of their mismatches with relapse rate and GVHD. We observed a tendency toward a higher incidence of GVHD and lower relapse rate in patients with at least one or more incompatibilities of mHas than in patients without incompatibilities. In particular, relapse rate was significantly lower in a group of patients grafted from HLA-identical sibling donors with mHa-incompatibilities (p=0.05). In addition, no relapse was observed in at least one or more mHa-incompatible patients who developed GVHD. We conclude that mHas contribute to the development of the graft-versus-leukemia effect and may potentiate the graft-versus-leukemia effect in incompatible patients who develop GVHD.

Keywords: accelerated phase; chronic myeloid leukaemia; non-myeloablative stem cell transplantation; donor leucocyte infusion; fluorescence in situ hybridization

Background and Objectives To estimate the minimum volume of processed blood necessary for the purpose of donor leucocyte infusion (DLI), we determined the number of CD3+ cells harvested by apheresis from normal donors and examined adverse events during the procedure. Materials and Methods Leukapheresis utilizing the COBE Spectra‘ was performed a total of 24 times from 12 normal donors. Blood counts were obtained and the number of CD3+ cells was evaluated before and after apheresis and in the peripheral blood products. Complications associated with the procedure were documented. Results Blood products contained, on average, 51·1% CD3+ cells. A linear correlation was found between the number of CD3+ cells collected and the processed blood volume, up to 12 000 ml (r = 0·930, P

Objective Although the graft-vs-leukemia (GVL) effect induced by donor leukocyte infusion (DLI) is thought to be mediated by T cells, their features, as well as target molecules, remain unknown. To characterize T cells that mediate the GVL effect on chronic myelogenous leukemia (CML), we studied T-cell repertoire in peripheral blood (PB) of two patients treated with DLI for relapsed CML after allogeneic bone marrow transplantation. Material and Methods Peripheral blood mononuclear cells (PBMCs) were obtained at 2-week intervals following DLI and examined for the presence of antigen-driven T-cell proliferation using complementarity-determining region (CDR) 3 size spectratyping of T-cell receptor β chain subfamilies. Results Both patients exhibited transient proliferation of a limited number of T cells at a certain point in time (day 132 for patient 1 and day 75 for patient 2) after DLI in association with a decrease in the proportion of Philadelphia chromosome (Ph)–positive cells. In patient 2, who showed expansion of a BV16 + T cell in PB, expansion of BV16 + T cells with a similar CDR3 motif containing QDR to that of PB was demonstrated in the bone marrow (BM) sampled on day 33 and in the buccal mucosal tissue, showing chronic graft-vs-host disease (GVHD) on day 138 after DLI. When PBMCs obtained from patient 2 in remission were cultured with cryopreserved CML cells for 2 weeks, proliferation of a BV16 + T cell with a CDR3 motif of QIR was induced in vitro. Conclusion These findings indicate that transient proliferation of a limited number of T cells detected in PB 3–5 months after DLI probably reflects the GVL response against CML cells and may serve as a marker for the appearance of the GVL effect induced by DLI.

To clarify the pathologic significance of granulocytes exhibiting the paroxysmal nocturnal haemoglobinuria (PNH) phenotype in patients with aplastic anaemia (AA), we examined peripheral blood from 100 patients with AA for the presence of granulocytes deficient in glycosylphosphatidylinositol (GPI)-anchored proteins using a sensitive flow cytometric assay. A significant increase in the frequency of CD55 + CD59 + CD11b + granulocytes (>0.003%) compared to normal individuals was observed in 31 of 35 (88.6%) patients with untreated AA at diagnosis. The proportions of patients showing increased PNH granulocytes in treated AA patients with a short ( 5 yr) disease duration were 68.6% (11/16) and 20.4% (10/49), respectively. When 19 patients showing increased frequency of PNH granulocytes before therapy were studied 6-12 months after antithymocyte globulin plus cyclosporin A therapy, the frequency decreased to 0.01-90% of pretreatment values in 15 recovering patients. These findings suggest that a relative increase in the number of PNH granulocytes is a common feature of AA at diagnosis, and that it may represent the presence of immunologic pressure to normal haematopoietic stem cells as a cause of AA.

To clarify the role of donor leukocyte infusion (DLI) in the treatment of leukemia relapsing after allo-BMT, data from 100 patients were collected from 46 facilities in Japan and analyzed with respect to the efficacy and adverse effects of donor leukocyte infusion. Complete remission was achieved in 11 of 12 (91%) patients with relapsed chronic myelogenous leukemia (CML) in chronic phase, three of 11 (27%) with CML in the acute phase, eight of 21 (38%) with acute myelogenous leukemia (AML), six of 23 (25%) with acute lymphoblastic leukemia (ALL) and five of 11 (45%) with myelodysplastic syndrome (MDS). The probability of remaining in CR at 3 years was 82% in CML patients in the chronic phase, but 0% in those with CML in the acute phase, 7% in those with AML, 0% with ALL and 33% with MDS. Acute GVHD (>/=2) developed in 31 of 89 (34%) patients with HLA-identical related donors and was fatal for seven (7%). Cytopenia developed in 21 of 94 (22%) with no associated fatalities. When the outcome of patients with CML in CP and MDS was analyzed, development of GVHD, cytopenia, or both, was associated with a higher GVL effect (15 of 16, 93%) than in those without adverse affects (one of 6, 17%). A leukocyte dose of 5 x 107/kg of recipient body weight appeared to be optimal as an initial dose of DLI. Given the relatively low incidence of acute GVHD and the similar GVL effect, DLI may be more beneficial to patients in Japan with recurrent leukemia than to those in Western countries. Bone Marrow Transplantation (2000) 26, 769-774.

To better characterize immunologic aberrations in aplastic anaemia (AA), we examined peripheral blood mononuclear cells (PBMC) of 67 patients with AA and other patients with various haematological diseases for the expression of heat shock protein 72 (hsp72), which is inducible in lymphocytes by various stressors including antigenic stimulation. When freshly obtained PBMC were examined using flow cytometry, the proportion of cells expressing hsp72 in cytoplasm was significantly higher in allogeneic marrow transplant recipients (22 ± 15%, mean ±standard deviation (SD)) and AA patients (17 ± 21%) than in normal individuals (6 ± 3%). When PBMC were tested after heat treatment, only the proportion of hsp72+ cells of AA patients (37 ± 30%) was significantly higher than that of the normal control (17 ± 11%). Dual fluorescence analysis of the PBMC revealed that the majority of hsp72+ cells was CD3+. For 28 untreated AA patients, the proportion of hsp72+ cells in those who later responded to cyclosporine (CyA) (62 ± 24%) was higher than that in non-responders (19 ± 13%). Immunoblotting analysis revealed predominant expression of hsp72 in T cells. These findings indicate that high inducibility of hsp72 in PBMC by heat treatment is an immunologic aberration characteristic of CyA-responsive AA and that this simple test may be useful for identifying a subset of AA patients responsive to immunosuppressive therapy.

We report a case of natural killer cell large granular lymphocytic (NK-LGL) leukaemia successfully treated with allogeneic peripheral blood stem cell transplantation (allo-PBSCT). The peripheral blood (PB) revealed an abnormal expansion of LGL that were CD3-, CD16- and CD56+, and had natural killer activity. In situ EBER-1 hybridization of the PB mononuclear cells showed the presence of Epstein-Barr virus (EBV) in the LGL as well as in lymphocytes infiltrating the tonsils and colon. Southern blotting with an EBV-terminal repetitive sequence probe demonstrated clonal proliferation of EBV+ cells. The patient received allo-PBSCT from his HLA-matched sister with a conditioning regimen involving the use of cyclophosphamide and fractionated total body irradiation. The patient promptly recovered trilineage haematopoiesis without graft-versus-host disease, and has been in complete remission without therapy for 10 months since allo-PBSCT, suggesting that allo-PBSCT could eradicate the NK-LGL leukaemic cells.

To assess the consequence of second BMT (BMT2) for leukemia relapse after allogeneic BMT, we analyzed the clinical course of 66 recipients who were treated by BMT2 in Japan. Diagnoses included 29 ANLL, 27 ALL, six CML and four MDS. Durations between the first BMT (BMT1) to relapse and BMT1 to BMT2 were 13.5 +/- 13.7 months and 17.4 +/- 13.9 months, respectively. Donors for BMT2 were replaced in 11 cases. Thirty-one patients were in CR (or CP) at BMT2. Earlier deaths were observed in those who received BMT2 within 12 months after BMT1, mostly caused by regimen-related toxicity and infections. Overall leukemia-free survival rate was 28% at 2 years and 16% at 4 years. Factors influencing the poor prognosis after BMT2 were early (

Predicting the treatment response of aplastic anaemia (AA) is essential when considering cyclosporine (CyA) therapy among several treatment options, because it requires at least 2 months determine whether the therapy is beneficial to a patient with AA. To identify the characteristics of patients with AA who are likely to respond to CyA therapy we retrospectively reviewed the clinical records and bone marrow smears of patients treated with CyA. Among 30 patients who received the therapy for at least 3 months within 1 year after diagnosis of AA, and who had not been exposed to antilymphocyte or antithymocyte globulin, 16 (53%) responded with disease remission. CyA-responsive patients had a significantly higher ratio of erythroblasts to granulocytes (E/G ratio) in the bone marrow at the time of diagnosis as compared with patients refractory to therapy (P = 0.004). Multivariate analysis revealed that a high E/G ratio ( > 0.6) was significantly associated with a good response to CyA (P = 0.03): 15 (83%) of the 18 patients with an E/G ratio > 0.6 responded, but only one (8%) of the 12 with an E/G ratio > or = 0.6 did. Although the presence of subclinical paroxysmal nocturnal haemoglobinuria was suspected from the relative erythroblastosis observed in the bone marrow of these patients, flow cytometric analysis of neutrophils in the peripheral blood failed to reveal neutrophils deficient for glycosyl-phosphatidylinositol (GPI) anchored membrane proteins in all but one case. Identification of the presence of relative erythroid hyperplasia in he bone marrow when AA is diagnosed may help to predict a favourable response to CyA therapy, and therefore facilitate the selection of optimal therapy for AA.

Hematopoietic function of some aplastic anemia (AA) patients is dependent on the administration of cyclosporine (CyA). To investigate whether certain HLA class II genes are associated with susceptibility to such CyA-dependent AA, we determined the HLA class II alleles of 59 AA patients treated with CyA. Among 26 patients successfully treated with CyA, 13 required a small dose of CyA to maintain stable hematopoiesis. Of these 13 AA patients, 10 shared an HLA class II haplotype of DRB1*1501-DQA1*0102-DQB1*0602. None of the 13 responders who obtained a sustained remission off CyA therapy possessed this haplotype. In the 10 patients who shared the HLA class II haplotype, single-strand conformation polymorphism analysis of each gene fragment of this haplotype failed to detect a polymorphism in the nucleotide sequence. When the AA patients were assessed for their likelihood to respond to CyA therapy, the response rate in patients with this haplotype (71%) was significantly higher than that of patients with another haplotype associated with HLA-DR2, DRB1*1502-DQA1*0103- DQB1*0601 (36%) and that of patients without HLA-DR2 (35%). These findings indicate that the CyA-dependent response of AA is closely related to an HLA class II haplotype of DRB1*1501-DQA1*0102-DQB1*0602 and suggest that, in AA patients with this haplotype, immune mechanisms play an important role in the pathogenesis of bone marrow failure.

Summary. We present an immunocompetent patient with transfusion-associated graft-versus-host disease (GVHD), in which chimaerism of peripheral blood lymphocytes was demonstrated by analysis of a highly polymorphic genome. The patient was treated successfully with anti-CD 3 monoclonal antibody, OKT3 and cyclosporin A. Although it is undoubtedly important to prevent transfusion-associated GVHD by irradiation of cellular blood components, intensive therapy with OKT3 and cyclosporin A in the early phase of onset may be effective for treatment of this potentially fatal condition. The mechanism of the effectiveness of this treatment for transfusion-associated GVHD is discussed.

Cyclosporine (CyA) therapy has been shown to be effective in some patients with aplastic anemia. In an attempt to characterize aplastic patients likely to benefit from CyA therapy, we examined bone marrow mononuclear cells (BMMC) obtained before therapy from 23 patients with aplastic anemia, who were treated with CyA alone. Expression of four myelosuppressive cytokines, including tumor necrosis factor (TNF), lymphotoxin, macrophage inflammatory protein-1 alpha (MIP-1 alpha), and interferon-gamma (IFN-gamma) was examined using polymerase chain reaction (PCR)-assisted messenger RNA (mRNA) amplification. mRNA for TNF, lymphotoxin, and MIP-1 alpha was readily detectable at variable levels in BMMC from normal and transfused controls as well as in BMMC from aplastic patients. In contrast, IFN-gamma mRNA was only demonstrable in BMMC from some patients with aplastic anemia, irrespective of a history of transfusions. Of 13 patients who responded to CyA therapy and achieved transfusion-independence, IFN-gamma mRNA was detected in 12 patients, whereas the mRNA was only detectable in 3 of 10 patients refractory to CyA therapy (P = .003, Fisher's exact test). Follow-up examination of BMMC obtained from seven CyA-responding patients after hematologic remission showed disappearance of IFN-gamma mRNA in four patients. These results suggest that detection of IFN- gamma gene expression in pretreatment BMMC from aplastic patients using PCR may be helpful in predicting a good response to CyA therapy.

We describe the clinical course of a 16 year old girl with aplastic anemia who was treated by syngeneic bone marrow transplantation. Engraftment was not obtained by simple infusion of bone marrow without immunosuppression. The patient received a high-dose cyclophosphamide and thoracoabdominal irradiation, followed by second marrow transplantation from the same donor. Incomplete but significant hematologic recovery was observed; however, marrow failure recurred 5 months after transplantation. Since donor and recipient pairs were genotypically identical, graft failure could not be attributed to immunological reactivity of recipient cells to donor non-HLA antigens. This case report implies that graft failure in some cases of aplastic anemia might be mediated by inhibitory cells resistant to cyclophosphamide and irradiation.

Mismatches of minor histocompatibility antigens (mHas) between HLA-identical stem cell donors and recipients are known as a major risk factor for graft-versus-host disease (GVHD). We determined the alleles of 5 polymorphic molecules including HA-1 and 4 adhesion molecules in 102 patients who had undergone stem cell transplantation from HLA-identical donors and investigated the association of their mismatches with the relapse rate and incidence of GVHD. We observed relapse rates of 16.1% in patients with at least one or more incompatibilities and 39.4% in patients without incompatibilities (p = 0.018). The respective relapse rates of patients with CD62L, HA-1, CD31 exon 563, CD31 exon 125 and 49b incompatibilities were 6.1%, 14.3%, 11.7%, 20% and 40%. Only patients with CD62L incompatibilities showed a lower relapse rate than patients who received a compatible graft. Since there was no difference between patients with and without incompatibilities as far as the appearance of acute GVHD (or = 2) was concerned, we conclude that the polymorphic CD62L molecule contributes to graft-versus-leukemia rather than the development of GVHD after HLA-identical stem cell transplantation.

The immune system of females is capable of recognizing and reacting against the male-specific minor histocompatibility antigen (mHA), HY. Thus, cytotoxic T-lymphocytes (CTLs) recognizing this antigen may be useful in eradicating leukemic cells of a male patient if they can be generated in vivo or in vitro from a human leukocyte antigen (HLA)-identical female donor. The HLA-A*0201-restricted HY antigen, FIDSYICQV, is a male-specific mHA. Using HLA-A2/HY peptide tetrameric complexes, we reveal a close association between the emergence of HY peptide-specific CD8(+) T cells in peripheral blood and molecular remission of relapsed BCR/ABL(+) chronic myelogenous leukemia in lymphoid blast crisis in a patient who underwent female-to-male transplantation. Assessment of intracellular cytokine levels identified T cells that produce interferon-gamma in response to the HY peptide during the presence of HY tetramer-positive T cells. These results indicate that transplant with allogeneic HY-specific CTLs has therapeutic potential for relapsed leukemia, and that expansion of such T cells may be involved in the development of a graft-versus-leukemia response against lymphoblastic leukemia cells.

To clarify graft-versus-leukemia effect of graft-versus-host disease, we studied 166 patients treated with allogeneic stem cell transplantation for hematologic malignancies. The cumulative incidence of relapse in patients with acute GVHD was significantly lower than that in patients without acute GVHD, but there was no similar GVL effect for chronic GVHD.

Background : The aim of this study was to investigate the efficacy and safety of high-dose chemotherapy (HDCT) for the treatment of patients with advanced testicular cancer. Methods: Fourteen patients were treated with high-dose carboplatin, etoposide and cyclophosphamide (with or without THP-adriamycin) followed by peripheral blood stem cell transplantation. The treatment was used for two refractory cases, a second relapse, and for consolidation after the first relapse in one case each. It was also used for nine cases as part of the first-line treatment following primary conventional-dose chemotherapy, and for one case as the first salvage for a late recurrent tumor of teratoma with malignant transformation. Results: The first two patients who received intensive pretreatment with cisplatin-based chemotherapy did not respond to HDCT. The two patients who were treated with HDCT as the first or second salvage therapy achieved successful outcomes. The results for the subsequent nine patients (consisting of two with stage IIIC, five with IIIB2, one with IIB, and one extragonadal seminoma) were two progressive disease, three no change and four partial remission. Only three are alive with NED following salvage surgery. Finally, a case of teratoma with malignant transformation did not respond well to two cycles of HDCT. There were no marked adverse reactions except one episode of severe neutropenic colitis. Conclusions: The results demonstrated the limited efficacy of HDCT even in cases with a good to intermediate risk rating according to classification by the International Germ Cell Cancer Collaborative Group. Because treatment for relapse after HDCT is extremely difficult, new HDCT regimens consisting of drugs that are not used in induction chemotherapy need to be established.

To clarify the role of dose escalation of donor leukocyte infusion (DLI) in the treatment of relapsed leukemia after allogeneic bone marrow transplant (BMT), data from 100 patients were collected from 46 facilities in Japan and analyzed with respect to indications and infused cell dose. Complete remission (CR) was achieved in 11 of 12 (91%) patients with relapsed chronic myelogenous leukemia (CML) in the chronic phase, 3 of 11 (27%) with CML in the acute phase, 8 of 21 (38%) with acute myelogenous leukemia (AML), 6 of 23 (25%) with acute lymphoblastic leukemia (ALL), and 5 of 11 (45%) with myelodysplastic syndrome (MDS). The probability of remaining in CR at 3 years was 82% in CML patients in the chronic phase, but 0% in those with CML in the acute phase, 7% in those with AML, 0% with ALL, and 33% with MDS. Acute graft-versus-host disease (GVHD) (> or = 2) developed in 31 of 89 (34%) patients with human leukocyte antigen identical related donors and was fatal for 7 (7%). A leukocyte dose of 1 x 10(7)/kg of recipient body weight with CML in the chronic phase, 3 x 10(7)/kg of recipient body weight with MDS, and 1 x 10(8)/kg of recipient body weight with acute leukemia appeared to be optimal as an initial dose of DLI. However, the minimal dose of leukocyte developing fatal GVHD was 7 x 10(7)/kg of recipient body weight. These suggest that a relatively small dose of DLI ranging from 1 x 10(7)/kg to 5 x 10(7)/kg of recipient body weight should be administered initially then the infused escalating dose 2 or 3 months later in patients with CML in the chronic phase and MDS. However, a large number of leukocytes around 1 x 10(8)/kg are needed to induce graft versus leukemia effects in patients with acute leukemia despite a 7% fatality in GVHD.

The experience at Kanazawa bone marrow transplantation team in harvesting marrow for allogeneic BMT from 66 normal donors is presented in detail. Six donors were less than 10 years old and three donors were older than 50 years old. No special difficulties were encountered with these donors. Median volume of harvesting marrow was 750ml, median duration of the anesthesia and the operation were two hours and 35 minutes and one hour and a half respectively. Fifty-nine % of the donors was associated with transient fever and 4.7% of the donors needed medication for pain of aspiration site. The median duration of hospitalisation after the operation was 4 days. Three donors needed further hospitalisation than 8 days after the operation for treatment of goat, bronchial asthma and lung tuberculosis. No major complications occurred in any of the donors.

Background: Induction of autologous graft-versus-host disease after peripheral blood stem cell transplantation has not been studied well. Objective: The purpose of this study was to analyze the factors that affect the development of autologous graft-versus-host disease. Methods: Nineteen patients with non-Hodgkin's lymphoma underwent peripheral blood stem cell transplantation followed by the administration of cyclosporin A for 28 days (group A) or 21 days (group B) and IFN-γ. Results: Autologous graft-versus-host disease failed to develop in the any of the group A patients, who did not receive total body irradiation for conditioning and underwent the transplantation with unmanipulated peripheral blood stem cells, although cytotoxic activity against autologous lymphocytes was detectable in peripheral blood mononuclear cells obtained from all of them after the transplantation. Autologous graft-versus-host disease developed in 2 of 4 patients in group A who received enriched CD34 + cells and in 7 of 11 patients who underwent conditioning with total body irradiation. Maculopapular erythema that was compatible with graft-versus-host disease developed on days 19 to 27 after the transplantation in these patients and resolved after 3 to 9 days without treatment. Conclusions: Either the depletion of regulatory cells from the graft by enrichment of CD34 + cells or the abolition of the autoregulation by including total body irradiation in the conditioning regimen may be effective in inducing autologous graft-versus-host disease after peripheral blood stem cell transplantation. Three weeks of cyclosporin A therapy appears to be sufficient to induce autologous graft-versus-host disease in recipients of peripheral blood stem cells. (J Allergy Clin Immunol 2000;106:S51-7.)

To determine whether the antigen-driven T-cell response is involved in the pathogenesis of aplastic anemia (AA), we examined the complementarity-determining region 3 (CDR3) size distribution of T-cell receptor (TCR) β-chain (BV) subfamilies in the bone marrow (BM) of untreated AA patients. AA patients who did not respond to immunosuppressive therapy and those who obtained unmaintained remission early after cyclosporine (CyA) or antithymocyte globulin (ATG) therapy exhibited essentially a normal CDR3 size pattern. In contrast, five patients who needed continuous administration of CyA to maintain remission exhibited a skewed CDR3 size pattern in a number (>40%) of BV subfamilies suggestive of clonal predominance. The skewing of CDR3 size distribution became less pronounced in one of the CyA-dependent patients when the patient achieved unmaintained remission after a 4-year therapy with CyA, whereas it persisted longer than 7 years in the other patient requiring maintenance therapy. Sequencing of BV15 cDNA for which the CDR3 size pattern exhibited apparent clonal predominance in all CyA-dependent patients showed high homology of the amino acid sequence of the CDR3 between two different patients. These findings indicate that antigen-driven expansion of T cells is involved in the pathogenesis of AA characterized by CyA-dependent recovery of hematopoiesis.

To determine whether administration of G-CSF induces phenotypic or functional changes in T cells, we examined peripheral blood T cells from normal individuals receiving G-CSF for activation antigen and adhesion molecule expression before and after G-CSF administration. G-CSF (10 microg/kg/day) was administered subcutaneously to 14 normal individuals for 3-5 days and their PBMC were serially analyzed with monoclonal Ab (mAb) directed to HLA-DR, CD45RO, CD45RA, CD25, CD122, CD95, CD11a, CD49d, CD44 and CD62L (L-selectin) coupled with anti-CD3 mAb. Among T cells positive for these antigens, only the proportion of T cells expressing L-selectin significantly decreased from 68% to 37% after 3-day G-CSF administration. When peripheral blood CD3+ T cells obtained before and after G-CSF administration were sorted into two populations depending on the expression of L-selectin and tested for their proliferative response to allogeneic B cells, the reactivity of L-selectin- cells to alloantigen stimulation was consistently lower than that of L-selectin+ cells regardless of the exposure to G-CSF. The decrease in the relative number of L-selectin+ cells induced by G-CSF administration may contribute to the unexpectedly low incidence of severe acute GVHD after allogeneic PBSC transplantation.

Background: Autologous graft-versus-host disease is inducible after autologous bone marrow transplantation by means of administration of cyclosporine. Objective: This study was performed to investigate the inducibility of autologous graft-versus-host disease after transplantation of peripheral blood stem cells (PBSCs). Methods: Two patients with non-Hodgkin's lymphoma in remission underwent PBSC transplantation followed by administration of cyclosporine and low-dose interferon-γ. Results: Although autologous graft-versus-host disease did not develop in either patient, T lymphocytes with cytotoxic activity against autologous lymphocytes appeared transiently in the early posttransplant period. Such autocytotoxic lymphocytes were not detectable in another patient who underwent PBSC transplantation who did not receive cyclosporine and interferon-γ. When CD4 + and CD8 + cells were sorted from the peripheral blood mononuclear cells of one of the two patients and tested for cytotoxicity against autologous lymphocytes, only CD8 + cells exhibited cytotoxic activity. Conclusions: The results indicate that administration of cyclosporine and interferon-γ after PBSC transplantation can induce autocytotoxic CD8 + T cells, even though it may not produce autologous graft-versus-host disease. It is unclear whether induction of such autocytotoxic T cells among patients with non-Hodgkin's lymphoma who undergo PBSC transplantation has any antilymphoma effect. (J Allergy Clin Immunol 1997;100:S65-9.)

HIA-DRB1*1501, a subtype of HLA-DR2, has been shown to be closely associated with a good response to cyclosporine (CyA) therapy in patients with aplastic anaemia (AA). To determine whether this DRB1 allele can also predict a response to antithymocyte globulin (ATG) therapy in AA patients, we analysed the results of HLA-DRB1 typing in 59 Japanese patients who received ATG within 2 years after diagnosis of AA and also in 52 patients treated with CyA. All patients were divided into three groups : those with DRB1*1501, those with DRB1*1502, and those without either of these two alleles (DR2 - ). The response rate to ATG in DRB1*1501 + patients (56%) was not significantly higher than that in DRB1*15102 + patients (47%) and in the other DR2- patients (54%). In contrast, the response rate to CyA therapy in DRB1*1501 + patients (92%) was significantly higher than that in the DRB1*1502 + (41%) and in DR2 - patients (57%). Multivariate analysis revealed that possessing DRB1*1501 was an independent factor significantly predictive of a good response to CyA. These results indicate that although identifying the DRB1*1501 allele in AA patients prior to therapy is predictive of a good response to CyA therapy, it does not have a predictive value for ATG therapy.

In October, 1989, the Tokai Marrow Donor Bank (TMDB) was established through the cooperation of patients' families, the branches of blood centers of Japanese Red Cross and the hematologists' group in Tokai Area (Aichi, Shizuoka, Gifu and Mie Prefectures) in Japan to facilitate the procurement of suitable marrow from unrelated volunteer donors for patients lacking related donors. The number of human leukocyte antigen (HLA)-A, B typed donors totaled 3,083 and the number of patients registered for donor search totaled 1,415 by June 1992, when the activities of TMDB were transferred to the newly created Japan Marrow Donor Program (JMDP), and 55 transplanations from unrelated donors facilitated by TMDD were performed.

Abstract 1180 Background: Although peripheral blood stem cell (PBSC) donation has been considered safe and less stressful, certain fetal or life-threatening acute (within 30 days of post donation) adverse events as well as late occurrence of hematological malignancies have been reported among donors. Since the Japan Marrow Donor Program requires the confirmation of safety and risk of PBSC donation at family donors prior to applying this technique for volunteer donors, the Japan Society for Hematopoietic Cell Transplantation (JSHCT) performed nation-wide consecutive pre-registration and follow up for PBSC family donors from 2000 to 2010. This time, we report the comprehensive outcome of this project. Methods: The JSHCT mandated the registration of every PBSC family donor at the donor registration center then, issued donor identification number to each donor. The society required every harvest center to observe the JSHCT standards for donor eligibility, stem cell mobilization and harvest. The society also required to notify it of any severe adverse events, the results of the day30 clinical and laboratory check and of the annual health check for five years. Findings: Among 3,264 pre-registered donors, 47 emergency reports were submitted for 5 years and it was revealed that acute unexpectedly severe adverse events such as interstitial pneumonitis or anginal attack occurred at 0.58 % of donors although no mortality cases within 30 days of post donation were found. The relationship between donors' basic information such as age or gender and clinical and laboratory abnormalities obtained from 2,882 day 30 reports was studied and it revealed the followings; the risk factor for fatigue, headache, insomunia, anorexia and nausea was female, the risk factors for prolongation of hospitalized period were older age, low body weight, high total dose of granulocyte colony stimulating factor (G-CSF), the presence of past or current health problems and the episode of past stem cell donation, the risk factors for thrombocytopenia were older age and high total dose of G-CSF, the risk factors for splenomegary were older age and high total dose of G-CSF, the risk factors for poor CD34+ cell mobilization were older age, female and the episode of past stem cell donation, the risk factor for over CD34+ stem cell mobilization was younger age. The information from 6,233 annual health checks from 1,708 donors for 5 years showed the followings; the incidence of non-malignant but significant health problems was 1.5%, the incidence of non hematological malignancies was 0.7%, the incidence of hematological malignancy was 0.06%. It was also confirmed that the incidence of hematological malignancies among PBSC donors was not high compared with that among retrospectively surveyed bone marrow family donors. Interpretation: The consecutive donor pre-registration and annual follow up system that sets strict standards for donor eligibility, cell mobilization and harvest is effective in preventing real life-threatening acute adverse events and also is useful to know the real figures on PBSC donors and to assure donor safety. Such a system should be applied to all hematopoietic stem cell (family and volunteer, bone marrow and peripheral blood) donors. Disclosures: No relevant conflicts of interest to declare.

In an attempt to characterize host-derived haemopoietic cells in mixed haemopoietic chimeras, we studied bone marrow cells from male patients with chronic myelocytic leukaemia (CML) transplanted with marrow grafts from female donors. Amplification of a Y chromosome-specific sequence (YDNA) in DNA from marrow mononuclear cells using polymerase chain reaction (PCR) revealed persistence of host cells in 2 of 3 patients studied. On the other hand, persistence of Ph1-positive cells was unable to be demonstrated in the marrow cells from these patients using reverse transcription PCR (RT-PCR) for detecting bcr-abl chimeric messenger RNA. RT-PCR sensitivity for detecting minimal Ph1-positive cells in a background of Ph1-negative cells proved better than that of the PCR for detecting male cells among female cells. When bone marrow progenitor cells from one of the documented mixed chimeras were analyzed after an in vitro colony assay using PCR, 2 of 12 erythroid burst-forming units (BFU-E) proved to be YDNA-positive, i.e., of host origin, whereas none of them was shown to be Ph1-positive, although 12 BFU-E analyzed in the marrow cells obtained pretransplant from the same patient were all YDNA- and Ph1-positive. These findings indicate that, in some marrow transplant recipients with CML, a small number of host-derived normal haemopoietic progenitor cells may persist following lethal chemoradiotherapy and allogeneic bone marrow transplantation despite the fact that Ph1-positive clones can be eradicated.

To characterize hematopoietic cells in mixed hematopoietic chimeras after allogeneic bone marrow transplantation (BMT), the authors examined the origin of progenies derived from hematopoietic progenitor cells of male recipients who received a marrow graft from female donors, by use of a Y-chromosome specific DNA (YDNA) probe in combination with an in vitro colony assay. Host-type hematopoietic cells were detected in cultured bone marrow mononuclear cells (BMMC) from 4 out of 6 patients studied, who were all in complete remission. In 2 patients of the mixed chimeras, the relative amount of host-derived YDNA from BMMC increased after methyl cellulose cultures for 14 days. Analysis of individual colonies derived from granulocyte-macrophage colony forming units (CFU-GM) from these mixed chimeras, including 2 patients with chronic myelogenous leukemia (CML), revealed approximately 30% of total colonies were host-type, although no evidence for the existence of residual Ph1 positive cells was obtained by using polymerase chain reaction for detecting bcr-abl chimeric messenger RNA in the 2 CML patients. These findings provide direct evidence that considerable numbers of host-derived normal hematopoietic progenitors survive and persist for a long term in a certain population of marrow recipients, after BMT following supralethal radiochemotherapy.

Background The safety and the risk in normal healthy BM/PB donors are serious concerns. Several retrospective surveys reported adverse events in healthy donors, but the real incidence of these events may be underestimated in related donors while it has been fully monitored in unrelated donors by BM/PB donor registries. Methods JSHCT has conducted a nation-wide consecutive pre-registration of related donors in Japan, for PB since April 2000 and for BM since April 2005. JSHCT mandatory requires all members to register every related donors and to report any severe adverse events in donors in the first 30 days and then annually for 5 years after donation. These early and late severe events were categorized into definitely severe; life-threatening, treatment-required or long-lasting and relatively severe to mild; transient and not treatment-required. Pre-registration to the JSHCT as a BM/PB donor is made into the conditions of personal-accident-insurance subscription. Results 1. The preceding retrospective survey on related BM donors by a governmental research committee in 2005 reported four A-events (0.07%) including one death and 21 B-events (0.35%) in 5921 donors. 2. 3975 PB donors (3264 before and 711 after April 2005) and 999 BM donors (after April 2005) have been registered to this prospective registration by the end of July 2007. 3. There has been no death in PB /BM donors. 4. 64 events (1.6%) have been reported in PB donors as early events. 11 A-events include interstitial pneumonitis(2), hypoxemia(2), ascites, SAH, retroperitoneal hematoma, anginal attack, precordial discomfort, vein thrombosis and cholangitis. 5. Four-B events (0.4%) have been reported in BM donors as early events. 6. Relatively severe early (acute) adverse events occurred in 1.39% of those donors who fulfilled donor eligibility standards of the JSHCT guideline (N=3097), and 3.85% of those with one or more exclusion criteria (N=78) (P=0.09) 7. 35 late adverse events were reported in 3167 PB in the five year observation period. Although these late events were not proved to be related to G-CSF administration or apheresis, the relationship could not be completely denied. These occurred in 1.10% of those fulfilled the JSHCT donor eligibility standards (N=3097) and in 1.28% of those with exclusion criteria (N=78)(P=0.88). 8. Late adverse events have not been reported in BM donors because of short observation period. Conclusion Both BM and PB collection in related donors were shown to be safe in general. But several serious adverse events were reported. Prospective donor registration has enabled us to accurately monitor the real incidence of adverse events and has been useful for transplant physicians to observe the safety guideline for BM /PB collection by JSHCT, which results in lowering the rate of avoidable adverse events in related donors with known risk factors. Our seven year experience suggests the importance and usefulness of the prospective registration system of related PB /BM donors, and international collaboration is required to clarify and to improve the safety and the risk of PB /BM donation both in related and in unrelated donors.

Mismatches of minor histocompatibility antigen (mHag) between HLA identical stem cell donor and host are known as a major risk factor for graft-versus-host disease (GVHD). We determined the alleles of 4 polymorphic molecules including HA-1 and 3 adhesion molecules; CD62L, CD31 codon563 and CD31 codon125 in 114 patients transplanted HLA identical stem cell grafts and investigated the association of mismatches as rates of relapse and GVHD. All 114 recipients underwent stem cell transplantation after myeloablative conditioning between 1985 and 2004. Risk ststus of the disease at SCT was standard (n=71) and high (n=43). After SCT, 36, 57 and 35 developed acute GVHD (≥2), chronic GVHD and relapsed, respectively. Our patients relapsed at rates of 14.3% and 38.0% with one or more and without incompatibilities (P=0.011). In standard risk group, the relapse rates of 20 and 51 patients with and without mHag incompatibilities were 5.0% and 39.2%, respectively (P=0.011). The relapse rates of patients with CD62L, CD31 codon563, HA-1, CD31 codon125 incompatibilities were 5.3%, 11.8%, 16.7%, 23.1% respectively. The frequency of acute GVHD (≥2) did not differ regardless of incompatibilities. The probability of 10 year survival rates were 84.0% with incompatibility and 54.3% without incompatibility patients (P=0.009). Our data suggests that incompatibilities of at least one of 4 polymorphic molecules contribute to GVL effect rather than to GVHD, resulting in prolonged survival after HLA identical SCT. Figure Figure

In April 2000, we created a system, which was cooperatively steered by The Japan Society for Hematopoietic Cell Transplantation (JSHCT) and G-CSF producing and/or selling companies, to catch up the types and the frequencies of acute and late severe adverse events (SAE) of peripheral blood stem cell (PBSC) donors among relatives in Japan. Every PBSC donor was registered to JSHCT and was given unique donor number before the PBSC donation. Every harvest center was mandatory required to observe the JSHCT Guideline for donor’s criteria and to submit the day 30 report as well as the immediate report of any severe SAE, and also to ask donors’ receiving annual health check for 5 years. This time, we report the acute SAE observed among 3,262 consecutive donors in 233 institutes and the late SAE reported through annual health check by the forth year of post PBSC harvest among 1,370 donors (2,849 times) who agreed with this work of the society. As of March 2005, 50 acute SAE out of 3,262 cases (1.5%) were reported, including anginal attack, vain thrombosis, retroperitoneal hematoma, subarachnoid hemorrhage, interstitial pneumonitis and others. Twenty-eight (2.0%) of late SAE were reported from 1,370 cases, including 1 hematological malignancy (acute myelogenous leukemia ), 8 other malignancies and others. To compare these acute and late SAE of PBSC donors to those of bone marrow (BM) donors, the questionnaires, a part of which was shared with The European Group of Blood and Marrow Transplantation (EBMT) were sent to 286 institutes of JSHCT and 191 institutes (67%) answered about 5,921 cases of bone marrow harvest from relatives and the comparative results were as followings: PBSC donors: BM donors, per 10,000; Death within 30 days = 0:1.7, SAE within 30days of post-harvest=153.0:35.5 (Definite SAE=21.5:6.8), Hematological malignancy at anytime of post donation = 3.1:2.9. On the other hand, the results obtained by EBMT, where the both surveys were retrospective, were as followings (PBSC donors: BM donors, per 10,000 ); Death within 30 days = 1.83:0.22, SAE within 30 days = 9.7:2.7, Hematological malignancy at anytime of post-donation =3.0:2.0. These results showed the followings; 1) the acute SAE might occur more frequently at PBSC donors, 2) the different frequency of acute SAE and of the mortality within 30 days of post-donation at PBSC donors between JSHCT side and EBMT side might reflect the difference of donor survey system; pre-registration with guideline for donor’s eligibility vs. retrospective survey, 3) the frequency of the occurrence of hematological malignancy was not necessarily high at PBSC donors. Although the case numbers studied at PBSC donors and BM donors in JSHCT and EBMT were different each other, no death within 30days was reported among PBSC donors of JSHCT so far and it might come from the pre-registration system of donors which made the surveillance for the safety of every donor possible and is recommended for both PBSC and BM donors at an international level in order to improve donor safety.

Mismatches of minor histocompatibility antigen ( mHag) between HLA identical stem cell donor and host are known as a major risk factor for graft-versus-host disease (GVHD). We determined alleles of 5 polymorphic molecules including HA-1 and 4 adhesion molecules for 107 patients stem cell transplanted from HLA identical donors and investigated the association of their mismatches with relapse rate and GVHD. All of 107 recipients received stem cell transplantation ( SCT) after myeloablative conditioning regimen from 1978 to 2002, 89 received marrow, 15 received PBSC and 3 received both. Stage of the disease at SCT was 63 in standard risk and 43 in high risk. After SCT, 37 developed acute GVHD( ≥2), 50 developed chronic GVHD and 31 relapsed during at least 2 years follow up period. We observed 16.1% of relapse rate in patients with at least one or more incompatibilities and 39.3% of relapse rate without incompatibilities (P= 0.018). Relapse rate of patients with CD62L, HA-1, CD31 at codon 563, CD31 at codon 125 and 49b incompatibilities are as followed 6.1%, 14.3%, 11.7%, 20% and 40% respectively. No difference of acute GVHD was observed in patients with and without incompatibilities. Among standard risk patients, 11 patients with HA-1 incompatibility showed 0% of relapse rate which was better than 45% (P= 0.003) in 47 patients without HA-1 incompatibility. In addition, 10 year survival rate was 100% in the former patients which was better than 52% of survival rate (p=0.03) in the latter patients. These data suggests that polymorphic molecules of HA-1 and CD62L contribute to GVL effect rather than the developing of GVHD which resulted in long term survivor after HLA identical stem cell transplantation. Mhag analysis of donor and host in addition to HLA antigen will help a patient to find a suitable donor in HLA identical family and voluntary donor. Figure Figure

Autologous mixed lymphocyte culture (AMLR) is an immunologic response with memory and specificity and plays a role in immune regulation. Effects of T cells activated by AMLR were studied in the regulation of in vitro erythropoiesis. AMLR-activated T cells were cocultured with autologous non-T, nonphagocytic peripheral blood mononuclear cells for assaying erythroid progenitor cells (BFU-E). T cells activated for 3 days in AMLR showed significant enhancement of in vitro colony growth by BFU-E. In contrast, activated T cells from day 7 AMLR caused significant suppression of BFU-E growth. Both enhancing and suppressing activities of AMLR-activated T cells were mediated by an la-positive and radiosensitive population within the OKT4+ subset. These observations suggest that AMLR-activated T cells may play a role in the immune-mediated regulation of in vitro erythropoiesis. It is also suggested that heterogeneous T-cell subsets may exert regulatory functions in the regulation of in vitro hematopoiesis.