Makoto Yazaki’s research while affiliated with Hoshigaoka Koseinenkin Hospital and other places

T cell lymphoblastic lymphoma (T-LBL) accounts for 30 % of all childhood non-Hodgkin's lymphomas (NHL) in Japan. Twenty-nine patients with T-LBL in stages III and IV were eligible for and enrolled in the JACLS NHL-T98 trial (1998-2002), and 72 patients with T-ALL were enrolled in the JACLS ALL-T97 trial (1997-2001). The 10-year overall survival (OS) (61.1 ± 11.5 %) and the 10-year event-free survival (EFS) (44.4 ± 11.7 %) of stage III LBL were lower than those of other diseases, and the OS and EFS were nearly the same when comparing stage IV LBL and ALL (OS: stage IV LBL, 80.0 ± 12.7 % vs. ALL, 80.2 ± 4.9 %; EFS: stage IV, LBL 70.0 ± 14.5 % vs. ALL, 70.7 ± 5.5 %). Outcomes were worse for stage III LBL than for stage IV LBL or T-ALL. Given that the treatment results of T-ALL and LBL stage IV did not differ when compared with previous reports, LBL stage III in Japanese children may differ from LBL stage III in children in other countries.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow disease. The condition is characterized by anemia that usually presents during infancy or early childhood and congenital malformation. Several reports show that DBA is associated with mutations in the ribosomal protein (RP) genes, RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, and RPS7. Recently, 5 and 12 patients with mutations in RPS10 and RPS26, respectively, were identified in a cohort of 117 DBA probands. Therefore, we screened the DBA patients who were negative for mutations in these DBA genes for mutations in RPS10 and RPS26. The present case report describes the identification of the first Japanese DBA patient with a novel mutation in RPS10.

Vitamin B12 deficiency in infants often presents with nonspecific hematological, gastrointestinal, and neurological manifestations. It is usually caused by inadequate intake, abnormal absorption, or congenital disorders of vitamin B12 metabolism, including transport disorders. We describe a vitamin B12-deficient infant with severe anemia who was breastfed. His mother had undiagnosed vitamin B12 deficiency having undergone total gastrectomy 18 years earlier. The infant developed normally after taking vitamin B12. It is important to suspect vitamin B12 deficiency in mothers who have undergone gastrectomy. Early diagnosis and treatment of vitamin B12 deficiency in infants is important and will help improve long-term prognosis.

Incidence and characteristics of early bacterial infection within 100 days after unrelated cord blood transplantation (UCBT) were assessed for 664 pediatric and 1208 adult recipients in Japan. Cumulative incidence of early bacterial infection at day 100 post-UCBT was 11% (95% confidence interval [CI], 8%-13%) for children and 21% (CI, 19%-24%) for adults (P < .0001). Early bacterial infection in adults had a significant impact on mortality (hazard ratio [HR] = 2.1, CI, 1.7-2.6; P < .0001), although no significant risk factors were identified. Multivariate analysis identified older age group (6-10, and 11-15 years versus 0-5 years of age) at transplant (HR = 2.0 and 2.7, CI, 1.1-3.5 and 1.4-4.9; P = .020 and .002, respectively) as an independent risk factor of early bacterial infection for children. Early bacterial infection in children did not have a significant impact on mortality when adjusted. Of 315 bacteremia, 74% were caused by Gram-positive microorganisms. Pneumonia occurred in 39 patients including 13 cases of Stenotrophomonas maltophilia pneumonia. Early bacterial infection had a negative effect on survival for adults and the median day of development was 10 days after transplant, suggesting that the prevention of bacterial infection in the very early post-UCBT phase is important.

Minor histocompatibility antigens (mHags) are the molecular targets of allo-immunity associated with major anti-tumor activities in hematopoietic stem cell transplantation (HSCT), but are also involved in the pathogenesis of graft-versus-host disease (GVHD). They are typically defined by the host’s SNPs that are not shared by the donor and immunologically recognized by cytotoxic T-cells isolated from the post-HSCT patients. However, despite their critical importance in transplantation medicine, fewer than 20 mHags have been identified during the past 20 years due to the lack of an efficient method for their isolation. Here we developed a novel method in which the large data set from the International HapMap Project can be directly used for genetic mapping of novel mHags. Concretely, immortalized B lymphoblastoid cell lines (LCLs) from a HapMap panel are grouped into mHag positive (mHag+) and negative (mHag−) subpanels according to their susceptibility to a cytotoxic T-cells (CTL) clone as determined by conventional chromium release cytotoxicity assays (CRAs), and the target mHag locus could be directly identified by association scan (indicated by χ2 statistic) using the highly qualified HapMap data set having over 3,000,000 SNP markers. The major concern about this approach arises from the risk of overfitting observed phenotypes to one or more incidental SNPs from this large number of the HapMap SNPs. To address this problem, we first estimated the maximum sizes of the test statistics under the null hypothesis (i.e., no associated SNPs within the HapMap set) empirically by simulating 10,000 case-control HapMap panels in different experimental conditions, and compared them with the expected size of test statistic values from the marker SNPs associated with the target SNP, assuming different linkage disequilibrium (LD), or values in between. Except for those mHags having very low minor allele frequencies (MAF) below ~0.05, the possibility of overfitting is progressively reduced as the number of LCLs increases, allowing for unique identification of the target locus in a broad range of values. To demonstrate the feasibility of this method, we tried to map the locus for HA-1H mHag, by actually immunophenotyping 58 LCLs from the JPT+CHB HapMap panel with CRAs using HLA-A*0206-restricted LCL (CTL-4B1). As expected, the genome-wide scan clearly indicated a unique association within the HMHA1 gene, showing a peak χ2 statistic of 52.8 (not reached in 100,000 permutations) at rs10421359. Next, we applied this method to mapping novel mHags recognized by HLA-B*4002-restricted CTL-3B6 and HLA-A*0206-restricted CTL-1B2, both of whose target mHags had not been identified. The peak in chromosome 19q13.3 for the CTL-3B6 set showed the theoretically maximum χ2 value of 50 (not reached in 100,000 permutations) at rs3027952, which was mapped within a small LD block of ~182kb containing a single gene, SLC1A5, as a candidate mHag gene. In fact, when expressed in HEK293T with HLA-B*4002 transgene, recipient-derived, but not donor-derived, SLC1A5 cDNA was able to stimulate interferon-γ secretion from CTL-3B6, indicating that SLC1A5 encodes the target mHag recognized by CTL-3B6. Conventional epitope mapping finally identified an undecameric peptide, AEATANGGLAL, which was further confirmed by epitope reconstitution assays. The target mHag locus for CTL-1B2 was identified at the peak (max χ2 = 44, not reached in 100,000 permutations) within a 598 kb block on chromosome 4q13.1, and coincides with the locus for a previously reported mHag, UGT2B17. Our epitope mapping by using UGT2B17 cDNA deletion mutants, prediction of candidate epitopes by HLA-binding algorithms and epitope reconstitution assays successfully identified a novel nonameric peptide, CVATMIFMI. Our results demonstrate how effectively the HapMap resources could be used for genetic mapping of clinically relevant human traits. This method may be also applied to disclosing other relevant human variations, if an accurate bioassay is applied to discriminate them. We anticipate our method based on the HapMap scan greatly accelerates isolation of novel mHags, which could be used for the development of selective allo-immune therapies to intractable blood cancers, circumventing potentially life-threatening GVHD, while harnessing its anti-tumor effects. Such knowledge on mHags should also promote our understanding of allo-immunity.

Minor histocompatibility antigens (mHags) are molecular targets of allo-immunity associated with hematopoietic stem cell transplantation (HSCT) and involved in graft-versus-host disease, but they also have beneficial antitumor activity. mHags are typically defined by host SNPs that are not shared by the donor and are immunologically recognized by cytotoxic T cells isolated from post-HSCT patients. However, the number of molecularly identified mHags is still too small to allow prospective studies of their clinical importance in transplantation medicine, mostly due to the lack of an efficient method for isolation. Here we show that when combined with conventional immunologic assays, the large data set from the International HapMap Project can be directly used for genetic mapping of novel mHags. Based on the immunologically determined mHag status in HapMap panels, a target mHag locus can be uniquely mapped through whole genome association scanning taking advantage of the unprecedented resolution and power obtained with more than 3 000 000 markers. The feasibility of our approach could be supported by extensive simulations and further confirmed by actually isolating 2 novel mHags as well as 1 previously identified example. The HapMap data set represents an invaluable resource for investigating human variation, with obvious applications in genetic mapping of clinically relevant human traits.

Umbilical cord blood (UCB) transplantation is limited by the low number of hematopoietic stem cells in UCB units, which results in a low engraftment rate in transplant recipients. Here, we measured the total nucleated cell count and CD34(+), CD3(+), CD4(+), CD8(+), CD14(+), and CD16(+)/56(+) cell doses in each UCB unit and evaluated their influence on engraftment and other outcomes in 146 recipients. Multivariate analysis showed a significant association between a higher incidence of successful engraftment and a dose of CD34(+) and CD8(+) cells above the median (1.4 x 10(5) and 15.7 x 10(5) cells/kg, respectively). Engraftment occurred 4 days earlier in patients who received UCB with more than the median dose of CD34(+) cells than those receiving UCB at or below the median. Stratification of the group according to CD34(+) cell dose revealed a significant influence of the CD8(+) cell dose on the time to achieve neutrophil engraftment in patients receiving a lower CD34(+) cell dose, whereas there was no significant influence in the patients receiving a higher CD34(+) cell dose. These results suggest that consideration of CD34(+) and CD8(+) cell doses in UCB units may improve the engraftment in recipients of UCB transplantation.

Background> The risk of rejection after umbilical cord blood (CB) transplantation (CBT) is substantially higher than that after allogeneic BMT or PBSCT. In CBT, higher total nucleated cell count and CD34+ cell dose are shown to be associated with significant rapid and proper engraftment. Although CD8+ or other accessory cells are shown to have positive association with engraftment in many experimental and clinical BMT, potential contribution of these cells to engraftment after CBT has not been examined comprehensively. We examined CB subpopulation doses, such as CD34+, CD3+, CD4+, CD8+, CD14+ and CD16/56+ cells, in cryopreserved CB unit by flowcytometry, and analyzed their influence on the risks of primary graft-failure, GVHD, non-relapse mortality and the probability of survival. Data on 146 patients [median age 15.0 years old (range, 0.2–74.0), median body weight 41.0 kg (range, 4.8–85.0)] who received CBT as their first transplant between 1995 and 2002 for the treatment of malignant (n=137), including advanced disease patients (n=100), and nonmalignant (n=9) diseases were evaluated. The HLA-disparity between CB and patient was 0/6: 35 (24.0%), 1/6: 65 (44.5%), 2/6: 45 (30.8%), and 3/6: 1 (0.7%). In a multivariate analysis, CD34+ cell dose at higher than the median (>1.4 × 105/kg) and CD8+ cell dose at higher than the median (>15.7 × 105/kg) were significantly associated with successful engraftment [Hazard ratio (HR), 0.56; 95% confidence interval (CI), 0.37–0.85, and HR, 0.65; 95% CI, 0.43–0.99, respectively]. In the subgroup of CD34+ cell higher than the median group, the median time to achieve neutrophil engraftment was comparable between CD8+ higher and lower than the median group (21 days and 22 days, P=0.11). In the subgroup analysis for patients who received lower than the median (1.4 × 105/kg) of CD34+ cells, the median time to achieve neutrophil engraftment in the patients received more CD8+ cells (>15.7 × 105/kg) was significantly faster than that in the patients received less CD8+ cells (21 days versus 25 days, P=0.0047). CD8+ cell dose in at least median and HLA-disparity more than 1 locus were significant factors associated with an increasing incidence of grade II–IV acute GVHD [HR, 2.05; 95% CI, 1.18–3.59 and HR, 2.41; 95% CI, 1.16–5.00, respectively]. We could not detect any association of higher CD34+ and CD8+ cell doses with improved survival. No significant impact of other CB subpopulations on transplant outcome was observed. Our findings and previous study reported by Wagner et al. (Blood.2002;100:1611–8) demonstrated the CD34+ cell dose in CB unit is a significant and definitive predictor of engraftment rate and kinetics after CBT, and suggested the threshold dose for successful engraftment is around 1.4–1.7 × 105/kg. Additionally, CD8+ cells may have a supportive role in engraftment following CBT with an insufficient CD34+ cell dose. To confirm the effect of these cell subpopulations on survival, further study with more monotonous background is warranted.

Between October 1981 and December 2000, 46 patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) underwent allogeneic hematopoietic stem cell transplantation (HSCT) in the Nagoya Blood and Marrow Transplantation Group. The median age was 28.5 years (range, 4-51 years). All but one patient achieved engraftment. Grade II-to-IV acute graft-versus-host disease (GVHD) developed in 32.5% of patients, and chronic GVHD developed in 40.5%. The incidences of relapse and treatment-related mortality (TRM) at 5 years were 65% and 26%, respectively. The estimated overall survival rate at 5 years was 23%. Univariate analysis showed that improved disease-free survival (DFS) was independently associated with complete remission (CR) at transplantation (39%), compared with non-CR (8%) (P = .023). Non-CR at transplantation was associated with a higher risk of relapse. Donor type, acute GVHD, and time from diagnosis to HSCT all had a significant effect on TRM. In a multivariate analysis, 9 months or more from diagnosis to HSCT was the only variable statistically significant for DFS (relative risk, 3.22; P = .01). This study demonstrates that allogeneic HSCT cures a significant population of patients with Ph+ ALL. Relapse is the major obstacle limiting the success of HSCT. Early transplantation during CR from donors, including unrelated persons or mismatched relatives, may offer improved long-term DFS.

To explore the potential efficacy of l-asparaginase treatment in acute myeloid leukaemia (AML) patients, we studied the in vitro resistance of French-American-British (FAB) subtypes of childhood AML to l-asparaginase using a methyl-thiazol-tetrazolium assay. We tested leukaemic cells obtained from 177 common acute lymphoblastic leukaemia (cALL) and 228 AML children at diagnosis. The median 70% lethal dose of l-asparaginase (LD70asp) (U/ml) was 0.46 in the cALL and 6.70 in the AML samples. The median LD70asp among each FAB subtype of AML was 0.76 (M0), 0.46 (M1), 10.00 (M2), 10.00 (M3), 1.18 (M4), 1.35 (M5) and 10.00 (M7). Type M3 samples had the highest LD70asp. The LD70asp of the M2 samples was significantly higher than that of the M1, M4 and M5 samples. When the LD70asp values were classified as low (0.016-0.159), intermediate (0.16-1.59) or high (1.6-10.00), the frequency of low, intermediate or high LD70asp among the M1 samples were similar to those among the cALL samples. In conclusion, cells from AML types M1, M4 and M5 were relatively sensitive to l-asparaginase, and M1 cells were as sensitive as those of cALL, suggesting that l-asparaginase treatment may be effective for these subtypes of AML.