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In Japan, unrelated bone marrow transplantations (U-BMT) for severe aplastic anemia (SAA) were first performed in 1993. Transplant regimens and patient selection have changed substantially since then. We aimed to determine the effect of these changes on transplant outcome. We retrospectively analyzed the outcome in patients with SAA who received U-BMT through the Japan Marrow Donor Program between 1993 and 2005. We selected 302 recipient-donor pairs in which molecular analysis of HLA-A, -B, -C, -DRB1, and DQB1 were performed. Patient ages ranged from 1 to 64 years (median, 17 years). Various preconditioning regimens were used by individual centers. Either tacrolimus with methotrexate or cyclosporine with methotrexate was used for the prophylaxis against graft-versus-host disease (GVHD) in 137 (45%) patients and 127 patients (42%), respectively. Of the 302 pairs, 104 (34%) were found to be matched at HLA-A, -B, -C, -DRB1, and DQB1; 97 (32%) were mismatched at a single HLA allele (23 HLA-A or -B, 42 HLA-C, 32 HLA-DRB1 or HLA-DQB1); 83 (28%) were mismatched at two HLA alleles (35 HLA-A or -B and HLA-C, 8 HLA-A or -B and HLA-DRB1 or -DQB1, 40 HLA-C and HLA-DRB1 or -DQB1); and 18 (6%) were mismatched at three HLA alleles. Currently, 210 of the 302 patients are alive with the median follow-up period of 722 days after transplantation. The incidence of graft failure was 2.2%; that of grade II/IV GVHD was 29.0%; that of III/IV acute GVHD was 13.8%; and that of chronic GVHD was 25.2%. Multivariate analysis revealed the following significant risk factors for survival: patients older than 15 years (RR, 1.98; range 1.17–3.35); HLA-A or -B + HLA-C or HLA-DQB1 or -DRB1 (RR 2.18; range 1.29–3.68); three loci mismatching (RR 3.14; range 1.47–6.69); prophylaxis against GVHD with tacrolimus with methotrexate (RR 0.45; range 0.28–0.73), ABO major mismatch (RR 1.67; range 1.05–2.63), and non-ATG-containing CY+TBI regimen (RR 2.17; range 1.16–4.03). Patients were divided into two cohorts based on years of transplantation and we compared patients transplanted within two time periods: 1993–2000 and 2001–2005. Five-year survival increased 55.9+/−4.6% in the 1993–2000 cohort (n=116) and 72.7+/−3.8% in the 2001–2005 cohort (n=186) (p=0.008). Patients and transplant characteristics that differed significantly between the two periods were patients’ age distribution, GVHD prophylaxis, HLA matching, and conditioning regimens. The percentage of patients older than 15 years was significantly larger in the recent period (p

No nucleophosmin mutations in pediatric acute myeloid leukemia with normal karyotype: a study of the Japanese Childhood AML Cooperative Study Group

Although most patients with acute lymphoblastic leukemia (ALL) having MLL rearrangements suffer poor treatment outcome, there exists a subgroup of patients with this disease who enjoy long-term disease-free survival. In order to verify our previously reported new classification of this type of leukemia predicting treatment outcome, we analyzed gene expression profiles of MLL-rearranged leukemic cell lines and acute myeloid leukemia (AML) samples along with the previously reported ALL samples. Hierarchical clustering and the weighted vote method showed that cell line samples formed a strong association with the poorer prognosis subgroup in the classification, and that AML samples had some relationship with the better prognosis subgroup. Highly expressed genes in the poorer prognosis subgroup combined with cell line samples compared with the better prognosis subgroup include genes related to tumor aggressiveness, such as SNCG, GRM4, and EDG4. These results support the significance of the previously described classification associated with prognosis. Large-scale clinical studies, therefore, may be worth trying on the basis of this classification.

BACKGROUND: A rapid clearance of leukemic blasts in the peripheral blood (PB) is well known to be correlated with a good prognosis in children with ALL. Most groups use a cut-off of Day 8 PB blasts as 1000/μ L. It is possible that patients with no blasts may have an even better outcome. In L99-15 study, we tested the utility of a cut-off of 0 (zero) blasts to stratify children with ALL. OBJECTIVE: To evaluate the significance of complete clearance of peripheral blasts after 7 days of PSL in children with ALL. PATIENTS AND METHODS: Children newly diagnosed as ALL between 1999 and 2003 were consecutively enrolled on the TCCSG L99-15 study. All patients initially received 7 days prednisolone 60mg/m2 monotherapy (without intrathecal therapy) and day 8 PB blasts were classified into 3 categories, 0, 1-999, 1000 or more/μL. Risk stratification was based on the age, initial white blood cell (WBC) count, immunophenotype, cytogenetics and the response to prednisolone. Induction treatment consisted of a standard 4-drug regimen in standard-risk group (SR) groups whereas cyclophosphamide was added in high-risk group (HR) and very high-risk group (HEX). After induction, SR and HR patients received BFM-type early intensification, consolidation, reinduction, and maintenance therapy. HEX patients received early intensification and intensive rotational consolidation therapy including AML-type consolidation, and no maintenance therapy. Total duration of therapy was 3 years for SR, 2 years for HR, and 1year for HEX patients. Allogeneic stem cell transplantation was indicated approximately in 50% of the HEX patients. RESULTS: Totally, 267 (35%) out of 770 children were categorized into SR, 317 (41%) into HR and 186 (24%) into HEX. Remission was obtained in 259 (97%) patients in SR, 311 (98%) in HR and 171 (92%) in HEX. Event-free survival (EFS) (SE) at 4 years was 80.5% (1.7%) in B precursor ALL (n = 669), and 66.0% (5.1%) in T ALL (n = 92). In B precursor ALL, patients with day 8 PB blasts as 0 were 129 (44%) out of 295 initial SR group, 85 (29%) out of 289 initial HR group, 6 (10%) out of 59 initial HEX group, and 4y-DFS was 91.1% (2.6%), 89.4% (3.6%), 83.3% (15.2%), respectively. In T ALL, patients with day 8 PB blasts as 0 were 22 (26%) out of 84, and 4y-DFS was 100% (0%). CONCLUSION: Patients with Day8 PB blasts as 0 (zero) constituted about 30% of all the cases with childhood ALL. Excellent outcome (4-year EFS of 90%) was obtained for those with 0 blasts. Initial Risk Groups WBC/age 1–6y 7–9y 10y– K = 1,000 μL −20K SR HR HR 20–50K HR HR HR 50–100K HR HR HEX 100k– HEX HEX HEX Final Risk Groups day 8 blast 0 1–999 1000– HEX/SCT: HEX and allogeneic stem cell transplantation Initial Risk Groups non-T/SR SR SR HR non-T/HR HR HR HEX non-T/HEX HR HEX HEX/SCT T-ALL HR HEX HEX/SCT

BACGROUND: Acquired aplastic anemia (AA) is thought to be an immune-mediated disease. Immunosuppressive therapy (IST) has been the treatment of choice for patients who did not have suitable donors. Previously, we had published promising results of IST for children with acquired AA. In the study, overall survival rate (OS) at 4 years was 83% in patients with vSAA and 92% in those with SAA/nonSAA. OBJECTIVES: Here we report follow-up results focusing on pediatric patients with relapse and clonal diseases, MDS/AML and PNH. PATIENTS and TREATMENT: From 1992 to 1997, 119 newly diagnosed children with acquired AA (median age; 9) entered AA-92 study. 50 vSAA patients were treated with ATG+CyA+mPSL+danazole +G-CSF, 36 SAA and 28 nonSAA patients were treated with ATG+CyA+mPSL+danazole +/−G-CSF. Complete remission (CR) was defined as a neutrophil count >1.5x10^9/L, a platelet count >100xx10^9/L, and a hemoblobin level of >11 g/dl. Partial response (PR) was defined as a neutrophil count >0.5x10^9/L, a platelet count >20x10^9/L, and a hemoglobin level of >8.0 g/dl. Relapse was indicated by the return of the PB counts to levels meeting the definition of SAA and/or the requirement for blood transfusion. Response rate was 71% at 6 months in vSAA patients, 65% in SAA/nonSAA patients, respectively. There was no statistical difference in 6-month response rate between G-CSF +/− treatments. No patient responded after 6 months. Therefore, 75 responders and 29 non-responders at 6 months were analyzed their OS, relapse rate (RR), and treatment-failure-free survival (TFFS). The median observation time of surviving patients is 118 months, ranging from 75 to 168 months. RESULTS: Among 119 patients, 38 patients received BMT and 17 patients died during an observation period. The OS was 81.9+−3.8% at 10 years, but has not reached plateau. Of the 75 6mo-responders, 23 patients relapsed and the RR was 33.6+−6.0% at 10 years. TFFS of the 75 was 67.4+−5.5%. Fourteen patients received 2nd ATG therapy and 5 of them responded. Eleven of the 23 patients with relapse received alternative donor BMT and 8 are alive. 10y-OS of these relapsed patients was 73.4+−9.4%. Nineteen of the 29 6mo-non-responders received alternative donor BMT and 15 are alive. There is no statistically significant difference in 10y-OS between the responders and the non-responders (89.1+−3.7% vs. 75.0+−8.2%, p=0.09). New clonal abnormalities appeared in 9 of 119 patients (10.0+−3.2%KM probability): monosomy 7(3 patients), trisomy 8(3 patients), trisomy 9, trisomy11, del (13)(1 patient each). There is no statistically difference in 10y-OS, RR, and incidence of clonal abnormality between randomized G-CSF+/− treatments. Clinical PNH with symptomatic hemolysis developed in 3 of the responder patients, 96, 105 and 138 months after the AA diagnosis, respectively. Among 65 surviving responders, 33(51%) have CR and 26(40%) PR at last follow-up time. CONCLUSIONS: Our data demonstrate that IST is effective for children with acquired AA, but relapse are common. Effective 2nd line treatment should be developed. PNH developed even in pediatric AA patients during long-term observation.

More than 70% of children with ALL enjoy a long-term event-free survival (EFS), however, the rest of them still suffer from a relapse and the treatment for relapsed ALL is not always successful. The treatment results of childhood ALL with a first relapse were retrospectively analyzed to determine prognostic factors. Of the 1336 children enrolled on the TCCSG L95-14 and L99-15 studies, diagnosed between 1995 and 2003, 224 suffered from a relapse before March 2005. The relapsed patients were treated differently according to the protocol of each institution. The potential prognostic factors examined were : the age (low; 1–9 years, high; over 10 years), white blood cell count (low; The sites of relapse were: isolated bone marrow (BM, n=165), BM with other sites (n=21) and extramedullary only (n=38). Ninety-nine relapses occurred in very early stage, 43 in early stage and 82 in late stage. Twenty-three patients received SCT before relapse according to the protocol. A second complete remission (2CR) was achieved in 175/220 patients (79%) and 74 of them are still in 2CR. Five-year EFS for all the relapsers was of 29±3% (mean±s.e.) and 5-year overall survival rate was 38±4%. In a subset of patients (n=126) with B-lineage phenotypes and with isolated BM relapse, who were treated with chemotherapy alone before relapse, we found 2 significant prognostic factors identified by a multivariate analysis : the age at initial diagnosis (EFS: age low; 35±5%, high; 8±8%, p=0.0016) and the timing of relapse (EFS: late; 39±8%, early; 24±9%, very early; 16±5%, p=0.0051). Gender, initial leukocyte count, or the use of SCT after obtaining 2CR was not related with patient’s outcome in a multivariate analysis. The importance of the timing of relapse was overwhelmingly shown, however, the significance of the age at the diagnosis was not demonstrated before. The results will be incorporated into our next protocols to relapsed patients with ALL.

Karyotypic abnormalities are associated with a prognosis of acute myeloid leukemia (AML). However, there are some patients who had poor prognosis among patients with the same karyotypic abnormalities. Recently, it is revealed that alterations of the genes including tyrosine kinases, lead to poor prognosis in AML. Genetic alterations associated with a poor prognosis remain to be clarified in karytotypic subgroups of AML. We performed the mutation analysis of FLT3, MLL, KIT, RAS and NPM in 158 pediatric AML patients who were enrolled in Japanese Childhood AML Cooperative Treatment Protocol, AML99, including 33 with normal karyotype, 46 with t(8;21), 7 with inv(16), 20 with 11q23 translocations, 13 with t(15;17), 10 with Down syndrome (DS) FAB-M7 and 29 other karyotypic abnormalities. In the 33 patients with normal karyotype, 9 FLT3-internal tandem duplication (ITD), 2 FLT3-D835 mutation (D835Mt), 8 MLL-partial tandem duplication (PTD), 2 KIT, 2 NRAS and 3 KRAS mutations were identified. FLT3-ITD and MLL-PTD were associated with a poor prognosis. Notably, NPM gene mutation was not identified in these patients. In the 46 patients with t(8;21), 2 FLT3-ITD, 1 D835Mt, 4 MLL-PTD, 8 KIT, 4 NRAS and 5 KRAS mutations were found. KIT mutations were associated with a poor prognosis. In the 7 patients with inv(16), 2 FLT3-D835Mt and 1 KIT mutations were identified. Only one patient with KIT mutation relapsed. In the 20 patients with 11q23 translocations, 1 D835Mt and 5 MLL-PTD were identified. MLL-PTD was associated with a poor prognosis in patients with 11q23 translocations. In the 13 patients with t(15;17), 3 FLT3-ITD and 3 D835Mt were found. FLT3-ITD was not associated with a poor prognosis. In the 10 patients with DS-M7, KIT and KRAS mutations were found in each one patient, showing no prognostic significance. In the 29 patients with other karyotypic abnormalities, 6 FLT3-ITD, 2 D835Mt, 4 MLL-PTD, 0 KIT, 2 NRAS and 2 KRAS mutations were found. FLT3-ITD and MLL-PTD were associated with a poor prognosis. Fifteen patients (9.5%) who had alterations of 2 of these 4 genes showed a poor prognosis. RAS gene mutations were found in 26 (16.5%) of 158 patients, but were not associated with the prognosis. These results suggest that a new different therapeutic strategy for the patients with these gene alterations in each subgroup, for example KIT mutations in t(8;21)-patients and MLL-PTD in patients with 11q23 translocation, is needed to improve the prognosis of pediatric AML.

From 1993 to 2003, 1223 patients, 0 to 61 year old, with high risk acute lymphoblastic leukemia underwent their first bone marrow transplantation through Japan Marrow Donor Program. Most transplants were performed between HLA serologically matched Japanese pairs with a few exceptions. Of all, 520 patients underwent transplantation in the first complete remission (1CR: median follow up 488 days), and their probability of 5 year overall survival (OS) by Kaplan Meier analysis was 58.4(standard error; SE 2.8) %. The 281 patients in the 2CR (452 days) showed OS 49.4(3.7) %, 75 at the third or further CR (249 days), OS 29.5(5.9) %, and 333 at relapse or non CR(162 days), OS 16.4(2.6) %. The outcome of the younger tended to be the better except that the oldest group 51–61 year of age did fair. The GVHD prophylaxes were cyclosporine and short term methotrexate (sMTX) in 681 patients, and tacrolimus plus sMTX in 461. Among the 1CR patients, the 5 year OS of 276 patients in cyclosporine group was 52.0% (SE.3.7%) and 217 tacrolimus group showed significantly better OS, 66.5% (SE 5.7%, P=0.03). The grade II to IV, or III to IV acute GVHD developed in 47.3% or 19.1% respectively without any age difference. Acute GVHD grade I might solely favor the patients in 1CR at 5 years or earlier post-transplant, and the survival of those in 2CR was minimally affected by grade 0 to III acute GVHD. On the other hand, the presence of chronic GVHD did not improve the survival rate of the patients in 1CR, whereas the patients in 2CR achieved better survival if they had limited or extensive chronic GVHD. The importance of HLA class I DNA type matching in Japanese population has been published (N Engl J Med. 1998 Oct 22). Patients with ALL did poorly between the pairs with DNA A-locus mismatch in 1CR and 2CR, B-locus mismatch in 2CR, DRB1-locus mismatch in 2CR. We concluded that, the unrelated donor marrow transplantation in Japan provided as good chance of survival for the patients with ALL as related donors did, despite poorer risk group they belong to. GVHD prophylaxis with tacrolimus might attain better outcome in 1CR patients, and chronic GVHD seemed to improve the OS for the patients in 2CR, but not for 1CR patients.

To find poor prognostic factors other than karyotypes is desired to improve the treatment outcome in pediatric acute myeloid leukemia (AML). Total three hundread and eighteen patients were enrolled in Japanese Childhood AML Cooperative Treatment Protocol, AML99, from January 2000 to December 2002. We performed the mutation analysis of FLT3 and MLL in 158 patients (13 of 29 with FAB-M3, 10 of 49 with Down syndrome, 135 of 240 with other type of AML) Patients with FAB-M3 and patients with Down syndrome were treated on different protocol. Seventeen (12.6%) of 135 patients had FLT3-internal tandem duplication (ITD). The differences between AML patients with or without FLT3-ITD were significant in 4-year overall survival (OS) (35.3% vs. 86.1%, p

Purpose: Recent multi-institutional studies have reported that children with Down syndrome (DS) and acute myeloid leukemia (AML) have a favorable outcome with less intensive chemotherapy. Based on our previous trial (Kojima, et al: Leukemia; 14:786,2000), the Japanese Childhood AML Cooperative Study Group conducted the AML-Down protocol study designed for children with DS and AML. Patients and Method: Between February 2000 and June 2004, 72 children (44 boys, 28 girls; median age, 1 year; range, 7 months to 7 years) were enrolled in this study. The median white blood cell count was 5,800/10−9 L. The median follow-up period was 3 years (range, 9 months to 5 years). Acute megakaryocytic leukemia (M7) was diagnosed most often (90%). The treatment regimen consisted of 5 cycles of Ara C 100mg/m2 (1-hour infusion) x 7 days, THP-ADR 25mg/m2 x 2 days, and etoposide 150mg/m2 x 3days. No prophylaxis against CNS leukemia was included. Results: Among the 72 children, 69 achieved complete remission (CR) after 1 to 2 cycles of induction therapy, with no deaths occurring during the induction period. One of 3 patients with induction failure achieved CR after another intensified chemotherapy. Eight patients relapsed during chemotherapy. One relapsed while off therapy and successfully entered a second remission after an intensified chemotherapy, followed by an allogeneic bone marrow transplant. There was no CNS relapse alone, although 1 patient relapsed in the bone marrow and CNS simultaneously. Eight relapsed patients and 2 refractory patients died without achieving a remission. The cause of death was pneumonia in 4 patients and disease progression in 7 patients. One patient died from pneumonia during the first CR. The CR rate, 3-year survival rate, and event-free survival (EFS) rate were 97.2%, 84.4%, and 83.0%, respectively. In a univariate analysis of factors that predict EFS, we found that the presence of monosomy 7 cytogenetic abnormality at diagnosis, and response to induction therapy were predictive factors for EFS. Neither age older than 2 years nor higher white blood cell count at diagnosis were statistically significant risk factors. Children with monosomy 7 had more adverse outcomes than those without monosomy 7 (41.7% vs 86.4%, p=0.02). Discussion: Our AML protocol specified for children with DS and AML does not include high-dose Arac and is much less intensive than other protocols used for treatment of these children. However, this less intensive regimen leads to an excellent outcome. In contrast to a previous study reported from CCG (Children’s Cancer Group) in the United States, age was not a significant risk factor. However, monosomy 7 is a poor prognostic factor in children with AML, whether or not they have DS. Our study strongly suggests that children with DS and AML can be treated successfully with a less intensive chemotherapy regimen that does not include high-dose Arac.

Therapy with all-trans retinoic acid (ATRA) for acute promyelocytic leukemia (APL) is the molecular target therapy, and chemotherapy combined with ATRA of recent studies, in which most patients were adults, have improved the prognosis of patients with APL. However, it is not thoroughly clarified how ATRA should be combined with chemotherapy, or whether such combination therapy is as effective for childhood APL as for adults. The Japanese Childhood AML Cooperative Study Group has conducted AML99-M3 study for childhood APL and we presented here the result of the study. Patients and Methods: From August 1997 to March 2004, 58 de novo APL children (31 boys and 27 girls) with median age of 11 years (11m to 16y) were enrolled in this study. Median follow-up period was 2.4 years. Diagnosis of APL was made on the hematological features and cytogenetic findings, which revealed 47 patients had t(15;17) translocation, nine had t(15;17) with additional abnormality, and two showed normal karyotype or other abnormality. Mean white blood cell and platelet counts at diagnosis were 20,510/μl and 36,000/μl, respectively. AML99-M3 protocol, which consisted of ATRA (5220 mg/m2 or more), anthracyclines (daunorubicin 135 mg/m2, pirarubicin 90 mg/m2, mitoxantrone 20 mg/m2, and aclarubicin 180 mg/m2) and cytarabine (69400 mg/m2), was characterized by the combination of ATRA, anthracyclines and cytarabine in not only induction but also consolidations, by intermittent administrations of ATRA in maintenance, and by intrathecal treatment at every consolidations. Results: Overall and event-free survival rates at three years were 92.1% and 90.5%, respectively. Of 58 patients, 56 (96%) achieved complete remission, and two (3.6%) relapsed at bone marrow during consolidation and after completion of protocol, respectively. In induction, exacerbation of coagulopathy in six patients, two of whom died of intracranial or pulmonary hemorrhage, and ATRA syndrome in three patients were principal complications, while ATRA-associated headache/nausea was the most frequent complication all over the protocol. In consolidations, prolonged bone marrow suppression was observed with 10.4 days on the average of neutrophil counts lower than 100/μl, during which one died of sepsis. Discussion: In recent studies adopting chemotherapy combined with ATRA for APL patients, overall survival rates have increased to 80 to 90% at three to five years, as is the case for this study. Moreover, as compared with recent studies, the lower relapse rate of this study may be attributed to the triple combination therapy (ATRA, anthracyclines and cytarabine) and to the intrathecal treatment. However, exacerbated coagulpathy and sepsis, two major life-threatening complications, remain to be solved. Although further observation is needed, this study suggests that combination therapy of ATRA with anthracyclines and cytarabine for childhood APL may be as effective as for adults, and that this combination therapy, if life-threatening complications were properly managed, would further improve the outcome for patients with APL.

Intensive chemotherapy has contributed to an improved survival in pediatric acute myeloid leukemia (AML). We here report the long term results of the two consecutive AML trials of Tokyo Children’s Cancer Study Group; TCCSG M91-13 and M96-14. Between August 1991 and September 1998, a total of 216 eligible children with newly diagnosed AML entered either of the two trials. In M91-13 trial, patients received 3-drug induction therapy consisted of cytarabine, mitoxantrone, and etoposide, 4 courses of intensive consolidation chemotherapy including 2 courses with high-dose cytarabine. Subsequently, patients received another 4 courses of high-dose cytarabine containing consolidation chemotherapy, or autologous bone marrow or peripheral blood stem cell transplantation (A-BMT/PBSCT) instead. Allogeneic bone marrow transplantation (allo-BMT) was recommended for patients with HLA-matched family donor. In M96-14 trial, the last two courses of consolidation chemotherapy were omitted from the chemotherapy arm, and patients with FAB M3 were treated with all-trans-retinoic acid containing regimen. The remission induction rate, as a whole, was 81% and probability of 5-year overall survival and event-free survival were 62%±6% (95% confidence interval) and 56%±6% by Kaplan-Meier method, respectively. There were no significant differences in survivals among the three types of post-remission therapy (chemotherapy (n = 126), 64%±8%; allo-BMT (n = 34), 76%±14%; A-BMT/PBSCT (n = 32), 80%±13%). Although the number of consolidation chemotherapy courses were reduced from 8 to 6 in M96-14 trial, overall survival rate of each trial was similar (61%±8% vs. 64%±10%, P = 0.48). The overall survival rates of patients with low risk features, namely, t(8;21), inv(16), t(15;17) or FAB M3, and Down syndrome, were significantly better than that of the other patients (75%±9% vs. 55%±8%, P = 0.002). These results suggest that intensive high-dose cytarabine containing treatment was very effective for childhood AML regardless of types of post-remission therapy. As the chemotherapy courses could be safely reduced in number without compromising the results in the latter study, further optimization of AML therapy is required.

Acute and chronic GVHD contribute to much of the morbidity and mortality associated with unrelated bone marrow transplantation (UBMT). Effective prevention of these complications is crucial for the success of UBMT. Cyclosporine(CSA) was introduced for the prophylaxis of GVHD and has been shown to be effective; similar findings have been shown for tacrolimus. However, there are no comparative studies between tacrolimus/MTX and CSA/MTX in patients with SAA who undergo UBMT. We compared tacrolimus/MTX with CSA/MTX in patients with SAA who received UBMT using a matched-pair analysis. Patients with acquired SAA who received UBMT between July 1997 and December 2002 through the Japan Marrow Donor Program were eligible for analysis. Recipients of UBMT were divided into two cohorts for comparison: those who received tacrolimus/MTX and those who received CSA/MTX. Because our previous studies identified that recipient age and conditioning regimen are the most important variables associated with treatment failure, we selected pairs of patients treated with either tacrolimus/MTX or CSA/MTX by matching these variables. We were able to identify 47 pairs that could be matched exactly for the recipient age and conditioning regimens. There was an imbalance of HLA-A, -B, and -DRB1 antigen mismatches, with 18 mismatched pairs in the tacrolimus group and 7 in the CSA group (p=0.005). Other variables were comparable between the two groups. Engraftment took place in 46 patients (98%) in the tacrolimus group and 43 patients (92%) in the CSA group. The median time to neutrophil recovery was the same in the two groups (tacrolimus group: 18.0 days, CSA group: 17.5 days). The cumulative incidence of grade 2 to 4 acute GVHD was 28.9% in the tacrolimus group and 32.6% in the CSA group (p=0.56). The estimate of chronic GVHD occurrence was 13.3% for the tacrolimus group and 36.0% for the CSA group (p=0.10). 8 patients died of transplantation related toxicities in the tacrolimus group and 22 patients in the CSA group (p=0.002). There was a higher frequency of deaths from thrombomicroangiopathy in the CSA group than in the tacrolimus group (6 vs 0: p=0.03). The 4-year survival rate was 82% for patients in the tacrolimus group and 52% for patients in the CSA group (p=0.001). Although a disadvantage existed in terms of HLA disparity in the tacrolimus group, there was no difference in the incidence of acute and chronic GVHD between the two groups. The present study shows the superiority of tacrolimus/MTX over CSA/MTX in overall survival because of lower incidence of regimen-related toxic deaths.

Risk stratified treatment of childhood AML is important to improve the patient’s QOL. Analysis of the Japan cooperative study ANLL91 revealed that response to induction therapy, age at onset, initial WBC counts, and cytogenetics were prognostic factors. Based on these results and reports from other pediatric AML clinical trials, the Japan cooperative study AML99 was planned to test the concept of risk stratified treatment. Patients were stratified into 3 risk categories: low (t(8;21) and WBC

High dose Ara-C is employed in the treatment of acute myeloid leukemia; however, its effect on ALL has not been evaluated systematically. We tested high dose Ara-C incorporated in an early phase with a randomized fashion in children with high-risk ALL. Patients diagnosed as ALL between 1999 and 2003 were consecutively enrolled on TCCSG L99-15 study. Risk stratification was based on the age, initial white blood cell count, immunophenotype, cytogenetics, and the response to prednisolone monotherapy. Totally, 317 out of 770 children (41%) were categorized in a high-risk group. A standard-risk group constituted 35% and a very high-risk group constituted 24% of all the patients. Induction treatment consisted of a standard 4-drug regimen plus cyclophosphamide. Remission was obtained in 310 patients (98%). After remission was obtained, patients were randomized to receive either early intensification regimen A or B: regimen A consisted of high-dose cytarabine (2g/m2 x 8 times with L-asparaginase); regimen B consisted of a combination of cyclophosphamide, cytarabine (75mg/m2 x 15 times), and 6-mercaptopurine. Then all the patients were given an identical treatment including high-dose methotrexate (3g/m2 x 3 times), reinduction therapy, late intensifications, and maintenance therapy. The treatment was completed 24 months after diagnosis. The number of patients randomized in each arm was 155 cases for each arm. Clinical features including immunophenotype, specific karyotype, and the response to prednisolone monotherapy were not different in patients allocated in each arm. The median follow-up for patients who survived at the time of the analysis was 3.2 years. Event-free survival (EFS) (SE) and overall survival (OS) (SE) at 4 years for the 310 patients were 78.4% (2.9%) and 88.9% (2.3%), respectively. EFS (SE) at 4 years for patients in regimen A and B were 77.0% (4.1%) and 79.7% (4.0%) while OS (SE) at 4 years in regimen A and B were 88.3% (3.2%) and 89.7% (3.2%). The site of relapse was not different in two arms. In 23 cases with T-cell phenotype, patients treated with regimen A did not have better EFS than the others. Similarly, in 25 cases with E2A-PBX1 rearrangement, patients treated with regimen A did not have better EFS than the others. Although no patients in either regimen died of infections, more patients in regimen A experienced bacterial sepsis: 13 cases in regimen A vs. 8 cases in regimen B. In conclusion, we did not find any benefit of employing early high-dose Ara-C in children with high-risk ALL.

In contrast to treatment for childhood severe aplastic anemia (SAA), there is no consensus on the treatment of childhood nSAA. Two thirds of patients progress to SAA, suggesting the need to consider early immunosuppressive therapy (IST). ATG+CsA was superior to ATG alone in a German randomized trial that enrolled both patients with SAA and nSAA. However, subgroup analysis revealed no advantage of adding CsA for patients with nSAA. These findings formed the basis for this prospective, randomized study comparing ATG alone with ATG+CsA for treatment of childhood nSAA. From 1997 to 2004, 62 children with newly diagnosed nSAA were enrolled. Interim analysis was done in April 2005. Patients were randomized to receive either horse ATG (Lymphoglobuline, 1.5vial/10kg/day for 5 days; n=31) or ATG+CsA (6mg/kg/day; n=31). CsA was added for non-responders at day+90 in ATG group. The endpoints were hematologic response at 6 months and failure-free survival (FFS). Complete response (CR) was defined as a neutrophil count > 1.5x 109/L, a platelet count > 100x109/L, and a hemoglobin level > 11.0g/dl. Partial response (PR) was defined as a neutrophil count > 1.0x 109/L, a platelet count > 30x109/L, and a hemoglobin level > 8.0g/dl. FFS was defined as survival with a response. Clinical profiles of two treatment groups were comparable. Median age was 11 years and 10 years in ATG+CsA group and ATG group, respectively. Median time from diagnosis to treatment was 20 days and 23 days in ATG+CsA group, and ATG group, respectively. After 6 months, CR was observed in ATG+CsA group in 5 patients (16%) and PR in 12 patients (39%). In ATG groups, 2 patients (6%) achieved CR and 13 patients (42%) achieved PR. However, 19 patients in the ATG group received additional CsA. There were 22 failures in ATG group and 14 failures in ATG+CsA group. A total of 10 patients who failed initial therapy (7 in ATG group and 3 in ATG+CsA group) received a second IST. Bone marrow transplant (BMT) was attempted in 4 non-responders in ATG group and 6 non-responders in ATG+CsA group. Requiring CsA was a cause of failure in 8 patients of ATG group. The probability of FFS was 54.8% in ATG+CsA group compared with 26.7% in the ATG group (p=0.001). There was 1 death in each group: 1 from BMT-related toxicity in ATG+CsA group and 1 from bacterimia in ATG group. The 5-year survival rate was 94.8% in ATG+CsA group and 96.7% in ATG group. Conclusion; the hematologic response rate at 6 months and overall survival rate are comparable between the two groups, however the combination of ATG and CsA is superior to ATG alone in term of FFS for children with nSAA.

Prior studies assessing the effects of HLA mismatching on outcomes of unrelated donor marrow transplantation (UBMT) have yielded conflicting results. Our previous study revealed that mismatching of HLA-A and -B antigens, and not of HLA-DRB1 antigen, was the most crucial risk factor for survival for patients with SAA who received UBMT (Kojima S et al, Blood100: 799, 2002). Because only the data of HLA-A, -B, and DRB1 were available, the role of HLA-C and DQB1 mismatching was not clarified in that study. The current study aimed to evaluate the effects of HLA-C and DQB1 mismatching. From Feb 1993 to April 2003, 260 consecutive patients with acquired aplastic anemia (AA) received UBMT through the Japan Marrow Donor Program. We selected 79 recipient-donor pairs in which molecular analysis of HLA-A, -B, -C, -DRB1, and DQB1 were performed. Patient ages ranged from 3 to 46 years (median, 15 years). Various preconditioning regimens were used by individual centers. In 69 patients (87%), cyclosporine and methotrexate were used for the prophylaxis against GVHD. Of the 79 pairs, 26 (33%) were found to be matched at HLA-A, -B, -C, -DRB1, and DQB1; 18 (23%) were mismatched at a single HLA antigen (6 HLA-A or -B, 7 HLA-C, 5 HLA-DRB1 or HLA-DQB1); 31 (39%) were mismatched at two HLA antigens (5 HLA- and HLA-DQB1, 18 HLA-A or -B and HLA-C or -DRB1 or -DQB1, 8 HLA-C and HLA-DRB1 or -DQB1); and 9 (11%) were mismatched at 3 HLA antigens. Transplant-related toxicities such as graft failure, grade 3 to 4 acute GVHD, and chronic GVHD were found in 7/74, 14/70, and 16/59, respectively. In a univariate analysis, any single HLA mismatching including HLA-A, -B, or -C did not increase the incidence of acute GVHD. Of 79 patients, 44 survived. The 5-year survival rate did not differ between recipients transplanted from a full-matched donor (68.5%) and those from any single HLA mismatched donors (66.7 to 80.0%). In addition, the 5-year survival rate was not worse (62.5%) in patients transplanted from HLA-C and HLA-DRB1 or -DQB1 mismatched donors. However, the 5-year survival rate was significantly worse (38.9%) in recipients who transplanted from HLA-A or -B mismatched and -C or -DRB1 or -DQB1 mismatched donors and it was 0% in patients who were mismatched at 3 antigens. In a multivariate analysis, the relative risk for HLA-A or -B and -C or -DRB1 or -DQB1 mismatching was 4.24 (95% CI, 1.6–11.5) and for 3 antigen mismatching, it was 20.0 (95% CI, 5.6–71.6). The current JMDP study showed that mismatching of a single HLA antigen did not result in increased mortality. However, in patients with HLA-A or -B mismatching, additional mismatching at HLA-C, -DRB1, or -DQB1 had a significant adverse effect on survival. Matching of HLA-C and -DQB1 should be incorporated into algorithms for unrelated donor selection.

The PTPN11 gene encodes SHP2, which is a protein tyrosine phosphatase functioning as signal transducer downstream to growth factors and cytokine receptors and its function is mediated, at least in part, through the Ras/Raf/ERK cascade in hematopoietic and non-hematopoietic cells. Recently, it has been reported that germline mutations in PTPN11 cause Noonan syndrome and a gain-of-function somatic mutations in PTPN11 has also been reported in juvenile myelomonocytic leukemia (JMML). To investigate the prevalence of mutations in PTPN11 in childhood acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), we screened the hot spot of PTPN11, exon 3, in 80 cell lines, including 14 AML, 5 chronic myeloid leukemia, 14 T-cell ALL and 47 B-precursor ALL cell lines, and 207 fresh samples, including 20 myelodysplastic syndrome (MDS), 3 JMML, 85 AML, 57 B-precursor ALL patients as well as 42 infant leukemia patients (6 AMLs, 35 ALLs, 1 mixed lineage leukemia), using polymerase chain reaction-single strand conformation polymorphism and direct sequencing analyses. In exon 3, mutations were observed in 2 of 14 (14.3%) AML cell lines (Asp61Val and Glu76Gly). A missense mutation (Ala72Val) was detected in 1 of 85 (1.2%) AML patients (a 9-year-old girl with FAB-M0). In 20 MDSs, a missense mutation (Asp61Val) (5%) was identified in a RAEB-T patient. In 3 JMMLs, 2 missense mutations (66.7%) were detected; one was Gly60Ala, and the other was Glu76Val. In 42 infant leukemia patients, we detected only one missense mutation (Ala72Asp) (2.4%) in an AML-M5b patient. However, we did not detect any mutations of exon 3 in 61 ALL cell lines and 92 ALL patients including 35 infant ALL patients. The screening for other coding regions is still undergoing although more than 90% of the reported mutations were clustered in exon 3. In conclusion, our data suggest that the PTPN11 mutations are involved in myeloid hematological malignancies rather than ALL.

Immunosuppressive therapy (IST) has been shown to be an effective therapy for aplastic anemia (AA) patients without HLA-matched family donors. However, a significant proportion of patients are refractory to IST. Although the efficacy of repeated IST and stem cell transplantation (SCT) from an alternative donor has been confirmed, a direct comparison between these two procedures has not yet been conducted. In this study, we prospectively compared the efficacy of these two treatments. Between Oct. 1997 and Apr. 2003, 231 patients (125 male, 106 female; median age, 9 y; range, 0 to 17 y) were enrolled in the AA-97 study. Analysis was performed in April 2004. A total of 103 very severe AA and 74 severe AA patients received first-line IST consisting of antithymocyte globulin (ATG; Lymphoglobulin), Cyclosporin (CyA) and methylprednisolone (Mepred), while 54 non-severe AA patients received IST consisting of ATG and Mepred with or without CyA. G-CSF was administered only to patients with very severe AA. Sixty patients (26%) were non-responders to first-line IST and were eligible for 2 nd -line therapies. Of these, 29 patients lacking suitable donors received 2 nd-line IST with ATG (Lymphoglobulin), CyA and Mepred. Four developed severe anaphylactoid reactions and could not complete the 2 nd-line treatment. Twenty-nine patients received SCT as follows: BMT from an unrelated donor (n=22), BMT from an HLA-mismatched family donor (n=4) and cord blood SCT from an unrelated donor (n=3). Two patients did not receive any 2 nd-line treatment. Median interval between 1 st -line IST and 2 nd-line treatment was 8 mo for the SCT group (range: 4–20 mo) and 7 mo for the IST group (range: 5–14 mo). One patient receiving 2 nd-line IST and 5 patients receiving SCT died of infections. The overall probability of survival at 5 y from the start of 2 nd-line treatment was 82 ± 10% in the SCT group and 97 ± 5% in the IST group (p:0.08 ). However, trilineage response to a 2 nd course of IST was seen in only 3 of 25 evaluable patients (12%), and 10 non-responders to 2 nd-line IST later received BMT from an unrelated donor as a 3 rd -line therapy. Blood counts normalized in all patients following SCT. Failure-free survival (FFS) was defined as survival with treatment response. The estimated FFS at 5 y was significantly better (p Conclusion: SCT from alternative donors offers a better chance of FFS than 2 nd-line IST in non-responders to 1 st -line IST. However, overall survival rate is not different between two approaches.

FLT3 is a type III receptor tyrosine kinase and this kinase mutation is most frequently observed in cases of acute myeloid leukemia (AML). Approximately 30% of adult AML patients have internal tandem duplication (ITD) of the juxtamembrane domain or point mutation of kinase domain (D835Mt) leading to the constitutive activation of downstream signaling pathways and aberrant cell growth. We examined FLT3-ITD and D835Mt in 160 pediatric AML patients treated on Japan pediatric AML cooperative treatment protocol, AML 99. This protocol is consisted of intensification of multi-drug chemotherapy and early application of stem cell transplantation (SCT) for the high risk patients (monosomy 7, Ph1, or delayed remission etc.). FLT3-ITD was found in 22 of 160 (13.8%) patients, including 5 of 15 (33.3%) patients with FAB-M3, 5 of 25 (20.0%) with M5, and 4 of 24 (16.7%) with M1. No specific chromosomal abnormalities were associated with ITD; 9 of 17 (52.9%) patients with ITD showed normal karyotype except for t(15;17). The D835Mt was found in 12 of 160 (7.5%) patients, including 4 of 15 (26.7%) patients with M3. The initial WBC count and the age at diagnosis in ITD or D835Mt patients were significantly higher than those in patients with wild type (WT) of FLT3 (p< 0.05). Except for M3, 12 of 17 (70.6%) patients with ITD received SCT (3 patients in 1st CR, 1 patient in 2nd CR, 3 patients in 1st relapse, although 4 patients in non-CR). Eight patients showed early relapse after SCT and died. No patients with D835Mt showed the induction failure or relapse. In M3, only one patient with FLT3-WT died in early induction phase. The overall survivals (OS) for patients with ITD, D835Mt and WT by Kaplan-Meier method were 33%, 100% and 80%, respectively (p

過去11年間に, 外子宮口より膣内へ脱出した懸垂型子宮筋腫8例と重症トリコモナス腔炎2例の細胞診に, 悪性細胞と誤認した剥離細胞を経験した. そこで, それらの症例の組織標本をもとに, 剥離細胞の母地組織を精査観察し, いわゆる変性した再生上皮に起因することを知った. よってかかる疾患の細胞診判定に際しては, 十分注意するとともに悪性の判定基準と比較検討する意味においても, 重要な参考所見として報告する.特に, 細胞診において, 再生上皮の変性に関する論文はほとんどみあたらず, その所見をまとめてみると, 次のごとくである.核所見では, 核腫大, 核の大小著明, ときに巨大裸核, 核小体著明, 細網または細穎粒状クロマチン, 不規則な類円形などが挙げられる. 細胞質所見では, 紡錘形, 辺縁融解と境界不鮮明, 異染性などがあり, 全体として化生細胞様, あるいは再生上皮様であり, なかには剥離細胞が腺腔様配列を呈することもある. そしてその成因は, おそらく腔内へ脱出した筋腫表面の剥離再生, 変性の繰り返しにより上皮の損傷, 修複が不完全に継続する結果, いわゆる悪性細胞に酷似した変性型再生上皮となり, その剥離細胞を検鏡する結果となる.

われわれは, 淋疾とトリコモナス膣炎との合併の1症例を経験した.患者は, 25歳女性, 初診時, 発熱, 下腹部痛, 帯下増加の主訴にて受診.骨盤腹膜炎症状を呈し膣分泌中にトリコモナス原虫, かつ頸管粘液中に双球菌を認めた.そこでグラム染色を行ったところ白血球に取り込まれたグラム陰性球菌を認め, さらにMicrocult GC培養にて陽性であることより, 淋疾トリコモナス合併例と診断した.膣スメァ (Pap.染色) では, 細胞質は, 細胞質空胞, 核周囲空胞, 細胞質好酸性化の変化を認め, 同時に核に軽度の変化がみられた.すなわち, 2核, 核腫大, 核小体増加, 核縁不整, 軽度クロマチンの増加が出現した.背景は炎症性細胞特に双球菌を食食した白血球ならびにトリコモナス原虫の出現をみた.膣スメァ (Pap.染色) では, 細胞質は, 細胞質空胞, 核周囲空胞, 細胞質好酸性化の変化を認め, 同時に核に軽度の変化がみられた.すなわち, 2核, 核腫大, 核小体増加, 核縁不整, 軽度クロマチンの増加が出現した.背景は炎症性細胞特に双球菌を食食した白血球ならびにトリコモナス原虫の出現をみた.

子宮頸癌の放射線感受性の有無に対する解明の足掛りとして, 照射早期における細胞診にて悪性細胞の分化度, 小型癌細胞, 細胞質内空胞, 封入細胞について検討を行った.研究対象は当院において放射線治療のみを行った子宮頸部扁平上皮癌患者で, 5年成績の明らかな生存11例, 局所再発3例の計14例であり, 次の結果を得た.1) 1, 500rad照射時の悪性細胞の分化度指数において, 照射前と比し右方移動を示した症例は14例中6例であったが, 予後および組織型における特徴は認められなかった.2) 500rad照射時における小型癌細胞の反応においても, 予後および組織型における統一的傾向は認められなかった.3) 細胞質内空胞のうち, 特にmultivacuolatetypeに注目したところ, 生存群に出現数が多く, 照射初期より出現するものは予後のよい傾向があった.4) 封入細胞は生存群に出現が多く, 予後判定の一助になることがわかった.

Primary carcinoma of the pancreas is mostly observed in individuals over 40 years of age, and has rarely been seen in children. A review of the world literature reveals about 60 cases of primary carcinoma of the pancreas in children 15 years of age and under [1–5].