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BACKGROUND Clonal evolution of malignancy is a complex process related to intratumoral heterogeneity, as recent studies have also demonstrated in rhabdomyosarcoma. The purpose of this study is to present a distinct clonal feature of a case with anaplastic embryonal type rhabdomyosarcoma (ERMS) using molecular analysis. METHODS A five-year-old girl developed a metastatic pelvic tumor. We cultured neoplastic cells isolated from the biopsy sample. Next, to characterize the current case, we analyzed the biopsy sample, autopsy sample, and established cell line using combined modalities, including histopathological, cytogenetic, and molecular assay. We also undertook the backtrack mutation-specific polymerase chain reaction to reveal clonal composition. RESULTS The histology of the biopsy sample was consistent with ERMS with focal anaplasia. We established a permanently growing cell line, ICH-ERMS-1, from the biopsy sample. On molecular analysis, the biopsied tissue revealed a missense mutation at codon 245 of TP53. In contrast, the autopsy tumor tissue and the cell line established from the biopsied tissue showed a missense mutation at codon 248. A backtrack study using mutation-specific polymerase chain reaction detected a TP53 codon 248 mutation in the original biopsy sample. All the specimens examined had a missense mutation at PTPN11 codon 69. CONCLUSIONS This study highlights intratumoral heterogeneity and distinct clonal change related to the functional context in our anaplastic ERMS case, supporting the concept of intratumoral heterogeneity and clonal evolution. It requires further case collection to reveal whether p14ARF-p53-MDM2 tumor suppressor pathway alteration, considered a late event in ERMS tumorigenesis, is responsible for anaplasia in ERMS.

We performed cytogenetic and molecular cytogenetic analyses of a primary cutaneous CD8-positive aggressive epidermotropic cytotoxic T-cell lymphoma, a rare type of primary cutaneous T-cell lymphoma. G-banded analysis at initial diagnosis and recurrence revealed complex karyotype and clonal evolution reflecting genomic instability that parallels the aggressive clinical course observed. Spectral karyotyping revealed numerous structural abnormalities. SNP array-based analysis of an initial diagnostic sample revealed numerous gains and losses of chromosomal material, including loss of short arm of the chromosome 17, to which TP53 is mapped. The molecular cytogenetics and array data of this case suggest genomic instability, particularly chromosomal instability and haploinsufficiency for TP53, the latter possibly giving rise to alteration of p14ARF-Mdm2-p53 tumor suppressor protein pathway, likely to be associated with unfavorable clinical course.

The 2008 World Health Organization classification proposed a new entity in childhood myelodysplastic syndrome, refractory cytopenia of childhood. However, it is unclear whether this morphological classification reflects clinical outcomes. We retrospectively reviewed bone marrow morphology in 186 children (median age 8 years; range 1–16 years) who were enrolled in the prospective study and received horse antithymocyte globulin and cyclosporine between July 1999 and November 2008. The median follow-up period was 87 months (range 1–146 months). Out of 186 patients, 62 (33%) were classified with aplastic anemia, 94 (49%) with refractory cytopenia of childhood, and 34 (18%) with refractory cytopenia with multilineage dysplasia. Aplastic anemia patients received granulocyte colony-stimulating factor more frequently and for longer durations than other patients (P

Background Management of the adverse effects of chemotherapy is essential to improve outcome of children with leukemia. Some genetic polymorphisms can predict treatment-related toxicity, and be used individually in dose modification of 6-mercaptopurine (6-MP) and methotrexate (MTX) in maintenance therapy for childhood acute lymphoblastic leukemia (ALL). We investigated associations between clinical course and candidate gene polymorphisms less evaluated in Japanese patients. Methods Fifty-three children who received maintenance chemotherapy were enrolled in this study. The scheduled dose of oral 6-MP was 40 mg/m2 daily and that of oral MTX was 25 mg/m2 weekly. The doses were adjusted according to white blood cell count (target range, 2.5–3.5 × 109/L) and aspartate aminotransferase and alanine aminotransferase level ( C than in the patients with wild homozygous genotype. The other analyzed polymorphisms were not associated with toxicity, 6-MP, or MTX dose. Conclusions Polymorphism of SLCO1B1 c.521 T > C could be a strong predictor of 6-MP dose reduction in maintenance chemotherapy in childhood ALL.

Bullous pemphigoid (BP) is an autoimmune skin disorder characterized by subepidermal blisters due to deposit of autoantibody against dermal basement membrane protein. It has been reported that BP can occur after allogeneic hematopoietic stem cell transplantation (HSCT). We describe a patient with BP having autoantibody against BP180 after unrelated-donor HSCT against T lymphoblastic leukemia. The patient was treated with steroid leading to complete resolution of BP, but T lymphoblastic leukemia progressed rapidly after steroid hormone treatment. Given that immunosuppressant may reduce graft-versus-tumor effect, immunomodulatory agents such as nicotinamide and tetracycline, erythromycin, and immunoglobulin may be appropriate as soon as typical blister lesions are seen after HSCT.

Relapse period is strongly associated with second relapse risk in relapsed acute lymphoblastic leukemia (ALL) in children. In this context, the treatment outcome of very late relapse should be better; however, data regarding very late relapse is limited. We retrospectively analyzed the outcomes of two consecutive Tokyo Children's Cancer Study Group (TCCSG) ALL trials (1995-2004) with a focus on late relapse, which was divided into two categories: late relapse (6-24 months from the end of therapy, n = 48) and very late relapse (>24 months from the end of therapy, n = 57). Forty-three patients (29 late relapse and 14 very late relapse) received allogeneic hematopoietic stem cell transplantation (HSCT) at second remission. The event-free survival (EFS) probabilities of late relapse and very late relapse were 54.5 ± 7.3 and 64.8 ± 6.8 % at 7 years, respectively (P = 0.36), and were not significantly different. However, the second relapse incidence of late relapse (34.7 ± 7.1 %) was higher than that of very late relapse (15.5 ± 5.1 %, P = 0.03). The second relapse risk was low for very late relapse ALL, which suggests that these patients should be treated without allogeneic HSCT.

The outcomes of children with relapsed acute myeloid leukemia (AML) are known to be poor, but remain obscure. We retrospectively analyzed 71 patients who had relapsed following first-line treatment under the AML99 protocol. We investigated the time and site of recurrence, response to re-induction therapy, and performance of hematopoietic stem cell transplantation (HSCT) in relapsed cases, and performed a multivariate analysis to identify prognostic factors. The 5-year overall-survival (OS) rate after relapse was 37 %. Of 71 patients, three died without any anti-leukemic therapy and two underwent allogeneic HSCT. The remaining 66 patients received re-induction chemotherapy, and 33 (50 %) achieved second CR (CR2). Twenty-two of 25 (88 %) late relapse patients and 11 of 41 (27 %) early relapse patients achieved CR2 (P < 0.001). Twenty-nine CR2 cases and 35 non-CR2 cases underwent allogeneic HSCT. The 5-year OS rate was significantly higher in patients who underwent HSCT in CR2 than those in non-CR2 (66 vs. 17 %, P < 0.000001). Multivariate analysis indicated that early relapse (P < 0.05) and the positivity of the FMS-like tyrosine kinase 3—internal tandem duplication (P < 0.05) were adverse prognostic factors for survival. In conclusion, the etiology of relapsed pediatric AML needs to be elucidated and effective chemotherapy should be administered to obtain CR2.

There is no standard treatment for adolescents aged 15 years or older with acute lymphoblastic leukemia (ALL), although this age group has been reported as having a poorer prognosis compared to younger patients. We retrospectively analyzed the outcomes of three consecutive Tokyo Children's Cancer Study Group ALL trials (1995-2006) of 373 patients aged 10 years or older, with particular focus on adolescents aged 15-18 years (older-adolescents n = 41), compared to those aged 10-14 years (younger-adolescents n = 332). The probability of event-free survival at 8 years was 67.5 ± 7.4 % for the older-adolescents and 66.5 ± 2.6 % for the younger-adolescents (p = 0.95). Overall survival was 70.7 ± 7.1 % for the older-adolescents and 74.3 ± 2.4 % for the younger-adolescents (p = 0.48). The differences between groups in relapse incidence, non-relapse mortality, and death rate during induction were not statistically significant, although the older-adolescents trended towards a higher frequency of having stem-cell transplantation during the first remission. In conclusion, our treatment strategy, which consists of intensive induction and block-type consolidation, provided improved outcomes for patients aged 15-18 years, comparable to those for patients aged 10-14 years.

This study analysed 65 children who were prospectively registered between 1999 and 2008 and fulfilled the World Health Organization 2008 criteria of refractory cytopenia of childhood (RCC). First-line therapy was determined by the treating physicians: 25 patients received immunosuppressive therapy (IST), 12 patients received haematopoietic stem cell transplantation (HSCT) and one patient received intensive chemotherapy. The remaining 27 patients were followed without treatment for more than 2 years (watch and wait; WW). In the WW group, 18 patients had stable disease without further intervention. Thirteen of 29 patients (45%) who ended up receiving IST showed response. The combination of ciclosporin and antithymocyte globulin was not shown to be superior to ciclosporin alone with regard to response rate or survival. Of 28 patients who ended up undergoing HSCT, 17 patients are alive in complete remission, whereas nine patients died mostly due to transplantation-related mortality. The 5-year overall survival for all patients was 82 ± 5%. Eight patients suffered from disease progression. Patients with monosomy 7 or multilineage-dysplasia had a significantly higher incidence of disease progression. This analysis revealed heterogeneity in the clinical course of RCC, varying from those who remained stable for long periods to those who progressed to advanced disease.

Background The pain associated with bone marrow aspiration and biopsy (BMAB) has an enormous impact on pediatric cancer patients and their families, but no specific reference standards for sedation and analgesia have been developed in Japan. To determine the problems associated with pain management during BMAB, a cross-sectional investigation was conducted. Methods A survey was sent in October 2011 to data managers in institutions belonging to the Tokyo Children's Cancer Study Group, addressing the non-pharmacological and pharmacological pain management for BMAB performed on pediatric cancer inpatients between January 2010 and December 2010. Results The eligible response rate was 41 of 57 institutions (71.9%). Non-pharmacological pain intervention was provided in 68% of surveyed institutions. All institutions provided pharmacological pain management. In most institutions, sedation/analgesia was performed by pediatric oncologists in a treatment room in the ward. Standards for pain management were developed and utilized in only four institutions. Other means of pain management were provided in various settings. Twelve institutions reported insufficient sedation/analgesia. In total, 80% of institutions reported some adverse events. Two serious adverse events were reported in cases of underlying or complicated conditions. No serious long-term consequences were reported. Conclusions Significant issues were identified regarding the efficacy and safety of pain management. Adverse events can occur in any institution. Children with underlying or complicated conditions are at high risk for serious adverse events. Therefore, adequate and systematic assessment, patient monitoring, preparation and treatment for adverse events, and cooperation with skilled specialists of pediatric oncology, anesthesiology, and intensive care are essential.

The aim of this study was to prospectively evaluate the PK and safety of ivBU in 25 Japanese children (median age six yr; range, five months-17 yr) as one of a combination of drugs in a pretransplant regimen. The patients had acute leukemia (n = 14), CML (2), JMML (5), solid tumors (2), chronic granulomatous disease (1), or metachromatic leukodystrophy (1). Five different dose schedules were used according to the patient's ABW:9 kg (1.0 mg/kg), 9 to16 (1.2 mg/kg), 16-23 (1.1 mg/kg),23-34 (0.95 mg/kg), and34 kg of BW (0.8 mg/kg). Each dose was given over two h, and sample blood was drawn at nine or 11 separate points for analysis by gas chromatography-mass spectrometry. The AUC varied from 796 to 1905 μmol min/L, and 19 of the 25 patients (76%) remained within the target range without dose adjustment. Two were diagnosed with engraftment failure. Hepatic VOD developed in four, and only one of these showed high AUC (1500 μmol min/L). Toxicities did not correlate with the BU level. Our data showed very similar PK to those in previous studies, and these dose schedules are applicable to Japanese children.

Backgrounds. Outcome of childhood malignancy has been improved mostly due to the advances in diagnostic techniques and treatment strategies. While methotrexate (MTX) related polymorphisms have been under investigation in childhood malignancies, many controversial results have been offered. Objectives. To evaluate associations of polymorphisms related MTX metabolisms and clinical course in childhood lymphoid malignancies. Method. Eighty-two acute lymphoblastic leukemia and 21 non-Hodgkin’s lymphoma children were enrolled in this study. Four single nucleotide polymorphisms in 2 genes (MTHFR (rs1801133/c.677C>T/p.Ala222Val and rs1801131/c.1298A>C/p.Glu429Ala) and SLCO1B1 (rs4149056/c.521T>C/p.V174A and rs11045879/c.1865+4846T>C)) were genotyped by Taqman PCR method or direct sequencing. Clinical courses were reviewed retrospectively. Results. No patient who had the AC/CC genotype of rs1801131 (MTHFR) had relapsed or died, in which distribution was statistically different among the AA genotype of rs1801131 (P=0.004). Polymorphisms of SLCO1B1 (rs11045879 and rs4149056) were not correlated with MTX concentrations, adverse events, or disease outcome. Conclusions. Polymorphisms of MTHFR (rs1801131) could be the plausive candidate for prognostic predictor in childhood lymphoid malignancies.

Summary The Tokyo Children's Cancer Study Group conducted a randomized controlled study to evaluate the effect of experimental early intensification using high-dose cytarabine and L-asparaginase in paediatric intermediate-risk (IR) acute lymphoblastic leukaemia (ALL). A total of 310 IR ALL patients were randomized to receive either experimental early intensification (n = 156) or standard early intensification including standard-dose cytarabine arm (n = 154) after induction therapy. The experimental arm consisted of high-dose cytarabine and L-asparaginase, while the standard arm consisted of standard-dose cytarabine, oral 6-mercaptopurine and cyclophosphamide. The probabilities of event-free survival at 8 years in the experimental and standard arms were 72·3 ± 3·7% and 77·5 ± 3·5%, respectively (P = 0·32). The 8-year overall survival rates for these two arms were 85·0 ± 3·0% and 86·9 ± 2·8%, respectively (P = 0·72). The frequency of infectious events was significantly higher in the experimental arm (66·4%) than in the standard arm (24·6%) (P

Summary With improvement in survival, it is important to evaluate the impact of treatment on secondary cancers in acute lymphoblastic leukaemia (ALL) survivors. A retrospective cohort study comprising 2918 children diagnosed with ALL and enrolled on Tokyo Children's Cancer Study Group (TCCSG) protocols between 1984 and 2005 was conducted to evaluate the incidence of secondary cancers and associated factors including treatment protocol, cranial irradiation and other characteristics of the primary ALL. Thirty-seven patients developed secondary cancers, including acute myeloid leukaemia (n = 11), myelodysplastic syndrome (n = 5), non-Hodgkin lymphoma (n = 2), brain tumours (n = 13) and other solid carcinomas (n = 6) within a median follow-up duration of 9·5 years. The cumulative incidence of any secondary cancers was 1·0% (95% confidence interval (CI), 0·7–1·4%) at 10 years and 2·4% (95% CI, 1·5–3·7%) at 20 years, respectively. Standardized incidence rate ratio of secondary cancers was 9·3 (95% CI, 6·5–12·8). Multivariate analyses showed an increased risk of secondary cancers associated with the recent treatment protocol and cranial irradiation. There was no evidence of a reduction in secondary cancer incidence despite marked decreases in cranial irradiation use in the recent protocols.

The prognostic value of WT1 mRNA expression in pediatric acute myeloid leukemia (AML) remains controversial. A sample of newly diagnosed (n = 158) AML patients from the Japanese Childhood AML Cooperative Treatment Protocol, AML 99, were simultaneously analyzed for WT1 expression, cytogenetic abnormalities and gene alterations (FLT3, KIT, MLL, and RAS). WT1 expression (including more than 2,500 copies/μgRNA) was detected in 122 of the 158 (77.8 %) initial diagnostic AML bone marrow samples (median 45,500 copies/μgRNA). Higher WT1 expression was detected in French American British (FAB)-M0, M3, M7 and lower expression in M4 and M5. Higher WT1 expression was detected in AML with inv(16), t(15;17) and Down syndrome and lower in AML with 11q23 abnormalities. Multivariate analyses demonstrated that FLT3-internal tandem duplication (ITD), KIT mutation, MLL-partial tandem duplication were correlated with poor prognosis; however, higher WT1 expression was not. FLT3-ITD was correlated with WT1 expression and prognosis. Furthermore, 74 WT1 expression after induction chemotherapy was analyzed. Higher WT1 expression after induction chemotherapy was significantly correlated with M1 or M2/M3 marrow, FLT3-ITD and poor prognosis. Multivariate analyses in 74 AML patients revealed that FLT3-ITD, MLL-PTD, and KIT mutations were associated with poor prognosis; however, NRAS Mutation, KRAS mutation and high WT1 expression (>10,000 copies/μgRNA) did not show poor prognosis. Our findings suggest that higher WT1 expression at diagnosis does not correlate with poor prognosis, but that WT1 expression after induction chemotherapy is considered to be a useful predictor of clinical outcome in pediatric AML.

Ischemic reperfusion (I/R) injury of the kidney is closely associated with delayed graft function, increased acute rejection, and late allograft dysfunction. Splenectomy reduced hepatic I/R injury by inhibiting leukocyte infiltration in the liver, release of TNF-α, cell apoptosis, and expression of caspase-3. Thus, we investigated the effects of splenectomy on renal I/R injury in the rat.Male Wistar rats were assigned to four groups: sham operation (sham group), sham operation+splenectomy (sham+SPLN group), right nephrectomy followed by clamping the left renal pedicle for 30min (I/R 30 group), and I/R 30+splenectomy (I/R 30+SPLN group). Renal function was determined by measuring the concentration of blood urea nitrogen (BUN) and serum creatinine (S-Cr). The serum level of tumor necrosis factor-α (TNF-α) was measured as the marker for inflammation. Left kidneys were obtained 24h after reperfusion. TUNEL assay was assessed for cell apoptosis. Spleens were obtained immediately (0-h group) and 3h after reperfusion (3-h group). The removed spleens were histologically evaluated.The BUN and S-Cr levels were significantly lower in the I/R 30+SPLN group than in the I/R 30 group (p0.05 for both). Apoptotic cells were significantly lower in the I/R 30+SPLN group than in the I/R 30 group. The serum level of TNF-α, which was increased after I/R, was significantly lower in the I/R 30+SPLN group than in the I/R 30 group (p0.05). Spleen weights were significantly lower in the 3-h group than in the 0-h group (p0.05).These results suggest that splenectomy reduces renal I/R injury, and this effect may occur by an anti-inflammatory pathway and inhibition of cell apoptosis.

Recent advances in unrelated cord blood transplantation (UCBT) and high-resolution typing of human leukocyte antigen (HLA) from an unrelated donor have increased choices in alternative donor/stem cell source selection. We assessed HLA-mismatched locus-specific comparison of the outcomes of 351 single-unit UCB and 1,028 unrelated bone marrow (UBM) adult recipients 16 years old or older at the time of transplantation who received first stem cell transplantation with myeloablative conditioning for acute leukemia or myelodysplastic syndromes. With adjusted analyses, HLA 0 to 2 mismatched UCBT showed similar overall mortality (relative risk [RR] = 0.85, 95% confidence interval [CI], 0.68-1.06; P = .149) compared with that of single-HLA-DRB1-mismatched UBMT. UCBT showed inferior neutrophil recovery (RR = 0.50, 95% CI, 0.42-0.60; P < .001), lower risk of acute graft-versus-host disease (RR = 0.55, 95% CI, 0.42-0.72; P < .001), and lower risk of transplantation-related mortality (RR = 0.68, 95% CI, 0.50-0.92; P = .011) compared with single-HLA-DRB1-mismatched UBMT. No significant difference was observed for risk of relapse (RR = 1.28, 95% CI, 0.93-1.76; P = .125). HLA 0 to 2 antigen-mismatched UCBT is a reasonable second alternative donor/stem cell source with a survival outcome similar to that of single-HLA-DRB1-mismatched or other 7 of 8 UBMT.

Summary Early T-cell precursor acute lymphoblastic leukaemia (ETP-ALL) is a recently identified subtype of T-ALL with distinctive gene expression and cell marker profiles, poor response to chemotherapy and a very high risk of relapse. We determined the reliability of restricted panel of cell markers to identify EPT-ALL using a previously classified cohort. Then, we applied the cell marker profile that best discriminated ETP-ALL to a cohort of 91 patients with T-ALL enrolled in the Tokyo Children’s Cancer Study Group L99-15 study, which included allogeneic stem cell transplantation (allo-SCT) for patients with poor prednisone response. Five of the 91 patients (5·5%) met the ETP-ALL criteria. There were no significant differences in presenting clinical features between these and the remaining 86 patients. Response to early remission induction therapy was inferior in ETP-ALL as compared with T-ALL. The ETP-ALL subgroup showed a significantly poorer event-free survival (4-year rate; 40%) than the T-ALL subgroup (70%, P = 0·014). Of note, three of four relapsed ETP-ALL patients survived after allo-SCT, indicating that allo-SCT can be effective for this drug-resistant subtype of T-ALL.

We report two patients with chronic granulomatous disease (CGD). The first patient presented with granulomatous colitis and pulmonary aspergillosis, and the second patient with liver abscess and restrictive pulmonary disorder. Both patients underwent allogeneic hematopoietic stem cell transplantation, the first from an HLA-matched sibling donor, and the second from an HLA-matched unrelated donor, after preconditioning with fludarabine, anti-thymocyte globulin, cyclophosphamide, and total-body irradiation of 3 Gy. The engraftment was prompt and the regimen-related toxicity was mild. The patients are able to return to their daily lives with full donor chimerism, although the second patient underwent a living-related-donor orthotopic liver transplantation from his mother for chronic liver graft-versus-host disease. The conditioning regimen we used was feasible and applicable to patients with CGD accompanied by inflammatory disease and severe infection.

Background Although the therapeutic outcome of acquired aplastic anemia has improved markedly with the introduction of immunosuppressive therapy using antithymocyte globulin and cyclosporine, a significant proportion of patients subsequently relapse and require second-line therapy. However, detailed analyses of relapses in aplastic anemia children are limited. Design and Methods We previously conducted two prospective multicenter trials of immunosuppressive therapy for children with aplastic anemia: AA-92 and AA-97, which began in 1992 and 1997, respectively. In this study, we assessed the relapse rate, risk factors for relapse, and the response to second-line treatment in children with aplastic anemia treated with antithymocyte globulin and cyclosporine. Results From 1992 to 2007, we treated 441 children with aplastic anemia with standard immunosuppressive therapy. Among the 264 patients who responded to immunosuppressive therapy, 42 (15.9%) relapsed. The cumulative incidence of relapse was 11.9% at 10 years. Multivariate analysis revealed that relapse risk was significantly associated with an immunosuppressive therapy regimen using danazol (relative risk, 3.15; P =0.001) and non-severe aplastic anemia (relative risk, 2.51; P =0.02). Seventeen relapsed patients received additional immunosuppressive therapy with antithymocyte globulin and cyclosporine. Eight patients responded within 6 months. Seven of nine non-responders to second immunosuppressive therapy received hematopoietic stem cell transplantation and five are alive. Eleven patients underwent hematopoietic stem cell transplantation directly and seven are alive. Conclusions In the present study, the cumulative incidence of relapse at 10 years was relatively low compared to that in other studies mainly involving adult patients. A multicenter prospective study is warranted to establish optimal therapy for children with aplastic anemia.

In aplastic anemia, predictive markers of response to immunosuppressive therapy have not been well defined. We retrospectively evaluated whether clinical and laboratory findings before treatment could predict response in a pediatric cohort from the multicenter AA-97 study in Japan. Between 1997 and 2006, 312 newly diagnosed children were enrolled and treated with a combination of antithymocyte globulin and cyclosporine. In multivariate analyses, lower white blood cell count was the most significant predictive marker of better response; patients with white blood cell count less than 2.0×10(9)/L showed a higher response rate than those with white blood cell count of 2.0×10(9)/L or more (P=0.0003), followed by shorter interval between diagnosis and therapy (P=0.01), and male sex (P=0.03). In conclusion, pre-treatment clinical and laboratory findings influence response to therapy. The finding that response rate worsens with increasing interval between diagnosis and treatment highlights the importance of prompt immunosuppressive therapy for patients with aplastic anemia.

We present a cytogenetically normal neonate who developed transient abnormal myelopoiesis. The blasts showed trisomy 21. In contrast, fibroblasts, and PHA-stimulated peripheral blood demonstrated normal diploid line on extensive karyotyping. Direct sequencing of the DNA derived from the peripheral blood at overt disease revealed splice site mutation in the boundary of GATA1 exon 2. The patient received three courses of chemotherapy leading to complete remission. During the complete remission, there was neither mutation of GATA1 exon 2 nor trisomy 21, confirming somatic nature of both abnormalities. The patient is now free from the disease 12 months after remission. This case emphasizes the significance of trisomy 21 as the cause of transient abnormal myelopoiesis in Down syndrome.

Tacrolimus (FK) and cyclosporine (CsA) have been shown to be effective in the prophylaxis of graft-versus-host disease (GVHD). However, no comparative studies have yet been conducted to examine the efficacy of FK/methotrexate (MTX) and CsA/MTX in patients with severe aplastic anemia (SAA) given unrelated donor bone marrow transplantation (U-BMT). We used matched-pair analysis to compare FK/MTX with CsA/MTX in patients with SAA who received U-BMT through the Japan Marrow Donor Program. Forty-seven pairs could be matched exactly for recipient age and conditioning regimens. Forty-five patients achieved engraftment in the FK group and 42 patients in the CsA group. The probability of grade II-IV acute GVHD (aGVHD) was 28.9% in the FK group and 32.6% in the CsA group (P=.558). The probability of chronic GVHD (cGVHD) was 13.3% in the FK group and 36.0% in the CsA group (P=.104). The 5-year survival rate was 82.8% in the FK group and 49.5% in the CsA group (P=.012). The study shows the superiority of FK/MTX over CsA/MTX in overall survival because of the lower incidence of transplantation-related deaths. A prospective randomized study comparing FK/MTX and CsA/MTX is warranted.

Background Although hematopoietic stem cell transplantation (HSCT) is offered as a curative therapy for pediatric myelodysplastic syndrome (MDS), it may cause severe complications and mortality. Several reports have shown the efficacy of immunosuppressive therapy (IST) in adult patients with refractory anemia (RA), but its safety and efficacy remains to be fully elucidated in childhood RA. Procedure Eleven children diagnosed with RA and enrolled on a prospective multicenter trial conducted by the Japanese Childhood MDS Study Group were eligible for analysis. If patients showed transfusion dependent or suffered from infection due to neutropenia, they received IST consisting of antithymocyte globulin (ATG), cyclosporine (CyA), and methylprednisolone (mPSL). Results Eight children received IST, 2 received only supportive therapy, and one underwent HSCT without IST. Five (63%) of eight children who received IST showed hematological response. Of note, one patient showed the disappearance of monosomy 7 after IST. Responders were significantly younger than non-responders (29 months vs. 140 months; P = 0.03). No severe adverse events related to IST were reported in this study. Of 6 children with chromosomal abnormalities who received IST, four showed hematological response. The probability of failure-free and overall survival at 5 years was 63 ± 17% and 90 ± 9% respectively. Conclusion IST is likely to be a safe and effective modality for childhood RA. Pediatr Blood Cancer 2009;53:1011–1015. © 2009 Wiley-Liss, Inc.

Purpose To improve the prognosis in children with newly diagnosed acute myeloid leukemia (AML) by introducing a dose-dense intensive chemotherapy regimen and an appropriate risk stratification system. Patients and Methods Two hundred forty children with de novo AML were treated with continuous cytarabine-based induction therapy and stratified to three risk groups based on the initial treatment response, age, and WBC at diagnosis and cytogenetics. All of the patients were treated with intensive consolidation chemotherapy including three or four courses of high-dose cytarabine. Allogeneic hematopoietic stem-cell transplantation (HSCT) was indicated for only the intermediate-risk patients with matched related donors and for all the high-risk subsets. Results Two hundred twenty-seven children (94.6%) achieved a complete remission (CR). Four children demonstrated induction death. The median follow-up of the live patients was 55 months (range, 37 to 73 months). The 5-year overall survival of all 240 children was 75.6% (95% CI, 70.3% to 81.4%) and event-free survival was 61.6% (95% CI, 55.8% to 68.1%). The 5-year disease-free survival in each risk group were 71.3% (95% CI, 63.4% to 80.2%) in the low-risk group (n = 112), 59.8% (95% CI, 50.6% to 70.7%) in the intermediate-risk group (n = 92), and 56.5% (95% CI, 39.5% to 80.9%) in the high-risk group (n = 23). Eight children died during the first CR, including four after HSCT. Conclusion A high survival rate, 75.6% at 5 years, was achieved for childhood with de novo AML in the AML99 trial. The treatment strategy was well tolerated with only 1.7% induction death rate and 3.5% remission death rate. Low-risk children were successfully treated with chemotherapy alone.

Treatment response has become one of the most important prognostic factors in childhood acute lymphoblastic leukemia. We evaluated the significance of the complete clearance of peripheral leukemic blasts on survival in children with acute lymphoblastic leukemia.Seven hundred and fifty-four children diagnosed with acute lymphoblastic leukemia, consecutively enrolled from 1999 to 2003 in the TCCSG L99-15 study, were eligible for analysis. Patients were stratified into three risk groups based on presenting features, such as age and the leukocyte count before starting the treatment, followed by reclassification into three categories 7 days after prednisolone monotherapy based on the peripheral blast count; 0/microL (Day8NoBlasts), 1-999/microL andor= 1,000/microL.After 7 days of prednisolone monotherapy, 249 patients (33%) were classified as Day8NoBlasts, 392 patients (52%) had blast counts of 1-999/microL, and 113 patients (15%) had blast countsor= 1,000/microL. The event-free survival for all patients was 79.6+/-1.6 (SE)% at 4 years, whereas that for patients with Day8NoBlasts was 90.4+/-2.0% (n=249) and the event-free survival for the other patients was 74.2+/-2.2% (n=504) (log rank p0.001). The event-free survival for Day8NoBlasts patients with B-lineage acute lymphoblastic leukemia and T-cell acute lymphoblastic leukemia was 89.8+/-2.1% (n=226) and 95.7+/-4.3% (n=23), respectively. In a multivariate analysis, age at diagnosis, the initial white blood cell count, immunophenotype, and gender did not remain as independent risk factors for treatment failure, whereas Day8NoBlasts and marked hyperdiploidy (more than 50 chromosomes) became statistically significant.Children with Day8NoBlasts constituted one third of all the cases with childhood acute lymphoblastic leukemia with an excellent outcome, and should be candidates for curative management with less intensive treatment.

Background. Mixed-lineage leukemia (MLL)-partial tandem duplication (PTD) is associated with poor prognosis in adult acute myeloid leukemia (AML), but its relationship to pediatric AML is unknown. Procedure. One hundred fifty-eight newly diagnosed AML patients, including 13 FAB-M3 and 10 Down syndrome (DS) patients, who were treated on the Japanese Childhood AML Cooperative Treatment Protocol AML 99 were analyzed for MLLPTD, as well as internal tandem duplication (ITD) and the kinase domain mutation (D835Mt) in the FLT3 gene. Results. We found MLL-PTD in 21 (13.3%) of 158 AML patients, but not in FAB-M3 or DS patients. The differences between patients with and without MLLPTD were significant for 3-year overall survival (OS) (56.3% vs. 83.2%, P ¼0.018), disease-free survival (DFS) (41.7% vs. 69.6%, P ¼0.010), and relapse rate (RR) (54.3% vs. 27.6%, P ¼0.0085) of 135 AML patients excluding the FAB-M3 and DS patients. Furthermore, ITD and D835Mt in the FLT3 gene were found in 17 (12.6%) and 8 (5.9%) of these 135 patients, respectively. The differences between patients with FLT3-ITD and the wild-type allele were significant for 3-year OS (35.3% and 84.3%, P

We retrospectively surveyed pediatric acute myeloid leukemia (AML) patients with multilineage dysplasia treated with the AML 99 and the Children's Cancer and Leukemia Study Group (CCLSG) AML 9805 protocols. We found only 9 AML patients (2.6%) with multilineage dysplasia among the 341 patients with newly diagnosed de novo AML. Eight of the 9 patients obtained complete remission (CR) following the intensive AML-oriented treatments. Three of 7 patients who underwent stem cell transplantation were alive in CR for more than 4 years, and the 2 patients treated only with chemotherapy were alive in CR for more than 30 months. We did not identify any particular chromosomal abnormalities or differentiation according to the French-American-British classification in these 9 patients. No reports have described AML with multilineage dysplasia in children, and the incidence of the disease is expected to be very low. We plan to conduct a prospective pathologic review to select cases with this disease entity in the next Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 protocol.

The AML1 gene is frequently rearranged by chromosomal translocations in acute leukemia. We identified that the LAF4 gene on 2q11.2-12 was fused to the AML1 gene on 21q22 in a pediatric patient having T-cell acute lymphoblastic leukemia (T-ALL) with t(2;21)(q11;q22) using the bubble PCR method for cDNA. The genomic break points were within intron 7 of AML1 and of LAF4, resulting in the in-frame fusion of exon 7 of AML1 and exon 8 of LAF4. The LAF4 gene is a member of the AF4/FMR2 family and was previously identified as a fusion partner of MLL in B-precursor ALL with t(2;11)(q11;q23), although AML1-LAF4 was in T-ALL. LAF4 is the first gene fused with both AML1 and MLL in acute leukemia. Almost all AML1 translocations except for TEL-AML1 are associated with myeloid leukemia; however, AML1-LAF4 was associated with T-ALL as well as AML1-FGA7 in t(4;21)(q28;q22). These findings provide new insight into the common mechanism of AML1 and MLL fusion proteins in the pathogenesis of ALL. Furthermore, we successfully applied bubble PCR to clone the novel AML1-LAF4 fusion transcript. Bubble PCR is a powerful tool for detecting unknown fusion transcripts as well as genomic fusion points.

Background Extramedullary infiltration (EMI) is an occasional clinical symptom in childhood acute myelogenous leukemia (AML), but there is considerable controversy regarding the prognostic significance of EMI in AML. Procedure We evaluated the frequency and prognostic significance of EMI at diagnosis of AML in children. Results Of 240 cases of de novo AML excluding children with Down syndrome and acute promyelocytic leukemia, 56 (23.3%) showed EMI at diagnosis. Patients with EMI had a higher initial WBC count and a higher proportion of M4/M5 morphological variants. The complete remission rate following induction chemotherapy was lower in patients with EMI. However, the overall survival and event-free survival did not differ between patients with and without EMI. A detailed analysis showed that patients with EMI with a WBC count at diagnosis of over 100 × 109/L or infiltration into the central nervous system are likely to have a poor prognosis. Conclusions CNS leukemia and EMI together with a WBC count of >100 × 109/L at diagnosis of AML are high risk factors for relapse, and alternative treatment approaches for patients with these characteristics should be explored. Pediatr Blood Cancer © 2006 Wiley-Liss, Inc.

In a retrospective review of the survival outcome of children with isolated central nervous system (CNS) relapse of acute lymphoblastic leukemia (ALL), we identified 79 patients with CNS relapse among the eligible patients enrolled in ALL trials of 3 Japanese pediatric oncology study groups (Japanese Children's Cancer and Leukemia Study Group [JCCLSG], Tokyo Children's Cancer Study Group [TCCSG], and Japan Association of Childhood Leukemia Study [JACLS]) between 1989 and 1999. CNS relapses were diagnosed as the first adverse event between 1991 and 1999. The median age at the time of CNS relapse was 5.0 years (range, 0.7-15.1 years). The duration of the first remission ranged from 1.4 to 54 months (median, 12.4 months), and the observation period after CNS relapse ranged from 1 to 131 months (median, 27 months). Overall, 75 of the 79 patients achieved a second complete remission, 44 of whom had second relapses in the following sites: CNS, 18 patients; bone marrow, 15 patients; combined sites, 8 patients; and testis, 2 patients. Rates of overall survival and event-free survival at 4 years were 43.7% +/- 5.8% (mean +/- SE) and 32.9% +/- 5.5%, respectively. The probability of remaining in second remission was significantly correlated with the leukocyte count (P= .005) and age (P= .02) at the initial diagnosis.

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.

Recent reports of myelodysplastic syndrome/acute myeloid leukemia (t-MDS/AML) developing after treatment with immunosuppressants and granulocyte colony-stimulating factor (G-CSF) has raised the question of whether previously unrecognized myelodysplastic features had been present or whether actual transformation had occurred. We undertook a multi-institutional study of 112 children with aplastic anemia diagnosed between 1976 and 1996 and then treated with immunosuppressants with or without G-CSF. In each case, bone marrow specimens were tested at study entry and every 6 months for 3 years to detect t-MDS/AML as defined by morphologic and molecular/cytogenetic criteria. As of December 2001, all eligible patients had been followed for a median of 3 years. Morphologic abnormalities were found in 17 cases. The patients in 4 of these cases had clonal cytogenetic abnormalities and received MDS diagnoses. The morphologic features of the patients with and without clonal cytogenetic abnormalities were indistinguishable. However, the mast cell content was lower in cases with cytogenetic abnormalities than in cases without them. An elucidation of the role of mast cells may provide information about the differences between aplastic anemia and MDS or about the transition of aplastic anemia to MDS.

We retrospectively analyzed results for 154 patients with acquired severe aplastic anemia who received bone marrow transplants between 1993 and 2000 from unrelated donors identified through the Japan Marrow Donor Program. Patients were aged between 1 and 46 years (median, 17 years). Seventy-nine donor-patient pairs matched at HLA-A, -B, and -DRB1 loci, as shown by DNA typing. Among the 75 mismatched pairs, DNA typing of 63 pairs showed that 51 were mismatched at 1 HLA locus (18 HLA-A, 11 HLA-B, 22 HLA-DRB1) and 12 were mismatched at 2 or more loci. Seventeen patients (11%) experienced either early or late graft rejection. The incidence of grade III/IV acute graft versus host disease and chronic graft versus host disease was 20% (range, 7%-33%) and 30% (range, 12%-48%), respectively. Currently, 99 patients are alive, having survived for 3 to 82 months (median, 29 months) after their transplantations. The probability of overall survival at 5 years was 56% (95% confidence interval, 34%-78%). Multivariate analysis revealed the following unfavorable factors: transplantation more than 3 years after diagnosis (relative risk [RR], 1.86; P = .02), patients older than 20 years (RR, 2.27; P = .03), preconditioning regimen without antithymocyte globulin (RR 2.28; P = .04), and HLA-A or -B locus mismatching as determined by DNA typing. Matching of HLA class I alleles and improvement of preparative regimens should result in improved outcomes in patients with severe aplastic anemia who receive transplants from unrelated donors.

BACKGROUND: Several conventional forms of immunosuppression have been shown to antagonize the efficacy of anti-CD154 monoclonal antibody- (mAb) based costimulatory molecule blockade immunotherapy. Our objective was to determine if allograft recipients treated with a conventional immunosuppressive regimen could be sequentially converted to anti-CD154 mAb monotherapy without compromising graft survival. METHODS: Outbred juvenile rhesus monkeys underwent renal allotransplantation from MHC-disparate donors. After a 60-day course of triple therapy immunosuppression with steroids, cyclosporine, and mycophenolate mofetil, monkeys were treated with: (1) cessation of all immunosuppression (control); (2) seven monthly doses of 20 mg/kg hu5C8 (maintenance), or; (3) 20 mg/kg hu5C8 on posttransplant days 60, 61, 64, 71, 79, and 88 followed by five monthly doses (induction+maintenance). Graft rejection was defined by elevation in serum creatinine>1.5 mg/dl combined with histologic evidence of rejection. RESULTS: Graft survival for the three groups were as follows: group 1 (control): 70, 75, >279 days; group 2 (maintenance): 83, 349, >293 days, and; group 3 (induction+maintenance): 355, >377, >314 days. Acute rejection developing in two of four monkeys after treatment with conventional immunosuppression was successfully reversed with intensive hu5C8 monotherapy. CONCLUSIONS: Renal allograft recipients can be successfully converted to CD154 blockade monotherapy after 60 days of conventional immunosuppression. An induction phase of anti-CD154 mAb appears to be necessary for optimal conversion. Therefore, although concurrent administration of conventional immunosuppressive agents including steroids and calcineurin inhibitors has been shown to inhibit the efficacy of CD154 blockade, sequential conversion from these agents to CD154 blockade appears to be effective.

PURPOSE: To determine the effects of eliminating initial lumbar punctures in 418 consecutively treated children with acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Patients were enrolled onto a trial conducted in central Japan between 1989 and 1992. Treatment consisted of standard four-drug induction therapy followed by a risk-based intensification phase, reinduction therapy, late intensification, and remission maintenance therapy (total of 104 weeks). The initial lumbar puncture, with an intrathecal injection of chemotherapy, was performed after 1 week of prednisolone sensitivity testing (day 8). End points included response to prednisolone, CNS status at the time of the day 8 lumbar puncture, subsequent adverse events in CNS and bone marrow, and event-free survival (EFS). RESULTS: The remission induction rate was 93.1% with a 6-year EFS rate (± SE) of 68.7% ± 2.4%, which is similar to historical results for patients who received their diagnostic lumbar puncture and first instillation of intrathecal chemotherapy on day 0. Overall, 84.5% of the patients had good responses to prednisolone, whereas 15.5% had poor responses. Clinical outcome was strikingly better for the good responders (6-year EFS, 74.1% ± 2.5% compared with 40.1% ± 6.4% for patients with poor responses), suggesting that omission of intrathecal chemotherapy did not alter the predictive value of drug sensitivity testing. Eighteen patients experienced CNS relapse as their first adverse event (cumulative risk, 5.1%; 95% confidence interval, 2.7% to 7.4%), coincident with reports from groups using conventional strategies of CNS clinical management. Bleeding into the CSF at the time of the day 8 lumbar puncture was apparent in 29 cases (8.1%), but leukemic blasts were identified in only two. CONCLUSION: Delay of the initial lumbar puncture and intrathecal injection of chemotherapy seems to be feasible in children with ALL. Further controlled evaluations are needed to establish the validity of this conclusion.

Factors influencing the outcome for 39 children with haematological malignancy who were subjected to a cord blood transplantation (CBT) from genotypically HLA-mismatched unrelated donors were analysed. This retrospective study included 21 children with acute lymphoblastic leukaemia, 15 with acute myelogenous leukaemia and one each with chronic myelogenous leukaemia, refractory anaemia with myelodysplastic syndrome (MDS) and juvenile myelomonocytic leukaemia (JMML). Those subjected to CBT during the first or second complete remission (CR) and MDS without blasts were assigned to the standard-risk (SR) group (n = 16). Patients in third or subsequent remission, relapse or partial remission with refractory leukaemia at the time of CBT were considered to be in advanced phase, and placed in the high-risk (HR) group (n = 11). JMML and the second CR after a relapse (n = 8), or bone marrow failure after a rejection (n = 3), following haematopoietic stem cell transplantation (HSCT) in the first CR were included in the high-risk group. Kaplan–Meier estimates for neutrophil and platelet recovery were 83·7 ± 12·2 at d 60 and 55·4 ± 16·6% at d 100 respectively. The incidence of grades II–VI acute graft-versus-host disease was 58·5 ± 16·8%. The Kaplan–Meier estimate for 3-year event-free survival (EFS) was 49·2 ± 16·6. From multivariate analysis, the most important factor influencing EFS was disease status at CBT: SR patients had a 3-year EFS of 75·0 ± 21·6%, compared with 29·6 ± 20·6% for those with HR disease (P = 0·013, RR 4·746, 95% CI 1·382–16·298). These data confirm that HLA-mismatched, unrelated CBT is a feasible procedure to cure a significant proportion of children with leukaemia, especially if conducted in a favourable phase of the disease.

In the era of 22-oxacalcitriol (OCT), newly synthesized 1α,25-dihydroxyvitamin D3 analogue, against secondary hyperparathyroidism, the indications of parathyroidectomy (PTx) has been restricted. Recent investigations on animal models have revealed the inhibitory effects on PTH secretion after OCT treatment, whereas there has been no evidence about human parathyroid glands. A 38-year-old man with a 19-year history of hemodialysis was performed PTx after the failure of OCT treatment. Expressions of proliferative nuclear cell antigen (PCNA), calcium-sensing receptor (CaSR), vitamin D receptor (VDR), p53 and p21WAF1/Cip1 were analyzed by Western blotting and immunohistochemistry on resected parathroid glands. We confirmed up-regulations of CaSR and VDR, which contribute the reduction of serum PTH, by OCT treatment. Concomitant up-regulation of p21WAF1/Cip1 but not p53, especially in nodular hyperplasia, can be considered to induce cell cycle arrest of the parathyroid cells, but not cytocidal effect of OCT.

Background. In our previously described primate renal allograft model, T cell ablation leads to long-term graft survival.The role of endothelial cell alteration in chronic rejection was examined in our model. Methods. Renal transplants were performed in rhesus monkeys using a T cell- depleting immunotoxin, FN18-CRM9. Sections from 10 rejected kidneys (5 acute and 7 chronic rejection) were examined after immunohistochemical staining for expression of endothelium-related proteins [von Willebrand factor (vWF), CD62P, and CD31], fibrinogen, and a macrophage marker (CD68). Glomerular staining for each antigen was graded on a semiquantitative scale. Results. Intense staining for vWF was consistently observed in glomerular endothelium, subendothelium, and mesangium in all kidneys removed due to chronic rejection. vWF staining was weak in kidneys showing acute rejection. The difference in glomerular staining was statistically significant. Staining for vWF in extraglomerular vessels was nearly identical in kidneys showing acute and chronic rejection. Expression of CD62P was increased in extraglomerular vessels in allografts with chronic rejection, but the glomeruli showed little or no staining. There was no significant difference in the glomerular staining for CD62P or CD31 in organs showing acute and chronic rejection. Fibrinogen staining of glomerular mesangium was seen in kidneys with chronic rejection. Macrophages (CD68+) infiltrating glomeruli were more numerous in kidneys showing chronic rejection. Conclusion. Increased glomerular deposition of vWF in renal allografts showing chronic rejection, without increased staining for CD62P or CD31, suggests increased constitutive secretion of vWF from endothelial cells as a component of the mechanism of chronic rejection in our model.

Background: As a first step to formulate a new treatment strategy for refractory acute lymphoblastic leukemia (ALL) in infants, clinical results and immunophenotypic and cytogenetic data were analyzed and compared with those from overseas. Methods: There were 62 infants with ALL who were treated between 1977 and 1995 at 30 institutions affiliated with the Tokyo Children’s Cancer Study Group. Clinical and laboratory data obtained from these infants (all under 1 year of age) were retrospectively studied. Results: The morphological diagnoses were FAB-L1 for 51 patients (82.2%) and FAB-L2 for 11 patients (17.8%). Hepatomegaly and splenomegaly were found in 40 (70.0%) and 40 patients (68.3%), respectively. The mean (~SEM) leukocyte count at diagnosis was 205 900~35 700 /μL. The involvement of the central nervous system was evident in nine of 36 patients who were subjected to lumbar puncture, while three of these nine patients were free of neurological symptoms at diagnosis. Thirty-one patients (55.4%) were CD10 negative and 14 (25.0%) were CD10 positive. Thirty-one of 47 patients (65.9%) exhibited chromosomal abnormalities, including 28 patients (59.6%) with 11q23 abnormalities. Rearrangements in the MLL gene were found in nine of 13 infants (69.2%) examined. Translocation of 11q23 and/or MLL gene rearrangement (11q23/MLL) was significantly associated with the absence of the CD10 antigen. Hyperleukocytosis of more than 50 000 /μL and 11q23/MLL gene rearrangements were related to a poor prognosis. The probability of an event-free survival in 62 infants was 13.1~4.8% at 48 months. Conclusions: New therapeutic strategies and large-scale cooperative prospective trials are needed to improve the prognosis of ALL in infants.

The natural course and the clinical significance of hepatitis G virus (HGV) infection were investigated in 106 pediatric patients who received chemotherapy for hematological malignancy or underwent bone marrow transplantation (BMT) using HGV-RNA and antibodies to the HGV-E2 protein (anti-E2). HGV markers were detected in 21 patients (19.8%; HGV-RNA in 19 and anti-E2 in 2). Longitudinal analysis of these HGV-infected patients showed that 1 had anti-E2 before the initial blood transfusion, 14 had persistent viremia, and 6 became clear of circulating HGV-RNA after completion of therapy, although 5 of the 6 HGV-cleared patients never developed anti-E2. Reactivation of HGV infection during chemotherapy was observed in two anti-E2–positive, HGV-RNA–negative patients; the reappearance of the same HGV strain was confirmed by phylogenetic analysis. Among BMT survivors without other known causes of liver dysfunction, HGV-RNA–positive patients had a higher peak serum alanine amino transferase (ALT) value than negative patients. Contrary to previous reports, immunosuppressed patients can apparently recover from HGV infection without detectable anti-E2 and some patients who supposedly recovered from HGV infection can nonetheless suffer exacerbation when subsequently immunosuppressed.

Background: T-lymphocyte depletion 7 days before transplantation with immunotoxin FN 18-CRM9 has resulted in tolerance to subsequent renal allografts. We tested the effect of giving immunotoxin on the day of the transplantation and evaluated its effect on rhesus monkey renal allograft survival, on antibody production, and on T-cell recovery. Methods: Major histocompatibility complex mismatched renal allografts were performed in rhesus monkeys. Immunotoxin was given starting on the day of transplantation, with and without prednisone and mycophenolate mofetil for 3 days. T-cell subsets and alloantibody levels were measured by flow cytometry. The ability of treated monkeys to develop antibody to tetanus, diphtheria, and xenoantibody was measured. Histology of renal transplants was read in a blinded manner. Results: Immunotoxin started on the day of transplantation resulted in prolonged allograft survival in all treatment groups. Graft loss between days 50 and 135 was most often due to interstitial nephritis. Later graft loss was due to chronic rejection. Monkeys had intact antibody responses to alloantigen, tetanus, diphtheria, and xenoantibody. Their CD4 cells recovered gradually over 6 months. Conclusions: Immunotoxin reliably prolongs renal allograft survival when started on the day of transplantation, but interstitial nephritis and chronic rejection limit the development of long-term tolerance. T-cell-dependent B-cell responses remain intact after treatment. (Surgery 1998;124:438-47.)

Purpose To characterize nonleukemic CD10-positive cells in the marrows of children with leukemia in remission or benign conditions. Patients and methods Seventeen children with acute leukemia in complete remission, 12 with solid tumors, and 17 with benign blood diseases were included in this study. Bone marrow cells were analyzed by multicolor flow-cytometry and polymerase chain reaction (PCR) to detect immunoglobulin gene rearrangement. The CD10-positive cells were purely sorted and examined by light microscopy and single cell hemopoietic progenitor assay. Results In patients with acute leukemia, CD10-positive cells were present in higher proportion after completion of therapy than during chemotherapy. They were also higher in the patients of preschool age than in the older age group with benign blood diseases and solid tumors. These CD10-positive cells were morphologically compatible with immature lymphocytes but some blast-like cells also occurred in this population. Most CD10-positive cells coexpressed CD19 and HLA-DR, although only 10 to 30% coexpressed CD20 and CD34. Although some CD10-positive cells expressed CD34, they did not make any colonies. PCR analysis did not show monoclonal bands in CD10-positive bone marrow cells in any patients in remission. Conclusion Marrow CD10-positive cells possess immature B-lymphocyte phenotype and are present in higher proportion in the marrows of children with acute leukemia in continuous complete remission after completion of therapy and children of preschool age than school-age children with benign diseases or solid tumors without marrow involvement. The clonality of these cells was excluded by PCR, which is a distinct point from CD10-positive ALL blasts.

The improved outcome of acquired aplastic anemia (AA) has revealed later complications, such as myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). We retrospectively analyzed 167 children with severe acquired AA. Eleven of 50 children treated with cyclosporin (CSA) and recombinant human granulocyte colony-stimulating factor (rhG-CSF ) developed MDS/AML; 8 of these were within 36 months of the diagnosis of AA, much earlier than previous reports. Six of the 11 children received rhG-CSF exceeding 10 μg/kg/d, and 9 received rhG-CSF therapy for over 1 year. Ten children showed monosomy 7 at diagnosis of MDS. All of the 11 children were administered both CSA and rhG-CSF. There was no development of MDS/AML among 41 children treated with either CSA or rhG-CSF or among 48 children who underwent bone marrow transplantation. A well-controlled clinical trial is warranted to determine whether therapeutic modalities affect the development of MDS/AML in children with severe acquired AA.

Mutations in Wilms tumor 1 (WT1) have been reported in 10–22 % of patients with cytogenetically normal acute myeloid leukemia (CN-AML), but the prognostic implications of these abnormalities have not been clarified in either adults or children. One hundred and fifty-seven pediatric AML patients were analyzed for WT1 mutations around hotspots at exons 7 and 9; however, amplification of the WT1 gene by the reverse transcriptase-polymerase chain reaction was not completed in four cases (2.5 %). Of the 153 evaluable patients, 10 patients (6.5 %) had a mutation in WT1. The incidence of WT1 mutations was significantly higher in CN-AML than in others (15.2 vs. 4.5 %, respectively, P = 0.03). Of the 10 WT1-mutated cases, eight (80 %) had mutations in other genes, including FLT3-ITD in two cases, FLT3-D835 mutation in two, KIT mutation in three, MLL-PTD in three, NRAS mutation in one, and KRAS mutation in two (in some cases, more than one additional gene was mutated). The incidences of KIT and FLT3-D835 mutations were significantly higher in patients with than in those without WT1 mutation. No significant differences were observed in the 3-year overall survival and disease-free survival; however, the presence of WT1 mutation was related to a poor prognosis in patients with CN-AML, excluding those with FLT3-ITD and those younger than 3 years.

Neither accurate incidence nor survival data for pediatric patients with hematological malignancies (HM) have been available in Japan to date. Incidence of patients under 20 years of age, who were diagnosed with HM from 2006 to 2010, and their two-year survival rate (2y-OS) were obtained from disease registry data maintained by the Japan Society of Pediatric Hematology (JSPH). A total of 5,287 cases of HM were identified during this period. Acute lymphoblastic leukemia (ALL, 46.6%) showed the highest incidence, followed by acute myeloid leukemia (AML, 16.7%), non-Hodgkin lymphoma (NHL, 11.9%), and histiocytosis (11.8%). ALL, AML and histiocytosis were common in younger patients aged 1-4, while NHL tended to occur more frequently in older patients aged 5-14. The 2y-OS of HM was 91.6%, with that for the most common B-precursor ALL rising to 96.2%. The 2y-OS for M3 AML, lymphoblastic-B-precursor or diffuse large B cell NHL, Hodgkin lymphoma, myeloproliferative disorders, and Langerhans cell histiocytosis was >95%. There were no gender differences in prognosis, while infants (88.0%) and adolescents aged 15-19 (90.6%) tended toward a poorer prognosis. This is the first report to describe incidence and survival times from the nationwide JSPH disease registry. More precise data with longer follow-up is needed.

CAPD歴6年の26歳男性に高リン血症に伴う腫瘍状石灰化 (tumoral calcinosis) が発生した. 保存的治療を試みたが高リン血症は改善せず, 疼痛などの症状が強くなったため外科的切除を施行した. 再発予防目的でサイクラーを使用しCAPD+NIPD (nightly intermittent peritoneal dialysis) 療法を施行したところ, 残存皮下石灰沈着は完全に消失し, 異所性石灰化の再発は認めない. 本症例で良好な経過が得られたのは食事指導のほかに, サイクラーの使用によって透析効率が改善されたことも一つの要因であると考えられた.

PATIENTS AND METHODS We report patients who were treated for non-Hodgkin's lymphoma (NHL) or Ki-1 antigen-positive (Ki-1) lymphoma with a T-8801 protocol that included etoposide (VP-16) and behenoylcytosine arabinoside. RESULTS Secondary acute myeloid leukemia (AML) developed in 5 of 38 NHL and Ki-1 lymphoma patients, and the cumulative risk at 4 years was 18.4%. The median time from the initiation of the chemotherapy to the development of AML was 21 months (range, 13-30). Four patients had a FAB M5 morphology, and one had FAB M2. In four of five examined cases, chromosomal alterations involving the long arm of chromosome 11 were demonstrated at the time of development of AML. None of the 46 NHL patients who we treated with another protocol (B-8801), using significantly higher cumulative doses of VP-16 than in the case of the patients with T-8801 and a different schedule of VP-16 administration, developed secondary AML. CONCLUSIONS The risk of secondary AML possibly related to the use of VP-16 given twice weekly.