Characterization of pediatric Philadelphia-negative B-cell precursor acute lymphoblastic leukemia with kinase fusions in Japan.

Recent studies revealed that a substantial proportion of patients with high-risk B-cell precursor acute lymphoblastic leukemia (BCP-ALL) harbor fusions involving tyrosine kinase and cytokine receptors, such as ABL1, PDGFRB, JAK2 and CRLF2, which are targeted by tyrosine kinase inhibitors (TKIs). In the present study, transcriptome analysis or multiplex reverse transcriptase-PCR analysis of 373 BCP-ALL patients without recurrent genetic abnormalities identified 29 patients with kinase fusions. Clinically, male predominance (male/female: 22/7), older age at onset (mean age at onset: 8.8 years) and a high white blood cell count at diagnosis (mean: 94 200/µl) reflected the predominance of National Cancer Institute high-risk (NCI-HR) patients (NCI-standard risk/HR: 8/21). Genetic analysis identified three patients with ABL1 rearrangements, eight with PDGFRB rearrangements, two with JAK2 rearrangements, three with IgH-EPOR and one with NCOR1-LYN. Of the 14 patients with CRLF2 rearrangements, two harbored IgH-EPOR and PDGFRB rearrangements. IKZF1 deletion was present in 16 of the 22 patients. The 5-year event-free and overall survival rates were 48.6±9.7% and 73.5±8.6%, respectively. The outcome was not satisfactory without sophisticated minimal residual disease-based stratification. Furthermore, the efficacy of TKIs combined with conventional chemotherapy without allogeneic hematopoietic stem cell transplantation in this cohort should be determined.

High expression of EVI1 and MEL1 is a compelling poor prognostic marker of pediatric AML.

EVI1 and MEL1 are homolog genes whose transcriptional activations by chromosomal translocations are known in small subsets of leukemia. From gene expression profiling data of 130 Japanese pediatric acute myeloid leukemia (AML) patients, we found that EVI1 and MEL1 were overexpressed in ~30% of patients without obvious translocations of these gene loci, and that their high expression was significantly associated with inferior survival. High EVI1 expression was detected mainly in myelomonocytic-lineage (designated as e-M4/M5 subtype) leukemia with MLL rearrangements and in megakaryocytic-lineage (designated as e-M7 subtype) leukemia, and its prognostic association was observed in the e-M4/M5 subtype but not in the e-M7 subtype. On the other hand, high MEL1 expression was detected in myelocytic-lineage (designated as e-M0/M1/M2 subtype) and e-M4/M5 subtype leukemia without MLL rearrangements, and its prognostic association was independent from the subtypes. Because of their subtype-dependent and mutually exclusive expression, a combined evaluation of their high expression enabled a clear distinction of patients with inferior survival (P<0.00001 in event-free survival (EFS) and overall survival (OS)). This association was confirmed by quantitative reverse transcription PCR analysis of an independent cohort of 81 patients (P=0.00017 in EFS, P=0.00028 in OS). We propose that the combined estimation of EVI1 and MEL1 expression will be an effective method to predict the prognosis of pediatric AML.

Early use of allogeneic hematopoietic stem cell transplantation for infants with MLL gene-rearrangement-positive acute lymphoblastic leukemia.

Sixty-two infants with MLL gene-rearrangement-positive acute lymphoblastic leukemia (MLL-r ALL) were treated with the MLL03 protocol of the Japanese Pediatric Leukemia/Lymphoma Study Group: short-course intensive chemotherapy followed by early allogeneic hematopoietic stem cell transplantation (HSCT) within 4 months of the initial induction. The 4-year event-free survival and overall survival rates were 43.2% (95% confidence interval (CI)=30.7-55.1%) and 67.2% (53.8-77.4%), respectively. A univariate analysis showed younger age (<90 days at diagnosis), central nervous system disease and poor response to initial prednisolone therapy significantly associated with poor prognosis (P<0.05). In a multivariate analysis, younger age at diagnosis tended to be associated with poor outcome (hazard ratio=1.969; 95% CI=0.903-4.291; P=0.088). Although the strategy of early use of HSCT effectively prevented early relapse and was feasible for infants with MLL-r ALL, the fact that substantial number of patients still relapsed even though transplanted in their first remission indicates the limited efficacy of allogeneic HSCT for infants with MLL-r ALL. Considering the risk of severe late effects, indications for HSCT should be restricted to specific subgroups with poor risk factors. An alternative approach incorporating molecular-targeted drugs should be established.

Markedly improved outcomes and acceptable toxicity in adolescents and young adults with acute lymphoblastic leukemia following treatment with a pediatric protocol: a phase II study by the Japan Adult Leukemia Study Group.

The superiority of the pediatric protocol for adolescents with acute lymphoblastic leukemia (ALL) has already been demonstrated, however, its efficacy in young adults remains unclear. The ALL202-U protocol was conducted to examine the efficacy and feasibility of a pediatric protocol in adolescents and young adults (AYAs) with BCR-ABL-negative ALL. Patients aged 15-24 years (n=139) were treated with the same protocol used for pediatric B-ALL. The primary objective of this study was to assess the disease-free survival (DFS) rate and its secondary aims were to assess toxicity, the complete remission (CR) rate and the overall survival (OS) rate. The CR rate was 94%. The 5-year DFS and OS rates were 67% (95% confidence interval (CI) 58-75%) and 73% (95% CI 64-80%), respectively. Severe adverse events were observed at a frequency that was similar to or lower than that in children treated with the same protocol. Only insufficient maintenance therapy significantly worsened the DFS (hazard ratio 5.60, P<0.001). These results indicate that this protocol may be a feasible and highly effective treatment for AYA with BCR-ABL-negative ALL.

Prognostic implications of CEBPA mutations in pediatric acute myeloid leukemia: a report from the Japanese Pediatric Leukemia/Lymphoma Study Group.

CCAAT/enhancer-binding protein alpha (CEBPA) mutations are a favorable prognostic factor in adult acute myeloid leukemia (AML) patients; however, few studies have examined their significance in pediatric AML patients. Here we examined the CEBPA mutation status and clinical outcomes of pediatric AML patients treated in the AML-05 study. We found that 47 (14.9%) of the 315 evaluable patients harbored mutations in CEBPA; 26 cases (8.3%) harbored a single mutation (CEBPA-single) and 21 (6.7%) harbored double or triple mutations (CEBPA-double). After excluding core-binding factor-AML cases, patients harboring CEBPA mutations showed better overall survival (OS; P=0.048), but not event-free survival (EFS; P=0.051), than wild-type patients. Multivariate analysis identified CEBPA-single and CEBPA-double as independent favorable prognostic factors for EFS in the total cohort (hazard ratio (HR): 0.47 and 0.33; P=0.02 and 0.01, respectively). CEBPA-double was also an independent favorable prognostic factor for OS (HR: 0.30; P=0.04). CEBPA-double remained an independent favorable factor for EFS (HR: 0.28; P=0.04) in the normal karyotype cohort. These results suggest that CEBPA mutations, particularly CEBPA-double, are an independent favorable prognostic factor in pediatric AML patients, which will have important implications for risk-stratified therapy.

Comparison of matched-sibling donor BMT and unrelated donor BMT in children and adolescent with acquired severe aplastic anemia.

From January 1991 to March 2007, 61 children and adolescent with acquired severe aplastic anemia received BMT in our institutions. We retrospectively compared the outcome of 30 cases of matched-sibling donor BMT (MSD-BMT) and 31 cases of unrelated donor BMT (URD-BMT). We observed one graft failure among MSD-BMT recipients and three graft failures among URD-BMT recipients, respectively. No patients in the MSD-BMT group developed grades II-IV acute GVHD compared with 11 of 30 patients (37%) in the URD-BMT group (P<0.001). One of 30 MSD-BMT recipients (3%) developed chronic GVHD compared with 8 of 30 URD-BMT recipients (27%) (P=0.013). The incidence of EBV and CMV reactivation was 11 of 20 URD-BMT recipients and 23 of 30, respectively. One patient in the URD-BMT group died of a motor accident 5.5 years after BMT. Ten-year OS was 100% in MSD-BMT recipients and 93.8% (95% CI, 81.9-100%) in URD-BMT recipients, respectively (P=0.252). Ten-year failure-free survival was 96.7% (95% CI, 90.2-100%) in the MSD-BMT group and 84.7% (95% CI, 70.2-99.2%) in the URD-BMT group, respectively (P=0.161).

Relationship between tacrolimus blood concentrations and clinical outcome during the first 4 weeks after SCT in children.

The relationship between tacrolimus concentration and acute GVHD is not well known, with few published data available for lower target levels. We hypothesized that lower levels of tacrolimus would correlate with higher incidence of acute GVHD and poorer prognosis. Receiver operator characteristic curves (ROC) were used to quantify tacrolimus blood levels as predictors of grade II-IV acute GVHD. A total of 97 pediatric patients with hematological malignancies met the study criteria. On the ROC, a cutoff of 7 ng/ml provided the best balance between sensitivity and specificity (62.8 vs 68.2%, respectively). Cumulative incidence of acute GVHD was 65.9% (range 58.5-73.3%) in patients with mean tacrolimus concentration of < or =7 ng/ml and 34.8% (range 27.8-41.8%) in patients with mean tacrolimus concentration of >7 ng/ml (P=0.002). Incidence of non-relapse mortality (NRM) was higher in patients with tacrolimus of < or =7 ng/ml (42.9%; range 35.6-50.2%) than in patients with tacrolimus of >7 ng/ml (28.3%; range 17.4-39.2%; P=0.008). This translated into better EFS in patients with tacrolimus of >7 ng/ml (48.9%; range 39.8-58.0%) than in patients with tacrolimus of < or =7 ng/ml (31.8%; range 25.0-38.6%; P=0.031). Multivariate analysis showed that tacrolimus concentration was significantly associated with clinical outcomes. Mean whole-blood level of tacrolimus as continuous infusion should be maintained at > or =7 ng/ml for pediatric patients.

Outcome of risk-based therapy for infant acute lymphoblastic leukemia with or without an MLL gene rearrangement, with emphasis on late effects: a final report of two consecutive studies, MLL96 and MLL98, of the Japan Infant Leukemia Study Group.

We evaluated the efficacy of a treatment strategy in which infants with acute lymphoblastic leukemia (ALL) were stratified by their MLL gene status and then assigned to different risk-based therapies. A total of 102 patients were registered on two consecutive multicenter trials, designated MLL96 and MLL98, between 1995 and 2001. Those with a rearranged MLL gene (MLL-R, n=80) were assigned to receive intensive chemotherapy followed by hematopoietic stem cell transplantation (HSCT), while those with germline MLL (MLL-G, n=22) were treated with chemotherapy alone. The 5-year event-free survival (EFS) rate for all 102 infants was 50.9% (95% confidence interval, 41.0-60.8%). The most prominent late effect was growth impairment, observed in 58.9% of all evaluable patients in the MLL-R group. This plan of risk-based therapy appears to have improved the overall prognosis for infants with ALL, compared with previously reported results. However, over half the events in patients with MLL rearrangement occurred before the instigation of HSCT, and that HSCT-related toxic events comprised 36.3% (8/22) of post-transplantation events, suggesting that further stratification within the MLL-R group and the development of more effective early-phase intensification chemotherapy will be needed before the full potential of this strategy is realized.

The seventh international childhood acute lymphoblastic leukemia workshop report: Palermo, Italy, January 29--30, 2005.

Between 1995 and 2004, six International Childhood Acute Lymphoblastic Leukemia (ALL) Workshop have been held, and the completion of several collaborative projects has established the clinical relevance and treatment options for several specific genetic subtypes of ALL. This meeting report summarizes the data presented in the seventh meeting and the discussion.

Pathology review for paediatric non-Hodgkin's lymphoma patients in Japan; a report from the Japan association of childhood leukaemia study (JACLS).

A central pathology review system with an immunophenotyping laboratory was established in Japan to support the clinical trial, the Japan Association of Childhood Leukaemia Study (JACLS) NHL-98, for patients with paediatric non-Hodgkin's lymphoma (NHL). Pathology samples from 155 clinically-suspected NHL cases were evaluated centrally initially using the Revised European-American Lymphoma (REAL) classification in a rapid review (within 2 weeks after surgery/biopsy) and then later at the consensus review (once a year). The samples were subsequently re-classified according to the new World Health Organisation (WHO) classification. After the pathology review, 96 (62%) patients were eligible for the study, and 58 of them (60%) had extra-nodal primaries. These NHL cases included B-cell lymphomas (precursor B-cell, 11; Burkitt, 18; diffuse large B-cell, 18; not otherwise specified, 3) and T/Natural Killer (NK)-cell lymphomas (precursor T-cell, 23; anaplastic large cell, 20; others, 3). There was excellent concordance in making the diagnoses (95/96, 99%) and typing (93/96, 97%) of NHL between the rapid and consensus reviews. Five cases, initially diagnosed as diffuse large B-cell lymphoma by the review, were re-classified as Burkitt lymphoma according to the immunocytochemical criteria by the WHO classification. A total of 59 (38%) cases were excluded from the study: they were Hodgkin lymphoma (7), leukaemias (11), reactive lymphoid hyperplasia (20), necrotizing lymphadenitis (7), no consensus diagnosis (1), insufficient materials (2), and others (11). This is the first report of the central pathology review from the paediatric NHL group study in Japan. Because various diseases, either neoplastic or reactive, mimicked NHL, clinically and histopathologically, the central pathology review system was critical and essential for patient enrollment and protocol assignment in our clinical trial. Through the two-step review system, highly reliable data were generated to support this study.

Flow cytometric determination of intracytoplasmic Wiskott-Aldrich syndrome protein in peripheral blood lymphocyte subpopulations.

We have produced a novel monoclonal antibody (mAb) directed against Wiskott-Aldrich syndrome protein (WASP) by immunizing mice with the recombinant protein. The mAb designated 5A5 is highly specific to WASP and suitable for Western blot analysis and immunoprecipitation. A flow cytometric assay using the 5A5 mAb identifies expression of intracytoplasmic WASP in lymphocytes from normal individuals. Double staining analysis with cell surface CD3, CD19, and CD56, and intracytoplasmic molecules revealed WASP expression in each subpopulation. With regard to WASP expression in patients with Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT), peripheral blood mononuclear cells (PBMCs) from nine patients and Epstein-Barr virus-transformed B-lymphoblastoid cell lines from seven patients examined did not show WASP expression by flow cytometric analysis. These results were confirmed by Western blot analysis. We conclude that WASP expression in lymphocyte subpopulations from patients with WAS and XLT can be more precisely evaluated by flow cytometry as compared with Western blot analysis. This flow cytometry method is important as a supplement to Western blots, but even more important as an alternative and powerful assay that can contribute to research on WASP as well as diagnosis in a clinical setting.

Prospective monitoring of the Epstein-Barr virus DNA by a real-time quantitative polymerase chain reaction after allogenic stem cell transplantation.

Epstein-Barr virus (EBV)-related lymphoproliferative disorder (LPD) is a serious complication of haematopoietic stem cell transplantation (HSCT). To clarify the frequency, natural course and risk factors for LPD, we prospectively monitored 38 allogeneic (allo)-HSCT patients, focusing on the use of anti-thymocyte globulin (ATG). We used a recently developed real-time polymerase chain reaction assay to monitor EBV genome load. The subjects consisted of 19 patients given ATG for conditioning and 19 patients not given ATG. Of the 19 patients given ATG, 47.4% (nine patients) had a significant increase in EBV genome load (10(2.5) copies/microg DNA). Of these nine patients, two developed LPD. Therefore, 10.5% of the patients receiving allo-HSCT with ATG developed LPD. In contrast, none of the 19 patients without ATG had a significantly increased EBV load. The increases in viral load were observed in the second or third month after HSCT. We found that the peak viral loads of LPD patients were > 10(4.0 ) copies/microg DNA. On the other hand, the viral loads of most patients with no symptoms were < 10(2.5) copies/microg DNA. In conclusion, routine monitoring of EBV load during the second and third months after transplantation may benefit patients undergoing HSCT with ATG. We propose that an EBV load > 10(2.5) copies/microg DNA is the reactivation of EBV, and that an EBV load > 10(4.0) copies/microg DNA is indicative of developing LPD.

Age-related changes in brain neuromagnetic responses to face perception in humans.

In order to investigate the effects of ageing on face perception, we studied the magnetic responses to face images in 15 young (19-38 years) and 10 elderly (51-81 years) subjects. Face-specific responses (160mF), which originate in the inferior occipitotemporal cortices, and face non-specific responses (100m), which originate in the primary visual cortices, were evoked in all subjects. Averaged peak latency of the 160mF in the elderly group (174.0+/-9.1 ms) was significantly longer (P<0.0005) than that in the young group (161.5+/-5.1 ms), while no inter-group difference was found in the 100m latency. There was a significant correlation between age and 160mF latency (+0.35 ms/year, R=0.747) suggesting age-related decline of face perception.

Unrelated donor marrow transplantation in children with severe aplastic anaemia using cyclophosphamide, anti-thymocyte globulin and total body irradiation.

We report a favourable outcome in 15 patients with severe aplastic anaemia (SAA) who were < 20 years of age and who underwent bone marrow transplantation (BMT) from a human leucocyte antigen (HLA)-matched unrelated donor. All patients were non-responders to intensive immunosuppressive therapy (IST) and were multiply transfused. The conditioning regimen consisted of cyclophosphamide (60 mg/kg/d, on d -4 and -3), anti-thymocyte globulin (2.5 mg/kg/d, on d -5 to -2) and total body irradiation (2.5 Gy x 2/d, on d -2 and -1). Patients received cyclosporine and methotrexate for prophylaxis of graft-versus-host disease (GVHD), except for the last four who received tacrolimus instead of cyclosporine. Donor/recipient pairs were identical for HLA class I and II antigens by serological typing, but four pairs were found to have a mismatch at the HLA-A, -B or -DRB1 locus by high-resolution typing. All patients achieved rapid engraftment and are alive at 2-86 months after transplantation (median follow-up, 51 months). Moderate to severe acute GVHD occurred in 5 out of 15 patients (33%); only one patient developed extensive chronic GVHD. Considering our encouraging results, unrelated donor transplantation for SAA is recommended as a salvage therapy in non-responders to IST.

Clinicopathologic characteristics of leukemia in Japanese children and young adults.

The aim of this study is to clarify the clinicopathologic characteristics of adolescent leukemia in Japan by retrospective analysis. Patients with acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), and myelodysplastic syndrome (MDS), consecutively diagnosed from 1986 to 1999, were enrolled. A total of 3,856 patients from 1 to 15 years of age and 1,803 patients from 15 to 29 years of age were eligible for this study. Demographically, the frequency of AML found was almost constant during the teenage years, whereas the frequency of ALL gradually decreased. The relative frequency of CML and MDS apparently started to increase in patients in their late teens. The relative frequency of M3 and t(15;17) gradually increased during adolescence. Among patients aged 1 to 4 years, M7 was the most frequent FAB subtype. Among patients aged 5 to 9 years, M2 and t(8;21) was the most frequent subtype. The percentage of T cell ALL increased in patients 5 to 9 years old, reaching 31.2% in the 20- to 24-year-old age group. The percentage of patients with hyperdiploidy over 50 chromosomes was highest (17.0%) in patients aged 1 to 4 and decreased to 3.9% in the older teens. The percentage of patients with the Ph1 chromosome increased from 9.9% in teens to 30.0% in patients in their late twenties. When comparing event-free survival (EFS) rates for ALL according to age, the estimated 7-year EFS rate was highest for patients aged 1 to 9 years (65.9%) and intermediate for patients aged 10 to 15 years (48.4%). However, the EFS rate was significantly worse for patients aged 15 to 19 years (19.4%) and 20 to 29 years (17.0%) (P = 0.024). On the other hand, the EFS rate for AML decreased with increasing age, although without statistical significance. The overall survival rates are approximate among all age groups. The results of the study indicate that there are considerable variations in biologic features of leukemia between children and young adults. The prognosis for adolescent leukemia may be improved by introducing pediatric trials, which take into account the prognostic biological features.