NUP98-NSD1 gene fusion and its related gene expression signature are strongly associated with a poor prognosis in pediatric acute myeloid leukemia.

The cryptic t(5;11)(q35;p15.5) creates a fusion gene between the NUP98 and NSD1 genes. To ascertain the significance of this gene fusion, we explored its frequency, clinical impact, and gene expression pattern using DNA microarray in pediatric acute myeloid leukemia (AML) patients. NUP98-NSD1 fusion transcripts were detected in 6 (4.8%) of 124 pediatric AML patients. Supervised hierarchical clustering analyses using probe sets that were differentially expressed in these patients detected a characteristic gene expression pattern, including 18 NUP98-NSD1-negative patients (NUP98-NSD1-like patients). In total, a NUP98-NSD1-related gene expression signature (NUP98-NSD1 signature) was found in 19% (24/124) and in 58% (15/26) of cytogenetically normal cases. Their 4-year overall survival (OS) and event-free survival (EFS) were poor (33.3% in NUP98-NSD1-positive and 38.9% in NUP98-NSD1-like patients) compared with 100 NUP98-NSD1 signature-negative patients (4-year OS: 86.0%, 4-year EFS: 72.0%). Interestingly, t(7;11)(p15;p15)/NUP98-HOXA13, t(6;11)(q27;q23)/MLL-MLLT4 and t(6;9)(p22;q34)/DEK-NUP214, which are known as poor prognostic markers, were found in NUP98-NSD1-like patients. Furthermore, another type of NUP98-NSD1 fusion transcript was identified by additional RT-PCR analyses using other primers in a NUP98-NSD1-like patient, revealing the significance of this signature to detect NUP98-NSD1 gene fusions and to identify a new poor prognostic subgroup in AML.

Evaluation of the safety and efficacy of liposomal amphotericin B (L-AMB) in children.

A multicenter, uncontrolled clinical study has been conducted to evaluate the safety, efficacy, and pharmacokinetics of liposomal amphotericin B (L-AMB) in children. In this article, the safety and efficacy of L-AMB are discussed. Subjects were diagnosed with invasive fungal infection (definitely diagnosed cases), possible fungal infection (clinically diagnosed cases), and febrile neutropenia with suspected fungal infection (febrile neutropenia cases). Of the 39 subjects treated with L-AMB, 18 received a definite (11) or clinical (7) diagnosis of invasive fungal infection. In these subjects, excluding one unevaluable subject, L-AMB was effective in nine out of 17 subjects(52.9%). Of 12 febrile neutropenia cases, improvement in clinical symptoms, etc., was observed for six but these were excluded from the efficacy analysis because they concomitantly used medications that may have affected efficacy. The causative fungus was identified in four out of 39 subjects and confirmed to be eliminated by treatment with L-AMB in one subject. Adverse events possibly related to L-AMB (adverse drug reactions) were reported in 36 out of 39 subjects (92.3%). The most commonad verse drug reaction was decreased potassium in 20 out of 39 subjects (51.3%), but all these subjects recovered with appropriate treatment, for example potassium supplementation.In a Japanese Phase II clinical study of adult patients, the incidence of adverse drug reactions was 95.3%(82/86 subjects) and the efficacy was 63.6% (42/66). Taken together, these data indicate that the safety and efficacy of L-AMB are almost the same in pediatric and adult patients.

Prospective study of a therapeutic regimen with all-trans retinoic acid and anthracyclines in combination of cytarabine in children with acute promyelocytic leukaemia: the Japanese childhood acute myeloid leukaemia cooperative study.

In childhood acute promyelocytic leukaemia (APL), the efficacy of therapy combining cytarabine with all-trans retinoic acid (ATRA) and anthracyclines remains unclear in terms of long-term prognosis. Between August 1997 and March 2004, 58 children with APL (median age: 11 years) were enrolled into an acute myeloid leukaemia (AML) study (AML99-M3) and followed up for a median time of 86 months. The regimen included ATRA and anthracyclines combined with cytarabine in both induction and consolidation. In induction, two patients died of haemorrhage and four patients developed retinoic acid syndrome. Of 58 patients, 56 (96·6%) achieved complete remission, two of whom relapsed in the bone marrow after 15 and 19 months respectively. Sepsis was a major complication, with an incidence of 5·6-10·9% in the consolidation blocks, from which all but one of patients recovered. Consequently, 7-year overall and event-free survival rates were 93·1% and 91·4% respectively, and cumulative incidence of relapse plateaued at 3·6% after 2 years. Follow-up survey of 54 patients revealed no patients with late cardiotoxicity or secondary malignancy, except one with asymptomatic prolongation of QTc interval. This study suggests that the combination of cytarabine with ATRA and anthracycline-based therapy may have useful implications in the perspective of long-term prognosis and late adverse effects for childhood APL.

Relapse of aplastic anemia in children after immunosuppressive therapy: a report from the Japan Childhood Aplastic Anemia Study Group.

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.

[Effectiveness of remission induction with high-dose cytarabine for relapsed or refractory pediatric acute leukemia].

We conducted a multicenter postmarketing study to investigate the efficacy and safety of reinduction therapy with a high-dose cytarabine-containing regimen for pediatric patients with relapsed or refractory acute leukemia. Seven of 13 patients (53.8%) with ALL achieved complete or partial remission, and only 1 of 6 patients (16.7%) with AML achieved partial remission. The frequent non-hematologic adverse events were gastrointestinal toxicities, such as vomiting, diarrhea and abdominal pain, as well as pyrexia and headache. Infection appeared in 9 of 20 (45%) patients. There were two death during reinduction therapy. One died of invasive bronchopulmonary aspergillosis, and the other died of intracranial hemorrhage and renal failure. These results indicated that a high-dose cytarabine regimen is effective as reinduction therapy in pediatric patients with relapsed ALL, and supportive care is essential to prevent or control treatment-related adverse events, such as infection.

Age-associated difference in gene expression of paediatric acute myelomonocytic lineage leukaemia (FAB M4 and M5 subtypes) and its correlation with prognosis.

Acute myeloid leukaemia, French-American-British M4 and M5 subtypes (AML-M4/M5) is frequently associated with MLL gene rearrangement and its incidence is relatively high among infants. Clinically, paediatric AML-M4/M5 has been considered as an intermediate or undefined prognostic group. In this study, we analysed gene expression of 40 paediatric AML-M4/M5 patients excluding inv(16) and t(8;21) patients, and found striking differences among the patients in an age-associated manner. In particular, most of the infants displayed very distinct gene expression. On the basis of this difference, we divided paediatric patients into three subgroups (A, B and C) with the average age of 0.3, 3.1 and 6.6 years old respectively. All subgroups included patients with MLL gene rearrangement as well as normal and other karyotypes. Surprisingly, gene expression signatures of MLL gene rearrangement differed substantially among these subgroups. In addition, subgroup C presented extremely poor outcome (3-year event-free survival 28%) whilst eight patients with MLL gene rearrangement in subgroup C had all relapsed within 18 months. These results suggest that age is an important factor contributing to the biology of AML-M4/M5 and the sub-grouping procedures developed in this study could be a powerful tool to identify unfavourable risk patients within paediatric AML-M4/M5.

Tandem duplications of MLL and FLT3 are correlated with poor prognoses in pediatric acute myeloid leukemia: a study of the Japanese childhood AML Cooperative Study Group.

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 MLL-PTD, 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 MLL-PTD were significant for 3-year overall survival (OS) (56.3% vs. 83.2%, P = 0.018), disease-free survival (DFS) (41.7% vs. 69.6%, P = 0.010), and relapse rate (RR) (54.3% vs. 27.6%, P = 0.0085) 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 < 0.0000001), DFS (40.0% and 66.9%, P < 0.003), and RR (52.4% and 30.3%, P < 0.005). Coduplication of both genes was found in only 3 (1.9%) patients. CONCLUSION: AML patients with FLT3-ITD, but not D835Mt, showed a poor prognosis. AML patients with MLL-PTD were also correlated with poor prognosis in this study.

Antithymocyte globulin and cyclosporine for treatment of 44 children with hepatitis associated aplastic anemia.

We analyzed the outcomes of 44 children with hepatitis associated aplastic anemia (HAA) who received immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporine (CsA). Fourteen (31.8%) patients achieved complete response and 17 (38.6%) achieved partial response, for an overall response rate of 70.4% after 6 months. Seven non-responders received bone marrow transplantation from an HLA-matched unrelated donor and 6 out of 7 are alive. The probability of overall survival at 10 years was 88.3+/-4.9%, which supports the role of IST with ATG and CsA as treatment of choice for children with HAA without an HLA identical sibling donor.

Serial morphologic observation of bone marrow in aplastic anemia in children.

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.

Detection of myelodysplastic syndrome/ acute myeloid leukemia evolving from aplastic anemia in children, treated with recombinant human G-CSF.

BACKGROUNDS AND OBJECTIVES: Recombinant human granulocyte colony-stimulating factor (G-CSF) has clear benefits in patients with severe neutropenia. However, recent reports of myelodysplastic syndrome/acute myeloid leukemia (t-MDS/AML) developing after treatment with immunosuppressants and G-CSF has raised concern over the use of this agent in patients with aplastic anemia. DESIGN AND METHODS: We undertook a multi-institutional, non-randomized study of 112 children given a diagnosis of aplastic anemia, and then treated with different 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, defined by stringent morphological and molecular/cytogenetic criteria. Incidence rates were calculated by the person-years statistical method. RESULTS: As of December 2001, all eligible patients had been followed for a median of 3 years, and the G-CSF (+) group had received a median total G-CSF dose of 30,100 micrograms altogether, administered over a median of 4 months. Only one case of MDS developed among the G-CSF (+) patients (n=81), compared with three in the group receiving other agents (n=31). This isolated case was not associated with monosomy 7, the cytogenetic abnormality most often linked to G-CSF treatment. Incidence rates of MDS in the two groups were not significantly different (3.8 vs. 22.4 per 1,000 patient-years at risk, p=0.125). There were no cases of overt AML in either cohort. INTERPRETATION AND CONCLUSIONS: G-CSF therapy did not increase the risk of t-MDS/AML development in children with aplastic anemia over a median follow-up of 3.7 years

Diamond-Blackfan anemia in Japan: clinical outcomes of prednisolone therapy and hematopoietic stem cell transplantation.

The epidemiology and treatment outcomes for Diamond-Blackfan anemia (DBA) were surveyed in a cohort of 54 children (M/F = 26:28) registered in Japan from 1988 to 1998. The annual incidence was 4.02 cases per million births, the median age at diagnosis was 60 days, and 59% of the cases presented by 3 months of age. Three patients had a familial occurrence. All patients received prednisolone (PSL), and cyclosporin A (CsA) was added to the therapy in 17 patients. Forty-seven patients received transfusions, and 13 underwent hematopoietic stem cell transplantation (HSCT). The cumulative probabilities of a medication-free or a transfusion-free state prior to HSCT were 36% and 69%, respectively, at more than 5 years after diagnosis. Thirteen patients were weaned from PSL therapy without HSCT, and CsA was not associated with weaning from therapy. Transfusion and medication were stopped at 249 days and 933 days after diagnosis in 34 and 13 patients, respectively, who achieved a state of independence. No initial findings predicted the treatment dependence. More than 20% of patients experienced sustained hemosiderosis and/or adverse effects of PSL. The ages and reticulocyte counts at diagnosis of the patients who underwent HSCT were lower than in the patients who did not. HSCT led to the highest success (85%) of all previous reports, even though 5 alternative donors were included in our study. Two cord blood transplants from unrelated donors failed. These findings suggest the need for developing an integral treatment strategy including selective HSCT for refractory DBA.

Solid, cystic and vanishing tumors of the pancreas.

Two girls with pancreatic tumors are reported. They were presented with abdominal pain, no history of abdominal injury and no tumor marker abnormalities. Imaging studies demonstrated that the tumors in two patients had both solid and cystic components. The tumors gradually regressed and finally disappeared without any treatment. There has been no evidence of recurrence in 5- and 4-year observation periods, respectively. Pancreatic tumor with solid and cystic components may rarely be self-limited.

[Pharmacokinetic and clinical studies on teicoplanin for sepsis by methicillin-cephem resistant Staphylococcus aureus in the pediatric and neonate field].

Pharmacokinetics, clinical efficacy and safety of teicoplanin (TEIC) were evaluated in pediatric and neonate patients with MRSA sepsis in the dosages approved in overseas. The administrated dose for pediatrics patients was 10 mg/kg once at hour 0, 12 and 24, followed by every 24 hours intervals. In neonates patients, first dose was 16 mg/kg, then 8 mg/kg every 24 hours intervals. 1. Pharmacokinetic results. All 17 patients (9 neonates and 8 pediatrics) who received TEIC were evaluated for pharmacokinetics. Trough concentrations were analyzed in 16 patients (9 neonates and 7 pediatrics) excluding one patient for lack of measurement of drug concentration at day 7. No patient with a concentration exceeding 60 micrograms/mL in peak or trough concentrations were reported. Mean concentrations in trough at day 3, 4 and 7 in neonates were 15.2, 14.7 and 17.8 micrograms/mL, and in pediatrics were 12.5, 12.2 and 13.1 micrograms/mL, respectively. These results were similar to those reported in foreign pediatrics and neonates patients. 2. Efficacy and safety results. Since no patient was excluded, all patients were evaluated for efficacy and safety. Microbiological efficacy as well as clinical cure were secondarily evaluated in 2 patients for whom MRSA was isolated from blood. Clinical efficacy rate was 76.5% (13/17) and number of cases in judgments of excellent, good, fairly improved and no change were 12, 1, 3 and 1 cases respectively. The patients for whom MRSA was isolated from blood were judged as MRSA eradicated case and cured without any additional anti-MRSA drugs. Adverse events were reported in 2 neonates and 3 pediatric patients. Possibly related adverse events to study drug (adverse drug reactions) were: 1 case of respiratory disorder, thrombocythemia, gamma-GTP increased, GOT increased and GPT increased in 3 pediatrics. These results suggest that an application of overseas dose regimen of TEIC for neonate and pediatrics is appropriate in Japan.

Outcome of children with relapsed acute myeloid leukemia following initial therapy under the AML99 protocol.

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.

WT1 mutation in pediatric patients with acute myeloid leukemia: a report from the Japanese Childhood AML Cooperative Study Group.

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.

RAS mutations are frequent in FAB type M4 and M5 of acute myeloid leukemia, and related to late relapse: a study of the Japanese Childhood AML Cooperative Study Group.

Mutations in RAS are frequent in acute myeloid leukemia (AML), and are thought to contribute to leukemogenesis in a subset of patients; however, their prognostic significance has not been firmly established. One hundred and fifty-seven pediatric patients with AML were analyzed for NRAS and KRAS mutations around hot spots at codons 12, 13, and 61. Twenty-nine patients (18.5%) had an activating mutation of RAS. We found KRAS mutations to be more frequent than NRAS mutations (18/29, 62.1% of patients with RAS mutation), in contrast to previous reports (18-40%). The frequency of RAS mutation was higher in French-American-British types M4 and M5 than other types (P = 0.02). There were no significant differences in other clinical manifestations or distribution in cytogenetic subgroups, or aberrations of other genes, including KIT mutation, FLT3-ITD, and MLL-PTD, between patients with and without RAS mutations. No significant differences were observed in the 3-year overall survival and disease-free survival; however, the presence of RAS mutation was related to late relapse. The occurrence of clinical events at relatively late period should be monitored for in AML patients with mutations in RAS.

High WT1 mRNA expression after induction chemotherapy and FLT3-ITD have prognostic impact in pediatric acute myeloid leukemia: a study of the Japanese Childhood AML Cooperative Study Group.

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.