ALBERT

Description: Prognosis of childhood AML has improved, but still one third of patients relapse. Conventional drugs not being used in AML yet might be useful to reduce the relapse rate. Preclinical and clinical studies suggest that methotrexate may be beneficial in AML. We previously showed (Rots et al., Eur J Cancer 2001, 37: 492) that in vitro methotrexate inhibits the key enzyme thymidylate synthase at least as effectively in AML as in ALL, if the exposure time is long instead of short. Older clinical studies with methotrexate at doses up to 300 mg/m2 showed responses in up to 40% of patients with AML. We therefore initiated a single agent methotrexate window study in children with relapsed AML, in the setting of the I-BFM-SG. Children and adolescents up to 21 years of age with relapsed AML with bone marrow (BM) involvement were eligible. Children with Down syndrome were not eligible. Because of the possibility of toxicity postponing reinduction chemotherapy known to be effective, and the experience with methotrexate given at 1 gr/m2 over 36 hours (10% given as a loading dose) in relapsed ALL, that regimen was chosen instead of higher-dose methotrexate. Folinic acid rescue started at 48 hours after the start of the methotrexate infusion for at least 2 doses at 15 mg/m2. Hyperhydration and urine alkalanisation were also required. Clinical response was determined by a day 7 BM examination and was arbitrarily defined as good in case of a relative reduction in BM leukemic blasts of at least 50%, as partial in case of a 10–50% reduction, as progressive disease in case of an increase of at least 10%, and as stable in others. In addition, peripheral blood (PB) was examined until the day of the BM aspiration. The study was designed according to the 2-stage method of Gehan. The chance to reject the drug with a good response rate of at least 30% is less than 5% in case of no good responder among the first cohort of 9 patients. Disappointingly, the first 9 patients did not show a single good responder. Moreover, 3 patients had progressive disease (all 3 early relapsed AML FAB type M0 or M7). Therefore, it was decided to close the study. At that time, 4 more patients had been enrolled. The 13 patients all had first relapsed AML, 6 early (within 1 year from initial diagnosis; 1 FAB type M0, 2 M4, 1 M5, 2 M7) and 7 late (2 FAB type M0, 1 M1, 1 M2, 1 M4, 1 M5, 1 M7). In total, 1 patient (late relapsed M7) showed a good response, 2 a partial response (both late relapsed AML, FAB type M1 and M2), and 5 progressive disease. Based on PB, responses were seen in all patients with circulating blasts. Five grade III/IV toxicities were reported in 4 patients (mucositis, transaminatitis, infection, erythema and vomiting). In conclusion, the response rate (23%, only 1 out of 13 with a good response) to single agent methotrexate at 1 gr/m2 over 36 hours given once was low in this cohort of children with first relapsed AML. The relatively low and single dose may have contributed to this result. However, 5 out of 13 patients had progressive disease based on BM examination. Future studies of the AML committee of the International BFM Study Group will focus on novel agents, such as clofarabine and gemtuzumab ozogamicin. This study did prove the feasability to perform such studies in an international setting.

Description: The farnesyltransferase inhibitor tipifarnib (Zarnestra™) was originally developed to target malignancies harbouring RAS mutations. In the first clinical studies with tipifarnib, in adults with leukemia, it was found that patients who responded did not harbour any RAS mutations, suggesting a different mechanism of response. In a previous study we showed that 18% of 150 untreated pediatric AML patients harbour mutations in RAS, of which 30% were CBF-AML. We now studied 44 untreated and 13 relapsed pediatric AML, as well as 22 untreated ALL samples for mutations in RAS, using D-HPLC and direct sequencing. In vitro tipifarnib resistance was determined by a 4-day MTT assay (concentration 0.016-51μM, kindly provided by Janssen Research). The LC50 value, the concentration at which 50% of cells are killed by tipifarnib, was used as a measure of resistance. Patient characteristics were; for untreated AML: 64% boys; median age 9.3 years; median WBC 74.8x109/L; FAB 2xM0, 2xM1, 8xM2, 3xM3, 16xM4, 8xM5, 5x unclassified; for relapsed AML: 77% boys; median age 4.0 years; median WBC 41.6x109/L; FAB 2xM0, 2xM2, 3xM4, 2xM5, 2xM7, 2x unclassified; for untreated ALL: 73%boys; median age 6.0 years; median WBC 10.2x109/L; 15 B-cell precursor (BCP) ALL and 7 T-ALL. We found RAS mutations in 14 (32%) untreated AML samples (N-RAS : 8 samples exon 1, 1 sample exon 2; K-RAS: 5 samples exon 1 mutations). In relapsed AML 2 samples showed an N-RAS exon 1 mutation (15.4%). In ALL 18.2% had a RAS mutation: an N-RAS exon 1 mutation was found in 2 patients (9.1%) and a K-RAS exon 1 mutation in another 2 patients (9.1%). The distribution of tipifarnib sensitivity was similar in RAS mutated- and non-mutated untreated AML patients [median LC50 RAS mutated 7.1μM (P25-P75: 6.0-9.6μM) vs. non-mutated 4.9μM (P25-P75 2.3-8.2μM); p=0.199]. When we compared N-RAS mutated samples with K-RAS mutated samples there was no statistically significant difference in sensitivity to tipifarnib (median LC50 [p25-p75] 3.2μM [2.9-3.9μM] and 4.9μM [3.7-23.1μM], p=0.20), and comparing them separately with non-mutated AML did not show differences in sensitivity to tipifarnib (p=0.172 and p=0.463 respectively). One out of 9 (11%) N-RAS mutated and 3 out of 5 (60%) K-RAS mutated samples had an LC50 value above the 75th percentile for non-mutated AML and were considered resistant. Within relapsed AML the 2 RAS mutated samples had LC50 values of 0.83 and 6.3μM, versus a median value of 6.9μM for non-mutated relapsed AML. In ALL, we found similar results [median LC50 RAS mutated 7.8μM (P25-75: 4.1-12.8μM) vs. non-mutated 17.4μM (P25-75: 4.5-22.9μM), p=0.3], but the groups were very small. In conclusion, primary pediatric AML and ALL samples withRAS mutations show similar distributions of tipifarnib sensitivity as samples withoutRAS mutations. Hence, some RAS mutated samples may be relatively in-vitro resistant to tipifarnib, and some non-mutated samples may be relatively sensitive. Therefore, clinical studies with these compounds should not be restricted to RAS-mutated leukemia. Further studies are necessary to determine the molecular targets of farnesyltransferase inhibitors.

Description: Purpose: Infectious complications remain a substantial cause of morbidity and mortality in patients with acute myeloid leukemia (AML). Candidate gene studies have identified common polymorphisms in genes of molecules of innate immunity as risk factors for susceptibility and/or outcome to a spectrum of pathogens in various populations. This study was performed to investigate whether common polymorphism in genes of innate immunity might influence the risk for serious infection during therapy for pediatric AML. Patients and Methods: Genotype analysis was performed in 168 children treated on clinical trial AML-BFM 93. Infectious events were categorized as microbiologically or clinically documented infections and as fever without an identifiable source. Candidate gene polymorphisms included tumor necrosis factor-alpha, interleukin 6 and 8 (IL6 and IL8), myeloperoxidase, chitotriosidase (CHIT), the low affinity Fc receptor 2A, and the toll like-receptors 2 and 4. Results: The 168 children treated according to clinical trial AML-BFM 93 experienced a total of 508 infectious episodes. Only three patients escaped significant infectious complication. One-hundred-and-ten patients had a least one microbiologically documented infection. Gram-positive bacteria were isolated in the bloodstream of 84 children, whereas Gram-negative bacteria were recovered from the bloodstream of 24 patients. There was an association between Gram-negative bacterial infection and each of the IL6 and CHIT genetic variants. The risk of bacteremia with Gram-negative organisms was significantly higher in patients with the G allele in the IL6 promoter at 174bp [22/123 (17.9%) vs 0/25 (0%); P=.022 (OR 11.31, 95% CI 1.41–90.6)] and in patients with the H allele (24-bp duplication in exon 10) of CHIT [13/56 (23.25) vs 11/112 (9.8%); P=.020 (OR 2.78; 95% CI 1.18–6.54)]. No significant association of the other variant genotypes with any of the clinical endpoints analyzed was observed. Conclusion: The data suggest that variant alleles of both IL6 and CHIT influence susceptibility to infection with Gram-negative bacteria in children undergoing therapy for AML. Further studies are warranted to confirm our findings and to investigate underlying mechanisms.

Description: Ara-C is a key drug in the treatment of AML and ALL. However, resistance may occur, when it is associated with treatment failure. Resistance might be circumvented by other deoxynucleoside analogs. We previously reported increased resistance to ara-C in childhood leukemia subgroups, e.g. in T-cell ALL and AML FAB type M1 and M2. We have now studied subgroup-related differences in sensitivity to ara-C analogs in 362 untreated childhood leukemia and 32 normal pediatric bone marrow samples; all obtained with informed consent. To analyze group-differences the Mann-Whitney-U test was used. In addition, cross-resistance patterns were determined within a group of 60 AML samples in which all 4 deoxynucleoside analogs had been tested successfully, applying the Pearson correlation test after log-transformation of the data. Statistical significance was set at p

Description: Background: Treatment for acute myelogenous leukemia (AML) is one of the most intensive treatment modalities in pediatric oncology. Infection-related mortality is particularly high among these patients during chemotherapy-induced neutropenia. Granulocyte colony-stimulating factor (G-CSF) expands the circulating pool of neutrophils by stimulating proliferation and maturation of myeloid progenitor cells. Although G-CSF is widely used for the prevention of infectious complications in cancer patients, no larger randomized study has evaluated the effect of G-CSF in pediatric cancer patients undergoing treatment for AML. Patients and Methods: The impact of G-CSF on hematopoetic recovery, infectious complications and five-year event-free survival (5-EFS) was prospectively evaluated in the multicenter clinical trial AML-BFM 98. Patients (inclusion criteria: primary diagnosis of AML, age 0–18 years) were randomized to receive or not to receive G-CSF (5 μg/kg/day) after induction 1 (cytarabine, idarubicin, etoposide; AIE) and induction 2 (high-dose cytarabine and mitoxantrone; HAM). Patients who had more than 5% blasts in the bone marrow on day 15 were excluded from randomization. Patients with Down Syndrome were included in the clinical trial, but received only 60% of idarubicin in induction 1 and no second induction (HAM). Results: Between July 1998 and June 2003, 546 patients entered the clinical trial AML-BFM 98 (Down Syndome: n=45). Three-hundred-and-twenty-seven patients were eligible for randomization. Hundred-fifty eight of these patients were randomized to receive G-CSF, and 169 patients not to receive G-CSF. Both groups did not differ in their clinical characteristics. The G-CSF group had a significantly shorter duration of neutropenia (ANC

Description: We have previously reported that FLT3 internal tandem duplications (FLT3/ITD) were detectable in 11.5% of 234 pediatric AML samples at initial diagnoses, and conferred a poor prognosis (Zwaan et al, Blood 2003). However, no data were available on relapsed pediatric AML and on paired initial-relapse samples. Currently we have studied genomic DNA of 99 relapsed and 42 paired initial-relapse pediatric AML samples for FLT3/ITD. Samples were obtained from the AML-BFM-SG, the Dutch Childhood Oncology Group, and by centers participating in the International BFM Relapsed AML 2001/01 study. Of the 99 relapsed patients the median age was 10.2 years and 71% were boys. Median time from diagnosis to first relapse was 14 months. Patient characteristics of the 42 pairs were at diagnosis: median age 7.7 years, sex 80% boys; at relapse: median age 14.3 years. Median time from diagnosis to first relapse was 14 months. ITDs were detected by PCR amplification including a fluorescent labeled primer. Fragments were separated by capillary electrophoresis on an ABI 3100 and analyzed using Genescan software. Of the 99 relapsed samples, 20 were ITD positive (20.2%), with ITDs varying in length from 18–129 bp. ITDs occurred in 15% of children

Description: In order to further improve survival of children undergoing therapy for acute myeloid leukemia (AML), the multicenter clinical trial AML-BFM 98 intensified chemotherapy for standard risk (SR) patients (pts.). In addition, this randomized trial prospectively evaluated whether 2 short cycles of chemotherapy resulted in better prognosis than a 6-week consolidation. Patients and Methods: Between July 1998 and June 2003, 461 pts. 〈 18 years with de novo AML were enrolled in the trial AML-BFM 98. The SR group consisted of 170 (37%) pts. (FABM1/M2 with Auer rods or M4eo with ≤ 5 % blasts in the day 15 bone marrow; all pts. with FAB M3). All other pts. (n=291) were considered as high-risk (HR) pts.. In contrast to trial AML-BFM 93, a 2nd induction (HAM) was included in the treatment strategy for SR pts. (excluding FAB M3) which consisted of high-dose cytarabine (3g/m/12h x3 days) and mitoxantrone (10mg/m/d x2 days). Both SR and HR pts. were then randomly assigned to receive a 6-week consolidation or two short cycles of therapy. Compared to the 6-week consolidation, the short cycles contained higher doses of cytarabine, but the same cumulative dose of anthracylines. All other therapy elements [first induction (AIE; cytarabine, idarubicin and etoposide), intensification (HAE; high dose cytarabine, etoposide), and maintenance therapy] were identical in studies 93 and 98. Results: Overall, 407 out of 461 (88%) pts. achieved remission (CR). Five-year survival, event-free survival (EFS) and disease-free survival (DFS) were 59%±3%, 49%±3% and 55%±3%, respectively. Estimated survival and pEFS were similar to those of study 93 (58%±2% and 50%±2%, p logrank .09 and .80). Analysis of SR pts. (FAB M3 excluded) showed that the additional application of HAM did not improve the prognosis of SR pts. compared to AML-BFM 93 [CR rate 92% vs. 91%, p(chi)=.78; 5-year pEFS 58%±5% vs. 66%±4%; p logrank =.24]. However, when comparing HAM in study 98 and the 2nd chemotherapy cycle in study 93, significantly more severe infections (grade 3/4) occurred with HAM. Overall treatment related mortality in CR was 4% in both HR and SR pts., which was similar to trial AML-BFM 93. In addition, the outcome of pts. randomized for the 6-week consolidation was similar to that of the short cycles (p logrank .81). However, morbidity was lower in the short cycle arm (6 vs. 11 deaths in CCR, 2 vs. 4 in SR pts.). Conclusion: Our results indicate that in SR pts. with AML, a more intensive chemotherapy consisting of HAM does not result in improved survival. Therefore, new treatment options should be considered in this patient group. At the same time, optimizing treatment using the less toxic therapy with short cycles and improvement of supportive care strategies might help to reduce treatment related mortality and improve outcome in children with AML.

Description: Monosomy 7 (−7) and deletion 7q [del(7q)] are rare in childhood AML and are considered to be associated with a poor outcome. We retrospectively analyzed data on 267 children with de novo AML or RAEB-T (PB or BM blasts 〉 20%) with −7 or del(7q) with or without additional cytogenetic aberrations (other). Patients were diagnosed between 1985 and 2003. Karyotypes showed −7 (n=90), −7 other (n=82), del(7q) (n=22), and del(7q) other (n=73). All 24 patients with RAEB-T had −7 +/− other. The median age at diagnosis was 7.6 years (range; 0–18.1) with no difference in age distribution between cytogenetic subgroups. The most common additional cytogenetic aberrations were inv(3)/t(3;3) in 19 patients (−7, n=17; del(7q), n=2) and favorable aberrations [t(8;21), inv(16), and t(15;17)] observed in 25 patients (−7, n=3; del(7q), n=22). The 5-year overall probability of survival for the whole group was 39%, SE=3%, and survival was higher in patients with del(7q) +/− other compared with −7 +/− other (52% vs. 30%). The survival of patients with del(7q) and favorable cytogenetic aberrations (n=22) was higher than that of other patients with del(7q) (75% vs. 46%, p=0.026). There was no significant difference in survival for patients with −7 and −7 other. Complete remission (CR) was obtained in 188 patients (−7 +/− other 62%; del(7q) +/− other 88%), of these, 67 received hematopoietic stem cell transplantation (HSCT) in CR1. There was no significant difference (Mantel-Byar test) in survival for patients who received HSCT in CR1 (49%, SE= 7%) and those who received chemotherapy only (51%, SE=5%, 54% for patients surviving at least the median time to HSCT). In conclusion childhood AML subgroups −7 and del(7q) differ in their associated cytogenetic aberrations and response to therapy.

Description: Acute megakaryoblastic leukemia (AMKL) in children without Down’s syndrome occurs in less than 10% of childhood AML. Data on clinical presentation, immunological and genetic features and treatment strategies are rare. Between 6/1987 and 7/2003 89 children with AMKL (m=46; f=43) were treated according to the AML-BFM trials 87 (n=19), 93 (n=39) and 98 (n=31). AML FAB M7 was centrally confirmed by immunophenotyping (n=86) or immunohistology (n=3). The median age at diagnosis (1.5 yrs) was significantly lower compared to other children with AML (total group n=1,149; median age 7.8 yrs; p 100,000/μl) was less frequent (13%) compared to other AML subtypes (21%; p