ALBERT

Description: Pediatric patients with core binding factor (CBF) acute myeloid leukemia (AML) [t(8;21) and inv(16)] typically have favorable outcome, yet 20–40% of patients will relapse, suggesting that additional events may impact disease response. Recently, mutations of the c-KIT receptor tyrosine kinase gene were implicated as a prognostic factor in adults with CBF AML. The prevalence and prognostic significance of c-KIT mutations in pediatric CBF AML has not been well established. We analyzed diagnostic specimens from 156 pediatric patients with CBF AML enrolled on serial AML protocols CCG 2891, 2961 and COG AAML03P1 with 3 different methodologies (direct sequencing, HPLC, and micro capillary electrophoresis) to identify mutations in c-KIT exons 8 and 17. Mutations were detected in 27 samples (17%); 13 (48%) involved exon 8, 13 (48%) involved exon 17, and 1 (4%) involved both regions. Of 15 inv(16) specimens with c-KIT mutations, 9 (60%) involved exon 8, 5 (33%) involved exon 17, and 1 (7%) affected both exons. In contrast, of 12 t(8;21) specimens with c-KIT mutations, 4 (33%) involved exon 8 whereas 8 (67%) affected exon 17. Clinical outcome data from 98 CBF AML patients enrolled on CCG 2961 was analyzed to determine the prognostic significance of c-KIT mutations in this patient population. CBF AML patients with c-KIT mutations had similar clinical characteristics (age, sex, race, presenting white blood cell count and blast percentage) to those CBF AML patients without c-KIT mutations. There was no difference in the rate of complete remission (CR) (c-KIT mutations, 88% vs. no mutation 92%; p=0.645), overall survival (OS) at 5 years from study entry (c-KIT mutation, 68% vs. no mutation 71%; p=0.733), or corresponding relapse risk (RR) from the time of CR (c-KIT mutations, 33% vs. no mutation 31%; p=0.92). The relative prognostic significance of the location of c-KIT mutation (i.e. exon 8 or exon 17) was also evaluated. No differences were found in CR rates (exon 8 mutation, 100% vs. exon 17 mutation, 75%; p= 0.467), OS from CR (exon 8 mutation, 86% vs. exon 17 mutation, 47%; p=0.088), or corresponding RR at 5 years from CR (exon 8 mutation, 29% vs. exon 17 mutation, 50%; p=0.517). This study demonstrates that the prevalence of c-KIT mutations in pediatric CBF AML patients enrolled on serial CCG/COG AML protocols is lower than that observed in adult studies. We found that c-KIT mutations lacked prognostic significance in pediatric CBF AML patients treated with intensive chemotherapy on CCG 2961, though there was a suggestion that patients with exon 17 mutations may have worse outcome. Outcome data from the entire cohort (CCG 2891, 2961 and COG AAML03P1) will be analyzed to determine whether this finding is significant with larger scale analysis.

Description: Evolution and progression of AML is considered a result of cooperation of multiple genetic and epigenetic alterations. FLT3 internal tandem duplication (ITD) is a common mutation in AML and is considered a secondary, proliferative event in the pathogenesis of AML. We used SNP/CGH microarrays to evaluate the mechanism of disease evolution in FLT3/ITD-positive AML. Diagnostic DNA from patients with FLT3/ITD (N=25), FLT3 activation loop mutation (FLT3/ALM, N=15) or with no FLT3 mutations (FLT3/WT, N=55) was evaluated for copy number and LOH alterations. 12/25 (48%) FLT3/ITD-positive patients had loss of heterozygosity (LOH) of a large segment of 13q arm downstream of the FLT3 gene without an associated chromosome loss, demonstrating a copy neutral LOH (CN-LOH) state. In contrast, CN-LOH was not detected in those with FLT3/ALM or FLT3/WT suggesting that FLT3/ITD is causally associated with evolution of CN-LOH. Single cell analysis of the FACS purified leukemic blasts from FLT3/ITD-positive samples that were heterozygous for 13q (no CN-LOH) by SNP/CGH arrays demonstrated the presence of 3 distinct molecular subtypes that included a subpopulation with CN-LOH, as well as heterozygous ITD and FLT3/WT, demonstrating oligoclonal nature of the leukemic blast population which prevented identification of CN-LOH. In patient samples with CN-LOH, CFU-GM and BFU-E colonies derived from CD34+/CD33- progenitors demonstrated heterozygous ITD, with no LOH or copy number alterations. In contrast, more differentiated, CD33+ leukemic blasts as well as CFU-GM colonies derived from CD34+/CD33+ progenitors had homozygous ITD and CN-LOH. This cumulative data demonstrates that CN-LOH of the 13q, as a result of gene conversion-type event mediates the pathogenesis of FLT3/ITD and is a secondary event in the evolution of high risk FLT3/ITD. Gene conversion, leading to haplo-insufficiency may be an important event in the evolution of AML.

Description: Approximately 40% of patients treated with chemotherapy for pediatric AML will relapse after initial treatment and 8–10% will die of toxic side effects of chemotherapy. This highly variable response to standardized treatment protocols is due in part to genetic variation among patients in the metabolism of chemotherapeutic agents and in the response to the DNA damage they induce. We have analyzed polymorphisms in the coding region of the DNA alkylation repair gene O6-Methylguanine-DNA Methyltransferase (MGMT) in 356 patients with de novo pediatric AML to determine whether naturally occurring MGMT variant alleles correlate with leukemia susceptibility, treatment efficacy, and the incidence of adverse effects of chemotherapy. The polymorphism rs2308321 (Ile143Val) has been shown to modify biochemical properties of the MGMT protein, with altered substrate specificity of the rare valine allele (G genotype) compared with the isoleucine allele (A genotype) (Margison et al, Carcinogenesis, 26;1473, 2005). All patients were treated on Children’s Cancer Group protocols 2941 and 2961. Outcomes were examined stratified by genotype. All patients received intensively timed induction therapy with IDA-DCTER (idarubicin, dexamethasone, Ara-C, thioguanine, etoposide and daunomycin) given on days 0 to 3 followed by DCTER given on days 10 to 13. On recovery of ANC and platelet counts, patients were randomized to consolidation therapy with a further course of IDA-DCTER/DCTER or IDA-FLAG (idarubicin, fludarabine, Ara-C and G-CSF). Patients received intensification with a single course of high dose Ara-C and L-asparaginase unless a family donor was available when allogeneic transplant was performed after busulfan and cyclophosphamide. Black children were significantly more likely to have a homozygous wild-type AA genotype than white children (p=0.002). When compared to a healthy control population, there was no correlation between MGMT polymorphisms and the risk of AML overall. However, in a case-case comparison of genetic sub-types of AML, children with a variant allele (AG or G/G genotype) were significantly more likely to have monosomy 7 than AA cases (9% vs 1%; p=0.01); in contrast, children with trisomy 8 were significantly more likely to have an AA genotype (8% vs 0%; p=0.028). The presence of at least one G allele was associated with a significant reduction in the incidence of relapse. There was no effect on treatment-related mortality (TRM). In a multivariate Cox model adjusted for race, the increased risk of relapse (RR) associated with the variant G allele remained significant (hazard ratio 1.72 95% CI 1.03–2.86; p=0.037). These results suggest that genotyping at the rs2308321 polymorphism in the MGMT gene could be useful in the risk stratification of patients with AML prior to therapy. A/A A/G+G/G N % ± 2 SE N % ± 2 SE p-value 5yr OS from end of course 1 280 54 ± 6 76 71 ± 11 0.031 5yr RR from end of course 1 280 43 ± 6 76 27 ± 11 0.014 5yr TRM from end of course 1 280 14 ± 4 76 14 ± 8 0.11

Description: Abstract 328 Hematopoietic cell transplantation (HCT) is utilized in the treatment of a subset of children with acute myeloid leukemia (AML). Although presence of minimal residual disease (MRD) has been associated with a high relapse rate in children with AML, the clinical implications of MRD prior to HCT have not previously been defined. We explored the clinical outcomes associated with the presence of morphologic or sub-morphologic disease prior to HCT. In the period of 1995-2007, 89 patients age 1-18 years received myeloablative conditioning and allogeneic HCT from related (N=36) or unrelated (N=53) donors at Fred Hutchinson Cancer Research Center. At the time of HCT, 63 patients received transplants in complete remission (CR) and 26 had morphologic disease. Among those in morphologic CR, 43 were in 1st CR, 17 in 2nd CR and 3 were in 3rd CR. All patients had bone marrow evaluation for MRD by multi-dimensional flow cytometry (MDF) 2-4 weeks prior to HCT. MDF testing showed that 10 of 63 patients (16%) who received transplants in morphologic CR had MRD levels ranging from 0.3% to 12% (median 3.7%) while the remaining 53 patients had no detectable MRD. The median follow up for the 38 survivors is 6 years (range 1 – 12). Actuarial overall survival at 3 years from HCT for patients who received HCT with and without morphologic disease was 12% vs. 59%, (p

Description: CEBPA mutations have been associated with improved outcome in adult acute myeloid leukemia (AML). We evaluated the prevalence and prognostic significance of CEBPA mutations in 847 children with AML treated on 3 consecutive pediatric trials. Two types of CEBPA mutations—N-terminal truncating mutations and in-frame bZip-domain mutations—were detected in 38 (4.5%) of 847 patients tested; 31 (82%) of 38 patients with mutations harbored both mutation types. Mutation status was correlated with laboratory and clinical characteristics and clinical outcome. CEBPA mutations were significantly more common in older patients, patients with FAB M1 or M2, and patients with normal karyotype. Mutations did not occur in patients with either favorable or unfavorable cytogenetics. Actuarial event-free survival at 5 years was 70% versus 38% (P = .015) with a cumulative incidence of relapse from complete remission of 13% versus 44% (P = .007) for those with and without CEBPA mutations. The presence of CEBPA mutations was an independent prognostic factor for improved outcome (HR = 0.24, P = .047). As CEBPA mutations are associated with lower relapse rate and improved survival, CEBPA mutation analysis needs to be incorporated into initial screening for risk identification and therapy allocation at diagnosis. The clinical trials in this study are registered at http://www.clinicaltrials.gov under NCT00002798 and NCT00070174.

Description: Nucleophosmin mutations (NPM-mu) result in aberrant cytoplasmic localization of the NPM protein and occur in 25–35% of adult AML. NPM-mu are most commonly found in cases with normal karyotype, and are frequently associated with FLT3/ITD mutations. NPM-mu have been associated with high remission induction rates and improved survival, especially in patients with normal karyotype that lack FLT3/ITD mutations. The incidence and clinical significance of NPM-mu in childhood AML are less well-characterized. The AIEOP in Italy reported NPM-mu in 7 of 107 (6.5%) children treated on its AML02 protocol, and a Taiwanese group reported NPM-mu in 1 of 47 (2.1%) of children. The prognostic significance of NPM-mu in childhood AML is not known. The purpose of this study was to determine the incidence and clinical significance of NPM-mu in two large cohorts of children with newly-diagnosed AML treated on U.S. cooperative group phase III clinical trials (CCG-2961 and POG-9421). Criteria for selection of study patients included enrollment on the therapeutic trial and availability of banked genomic DNA (for CCG-2961) or RNA (for POG-9421). 919 patients met these criteria (566 from CCG-2961, 353 from POG-9421). For the genomic DNA samples, exon 12 of the nucleophosmin gene was directly amplified by PCR. The RNA samples were reverse transcribed to cDNA prior to PCR amplification. Mutations were detected using SSCP gel electrophoresis and confirmed by direct sequencing. The incidence of NPM-mu was 8.8% (9.5% for CCG, 7.7% for POG). As in prior reports, all of the mutations consisted of 4-bp insertions that resulted in changes in the 2 trytophan residues at AA positions 288 and 290 (important for nuclear localization). Only 48% of the mutations were of the “A” type (compared to 70–80% in adult AML), and 36% were novel mutations. FLT3/ITD mutations were more common in NPM-mu than NPM-wild type (wt) patients (17% vs. 9%, p=0.0381). NPM-mu patients were older than NPM-wt (median age 12 vs. 9 years, p=0.018). NPM-mu was particularly uncommon in children less than 3 years (1 mutation in 178 patients). Females accounted for 62% of the NPM-mut patients vs. 46% of the NPM-wt patients (p=0.0154). 73% of NPM-mut patients had normal cytogenetics, vs. 25% of NPM-wt patients (p

Description: Molecular alterations of the WT1 gene have been associated with clinical outcome in adult AML. We evaluated the prevalence and prognostic significance of WT1 mutations in a cohort of 842 pediatric patients treated on pediatric AML trials CCG-2941, CCG-2961, or COG-AAML03P1. Exons 6–10 of the WT1 gene were evaluated by microcapillary electrophoresis and direct sequencing. Of the 842 samples diagnostic specimens analyzed, 68 (8%) contained mutations in exon 7 (n=62), exon 8 (n= 5), or exon 9 (n=1). Correlation analyses were done to determine whether the presence of WT1 mutations is associated with laboratory and disease characteristics and clinical outcome. There were no differences in sex, race, median diagnostic blast %, or median diagnostic WBC count between samples from patients carrying WT1 mutations and those from patients who did not; however, such mutations were less common in the younger patients (age, 0–2 years; p

Description: CCAAT/enhancer binding protein-α (C/EBPα) is a transcription factor that regulates granulocytic differentiation. Molecular alterations of the CEBPα gene which alter its function have been identified in AML; their presence may have prognostic implications in AML. We evaluated the prevalence and prognostic significance of CEBPα mutations in a cohort of 428 pediatric AML patients treated on pediatric AML trials CCG-2941 and CCG-2961. Initial screening of 100 samples by direct sequencing of the entire coding region of the CEBPα gene identified mutations in 8/100 (8%) of the patients tested. All 8 mutations were insertional or deletional mutations. A multiplex micro-capillary electrophoresis was devised to facilitate further screening of an additional 328 samples. Of the 428 samples analyzed, 65 mutations were identified in 57 patients (13%). 16/65 mutations (25%) were insertions or deletions in the N-terminal region, 14 of which would cause a frameshift resulting in a premature stop codon. 32/65 (49%) mutations involved the second transactivation domain (TAD2); except for one case, all were 6 bp insertions, and except for one case, all occurred at the same location. 17/65 (26%) mutations were in-frame insertions (N=16) or deletions (N=1) in the basic region leucine zipper (bZip) domain. This resulted in the insertion of one to twelve amino acids, or the deletion of six amino acids, which would be expected to disrupt the leucine zipper. 6/57 (11%) patients with mutations harbored more than one distinct mutation; all such instances of combined mutations included at least one mutation in the N-terminal region coupled with at least one mutation in the bZip domain. Presence of mutations was correlated with laboratory and clinical characteristics and clinical outcome. There were no differences in median age, sex, race, median diagnostic blast % or median diagnostic WBC between those with and without CEBPα mutations, although such mutations were less common in the younger patients between ages of 0–2 years (p=0.02). CEBPα mutations were more common in those with normal karyotype (38% vs. 21%, p=0.03) and less common in those with 11q23 abnormalities (8% vs. 26%, p=0.01). Of the 57 patients with CEBPα mutations, 15% and 10% had t(8;21) and inv(16), respectively, no different than those without mutations. Four of the 57 patients with CEBPα mutations had FLT3/ITD (7%) compared to 41/363 (11%) of the CEBPα-WT patients (p=0.46). Response to induction therapy was similar for those with and without mutations (91% vs. 86%, p=0.43). Actuarial overall survival (OS) from study entry was 63% for those with and 52% for those without CEBPα mutations (P=0.12). Of those who achieved an initial remission, cumulative incidence of relapse from remission was 25% vs. 39% for those with and without CEBPα mutations (p=0.069) with a corresponding OS at 5 years from remission of 67% vs. 58%, (p=0.27). CEBPα mutations are frequent events in pediatric AML and are highly associated with normal karyotype. Analysis of a larger cohort of patients may determine whether those with CEBPα mutations have a lower relapse rate and improved survival.

Description: FLT3 internal tandem duplication (FLT3/ITD) is a common somatic mutation in acute myeloid leukemia (AML) with significant variation in the position, length, and number of duplications of the FLT3 gene. We evaluated these physical characteristics in FLT3/ITD-positive patients who were treated on CCG-2941/2961 and correlated them with clinical outcome. Fiftynine of 77 FLT3/ITD-positive patients (77%) had a single ITD, 16 (21%) had 2 ITDs, and 2 (3%) had 3 ITDs. The length of the duplicated region varied from 6 to 51 amino acids, and in all cases amino acid residues Y591–Y597 were duplicated. Structural analysis demonstrated that Y591–Y597 encodes the switch and zipper regions of the juxtamembrane domain of FLT3. In addition, 24 of 77 patients (31%) had duplication of the critical STAT5 docking sites Y589/591. Patients with longer ITDs had a worse relapse-free survival (19% vs 51%, P = .035), while the presence of more than 1 ITD was not clinically significant. Physical characteristics including the length of FLT3/ITD may influence FLT3 activation state by altering its structure and may impact response to therapy.

Description: We previously demonstrated that the presence of residual leukemic blasts (MRD) in patients in morphologic remission was predictive of subsequent leukemic relapse. More recently, we developed a four-color multidimensional flow cytometric method using standardized rather than patient specific panels, distinguishing the abnormal cells based on “difference from normal”. The feasibility of utilizing this method, which does not require a diagnostic specimen and is applicable even if the phenotype of the leukemic clone changes was tested in a blinded study as part of the recently completed COG AML pilot AAML03P1 that enrolled 341 patients. 302/341 patients in the study consented to participate in the biology portion of the study. 223 (74%) had evaluable specimens at the end of induction I, 191 (87%) of which achieved a morphologic CR at the end of induction I. Of the 191 patients in CR at the end of induction I, 48 (25%) had evidence of MRD. The level of MRD ranged from 0.02% to 3% with 70% of patients having an MRD level between 0.1% to 1%. Relapse risk was assessed in those with and without MRD. Those with evidence of MRD were at significantly higher risk of subsequent relapse, with a relapse-free survival (RFS) from end of induction I of 36% for those with MRD compared to 70% for those without MRD (p 〈 0.001). The corresponding overall survival at 2 years from induction I was 63% and 86% for those with and without MRD (p=0.003). 181 patients who were in morphologic CR at the end of induction II had evaluable samples for MRD analysis. MRD was detected in 36/181 patients (20%) and presence of MRD was associated with a RFS from end of induction II of 34% compared to that of 70% in those without MRD (p