Pathologic, cytogenetic, and molecular features of acute myeloid leukemia with megakaryocytic differentiation: A report from the Children's Oncology Group.

BACKGROUND: Acute myeloid leukemia (AML) with megakaryocytic differentiation (AMkL) is a rare subtype of AML more common in children. Recent literature has identified multiple fusions associated with this type of leukemia. METHODS: Morphology, cytogenetics, and genomic sequencing were assessed in patients from Children's Oncology Group trials AAML0531 and AAML1031 with central-pathology review confirmed non-Down syndrome AMkL. The 5-year event-free survival (EFS), overall survival (OS), and RR were evaluated in these AMkL subcategories. RESULTS: A total of 107 cases of AMkL (5.5%) were included. Distinct fusions were identified in the majority: RBM15::MRTFA (20%), CBFA2T3::GLIS2 (16%), NUP98 (10%), KMT2A (7%), TEC::MLLT10 (2%), MECOM (1%), and FUS::ERG (1%); many of the remaining cases were classified as AMkL with (other) myelodysplasia-related changes (MRC). Very few cases had AML-associated somatic mutations. Cases with CBFA2T3::GLIS2 were enriched in trisomy 3 (p = .015) and the RAM phenotype, with associated high CD56 expression (p < .001). Cases with NUP98 fusions were enriched in trisomy 6 (p < .001), monosomy 13/del(13q) (p < .001), trisomy 21 (p = .026), and/or complex karyotypes (p = .026). While different 5-year EFS and OS were observed in AMkL in each trial, in general, those with CBFA2T3::GLIS2 or KMT2A rearrangements had worse outcomes compared to other AMkL, while those with RBM15::MRTFA or classified as AMkl-MRC fared better. AMkL with NUP98 fusions also had poor outcomes in the AAML1031 trial. CONCLUSION: Given the differences in outcomes, AMkL classification by fusions, cytogenetics, and morphology may be warranted to help in risk stratification and therapeutic options.

Prognostic significance of chromosomal abnormalities at relapse in children with relapsed acute myeloid leukemia: A retrospective cohort study of the Relapsed AML 2001/01 Study.

BACKGROUND: In addition to treatment response, cytogenetic and molecular aberrations are the most important prognostic factors in children with de novo acute myeloid leukemia (AML). However, little is known about cytogenetics at the time of relapse. METHODS: This international study analyzed the prognostic value of cytogenetic profiles and karyotypic changes in pediatric relapsed AML in relation to the probability of event-free (pEFS) and overall survival (pOS). For this purpose, cytogenetic reports from all patients registered on the Relapsed AML 2001/01 Study were reviewed and classified. RESULTS: Cytogenetic information at relapse was available for 403 (71%) of 569 registered patients. Frequently detected aberrations at relapse were t(8;21)(q22;q22) (n = 60) and inv(16)(p13.1q22)/t(16;16)(p13.1;q22) (n = 24), both associated with relatively good outcome (4-year pOS 59% and 71%, respectively). Monosomy 7/7q-, t(9;11)(p22;q23), t(10;11)(p12;q23), and complex karyotypes were associated with poor outcomes (4-year pOS 17%, 19%, 22%, and 22%, respectively). Of 261 (65%) patients for whom cytogenetic data were reliable at both diagnosis and relapse, pEFS was inferior for patients with karyotypic instability (n = 128, 49%), but pOS was similar. Unstable karyotypes with both gain and loss of aberrations were associated with inferior outcome. Early treatment response, time to relapse, and cytogenetic profile at time of relapse were the most important prognostic factors, both outweighing karytoypic instability per se. CONCLUSION: The cytogenetic subgroup at relapse is an independent risk factor for (event-free) survival. Cytogenetic assessment at the time of relapse is of high importance and may contribute to improved risk-adapted treatment for children with relapsed AML.

Phase 1 trial, pharmacokinetics, and pharmacodynamics of dasatinib combined with crizotinib in children with recurrent or progressive high-grade and diffuse intrinsic pontine glioma.

BACKGROUND: Progressive/recurrent high-grade and diffuse intrinsic pontine gliomas (DIPGs) are fatal. Treatments targeting molecular pathways critical for these cancers are needed. METHODS: We conducted a phase 1 study (rolling-six design) to establish the safety and maximum tolerated dose (MTD) of dasatinib, an oral platelet-derived growth factor receptor A (PDGFRA) inhibitor, and crizotinib, an oral c-Met inhibitor, in such patients. Pharmacokinetics of both agents were performed. Biomarkers of cellular pathway activation in peripheral-blood mononuclear cells (PBMC) were evaluated before and after administration of dasatinib. PDGFRA and MET amplification, and PDGFRA mutations were studied in tumor samples. RESULTS: Twenty-five patients were enrolled in this study (median age: 11.9 years). Eleven patients had DIPG. Glioblastoma accounted for 40% of cases. Dasatinib at 50 mg/m2 and crizotinib at 130 mg/m2 or 100 mg/m2 were poorly tolerated when administered twice daily. Drug administration was then switched to once daily. Dasatinib administered at 50 mg/m2 and crizotinib at 215 mg/m2 once daily was the MTD. Dose-limiting toxicities consisted of diarrhea, fatigue, proteinuria, hyponatremia, rash, and grade 4 neutropenia. Only two patients received therapy for at least 6 months. No objective radiologic responses were observed. Pharmacokinetics of dasatinib and crizotinib were comparable to previous studies. A statistically significant decrease in the ratio of p-AKT/total AKT in PBMC occurred after dasatinib administration. PDGFRA and MET amplification were found in four and two cases, respectively. Only one of 10 tumors harbored a PDGFRA mutation. CONCLUSIONS: This drug combination was poorly tolerated and its activity was minimal. We do not recommend further testing of this combination in children.

The genetic basis of early T-cell precursor acute lymphoblastic leukaemia.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

Heterogeneous cytogenetic subgroups and outcomes in childhood acute megakaryoblastic leukemia: a retrospective international study.

Comprehensive clinical studies of patients with acute megakaryoblastic leukemia (AMKL) are lacking. We performed an international retrospective study on 490 patients (age ≤18 years) with non-Down syndrome de novo AMKL diagnosed from 1989 to 2009. Patients with AMKL (median age 1.53 years) comprised 7.8% of pediatric AML. Five-year event-free (EFS) and overall survival (OS) were 43.7% ± 2.7% and 49.0% ± 2.7%, respectively. Patients diagnosed in 2000 to 2009 were treated with higher cytarabine doses and had better EFS (P = .037) and OS (P = .003) than those diagnosed in 1989 to 1999. Transplantation in first remission did not improve survival. Cytogenetic data were available for 372 (75.9%) patients: hypodiploid (n = 18, 4.8%), normal karyotype (n = 49, 13.2%), pseudodiploid (n = 119, 32.0%), 47 to 50 chromosomes (n = 142, 38.2%), and >50 chromosomes (n = 44, 11.8%). Chromosome gain occurred in 195 of 372 (52.4%) patients: +21 (n = 106, 28.5%), +19 (n = 93, 25.0%), +8 (n = 77, 20.7%). Losses occurred in 65 patients (17.5%): -7 (n = 13, 3.5%). Common structural chromosomal aberrations were t(1;22)(p13;q13) (n = 51, 13.7%) and 11q23 rearrangements (n = 38, 10.2%); t(9;11)(p22;q23) occurred in 21 patients. On the basis of frequency and prognosis, AMKL can be classified to 3 risk groups: good risk-7p abnormalities; poor risk-normal karyotypes, -7, 9p abnormalities including t(9;11)(p22;q23)/MLL-MLLT3, -13/13q-, and -15; and intermediate risk-others including t(1;22)(p13;q13)/OTT-MAL (RBM15-MKL1) and 11q23/MLL except t(9;11). Risk-based innovative therapy is needed to improve patient outcomes.

Phenotype in combination with genotype improves outcome prediction in acute myeloid leukemia: a report from Children's Oncology Group protocol AAML0531.

Diagnostic biomarkers can be used to determine relapse risk in acute myeloid leukemia, and certain genetic aberrancies have prognostic relevance. A diagnostic immunophenotypic expression profile, which quantifies the amounts of distinct gene products, not just their presence or absence, was established in order to improve outcome prediction for patients with acute myeloid leukemia. The immunophenotypic expression profile, which defines each patient's leukemia as a location in 15-dimensional space, was generated for 769 patients enrolled in the Children's Oncology Group AAML0531 protocol. Unsupervised hierarchical clustering grouped patients with similar immunophenotypic expression profiles into eleven patient cohorts, demonstrating high associations among phenotype, genotype, morphology, and outcome. Of 95 patients with inv(16), 79% segregated in Cluster A. Of 109 patients with t(8;21), 92% segregated in Clusters A and B. Of 152 patients with 11q23 alterations, 78% segregated in Clusters D, E, F, G, or H. For both inv(16) and 11q23 abnormalities, differential phenotypic expression identified patient groups with different survival characteristics (P<0.05). Clinical outcome analysis revealed that Cluster B (predominantly t(8;21)) was associated with favorable outcome (P<0.001) and Clusters E, G, H, and K were associated with adverse outcomes (P<0.05). Multivariable regression analysis revealed that Clusters E, G, H, and K were independently associated with worse survival (P range <0.001 to 0.008). The Children's Oncology Group AAML0531 trial: clinicaltrials.gov Identifier: 00372593.

NUP98/NSD1 and FLT3/ITD coexpression is more prevalent in younger AML patients and leads to induction failure: a COG and SWOG report.

NUP98/NSD1 has recently been reported in association with poor outcome in acute myeloid leukemia (AML). Previous studies also observed a high overlap between NUP98/NSD1 and FLT3/ITD, raising the question as to whether the reported poor outcome is due to NUP98/NSD1 or caused by the co-occurrence of these 2 genetic lesions. We aimed to determine the prognostic significance of NUP98/NSD1 in the context of FLT3/ITD AML. A total of 1421 patients enrolled in 5 consecutive Children's Oncology Group/Children's Cancer Group and SWOG trials were evaluated. NUP98/NSD1 was found in 15% of FLT3/ITD and 7% of cytogenetically normal (CN)-AML. Those with dual FLT3/ITD and NUP98/NSD1 (82% of NUP98/NSD1 patients) had a complete remission rate of 27% vs 69% in FLT3/ITD without NUP98/NSD1 (P < .001). The corresponding 3-year overall survival was 31% vs 48% (P = .011), respectively. In CN-AML, patients with concomitant NUP98/NSD1 and FLT3/ITD had a worse outcome than those harboring NUP98/NSD1 only. In multivariate analysis, the dual NUP98/NSD1 and FLT3/ITD remained an independent predictor of poor outcome, and NUP98/NSD1 without FLT3/ITD lost its prognostic significance. Our study demonstrates that it is the interaction between NUP98/NSD1 and FLT3/ITD that determines the poor outcome of patients with NUP98/NSD1 disease.

miR-155 expression and correlation with clinical outcome in pediatric AML: A report from Children's Oncology Group.

BACKGROUND: Aberrant expression of microRNA-155 (miR-155) has been implicated in acute myeloid leukemia (AML) and associated with clinical outcome. PROCEDURE: We evaluated miR-155 expression in 198 children with normal karyotype AML (NK-AML) enrolled in Children's Oncology Group (COG) AML trial AAML0531 and correlated miR-155 expression levels with disease characteristics and clinical outcome. Patients were divided into quartiles (Q1-Q4) based on miR-155 expression level, and disease characteristics were then evaluated and correlated with miR-155 expression. RESULTS: MiR-155 expression varied over 4-log10-fold range relative to its expression in normal marrow with a median expression level of 0.825 (range 0.043-25.630) for the entire study cohort. Increasing miR-155 expression was highly associated with the presence of FLT3/ITD mutations (P < 0.001) and high-risk disease (P < 0.001) and inversely associated with standard-risk (P = 0.008) and low-risk disease (P = 0.041). Patients with highest miR-155 expression had a complete remission (CR) rate of 46% compared with 82% in low expressers (P < 0.001) with a correspondingly lower event-free (EFS) and overall survival (OS) (P < 0.001 and P = 0.002, respectively). In a multivariate model that included molecular risk factors, high miR-155 expression remained a significant independent predictor of OS (P = 0.022) and EFS (0.019). CONCLUSIONS: High miR-155 expression is an adverse prognostic factor in pediatric NK-AML patients. Specifically, high miR-155 expression not only correlates with FLT3/ITD mutation status and high-risk disease but it is also an independent predictor of worse EFS and OS.

Prognostic factors in children with acute myeloid leukaemia and excellent response to remission induction therapy.

Minimal residual disease (MRD) is a strong prognostic factor in children and adolescents with acute myeloid leukaemia (AML) but nearly one-quarter of patients who achieve MRD-negative status still relapse. The adverse prognostic factors among MRD-negative patients remain unknown. We analysed the AML02 study cohort to identify demographic and genetic prognostic factors. Among the presenting features, certain 11q23 abnormalities, such as t(6;11) and t(10;11), acute megakaryoblastic leukaemia without the t(1;22), and age ≥10 years were associated with inferior outcome in patients who had MRD-negative status after either remission induction I or II. By contrast, those with rearrangement of CBF genes had superior outcome. Our study identifies patient populations for whom close post-remission MRD monitoring to detect and treat emerging relapse and adjustment in treatment intensity might be indicated.

Acute myeloid leukaemia (AML) with t(6;9)(p23;q34) is associated with poor outcome in childhood AML regardless of FLT3-ITD status: a report from the Children's Oncology Group.

Acute myeloid leukaemia (AML) with t(6;9)(p23;q34) is a rare subtype associated with FLT3-internal tandem duplication (ITD) and poor outcomes. The clinical outcomes of paediatric patients with t(6;9) with and without FLT3-ITD treated on six consecutive cooperative trails were evaluated. In contrast to patients without t(6;9), those with t(6;9) had a significantly lower complete remission rate, higher relapse rate (RR), and poor overall survival (OS). Within t(6;9) patients, those with and without FLT3-ITD had an OS of 40% and 27% respectively (P > 0·9), demonstrating that t(6;9) is a high-risk cytogenetic feature in paediatric AML and its clinical impact is independent of the presence of FLT3-ITD.

Correlation of CD33 expression level with disease characteristics and response to gemtuzumab ozogamicin containing chemotherapy in childhood AML.

CD33 is expressed on the majority of acute myeloid leukemia (AML) leukemic blasts and is the target for gemtuzumab ozogamicin (GO), a toxin-conjugated anti-CD33 mAb. In the present study, we quantified the CD33 mean fluorescent intensity of leukemic blasts prospectively in 619 de novo pediatric AML patients enrolled in Children's Oncology Group GO-containing clinical trials and determined its correlation with disease characteristics and clinical outcome. CD33 expression varied more than 2-log fold; a median mean fluorescent intensity of 129 (range, 3-1550.07) was observed. Patients were divided into 4 quartiles, quartiles 1-4 (Q1-4) based on expression and disease characteristics and clinical response defined across quartiles. High CD33 expression was associated with high-risk FLT3/ITD mutations (P < .001) and was inversely associated with low-risk disease (P < .001). Complete remission (CR) rates were similar, but patients in Q4 had significantly lower overall survival (57% ± 16% vs 77% ± 7%, P = .002) and disease-free survival from CR (44% ± 16% vs 62% ± 8%, P = .022). In a multivariate model, high CD33 expression remained a significant predictor of overall survival (P = .011) and disease-free survival (P = .038) from CR. Our findings suggest that CD33 expression is heterogeneous within de novo pediatric AML. High expression is associated with adverse disease features and is an independent predictor of inferior outcome. The correlation between CD33 expression and GO response is under investigation. These studies are registered at www.clinicaltrials.gov as NCT00070174 and NCT00372593.

Residual disease detected by multidimensional flow cytometry signifies high relapse risk in patients with de novo acute myeloid leukemia: a report from Children's Oncology Group.

Early response to induction chemotherapy is a predictor of outcome in acute myeloid leukemia (AML). We determined the prevalence and significance of postinduction residual disease (RD) by multidimensional flow cytometry (MDF) in children treated on Children's Oncology Group AML protocol AAML03P1. Postinduction marrow specimens at the end of induction (EOI) 1 or 2 or at the end of therapy from 249 patients were prospectively evaluated by MDF for RD, and presence of RD was correlated with disease characteristics and clinical outcome. Of the 188 patients in morphologic complete remission at EOI1, 46 (24%) had MDF-detectable disease. Those with and without RD at the EOI1 had a 3-year relapse risk of 60% and 29%, respectively (P < .001); the corresponding relapse-free survival was 30% and 65% (P < .001). Presence of RD at the EOI2 and end of therapy was similarly predictive of poor outcome. RD was detected in 28% of standard-risk patients in complete remission and was highly associated with poor relapse-free survival (P = .008). In a multivariate analysis, including cytogenetic and molecular risk factors, RD was an independent predictor of relapse (P < .001). MDF identifies patients at risk of relapse and poor outcome and can be incorporated into clinical trials for risk-based therapy allocation. This study was registered at www.clinicaltrials.gov as NCT00070174.

Diagnosis and management of acute myeloid leukemia in children and adolescents: recommendations from an international expert panel.

Despite major improvements in outcome over the past decades, acute myeloid leukemia (AML) remains a life-threatening malignancy in children, with current survival rates of ∼70%. State-of-the-art recommendations in adult AML have recently been published in this journal by Döhner et al. The primary goal of an international expert panel of the International BFM Study Group AML Committee was to set standards for the management, diagnosis, response assessment, and treatment in childhood AML. This paper aims to discuss differences between childhood and adult AML, and to highlight recommendations that are specific to children. The particular relevance of new diagnostic and prognostic molecular markers in pediatric AML is presented. The general management of pediatric AML, the management of specific pediatric AML cohorts (such as infants) or subtypes of the disease occurring in children (such as Down syndrome related AML), as well as new therapeutic approaches, and the role of supportive care are discussed.

t(6;9)(p22;q34)/DEK-NUP214-rearranged pediatric myeloid leukemia: an international study of 62 patients.

Acute myeloid leukemia with t(6;9)(p22;q34) is listed as a distinct entity in the 2008 World Health Organization classification, but little is known about the clinical implications of t(6;9)-positive myeloid leukemia in children. This international multicenter study presents the clinical and genetic characteristics of 62 pediatric patients with t(6;9)/DEK-NUP214-rearranged myeloid leukemia; 54 diagnosed as having acute myeloid leukemia, representing <1% of all childhood acute myeloid leukemia, and eight as having myelodysplastic syndrome. The t(6;9)/DEK-NUP214 was associated with relatively late onset (median age 10.4 years), male predominance (sex ratio 1.7), French-American-British M2 classification (54%), myelodysplasia (100%), and FLT3-ITD (42%). Outcome was substantially better than previously reported with a 5-year event-free survival of 32%, 5-year overall survival of 53%, and a 5-year cumulative incidence of relapse of 57%. Hematopoietic stem cell transplantation in first complete remission improved the 5-year event-free survival compared with chemotherapy alone (68% versus 18%; P<0.01) but not the overall survival (68% versus 54%; P=0.48). The presence of FLT3-ITD had a non-significant negative effect on 5-year overall survival compared with non-mutated cases (22% versus 62%; P=0.13). Gene expression profiling showed a unique signature characterized by significantly higher expression of EYA3, SESN1, PRDM2/RIZ, and HIST2H4 genes. In conclusion, t(6;9)/DEK-NUP214 represents a unique subtype of acute myeloid leukemia with a high risk of relapse, high frequency of FLT3-ITD, and a specific gene expression signature.

The prognostic effect of high diagnostic WT1 gene expression in pediatric AML depends on WT1 SNP rs16754 status: report from the Children's Oncology Group.

BACKGROUND: WT1 is aberrantly over-expressed in most cases of AML. We recently demonstrated that WT1 SNP rs16754 correlates with favorable outcome and high diagnostic WT1 expression in childhood AML. We examined the clinical correlates of diagnostic WT1 expression within a contemporary COG trial and determined whether its prognostic impact differs between SNP+ and SNP- patients. PROCEDURE: WT1 mRNA expression was measured via qRT-PCR in diagnostic specimens obtained from 225 patients enrolled on COG-AAML03P1. Direct sequencing of WT1 exon 7 was performed to determine SNP rs16754 genotype. WT1 expression was correlated with disease characteristics, SNP status, and outcome. RESULTS: Patients were categorized into four groups (quartiles: Q1 through Q4) based on diagnostic WT1 expression for analysis. FLT3/ITD (P = 0.017) and WT1 mutations (P < 0.001) both occurred more frequently in patients with the highest WT1 expression. SNP rs16754 frequency did not vary significantly among the quartiles. When all patients were considered, survival outcomes were similar between quartiles. However, when only SNP- patients (n = 150) were analyzed, those with highest WT1 expression (Q4) had the poorest OS (51% vs. 72% for Q1-Q3, P = 0.006) and EFS (35% vs. 54% for Q1-Q3, P = 0.031). Among SNP+ patients (n = 75), survival did not vary significantly between WT1 expression quartiles. CONCLUSION: Although WT1 expression was not prognostic when all patients were considered together, stratifying patients by SNP rs16754 genotype revealed significant differences in outcome. In SNP- patients, high WT1 expression predicted decreased survival in univariate, but not multivariate, analysis, due to a preponderance of high-risk cyto/molecular abnormalities in the highest expression quartile.

BIRC5 (survivin) splice variant expression correlates with refractory disease and poor outcome in pediatric acute myeloid leukemia: a report from the Children's Oncology Group.

BACKGROUND: The inhibitor-of-apoptosis protein survivin, encoded by BIRC5, regulates apoptosis, cell division and proliferation. Several survivin splice variants have been described however, the prognostic significance of their expression has not been well defined in pediatric acute myeloid leukemia (AML). PROCEDURE: Quantitative expression analyses of BIRC5 mRNA (n = 306) and survivin transcript splice variants (n = 90) were performed on diagnostic bone marrow samples from children with de novo AML treated on the clinical trials CCG-2961 and AAML03P1, then correlated with disease characteristics and clinical outcome. RESULTS: Total BIRC5 expression did not correlate with clinical outcome. Fragment length analysis and sequencing of the entire BIRC5 transcript demonstrated three splice variants. The most prominent product, wild-type survivin, was expressed in all samples tested. Two minor transcripts were present in 90 patients treated on CCG-2961; survivin-2B and a novel variant, survivin-ΔEx2, characterized by deletion of BIRC5 exon II. A high 2B/ΔEx2 expression ratio (≥1) correlated with increased diagnostic WBC count, monocytic phenotype, +8 cytogenetics, lower complete remission (45% [n = 10] vs. 88% [n = 59], P < 0.001) and higher induction failure rates (23% [n = 5] vs. 3% [n = 2], P = 0.009). Consistent with this poor induction response, patients with a 2B/ΔEx2 ratio ≥1 had inferior 5-year survival rates (OS 36% vs. 60%, P = 0.011; EFS 23% vs. 53% at 5 years, P = 0.001) and appear to have increased relapse risk (P = 0.056). Subset analyses suggest that relative over-expression of 2B, rather than under-expression of ΔEx2 determines clinical response. CONCLUSIONS: High survivin-2B/ΔEx2 ratios are associated with refractory disease and inferior survival in childhood AML. Survivin splice variant expression warrants prospective evaluation in clinical trials.