Risk stratification for acute myeloid leukemia (AML) uses molecular and cytogenetic abnormalities identified at diagnosis. Response to therapy informs risk, and morphology continues to be used more frequently than flow cytometry. Herein, the largest cohort of pediatric patients prospectively assessed for measurable residual disease (MRD) by flow cytometry (N = 784) is reported. The "difference from normal" (ΔN) technique was applied: 31% of all patients tested positive (AML range, 0.02% to 91%) after the first course of treatment on Children's Oncology Group study AAML0531. Detection of MRD following initial chemotherapy proved the strongest predicator of overall survival (OS) in univariable and multivariable analyses, and was predictive of relapse risk, disease-free survival, and treatment-related mortality. Clearance of MRD after a second round of chemotherapy did not improve survival. The morphologic definition of persistent disease (>15% AML) failed 27% of the time; those identified as MRD- had superior outcomes. Similarly, for patients not achieving morphologic remission (>5% blasts), 36% of patients were MRD- and had favorable outcomes compared with those who were MRD+ (P < .001); hence an increase in myeloid progenitor cells can be favorable when ΔN classifies them as phenotypically normal. Furthermore, ΔN reclassified 20% of patients in morphologic remission as having detectable MRD with comparable poor outcomes. Retrospective analysis using the relapse phenotype as a template demonstrated that 96% of MRD- patients had <0.02% of the relapse immunophenotype in their end of induction 1 marrow. Thus, the detection of abnormal myeloid progenitor cells by ΔN is both specific and sensitive, with a high predictive signal identifiable early in treatment. This trial was registered at www.clinicaltrials.gov as #NCT00372593.

<p><strong>PURPOSE: </strong>Increased CD123 surface expression has been associated with high-risk disease characteristics in adult acute myeloid leukemia (AML), but has not been well-characterized in childhood AML. In this study, we defined CD123 expression and associated clinical characteristics in a uniformly treated cohort of pediatric patients with newly diagnosed AML enrolled on the Children's Oncology Group AAML1031 phase III trial (NCT01371981).</p>

<p><strong>MATERIALS AND METHODS: </strong>AML blasts within diagnostic bone marrow specimens (n = 1,040) were prospectively analyzed for CD123 protein expression by multidimensional flow cytometry immunophenotyping at a central clinical laboratory. Patients were stratified as low-risk or high-risk on the basis of (1) leukemia-associated cytogenetic and molecular alterations and (2) end-of-induction measurable residual disease levels.</p>

<p><strong>RESULTS: </strong>The study population was divided into CD123 expression-based quartiles (n = 260 each) for analysis. Those with highest CD123 expression (quartile 4 [Q4]) had higher prevalence of high-risk rearrangements and -ITD mutations ( &lt; .001 for both) and lower prevalence of low-risk t(8;21), inv(16), and mutations ( &lt; .001 for all). Patients in lower CD123 expression quartiles (Q1-3) had similar relapse risk, event-free survival, and overall survival. Conversely, Q4 patients had a significantly higher relapse risk (53% 39%, &lt; .001), lower event-free survival (49% 69%, &lt; .001), and lower overall survival (32% 50%, &lt; .001) in comparison with Q1-3 patients. CD123 maintained independent significance for outcomes when all known contemporary high-risk cytogenetic and molecular markers were incorporated into multivariable Cox regression analysis.</p>

<p><strong>CONCLUSION: </strong>CD123 is strongly associated with disease-relevant cytogenetic and molecular alterations in childhood AML. CD123 is a critical biomarker and promising immunotherapeutic target for children with relapsed or refractory AML, given its prevalent expression and enrichment in patients with high-risk genetic alterations and inferior clinical outcomes with conventional therapy.</p>

<p>Bi-allelic CEBPA mutations are associated with favorable outcomes in AML. We evaluated the clinical and biologic implications of CEBPA-bZip mutations in childhood/young adult newly diagnosed AML. CEBPA-bZip mutation status was determined in 2,958 AML patients enrolled on COG trials (NCT00003790, NCT0007174, NCT00372593, NCT01379181). Next generation sequencing (NGS) was performed in 1,863 patients, 107 with CEBPA mutations, to characterize the co-occurring mutations. CEBPA mutational status was correlated with disease characteristics and clinical outcomes. CEBPA-bZip mutations were identified in 160/2958 (5.4%) patients, with 132 (82.5%) harboring a second CEBPA mutation (CEBPA-dm) and 28 (17.5%) with a single CEBPA-bZip only. The clinical and laboratory features of the two CEBPA cohorts were very similar. CEBPA-dm and CEBPA-bZip patients experienced identical event-free survival (EFS) of 64% and similar overall survival (OS) of 81% and 89%, respectively (p=0.259); this compared favorably to EFS and OS in CEBPA wild type (CEBPA-WT) of 46% and 61%, respectively (both p&lt;0.001). Transcriptome analysis demonstrated similar expression profiles for CEBPA-bZip and CEBPA-dm cases. Comprehensive NGS of CEBPA-mutant cases identified co-occurring CSF3R and GATA2 mutations in 13.1% and 21.5% of patients, respectively. Patients with dual CEBPA/CSF3R mutations had an EFS of 17% vs. 63% for CEBPA-mutant/CSF3R-WT (p&lt;0.001) with a corresponding relapse rate (RR) of 83% vs. 22%, respectively (p&lt;0.001); GATA2 co-occurrence did not impact outcome. CEBPA bZip domain mutations are associated with favorable clinical outcomes, regardless of mono or bi-allelic status. Co-occurring CSF3R and CEBPA mutations are associated with a high RR and nullifies the favorable prognostic impact of CEBPA mutations.</p>

<p><strong>PURPOSE: </strong>A cryptic inv(16)(p13.3q24.3) encoding the CBFA2T3-GLIS2 fusion is associated with poor outcome in infants with acute megakaryocytic leukemia. We aimed to broaden our understanding of the pathogenesis of this fusion through transcriptome profiling.</p>

<p><strong>EXPERIMENTAL DESIGN: </strong>Available RNA from children and young adults with de novo AML (N=1,049) underwent transcriptome sequencing (mRNA and miRNA). Transcriptome profiles for those with the CBFA2T3-GLIS2 fusion (N=24) and without (N=1,025) were contrasted to define fusion-specific miRNAs, genes, and pathways. Clinical annotations defined distinct fusion-associated disease characteristics and outcomes.</p>

<p><strong>RESULTS: </strong>The CBFA2T3-GLIS2 fusion was restricted to infants &lt; 3 years-old (p&lt;0.001) and presence of this fusion was highly associated with adverse outcome (p&lt;0.001) across all morphological classifications. Further, there was a striking paucity of recurrent cooperating mutations and transduction of cord blood stem cells with this fusion was sufficient for malignant transformation. CBFA2T3-GLIS2 positive cases displayed marked up-regulation of genes with cell membrane/extracellular matrix localization potential, including NCAM1 and GABRE. Additionally, miRNA profiling revealed significant over-expression of mature miR-224 and miR-452, which are intronic miRNAs transcribed from the GABRE locus. Gene-set enrichment identified dysregulated Hippo, TGFβ, and hedgehog signaling, as well as NCAM1 (CD56) Interaction pathways. Therapeutic targeting of fusion-positive leukemic cells with CD56-directed ADC caused significant cytotoxicity in leukemic blasts.</p>

<p><strong>CONCLUSIONS: </strong>The CBFA2T3-GLIS2 fusion defines a highly refractory entity limited to infants that appears to be sufficient for malignant transformation. Transcriptome profiling elucidated several highly targetable genes and pathways, including the identification of CD56, providing a highly plausible target for therapeutic intervention.</p>

<p><strong>PURPOSE: </strong> mutations (+) are common in core binding factor (CBF) AML and have been associated with varying prognostic significance. We sought to define the functional and clinical significance of distinct mutations in CBF pediatric AML.</p>

<p><strong>EXPERIMENTAL DESIGN: </strong>Following transfection of exon 17 (E17) and exon 8 (E8) mutations into HEK293 and Ba/F3 cells, KIT phosphorylation, cytokine independent growth, and response to tyrosine kinase inhibitors (TKI) were evaluated. Clinical outcomes of patients treated on COG AAML0531 (NCT01407757), a phase III study of gemtuzumab ozogamicin (GO), were analyzed according to mutation status (+ vs wild type (-)) and mutation location (E8 vs. E17).</p>

<p><strong>RESULTS: </strong> mutations were detected in 63/205(31%) patients; 22 (35%) involved only E8, 32(51%) only E17, 6(10%) both exons, and 3(5%) alternative exons. Functional studies demonstrated that E17, but not E8, mutations result in aberrant KIT phosphorylation and growth. TKI exposure significantly impacted growth of E17, but not E8, transfected cells. + CBF AML patients had comparable overall survival (OS) to that of - (78%, vs. 81%, p=0.905) but higher relapse rates (RR 43% vs. 21%, p=0.005). E17 + outcomes were inferior to patients [disease free survival (DFS) 51% vs. 73%, p=0.027; RR 21% vs. 46%, p=0.007)] although GO abrogated this negative prognostic impact. E8 mutations lacked significant prognostic impact and GO failed to significantly improve outcome.</p>

<p><strong>CONCLUSIONS: </strong>E17 mutations impact prognosis in CBF AML, as well as response to GO and TKIs, thus clinical trials utilizing both agents should be considered for + patients.</p>

<p>Gemtuzumab-ozogamicin (GO), a humanized-anti-CD33 antibody linked with the toxin-calicheamicin-γ is a reemerging and promising drug for AML. Calicheamicin a key element of GO, induces DNA-damage and cell-death once the linked CD33-antibody facilitates its uptake. Calicheamicin efflux by the drug-transporter PgP-1 have been implicated in GO response thus in this study, we evaluated impact of ABCB1-SNPs on GO response. Genomic-DNA samples from 942 patients randomized to receive standard therapy with or without addition of GO (COG-AAML0531) were genotyped for ABCB1-SNPs. Our most interesting results show that for rs1045642, patients with minor-T-allele (CT/TT) had better outcome as compared to patients with CC genotype in GO-arm (Event-free survival-EFS: p = 0.022; and risk of relapse-RR, p = 0.007). In contrast, no difference between genotypes was observed for any of the clinical endpoints within No-GO arm (all p &gt; 0.05). Consistent results were obtained when genotype groups were compared by GO and No-GO arms. The in vitro evaluation using HL60-cells further demonstrated consistent impact of rs1045642-T-allele on calicheamicin induced DNA-damage and cell-viability. Our results show the significance of ABCB1 SNPs on GO response in AML and warrants the need to investigate this in other cohorts. Once validated, ABCB1-SNPs in conjunction with CD33-SNPs can open up opportunities to personalize GO-therapy.</p>

<p>Purpose Gemtuzumab ozogamicin (GO), a CD33-targeted immunoconjugate, is a re-emerging therapy for acute myeloid leukemia (AML). CD33 single nucleotide polymorphism rs12459419 C&gt;T in the splice enhancer region regulates the expression of an alternatively spliced CD33 isoform lacking exon2 (D2-CD33), thus eliminating the CD33 IgV domain, which is the antibody-binding site for GO, as well as diagnostic immunophenotypic panels. We aimed to determine the impact of the genotype of this splicing polymorphism in patients with AML treated with GO-containing chemotherapy. Patients and Methods CD33 splicing single nucleotide polymorphism was evaluated in newly diagnosed patients with AML randomly assigned to receive standard five-course chemotherapy alone (No-GO arm, n = 408) or chemotherapy with the addition of two doses of GO once during induction and once during intensification (GO arm, n = 408) as per the Children's Oncology Group AAML0531 trial. Results The rs12459419 genotype was CC in 415 patients (51%), CT in 316 patients (39%), and TT in 85 patients (10%), with a minor allele frequency of 30%. The T allele was significantly associated with higher levels of D2-CD33 transcript ( P &lt; 1.0E(-6)) and with lower diagnostic leukemic cell surface CD33 intensity ( P &lt; 1.0E(-6)). Patients with the CC genotype had significantly lower relapse risk in the GO arm than in the No-GO arm (26% v 49%; P &lt; .001). However, in patients with the CT or TT genotype, exposure to GO did not influence relapse risk (39% v 40%; P = .85). Disease-free survival was higher in patients with the CC genotype in the GO arm than in the No-GO arm (65% v 46%, respectively; P = .004), but this benefit of GO addition was not seen in patients with the CT or TT genotype. Conclusion Our results suggest that patients with the CC genotype for rs12459419 have a substantial response to GO, making this a potential biomarker for the selection of patients with a likelihood of significant response to GO.</p>

<p><strong>PURPOSE: </strong>The FLT3 cell-surface receptor tyrosine kinase (CD135) is expressed in a majority of both acute lymphoid leukemia (ALL) and myeloid leukemia (AML). However, the prognostic significance of CD135 expression in AML remains unclear. We therefore evaluated the association between FLT3 surface expression and disease characteristics and outcomes in pediatric patients with AML.</p>

<p><strong>EXPERIMENTAL DESIGN: </strong>We analyzed FLT3 receptor expression on AML blasts by multi-dimensional flow cytometry and its association with disease characteristics, clinical outcomes, and FLT3 transcript level in 367 children with AML treated on the Children's Oncology Group trial AAML0531.</p>

<p><strong>RESULTS: </strong>There was high variability in blast CD135 cell-surface expression across specimens. CD135 expression measure by flow cytometry was not correlated with FLT3 transcript expression determined by quantitative RT-PCR. Overall, CD135 expression was not significantly different for patients with FLT3/WT and those with FLT3/ITD and FLT3/ALM (p=0.25). High cell-surface CD135 expression was associated with FAB M5 subtype (p&lt;0.001), KMT2A rearrangements (p=0.009) and inversely associated with inv(16)/t(16;16) (p&lt; 0.001). Complete remission rate, overall survival, disease-free survival, and relapse rates were not significantly different between patients with low and high CD135 expression.</p>

<p><strong>CONCLUSIONS: </strong>FLT3 cell-surface expression did not vary by FLT3 mutational status, but high FLT3 expression was strongly associated with KMT2A rearrangements. Our study found that there was no prognostic significance of FLT3 expression in pediatric AML.</p>

<p><strong>PURPOSE: </strong>Gemtuzumab ozogamicin (GO), a calicheamicin-conjugated mAb against CD33, has been used in the treatment of acute myeloid leukemia (AML). We evaluated the impact of the addition of GO to standard chemotherapy and hematopoietic stem cell transplant (HCT) in patients with FLT3/ITD.</p>

<p><strong>EXPERIMENTAL DESIGN: </strong>We analyzed children with FLT3/ITD-positive AML (n = 183) treated on two consecutive Children's Oncology Group AML trials (NCT00070174 and NCT00372593). Outcomes were assessed for FLT3/ITD patients receiving standard chemotherapy with or without GO (GO vs. No-GO, respectively), and the impact of consolidation HCT for high-risk FLT3/ITD patients [high FLT3/ITD allelic ratio (ITD-AR)].</p>

<p><strong>RESULTS: </strong>For all FLT3/ITD patients, complete remission (CR) rates for the GO versus No-GO cohorts were identical (64% vs. 64%; P = 0.98). Relapse rate (RR) after initial CR was 37% for GO recipients versus 59% for No-GO recipients (P = 0.02), disease-free survival (DFS) was similar (47% vs. 41%; P = 0.45), with higher treatment-related mortality (TRM) in GO recipients (16% vs. 0%; P = 0.008). Among high-risk FLT3/ITD patients with high ITD-AR, those who received HCT in first CR with prior exposure to GO had a significant reduction in RR (15% vs. 53%; P = 0.007), with a corresponding DFS of 65% versus 40% (P = 0.08), and higher TRM (19% vs. 7%; P = 0.08).</p>

<p><strong>CONCLUSIONS: </strong>CD33 targeting with HCT consolidation may be an important therapeutic strategy in high-risk FLT3/ITD AML and its efficacy and associated toxicity warrant further investigation. Clin Cancer Res; 1-7. ©2015 AACR.</p>