High-risk Pediatric Acute Myeloid Leukemia Research Articles

Impact of Molecular Risk on Efficacy of Blood and Marrow Transplantation for Pediatric Acute Myeloid Leukemia: A Report from the Children's Oncology Group

Outcomes for children and adolescents with newly diagnosed acute myeloid leukemia (AML) have only shown modest improvement over the past three decades. Additionally, outcomes for relapsed disease continue to be poor. As a result, there is a pressing need to optimize consolidation therapy to ensure deep and persistent remissions and prevent future relapse. In particular, the utility of bone and marrow transplant (BMT) for consolidation in pediatric AML has been debated for decades. A prior Children's Oncology Group (COG) study risk-stratified patients on the basis of cytogenetic alterations and demonstrated that consolidative BMT performed in first complete remission (CR1) improved long-term outcomes specifically in patients diagnosed with intermediate-risk disease, but found no survival benefit in patients with either low- or high-risk disease. Since then, whole genome and transcriptome sequencing have uncovered a large number of somatic mutations and cryptic fusions that carry prognostic significance in pediatric AML. Combining these molecular features with traditional cytogenetic risk stratification results in the re-classification of a significant number of patients. While this cytomolecular risk stratification is now being used in contemporary clinical trials (e.g., AAML1831/NCT04293562), the merits of consolidative BMT have not been rigorously re-assessed using these newly defined risk strata. To address this gap, we analyzed clinical and biological data from patients enrolled on COG phase 3 clinical trials AAML0531/NCT00372593 and AAML1031/NCT01371981. We filtered the resulting 2,024 patients to include those who had sufficient diagnostic data to ascertain their cytomolecular risk stratification. Additional inclusion criteria were achieving CR1; completing induction I, induction II, and intensification I; and undergoing BMT after intensification I, which was the recommended time point for BMT on both protocols. Our final analysis included 1,004 patients who met all inclusion criteria. BMT versus chemotherapy alone in CR1 was recommended based on indications provided by AAML0531 or AAML1031, which differ from one another (e.g., AAML1031 incorporated measurable residual disease). Clinical outcomes were assessed using Kaplan-Meier analysis and differences between groups were determined using log-rank testing. Clinical outcomes were superior for patients in the cytomolecular high-risk cohort who underwent BMT in CR1 relative to those who received chemotherapy alone. Specifically, high-risk patients who underwent BMT had a significantly improved five-year OS of 60.8 ± 9.3% (versus 41.3 ± 7.7%; p = 0.002) ( Fig. 1A) and DFS of 55.9 ± 9.4% (versus 30.6 ± 7.2%; p < 0.001) (data not shown). We also observed a decreased incidence of relapse in high-risk patients who received BMT, with a RR of 31.8 ± 8.8% relative to 64.5 ± 7.5% for patients in the chemotherapy only group (p < 0.001) ( Fig. 1B). Conversely, there was no difference in any of these outcome measures for patients in the low- and intermediate-risk groups who underwent BMT in CR1 versus receiving chemotherapy alone. Subsequent and ongoing analysis preliminarily indicates that while a subset of high-risk cytomolecular subtypes benefit from BMT in CR1, others do not. Increased availability of high-throughput sequencing has facilitated the identification of numerous molecular features that have prognostic importance in pediatric AML, leading to more accurate risk stratification and opportunities to better allocate patients to appropriate risk-adapted therapy. In this study, we demonstrate that incorporation of these molecular features into the traditional risk stratification schema significantly impacts the clinical indications for BMT as consolidation therapy in pediatric AML. Whereas BMT was previously found to benefit only those patients with intermediate-risk disease, our data suggest that patients with contemporary high-risk cytomolecular alterations benefit significantly from consolidative BMT. Notably, our current analysis does not yet address the potential bias that patients who reach BMT systematically differ from those patients who did not reach BMT (e.g., toxicity that precludes BMT), which we will address with multivariable analysis using BMT as a time varying exposure. These findings support the need for further prospective studies evaluating the role of BMT in high-risk pediatric AML.

Post Hematopoietic Cell Transplantation Maintenance Therapy with Low-Dose Azacitidine in a Pediatric Population with High-Risk Myeloid Neoplasms

Patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) undergoing allogeneic hematopoietic cell transplantation (HCT) remain at risk for relapse. Children with relapsed AML and high-risk features including prior HCT, early relapse, and high-risk cytomolecular features experience survival of ≈30%. Given the even more dismal survival and limited therapy options for post-HCT relapse, azacitidine (aza) has been explored as post-HCT maintenance therapy based on potential disease targeting activity and enhancement of graft versus leukemia. Studies to date suggest that this approach is safe with signals of favorable survival. A phase 3 trial in adults did not show a survival benefit, although results were confounded by a high rate of withdrawal and a minority of patients received all intended cycles. We performed a descriptive analysis of all patients with high-risk myeloid neoplasms who underwent allogeneic HCT at Seattle Children's Hospital (SCH) from November 2017 to January 2023 and received post-HCT maintenance aza. Patients were considered high-risk if they had undergone prior HCT, measurable residual disease at HCT, ≥CR2, or high-risk cytomolecular features. Patients received myeloablative conditioning according to standard protocols. All patients were in remission prior to aza initiation. Aza 36-50 mg/m 2 was administered as IV or subcutaneous infusions daily for 5 consecutive days on a 28-day cycle with intention to continue therapy with cycles beginning through 1-year post-HCT. Nineteen patients began aza therapy at a median of 109 days (range 66-263) post-HCT. Demographics and disease information plus the primary high-risk feature for consideration of post-HCT aza are reported in Table 1. Following aza initiation, 9 (47.4%) patients continued subsequent aza cycles at their referring institution with concurrent remote management by SCH as needed. Median post-HCT follow-up was 30.4 months (range 7-66). The median number of completed aza cycles was 8 (range 3-12) and 14 (73.7%) patients received all intended cycles through 1 year following HCT. In 3 patients, aza cycles were delayed >4 weeks due to non-neutropenic fever or infection; de-escalation to 36 mg/m 2 for subsequent courses in the 2 patients who were receiving 50 mg/m 2 led to no further delays. There were no delays due to marrow suppression, no increased transfusion needs, and no hospitalizations attributed to aza. Fourteen (73.7%) patients had grade II acute graft versus host disease (GVHD) prior to initiation of aza. Two (10.5%) of these patients subsequently developed chronic oral GVHD, 1 diagnosed incidentally during their last aza cycle and 1 diagnosed after completion of aza. Two (10.5%) patients experienced ≤ grade II acute GVHD flares while tapering systemic immunosuppressive treatment (IST) and receiving aza, prompting escalation of same systemic IST (n=1) or addition of topical IST (n=1). Seventeen of 18 patients who were in remission at 1-year post-HCT were tapering or off IST. Five (26.3%) patients discontinued aza before 1 year post-HCT: 2 due to relapse, 2 per family preference, and 1 for inflammatory process with persistent dual chimerism from double cord HCT. Three-year estimates for overall and relapse free survival were 92% (95% CI 54-99) and 76% (95% CI 46-91), respectively, and cumulative incidence of relapse was 24% (95% CI 7-48) (Figure 1). The 6 patients with history of prior HCT are all are alive with ongoing remission at ≥3 years following second HCT with aza maintenance. Our experience demonstrates that post-HCT maintenance therapy with low-dose aza for high-risk pediatric AML/ MDS is tolerable with limited toxicity and without significant impact on marrow suppression or GVHD. The ability to successfully implement a post-HCT aza regimen in partnership with local institutions allowed families to return home, potentially reducing emotional hardship and financial toxicity due to travel/ housing and supporting feasibility. Outcomes in this cohort were improved compared to historical controls; this was especially notable in patients undergoing 2 nd HCT and in patients ≥CR2. Our findings suggest a potential role for post-HCT low-dose aza in children with high-risk AML/ MDS but further prospective pediatric studies are needed to define optimal dosing and evaluate for benefit.

Immune profiling after allogeneic hematopoietic cell transplantation in pediatric acute myeloid leukemia.

Although allogeneic hematopoietic cell transplant (allo-HCT) is curative for high-risk pediatric acute myeloid leukemia (AML), disease relapse remains the primary cause of posttransplant mortality. To identify pressures imposed by allo-HCT on AML cells that escape the graft-versus-leukemia effect, we evaluated immune signatures at diagnosis and posttransplant relapse in bone marrow samples from 4 pediatric patients using a multimodal single-cell proteogenomic approach. Downregulation of major histocompatibility complex class II expression was most profound in progenitor-like blasts and accompanied by correlative changes in transcriptional regulation. Dysfunction of activated natural killer cells and CD8+ T-cell subsets at relapse was evidenced by the loss of response to interferon gamma, tumor necrosis factor α signaling via NF-κB, and interleukin-2/STAT5 signaling. Clonotype analysis of posttransplant relapse samples revealed an expansion of dysfunctional T cells and enrichment of T-regulatory and T-helper cells. Using novel computational methods, our results illustrate a diverse immune-related transcriptional signature in posttransplant relapses not previously reported in pediatric AML.

Pediatric Acute Myeloid Leukemia with Co-Occurring BCR::ABL and CBFA2T3::GLIS2 Dual Fusion with Deep Response to FOLR1-Targeting Antibody Drug Conjugate Stro-002 and Tyrosine Kinase Inhibitor

Introduction: Pediatric acute myeloid leukemia (AML) is characterized by significant cytogenetic and molecular heterogeneity. AML with CBFA2T3::GLIS2 fusion (CBF::GLIS) involving infants and very young children is a rare but uniformly refractory disease with high mortality. Laboratory data demonstrates that this fusion is sufficient for leukemic transformation without recurrent secondary variants. These studies have also linked CBF::GLIS fusion to induction of folate receptor alpha (FOLR1) expression in transformed cells (Meshinchi et al, 2020). Incidence of Ph+ de novo (BCR::ABL minor breakpoint, p190 hybrid) disease ranges from 0.5-3% of all AML cases across the age spectrum (Soupir et al, 2017). We, for the first-time, report dual existence of both fusions in a child with AML and discuss novel interventions including combination of a BCR::ABL tyrosine kinase inhibitor (TKI) and antifolate therapy consisting of methotrexate (MTX) and FLOR1-specific antibody-drug conjugate (ADC) STRO-002 (Sutro Biopharma) based on available data regarding its in-vitro preclinical activity in CBF::GLIS disease (Meshinchi et al, ASH abstract, 2021). Case: A 2-year-old girl of Vietnamese descent received a bone marrow examination for bicytopenia and macrocytosis. Flow cytometry confirmed the poor prognostic RAM immunophenotype AML (CD45 dim, HLA-DR negative, CD38 dim, CD56 bright) and FOLR1 expression. Marrow aspirate smears showed numerous hypercellular spicules and blasts with scant cytoplasm and some blebbing but no granules or Auer rods. Marrow exhibited 100% cellularity. CNS was not involved. Review of diagnostic genomic profile mid induction showed a BCR::ABL minor breakpoint fusion as well as CBF::GLIS fusion. The child began AML induction on COG AAML1831 trial randomized to the experimental arm with CPX-351 and gemtuzumab ozogamicin (GO). Due to lack of response to AML therapy at end of Induction (EOI) I, she was taken off protocol and started on modified ALL regimen for Induction II consisting of cytarabine, low dose weekly methotrexate and bimonthly peg-asparaginase with addition of an oral TKI, dasatinib. EOI II marrow examination showed improvement with reduction in blasts (11%) and detectable BCR::ABL but undetectable CBF::GLIS by Q-RT-PCR. Induction III included low dose weekly methotrexate, cytarabine, GO, and dasatinib. EOI III marrow examination showed complete morphologic remission (CR) with no evidence of minimal residual disease (MRD) or fusion transcripts of either clone. Due to availability of a matched sibling donor, the child underwent hematopoietic stem cell transplantation (HSCT); however, was noted to have 3% blasts on day +83 marrow examination despite previously commencing dasatinib post-transplant prophylaxis. BCR::ABL and CBF::GLIS transcripts were again detectable at low levels. Dasatinib dosage was maximized and weekly MTX and bimonthly peg-asparaginase were reintroduced, but the marrow disease burden increased significantly to 78% blasts positive only for CBF::GLIS fusion. Given availability of FOLR1 directed ADC on single-patient compassionate use basis, patient received single agent STRO-001 on a bi-monthly basis. With rising ANC, evaluation of marrow after 2nd STRO-002 dose remarkably showed CR with MRD negative remission by flow and molecular studies. This time remission was consolidated with monthly STRO-002 and donor lymphocyte infusions (DLI). Currently, patient remains in MRD-negative remission after the 3 of 4 planned STRO-002/DLI maintenance. Patient has maintained a 100% Lansky play score and tolerated the regimen with only prolonged initial cytopenia requiring supportive care as a toxicity. Plan is to continue TKI and monthly STRO-002 as maintenance therapy. Conclusion: Complete genomic sequencing has revolutionized pediatric AML categorization. It is quite conceivable that (BCR::ABL, CBF::GLIS) dual fusion disease will become a more common finding in future. Although CBF::GLIS AML is uniformly refractory to conventional therapy, targeted therapy with FOLR1-directed STRO-002 was highly effective in eliminating CBF::GLIS clone in this patient, and combination with DLI appears to be feasible. Long-term follow-up is required to assess durability of remission. Additional testing of this approach in a larger patient population is needed to determine the role of STRO-002 in this high-risk pediatric AML population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Allogeneic hematopoietic stem cell transplantation for pediatric acute myeloid leukemia in first complete remission: a meta-analysis

Identification of pediatric patients with acute myeloid leukemia (AML) candidates to receive allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR1) is still a matter of debate. Currently, transplantation is reserved to patients considered at high risk of relapse based on cytogenetics, molecular biology, and minimal residual disease (MRD) assessment. However, no randomized clinical trial exists in the literature comparing transplantation with other types of consolidation therapy. Here, we provide an up-to-date meta-analysis of studies comparing allo-HSCT in CR1 with chemotherapy alone as a post-remission treatment in high-risk pediatric AML. The literature search strategy identified 10 cohorts from 9 studies performing as-treated analysis. The quantitative synthesis showed improved overall survival (OS) (relative risk, 1.15; 95% confidence interval [CI], 1.06–1.24; P = 0.0006) and disease-free survival (relative risk, 1.31; 95% CI, 1.17–1.47; P = 0.0001) in the allo-HSCT group, with increased relapse rate in the chemotherapy group (relative risk, 1.26; 95% CI, 1.07–1.49; P = 0.006). Sensitivity analysis including prospective studies alone and excluding studies that reported the comparison only on intermediate-risk patients confirmed the benefit of allo-HSCT on OS. Further research should focus on individualizing allo-HSCT indications based on molecular stratification and MRD monitoring.

Outcomes of intensification of induction chemotherapy for children with high-risk acute myeloid leukemia: A report from the Children's Oncology Group.

High-risk pediatric acute myeloid leukemia confers a poor prognosis, and alternative strategies are needed to improve outcomes. We hypothesized that intensifying induction on the AAML1031 clinical trial would improve outcomes compared to the predecessor trial AAML0531. Patients on AAML0531 received cytarabine (1600mg/m2 )/daunorubicin (150mg/m2 )/etoposide (ADE) for induction II and patients on AAML1031 received mitoxantrone (48mg/m2 )/cytarabine (8000mg/m2 ) (MA). Stem cell transplant (SCT) conditioning included busulfan/cyclophosphamide on AAML0531, whereas AAML1031 used busulfan/fludarabine and liberalized donor eligibility. Patients were included in this analysis if they met high-risk criteria common to the two trials by cytogenics or poor disease response after induction I ADE. MA provided no benefit over ADE at: induction II response (complete response [CR]: 64% vs. 62%, p=.87; measurable residual disease [MRD]+: 57% vs. 46%, p=.34); or intensification I response (CR: 79% vs. 94%, p=.27; MRD+: 27% vs. 20%, p=1.0). When considered with altered SCT approach, MA did not improve 5-year disease-free survival (24% ± 9% vs. 18% ± 15%, p=.63) or 5-year overall survival (35% ± 10% vs. 38% ± 18%, p=.66). MA was associated with slower neutrophil recovery (median 34 vs. 27days, p=.007) and platelet recovery (median 29 vs. 24.5days, p=.04) and longer hospital stay (32 vs. 28days, p=.01) during induction II. Intensification of induction II did not improve treatment response or survival, but did increase toxicity and resource utilization. Alternative strategies are urgently needed to improve outcomes for pediatric patients with high-risk acute myeloid leukemia (trials registered at clinicaltrials.gov NCT01371981, NCT00372593).

Combined Treatment with JQ1 and Curcumin Synergically Modulates Apoptosis, Cell Cycle Features, Micrornas and Gene Expression in High-Risk Pediatric Acute Myeloid Leukemia (AML)

BackgroundDespite recent progresses in the treatment of childhood AML, the outcome of high-risk patients (carrying selected molecular aberrations and/or having high levels of minimal residual disease at the end of therapy) remains dismal. In particular, patients harboring FLT3-ITD or some translocations involving MLL (such as MLL-AF10 or MLL AF4) still have a poor prognosis, mainly due to leukemia recurrence.A key feature of FLT3-ITD and MLL-rearranged leukemia is cMYC overexpression, a well-known oncogene often involved in cancer. JQ1 is a BET inhibitor, preventing cMYC expression through the binding to the BET bromodomain protein, BRD4. Moreover, Curcumin is a compound able to induce epigenetic changes, including inhibition of histone-acetyltransferases (HATs), resulting in a decrease of acetylated lysine residues, a target for BRD4 bound on DNA.AimWe evaluated whether the combination of JQ1 and Curcumin has a potential synergic effect on AML cell lines or primary blasts harboring FLT3-ITD and MLL-rearrangement. These experimental data could be instrumental for developing novel treatment options alternative or complementary to standard chemotherapy.MethodsCell lines with FLT3-ITD and/or MLL fusion proteins used in this study were: MV-4-11, THP1, MOLM-13 and RS-4-11 [ALL cell line harboring t(4;11)]. Cells were all treated with JQ1 [250nM], while the dose of Curcumin varied according to the MLL-rearrangement (5μM for MV-4-11 and RS-4-11 carrying MLL-AF4 rearrangement and 10μM for THP1 and MOLM-13 harboring MLL-AF9 rearrangement). After 48 hours of either single-drug or combined treatment (J+C), western blot, XTT assay, RT-qPCR and Annexin V FACS analysis were carried out to assess proliferation rates, apoptosis and expression of their related genes, as well as the expression of cMYC and AcH3K14. Capacity to form colonies after either single or combined drug exposure was tested on bone marrow derived CD34+ cells from 4 patients with either MLL-rearranged acute leukemia (AML MLL-AF9; ALL MLL-AF4; AML MLL-AF4) or FLT3-ITD using Methocult media.miRNA expression profile of 20 BM samples of either FLT3-ITD or MLL-rearranged AML collected at both diagnosis (Dx) and at relapse (Rel) was assessed with Nanostring platform. Results were compared with those obtained analyzing BM samples of 8 healthy controls (HCs) and of 12 patients with the same molecular lesions who did not relapse (NRel). Expression levels of the modulated miRNAs were also evaluated on the cell lines reported above. A supplemental miRNA profiling analysis was conducted comparing cells harboring FLT3-ITD with those carrying MLL-rearrangements with respect to HCs.ResultsIn comparison with treatment with either of the 2 agents, the J+C combination resulted into to a synergic effect on apoptosis rate of MOLM-13 cells and of RS-4-11 cells. In particular, the apoptosis rates of the former cell line were 16% with JQ1, 10% with Curcumin and 56% with J+C, while those of the latter cell line were 20% with JQ1, 4% with Curcumin and 52% with J+C, respectively. The XTT metabolic assay showed significant reduction in 24 and 72-hour proliferation rates after both single-agent and combined treatments in all cell lines compared to DMSO (2WayANOVA; P<0.05)RT-qPCR and western blot experiments showed an increased expression of BCL2, cleaved CASPASE-3, cleaved PARP1 and p21 levels, as well as a decrease of cMYC and AcH3K14. Single-agent and combined treatment also resulted into a significant decrease of number of colonies in all the 4 primary cultured patient blasts (Fisher's exact t-test; P<0.05). 118 miRNAs were found to be significantly modulated in samples of Rel and NRel groups compared with those of HCs, while 18 miRNAs were found to be modulated in either MLL-rearranged or FLT3-ITD AML as described in the Figure1, 2, and Table 1, 2. We also noticed an opposite expression trend for miR-99 and miR-34a in MLL-AF4 AML and MLL-AF9 AML, and significant modulations of expression after single-agent and J+C treatment for miR-99, miR-34a, miR-451 and miR-196a in our cell lines.ConclusionsOur data demonstrate that J+C exert an intense synergic effect on AML cell lines and primary blasts, leading to increased apoptosis, reduced proliferation rate, modulation in expression of some key genes, such as cMYC, BCL2 and AcH3K14. We also identified distinct miRNA signatures in patients collected at relapse as compared to those obtained at diagnosis or HCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparison of FLU-BU12 Conditioning with the Standard BU-CY Myeloablative Regimen in High-Risk Pediatric ACUTE Myeloid Leukemia Patients Undergoing Allogeneic STEM Cell Transplantation

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for high-risk acute myeloid leukemia (AML).The preparative regimen consisting of busulfan and cyclophosphamide (BuCy) is considered as one of the classical myeloablative conditioning regimens (MAC); however, it is associated with significant early and long-term toxicities, leading to a high rate of transplant-related mortality (TRM). P urpose: To compare toxicities and outcomes of BuCy and FluBu12 conditioning in the pediatric population. Methods: We retrospectively analyzed 71 pediatric high-risk AML patients in CR1/2 (n=51, 71,8%) and R/R disease (n=20, 28,2%) received allo-HSCT from MSD (n=16, 22,5%), MUD (n=43, 60,6%) and Haplo donor (n=12, 16,9%). BuCy and FluBu conditioning regimens were used in 47 (66,2%) and 24 (33,8%) patients respectively. Median age was 6 years (0-17). GVHD prophylaxis was PTCy±CNI±m-TOR inhibitor in 32 (45%) or ATG±CNI in 39 (55%) patients. The primary end points were TRM, relapse-free survival (RFS), graft-versus-host disease (GVHD) free, relapse free survival (GRFS) and overall survival (OS). Secondary end points included neutrophil engraftment, sinusoidal obstruction syndrome (SOS), acute and chronic GVHD. Patient were censored at the time of death or last follow-up. Probabilities of OS, RFS and GRFS were estimated using Kaplan-Meier curves. TRM was defined as any death that occurred in the absence of disease relapse; relapse was a competing risk for this event. Results: Engraftment rate was 91% and 87,5% in patients received BuCy and FluBu, respectively (p=0,4). There was a trend to lower day 100 TRM after FluBu (4,1% vs 14,9% after BuCy, p=0,07). Overall survival at 2 years did not differ as well (BuCy 48,9%, FluBu 56%, p=0,5). BuCy showed borderline higher RFS at 2 years (70,2% vs 52%, p=0,06). The composite endpoint GRFS did not differ between two study cohorts being 31,8% and 29,2% at 2 years for BuCy and FluBu (p=0,7). We observed a tendency towards a higher incidence of III0-IV0 aGVHD and cGVHD following BuCy when compared with FluBu: 57,1% vs 41% (p=0,06) and 46,5% vs 28,5% respectively (p=0,2). No significant differences were found between the BuCy and FluBu groups in risk of SOS (6% and 8%, respectively). Conclusions: The optimal conditioning regimen for children with AML is still a matter of debate. Our results suggest that FluBu represents a valid myeloablative regimen, able to provide lower TRM, aGVHD and cGVHD. This conditioning might become an alternative approach in patients with a high risk of severe post-transplant complications. Disclosures No relevant conflicts of interest to declare.

Genome and Transcriptome Profiling of Monosomy 7 AML Defines Novel Risk and Therapeutic Cohorts

Monosomy 7 (mono7) alterations in acute myeloid leukemia (AML) are associated with poor outcome and disease progression. Through advancements in multi-omic approaches, more specific treatment strategies may be available for high-risk cohorts. Here we describe pediatric cases of AML with mono7, co-occurring fusions, associated outcome, and potential therapeutic treatments. Of the 2200 patients treated in 3 consecutive Children's Oncology Group protocols (AAML03P1, AAML0531, and AAML1031), 45 patients (2%) had karyotypic evidence of mono7 with full complement of clinical data for analysis. RNA sequencing was available for 37 and whole genome sequencing (WGS) for 8 cases. Fusions were identified using TransAbyss, STARfusion, and Cicero algorithms, while structural variants were analyzed by CREST in the WGS samples. Differential expression comparing mono7 AML (N=28) vs. other AML (N=1064) was performed and epigenetic profiling was evaluated using Illumina's EPIC array (N=1025, N=79 normal bone marrow controls). Of the 45 patients with mono7, 22 had additional karyotypic alterations including copy number variants in 7 and translocations in 15 (6 had confirmatory evidence by RNA seq). RNA seq also identified 9 additional cryptic fusions. In total, the cohort contains 5 cases (11.1%) with KMT2A fusions, 3 (6.7%) with CBF fusions, 7 (15.7%) with 3q26 alterations, and 7 (15%) with copy number alterations (Fig. 1A). In 14 patients (31%), mono7 was the sole karyotypic alteration. In 28 patients with ribo-depleted RNA seq data, cryptic fusions involving the ALK gene were identified in 4 patients (14.3%) with ALK fused to either SPTBN1 (n=3) or RANBP2 (n=1) genes. Cryptic ALK fusions were not detected in the 1064 cases of other AML (p=1.5x10-37), suggesting a unique association between ALK fusions and mono7 and potential genomic cooperation. A differential expression analysis compared patients with mono7 (N=28) to all other AML (N=1064). This analysis identified 1547 dysregulated genes. Of these, MECOM (MDS1/EVI1 COMplex) was identified as the top upregulated gene (logFC 7.24; p=4.4x10-74) with a median expression 5.45 TPM (range 0-89.2 TPM) in patients with mono7 vs. 0.013 TPM in other AML patients. Evaluation of outcome based on MECOM expression demonstrated that patients with high MECOM expression (greater than median) had a 3-yearOS of 22%±20% compared to that of 68%±20% with low MECOM expression, (p=0.026, Fig. 1B) All patients with 3q26 variants had elevated MECOM expression (n=7). In addition, 11 patients without 3q26 alterations/MECOM fusions had MECOM overexpression. Given lack of underlying genomic etiology, we sought to determine whether epigenetic factors might mediate MECOM expression. A panel of 6 CpGs was sufficient to distinguish hematopoietic stem cells (HSCs) from granulocyte-monocyte progenitors (GMPs). HSC-like hypermethylation of these CpGs was strongly associated with high MECOM expression. Further, there was high correlation between total MECOM expression and the methylation status of a CpG island proximal to the short EVI1 transcript variant of MECOM (Spearman's rho = -0.51, p &amp;lt; 0.001, Fig. 1C), suggesting a regulatory underpinning for permissive expression of EVI1. The stem-like epigenetic signature and concordant high MECOM expression of poor-prognosis mono7 AML are consistent with a "stemness" signature and portend poor survival (Fig. 1D). Stemness mRNA signatures have been implicated in high-risk pediatric AML (Smith JL, ASH 2017) and the epigenetic signatures of these genes further corroborate cell of origin as an independently prognostic factor even within high-risk AML. Focused interrogation of the MECOM locus thru integration of the RNA Seq and WGS identified allele specific MECOM expression, adding additional potential mechanism of modulation to MECOM expression. Structural variant analysis confirmed no other CNVs on chromosome 3, indicating cis dysregulation of MECOM. Here, by interrogation of the genome, phenome, transcriptome, and epigenome of mono7 AML a substantial phenotypic and prognostic heterogeneity exists defining a cohort of patients, regulated by genomic and epigenomic alterations. Further, discovery of cryptic ALK fusions in mono7 present a target for ALK inhibitors (FDA approved for non-small cell lung cancer) in this high-risk population. Disclosures No relevant conflicts of interest to declare.

The Molecular Characteristics and Clinical Relevance of NUP98-Other Translocations in Pediatric Acute Myeloid Leukemia

Background Cytogenetic and molecular aberrations are important prognostic factors in pediatric acute myeloid leukemia (AML). NUP98 translocations with more than 30 different partner genes have been identified in pediatric AML. The 2 most common fusions, NUP98-NSD1 and NUP98-KDM5A, have been shown to have distinct characteristics and are both associated with adverse outcomes. Although NUP98 fusions with less common fusion partners have been identified, the biological and clinical implications of these variants are unknown. Methods To determine the biological and clinical implications of the less common "other" NUP98 translocations (NUP98-X), we evaluated the clinical characteristics and transcriptome and genome sequencing data from 2396 children and young adults with AML within 4 consecutive Children's Cancer Group (CCG) and Children's Oncology Group (COG) trials CCG-2961, AAML03P1, AAML0531 and AAML1031. All NUP98-X translocations were confirmed by RNA sequencing. Results Of the 2396 patients screened, 164 patients (6.8%) had a NUP98 translocation. We identified 20 patients with a NUP98-X fusion (0.83%) and compared them with those with NUP98-NSD1 (n=110, 4.5%), NUP98-KDM5A(n=34, 1.4%), and a reference cohort without NUP98 translocations (n=2232). Translocation partners identified in the NUP98-X group were HOXA9 (n=4), HOXD13 (n=3), PHF15 (n=2), PHF23 (n=2), and single cases of BPTF, BRWD3, DDX10, HMGB3, HOXA13, KAT7, PRRX1, SET and TOP1. Besides the distinct characteristics of NUP98-NSD1 and NUP98-KDM5A, the NUP98-X group showed high inter-patient variance in clinical characteristics compared to our reference cohort. NUP98-X patients showed a clear bimodal age distribution with half of the patients being in the older age category and a similar number in the category &amp;lt;3 years (Figure 1). White blood cell count and blast percentages were in line with those of the reference cohort. We investigated the co-occurrence of common pediatric AML mutations within our cohort (Figure 2). There was a lack of additional driving mutations with the exception of WT1 mutations that are significantly more prevalent in the NUP98-X group compared to the reference cohort (25% vs 8%, p=0.018). NUP98-X patients with a WT1 mutation have a median age of 16.3 years compared to 2.3 years for those without a WT1 mutation. Also, in contrast to NUP98-NSD1 translocated patients, the co-occurrence of FLT3-ITD did not occur in NUP98-X translocated patients. Further, NUP98-X patients had a more varied transcriptome profile than did patients with NSD1 or KDM5A translocations (Smith J, et al. ASH abstract 2020). We evaluated the impact of NUP98-X translocations in response to the initial induction therapy. The morphological complete remission (CR) rate after course 1 was 65% in the NUP98-X cohort versus 76% in the reference cohort (p=0.266). NUP98-NSD1 patients had an inferior CR rate (36%, p&amp;lt;0.001), but the CR rate in NUP98-KDM5A patients was similar (77%) to the reference cohort. Of all NUP98-X patients, 11% underwent stem cell transplantation (SCT), compared to 17% in the reference cohort, 40% in the NUP98-NSD1 and 23% in the NUP98-KDM5A cohort. Outcome analysis for NUP98-X patients demonstrated that despite differences in disease characteristics, they have similar adverse outcomes as patients with NSD1 or KDM5A translocations (Figure 3). Overall survival (OS) at 5 years from diagnosis was 35% compared to 65% in the group without NUP98 translocations (p=0.006, Figure 3). Event-free survival was 35% for NUP98-X patients, compared to 48% for those without NUP98 translocations (p=0.282). These adverse outcome rates are comparable to those reported in studies on high-risk pediatric AML patients. Conclusion NUP98-X translocated pediatric AML patients represent a rare cohort with a high variability in both translocation partners and other clinical characteristics. Despite this heterogeneity, the OS of these patients is comparable to high-risk pediatric AML patients, which justifies a high-risk stratification of these patients and emphasizes the need for developing new treatment strategies. Further research within large patient cohorts is needed to investigate the biologic and clinical characteristics of these rare translocations and potential differences between the different NUP98-X fusion partners. Disclosures Kaspers: AbbVie: Ended employment in the past 24 months; Helsinn Healthcare: Ended employment in the past 24 months; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Janssen R&amp;D: Ended employment in the past 24 months. Cooper:Celgene: Other: Spouse was an employee of Celgene (through August 2019).

Genotype-outcome correlations in pediatric AML: the impact of a monosomal karyotype in trial AML-BFM 2004

We conducted a cytogenetic analysis of 642 children with de novo acute myeloid leukemia (AML) treated on the AML-Berlin-Frankfurt-Münster (BFM) 04 protocol to determine the prognostic value of specific chromosomal aberrations including monosomal (MK+), complex (CK+) and hypodiploid (HK+) karyotypes, individually and in combination. Multivariate regression analysis identified in particular MK+ (n=22) as a new independent risk factor for poor event-free survival (EFS 23±9% vs 53±2% for all other patients, P=0.0003), even after exclusion of four patients with monosomy 7 (EFS 28±11%, P=0.0081). CK+ patients without MK had a better prognosis (n=47, EFS 47±8%, P=0.46) than those with MK+ (n=12, EFS 25±13%, P=0.024). HK+ (n=37, EFS 44±8% for total cohort, P=0.3) influenced outcome only when t(8;21) patients were excluded (remaining n=16, EFS 9±8%, P<0.0001). An extremely poor outcome was observed for MK+/HK+ patients (n=10, EFS 10±10%, P<0.0001). Finally, isolated trisomy 8 was also associated with low EFS (n=16, EFS 25±11%, P=0.0091). In conclusion, monosomal karyotype is a strong and independent predictor for high-risk pediatric AML. In addition, isolated trisomy 8 and hypodiploidy without t(8;21) coincide with dismal outcome. These results have important implications for risk stratification and should be further validated in independent pediatric cohorts.

Molecular Profiling of High-Risk Pediatric Acute Myeloid Leukemia

Background: Several landmark genomic profiling studies have dramatically advanced our understanding of the origin, progression and clonal evolution of adult acute myeloid leukemia (AML) and directly impacted clinical care. However, very little is known about the mutational landscape of pediatric AML, a distinct entity that shares few genetic and clinical characteristics with adult AML. To investigate potential drivers of high-risk pediatric AML, comprehensive genomic profiling was performed on high-risk AML samples as part of a prospective clinical next-generation sequencing program.Methods:Samples obtained from patients with known high-risk features at diagnosis or with refractory or relapsed AML were selected for molecular profiling. Comprehensive testing included whole-exome sequencing (WES) of matched tumor (bone marrow or chloroma tissue) and normal tissue (peripheral blood or buccal swab) samples and transcriptome analysis (RNAseq). Targeted sequencing of 467 cancer-associated genes was used when tumor tissue was limited. Sequencing was performed on Illumina's HiSeq 2500 with 150X and 500X average coverage for WES and targeted sequencing, respectively. Variants were filtered to select alterations in cancer-related genes or genes relevant for patient care.Results:Fifteen patients with AML (mean age 7.7 yrs; range 0.75-19 yrs) met high-risk criteria (high-risk features at diagnosis = 4, relapsed disease = 8, refractory disease = 3) and were selected for profiling. WES and RNAseq were performed on 11 samples, WES only on 3 samples and targeted DNA sequencing on 1 sample. The median number of variants was 60 (range 14- 5950) per case. After filtering, 54 mutations were identified in 35 genes with a mean of 3.6 mutated genes per patient sample (range 0-14); two samples only carried a fusion gene with no other genetic alterations. At least one driver genetic alteration was detected in each patient sample. Thirteen samples carried mutations in at least one gene known to be altered in AML (e.g. IDH1, WT1, TP53, NRAS) (mean, 2; range, 1-6) and 5 samples carried novel mutations in 15 genes not previously implicated in AML (e.g. CARD9, CHD9, Axin1). Mutations in 11 AML related genes were detected in more than one sample including NRAS in 4, TP53 in 3 and KRAS, PTPN11 PHF6, JAK3 in 2 samples each; genes not previously implicated in AML were only mutated in single patients. Of note, mutations in genes encoding members of the RAS pathway occurred in 60% of cases (9/15 samples). RNAseq identified gene fusions in 7/11 samples (63%). Four fusions involving KMT2A and core binding factor genes were also detected by FISH while three fusions were detected by RNAseq only: NUP98-NSD1 in two patients and CBFA2T3-GLIS2 in one patient. Samples carrying driver gene fusions had the lowest number of mutated genes (0-1) compared to samples lacking a gene fusion (1-5 mutated genes), with one exception of a patient with history of infant ALL who later developed KMT2A-AFF1AML with the highest number of mutated genes (n=14). There was no correlation between the number of mutated genes and age, clinical characteristics, initial risk classification at diagnosis or intensity of therapy prior to sequencing.Conclusion:Our study provides an initial overview of the genetic alterations that characterize high-risk, chemo-resistant pediatric AML. Analysis of the data highlights the overall low genetic complexity of high-risk AML despite the aggressive clinical behavior and exposure to intense chemotherapy, including stem cell transplant. Of interest, similar to adult AML, we found that mutations leading to aberrant activation of the RAS pathway were also very frequent in our cohort of pediatric high-risk AML, while genes typically mutated early in the process of leukemogenesis in adult AML, such as NPM1, DNMT3A, FLT3, IDH1, IDH2 were not affected. Such findings suggest that distinct, age-specific mechanisms of leukemogenesis might exist. Furthermore, our data also highlights the important role of RNA sequencing in complementing current standard diagnostic tools, allowing the identification of driver fusion genes in samples for which no other driver event is detected. Larger studies, preferably including diagnostic samples and utilizing broader approaches, are needed to better understand the mechanisms responsible for the initiation and progression of childhood AML. DisclosuresNo relevant conflicts of interest to declare.

A recurrent immunophenotype at diagnosis independently identifies high-risk pediatric acute myeloid leukemia: a report from Children’s Oncology Group

A recurrent immunophenotype at diagnosis independently identifies high-risk pediatric acute myeloid leukemia: a report from Children’s Oncology Group

A Recurrent Immunophenotype at Diagnosis Independently Identifies High Risk Pediatric AML: A Report from the Children's Oncology Group Trial AAML0531

Background: Risk stratification of pediatric patients with acute myeloid leukemia (AML) combines cytogenetics and molecular markers at diagnosis with detection of measurable residual disease (MRD) by multidimensional flow cytometry (MDF) after initial induction chemotherapy (EOI1). However, using MDF to distinguish between favorable and poor risk groups by diagnostic immunophenotype has not been sufficiently reproducible for clinical implementation.Objective: We sought to characterize a recurrent, unique diagnostic immunophenotype identified among patients treated on Children's Oncology Group trial AAML0531 and determine the clinical characteristics, frequency, and outcome of patients with this immunophenotype in a large cohort.Patients and Methods: Of the 1022 newly diagnosed pediatric patients with de novo AML who enrolled in protocol AAML0531 (exclusive of patients with promyelocytic leukemia and Down syndrome), 812 who submitted a specimen for MDF at diagnosis and gave consent to MRD testing were eligible for the study.Results: A recurrent diagnostic phenotype was identified by a combination of high intensity CD56 expression, a lack of HLA-DR, dim to negative CD38 expression and dim to negative CD45 expression (designated as RAM phenotype). At diagnosis 19 patients (2.3%) expressed this phenotype and MRD was subsequently detected by MDF in 16/19 patients at EOI1 at a level of 0.02%-41% (median 0.3). Eleven patients with this phenotype submitted bone marrow specimens for MDF analysis at all 3 additional monitoring time points during therapy and in 8 patients MDF analysis revealed disease persistence throughout treatment. The experimental cohort (RAM cohort, N=19) and 2 control cohorts, one consisting of all other patients with CD56 positive AML (CD56+ control, N=166) and the other of patients with CD56 negative AML (CD56- control, N=627), were analyzed for clinical outcomes.Patients in the RAM cohort were significantly younger with a median age at diagnosis of 1.26 years [range 0.75-16.89] than those in the CD56+ control (median 7.57 years [range 0.01-29.8], p=0.002) and the CD56- control (median 10.64 [range 0.02-23.94], p<0.001). All patients in the RAM cohort were classified as standard risk per study protocol compared to 55% (p<0.001) for the CD56+ control and 46% (p<0.001) for the CD56- control. Patients in the RAM cohort were FLT3-ITD- and wild type for CEBPA, NPM1, and WT1. Of them, 7 had normal cytogenetics, 1 had trisomy 8, and 11 had other cytogenetic abnormalities not associated with prognostic subgroups, including the 11q23/MLL rearrangement. According to morphology, patients in the RAM cohort were more often classified as having megakaryoblastic AML (38%) compared to the CD56+ control (2.0%, p<0.001) and the CD56- control (4.0%, p<0.001).MDF analysis detected MRD in 84% of RAM cohort compared to 29% (p<0.001) for the CD56+ control and 33% (p<0.001) for the CD56- control cohorts. MRD detected at EOI1 was compared to morphologic complete remission (CR). Morphologic CR at EOI1 was achieved in 11 (58%) patients in the RAM cohort (58%) versus 71% of patients in the CD56+ (p=0.250) and 74% of patients in the CD56- (p=0.116) control cohorts. Patients in the RAM cohort had a 3-year event free survival (EFS) of 16% versus 52% for those in the CD56+ (p=0.003) and 51% for those in the CD56- (p<0.001) control cohorts. The 3-year overall survival (OS) for patients in the RAM cohort was 26% versus 66% for those in the CD56+ (p<0.001) and 70% for those in the CD56- (p<0.001) control cohorts. Multivariable Cox regression analysis revealed that the diagnostic RAM immunophenotype was an independent predictor of adverse outcome (hazard ratios [and 95% confidence intervals]: OS= 3.4 (1.84 - 6.18), relapse risk from CR=4.97 (2.67 - 9.24), DFS from CR=4.27 (2.04 - 8.95)).Conclusions: A definitive diagnostic phenotype is identified that is highly predictive of poor outcome in pediatric AML. The survival of patients with the RAM phenotype is similar to or worse than those with high-risk molecular features. It occurs in 10% of infants (<1 year old) with AML who lack known risk factors, do not respond to standard therapy, and have extremely poor outcomes. This phenotype can be identified rapidly at diagnosis, allowing clinicians to predict failure before initiating therapy, and thereby allowing administration of more effective alternative therapies and minimizing exposure to non-efficacious treatment. DisclosuresBrodersen:Hematologics Inc.: Employment. Menssen:Hematologics Inc.: Employment. Pardo:Hematologics Inc.: Employment. Voigt:Hematologics Inc.: Employment. Loken:Hematologics Inc.: Equity Ownership.

The Biology and Targeting of FLT3 in Pediatric Leukemia.

Despite remarkable improvement in treatment outcomes in pediatric leukemia over the past several decades, the prognosis for high-risk groups of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), as well as for relapsed leukemia, remains poor. Intensification of chemotherapy regimens for those at highest risk has improved success rates, but at the cost of significantly increased morbidity and long-term adverse effects. With the success of imatinib in Philadelphia-chromosome-positive leukemia and all-trans retinoic acid in acute promyelocytic leukemia, the quest to find additional molecularly targeted therapies has generated much excitement over recent years. Another such possible target in pediatric acute leukemia is FMS-like tyrosine kinase 3 (FLT3). FLT3 aberrations are among the most frequently identified transforming events in AML, and have significant clinical implications in both high-risk pediatric AML and in certain high-risk groups of pediatric ALL. Therefore, the successful targeting of FLT3 has tremendous potential to improve outcomes in these subsets of patients. This article will give an overview of the molecular function and signaling of the FLT3 receptor, as well as its pathogenic role in leukemia. We review the discovery of targeting FLT3, discuss currently available FLT3 inhibitors in pediatric leukemia and results of clinical trials to date, and finally, consider the future promise and challenges of FLT3 inhibitor therapy.

Comparable survival for pediatric acute myeloid leukemia with poor-risk cytogenetics following chemotherapy, matched related donor, or unrelated donor transplantation

We sought to better define the role of hematopoietic cell transplantation (HCT) in first remission (CR1) for high-risk pediatric acute myeloid leukemia (AML). Outcomes were compared among patients aged less than 21 years with cytogenetically defined poor-risk AML treated with chemotherapy, matched related (MRD), or unrelated donor (URD) transplantation in CR1. Poor-risk cytogenetics was defined as monosomy 7/del7q, monosomy 5/del 5q, abnormalities of 3q, t(6;9)(p23;q34), or complex karyotype. Included are patients treated on Children's Oncology Group trials or reported to the Center for International Blood and Marrow Transplant Research from 1989 to 2006. Of the 233 patients, 123 received chemotherapy, 55 received MRD HCT, and 55 received URD HCT. The 5-year overall survival from the time of consolidation chemotherapy or transplant conditioning was similar: chemotherapy (43% ± 9%), MRD (46% ± 14%), or URD (50% ± 14%), P = 0.99. Similarly, multivariate analysis demonstrated no significant differences in survival [(reference group = chemotherapy); MRD HR 1.08, P = 0.76; URD HR 1.13, P = 0.67] despite lower relapse risk with URD HCT (HR = 0.43, P = 0.01). Our findings do not provide support for the preferential use of HCT over chemotherapy alone for children with cytogenetically defined poor-risk AML in CR1.

Allogeneic Hematopoietic Cell Transplantation in First Remission Abrogates Poor Outcomes Associated with High-Risk Pediatric Acute Myeloid Leukemia

Despite remission rates of approximately 85% for children diagnosed with acute myeloid leukemia (AML), greater than 40% will die from relapsed disease. Patients with poor-risk molecular/cytogenetics and/or inadequate response to up-front therapy are typically considered high-risk (HR) and historically have poor outcomes with chemotherapy alone. We investigated whether allogeneic hematopoietic cell transplantation (allo-HCT) with best available donor in first remission (CR1) would abrogate the poor outcomes associated with HR AML in children and young adults treated with chemotherapy. We reviewed the outcomes of 50 consecutive children and young adults (ages 0 to 30 years) with AML who received a myeloablative allo-HCT between 2001 and 2010. Thirty-six patients (72%) were HR, defined as having FLT3-ITD mutations, 11q23 MLL rearrangements, chromosome 5 or 7 abnormalities, induction failure, and/or having persistent disease. The majority of patients received cyclophosphamide and total body irradiation conditioning, and graft-versus-host-disease (GVHD) prophylaxis was cyclosporine based. Transplantation outcomes for HR patients were compared to standard-risk patients, with no significant differences observed in overall survival (72% versus 78%, P = .72), leukemia-free survival (69% versus 79%, P = .62), relapse (11% versus 7%, P = .71), or treatment-related mortality (17% versus 14%, P = .89). Children and young adults with HR-AML have comparable outcomes to standard-risk patients following allo-HCT in CR1.

Successful cell‐mediated cytokine‐activated immunotherapy for relapsed acute myeloid leukemia after hematopoietic stem cell transplantation

Acute myeloid leukemia (AML) is an extremely aggressive disease with a high relapse rate even after allogeneic hematopoietic stem cell transplantation (HSCT). We report the successful outcome of cell-mediated cytokine-activated immunotherapy in a high-risk pediatric AML patient who relapsed shortly after allogeneic HSCT. Donor lymphocyte infusion along with interferon induced a graft-versus-leukemia effect, presenting as a reversible episode of graft-versus-host disease, which led to stable complete donor chimerism and total eradication of AML for over 24 months, at the time of this report. The curative potential of immunotherapy in hematological malignancies is discussed.