Childhood AML Research Articles

Super enhancer related gene ANP32B promotes the proliferation of acute myeloid leukemia by enhancing MYC through histone acetylation

BackgroundAcute myeloid leukemia (AML) is a malignancy of the hematopoietic system, and childhood AML accounts for about 20% of pediatric leukemia. ANP32B, an important nuclear protein associated with proliferation, has been found to regulate hematopoiesis and CML leukemogenesis by inhibiting p53 activity. However, recent study suggests that ANP32B exerts a suppressive effect on B-cell acute lymphoblastic leukemia (ALL) in mice by activating PU.1. Nevertheless, the precise underlying mechanism of ANP32B in AML remains elusive.ResultsSuper enhancer related gene ANP32B was significantly upregulated in AML patients. The expression of ANP32B exhibited a negative correlation with overall survival. Knocking down ANP32B suppressed the proliferation of AML cell lines MV4-11 and Kasumi-1, along with downregulation of C-MYC expression. Additionally, it led to a significant decrease in H3K27ac levels in AML cell lines. In vivo experiments further demonstrated that ANP32B knockdown effectively inhibited tumor growth.ConclusionsANP32B plays a significant role in promoting tumor proliferation in AML. The downregulation of ANP32B induces cell cycle arrest and promotes apoptosis in AML cell lines. Mechanistic analysis suggests that ANP32B may epigenetically regulate the expression of MYC through histone H3K27 acetylation. ANP32B could serve as a prognostic biomarker and potential therapeutic target for AML patients.

Long-term experience in treatment of acute promyelocytic leukemia in Mexican children in a tertiary care hospital.

Acute promyelocytic leukemia (APL) is a rare myeloid leukemia subtype affecting adult and pediatric populations. APL constitutes 15-20% of all childhood AML in Latin America, compared to 7% in the non-Latino population. This leukemia has unique characteristics, such as its association with chromosomal translocations involving the retinoid acid receptor α (RARA) gene on chromosome 17. In addition, APL is also distinct from other AML subtypes due to its response to all-trans-retinoic acid (ATRA), which induces terminal granulocytic differentiation of blasts. Overall 5-year survival rates are generally reported to be greater than 80%. A study was conducted from January 2008 to December 2022 applying the IC-APL 2006 treatment protocol. This case series reports the clinical results of 22 children with APL. In all cases, the diagnosis was made by bone marrow aspiration and evaluation of the t(15:17) or t(11:17) transcripts. We identified 22 patients with APL, of whom 10 were female and 12 were male. Twelve patients debuted with coagulation abnormalities. The doses of anthracyclines varied according to the risk, with an average of 496.8 mgm2. The cardiological assessment was performed before and after chemotherapy, finding 2/22 patients with moderate sisto-diastolic dysfunction and one with mild pulmonary insufficiency at the end of treatment. There were 6/22 patients with complications related to ATRA treatment, the most frequent being pseudotumor cerebri. All complications were transitory and treated immediately without complications. In this series of cases, an overall survival of 90.6% and a relapse-free survival of 90.6% were recorded. The follow-up mean was 9.1 ± 3.8 years. APL is a highly curable disease when combined with ATRA and anthracyclines. In this series of cases, good long-term results were observed with the IC-APL 2006 protocol. However, in Latin America, the availability of drugs such as arsenic trioxide as the first line of treatment is an unresolved challenge.

Targeting HDAC9 Contributes to Degradation of MLL Fusion Oncoproteins in KMT2A-Rearranged Acute Myeloid Leukemia

Acute myeloid leukemia with MLL rearrangement (MLLr-AML) is a highly aggressive subtype of AML. Existing treatment strategies are nonspecific and ineffective. The MLL fusion gene is one of the driver oncogenes in leukemia and is central to the pathogenesis of MLLr-AML, and more research has recently begun to focus on targeting MLL fusion proteins. Targeting MLL fusion proteins has been shown to have a tumor suppressor effect, demonstrating efficacy in preclinical models of MLLr-AML, and in 2022 Scott A Armstrong ‘s team suggested that degradation of MLL-AF9 can cause an increase in cell differentiation and apoptosis, confirming the efficacy of targeting MLL fusions. However, specific targets that degrade MLL fusion proteins as well as clinically translatable targeted therapies are still lacking and need to be further explored. Study confirmed that MLL-rearranged ALL cells are highly sensitive to the broad-spectrum HDAC inhibitor panobinostat. A recent clinical study by Mireia Camós’ team proposed that HDAC9 has a significantly high expression in childhood MLL-rearranged B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and AML, but that article did not address any mechanistic studies. The potential value of HDAC9 in adult lymphoid hematologic diseases has been gradually revealed, however, the expression profile and precise molecular mechanisms of HDAC9 in adult myeloid hematologic tumors, have not been reported, which deserves to be further explored. In order to investigate the expression and role of HDAC9 in MLLr-AML, we analyzed the second-generation sequencing data from the Beat AML dataset and found that HDAC9 has a significantly high expression in MLLr-AML, while only HDAC9 is characterized by this feature in HDAC family molecules. Further we analyzed the data of 300 adult AML specimens in our hospital (including 20 MLLr-AML patients), and the results further confirmed that only HDAC9 among the HDAC family had a significantly higher expression level in the MLLr-AML patient group than that in the Non-MLLr-AML patient group (P<0.001), and the high expression of HDAC9 in the MLLr-AML patient group was a common phenomenon and not limited to a particular class of MLL chaperone molecules. Further studies revealed that overexpression of MLL fusion proteins in HEK293T could cause up-regulation of HDAC9 protein levels, and this experiment clarified the direct correlation between the MLL fusion genes and the high expression of HDAC9. The HDAC IIa inhibitor (TMP-269), which can target HDAC9, was significantly more sensitive in MLLr-AML cell lines than in Non-MLLr-AML cell lines, and also that MLLr-AML cell survival was dependent on HDAC9, and that knockdown of HDAC9 induced an increase in apoptosis and a slowdown in proliferation in MLLr-AML cells. We subsequently demonstrated that knockdown of HDAC9 downregulates the expression of MLL fusion proteins through degradation of MLL proteins. Furthermore, when exploring therapeutic options for targeting MLLr-AML, we found that combining the BCL-2 inhibitor Venetoclax (VEN) with an MLL-Menin specific inhibitor (MEN1i) specifically downregulated the expression of HDAC9 and had a favorable synergistic killing effect on MLLr-AML in both in vitro and in vivo models, further suggesting an important role of HDAC9 in the treatment of MLLr-AML. To sum up, through this study, we can shed light on the role of specific HDAC molecules in MLLr-AML and provide a new potential target for the degradation of MLL fusion protein to eradicate MLLr-AML.

Homoharringtonine-Based Induction Regimen Improved the Remission Rate and Survival Rate in Chinese Childhood AML: A Report From the CCLG-AML 2015 Protocol Study.

Homoharringtonine (HHT) is commonly used for the treatment of Chinese adult AML, and all-trans retinoic acid (ATRA) has been verified in acute promyelocytic leukemia (APL). However, the efficacy and safety of HHT-based induction therapy have not been confirmed for childhood AML, and ATRA-based treatment has not been evaluated among patients with non-APL AML. This open-label, multicenter, randomized Chinese Children's Leukemia Group-AML 2015 study was performed across 35 centers in China. Patients with newly diagnosed childhood AML were first randomly assigned to receive an HHT-based (H arm) or etoposide-based (E arm) induction regimen and then randomly allocated to receive cytarabine-based (AC arm) or ATRA-based (AT arm) maintenance therapy. The primary end points were the complete remission (CR) rate after induction therapy, and the secondary end points were the overall survival (OS) and event-free survival (EFS) at 3 years. We enrolled 1,258 patients, of whom 1,253 were included in the intent-to-treat analysis. The overall CR rate was significantly higher in the H arm than in the E arm (79.9% v 73.9%, P = .014). According to the intention-to-treat analysis, the 3-year OS was 69.2% (95% CI, 65.1 to 72.9) in the H arm and 62.8% (95% CI, 58.7 to 66.6) in the E arm (P = .025); the 3-year EFS was 61.1% (95% CI, 56.8 to 65.0) in the H arm and 53.4% (95% CI, 49.2 to 57.3) in the E arm (P = .022). Among the per-protocol population, who received maintenance therapy, the 3-year EFS did not differ significantly across the four arms (H + AT arm: 70.7%, 95% CI, 61.1 to 78.3; H + AC arm: 74.8%, 95% CI, 67.0 to 81.0, P = .933; E + AC arm: 72.9%, 95% CI, 65.1 to 79.2, P = .789; E + AT arm: 66.2%, 95% CI, 56.8 to 74.0, P = .336). HHT is an alternative combination regimen for childhood AML. The effects of ATRA-based maintenance are comparable with those of cytarabine-based maintenance therapy.

RUNX1 Mutations Have Distinct Prognostic and Clinical Implication in Pediatric AML: Results of the AML-BFM Study Group

Background: Runt-related transcription factor 1 (RUNX1) critically regulates hematopoiesis and contributes to leukemogenesis when mutated. In adult AML, RUNX1 mutations (RUNX1mut) have been identified as negative prognosticator associated with distinct clinicopathological features. These findings prompted the inclusion of RUNX1mut into the recent WHO classification as a new provisional entity in AML. However, the significance of RUNX1mut in pediatric AML remained to be validated. Methodology: To determine the impact of RUNX1mut (OMIM No. 151385) in pediatric AML, a German cohort of 512 AML patients (age <18 years) was retrospectively analyzed. Patients with diagnosis between 2015 and 2021 were enrolled to either the AMLR12 or AMLR17 register of the AML-BFM study group (Essen, Germany). Promyelocytic leukemia, Down-syndrome, and treatment related AML were excluded. Mutational screening of 52 different leukemia-specific genes was performed by next-generation sequencing (TruSight Myeloid panel, Illumina, San Diego, California, USA). RUNX1mut were confirmed by PCR and subsequent Sanger sequencing. Probability of overall survival (OS) and event-free survival (EFS) was determined using the Kaplan-Meyer method and log-rank test. Cox model was used for uni- and multivariable regression analyses. Results: In this cohort of childhood AML, 23 of 512 patients (4.5%) harbored 24 different RUNX1mut. RUNX1mut was associated with older average age at diagnosis (11.5 vs. 8.3 years, p = 0.01*) and male gender (63% vs. 50%, p = 0.06). Compared with non-mutated (wt) samples, RUNX1mut was more frequently allocated to myelomonocytic AML (FAB M4, 30% vs. 18%, p = 0.09), followed by AML with minimal differentiation and without maturation (FAB M0, 4.3% vs. 1.5%, p = 0.3 and FAB M1, 22% vs. 14%, p = 0.2), however without significance. Cytogenetically, RUNX1mut tends to correlate with -7/7q, (13% vs. 5%, p = 0.14) and most frequently accompanied by t(8;21) and trisomy 8, showing almost equal distribution (13% vs. 12%, p = 0.75 and 13% vs. 16%, p = 1.0). Together, except of -7/7q, no correlations between RUNX1mut and recurrent cytogenetic aberrations were observed. In the molecular analysis, FLT3-ITD was found to be the most frequently co-occurring mutation and significantly correlated with RUNX1mut (44% vs. 23%, p = 0.02*). Additionally, RUNX1mut was associated with WT1 mutations (26.1% vs. 12.4%, p = 0.1), but mutually exclusive of KIT, KRAS and NPM1 aberrations (10.8% vs. 0%, p = 0.15, 10.2% vs. 0%, p = 0.15 and 9.2% vs. 0%, p = 0.25). Contrary to reports on RUNX1mut in adult AML, we could not confirm exclusiveness of the recurrent aberration CEBPA (13% vs. 6.2%, p = 0.18). A broad range of co-occurring mutations, cooperating with RUNX1mut was found (Figure 1 a). Overall, the number of mutations was significantly increased in RUNX1mut group compared with the RUNX1wt group (average number 2.9 (range 1-4) vs. 2.0 (range 1-6), p < 0.001***). Paired analyses of initial and relapse samples in 8 cases suggest instability of RUNX1mut which was present only at relapse in 5 out of 8 cases and at initial diagnosis in a single case. Together, these results point to RUNX1 being a late rather than an early event in the pathogenesis of pediatric AML. Noteworthy, RUNX1mut did not have an adverse effect on 5-year OS (83.3±8.9% vs. 78.2±2.1%, p = 0.33) (Figure 1 b) or EFS (53.5±11% vs. 61.6±2.5%, p = 0.53) in pediatric AML and was not correlated with a worse response rate. This was supported in multivariable analyses, where RUNX1mut was found to have no effect on OS or EFS independent of other prognostic relevant covariates. In subset analysis, the co-occurring mutations FLT3-ITD and WT1 had no additional prognostic impact on RUNX1mut AML. Same pattern without prognostic difference of RUNX1mut for OS and EFS was determined in subset analysis for stem cell transplantation, age, number of mutation and risk groups. Together, these results failed to establish any prognostic value of RUNX1mut in childhood AML. Conclusion: This is the most comprehensive study assessing the impact of RUNX1mut in a large pediatric cohort. Unlike adult AML (Gaidzik et al., 2011 and 2016), our results provide evidence for a different clinicopathologic impact of RUNX1mut in pediatric AML, arguing against its classification as a distinct entity in childhood AML. These results may broaden the perspective on the pathological significance of RUNX1mut for leukemogenesis in pediatric AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical and Functional Implications of MYC Variants As a New Class of Pathogenic Variants in AML

MYC, a well-known oncogene, is implicated in a majority of cancers, but there is little data on its role in myeloid malignancies. We recently described a novel internal tandem duplication of MYC gene (MYC-ITD) in a subset of patients with AML. As the prevalence of MYC aberrations have not been well documented in AML, we interrogated the genome, transcriptome and associated clinical and laboratory data from the largest cohort of pediatric AML patients to define the prevalence, frequency of co-occurring cytomolecular alterations and outcomes associated with MYC variants. Mutation Profiling: Diagnostic specimens from 1,806 patients with AML (age 0-29 years) enrolled on COG Phase III trials were retrospectively interrogated using contemporary NGS techniques or a PCR based "Tandem Repeat" assay for MYC-ITD. Conventional karyotyping and mutational analysis, including NGS, were used to determine additional cytogenetic and molecular abnormalities. Of the 1,806 patients studied, MYC aberrations were identified in 58 patients (3.2%) with 50% (N=29) harboring MYC-ITD, 46.5% (N=27) with a SNV or indel (MYCSNV/indel) and 2 patients (3.5%) with dual MYC-ITD and SNV. For MYC-ITD, tandem duplications occurred in exon 3 (Max binding domain) with ITD lengths ranging from 252-588 bp. The majority of MYCSNV/indel (77%) were identified at the P72-P78 hotspot with 33.3% (N=9) harboring the P74>L mutation and 18.5% (N=5) having the T73>N mutation. Functional Studies: Given the novelty of the ITD variant, we initially characterized the functional properties of MYC-ITD in vitro. Similar to wild-type (MYCWT), MYC-ITD is localized to the nucleus and requires Max to bind to specific DNA sequences (E-boxes). However, in contrast to MYCWT, MYC-ITD fails to recognize sequences containing a single E-box in vitro, but exhibits higher affinity toward sequences with two or more E-boxes. MYC-ITD is consistently more efficient in activating promoters with multiple E-boxes than MYCWT. Thus, MYC-ITD may possess different DNA-binding and protein-interacting properties, potentially leading to altered transcriptional regulation and biological output. Given the alterations in DNA binding properties, we evaluated the functional consequences of MYC-ITD in our cord blood stem cell/endothelial cell (EC) co-culture system. Normal cord blood stem cells were transduced with MYC-ITD vs. MYCWT and grown in an EC co-culture system. MYC-ITD transduced cord blood stem cells showed significantly higher proliferation compared to MYCWT or control GFP-transduced cells with proliferation advantage of 37-and 45-fold after 14 and 21 days in co-culture, respectively (p<0.001). Clinical Impact: Analysis of cooperating cytomolecular lesions revealed a striking difference between MYC-ITD and MYCSNV/indel. MYC-ITD was highly enriched in core-binding factor (CBF) AML found in 86% (N=25)[(N=18, 62%) with inv(16) and (N=7, 24%) with t(8;21)] (Figure 1A), vs. 26% in the MYCWT cohort and had a paucity of somatic mutations. Conversely, MYCSNV/indel was enriched in NUP98-NSD1 fusions (N=7, 27%), FLT3-ITD (N=10, 38%) and NPM1 mutations (N=9, 33%) (Figure 1A), which was significantly higher than the WT cohort. Patients with MYC-ITD had a morphologic CR rate after course 1 of 92.3% vs. 80% (p=0.249) in patients with MYCSNV/indel with a corresponding relapse rate of 24% and 52% (p=0.046). Lower CR and higher relapse reflects the higher proportion of patients with high-risk lesions (FLT3-ITD, NUP98-NSD1 and WT1) in MYCSNV/indel and favorable CBF in MYCITD. Those with MYC-ITD and MYCSNV/indel had a 5-year event free survival (EFS) of 70% vs. 38.5%, respectively (p=0.018) with a corresponding overall survival (OS) of 76% and 47% (p=0.023) similar to patients with favorable or high risk disease (Figure 1B). Conclusion: Here we define MYC aberrations as a new class of pathogenic variants in childhood AML. We demonstrate functional and clinical characterization of a novel MYC-ITD and contrast this novel variant to that of established pathogenic MYCSNV/indel. We show the high prevalence of co-occurring core-binding factor fusions with MYC-ITD, and high frequency of co-occurring NUP98-NSD1, FLT3-ITD and NPM1 mutations in MYCSNV/indel. Patients with MYC-ITD have superior clinical outcomes while patients with MYCSNV/indel have clinical outcomes similar to high-risk patients, likely due to the associations with the disease associated fusions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High CX3CR1 expression predicts poor prognosis in paediatric acute myeloid leukaemia undergoing hyperleukocytosis.

Paediatric AML patients with hyperleukocytosis have a poor prognosis and higher early mortality. Therefore, more studies are needed to explore relevant prognostic indicators and develop effective prevention strategies for this type of childhood AML. All original data were obtained from the TARGET database. First, we explored meaningful differentially expressed genes (DEGs) between the hyperleukocytosis group and the non-hyperleukocytosis group. Next, we screened and identified valuable target genes using univariate Cox regression, Cytoscape software, and Kaplan-Meier survival curves. Finally, the coexpressed genes, functional networks, and immune-related activities associated with the target gene were deeply analysed by the GeneMANIA, LinkedOmics, GEPIA2021, TISIDB, and GSCA databases. We selected 1229 DEGs between the hyperleukocytosis group and the non-hyperleukocytosis group in paediatric AML patients. Among them, 495 DEGs were significantly linked with the overall survival of paediatric AML patients. Further, we discovered that CX3CR1 was a promising target gene. Meanwhile, we identified CX3CR1 as an independent prognostic predictor. Besides, we showed that CX3CR1 had strong physical interactions with CX3CL1. Additionally, functional network analysis suggested that CX3CR1 and its coexpressed genes modulated immune response pathways. Subsequent analysis found that immune cells with a high median value of CX3CR1 were monocytes, resting NK cells and CD8 T cells. Finally, we observed that CX3CR1 expression correlated with infiltrating levels of immune cells and immune signatures. Elevated CX3CR1 expression may be an adverse prognostic indicator in paediatric AML patients undergoing hyperleukocytosis. Moreover, CX3CR1 may serve as an immunotherapeutic target for AML with hyperleukocytosis in children.

Abstract 3479: UBTF tandem duplications (UBTF-TD) in childhood AML: Enrichment in FLT3-ITD and association with clinical outcome

Abstract Childhood AML is an aggressive disease with high rates of failures and poor survival. We have demonstrated that the molecular landscape of AML in children is distinct, and co-occurrence of variants modulate outcomes. Recent discovery of tandem duplication (TD) of the UBTF gene in AML, with enrichment in FLT3-ITD has implicated yet another mutation whose cooperation with FLT3-ITD may modify outcome. Here, we provide a comprehensive evaluation of UBTF-TD in de novo AML and define its clinical implications within FLT3-ITD patients. Initial interrogation of transcriptome data from 1,158 children enrolled on COG AAML1031 identified 50 cases of UBTF-TD (4.3%). Overwhelming majority of UBTF-TD cases were observed in FLT3-ITD cases (77%), vs. that of 1.2% in those without FLT3-ITD (p&amp;lt;0.001). Given extreme enrichment of UBTF-TD in FLT3-ITD, we inquired whether cooperation of UBTF-TD and FLT3-ITD creates a distinct clinical entity. To this end we screened diagnostic DNA from 400 FLT3-ITD patients treated on three consecutive CCG/COG trials (COG AAML1031, COG AAML0531, and CCG-2961) by PCR and fragment analysis. UBTF-TD was identified in 61 FLT3-ITD cases (15.3%). The data presented here forth focuses on evaluation of implications of UBTF-TD in FLT3-ITD positive patients only. Within the FLT3-ITD patients, initial correlation of UBTF-TD with demographics, disease characteristics, and associated genomic variants was conducted. Patients with and without UBTF-TD had a similar median age at diagnosis (p=0.322), lower diagnostic WBC (p=0.010) and higher marrow blast % (p&amp;lt;0.001). There was a stark paucity of cooperating variants that commonly co-occur with FLT3-ITD, with a single NPM1 mutation (1.6% vs. 29%, p&amp;lt;0.001) and no NUP98 fusions (0% vs. 23%, p&amp;lt;0.001). There was a significant enrichment of WT1 mutations, with 45% UBTF-TD patients with a WT1 mutation (FLT3-ITD/UBTF-TD/WT1), vs. 11% in UBTF-WT (p&amp;lt;0.001). Trisomy 8 (Tri8) was seen in 15% of UBTF-TD. Patients with UBTF-TD had a lower CR rate (44% vs. 60%, p = 0.018), and Higher MRD rate (38% vs. 21%, p&amp;lt;0.001). Patients with and without UBTF-TD had an EFS of 28% vs. 42% (p=0.047) with a corresponding OS of 40% and 57% (p=0.019). Given enrichment of WT1 mutations and Tri8 in patients with UBTF-TD, we studied the outcome UBTF-TD patients in the context of these two variants. FLT3-ITD/UBTF-TD/WT1 patients had a 5-year EFS of 17% vs. 38% for similar patients without WT1 mutations (p=0.0062). Patients with UBTF-TD with additional Tri8 had a similarly poor outcome with an EFS of 23% with a corresponding OS of 33%, providing a distinct high risk UBTF-TD cohort (+WT1 or Tri8), whereas the remaining UBTF-TD patients had a more favorable outcome with EFS and OS of 64% and 86%, respectively (p&amp;lt;0.0001, and p&amp;lt;0.0001). UBTF-TD is a novel genomic entity with high enrichment in patients with FLT3-ITD and a distinct clinical outcome driven by cooperating WT1 mutation and Tri8. Citation Format: Leila Robinson, Amanda Leonti, Todd A. Alonzo, Yi-Cheng Wang, Michele S. Redell, Rhonda E. Ries, Jenny L. Smith, Tiffany A. Hylkema, Quy Le, E Anders Kolb, Richard Aplenc, Xiaotu Ma, Jeffrey Klco, Katherine Tarlock, Soheil Meshinchi. UBTF tandem duplications (UBTF-TD) in childhood AML: Enrichment in FLT3-ITD and association with clinical outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3479.

Straight to the Point-The Novel Strategies to Cure Pediatric AML.

Although the outcome has improved over the past decades, due to improved supportive care, a better understanding of risk factors, and intensified chemotherapy, pediatric acute myeloid leukemia remains a life-threatening disease, and overall survival (OS) remains near 70%. According to French-American-British (FAB) classification, AML is divided into eight subtypes (M0–M7), and each is characterized by a different pathogenesis and response to treatment. However, the curability of AML is due to the intensification of standard chemotherapy, more precise risk classification, improvements in supportive care, and the use of minimal residual disease to monitor response to therapy. The treatment of childhood AML continues to be based primarily on intensive, conventional chemotherapy. Therefore, it is essential to identify new, more precise molecules that are targeted to the specific abnormalities of each leukemia subtype. Here, we review abnormalities that are potential therapeutic targets for the treatment of AML in the pediatric population.

KMT2A Partial Tandem Duplications (KMT2A-PTD) Is a Rare, but Recurrent Genomic Event in Childhood AML and Associated with High Rate of Co-Occurring FLT3 Mutations

KMT2A partial tandem duplication (KMT2A-PTD), characterized by a large internal duplication spanning 6-8 exons, has been documented in adult AML with a prevalence of 3-10% and is associated with poor outcomes. Despite the high prevalence of KTM2A fusions and known associated outcomes, prevalence and clinical implications of KMT2A-PTD has not been well characterized in childhood AML. We interrogated the transcriptome and associated clinical and genomic data from pediatric AML patients treated on COG AAML1031 to define the prevalence of KMT2A-PTD, frequency of co-occurring genetic mutations and outcomes associated with this variant.Ribo-depleted RNA seq data from 1,294 patients with AML (age 0-29 years) enrolled on COG AAML1031 with available cytogenetic, molecular, and clinical data were utilized. Gene fusions, internal tandem duplications (ITD) and partial tandem duplications (PTD) were detected by Cicero. Conventional karyotyping and mutational analysis including NGS were used to determine additional cytogenetic and molecular abnormalities. Response was measured by 5-year overall survival (OS), event-free survival (EFS), disease-free survival (DFS) and relapse risk (RR).Of the 1,294 patients studied, KMT2A-PTD was identified in 20 patients (1.5%). Evaluation of karyotype demonstrated striking paucity of karyotypic alterations with 75% with normal karyotype; 3 cases (15%) of trisomy 11 were identified. In contrast to KMT2A-rearranged (KMT2A-r) AML, which has the highest prevalence in young patients (median age 3.3 years), the median age with KMT2A-PTD was 13.4 years (p<0.0001). Further analysis demonstrated a high rate of co-occurrence of FLT3 mutations; 15 patients (75%) had FLT3-ITD (N=13; 65%) and/or FLT3 activating mutations (N=5; 25%). The rate of co-occurring FLT3-ITD was significantly higher in the KMT2A-PTD cohort compared to patients without KMT2A-PTD (65% vs. 19%, p<0.001). Prevalence of KMT2A-PTD in patients with FLT3-ITD was 5.7%. Additional co-occurring genetic mutations were identified in WT1 (20%) and the RAS pathway. There was a striking enrichment of mutations that are commonly associated with AML in older adults with 8/20 patients (40%) harboring mutations in IDH1/IDH2, U2AF1, and TP53, far in excess of what is seen in other pediatric AML patients (Figure 1A).We evaluated the significance of KMT2A-PTD on clinical outcome. Patients with KMT2A-PTD had a poor response to induction therapy with a morphologic CR rate of 45% and rate of residual disease (MRD) positivity by flow cytometry after initial course of therapy was 40%. Additionally, high rates of relapse were noted for patients with KMT2A-PTD with a RR of 53%. EFS for patients with and without KMT2A-PTD was 39% vs. 46%, respectively (p=0.54) with a corresponding OS of 58% and 64% (p=0.61). Outcome analysis for the KMT2A-PTD cohort demonstrated dual KMT2A-PTD/FLT3-ITD patients had an EFS of 46% compared to 29% for KMT2A-PTD/non-ITD patients (p=0.62, Figure 1B). We inquired whether hematopoietic stem cell transplant (HSCT) may modify outcomes in patients with KMT2A-PTD. Of the 20 patients with KMT2A-PTD, 7 (35%) proceeded to HSCT in CR1, with the indication in 6 of 7 patients due to FLT3-ITD high allelic ratio (HAR, ≥0.4). Stem cell transplant recipients had a DFS of 83% vs. 0% for those who continued on protocol chemotherapy (p=0.002, Figure 1C) with corresponding OS of 100% vs. 43%, respectively (p=0.05).In this large cohort of childhood AML patients treated on AAML1031, we identified a small subset of patients with KMT2A-PTD and show the high prevalence of co-occurring FLT3 mutations. Although KMT2A-PTD patients with and without FLT3-ITD had similar outcomes, the cohort who received HSCT in CR1 (most with FLT3-ITD) experienced improved outcomes compared to the rest of the cohort, suggesting that intensification of therapy may benefit this group of patients overall. Further research into KMT2A-PTD in pediatric AML will guide future risk-adapted therapy and enhance understanding of biologic implications of this lesion, including whether altered KMT2A may serve as a therapeutic target as it may for KMT2A-r AML. [Display omitted] DisclosuresPollard: Syndax: Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Membership on an entity's Board of Directors or advisory committees.

Significant Improvements in Survival for Patients with t(6;9)(p23;q34)/DEK-NUP214 in Contemporary Trials with Intensification of Therapy: A Report from the Children's Oncology Group

The occurrence of t(6;9)(p22;q34)/DEK-NUP214 is a rare subtype of pediatric AML that commonly co-occurs with FLT3-ITD mutations and is associated with poor outcomes, regardless of FLT3-ITD status. With increased recognition of the inferior prognostic impact of FLT3-ITD and to a lesser degree t(6;9), and with early identification of these lesions, these patients have been allocated to high risk intensive therapy on more contemporary clinical trials. Therefore we sought to interrogate the outcome of children with t(6;9) AML to determine if intensification of therapy, specifically the use of hematopoietic stem cell transplant (HSCT) in CR1, and FLT3 inhibitors for ITD+ patients, may have improved outcomes for this high risk group of patients.We evaluated the outcomes of all patients with t(6;9) detected by karyotype analysis on pediatric patients with de novo AML enrolled on the previous 7 Children's Oncology Group (COG) or its predecessor (POG or CCG) trials over 31 years (1988-2017). A total of 66 cases of t(6;9) AML were identified from CCG-2861 (n=2), CCG-2891 (n=5), POG-9421 (n=7), CCG-2961 (n=10), AAML03P1 (n=7), AAML0531 (n=17), and AAML1031 (n=18). For all studies prior to 2005 (CCG-2861, CCG-2891, POG-9421, CCG-2961) neither t(6;9) nor FLT3-ITD was used as a prognostic factor to adjust treatment, while on AAML0531 post an amendment in 2008 all patients on AAML1031 with high allelic ratio (HAR; >0.4) FLT3-ITD were allocated to HSCT in CR1, which continued on AAML1031 and this group of patients also received sorafenib.Among all 66 t(6;9) patients identified, 45 (68%) had known data for ITD. Among these, 69% (n=31) harbored a co-occurring FLT3-ITD mutation (AR range 0.07-13.35), while 31% (n=14) were FLT3-ITD negative. In the early phase of trials prior to recognition of FLT3-ITD (1988-2002; CCG-2861, CCG-2891, POG-9421, and CCG-2961), the 5-year event-free survival (EFS) and overall survival (OS) for all t(6;9) patients was dismal at 14% and 18%, respectively (Figure 1). Trials from 2006-2010 (AAML03P1, AAML0531), evaluated the addition of gemtuzumab ozogamicin (GO) to chemotherapy as well as the use of HSCT in CR1 for patients with matched related donors, and the 5-year EFS and OS for patients with t(6;9) improved to 48% and 58% (p<0.001 compared to 1998-2002, Figure 1). Among this group of patients, only 16% received HSCT in CR1. The prevalence and clinical implications of FLT3-ITD in childhood AML was reported early and was incorporated as a risk stratifying biomarker following an amendment to 0531 and for all FLT3-ITD patients with HAR disease on AAML1031; this cohort was considered high risk and allocated to intensified therapy, which in many cases significantly impacted the treatment of patients t(6;9) given the significant overlap. In the most recent trial (AAML1031, 2011-2017), the 5-yr EFS and OS for patients with t(6;9) was excellent at 71% and 94%, which was significantly improved compared to other trials (p<0.001; Figure 1). Among the 18 t(6;9) patients on 1031, 72% (n=13) were allocated to HSCT in CR1, the indication for 7 was HAR FLT3-ITD and they also received sorafenib and for 6 was presence of residual disease after induction 1 (4 with LAR FLT3-ITD, 2 ITD negative). The EFS and OS for the t(6;9)/FLT3-ITD HAR and t(6;9)/FLT3-ITD LAR/neg ITD groups who both received HSCT in CR1 were comparable and very good (EFS: 87.5% vs 100%, OS: 87.5% vs 100%, p=NS for both).We show that over the past 3 decades, outcomes of patients with t(6;9) AML have improved significantly on upfront trials. While general improvements in AML treatment and supportive care are an important aspect, our data with excellent EFS on AAML1031 suggest that the intensification of upfront therapy with HSCT, which occurred for many t(6;9) patients due to their co-occurrence with FLT3-ITD, was critical to these improvements. On the current COG phase III AAML1831 trial, all patients with t(6;9) regardless of ITD status are considered high risk and allocated to HSCT in CR1, while those with a FLT3 mutation also receive gilteritinib. Evaluation of the outcome of t(6;9) patients with this therapy will be important as while FLT3-ITD patients have been an example of a group whose poor outcomes have been shown to be abrogated with intensified therapy, it appears that t(6;9) patients may have benefitted from their co-occurrence pattern and are also a group whose outcomes can be significantly improved with an intensified upfront therapeutic approach. [Display omitted] DisclosuresPollard: Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees.

Functional Consequence and Therapeutic Targeting of Cryptic ALK Fusions (ALK fus) in Monosomy7 AML

Monosomy 7 (Mono7) AML is one of the most established adverse prognostic markers in AML with over four decades of data on response and outcome in varied clinical trials. However, despite intensive and myeloablative therapies, outcome for this subtype of AML remains dismal with no meaningful advances in therapy in decades. As part of our recently completed discovery efforts in childhood AML (TARGET Pediatric AML; TpAML), we interrogated the transcriptome of 1068 children and young adults treated on COG AAML1031, including 28 cases of Mono7. We discovered novel cryptic fusions involving the Anaplastic Tyrosine Kinase (ALK) gene that were exclusively seen in 4 of 28 Mono7 patients (14.3%) and not detected in the remaining 1064 patients (p<0.001). These ALK fusions (ALK fus) included SPTBN1-ALK (n=3) or RANBP2-ALK (n=1) (figure 1A) and all 4 cases included the entire kinase domain of the ALK gene. Expression of ALK transcript was evaluated in Mono7 patients with and without ALK fus to determine whether these fusions lead to induction/upregulation of ALK. As demonstrated in figure 1B, the only patients with any ALK expression were those with ALK fus positive Mono7.Expression of the resultant onco-proteins of the ALK fus was confirmed by transfection of parental HEK293T cells with the oncofusion and western blot analysis of the protein lysates. As shown in the western blot in figure 1C, immunoblotting of the lysates from ALK fus cells with ALK antibody confirmed expression of the appropriate ALK+ fusion oncoprotein. Under experimental conditions, fusion involving SPTBN1-ALK appeared to generate higher levels of the fusion onco-protein compared to that of RANBP2-ALK fusion. Confirmation of the fusion onco-protein expression further validates the possibility of the functional significance and potential targetability of these fusions.The ALK gene codes for a receptor tyrosine kinase belonging to a family of protein kinases linked to unregulated cell growth which play a key role in CNS development. Alterations of the ALK gene are commonly seen in Non-Small Cell Lung Cancer (NSCLC), Anaplastic Large-Cell Lymphomas, and Neuroblastomas. We further inquired about the functionality of the ALK fus and whether they may confer cytokine-independent proliferation. The ALK fus were cloned from patient samples into pCSII, a direct GFP-tagged lentiviral backbone under the Ef1α promoter; followed by Sanger sequencing to verify the insert. IL-3 dependent Ba/F3 cells were transduced with the GFP tagged lentiviral vectors containing the SPTBN1-ALK or RANBP2-ALK fusion transcripts.Transduced cells were sorted to GFP homogeneity and growth parameters evaluated post-IL-3 withdrawal. In contrast to the parental line, which rapidly died in the absence of cytokines, cells expressing either SPTBN1-ALK or RANBP2-ALK sustained growth and rapidly proliferated in cytokine-free media, suggesting a conferred transformation event by the fusions, figure 1D. SPTBN1-ALK fusion appears to show more transforming potential with more rapid cytokine-free proliferation compared to that of RANBP2-ALK fusion. The observed differential growth pattern with the two ALK fus correlates with the differential expression of the two oncoproteins as presented above.We then sought to evaluate the efficacy of Crizotinib as a potential therapy for aberrant ALK fus positive Mono7 AML. Crizotinib has been FDA-approved and demonstrated efficacy in treating ALK positive NSCLC. ALK fus -positive cells were treated with varying doses of Crizotinib in cytokine-free media and cell viability and cell death were determined with the Promega Cell Titer Glow assay after 48 hours in culture. Fusion positive cells show sensitivity to Crizotinib with IC50s of 144nM and 95nM for the SPTBN1-ALK or RANBP2-ALK fusions, respectively.Here we demonstrate experimental data that when newly discovered cryptic ALK fus in Mono7 AML are functional (translation to fusion onco-protein, conferring cytokine independence) and show susceptibility to the ALK inhibitor crizotinib. This data may support effective therapeutic targeting of a subset of this highly refractory AML. [Display omitted] DisclosuresHylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. OffLabel Disclosure:Crizotinib is a first-generation ALK inhibitor that is FDA-approved for the treatment of Non-Small Cell Lung Cancer, Anaplastic Lymphoma, and Neuroblastoma.

Fanconi Anaemia, Childhood Cancer and the BRCA Genes.

Fanconi anaemia (FA) is an inherited chromosomal instability disorder characterised by congenital and developmental abnormalities and a strong cancer predisposition. In less than 5% of cases FA can be caused by bi-allelic pathogenic variants (PGVs) in BRCA2/FANCD1 and in very rare cases by bi-allelic PGVs in BRCA1/FANCS. The rarity of FA-like presentation due to PGVs in BRCA2 and even more due to PGVs in BRCA1 supports a fundamental role of the encoded proteins for normal development and prevention of malignant transformation. While FA caused by BRCA1/2 PGVs is strongly associated with distinct spectra of embryonal childhood cancers and AML with BRCA2-PGVs, and also early epithelial cancers with BRCA1 PGVs, germline variants in the BRCA1/2 genes have also been identified in non-FA childhood malignancies, and thereby implying the possibility of a role of BRCA PGVs also for non-syndromic cancer predisposition in children. We provide a concise review of aspects of the clinical and genetic features of BRCA1/2-associated FA with a focus on associated malignancies, and review novel aspects of the role of germline BRCA2 and BRCA1 PGVs occurring in non-FA childhood cancer and discuss aspects of clinical and biological implications.

Clinical Features and Prognostic Impact of RUNX1 and PTPN11 Mutations in Pediatric Acute Myeloid Leukemia—the Jccg Study, JPLSG AML-05-

IntroductionAcute myeloid leukemia (AML) is a complex disease caused by various genetic alterations. Many acquired gene mutations, RUNX1, NPM1, DNMT3A, TET2, and IDH1/2, have shown prognostic significance in patients with AML. However, not much is known about the clinical implications of these mutations in pediatric AML patients because of their low frequency. Currently, RUNX1 mutations have been added to the adverse risk group in adult AML according to the European Leukemia Net recommendations of 2017. Moreover, some reports have suggested that PTPN11 mutations may be a poor prognostic marker in adult AML. However, the prognoses of pediatric AML patients with RUNX1 or PTPN11 mutations also remain uncertain. Thus, we investigated these mutations and their correlation with other gene aberrations to verify their prognostic impact on pediatric AML patients.MethodsRUNX1 mutations were analyzed using next-generation sequencing in 503 pediatric patients with de novo AML (0-17 years) who participated in AML99 clinical trial by the Japanese Childhood AML Cooperative Study conducted between 2000 and 2002 or AML-05 trial by the Japanese Pediatric Leukemia/Lymphoma Study Group conducted between 2006 and 2010. PTPN11 (exons 2, 3, 4, and 13) mutations were analyzed using Sanger sequencing in 369 patients who participated in AML-05 trial.Results and DiscussionRUNX1 mutations occurred at a frequency of 2.6% (13/503) in pediatric patients with de novo AML with 54% being frameshift/nonsense mutations and 46% being missense mutations. Missense variants were considered as mutations if they were not reported in the 1000 Genome database or dbSNP150. Six of 13 mutations were found in patients with normal karyotype. KMT2A -partial tandem duplications (KMT2A -PTD) were frequently found in patients with RUNX1 mutations (P < 0.001). Conversely, RUNX1 mutations were mutually exclusive with mutations in NPM1 and CEBPA . Interestingly, RUNX1 mutations were highly prevalent in patients with non-complete remission (non-CR; 5/13, 38% vs. 50/490, 10%; P= 0.009). PTPN11 mutations were identified in 21 (5.7%) of 369 pediatric AML patients and all were heterozygous point mutations leading to translational changes. PTPN11 mutations were frequently observed in patients with M0 subtype (3/7, 43%, P < 0.01) and RUNX1 mutations (3/9, 33%, P= 0.01). Interestingly, PTPN11 mutations were mutually exclusive with inv(16) and fusions of NUP98-NSD1, and CBFA2T3-GLIS2 and nearly mutually exclusive with other tyrosine kinase pathway genes, such as FLT3 -ITD, NRAS, KRAS, and KIT. Although PTPN11 mutations were mutually exclusive with BCOR/BCORL1 mutations, they were associated with mutations in cohesin complex component genes (P= 0.02). Overall survival (OS) and event-free survival (EFS) of patients with RUNX1 mutations were significantly worse than those of patients without RUNX1 mutations (5-year OS, 40% vs. 71%, P= 0.003; 5-year EFS, 23% vs. 55%, P= 0.002).The results of univariate and multivariate analyses were assessed using Cox regression analyses. FLT3 -ITD and some cytogenetic groups, such as t(8;21), inv(16), 5q deletion, monosomy 7, and t(16;21), were used as explanatory variables in the multivariate analysis. RUNX1 mutations were significantly associated with inferior OS (univariate: HR = 3.221, 95% CI: 1.550-6.695, P= 0.002; multivariate: HR = 2.103, 95% CI: 1.012-4.368, P= 0.046) in the univariate and multivariate analysis. Although EFS was significantly worse in patients with PTPN11 mutations than in those without (5-year EFS, 26% vs. 57%; P= 0.01), no significant difference was observed in OS. Remarkably, the number of patients receiving stem cell transplantation (SCT) revealed a significant difference between patients with and without PTPN11 mutations (71% vs. 46%; P= 0.03). These results indicate that most patients with PTPN11 mutations were rescued using SCT.ConclusionRUNX1 mutations were associated with non-CR and KTM2A -PTD and were related to adverse outcomes consistent with previous reports of adult AML patients. Although earlier reports did not conclude the prognostic impact of RUNX1 mutations because the number of pediatric patients was limited, our study had a considerable number of patients to confirm the prognostic impact of RUNX1 mutations. Furthermore, PTPN11 mutations were also related to adverse outcomes for EFS in pediatric AML patients. DisclosuresNo relevant conflicts of interest to declare.

Gene Expression Analysis of Pediatric Acute Myeloid Leukemia Identified a Hyperactive ASXL1/BAP1 Axis Linked with Poor Prognosis and over Expressed Epigenetic Modifiers

Genetic aberrations in the epigenome are rare in pediatric AML, hence expression data in epigenetic regulation and its downstream effect is lacking in childhood AML. Our pilot study screened epigenetic modifiers and its related oncogenic signal transduction pathways concerning clinical outcomes in a small cohort of pediatric AML in KSA. RNA from diagnostic BM biopsies (n = 35) was subjected to expression analysis employing the nCounter Pan-Cancer pathway panel. The patients were dichotomized into low ASXL1 (17/35; 49%) and high ASXL1 (18/35; 51%) groups based on ROC curve analysis. Age, gender, hematological data or molecular risk factors (FLT3 mutation/molecular fusion) exposed no significant differences across these two distinct ASXL1 expression groups (P > 0.05). High ASXL1 expression showed linkage with high expression of other epigenetic modifiers (TET2/EZH2/IDH1&2). Our data showed that high ASXL1 mRNA is interrelated with increased BRCA1 associated protein-1 (BAP1) and its target gene E2F Transcription Factor 1 (E2F1) expression. High ASXL1 expression was associated with high mortality {10/18 (56%) vs. 1/17; (6%) P < 0 .002}. Low ASXL1 expressers showed better OS {740 days vs. 579 days; log-rank P= < 0.023; HR 7.54 (0.98-54.1)}. The association between high ASXL1 expression and epigenetic modifiers is interesting but unexplained and require further investigation. High ASXL1 expression is associated with BAP1 and its target genes. Patients with high ASXL1 expression showed poor OS without any association with a conventional molecular prognostic marker.

Modifiable and Non-Modifiable Factors Affecting the Risk of Childhood Leukemia: An Overview of Meta-Analysis

Background: Numerous meta-analyses of childhood leukemia have been published, but no comprehensive study has examined the factors influencing childhood leukemia. We aimed to scrutinize the modifiable and non-modifiable factors affecting the risk of childhood leukemia. Materials and Methods: In this overview, two independent researchers screened the articles studying the effect of modifiable and non-modifiable factors on the childhood leukemia from the related databases, including Scopus, EMBASE, Cochrane, Web of Science, and Medline, without time and language restrictions from inception up to March 10, 2020. Results: Eight meta-analyses were included in this overview. Breastfeeding compared with non-breastfeeding was associated with 9% reduction in the risk of childhood leukemia (OR = 0.91). The children with the birth weight of ≥4,000 g were at a higher risk of acute lymphoblastic leukemia compared with those with low birth weight (OR = 1.26, 95% CI = 1.17, 1.37). There was a significant relationship between childhood leukemia and residential pesticide exposure (SOR=1.57). There was a relationship between influenza during pregnancy and higher risk of ALL (POR=3.64; 95% CI = 1.34-9.90), and childhood leukemia (POR=1.77). The childhood AML had a statistically significant relationship with no alcohol consumption during pregnancy. The SOR of childhood ALL had an association with paternal smoking. A statistical relationship exists between magnetic field intensity of ≥0.4 μT and childhood leukemia. The exposure to NO2 and benzene exhibited the OR of 1.64 (95% CI = 0.91-2.95) and 1.21 (95% CI = 0.97-1.52), respectively. Conclusion: Decreased breastfeeding, high birth weight, viral infections during pregnancy, alcohol consumption, maternal exposure to direct and indirect smoking, and exposure to electromagnetic fields and airborne pollutants are found to be significant risk factors for childhood leukemia.

Improved Outcome in Pediatric AML - the AML-BFM 2012 Study

Introduction Over the past 40 years, therapy intensification has continuously contributed to an improvement in the prognosis of children with AML. Hence, the AML-BFM 2004 trial resulted in a 5-year overall survival rate (OS) of 70%. Similar outcomes backing this claim were obtained by other study groups. During the prospective, randomized AML-BFM 2012 trial, purine analogue clofarabine (C) was introduced to the therapy regimen. Prev. synergistic effects of C combined with cytarabine (A) have been observed in relapsed/refractory ped. AML. It was meant to further increase the antileukemic efficacy in the 1st induction of de novo AML. Clofarabine (C), liposomal daunorubicin (Dx; L-DNR), and cytarabine (A) were randomized against to the stand. induction course of cytarabine, etoposide (E), and L-DNR. The implementation of a stand. stem cell transplantation (SCT) indication for patients with HR AML was another progress. The trials' primary obj. was the improvement of event-free survival (EFS) in ped. AML. Patients and Methods AML-BFM 2012 was an open, interventional, multi-center, prospective, randomized clinical trial for patients with de-novo childhood AML (age&amp;lt;18 years), comparing the use of clofarabine (CDxA) and etoposide (ADxE) in the 1st induction course. Recruitment began in 02/15. Since 11/17 the auxiliary drug, L-DNR, was no longer commercially available. With the use of CDxA being discontinued, this resulted in both the CDxA and ADxE arm being replaced with the alternative stand. arm, AIE. Upon permanent unavailability of the L-DNR, the study was terminated early. The total no. of recruited patients was 164, instead of the initially planned n=500. Results With a 3-yr OS of 82 ± 3% &amp; 3-yr EFS of 69 ± 4%, respectively, children treated by AML-BFM 2012 have an excellent outcome (median FU: 3.03 yrs.), which is superior to previous trials. The outcome did not significantly differ between ADxE and CDxA. In the study n=9 early deaths (EDs; defined as death &amp;lt;d42) were observed, in n=4 no treatment was initiated before the death. N=2 died from treatment-related mortality (TRM; 1,2%) in CR; in another 6 patient's death was assoc. with toxicities or complications from allogeneic SCT (3,7%). Severe infections were the most common complications; 32.8% of all reported infections were observed during the 1st or 2nd course. Prompted by the skin toxicity levels relating to C found in lit., upon closer observation, 11 events in total were categorized to the CTCAE grade ≥3 in both the 1st &amp; 2nd cycle. 6/11 events were assoc. with C. Two statistical simulations were used to determine the potential significance of the study's primary obj., if 500 patients had been enrolled. In both simulations, the same progress was assumed for the patient outcome, only extrapolated to the required no. of cases. In the 1st simulation, the actual randomization compliance was retained in favor of the stand. arm ("as treated" simulation). In the 2nd calculation, therapy groups were weighted according to the planned intent-to-treat ratio of 1:1 ("intent to treat" simulation). Various simulated case nos. were translated into whole nos. to fulfill the criteria for the Kaplan-Meier analysis. Neither calculation shows a major effect on EFS or OS for either arm. Conclusion The treatment regimen used in AML-BFM 2012 has an excellent overall outcome, which exceeded outcomes from previous trials. The outcome is not only a result of the intro. of C, despite its potential effectivity in ped. AML. The results are closely linked to a better risk group stratification, improvements of allogeneic SCT in HR patients, and better supportive care. Since L-DNR was successfully evaluated in ped. AML in first and second-line treatment (AML-BFM Study 2004 (Creutzig et al. 2013) / Int. Relapsed AML 2001/01 (Kaspers et al. 2013)), incl. a possible reduction of anthracycline-induced cardiomyopathies, the unavailability of L-DNR due to industrial reasons resulted in severe damage in the treatment devel. in children with malignancies. More early clinical trials are vital to further examine other neoadjuvant drug combs. that replace L-DNR. In summary, AML-BFM 2012 showed an improvement of EFS and OS. Forced termination of clinical trials for commercial reasons is ethically dubious. Pharmaceutical companies need to act responsibly regarding clinical trials involving sick children. The introduction of C as the first-line treatment of ped. AML should be considered feasible. Disclosures Reinhardt: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; CLS Behring: Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding. OffLabel Disclosure: Clofarabine, Etoposide, liposomal Daunorubicine using in AML frontline treatment in children

Newly Diagnosed Childhood AML Patients Treated with Bortezomib Show Superior Survival If CD74 Is Expressed: A Report of 991 Patients from the Children's Oncology Group AAML1031 Protocol

Introduction: CD74 is a type II transmembrane protein expressed on antigen-presenting cells and an MHC class II chaperone. It has been associated with tumor progression and metastasis in solid tumors, and its expression has been suggested to serve as a prognostic factor in many cancers, with higher relative expression associated with oncogenesis. As the expression of CD74 has been associated with response to bortezomib in multiple myeloma, we inquired whether such correlation might be seen in pediatric AML. We prospectively evaluated CD74 expression by difference from normal (ΔN) flow cytometry and correlated expression with clinical characteristics and outcome as part of Children's Oncology Group protocol AAML1031 that randomized patients younger than 30 years of age with de novo AML to standard treatment with (Arm B) or without (Arm A) bortezomib. The addition of bortezomib to standard chemotherapy increased toxicity but did not improve survival. Given the association of CD74 with B-lymphoid neoplasms and bortezomib's known efficacy in B-cell neoplasms and multiple myeloma, we hypothesized that within Arm B, the patients with CD74 expression would have a more favorable outcome. Methods: In total, 1,139 newly diagnosed pediatric patients with de novo AML were randomized to standard chemotherapy (n=561, Arm A) or standard chemotherapy with bortezomib (n=578, Arm B). All patients received the identical chemotherapy backbone with either four intensive chemotherapy courses or three courses followed by allogeneic hematopoietic stem cell transplantation for high-risk patients. To qualify for this correlative study, 991 patients satisfied 2 criteria: (1) submitting a bone marrow aspirate for ΔN at diagnosis and (2) providing consent for correlative biology studies. All diagnostic specimens were centrally and prospectively evaluated for the expression of CD74 by ΔN. AML was considered to be CD74-positive if the MFI was more than two times above background autofluorescence and more than 40% of the leukemia was above background autofluorescence. Results: Among 991 patients, 263 were CD74-positive (26.5%) by ΔN, with similar prevalence in Arm A (27.9%) and Arm B (25.2%). Correlation of CD74 expression with clinical characteristics showed that those with CD74 expression had higher median age (p&amp;lt;0.001), lower median WBC (p&amp;lt;0.001), higher prevalence of low risk protocol status (p=0.039), lower frequency of CEBPA mutation (p=0.039), inv(16) (p=0.001), and KMT2A rearrangements (p=0.002), and were enriched for t(8;21) (p&amp;lt;0.001) and t(6;9) (p=0.014) fusions. All these features retained significance when patients were sub analyzed by respective treatment arms. CD74-positive patients had a higher morphologic CR rate (p=0.016), however, measurable residual disease by flow cytometry was not significant in the entire cohort or in the sub analysis of treatment arms (p=0.155). CD74-positive patients showed superior 5-year OS (70.6% vs 61.8%, p=0.003) and EFS survival (51.2% vs 43.1%, p=0.007) compared to those who were CD74-negative. For patients in Arm A (no bortezomib), the differences in OS (66.1% vs 61.0%, p=0.138) and EFS (48.9% vs 41.7% p=0.088) were not significant between those that were CD74-positive and those that were CD74-negative (Figure 1). However, patients in Arm B receiving bortezomib that were CD74-positive showed a significant improvement in OS (75.3% vs 62.5%, p=0.006) and EFS (53.6% vs 44.3%, p=0.028) compared to those who were CD74-negative (Figure 1). Comparison of the outcomes for CD74-positive patients with and without bortezomib exposure showed a difference in OS of 66.1% vs. 75.3% for those in Arm A vs. Arm B but did not reach significance (p=0.155). Multivariable analysis for OS yielded a hazard ratio of 0.67 (95% CI: 0.44 - 1.02) and p=0.061, approaching, but not reaching, statistical significance. Conclusions: These data demonstrate that CD74 expression is associated with more favorable disease characteristics and survival. Patients receiving bortezomib that were CD74-positive showed a superior response to therapy compared to patients who did not express CD74, by both OS and EFS, suggesting that CD74-positive childhood AML patients stand to benefit from bortezomib therapy. Bortezomib may induce a mechanistic response in CD74-positive AMLs similar to that in bortezomib-treated B-cell neoplasms and/or multiple myeloma, where bortezomib has proven to be beneficial. Disclosures Cooper: Celgene: Other: Spouse was an employee of Celgene (through August 2019). Pollard:Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

A Monocentric Clinical Observation of Haploidientical Transplantation in 27 Patients with Acute Megakaryocytic Leukemia

Background Acute Megakaryocytic Leukemia (AMKL) accounts for approximately 10% of childhood AML and 1% of adult AML cases. Although AMKL with Down syndrome has a good prognosis, the prognosis for non-DS-AMKL is quite poor with a 3-years survival rate of less than 40%. At present, only allogeneic HSCT is curative. In our study, we followed 27 patients with AMKL who received a modified Bu/Cy/Mel protocol and underwent hematopoietic stem cell transplantation. The patients were then followed up with monocentric clinical observation. Methods From August 2015 to July 2020, 27 AMKL patients (14 males, 13 females, all non-Down-AMKL) with a median age of 2 years (range: 1-9 years) were continuously treated in our hospital (Hebei Yanda Lu Daopei Hospital) including 18 cases of chromosome abnormality including 3 cases of CBFA2T3/GLIS2 gene fusion (11.1%). Gene mutation included WT1(8), EVI1(5), JAK2(5): 18.51%, TET2(4), ASXL1(4), PTPN11(4): 14.81%, GATA1(2), GATA2(2): 7.4%. Bone marrow status before transplantation: 19 complete remission (CR) cases, 3 partial response (PR) cases, and 5 no response (NR) cases. There were 24 haploidentical transplants (including one patient going through 3 transplants), one transplant from a HLA-identical sibling donor and two from matched unrelated donors. Source of donor stem cells: parents in 23 cases, brothers in 2 cases, non-blood relationship in 2 cases, and none from offspring. HLA matching between the donor and recipient: 21 cases with HLA 5/10, 6 cases with ≥HLA 6/10. Haplo stem cells all came from BM+PBSC, with a median MNC input of 20.32x108/kg (10.1-26.7), CD34+ 11.68106/kg (4.05-22.44), and CD3+ 4.66x108/kg (2.11-11.29). Pre-treatment protocol: 27 patients received a modified Bu/Cy/Mel and were treated with graft-versus-host disease (GVHD) to prevent CsA+MMF+sMTX. Results During a median follow-up period of 10 months (range: 2-48 months), the 27 patients remained alive, with a median of +13 days (range: 9-21 days) to leukocyte transplantation and a median of +9 days (range: 4-38 days) to platelet transplantation. One month following transplantation, the bone marrow of all patients was 100% donor type. The overall survival (OS) rate was 63.0%, event-free survival (EFS) was 59.3%. OS was 84.2%, 33.3%, and 0% in the CR, PR, and NR groups, respectively (P&amp;lt;0.001). There were 7 cases of relapse and 10 deaths, among which 4 were caused by GVHD and 5 due to relapse. Incidence of acute GVHD (aGVHD) was 59.25% Grade I-IV and 18.51% Grade III-IV. Incidence of chronic GVHD (cGVHD) was 56%, with cystitis accounting for 14.81% of cases, 29.62% cytomegalovirus (CMV), 3.7% EBV, 0% thrombotic microangiopathy (TMA) and 18.51% infection. Conclusion For high-risk AMKL patients with poor long-term prognosis, the application of a modified Bu/Cy/Mel pretreatment significantly improves the efficacy of haploid transplantation without increasing pretreat-related death. For non-Down syndrome AMKL, we conclude that it is feasible to perform hematopoietic stem cell transplantation in CR status patients. Additional studies and multicenter and large-scale clinical studies with long-term follow-up are still needed to confirm these results. Disclosures No relevant conflicts of interest to declare.

3098 – THE ROLE OF GATA2 IN HSC GENERATION THROUGH ENDOTHELIAL-TO-HEMATOPOIETIC TRANSITION

GATA2 is the most frequently mutated gene in childhood AML. These patients have germline mutations in one allele of GATA2 and have a high predisposition for MDS/AML. Also, in mouse, Gata2 is required for embryonic HSC generation and survival and Gata2 haploinsufficiency results in a severe reduction in the formation of HSCs. The first HSCs are formed at embryonic day (E)10.5 through a process called endothelial-to-hematopoietic transition (EHT) from specialized hemogenic endothelial cells of the dorsal aorta in the aorta-gonad-mesonephros (AGM) region. As all patients suffering from GATA2 haploinsufficiency syndromes have innate GATA2 mutations we want to understand how Gata2 haploinsufficiency affects embryonic HSC generation. Therefore, we sorted phenotypic HSPCs (CD31+cKit+ cells) from WT and Gata2+/- E11 AGMs and performed transcriptome analysis. Surprisingly, we found that the hematopoietic transcriptional program is not abrogated in Gata2+/- mouse E11 CD31+cKit+ cells. However, Gata2+/- HSPCs still express the endothelial program indicating that the hematopoietic cells are stuck in the endothelium and cannot complete EHT. We found that Gfi1b is downregulated in Gata2+/- HSPCs. As Gfi1b is known to be required for downregulation of the endothelial program for EHT during primitive hematopoiesis, we hypothesize that Gata2 directly regulates Gfi1b and that this regulation is required for normal EHT. We are currently studying if a specific HSPC subtype can overcome Gata2 haploinsufficiency and will still undergo EHT as this may have implications for the hematopoietic cells that eventually populate the hematopoietic tissues.