Pediatric AML Research Articles

Evaluation of Cancer Testis Antigens (CTAs) As Immuno-Therapeutic Targets in Pediatric AML

The Cancer Testis Antigen (CTA) superfamily of genes have long emerged as potential targets for immunotherapy and response monitoring due to their relative restrictive expression in testis and variable expression in some cancers. Much of the data on CTA expression in cancer have been in solid tumors with little data on CTA expression in leukemias. Identification of leukemia associated antigens (LAA) may provide attractive targets for immunotherapy or monitoring of residual disease.In order to discover AML associated CTAs, we performed transcriptome sequencing on 446 de novo pediatric AML samples and 20 normal bone marrows (NBM) from healthy donors and interrogated the expression of 245 candidate CTAs obtained from the CT database (http://www.cta.lncc.br). Differential expression using Limma (https://bioconductor.org/packages/release/bioc/html/limma.html) identified 32 CTA genes to be significantly differentially expressed with 7 being up-regulated compared to NBM and 15 genes with expression in NBM which are silenced in AML. The upregulated genes include: CCNA1, PRAME , CT45A5, IGF2BP3, SPAG6, TEX14, and ZNF165 and are collectively expressed in 330 of 446 patients (75%) with the highest contributors being PRAME and CCNA1 (Fig. 1A, Fig. 1B) . These 2 CTAs are expressed in every fusion group and constitute 65% of the total cohort, with a notable enrichment in core binding factor (CBF) patients (80%). High CCNA1 expression are present in 2/3 of patients with Inv(16), 71% of patients with a NUP98 fusion, 88% of DEK - NUP214 carriers, and 64% of cytogenetic normal cohort. PRAME is expressed in 79% of patients with a RUNX1 - RUNX1T1 fusion, 57% of CBFA2T3 - GLIS2 positives, and 20% of Inv(16) patients. While the oncoprint (Fig. 1A) shows that most patients express 1 or 2 CTAs, with some genes showing exclusivity, patients with a KMT2A fusion tend to express multiple CTAs. As a group, 77% of patients with KMT2A fusions express at least 1 CTA.To determine the underlying mechanism of dysregulated CTA expression in AML, we inquired whether promoter methylation status might be associated with CTA expression. We correlated the expression level of the 7 upregulated CTA genes with the methylation of their corresponding promoters. Overall there was a strong signal demonstrating anti-correlation of expression of the above mentioned genes with their promoter methylation. In specific, for PRAME, differences in methylation at the region chr22:22899991-22901267 explain over 87% of the variation in expression, and this locus is normally methylated in NBM (Fig. 1C). Likewise, for CT45A5, it's normally methylated in NBM, and demethylation of this locus explains about 61% of the variance in expression (Fig. 1C).Additionally, we evaluated the clinical implications of CTA expression, and demonstrated that CCNA1 expression was significantly associated with clinical outcome. The range of expression for CCNA1 in the AML samples was 0-561.4 TPM (mean = 31.8 TPM), whereas expression for NBMs ranged from 0.3-2.5 TPM (mean = 0.9) (p=8.45E-08). After binning expression values to determine the best cutpoint for high and low expressors of CCNA1, patients classified with high expression had statistically significant worse event-free (EFS, Fig.1D) and overall survival (OS) (p=<0.0001, p=0.0393 respectively, Log-Rank test). As a sub-group, patients with KMT2A rearrangements and high CCNA1 expression also show prognostic significance with correlation of poor outcome (EFS, p=0.0009, Fig. 1E). In addition, cytogenetically normal (CN-AML) patients with FLT3 ITD had a much higher overall expression of CCNA1 than did CN-AML without FLT3 ITD (p=0.009, Mann-Whitney test).In this analysis we identify candidate CTA genes that are cumulatively expressed in the majority of AML cases and can provide novel therapeutic options by targeting with modified T-cell receptor therapies. They can also be used as biomarkers for response monitoring. Further, as aberrant expression of these genes are regulated by altered methylation, use of hypomethylating agents might enhance therapeutic efficacy by increasing target expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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.

Fusion Transcripts without Corresponding Cytogenetic Abnormalities in Acute Myeloid Leukemia: Implications for AML Pathogenesis

Chromosomal translocations and inversions that create fusion genes are important causal events in AML. 7 recurrent fusion genes define AML entities in the 2016 revision of the WHO classification of hematologic malignancies. We have developed a comprehensive diagnostic AML platform based on next-generation mRNA sequencing (RNAseq). The adaptable integrated bioinformatic pipeline HAMLET (Human AML Expedited Transcriptomics) analyses the raw data simultaneously for fusion transcripts and for sequence variants and expression levels of genes with relevance to AML pathogenesis, prognosis, or actionability.Poly(A)+ RNA from 100 cryopreserved AML samples with a blast count of 10-99% (median: 75%) from 96 patients (3 diagnosis-relapse pairs, 1 de novo AML and subsequent tAML) was sequenced at a depth of 59x106 paired-end reads/sample with median read length of 126 bp in an ISO17025-accredited Illumina HiSeq 2500 pipeline. Fusion transcripts were called by STAR-Fusion and FusionCatcher. HAMLET detected the recurrent fusion transcripts RUNX1-RUNX1T1, CBFB-MYH11, PML-RARA, ERG-FUS, DEK-NUP214, and KMT2A-MLLT3 plus the variants KMT2A-MLLT4, KMT2A-MLLT1, and KMT2A-MLLT6 with 100% sensitivity and 100% specificity compared to metaphase cytogenetics, FISH, and RT-PCR (Griffioen, ASH 2016).In addition, HAMLET identified fusion transcripts from 54 combinations of genes in 57 AML samples that lacked corresponding chromosomal aberrations by metaphase cytogenetics. 14 “unusual” fusions involved genes located on different chromosomes; 40 fusions represented intrachromosomal events. 8 fusions were detected recurrently, i.e. in at least 2 cases (Fig. A). 32 AML carried at least 2 unusual fusions, with a maximum of 9 different fusions in an AML with a complex karyotype (Fig. B). Unusual fusions were not correlated with classical recurrent AML fusion genes. The 5 most prevalent fusions and an ETV6-LYN fusion were successfully validated in a total of 18 AML by RT-PCR. The only fusion that could not be validated was a YWHAZ-KLF10 fusion (2 cases).The most prevalent RT-PCR-validated fusion (seen in 30 AML cases) has been described in ALL (Roberts, 2012; Safavi, 2015) and was an intrachromosomal TPM4-KLF2 fusion between the tropomyosin 4 and an alternative reading frame (ARF) of the transcription factor Kruppel-like factor 2 genes. Unexpectedly, we detected this fusion in all 3 tested normal bone marrows (BM) but not in EBV-transformed B cells. The hitherto unvalidated C15orf57-CBX3 fusion may be generated by retrotransposition (Schrider, 2013) and was present in 18 AML as well as in 2/3 normal BM and 2/3 EBV B-cells with CBX3 in ARF. The intrachromosomal NCOR2-UBC fusion (9 AML) is listed in the database ChimerDB without further characterization and involves the genes of Nuclear corepressor 2 and Ubiquitin C in the normal reading frame (NRF). The NUP98-NSD1 fusion (3 AML) with NSD1 in NRF is frequently found in hematological malignancies including AML and originates from a cryptic t(5;11) due to telomeric positions of both genes. The PIM3-SCO2 fusion between the serine/threonine kinase PIM3 and an ARF of Cytochrome C oxidase assembly protein can originate from a 0.6 Mb inv(22) in chronic neutrophilic leukemia (Menezes, 2013) and occurs frequently in pediatric AML (Kuhn, 2013). The well-characterized ETV6-LYN fusion (1 AML) fusing the TEL1 oncogene to the SRC family kinase LYN in NRF originates from a complex translocation with inversion involving chromosomes 8 and 12 (Telford, 2016) and has oncogenic activity by activation of STAT5 (Takeda, 2011).Fusion transcripts without recognizable chromosomal abnormalities on metaphase cytogenetics occur frequently in AML and involve actionable targets. The upstream sequence can be fused to the normal or alternative reading frame of the downstream coding sequences and involve potential or validated proto-oncogenes. Apart from cryptic translocations or inversions, trans-splicing is suggested as an alternative mechanism by the presence of some fusion genes in non-malignant hematopoietic cells. Trans-splicing without DNA rearrangement can occur physiologically and alter cellular development towards potential premalignant differentiation (Yuan, 2013). Increasing use of RNAseq technology in routine AML diagnostics, e.g. by HAMLET, will permit to unravel the prognostic significance of unexpected fusion transcripts and to guide novel targeted therapies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Error-Corrected Sequencing of Cord Bloods Identifies Pediatric AML-Associated Clonal Hematopoiesis

Background:Recently, several studies have used next-generation sequencing (NGS) to characterize an age-specific increase in the variant allele frequencies (VAF) and number of somatic mutations in AML-associated genes in adults without hematological malignancies. Termed Clonal Hematopoiesis of Indeterminate Significance (CHIP), this phenomenon is defined as being >2% VAF and associated with an increased risk of hematologic malignancy later in life. The threshold of 2% VAF is arbitrarily defined because mutations below the threshold could not be reliably called due to the inherent error rate of sequencing. In light of this, the lab has previously developed error-corrected sequencing (ECS) to identify point mutations and small indels from adult blood and marrow samples at VAF as low as 0.01%. Moving on to pediatric samples, the most commonly mutated genes in pediatric AML vary substantially from adult AML, and the prevalence of clonal hematopoiesis in pediatric AML-specific genes in early childhood is unknown. Using ECS, we investigated the prevalence of clonal hematopoiesis in 94 adult and pediatric AML-associated genes in 31 random human umbilical cord blood (UCB) samples. We hypothesized clonal mutations in cord blood would be rare because of the age-related linear acquisition of mutations observed in older individuals, but the genes in question had not been previously assessed.Methods:Random cord blood samples were obtained from the St. Louis Cord Blood Bank (http://www.slcbb.org/). ECS libraries from extracted genomic DNA were prepared according to established protocol. A total of 1602 amplicons were targeted (total queried genome space ~270kb) using an Illumina panel designed to enrich 94 genes frequently mutated in both adult and pediatric AML. The libraries were sequenced on the Illumina HiSeq 3000 platform and raw sequence data was analyzed using a validated bioinformatic pipeline established in the lab. A subset of called mutations was secondarily validated using the QX200 droplet digital PCR platform (Bio-Rad) in the Druley lab.Results and Conclusions:We found that 18.2% of cord blood samples harbor validated somatic mutations with VAFs ranging from 0.002 - 0.006. With ECS, we showed that the prevalence of clonal hematopoiesis in younger population is much higher than previously expected. These results suggest that clonal hematopoiesis is likely a stochastic process that happens in everyone as early as fetal development, yet the incidence of pediatric leukemia is estimated to be 7.7 cases per million children.Future work:In order to rule out the possibility of maternal blood contamination of the UCB, we are flow sorting CD34+ cells from the cord blood samples and screening for the mutations. DisclosuresNo relevant conflicts of interest to declare.

Functional Evaluation and Clinical Implications of FLT3 J uxtamembrane Domain Short Variants in Pediatric AML

Introduction: Somatic FLT3 activating mutations are common in AML and occur as internal tandem duplications (FLT3 /ITD) within the juxtamembrane domain (JMD) or in the activating loop of the tyrosine kinase domain (FLT3 /ALM). There is considerable variability in the length of the ITD, varying from 6 to >300 bp. We, and others, have previously demonstrated that ITD length is associated with outcome, where those with longer ITDs based on median ITD length (>48bp) had adverse outcome. However, the underlying etiology of this association has not been established. We hypothesized that longer ITDs are more likely to disrupt the auto-inhibitory properties of the JMD, leading to enhanced receptor phosphorylation and autonomous activation. We aimed to define a biologic threshold for ITD-associated relapse risk based on the length of ITD that causes maximal activation. We also evaluated the functional properties of newly identified single nucleotide variants (SNVs) within the FLT3 JMD.Methods: To evaluate the impact of FLT3 /ITD length on receptor auto-phosphorylation, FLT3 /ITD clones with a variety of ITD lengths (6, 12, 18, 24, 30, 36, 42, 48, 54 bps) were engineered and expressed in HEK293 cells. Semi-quantitative immunoblotting was used to evaluate the level of phosphorylated FLT3 (pFLT3), and comparison of pFLT3 to non-pFLT3 was calculated and presented as fold change (FC) of pFLT3:FLT3 for the different ITD lengths. We also assessed the functionality of newly discovered JMDSNVs in a similar manner, and further evaluated their response to FLT3 inhibitors. For evaluation of the clinical impact of ITD length, of the 1,022 patients treated on AAML0531, FLT3 /ITD data including ITD length as determined by fragment length analysis and allelic ratio was available from 144 patients and was used for clinical correlation with ITD length.Results: Initial evaluation of the activation potential of the different ITD lengths showed a correlation between ITD length and FLT3 phosphorylation (Fig 1A). A modest autonomous FLT3 phosphorylation was observed at 6 and 12 bp, with incremental increase in 18bp and 24bp. Maximal pFLT3 of FC was observed at 30 bp, with no significant increases detected with longer ITDs. Therefore, a biologic threshold of 30 bp was used to evaluate the clinical outcomes of patients with short ITDs (S-ITD; <30 bps) and long ITDs (L-ITD; ≥30 bps). Among the 144 FLT3 /ITD+ patients, n=37 had S-ITDs and n=107 had L-ITDs. Overall, based on cooperating cytogenetic and molecular features, a higher number of patients with S-ITDs were considered as low risk as compared to L-ITDs (36% vs. 13%; p=0.003), with a higher incidence of t(8;21) in the S-ITD cohort. There was no correlation observed between ITD length and allelic ratio (Pearson correlation r=0.54). Evaluation of clinical outcome based on ITD length as defined above demonstrated that those with L-ITD had a 5-year overall survival of 47±13% compared to 83±16% in those with S-ITD (p=0.004; Fig1B).We additionally evaluated the functional significance of newly discovered FLT3 SNVs in the JMD. A total of 20 unique mutations affecting 13 distinct codons were identified. We evaluated the effect of each amino acid variant on FLT3 activation as measured by aberrant pFLT3. Six of the 20 mutations resulted in varying degrees of autonomous pFLT3 (E573D, L576R, V592A, F594Y, F594C, Y559C). Notably, the V592A mutation resulted in potent autonomous receptor activation, with a pFLT3 level greater than that observed the D835 FLT3/ ALM mutation. All variants resulting in autonomous pFLT3 were found to be highly sensitive to the FLT3 inhibitor crenolanib, with IC50s for inhibition of pFLT3 ranging between 1.3-5.6 nMConclusion: The JMD is a critical region of FLT3, and disruption through ITDs or SNVs can result in profound dysregulation of the receptor and downstream signaling, thus serving as oncogenic events. We show that longer ITDs result in more potent FLT3 receptor activation and are associated with inferior clinical outcome. FLT3 /ITD and ALMs have recently been shown to be excellent targets for therapeutic intervention with FLT3 inhibitors. Further investigation on the use FLT3 inhibitor therapy should focus on those patients who may derive the most therapeutic benefit from these agents, which may include those with highly damaging JMD variants. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Functional Properties of Exon 17 KIT Mutations in Pediatric CBF AML Predict for Inferior Outcome and Enhanced Response to Tyrosine Kinase Inhibition: A Report from the Children's Oncology Group

Introduction: KIT mutations are detected in approximately 20-30% of core binding factor (CBF, t(8;21) and inv(16)/t(16;16)) AML and have been associated with outcome and response to tyrosine kinase inhibitors (TKI). Mutations tend to localize to 2 regions- the receptor's kinase activation loop in exon 17 (E17) and a region of the extracellular domain in exon 8 (E8). The prognostic significance of the 2 mutations has varied in previous series. In this analysis, the functional significance of E8) and E17 KIT mutations in vitro and their response to TKIs were evaluated. We hypothesized that activating variants might be associated with inferior clinical outcome and be more susceptible to KIT-targeted agents.Methods: COG AAML0531 enrolled 1,070 patients, of which 800 underwent targeted next generation sequencing of the entire KIT gene coding sequence to identify somatic KIT mutations. To evaluate the functional impact of the most common KIT mutations, E8 (D419G, D419indel) and E17 (D816V, N822K) mutations were engineered and transduced into HEK293 cells for evaluation of KIT phosphorylation (pKIT) by immunoblotting and into Ba/F3 cells for cytokine independent proliferation studies. Mutations that resulted in pKIT were evaluated for response to the TKIs dasatinib and crenolanib. We further correlated our mutational and functional analysis to clinical outcome data from COG AAML0531. We analyzed outcomes by treatment arms (no-gemtuzumab ozogamicin (no-GO) vs. GO), focusing on the GO arm given the potentially favorable impact of GO on CBF AML response.Results: A total of 247 CBF patients were enrolled, with 218 undergoing screening for somatic KIT mutations. Mutations were detected in 55 (25%) patients; 27 (49%) in E8, 26 (47%) in E17, and 2 (4%) involved both exons. Among the 109 patients with CBF AML treated on no-GO arm, KIT mutations were detected in 29/109 (27%); 14 (48%) in E8, 13 (45%) in E17, and 2 (7%) involved both exons. E8 mutations were a combination of missense mutations or 3-9 bp indels that maintained reading frame, while E17 variants were primarily missense mutations involving the D816 or N822 codons, while. Cells transduced with E8 mutations showed baseline receptor phosphorylation and no IL-3 independent proliferation. In contrast, E17 mutations showed autonomous receptor phosphorylation and IL-3 independent proliferation, with D816V showing more robust cellular proliferation and higher levels of pKIT (Fig1A).We further evaluated the cytotoxic effect of 2 TKIs in engineered cells with activating KIT mutations and KIT wild-type (WT). Neither parental line nor cells expressing KIT -WT had any response to the two TKIs with IC50s > 10,000μM, however cells transduced with the E17 variants D816V or N822K were exquisitely sensitive to the TKIs. Following exposure to dasatinib and crenolanib, N822K+ cells had an IC50 of 3.8nM and 28.3nM respectively, with D816V+ cells demonstrating similar IC50s of 13nM and 14.3nM respectively (Fig 1B).Given the differential activating potential of E8 vs. E17 variants, we evaluated the clinical implications of the mutations in the 2 regions among CBF patients. On the no-GO arm, E8 KIT mutations lacked clinical significance, with an overall survival (OS) of 81±20% vs. 80±9% (p=0.91) for patients with and without E8 mutations, with a corresponding disease-free survival (DFS) of 57±26% vs. 62±11% (p=0.75) and relapse risk (RR) of 43±28% vs. 36±11% (p=0.59) respectively. In contrast, activating E17 mutations were associated with adverse outcome with an OS of 56±27% vs. 85±7% for those with and without E17 mutations (p=0.02; Fig 1C), with a corresponding DFS 33±27% vs. 65±11% (p=0.05) and a RR of 67±30% vs. 32±11% (p=0.03) respectively. Interestingly, when we evaluated outcomes on the GO arm, patients with E17 mutations had an OS of 77±23% vs. 83±8% for KIT-WT (p=0.62), suggesting that GO may abrogate the adverse prognostic impact of E17 mutations.Conclusions: Somatic E17 KIT mutations lead to autonomous receptor phosphorylation and cellular proliferation and are associated with inferior clinical outcome when treated with conventional chemotherapy alone. This is in contrast to E8 mutations, which lack functional significance. KIT E17 mutations are uniquely sensitive to the cytotoxic effects of KIT inhibition. Consideration of TKI therapy for at least this subset of pediatric KIT + CBF AML is prudent given their inferior prognosis and this preclinical correlate. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genetic Heterogeneity of t(8;21)(q22;q22.1) Acute Myeloid Leukemia Revealed By High-Throughput Targeted Sequencing

Background: Recently, a comprehensive high-throughput sequencing study of pediatric and adult core-binding factor (CBF) AML [t(8;21), n=85; inv(16), n=80] provided novel aspects of the genetic heterogeneity and disease biology of t(8;21) AML (Faber et al. Nat Genet 2016). In addition to well-established variants in genes encoding for proteins in tyrosine kinase (TK)/RAS signaling, epigenetic regulation (ER), and in the cohesin complex (CC), novel candidate genes were identified.Aims: We aimed to validate and to further extend recent findings by comprehensive profiling of the mutational spectrum of t(8;21) AML using a high-throughput targeted sequencing (HTS) approach.Methods: HTS was performed of the entire coding region of 244 genes with evidence in hematological malignancies. Pretreatment peripheral blood or bone marrow specimens of 143 adult t(8;21) AML patients (pts) were analyzed. 114/143 pts were enrolled in seven prospective AMLSG treatment trials. Libraries (total probes size: 1.36 Mbp) were prepared using SureSelectXT custom solutions (Agilent). Paired-end sequencing was carried out on a HiSeq platform (Illumina).Results: A mean on-target sequencing depth of 900x was obtained for all pts. Coding variants without known SNP annotation and with a minimum variant allele fraction (VAF) of 5% were called. In sum 756 mutations (missense: 70%; indels: 19%; nonsense: 9%; other: 2%) were detected with an average of 5.3 per pt (SD: ±2.5). 99% of pts harbored at least 1 mutation and 90% 3 or more. In line with previous studies, activating mutations in TK/RAS signaling pathways represented recurrent events: KIT (34/143; 24%), NRAS (24/143; 17%), FLT3 (15/143; 10%; point mutations only), and KRAS (4/143; 3%).In addition to these mutations conferring proliferative clonal advantage, a striking enrichment of mutations was observed in genes involved in histone modification and DNA methylation, ASXL2 (22/143; 15%), ASXL1 (21/143; 15%), KDM6A (14/143; 10%), EZH2 (11/143; 8%), CREBBP (8/143; 6%), SRCAP (8/143; 6%), TET2 (19/143; 13%) and DNMT3A (9/143; 6%), emphasizing their contribution to impairing differentiation in this leukemia subtype. Further, frequent aberrations were found in the cohesin genes that are involved in sister chromatid segregation during mitosis and DNA repair: RAD21 (17/143; 12%), SMC1A (10/143; 7%), STAG2 (5/143; 3%), and SMC3 (5/143; 3%). In general, mutations in genes belonging to the same functional subgroups were rarely co-occurring suggesting functional redundancy without additional synergistic effect. We also identified recurrent variants in previously detected candidate genes outside these functional clusters. A remarkably high incidence of loss of function mutations was found in the ZBTB7A gene(27/143; 19%), encoding for a transcription factor guiding hematopoietic lineage fate. Recurrent mutations were also observed in CCND2 (14/143; 10%) , involved in hematopoietic cell proliferation, MGA (11/143; 8%), a negative regulator of MYC, as well as DHX15 (10/143; 7%), associated with ribosome biogenesis and spliceosome function. When focusing on the clonal hierarchy we found that the median VAF in genes belonging to ER and CC (0.31; range 0.3-0.91; 0.35, range 0.05-0.96; respectively) was higher than in genes associated with TK/RAS signaling (0.16, range 0.05-0.53) suggesting that mutations affecting the epigenetic state and differentiation occur earlier than those impairing proliferation during development of t(8;21) AML.Conclusion: Using a comprehensive sequencing approach we could further delineate the molecular pattern of t(8;21) AML that despite shared genetic lesions is remarkably different to inv(16) AML. Here, mutation clusters in genes involved in TK/RAS signaling, ER and CC were confirmed as well as novel t(8;21)-associated gene mutations such as in ZBTB7A and CCND2 that play an essential role in regulation of hematopoietic cell proliferation and differentiation. Further analyses including sample size extension as well as correlation of findings with clinical parameters are ongoing. DisclosuresDöhner:Astex Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Honoraria; Arog Pharmaceuticals: Honoraria, Research Funding; Pfizer: Research Funding; Abbvie: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Research Funding; Boehringer Ingelheim: Research Funding; Seattle Genetics: Honoraria; Agios: Honoraria; Amgen: Honoraria; Celator: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Dohner:Novartis: Honoraria, Research Funding.

Recurrent Copy Number Variants Are Highly Prevalent in Acute Myeloid Leukemia

Introduction: Large chromosomal copy number variants (CNVs) detected by conventional karyotyping are common in AML; however, small segmental CNVs or copy-neutral loss of heterozygosity (CN-LOH) are not detected by conventional techniques and require sequence-based approaches. Specifically, acquired CN-LOH occurs through duplication of genetic material and subsequent loss of the reciprocal chromosome resulting in a hemizygous state. CN-LOH has been demonstrated in AML, mostly occurring in regions of known recurrent mutations (ex: FLT3 , JAK2 , TET2 ), but its overall prevalence and involvement in AML pathogenesis has yet to be determined. Initial studies regarding the significance of CNVs suggest possible prognostic impact. We aimed to explore the prevalence of segmental CNVs and CN-LOH in a cohort of pediatric and adult AML specimens that underwent clinical comprehensive genomic profiling (CGP) (FoundationOne Heme Sequencing).Methods: CGP for targeted DNA and RNA sequencing was performed on 932 patients with AML (n=179 pediatric age ≤ 18 years, n=753 adult age ≥ 19 years). Specimen sites included bone marrow, peripheral blood, and biopsy of extramedullary disease. Following strict quality control (QC) measures to exclude any samples with transplant history, contamination, low purity, and lack of aneuploidy, all level CNV and LOH events were reported using a proprietary algorithm. A total of 323 samples met QC standards for LOH events, with all level copy number analyses available for 236 (73%) patients. We analyzed the loci of 91 genes to determine the type and prevalence of CNV events for amplifications (CNA; CN>2), deletions (CND; CN<2), CN-LOH, copy-loss LOH (CL-LOH) events at the genomic or chromosome level excluding sex chromosomes.Results: Among the 323 samples (n=59 pediatric, n=264 adult), the prevalence of LOH events was 91% (294). Identified LOH events included single gene LOH or chromosomal LOH, including arm or whole chromosome. Chromosomal events occurred in 172 (53%) of patients, and most frequently involved the following arms: 7q (n=59), 7p (n=57), 13q (n=25), 18q (n=23), 18p (n=22), 5q (n=19), and 17p (n=19). Among patients with chromosomal LOH events, 131 (76%) had events in the 7 chromosomal arms with the highest frequency (Fig 1A). Genomic LOH events were detected in 287 (89%) patients, and were detected in all 91 of the genes analyzed, with a frequency of 1-58. Further, CN-LOH events were detected in 162 (50%) patients occurring in 89 (98%) of genes analyzed (Fig 1B), with a frequency of 1-17. Among the 20 genes with highest frequency of LOH, 11 of them occurred at the 7q and 13q regions, which is consistent with previous findings that these are areas of frequent genomic losses. LOH, including CN-LOH, events overall commonly occurred in genes located in the 5q, 7p, 13q, 16q, and 17p regions. Recurrent LOH events in 3 of the most commonly affect genes occurred in approximately 31% of patients (EZH2 , n=103, BRAF , n=102, and KMT2C , n=99), all located on 7q. CN-LOH events were detected in oncogenes such as CDK6 , MET , FLT3 , JAK2 , ABL1 .Among the 236 samples evaluable for CNA assessment, 217 (92%) had at least one CNA or CND detected. CNAs were detected in 163 patients (69%) and occurred in all 91 of the genes analyzed (Fig 1C). Among samples with CNAs detected, the median number of somatically acquired CNAs was 5 (range 1-91). Recurring CNAs were detected across all chromosomes, but were most common in genes located on chromosome 1q, 8, 11q, 13q, 17q, 19, and 21q. CNDs were detected in 165 patients (70%) and occurred in 83 of genes (91%) analyzed. Among samples with CNDs detected, the median number of CNDs was 6 (range 1-58).Conclusion: CNVs and LOH events were commonly detected in this sample of 323 AML specimens. We show that LOH is highly prevalent, with genomic events detected in 89% of cases analyzed and CN-LOH events specifically detected in 50%. We found highly recurrent genes located at a few distinct chromosomal loci, suggesting some regions may be more prone to chromosomal instability. Future research in AML is needed to understand the mechanisms behind genomic and large-scale chromosomal disruptions, including CN-LOH. Identification of segmental CNVs and CN-LOH may identify cryptic variants that might aid in more appropriate diagnosis, management, disease monitoring, as well aid in the interpretation of mutation data as these events might alter the variant allele fraction of sequence variants. [Display omitted] DisclosuresHe:Foundation Medicine: Employment, Other: stock. Sun:Foundation Medicine: Employment, Other: Shareholder. Severson:Foundation Medicine: Employment, Other: Stock. Bailey:Foundation Medicine: Employment, Other: stock. Morley:Foundation Medicine: Employment, Other: Stock. Balasubramanian:Foundation Medicine: Employment, Other: stock. Erlich:Foundation Medicine: Employment, Other: stock. Lipson:Foundation Medicine: Employment, Other: stock. Otto:Foundation Medicine: Employment, Other: stock. Vergillo:Foundation Medicine: Employment, Other: stock. Ross:Foundation Medicine Inc: Employment, Other: stockholder. Mughal:Foundation Medicine Inc: Other: stockholder. Stephens:Foundation Medicine: Employment, Other: stock. Miller:Foundation Medicine Inc: Employment, Other: stockholder. He:Foundation Medicine Inc: Employment, Other: stockholder.

Molecular Characteristic of Acute Leukemias with t(16;21)/Fus-Erg

T(16;21)(p11;q22) is a rare but recurrent translocation in acute leukemias and in some types of solid tumors. The translocation leads to the formation of a FUS-ERG chimeric gene with the expression of multiple types of FUS-ERG chimeric transcripts. The prognosis of AML with t(16;21)(p11;q22) is poor in children and even worse in adults. T(16;21)(p11;q22) is also shown to be accompanied by mutations, the most frequently mutated gene was RUNX1 . We aimed to examine the mutational landscape of pediatric AML with t(16;21)(p11;q22) using an NGS-based approach.The mutational status was analyzed with the TruSight Myeloid Panel (Illumina, San Diego, CA, USA) for the primary, remissionand relapse samples. All mutations were validated by Sanger sequencing.Two mutations (DNMT3A p.E30A and ASXL1 p.G652S) were found in pt. 1 initially and in the first relapse and in the second relapse. The frequency of the alternative variant correlated to the blast count and number of cells with the FUS rearrangement measured by FISH. These mutations also persisted in the first remission, when no blast cells were present and there was not a detectable level of the FUS-ERG chimeric gene and transcript. Interestingly, in the third relapse, the DNMT3A p.E30A mutation disappeared and ASXL1 p.G652S was still present. The same mutation was observed in transplant sample of patient's father who was a donor for the second HSCT.BCOR p.P1401L and RUNX1 p.S141W mutations were present in pt. 2 initially. BCOR p.P1401L was also found in the first remission. The patient was transplanted in the first remission from HLA10/10-matched unrelated donor, but after 17 months, a relapse occurred. Here, we observed a BCOR p.P1401L mutation, no RUNX1 p.S141W mutation and a new mutation ASXL1 p.E1102D. The ASXL1 p.E1102D mutation had an alternative allele frequency that was much higher than the current blast count (44% vs 12%). This mutation was not present initially and also it disappeared after HSCT2 and its donor origin was suspected. Direct sequencing of the donor material (male, 28 y. o.) revealed ASXL1 p.E1102D. And BCOR p.P1401L mutation was also found in transplant sample of patient's mother who was a donor for the second HSCT.We suggest that epigenetic regulator's somatic mutation DNMT3A p.E30A in pt. 1 could be the first event preceding the full transformation by t(16;21)(p11;q22)/ FUS-ERG as it persist during remission (without a chimeric gene or transcript detectable).Findings of epigenetic regulators' mutations (ASXL1 p.G652S in pt.1 and BCOR p.P1401L in pt. 2) that are probably inherited, potentially pathogenic by computational analysis and estimated to be preleukemic in adults raise the questions if they have some influence on the emergence of primary disease and if their persistence in hematopoietic donors cells also contribute to the transplant function. It also remains to be confirmed whether unrelated unrelated donor-derived mutations (ASXL1 p.E1102D in pt. 2) actually contribute to the post-transplant relapse of the original disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Epidemiology, Cytogenetic and Molecular Abnormalities and Outcome of Childhood Acute Myeloid Leukemia- an Israeli Retrospective Multicenter Study

IntroductionAcute myeloid leukemia (AML) contributes to 18% of childhood leukemia cases and about 50% of all deaths of pediatric leukemia in the United States .In high-income countries, intensive therapy has increased survival rates to 60-70% . Contributing factors to the improved survival include refinement in the diagnosis of AML and advancement in therapeutic approaches.In light of the ethnic diversity in the Israeli population, we reviewed patient's data and investigate whether the clinical, biologic and molecular features differ between Israeli Arab and Jewish AML patients.Patients and MethodsWe performed a retrospective epidemiological and clinical study including patients younger than 18 years of age, treated at all six Israeli pediatric oncology centers between 2000-2015. Down syndrome and secondary AML were excluded . The clinical characteristics such as gender, age, subtype of FAB, leukocyte count at diagnosis, cytogenetics and molecular markers were analyzed in correlation with their prognosis between different characteristics groups and different ethnicity. Survival analyses were calculated by Kaplan-Meier survival curves and the log rank test. Multivariate analyses on categorized data were performed using Cox proportional hazards model.ResultsWe analyzed 175 patients, 46% females and 54%, males, 15 % younger than 2 years old, 35% from 2 to 10 years and 50% older than 10 years (median age 9.3±5.6 years ). According to FAB subtype, the majority subtypes were M2 and M4 for 26% and 27% respectively , 40% of patients presented WBC count below 10 X 109/L at diagnosis while 13% of patients higher than 100 X 109/L. The most common cytogenetic abnormalities in these children, including t(8;21)(q22;q22), inv(16)(p13.1q22) and 11q23/MLL-rearranged abnormalities were detected, with the occurrence of 18% , 8% and 13% respectively.Acute promyelocytic leukemia (APL) was diagnosed in 14% of the children. Molecular data such as FLT3 were not evaluated due to small numbers.No statistical difference was observed regarding ethnicity as to age, white blood cell (WBC) and central nervous system (CNS) involvement. However, the Arab patients presented with a significantly higher incidence of M2 (42% vs 23% p=0.035), while the Jewish population demonstrated significantly higher incidence of M4 (33% vs. 10% p=0.011). The high incidence of M3 was common to both ethnic groups. In addition, cytogenetic subtype distribution was similar in both ethnic groups.Out of 175 patients, 114 patients were treated in accordance with the same protocol, namely BFM98 . The demographic characteristics of these 114 pts, as well as their WBC, FAB and cytogenetics were distributed similarly to the whole cohort.Among the 114 AML patients, 105 pts achieved complete remission (CR) (92%) after one course of chemotherapy. Of those who achieved CR after 2 induction cycles, 62 pts are alive and free of disease, 38 pts relapsed, 4 pts died in CR (3 due to bacterial infection and 1 due to post transplant lymphoproliferative disease) , one patient developed myelodysplastic syndrome after treatment for APL . Six patients did not go into remission after two induction phases. Three patients had early death (within six weeks of diagnosis), all due to bleeding- 2 were APL and one with MLL split and hyperleukocytosis.The median follow-up was 6.3 years (0.7y-16y), with a 5-year event free survival (EFS) and overall survival (OS) of 54% and 67% respectively.There was no prognostic significance in EFS and OS related to gender, age and ethnicity.As to risk stratification, there was a statistical difference in EFS and OS in the standard risk (SR) and the high risk (HR) group: 5 year EFS SR group 83% and 39% for HR group (p<0.0001) 5 year OS for SR group 98% and 52% for HR group (p<0.0001).ConclusionsIn this retrospective study of pediatric AML patients in Israel,the clinical, laboratory and cytogenetic characteristics were not significantly different between Jewish and Arab patients, with a similar outcome. We report a 14% incidence of APL - an incidence that is higher than that reported for the European and American populations. It may be of interest to further investigate this increased rate of APL in the Israeli population and possibly identify underlying genetic predisposing factors.The pts treated according to AML-BFM-98 protocol without pCRT have a 5y EFS and OS of 54% and 67%, respectively. These outcomes are similar to those reported for BFM98. DisclosuresNo relevant conflicts of interest to declare.

A New Highly Sensitive Realtime PCR Assay with Faster Turnaround Time for Detecting IDH1 and IDH2 mutations in Acute Myeloid Leukemia (AML) Patients

Introduction:Genetic profiling of AML reveals distinct molecular subgroups that inform disease classification and prognostic stratification (Tayyab M et al., J. Cancer Sci. Ther. 2014). Studies have demonstrated, that the Isocitrate Dehydrogenase Isoenzymes (IDH1/2), regulating cellular metabolism co-operate to result in AML (Papaemmanuil E et al., N. Engl. J. Med. 2016). Both IDH1/2 mutations produce neomorphic activity resulting in the overproduction of an onco-metabolite 2-hydroxyglutarate (2HG) (Gross S et al., J Exp Med. 2010). IDH1 mutations affect codon 132 and IDH2 mutations affect codon 140 or 172. IDH1 mutations have been reported to occur in 6-9% of adult and 1% of pediatric AML cases respectively, while IDH2 mutations have been reported to occur in 8-12% of adult and 1-2% of pediatric AML cases respectively (Abbas, S, et al., Blood. 2010; Andersson AK et al., Leukemia. 2011). The objective of this study was to conduct workflow analysis of the Abbott RealTime IDH1 and IDH2 assays in comparison to the Sanger based sequencing assays for IDH1/2 in blood/bone marrow specimens from subjects with advanced hematological malignancies. Next Generation Sequencing (NGS) was utilized to resolve samples with discordant results.Methods:This study was approved by Medical College of Wisconsin Institutional Review Board. The clinical specimens used were blinded during the study. Blood or bone marrow aspirates from 100 AML subjects sent to the BloodCenter of Wisconsin (BCW) were tested for IDH1/2 with four different assays (Abbott RealTime IDH1, Abbott RealTime IDH2 assays, the BCW's Polymerase Chain Reaction (PCR) based Sanger sequencing assay and NGS HemeOnc panel (contains 30 genes including IDH1/2). Abbott RealTime IDH1 and IDH2 assays are qualitative in vitro PCR tests wherein, the IDH1 assay detects five IDH1 mutations (R132H, R132C, R132L, R132G and R132S) and the IDH2 assay detects nine IDH2 mutations (R140Q, R140L, R140G, R140W, R172K, R172M, R172G, R172S and R172W).The BCW's PCR based Sanger sequencing assay and NGS HemeOnc panel detect mutations in exon 4 of IDH1/2 with a sensitivity limit of 20% and 10% respectively. In this study NGS data was analyzed for only IDH1 and IDH2 .DNA was extracted from 100 clinical specimens either by the Abbott m Sample preparation system or by the Qiagen DNA extraction method and tested using the Abbott RealTime IDH1 and IDH2 assays. Additionally, a set of commercially available controls (IDH1 R132H/ IDH2 R172K, IDH1 R132C/ IDH2 R140Q) with different mutation levels (20%, 5%, 1% and 0.25%) were tested using the above mentioned four assaysResults:The Abbott RealTime IDH1 and IDH2 assays demonstrated 100% sensitivity and 95% specificity compared to the BCW Sanger sequencing assay. Five out of 100 samples with low level IDH2 mutations were missed by BCW Sanger sequencing assay but were detected as mutation positive by Abbott RealTime IDH2 assay. These five discordant samples with low level IDH2 mutations were subsequently tested with the BCW's NGS assay and two of the five samples were detected as positive by the NGS assay (Table 1). Additionally, turnaround time (TAT) analysis demonstrated that the results from the Abbott RealTime IDH1/2 assays were available in 2 business days as compared to 10 days for the BCW Sanger sequencing assay and 21 days for the BCW NGS HemOnc panel.Testing of the mixture of IDH1 and IDH2 mutations with differing mutation levels (20%, 5%, 1% and 0.25%) resulted in the Abbott RealTime IDH1 and IDH2 assay reliably detecting the mutations down to the 1% level (with some variability at 0.25%) (Table2).Conclusions:This study demonstrated that the Abbott RealTime IDH1 and IDH2 assays detected IDH1 and IDH2 mutations at 1% mutation level in AML patients. The Abbott assays were highly sensitive (1%) compared to the PCR based Sanger sequencing and NGS HemeOnc panel, which have sensitivity limits of 20% and 10% respectively. The higher sensitivity and relatively fast TAT of the Abbott assays may potentially improve the ability to diagnose new and relapsed patients in settings of lower disease burden. The relatively fast TAT could facilitate clinicians in making a quicker decision about therapeutic treatment recommendations for afflicted patients. [Display omitted] DisclosuresDash:Abbott Molecular Inc.: Other: I do not have financial relationship, however wanted to mention here is that this study was sponsored by Abbott Molecular Inc which was performed in BloodCenter of Wisconsin.

Outcomes Based on CNS Disease Status in Pediatric Acute Myeloid Leukemia and the Role of Peripheral Blood Contamination in Diagnostic Lumbar Punctures; A Report from the Children's Oncology Group Studies AAML0531 and AAML1031

The diagnosis and treatment of CNS leukemia in AML differs between pediatric and adult oncology. Adult AML patients generally receive no prophylactic intrathecal (IT) treatment. Here we evaluate an approach utilized on 2 recent Children's Oncology Group (COG) clinical trials for pediatric AML which included CNS prophylaxis and a low threshold to initiate CNS leukemia directed therapy.A total of 2119 patients with de novo AML were evaluable from COG trials AAML0531 and AAML1031. Patients with FLT3/ITD on AAML1031 are excluded as that cohort remains eligible for ongoing accrual. The median (range) follow-up time was 7.1 (0 - 10.5) years for AAML0531 and 2.5 (0 - 5.7) years for AAML1031. All patients received IT cytarabine once with each induction and intensification cycle (except for one of the intensification cycles with high dose cytarabine). An LP to determine CNS status was performed at start of induction therapy. CNS positive (CNSpos) leukemia was treated with twice weekly IT cytarabine until 2 negative CSF samples were obtained (minimum 4 and maximum 6 ITs). CNSpos was defined similarly on both trials (Table 1). In order to understand the role of traumatic LP in CNS status and outcomes, CNS status in this analysis was further classified according to criteria used for ALL (Table 1). Comparing the ALL criteria to the AML CNS definition, CNS1 patients are CNS negative (CNSneg) and CNS3 patients are CNSpos, however among patients with CNS2 by ALL criteria, the AML CNS definition does not take into account the number of CSF WBCs in an atraumatic LP and also uses a higher CSF RBC number (100 versus 10) to require use of the Steinherz/Bleyer algorithm.The rate of CNS relapse (isolated CNS and combined CNS and bone marrow) was evaluated separately on each trial since the protocols differed in number of chemotherapy courses and SCT allocation. On AAML0531, the 5 year CNS relapse rate was lower for CNS1 (4.5 ± 1.7%) compared to CNS2 (9.1 ± 4.5%) or CNS3 (17.7 ±9.4%), P<0.001. This finding was replicated in the AAML1031 outcomes; CNS relapse rates were 4.9 ± 1.8% (CNS1), 11.7 ± 5.0% (CNS2) and 19.3 ±8.8% (CNS3), P<0.001. Patients who were CNSpos had a higher 5 year CNS relapse rate on both studies (CNSpos 13.2 ± 5.1% vs. CNSneg 4.9 ± 1.7% on AAML0531, P<0.001; CNSpos 12.9 ± 4.3 % vs. CNSneg 5.6 ± 1.8% on AAML1031, P<0.001). Event free survival (combined AAML0531 and AAML1031) differed by CNS category (CNS1 51.2%, CNS2 43.5%, CNS3 37.7%, P<0.001) and AML CNS group (CNSneg 41.2%, CNSpos 49.9%, P<0.001), but overall survival did not differ significantly.We evaluated the role of peripheral blood contamination. For patients with CSF RBC ≥100, the majority of patients were classified by ALL criteria as CNS2 or CNS3, and the rate of CNS3 disease increased approximately two fold compared to those with CSF RBC <100 (Fig 1a). When considering the COG AML definition, CNSpos rate was substantially higher among those with CSF RBC 10-99 (41%) compared to those with CSF RBC <10 (24%) or RBC ≥100 (27%; Fig 1b). This difference is likely due to the presence of peripheral blood blasts without “correction” for this blood contamination because the Steinherz/Bleyer algorithm was not used to determine CNSpos when CSF RBC <100.The median blood WBC count was significantly lower in patients with CNS1 (14,650; 95% CI 13,200-15,900) compared to CNS2 (46,200; 95% CI 40,500-53,700) or CNS3 (32,400; 95% CI 21,900-43,500). Further, when this analysis was restricted to patients with an atraumatic tap (CSF RBC <10), those with CNS1 still had significantly lower WBC compared to those with CNS2 or CNS3 (median WBC [95% CI]: CNS1 15,100 [13,400-16,800]; CNS2 53,700 [45,800-62,000]; CNS3 35,500 [21,000-50,100]). This suggests that the biologic drivers resulting in leukocytosis may also increase the risk of CNS leukemia.This analysis demonstrates that CNS disease in AML is associated with a higher CNS relapse risk but caution must be used in interpreting disease status at diagnosis. Due to the worse prognosis associated with CNS disease, we advocate continued use of diagnostic lumbar punctures and intensified treatment for pediatric patients with CNS disease. However, we also speculate that deferring the initial diagnostic lumbar puncture until clearance of peripheral blasts (which usually occurs by day 5-8 of induction) may minimize the risk of false positive diagnosis of CNS disease resulting from high peripheral WBC and blast contamination of CSF samples. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cytogenetics of Pediatric Acute Myeloid Leukemia: A Review of the Current Knowledge.

Pediatric acute myeloid leukemia is a rare and heterogeneous disease in relation to morphology, immunophenotyping, germline and somatic cytogenetic and genetic abnormalities. Over recent decades, outcomes have greatly improved, although survival rates remain around 70% and the relapse rate is high, at around 30%. Cytogenetics is an important factor for diagnosis and indication of prognosis. The main cytogenetic abnormalities are referenced in the current WHO classification of acute myeloid leukemia, where there is an indication for risk-adapted therapy. The aim of this article is to provide an updated review of cytogenetics in pediatric AML, describing well-known WHO entities, as well as new subgroups and germline mutations with therapeutic implications. We describe the main chromosomal abnormalities, their frequency according to age and AML subtypes, and their prognostic relevance within current therapeutic protocols. We focus on de novo AML and on cytogenetic diagnosis, including the practical difficulties encountered, based on the most recent hematological and cytogenetic recommendations.

Aberrantly low STAT3 and STAT5 responses are associated with poor outcome and an inflammatory gene expression signature in pediatric acute myeloid leukemia

The relapse rate for children with acute myeloid leukemia is nearly 40% despite aggressive chemotherapy and often stem cell transplant. We sought to understand how environment-induced signaling responses are associated with clinical response to treatment. We previously reported that patients whose AML cells showed low G-CSF-induced STAT3 activation had inferior event-free survival compared to patients with stronger STAT3 responses. Here, we expanded the paradigm to evaluate multiple signaling parameters induced by a more physiological stimulus. We measured STAT3, STAT5 and ERK1/2 responses to G-CSF and to stromal cell-conditioned medium for 113 patients enrolled on COG trials AAML03P1 and AAML0531. Low inducible STAT3 activity was independently associated with inferior event-free survival in multivariate analyses. For inducible STAT5 activity, those with the lowest and highest responses had inferior event-free survival, compared to patients with intermediate STAT5 responses. Using existing RNA-sequencing data, we compared gene expression profiles for patients with low inducible STAT3/5 activation with those for patients with higher inducible STAT3/5 signaling. Genes encoding hematopoietic factors and mitochondrial respiratory chain subunits were overexpressed in the low STAT3/5 response groups, implicating inflammatory and metabolic pathways as potential mechanisms of chemotherapy resistance. We validated the prognostic relevance of individual genes from the low STAT3/5 response signature in a large independent cohort of pediatric AML patients. These findings provide novel insights into interactions between AML cells and the microenvironment that are associated with treatment failure and could be targeted for therapeutic interventions.

Serial Evaluation of T cell Subsets in Paediatric Acute Myeloid Leukaemia- a Prospective Study.

There is paucity of data regarding T-cells in paediatric AML patients. The aim of this prospective study was to evaluate trend of T-cell subset during disease course of paediatric AML patients and to see its correlation with patient characteristics and survival outcome. T-cell subsets (CD3, CD4 and CD8) were evaluated by flow-cytometry at diagnosis, post-induction, post-treatment completion, at 3months and 6months post-treatment completion, and relapse in 29 pediatric AML patients. Trend of T-cells was plotted between group A (those in continuous remission) and group B (those who relapsed) patients. Patients with high WBC count had significantly higher number of CD3, CD4 and CD8 cell. Baseline Tcell subsets did not affect CR, EFS and OS; however, higher than median CD4 count predicted improved DFS [58% vs 25%; HR = 0.306 (0.10-0.93); P = 0.037]. On serial follow-up from post-induction till 3months after completion of therapy, there was no difference in the absolute values of T cell subsets between group A and B patients. Our study demonstrated T cell subsets are increased in AML subjects with high WBC count. CD4 cells have a positive impact on DFS. Serial follow-up has no impact on T cell subsets. Further studies in larger patient cohorts are needed to evaluate if CD4 population may serve as an immune biomarker for AML.

Targeting EZH2-mediated methylation of histone 3 inhibits proliferation of pediatric acute monocytic leukemia cells in vitro

ABSTRACT Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and a catalytic subunit of the polycomb repressive complex 2 (PRC2) that catalyzes the mono-, di-, and tri-methylation of histone H3 at Lys 27 (H3K27me3) to facilitate chromatin-remodeling and gene-silencing functions. Previous reports showed a significant association of EZH2 aberrations in pediatric cancers, such as soft tissue sarcomas and glioblastoma. Recent reports in human subjects and animal models have also suggested a central role of EZH2 in the induction and progression of acute myeloid leukemia. In this study, we aimed to investigate the molecular status of EZH in cell lines derived from distinct pediatric leukemia to assess the efficacy of targeting EZH2 to suppress cancer cell survival and proliferation. Our results showed that EZH2 protein is overexpressed in the pediatric monocytic cell-line THP-1, but not in other leukemia-derived cell lines MV4;11 and SEM. Screening a panel of methyltransferase inhibitors revealed that three inhibitors; GSK126, UNC1999 and EPZ-5687 are the most potent inhibitors that suppressed EZH2 activity selectively on lysine 27 which resulted in increased apoptosis and inhibition of AKT and ERK protein phosphorylation in THP-1 cells. Our data demonstrated a significant increase in apoptosis in cells treated with drug combination (EZH2i and selinexor) compared to EZH2i inhibitors alone. Taken together, our data provide initial evidence that targeting EZH2 is a promising therapeutic strategy for the treatment of subtypes of pediatric AML. Also, combining EZH2 inhibitors with selinexor may increase the treatment efficacy in these patients.

Multidrug Resistant Infections in Paediatric AML: An Ominous Sign of the Times to Come

Multidrug Resistant Infections in Paediatric AML: An Ominous Sign of the Times to Come

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.

Redirecting the Immune Microenvironment in Acute Myeloid Leukemia.

Simple SummaryDespite remarkable progress in the outcome of childhood acute myeloid leukemia (AML), risk of relapse and refractory diseases remains high. Treatment of the chemo-refractory disease is restricted by dose-limiting therapy-related toxicities which necessitate alternative tolerable efficient therapeutic modalities. By disrupting its immune environment, leukemic blasts are known to gain the ability to evade immune surveillance and promote disease progression; therefore, many efforts have been made to redirect the immune system against malignant blasts. Deeper knowledge about immunologic alterations has paved the way to the discovery and development of novel targeted therapeutic concepts, which specifically override the immune evasion mechanisms to eradicate leukemic blasts. Herein, we review innovative immunotherapeutic strategies and their mechanisms of action in pediatric AML.Acute myeloid leukemia is a life-threatening malignant disorder arising in a complex and dysregulated microenvironment that, in part, promotes the leukemogenesis. Treatment of relapsed and refractory AML, despite the current overall success rates in management of pediatric AML, remains a challenge with limited options considering the heavy but unsuccessful pretreatments in these patients. For relapsed/refractory (R/R) patients, hematopoietic stem cell transplantation (HSCT) following ablative chemotherapy presents the only opportunity to cure AML. Even though in some cases immune-mediated graft-versus-leukemia (GvL) effect has been proven to efficiently eradicate leukemic blasts, the immune- and chemotherapy-related toxicities and adverse effects considerably restrict the feasibility and therapeutic power. Thus, immunotherapy presents a potent tool against acute leukemia but needs to be engineered to function more specifically and with decreased toxicity. To identify innovative immunotherapeutic approaches, sound knowledge concerning immune-evasive strategies of AML blasts and the clinical impact of an immune-privileged microenvironment is indispensable. Based on our knowledge to date, several promising immunotherapies are under clinical evaluation and further innovative approaches are on their way. In this review, we first focus on immunological dysregulations contributing to leukemogenesis and progression in AML. Second, we highlight the most promising therapeutic targets for redirecting the leukemic immunosuppressive microenvironment into a highly immunogenic environment again capable of anti-leukemic immune surveillance.

A multimodal genomics approach to diagnostic evaluation of pediatric hematologic malignancies

Detection of somatic genetic drivers is important for risk stratification and treatment selection in pediatric leukemias; however, newly recognized genetic markers may not be detected by routine karyotyping and fluorescence in situ hybridization (FISH). To identify the combination of assays that provides the highest detection rate for clinically significant molecular abnormalities, we tested 160 B- lymphoblastic leukemia (B-ALL) by karyotyping, FISH, chromosomal microarray analysis (CMA) and the custom next-generation sequencing (NGS) panel, OncoKidsⓇ. In addition, we tested 40 myeloid malignancies with karyotyping, chromosomal microarray analysis (CMA), and OncoKidsⓇ; 36/40 myeloid malignancies were also tested with FISH. In B-ALL, individual testing methods had the following diagnostic yields for the key genetic drivers: karyotype 34%; basic FISH panel 45%; FISH panel with IGH and CRLF2 probes 65%; CMA 48%; OncoKidsⓇ 39%. CMA and OncoKidsⓇ testing allowed detection of key genetic drivers in 42% of the samples that remained unknown upon testing by conventional methods. In myeloid malignancies, OncoKidsⓇ had the highest yield for detection of both primary and secondary DNA mutations and RNA fusions. Our data highlights the complementarity between CMA and NGS and conventional cytogenetics/FISH in pediatric leukemia diagnostics. Due to rapid turn-around-time, FISH may be useful as an initial screening method in B-ALL. Our data also suggests NGS testing with a comprehensive panel, despite a longer turnaround time, is a good alternative to karyotyping and FISH in pediatric AML due to its superior detection rate.