ALBERT

Description: BACKGROUND: We have previously shown that one target of hyper-methylation in AML is the promoter of the tumour suppressor and stress-response mediator Growth Arrest and DNA Damage inducible 45A (GADD45A) (GADD45AmeHI; 42% of AML). In mice Gadd45a has recently been shown to play a critical role in HSC stress responses. Gadd45a deficiency leads to enhanced HSC self-renewal, DNA damage accumulation in HSC, increased susceptibility to leukemogenesis, and impairment in HSC apoptosis after genotoxic exposure (Chen et al, Blood 2014). These findings suggest that hypermethylation of the GADD45A gene may play an important role in the altered properties of HSC, leukaemic initiation and progression. Promoter hypermethylation of this gene defines a patient group with poor survival on standard therapy (Perugini et al, Leukaemia 2012). To explore further the molecular basis of the GADD45AmeHI patient group weperformed genetic profiling of diagnosis samples using a Sequenom multiplex mutation panel, or using whole exome sequencing for broader coverage (n=95 patients).Sequenom MassARRAY was used for quantitative detection of GADD45A promoter methylation in patient samples. For a cohort of matched diagnosis and relapse samples we used CpG methylation data for GADD45A determined by ERRBS (Akalin et al, PLoSGenetics 2012). Response to cytotoxic drugs and assessment of drug combinations with 5-Aza-deoxycytidine (decitabine, DAC) and anthracycline (Daunorubicin, DNR) was performed in AML cell lines, and with primary leukemic cell populations. RESULTS: The association of the GADD45AmeHI patient group with poor outcome was validated in an independent AML patient cohort of 48 patients from the Alfred Hospital, Melbourne, Australia (p=0.003; HR3.35). Whole exome sequencing and Sequenom multiplex analysis of 95 AML patients revealed a striking co-occurrence of the GADD45AmeHI phenotype with mutations in IDH1, IDH2, and TET2 (p

Description: Introduction - AML is a complex group of malignancies, with heterogeneity in morphology, cytogenetics, molecular characteristics, aggressiveness and importantly, in its response to treatment and survival outcomes. Next generation sequencing by the Cancer Genome Atlas Research Network analysed 200 primary AML cases and identified 23 genes that display recurrent somatic mutations at varying frequency in AML (NEJM 368(22):2059-2074). Defects in DNA repair are frequently identified in treatment-related AML and inherited mutations in genes of DNA repair pathways predispose patients to myeloid malignancies. For example, biallelic mutations in FANC genes, which cause the recessive heritable bone marrow failure syndrome Fanconi Anaemia (FA) are associated with high risk of progression to AML and other cancers (Kutler et al.Blood, 101:1249-1256), suggesting a potential involvement of FANC gene mutations in AML pathogenesis. Methods - In this study we present a two-stage approach to gene discovery in AML: initial unbiased whole genome sequence (WGS) and whole exome sequence (WES) analysis of tumour DNA from a cytogenetically normal AML case at diagnosis and relapse, and corresponding germ-line DNA (prepared from mesenchymal stromal cells). Potential oncogenic mutations and changes associated with disease progression were identified. WES of a further 96 diagnostic AML samples further defined recurrent mutations and allowed identification of affected functional groups and networks in AML. Results – WGS and WES were performed on diagnosis, non-haematopoietic and relapse samples from an index AML patient. Somatic SNVs and indels unique to the tumour samples include a number of variants in genes previously reported as recurrently somatically mutated in AML including FLT3, WT1 and IDH2. Somatic mutations in genes not previously associated with AML were also identified including a mutation in FANCD2 (p.S1412N) present in the index AML tumour DNA at diagnosis and at relapse. Variants in genes recurrently mutated at low frequency in AML can also be disease drivers, however separating such genes from the background level of mutation in AML requires analysis across multiple samples, and sequencing studies to determine recurrence and/or mutations in proteins involved in the same functional pathway or complex. STRING-db v9.05 (Franceschini et al. NAR, 2013(41), Database issue) was used to identify a larger network of proteins, including and associated with the FANC genes, involved in homologous recombination-mediated DNA repair. Known somatic mutations from other AML studies were mapped onto this network; as shown in Figure 1 multiple genes in this extended network are affected by somatic mutation in AML suggesting a potential role in pathogenesis. Analysis of our WES data from diagnosis samples from a further 96 Australian AML cases identified an additional two somatic mutations in genes from the extended STRING-db v9.05 FANC network. In total we identified 18 mutations in the 16 classified FANC genes and 8 variants in the BLM complex as shown in Figure 2. Two of the germline FANC gene mutations, FANCM-Q13333fs and FANCD2-R926X, are known pathogenic mutations in FA. Patients with mutations in the 8 FANC genes of the core complex form a distinct subset from those with mutations in the other 8 FANC genes. 5 of the 8 patients with mutations in the BLM complex also form a separate group while BLM complex mutations are present in 2 patients that also have FANC mutations. For the two patients with acquired changes the allele frequency for these FANC mutations is greater than 25% suggesting an early origin in disease. Discussion. Our findings suggest that germline and somatic mutations affecting function of the FANC DNA repair pathway may be a recurrent abnormality in AML, potentially contributing to leukaemogenesis. FANC/BLM gene mutations frequently co-exist with mutations in DNMT3A and DNMT1; 46% of the patients with DNMT3A/DNMT1 mutations are also mutant for FANC or BLM complex genes representing significant over-representation (p = 0.021). Within the group of FANC and BLM patients there is also significant under-representation of FLT3-ITD mutations and mutations in N-RAS and K-RAS (p = 0.051), raising the possibility that defects in homologous DNA repair may favour cooperation with alternative signalling pathways. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: We have previously reported that hypermethylationof the GADD45A promoter (GADD45AmeHI) occurs frequently in AML at a specific CpG residue (CpG1) and associates with poor overall survival for patients on standard chemotherapy (Perugini et al, Leukemia 2013). Sequenom multiplex analysis of 195 AML patients revealed a co-occurrence of GADD45AmeHI with recurrent mutations at conserved residues in IDH1 and IDH2 (p

Description: The role of metabolic dysfunction in the initiation and progression of AML is still to be determined. Next-generation sequencing studies in AML have identified somatic mutations in mitochondrially encoded subunits of the mitochondrial respiratory chain (MRC; N Engl J Med, 2013; 368:2059). Recurrent somatic mutations in nuclear MRC genes are yet to be reported, but recurrent founding mutations occur in nuclear genes encoding the TCA cycle enzymes Isocitrate Dehydrogenase 1 and 2 (IDH1, IDH2). IDH1 and IDH2 mutant AML samples display sensitivity to BCL-2 inhibition (Nat Med, 2015; 21:178), and reduced proliferative capacity has been observed for IDH1 mutant samples following glutaminase inhibition (Exp Hematol, 2014; 42:247). In glioma, IDH2 mutant tumors show enrichment of oxidative phosphorylation (OXPHOS) related gene sets (J Exp Clin Cancer Res, 2016;35:86). Additionally, germline succinate dehydrogenase (MRC Complex-II) mutations have been associated with hereditary paragangliomas (Science, 2000; 287:848). We therefore investigated the nature and frequency of rare germline and somatic mutations affecting nuclear MRC genes in an adult AML cohort, and profiled the metabolic phenotypes of primary BM AML samples. Whole-exome sequencing was performed on 145 diagnosis (Dx) adult BM AML samples. Variants were filtered against dbSNP137, 1000 genome and NHLBI-ESP with a focus on rare germline/somatic variants with a minor allele frequency 〈 0.005. A total of 62 variants affecting the 39 nuclear MRC Complex-I (CI) genes were found in 52 samples. Across the MRC (85 nuclear genes), we identified 140 variants in 95 samples. Confirmation of the germline/somatic status of identified variants is ongoing, with 20 / 21 variants tested being germline in origin. Case-control burden analysis (Am J Hum Genet, 2013; 92:841) was performed to compare variants in the AML cohort with those in an ethnically matched healthy control cohort (n = 329; PLoS Genet, 2011; 7:e1001372). This showed enrichment in the AML cases for variants across all 5 MRC complexes (p = 0.04; Burden analysis, Bonferroni adjusted), and for variants affecting the β-complex of CI (14 genes; p = 0.01; Burden analysis, Bonferroni adjusted). We further investigated the association of CI variants with genes commonly mutated in AML. This revealed a significant under-representation of IDH1 mutations in AML with CI variants (p = 0.036; Fisher's exact; Figure 1). Additionally, for a panel of AML samples (n = 117), we used the BioMark (Fluidigm) qRT-PCR platform to profile the expression of all nuclear CI genes and a panel of AML oncogenes and tumor suppressors. This revealed a significant negative correlation (p 〈 0.05; Pearson's correlation, Bonferroni adjusted) between the expression of all nuclear CI genes and that of IDH2, which is independent of underlying mutation or genetic alterations, and which is not observed for IDH1 expression. Finally, Seahorse metabolic profiling showed that IDH1 mutant samples were found to be utilizing their glycolytic pathway at maximal capacity with no glycolytic reserve while simultaneouslydisplaying significantly reduced glycolytic rates (p〈 0.05; T-test, Bonferroni adjusted) when compared to IDH2 mutant AML samples, AML samples lacking recurrent AML mutations (WT-AML), and normal bone marrow (NBM) controls. Both IDH1 and IDH2 mutant samples showed significantly deficient OXPHOS reserve capacity when compared to NBM and WT-AML samples (p 〈 0.05; T-test, Bonferroni adjusted). Ongoing investigations will determine the metabolic phenotype of CI mutant samples. In summary, we observed significant enrichment of rare variants affecting the MRC and CI genes in an adult AML cohort compared to healthy controls, highlighting a potential role for altered cellular energetics in AML pre-disposition. The significant under-representation of somatic IDH1 mutations in AML with rare germline/somatic CI mutations raises the possibility that these CI variants induce functional consequences that mimic those associated with somatic IDH1 mutation. The distinct glycolytic profiles of IDH1 and IDH2 mutant samples, and the selective negative correlation of CI gene expression with IDH2 mRNA expression, suggests that distinct metabolic phenotypes maybe associated with perturbations to IDH1 and IDH2. Ongoing investigations will compare the metabolic phenotype of samples with CI variants to that of IDH1 and IDH2 mutant samples. Disclosures Gill: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Background:The small molecule BCL-2 inhibitor Venetoclax (VEN) has emerged as a promising frontline therapy in combination with low dose cytarabine (LDAC) or hypomethylating agents in older patients with acute myeloid leukemia (AML)(DiNardo et al, Lancet Oncol 2018, Blood 2019; Wei et al,JCO 2019). The anti-leukemic activity of single agent VEN as monotherapy in newly diagnosed AML is unknown. This information would be useful for studies considering VEN as a maintenance therapy. We have previously presented preliminary outcomes from a phase 1b/2 study: Chemotherapy and Venetoclax in Elderly AML Trial (CAVEAT), which incorporated a 7-day single agent VEN run-in phase prior to combination with chemotherapy (Wei et al, ASH 2018). The study procedures incorporated a bone marrow assessment performed at study screening and on day 8, prior to commencement of chemotherapy (Figure 1A). This allowed us to explore the single agent activity of venetoclax on bone marrow blasts in treatment naïve patients with AML and to assess molecular dynamics of response. Methods:The CAVEAT trial included 51 de novo or secondary patients with AML aged ≥65 years and considered fit for intensive chemotherapy. The study enrolled patients to 5 VEN dose cohorts (50-100-200-400-600mg). VEN was administered on days 1-14 during induction. Chemotherapy commenced on day 8, with a 7-day VEN monotherapy pre-phase. Molecular studies:a 54-gene myeloid NGS panel (Illumina TruSight) and MassARRAY MALDI-TOF mass spectrometry was performed on DNA extracted from bone marrow aspirates. FLT3-ITD testing was by fragment analysis, PCR and capillary electrophoresis. Minimal residual disease (MRD) testing was performed by digital droplet PCR for IDH mutations (assay sensitivity: 10-4-10-5). Results: A total of 46 patients had paired BM assessments at screening and on day 8 for comparison. A ≥50% relative reduction in BM blasts was recorded in 13/46 (28%) patients. Figure 1B shows the relative BM blast reduction stratified by molecular subgroups. Patients with NPM1(n=9) or IDH2 (n=8) mutation achieved greater BM blast reductions (median of 56% and 55%, respectively) than IDH1 (n=6), TP53 (n=10) or FLT3-ITD (n=5) mutant cases (median of 37%, 17% and 7%, respectively). Three NPM1 mutant cases with

Description: Introduction Molecular MRD assays targeting NPM1 mutant (mut) transcripts have an established role for monitoring treatment efficacy in patients with NPM1mut AML. Approximately 25% of NPM1mut patients show persistent MRD level in the bone marrow (BM) at the end of treatment (EOT), which is associated with a higher risk of relapse (Ivey, NEJM 2016; Kronke, JCO 2011). Molecular persistence at low copy number (MP-LCN) is defined by the European LeukemiaNet (ELN) as MRD positivity in patients in morphological complete remission (CR) with 6 months, 31 (39%) had MF and 13 (16%) had morphologic relapse without preceding MF (due to rapid relapse [n=8], lack of monitoring [n=3], extramedullary or NPM1 wild-type relapse [n=1 each]) (Figure 1). Nine patients (11%) had persisting MP-LCN (included transient MRD negativity): median interval between EOT to the last known positive sample was 12 mo (range 3-32), and all patients currently remain alive and without morphologic relapse by the data cut-off date July 13, 2020. Next, we examined the variables associated with MF and/or morphologic relapse. In univariate analysis, factors associated (p

Description: The assessment of circulating tumor DNA (ctDNA), released by tumor cells undergoing apoptosis or necrosis, has established utility in solid tumors due to the advantage of a non-invasive "liquid biopsy" replacing multiple site-specific biopsies. However, its role in acute myeloid leukemia (AML) is uncertain, where a significant proportion of variants detected in the bone marrow (BM) may not be detected in ctDNA (Short, Blood Adv 2020). We have previously demonstrated the possibility of comprehensive genomic characterization of lymphoid malignancy from ctDNA using a single targeted next generation sequencing (NGS) hybridization-based panel (Blombery, BJH 2017). We aimed to assess the performance of this same genomic approach in ctDNA and to compare it against BM in AML. In addition, we aimed to assess the integration of a sensitive variant caller (Mutect2; Benjamin, bioRxiv 2019) to the bioinformatics suite in an attempt to improve low-level variant detection. Nineteen patients were identified from sequential patients with AML treated at our institutions where paired ctDNA and BM aspirate DNA were available. ctDNA was analyzed using a hybridization-based NGS panel targeting genes recurrently mutated in hematological malignancy followed by a suite of bioinformatics tools including HaplotypeCaller (GATK)/Mutect2 (GATK) for variant calling, CNSpector/CNSpectorX (Markham, Sci Reports 2019) for copy number variation (CNV) assessment and GRIDSS (Cameron, Genome Res 2017) for structural variant detection. The cohort clinical details are summarized in Table 1; none had documented extramedullary disease at time of collection. A total of 66 unique variants in 27 genes were detected, summarized in Figure 1. Median number of variants detected was 3 per patient sample, including NPM1 (n=6), IDH1/2 (n=4) and FLT3 point mutation (n=2). Three patient samples had FLT3-ITD detected by fragment length analysis; none were detected by the NGS panel in either ctDNA or BM. Variant allele frequency (VAF) from both compartments were highly correlated (R2 0.87). Higher VAFs in ctDNA were more commonly observed for kinase activating mutations (12/17 variants) and TP53 (5/6). Using HaplotypeCaller alone, 58 and 61 variants were detected in the BM and ctDNA, respectively. Of the 2 variants "specific" to the BM, both (IDH1 and NRAS) were called by Mutect2 in ctDNA and confirmed by visual inspection of sequence read alignments. Of the 5 variants "specific" to the ctDNA, 4 were detected in the BM at low VAF: CBL (n=2), KRAS and TET2. One discrepant case was patient #15 with prior breast cancer: KRAS G13D 21% in ctDNA but absent in the BM. Analysis by Mutect2 additionally detected 3 variants not called by HaplotypeCaller: NRAS and KIT in both ctDNA and BM, and TP53 P278R (VAF 6%) specific to the ctDNA in patient #9 with normal karyotype AML without history of prior malignancy. Overall, 3/5 variants in ctDNA and 6/6 in BM with low VAF were kinase activating mutations. We then performed genome-wide alignment of off-target reads to generate a low-resolution digital karyotype and CNV from ctDNA (Figure 2) which was compared with CNV and conventional karyotyping from BM. CNVs were detected in ctDNA in 10/11 patients with abnormal karyotype (Figure 1). Of these, 6/7 with non-complex abnormal karyotypes had consistent ctDNA CNVs including (i) 3 patients (#1, #10 and #15) with either rearranged/amplified KMT2A by FISH were all found to have gains at 11q23.3-qter, and (ii) 1 patient (#4) had 2 marker chromosomes of unknown origin on karyotyping which were resolved as additional copies of 4p using ctDNA sequencing. Although no CNVs were detected in all 8 patients with normal karyotype, analysis of B-allele frequency from ctDNA revealed one patient (#5) with copy neutral loss of heterozygosity (CN-LOH) in 7q (with a concurrent EZH2 mutation 〉95% VAF). In summary, we have demonstrated the ability to detect sequence variants, perform low-resolution digital karyotyping, CNV detection and CN-LOH detection from ctDNA in AML using a single hybridization based NGS assay. When using this approach, we show a high degree of concordance for both sequence variant and CNV detection, supporting the use of ctDNA as an alternative to BM (e.g. in cases of dry tap, hypocellular AML or failed karyotyping). Finally, using a sensitive variant caller, additional mutations were able to be detected in the ctDNA, the significance of which require evaluation in future studies. Disclosures Tiong: Amgen: Consultancy, Honoraria; Pfizer: Consultancy; Servier: Consultancy. Wilson:Illumina: Other: Illumina Diagnostic Genomics Scholarship. Bajel:Novartis: Honoraria; Astellas: Honoraria; Abbvie: Honoraria; Amgen: Honoraria, Speakers Bureau; Pfizer: Honoraria. Blombery:Invivoscribe: Honoraria; Novartis: Consultancy; Janssen: Honoraria; Amgen: Consultancy.

Description: Introduction Midostaurin is the only approved FLT3 inhibitor for combination with intensive induction and consolidation chemotherapy in newly diagnosed patients with FLT3 mutant AML. The FLT3 inhibitor, sorafenib, was investigated in the randomized SORAML trial (Röllig, Lancet Onc 2015), in combination with intensive chemotherapy (IC) for newly diagnosed adults with AML