ALBERT

Description: AML is heterogeneous group of diseases with variable clinical outcomes. While cytogenetics, molecular markers and gene expression profiling can help to classify these patients, they still cannot fully explain the biology and clinical outcomes of the disease. Epigenetic gene deregulation is a hallmark of cancer and our preliminary data suggest that epigenetic signatures are critical determinants of cellular phenotype in AML. Therefore, we hypothesized that aberrant epigenetic regulation of genes would provide critical insight into the biological complexity of AML and identify new and clinically relevant disease subtypes. We studied genome wide DNA methylation in a cohort of 295 patients from HOVON multicenter clinical trials using the HELP assay, which measures with 〉95% accuracy the abundance of DNA methylation at ~50,000 CpG sites covering ~13,000 promoter regions. Median follow-up was 18.2 months (range=0.1–214.5); median age: 48.1 years (range=15.8–75). Unsupervised analysis using hierarchical clustering (Pearson correlation distance with Ward’s clustering method) segregated the AMLs into 16 well-defined epigenetic clusters. Cluster 1 consisted 100% of patients with acute promyelocytic leukemia (n=6); 100% of cluster 4 harbored CEBPA mutations (n=14); 21/23 patients in cluster 6 carried an inv(16); clusters 7 and 9 were enriched for cases carrying the NPM1 mutation (#7: 80% NPM1+ and #9: 96%) and cluster 12 was enriched for t(8;21) AMLs (18/23). Most of the clusters however define previously unknown biological entities. Next we used a supervised analysis and identified the differentially methylated genes and gene networks that define each cluster, which revealed previously unknown biological differences among these patients. Moreover, Kaplan-Meier survival analysis revealed significant differences in event-free survival (EFS) and overall survival (OS) for the 8 clusters that consisted of 〉20 patients (clusters 5, 6, 7, 8, 9, 11, 12, and 14), which includes clusters that represent previously unidentified AML subtypes. The inv(16) and t(8;21) containing clusters (i.e. #6 and #12) demonstrated a 2-year EFS of 48% and 58%, respectively, compared to 2-year EFS ranging from 19%–44% for all other clusters (p=0.002 by log-rank test) and a 2-year OS of 70% and 61%, respectively, compared to 2-year OS ranging from 25%–50% for all other clusters (p=0.008 by log-rank test). After adjustment for age, cytogenetic risk, NPM1 mutation, and FLT3-itd status in a multivariate cox proportional hazards regression model, differences in EFS and OS remained between clusters i.e. multivariate analysis (utilizing cluster 12 as reference) showed that clusters 9, 5, 8 and 11 demonstrated hazard ratios for poor events of 3.2 (95% CI=1.0, 10.6; p=0.06), 3.2 (95% CI=1.1, 9.1; p=0.03), 3.4 (95% CI=1.2, 9.8; p=0.03) and 3.6 (95% CI=1.2, 10.3; p=0.02), respectively. Similarly, clusters 9, 8 and 11 demonstrated hazard ratios for mortality of 4.7 (95% CI=1.1, 19.8; p=0.03), 4.1 (95% CI=1.1, 15.2; p=0.03) and 3.7 (95% CI=1.0, 13.6; p=0.05), respectively. Interestingly none of these clusters could be entirely explained by any of the known molecular or cytogenetic markers. Clusters 9 and 5 consisted mainly of cases with normal karyotypes, while #8 and #11 grouped cases with a variety of karyotypes. Furthermore, cluster 9 was associated with a worse outcome despite the fact that 24/25 cases were NPM1+, only 11 of which also presented the poor risk association with FLT3-itd. An analysis restricted to the 125 cases with normal karyotype (NK-AML) segregated them into 2 main clusters, one enriched for NPM1+ cases (81.9%) and the other not (29.6% NPM1+) (Fisher exact test: p-value

Description: Although the clinical outcome for patients with acute myeloid leukemia (AML) has improved over the years, failure to maintain complete remission remains a major problem with current standard treatments. The development of individually tailored and patient-specific therapy could potentially significantly improve therapeutic efficacy. In particular we are interested in better understanding the biological features associated with aberrant expression of the EVI1 oncogene, which we previously showed is associated with a poor prognosis. Two different EVI1 transcripts have been identified, i.e. a short form (E) and a long form called MDS1-EVI1 (ME) encoding respectively, a 140 kDa and 170 kDa protein. In EVI1 positive AMLs a distinction can be made between patients that express both EVI1 transcripts (E+/ME+) and cases that express the short form solely (E+), since the latter group is exclusively associated with 3q26 chromosomal abnormalities. EVI1 is a nuclear zinc-finger transcriptional repressor oncoprotein that is known to interact with several epigenetic regulators, e.g. HDACs, CtBPs, histone methyl transferases and MBD3. Since EVI1 presumably mediates its effects through aberrant transcriptional repression, we hypothesize that its aberrant expression results in aberrant epigenetic programming of leukemia cells, which might provide an opportunity for epigenetic-targeted therapy in these patients. In order to determine whether EVI1 over-expressing (EVI1+) AMLs display aberrant epigenetic programming we performed HELP (HpaII tiny fragment enrichment by ligation-mediated PCR) DNA methylation assays in 26 EVI1+ AMLs and 8 CD34+ normal bone marrow controls (NBM). Our HELP assay measured the abundance of DNA methylation at ~50,000 CpG sites covering ~13,000 promoter regions. Single locus validation assays using Sequenom Epityping showed that HELP was 〉95% accurate in quantifying CpG methylation. We found that unsupervised analysis using hierarchical clustering (Pearson correlation distance with Ward’s clustering method) readily separated the EVI1+ AMLs from NBMs. Supervised analysis comparing EVI1+ to NBM identified 303 promoter sequences as being differently methylated (P1.5). Remarkably, 80% of these genes were hypermethylated in EVI1+ patients, while only 20% of genes were hypomethylated. The hypermethylated profile included genes associated with cell death (Caspase-2, MAD1L1) and cell cycle (TNF, JARID1B). The 26 EVI1+ leukemias further segregated into two distinct subgroups in unsupervised analysis: one cluster (n=14) was highly enriched for E+ AML cases carrying 3q26 abnormalities (n=7) while the other one (n=12) mainly harbored the E+/ME+ AMLs (n=10). Supervised analysis of these two EVI1+ clusters revealed that the 3q26-enriched group featured 122-gene signature (P1.5) consisting entirely of hypermethylated genes. When each of the individual EVI1 clusters was independently compared to the NBM samples using supervised analysis we found that the 3q26-enriched group contained a significantly more methylated gene signature containing 429 hypermethylated and 47 hypomethylated HpaII fragments (P1.5). Pathway analysis of the promoter regions differentially methylated in the 3q26-enriched AML group included genes involved in protein degradation and cellular response to therapeutics. In contrast, the E+/ME+ enriched group showed a more balanced distribution of differential methylation when compared to the NBMs (226 hypermethylated and 158 hypomethylated genes). Taken together, our data show that EVI1 overexpression is associated with specific alterations in epigenetic programming vs. normal CD34+ cells. Even more remarkably, we showed that EVI1+ AMLs form two epigenetically distinct AML subtypes. Specifically, the 3q26 subgroup, short EVI1+ isoform AMLs display marked hypermethylation vs. the MDS1-EVI1 expressing patients, involving aberrant methylation of different pathways. This shows that the two forms of EVI1+ AMLs become aberrantly programmed in different ways and are biologically distinct entities, and further suggest distinct mechanisms of action for the different EVI1 isoforms. The marked hypermethylation profile of the short EVI1 isoform AMLs suggests that these patients might benefit from treatment with DNA methyltransferase inhibitors.

Description: Abstract 1578 While self renewal is an essential feature for the maintenance of both normal hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs), very little is known about the underlying molecular pathways. Here we report a critical functional interplay between Bmi1 and Hox in establishment of HSCs and LSCs. Using Bmi1-/- bone marrow cells, we observe that leukemia-associated fusion proteins have distinctive Bmi1 requirements. AML1-ETO (AE) and PLZF-RARα (PR) fail to transform Bmi1-/- primary hematopoietic cells, and induce expression of p16/Arf leading to oncogene-induced senescence (OIS). In contrast, MLL-AF9 driving expression of multiple Hox genes can bypass oncogene-induced senescence and exhibits modest Bmi1-dependence for establishment of LSCs, which can induce leukemia upon serial transplants. Since members of Hox genes with proclaimed self-renewal property are specifically up-regulated by MLL fusions in patient samples and our murine models, we asked the question if these Hox genes may partly compensate the functions associated with the loss of Bmi1. To this end, we generated compound Bmi1-/-Hoxa9-/- mice, which have even more compromised hematopoietic stem cell/progenitor compartments than those of Bmi1-/- or Hoxa9-/- mice. Bmi1-/-Hoxa9-/- mice have a greater than eight-fold reduction in the absolute number of Lin-Sca+kit+ (LSK) in the bone marrow as compared to Bmi1-/- mice and a very significant forty-fold reduction for long term hematopoietic stem cells (LT-HSC). More importantly, while MAF9 is able to transform wild type, Bmi1-/- and Hoxa9-/-, it fails to transform Bmi1-/-Hoxa9-/- cells for establishment of LSCs, which can however be resurrected by re-expression of either Bmi1 or Hoxa9, indicating a critical functional interplay between these protein in development of MLL LSCs. Consistent with the known function of Bmi1 in suppressing cellular senescence and the expression of p16/Arf loci, we showed that Hoxa9 alone can also inhibit replicative senescence and Ras-induced senescence in primary human fibroblast. Forced expression of Hoxa9 can suppress p16/Arf expression, as well as cellular senescence induced by AE and PR in Bmi1-/- cells. Together, these results reveal a previously unrecognized functional interplay between Hox and Bmi1 in regulating cell senescence and development of LSCs induced by fusion proteins, which also suggests that synergistic targeting of both molecules may be required for certain LSCs. Disclosures: No relevant conflicts of interest to declare.

Description: The proto-oncogene EVI1 (ecotropic viral integration site-1), located on chromosome band 3q26, is aberrantly expressed in human acute myeloid leukemia (AML) with 3q26 rearrangements. In the current study, we showed, in a large AML cohort carrying 11q23 translocations, that ∼ 43% of all mixed lineage leukemia (MLL)–rearranged leukemias are EVI1pos. High EVI1 expression occurs in AMLs expressing the MLL-AF6, -AF9, -AF10, -ENL, or -ELL fusion genes. In addition, we present evidence that EVI1pos MLL-rearranged AMLs differ molecularly, morphologically, and immunophenotypically from EVI1neg MLL-rearranged leukemias. In mouse bone marrow cells transduced with MLL-AF9, we show that MLL-AF9 fusion protein maintains Evi1 expression on transformation of Evi1pos HSCs. MLL-AF9 does not activate Evi1 expression in MLL-AF9–transformed granulocyte macrophage progenitors (GMPs) that were initially Evi1neg. Moreover, shRNA-mediated knockdown of Evi1 in an Evi1pos MLL-AF9 mouse model inhibits leukemia growth both in vitro and in vivo, suggesting that Evi1 provides a growth-promoting signal. Using the Evi1pos MLL-AF9 mouse leukemia model, we demonstrate increased sensitivity to chemotherapeutic agents on reduction of Evi1 expression. We conclude that EVI1 is a critical player in tumor growth in a subset of MLL-rearranged AMLs.

Description: Background After European Medicines Agency (EMA) approval of axicabtagene ciloleucel and tisagenlecleucel for the treatment of relapsed/refractory (r/r) high-grade lymphoma in 2018, England was one of the first European countries granting fully funded access to these CD19 CAR-T therapies. Both products are available through the National Health Service England (NHSE) Cancer Drug Fund until their cost-effectiveness has been determined. The NHSE CAR-T program has been set up in a structure aiming to implement robust and transparent criteria for patient selection and to ensure equity of treatment access: CAR-T slots are approved by a weekly National CAR-T Clinical Panel (NCCP), consisting of independent clinical experts, patient representatives, and delegates from each CAR-T centre; treatment is delivered in 7 geographically spread commissioned CAR-T centres (Birmingham, Bristol, King's College Hospital London, University Hospital London, The Christie Manchester, Manchester Royal Infirmary, Newcastle). Here, we report prospective data on the first 122 lymphoma patients approved by the NCCP. Methods Patients with r/r high-grade lymphoma referred to the NCCP between December 2018 and July 2019 and deemed eligible for treatment with CD19 CAR-T were analysed. Eligibility was assessed in the CAR-T centre's tumor board, based on organ function and fitness (performance status 0/1), absence of active CNS disease, and biopsy confirmation of r/r high-grade lymphoma. The final decision on patient eligibility was made by consensus through the NCCP independent clinical panel. CAR-T product selection for each patient was done by the CAR-T centre, mainly on the basis of manufacturing slot availability. Results 122 patients were approved for treatment with CD19 CAR-T therapy by the panel. CAR-T centres selected 76 patients for axicabtagene ciloleucel and 46 for tisagenlecleucel. Patients' median age was 56 years (range 18-75). 62% were male. 87 (71%) patients had de novo diffuse large B-cell lymphoma, 29 (24%) transformed lymphoma (23 from follicular- and 6 from marginal zone lymphoma), and 6 (5%) primary mediastinal B-cell lymphoma. 96 (79%) patients had biopsy confirmation of disease prior to submission. 71 (58%) patients had received 2 prior lines of therapy for high-grade lymphoma, 51 (42%) patients 3 or more treatment lines (maximum 6). 5 patients had previous allogeneic, 19 previous autologous transplant. 88% of patients (107/122) were refractory to the last line of treatment (stable- or progressive disease (PD) or relapse within 6 months). Among 122 patients, 112 completed leukapheresis, 3 are awaiting the procedure, and 7 patients did not proceed (6 due to PD, 1 opted for radical radiotherapy). 57 of 112 patients were infused at the time of abstract submission, 42 are awaiting CAR-T infusion. 10 patients did not proceed to infusion due to disease progression and clinical deterioration (3 with CNS relapse), 2 due to manufacturing failure. One patient achieved a complete response following bridging therapy and is currently monitored. 84% (88/105) patients received bridging therapy between the time of NCCP approval and CAR-T infusion (median 64 days), 62 had chemotherapy, 9 radiotherapy, and 17 steroids only. Details on bridging therapy, treatment-related toxicities and outcomes will be provided at the meeting, by which time approximately 62 patients will have completed their 3 months PET response assessment. Conclusion NHSE has successfully implemented a national structure for providing licenced CAR-T products in England, enabling equity of access and oversight on capacity and patient outcomes, which can serve as a model for newly licenced, cost-intense and complex cell- and gene therapies in the future. The prospective and centralised nature of this dataset offers a true reflection of the real-world patient population undergoing CAR-T therapy in England. Disclosures Kuhnl: Kite Gilead: Honoraria. Roddie:Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Novartis: Consultancy. Menne:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kite/Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Kyowa Kirin: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Daiichi Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau. Sanderson:Kite/Gilead: Honoraria. Osborne:Novartis: Other: Travel; Pfizer: Honoraria, Speakers Bureau; MSD: Consultancy; Takeda: Consultancy, Honoraria, Other: Travel, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Speakers Bureau; Servier: Consultancy; Gilead: Consultancy. Radford:AstraZeneca: Equity Ownership, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; ADC Therapeutics: Consultancy, Research Funding; GSK: Equity Ownership; Seattle Genetics: Consultancy, Honoraria. Patten:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Roche: Honoraria, Research Funding. O'Reilly:Kite Gilead: Honoraria. Bloor:Abvie, Gilead, Novartis, Autolus, Celgene, etc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational grant. Rowntree:Novartis: Consultancy. Bowles:Abbvie: Research Funding; Janssen: Research Funding. Collins:Gilead: Consultancy, Honoraria. McMillan:BMS: Honoraria; Celgene: Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau; Gilead: Honoraria; Novartis: Honoraria; Sandoz: Honoraria; Pfizer: Honoraria, Research Funding; MSD: Honoraria.

Description: Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) were recently demonstrated in acute myeloid leukemia (AML), but their prevalence and prognostic impact remain to be explored in large extensively characterized AML series, and also in various other hematologic malignancies. Here, we demonstrate in 893 newly diagnosed cases of AML mutations in the IDH1 (6%) and IDH2 (11%) genes. Moreover, we identified IDH mutations in 2 JAK2 V617F myeloproliferative neoplasias (n = 96), a single case of acute lymphoblastic leukemia (n = 96), and none in chronic myeloid leukemias (n = 81). In AML, IDH1 and IDH2 mutations are more common among AML with normal karyotype and NPM1mutant genotypes. IDH1 mutation status is an unfavorable prognostic factor as regards survival in a composite genotypic subset lacking FLT3ITD and NPM1mutant. Thus, IDH1 and IDH2 mutations are common genetic aberrations in AML, and IDH1 mutations may carry prognostic value in distinct subtypes of AML.

Description: The EVI1 (ecotropic virus integration-1) gene plays an important role in hematopoiesis especially in megakaryocyte development. The MDS1 gene is located upstream of EVI1, and its function is currently unknown. Normally the MDS1/EVI1 intergenic splice variant is co-expressed with EVI1. In adult acute myeloid leukemia (AML) overexpression of EVI1 (EVI1+) can be found in patients with chromosome 3q26-rearrangements. Often, these patients do not co-express MDS1/EVI1. Recently high EVI1 expression was also discovered in a separate subgroup of patients that did not have 3q26-rearrangements. Occasionally, they did not show overexpression of MDS1/EVI1. In these patients cryptic inversions of chromosome 3 were identified with fluorescence in situ hybridization (FISH). Of interest, EVI1+ was found to be an independent poor prognostic marker in adult AML (Lugthart et al, Blood 2008). In pediatric AML, 3q26-rearrangements are rare and the role of EVI1 is unknown. In this study, we investigated the frequency and clinical relevance of EVI1+ in pediatric AML. EVI1 expression was analyzed in 233 pediatric AML patients, of whom microarray gene expression profiling data were available. EVI1+ was found in 25 pediatric AML patients (11%), and confirmed with real-time quantitative PCR. This included 13/49 (26%) patients with MLL-rearranged AML: 5/22 (23%) cases with t(9;11); and all (n=4) cases with t(6;11). Moreover, EVI1+ was found in 4/7 (57%) cases with AML M7; in 2/3 (66%) cases with AML M6; in both cases with monosomy 7; in 1/43 (2%) cases with normal cytogenetics; in 2 patients with random cytogenetics, and in 1 patient with a cytogenetic failure. EVI1+ was not found in the t(8;21), inv(16) and t(15;17) subgroups. 3/25 EVI1+ patients lacked the MDS/EVI1 transcript, but no cryptic 3q26-rearrangements were detected with FISH. Molecular analysis showed that one patient had a CEBPα mutation; one patient had an FLT3-ITD; and 3 patients showed a mutation in the RAS oncogene. EVI+ was not correlated with sex or white blood cell count. However, the frequency in children younger than 10 years old was twice as high when compared to older children (14% vs 7%, p=0.12). Survival analysis was restricted to the subset of patients who were treated using uniform DCOG and BFM treatment protocols (n=204). In this cohort, EVI1+ patients had a worse 5-years event-free survival (pEFS) compared to patients without EVI1+ (30 vs. 43%, p=0.02). However, multivariate analysis, including cytogenetics (favorable [t(8;21, inv(16), t(15;17)] vs. other), FLT3-ITD, age and WBC, showed that EVI1+ was not an independent prognostic factor for survival. Moreover, within the unfavorable/normal cytogenetic subgroup, there was no difference in outcome between patients with and without EVI1+. We conclude that EVI1+ is found in ~10% of pediatric AML, and highly correlated with specific unfavorable cytogenetic (MLL-rearrangements) and morphologic (FAB M6/7) subtypes. In contrast to adult AML, no 3q26-rearrangements or cryptic inversions were found, and EVI1+ was not an independent prognostic factor. This difference in prognostic relevance may be due to differences in treatment. Alternatively, these results may indicate that EVI1 plays a different role in disease biology between adult and pediatric AML. This is at least suggested by the lack of 3q26 aberrations in pediatric AML.

Description: Inappropriate expression of EVI1 (ecotropic virus integration-1), in particular splice form EVI1-1D, through chromosome 3q26 lesions or other mechanisms has been implicated in the development of high-risk acute myeloid leukemia (AML). To validate the clinical relevance of EVI1-1D, as well as of the other EVI1 splice forms and the related MDS1/EVI1 (ME) gene, real-time quantitative polymerase chain reaction was performed in 534 untreated adults with de novo AML. EVI1-1D was highly expressed in 6% of cases (n = 32), whereas 7.8% were EVI1+ (n = 41) when all splice variants were taken into account. High EVI1 predicted a distinctly worse event-free survival (HR = 1.9; P = .002) and disease-free survival (HR = 2.1, P = .006) following multivariate analysis. Importantly, we distinguished a subset of EVI1+ cases that lacked expression of ME (EVI1+ME−; n = 17) from cases that were ME+ (EVI1+ME+; n = 24). The atypical EVI1+ME− expression pattern exhibited cytogenetically detectable chromosomal 3q26 breakpoints in 8 cases. Fluorescence in situ hybridization revealed 7 more EVI1+ME− cases that carried cryptic 3q26 breakpoints, which were not found in the EVI1+ME+ group. EVI1+ME− expression predicts an extremely poor prognosis distinguishable from the general EVI1+ AML patients (overall survival [OS]: P 〈 .001 and event-free survival [EFS]: P = .002). We argue that EVI1/ME quantitative expression analysis should be implemented in the molecular diagnostic procedures of AML.

Description: DNA methylation patterns are frequently dysregulated in cancer, although little is known of the mechanisms through which specific gene sets become aberrantly methylated. The ecotropic viral integration site 1 (EVI1) locus encodes a DNA binding zinc-finger transcription factor that is aberrantly expressed in a subset of acute myeloid leukemia (AML) patients with poor outcome. We find that the promoter DNA methylation signature of EVI1 AML blast cells differs from those of normal CD34+ bone marrow cells and other AMLs. This signature contained 294 differentially methylated genes, of which 238 (81%) were coordinately hypermethylated. An unbiased motif analysis revealed an overrepresentation of EVI1 binding sites among these aberrantly hypermethylated loci. EVI1 was capable of binding to these promoters in 2 different EVI1-expressing cell lines, whereas no binding was observed in an EVI1-negative cell line. Furthermore, EVI1 was observed to interact with DNA methyl transferases 3A and 3B. Among the EVI1 AML cases, 2 subgroups were recognized, of which 1 contained AMLs with many more methylated genes, which was associated with significantly higher levels of EVI1 than in the cases of the other subgroup. Our data point to a role for EVI1 in directing aberrant promoter DNA methylation patterning in EVI1 AMLs.