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Clinical outcome of patients with acute myeloid leukemia (AML) is associated with cytogenetic and molecular factors and patient demographics (e.g., age and race). We compared survival of 25,523 non-Hispanic Black and White adults with AML using Surveillance Epidemiology and End Results (SEER) Program data and performed mutational profiling of 1,339 patients with AML treated on frontline Alliance for Clinical Trials in Oncology (Alliance) protocols. Black patients had shorter survival than White patients, both in SEER and in the setting of Alliance clinical trials. The disparity was especially pronounced in Black patients NPM1 and more IDH2 mutations in younger Black patients. Overall survival of younger Black patients was adversely affected by IDH2 mutations and FLT3-ITD, but, in contrast to White patients, was not improved by NPM1 mutations.Significance:We show that young Black patients have not benefited as much as White patients from recent progress in AML treatment in the United States. Our data suggest that both socioeconomic factors and differences in disease biology contribute to the survival disparity and need to be urgently addressed.See related commentary by Vyas, p. 540.This article is highlighted in the In This Issue feature, p. 521

Supplementary Methods, Supplementary References Supplementary Table S1: Inherited regions of homozygosity in >1% of 1,798 non-leukemic individuals Supplementary Table S2: Acquired uniparental disomies detected in 425 cytogenetically normal AML patients Supplementary Table S3. Associations between recurrent UPDs with pretreatment patient characteristics for patients with cytogenetically normal acute myeloid leukemia Supplementary Table S5. Variant allele fraction of gene mutations that co-occurred with UPDs Supplementary Table S6. Allelic ratio of FLT3 internal tandem duplications that co-occurred with UPDs Supplementary Table S7: Copy number gains and losses in 425 patients with cytogenetically normal acute myeloid leukemia

Purpose:Uniparental disomy (UPD) is a way cancer cells duplicate a mutated gene, causing loss of heterozygosity (LOH). Patients with cytogenetically normal acute myeloid leukemia (CN-AML) do not have microscopically detectable chromosome abnormalities, but can harbor UPDs. We examined the prognostic significance of UPDs and frequency of LOH in patients with CN-AML.Experimental Design: We examined the frequency and prognostic significance of UPDs in a set of 425 adult patients with de novo CN-AML who were previously sequenced for 81 genes typically mutated in cancer. Associations of UPDs with outcome were analyzed in the 315 patients with CN-AML younger than 60 years.Results:We detected 127 UPDs in 109 patients. Most UPDs were large and typically encompassed all or most of the affected chromosome arm. The most common UPDs occurred on chromosome arms 13q (7.5% of patients), 6p (2.8%), and 11p (2.8%). Many UPDs significantly cooccurred with mutations in genes they encompassed, including 13q UPD with FLT3-internal tandem duplication (FLT3-ITD; P < 0.001), and 11p UPD with WT1 mutations (P = 0.02). Among patients younger than 60 years, UPD of 11p was associated with longer overall survival (OS) and 13q UPD with shorter disease-free survival (DFS) and OS. In multivariable models that accounted for known prognostic markers, including FLT3-ITD and WT1 mutations, UPD of 13q maintained association with shorter DFS, and UPD of 11p maintained association with longer OS.Conclusions:LOH mediated by UPD is a recurrent feature of CN-AML. Detection of UPDs of 13q and 11p might be useful for genetic risk stratification of patients with CN-AML.

Mutations detected in 425 CN-AML patients

Acute myeloid leukemia (AML) is a molecularly complex disease characterized by heterogeneous tumor genetic profiles and involving numerous pathogenic mechanisms and pathways. Integration of molecular data types across multiple patient cohorts may advance current genetic approaches for improved subclassification and understanding of the biology of the disease. Here, we analyzed genome-wide DNA methylation in 649 AML patients using Illumina arrays and identified a configuration of 13 subtypes (termed “epitypes”) using unbiased clustering. Integration of genetic data revealed that most epitypes were associated with a certain recurrent mutation (or combination) in a majority of patients, yet other epitypes were largely independent. Epitypes showed developmental blockage at discrete stages of myeloid differentiation, revealing epitypes that retain arrested hematopoietic stem-cell-like phenotypes. Detailed analyses of DNA methylation patterns identified unique patterns of aberrant hyper- and hypomethylation among epitypes, with variable involvement of transcription factors influencing promoter, enhancer, and repressed regions. Patients in epitypes with stem-cell-like methylation features showed inferior overall survival along with up-regulated stem cell gene expression signatures. We further identified a DNA methylation signature involving STAT motifs associated with FLT3-ITD mutations. Finally, DNA methylation signatures were stable at relapse for the large majority of patients, and rare epitype switching accompanied loss of the dominant epitype mutations and reversion to stem-cell-like methylation patterns. These results show that DNA methylation-based classification integrates important molecular features of AML to reveal the diverse pathogenic and biological aspects of the disease.

Purpose:Uniparental disomy (UPD) is a way cancer cells duplicate a mutated gene, causing loss of heterozygosity (LOH). Patients with cytogenetically normal acute myeloid leukemia (CN-AML) do not have microscopically detectable chromosome abnormalities, but can harbor UPDs. We examined the prognostic significance of UPDs and frequency of LOH in patients with CN-AML.Experimental Design: We examined the frequency and prognostic significance of UPDs in a set of 425 adult patients with de novo CN-AML who were previously sequenced for 81 genes typically mutated in cancer. Associations of UPDs with outcome were analyzed in the 315 patients with CN-AML younger than 60 years.Results:We detected 127 UPDs in 109 patients. Most UPDs were large and typically encompassed all or most of the affected chromosome arm. The most common UPDs occurred on chromosome arms 13q (7.5% of patients), 6p (2.8%), and 11p (2.8%). Many UPDs significantly cooccurred with mutations in genes they encompassed, including 13q UPD with FLT3-internal tandem duplication (FLT3-ITD; P < 0.001), and 11p UPD with WT1 mutations (P = 0.02). Among patients younger than 60 years, UPD of 11p was associated with longer overall survival (OS) and 13q UPD with shorter disease-free survival (DFS) and OS. In multivariable models that accounted for known prognostic markers, including FLT3-ITD and WT1 mutations, UPD of 13q maintained association with shorter DFS, and UPD of 11p maintained association with longer OS.Conclusions:LOH mediated by UPD is a recurrent feature of CN-AML. Detection of UPDs of 13q and 11p might be useful for genetic risk stratification of patients with CN-AML.

Alterations in global DNA methylation patterns are a major hallmark of cancer and represent attractive biomarkers for personalized risk stratification. Chronic lymphocytic leukemia (CLL) risk stratification studies typically focus on time to first treatment (TTFT), time to progression (TTP) after treatment, and overall survival (OS). Whereas TTFT risk stratification remains similar over time, TTP and OS have changed dramatically with the introduction of targeted therapies, such as the Bruton tyrosine kinase inhibitor ibrutinib. We have shown that genome-wide DNA methylation patterns in CLL are strongly associated with phenotypic differentiation and patient outcomes. Here, we developed a novel assay, termed methylation-iPLEX (Me-iPLEX), for high-throughput quantification of targeted panels of single cytosine guanine dinucleotides from multiple independent loci. Me-iPLEX was used to classify CLL samples into 1 of 3 known epigenetic subtypes (epitypes). We examined the impact of epitype in 1286 CLL patients from 4 independent cohorts representing a comprehensive view of CLL disease course and therapies. We found that epitype significantly predicted TTFT and OS among newly diagnosed CLL patients. Additionally, epitype predicted TTP and OS with 2 common CLL therapies: chemoimmunotherapy and ibrutinib. Epitype retained significance after stratifying by biologically related biomarkers, immunoglobulin heavy chain mutational status, and ZAP70 expression, as well as other common prognostic markers. Furthermore, among several biological traits enriched between epitypes, we found highly biased immunogenetic features, including IGLV3-21 usage in the poorly characterized intermediate-programmed CLL epitype. In summary, Me-iPLEX is an elegant method to assess epigenetic signatures, including robust classification of CLL epitypes that independently stratify patient risk at diagnosis and time of treatment.

Chronic Lymphocytic Leukemia (CLL) has a complex pattern of driver mutations and much of its clinical diversity remains unexplained. We devised a method for simultaneous subgroup discovery across multiple data types and applied it to genomic, transcriptomic, DNA methylation and ex-vivo drug response data from 217 Chronic Lymphocytic Leukemia (CLL) cases. We uncovered a biological axis of heterogeneity strongly associated with clinical behavior and orthogonal to the known biomarkers. We validated its presence and clinical relevance in four independent cohorts (n=547 patients). We find that this axis captures the proliferative drive (PD) of CLL cells, as it associates with lymphocyte doubling rate, global hypomethylation, accumulation of driver aberrations and response to pro-proliferative stimuli. CLL-PD was linked to the activation of mTOR-MYC-oxidative phosphorylation (OXPHOS) through transcriptomic, proteomic and single cell resolution analysis. CLL-PD is a key determinant of disease outcome in CLL. Our multi-table integration approach may be applicable to other tumors whose inter-individual differences are currently unexplained.

BackgroundExportin 1 (XPO1/CRM1) is a key mediator of nuclear export with relevance to multiple cancers, including chronic lymphocytic leukemia (CLL). Whole exome sequencing has identified hot-spot somaticXPO1point mutations which we found to disrupt highly conserved biophysical interactions in the NES-binding groove, conferring novel cargo-binding abilities and forcing cellular mis-localization of critical regulators. However, the pathogenic role played by change-in-functionXPO1mutations in CLL is not fully understood.MethodsWe performed a large, multi-center retrospective analysis of CLL cases (N = 1286) to correlate nonsynonymous mutations inXPO1(predominantly E571K or E571G;n = 72) with genetic and epigenetic features contributing to the overall outcomes in these patients. We then established a mouse model with over-expression of wildtype (wt) or mutant (E571K or E571G)XPO1restricted to the B cell compartment (Eµ-XPO1). Eµ-XPO1 mice were then crossed with the Eµ-TCL1 CLL mouse model. Lastly, we determined crystal structures of XPO1 (wt or E571K) bound to several selective inhibitors of nuclear export (SINE) molecules (KPT-185, KPT-330/Selinexor, and KPT-8602/Eltanexor).ResultsWe report that nonsynonymous mutations in XPO1 associate with high risk genetic and epigenetic features and accelerated CLL progression. Using the newly-generated Eµ-XPO1 mouse model, we found that constitutive B-cell over-expression of wt or mutantXPO1could affect development of a CLL-like disease in aged mice. Furthermore, concurrent B-cell expression ofXPO1with E571K or E571G mutations andTCL1accelerated the rate of leukemogenesis relative to that of Eµ-TCL1 mice. Lastly, crystal structures of E571 or E571K-XPO1 bound to SINEs, including Selinexor, are highly similar, suggesting that the activity of this class of compounds will not be affected byXPO1mutations at E571 in patients with CLL.ConclusionsThese findings indicate that mutations inXPO1at E571 can drive leukemogenesis by priming the pre-neoplastic lymphocytes for acquisition of additional genetic and epigenetic abnormalities that collectively result in neoplastic transformation.

Clinical outcome of patients with acute myeloid leukemia (AML) is associated with cytogenetic and molecular factors and patient demographics (e.g., age and race). We compared survival of 25,523 non-Hispanic Black and White adults with AML using Surveillance Epidemiology and End Results (SEER) Program data and performed mutational profiling of 1,339 patients with AML treated on frontline Alliance for Clinical Trials in Oncology (Alliance) protocols. Black patients had shorter survival than White patients, both in SEER and in the setting of Alliance clinical trials. The disparity was especially pronounced in Black patients Significance: We show that young Black patients have not benefited as much as White patients from recent progress in AML treatment in the United States. Our data suggest that both socioeconomic factors and differences in disease biology contribute to the survival disparity and need to be urgently addressed. See related commentary by Vyas, p. 540. This article is highlighted in the In This Issue feature, p. 521

Paramutations represent directed and meiotically-heritable changes in gene regulation leading to apparent violations of Mendelian inheritance. Although the mechanism and evolutionary importance of paramutation behaviors remain largely unknown, genetic screens in maize (Zea mays) identify five components affecting 24 nucleotide RNA biogenesis as required to maintain repression of a paramutant purple plant1 (pl1) allele. Currently, the RNA polymerase IV largest subunit represents the only component also specifying proper development. Here we identify a chromodomain helicase DNA-binding 3 (CHD3) protein orthologous to Arabidopsis (Arabidopsis thaliana) PICKLE as another component maintaining both pl1 paramutation and normal somatic development but without affecting overall small RNA biogenesis. In addition, genetic tests show this protein contributes to proper male gametophyte function. The similar mutant phenotypes documented in Arabidopsis and maize implicate some evolutionarily-conserved gene regulation while developmental defects associated with the two paramutation mutants are largely distinct. Our results show that a CHD3 protein responsible for normal plant ontogeny and sperm transmission also helps maintain meiotically-heritable epigenetic regulatory variation for specific alleles. This finding implicates an intersection of RNA polymerase IV function and nucleosome positioning in the paramutation process., Author summary Genes are switched “on” and “off” during normal development by regulating DNA accessibility within the chromosomes. How certain gene variants permanently maintain “off” states from one generation to the next remains unclear, but studies in multiple eukaryotes implicate roles for specific types of small RNAs, some of which define cytosine methylation patterns. In corn, these RNAs come from at least two RNA polymerase II-derived complexes sharing a common catalytic subunit (RPD1). Although RPD1 both controls the normal developmental switching of many genes and permanently maintains some of these “off” states across generations, how RPD1 function defines heritable DNA accessibility is unknown. We discovered that a protein (CHD3a) belonging to a group known to alter nucleosome positioning is also required to help maintain a heritable “off” state for one particular corn gene variant controlling both plant and flower color. We also found CHD3a necessary for normal plant development and sperm transmission consistent with the idea that proper nucleosome positioning defines evolutionarily-important gene expression patterns. Because both CHD3a and RPD1 maintain the heritable “off” state of a specific gene variant, their functions appear to be mechanistically linked.

Epigenetic changes during B cell differentiation generates distinct DNA methylation signatures specific for B cell subsets, including memory B cells (MBCs) and plasma cells (PCs). Waldenström's macroglobulinemia (WM) is a complex B cell malignancy uniquely comprised of a mixture of lymphocytic and plasmacytic phenotypes. Here we integrated genome-wide DNA methylation, transcriptome, mutation and other phenotypic features of tumor cells from 35 MYD88-mutated WM patients in relation to normal plasma and B cell subsets. We discovered that WM patients naturally segregate into two groups according to DNA methylation patterns, related to normal MBC and PC profiles, and reminiscent of other memory and plasma cell-derived malignancies. Concurrent analysis of DNA methylation changes in normal and WM development were used to capture tumor-specific events, highlighting a selective reprogramming of enhancer regions in MBC-like WM and repressed and heterochromatic regions in PC-like WM. MBC-like WM hypomethylation was enriched in motifs belonging to PU.1, TCF3 and OCT2 transcription factors and involved elevated MYD88/TLR pathway activity. PC-like WM displayed marked global hypomethylation and selective overexpression of histone genes. Finally, WM subtypes exhibited differential genetic, phenotypic and clinical features. MBC-like WM harbored significantly more clonal CXCR4 mutations (P=0.015), deletion 13q (P=0.006), splenomegaly (P=0.02) and thrombocytopenia (P=0.004), while PC-like WM harbored more deletion 6q (P=0.012), gain 6p (P=0.033), had increased frequencies of IGHV3 genes (P=0.002), CD38 surface expression (P=4.1e-5), and plasmacytic differentiation features (P=0.008). Together our findings illustrate a novel approach to subclassify WM patients using patterns of DNA methylation and reveal divergent molecular signatures among WM patients.

Genetic profiling of leukemic cells forms the basis for risk stratification in acute myeloid leukemia (AML). Genetic markers in AML are used to make recommendations for distinct treatment approaches, such as remission consolidation with chemotherapy rather than stem cell transplant for patients with favorable risk genetics as defined by the current guidelines from the European LeukemiaNet (ELN). Yet, several limitations remain, such as overall rarity of many mutations, hierarchical complexity in cases with multiple mutations, conflicting reports of associations with outcomes for some genetic markers, and the absence of markers with prognostic significance in some patients. We have recently described genome-wide DNA methylation signatures that underlie biological features of AML cells and their utility to classify patients [Giacopelli et al. Genome Res. 2021;31:747]. The additional value of epigenetic information for risk assessment in AML in the context of current genetic and other clinical prognostic markers remains largely unexplored. In this study, we have first developed a targeted approach for assessment of DNA methylation-based signatures and employed it to classify 1,262 patients with de novo AML enrolled onto the Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology studies. We successfully classified 87.5% of patients into one of 13 DNA methylation subgroups, termed 'epitypes' (Figure 1A,B). We found that epitypes are composed of a majority of patients with a specific genetic alteration (or a unique combination of alterations) in 9 of 13 epitypes. However, we also identified subgroups of patients that lack these highly recurrent alterations, and, instead, represent an epigenetic phenocopy of the dominant genetic feature (epiphenocopy). Epiphenocopies within epitypes were often enriched in specific lower frequency mutations, suggesting convergence of biological function(s) for these rare mutations. Epiphenocopying was also exhibited by patients displaying a DNA methylation signature involving hypomethylation of STAT DNA sequence motifs (termed the STAT hypomethylation signature, SHS) that mimicked FLT3-ITD mutations. Epitype and SHS DNA methylation signatures affected clinical outcomes separately to ELN risk groups (P To broadly examine the prognostic power of DNA methylation signatures, we combined methylation-based classifications into a knowledge bank containing a compendium of other prognostic markers. Using a recently developed machine-learning approach [Gerstung et al. Nat Genet. 2017;49(3):332], we found that DNA methylation retained a high degree of importance for clinical outcomes, including overall survival (Figure 1F). Specifically, SHS and 6 epitypes were the most significant features negatively associated with overall survival along with age (P Our study demonstrates that DNA methylation signatures advance our understanding of the biology of AML and improve risk stratification through the identification of patients with epiphenocopies that mimic genetic mutations and other biological features. Use of DNA methylation signatures may lead to more effective assignment of patients to existing and novel therapeutic approaches. Support: U10CA180821, U10CA180882, U24CA196171; https://acknowledgments.alliancefound.org; ClinicalTrials.gov Identifiers: NCT00048958 (8461), NCT00899223 (9665), and NCT00900224 (20202) Figure 1 Figure 1. Disclosures Blachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Blum: Abbvie: Honoraria; AmerisourceBergen: Honoraria; Celyad Oncology: Research Funding; Xencor: Research Funding; Nkarta: Research Funding; Forma Therapeutics: Research Funding; Leukemia and Lymphoma Society: Research Funding; Syndax: Honoraria. Stone: Agios Pharmaceuticals Inc, Novartis;: Research Funding; ACI Clinical, Syntrix Pharmaceuticals, Takeda Oncology: Other: Data Safety & Monitoring; AbbVie Inc, Actinium Pharmaceuticals Inc, Aprea Therapeutics, BerGenBio ASA, ElevateBio, Foghorn Therapeutics, GEMoaB, GlaxoSmithKline, Innate Pharma, Syndax Pharmaceuticals Inc, Syros Pharmaceuticals Inc, Takeda Oncology: Other: Advisory Committee. Eisfeld: Karyopharm (spouse): Current Employment. Byrd: Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Newave: Membership on an entity's Board of Directors or advisory committees.

Epigenetic changes during B-cell differentiation generate distinct DNA methylation signatures specific for B-cell subsets, including memory B cells (MBCs) and plasma cells (PCs). Waldenström macroglobulinemia (WM) is a B-cell malignancy uniquely comprising a mixture of lymphocytic and plasmacytic phenotypes. Here, we integrated genome-wide DNA methylation, transcriptome, mutation, and phenotypic features of tumor cells from 35 MYD88-mutated WM patients in relation to normal plasma and B-cell subsets. Patients naturally segregate into 2 groups according to DNA methylation patterns, related to normal MBC and PC profiles, and reminiscent of other memory and PC-derived malignancies. Concurrent analysis of DNA methylation changes in normal and WM development captured tumor-specific events, highlighting a selective reprogramming of enhancer regions in MBC-like WM and repressed and heterochromatic regions in PC-like WM. MBC-like WM hypomethylation was enriched in motifs belonging to PU.1, TCF3, and OCT2 transcription factors and involved elevated MYD88/TLR pathway activity. PC-like WM displayed marked global hypomethylation and selective overexpression of histone genes. Finally, WM subtypes exhibited differential genetic, phenotypic, and clinical features. MBC-like WM harbored significantly more clonal CXCR4 mutations (P = .015), deletion 13q (P = .006), splenomegaly (P = .02), and thrombocytopenia (P = .004), whereas PC-like WM harbored more deletion 6q (P = .012), gain 6p (P = .033), had increased frequencies of IGHV3 genes (P = .002), CD38 expression (P = 4.1e-5), and plasmacytic differentiation features (P = .008). Together, our findings illustrate a novel approach to subclassify WM patients using DNA methylation and reveal divergent molecular signatures among WM patients.

Background Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor survival. Recurrent genetic aberrations, such as chromosomal rearrangements and gene mutations, are currently used for patient classification and prognosis, and form the basis of our current understanding of pathogenic mechanisms. However, these markers incompletely predict disease behavior and outcomes. Alterations in DNA methylation patterns are a major hallmark of cancer and recent studies have demonstrated differential global DNA methylation patterns among AML patients. Here we sought to define a novel approach to classify AML patients using genome-wide DNA methylation patterns and to uncover the biological basis and features associated with these epigenetic patterns by integrating mutation, gene expression, and functional data. Methods We analyzed genome-wide DNA methylation data from Illumina arrays on 649 AML cases combined from the Beat AML consortium housed at the Oregon Health & Science University (OHSU) (n=226), The Ohio State University (n=27), TCGA (n=190), and other published studies (n=206). Small molecule inhibitor response data was obtained from the Beat AML consortium. RNA sequencing and survival analyses were performed by Kaplan-Meier analysis in the TCGA and OHSU cohorts. Results K-mediods based clustering on the 500 most-variable CpGs identified 13 distinct subtypes, we termed AML 'epitypes' (Fig. 1A). Comparative analyses incorporating normal hematopoietic cells revealed that AML epitypes retain unique combinations of biological features including the degree of myeloid development, chromatin landscapes, transcription factor binding and gene expression patterns/pathways. Eleven of 13 epitypes were associated with somatic genetic aberrations, i.e. mutations (or combinations thereof) in NPM1, IDH1/2, TET2, DNMT3A, CEBPA, and chromosomal rearrangements t(8;21), t(15;17) and inv(16) accounted for the majority of patients within each respective epitype. The remaining two epitypes were not associated with dominant genetic events, rather they clustered closely with normal CD34+ hematopoietic stem and progenitor cells. These 'stem-like' epigenetic patterns accounted for 27% of patients and showed limited correlation to other leukemic stem cell gene expression signatures. Stem-like epitypes demonstrated broad resistance to a range of small molecule inhibitors. Differential gene expression analysis between normal cells and epitypes revealed enrichment of the JAK-STAT pathway along with other inflammatory pathways, such as interleukin and interferon signaling, selectively in the stem-like epitypes. Patients with stem-like epitypes exhibited decreased overall survival relative to others independent of age (P We next investigated the impact of STAT pathway activation on AML epigenetic patterns. FLT3 mutations are known to selectively activate the STAT pathway in AML and are associated with inferior outcomes. Controlling for the distribution of FLT3 mutations across epitypes, we uncovered a hypomethylation signature enriched in STAT transcription factor binding and FLT3 mutations which we have termed the 'STAT hypomethylation signature' (SHS). SHS-positivity was not restricted to FLT3 mutations, as 34% of SHS+ AMLs lacked FLT3 mutations and, conversely, SHS was absent in 24% of FLT3 mutated patients. SHS-positivity did not impact outcome in the stem-like epitypes; however, within non-stem-like epitypes, SHS+ patients displayed significantly poorer outcomes (P=0.021; Fig 1C). Conclusion Applying our integrative approach of using global DNA methylation profiles to subclassify AML patients, we have uncovered new insights into potential pathogenic mechanisms by which genetic and epigenetic aberrations may contribute to discrete avenues of AML development. Distinct global DNA methylation patterns are commonly driven by genetic aberrations, while others were associated with stem cell-like features and inflammatory pathways. The identification of JAK/STAT pathway upregulation in the stem-like epitype and a subset of non-stem-like samples highlights that nearly half of AML patients exhibit evidence of JAK/STAT activation and experience poor outcomes. Disclosures Druker: Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; Bristol-Myers Squibb: Patents & Royalties, Research Funding; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Celgene: Consultancy; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Beat AML LLC: Other: Service on joint steering committee; Cepheid: Consultancy, Honoraria; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Gilead Sciences: Other: former member of Scientific Advisory Board; CureOne: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; Monojul: Other: former consultant; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees . Tyner:Petra: Research Funding; Janssen: Research Funding; Aptose: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Takeda: Research Funding; Syros: Research Funding; Array: Research Funding; AstraZeneca: Research Funding; Array: Research Funding; Incyte: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Seattle Genetics: Research Funding; AstraZeneca: Research Funding; Syros: Research Funding; Seattle Genetics: Research Funding; Constellation: Research Funding; Aptose: Research Funding; Incyte: Research Funding; Agios: Research Funding; Petra: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Agios: Research Funding. Byrd:Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Acerta: Research Funding; BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Genentech: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau.

Monoclonal B cell lymphocytosis (MBL) has been shown to be the precursor condition that precedes overt diagnosis of chronic lymphocytic leukemia (CLL). Whereas CLL is classified with greater than or equal to 5 × 109/L B lymphocytes in the peripheral blood, MBL is a clonal expansion of B-cells below this threshold. MBL can be further divided into high- or low-count based on whether the B-cell count is above or below 0.5 × 109/L. Approximately 10% of the population over 40 is estimated to have MBL, increasing to >50% over the age of 90. While low-count MBL is unlikely to progress, 1-2% of high-count (HC) MBL individuals progress to CLL requiring therapy per year. It is debatable if all patients with detectable MBL should be classified as an entity requiring monitoring by a hematologist, especially for low-count MBL. In addition, the diagnosis of leukemia is distressing to patients; therefore, it is important to identify HC MBL patients that are more likely to progress to disease requiring treatment and thus should be monitored more closely. As the majority of MBL cases phenotypically resemble CLL, established prognostic markers including recurrent chromosomal aberrations, beta-2 microglobulin levels, and the mutational status of the Immunoglobulin heavy-chain variable region locus (IGHV) have been shown to predict time to treatment (TTT) and overall survival (OS) in a large retrospective study of MBL1. CLL patients can also be divided into three distinct epigenetic subtypes that reflect progressive DNA methylation changes that occurs during B cell development. These 'epitypes' termed low-programmed (LP), intermediate-programmed (IP), and high-programmed (HP) independently predict clinical outcomes irrespective of disease stage and treatment2. LP-CLL patients follow a generally unfavorable clinical course compared to the more indolent HP-CLL patients, while IP CLL patients display an intermediate outcome. Here we sought to determine if epitype forecasts progression to CLL and eventual clinical outcome for individuals with MBL. We analyzed 66 individuals diagnosed with HC MBL at the Mayo Clinic with a median follow-up of 6.3 years. Developmental epitype was determined using our novel Methylation-iPLEX technique that interrogates 34 CpGs and assigns epitype using a random forest model2. Seventy-seven percent of the MBL cases were assigned to one of the three epitypes: 42.4% HP, 19.7% IP, and 15.2% LP. The residual 23% remained unclassified due to ambiguous (low confidence) epigenetic patterns or insufficient purity (Figure 1A). The overall proportion of HP and IP epitypes in MBL were significantly greater than proportions observed in CLL cohorts (P In this study we evaluated a cohort of 66 HC MBL cases and determined that classification using developmental DNA methylation epitypes can be employed in HC MBL to aid in risk stratification. HC MBL patients classified as LP are more likely to progress to requiring treatment and have a significantly reduced OS. The epigenetic classification may help clinicians decide how closely and frequently a HC MBL individual needs to be monitored. Figure 1: (A) Breakdown of the epigenetic subtype assigned to 66 HC MBL samples. (B) Kaplan-Meier analysis of time to treatment and (C) overall survival of MBL patients separated by epitype. 1. Parikh, S. A. et al. Outcomes of a large cohort of individuals with clinically ascertained high-count monoclonal B-cell lymphocytosis. Haematologica103, e237-e240 (2018). 2. Giacopelli, B. et al. Developmental subtypes assessed by DNA methylation-iPLEX forecast the natural history of chronic lymphocytic leukemia. Blood blood.2019000490 (2019). doi:10.1182/blood.2019000490 Disclosures Byrd: Ohio State University: Patents & Royalties: OSU-2S; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding. Shanafelt:Patent: Patents & Royalties: US14/292,075 on green tea extract epigallocatechin gallate in combination with chemotherapy for chronic lymphocytic leukemia; Merck: Research Funding; Polyphenon E International: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Glaxo-SmithKline: Research Funding; Abbvie: Research Funding; Cephalon: Research Funding; Celgene: Research Funding. Parikh:Genentech: Honoraria; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding. Kay:Agios: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board.

Introduction: AML is a heterogeneous disease with diverse patient outcomes. During the last 4 decades, several cytogenetic and molecular markers have been used for risk stratification of AML pts and to guide therapeutic decisions. In 2017, Gerstung et al. (Nat Genet 2017;49:332) used a knowledge bank (KB; i.e., combination of clinical, outcome, cytogenetic and sequencing data of 111 genes from 1,540 AML pts) to generate an algorithm that is able to predict likelihoods for remission, relapse, and mortality in AML pts. The prognostic value of the established KB algorithm was validated in two independent but smaller pt cohorts (Gerstung et al.: n=186 pts; Huet et al. Blood 2018;132:865: n=155 pts). Aims: The aim of our study was to validate the prognostic relevance of the KB algorithm both in our entire large independent adult pt cohort and in age subgroups (i.e., younger [ Methods: We analyzed 1,617 pts (median age: 53 y; 1,048 aged Results: We used the 3-y overall survival (OS) rates to compare the KB algorithm prediction with the actual outcome. In the whole cohort, we found the area under the receiver operating characteristic curve (AUC) to be AUCKB = 0.79 (Figure 1A). Of note, AUC = 1.00 means perfect prediction ability whereas AUC = 0.50 denotes lack of prediction ability equal to that of random chance. Concerning other clinical endpoints, we found that the KB approach had the highest AUC for predicting non-remission death (i.e., pts who died without achieving a CR1, AUCKB = 0.84), followed by relapse death (i.e., pts who died after relapse, AUCKB = 0.69), and non-relapse death (i.e., pts who died in CR1, AUCKB = 0.61). Analysis of the 3-y OS in the subgroup of younger pts yielded similar results with an AUCKB = 0.78. Older pts are known to have poorer prognosis and risk stratification is more difficult for this cohort, but, we still found an AUCKB = 0.79 for the KB approach. Next, we compared the predictive value of the KB approach with the current ELN classification and found that KB outperformed the ELN classification in the whole cohort (AUCKB = 0.79 vs AUCELN = 0.53, P Conclusions: Our analysis of a large cohort of 1,617 pts with de novo AML treated with intensive chemotherapy validated the prognostic value of the recently published KB algorithm for the 3-y OS endpoint. Although we found that the KB approach had a high predictive relevance for non-remission death, the AUCs for relapse death and non-relapse death were lower. We also showed that the KB approach had a better predictive value than the current ELN classification but the differences in the AUCs were smaller when ED pts were excluded. Support: CA233338, U24CA196171, U10CA180821, U10CA180882. https://acknowledgments.alliancefound.org Disclosures Kolitz: Boeringer-Ingelheim: Research Funding; Astellas: Research Funding; Roche: Research Funding. Powell:Jazz Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Research Funding; Rafael Pharmaceuticals: Consultancy, Research Funding; Novartis: Consultancy, Speakers Bureau; Janssen: Research Funding. Stone:AbbVie, Actinium, Agios, Argenx, Arog, Astellas, AstraZeneca, Biolinerx, Celgene, Cornerstone Biopharma, Fujifilm, Jazz Pharmaceuticals, Amgen, Ono, Orsenix, Otsuka, Merck, Novartis, Pfizer, Sumitomo, Trovagene: Consultancy; Argenx, Celgene, Takeda Oncology: Other: Data and Safety Monitoring Board/Committee: ; Novartis, Agios, Arog: Research Funding. Byrd:Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Genentech: Research Funding; Genentech: Research Funding; BeiGene: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau.

The characteristic clinical heterogeneity observed in chronic lymphocytic leukemia (CLL) reflects its biological diversity and as a consequence, better understanding of the biology of this disease has led to the identification of a number of useful prognostic tools. Advances in massively parallel sequencing technologies have unearthed genetic and epigenetic heterogeneity and reinforced the concept that specific genetic lesions and the development of genomic complexity can modulate the pathology of CLL. In addition, DNA methylation profiling has shown that CLL patients can be grouped into at least three clinically relevant epigenetic subgroups with distinct outcomes. However, despite this explosion in knowledge, defining the prognosis of individual CLL patients remains a significant clinical challenge. Consequently, there is a need to identify prognostic tests that can provide reliable personalized risk assessments. We have recently shown that a subset of CLL patients manifest telomere dysfunction, which drives genomic instability and clonal evolution. We went on to show an association between short, dysfunctional telomeres and 'high-risk' genetic lesions1,2. Importantly, longitudinal analysis confirmed that tumor cell telomere length remained remarkably stable throughout the course of the disease suggesting that the telomere length is fixed at an early point in disease pathogenesis making it a reliable predictor of both clinical progression and overall survival (OS) regardless of when the measurement is made. In this study, we generated telomere length profiles in 224 newly diagnosed, early stage CLL patients seen at the Mayo clinic in Rochester using high-throughput single telomere length analysis (HT-STELA). We then tested the ability of telomere length to predict time to treatment (TTT) and OS and compared its predictive power with DNA methylation profiling and CLL-IPI scores. Patients with a mean telomere length inside the fusogenic range (IFR) had a significantly shorter time to treatment (P2.17kb), patients with the shortest telomeres had a significantly worse OS (P = 0.034; HR = 2.83). Telomere length was also strongly associated with CLL-IPI score; the CLL-IPI low-risk group (CLL-IPI 1) had the longest telomeres and the CLL-IPI high-risk group (CLL-IPI 4) had the shortest telomeres (P References Lin TT et al. Blood. 2010;116(11):1899-907. Britt-Compton et al. Leukemia. 2012; 26(4):826-30. Disclosures Norris: Cardiff University: Patents & Royalties: Telomere measurement patent. Shanafelt:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Pharmacyclics: Research Funding; Jansen: Research Funding. Kay:Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees. Fegan:Napp: Honoraria; Gilead Sciences, Inc.: Honoraria; Roche: Honoraria; Abbvie: Honoraria; Janssen: Honoraria. Baird:Cardiff University: Patents & Royalties: Telomere measurement patents. Pepper:Cardiff University: Patents & Royalties: Telomere measurement patents.

Epigenetic alterations are universal in cancer and are important in establishing the malignant phenotype. Dissection of the factors that shape the tumor-specific epigenome may reveal insight into key aspects of tumorigenesis and therapeutic resistance. In chronic lymphocytic leukemia (CLL), we have previously found that broad changes in epigenetic patterns co-occur with the evolution of genetic alterations. We have also uncovered that aberrant patterning of DNA methylation in CLL involves excessive activity of a defined group of transcription factors (TFs), including the early growth response (EGR) TF family. Recent work has further revealed that recurrent mutations in EGR2 are associated with exceptionally poor clinical outcomes in CLL. The basis for the adverse association of EGR2 mutations in CLL is unclear. To explore the role of EGR2 mutations in CLL, we initially performed genome-wide DNA methylation analysis using Illumina arrays on CLL patients harboring EGR2 mutations (n=27) compared to EGR wild-type cases (n=265). We found that the three most common recurrent mutations, occurring at amino acid positions E356K, H384N and D411H within the DNA binding domain, are each associated with an exclusive subset of tumor-specific hypomethylated CpG sites. A search for TF sequence motifs at these loci revealed a strong enrichment of novel derivative EGR2 motifs that differ only marginally (usually by a single nucleotide) from the canonical EGR2 recognition sequence. Each recurrent mutation led to specific enrichment of a different derivative EGR2 motif. Furthermore, the canonical (wild-type) recognition sequence was not enriched, suggesting that mutations re-localize binding activity to derivate sequence motifs rather than simply altering binding affinity. Luciferase enhancer, proximity ligation and electrophoretic mobility shift assays confirmed that each EGR2 mutant protein specifically binds and enhances transcriptional activity only when the matched EGR2 derivative recognition motif is present. These results establish that derivative motif sequences may function as novel cryptic enhancers in the presence of the cognate EGR2 mutant TF. We performed multiomics profiling (DNA methylome, ATAC-seq, ChIP-seq and RNA-seq) to examine the nature of the epigenetic reconfiguration and the phenotypic impact of individual EGR2 mutations. Whole genome bisulfite sequencing of E356K- and H384N-mutated CLL samples (n=4 each) was used to reveal the full complement of recurrent differentially methylated regions (DMRs) across the genome, and recapitulated the mutually-exclusive pattern of DMRs between mutations. Overlaying DMRs with data from ChIP-seq and ATAC-seq experiments in the same samples revealed the nature of EGR2 mutation-specific chromatin reconfiguration to be remarkably mutation-specific. For E356K, hypomethylated DMRs are often associated with foci of accessible chromatin, EGR2 binding, and flanked by gains of H3K4me1 and H3K27ac, indicative of the acquisition of active enhancer function. Conversely, H384N mutations generated fewer DMRs and mainly directed the deposition of H3K4me1 only, indicative of gain of poised enhancers at these loci. RNA-sequencing analyses revealed that a subset of epigenetically reconfigured regions was associated with mutation-specific altered gene expression, and differences were virtually always associated with proximal gene activation. E356K and H384N mutations displayed highly differential gene expression patterns, with E356K exhibiting a greater impact on gene expression. Integrated analyses indicated that E356K mutations may specifically involve activated Notch signaling, revealed by the aberrant activation of Notch target genes and the mutual exclusivity of NOTCH1 mutations, further highlighted by enriched co-mutation of NOTCH1 in H384N-mutated CLL. Together these findings provide an exceptional example of the precise role that a singular TF may play in programming the epigenetic landscape. As there are no known TFs that naturally bind derivative EGR2 motifs, these mutant proteins provide insight into aberrant enhancer generation and the phenotypic impact of (re)directed TF binding in a human disease setting. Although these recurrent mutations are presently only known in CLL, these findings provide insight into the mechanisms that may surround other gain-of-function TF activity in various malignancies. Disclosures Kipps: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Genentech Inc: Consultancy, Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees.

Paramutations represent locus-specific trans-homolog interactions affecting the heritable silencing properties of endogenous alleles. Although examples of paramutation are well studied in maize (Zea mays), the responsible mechanisms remain unclear. Genetic analyses indicate roles for plant-specific DNA-dependent RNA polymerases that generate small RNAs, and current working models hypothesize that these small RNAs direct heritable changes at sequences often acting as transcriptional enhancers. Several studies have defined specific sequences that mediate paramutation behaviors, and recent results identify a diversity of DNA-dependent RNA polymerase complexes operating in maize. Other reports ascribe broader roles for some of these complexes in normal genome function. This review highlights recent research to understand the molecular mechanisms of paramutation and examines evidence relevant to small RNA-based modes of transgenerational epigenetic inheritance.

Aberrant DNA methylation patterning occurs in many cancers, including chronic lymphocytic leukemia (CLL). Methylation programming changes as B cells differentiate from immature progenitors to mature B cells. CLL clones originate from a continuum of B cell maturation states, which differ in their "methylome." Emerging evidence suggests that aberrant methylation programming occurs at numerous genes that are differentially expressed in IGHV-defined CLL subtypes. Nuclear receptor interacting protein 1 (NRIP1) reproducibly shows significant gene expression differences in IGHV unmutated (U-CLL) and mutated CLL (M-CLL), with lower transcript levels observed in U-CLL cells. Low expression of NRIP1 is associated with poorer overall survival and time-to-treatment. NRIP1 encodes a cofactor that interacts with various nuclear receptors and other proteins to regulate transcription of genes involved in cellular proliferation, survival, metabolism, inflammation, and other processes. Published data reveal several CpG dinucleotides near the NRIP1 promoter that undergo increasing methylation during the maturation and differentiation of normal peripheral blood B cells (NBC), concordant with a stepwise decrease in transcript levels of NRIP1 over the course of maturation. Interestingly, our analysis of publically available expression data revealed significantly lower NRIP1 expression in U-CLL cells (the less mature CLL subtype) than NBC, while M-CLL cells (the more mature CLL subtype) express levels comparable to NBC. Given these observations, our objective was to interrogate the promoter and regulatory regions at the NRIP1 locus for differences in CpG methylation in genomic DNA isolated from U-CLL, M-CLL, and NBC. We hypothesized that the NRIP1 locus is more highly methylated in U-CLL than in M-CLL and NBC, and that this increased methylation represses NRIP1 gene transcription, contributing to its aberrantly low expression in U-CLL compared to M-CLL and NBC. We bisulfite-converted genomic DNA from negatively-selected CD19+ treatment-naïve CLL samples (n=74 U-CLL, n=37 M-CLL) and NBC from healthy donors (n=5). We PCR-amplified regions in or near the CpG island of NRIP1 and used Agena Bioscience's EpiTyper kit to prepare the samples for analysis on a MassARRAY spectrometer. EpiTyper software was used to detect mass differences that indicate either non-methylated or methylated CpG-containing fragments, and to calculate their relative frequency in each sample. For each CpG locus that was assayed, we fit a beta-regression model to detect differential methylation between M-CLL and U-CLL. We also computed Pearson correlation coefficients comparing methylation levels to gene expression levels. We assessed 47 CpGs mapping to the promoter and nearby regulatory elements of NRIP1 for methylation frequency and correlation with NRIP1 expression. From our comparison of methylation frequency in U-CLL and M-CLL, 3 adjacent CpG loci were significant at false discovery rate (FDR) =1% (CpG 58, p=0.00016; CpG 144, p=0.00062; and CpG 155, p=0.00287). The three most significant correlation coefficients arose from the same three CpG loci (CpG 58, R=0.677, p=2.2e-16; CpG 144, R=0.539, p=7.16e-10; CpG 155, R=0.651, p=5.77e-15). Unexpectedly, all 3 loci were methylated at higher levels in M-CLL and at lower levels in U-CLL (on average), and expression was positively correlated with methylation levels. These CpGs map near a documented binding site for the repressive transcription factor RUNX3, prompting us to hypothesize that differential methylation in U-CLL versus M-CLL may affect the binding affinity of RUNX3 and contribute to the robust NRIP1 expression differences in these CLL subtypes. Future work will test this hypothesis using gel-shift and luciferase reporter assays followed by chromatin immunoprecipitation experiments. Few studies have focused on aberrant methylation patterning in CLL and the consequences of deregulated gene expression. We are the first to look at NRIP1 for differences in DNA methylation across IGHV subtypes. Future studies aimed at understanding how methylation at this locus affects NRIP1 transcription, as well as how levels of NRIP1 affect cell survival pathways, will enhance our understanding of the role of this gene in CLL, and the contributions of deregulated methylation to disease pathophysiology. Disclosures No relevant conflicts of interest to declare.

In chronic lymphocytic leukemia (CLL), it is recognized that germinal center experience by the cell-of-origin has an influence on the malignant phenotype and impacts clinical behavior. Measurement of the mutational status of the IGHV locus is a commonly used approach that subgroups patients according to this natural history. Other biomarkers correlating with these subgroups, such as ZAP70 expression and ZAP70 DNA methylation, are also used. Recently, we and others have found vast differences in global DNA methylation profiles among CLL cases that classifies distinct subgroups related to their natural history. Here we systematically evaluate various markers of natural history of the cell-of-origin for their ability to discriminate clinical outcomes. IGHV mutational status and ZAP70 expression are currently the most widely used prognostic markers of germinal center experience. These markers are generally concordant, with IGHV-unmutated associating with ZAP70 positivity. To compare their relative prognostic impact, we selected CLL cases from a large sample cohort collected as a prospective study maintained by the CLL Research Consortium that were discordant (IGHV-mutated and ZAP70(+) or IGHV-unmutated and ZAP70(-); n=192). We found a significant difference in time-to-first-treatment (TTFT; P=0.002) and overall survival (OS; P=0.032) between discordant groups, with curves separating according to IGHV as opposed to ZAP70 (Fig.1A). DNA methylation-based classification of CLL according to their natural history has not been compared to IGHV status. This classification strategy subgroups the majority of cases into low-programmed CLL (LP-CLL) or high-programmed (HP-CLL) subgroups that generally correlate with IGHV-unmutated/ZAP70(+) and IGHV-mutated/ZAP70(-), respectively, along with a minority of cases in an intermediate (IP-CLL) subgroup. We used our previously-described DNA methylation assay, based on MassARRAY interrogating 7 genomic loci including ZAP70, to group the 192 samples with discordant IGHV/ZAP70 results with 327 samples analyzed previously from the same cohort. DNA methylation (LP-CLL vs. HP-CLL) outperformed IGHV classification (unmutated vs. mutated) in terms of separating TTFT curves (n=432; LP-CLL vs. HP-CLL median difference=4.8 years, unmutated vs. mutated IGHV median difference=2.9 years) and OS curves (n=491; LP-CLL vs. HP-CLL median difference=12.9 years, unmutated vs. mutated IGHV median difference=6.8 years). To evaluate whether DNA methylation subgroups provide additional prognostic information to IGHV, we compared TTFT and OS within IGHV subtypes. We found that the ability of DNA methylation subgrouping to separate OS curves was maintained in the IGHV-unmutated subtype (P0.5 for all). Further investigation of the features of DNA methylation subgroups as defined revealed a strong bias in the frequency of several recurrent adverse prognostic somatic mutations. In particular 17p deletions and EGR2, SF3B1 and XPO1 mutations occurred frequently within the LP-CLL subgroup. We next explored whether the presence of each mutation correlated with clinical outcome within its corresponding DNA methylation subgroup, and found heterogenous results. EGR2 mutations separated TTFT in the LP-CLL subgroup (P=0.0009), but 17p, XPO1 and SF3B1 did not (P>0.3). This suggests that the cell-of-origin should be taken into account to accurately assess the clinical impact of somatic mutations. In summary, we demonstrate that among discordant cases for IGHV/ZAP70, IGHV appears to be a stronger prognostic factor for both TTFT and OS. When then evaluating the relative importance of DNA methylation subgrouping and IGHV status on TTFT and OS, we find that DNA methylation subgrouping is the more powerful prognostic factor. Among the markers of the cell-of-origin, DNA methylation is a strong tool for risk-stratifying patients and should be considered in development of risk prediction models. Figure 1. A) Kaplan-Meier analysis of TTFT of IGHV-mutated and ZAP70(+) versus IGHV-unmutated and ZAP70(-) (P=0.002, median difference=4.4 years). B) Kaplan-Meier analysis of OS in patients with unmutated IGHV separated by DNA methylation subgroup (P Disclosures Wierda: Acerta: Research Funding; Novartis: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Abbvie: Research Funding. Brown:Infinity: Consultancy; Janssen: Consultancy; Gilead Sciences: Consultancy; Roche/Genentech: Consultancy; Celgene: Consultancy; Sun BioPharma: Consultancy; Pfizer: Consultancy; Abbvie: Consultancy. Kipps:Roche: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding.