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PURPOSE Persistent molecular disease (PMD) after induction chemotherapy predicts relapse in AML. In this study, we used whole-exome sequencing (WES) and targeted error-corrected sequencing to assess the frequency and mutational patterns of PMD in 30 patients with AML. MATERIALS AND METHODS The study cohort included 30 patients with adult AML younger than 65 years who were uniformly treated with standard induction chemotherapy. Tumor/normal WES was performed for all patients at presentation. PMD analysis was evaluated in bone marrow samples obtained during clinicopathologic remission using repeat WES and analysis of patient-specific mutations and error-corrected sequencing of 40 recurrently mutated AML genes (MyeloSeq). RESULTS WES for patient-specific mutations detected PMD in 63% of patients (19/30) using a minimum variant allele fraction (VAF) of 2.5%. In comparison, MyeloSeq identified persistent mutations above 0.1% VAF in 77% of patients (23/30). PMD was usually present at relatively high levels (>2.5% VAFs), such that WES and MyeloSeq agreed for 73% of patients despite differences in detection limits. Mutations in DNMT3A, ASXL1, and TET2 (ie, DTA mutations) were persistent in 16 of 17 patients, but WES also detected non-DTA mutations in 14 of these patients, which for some patients distinguished residual AML cells from clonal hematopoiesis. Surprisingly, MyeloSeq detected additional variants not identified at presentation in 73% of patients that were consistent with new clonal cell populations after chemotherapy. CONCLUSION PMD and clonal hematopoiesis are both common in patients with AML in first remission. These findings demonstrate the importance of baseline testing for accurate interpretation of mutation-based tumor monitoring assays for patients with AML and highlight the need for clinical trials to determine whether these complex mutation patterns correlate with clinical outcomes in AML.

Acute myeloid leukemia (AML) patients rarely have long first remissions (> 5 years) after standard-of-care chemotherapy, unless classified as favorable risk at presentation. Identification of the mechanisms responsible for long vs. more typical, short remissions may help to define prognostic determinants for chemotherapy responses. Using exome sequencing, RNA sequencing and functional immunologic studies, we characterized 28 Normal Karyotype (NK)-AML patients with >5 year first remissions after chemotherapy (Long First Remissions, LFR) and compared them to a well-matched group of 31 NK-AML patients who relapsed within 2 years (Standard First Remissions, SFR). Our combined analyses indicated that genetic risk profiling at presentation (as defined by ELN 2017 Criteria) was not sufficient to explain the outcomes of many SFR cases. Single cell RNA-sequencing studies of 15 AML samples showed that SFR AML cells differentially expressed many genes associated with immune suppression. The bone marrow of SFR cases had significantly fewer CD4+ Th1 cells; these T cells expressed an exhaustion signature and were resistant to activation by T-cell receptor stimulation in the presence of autologous AML cells. T cell activation could be restored by removing the AML cells, or blocking the inhibitory MHC Class II receptor, LAG3. Most LFR cases did not display these features, suggesting that their AML cells were not as immunosuppressive. These findings were confirmed and extended in an independent set of 50 AML cases representing all ELN 2017 risk groups. AML cell-mediated suppression of CD4+ Tcell activation at presentation is strongly associated with unfavorable outcomes in AML patients treated with standard chemotherapy.

The malignant Hodgkin and Reed Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) are scarce in affected lymph nodes, creating a challenge to detect driver somatic mutations. As an alternative to cell purification techniques, we hypothesized that ultra-deep exome sequencing would allow genomic study of HRS cells, thereby streamlining analysis and avoiding technical pitfalls. To test this, 31 cHL tumor/normal pairs were exome sequenced to ∼1000x median depth of coverage. An orthogonal error-corrected sequencing approach verified >95% of the discovered mutations. We identified mutations in genes novel to cHL including: CDH5 and PCDH7; novel mutations in IL4R, and a novel pattern of recurrent mutations in pathways regulating Hippo signaling. This study provides proof-of-principle that ultra-deep exome sequencing can be utilized to identify recurrent mutations in HRS cells, allowing for the analysis for clinically relevant genomic variants in large cohorts of cHL patients.

BACKGROUND: As consolidation therapy for acute myeloid leukemia (AML), allogeneic hematopoietic stem-cell transplantation provides a benefit in part by means of an immune-mediated graft-versus-leukemia effect. We hypothesized that the immune-mediated selective pressure imposed by allogeneic transplantation may cause distinct patterns of tumor evolution in relapsed disease. METHODS: We performed enhanced exome sequencing on paired samples obtained at initial presentation with AML and at relapse from 15 patients who had a relapse after hematopoietic stem-cell transplantation (with transplants from an HLA-matched sibling, HLA-matched unrelated donor, or HLA-mismatched unrelated donor) and from 20 patients who had a relapse after chemotherapy. We performed RNA sequencing and flow cytometry on a subgroup of these samples and on additional samples for validation. RESULTS: On exome sequencing, the spectrum of gained and lost mutations observed with relapse after transplantation was similar to the spectrum observed with relapse after chemotherapy. Specifically, relapse after transplantation was not associated with the acquisition of previously unknown AML-specific mutations or structural variations in immune-related genes. In contrast, RNA sequencing of samples obtained at relapse after transplantation revealed dysregulation of pathways involved in adaptive and innate immunity, including down-regulation of major histocompatibility complex (MHC) class II genes (HLA-DPA1, HLA-DPB1, HLA-DQB1, and HLA-DRB1) to levels that were 3 to 12 times lower than the levels seen in paired samples obtained at presentation. Flow cytometry and immunohistochemical analysis confirmed decreased expression of MHC class II at relapse in 17 of 34 patients who had a relapse after transplantation. Evidence suggested that interferon-γ treatment could rapidly reverse this phenotype in AML blasts in vitro. CONCLUSIONS: AML relapse after transplantation was not associated with the acquisition of relapse-specific mutations in immune-related genes. However, it was associated with dysregulation of pathways that may influence immune function, including down-regulation of MHC class II genes, which are involved in antigen presentation. These epigenetic changes may be reversible with appropriate therapy. (Funded by the National Cancer Institute and others.)

Key Points Novel RARG-CPSF6 fusion in an AML case with promyelocytic features and no evidence of PML-RARA or X-RARA fusion. Gene fusions involving RARG can initiate AML with promyelocytic morphological features.

Background: A normal karyotype is the most common cytogenetic finding in acute myeloid leukemia cells ("NK-AML") and has highly variable and difficult to predict outcomes at presentation. Refinements introduced with the ELN 2017 Criteria (presence or absence of mutations in NPM1, FLT3-ITD, and/or biallelic CEBPA mutations) have improved--but not completely solved--up-front risk classification. NK-AML patients with very long first remissions after chemotherapy only are rare, and their genetic and epigenetic characteristics have not yet been definitively evaluated, but could potentially provide clues regarding determinants that affect chemotherapy responses. Results: Using exome sequencing, RNA-sequencing, and functional studies, we performed an in-depth characterization of 28 de novo adult NK-AML patients with >5 year, Long First Remissions (LFR) after chemotherapy only (7+3 with HiDAC consolidation). As comparators, we studied a well-matched group of 31 NK-AML patients who relapsed within 2 years (Standard First Remissions, SFR). None of the patients in either group received an allogeneic transplant in first remission. Bulk RNA sequencing and targeted investigation of cell type proportions and pathways expected to be relevant for chemotherapy sensitivity (i.e. cell cycle, DNA repair, or AraC responsiveness) revealed no significant differences between LFR and SFR AML samples. However, single cell RNA sequencing (sc-RNAseq) analyses of genetically-defined AML cells from 8 LFR and 7 SFR cases showed that SFR AML cells expressed significantly higher levels of stem cell markers, and of several immunomodulatory genes (MHC class II, CD200, MRC1). Combined sc-RNAseq and flow cytometry analyses of the AML bone marrow samples from LFR patients revealed well-preserved CD4 and CD8 populations that responded robustly to TCR-mediated activation, achieved through CD3/28 bead stimulation. In contrast, SFR AML marrows had reduced numbers of immune cells (particularly of CD4 Th1 cells) and their residual T cells expressed an exhaustion signature. TCR-mediated T cell activation was blocked in the SFR samples by the presence of the AML cells in the bone marrow samples: removing the AML cells released this blockade. Evaluation of T cell immune checkpoints and their putative ligands revealed LAG3 to be a leading candidate for immune suppression, because of its high expression in SFR T cells, and because the genes encoding its major ligands (MHC Class II) were highly expressed in SFR AML cells. Indeed, blocking the inhibitory receptor LAG-3 was able to reverse the AML cell-mediated T cell suppression of most SFR cases. To validate and extend these findings, we performed the T cell activation assay in an independent cohort of 50 AML cases representing all ELN risk groups. The ability of marrow derived CD4+ T cells to activate correlated strongly with ELN groups. Importantly, for the ELN intermediate patients, the immune activation assay stratified these patients in 2 groups with very different relapse free survival (RFS): the T cell "activators" had an RFS of 1200 days, while the "non-activators" had RFS of 146 days (Mantel-Cox Log-rank = 0.02). A multivariate analysis of covariates at diagnosis, including sex, age, ELN risk category, WBC count, bone marrow blast count at presentation, and mutation status (for DNMT3A, NPM1, FLT3, NRAS, WT1, TET2, CEBPA, PTPN11, IDH1, IDH2, RUNX1, RAD21, SMC1A, and SRSF2) identified none that were predictive of CD4 T cell activation status at presentation. Conclusions: These data strongly suggest that most AML cases have an immunosuppressive phenotype at presentation, mediated by inhibitory T cell receptors (e.g. LAG3) via ligands that are expressed by AML cells (e.g. MHC Class II proteins). The absence of this immunosuppressive phenotype was strongly correlated with favorable clinical outcomes with standard chemotherapy. The ability of CD4 T cells to be activated via the TCR in the presence of AML cells at presentation (which indirectly measures AML cell-mediated immunosuppression) is a novel biomarker for risk assessment that is currently not captured by any other well-recognized covariate (especially for ELN intermediate risk cases), and should be further evaluated for its predictive potential in prospective clinical trials. Disclosures Eisfeld: Karyopharm (spouse): Current Employment. Retting: BioLineRx Ltd.: Research Funding. Uy: GlaxoSmithKline: Consultancy; Agios: Consultancy; Novartis: Consultancy; Genentech: Consultancy; AbbVie: Consultancy; Macrogenics: Research Funding; Astellas: Honoraria, Speakers Bureau; Jazz: Consultancy. Spencer: Wugen, Inc.: Consultancy, Other: Stock options.

Background There is growing evidence that genomics-based assessment of persistent molecular disease (PMD) may be a useful risk stratification tool for patients with acute myeloid leukemia (AML). However, there is no consensus about the optimal approach for detection and monitoring of leukemia-associated mutations (LAMs) for PMD testing. One approach is whole exome sequencing (WES), which provides a comprehensive assessment of the clonal architecture by identifying LAMs across the exome at diagnosis, which can then be measured in follow-up samples. Another approach is to use highly sensitive targeted gene sequencing (TGS) to detect persistent LAMs even at very low levels, although detection is limited to genes interrogated by the panel. Although both of approaches have been shown to predict outcome in retrospective studies, there are substantial differences in the number of LAMs identified and the limit of detection, and a head-to-head comparison has not yet been reported. Here, we use a defined cohort enrolled in a prospective clinical trial to compare PMD results from deep WES to error-corrected TGS. Methods Cohort: Patients were age 18-60 with de novo AML classified as intermediate risk by European Leukemia Net criteria, who achieved a morphologic remission after undergoing standard induction therapy. All patients were enrolled in a prospective clinical trial (NCT02756962). Sequencing: Deep WES was performed using DNA from normal tissue (buccal swab or skin) and pre- and post-induction (~day 30) bone marrow (BM) samples to achieve an average coverage depth of ~600x. LAMs identified via paired tumor/normal analysis of the pre-induction sample were queried in the post-induction sample for assessment of PMD. PMD testing by TGS was performed using error-corrected sequencing of 40 genes recurrently mutated AML genes to an average error-corrected coverage depth of ~4500x. Definitions: Based on previously published work, we used two separate variant allele frequency (VAF) cutoffs to define PMD. For WES, PMD+ was defined as ≥1 LAM with a VAF >2.5% (Klco JAMA 2015). For TGS, PMD+ was defined as having ≥1 LAM with a VAF >0.5% (Duncavage NEJM 2018). LAMs were sub-classified per Table 1. TGS mutations not identified as LAMs by WES at diagnosis were excluded from the primary analysis. Results 31 patients were studied. LAMs are summarized in Table 1. 20 patients (65%) were PMD+ by WES after induction, and 22 patients (71%) were PMD+ by TGS. The concordance between WES and TGS was 81% (25/31) (Table 2). Two patients were PMD+ by WES only, due to the persistence of LAMs in the exome space, but not in the targeted panel. Four patients were PMD+ by TGS only, due to the presence of recurrent mutations at VAFs below the detection limit of WES (range: 0.59 - 1.90%). Two patients were PMD+ by both assays, but due to different mutations. All other patients who were PMD+ by both assays had at least one overlapping mutation. Analysis of the mutations that persisted after therapy in both assays showed that 26% of patients (8/31) were PMD+ by TGS because of mutations in DNMT3A, TET2, or ASXL1 (i.e., "DTA" mutations). All of these patients were also PMD+ by WES, with 7 of the 8 patients having ≥1 additional non-DTA mutation (median: 3, range: 1 - 7). In an exploratory analysis, 22 additional mutations in 13 patients were identified by TGS that were not detected by WES on the diagnostic sample. This included two patients who were PMD-, but who had new mutations in TET2 and DNMT3A, respectively, likely representing selection for ancestral clones that were unrelated to the AML founding clone. Conclusion Concordance between WES and TGS-based PMD assessment was high. Discordant results were generally driven by non-recurrent mutations detected by WES, and low-level mutations detected by the high coverage, error-corrected TGS. Although isolated DTA mutations were common on TGS, WES analysis showed additional LAMs accompanied these variants in most cases, indicating the persistence of an ancestral leukemic clone that may provide useful prognostic information. We also observed new, low-level mutations that emerged after therapy in 42% of patients, some of which were not part of the leukemic clone identified at diagnosis. This indicates that use of highly sensitive PMD approaches may be challenging without pre-induction mutation testing, which is required to understand the relevance of markers of persistent molecular disease. Disclosures Jacoby: AbbVie: Research Funding; Takeda: Consultancy; Jazz Pharmaceuticals: Research Funding.

Background: Previous studies indicate that mutations in signaling (e.g., receptor tyrosine kinases and RAS pathway members) and transcription factor genes are more common in secondary acute myeloid leukemia (sAML) than myelodysplastic syndrome (MDS), suggesting a role in disease progression. However, our understanding of the timing and order of mutation acquisition in these genes remains incomplete without analyzing paired MDS and sAML samples from the same patient. Defining the role of signaling gene mutations during progression should provide biologic insight into clonal evolution and help define prognostic markers for MDS progression. Methods: We banked paired MDS and sAML (and matched skin) samples from 44 patients (median time to progression: 306 days, range 21-3568). We sequenced 44 sAML (+ skin) samples for 285 recurrently mutated genes (RMGs) and 12 samples were selected for enhanced whole genome sequencing (eWGS, genome with deep exome coverage) of MDS and sAML samples (+ skin) to determine clonal hierarchy. Somatic mutations in these 12 samples were validated with high coverage error-corrected sequencing, and clonality was defined in MDS and sAML samples using mutation variant allele frequencies (VAFs). Additionally, error-corrected sequencing for all sAML RMG mutations, plus 40 additional genes, was performed on 43 of the MDS samples. Single cell DNA sequencing (scDNAseq, Mission Bio) was performed on 6 samples. Results: We identified 32 signaling gene mutations in 15 of the 44 sAML samples, with only 11 of 32 mutations (34%) detected in the initial, paired MDS sample (limit of detection; We next asked if low-level ( Finally, we observed that several MDS (n=6) and sAML (n=10) samples had multiple signaling gene mutations, and it was not always clear whether they occurred in the same subclone. We performed scDNAseq of 6 sAML samples with multiple signaling gene mutations (2-4/case). In 5 of 6 cases the signaling gene mutations did not occur in the same subclone. One sample contained 2 subclones with a NRAS and a PTPN11 mutation, with a separate subclone harboring an additional NRAS mutation. In sum, the co-occurrence of two signaling gene mutations in the same subclone is rare, indicating that the presence of multiple signaling gene mutations may be functionally redundant or detrimental to leukemia cells. Conclusions: Rare cells containing signaling gene mutations are present in nearly half of MDS patients who progress to sAML. The high frequency of signaling gene mutations and diverse patterns of clonal evolution (including the loss of one mutation and acquisition of another), suggest that signaling genes are a major driver of progression to sAML. The paucity of subclones with multiple signaling gene mutations suggests a therapeutic vulnerability for mutant cells. Disclosures DiPersio: Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Jacoby:AbbVie: Research Funding; Jazz Pharmaceuticals: Research Funding.

Item does not contain fulltext OBJECTIVE: In many rheumatoid arthritis (RA) patients, disease is controlled with anti-tumor necrosis factor (anti-TNF) biologic therapies. However, in a significant number of patients, the disease fails to respond to anti-TNF therapy. We undertook the present study to examine the hypothesis that rare and low-frequency genetic variants might influence response to anti-TNF treatment. METHODS: We sequenced the coding region of 750 genes in 1,094 RA patients of European ancestry who were treated with anti-TNF. After quality control, 690 genes were included in the analysis. We applied single-variant association and gene-based association tests to identify variants associated with anti-TNF treatment response. In addition, given the key mechanistic role of TNF, we performed gene set analyses of 27 TNF pathway genes. RESULTS: We identified 14,420 functional variants, of which 6,934 were predicted as nonsynonymous 2,136 of which were further predicted to be "damaging." Despite the fact that the study was well powered, no single variant or gene showed study-wide significant association with change in the outcome measures disease activity or European League Against Rheumatism response. Intriguingly, we observed 3 genes, of 27 with nominal signals of association (P < 0.05), that were involved in the TNF signaling pathway. However, when we performed a rigorous gene set enrichment analysis based on association P value ranking, we observed no evidence of enrichment of association at genes involved in the TNF pathway (Penrichment = 0.15, based on phenotype permutations). CONCLUSION: Our findings suggest that rare and low-frequency protein-coding variants in TNF signaling pathway genes or other genes do not contribute substantially to anti-TNF treatment response in patients with RA.

To the editor: Quantitative response evaluation in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDSs) relies on the morphologic quantification of bone marrow (BM) blasts. This process is subject to the operator-dependent quality of BM collection and the interobserver variability

BackgroundThe molecular determinants of clinical responses to decitabine therapy in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) are unclear. MethodsWe enrolled 84 adult patients with AML or MDS in a single-institution trial of decitabine to identify somatic mutations and their relationships to clinical responses. Decitabine was administered at a dose of 20 mg per square meter of body-surface area per day for 10 consecutive days in monthly cycles. We performed enhanced exome or gene-panel sequencing in 67 of these patients and serial sequencing at multiple time points to evaluate patterns of mutation clearance in 54 patients. An extension cohort included 32 additional patients who received decitabine in different protocols. ResultsOf the 116 patients, 53 (46%) had bone marrow blast clearance (

Traditional response criteria in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are based on bone marrow morphology and may not accurately reflect clonal tumor burden in patients treated with non-cytotoxic chemotherapy. We used next-generation sequencing of serial bone marrow samples to monitor MDS and AML tumor burden during treatment with epigenetic therapy (decitabine and panobinostat). Serial bone marrow samples (and skin as a source of normal DNA) from 25 MDS and AML patients were sequenced (exome or 285 gene panel). We observed that responders, including those in complete remission (CR), can have persistent measurable tumor burden (that is, mutations) for at least 1 year without disease progression. Using an ultrasensitive sequencing approach, we detected extremely rare mutations (equivalent to 1 heterozygous mutant cell in 2000 non-mutant cells) months to years before their expansion at disease relapse. While patients can live with persistent clonal hematopoiesis in a CR or stable disease, ultimately we find evidence that expansion of a rare subclone occurs at relapse or progression. Here we demonstrate that sequencing of serial samples provides an alternative measure of tumor burden in MDS or AML patients and augments traditional response criteria that rely on bone marrow blast percentage.

Familial clustering of myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML) can be caused by inherited factors. We screened 59 individuals from 17 families with 2 or more biological relatives with MDS/AML for variants in 12 genes with established roles in predisposition to MDS/AML, and identified a pathogenic germ line variant in 5 families (29%). Extending the screen with a panel of 264 genes that are recurrently mutated in de novo AML, we identified rare, nonsynonymous germ line variants in 4 genes, each segregating with MDS/AML in 2 families. Somatic mutations are required for progression to MDS/AML in these familial cases. Using a combination of targeted and exome sequencing of tumor and matched normal samples from 26 familial MDS/AML cases and asymptomatic carriers, we identified recurrent frameshift mutations in the cohesin-associated factor PDS5B, co-occurrence of somatic ASXL1 mutations with germ line GATA2 mutations, and recurrent mutations in other known MDS/AML drivers. Mutations in genes that are recurrently mutated in de novo AML were underrepresented in the familial MDS/AML cases, although the total number of somatic mutations per exome was the same. Lastly, clonal skewing of hematopoiesis was detected in 67% of young, asymptomatic RUNX1 carriers, providing a potential biomarker that could be used for surveillance in these high-risk families.

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation is the only curative treatment for patients with myelodysplastic syndrome (MDS). The molecular predictors of disease progression after transplantation are unclear. METHODS: We sequenced bone marrow and skin samples from 90 adults with MDS who underwent allogeneic hematopoietic stem-cell transplantation after a myeloablative or reduced-intensity conditioning regimen. We detected mutations before transplantation using enhanced exome sequencing, and we evaluated mutation clearance by using error-corrected sequencing to genotype mutations in bone marrow samples obtained 30 days after transplantation. In this exploratory study, we evaluated the association of a mutation detected after transplantation with disease progression and survival. RESULTS: Sequencing identified at least one validated somatic mutation before transplantation in 86 of 90 patients (96%); 32 of these patients (37%) had at least one mutation with a maximum variant allele frequency of at least 0.5% (equivalent to 1 heterozygous mutant cell in 100 cells) 30 days after transplantation. Patients with disease progression had mutations with a higher maximum variant allele frequency at 30 days than those who did not (median maximum variant allele frequency, 0.9% vs. 0%; P

Targeted sequencing informs the evaluation of normal karyotype cytopenic patients for low-grade myelodysplastic syndrome

DNMT3A mutations occur in ~25% of acute myeloid leukemia (AML) patients. The most common mutation, DNMT3AR882H, has dominant negative activity that reduces DNA methylation activity by ~80% in vitro. To understand the contribution of DNMT3A-dependent methylation to leukemogenesis, we performed whole-genome bisulfite sequencing of primary leukemic and non-leukemic cells in patients with or without DNMT3AR882 mutations. Non-leukemic hematopoietic cells with DNMT3AR882H displayed focal methylation loss, suggesting that hypomethylation antedates AML. Although virtually all AMLs with wild-type DNMT3A displayed CpG island hypermethylation, this change was not associated with gene silencing, and was essentially absent in AMLs with DNMT3AR882 mutations. Primary hematopoietic stem cells expanded with cytokines were hypermethylated in a DNMT3A-dependent manner, suggesting that hypermethylation may be a response to, rather than a cause of, cellular proliferation. Our findings suggest that hypomethylation is an initiating phenotype in AMLs with DNMT3AR882, while DNMT3A-dependent CpG island hypermethylation is a consequence of AML progression.

SummaryThe relationships between clonal architecture and functional heterogeneity in acute myeloid leukemia (AML) samples are not yet clear. We used targeted sequencing to track AML subclones identified by whole-genome sequencing using a variety of experimental approaches. We found that virtually all AML subclones trafficked from the marrow to the peripheral blood, but some were enriched in specific cell populations. Subclones showed variable engraftment potential in immunodeficient mice. Xenografts were predominantly comprised of a single genetically defined subclone, but there was no predictable relationship between the engrafting subclone and the evolutionary hierarchy of the leukemia. These data demonstrate the importance of integrating genetic and functional data in studies of primary cancer samples, both in xenograft models and in patients.

The persistence of leukemic mutation(s) in AML patients who have achieved a morphologic complete remission (CR) after intensive induction chemotherapy is a strong predictor of early relapse and reduced overall survival (OS) (Klco JAMA, 2015; Morita, J Clin Oncol 2018; Jongen-Lavrencic, NEJM, 2018). There is no clinical consensus as to the optimal consolidation therapy for the ~50% of patients with intermediate-risk AML. The median relapse-free survival (RFS) for patients ≤60 years with ELN intermediate-risk disease is 0.8 years to 1.2 years, with a median OS of 1.2-2.1 years (Mrozek, J Clin Oncol, 2012). We have shown that intermediate-risk patients who clear all leukemia-associated mutations (LAMs) to a variant allele fraction (VAF) of Here, we report the development of a pipeline to prospectively determine the persistence of LAMs after remission-induction, and return results in a clinically actionable time-frame. We perform enhanced exome sequencing (EES) of paired skin or buccal swab (normal tissue) and bone marrow DNA to comprehensively identify all LAMs at diagnosis (Day 0) and to assess their clearance post-induction (~Day 30). EES data are generated using a CLIA-compliant assay in the CLIA-licensed environment (CLE) lab at the McDonnell Genome Institute, and results are returned to the treating physician. Intermediate risk patients ≤60 years with clearance of all LAMs (VAFs For the 23 patients sequenced to date, the mean turnaround time to issue sequencing results to the treating physician was 24 days from the time of the remission biopsy. All 23 patients had detectable LAMs at presentation (mean 28 per patient, range, 6 to 43) that could be used to track persistent disease in the day 30 remission sample. Eleven patients (48%) cleared all LAMs and received HiDAC only (Cohort A). There was no flow cytometric evidence of residual AML in Cohort A. Twelve patients (52%) had persistent LAMs (Cohort B, investigator's choice). The number of persistent leukemia-associated variants present in Cohort B ranged between 1 and 14. Surprisingly, 9 of the 12 patients with persistent LAMs by sequencing had no flow cytometric evidence of residual leukemia. Seven of 12 patients on the investigator's choice arm have received an alloHCT, and none have relapsed to date. The median follow-up for all subjects is 378 days (range, 59-683). Neither the median RFS (Fig. 1A) nor the median OS (Fig. 1B) has been reached for either cohort. While preliminary, these results suggest that patients who clear all LAMs to a VAF of In summary, identifying persistent LAMs after induction chemotherapy is feasible in an actionable time-frame. Early data suggest that using LAM clearance post-induction may improve current risk-stratification for intermediate-risk AML. Accrual of patients and continued follow-up are ongoing. Disclosures Jacoby: NovoNordisk: Consultancy; Celgene: Speakers Bureau. Loken:Hematologics, Inc: Employment, Equity Ownership. Schroeder:Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Uy:GlycoMimetics: Consultancy; Curis: Consultancy. Vij:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: 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, Research Funding. Kahl:Gilead: Consultancy; AstraZeneca: Consultancy; Genentech: Consultancy; CTI: Consultancy; ADC Therapeutics: Consultancy; Abbvie: Consultancy; Seattle Genetics: Consultancy; Acerta: Consultancy; Juno: Consultancy; Celgene: Consultancy.

We performed an integrated genomic, transcriptomic and proteomic characterization of 373 endometrial carcinomas using array- and sequencing-based technologies. Uterine serous tumours and ~25% of high-grade endometrioid tumours had extensive copy number alterations, few DNA methylation changes, low oestrogen receptor/progesterone receptor levels, and frequent TP53 mutations. Most endometrioid tumours had few copy number alterations or TP53 mutations, but frequent mutations in PTEN, CTNNB1, PIK3CA, ARID1A and KRAS and novel mutations in the SWI/SNF chromatin remodelling complex gene ARID5B. A subset of endometrioid tumours that we identified had a markedly increased transversion mutation frequency and newly identified hotspot mutations in POLE. Our results classified endometrial cancers into four categories: POLE ultramutated, microsatellite instability hypermutated, copy-number low, and copy-number high. Uterine serous carcinomas share genomic features with ovarian serous and basal-like breast carcinomas. We demonstrated that the genomic features of endometrial carcinomas permit a reclassification that may affect post-surgical adjuvant treatment for women with aggressive tumours., National Institutes of Health (U.S.) (Grant 5U24CA143799-04), National Institutes of Health (U.S.) (Grant 5U24CA143835-04), National Institutes of Health (U.S.) (Grant 5U24CA143840-04), National Institutes of Health (U.S.) (Grant 5U24CA143843-04), National Institutes of Health (U.S.) (Grant 5U24CA143845-04), National Institutes of Health (U.S.) (Grant 5U24CA143848-04), National Institutes of Health (U.S.) (Grant 5U24CA143858-04), National Institutes of Health (U.S.) (Grant 5U24CA143866-04), National Institutes of Health (U.S.) (Grant 5U24CA143867-04), National Institutes of Health (U.S.) (Grant 5U24CA143882-04), National Institutes of Health (U.S.) (Grant 5U24CA143883-04), National Institutes of Health (U.S.) (Grant 5U24CA144025-04), National Institutes of Health (U.S.) (Grant U54HG003067-11), National Institutes of Health (U.S.) (Grant U54HG003079-10), National Institutes of Health (U.S.) (Grant U54HG003273-10)

We report the findings from a patient who presented with a concurrent mediastinal germ cell tumor (GCT) and acute myeloid leukemia (AML). Bone marrow pathology was consistent with a diagnosis of acute megakaryoblastic leukemia (AML M7), and biopsy of an anterior mediastinal mass was consistent with a nonseminomatous GCT. Prior studies have described associations between hematological malignancies, including AML M7 and nonseminomatous GCTs, and it was recently suggested that a common founding clone initiated both cancers. We performed enhanced exome sequencing on the GCT and the AML M7 from our patient to define the clonal relationship between the two cancers. We found that both samples contained somatic mutations in PTEN (C136R missense) and TP53 (R213 frameshift). The mutations in PTEN and TP53 were present at ∼100% variant allele frequency (VAF) in both tumors. In addition, we detected and validated five other shared somatic mutations. The copy-number analysis of the AML exome data revealed an amplification of Chromosome 12p. We also identified a heterozygous germline variant in FANCA (S858R), which is known to be associated with Fanconi anemia but is of uncertain significance here. In summary, our data not only support a common founding clone for these cancers but also suggest that a specific set of distinct genomic alterations (in PTEN and TP53) underlies the rare association between GCT and AML. This association is likely linked to the treatment resistance and extremely poor outcome of these patients. We cannot resolve the clonal evolution of these tumors given limitations of our data.

A variety of microbial communities and their genes (the microbiome) exist throughout the human body, with fundamental roles in human health and disease. The National Institutes of Health (NIH)-funded Human Microbiome Project Consortium has established a population-scale framework to develop metagenomic protocols, resulting in a broad range of quality-controlled resources and data including standardized methods for creating, processing and interpreting distinct types of high-throughput metagenomic data available to the scientific community. Here we present resources from a population of 242 healthy adults sampled at 15 or 18 body sites up to three times, which have generated 5,177 microbial taxonomic profiles from 16S ribosomal RNA genes and over 3.5 terabases of metagenomic sequence so far. In parallel, approximately 800 reference strains isolated from the human body have been sequenced. Collectively, these data represent the largest resource describing the abundance and variety of the human microbiome, while providing a framework for current and future studies.

Alterations in DNA methylation have been implicated in the pathogenesis of myelodysplastic syndromes (MDS), although the underlying mechanism remains largely unknown. Methylation of CpG dinucleotides is mediated by DNA methyltransferases, including DNMT1, DNMT3A and DNMT3B. DNMT3A mutations have recently been reported in patients with de novo acute myeloid leukemia (AML), providing a rationale for examining the status of DNMT3A in MDS samples. In this study, we report the frequency of DNMT3A mutations in patients with de novo MDS, and their association with secondary AML. We sequenced all coding exons of DNMT3A using DNA from bone marrow and paired normal cells from 150 patients with MDS and identified 13 heterozygous mutations with predicted translational consequences in 12/150 patients (8.0%). Amino acid R882, located in the methyltransferase domain of DNMT3A, was the most common mutation site, accounting for 4/13 mutations. DNMT3A mutations were expressed in the majority of cells in all tested mutant samples regardless of myeloblast counts, suggesting that DNMT3A mutations occur early in the course of MDS. Patients with DNMT3A mutations had worse overall survival compared with patients without DNMT3A mutations (P=0.005) and more rapid progression to AML (P=0.007), suggesting that DNMT3A mutation status may have prognostic value in de novo MDS.

BACKGROUND The genetic alterations responsible for an adverse outcome in most patients with acute myeloid leukemia (AML) are unknown. METHODS Using massively parallel DNA sequencing, we identified a somatic mutation in DNMT3A, encoding a DNA methyltransferase, in the genome of cells from a patient with AML with a normal karyotype. We sequenced the exons of DNMT3A in 280 additional patients with de novo AML to define recurring mutations. RESULTS A total of 62 of 281 patients (22.1%) had mutations in DNMT3A that were predicted to affect translation. We identified 18 different missense mutations, the most common of which was predicted to affect amino acid R882 (in 37 patients). We also identified six frameshift, six nonsense, and three splice-site mutations and a 1.5-Mbp deletion encompassing DNMT3A. These mutations were highly enriched in the group of patients with an intermediate-risk cytogenetic profile (56 of 166 patients, or 33.7%) but were absent in all 79 patients with a favorable-risk cytogenetic profile (P

Importance Tests that predict outcomes for patients with acute myeloid leukemia (AML) are imprecise, especially for those with intermediate risk AML. Objectives To determine whether genomic approaches can provide novel prognostic information for adult patients with de novo AML. Design, Setting, and Participants Whole-genome or exome sequencing was performed on samples obtained at disease presentation from 71 patients with AML (mean age, 50.8 years) treated with standard induction chemotherapy at a single site starting in March 2002, with follow-up through January 2015. In addition, deep digital sequencing was performed on paired diagnosis and remission samples from 50 patients (including 32 with intermediate-risk AML), approximately 30 days after successful induction therapy. Twenty-five of the 50 were from the cohort of 71 patients, and 25 were new, additional cases. Exposures Whole-genome or exome sequencing and targeted deep sequencing. Risk of identification based on genetic data. Main Outcomes and Measures Mutation patterns (including clearance of leukemia-associated variants after chemotherapy) and their association with event-free survival and overall survival. Results Analysis of comprehensive genomic data from the 71 patients did not improve outcome assessment over current standard-of-care metrics. In an analysis of 50 patients with both presentation and documented remission samples, 24 (48%) had persistent leukemia-associated mutations in at least 5% of bone marrow cells at remission. The 24 with persistent mutations had significantly reduced event-free survival vs the 26 who cleared all mutations (median [95% CI]: 6.0 months [95% CI, 3.7-9.6] for persistent mutations vs 17.9 months [95% CI, 11.3-40.4] for cleared mutations, log-rank P P P = .003; HR, 2.86 [95% CI, 1.39-5.88], P = .004). Among the 32 patients with intermediate cytogenetic risk, the 14 patients with persistent mutations had reduced event-free survival compared with the 18 patients who cleared all mutations (median [95% CI]: 8.8 months [95% CI, 3.7-14.6] for persistent mutations vs 25.6 months [95% CI, 11.4-not estimable] for cleared mutations, log-rank P = .003; HR, 3.32 [95% CI, 1.44-7.67], P = .005) and reduced overall survival (median [95% CI]: 19.3 months [95% CI, 7.5-42.3] for persistent mutations vs 46.8 months [95% CI, 22.6-not estimable] for cleared mutations, log-rank P = .02; HR, 2.88 [95% CI, 1.11-7.45], P = .03). Conclusions and Relevance The detection of persistent leukemia-associated mutations in at least 5% of bone marrow cells in day 30 remission samples was associated with a significantly increased risk of relapse, and reduced overall survival. These data suggest that this genomic approach may improve risk stratification for patients with AML.

Summary Heterozygous somatic mutations in the spliceosome gene U2AF1 occur in ∼11% of patients with myelodysplastic syndromes (MDS), the most common adult myeloid malignancy. It is unclear how these mutations contribute to disease. We examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a doxycycline-inducible transgenic mouse model. Mice expressing mutant U2AF1(S34F) display altered hematopoiesis and changes in pre-mRNA splicing in hematopoietic progenitor cells by whole transcriptome analysis (RNA-seq). Integration with human RNA-seq datasets determined that common mutant U2AF1-induced splicing alterations are enriched in RNA processing genes, ribosomal genes, and recurrently mutated MDS and acute myeloid leukemia-associated genes. These findings support the hypothesis that mutant U2AF1 alters downstream gene isoform expression, thereby contributing to abnormal hematopoiesis in patients with MDS.

Induced pluripotent stem cells (iPSCs) have tremendous potential as a tool for disease modeling, drug testing, and other applications. Since the generation of iPSCs "captures" the genetic history of the individual cell that was reprogrammed, iPSC clones (even those derived from the same individual) would be expected to demonstrate genetic heterogeneity. To assess the degree of genetic heterogeneity, and to determine whether some cells are more genetically "fit" for reprogramming, we performed exome sequencing on 24 mouse iPSC clones derived from skin fibroblasts obtained from two different sites of the same 8-week-old C57BL/6J male mouse. While no differences in the coding regions were detected in the two parental fibroblast pools, each clone had a unique genetic signature with a wide range of heterogeneity observed among the individual clones: a total of 383 iPSC variants were validated for the 24 clones (mean 16.0/clone, range 0-45). Since these variants were all present in the vast majority of the cells in each clone (variant allele frequencies of 40-60% for heterozygous variants), they most likely preexisted in the individual cells that were reprogrammed, rather than being acquired during reprogramming or cell passaging. We then tested whether this genetic heterogeneity had functional consequences for hematopoietic development by generating hematopoietic progenitors in vitro and enumerating colony forming units (CFUs). While there was a range of hematopoietic potentials among the 24 clones, only one clone failed to differentiate into hematopoietic cells; however, it was able to form a teratoma, proving its pluripotent nature. Further, no specific association was found between the mutational spectrum and the hematopoietic potential of each iPSC clone. These data clearly highlight the genetic heterogeneity present within individual fibroblasts that is captured by iPSC generation, and suggest that most of the changes are random, and functionally benign.

HOX genes are highly expressed in many acute myeloid leukemia (AML) samples, but the patterns of expression and associated regulatory mechanisms are not clearly understood. We analyzed RNA sequencing data from 179 primary AML samples and normal hematopoietic cells to understand the range of expression patterns in normal versus leukemic cells. HOX expression in AML was restricted to specific genes in the HOXA or HOXB loci, and was highly correlated with recurrent cytogenetic abnormalities. However, the majority of samples expressed a canonical set of HOXA and HOXB genes that was nearly identical to the expression signature of normal hematopoietic stem/progenitor cells (HSPCs). Transcriptional profiles at the HOX loci were similar between normal cells and AML samples, and involved bidirectional transcription at the center of each gene cluster. Epigenetic analysis of a subset of AML samples also identified common regions of chromatin accessibility in AML samples and normal CD34+ cells that displayed differences in methylation depending on HOX expression patterns. These data provide an integrated epigenetic view of the HOX gene loci in primary AML samples, and suggest that HOX expression in most AML samples represents a normal stem cell program that is controlled by epigenetic mechanisms at specific regulatory elements.

Human chromosome 7 has historically received prominent attention in the human genetics community, primarily related to the search for the cystic fibrosis gene and the frequent cytogenetic changes associated with various forms of cancer. Here we present more than 153 million base pairs representing 99.4% of the euchromatic sequence of chromosome 7, the first metacentric chromosome completed so far. The sequence has excellent concordance with previously established physical and genetic maps, and it exhibits an unusual amount of segmentally duplicated sequence (8.2%), with marked differences between the two arms. Our initial analyses have identified 1,150 protein-coding genes, 605 of which have been confirmed by complementary DNA sequences, and an additional 941 pseudogenes. Of genes confirmed by transcript sequences, some are polymorphic for mutations that disrupt the reading frame.

Summary Recent genomic analyses of pathologically defined tumor types identify "within-a-tissue" disease subtypes. However, the extent to which genomic signatures are shared across tissues is still unclear. We performed an integrative analysis using five genome-wide platforms and one proteomic platform on 3,527 specimens from 12 cancer types, revealing a unified classification into 11 major subtypes. Five subtypes were nearly identical to their tissue-of-origin counterparts, but several distinct cancer types were found to converge into common subtypes. Lung squamous, head and neck, and a subset of bladder cancers coalesced into one subtype typified by TP53 alterations, TP63 amplifications, and high expression of immune and proliferation pathway genes. Of note, bladder cancers split into three pan-cancer subtypes. The multiplatform classification, while correlated with tissue-of-origin, provides independent information for predicting clinical outcomes. All data sets are available for data-mining from a unified resource to support further biological discoveries and insights into novel therapeutic strategies.

A 77 year-old man initially presented in July 2013 with anemia, splenomegaly, and constitutional symptoms. A bone marrow biopsy revealed a hypercellular marrow with megakaryocytic hyperplasia and atypia and mild reticulin fibrosis, consistent with a diagnosis of primary myelofibrosis (PMF). Cytogenetics revealed a normal karyotype. JAK2 V617F testing was negative. Initiation of treatment with the JAK inhibitor ruxolitinib led to marked symptomatic improvement. The patient was then enrolled in a Phase 2 study with the anti-lysyl oxidase-like-2 (LOXL2) monoclonal antibody simtuzumab, administered via IV infusion every two weeks (while continuing ruxolitinib). He tolerated the simtuzumab infusions well initially, but with the 11th and 12th infusions experienced rigors, hypotension, and hypoxia. This occurred ~8 months after his initial PMF diagnosis. A repeat bone marrow biopsy revealed large aggregates of mast cells comprising 30-40% of the marrow cellularity (with 16% mast cells enumerated on the aspirate). A subset of the mast cells exhibited spindled morphology, and CD25 co-expression was demonstrated by flow cytometry in a subset of CD117-positive cells. Testing for the KITD816V mutation was positive. Tryptase levels were significantly elevated (375 ng/mL). These findings were consistent with a diagnosis of aggressive systemic mastocytosis with an associated hematologic non-mast cell lineage disorder (ASM-AHNMD). Compassionate-use approval for the KIT inhibitor midostaurin was obtained, and treatment with midostaurin (in addition to continuation of ruxolitinib) was initiated. The patient initially reported symptomatic improvement with midostaurin treatment, but after several months his symptoms began to worsen, with a corresponding increase in tryptase (875 ng/mL). A repeat bone marrow biopsy revealed overt evolution to mast cell leukemia (MCL) with > 90% mast cell involvement. Based on the presence of an IDH2mutation identified on a clinical next-generation sequencing assay, the patient was evaluated for a clinical trial with the mutant IDH2 inhibitor AG-221. Unfortunately, the patient decompensated and expired before he could enroll in the study. To identify contributing driver mutations and to delineate clonal hierarchy associated with disease initiation and progression in this unique case of PMF with concomitant ASM, exome sequencing was performed on serial samples obtained at the following disease stages:PMF diagnosis (pre-ruxolitinib) (Day 0)PMF on ruxolitinib (before ASM diagnosis) (Day 181)ASM diagnosis (post-anti-LOXL2 antibody, pre-midostaurin) (Day 394)Progression to MCL (on midostaurin) (Day 519)Matched skin (normal) sample Likely driver mutations in IDH2 and SRSF2 were identified at ~40-50% variant allele frequency (VAF) in all samples and were therefore likely present in the founding clone. The KIT D816V mutation was found at 23% VAF at Day 0, then ~40% VAF in all other samples, suggesting it was present in a daughter subclone of the IDH2/SRSF2-containing clone that became dominant over time with disease progression. These findings also suggest that targeting KIT with midostaurin would be unlikely to eradicate the founding clone. Rather, selective targeting of IDH2 and/or SRSF2 could potentially ameliorate both diseases (PMF and ASM/MCL). To provide pre-clinical evidence of the potential utility of targeting IDH2, peripheral blood mononuclear cells obtained at the time of ASM diagnosis were plated in liquid culture in the presence or absence of the mutant IDH2 inhibitor AGI-6780. After 14 days in culture, the differentiation status of the cultured cells was examined by mass cytometry (CyTOF). Treatment with AGI-6780 resulted in a marked enhancement of myeloid differentiation (denoted by CD15 and CD66b expression) along with a corresponding decrease in CD34+ progenitor cells. These effects were not seen in cells cultured in the absence of AGI-6780. These results are consistent with prior studies in acute myeloid leukemia indicating that the beneficial effects of mutant IDH2 inhibition are likely related to inducing differentiation of primitive cells. In summary, this study highlights the capacity of serial genomic analysis to define the clonal architecture that drives disease initiation and evolution, and to distinguish founding vs subclonal mutations to identify the most promising targets for therapeutic intervention. Disclosures Oh: Janssen: Research Funding; CTI: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding.

The myelodysplastic syndromes (MDS) are the most common cause of bone marrow failure in adults, with an incidence of 40,000 cases per year. Next-generation sequencing of candidate genes has led to major advances in the description of the genetic landscape of MDS, identifying recurrently mutated genes and cellular pathways involved in disease pathogenesis. However, use of targeted panels indicates more comprehensive, unbiased sequencing techniques may yet identify additional recurrently mutated genes or cellular pathways important in MDS. Diseases caused by defects in telomere maintenance (telomeropathies) are variable in clinical and genetic presentation but often involve bone marrow failure. We hypothesized that acquired mutations in telomerase maintenance genes may be a recurrent event in MDS. This is significant as identification of recurrent somatic mutations in telomerase maintenance genes would provide further insight into MDS pathogenesis and identify a potential druggable pathway for MDS patients, as novel agents targeting the telomerase pathway are currently in clinical development. First, we identified three adults who presented in middle age with MDS and idiopathic pulmonary fibrosis (IPF), two of which also had a family history of IPF. All three patients had shortened telomeres ( Table Table. Disclosures Jacoby: Sunesis: Research Funding; Quintiles: Consultancy; Celgene: Speakers Bureau.

Background: MDS is composed of a dominant founding clone and typically one or more subclones that descend from the founding clone. Using whole genome sequencing (WGS) of secondary AML patients who were initially diagnosed with MDS, we previously showed that AML progression always coincided with expansion of at least one subclone. However, this emergent subclone was not always detectable in the antecedent MDS samples using high-coverage sequencing. It is currently unclear whether these emerging secondary AML-specific clones represent new mutations acquired in cells during treatment or selection of a rare subclone that was below the level of detection in the diagnostic MDS sample. Identification of such pre-existing subclones at MDS diagnosis could provide important prognostic information. Using an ultra-sensitive, error-corrected sequencing approach coupled with high coverage depths, we sought to determine whether secondary AML-specific mutations could be detected in antecedent MDS samples. Methods: We studied 4 MDS patients who eventually progressed to secondary AML (mean 601 days to transformation; range 131-955) that had DNA from initial MDS banking, secondary AML, and skin (as a normal control). DNA from the secondary AML sample was whole genome (3 cases) or exome sequenced (1 case). Custom enrichment panels were designed to target somatic mutations identified in the secondary AML samples and used to sequence the prior MDS samples. All 4 MDS cases had at least one subclone present in the secondary AML sample that was not detected by standard panel-based re-sequencing of the initial MDS sample (539x average coverage). We designed custom molecular barcode-based reagents to perform high-sensitivity, error-corrected sequencing targeting mutations that were detected only at progression to secondary AML. These mutation sites were then sequenced to ultra-deep coverage in prior MDS samples. To determine the sensitivity and specificity of the assay, we first sequenced known dilutions of cell line DNA and showed a sensitivity of 75% to detect variant allele fractions (VAFs) of 0.06% (one mutant cell in 800 cells) while maintaining a specificity of >99.9%. Only reads with unique molecular barcodes that were seen at least three times and had >90% identical reads within a read family were considered. Samples were sequenced to an average total coverage depth of 187,000x (range 107k-474k fold) corresponding to 10,398 unique molecular barcodes of which 3,549 passed filtering (at least two unique read families were required to call a variant), resulting in a mean predicted maximum VAF sensitivity of 0.05% (one mutant cell in 1,000 cells). Results: Using error-corrected barcode sequencing all 4 MDS cases had a subset of mutations detected that were originally only detected in the secondary AML; the mean VAF of detected mutations was 0.25% (range 0.02% to 3.9%) in the antecedent MDS samples, corresponding to 1 mutant cell in 200 cells. However, not all of the 'secondary AML-specific' mutations were detected in MDS samples. On average, only 17% (range 10-39%) of the 'secondary AML-specific' subclone mutations were detected in each MDS sample. This suggests that while some mutations pre-exist in an ancestral subclone in the MDS sample, others are acquired during the intervening months to years between MDS and progression to secondary AML. As an example, in two patients who were given decitabine prior to secondary AML progression, mutations detected in the secondary AML subclone that were not detected in the antecedent MDS sample using error-corrected barcode sequencing were more frequently C->G transversions compared to commonly acquired C->T transitions seen in MDS samples (p=.005; Mantel-Haenszel test). Increased C->G transversions are associated with DNA damage induced by decitabine, suggesting that in these two patients an ancestral subclone pre-existed in MDS that acquired additional decitabine-induced mutations during treatment (i.e., C->G transversions). Conclusion: Collectively, the data indicate that very rare subclones pre-exist at MDS diagnosis. These pre-existing subclones can subsequently give rise to the dominant clone at progression to secondary AML. Additional mutations -- some induced by chemotherapy treatment --- are also acquired in ancestral subclones that pre-exist in MDS samples. The combination of subclonal mutations may ultimately contribute to disease progression. Disclosures Jacoby: Sunesis: Research Funding; Quintiles: Consultancy; Celgene: Speakers Bureau.

Background: MDS is a genetically complex, oligoclonal disease consisting of a founding clone and typically one or more subclones derived from the founding clone. Previously we have shown that in MDS patients treated with chemotherapy, a minor subclone present at diagnosis can expand during disease progression from MDS to secondary AML, highlighting the clinical implications of clonal heterogeneity. Whether a similar pattern of clonal evolution occurs in MDS patients that relapse following allogeneic hematopoietic stem cell transplant (alloHSCT) is not known. Methods: We identified 9 MDS patients who progressed after receiving either a myeloablative (n=3) or reduced-intensity (n=6) alloHSCT (median time to progression 309 days, range 98-881). We performed enhanced exome sequencing (EES) to define the clonal architecture of 23 tumor samples at the following clinical landmarks (with matched skin as a source of normal DNA): diagnosis, Results: Averaged sequencing coverage depth was 246x for tumors subjected to EES; 537x for validation sequencing, and 24,150x total and 5,180x unique for the ultra-deep sequencing. In all cases, we observed that mutations found in the diagnostic founding clone were always detected at relapse. However, using SNVs, INDELs, and copy number analysis, we show that the dominant clone at relapse was often derived from a population that was subclonal at presentation. We observed the following, non-mutually exclusive patterns of clonal evolution at relapse: i) A subclone expanded or emerged and became the dominant clone at relapse as compared to presentation (n=6). In 2 of these cases, the subclone contained mutations that were not detected at presentation even via ultra-deep sequencing. ii) A subclone was cleared with alloHSCT (defined as VAF Finally, we used ultra-deep sequencing to determine if mutations (i.e., tumor cells) could be detected at day 30 post-alloHSCT in 7 of the 8 patients with no evidence of disease, who had available data. Mutations were detected in 6 of 7 patients. The average detectable mutation VAF per patient was 0.37% (ranged from 0.04% to 0.93%)(i.e., 1 mutant cell in 135). Conclusion: Complex clonal dynamics and clonal evolution are observed at relapse post-alloHSCT for MDS. Although minor subclones rise and may become the dominant clone at relapse, mutations present in the dominant (i.e., founding) clone of the diagnostic MDS sample pre-alloHSCT are always detected at relapse. This is similar to the pattern of clonal evolution previously observed for MDS progression to secondary AML following chemotherapy. These observations have implications for targeted therapy and tumor burden monitoring. Ultra-deep sequencing can detect persistent or emerging mutations at early time-points post-alloHSCT that are associated with subsequent relapse. The predictive value of detecting persistent mutations early after post-alloHSCT merits testing in future studies. Disclosures Jacoby: Quintiles: Consultancy; Sunesis: Research Funding; Celgene: Speakers Bureau. DiPersio:Incyte Corporation: Research Funding.

Background: Composite lymphomas (CLs) describe the rare situation when two morphologically distinct lymphomas occur in the same patient. Prior studies have supported a shared origin of CLs based on shared chromosomal translocations and/or the presence of shared immunoglobulin rearrangements. We used exome sequencing to examine somatic events in two cases of CLs. We hypothesized that each lymphoma will have shared and unique mutations, consistent with the presence of a common lymphoma precursor. Further, we postulated that the shared mutations represent potential initiating events in lymphomagenesis. Finally, because each case includes a Hodgkin (HL) and non-Hodgkin lymphoma (NHL), we hypothesize that the somatic mutations found in HL may offer insight into genomic drivers of HL. Clinical Synopsis:Case 1 was a 57-year-old man who presented with abdominal pain associated with a small bowel obstruction (SBO) and lymphadenopathy (LAD). Pathology showed DLBCL (diffuse large B-cell lymphoma, non-germinal center subtype; stage IV). He was treated with 6 cycles of R-CHOP with complete response. Seven months after the completion of R-CHOP, he presented with left neck LAD. An excisional biopsy revealed classical HL (stage II). Hodgkin and Reed-Sternberg (HRS) cells comprised 10% of the sample. He was treated with 3 cycles of ABVD and involved-field radiation therapy. Case 2 was a 66-year-old man with a history of solitary LAD who presented with increasing LAD, night sweats, and a chest wall mass. An excisional biopsy revealed both follicular lymphoma (FL; grade I-II) and classical HL in distinct areas of the lymph node. HRS cells comprised 30% of the sample. FISH studies were positive for t(14;18). He was treated with ABVD for 6 cycles with partial response. Upon progression, a repeat lymph node biopsy showed persistent HL. He was treated with bendamustine and rituximab for 4 cycles with progressive disease. Methods: We performed whole exome sequencing on formalin fixed paraffin embedded tumor samples and matched normal skin samples. We used our standard somatic variant calling pipeline to call somatic variants. SNV (single nucleotide variant) and indel calls were filtered for basic quality metrics and, using in-house software, were processed through a Bayesian classifier to remove false positive somatic events. Filtered variants were manually reviewed to further verify somatic status. Somatic variants were validated using Ampliseq in Case 1. Results: Sequencing resulted in >90% of the target regions with at least 20x in all samples. The mean depth of the NHL and normal samples in Case 1 was >65x. To account for the low malignant tumor cellularity of HL, the HL sample of Case 1 was sequenced to a mean depth of 310x. The mean depth for all samples in Case 2 was >100x (FL = 188x; normal = 104x; HL = 215x). After filtering, we identified 60 and 133 variants in 57 and 101 genes in Case 1 and Case 2, respectively. In Case 1, we identified three sites inTP53, TNFRSF14, and RASAL2 that were shared. We also identified variants in HIST1H2AG, KMT2D, and STAT3 unique to the DLBCL sample, and two mutations in PTPRT unique to the HL sample of Case 1 (Figure 1). In Case 2, we identified shared variants in TNFRSF14 and HIST1H2BF. We also identified two distinct BCL10 mutations in the FL and HL samples of Case 2 (Figure 1). Conclusions: From the shared somatic mutations identified, we infer that a shared lymphoma precursor for each case is likely, and the shared mutations may be early initiating events. In both cases, a shared nonsense mutation was found at TNFRSF14. TNFRSF14 is recurrently mutated in NHL, and recurrent deletions involving TNFRSF14 have been described in HL. Further work is needed to understand how TNFRSF14 alterations drive HL vs. NHL. Finally, it is intriguing that the HL samples contain independent mutations in PTPRT and BCL10. PTPRT is a known cancer gene, and BCL10 has been shown to be associated with the pathogenesis of B-cell NHLs. These data provide new hypotheses for loci involved in HL pathogenesis. Figure 1 Variant allele frequencies (VAFs) in CL samples from Case 1 (A) and Case 2 (B). (A) shows the VAFs of confirmed variants in the DLBCL vs. HL. (B) shows VAFs of FL vs. HL. Variants of note are highlighted in black. The lack of separation of sites in Case 2 may be the result of some admixture of the two lymphomas, which were sequenced from cores taken from the same lymph node. Figure 1. Variant allele frequencies (VAFs) in CL samples from Case 1 (A) and Case 2 (B). (A) shows the VAFs of confirmed variants in the DLBCL vs. HL. (B) shows VAFs of FL vs. HL. Variants of note are highlighted in black. The lack of separation of sites in Case 2 may be the result of some admixture of the two lymphomas, which were sequenced from cores taken from the same lymph node. Disclosures No relevant conflicts of interest to declare.

The Cancer Genome Atlas (TCGA) Research Network has profiled and analyzed large numbers of human tumors to discover molecular aberrations at the DNA, RNA, protein and epigenetic levels. The resulting rich data provide a major opportunity to develop an integrated picture of commonalities, differences and emergent themes across tumor lineages. The Pan-Cancer initiative compares the first 12 tumor types profiled by TCGA. Analysis of the molecular aberrations and their functional roles across tumor types will teach us how to extend therapies effective in one cancer type to others with a similar genomic profile., National Cancer Institute (U.S.), National Human Genome Research Institute (U.S.)

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome, an acquired clonal abnormality resulting from translocation of chromosomes 9 and 22, and the generation of the BCR–ABL fusion oncogene. The development of tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of CML, as TKI therapy leads to inhibition of BCR–ABL activity, suppression of the BCR–ABL-containing clone and restoration of normal hematopoiesis in the vast majority of cases.

The myelodysplastic syndromes are a group of hematologic disorders that often evolve into secondary acute myeloid leukemia (AML). The genetic changes that underlie progression from the myelodysplastic syndromes to secondary AML are not well understood.We performed whole-genome sequencing of seven paired samples of skin and bone marrow in seven subjects with secondary AML to identify somatic mutations specific to secondary AML. We then genotyped a bone marrow sample obtained during the antecedent myelodysplastic-syndrome stage from each subject to determine the presence or absence of the specific somatic mutations. We identified recurrent mutations in coding genes and defined the clonal architecture of each pair of samples from the myelodysplastic-syndrome stage and the secondary-AML stage, using the allele burden of hundreds of mutations.Approximately 85% of bone marrow cells were clonal in the myelodysplastic-syndrome and secondary-AML samples, regardless of the myeloblast count. The secondary-AML samples contained mutations in 11 recurrently mutated genes, including 4 genes that have not been previously implicated in the myelodysplastic syndromes or AML. In every case, progression to acute leukemia was defined by the persistence of an antecedent founding clone containing 182 to 660 somatic mutations and the outgrowth or emergence of at least one subclone, harboring dozens to hundreds of new mutations. All founding clones and subclones contained at least one mutation in a coding gene.Nearly all the bone marrow cells in patients with myelodysplastic syndromes and secondary AML are clonally derived. Genetic evolution of secondary AML is a dynamic process shaped by multiple cycles of mutation acquisition and clonal selection. Recurrent gene mutations are found in both founding clones and daughter subclones. (Funded by the National Institutes of Health and others.).

The Human Microbiome Project will establish a reference data set for analysis of the microbiome of healthy adults by surveying multiple body sites from 300 people and generating data from over 12,000 samples. To characterize these samples, the participating sequencing centers evaluated and adopted 16S rDNA community profiling protocols for ABI 3730 and 454 FLX Titanium sequencing. In the course of establishing protocols, we examined the performance and error characteristics of each technology, and the relationship of sequence error to the utility of 16S rDNA regions for classification- and OTU-based analysis of community structure. The data production protocols used for this work are those used by the participating centers to produce 16S rDNA sequence for the Human Microbiome Project. Thus, these results can be informative for interpreting the large body of clinical 16S rDNA data produced for this project.

Hematopoietic cells from patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) contain gene mutations that are variably distributed between the founding clone and daughter subclone(s). Traditional response criteria in MDS and AML are based on bone marrow morphology and may not accurately reflect antitumor activity and clinical benefit in patients treated with hypomethylating agents. We used digital sequencing of serial bone marrow samples to monitor tumor burden and to characterize the changes in the clonal structure of MDS and AML that occur during treatment with epigenetic therapy. We hypothesized that digital sequencing may provide an alternative measure of antitumor activity and identify the persistence or emergence of resistant clones during treatment which mediate disease relapse. We conducted a phase I/II study in older adults (age ≥ 60) with advanced MDS (IPSS ≥ 1.5) or AML. Subjects received a combination of decitabine 20 mg/m2 on d1-5 with the histone deacetylase inhibitor, panobinostat 10-40 mg po 3x/week every 28 days for up to 12 cycles. Serial bone marrow samples were collected for digital sequencing at baseline, after every 2 cycles of treatment and at the time of relapse. A total of 52 patients, 14 with MDS and 38 with AML were enrolled in this study. For AML patients, 10% achieved a complete remission (CR+CRi) with an additional 18% of patients achieving a morphologic leukemia-free state (mLFS) using IWG response criteria. For patients with MDS, 14% achieved a CR and 21% achieved a marrow CR. We identified 9 MDS and 16 AML patients that had banked, paired bone marrow and skin (as a source of normal DNA) samples and a somatic mutation in at least 1 of 54 recurrently mutated MDS/ AML genes. DNA was enriched for 285 genes commonly mutated in MDS and AML (n=24 patients) or whole exome probes spiked-in with the 285 genes (enhanced exome sequencing; EES) (n=7 patients), and sequenced on a HiSeq2000 instrument with 2x101bp reads. We detected an average of 4.9 SNVs and indels per patient (range 1-15) when only the 285 gene panel was used, compared to 27.4 mutations per patient (range 9-43) using EES. Ten genes were mutated in at least 3 pre-study samples. The presence of a TP53 mutation (N=8) was associated with a trend towards achieving a response (p=0.09). We then analyzed variant allele frequencies (VAF) of mutations in serial samples. We observed five distinct patterns that were associated with different clinical responses, including i) AML patients achieving a CR+CRi (n=2): mutation VAFs were undetectable by cycle 2 using standard sequencing, ii) AML with mLFS (n=2): mutation VAFs remained detectable but decreased to 10%, and v) AML with treatment failure (n=5): mutation VAFs were essentially unchanged and remained >30%. We observed responding patients can have persistent measurable clonal hematopoiesis for at least one year without disease progression. Sequencing also revealed selective AML subclone clearance in a patient with treatment failure, nominating a set of mutations that may mark super-responder clones. We observed that the blast percentage decreases prior to mutation VAFs in some patients, suggesting that the differentiation of blasts could falsely underestimate tumor burden. Finally, sequencing revealed that tumor burden can be measured even in patients achieving a CR. Using an ultra-sensitive barcode sequencing approach, we sequenced 1 MDS and 1 AML patient achieving a clinical and molecular CR (based on standard sequencing). We detected extremely rare TP53 mutations months to years prior to disease relapse (VAFs = 0.23% in MDS and 0.05% in AML during a CR - equivalent to a sensitivity of 1 in 2000 heterozygous mutant cells). While patients can live with persistent clonal hematopoiesis in a CR or stable disease, ultimately we find evidence that expansion of a rare subclone drives relapse or progression from MDS to secondary AML. Digital sequencing provides an alternative measure of disease response which may augment traditional clinical response criteria and should be explored in future clinical trials. Disclosures Uy: Novartis: Research Funding. Off Label Use: Panobinostat in MDS/AML. Duncavage:Cofactor Genomics: Consultancy; DI&P Consulting: Consultancy. Jacoby:Sunesis: Research Funding; Novo Nordisk: Consultancy. Abboud:Teva Phamaceutical: Research Funding.

Introduction: The clinical diagnosis of myelodysplastic syndrome (MDS) relies on the presence of persistent cytopenias, not otherwise explained, and evidence of morphologic dysplasia in the bone marrow. Low grade MDS (bone marrow blasts Methods: We screened patients who presented for evaluation of MDS between 2002 and 2014 that had consented for sequencing studies and had banked samples. Patients were selected based on 1) World Health Organization defined cytopenia (WBC Results: Thirty-eight patients met the selection criteria, and 30 of these had bone marrow aspirates available for morphologic review and were included in the study. A mean unique coverage depth of 913x was achieved for targeted genes and all reported variants had >50x coverage, variant allele fractions (VAFs) >3%, and minor allele frequencies (MAFs) < 1% in any population. Of the 30 sequenced cases, 25 had a somatic mutation in at least one gene (mean 3.3 mutations/case, range 1-10 mutations/case). The most commonly mutated gene was TET2 (7 cases), followed by ASXL1 (5 cases), EZH2 (4 cases), SRSF2 (4 cases), and U2AF1 (4 cases). Of the 285 sequenced genes, 44 were mutated in at least one case, and 14 were mutated in 2 or more cases. The mean VAF (variant reads/total reads) of detected mutations was 27% (range 3-98%). Morphologic review demonstrated definitive dysplasia (≥10% of cells in least one lineage) made by two pathologists in 18 of 30 cases (supporting the clinical diagnosis of MDS), no dysplasia in 6 of 30 cases, and equivocal dysplasia (where hematopathologists did not agree that dysplasia was ≥10%) in 6 of 30 cases. Thirteen of 18 cases (72%) with definitive dysplasia had a mutation, 5/6 cases (83%) without dysplasia had mutations, and 6/6 (100%) cases with equivocal dysplasia harbored somatic mutations. The mean VAF of mutations was 17.5% in cases without dysplasia, 29% in cases with equivocal dysplasia, and 28% in cases with definitive dysplasia. All of these groups included mutations in canonical MDS genes such as TET2, DNMT3A, SRSF2, RUNX1, and EZH2. Conclusions: In this cohort of 30 cytopenic patients with normal cytogenetics, 80% harbored a somatic mutation in at least one myeloid-associated gene. Somatic mutations were detected in 5 of 6 cases without definitive dysplasia ( Disclosures Duncavage: DI&P Consulting: Consultancy; Cofactor Genomics: Consultancy. Jacoby:Sunesis: Research Funding; Novo Nordisk: Consultancy.

We previously identified recurrent mutations in the de novo DNA methyltransferase DNMT3A in patients with acute myeloid leukemia (AML). The most common DNMT3A mutation in AML (R882H) creates a dominant negative protein that reduces DNA methylation activity by ~80% in AML cells, and causes canonical patterns of DNA hypomethylation in the AML genome (Russler-Germain et al, Cancer Cell 2014). Approaches to restore DNMT3A activity in these AML genomes may be therapeutically relevant, but only if remethylation can return these genomes to their native methylation state. To begin to address whether DNA methylation can be restored in hematopoietic cells that are deficient for Dnmt3a, we performed an Òadd-backÓ experiment using a transgenic mouse model system. First, to characterize the genome-wide effect of Dnmt3a loss on DNA methylation in hematopoietic cells, we carried out whole-genome bisulfite sequencing (WGBS) on the total bone marrow cells of wild type (WT) mice, vs. marrow derived from littermates homozygous for a germline Dnmt3a null mutation (Dnmt3a-/-, Okano et al Cell, 1999), which have overtly normal hematopoiesis. Total bone marrow cells from Dnmt3a-/- mice have a canonical pattern of DNA hypomethylation at specific CpG residues and regions in the genome; many of the CpGs are virtually unmethylated in specific regions, suggesting that the normal Dnmt3a-dependent methylation ÒmarkÓ was added in stem/progenitor cells, and then maintained in all lineages. To define the timing and specificity of remethylation in Dnmt3a-/- mice, we crossed heterozygotes from this line with transgenic mice containing a tetracycline-inducible human wild type DNMT3A cDNA (DNMT3A Tg mice), and a second transgenic mouse containing the rtTA coactivator, expressed from the Rosa26 locus. When DNMT3A Tg+, rtTA+ mice are fed Doxycycline (Dox) chow for one week, WT human DNMT3A is expressed in the marrow at a level ~4 times higher that of endogenous murine Dnmt3a. Since Dnmt3a-/- mice die of severe runting at about three weeks of age, we harvested the marrow cells from Dnmt3a-/-, DNMT3A Tg+, rtTA+mice at 2 weeks of age, and transplanted them into lethally irradiated C57Bl/6 recipients. The marrow was allowed to engraft for four weeks. Half of the mice were then given Dox chow, and half were untreated. Whole bone marrow was isolated from pairs of mice (+ vs. -Dox), DNA was purified, and then subjected to whole genome bisulfite sequencing (WGBS). WGBS produced methylation data on >93% of the CpGs in the mouse reference sequence with a median coverage of 10-12x per sample. Differential methylation analysis was performed on 2 kb tiled windows across the whole genome, revealing 108, 797 differentially methylated regions (DMRs) that were hypomethylated (Table 1). Dnmt3a-/-, DNMT3A Tg+, rtTA+bone marrow from mice without Dox (i.e. no DNMT3A was expressed) demonstrated no evidence for remethylation at any time after transplant. However, if mice were treated with Dox for only 2 weeks, 59% of these DMRs were remethylated, increasing to 70% at 4 weeks, and 83% by week 9. Data from weeks 12 and 24 are pending. Patterns of remethylation for a subset of 560 differentially methylated CpGs (DMCpGs) are shown in Figure 1. These CpGs all had methylation values of >= 90% in WT mouse bone marrow cells, and 66% methylated at a time point, yellow = 33%-66%, and green = < 33%). Most CpGs are remethylated after only 2 weeks of DNMT3A expression, but some are delayed, and become remethylated later (yellow at week 2, changing to red at 4 or 9 weeks). A small subset of these CpGs remethylate very slowly, if at all (green). Without the induction of DNMT3A (-Dox, lower panel), little or no remethylation is detected. Inspection of specific regions of the genome showed that regions that are normally unmethylated in the bone marrow cells of WT mice are rarely methylated by adding back human DNMT3A in this system (data not shown), showing that remethylation is highly specific. Although the mechanisms involved in specifying remethylation patterns are not yet clear, these data may have important implications for therapeutically restoring DNMT3A activity in AML patients with DNMT3A mutations that reduce its activity. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia is heterogeneous with respect to clinical outcome and molecular pathogenesis. Approximately 20% of AML cases are refractory to induction chemotherapy, and about 50% of patients ultimately relapse within a time interval that ranges from months to years. At the molecular level, diverse chromosomal abnormalities and genetic mutations have been observed across patients1. Although several clinical factors (age, white blood cell count), cytogenetic aberrations (t[15;17] translocation, loss of chromosome 5) 2-4, and genetic mutations (DNMT3A, FLT3) have been associated with differences in survival 5,6, these factors are of limited prognostic utility. Moreover, few studies have integrated sequence data with clinical and cytogentic factors to build predictive models of patient outcome. Here, we sought to identify genomic predictors of refractory disease or early relapse. We used whole genome and exome sequencing to analyze the genomes of 71 adult de novo AML patients treated with anthracycline and cytarabine-based induction chemotherapy. Of these, 34 had refractory disease or relapsed within 6 months, 12 relapsed in 6-12 months, and 25 had a long first remission (>12 months). We also developed an enhanced exome sequencing (EES) approach to identify and follow leukemia-associated variants over time. In 12 additional patients that achieved morphologic remission after induction chemotherapy, we used EES to identify and track variants at time of diagnosis, time of morphologic remission (roughly 30 days later), and a final time point corresponding to eventual relapse (n=8) or extended remission (n=4). No novel coding or non-coding variants present at the time of diagnosis were found to be predictive of refractory disease or early relapse. Using EES, however, we were able to detect leukemia-associated variants in the initial remission bone marrow in all eight patients who eventually relapsed. One persistent leukemia-associated variant was also detected in one patient still in remission, but all other variants in that patient were eliminated. We also detected 64 somatic variants that became enriched following chemotherapy, but were not detected in the original leukemic cells. These may represent relapse-specific variants or oligoclonal hematopoiesis after bone marrow recovery. Overall, our data suggest that the persistence of leukemia-associated variants after bone marrow recovery from cytotoxic therapy is strongly correlated with relapse, and may be used to complement more traditional, morphologic measures of leukemic cell clearance. 1. Cancer Genome Atlas Research N. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. The New England Journal of Medicine 2013;368:2059-74. 2. Byrd JC, Mrozek K, Dodge RK, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 2002;100:4325-36. 3. Grimwade D, Hills RK, Moorman AV, et al. Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood 2010;116:354-65. 4. Schlenk RF, Dohner K, Krauter J, et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. The New England Journal of Medicine 2008;358:1909-18. 5. Kihara R, Nagata Y, Kiyoi H, et al. Comprehensive analysis of genetic alterations and their prognostic impacts in adult acute myeloid leukemia patients. Leukemia 2014;28:1586-95. 6. Ley TJ, Ding L, Walter MJ, et al. DNMT3A mutations in acute myeloid leukemia. The New England Journal of Medicine 2010;363:2424-33. Citation Format: Jeffery M. Klco, Christopher A. Miller, Malachi Griffith, Allegra Petti, David H. Spencer, Shamika Ketkar-Kulkarni, Lukas D. Wartman, Matthew Christopher, Tamara L. Lamprecht, Jacqueline E. Payton, Jack Baty, Sharon E. Heath, Obi L. Griffith, Dong Shen, Jasreet Hundal, Gue Su Chang, Robert S. Fulton, Michelle O'laughlin, Catrina Fronick, Vincent Magrini, Ryan Demeter, David E. Larson, Shashikant Kulkarni, Bradley A. Ozenberger, John S. Welch, Matthew J. Walker, Timothy A. Graubert, Peter Westervelt, Jerald P. Radich, Daniel C. Link, Elaine R. Mardis, John F. DiPersio, Richard K. Wilson. Genomic approaches for risk assessment in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr PR11.

Acute myeloid leukemia is heterogeneous with respect to clinical outcome and molecular pathogenesis. Approximately 20% of AML cases are refractory to induction chemotherapy, and about 50% of patients ultimately relapse within a time interval that ranges from months to years. At the molecular level, diverse chromosomal abnormalities and genetic mutations have been observed across patients1. Although several clinical factors (age, white blood cell count), cytogenetic aberrations (t[15;17] translocation, loss of chromosome 5) 2-4, and genetic mutations (DNMT3A, FLT3) have been associated with differences in survival 5,6, these factors are of limited prognostic utility. Moreover, few studies have integrated sequence data with clinical and cytogentic factors to build predictive models of patient outcome. Here, we sought to identify genomic predictors of refractory disease or early relapse. We used whole genome and exome sequencing to analyze the genomes of 71 adult de novo AML patients treated with anthracycline and cytarabine-based induction chemotherapy. Of these, 34 had refractory disease or relapsed within 6 months, 12 relapsed in 6-12 months, and 25 had a long first remission (>12 months). We also developed an enhanced exome sequencing (EES) approach to identify and follow leukemia-associated variants over time. In 12 additional patients that achieved morphologic remission after induction chemotherapy, we used EES to identify and track variants at time of diagnosis, time of morphologic remission (roughly 30 days later), and a final time point corresponding to eventual relapse (n=8) or extended remission (n=4). No novel coding or non-coding variants present at the time of diagnosis were found to be predictive of refractory disease or early relapse. Using EES, however, we were able to detect leukemia-associated variants in the initial remission bone marrow in all eight patients who eventually relapsed. One persistent leukemia-associated variant was also detected in one patient still in remission, but all other variants in that patient were eliminated. We also detected 64 somatic variants that became enriched following chemotherapy, but were not detected in the original leukemic cells. These may represent relapse-specific variants or oligoclonal hematopoiesis after bone marrow recovery. Overall, our data suggest that the persistence of leukemia-associated variants after bone marrow recovery from cytotoxic therapy is strongly correlated with relapse, and may be used to complement more traditional, morphologic measures of leukemic cell clearance. 1. Cancer Genome Atlas Research N. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. The New England Journal of Medicine 2013;368:2059-74. 2. Byrd JC, Mrozek K, Dodge RK, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 2002;100:4325-36. 3. Grimwade D, Hills RK, Moorman AV, et al. Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood 2010;116:354-65. 4. Schlenk RF, Dohner K, Krauter J, et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. The New England Journal of Medicine 2008;358:1909-18. 5. Kihara R, Nagata Y, Kiyoi H, et al. Comprehensive analysis of genetic alterations and their prognostic impacts in adult acute myeloid leukemia patients. Leukemia 2014;28:1586-95. 6. Ley TJ, Ding L, Walter MJ, et al. DNMT3A mutations in acute myeloid leukemia. The New England Journal of Medicine 2010;363:2424-33. This abstract is also presented as a poster at the Translation of the Cancer Genome conference. Citation Format: Jeffery M. Klco, Christopher A. Miller, Malachi Griffith, Allegra Petti, David H. Spencer, Shamika Ketkar-Kulkarni, Lukas D. Wartman, Matthew Christopher, Tamara L. Lamprecht, Jacqueline E. Payton, Jack Baty, Sharon E. Heath, Obi L. Griffith, Dong Shen, Jasreet Hundal, Gue Su Chang, Robert S. Fulton, Michelle O'laughlin, Catrina Fronick, Vincent Magrini, Ryan Demeter, David E. Larson, Shashikant Kulkarni, Bradley A. Ozenberger, John S. Welch, Matthew J. Walker, Timothy A. Graubert, Peter Westervelt, Jerald P. Radich, Daniel C. Link, Elaine R. Mardis, John F. DiPersio, Richard K. Wilson. Genomic approaches for risk assessment in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr PR03.

SummaryMost mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is “captured” as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that often progress to chemotherapy-resistant secondary acute myeloid leukemia (sAML). We used whole-genome sequencing to perform an unbiased comprehensive screen to discover the somatic mutations in a sample from an individual with sAML and genotyped the loci containing these mutations in the matched MDS sample. Here we show that a missense mutation affecting the serine at codon 34 (Ser34) in U2AF1 was recurrently present in 13 out of 150 (8.7%) subjects with de novo MDS, and we found suggestive evidence of an increased risk of progression to sAML associated with this mutation. U2AF1 is a U2 auxiliary factor protein that recognizes the AG splice acceptor dinucleotide at the 3' end of introns, and the alterations in U2AF1 are located in highly conserved zinc fingers of this protein. Mutant U2AF1 promotes enhanced splicing and exon skipping in reporter assays in vitro. This previously unidentified, recurrent mutation in U2AF1 implicates altered pre-mRNA splicing as a potential mechanism for MDS pathogenesis.