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Progression from myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (AML) is associated with the acquisition and expansion of subclones. Our understanding of subclone evolution during progression, including the frequency and preferred order of gene mutation acquisition, remains incomplete. Sequencing of 43 paired MDS and secondary AML samples identified at least one signaling gene mutation in 44% of MDS and 60% of secondary AML samples, often below the level of standard sequencing detection. In addition, 19% of MDS and 47% of secondary AML patients harbored more than one signaling gene mutation, almost always in separate, coexisting subclones. Signaling gene mutations demonstrated diverse patterns of clonal evolution during disease progression, including acquisition, expansion, persistence, and loss of mutations, with multiple patterns often coexisting in the same patient. Multivariate analysis revealed that MDS patients who had a signaling gene mutation had a higher risk of AML progression, potentially providing a biomarker for progression.Significance:Subclone expansion is a hallmark of progression from MDS to secondary AML. Subclonal signaling gene mutations are common at MDS (often at low levels), show complex and convergent patterns of clonal evolution, and are associated with future progression to secondary AML.See related article by Guess et al., p. 316 (33).See related commentary by Romine and van Galen, p. 270.This article is highlighted in the In This Issue feature, p. 265

Supplementary Figure from Convergent Clonal Evolution of Signaling Gene Mutations Is a Hallmark of Myelodysplastic Syndrome Progression

Supplementary Table from Convergent Clonal Evolution of Signaling Gene Mutations Is a Hallmark of Myelodysplastic Syndrome Progression

Supplementary Data from Convergent Clonal Evolution of Signaling Gene Mutations Is a Hallmark of Myelodysplastic Syndrome Progression

The proliferative burst of B lymphocytes is essential for antigen receptor repertoire diversification during the development and selective expansion of antigen-specific clones during immune responses. High proliferative activity inevitably promotes oncogenesis, the risk of which is further elevated in B lymphocytes by endogenous gene rearrangement and somatic mutations. However, B-cell–derived cancers are rare, perhaps owing to putative intrinsic tumor-suppressive mechanisms. We show that c-MYC facilitates B-cell proliferation as a protumorigenic driver and unexpectedly coengages counteracting tumor suppression through its downstream factor TFAP4. TFAP4 is mutated in human lymphoid malignancies, particularly in >10% of Burkitt lymphomas, and reduced TFAP4 expression was associated with poor survival of patients with MYC-high B-cell acute lymphoblastic leukemia. In mice, insufficient TFAP4 expression accelerated c-MYC–driven transformation of B cells. Mechanistically, c-MYC suppresses the stemness of developing B cells by inducing TFAP4 and restricting self-renewal of proliferating B cells. Thus, the pursuant transcription factor cascade functions as a tumor suppressor module that safeguards against the transformation of developing B cells.

Mutations in the DNA methyltransferase 3A ( DNMT3A ) gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent DNMT3A mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ∼80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine Dnmt3a −/− bone marrow cells. To determine whether the hypomethylation phenotype of Dnmt3a −/− hematopoietic cells is reversible, we developed an inducible transgene to restore expression of DNMT3A in transplanted bone marrow cells from Dnmt3a −/− mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of DNMT3A addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring DNMT3A expression can alter the epigenetic “state” created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function DNMT3A mutations.

Progression from myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (AML) is associated with the acquisition and expansion of subclones. Our understanding of subclone evolution during progression, including the frequency and preferred order of gene mutation acquisition, remains incomplete. Sequencing of 43 paired MDS and secondary AML samples identified at least one signaling gene mutation in 44% of MDS and 60% of secondary AML samples, often below the level of standard sequencing detection. In addition, 19% of MDS and 47% of secondary AML patients harbored more than one signaling gene mutation, almost always in separate, coexisting subclones. Signaling gene mutations demonstrated diverse patterns of clonal evolution during disease progression, including acquisition, expansion, persistence, and loss of mutations, with multiple patterns often coexisting in the same patient. Multivariate analysis revealed that MDS patients who had a signaling gene mutation had a higher risk of AML progression, potentially providing a biomarker for progression.Significance:Subclone expansion is a hallmark of progression from MDS to secondary AML. Subclonal signaling gene mutations are common at MDS (often at low levels), show complex and convergent patterns of clonal evolution, and are associated with future progression to secondary AML.See related article by Guess et al., p. 316 (33).See related commentary by Romine and van Galen, p. 270.This article is highlighted in the In This Issue feature, p. 265

Kdm6a/Utx, a gene on the X chromosome, encodes a histone K27me3 demethylase that has an orthologue on the Y chromosome (Uty)[1]. We previously identified inactivating mutations of Kdm6a in approximately 50% of mouse acute promyelocytic leukemia samples; however, somatic mutations of KDM6A are more rare in human AML samples, ranging in frequency from 2-15% in different series of patients, where their role in pathogenesis is not yet clear. In this study, we show that female Kdm6aflox/flox mice (with allele inactivation initiated by Vav1-Cre in hematopoietic stem and progenitor cells (HSPCs) have a sex-specific phenotype that emerges with aging, with features resembling a myelodysplastic syndrome (MDS). Female Kdm6a-knockout (KO) mice have an age-dependent expansion of their HSPCs with aberrant self-renewal, but they did not differentiate normally into downstream progeny. These mice became mildly anemic and thrombocytopenic, but did not develop overt leukemia, or die from these cytopenias. ChIP-seq and ATAC-seq studies showed only minor changes in H3K27me3, H3K27ac, H3K4me, H3K4me3 and chromatin accessibility between Kdm6a-WT and Kdm6a-KO mice. Utilizing scRNA-seq, Kdm6a loss was linked to the transcriptional repression of genes that mediate hematopoietic cell fate determination. These data demonstrate that Kdm6a plays an important role in normal hematopoiesis, and that its inactivation may contribute to AML pathogenesis by altering the epigenetic state of HSPCs.

Significance Mutations that reduce the function of the de novo DNA methyltransferase, DNMT3A, are very common in patients with clonal hematopoiesis and hematopoietic malignancies. Restoring the function of DNMT3A in mouse hematopoietic cells deficient for this protein corrects the DNA methylation defect in an ordered fashion, and partially restores abnormalities in gene expression and myeloid population skewing. These data may be relevant for therapeutic strategies designed to restore DNMT3A activity in patients with diseases caused by mutations in this gene., Mutations in the DNA methyltransferase 3A (DNMT3A) gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent DNMT3A mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ∼80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine Dnmt3a−/− bone marrow cells. To determine whether the hypomethylation phenotype of Dnmt3a−/− hematopoietic cells is reversible, we developed an inducible transgene to restore expression of DNMT3A in transplanted bone marrow cells from Dnmt3a−/− mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of DNMT3A addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring DNMT3A expression can alter the epigenetic “state” created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function DNMT3A mutations.

Mechanisms of in vivo AML response and resistance to chemotherapy remain poorly characterized. To determine whether response to lenalidomide might share features with response to other forms of chemotherapy, we applied immunohistochemistry and gene panel sequencing to serial samples from a patient

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.

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.

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

The gene that encodes de novo DNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia genomes. Point mutations at position R882 have been shown to cause a dominant negative loss of DNMT3A methylation activity, but 15% of DNMT3A mutations are predicted to produce truncated proteins that could either have dominant negative activities or cause loss of function and haploinsufficiency. Here, we demonstrate that 3 of these mutants produce truncated, inactive proteins that do not dimerize with WT DNMT3A, strongly supporting the haploinsufficiency hypothesis. We therefore evaluated hematopoiesis in mice heterozygous for a constitutive null Dnmt3a mutation. With no other manipulations, Dnmt3a+/- mice developed myeloid skewing over time, and their hematopoietic stem/progenitor cells exhibited a long-term competitive transplantation advantage. Dnmt3a+/- mice also spontaneously developed transplantable myeloid malignancies after a long latent period, and 3 of 12 tumors tested had cooperating mutations in the Ras/MAPK pathway. The residual Dnmt3a allele was neither mutated nor downregulated in these tumors. The bone marrow cells of Dnmt3a+/- mice had a subtle but statistically significant DNA hypomethylation phenotype that was not associated with gene dysregulation. These data demonstrate that haploinsufficiency for Dnmt3a alters hematopoiesis and predisposes mice (and probably humans) to myeloid malignancies by a mechanism that is not yet clear.

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

Summary Tumor suppressor p53 regulates transcription of stress-response genes. Many p53 targets remain undiscovered because of uncertainty as to where p53 binds in the genome and the fact that few genes reside near p53-bound recognition elements (REs). Using chromatin immunoprecipitation followed by exonuclease treatment (ChIP-exo), we associated p53 with 2,183 unsplit REs. REs were positionally constrained with other REs and other regulatory elements, which may reflect structurally organized p53 interactions. Surprisingly, stress resulted in increased occupancy of transcription factor IIB (TFIIB) and RNA polymerase (Pol) II near REs, which was reduced when p53 was present. A subset associated with antisense RNA near stress-response genes. The combination of high-confidence locations for p53/REs, TFIIB/Pol II, and their changes in response to stress allowed us to identify 151 high-confidence p53-regulated genes, substantially increasing the number of p53 targets. These genes composed a large portion of a predefined DNA-damage stress-response network. Thus, p53 plays a comprehensive role in regulating the stress-response network, including regulating noncoding transcription.

Background: Expansion of one or more subclone occurs during progression from myelodysplastic syndrome (MDS) to secondary acute myeloid leukemia (sAML). Existing data suggest that acquired mutations in myeloid transcription factor (e.g., RUNX1, CEBPA, WT1) and signaling genes (e.g., receptor tyrosine kinases or RAS pathway genes) contribute to clonal evolution and the rising blast count that defines progression to sAML. While signaling gene (SG) mutations are typically acquired later in disease progression, our understanding of when transcription factor (TF) mutations occur, in what clone they occur (e.g. founding clone or subclone), and whether TF-mutated clones undergo further clonal evolution remains incomplete. This is largely due to the limited number of paired MDS and sAML samples analyzed, the limitation of current sequencing technology and the lack of serial samples, and incomplete characterization of tumor clonality. Methods: We banked paired MDS and sAML (plus skin) samples from 44 patients who progressed from MDS to sAML (median time to progression 306 days, range 21-3568). We sequenced sAML and skin samples for 285 recurrently mutated genes (RMGs) and genotyped the paired MDS sample in patients with TF and/or SG mutations. Twelve patients were selected for enhanced whole genome sequencing (eWGS) of MDS and sAML samples (plus skin) to characterize tumor clonality. Somatic mutations were validated using error-corrected sequencing and clones were identified in MDS and sAML samples using mutation variant allele frequencies (VAFs). We tracked clonal evolution by sequencing serial samples between MDS and sAML. Results: The frequency of both TF and SG mutations were elevated in the 44 sAML patients compared to our previously sequenced cohort of 150 independent de novo MDS patients (signaling: 36% vs. 15%, transcription factor: 30% vs. 11%, respectively, p We next addressed whether TF mutations were present in a founding clone or subclone at MDS. As the accuracy of discriminating founding clones and subclones increases with the number of detected mutations, we performed eWGS on 12 pairs of MDS and sAML samples to determine if TF and SG mutations were subclonal and how subclones evolved during progression. We validated a median of 596 (range: 305-1382) mutations per MDS sample and 582 (range 305-1373) mutations per sample at sAML. Mutation VAFs were used to cluster mutations and identify clones. A median of 4 clones (range: 2-6) were detected at MDS compared to 5 (range: 2-8) at sAML, with no patient showing a decrease in the number of clones at progression. When combined with cytogenetic/copy number alterations, 11/12 cases showed the expansion of a subclone during progression. Nine SG and 8 TF mutations (n=17 total) were detected in the 12 sAML samples. In total, 16/17 (94%) TF or SG mutations occurred in subclones, including 14 in subclones that expanded during progression to sAML. Only 1 TF mutation occurred in a founding clone, and 3/4 TF-mutated subclones gave rise to a new subclone, 2 containing a new RMG. Conclusions: Data from 44 patients shows that nearly half of sAML patients have transcription factor and/or signaling gene mutations. While both are enriched at sAML compared to MDS, TF and SG mutations show different timing of mutation acquisition. TF mutations are often present at MDS in subclones indicating that they may serve as a biomarker at MDS for later progression. In comparison, SG mutations are only rarely detected at MDS and are acquired between MDS and sAML. Additionally, for cells containing both TF and SG mutations, the TF mutation is typically acquired first. Despite these differences in acquisition, both TF and SG mutations occur in subclones that often expand and continue to clonally evolve during disease progression, suggesting that they may contribute to the rise in blast count. Disclosures No relevant conflicts of interest to declare.

Biochemical assessment of channel structure/function is incredibly challenging. Developing computational tools that provide these data would enable translational research, accelerating mechanistic experimentation for the bench scientist studying ion channels. Starting with the premise that protein sequence encodes information about structure, function and evolution (SF&E), we developed a unified framework for inferring SF&E from sequence information using a knowledge-based approach. The Gestalt Domain Detection Algorithm-Basic Local Alignment Tool (GDDA-BLAST) provides phylogenetic profiles that can model, ab initio, SF&E relationships of biological sequences at the whole protein, single domain and single-amino acid level.1,2 In our recent paper,4 we have applied GDDA-BLAST analysis to study canonical TRP (TRPC) channels1 and empirically validated predicted lipid-binding and trafficking activities contained within the TRPC3 TRP_2 domain of unknown function. Overall, our in silico, in vitro, and in vivo experiments support a model in which TRPC3 has signal-sensing antennae which are adorned with lipid-binding, trafficking and calmodulin regulatory domains. In this Addendum, we correlate our functional domain analysis with the cryo-EM structure of TRPC3.3 In addition, we synthesize recent studies with our new findings to provide a refined model on the mechanism(s) of TRPC3 activation/deactivation.

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.

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.

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.

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.

One of the major challenges in the genomic era is annotating structure/function to the vast quantities of sequence information now available. Indeed, most of the protein sequence database lacks comprehensive annotation, even when experimental evidence exists. Further, within structurally resolved and functionally annotated protein domains, additional functionalities contained in these domains are not apparent. To add further complication, small changes in the amino-acid sequence can lead to profound changes in both structure and function, underscoring the need for rapid and reliable methods to analyze these types of data. Phylogenetic profiles provide a quantitative method that can relate the structural and functional properties of proteins, as well as their evolutionary relationships. Using all of the structurally resolved Src-Homology-2 (SH2) domains, we demonstrate that knowledge-bases can be used to create single-amino acid phylogenetic profiles which reliably annotate lipid-binding. Indeed, these measures isolate the known phosphotyrosine and hydrophobic pockets as integral to lipid-binding function. In addition, we determined that the SH2 domain of Tec family kinases bind to lipids with varying affinity and specificity. Simulating mutations in Bruton's tyrosine kinase (BTK) that cause X-Linked Agammaglobulinemia (XLA) predict that these mutations alter lipid-binding, which we confirm experimentally. In light of these results, we propose that XLA-causing mutations in the SH3-SH2 domain of BTK alter lipid-binding, which could play a causative role in the XLA-phenotype. Overall, our study suggests that the number of lipid-binding proteins is drastically underestimated and, with further development, phylogenetic profiles can provide a method for rapidly increasing the functional annotation of protein sequences.

Recent whole genome sequencing efforts by our group and others have identified recurrent mutations in the DNA methyltransferase DNMT3A in various hematologic malignancies, including acute myeloid leukemia (AML), MPN, MDS, CMML, and T-ALL. The most common point mutation in AML, R882H, is nearly always heterozygous in AML, and has been demonstrated to have a dominant negative effect on the methylation activity of the remaining wild-type allele (Russler-Germain et al. Cancer Cell 2014). However, other heterozygous mutations produce frameshifts, premature stop codons, or deletions of the entire coding sequence of the gene, strongly suggesting that these mutations lead to simple haploinsufficiency for DNMT3A. In order to test the hypothesis that Dnmt3a haploinsuffiency may initiate AML, we performed a long-term tumor watch comparing wild-type mice (Dnmt3a+/+) to mice carrying one wild-type Dnmt3a allele and one knockout allele that contains a neomycin-resistance cassette inserted into the sequence coding for the catalytic domain of the protein, producing a true null allele (Dnmt3a+/- mice). At 6 weeks of age, Dnmt3a+/- mice have normal hematopoiesis, with no detectable differences from wild-type littermates in myeloid, lymphoid, erythroid, or stem/progenitor populations in the bone marrow or spleen. However, after 1.5 years of age, 15/43 Dnmt3a+/- mice (35%) became moribund and were euthanized for pathologic evaluation (Figure 1). Affected mice generally exhibited massive splenomegaly with myeloid infiltrates in spleen, liver, and other extramedullary organs. At conclusion of the tumor watch at two years, all remaining mice were euthanized, and similar pathologic findings were observed in an additional 9 Dnmt3a+/- mice, for an overall disease penetrance of 24/43 (56%). Flow cytometric evaluation of splenic tumors demonstrated positivity for the myeloid markers Gr-1 and CD11b, and the presence of a subpopulation of cells double-positive for the mature myeloid marker Gr-1 and the progenitor cell marker CD34. Based on flow cytometric and morphologic findings, 16 splenic tumors were definitively classified according to the Bethesda criteria and were found to exhibit myeloid proliferation/MPD (11/16), myeloid leukemia with maturation (2/16), MPD-like myeloid leukemia (2/16), or myeloid sarcoma (1/16). 6 of 16 tumors tested were able to successfully engraft and lead to lethal disease in sublethally irradiated wild-type recipients, providing further evidence that these tumors represent transplantable, cell-autonomous myeloid malignancies. Analysis of exome sequencing data from 5 primary tumor samples is ongoing. One MPD-like myeloid leukemia was engrafted into four separate wild-type recipients, and all 5 tumors were sequenced. Collectively, this set of samples was found to contain mutations in the Ras/MAPK pathway, including the canonical gain-of-function mutation Kras G12C, a Ptpn11 E76K mutation, and a missense mutation in the tumor suppressor Neurofibromatosis 1 (Nf1). A second, independent primary tumor was also found to contain an activating Kras G13D mutation. Examination of the Dnmt3a locus in all 5 sequenced samples revealed no evidence of mutations or loss of heterozygosity of the residual wild-type Dnmt3a allele. Importantly, 9/51 AML samples with DNMT3A mutations in the TCGA AML cohort also contained activating NRAS or KRAS mutations. These data strongly suggest that Ras/MAPK pathway mutations can cooperate with Dnmt3a haploinsufficiency to induce AML in C57Bl/6 mice and in humans. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.