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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.

Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥ 1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥ 15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.

DNA methylation has been functionally implicated in X-inactivation, genomic imprinting, and silencing of transposable elements. DNA methylation also has a complex regulatory relationship with gene expression. Canonically, methylation around the promoters of tumor-suppressor genes induces gene-silencing, thereby representing a hit in the two-hit hypothesis for the development of cancer. Unfortunately, profiling studies conducted to determine how aberrant methylation may contribute to cancer progression are confounded by heterogeneity in the original clinical sample. Thus, though studies in patients with Acute Myeloid Leukemia (AML), Diffuse Large B-cell Lymphoma (DLBCL), and Chronic Lymphocytic Leukemia (CLL) have found that variation in detected methylation values from patients at diagnosis correlates with prognosis following therapy, they do not address which subclonal methylation events contribute to cancer progression. To address this concern, we developed a novel computational method to deconvolve the bisulfite sequencing data from a sample into its major methylation profiles and their respective prevalence in the sample. Our method, based on a modified Hidden Markov Model, effectively models the autocorrelations found in methylation data and outperforms existing algorithms. Our method was validated across a wide range of mixture simulations, where bisulfite sequencing reads from various different cell types were subsampled to form test samples that could be deconvolved. We were able to accurately (98%) distinguish distinct methylation patterns corresponding to the expected underlying subpopulations, such as for CD14 and CD22 in mixtures of germinal center B-cells and monocytes and for CD4 and CD8A in mixtures of CD4+ T-lymphocytes and CD8+ T-lymphocytes. These patterns also recapitulated differentially methylated regions (DMRs) identified by an independent DMR-caller. Given that our method does not rely on cell-type specific parameters and is therefore robust to all samples, to further validate and demonstrate the applicability of our method, we conducted Agilent Methyl-Seq on 5 primary DLBCL samples procured by the Lymphoma Core at the Siteman Cancer Center. As a positive control, our method identified differential methylation profiles at loci expected to differ from underlying CD19+ and CD4+ cells, which comprise a large majority of each sample. Our method also identified distinct methylation profiles not found in reference profiles from normal cell-types, suggesting these methylation profiles may be specific to DLBCL. To further validate these findings, we used single-cell bisulfite-sequencing at ten loci to demonstrate that the methylation profiles predicted by our method from the original sample are found in individual cells. We found several methylation patterns that only existed in a subset of CD19+ cells, which may represent distinct epigenetic subclones of DLBCL. Using our novel computational method, we next profiled the subclonal epigenetic architecture of publicly available (dbGaP) paired samples from patients with AML (n=137) at diagnosis and following therapy. We were able to not only identify subclonal methylation profiles that were specific to cancer but also find profiles at higher prevalence in patients at relapse compared to diagnosis. These methylation profiles, which were enriched for genes in cancer pathways as seen by Gene Set Enrichment Analysis, may confer fitness advantages for a cancer subclone to expand. We are currently conducting additional analyses to characterize the epigenetic regulatory circuits that contribute to our observed increase in subclonal fitness. In summary, we have developed a robust method to identify subclonal methylation changes that may contribute to cancer progression and prognosis, as seen in AML, and may lead to new avenues for improving treatment for patients with leukemia or lymphoma. Disclosures No relevant conflicts of interest to declare.

Alterations to the epigenetic landscape of diffuse large B-cell lymphoma (DLBCL) play a fundamental role in deregulating genes involved in normal lymphocyte differentiation. To determine whether targeted epigenetic therapy could reverse these pathogenic chromatin changes and suppress the expression of a lymphoma oncogene, we focused on BCL6, a transcriptional repressor whose aberrant expression is tightly linked to DLBCL proliferation and survival. We fused zinc-finger (ZF) domains specific for regulatory regions in the BCL6 locus to a repressive epigenetic modifier, the Kruppel-associated box (KRAB) repressor domain. Distinct ZF-KRAB fusions repressed the local chromatin landscape, suppressed BCL6 expression, significantly impaired DLBCL growth, and caused widespread cell death in a BCL6-dependent manner. Importantly, expression of ectopic BCL6 protein rescued ZF-KRAB-induced cell death, demonstrating the modifiers' specificity. We show that sequence-specific epigenetic modifiers can alter oncogene expression and induce apoptosis in cancer cells, underscoring their potential for future development as targeted epigenetic protein therapies.

Innate lymphoid cells (ILCs) serve as sentinels in mucosal tissues, sensing release of soluble inflammatory mediators, rapidly communicating danger via cytokine secretion, and functioning as guardians of tissue homeostasis. Although ILCs have been studied extensively in model organisms, little is known about these “first responders” in humans, especially their lineage and functional kinships to cytokine-secreting T helper (Th) cell counterparts. Here, we report gene regulatory circuitries for four human ILC–Th counterparts derived from mucosal environments, revealing that each ILC subset diverges as a distinct lineage from Th and circulating natural killer cells, but shares circuitry devoted to functional polarization with their Th counterparts. Super-enhancers demarcate cohorts of cell identity genes in each lineage, uncovering new modes of regulation for signature cytokines, new molecules that likely impart important functions to ILCs, and potential mechanisms for autoimmune disease SNP associations within ILC–Th subsets.

Short tandem repeat (STR) analysis has emerged as the method of choice for testing to resolve specimen source contamination and identity problems that arise in surgical pathology. We studied a series of consecutive cases referred for STR typing during a 5-year period to document the usefulness of the approach and to describe the broadening scope of testing. The series demonstrates that STR-based typing can be applied in virtually any setting in which specimen source confirmation is requested, that STR-based typing is informative in 92% of cases, but that exceptions occasionally arise that complicate test interpretation. The series also demonstrates that in addition to traditional uses of STR typing, testing is now performed in the absence of any direct indication that a specimen mix-up or contamination may have occurred, namely, when the pathologic findings are unexpected or the clinical setting is atypical. The case series underscores the ability of STR testing to detect errors that cannot be captured by current laboratory protocols, a finding that has important implications for patient safety.

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

The t(8;21)(q22;q22) translocation, present in approximately 5% of adult acute myeloid leukemia (AML) cases, produces the AML1/ETO (AE) fusion protein. Dysregulation of the Pit/Oct/Unc (POU) domain-containing transcription factor POU4F1 is a recurring abnormality in t(8;21) AML. In this study, we showed that POU4F1 overexpression is highly correlated with, but not caused by, AE. We observed that AE markedly increases the self-renewal capacity of myeloid progenitors from murine bone marrow or fetal liver and drives the expansion of these cells in liquid culture. POU4F1 is neither necessary nor sufficient for these AE-dependent properties, suggesting that it contributes to leukemia through novel mechanisms. To identify targets of POU4F1, we performed gene expression profiling in primary mouse cells with genetically defined levels of POU4F1 and identified 140 differentially expressed genes. This expression signature was significantly enriched in human t(8;21) AML samples and was sufficient to cluster t(8;21) AML samples in an unsupervised hierarchical analysis. Among the most highly differentially expressed genes, half are known AML1/ETO targets, implying that the unique transcriptional signature of t(8;21) AML is, in part, attributable to POU4F1 and not AML1/ETO itself. These genes provide novel candidates for understanding the biology and developing therapeutic approaches for t(8;21) AML.

To define the factors that modulate regulatory T (Treg) cells in the tumor setting, we cocultured various tumor cells with either purified Treg cells, or with unfractionated splenocytes. We found that Treg expansion occurred only with unfractionated splenocytes, suggesting that accessory cells and/or factors produced by them play an essential role in tumor-induced Treg expansion. We performed gene expression profiling on tumor-associated Treg cells to identify candidate signaling molecules and studied their effects on tumor-induced Treg expansion. We inadvertently discovered that interleukin (IL)-12 treatment blocked Treg expansion in an IL-12 receptor–dependent fashion. Additional studies showed that IL-12 acts by stimulating IFN-γ mediated inhibition of Treg cell proliferation, which may partially account for the antitumor effects of IL-12. Furthermore, IL-12 treatment was found to decrease IL-2 production, which may lead to IFN-γ–independent inhibition of Treg cells, as IL-2 is required for their survival and expansion. Mechanistic studies revealed that IFN-γ signaling directly causes cell cycle arrest in Treg cells. This study shows that an IL-12–IFN-γ axis can suppress tumor-induced Treg proliferation. This mechanism may counteract the ability of Treg cells to promote tumor growth in vivo. [Cancer Res 2009;69(22):8700–9]

Cytogenetic analysis of acute myeloid leukemia (AML) cells has accelerated the identification of genes important for AML pathogenesis. To complement cytogenetic studies and to identify genes altered in AML genomes, we performed genome-wide copy number analysis with paired normal and tumor DNA obtained from 86 adult patients with de novo AML using 1.85 million feature SNP arrays. Acquired copy number alterations (CNAs) were confirmed using an ultra-dense array comparative genomic hybridization platform. A total of 201 somatic CNAs were found in the 86 AML genomes (mean, 2.34 CNAs per genome), with French-American-British system M6 and M7 genomes containing the most changes (10–29 CNAs per genome). Twenty-four percent of AML patients with normal cytogenetics had CNA, whereas 40% of patients with an abnormal karyotype had additional CNA detected by SNP array, and several CNA regions were recurrent. The mRNA expression levels of 57 genes were significantly altered in 27 of 50 recurrent CNA regions

Acute promyelocytic leukemia (APL) is characterized by the t(15;17) chromosomal translocation, which results in fusion of the retinoic acid receptor alpha (RARA) gene to another gene, most commonly promyelocytic leukemia (PML). The resulting fusion protein, PML-RARA, initiates APL, which is a subtype (M3) of acute myeloid leukemia (AML). In this report, we identify a gene expression signature that is specific to M3 samples; it was not found in other AML subtypes and did not simply represent the normal gene expression pattern of primary promyelocytes. To validate this signature for a large number of genes, we tested a recently developed high throughput digital technology (NanoString nCounter). Nearly all of the genes tested demonstrated highly significant concordance with our microarray data (P < 0.05). The validated gene signature reliably identified M3 samples in 2 other AML datasets, and the validated genes were substantially enriched in our mouse model of APL, but not in a cell line that inducibly expressed PML-RARA. These results demonstrate that nCounter is a highly reproducible, customizable system for mRNA quantification using limited amounts of clinical material, which provides a valuable tool for biomarker measurement in low-abundance patient samples.

Patients with acute myeloid leukemia (AML) frequently fail chemotherapy due to refractory disease, relapse, or toxicity. Among older AML patients (age > 60 years), there are few long-term survivors. Lenalidomide is a candidate for study in AML based on its clinical activity in a related disorder, myelodysplastic syndrome (MDS), with the 5q− chromosomal abnormality. We report induction of sustained morphologic and cytogenetic complete remission in 2 older AML patients treated with high-dose, single-agent lenalidomide; each patient had trisomy 13 as the sole cytogenetic abnormality. We show for the first time that lenalidomide has clinical activity in this poor-risk cytogenetic subset of AML. The clinical trials described in this paper have been registered with www.clinicaltrials.gov under identifiers NCT00466895 and NCT00546897.

Lay Summary Acute myeloid leukemia is a highly malignant hematopoietic tumor that affects about 13,000 adults yearly in the United States. The treatment of this disease has changed little in the past two decades, since most of the genetic events that initiate the disease remain undiscovered. Whole genome sequencing is now possible at a reasonable cost and timeframe to utilize this approach for unbiased discovery of tumor-specific somatic mutations that alter the protein-coding genes. Here we show the results obtained by sequencing a typical acute myeloid leukemia genome and its matched normal counterpart, obtained from the patient’s skin. We discovered 10 genes with acquired mutations; two were previously described mutations thought to contribute to tumor progression, and 8 were novel mutations present in virtually all tumor cells at presentation and relapse, whose function is not yet known. Our study establishes whole genome sequencing as an unbiased method for discovering initiating mutations in cancer genomes, and for identifying novel genes that may respond to targeted therapies. We used massively parallel sequencing technology to sequence the genomic DNA of tumor and normal skin cells obtained from a patient with a typical presentation of FAB M1 Acute Myeloid Leukemia (AML) with normal cytogenetics. 32.7-fold ‘haploid’ coverage (98 billion bases) was obtained for the tumor genome, and 13.9-fold coverage (41.8 billion bases) was obtained for the normal sample. Of 2,647,695 well-supported Single Nucleotide Variants (SNVs) found in the tumor genome, 2,588,486 (97.7%) also were detected in the patient’s skin genome, limiting the number of variants that required further study. For the purposes of this initial study, we restricted our downstream analysis to the coding sequences of annotated genes: we found only eight heterozygous, non-synonymous somatic SNVs in the entire genome. All were novel, including mutations in protocadherin/cadherin family members (CDH24 and PCLKC), G-protein coupled receptors (GPR123 and EBI2), a protein phosphatase (PTPRT), a potential guanine nucleotide exchange factor (KNDC1), a peptide/drug transporter (SLC15A1), and a glutamate receptor gene (GRINL1B). We also detected previously described, recurrent somatic insertions in the FLT3 and NPM1 genes. Based on deep readcount data, we determined that all of these mutations (except FLT3) were present in nearly all tumor cells at presentation, and again at relapse 11 months later, suggesting that the patient had a single dominant clone containing all of the mutations. These results demonstrate the power of whole genome sequencing to discover novel cancer-associated mutations.

B cell lymphomas (BCLs) are characterized by widespread deregulation of gene expression compared with their normal B cell counterparts. Recent epigenomic studies defined cis-regulatory elements (REs) whose activities are altered in BCL to drive some of these pathogenic expression changes. During transformation, multiple mechanisms are employed to alter RE activities, including perturbations in the function of chromatin modifiers, which can lead to revision of the B cell epigenome. Inherited and somatic variants also alter RE function via disruption of transcription factor (TF) binding. Aberrant expression of noncoding RNAs (ncRNAs) deregulates genes involved in B cell differentiation via direct repression and post-transcriptional targeting. These discoveries have established epigenetic etiologies for B cell transformation that are being exploited in novel therapeutic approaches.

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.

To identify genes that are commonly dysregulated in a murine model of acute promyelocytic leukemia (APL), we first defined gene expression patterns during normal murine myeloid development; serial gene expression profiling studies were performed with primary murine hematopoietic progenitors that were induced to undergo myeloid maturation in vitro with G-CSF. Many genes were reproducibly expressed in restricted developmental “windows,” suggesting a structured hierarchy of expression that is relevant for the induction of developmental fates and/or differentiated cell functions. We compared the normal myeloid developmental transcriptome with that of APL cells derived from mice expressing PML-RARα under control of the murine cathepsin G locus. While many promyelocyte-specific genes were highly expressed in all APL samples, 116 genes were reproducibly dysregulated in many independent APL samples, including Fos, Jun, Egr1, Tnf, and Vcam1. However, this set of commonly dysregulated genes was expressed normally in preleukemic, early myeloid cells from the same mouse model, suggesting that dysregulation occurs as a “downstream” event during disease progression. These studies suggest that the genetic events that lead to APL progression may converge on common pathways that are important for leukemia pathogenesis.

The presynaptic protein alpha-synuclein has been implicated in both neuronal plasticity and neurodegenerative disease, but its normal function remains unclear. We described the induction of an amphipathic alpha-helix at the N terminus (exons 2-4) of alpha-synuclein upon exposure to phospholipid vesicles, and hypothesized that lipid-binding might serve as a functional switch by stabilizing alpha-synuclein in an active (alpha-helical) conformation. Others have shown that alpha and beta-synucleins inhibit phospholipase D (PLD), an enzyme involved in lipid-mediated signaling cascades and vesicle trafficking. Here, we report that all three naturally occurring synuclein isoforms (alpha, beta, and gamma-synuclein) are similarly effective inhibitors of PLD2 in vitro, as is the Parkinson's disease-associated mutant A30P. The PD-associated mutant A53T, however, is a more potent inhibitor of PLD2 than is wild-type alpha-synuclein. We analyze mutations of the alpha-synuclein protein to identify critical determinants of human PLD2 inhibition in vitro. Deletion of residues 56-102 (exon 4) decreases PLD2 inhibition significantly; this activity of exon 4 may require adoption of an alpha-helical conformation, as mutations that disrupt alpha-helicity also abrogate inhibition. Deletion of C-terminal residues 130-140 (exon 6) completely abolishes inhibitory activity. In addition, PLD2 inhibition is blocked by phosphorylation at serine 129 or at tyrosine residues 125 and 136, or by mutations that mimic phosphorylation at these sites. We conclude that PLD2 inhibition by alpha-synuclein is mediated by a lipid-stabilized alpha-helical structure in exon 4 and also by residues within exon 6, and that this inhibition can be modulated by phosphorylation of specific residues in exons 5 and 6.

The structure of broken DNA ends is a critical determinant of the pathway used for DNA double-strand break (DSB) repair. Here, we develop an approach involving the hairpin capture of DNA end structures (HCoDES), which elucidates chromosomal DNA end structures at single-nucleotide resolution. HCoDES defines structures of physiologic DSBs generated by the RAG endonuclease, as well as those generated by nucleases widely used for genome editing. Analysis of G1 phase cells deficient in H2AX or 53BP1 reveals DNA ends that are frequently resected to form long single-stranded overhangs that can be repaired by mutagenic pathways. In addition to 3′ overhangs, many of these DNA ends unexpectedly form long 5′ single-stranded overhangs. The divergence in DNA end structures resolved by HCoDES suggests that H2AX and 53BP1 may have distinct activities in end protection. Thus, the high-resolution end structures obtained by HCoDES identify features of DNA end processing during DSB repair.

alpha-Synuclein is a highly conserved presynaptic protein with probable roles in normal synaptic development and plasticity as well as neurodegenerative disease, although its molecular function is not yet clear. To identify potential protein binding partners of alpha-synuclein, we performed co-immunoprecipitations using a monoclonal antibody (H3C) against its C-terminus. More than 20 detectable proteins were specifically co-immunoprecipitated from zebra finch and mouse forebrain extracts. One of these, with relative mobility of 55 kDa, was identified through microsequencing as a mixture of alpha- and beta-tubulin. Tubulin was specifically recovered from a mouse forebrain cytosolic extract by a GST/alpha-synuclein fusion protein immobilized on glutathione-Sepharose beads. In the converse experiment, alpha-synuclein bound to a column prepared from purified bovine brain tubulin immobilized upon CNBr-Sepharose. alpha-Synuclein does not appear to bind assembled microtubules, however, as alpha-synuclein did not pellet with polymerized microtubules in a standard assay for microtubule-associated proteins. Likewise, when a fusion construct of alpha-synuclein and green fluorescent protein (GFP) was expressed in African green monkey kidney epithelial (CV-1) cells, the fusion protein did not colocalize with endogenous microtubules. We conclude that alpha-synuclein may interact specifically with heterodimeric tubulin, but not microtubules, in the neuronal cytosol.

Altered DNA methylation is a well-known feature of acute myeloid leukemia (AML) genomes, but the mechanisms underlying these changes and their relevance for AML pathogenesis are unclear. We previously showed that DNMT3A is the predominant de novo methyltransferase expressed in AML cells, and that the DNMT3AR882H mutation in AML creates a dominant negative protein that reduces in vitro DNA methylation activity by ~80%. Since DNMT3A provides themajority of the methylation activity in AML cells, we hypothesized that AML samples with and without DNMT3AR882H could reveal novel insights about the role of this enzyme in AML initiation and progression. We performed whole-genome bisulfite sequencing (WGBS) of 38 primary human AML samples and 17 normal human hematopoietic cell samples, as well as a remission sample from a patient with a persistent DNMT3AR882H mutation, and blood samples from a non-leukemic patient with a constitutional DNMT3AR882H mutation. We first identified 3,848 differentially methylated regions ('DMRs') between DNMT3AR882H and DNMT3AWT AMLs, virtually all of which were hypomethylated in the DNMT3AR882H AMLs. Further, 28% (1,087/3,848) of these DMRs were also hypomethylated when compared to CD34 cells, implying that these regions are truly hypomethylated in the AML cells with the R882H mutation. In contrast, 72% (2,759/3,848) of the DMRs were unmethylated in bothDNMT3AR882H AMLs and CD34 cells, but were hypermethylated in the DNMT3AWT AML samples. These loci were associated with CpG dense regions, suggesting that they represent abnormal CpG island hypermethylation that occurs only in AML samples with wild-type DNMT3A. Analysis of 21 additional primary AML samples with wild-type DNMT3A identified 4,912 hypermethylated regions compared to CD34 cells, of which 4,544 (92%) were significantly less methylated in DNMT3AR882H AMLs, implying that functional DNMT3A mediates abnormal CpG island hypermethylation in AML. WGBS analysis of two non-leukemic hematopoietic samples with DNMT3AR882H mutations was also performed to understand the direct effects of DNMT3AR882H in non-transformed myeloid cells. These samples included peripheral blood (PB) neutrophils and monocytes from a newly identified 9-year old patient with an overgrowth syndrome and developmental delay (Tatton-Brown et. al., Nature Genetics 2014), who was found to have a heterozygous DNMT3AR882H mutation in all skin and peripheral blood cells. His CBC was normal, and he had no evidence of clonal hematopoiesis by exome sequencing. We identified 2,051 DMRs in his PB myeloid cells, all of which were hypomethylated compared to control PB myeloid cells from his healthy 13-year old brother (and also normal CD34 cells), demonstrating that DNMT3AR882H directly causes focal methylation loss. We also performed WGBS on cells expanded from single stem/progenitor cells from an AML patient with a persistent DNMT3AR882H mutation during remission. Expanded cells with DNMT3AR882H were hypomethylated relative to wild-type DNMT3A cells expanded from the same sample. The majority of the hypomethylated regions were also present in the patient's AML cells, implying that DNMT3AR882H-associated hypomethylation in pre-leukemic cells is maintained during AML progression. These findings demonstrate that DNMT3AR882H-associated hypomethylation precedes leukemia development, and may therefore represent an important initiating phenotype for AML. Our data also suggest that the abnormal hypermethylation of CpG islands in AML cells is DNMT3A-dependent, and must occur during disease progression. This hypermethylation is absent in AMLs with DNMT3AR882H, revealing that it is not required for leukemia progression. We therefore propose a model where DNMT3A-dependent DNA methylation in AML cells acts as a 'brake' that prevents abnormal self-renewal; the abnormal CpG island hypermethylation in DNMT3AWT AMLs may be an adaptive response that is ultimately overcome during leukemia progression. The absence of this 'braking' activity in AMLs with DNMT3AR882H may contribute directly to leukemia initiation. The restoration of DNMT3A activity in AML cells with the DNMT3AR882H mutation is therefore a therapeutic goal. Disclosures Spencer: Cofactor Genomics: Consultancy.

Background Biomarkers are required for pre-symptomatic diagnosis, treatment, and monitoring of neurodegenerative diseases such as Alzheimer's disease. Cerebrospinal fluid (CSF) is a favored source because its proteome reflects the composition of the brain. Ideal biomarkers have low technical and inter-individual variability (subject variance) among control subjects to minimize overlaps between clinical groups. This study evaluates a process of multi-affinity fractionation (MAF) and quantitative label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) for CSF biomarker discovery by (1) identifying reparable sources of technical variability, (2) assessing subject variance and residual technical variability for numerous CSF proteins, and (3) testing its ability to segregate samples on the basis of desired biomarker characteristics.

One of the goals of personalized medicine is to utilize biomarkers to sub-classify patients into risk groups that can be used to guide recommendations for therapy. In addition to classical risk factors, gene signatures and genomics are being developed as a means to biologically characterize tumors and to stratify patients according to the risk associated with the specific molecular aberrations present in their disease. Gene signatures and genomics are currently being investigated as a personalized medicine strategy for many cancers, but have been studied most extensively in breast cancer. In this disease, the results of genetic signatures have come to influence current recommendations for adjuvant chemotherapy for appropriately selected patient populations. In this chapter, we will review the use of gene signatures and genomics in the development of personalized oncology, with an emphasis on applications for breast cancer.

B-lineage cells reconcile the competing needs of proliferation and genome stability., DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre–B cell receptor (pre–BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre–BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre–B cells. Here, we show that RAG DSBs inhibit pre–BCR signals through the ATM- and NF-κB2–dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor, resulting in suppression of pre–BCR signaling. This regulatory circuit prevents the pre–BCR from inducing additional Igl chain gene rearrangements and driving pre–B cells with RAG DSBs into cycle. We propose that pre–B cells toggle between pre–BCR signals and a RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes.

While alpha- and beta-synuclein largely overlap in their expression in the vertebrate brain, only alpha-synuclein accumulates in the fibrillar aggregates typical of Parkinson's disease. It is thus critical to have immunological reagents that distinguish between these two protein isoforms. The monoclonal antibody Syn-1 (Transduction Labs) has been frequently used for the specific detection of alpha-synuclein. In this report, the epitope for Syn-1 is localized within residues 91-99 of human alpha-synuclein. Sequence differences exist in this domain that account for the specificity of Syn-1 for alpha- versus beta-synuclein. However, Syn-1 also displays reactivity with additional species (approximately 45 kDa) in brain homogenates from both wild-type and alpha-synuclein null mice, indicating a potential for cross-reactivity with a protein species that is unrelated to alpha-synuclein in brain tissue or extracts.

Purpose: Cervical tumor response on posttherapy 2[18F]fluoro-2-deoxy-d-glucose-positron emission tomography (FDG-PET) is predictive of survival outcome. The purpose of this study was to use gene expression profiling to identify pathways associated with tumor metabolic response. Experimental Design: This was a prospective tissue collection study for gene expression profiling of 62 pretreatment biopsies from patients with advanced cervical cancer. Patients were treated with definitive radiation. Fifty-three patients received concurrent chemotherapy. All patients underwent a pretreatment and a 3-month posttherapy FDG-PET/computed tomography (CT). Tumor RNA was harvested from fresh frozen tissue and hybridized to Affymetrix U133Plus2 GeneChips. Gene set enrichment analysis (GSEA) was used to identify signaling pathways associated with tumor metabolic response. Immunohistochemistry and in vitro FDG uptake assays were used to confirm our results. Results: There were 40 biopsies from patients with a complete metabolic response (PET-negative group) and 22 biopsies from patients with incomplete metabolic response (PET-positive group). The 3-year cause-specific survival estimates were 98% for the PET-negative group and 39% for the PET-positive group (P < 0.0001). GSEA identified alterations in expression of genes associated with the PI3K/Akt signaling pathway in patients with a positive follow-up PET. Immunohistochemistry using a tissue microarray of 174 pretreatment biopsies confirmed p-Akt as a biomarker for poor prognosis in cervical cancer. The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 inhibited FDG uptake in vitro in cervical cancer cell lines. Conclusions: Activation of the PI3K/Akt pathway is associated with incomplete metabolic response in cervical cancer. Targeted inhibition of PI3K/Akt may improve response to chemoradiation. Clin Cancer Res; 18(5); 1464–71. ©2012 AACR.

Monitoring BCR-ABL transcript levels in patients on TKI therapy using real-time quantitative PCR is standard of care in the management of CML, and achieving sensitivity to 4.5 logs below baseline is important in patients being considered for TKI discontinuation. In order to accurately assess treatment response, it is essential to ensure that variability in testing methodologies is tightly controlled. This has resulted in substantial efforts to standardize BCR-ABL molecular measurement across laboratories by introducing an International Scale (IS). Here we describe the development and analytical evaluation of Xpert® BCR-ABL Ultra, an automated cartridge-based BCR-ABL monitoring assay incorporating lot-specific standardization to the International Scale as defined by WHO. The BCR-ABL Ultra cartridge simplifies the generation of highly accurate and reproducible assay results by integrating nucleic acid extraction, target amplification, and quantification of the BCR-ABL b3a2 or b2a2 transcripts relative to ABL in peripheral blood specimens. The assay turn-around-time is approximately 2.5 hours and has a %BCR-ABL/ABL(IS) dynamic range of ~6 logs, including two key milestones for the assessment of clinical response at ~10% (IS) (SD=~0.08Log) and ~0.0033% (IS) (SD=~0.25Log) (CMR4.5). Lot-to-lot standardization is achieved using a secondary standard panel of controls derived from and aligned to the first WHO international genetic reference panel. The secondary standards are produced at Cepheid as part of the assay kit manufacturing process and applied to every production lot of cartridges in order to improve assay reliability and reporting comparability. Analytical sensitivity studies using spiked-in CML cell lines demonstrated a limit of detection (LoD) and limit of quantification (LoQ) of 0.003% BCR-ABL/ABL (IS) with a limit of blank (LoB) of 0.00017%. In an analytical specificity study, assay results were negative in 100% of 70 known CML-negative samples, including 20 AML or ALL patients and 50 healthy donors. A study was further conducted to estimate the lower limit of clinical sensitivity using blood from 12 CML patients on TKI therapy who had achieved and maintained MMR with reporting below 0.05% (IS). Eleven out of twelve low CML subjects were detected in at least 19 out of 20 replicates tested per subject over a range of 0.038% (SD=~0.17Log) to 0.0011% (IS) (SD=~0.4Log). In conclusion, the Xpert BCR-ABL Ultra assay demonstrates sensitivity in the range necessary to make clinical decisions regarding the continuation of TKI therapy in patients achieving CMR4.5, and every production lot of cartridges is assigned a unique conversion factor guaranteeing alignment to the WHO International Scale. Disclosures Uy: Novartis: Research Funding.

Assays used to monitor BCR-ABL levels in CML patients on TKI therapy should be sensitive enough to reliably detect BCR-ABL transcript levels at 4.5 logs below baseline for optimal clinical utility. Here we describe the clinical comparison of Xpert® BCR-ABL Ultra (V2) and Xpert® BCR-ABL Monitor (V1). V1 uses 200uL of peripheral blood. V2 uses 4mLs to enhance sensitivity. Both assays are currently standardized to the IS. V2 was compared to V1 using prospectively acquired CML patient specimens (N=165) in EDTA, NaCitrate, and PAXgene for a potential 495 paired comparisons (Phase I). Linear regression, Bland-Altman, and concordance at MMR and CMR4.0 analyses were performed. Using blinded precision materials (Ph+ cultured cells containing BCR-ABL e14a2 diluted into Ph- normal donor blood to produce specimens at targeted BCR-ABL1 transcript levels spanning the dynamic range from 10% to 0.001% IS), V1 and V2 were repeat tested by two operators at each of 4 sites 8 times on different days (N = 2 x 8 x 4 = 64 replicates per day; 5 sites participated on Day 1, N=80), for a total N = 272 (Phase II). 96.2% of V1, 93.7% of V2, and 99.6% of external control specimens were successful on the first attempt. Across all types of anticoagulant, linear regression shows a slope of 1.083 +/- 0.028 (95% CI) with intercept of 0.31 +/- 0.032 (95% CI) and an R2 = 94.5% for V2 log10 BCR-ABL/ABL% IS (Y) versus V1 log10 BCR-ABL/ABL% IS (X). Bland-Altman analyses reveal a mean bias across the dataset of 0.27. Positive Percent Agreement (PPA) for V2 vs. V1 was 96.4% (298/309) and Negative Percent Agreement (NPA) for V2 vs V1 was 66.9% (87/130). 39/43 specimens detected by V2 but not V1 were below 0.01% (IS). Concordance studies for MMR showed an Overall Percent Agreement (OPA = [# comparisons with both assays 0.01%]/total comparisons) > 97.0% for all comparisons and 98.0%, 93.9%, and 89.4%, respectively, for EDTA, NaCitrate, and PAXgene for CMR4.0. In Phase II, 100% (N=203) of samples at 1%-0.01% were positive by V2; 50.8% (32/63) at 0.001% were positive by V2. In conclusion, the Xpert BCR-ABL Ultra (V2) assay demonstrates concordance with version 1 of the Xpert BCR-ABL assay across a wide dynamic range in both clinical and contrived specimens. Higher discordance is noted in the range 0.01-0.001% (IS) likely due to the enhanced sensitivity of BCR-ABL Ultra (V2). Disclosures Mauro: Novartis Pharmaceutical Corporation: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Pfizer: Consultancy. Pinilla-Ibarz:Pfizer: Consultancy, Other: Consulting & Advisory Role, Research Funding, Speakers Bureau; ARIAD Pharmaceuticals, Inc.: Consultancy, Other: Consulting & Advisory Role, Research Funding; Novartis: Consultancy, Other: Consulting & Advisory Role, Research Funding; BMS: Consultancy, Honoraria, Other: Consulting & Advisory Role, Speakers Bureau; Teva: Consultancy, Speakers Bureau. Uy:Novartis: 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.

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.

Background Pilocytic Astrocytomas (PAs) are common low-grade central nervous system malignancies for which few recurrent and specific genetic alterations have been identified. In an effort to better understand the molecular biology underlying the pathogenesis of these pediatric brain tumors, we performed higher-order transcriptional network analysis of a large gene expression dataset to identify gene regulatory pathways that are specific to this tumor type, relative to other, more aggressive glial or histologically distinct brain tumours. Methods RNA derived from frozen human PA tumours was subjected to microarray-based gene expression profiling, using Affymetrix U133Plus2 GeneChip microarrays. This data set was compared to similar data sets previously generated from non-malignant human brain tissue and other brain tumour types, after appropriate normalization. Results In this study, we examined gene expression in 66 PA tumors compared to 15 non-malignant cortical brain tissues, and identified 792 genes that demonstrated consistent differential expression between independent sets of PA and non-malignant specimens. From this entire 792 gene set, we used the previously described PAP tool to assemble a core transcriptional regulatory network composed of 6 transcription factor genes (TFs) and 24 target genes, for a total of 55 interactions. A similar analysis of oligodendroglioma and glioblastoma multiforme (GBM) gene expression data sets identified distinct, but overlapping, networks. Most importantly, comparison of each of the brain tumor type-specific networks revealed a network unique to PA that included repressed expression of ONECUT2, a gene frequently methylated in other tumor types, and 13 other uniquely predicted TF-gene interactions. Conclusions These results suggest specific transcriptional pathways that may operate to create the unique molecular phenotype of PA and thus opportunities for corresponding targeted therapeutic intervention. Moreover, this study also demonstrates how integration of gene expression data with TF-gene and TF-TF interaction data is a powerful approach to generating testable hypotheses to better understand cell-type specific genetic programs relevant to cancer.

Context Whole-genome sequencing is becoming increasingly available for research purposes, but it has not yet been routinely used for clinical diagnosis. Objective To determine whether whole-genome sequencing can identify cryptic, actionable mutations in a clinically relevant time frame. Design, Setting, and Patient We were referred a difficult diagnostic case of acute promyelocytic leukemia with no pathogenic X-RARA fusion identified by routine metaphase cytogenetics or interphase fluorescence in situ hybridization (FISH). The case patient was enrolled in an institutional review board–approved protocol, with consent specifically tailored to the implications of whole-genome sequencing. The protocol uses a “movable firewall” that maintains patient anonymity within the entire research team but allows the research team to communicate medically relevant information to the treating physician. Main Outcome Measures Clinical relevance of whole-genome sequencing and time to communicate validated results to the treating physician. Results Massively parallel paired-end sequencing allowed identification of a cytogenetically cryptic event: a 77-kilobase segment from chromosome 15 was inserted en bloc into the second intron of the RARA gene on chromosome 17, resulting in a classic bcr3 PML-RARA fusion gene. Reverse transcription polymerase chain reaction sequencing subsequently validated the expression of the fusion transcript. Novel FISH probes identified 2 additional cases of t(15;17)–negative acute promyelocytic leukemia that had cytogenetically invisible insertions. Whole-genome sequencing and validation were completed in 7 weeks and changed the treatment plan for the patient. Conclusion Whole-genome sequencing can identify cytogenetically invisible oncogenes in a clinically relevant time frame.

Most patients with acute myeloid leukaemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level. To determine the mutational spectrum associated with relapse, we sequenced the primary tumour and relapse genomes from eight AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to define clonality and clonal evolution patterns precisely at relapse. In addition to discovering novel, recurrently mutated genes (for example, WAC, SMC3, DIS3, DDX41 and DAXX) in AML, we also found two major clonal evolution patterns during AML relapse: (1) the founding clone in the primary tumour gained mutations and evolved into the relapse clone, or (2) a subclone of the founding clone survived initial therapy, gained additional mutations and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific versus primary tumour mutations in all eight cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped, in part, by the chemotherapy that the patients receive to establish and maintain remissions.

Purpose: Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive sarcomas with variable patient survival and few known prognostically relevant genomic biomarkers. To identify survival-associated genomic biomarkers, we performed high-resolution array-based comparative genomic hybridization (aCGH) on a large set of MPNSTs. Experimental Design: Candidate gene alterations identified by aCGH in 38 MPNSTs were validated at the DNA, RNA, and protein levels on these same tumors and an independent set of 87 MPNST specimens. Results: aCGH revealed highly complex copy number alterations, including both previously reported and completely novel loci. Four regions of copy number gain were associated with poor patient survival. Candidate genes in these regions include SOX5 (12p12.1), NOL1 and MLF2 (12p13.31), FOXM1 and FKBP1 (12p13.33), and CDK4 and TSPAN31 (12q14.1). Alterations of these candidate genes and several others of interest (ERBB2, MYC and TP53) were confirmed by at least 1 complementary methodology, including DNA and mRNA quantitative real-time PCR, mRNA expression profiling, and tissue microarray-based fluorescence in situ hybridization and immunohistochemistry. Multivariate analysis showed that CDK4 gain/amplification and increased FOXM1 protein expression were the most significant independent predictors for poor survival in MPNST patients (P < 0.05). Conclusions: Our study provides new and independently confirmed candidate genes that could serve as genomic biomarkers for overall survival in MPNST patients. Clin Cancer Res; 17(7); 1924–34. ©2011 AACR.

Previous studies in our laboratory demonstrated that the azurophil granule protease neutrophil elastase (NE) cleaves promyelocytic leukemia–retinoic acid receptor (PML-RAR)α (PR), the fusion protein that initiates acute promyelocytic leukemia (APL). Further, NE deficiency reduces the penetrance of APL in a murine model of this disease. We therefore predicted that NE-mediated PR cleavage might be important for its ability to initiate APL. To test this hypothesis, we generated a mouse expressing NE-resistant PR. These mice developed APL indistinguishable from wild-type PR, but with significantly reduced latency (median leukemia-free survival of 274 days vs 473 days for wild-type PR, P < .001). Resistance to proteolysis may increase the abundance of full-length PR protein in early myeloid cells, and our previous data suggested that noncleaved PR may be less toxic to early myeloid cells. Together, these effects appear to increase the leukemogenicity of NE-resistant PR, contrary to our previous prediction. We conclude that NE deficiency may reduce APL penetrance via indirect mechanisms that are still NE dependent.

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML). It is characterized by the t(15;17)(q22;q11.2) chromosomal translocation that creates the promyelocytic leukemia-retinoic acid receptor α (PML-RARA) fusion oncogene. Although this fusion oncogene is known to initiate APL in mice, other cooperating mutations, as yet ill defined, are important for disease pathogenesis. To identify these, we used a mouse model of APL, whereby PML-RARA expressed in myeloid cells leads to a myeloproliferative disease that ultimately evolves into APL. Sequencing of a mouse APL genome revealed 3 somatic, nonsynonymous mutations relevant to APL pathogenesis, of which 1 (Jak1 V657F) was found to be recurrent in other affected mice. This mutation was identical to the JAK1 V658F mutation previously found in human APL and acute lymphoblastic leukemia samples. Further analysis showed that JAK1 V658F cooperated in vivo with PML-RARA, causing a rapidly fatal leukemia in mice. We also discovered a somatic 150-kb deletion involving the lysine (K)-specific demethylase 6A (Kdm6a, also known as Utx) gene, in the mouse APL genome. Similar deletions were observed in 3 out of 14 additional mouse APL samples and 1 out of 150 human AML samples. In conclusion, whole genome sequencing of mouse cancer genomes can provide an unbiased and comprehensive approach for discovering functionally relevant mutations that are also present in human leukemias.

Natural killer (NK) cells were identified by their ability to kill target cells without previous sensitization. However, without an antecedent “arming” event, NK cells can recognize, but are not equipped to kill, target cells. How NK cells become armed in vivo in healthy hosts is unclear. Because latent herpesviruses are highly prevalent and alter multiple aspects of host immunity, we hypothesized that latent herpesvirus infection would arm NK cells. Here we show that NK cells from mice latently infected with Murid herpesvirus 4 (MuHV-4) were armed as evidenced by increased granzyme B protein expression, cytotoxicity, and interferon-γ production. NK-cell arming occurred rapidly in the latently infected host and did not require acute viral infection. Furthermore, NK cells armed by latent infection protected the host against a lethal lymphoma challenge. Thus, the immune environment created by latent herpesvirus infection provides a mechanism whereby host NK-cell function is enhanced in vivo.

The full complement of DNA mutations that are responsible for the pathogenesis of acute myeloid leukemia (AML) is not yet known.We used massively parallel DNA sequencing to obtain a very high level of coverage (approximately 98%) of a primary, cytogenetically normal, de novo genome for AML with minimal maturation (AML-M1) and a matched normal skin genome.We identified 12 acquired (somatic) mutations within the coding sequences of genes and 52 somatic point mutations in conserved or regulatory portions of the genome. All mutations appeared to be heterozygous and present in nearly all cells in the tumor sample. Four of the 64 mutations occurred in at least 1 additional AML sample in 188 samples that were tested. Mutations in NRAS and NPM1 had been identified previously in patients with AML, but two other mutations had not been identified. One of these mutations, in the IDH1 gene, was present in 15 of 187 additional AML genomes tested and was strongly associated with normal cytogenetic status; it was present in 13 of 80 cytogenetically normal samples (16%). The other was a nongenic mutation in a genomic region with regulatory potential and conservation in higher mammals; we detected it in one additional AML tumor. The AML genome that we sequenced contains approximately 750 point mutations, of which only a small fraction are likely to be relevant to pathogenesis.By comparing the sequences of tumor and skin genomes of a patient with AML-M1, we have identified recurring mutations that may be relevant for pathogenesis.

Antiapoptotic BCL2 family members have been implicated in the pathogenesis of acute myelogenous leukemia (AML), but the functional significance and relative importance of individual proteins (e.g., BCL2, BCL-XL, and myeloid cell leukemia 1 [MCL1]) remain poorly understood. Here, we examined the expression of BCL2, BCL-XL, and MCL1 in primary human hematopoietic subsets and leukemic blasts from AML patients and found that MCL1 transcripts were consistently expressed at high levels in all samples tested. Consistent with this, Mcl1 protein was also highly expressed in myeloid leukemic blasts in a mouse Myc-induced model of AML. We used this model to test the hypothesis that Mcl1 facilitates AML development by allowing myeloid progenitor cells to evade Myc-induced cell death. Indeed, activation of Myc for 7 days in vivo substantially increased myeloid lineage cell numbers, whereas hematopoietic stem, progenitor, and B-lineage cells were depleted. Furthermore, Mcl1 haploinsufficiency abrogated AML development. In addition, deletion of a single allele of Mcl1 from fully transformed AML cells substantially prolonged the survival of transplanted mice. Conversely, the rapid lethality of disease was restored by coexpression of Bcl2 and Myc in Mcl1-haploinsufficient cells. Together, these data demonstrate a critical and dose-dependent role for Mcl1 in AML pathogenesis in mice and suggest that MCL1 may be a promising therapeutic target in patients with de novo AML.

Activating mutations in tyrosine kinase (TK) genes (eg, FLT3 and KIT) are found in more than 30% of patients with de novo acute myeloid leukemia (AML); many groups have speculated that mutations in other TK genes may be present in the remaining 70%. We performed high-throughput resequencing of the kinase domains of 26 TK genes (11 receptor TK; 15 cytoplasmic TK) expressed in most AML patients using genomic DNA from the bone marrow (tumor) and matched skin biopsy samples (“germline”) from 94 patients with de novo AML; sequence variants were validated in an additional 94 AML tumor samples (14.3 million base pairs of sequence were obtained and analyzed). We identified known somatic mutations in FLT3, KIT, and JAK2 TK genes at the expected frequencies and found 4 novel somatic mutations, JAK1V623A, JAK1T478S, DDR1A803V, and NTRK1S677N, once each in 4 respective patients of 188 tested. We also identified novel germline sequence changes encoding amino acid substitutions (ie, nonsynonymous changes) in 14 TK genes, including TYK2, which had the largest number of nonsynonymous sequence variants (11 total detected). Additional studies will be required to define the roles that these somatic and germline TK gene variants play in AML pathogenesis.

Somatic mutations in JAK2 are frequently found in myeloproliferative diseases, and gain-of-function JAK3 alleles have been identified in M7 acute myeloid leukemia (AML), but a role for JAK1 in AML has not been described. We screened the entire coding region of JAK1 by total exonic resequencing of bone marrow DNA samples from 94 patients with de novo AML. We identified 2 novel somatic mutations in highly conserved residues of the JAK1 gene (T478S, V623A), in 2 separate patients and confirmed these by resequencing germ line DNA samples from the same patients. Overexpression of mutant JAK1 did not transform primary murine cells in standard assays, but compared with wild-type JAK1, JAK1T478S, and JAK1V623A expression was associated with increased STAT1 activation in response to type I interferon and activation of multiple downstream signaling pathways. This is the first report to demonstrate somatic JAK1 mutations in AML and suggests that JAK1 mutations may function as disease-modifying mutations in AML pathogenesis.

Background Biomarkers are required for pre-symptomatic diagnosis, treatment, and monitoring of neurodegenerative diseases such as Alzheimer's disease. Cerebrospinal fluid (CSF) is a favored source because its proteome reflects the composition of the brain. Ideal biomarkers have low technical and inter-individual variability (subject variance) among control subjects to minimize overlaps between clinical groups. This study evaluates a process of multi-affinity fractionation (MAF) and quantitative label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) for CSF biomarker discovery by (1) identifying reparable sources of technical variability, (2) assessing subject variance and residual technical variability for numerous CSF proteins, and (3) testing its ability to segregate samples on the basis of desired biomarker characteristics. Methods/Results Fourteen aliquots of pooled CSF and two aliquots from six cognitively normal individuals were randomized, enriched for low-abundance proteins by MAF, digested endoproteolytically, randomized again, and analyzed by nano-LC-MS. Nano-LC-MS data were time and m/z aligned across samples for relative peptide quantification. Among 11,433 aligned charge groups, 1360 relatively abundant ones were annotated by MS2, yielding 823 unique peptides. Analyses, including Pearson correlations of annotated LC-MS ion chromatograms, performed for all pairwise sample comparisons, identified several sources of technical variability: i) incomplete MAF and keratins; ii) globally- or segmentally-decreased ion current in isolated LC-MS analyses; and iii) oxidized methionine-containing peptides. Exclusion of these sources yielded 609 peptides representing 81 proteins. Most of these proteins showed very low coefficients of variation (CV

Abstract 524 Understanding epigenetic mechanisms of gene regulation will provide an unprecedented opportunity for therapeutic intervention in cancer because, unlike genetic lesions, pathogenic changes to the epigenome are reversible. Non-Hodgkin Lymphoma (NHL), which strikes 70,000 Americans annually, is characterized by deregulated expression of large gene cohorts that mediate unchecked cell growth, the molecular basis of which remains poorly understood. Recently, recurrent somatic mutations were identified in chromatin modifier genes (EZH2, MLL2, EP300) in ∼30% of NHL, suggesting that epigenetic dysregulation may be a common mechanism for widespread gene expression changes. Indeed, previous studies have demonstrated evidence of aberrant DNA methylation in lymphoma. Moreover, NHL-specific regulatory elements are attractive candidates for therapeutic targeting, because reversal of their aberrant epigenetic profile would restore normal gene expression, while leaving normal cells intact. In this study, we performed integrative epigenomic, genomic, and transcriptomic profiling of purified CD19+ B cells from excisional lymph node biopsies and peripheral blood (PB, when available and free of circulating lymphoma) in 84 patients with NHL (Diffuse Large B cell lymphoma (DLBCL) N=34, Follicular Lymphoma (FL) N=38, Marginal Zone/Mantle Cell/Chronic Lymphocytic Leukemia (MZL/MCL/CLL) N=12), 20 patients undergoing elective tonsillectomy (tonsil B cells), and 40 healthy volunteers (PB). To enrich for key changes in the NHL epigenome, crosslinked chromatin was subjected to formaldehyde-assisted isolation of regulatory elements (FAIRE), for highly active histone-depleted regions, and chromatin immunoprecipitation (ChIP) for several activating (H3K4me1, H3K27ac, H3K9/14ac), and repressive (H3K27me3) histone marks, followed by high throughput sequencing (FAIRE/ChIP-Seq). To identify genes regulated by putative NHL-specific regulatory elements, we profiled gene expression (mi/mRNA) by microarray and, in primary tumors, by RNA-Seq to detect novel isoforms, translocations, and mutations, such as those in epigenetic modifier genes. In order to analyze this complex dataset, we created a global, integrative algorithm to evaluate the regulatory potential of a given epigenetic element, or set of elements, based on a step-wise approach. All regulatory elements were ranked according to several parameters, including distance to transcription start sites (TSS), gene expression level, chromatin state, distance to CTCF sites, genomic copy number, degree of sequence conservation, association with mutations in epigenetic modifiers, and most importantly, NHL-specificity and recurrence (number of samples). We first established the NHL-specificity of each regulatory element by comparison to matched PBB, if available, or to healthy donor B cells, identifying thousands of regulatory elements that are differentially enriched (“gained” or “lost”) in at least one primary NHL sample. We further assessed the likely role of each element by its genomic, epigenomic, and transcriptomic contexts. Gained elements were significantly associated with higher levels of gene expression in NHL compared to normal B cells (p Disclosures: No relevant conflicts of interest to declare.

Abstract 404 To characterize the genomic events associated with distinct subtypes of AML, we used whole genome sequencing to compare 24 tumor/normal sample pairs from patients with normal karyotype (NK) M1-AML (12 cases) and t(15;17)-positive M3-AML (12 cases). All single nucleotide variants (SNVs), small insertions and deletions (indels), and cryptic structural variants (SVs) identified by whole genome sequencing (average coverage 28x) were validated using sample-specific custom Nimblegen capture arrays, followed by Illumina sequencing; an average coverage of 972 reads per somatic variant yielded 10,597 validated somatic variants (average 421/genome). Of these somatic mutations, 308 occurred in 286 unique genes; on average, 9.4 somatic mutations per genome had translational consequences. Several important themes emerged: 1) AML genomes contain a diverse range of recurrent mutations. We assessed the 286 mutated genes for recurrency in an additional 34 NK M1-AML cases and 9 M3-AML cases. We identified 51 recurrently mutated genes, including 37 that had not previously been described in AML; on average, each genome had 3 recurrently mutated genes (M1 = 3.2; M3 = 2.8, p = 0.32). 2) Many recurring mutations cluster in mutually exclusive pathways, suggesting pathophysiologic importance. The most commonly mutated genes were: FLT3 (36%), NPM1 (25%), DNMT3A (21%), IDH1 (18%), IDH2 (10%), TET2 (10%), ASXL1 (6%), NRAS (6%), TTN (6%), and WT1 (6%). In total, 3 genes (excluding PML-RARA) were mutated exclusively in M3 cases. 22 genes were found only in M1 cases (suggestive of alternative initiating mutations which occurred in methylation, signal transduction, and cohesin complex genes). 25 genes were mutated in both M1 and M3 genomes (suggestive of common progression mutations relevant for both subtypes). A single mutation in a cell growth/signaling gene occurred in 38 of 67 cases (FLT3, NRAS, RUNX1, KIT, CACNA1E, CADM2, CSMD1); these mutations were mutually exclusive of one another, and many of them occurred in genomes with PML-RARA, suggesting that they are progression mutations. We also identified a new leukemic pathway: mutations were observed in all four genes that encode members of the cohesin complex (STAG2, SMC1A, SMC3, RAD21), which is involved in mitotic checkpoints and chromatid separation. The cohesin mutations were mutually exclusive of each other, and collectively occur in 10% of non-M3 AML patients. 3) AML genomes also contain hundreds of benign “passenger” mutations. On average 412 somatic mutations per genome were translationally silent or occurred outside of annotated genes. Both M1 and M3 cases had similar total numbers of mutations per genome, similar mutation types (which favored C>T/G>A transitions), and a similar random distribution of variants throughout the genome (which was affected neither by coding regions nor expression levels). This is consistent with our recent observations of random “passenger” mutations in hematopoietic stem cell (HSC) clones derived from normal patients (Ley et al manuscript in preparation), and suggests that most AML-associated mutations are not pathologic, but pre-existed in the HSC at the time of initial transformation. In both studies, the total number of SNVs per genome correlated positively with the age of the patient (R2 = 0.48, p = 0.001), providing a possible explanation for the increasing incidence of AML in elderly patients. 4) NK M1 and M3 AML samples are mono- or oligo-clonal. By comparing the frequency of all somatic mutations within each sample, we could identify clusters of mutations with similar frequencies (leukemic clones) and determined that the average number of clones per genome was 1.8 (M1 = 1.5; M3 = 2.2; p = 0.04). 5) t(15;17) is resolved by a non-homologous end-joining repair pathway, since nucleotide resolution of all 12 t(15;17) breakpoints revealed inconsistent micro-homologies (0 – 7 bp). Summary: These data provide a genome-wide overview of NK and t(15;17) AML and provide important new insights into AML pathogenesis. AML genomes typically contain hundreds of random, non-genic mutations, but only a handful of recurring mutated genes that are likely to be pathogenic because they cluster in mutually exclusive pathways; specific combinations of recurring mutations, as well as rare and private mutations, shape the leukemia phenotype in an individual patient, and help to explain the clinical heterogeneity of this disease. Disclosures: Westervelt: Novartis: Speakers Bureau.

Context The identification of patients with inherited cancer susceptibility syndromes facilitates early diagnosis, prevention, and treatment. However, in many cases of suspected cancer susceptibility, the family history is unclear and genetic testing of common cancer susceptibility genes is unrevealing. Objective To apply whole-genome sequencing to a patient without any significant family history of cancer but with suspected increased cancer susceptibility because of multiple primary tumors to identify rare or novel germline variants in cancer susceptibility genes. Design, Setting, and Participant Skin (normal) and bone marrow (leukemia) DNA were obtained from a patient with early-onset breast and ovarian cancer (negative for BRCA1 and BRCA2 mutations) and therapy-related acute myeloid leukemia (t-AML) and analyzed with the following: whole-genome sequencing using paired-end reads, single-nucleotide polymorphism (SNP) genotyping, RNA expression profiling, and spectral karyotyping. Main Outcome Measures Structural variants, copy number alterations, single-nucleotide variants, and small insertions and deletions (indels) were detected and validated using the described platforms. Results Whole-genome sequencing revealed a novel, heterozygous 3-kilobase deletion removing exons 7-9 of TP53 in the patient's normal skin DNA, which was homozygous in the leukemia DNA as a result of uniparental disomy. In addition, a total of 28 validated somatic single-nucleotide variations or indels in coding genes, 8 somatic structural variants, and 12 somatic copy number alterations were detected in the patient's leukemia genome. Conclusion Whole-genome sequencing can identify novel, cryptic variants in cancer susceptibility genes in addition to providing unbiased information on the spectrum of mutations in a cancer genome.

Abstract 99 Whole genome sequencing with next generation technologies represents a new, unbiased approach for discovering somatic variations in cancer genomes. Our group recently reported the DNA sequence and analysis of the genomes of two patients with normal karyotype acute myeloid leukemia (AML). Improvements in next generation sequencing technologies (principally, paired-end sequencing) led us to reevaluate the first case (Ley et al, Nature 456:66–72, 2008) with deeper sequence coverage. We discovered a novel frameshift mutation in DNMT3A, one of the three genes in humans (DNMT1, DNMT3A, and DNMT3B) that encodes a DNA methyltransferase that catalyzes the addition of methyl groups to cytosine within CpG dinucleotides. We then sequenced all the coding exons of this gene in 280 additional de novo cases of AML to define recurring mutations. 62/281 de novo AML cases (22%) had mutations with translational effects in the DNMT3A gene. 18 different missense mutations were identified, the most common of which was at amino acid R882 (37 cases). Frameshifts (n=6), nonsense mutations (n=6), splice site mutations (n=3), and a 1.5 Mbp deletion that included the DNMT3A gene were also identified. DNMT3A mutations were highly enriched in cases with intermediate risk cytogenetics (56/166=33.7%; p Disclosures: Westervelt: Novartis: Honoraria; Celgene: Honoraria, Speakers Bureau. DiPersio:Genzyme: Honoraria.