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In many areas of oncology, we lack sensitive tools to track low-burden disease. Although cell-free DNA (cfDNA) shows promise in detecting cancer mutations, we found that the combination of low tumor fraction (TF) and limited number of DNA fragments restricts low-disease-burden monitoring through the prevailing deep targeted sequencing paradigm. We reasoned that breadth may supplant depth of sequencing to overcome the barrier of cfDNA abundance. Whole-genome sequencing (WGS) of cfDNA allowed ultra-sensitive detection, capitalizing on the cumulative signal of thousands of somatic mutations observed in solid malignancies, with TF detection sensitivity as low as 10−5. The WGS approach enabled dynamic tumor burden tracking and postoperative residual disease detection, associated with adverse outcome. Thus, we present an orthogonal framework for cfDNA cancer monitoring via genome-wide mutational integration, enabling ultra-sensitive detection, overcoming the limitation of cfDNA abundance and empowering treatment optimization in low-disease-burden oncology care. A new approach for whole-genome sequencing of plasma circulating tumor DNA allows for dynamic monitoring of disease burden and ultra-sensitive detection of minimal residual disease.

DNA methylation patterns in the genome both reflect and help to mediate transcriptional regulatory processes. The digital nature of DNA methylation, present or absent on each allele, makes this assay capable of quantifying events in subpopulations of cells, whereas genome-wide chromatin studies lack the same quantitative capacity. Testing DNA methylation throughout the genome is possible using whole-genome bisulfite sequencing (WGBS), but the high costs associated with the assay have made it impractical for studies involving more than limited numbers of samples. We have optimized a new transposase-based library preparation assay for the Illumina HiSeq X platform suitable for limited amounts of DNA and providing a major cost reduction for WGBS. By incorporating methylated cytosines during fragment end repair, we reveal an end-repair artifact affecting 1%–2% of reads that we can remove analytically. We show that the use of a high (G + C) content spike-in performs better than PhiX in terms of bisulfite sequencing quality. As expected, the loci with transposase-accessible chromatin are DNA hypomethylated and enriched in flanking regions by post-translational modifications of histones usually associated with positive effects on gene expression. Using these transposase-accessible loci to represent the cis-regulatory loci in the genome, we compared the representation of these loci between WGBS and other genome-wide DNA methylation assays, showing WGBS to outperform substantially all of the alternatives. We conclude that it is now technologically and financially feasible to perform WGBS in larger numbers of samples with greater accuracy than previously possible.

The biological role of extracellular vesicles (EVs) in diffuse large B-cell lymphoma (DLBCL) initiation and progression remains largely unknown. We characterized EVs secreted by 5 DLBCL cell lines, a primary DLBCL tumor, and a normal control B-cell sample, optimized their purification, and analyzed their content. We found that DLBCLs secreted large quantities of CD63, Alix, TSG101, and CD81 EVs, which can be extracted using an ultracentrifugation-based method and traced by their cell of origin surface markers. We also showed that tumor-derived EVs can be exchanged between lymphoma cells, normal tonsillar cells, and HK stromal cells. We then examined the content of EVs, focusing on isolation of high-quality total RNA. We sequenced the total RNA and analyzed the nature of RNA species, including coding and noncoding RNAs. We compared whole-cell and EV-derived RNA composition in benign and malignant B cells and discovered that transcripts from EVs were involved in many critical cellular functions. Finally, we performed mutational analysis and found that mutations detected in EVs exquisitely represented mutations in the cell of origin. These results enhance our understanding and enable future studies of the role that EVs may play in the pathogenesis of DLBCL, particularly with regards to the exchange of genomic information. Current findings open a new strategy for liquid biopsy approaches in disease monitoring.

SummaryChanges in DNA methylation are required for the formation of germinal centers (GCs), but the mechanisms of such changes are poorly understood. Activation-induced cytidine deaminase (AID) has been recently implicated in DNA demethylation through its deaminase activity coupled with DNA repair. We investigated the epigenetic function of AID in vivo in germinal center B cells (GCBs) isolated from wild-type (WT) and AID-deficient (Aicda−/−) mice. We determined that the transit of B cells through the GC is associated with marked locus-specific loss of methylation and increased methylation diversity, both of which are lost in Aicda−/− animals. Differentially methylated cytosines (DMCs) between GCBs and naive B cells (NBs) are enriched in genes that are targeted for somatic hypermutation (SHM) by AID, and these genes form networks required for B cell development and proliferation. Finally, we observed significant conservation of AID-dependent epigenetic reprogramming between mouse and human B cells.

The Illumina HiSeq X platform has helped to reduce the cost of whole genome sequencing substantially, but its application for bisulphite sequencing is not straightforward. We describe the optimization of a library preparation and sequencing approach that maximizes the yield and quality of sequencing, and the elimination of a previously unrecognized artefact affecting several percent of bisulphite sequencing reads.

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

MicroRNAs (miRNAs) are evolutionarily conserved, 18- to 25-nucleotide, non–protein coding transcripts that posttranscriptionally regulate gene expression during development. miRNAs also occur in postmitotic cells, such as neurons in the mammalian central nervous system, but their function is less well characterized. We investigated the role of miRNAs in mammalian midbrain dopaminergic neurons (DNs). We identified a miRNA, miR-133b, that is specifically expressed in midbrain DNs and is deficient in midbrain tissue from patients with Parkinson's disease. miR-133b regulates the maturation and function of midbrain DNs within a negative feedback circuit that includes the paired-like homeodomain transcription factor Pitx3. We propose a role for this feedback circuit in the fine-tuning of dopaminergic behaviors such as locomotion.

Exosomes are membrane-bound vesicles that can be exchanged between cells and have been shown to modify the tumor microenvironment and contribute to progression of solid tumors. Little research has been done on exosomes in lymphoma and their potential biological role in tumor initiation and progression remains unknown. We characterized exosomes secreted by six DLBCL cell lines, four primary DLBCL tumors, and two normal control B cell samples. We optimized their purification and studied their nucleic acid content. We then determined that tumor-derived exosomes can be exchanged not only between tumor and immune cells, as has been shown before, but also between lymphoma cells with various biological consequences. Finally, we sequenced exosomal RNAs in order to better understand their function and role in lymphoma biology. We used serial ultracentrifugation to purify exosomes and microvesicles from six DLBCL cell lines (LY1, LY3, LY7, LY18, HBL1, TMD8). We isolated exosomes from four primary DLBCLs and two control tonsillar samples enriched for CD20+ B cells using the Total Exosome Isolation from Cell Culture Kit (Invitrogen). We found that the six DLBCL cell lines secrete large quantities of CD63+ exosomes. These exosomes also express the B-cell specific marker CD20, but not T cell marker CD3, which could enable tracing of the cell of origin and differential purification. Exosomes are known to contain small RNAs, which can be extracted with different efficiency depending on the isolation method used. We tested three approaches for nucleic acid isolation: Direct-zol™ and TRIzol® methods which were both followed by column purification, and miRCURY™ RNA isolation kit (Exiqon). We detected no traceable quantities of DNA in exosomal contents. The miRCURY™ RNA isolation kit yielded the highest amounts of RNAs of the techniques used and preserved sizes from 4-150 nucleotides (nt) in length with equal efficiency. Exosomes contained predominantly small RNAs with 6-80 nt species which constituted 9-28% of isolated RNA. We then prepared cDNA libraries using TruSeq Stranded Total kit (Illumina) and sequenced libraries using 50 bp Paired End approach on Illumina HiSeq 2000. We developed a small RNA-seq Analysis Pipeline that included QC, removing Illumina TruSeq adapters, mapping the reads, summarizing the data, and assigning reads to annotated genes. Our results indicate that exosomal RNAs contain protein coding RNAs (50-60%) and antisense RNAs (20-30%) as the most common biotypes. We also evaluated the exchange of exosomal content between DLBCL cell lines (LY1, LY3). The cells were stained with SYTO® RNASelect™ dye (Molecular Probes Inc) and cultured for 48 hours. The supernatant was collected, filtered, and cultured with unstained cells from the alternate DLBCL cell line for 48 hours. We observed uptake of LY1 exosomes by LY3 cells and LY3 exosomes by LY1 cells, as evidenced by SYTO® RNASelect™ dye transfer and CD63 positivity on FACS analysis. In addition, we tested the effect of exosomal exchange on resistance to doxorubicin, given the varied susceptibilities of LY1 and LY3 cell lines to this drug which is a part of standard frontline treatment for DLBCL. After culturing cells with filtered supernatant from the alternate cell line for 48 hours, we treated with doxorubicin and assessed for cell viability at 48 hours. We also studied the effect of exosome uptake on proliferation and cell cycle of recipient cells. Our results indicate that exosomal exchange between tumor cells and from tumor to normal B cells can modulate fundamental biological processes including drug resistance as well as cell proliferation, survival, and induction of apoptosis. We believe this is related to incorporation of exosomal RNA with resulting effects on the transcriptome of recipient cells. RNA sequencing analysis will aid in the design of additional functional studies in order to further elucidate the biological role of exosomes in DLBCL. Disclosures No relevant conflicts of interest to declare.

Treatment failure in Acute Myeloid Leukemia (AML) is attributed in many cases to relapsed disease. Relapsed AML is a fundamental clinical challenge since most patients have poor clinical outcomes. The exact biological basis of AML relapse remains unclear. Genetic clonal evolution is widely believed to underlie the emergence of chemotherapy resistant clones. However, only limited, predominantly non-overlapping, somatic mutations and copy number aberrations were found to occur upon AML relapse. Furthermore, in a subset of cases, no relapse specific somatic mutations or copy number aberrations were identified. This suggests a role for other mechanisms in relapsed AML. We hypothesize that epigenetic plasticity and deregulation contributes to the pathogenesis of relapse in AML. To explore this notion, we performed a genome scale epigenetic and genetic analysis of thirty-nine paired diagnosis and relapsed AML human patient samples using exome capture, RNA-seq and ERRBS for DNA methylation sequencing. Exome capture was performed on each patient’s germline DNA as well. Exome capture revealed only a limited number of known recurrent somatic mutations acquired upon disease relapse, in agreement with previous reports. In contrast, upon disease relapse we identified thousands of statistically significant changes in cytosine methylation patterns. Globally, the majority of patients (85%) displayed striking predominance of DNA hypermethylation (p= 1.00433e-05, binomial test for equality of proportions) upon disease relapse. Notably a smaller set of patients displayed the opposite epigenetic phenotype with prominent loss of cytosine methylation. While differential methylation in the hypermethylated group of patients localized predominantly to CpG islands, the majority of differential methylation in the hypomethylated group localized to regions lacking both CpG islands and shores. In spite of these two distinct overall cytosine methylation patterns, the majority of differentially methylated cytosines are located in intergenic regions in all cases, and a subset of promoters were hypermethylated in almost all patients at relapse. A pathway analysis indicated that the commonly hypermethylated gene promoters at relapse are involved in the Hedghog, Wnt and calcium signaling pathways (p In order to determine whether epigenetic events linked to AML relapse could be modeled experimentally we performed a pilot study of a human AML xenograft in immunocompromised mice. Engrafted mice were treated with Ara-C at a clinically relevant dose (60mg/Kg; n=2) or vehicle alone (n=3) for five consecutive days. Human AML cells were collected at various timepoints including 28 days after Ara-C treatment where the AML had frankly relapsed in mice. Cytosine methylation profiles obtained through ERRBS revealed predominantly hypermethylated cytosines when compared to the xenotransplanted diagnostic sample (72% hypermethylated versus 28% hypomethylated). Remarkably, there was a strong overlap with gene promoters that are also aberrantly methylated in relapsed AML patients (p We conclude that there are epigenetically distinct forms of relapsed AML. Nonetheless, there is convergent epigenetic regulation of specific gene pathways that may contribute to relapsed AML pathogenesis and xenotransplanted AML mice can serve as experimental models for further study. Finally, the genomic distribution of reprogrammed methylation suggests a role for epigenetic plasticity at distal regulatory elements. Whereas it remains unclear whether these changes represent clonal selection, their extensive and dynamic range suggest that exposure to chemotherapy may alter the fidelity of mechanisms that control cytosine methylation distribution thus permitting widespread and distant epigenetic reprogramming and contributing to disease relapse. Disclosures: No relevant conflicts of interest to declare.