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The standard treatment approach for stage II/III rectal cancer is neoadjuvant chemoradiation therapy (nCRT) followed by surgery. In recent years, new treatment approaches have led to higher rates of complete tumor eradication combined with organ-preservation strategies. However, better tools are still needed to personalize therapy for the individual patient. In this prospective observational study, we analyzed colon-derived cell-free (cf)DNA (c-cfDNA) using a tissue-specific DNA methylation signature, and its association with therapy outcomes. Analyzing plasma samples (n = 303) collected during nCRT from 37 patients with locally advanced rectal cancer (LARC), we identified colon-specific methylation markers that discriminated healthy individuals from patients with untreated LARC (area under the curve, 0.81; 95% confidence interval, 0.70-0.92; P 0.0001). Baseline c-cfDNA predicted tumor response, with increased levels linked to larger residual cancer. c-cfDNA measured after the first week of therapy identified patients with maximal response and complete cancer eradication, who had significantly lower c-cfDNA compared with those who had residual disease (8.6 vs 57.7 average copies/mL, respectively; P = 0.013). Increased c-cfDNA after one week of therapy was also associated with disease recurrence. Methylation-based liquid biopsy can predict nCRT outcomes and facilitate patient selection for escalation and de-escalation strategies.

The use of cardiopulmonary bypass (CPB) is thought to cause delayed cardiac damage. DNA methylation-based liquid biopsies are novel biomarkers for monitoring acute cardiac cell death. We assessed cell-free DNA molecules as markers for cardiac damage after open-heart surgery. Novel cardiomyocyte-specific DNA methylation markers were applied to measure cardiac cfDNA in the plasma of 42 infants who underwent open-heart surgery. Cardiac cfDNA was elevated following surgery, reflecting direct surgery-related tissue damage, and declined thereafter in most patients. The concentration of cardiac cfDNA post-surgery correlated with the duration of CPB and aortic cross clamping. Strikingly, cardiac cfDNA at 6 h predicted duration of mechanical ventilation and maximal vasoactive-inotropic score better than did maximal troponin levels. Cardiac cfDNA reveals heart damage associated with CPB, and can be used to monitor cardiac cell death, to predict clinical outcome of surgery and to assess performance of cardioprotective interventions.

PDF file - 3639K, Representative images of pancreatic lesions in wild type mice implanted with DMBA, or in Pdx1-Cre; LSL-Kras G12D mice.

PDF file - 56K, Proliferation rate analysis.

PDF file - 39K, Pathologic score of PanIN lesions in Pdx1-Cre; LSL-KRAS G12D mice, wild type (n=4 mice) or heterozygous (n=7) for the phospho-rpS6 mutant.

PDF file - 66K

PDF file - 44K, Staining for γH2AX (brown) in sections of the intestine from a CK1α-deficient mouse

PDF file - 47K, Mosaic phosphorylation of rpS6 in wild type islets.

Radiotherapy has an important role in the treatment of brain metastases (BrM) but carries risk of short and/or long-term toxicity, termed radiation-induced neurotoxicity (RIN). As the diagnosis of RIN is crucial for correct pa-tient management, there is an unmet need for reliable biomarkers for RIN. The aim of this proof-of concept study was to determine the utility of brain-derived circulating free DNA (BncfDNA), identified by specific methylation patterns for neurons, astrocytes, and oligodendrocytes, as RIN biomarkers. Twenty-four patients with BrM were monitored clinically and radiologically before, during and after brain radiotherapy, and blood for BncfDNA analysis (98 samples) was concurrently collected. Sixteen patients were treated with whole brain radiotherapy and eight patients with stereotactic radiosurgery. During follow-up nine RIN events were detected, and all correlated with significant increase in BncfDNA levels compared to baseline. Additionally, resolution of RIN correlated with de-crease in BncfDNA. Changes in BncfDNA were independent of tumor response. Elevated BncfDNA levels reflects brain cell injury incurred by radiotherapy, further research is needed to establish BncfDNA as a novel plasma-based biomarker for RIN.

SummaryChronological age prediction from DNA methylation sheds light on human aging, indicates poor health and predicts lifespan. Current clocks are mostly based on linear models from hundreds of methylation sites, and are not suitable for sequencing-based data.We present GP-age, an epigenetic clock for blood, that uses a non-linear cohort-based model of 11,910 blood methylomes. Using 30 CpG sites alone, GP-age outperforms state-of-the-art models, with a median accuracy of ~2 years on held-out blood samples, for both array and sequencing-based data. We show that aging-related changes occur at multiple neighboring CpGs, with far-reaching implications on aging research at the cellular level. By training three independent clocks, we show consistent deviations between predicted and actual age, suggesting individual rates of biological aging.Overall, we provide a compact yet accurate alternative to array-based clocks for blood, with future applications in longitudinal aging research, forensic profiling, and monitoring epigenetic processes in transplantation medicine and cancer.Graphical abstractMachine learning analysis of a large cohort (~12K) of DNA methylomes from bloodA 30-CpG regression model achieves a 2.1-year median error in predicting ageImproved accuracy (≥1.75 years) from sequencing data, using neighboring CpGsPaves the way for easy and accurate age prediction from blood, using NGS dataMotivationEpigenetic clocks that predict age from DNA methylation are a valuable tool in the research of human aging, with additional applications in forensic profiling, disease monitoring, and lifespan prediction. Most existing epigenetic clocks are based on linear models and require hundreds of methylation sites. Here, we present a compact epigenetic clock for blood, which outperforms state-of-the-art models using only 30 CpG sites. Finally, we demonstrate the applicability of our clock to sequencing-based data, with far reaching implications for a better understanding of epigenetic aging.

A major hypothesis for the etiology of type 1 diabetes (T1D) postulates initiation by viral infection, leading to double-stranded RNA (dsRNA)-mediated interferon response; however, a causal virus has not been identified. Here we use a mouse model, corroborated with human data, to demonstrate that endogenous dsRNA in beta-cells can lead to a diabetogenic immune response, thus identifying a virus-independent mechanism for T1D initiation. We found that disruption of the RNA editing enzyme ADAR in beta-cells triggers a massive interferon response, islet inflammation and beta-cell failure, with features bearing striking similarity to early-stage human T1D. Glycolysis via calcium enhances the interferon response, suggesting an actionable vicious cycle of inflammation and increased beta-cell workload.One sentence summaryAdar inactivation in beta-cells triggers a glucose-dependent interferon response causing insulitis and diabetes

Type 2 diabetes (T2D) is associated with compromised identity of insulin-producing pancreatic islet beta (β) cells, characterized by inappropriate production of other islet cell-enriched hormones. Here we examined how hormone misexpression was influenced by the MAFA and MAFB transcription factors, closely related proteins that maintain islet cell function. Mice specifically lacking MafA in β cells demonstrated broad, population-wide changes in hormone gene expression with an overall gene signature closely resembling islet gastrin (Gast)-positive cells generated under conditions of chronic hyperglycemia and obesity. A human β cell line deficient in MAFB, but not one lacking MAFA, also produced a gastrin (GAST)-positive gene expression pattern. In addition, GAST was detected in human T2D β cells with low levels of MAFB. Moreover, evidence is provided that human MAFB can directly repressGASTgene transcription. These results support a novel, species-specific role for MafA and MAFB in maintaining adult mouse and human β cell identity, respectively, by repressing expression of Gast/GAST and other non-β cell hormones.

Circulating cell-free DNA (cfDNA) fragments are a biological analyte with extensive utility in diagnostic medicine. Understanding the source of cfDNA and mechanisms of release is crucial for designing and interpreting cfDNA-based liquid biopsy assays. Using cell type-specific methylation markers as well as genome-wide methylation analysis, we determined that megakaryocytes, the precursors of anuclear platelets, are major contributors to cfDNA (∼26%), while erythroblasts contribute 1-4% of cfDNA in healthy individuals. Surprisingly, we discovered that platelets contain genomic DNA fragments originating in megakaryocytes, contrary to the general understanding that platelets lack genomic DNA. Megakaryocyte-derived cfDNA is increased in pathologies involving increased platelet production (Essential Thrombocythemia, Idiopathic Thrombocytopenic Purpura) and decreased upon reduced platelet production due to chemotherapy-induced bone marrow suppression. Similarly, erythroblast cfDNA is reflective of erythrocyte production and is elevated in patients with Thalassemia. Megakaryocyte- and erythroblast-specific DNA methylation patterns can thus serve as novel biomarkers for pathologies involving increased or decreased thrombopoiesis and erythropoiesis, which can aid in determining the etiology of aberrant levels of erythrocytes and platelets.GRAPHICAL ABSTRACT

IntroductionClassically, cancer therapy approaches lead to tumor cell death or an alternative fate such as senescence. Senescent cells remain viable but without proliferation markers. Senescent cells secrete factors with diverse actions that influence surrounding cells, the extracellular matrix (ECM) and the immune system, a phenomenon termed the senescence-associated secretory phenotype (SASP). Cyclin-dependent kinase 4/6 inhibitors (CDKi) are game-changers in the therapy of metastatic hormone receptor-positive, HER2-negative breast cancer. A potential mechanism of the action of these agents is induction of senescence in breast tumor cells, going beyond cell cycle arrest. However, senescence can be studied mainly using invasive biopsies, and therefore the prevalence and importance of senescence in patients are largely unknown. Moreover, only few circulating biomarkers exist to predict activity or measure CDKi effects. In order to develop a correlative liquid biopsy for CDKi activity, we have analyzed epigenetic (methylation) changes following therapy-induced senescence in vitro. We sought to identify (un)methylated loci that will be affected by different approaches and later can be discovered in circulating cell-free DNA. MethodsWe have treated luminal breast cancer MCF-7 cells with Doxorubicin, CDKi (Palbociclib) or by irradiation. This protocol resulted in up to 80% of cells with senescence-related beta-galactosidase activity. DNA methylation was profiled using Illumina Infinium MethylationEPIC 850K BeadChip. Differentially methylated loci (mDNA) were identified using GenomeStudio and Minfi. Analyses were done using GREAT, CSGene, and Reactome databases. Methylation age was analyzed using the Horvath Methylation Calculator. Statistical significance was defined as p ResultsSignificantly differentially methylated sites (in comparison to untreated cells) were revealed as following: 9111 sites in Doxorubicin treated cells, 3828 sites in Palbociclib treated cells, and 694 sites in irradiated cells. These loci comprise 1%, 0.45%, and 0.08% of analyzed methylation sites, respectively. 324 loci were similarly changed following the three treatment options ('common sites'). We found that the 'geographic' distribution of intragenic and intergenic methylation sites (5' UTR, gene body etc.) in all treated cells was similar. Gene set over-representation analysis revealed that sites associated with genes of the 'collagen metabolic process' set were significantly altered in drug-treated cells. Pathway investigation of the 324 'common sites' revealed that ECM-related 'focal adhesion assembly' is the most significant pathway involved. Specific analysis of senescence-related gene sets showed that 15.1% (76/503) of senescence genes changed after Doxorubicin treatment, 6.2% (31/503) after Palbociclib and 1% (5/503) after irradiation. Pathway analysis of these genes showed that drugs affected the 'oxidative stress-induced senescence' pathway, while irradiated cells had SASP-related genes affected.Despite the above mentioned methylation changes, age calculation based on methylation clock showed that all samples had similar age (6), regardless of manipulation. ConclusionSingle treatment of MCF-7 cells with known senescence inducers results in changes in methylation patterns. A significant number of common loci changed following all types of treatments, suggesting them as potential surrogate loci of senescence. Also, we have reproduced the well-known interplay between collagen and senescence/SASP.This preliminary data sheds light on epigenetic changes following treatment-induced senescence. Further studies are needed to validate whether these methylation changes can be found in vivo in tumors and in patients’ cell-free DNA following therapy with CDKi. Citation Format: Albert Grinshpun, Anatoli Kustanovich, Joshua Moss, Yael Gabai, Yuval Dor, Ittai Ben-Porath. Senescence-related methylation changes following therapy as potential biomarker for CDK4/6 inhibitor activity [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-43.

Circulating tumour-derived DNA (ctDNA) carries the genetic and epigenetic characteristics of the tumour from which it is derived and can give information about the biology and tissue origins of the underlying tumour. DNA methylation is an epigenetic mark that is specific to individual tissues and, as methylation profiles are disrupted in tumours, they can indicate the tissue of origin and cancer type of ctDNA. We have developed a set of methylation biomarkers for detecting breast cancer in plasma cell-free DNA (cfDNA). First, we mined publicly available methylation datasets to create synthetic methylation profiles that were modelled to reflect cfDNA from healthy subjects and cancer patients. These profiles were restricted to the most differentially methylated CpGs between breast tumour samples and haematopoietic cells. Regularised logistic regression models were trained using 10-fold cross-validation on synthetic cfDNA datasets with distinct fractions of breast tumour DNA spiked in silico into healthy cfDNA with the addition of 10% of a mix of different tissues. Initial validation with synthetic cfDNA permitted detection of breast cancer-derived DNA with as little as 0.25% tumour DNA spiked in silico into healthy subject cfDNA with an area under ROC curve (AUC) of 0.63. Performances of classifiers increased with increased fractions of spike-in tumour DNA (AUCs 0.77 and 0.93 at tumour DNA fractions 0.5% and 1% respectively). We then combined the most discriminative CpG markers from our models with methylation markers of breast cancer that had already been published to obtain a single marker set. In vitro testing of MCF-7 breast cancer cell line DNA spiked into leukocyte DNA showed highly significant correlation for individual markers between laboratory-measured and published methylation data for MCF-7 and leukocytes (R > 0.89, P < 2.2 × 10−16). These preliminary data indicate promising results for detection of breast cancer cell line DNA using this methylation marker set, which now require testing in cfDNA from breast cancer patients and healthy controls.

DNA methylation is a fundamental epigenetic mark that governs chromatin organization, cell identity, and gene expression. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 207 healthy tissue samples.Replicates of the same cell-type are >99.5% identical, demonstrating robustness of cell identity programs to genetic variation and environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny, and identifies methylation patterns retained since gastrulation. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hyper-methylated loci are rare and are enriched for CpG islands, polycomb targets, and CTCF binding sites, suggesting a novel role in shaping cell type-specific chromatin looping. The atlas provides an essential resource for interpretation of disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.Summary paragraphDNA methylation, a fundamental epigenetic mark, governs chromatin organization and gene expression1, thus defining the molecular identity of cells and providing a window into developmental processes with wide-ranging physiologic and clinical ramifications. Current DNA methylation datasets have limitations, typically including only a fraction of methylation sites, many from cell lines that underwent massive changes in culture or from tissues containing unspecified mixtures of cells2–6.We present a human methylome atlas based on deep whole-genome bisulfite sequencing of 39 sorted, primary cell types and use this dataset to address fundamental questions in developmental biology, physiology and pathology. Biological replicates are >99.5% identical, demonstrating unappreciated robustness to genetic variation and environmental perturbations. Clustering recapitulates key elements of tissue ontogeny, identifying methylation patterns retained since gastrulation. Loci uniquely unmethylated in individual cell types identify novel transcriptional enhancers and are enriched for tissue-specific transcription factors binding motifs. In contrast, loci uniquely hyper-methylated in specific cell types are rare, enriched for CpG islands and polycomb targets, and overlap CTCF binding sites, suggesting a novel role in shaping cell-type-specific chromatin looping. Finally, the atlas facilitates fragment-level deconvolution of tissue and plasma methylomes across thousands of cell-type specific regions to quantify their individual components at unprecedented resolution.The human cell-type-specific methylation atlas provides an essential resource for studying gene regulation by defining cell-type-specific distal enhancers and regulators of 3D organization, for identifying pathological changes in DNA methylation, and for the interpretation of methylation-based liquid biopsies.A deep methylation atlas of 39 human cell types, sorted from healthy samplesMethylomes record developmental history of cellsThousands of novel cell type-specific methylation markersHypo-methylation uncovers cell type-specific regulatory map of distal enhancersHyper-methylation across CTCF sitesCell type-specific biomarkers facilitate fragment-level deconvolution of tissues and cfDNA

Background: Cell-free DNA (cfDNA) molecules circulating in blood are emerging as important non-invasive biomarkers for monitoring physiological and pathological processes. Tumor-derived circulating cell-free DNA (cfDNA) is present in the plasma of individuals with cancer. Assays aimed at detecting common cancer mutations in cfDNA are being developed for the detection of multiple cancer types. In breast cancer, however, such assays have failed to detect the disease at a sensitivity relevant for clinical use, in part due to the absence of multiple common mutations that can be co-detected in plasma. Unlike individual mutations that exist in only a subset of tumors, unique DNA methylation patterns are universally present in cells of a common type and therefore may be ideal biomarkers. Here we describe the detection and quantification of breast derived cfDNA, using a breast-specific DNA methylation signature. Methods: In order to identify regions of the genome with unique methylation patterns in breast epithelium, we compared genome-wide methylation profiles of normal breast tissue and breast cancer to genome-wide methylation profiles of other normal and cancerous tissues and aimed to identify CpG sites that are uniquely hypermethylated or hypomethylated in the breast. By quantifying the proportion of molecules in a sample that harbored 3 chosen breast-specific methylation sites, we could obtain an estimate of the proportion of the sample derived from breast epithelial cells. We have collected plasma prospectively from healthy women, metastatic breast cancer patients and patients with localized (stage II-III) breast cancer who were assigned to preoperative chemotherapy at our cancer institute. In the latter cohort, samples were collected before and throughout treatment until surgery. The trial was approved by the Institutional Ethics Committee and all patients gave written informed consent. Results: All three markers clearly distinguished between the plasma of healthy donors (n=64) and patients with metastatic disease (n=14, p-value Conclusion: A breast-unique epigenetic signature in circulating cell-free DNA can be used as a universal biomarker for breast cancer, to allow sensitive detection and monitoring of localized breast cancer treatment outcomes. Further research will aid to evaluate this promising and affordable method in additional clinical scenarios. Citation Format: Albert Grinshpun, Aviad Zick, Joshua Moss, Einat Carmon, Myriam Maoz, Bracha Ohana, Ofri Abraham, Lili Germansky, Benjamin Glaser, Ruth Shemer, Yuval Dor, Beatrice Uziely. Non-personalized detection of circulating breast-derived DNA for monitoring of preoperative breast cancer treatment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-10.

Context There is an unmet need for biomarkers of pancreatic beta-cell death to improve early diagnosis of type 1 diabetes, enroll subjects into clinical trials, and assess treatment response. To address this need, several groups developed assays measuring insulin deoxyribonucleic acid (DNA) with unmethylated CpG sites in cell-free DNA. Unmethylated insulin DNA should be derived predominantly from beta-cells and indicate ongoing beta-cell death. Objective To assess the performance of three unmethylated insulin DNA assays. Design and Participants Plasma or serum samples from 13 subjects undergoing total pancreatectomy and islet autotransplantation were coded and provided to investigators to measure unmethylated insulin DNA. Samples included a negative control taken post-pancreatectomy but pretransplant, and a positive control taken immediately following islet infusion. We assessed technical reproducibility, linearity, and persistence of detection of unmethylated insulin DNA for each assay. Results All assays discriminated between the negative sample and samples taken directly from the islet transplant bag; 2 of 3 discriminated negative samples from those taken immediately after islet infusion. When high levels of unmethylated insulin DNA were present, technical reproducibility was generally good for all assays. Conclusions The measurement of beta cell cell-free DNA, including insulin, is a promising approach, warranting further testing and development in those with or at-risk for type 1 diabetes, as well as in other settings where understanding the frequency or kinetics of beta cell death could be useful.

BackgroundSchizophrenia is a common, severe and debilitating psychiatric disorder. Despite extensive research there is as yet no biological marker that can aid in its diagnosis and course prediction. This precludes early detection and intervention. Imaging studies suggest brain volume loss around the onset and over the first few years of schizophrenia, and apoptosis has been proposed as the underlying mechanism. Cell free DNA fragments (cfDNA) are released into the bloodstream following cell death. Tissue-specific methylation patterns allow the identification of the tissue origins of cfDNA.MethodsWe developed a cocktail of brain specific DNA methylation markers, and used it to assess the presence of brain-derived cfDNA in the plasma of patients with a first psychotic episode.ResultsWe detected significantly elevated neuron- (p=0.0013), astrocyte- (p=0.0016), oligodendrocyte- (p=0.0129) and whole brain-derived (p=0.0012) cfDNA in the plasma of patients during their first psychotic episode (n=29), compared with healthy controls (n=31). Increased cfDNA levels were not correlated with psychotropic medications use. Area Under the Curve (AUC) was 0.77, with 65% sensitivity at 90% specificity in patients with a psychotic episode.ConclusionsPotential interpretations of these findings include increased brain cell death, disruption of the blood brain barrier or a defect in clearance of material from dying brain cells. Brain-specific cfDNA methylation markers can potentially assist early detection and monitoring of schizophrenia and thus allow early intervention and adequate therapy.

DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes1. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells2–5. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.