Phenotypic plasticity is a key mechanism of metastatic progression and cancer therapy resistance. This hallmark of human malignancies is enabled by highly conserved epigenetic mechanisms that control gene expression. Functional alterations in DNA methylation and histone post-translational modifications have been extensively described as drivers of metastatic dissemination and therapy resistance. Pharmacological inhibitors of epigenetic enzymes can revert these alterations, thereby stopping cancer progression and counteracting the emergence of resistant clones. Despite promising pre-clinical evidence, the clinical implementation of epigenetic therapies in solid cancers has led to disappointing results. Several factors can explain these challenges, including the lack of rational combinations. Notably, response to epigenetic treatments can be heterogeneous and short-lived. A liquid biopsy technology that allows the measure of specific epigenetic alterations enables patient selection and therapy monitoring, leading to the development of precision epigenetic therapies. In this review, we discuss the state of the art of this emerging treatment modality, and we identify key challenges that need to be overcome to reach the full potential of this new therapeutic concept.

BACKGROUND:Aggressive Variant Prostate Cancers (AVPCs) are incurable malignancies. Platinum-based chemotherapies are used for the palliative treatment of AVPC. The Polycomb Repressive Complex 2 (PRC2) promotes prostate cancer progression via histone H3 Lysine 27 tri-methylation (H3K27me3). EZH2 encodes the catalytic subunit of PRC2. A recently developed nucleosome capture technology (Nu.QⓇ).measures H3K27me3 levels in biological fluids. EZH2 inhibitors (EZH2i) are being tested in clinical trials. We hypothesize that epigenetic reprogramming via EZH2i improves the efficacy of Carboplatin in AVPC and that EZH2i activity can be measured via both cellular- and cell-free nucleosomal H3K27me3 (cf-H3K27me3) levels. METHODS:We studied the expression of PRC2 genes in clinical prostate cancer cohorts (bioinformatics). We determined the effect of EZH2i on cellular- and cf-H3K27me3 levels. We measured dose-dependent effects of Carboplatin with/without EZH2i on AVPC cell viability (IC50). We used RNA-Seq to study how EZH2i modulates gene expression in AVPC cells. RESULTS:PRC2 genes were significantly up-regulated in AVPC vs other prostate cancer types. EZH2i reduced both cellular and cf-H3K27me3 levels. EZH2i significantly reduced Carboplatin IC50. EZH2i reduced the expression of DNA repair genes and increased the expression of p53-dependent pro-apoptotic factors. CONCLUSIONS:EZH2i plus Carboplatin is a promising combination treatment for AVPC.

Abstract CAIRE is a phase 2 umbrella study which assessed activity of combined anti-EZH2 tazemetostat and anti-PDL1 durvalumab (T+D) in advanced pretreated pancreatic, colorectal and sarcoma cancer patients. The are currently no plasma biomarkers for anti-EZH2 treatment monitoring or outcome. EZH2 catalyzes tri-methylation of Lysine 27 (K27me3) on nucleosomal histone 3 (H3, H3K27me3). Circulating, cell free H3K27me3 modified nucleosomes are thus a potential biomarker for tazemetostat activity. We evaluated the concentration of circulating H3K27me3 modified nucleosomes normalized to H3.1 nucleosomes (the most common isoform of H3) in T+D treated patients, irrespective of the tumor primary site, to determine target engagement during anti-EZH2 treatment. Plasma from patients receiving T+D was collected before the first cycle administration (C1D1), on the following two cycles administration (C2D1, C3D1) and at end of treatment (EOT). H3.1 and H3K27me3 containing nucleosomes were quantified by Nu.Q®-H3.1 and Nu.Q®-H3K27me3 Chemiluminescent Immunoassays. Concentrations are reported as ng/ml and the comparison of their median values at the different timepoints were tested with Kruskal-Wallis and Mann Whitney (independent samples) or Wilcoxon tests (paired samples), with SciPy.stat package (1.6.2). Bonferroni correction was applied to multiple comparisons. Of 197 total samples, 191 were evaluable for both biomarkers (6 higher than top standard after 5x dilution): 69 at C1D1, 63 at C2D1, 50 at C3D1 and 9 at EOT. For 58 patients paired C1D1 and C1D2 levels were available, 46 from C1D1 to C3D1 and 8 from C1D1 to EOT. The normalized Nu.Q®-H3K27me3 C1D1 median value (0.56) was significantly higher than C2D1 (0.31, p: e-12), C3D1 (0.31, p: e-12) and EOT ones (0.28, p: 0.001). Conversely, Nu.Q®-H3.1 levels were similar from C1D1 to C3D1 (range of the medians: 122.16-133.25 ng/ml) and increased at EOT (290.09 ng/ml), but with no significant difference between pairwise timepoints. Regarding paired-samples comparison, the normalized Nu.Q®-H3K27me3 was significantly higher at C1D1 than at C2D1 (p: e-8) and C3D1 (p: e-9) and at EOT (p: 0.02). Conversely, H3.1 concentration was significantly higher at EOT than at C1D1 (p: 0.02), at C2D1 (p: 0.02) and C3D1 (p: 0.05).In conclusion, we described for the first time that normalized circulating nucleosomal H3K27me3 values significantly decrease during T-based treatment in metastatic solid tumor patients, irrespective of the primary disease site, supporting its potential role as a pharmacodynamic biomarker for EZH2 inhibition. Moreover, total nucleosomal H3.1 seems to represent a surrogate of disease burden in metastatic pancreatic, colorectal and sarcoma cancers, as suggested previously in other hematological malignancies. Citation Format: Francesca Salani, Mark Eccleston, Lola-Jade Palmieri, Simon Pernot, Sophie Cousin, Gianluca Masi, Francesco Crea, Antoine Italiano. Circulating H3K27me3 modified nucleosomes as a biomarker to monitor anti EZH2-based treatment in advanced solid tumour patients: Translational analyses from CAIRE trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5128.

Background EZH2 promotes aggressive-variant prostate cancer (AVPC) progression via histone H3-Lysine-27 tri-methylation (H3K27me3). We hypothesize that epigenetic reprogramming via EZH2 inhibitors (EZH2i) improves the efficacy of chemotherapy in AVPC. Methods We studied the expression of EZH2 in clinical prostate cancer cohorts (bioinformatics). We determined the effect of EZH2i on both cellular- and cell-free-H3K27me3 levels. We measured effects of carboplatin with/without EZH2i on AVPC cell viability (IC50). We studied how EZH2i modulate gene expression (RNA Seq). Results EZH2 was significantly up-regulated in AVPC vs other prostate cancer types. EZH2i reduced both cellular and cell free-H3K27me3 levels. EZH2i significantly reduced carboplatin IC50. EZH2i reduced the expression of DNA repair and increased the expression of pro-apoptotic genes. Article Highlights ### Competing Interest Statement The authors have declared no competing interest.

During cell death, nucleosomes, the basic structural unit of chromatin, are released into the blood stream and elevated levels have been found in the plasma of patients with solid cancers. In this study, we demonstrate an increase in cell free circulating H3.1-nucleosomes levels in plasma samples from patients with hematological malignancy, non-Hodgkin lymphoma (NHL), relative to healthy donors. As histone post-translational modifications (PTMs) of circulating nucleosomes are described as potential biomarkers of various solid cancers, we investigated the epigenetic profile of nucleosomes from NHL patients following nucleosome enrichment (Nu.Q® capture) combined with mass spectrometry. Eight histones PTMs, including the acetylation of histone H3 at lysine 9, 14 and 18 as well as the methylation state of histone H3 at lysine 9, 27 and 36, were identified at a higher level in the plasma of NHL patients compared to healthy donors. These results were confirmed in a larger clinical cohort by immunoassay. Subsequently, the temporal profile of these histone PTMs in NHL patients undergoing treatment course highlighted the potential use of these new biomarkers to monitor treatment response and/or disease progression. Our results substantiate that levels of H3.1-nucleosomes are particularly elevated in NHL patients and may be a useful diagnostic tool. Moreover, our work emphasizes the crucial roles of the epigenetic marks present on circulating nucleosomes to detect and monitor tumor progression and/or treatment response of non-Hodgkin Lymphoma.

Background: Predictive biomarkers for advanced hepatocellular carcinoma are lacking. EZH2 drives sorafenib resistance through H3K27me3 and is counteracted by SETD2, which catalyzes H3K36me3. The authors tested the predictive power of circulating H3K27me3 and H3K36me3 in advanced hepatocellular carcinoma patients treated with sorafenib. Methods: A total of 80 plasma samples were tested for histone variants by ELISA. Changes from baseline to best response or progressive disease were correlated with patient survival. Results: A higher EZH2/SETD2 ratio predicted worse prognosis in this setting. H3K27me3 and H3K36me3 decreased from baseline to best response. The H3K27me3/H3K36me3 ratio increased from baseline to progressive disease. Higher ratios at best response were associated with shorter progression-free survival. Conclusion: The authors suggest that circulating H3K27me3/H3K36me3 ratio level acts as a predictive biomarker for sorafenib treatment outcomes in patients with advanced hepatocellular carcinoma.

The severity of coronavirus disease 2019 (COVID-19) varies significantly with cases spanning from asymptomatic to lethal with a subset of individuals developing Severe Acute Respiratory Syndrome (SARS) and death from respiratory failure. To determine whether global nucleosome and citrullinated nucleosome levels were elevated in COVID-19 patients, we tested two independent cohorts of COVID-19 positive patients with quantitative nucleosome immunoassays and found that nucleosomes were highly elevated in plasma of COVID-19 patients with a severe course of the disease relative to healthy controls and that both histone 3.1 variant and citrullinated nucleosomes increase with disease severity. Elevated citrullination of circulating nucleosomes is indicative of neutrophil extracellular trap formation, neutrophil activation and NETosis in severely affected individuals. Importantly, using hospital setting (outpatient, inpatient or ICU) as a proxy for disease severity, nucleosome levels increased with disease severity and may serve as a guiding biomarker for treatment. Owing to the limited availability of mechanical ventilators and extracorporal membrane oxygenation (ECMO) equipment, there is an urgent need for effective tools to rapidly assess disease severity and guide treatment selection. Based on our studies of two independent cohorts of COVID-19 patients from Belgium and Germany, we suggest further investigation of circulating nucleosomes and citrullination as biomarkers for clinical triage, treatment allocation and clinical drug discovery.

e13534 Background: In liquid biopsy, circulating tumor DNA (ctDNA) is more fragmented than background cell free DNA, peaking at 147bp (equivalent to a mono-nucleosome) instead of 165bp (nucleosome with an additional 20bp of linker DNA). Isolation of these shorter cell free DNA fragments from longer, extracted cell-free DNA improves detection of ctDNA as demonstrated by enrichment of tumor specific mutations. Nuclease protection of the additional 20bp of linker DNA, conferred by bound linker proteins such as Histone 1, would account for the 165bp peak in host background DNA. We hypothesised that extracting intact nucleosomes with linker DNA using a novel, H1 antigen based, immunocapture approach would enrich the ctDNA fraction in the remaining nucleosomes. Methods: We expressed H1.0 protein in E. coli and following extraction, purification and chemically immobilised it to tosyl-activated magnetic beads. The beads were first used to immunodeplete mono-nucleosomes from HeLa cell digests and the level of immunocaptured nucleosomes was determined by immunoassay targeting intact nucleosomes. The level of nucleosome levels determined before and after depletion was further determined by ELISA targeting H3.1 containing intact nucleosomes. DNA was extracted from the H1 immunocaptured “long” nucleosomes and size profiles compared with the remaining nucleosomes in the supernatant by BioAnalyzer. Then, the method was applied to clinical plasma samples and the size distribution of NGS Libraries (Illumina system) prepared from five colorectal cancer and three healthy samples, their immune depleted supernatants and the immunocaptured nucleosomes were then compared. Enrichment of specific genomic regions was also evaluated. Results: We observed relative enrichment of nucleosomes with short DNA in supernatants following H1 immuno-depletion of the cancer samples as evidenced by a change in size distribution by Bioanalyzer and NGS-sequencing. We also observed potential enrichment of TSS is the H1 immunocaptured nucleosomes consistent with linker DNA positioning of TF binding sites. Conclusions: Histone 1.0 has the highest affinity of H1 mammalian isoforms and successfully immunodepleted plasma samples containing cell free circulating nucleosomes with DNA longer that 147bp. Immobilized H1.0 effectively formed a pseudo-chromatosome by binding to free linker DNA or displacing endogenous H1 and other linker associated proteins. H1 antigen based immuno-depletion offers a simple way to enrich tumour derived nucleosomes and thus cell free DNA.

e20078 Background: There are over 700,000 new cases of non-Hodgkin lymphoma (NHL), acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL) diagnosed globally each year and approximately 415,000 deaths. The non-specific symptoms of lymphoma and leukemia often delay diagnosis. We investigated the circulating levels of intact nucleosomes containing the histone H3.1 isoform (Nu.Q-H3.1) in a variety of solid tumors, NHL, AML, ALL, and in healthy subjects. Methods: We measured levels of Nu.Q-H3.1 in plasma samples taken from 62 healthy volunteers (mean age = 45 yrs) and 329 patients diagnosed with cancer (mean age = 56 yrs), including 25 patients diagnosed with each of cancer of the bladder, bone, brain, oesophagus, cervix, skin, head & neck or melanoma, 21 patients diagnosed with uterine cancer as well as with NHL (n = 25), AML (n = 25) and ALL (n = 8). The cohort included samples taken from patients at diagnosis and at relapse. Whole blood samples were collected in EDTA plasma tubes, double-centrifuged at 1500 rcf for 15 minutes within 2 hrs of blood draw, after which plasma was transferred to a cryotube and frozen immediately until analysis. Plasma samples (20µl) were analyzed in duplicate for Nu.Q-H3.1 using an ELISA method developed and validated to CLSI guidelines. Results: We observed elevated levels of Nu.Q-H3.1 in the patients diagnosed with various cancers. Only 14 of 271 patients with a solid tumor had levels > 200ng/ml. In contrast the median Nu.Q-H3.1 levels observed for NHL, AML and ALL were 276, 284 and 585ng/ml respectively. The median nucleosome level in 62 healthy subjects was 40ng/ml; the highest level was 198ng/ml. The AUC for all patients diagnosed with NHL, AML or ALL (n = 54) vs healthy volunteers was 91% with a sensitivity of 74% at 95% specificity. The AUC for the subset of patients newly diagnosed with NHL, AML or ALL (n = 31) vs healthy volunteers was 92% with a sensitivity of 81% at 95% specificity. Conclusions: Elevated nucleosome levels have been reported for a number of diseases. Our early results indicate that levels of Nu.Q-H3.1 are particularly elevated in haematological malignancies and may be a useful diagnostic tool warranting further study.

Immunotherapies are revolutionizing the clinical management of a wide range of cancers. However, intrinsic or acquired unresponsiveness to immunotherapies does occur due to the dynamic cancer immunoediting which ultimately leads to immune escape. The evolutionarily conserved histone modifier enhancer of zeste 2 (EZH2) is aberrantly overexpressed in a number of human cancers. Accumulating studies indicate that EZH2 is a main driver of cancer cells’ immunoediting and mediate immune escape through downregulating immune recognition and activation, upregulating immune checkpoints and creating an immunosuppressive tumor microenvironment. In this review, we overviewed the roles of EZH2 in cancer immunoediting, the preclinical and clinical studies of current pharmacologic EZH2 inhibitors and the prospects for EZH2 inhibitor and immunotherapy combination for cancer treatment.

Aggressive variants of prostate cancer (AVPC) are a subtype of metastatic castration resistant prostate cancer (mCRPC), which express no androgen receptor (AR) and are currently incurable. The main AVPC subtypes are neuroendocrine prostate cancer (NEPC) and anaplastic prostate cancer (AR-, NEPC-). EZH2 is an epigenetic regulator that mediates gene silencing via histone H3 Lys27 trimethylation (H3K27me3). EZH2 acts as an oncogene in several malignancies. Three pharmacologic EZH2 inhibitors (Tazemetostat, GSK-126, and CPI-1205) are in clinical trials. Volition Nu.Q kits enable the detection of histone variants in biological fluids. We hypothesize that EZH2 could be a viable therapeutic target for AVPC. We queried the expression of EZH2 in a database comprising 444 mCRPC clinical samples. Our results show that higher EZH2 expression is positively correlated with NEPC features (p<<0.0001, T test), and negatively correlated with AR activity (p< 4.57e-11). Higher EZH2 expression predicts shorter overall survival (p< 0.0276, log-rank test). The three EZH2 inhibitors induce a dose-dependent inhibition of cell proliferation in two AVPC cell lines (DU-145, PC-3). GSK-126 is the most potent inhibitor. Total histone H3 and H3K27me3 are measurable in the supernatant from AVPC cell lines (Nu.Q assay). Treatment with EZH2 inhibitors induces a measurable reduction in H3K27me3. We are now testing the interaction between GSK-126 and chemotherapy drugs employed for the treatment of AVPC. We are also testing Nu.Q kits in blood samples from patient-derived xenografts. This information will be useful to develop tailored epigenetic therapies for AVPC patients.

Introduction: Successful adoption of stool based CRC screening across Europe is straining limited colonoscopy capacity. We have developed a Triage test to reduce unnecessary colonoscopies (no findings or low risk adenomas) in FIT positive individuals. The CE marked Nu.Q ™ Colorectal Cancer Screening Test is designed to reduce unnecessary colonoscopies by ≥ 20% whilst maintaining sensitivity for CRC ≥ 90%. We now report Triage test performance by stage in colorectal cancer, high, medium and low risk adenomas and establish intra lab reproducibility. Methods: Blinded serum samples from 1961 FIT positive individuals (>200ng/mL) prospectively recruited from the Danish National CR screening program were analyzed (10uL in duplicate per assay) for circulating nucleosomes containing methylated DNA and normalized to total circulating nucleosomes using Nu.Q™ immunoassays. The results were applied to locked algorithm developed in a training set (n = 1907). The Nu.Q™ Triage test calls (recommended colonoscopy or not) were compared to colonoscopy diagnosis prior to unblinding and evaluated relative to an increased FIT cut off of 200ng/mL as an alternate approach to reduce colonoscopy demand. Intra laboratory reproducibility was established using a 200 sample subset analysed in Volition's laboratory and a commercial clinical pathology Laboratory (Laboratoire CERBA, Paris). Results: In the training set, the Nu.Q™ Colorectal Cancer Screening Triage test identified a low-risk subset of 477 individuals (25%) for whom colonoscopy could be avoided whilst detecting 96.6% of CRCs and 88.5% of High Risk Adenomas (HRA). In the validation set, a potential 28.6% reduction in colonoscopies was achieved albeit with a slightly lower overall sensitivity for cancer (91.2%) and high risk adenoma (83.9%). Sensitivity for "screen relevant neoplasia" (CRC and high risk adenomas combined) was 86% compared to 80% at an increased FIT cut off at 200ng/mL. Referral rate for medium and low risk adenoma and clean bowel was reduced to 67.7% (compared to 64.5% at 200ng/mL FIT cut off) with a clear trend of increasing referral reductions for medium and low risk adenomas (77.6% and 68.4% referral rates respectively) and particularly in clean bowel (63.4% referral rate) compared to high risk adenomas and CRC (83.9% and 91.2% referral rate respectively). Intra laboratory reproducibility was over 93%. Conclusion: CONCLUSIONS: Combining a single, age adjusted NuQ® blood score with FIT score could reduce unnecessary colonoscopies in FIT positive individuals with minimal reduction in sensitivity and considerably improved performance relative to an increased FIT threshold from 100ng/mL to 200ng/mL. Screening throughput and overall detection of screen relevant neoplasia could therefore be increased where colonoscopy capacity is a limiting factor.

Abstract Genome wide epigenetic signals are altered in cancer cells. In addition, circulating nucleosome bound DNA fragments contain the same mutations as matched cancer tissue samples suggesting a tumor origin for at least some circulating nucleosomes. We have developed ELISA tests for circulating nucleosomes (NuQ®) and show that the profile of epigenetic features, including histone modifications and variants, DNA modifications and adducts between nucleosomes and non-histone proteins can be correlated with clinical disease and overcomes a major limitation of simple nucleosome quantification for diagnostic and prognostic use. Validation of this approach in a retrospective cohort of 4800 patients presenting with symptoms of Colorectal diseases generated a diagnostic NuQ® panel with sensitivity of 81% at a specificity of 80% in an age adjusted linear regression model for the detection of Colorectal cancer (CRC). In a subsequent pilot prospective study of 58 subjects, a sensitivity of 91% at 90% specificity was achieved for the detection of CRC. We now present preliminary Nucleosomics® data from the first 2000 individuals of a 14 000 asymptomatic, average risk cohort as part of a prospective clinical comparison with a national Fecal Immunological Screening (FIT) program in Denmark. Successful validation of this approach would provide an affordable, accessible and easily implemented blood based testing alternative to address relatively low compliance of fecal testing. Note: This abstract was not presented at the conference. Citation Format: Marielle Herzog, Mark Eccleston, Dorian Pamart, Brieuc Cuvelier, Ib Jarle Christensen, Hans Jørgen Nielson, Jake Micallef, Jason Terrell. Blood-based epigenetic profiling of circulating cell free nucleosomes in 2000 FIT positive individuals: A novel approach to colorectal cancer detection. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B02.

We have previously demonstrated differential nucleosome patterns in samples from a retrospective cohort of 4800 patients presenting with symptoms of Colorectal diseases. A diagnostic NuQ® panel was identified with sensitivity of 81% at a specificity of 80% in an age adjusted Linear Discriminant Analysis (LDA) model for the detection of Colorectal cancer (CRC). The success of stool based screening for CRC adopted across Europe has placed significant strain on limited colonoscopy capacity. The aim of the study was to evaluate combined NuQ® blood score and numeric FIT score as a triage approach for positive Fecal Immune Tests (FIT) in an average risk population i.e. to identify individuals with low risk adenomas or no findings on colonoscopy. We have developed ELISAs for specific epigenetic features of circulating nucleosomes (NuQ®) including histone modifications and variants, DNA modifications and nucleosome-protein adducts and shown that the epigenetic profiles can be correlated with disease. Serum samples were collected from a training cohort consisting of approximately 1900 FIT positive individuals with colonoscopic confirmation of diagnosis. 10 µl serum samples were analyzed using NuQ® ELISA blood tests and an algorithm developed by Linear Discriminant Analysis (LDA) was used to identify individuals with a false positive FIT result. A single, age adjusted NuQ® assay for nucleosome associated methylated DNA (normalized to total nucleosomes) combined with FIT score can reduce the need for unnecessary colonoscopies whilst maintaining sensitivity for CRC. For example, a 25% reduction in colonoscopies is associated with 96.6% sensitivity for CRC and 88.5% sensitivity for HRA. A single, age adjusted NuQ® blood score with FIT score could reduce non screen-relevant colonoscopies in FIT positive individuals with minimal reduction in cancer detection. This test has potential applications to reduce unnecessary colonoscopies and thereby ease the pressure on colonoscopy capacity constraints or, alternatively, to detect more cancers in a screening programme by increasing the throughput of screening subjects.

Researchers working in industrial laboratories as well as in academic laboratories discussed topics related to the use of extracellular nucleic acids in different fields. These included areas like non-invasive prenatal diagnosis, the application of different methods for the analysis and characterization of patients with benign and malignant diseases and technical aspects associated with extracellular nucleic acids. In addition, the possibilities and chances for a cooperation of researchers working in different worlds, i.e. academia and industry, were discussed.

To improve the prognosis of patients with pancreatic cancer, new biomarkers are required for earlier, pre-symptomatic diagnosis. Epigenetic mutations take place at the earliest stages of tumorigenesis and therefore offer new approaches for detecting and diagnosing disease. Nucleosomes are the repeating subunits of DNA and histone proteins that constitute human chromatin. Because of their release into the circulation, intact nucleosome levels in serum or plasma can serve as diagnostic disease biomarkers, and elevated levels have been reported in various cancers. However, quantifying nucleosomes in the circulation for cancer detection has been challenging due to nonspecific elevation in sera of patients with benign diseases. Here, we report for the first time differential, disease-associated epigenetic profiles of intact cell-free nucleosomes (cfnucleosomes) containing specific DNA and histone modifications as well as histone variants circulating in the blood. The study comprised serum samples from 59 individuals, including 25 patients with resectable pancreatic cancer, 10 patients with benign pancreatic disease, and 24 healthy individuals using Nucleosomics®, a novel ELISA method.