Epigenetic profiles of elevated cell free circulating H3.1 nucleosomes as potential biomarkers for non-Hodgkin lymphoma.

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.

Circulating H3K27 Methylated Nucleosome Plasma Concentration: Synergistic Information with Circulating Tumor DNA Molecular Profiling.

The molecular profiling of circulating tumor DNA (ctDNA) is a helpful tool not only in cancer treatment, but also in the early detection of relapse. However, the clinical interpretation of a ctDNA negative result remains challenging. The characterization of circulating nucleosomes (carrying cell-free DNA) and associated epigenetic modifications (playing a key role in the tumorigenesis of different cancers) may provide useful information for patient management, by supporting the contributive value of ctDNA molecular profiling. Significantly elevated concentrations of H3K27Me3 nucleosomes were found in plasmas at the diagnosis, and during the follow-up, of NSCLC patients, compared to healthy donors (p-value < 0.0001). By combining the H3K27Me3 level and the ctDNA molecular profile, we found that 25.5% of the patients had H3K27Me3 levels above the cut off, and no somatic alteration was detected at diagnosis. This strongly supports the presence of non-mutated ctDNA in the corresponding plasma. During the patient follow-up, a high H3K27Me3-nucleosome level was found in 15.1% of the sample, despite no somatic mutations being detected, allowing the identification of disease progression from 43.1% to 58.2% over molecular profiling alone. Measuring H3K27Me3-nucleosome levels in combination with ctDNA molecular profiling may improve confidence in the negative molecular result for cfDNA in lung cancer at diagnosis, and may also be a promising biomarker for molecular residual disease (MRD) monitoring, during and/or after treatment.

Monitoring plasma nucleosome concentrations to measure disease response and progression in dogs with hematopoietic malignancies.

BACKGROUND: Hematopoietic malignancies are extremely common in pet dogs and represent nearly 30% of the malignancies diagnosed in this population each year. Clinicians commonly use existing tools such as physical exam findings, radiographs, ultrasound and baseline blood work to monitor these patients for treatment response and remission. Circulating biomarkers, such as prostate specific antigen or carcinoembryonic antigen, can be useful tools for monitoring treatment response and remission status in human cancer patients. To date, there has a been a lack of useful circulating biomarkers available to veterinary oncology patients. METHODS: Circulating plasma nucleosome concentrations were evaluated at diagnosis, throughout treatment and during remission monitoring for 40 dogs with lymphoma, acute myelogenous leukemia and multiple myeloma. Additionally, C-reactive protein and thymidine kinase-1 levels were recorded. RESULTS: Plasma nucleosome concentrations were significantly higher at diagnosis and progressive disease than they were when dogs were in remission. All but two dogs had plasma nucleosome concentrations that returned to the low range during treatment. These two dogs had the shortest progression free and overall survival times. Dogs with the highest plasma nucleosome concentrations had a significantly shorter first progression free survival than dogs with lower plasma nucleosome concentrations at diagnosis. Plasma nucleosome concentrations correlated better with disease response and progression than either thymidine kinase or C reactive protein. CONCLUSIONS: Plasma nucleosome concentrations can be a useful tool for treatment monitoring and disease progression in dogs with hematopoietic malignancies.

A persistent neutrophil-associated immune signature characterizes post-COVID-19 pulmonary sequelae.

Interstitial lung disease and associated fibrosis occur in a proportion of individuals who have recovered from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection through unknown mechanisms. We studied individuals with severe coronavirus disease 2019 (COVID-19) after recovery from acute illness. Individuals with evidence of interstitial lung changes at 3 to 6 months after recovery had an up-regulated neutrophil-associated immune signature including increased chemokines, proteases, and markers of neutrophil extracellular traps that were detectable in the blood. Similar pathways were enriched in the upper airway with a concomitant increase in antiviral type I interferon signaling. Interaction analysis of the peripheral phosphoproteome identified enriched kinases critical for neutrophil inflammatory pathways. Evaluation of these individuals at 12 months after recovery indicated that a subset of the individuals had not yet achieved full normalization of radiological and functional changes. These data provide insight into mechanisms driving development of pulmonary sequelae during and after COVID-19 and provide a rational basis for development of targeted approaches to prevent long-term complications.

NETosis and Nucleosome Biomarkers in Septic Shock and Critical COVID-19 Patients: An Observational Study.

Background: Neutrophil extracellular traps' (NETs') formation is a mechanism of defense that neutrophils deploy as an alternative to phagocytosis, to constrain the spread of microorganisms. Aim: The aim was to evaluate biomarkers of NETs' formation in a patient cohort admitted to intensive care unit (ICU) due to infection. Methods: Forty-six septic shock patients, 22 critical COVID-19 patients and 48 matched control subjects were recruited. Intact nucleosomes containing histone 3.1 (Nu.H3.1), or citrullinated histone H3R8 (Nu.Cit-H3R8), free citrullinated histone (Cit-H3), neutrophil elastase (NE) and myeloperoxidase (MPO) were measured. Results: Significant differences in Nu.H3.1 and NE levels were observed between septic shock and critical COVID-19 subjects as well as with controls (p-values < 0.05). The normalization of nucleosome levels according to the neutrophil count improved the discrimination between septic shock and critical COVID-19 patients. The ratio of Nu.Cit-H3R8 to Nu.H3.1 allowed the determination of nucleosome citrullination degree, presumably by PAD4. Conclusions: H3.1 and Cit-H3R8 nucleosomes appear to be interesting markers of global cell death and neutrophil activation when combined. Nu.H3.1 permits the evaluation of disease severity and differs between septic shock and critical COVID-19 patients, reflecting two distinct potential pathological processes in these conditions.

Liquid Biopsy in Glioblastoma.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Despite recent advances in therapy modalities, the overall survival of GBM patients remains poor. GBM diagnosis relies on neuroimaging techniques. However, confirmation via histopathological and molecular analysis is necessary. Given the intrinsic limitations of such techniques, liquid biopsy (mainly via blood samples) emerged as a non-invasive and easy-to-implement alternative that could aid in both the diagnosis and the follow-up of GBM patients. Cancer cells release tumoral content into the bloodstream, such as circulating tumor DNA, circulating microRNAs, circulating tumor cells, extracellular vesicles, or circulating nucleosomes: all these could serve as a marker of GBM. In this narrative review, we discuss the current knowledge, the advantages, and the disadvantages of each circulating biomarker so far proposed.

Differential Biological Effects of Dietary Lipids and Irradiation on the Aorta, Aortic Valve, and the Mitral Valve.

Aims: Dietary cholesterol and palmitic acid are risk factors for cardiovascular diseases (CVDs) affecting the arteries and the heart valves. The ionizing radiation that is frequently used as an anticancer treatment promotes CVD. The specific pathophysiology of these distinct disease manifestations is poorly understood. We, therefore, studied the biological effects of these dietary lipids and their cardiac irradiation on the arteries and the heart valves in the rabbit models of CVD. Methods and Results: Cholesterol-enriched diet led to the thickening of the aortic wall and the aortic valve leaflets, immune cell infiltration in the aorta, mitral and aortic valves, as well as aortic valve calcification. Numerous cells expressing α-smooth muscle actin were detected in both the mitral and aortic valves. Lard-enriched diet induced massive aorta and aortic valve calcification, with no detectable immune cell infiltration. The addition of cardiac irradiation to the cholesterol diet yielded more calcification and more immune cell infiltrates in the atheroma and the aortic valve than cholesterol alone. RNA sequencing (RNAseq) analyses of aorta and heart valves revealed that a cholesterol-enriched diet mainly triggered inflammation-related biological processes in the aorta, aortic and mitral valves, which was further enhanced by cardiac irradiation. Lard-enriched diet rather affected calcification- and muscle-related processes in the aorta and aortic valve, respectively. Neutrophil count and systemic levels of platelet factor 4 and ent-8-iso-15(S)-PGF2α were identified as early biomarkers of cholesterol-induced tissue alterations, while cardiac irradiation resulted in elevated levels of circulating nucleosomes. Conclusion: Dietary cholesterol, palmitic acid, and cardiac irradiation combined with a cholesterol-rich diet led to the development of distinct vascular and valvular lesions and changes in the circulating biomarkers. Hence, our study highlights unprecedented specificities related to common risk factors that underlie CVD.

Serial profiling of cell-free DNA and nucleosome histone modifications in cell cultures.

Recent advances in basic research have unveiled several strategies for improving the sensitivity and specificity of cell-free DNA (cfDNA) based assays, which is a prerequisite for broadening its clinical use. Included among these strategies is leveraging knowledge of both the biogenesis and physico-chemical properties of cfDNA towards the identification of better disease-defining features and optimization of methods. While good progress has been made on this front, much of cfDNA biology remains uncharted. Here, we correlated serial measurements of cfDNA size, concentration and nucleosome histone modifications with various cellular parameters, including cell growth rate, viability, apoptosis, necrosis, and cell cycle phase in three different cell lines. Collectively, the picture emerged that temporal changes in cfDNA levels are rather irregular and not the result of constitutive release from live cells. Instead, changes in cfDNA levels correlated with intermittent cell death events, wherein apoptosis contributed more to cfDNA release in non-cancer cells and necrosis more in cancer cells. Interestingly, the presence of a ~ 3 kbp cfDNA population, which is often deemed to originate from accidental cell lysis or active release, was found to originate from necrosis. High-resolution analysis of this cfDNA population revealed an underlying DNA laddering pattern consisting of several oligo-nucleosomes, identical to those generated by apoptosis. This suggests that necrosis may contribute significantly to the pool of mono-nucleosomal cfDNA fragments that are generally interrogated for cancer mutational profiling. Furthermore, since active steps are often taken to exclude longer oligo-nucleosomes from clinical biospecimens and subsequent assays this raises the question of whether important pathological information is lost.

A novel proteomics approach to epigenetic profiling of circulating nucleosomes.

Alteration of epigenetic modifications plays an important role in human cancer. Notably, the dysregulation of histone post-translational modifications (PTMs) has been associated with several cancers including colorectal cancer (CRC). However, the signature of histone PTMs on circulating nucleosomes is still not well described. We have developed a fast and robust enrichment method to isolate circulating nucleosomes from plasma for further downstream proteomic analysis. This method enabled us to quantify the global alterations of histone PTMs from 9 CRC patients and 9 healthy donors. Among 54 histone proteoforms identified and quantified in plasma samples, 13 histone PTMs were distinctive in CRC. Notably, methylation of histone H3K9 and H3K27, acetylation of histone H3 and citrullination of histone H2A1R3 were upregulated in plasma of CRC patients. A comparative analysis of paired samples identified 3 common histone PTMs in plasma and tumor tissue including the methylation and acetylation state of lysine 27 of histone H3. Moreover, we highlight for the first time that histone H2A1R3 citrulline is a modification upregulated in CRC patients. This new method presented herein allows the detection and quantification of histone variants and histone PTMs from circulating nucleosomes in plasma samples and could be used for biomarker discovery of cancer.

Circulating Nucleosomes as Potential Markers to Monitor COVID-19 Disease Progression.

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.

Circulating cell-free nucleosomes as biomarker for kidney transplant rejection: a pilot study.

BACKGROUND: There is an unmet need for noninvasive markers specific for kidney transplant rejection. Such a marker may eventually overcome the need for a transplant biopsy. In this pilot study, the potential of circulating cell-free nucleosomes (CCFN) to serve as a biomarker for kidney transplant rejection was evaluated. METHODS: Forty de novo kidney transplant recipients were prospectively followed as part of a randomized, controlled clinical trial. Total CCFN (H3) and CCFN with the histone modifications H3K36me3 and H3 citrulline were measured in patients at four fixed time points: before transplantation and on days 3-6, 30 and 180 after kidney transplantation. In addition, serum collected at times of transplant rejection (n = 14) was analyzed. CCFN were measured with a Nu.Q™ Assay kit (VolitionRx), an ELISA-based assay using antibodies directed against nucleosomes. RESULTS: For total CCFN (H3), H3K36me3, and H3 citrulline, the same pattern was seen over time: Concentrations were elevated shortly after transplantation (day 3-6) followed by a decline reaching baseline (pre-transplantation) values at days 30 and 180. At times of acute rejection, the median concentration of total CCFN (H3) was significantly higher compared to the stable situation (day 30): 4309 (3435-5285) versus 2885 (1668-3923) ng/mL, p < 0.05, respectively. Total CCFN (H3) had an acceptable ability to discriminate rejection from no rejection (AUC-ROC = 0.73) with a negative predictive value of 92.9%. For both histone modifications (H3K36me3 and H3 citrulline), there was no significant difference between episodes of acute rejection and the stable situation (day 30). CONCLUSION: In this pilot study, total CCFN (H3) concentrations are increased at times of acute kidney transplant rejection. The high negative predictive value implies that whenever a patient experiences loss of renal transplant function and the total CCFN (H3) is not increased, causes other than acute rejection should be considered. Clinical implementation of total CCFN (H3) measurement may avoid unnecessary and potentially harmful kidney transplant biopsies.

NETosis Markers in Pregnancy: Effects Differ According to Histone Subtypes.

NETosis is an innate immune response occurring after infection or inflammation: activated neutrophils expel decondensed DNA in complex with histones into the extracellular environment in a controlled manner. It activates coagulation and fuels the risk of thrombosis. Human pregnancy is associated with a mild proinflammatory state characterized by circulatory neutrophil activation which is further increased in complicated pregnancies, placenta-mediated complications being associated with an increased thrombotic risk. This aberrant activation leads to an increased release of nucleosomes in the blood flow. The aim of our study was to initially quantify nucleosome-bound histones in normal pregnancy and in placenta-mediated complication counterpart. We analyzed the role of histones on extravillous trophoblast function. Circulating nucleosome-bound histones H3 (Nu.QH3.1, Nu.QH3PanCit, Nu.QH3K27me3) and H4 (Nu.QH4K16Ac) were increased in complicated pregnancies. In vitro using the extravillous cell line HTR-8/SVNeo, we observed that free recombinant H2B, H3, and H4 inhibited migration in wound healing assay, but only H3 also blocked invasion in Matrigel-coated Transwell experiments. H3 and H4 also induced apoptosis, whereas H2B did not. Finally, the negative effects of H3 on invasion and apoptosis could be restored with enoxaparin, a low-molecular-weight heparin (LMWH), but not with aspirin. Different circulating nucleosome-bound histones are increased in complicated pregnancy and this would affect migration, invasion, and induce apoptosis of extravillous trophoblasts. Histones might be part of the link between the risk of thrombosis and pregnancy complications, with an effect of LMWH on both.

Evaluation of nucleosome concentrations in healthy dogs and dogs with cancer.

INTRODUCTION: Nucleosomes consist of small fragments of DNA wrapped around a histone octamer core. Diseases such as cancer or inflammation lead to cell death, which causes fragmentation and release of nucleosomes into the blood. The Nu.Q™ technology measures circulating nucleosome levels and exploits the different compositions of cancer derived nucleosomes in blood to detect and identify cancer even at early stages. The objectives of this study are to identify the optimal sample type for the Nu.Q™ H3.1 assay and to determine if it can accurately detect nucleosomes in the blood of healthy canines as well as those with cancer. MATERIALS AND METHODS: Blood samples from healthy canine volunteers as well as dogs newly diagnosed with lymphoma were used. The blood was processed at a variety of times under a variety of conditions to determine the most reliable sample type and conditions, and to develop an appropriate processing strategy to ensure reliably accurate results. RESULTS: Nucleosomes could be detected using a variety of sample collection and processing protocols. Nucleosome signals were highest in EDTA plasma and serum samples and most consistent in plasma. Samples should be processed within an hour of collection. Experiments showed that samples were able to withstand several freeze thaw cycles. Processing time and tcollection tube type did affect nucleosome detection levels. Finally, significantly elevated concentrations of nucleosomes were seen in a small cohort of dogs that had been newly diagnosed with lymphoma. CONCLUSIONS: When samples are collected and processed appropriately, the Nu.Q™ platform can reliably detect nucleosomes in the plasma of dogs. Further testing is underway to validate and optimize the Nu.Q™ platform for veterinary use.

A new nucleosomic-based model to identify and diagnose SSc-ILD.

BACKGROUND: Systemic sclerosis (SSc) is a rare connective tissue disease associated with rapid evolving interstitial lung disease (SSc-ILD), driving its mortality. Specific biomarkers associated with the evolution of the lung disease are highly needed. We aimed to identify specific biomarkers of SSc-ILD to predict the evolution of the disease. Nucleosomes are stable DNA/protein complexes that are shed into the blood stream making them ideal candidates for biomarkers. METHODS: We studied circulating cell-free nucleosomes (cf-nucleosomes) in SSc patients, 31 with ILD (SSc-ILD) and 67 without ILD. We analyzed plasma levels for cf-nucleosomes and investigated whether global circulating nucleosome levels in association with or without other biomarkers of interest for systemic sclerosis or lung fibrosis (e.g., serum growth factors: IGFBP-1 and the MMP enzyme: MMP-9), could be suitable potential biomarkers for the correct identification of SSc-ILD disease. RESULTS: We found that H3.1 nucleosome levels were significantly higher in patients with SSc-ILD compared SSc patients without ILD (p < 0.05) and levels of MMP-9 were significantly increased in patients with SSc-ILD compared to SSc patients without ILD (p < 0.05). Conversely, IGFBP-1 was significantly reduced in patients with SSc-ILD compared to SSc without ILD (p < 0.001). The combination of cf-nucleosomes H3.1 coupled to MMP-9 and IGFBP-1 increased the sensitivity for the differential detection of SSc-ILD. High levels of accuracy were reached with this combined model: its performances are strong with 68.4% of positive predictive value and 77.2% of negative predictive value for 90% of specificity. With our model, we identified a significant negative correlation with FVC % pred (r = -0.22) and TLC % pred (r = -0.31). The value of our model at T1 (baseline) has a predictive power over the Rodnan score at T2 (after 6-18 months), showed by a significant linear regression with R2 = 19% (p = 0.013). We identified in the sole group of SSc-ILD patients a significant linear regression with a R2 = 54.4% with the variation of DLCO between T1 and T2 (p < 0.05). CONCLUSION: In our study, we identified a new blood-based model with nucleosomic biomarker in order to diagnose SSc-ILD in a SSc cohort. This model is correlated with TLC and FVC at baseline and predictive of the skin evolution and the DLCO. Further longitudinal exploration studies should be performed in order to evaluate the potential of such diagnostic and predictive model.

Circulating cell-free nucleosomes as biomarkers for early detection of colorectal cancer.

The aim was to evaluate serum levels of circulating cell-free nucleosomes (ccfn) containing a variety of epigenetic signals including 5-methylcytosine DNA, histone modifications H3K9Me3, H3K9Ac, H3S10PO4, H3K36Me3, H4K20Me3, H4PanAc and pH2AX, nucleosome variant H2AZ and nucleosome adducts with HMGB1 and EZH2 as well as ccfn per se, in addition to develop and evaluate predictor models based on the above mentioned ccfn and including serum levels of carcinoembryonic antigen (CEA), in early detection of colorectal cancer (CRC). Blood-samples were collected from 4,105 individuals undergoing colonoscopy. Serum levels of ccfn and CEA were determined using enzyme-linked immunosorbent assays platforms. Individual assessment of levels of ccfn showed area under the receiver operating characteristic curve (AUCROC) = 0.525-0.576 in discrimination of individuals with CRC from individuals with non-malignant findings. Predictor models including ccfn containing 5-methylcytosine DNA, CEA, age and gender improved results (AUCROC = 0.736, sensitivity = 0.37 at specificity = 0.90). Further improvement was achieved in discrimination of individuals with CRC from individuals with clean colorectum (AUCROC = 0.840, sensitivity = 0.57 at specificity = 0.90). The levels of ccfn among patients with CRC appeared to be stage-independent. In conclusion, the performance of the developed predictor models is potentially promising in early detection of CRC.

Circulating nucleosomes as new blood-based biomarkers for detection of colorectal cancer.

BACKGROUND: Colonoscopy is currently widely accepted as the gold standard for detection of colorectal cancer (CRC) providing detection of up to 95% of pre-cancerous lesions during the procedure. However, certain limitations exist in most countries including cost and access to the procedure. Moreover, colonoscopy is an invasive technique with risk inherent to the endoscopic procedure. For this reason, alternative screening tests, in particular, fecal occult blood-based tests, have been widely adopted for frontline screening. Limited compliance to colonoscopy and fecal screening approaches has prompted research on blood-based tests as an alternative approach to identifying individuals at risk who could then be referred for colonoscopy. Increased total levels of nucleosomes in the blood have been associated with tumor burden and malignancy progression. Here, we report for the first time, CRC-associated epigenetic profiles of circulating cell-free nucleosomes (cf-nucleosomes). METHODS: Levels of 12 epigenetic cf-nucleosome epitopes were measured in the sera of 58 individuals referred for endoscopic screening for CRC. RESULTS: Multivariate analysis defined an age-adjusted panel of four cf-nucleosomes that provided an AUC of 0.97 for the discrimination of CRC from healthy controls with high sensitivity at early stages (sensitivity of 75 and 86 at 90% specificity for stages I and II, respectively). A second combination of four cf-nucleosome biomarkers provided an AUC of 0.72 for the discrimination of polyps from the healthy group. CONCLUSIONS: This study suggests that a combination of different cf-nucleosome structures analyzed in serum samples by a simple ELISA is a promising approach to identify patients at risk of CRC.

Pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA or histone modification H3K9Me3.

AIM: To evaluate pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA (5mC) or histone modification H3K9Me3 (H3K9Me3). MATERIALS AND METHODS: Six studies were designed to assess the possible influence of pre-analytical variables. Study 1: influence of stasis and contamination with white-cells and platelets. Study 2: influence of within-day variations. Study 3: influence of day-to-day variation. Study 4: influence of temperature during handling and storage, and of neoplastic disease. Study 5: influence of colonoscopy. Study 6: influence of the surgical trauma. 5mC and H3K9Me3 measurements were performed using enzyme-linked immunosorbent assays. RESULTS: Stasis, white-cell and platelet contamination, within-day variations, varying storage time before centrifugation, colonoscopy, and surgical trauma had no significant influence on levels of 5mC or H3K9Me3. Day-to-day variations of 12.7% and 11.5% (intra-individual) and 98.1% and 60.8% (inter-individual) were shown for 5mC and H3K9Me3, respectively. Levels of 5mC or H3K9Me3 were significantly higher in samples stored at room temperature until centrifugation compared to samples stored on ice. Patients with cancer had significantly lower levels of 5mC or H3K9Me3 compared to levels in healthy individuals. CONCLUSION: Levels of 5mC or H3K9Me3 appear stable in most pre-analytical settings if blood samples are stored at room temperature until centrifugation.

Circulating nucleosomes as epigenetic biomarkers in pancreatic cancer.

BACKGROUND: 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. RESULTS: Multivariate analysis defined a panel of five serum cfnucleosome biomarkers that gave an area under the curve (AUC) of 0.95 for the discrimination of pancreatic cancer from healthy controls, which was superior to the diagnostic performance of the common pancreatic tumor biomarker, carbohydrate antigen 19-9 (CA 19-9) with an AUC of 0.87. Combining CA 19-9 with a panel of four cfnucleosome biomarkers gave an AUC of 0.98 with an overall sensitivity of 92 % at 90 % specificity. CONCLUSIONS: The present study suggests that global epigenetic profiling of cfnucleosomes in serum using a simple NuQ(®) immunoassay-based approach can provide novel diagnostic biomarkers in pancreatic cancer.

Novel serum nucleosomics biomarkers for the detection of colorectal cancer.

BACKGROUND: To improve prognosis of patients with colorectal cancer, powerful blood-based biomarkers enabling for early detection are needed. As genome-wide DNA hypomethylation is associated with carcinogenesis, and cell-free DNA, thought to be of tumor origin, is found in the circulation of patients with cancer, we investigated the relevance of 5-methylcytosine-modified DNA present in cell-free circulating nucleosomes as a serum biomarker using a convenient enzyme-linked immunosorbent assay (ELISA) technique. MATERIALS AND METHODS: Serum samples from 90 individuals [24 with colorectal cancer (CRC), 10 with benign colorectal diseases (BCD) and 56 healthy controls (HC)] were tested for the differential diagnostic performance of a novel ELISA for nucleosome-bound methylated DNA. Methodical features, including intra- and interassay imprecision, were tested using serum pools. To minimize interassay variability, values were transformed to adjusted optical densities and robust statistics were applied for clinical evaluation. Findings were later re-evaluated on a set of 113 patients (49 CRC, 26 BCD and 38 HC). RESULTS: Intra- and interassay reproducibility were 3.4% and 15.3%, respectively. Levels of circulating methylated DNA were significantly decreased in CRC and BCD when compared to HC (p<0.05), although there was no difference between BCD and CRC. For discrimination of CRC from HC, the area under the curve in receiver operating characteristic curve was 0.78 and sensitivities were 33% at 95% specificity and 75% at 70% specificity, respectively. The findings were generally confirmed when validated in the second set of patients. CONCLUSION: Reduced methylation of DNA on circulating nucleosomes detected by ELISA can potentially serve as a diagnostic tool in patients with CRC.

The histone demethylase Kdm3a is essential to progression through differentiation.

Histone demethylation has important roles in regulating gene expression and forms part of the epigenetic memory system that regulates cell fate and identity by still poorly understood mechanisms. Here, we examined the role of histone demethylase Kdm3a during cell differentiation, showing that Kdm3a is essential for differentiation into parietal endoderm-like (PE) cells in the F9 mouse embryonal carcinoma model. We identified a number of target genes regulated by Kdm3a during endoderm differentiation; among the most dysregulated were the three developmental master regulators Dab2, Pdlim4 and FoxQ1. We show that dysregulation of the expression of these genes correlates with Kdm3a H3K9me2 demethylase activity. We further demonstrate that either Dab2 depletion or Kdm3a depletion prevents F9 cells from fully differentiating into PE cells, but that ectopic expression of Dab2 cannot compensate for Kdm3a knockdown; Dab2 is thus necessary, but insufficient on its own, to promote complete terminal differentiation. We conclude that Kdm3a plays a crucial role in progression through PE differentiation by regulating expression of a set of endoderm differentiation master genes. The emergence of Kdm3a as a key modulator of cell fate decision strengthens the view that histone demethylases are essential to cell differentiation.