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.

The Open University's first one-day symposium on treatment-emergent neuroendocrine prostate cancer.

The Open University's first one-day symposium on treatment-emergent neuroendocrine prostate cancer attracted world-leading figures, early career researchers and industry colleagues. The symposium proved insightful into the 'real-world' impact and current problems faced in the diagnosis and treatment of neuroendocrine prostate cancer. It was important for this meeting to take place as the incidence of neuroendocrine prostate cancer is increasing due to the widespread use of next-generation androgen deprivation drugs. The symposium discussions proposed new molecularly driven deadlines to accelerate research and improved the treatment of this deadly and poorly recognized malignancy.

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.

Serological biomarkers in triage of FIT-positive subjects?

FIT-based colorectal cancer screening has been implemented in many countries including Denmark, where 916 colorectal cancer and 4468 high- or medium-risk adenoma patients were identified within April-December 2014, among 16,806 subjects with a positive FIT test. Screening increases the overall requirements for colonoscopy, which may challenge the current capacity. Some countries have increased their initial FIT cut-off level in order to comply with lack of colonoscopy capacity. Many patients with neoplasia will not be detected, however, by using increased FIT cut-off levels. The number of patients with neoplastic lesions missed by increased cut-off levels appears to be much higher than expected. Therefore, tests that identify those patients missed by increased FIT cut-off levels must be developed. Preliminary results of determination of one of several biomarker entities currently under investigation show that nucleosome blood tests may be one option for identifying some of these patients. Implementation of a triage test consisting of FIT, blood-based biomarkers and plus/minus colonoscopy is suggested to identify subjects with FIT levels between the initial and the increased cut-off level that must be offered colonoscopy. In addition, triage may reduce the frequency of unnecessary colonoscopies by 25%.

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.

Development of NuQ nucleosome blood tests for the detection of colon cancer.

Dr Jake Micallef speaks to Hannah L Wilson, Commissioning Editor: Dr Micallef has 20 years of experience in research and development and in the management of early-stage biotechnical companies, including the manufacture of biotechnology products and the establishment of manufacturing operations. Dr Micallef gained this experience while working for WHO over a 10-year period from 1985. While working for WHO, Dr Micallef developed new diagnostic products in the areas of reproductive health and cancer. In 1990 he commenced development of a new diagnostic technology platform for WHO that was launched in 1992 and supported 13 tests. Dr Micallef also initiated and implemented in-house manufacture (previously outsourced to Abbott Diagnostics Inc., Dartford, UK) and worldwide distribution of these products for WHO. In 1990, he started a 'not-for-profit' WHO company, Immunometrics Ltd (London, UK), which marketed and distributed those diagnostic products worldwide. In 1999 Dr Micallef studied for an MBA and went on to co-found Gene Expression Technologies Ltd (London, UK) in 2001 where he successfully lead the development of the chemistry of the GeneICE technology and implemented the manufacture of GeneICE molecules. He also played a major role in business development and procured a GeneICE contract with Bayer Pharmaceuticals (Leverkusen, Germany). From 2004 to 2007, he taught 'science and enterprise' to science research workers from four universities at CASS Business School (London, UK) before joining Cronos Therapeutics (London, UK) in 2004. In 2006 Cronos was listed in the UK on AIM, becoming ValiRx. Dr Micallef continued to work as Technical Officer for ValiRx, where he in-licensed the Hypergenomics and Nucleosomics technologies and co-founded ValiBio SA (Namur, Belgium), which is now Belgian Volition SA, a subsidiary of Singapore Volition. Dr Micallef was educated at King's College London (UK; BSc, Biology and Chemistry, 1977; PhD Physical Chemistry, 1981), St Thomas' Hospital Medical School, London (UK; MSc Chemical Pathology, 1985) and Imperial College Management School (UK; MBA, 2000).

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.