Circulating Cell Free DNA and Citrullinated Histone H3 As Useful Biomarkers of NETosis In Endometrial Cancer.

Abstract Background. Cancer mortality is mainly caused by organ failure and thrombotic events. It has been demonstrated that NETosis, a chromatin release mechanism implemented by neutrophils, may contribute to these lethal systemic effects. Our aim was to investigate NETosis biomarkers in endometrial cancer (EC).Methods. The experiments were conducted on 21 healthy subject (HS) with no gynecological conditions, and on 63 EC patients. To assess the presence of NETosis features IHC and IF was performed using antibodies against citrullinated histone H3 (citH3), neutrophil elastase (NE) and H2B. Serum levels of cell free DNA (cfDNA), cell free mitochondrial DNA (cfmtDNA) and citH3 were measured by qPCR using one microliter of deactivated serum, and by ELISA assay respectively. Fragmentation pattern of serum cfDNA was analyzed using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Chips. Receiver operating characteristic (ROC) analysis was used to identify a cut off for cfDNA and cfmtDNA values able to discriminate between ECs and HSs. Correlation analysis and multiple correspondance analysis (MCA), between cfDNA, mitcfDNA, citH3 and blood parameters were used to identify the potential association among serum parameters in EC grades. Results. We demonstrated the presence of NETosis features in tissues from all EC grades. Serum cfDNA and cfmtDNA levels discriminate ECs from HSs and a direct correlation between citH3 and cfDNA content and an inverse correlation between cfmtDNA and citH3 in EC sera was observed, not detectable in HSs. MCA indicates cfDNA, cfmtDNA and citH3 as features associated to G1 and G2 grades. A correlation between increased levels of cfDNA, citH3 and inflammation features was found. Finally, serum nucleosomal cfDNA fragmentation pattern varies in EC sera and correlates with increased levels of cfDNA, citH3, lymphocytes and fibrinogen.Conclusion. Our data highlight the occurrence of NETosis in EC and indicate serum cfDNA and citH3 as noninvasive biomarkers of tumor-induced systemic effects in endometrial cancer.

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Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS

Nature Communications ◽

10.1038/s41467-021-24109-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

A. Rose Brannon ◽

Gowtham Jayakumaran ◽

Monica Diosdado ◽

Juber Patel ◽

Anna Razumova ◽

...

Keyword(s):

Cancer Patients ◽

De Novo ◽

Low Frequency ◽

High Sensitivity ◽

Clinical Analysis ◽

De Novo Mutation ◽

Cell Free Dna ◽

Free Dna ◽

Somatic Alterations ◽

Mutation Profiling

AbstractCirculating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.

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Neutrophil Extracellular Traps May Contribute to the Pathogenesis in Adult-onset Still Disease

The Journal of Rheumatology ◽

10.3899/jrheum.181058 ◽

2019 ◽

Vol 46 (12) ◽

pp. 1560-1569 ◽

Cited By ~ 4

Author(s):

Mi-Hyun Ahn ◽

Jae Ho Han ◽

Young-Jun Chwae ◽

Ju-Yang Jung ◽

Chang-Hee Suh ◽

...

Keyword(s):

Immunohistochemical Analysis ◽

Serum Levels ◽

Neutrophil Extracellular Traps ◽

Polymorphonuclear Neutrophils ◽

Adult Onset ◽

Cell Free Dna ◽

Extracellular Traps ◽

Free Dna ◽

Still Disease

Objective.Release of neutrophil extracellular traps (NET) has been described as an effector mechanism of polymorphonuclear neutrophils in several inflammatory diseases. Thus, this study was performed to evaluate the role of NET in the pathogenesis of adult-onset Still disease (AOSD).Methods.We determined the serum levels of NET molecules and investigated their associations with clinical disease activities in patients with AOSD. Further, we analyzed the differences in the NETosis response in AOSD patients compared to healthy controls (HC). To explore the in vivo involvement of NET in AOSD, we performed immunohistochemical analysis of skin and lymph node (LN) biopsies for proteins related to NET in patients with active AOSD.Results.Serum levels of cell-free DNA, myeloperoxidase (MPO)-DNA complex, and α-defensin were significantly increased in patients with AOSD compared to HC. Serum levels of the NET molecules, cell-free DNA, MPO-DNA, and α-defensin were correlated with several disease activity markers for AOSD. In followup of patients with AOSD after treatment with corticosteroid, the levels of cell-free DNA and α-defensin decreased significantly. On immunohistochemistry, neutrophil elastase–positive and MPO-positive inflammatory cells were detected in skin and LN of patients with AOSD, and were expressed in fiber form in the lesions. The serum from patients with active AOSD induced NETosis in neutrophils from HC. NET molecules induced interleukin 1β production in monocytes, representing a novel mechanism in the pathogenesis of AOSD.Conclusion.The findings presented here suggest that NET may contribute to the inflammatory response and pathogenesis in AOSD.

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Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions

Diagnostics ◽

10.3390/diagnostics10040234 ◽

2020 ◽

Vol 10 (4) ◽

pp. 234 ◽

Cited By ~ 2

Author(s):

Eun Young Lee ◽

Eun-Ju Lee ◽

Hana Yoon ◽

Dong Hyeon Lee ◽

Kwang Hyun Kim

Keyword(s):

Cost Efficiency ◽

High Sensitivity ◽

Storage Conditions ◽

Sample Storage ◽

Sample Collection ◽

Cell Free Dna ◽

Free Dna ◽

Zymo Research ◽

Free Circulating Dna ◽

Commercial Kits

Urinary cell-free DNA (cfDNA) is an attractive body fluid for liquid biopsy. In this study, we compared the efficiencies of four commercial kits for urinary cell-free DNA (cfDNA) isolation and of various sample storage conditions. Urinary cfDNA was isolated from 10 healthy individuals using four commercial kits: QIAamp Circulating Nucleic Acid Kit (QC; Qiagen), MagMAX™ Cell-Free DNA Isolation Kit (MM; Applied Biosystems), Urine Cell-Free Circulating DNA Purification Midi Kit (NU; Norgen Biotek), and Quick-DNA™ Urine Kit (ZQ; Zymo Research). To assess the isolation efficiency, an Agilent 2100 Bioanalyzer with High Sensitivity DNA chips was used, and cfDNA yield was defined as the amount of cfDNA obtained from 1 mL of urine. MM and QC provided the highest cfDNA yield in the 50–300 bp range, and MM and NU gave the highest cfDNA yield in the 50–100 bp range. In particular, the NU kit was efficient for isolation of more fragmented cfDNA in the range of 50–100 bp with the lowest cellular genomic DNA contamination. ZQ had the best cost-efficiency for isolating the same amount of urinary cfDNA. Samples stored at −70 °C with the addition of 10 mM EDTA resulted in the highest cfDNA yield 3 months after sample collection.

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The amount of circulating cell-free DNA present in plasma of endometrial cancer patients is associated with surgical stage, histology and MRI-defined tumor volume

Gynecologic Oncology ◽

10.1016/j.ygyno.2019.04.139 ◽

2019 ◽

Vol 154 ◽

pp. 58

Author(s):

C.W. Ashley ◽

A. Stylianou ◽

J. Nincevic ◽

Y. Lakhman ◽

M.M. Leitao ◽

...

Keyword(s):

Endometrial Cancer ◽

Cancer Patients ◽

Tumor Volume ◽

Cell Free Dna ◽

Free Dna ◽

Circulating Cell Free Dna

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Longitudinal monitoring of cell-free DNA to predict for transarterial chemo-embolization failure in hepatocellular carcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e14544 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e14544-e14544

Author(s):

Joanne Evans ◽

Caroline Ward ◽

Madhava Pai ◽

Rohini Sharma

Keyword(s):

Hepatocellular Carcinoma ◽

Early Stage ◽

High Sensitivity ◽

Cost Effective ◽

Standard Of Care ◽

Intermediate Stage ◽

Cell Free Dna ◽

Early Stage Disease ◽

Free Dna ◽

Synthetic Function

e14544 Background: Transarterial chemo-embolization (TACE) is the standard of care for patients with intermediate stage hepatocellular carcinoma (HCC) with adequate synthetic function, and as bridging treatment in early stage disease. Survival outcomes are heterogeneous, and improved biomarkers are needed for detection of early relapse. Current practice relies on monitoring of alpha-foetoprotein (AFP) and serial imaging. Circulating cell-free DNA (cfDNA) is a compelling emergent biomarker. Here, we describe the first study of cfDNA as prognostic biomarker in HCC following TACE. Methods: 34 patients treated with TACE (2012-2018) who had full demographic information and longitudinal stored plasma available were identified. Samples were retrieved and cfDNA extracted using the QIAamp Circulating Nucleic Acid Kit. cfDNA yields were quantified by high-sensitivity Qubit analysis. Results: 74% of patients were male, 66% had Childs Pugh A disease, and 56% of patients secreted AFP ( > 10ng/mL). Aetiologies of liver diseases were varied: viral hepatitis (44%), alcoholic or non-alcoholic steatohepatitis/cirrhosis (29%), hereditary or auto-immune (12%), and unexplained (15%). 79% of patients saw a fall in cfDNA titre following TACE. Where no reduction was seen, patients had a poorer median overall survival than those who did: 20 months (range 5.13 – 66.7 months) compared to 37 months (9.9 – 79.3 months). Of note, 44% of patients were AFP non-secretors. Here, cfDNA had a clear advantage in disease monitoring, with mean cfDNA titres in this group falling post treatment: from 1605.1 (range 185.9 – 6830) ng/mL to 693.7 (range undetectable - 2300) ng/mL and rising again with subsequent progression (mean of 1329.2 - range 616 – 3341 - ng/mL). Conclusions: These findings support a cost-effective monitoring role for plasma cfDNA analysis following treatment with TACE, with an added advantage in AFP non-secretors. Prospective validation is suggested to fully assess clinical utility.

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High Sensitivity of Plasma Cell-Free DNA Genotyping in Cases With Evidence of Adequate Tumor Content

JCO Precision Oncology ◽

10.1200/po.20.00420 ◽

2021 ◽

pp. 921-930

Author(s):

Catherine B. Meador ◽

Marina S. D. Milan ◽

Emmy Y. Hu ◽

Mark M. Awad ◽

Michael S. Rabin ◽

...

Keyword(s):

Plasma Cell ◽

Nsclc Patient ◽

False Negative ◽

High Sensitivity ◽

Driver Mutation ◽

Driver Mutations ◽

Resistance Mutations ◽

Assay Sensitivity ◽

Cell Free Dna ◽

Free Dna

PURPOSE Plasma cell-free DNA (cfDNA) sequencing is a compelling diagnostic tool in solid tumors and has been shown to have high positive predictive value. However, limited assay sensitivity means that negative plasma genotyping, or the absence of detection of mutation of interest, still requires reflex tumor biopsy. METHODS We analyzed two independent cohorts of patients with advanced non–small-cell lung cancer (NSCLC) with known canonical driver and resistance mutations who underwent plasma cfDNA genotyping. We measured quantitative features, such as maximum allelic frequency (mAF), as clinically available measures of cfDNA tumor content, and studied their relationship with assay sensitivity. RESULTS In patients with EGFR-mutant NSCLC harboring EGFR T790M, detection of driver mutation at > 1% AF conferred a sensitivity of 97% (368/380) for detection of T790M across three cfDNA genotyping platforms. Similarly, in a second cohort of patients with EGFR or KRAS driver mutations, when the mAF of nontarget mutations was > 1%, sensitivity for driver mutation detection was 100% (43/43). Combining the two NSCLC patient cohorts, the presence of nontarget mutations at mAF > 1% predicts for high sensitivity (> 95%) for identifying the presence of the known driver mutation, whereas mAF of ≤ 1% confers sensitivity of only 26%-54% across platforms. Focusing on 21 false-negative cases where the driver mutation was not detected on plasma next-generation sequencing, other mutations (presumably clonal hematopoiesis) were detected at ≤ 1% AF in 14 (67%). CONCLUSION Plasma cfDNA genotyping is highly sensitive when adequate tumor DNA content is present. The likelihood of a false-negative cfDNA genotyping result is low in a sample with evidence of > 1% tumor content. Bioinformatic approaches are needed to further optimize the assessment of cfDNA tumor content in plasma genotyping assays.

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Post-biopsy cell-free DNA from blood: an open window on primary prostate cancer genetics and biology

10.21203/rs.3.rs-147849/v1 ◽

2021 ◽

Author(s):

Marinella Corbetta ◽

Chiara Chiereghin ◽

Ilaria De simone ◽

Giulia Maria Emilia Antonietta Soldà ◽

Monica Zuradelli ◽

...

Keyword(s):

Prostate Cancer ◽

Cancer Detection ◽

Prostate Biopsy ◽

Somatic Mutations ◽

Cancer Genetics ◽

High Sensitivity ◽

Patient Specific ◽

Cell Free Dna ◽

Primary Tumors ◽

Free Dna

Abstract BackgroundCirculating cell-free DNA (ccfDNA), released from normal and cancerous cells, is a promising biomarker for cancer detection as in neoplastic patients it is enriched in tumor-derived DNA (ctDNA). ctDNA contains cancer-specific mutations and epigenetic modifications, which can have diagnostic/prognostic value. However, in primary tumors, and in particular in localized prostate cancer (PCa), the fraction of ctDNA is very low and conventional strategies to study ccfDNA are unsuccessful.MethodsccfDNA was isolated from plasma before and after prostate biopsy by using the Maxwell RSC ccfDNA Plasma Kit and quantitatively and qualitatively analyzed by a fluorometer and by the Agilent High Sensitivity D5000 ScreenTape System. Somatic mutations were searched for by targeted RNAseq on prostate biopsies, sequencing libraries were prepared using the TruSight RNA Pan-Cancer Panel from Illumina. Detection of selected somatic mutations in ccfDNA was performed by ultra-deep sequencing of amplicons, using the NEBNext® Ultra™ II DNA Library Prep Kit for library preparation. All libraries were sequenced on the NextSeq550 platform.ResultsHere we demonstrate that prostate biopsy, by causing multiple injuries to the organ, leads to a significant increase in plasma concentration of ccfDNA (P < 0.0024) in primary PCa patients. By calculating the minor allele fraction at patient-specific somatic mutations pre- and post-biopsy, we show that ctDNA is significantly enriched (from 3.9 to 164 fold) after biopsy, representing a transient “molecular window” to access and analyze ctDNA. Moreover, we show that newly released ccfDNA contains a larger fraction of di-, tri- and multi-nucleosome associated DNA fragments. This feature could be exploited to further enrich prostate-derived ccfDNA and to analyze epigenetic markers.ConclusionOur data represent a proof-of-concept that liquid tumor profiling from peripheral blood performed just after the biopsy procedure can open a “valuable molecular metastatic window” giving access to the tumor genetic asset, thus providing an opportunity for early cancer detection and individual genomic profiling in the view of PCa precision medicine.

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Properties and Application of Cell-Free DNA as a Clinical Biomarker

International Journal of Molecular Sciences ◽

10.3390/ijms22179110 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9110

Author(s):

Felipe Silva de Miranda ◽

Valério Garrone Barauna ◽

Leandro dos Santos ◽

Gustavo Costa ◽

Paula Frizera Vassallo ◽

...

Keyword(s):

Clinical Practice ◽

Venous Blood ◽

Endothelial Damage ◽

Extracellular Dna ◽

Release Mechanism ◽

Blood Collection ◽

Cell Free Dna ◽

Molecular Features ◽

Free Dna ◽

Clinical Biomarker

Biomarkers are valuable tools in clinical practice. In 2001, the National Institutes of Health (NIH) standardized the definition of a biomarker as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. A biomarker has clinical relevance when it presents precision, standardization and reproducibility, suitability to the patient, straightforward interpretation by clinicians, and high sensitivity and/or specificity by the parameter it proposes to identify. Thus, serum biomarkers should have advantages related to the simplicity of the procedures and to the fact that venous blood collection is commonplace in clinical practice. We described the potentiality of cfDNA as a general clinical biomarker and focused on endothelial dysfunction. Circulating cell-free DNA (cfDNA) refers to extracellular DNA present in body fluid that may be derived from both normal and diseased cells. An increasing number of studies demonstrate the potential use of cfDNA as a noninvasive biomarker to determine physiologic and pathologic conditions. However, although still scarce, increasing evidence has been reported regarding using cfDNA in cardiovascular diseases. Here, we have reviewed the history of cfDNA, its source, molecular features, and release mechanism. We also show recent studies that have investigated cfDNA as a possible marker of endothelial damage in clinical settings. In the cardiovascular system, the studies are quite new, and although interesting, stronger evidence is still needed. However, some drawbacks in cfDNA methodologies should be overcome before its recommendation as a biomarker in the clinical setting.

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