scholarly journals Epigenetic Biomarkers in Cell-Free DNA and Applications in Liquid Biopsy

Genes ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 32 ◽  
Author(s):  
Wanxia Gai ◽  
Kun Sun
Keyword(s):  
Cancer Diagnosis ◽  
Liquid Biopsy ◽  
Prenatal Testing ◽  
Digital Pcr ◽  
Liquid Biopsies ◽  
Sequencing Technologies ◽  
Epigenetic Biomarkers ◽  
Free Circulating Dna ◽  
Epigenetic Biomarker ◽  
Fragmentation Patterns

Cell-free circulating DNA (cfDNA) in plasma has gained global interest as a diagnostic material for noninvasive prenatal testing and cancer diagnosis, or the so-called “liquid biopsy”. Recent studies have discovered a great number of valuable genetic and epigenetic biomarkers for cfDNA-based liquid biopsy. Considering that the genetic biomarkers, e.g., somatic mutations, usually vary from case to case in most cancer patients, epigenetic biomarkers that are generalizable across various samples thus possess certain advantages. In this study, we reviewed the most recent studies and advances on utilizing epigenetic biomarkers for liquid biopsies. We first reviewed more traditional methods of using tissue/cancer-specific DNA methylation biomarkers and digital PCR or sequencing technologies for cancer diagnosis, as well as tumor origin determination. In the second part, we discussed the emerging novel approaches for exploring the biological basis and clinical applications of cfDNA fragmentation patterns. We further provided our comments and points of view on the future directions on epigenetic biomarker development for cfDNA-based liquid biopsies.

scholarly journals High Clinical Value of Liquid Biopsy to Detect Circulating Tumor Cells and Tumor Exosomes in Pancreatic Ductal Adenocarcinoma Patients Eligible for Up-Front Surgery

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1656 ◽  
Author(s):  
Etienne Buscail ◽  
Catherine Alix-Panabières ◽  
Pascaline Quincy ◽  
Thomas Cauvin ◽  
Alexandre Chauvet ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Liquid Biopsy ◽  
Neoadjuvant Treatment ◽  
Digital Pcr ◽  
Portal Blood ◽  
Ductal Adenocarcinoma ◽  
Clinical Value ◽  
Liquid Biopsies

Purpose: Expediting the diagnosis of pancreatic ductal adenocarcinoma (PDAC) would benefit care management, especially for the start of treatments requiring histological evidence. This study evaluated the combined diagnostic performance of circulating biomarkers obtained by peripheral and portal blood liquid biopsy in patients with resectable PDAC. Experimental design: Liquid biopsies were performed in a prospective translational clinical trial (PANC-CTC #NCT03032913) including 22 patients with resectable PDAC and 28 noncancer controls from February to November 2017. Circulating tumor cells (CTCs) were detected using the CellSearch® method or after RosetteSep® enrichment combined with CRISPR/Cas9-improved KRAS mutant alleles quantification by droplet digital PCR. CD63 bead-coupled Glypican-1 (GPC1)-positive exosomes were quantified by flow cytometry. Results: Liquid biopsies were positive in 7/22 (32%), 13/22 (59%), and 14/22 (64%) patients with CellSearch® or RosetteSep®-based CTC detection or GPC1-positive exosomes, respectively, in peripheral and/or portal blood. Liquid biopsy performance was improved in portal blood only with CellSearch®, reaching 45% of PDAC identification (5/11) versus 10% (2/22) in peripheral blood. Importantly, combining CTC and GPC1-positive-exosome detection displayed 100% of sensitivity and 80% of specificity, with a negative predictive value of 100%. High levels of GPC1+-exosomes and/or CTC presence were significantly correlated with progression-free survival and with overall survival when CTC clusters were found. Conclusion: This study is the first to evaluate combined CTC and exosome detection to diagnose resectable pancreatic cancers. Liquid biopsy combining several biomarkers could provide a rapid, reliable, noninvasive decision-making tool in early, potentially curable pancreatic cancer. Moreover, the prognostic value could select patients eligible for neoadjuvant treatment before surgery. This exploratory study deserves further validation.

The role of liquid biopsies in prostate cancer management

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Chi-Ju Kim ◽  
Liang Dong ◽  
Sarah Amend ◽  
Yoon-Kyoung Cho ◽  
Kenneth Pienta
Keyword(s):  
Prostate Cancer ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Cancer Diagnosis ◽  
Liquid Biopsy ◽  
Liquid Biopsies ◽  
Cancer Management ◽  
The Common ◽  
Prostate Cancer Management

Liquid biopsy has emerged as a complement to invasive tissue biopsy to guide cancer diagnosis and treatment. The common liquid biopsy biomarkers are circulating tumor cells (CTCs), extracellular vesicles (EVs),...

Identifying the tissues-of-origin of circulating cell-free DNAs is a promising way in noninvasive diagnostics

2020 ◽  
Author(s):  
Xiaoqing Peng ◽  
Hong-Dong Li ◽  
Fang-Xiang Wu ◽  
Jianxin Wang
Keyword(s):  
Disease Risk ◽  
Parasitic Infection ◽  
Prenatal Testing ◽  
Methylation Pattern ◽  
Fragmentation Pattern ◽  
Dna Fragments ◽  
Noninvasive Diagnostics ◽  
Sequencing Technologies ◽  
Comprehensive Picture ◽  
Fragmentation Patterns

Abstract Advances in sequencing technologies facilitate personalized disease-risk profiling and clinical diagnosis. In recent years, some great progress has been made in noninvasive diagnoses based on cell-free DNAs (cfDNAs). It exploits the fact that dead cells release DNA fragments into the circulation, and some DNA fragments carry information that indicates their tissues-of-origin (TOOs). Based on the signals used for identifying the TOOs of cfDNAs, the existing methods can be classified into three categories: cfDNA mutation-based methods, methylation pattern-based methods and cfDNA fragmentation pattern-based methods. In cfDNA mutation-based methods, the SNP information or the detected mutations in driven genes of certain diseases are employed to identify the TOOs of cfDNAs. Methylation pattern-based methods are developed to identify the TOOs of cfDNAs based on the tissue-specific methylation patterns. In cfDNA fragmentation pattern-based methods, cfDNA fragmentation patterns, such as nucleosome positioning or preferred end coordinates of cfDNAs, are used to predict the TOOs of cfDNAs. In this paper, the strategies and challenges in each category are reviewed. Furthermore, the representative applications based on the TOOs of cfDNAs, including noninvasive prenatal testing, noninvasive cancer screening, transplantation rejection monitoring and parasitic infection detection, are also reviewed. Moreover, the challenges and future work in identifying the TOOs of cfDNAs are discussed. Our research provides a comprehensive picture of the development and challenges in identifying the TOOs of cfDNAs, which may benefit bioinformatics researchers to develop new methods to improve the identification of the TOOs of cfDNAs.

scholarly journals Epigenetic Biomarkers of Renal Cell Carcinoma for Liquid Biopsy Tests

2021 ◽  
Vol 22 (16) ◽  
pp. 8846
Author(s):  
Raimonda Kubiliute ◽  
Sonata Jarmalaite
Keyword(s):  
Dna Methylation ◽  
Liquid Biopsy ◽  
Renal Cell ◽  
Morphological Changes ◽  
Diagnostic Tools ◽  
Liquid Biopsies ◽  
Non Invasive ◽  
Epigenetic Biomarkers ◽  
Treatment Changes

Renal cell carcinomas (RCC) account for 2–3% of the global cancer burden and are characterized by the highest mortality rate among all genitourinary cancers. However, excluding conventional imagining approaches, there are no reliable diagnostic and prognostic tools available for clinical use at present. Liquid biopsies, such as urine, serum, and plasma, contain a significant amount of tumor-derived nucleic acids, which may serve as non-invasive biomarkers that are particularly useful for early cancer detection, follow-up, and personalization of treatment. Changes in epigenetic phenomena, such as DNA methylation level, expression of microRNAs (miRNAs), and long noncoding RNAs (lncRNAs), are observed early during cancer development and are easily detectable in biofluids when morphological changes are still undetermined by conventional diagnostic tools. Here, we reviewed recent advances made in the development of liquid biopsy-derived DNA methylation-, miRNAs- and lncRNAs-based biomarkers for RCC, with an emphasis on the performance characteristics. In the last two decades, a mass of circulating epigenetic biomarkers of RCC were suggested, however, most of the studies done thus far analyzed biomarkers selected from the literature, used relatively miniature, local, and heterogeneous cohorts, and suffered from a lack of sufficient validations. In summary, for improved translation into the clinical setting, there is considerable demand for the validation of the existing pool of RCC biomarkers and the discovery of novel ones with better performance and clinical utility.

scholarly journals Denaturation-Enhanced Droplet Digital PCR for Liquid Biopsies

2018 ◽  
Vol 64 (12) ◽  
pp. 1762-1771 ◽  
Author(s):  
Mariana Fitarelli-Kiehl ◽  
Fangyan Yu ◽  
Ravina Ashtaputre ◽  
Ka Wai Leong ◽  
Ioannis Ladas ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Gene Copy Number ◽  
Fold Increase ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Gene Copy ◽  
Clinical Samples ◽  
Liquid Biopsies ◽  
Patients With Cancer ◽  
Free Circulating Dna

Abstract BACKGROUND Although interest in droplet-digital PCR technology (ddPCR) for cell-free circulating DNA (cfDNA) analysis is burgeoning, the technology is compromised by subsampling errors and the few clinical targets that can be analyzed from limited input DNA. The paucity of starting material acts as a “glass ceiling” in liquid biopsies because, irrespective how analytically sensitive ddPCR techniques are, detection limits cannot be improved past DNA input limitations. METHODS We applied denaturation-enhanced ddPCR (dddPCR) using fragmented genomic DNA (gDNA) with defined mutations. We then tested dddPCR on cfDNA from volunteers and patients with cancer for commonly-used mutations. gDNA and cfDNA were tested with and without end repair before denaturation and digital PCR. RESULTS By applying complete denaturation of double-stranded DNA before ddPCR droplet formation the number of positive droplets increased. dddPCR using gDNA resulted in a 1.9–2.0-fold increase in data-positive droplets, whereas dddPCR applied on highly-fragmented cfDNA resulted in a 1.6–1.7-fold increase. End repair of cfDNA before denaturation enabled cfDNA to display a 1.9–2.0-fold increase in data-positive signals, similar to gDNA. Doubling of data-positive droplets doubled the number of potential ddPCR assays that could be conducted from a given DNA input and improved ddPCR precision for cfDNA mutation detection. CONCLUSIONS dddPCR is a simple and useful modification in ddPCR that enables extraction of more information from low-input clinical samples with minor change in protocols. It should be applicable to all ddPCR platforms for mutation detection and, potentially, for gene copy-number analysis in cancer and prenatal screening.

scholarly journals Clinical Utility of Liquid Biopsy-Based Actionable Mutations Detected via ddPCR

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 906
Author(s):  
Irina Palacín-Aliana ◽  
Noemí García-Romero ◽  
Adrià Asensi-Puig ◽  
Josefa Carrión-Navarro ◽  
Víctor González-Rumayor ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Clinical Utility ◽  
Cancer Genetics ◽  
High Sensitivity ◽  
Digital Pcr ◽  
Liquid Biopsies ◽  
Cancer Subtypes ◽  
Cancer Management ◽  
Main Challenge ◽  
Therapeutic Decisions

Cancer is one of the leading causes of death worldwide and remains a major public health challenge. The introduction of more sensitive and powerful technologies has permitted the appearance of new tumor-specific molecular aberrations with a significant cancer management improvement. Therefore, molecular pathology profiling has become fundamental not only to guide tumor diagnosis and prognosis but also to assist with therapeutic decisions in daily practice. Although tumor biopsies continue to be mandatory in cancer diagnosis and classification, several studies have demonstrated that liquid biopsies could be used as a potential tool for the detection of cancer-specific biomarkers. One of the main advantages is that circulating free DNA (cfDNA) provides information about intra-tumoral heterogeneity, reflecting dynamic changes in tumor burden. This minimally invasive tool has become an accurate and reliable instrument for monitoring cancer genetics. However, implementing liquid biopsies across the clinical practice is still ongoing. The main challenge is to detect genomic alterations at low allele fractions. Droplet digital PCR (ddPCR) is a powerful approach that can overcome this issue due to its high sensitivity and specificity. Here we explore the real-world clinical utility of the liquid biopsy ddPCR assays in the most diagnosed cancer subtypes.

Greater than two coexisting mutations in KRAS and NRAS identified in the circulating tumor DNA fraction of liquid biopsies by NGS and confirmed with ddPCR.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15563-e15563
Author(s):  
Hala Boulos ◽  
Robert Tell ◽  
Nike Beaubier ◽  
Richard Blidner
Keyword(s):  
Liquid Biopsy ◽  
Detection Threshold ◽  
Treatment Resistance ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Precision Oncology ◽  
Single Nucleotide Variants ◽  
Liquid Biopsies ◽  
Low Frequencies ◽  
Tumor Dna

e15563 Background: Liquid biopsies are increasingly utilized as a non-invasive tool in precision oncology to assess tumor mutational profiles in order to select targeted therapies, detect treatment resistance, and monitor disease progression in cancer patients. Additionally, liquid biopsies may provide a more comprehensive representation of tumor heterogeneity than standard tissue biopsies. However, limitations such as scarcity of circulating tumor DNA (ctDNA) and/or variants at low frequencies can be technically challenging to detect by next-generation sequencing (NGS) assays. Here, we use NGS to detect greater than two KRAS/NRAS mutations coexisting in single samples at low variant allele frequencies (VAFs). Methods: The Tempus xF liquid biopsy NGS assay is designed to detect actionable oncologic targets spanning 105 genes in plasma. The assay was validated to reliably detect single-nucleotide variants at 0.25% VAF, indels and copy number variants at 0.5% VAF, and fusions at 1% VAF with 96.2%-100% specificity and 97.4%-100% sensitivity. Pre-designed digital PCR assays were modified to measure 10ng of cell-free DNA (cfDNA) on a droplet-digital PCR (ddPCR) platform. Results: Overall, we report 100% positive predictive value and high correlation between ddPCR results and xF VAF, as well as in individual variants, such as KRAS G12D. Unexpectedly, we detected more than two coexisting KRAS/NRAS mutations at a low VAF in the plasma samples. To orthogonally confirm these results, ddPCR was deployed to independently measure the presence of each cfDNA variant with a sensitivity of 0.09% VAF. Subsequent ddPCR analysis of all targeted variants were concordant with NGS results. Conclusions: The occurrence of multiple KRAS and NRAS mutations in a single sample is quite uncommon and may be falsely interpreted as an NGS artifact. However, verification of this phenomenon by ddPCR confirmed the validity of the NGS liquid biopsy approach. These results highlight the capability of the Tempus xF assay to detect low-frequency variants, including those that fall below the validated detection threshold, which is essential for the diagnosis of early disease.

scholarly journals Circular RNAs in body fluids as cancer biomarkers: the new frontier of liquid biopsies

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Sumeng Wang ◽  
Ke Zhang ◽  
Shanyue Tan ◽  
Junyi Xin ◽  
Qianyu Yuan ◽  
...  
Keyword(s):  
Cancer Diagnosis ◽  
Liquid Biopsy ◽  
Rapid Development ◽  
Expression Patterns ◽  
Body Fluids ◽  
Circular Rnas ◽  
Liquid Biopsies ◽  
Cancer Dataset ◽  
Widespread Application ◽  
Low Sensitivity

AbstractCancer is a leading cause of death worldwide, particularly because of its high mortality rate in patients who are diagnosed at late stages. Conventional biomarkers originating from blood are widely used for cancer diagnosis, but their low sensitivity and specificity limit their widespread application in cancer screening among the general population. Currently, emerging studies are exploiting novel, highly-accurate biomarkers in human body fluids that are obtainable through minimally invasive techniques, which is defined as liquid biopsy. Circular RNAs (circRNAs) are a newly discovered class of noncoding RNAs generated mainly by pre-mRNA splicing. Following the rapid development of high-throughput transcriptome analysis techniques, numerous circRNAs have been recognized to exist stably and at high levels in body fluids, including plasma, serum, exosomes, and urine. CircRNA expression patterns exhibit distinctly differences between patients with cancer and healthy controls, suggesting that circRNAs in body fluids potentially represent novel biomarkers for monitoring cancer development and progression. In this study, we summarized the expression of circRNAs in body fluids in a pan-cancer dataset and characterized their clinical applications in liquid biopsy for cancer diagnosis and prognosis. In addition, a user-friendly web interface was developed to visualize each circRNA in fluids (https://mulongdu.shinyapps.io/circrnas_in_fluids/).

scholarly journals The detection of specific hypermethylated WIF1 and NPY genes in circulating DNA by crystal digital PCR™ is a powerful new tool for colorectal cancer diagnosis and screening

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Alexis Overs ◽  
Mylène Flammang ◽  
Eric Hervouet ◽  
Laurent Bermont ◽  
Jean-Luc Pretet ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Point Of View ◽  
University Hospital ◽  
Liquid Biopsies ◽  
Tissue Samples ◽  
Mutation Status ◽  
Tumor Tissues ◽  
Tumor Stages

Abstract Background In oncology, liquid biopsy is of major relevance from theranostic point of view. The searching for mutations in circulating tumor DNA (ctDNA) in case of colorectal cancers (CRCs) allows the optimization of patient care. In this context, independent of mutation status biomarkers are required for its detection to confirm the presence of ctDNA in liquid biopsies. Indeed, the hypermethylation of NPY and WIF1 genes appear to be an ideal biomarker for the specific detection of ctDNA in CRCs. The objective of this work is to develop the research of hypermethylation of NPY and WIF1 by Crystal Digital PCR™ for the detection of ctDNA in CRCs. Methods Detection of hypermethylated NPY and WIF1 was developed on Cristal digital PCR™. Biological validation was performed from a local cohort of 22 liquid biopsies and 23 tissue samples from patients with CRC. These patients were treated at the University Hospital of Besancon (France). Results The local cohort study confirmed that NPY and WIF1 were significantly hypermethylated in tumor tissues compared to adjacent non-tumor tissues (WIF1 p < 0.001; NPY p < 0.001; non-parametric Wilcoxon paired-series test). Histological characteristics, tumor stages or mutation status were not correlated to the methylation profiles. On the other hand, hypermethylation of NPY or WIF1 in liquid biopsy had a 95.5% [95%CI 77–100%] sensitivity and 100% [95%CI 69–100%] specificity. Conclusion Using Crystal digital PCR™, this study shows that hypermethylation of NPY and WIF1 are constant specific biomarkers of CRCs regardless of a potential role in carcinogenesis.

Liquid Biopsy: General Concepts

2019 ◽  
Vol 63 (6) ◽  
pp. 449-455 ◽  
Author(s):  
Geoffroy Poulet ◽  
Joséphine Massias ◽  
Valerie Taly
Keyword(s):  
Liquid Biopsy ◽  
Tumor Heterogeneity ◽  
Invasive Procedure ◽  
Digital Pcr ◽  
Cancer Tissue ◽  
Minimally Invasive Procedure ◽  
Cancer Evolution ◽  
Sequencing Technologies ◽  
Standardization Of Procedures ◽  
Generation Sequencing

Liquid biopsy provides the opportunity of detecting, analyzing and monitoring cancer in various body effluents such as blood or urine instead of a fragment of cancer tissue. It is composed of different biological matrices such as circulating tumor cells (CTCs), cell free nucleic acids, exosomes or tumors “educated platelets.” In addition to representing a non- or minimally invasive procedure, it should represent a better view of tumor heterogeneity and allows for real-time monitoring of cancer evolution. Recent technological and molecular advances, greatly facilitated by the use of microfluidics in many cases, have permitted large progresses both in our ability to purify and analyze liquid biopsy components. In particular, the great developments of droplet-based digital PCR and the various optimizations of next generation sequencing technologies are central to the several validations of CTC-free DNA as a strong cancer biomarker. However, complete adoption of liquid biopsy in clinics will require pursuing recent efforts in the standardization of procedures both on the pre-analytical and analytical aspects.

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