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Circulating cell-free DNA (cfDNA) and circulating tumor cells (CTCs). Figure illustrates the origin of circulating cfDNA and CTCs. Circulating cfDNA may be released by apoptotic and necrotic cells, as well as through the secretion of living cells. In cancer patients, the fraction of tumor-derived cfDNA comprises circulating tumor DNA (ctDNA).
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Over the past decade, the advancements in massively parallel sequencing have provided a new paradigm in biomedical research to uncover the genetic basis of human diseases. Integration of ‘omics information has begun transforming clinical management of cancer patients in terms of diagnostics and treatment options, giving rise to the era of precision...
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There is a strong demand for the identification of new biomarkers in colorectal cancer (CRC) diagnosis. Among all liquid biopsy analysts, cell-free circulating DNA (cfDNA) is probably the most promising tool with respect to the identification of minimal residual diseases, assessment of treatment response and prognosis, and identification of resista...
Precision medicine in the clinical management of cancer may be achieved through the diagnostic platform called “liquid biopsy”. This method utilizes the detection of biomarkers in blood for prognostic and predictive purposes. One of the latest blood born markers under investigation in the field of liquid biopsy in cancer patients is circulating tum...
Background
The accurate microscopic diagnosis of lung cancer has become insufficient due to the concept of personalized medicine. Tissue samples are used not only for microscopic diagnosis but also for the assessment of the different targets. Biopsies are performed in 80% of the patients and they are not sufficient for molecular diagnosis in 30% of...
In oncology, liquid biopsy is used in the detection of next-generation analytes, such as tumor cells, cell-free nucleic acids and exosomes in peripheral blood and other body fluids from cancer patients. It is considered one of the most advanced non-invasive diagnostic systems to enable clinically relevant actions and implement precision medicine. M...
Citations
... Diagnostic approach of cancer is based on qualitative, with methylation profiles and different gene mutations being the main focus, and quantitative assessment, [69,70]. CfDNA and other nucleic acid biomarkers diagnostic accuracy in HCC detection has been a field of research in the part, with promising results [70][71][72][73][74][75]. Apart from the diagnosis of cancer, those biomarkers have been used for the evaluation of biological activity and metastatic profile of the tumor [73]. ...
Donor-derived cell-free DNA (Dd-cfDNA) is a novel biomarker with many diagnostic applications in various areas of medicine and particularly transplantation. This biomarker is derived from donor cells that have undergone apoptosis or cell death and thus reflects possible graft damage. Regarding the field of liver transplantation, dd-cfDNA can contribute to the diagnosis of complications that include signs of rejection or other types of possible graft injury. Measurements of dd-cfDNA also depend on the graft’s size and origin; therefore, these data should be considered for the estimation and explanation of dd-cfDNA values. Despite the utility of this novel diagnostic technique, it comes with some limitations and application exclusions, such as cases where there is a blood relation between the donor and recipient. Combination of dd-cfDNA evaluation with the assessment of other currently used biomarkers, such as liver enzymes, or other novel biomarkers can result to high diagnostic value.
... Although, in other neoplasms, the use of ctDNA is already more consolidated, in HCC, its use is still gaining ground [76]. Patients with HCC naturally have a cfDNA concentration 3-4 times higher than those of chronic hepatitis patients (53) and almost 20 times those of healthy patients [77][78][79][80]. ...
Simple Summary
The liver is the world’s sixth most common primary tumor site, accountable for nearly 5% of all cancers and over 8% of cancer-related deaths. Hepatocellular carcinoma (HCC) is the predominant type of liver cancer, accounting for about 75% of all primary liver tumors. One of the major therapeutic tools for this disease is liver transplantation. This therapeutic modality, as with the others, faces the obstacle of tumor recurrence, in addition to graft rejection. In this context, cell-free DNA is presented as a new tool for decision-making. In this article, we summarize the main aspects of this new tool, exploring its strengths and weaknesses in the treatment of HCC.
Abstract
The liver is the world’s sixth most common primary tumor site, responsible for approximately 5% of all cancers and over 8% of cancer-related deaths. Hepatocellular carcinoma (HCC) is the predominant type of liver cancer, accounting for approximately 75% of all primary liver tumors. A major therapeutic tool for this disease is liver transplantation. Two of the most significant issues in treating HCC are tumor recurrence and graft rejection. Currently, the detection and monitoring of HCC recurrence and graft rejection mainly consist of imaging methods, tissue biopsies, and alpha-fetoprotein (AFP) follow-up. However, they have limited accuracy and precision. One of the many possible components of cfDNA is circulating tumor DNA (ctDNA), which is cfDNA derived from tumor cells. Another important component in transplantation is donor-derived cfDNA (dd-cfDNA), derived from donor tissue. All the components of cfDNA can be analyzed in blood samples as liquid biopsies. These can play a role in determining prognosis, tumor recurrence, and graft rejection, assisting in an overall manner in clinical decision-making in the treatment of HCC.
... ОбСУждЕНИЕ К настоящему времени в научной литературе опубликован ряд исследований, посвященных выявлению мутаций, характерных для опухолевой ткани конкретных пациентов, в соответствующих препаратах цДНК и продемонстрирована принципиальная возможность мониторинга течения заболевания в отдельных случаях. Аналогичные исследования выполнены и в отношении генетически гетерогенных типов опухолей, таких как ГЦК [15]. Например, при ультраглубоком (5500×) секвенировании образцов цДНК, полученных из плазмы и сыворотки 8 пациентов с ГЦК, подвергнутых хирургическому лечению, с использованием специально разработанной для ГЦК таргетной панели на 58 генов [16] достоверное присутствие мутантного аллеля хотя бы одного из исследованных генов было выявлено лишь в 4 (50 %) случаях ГЦК, причем все они были ассоциированы с инфекцией вирусами гепатита В или С. В 2 случаях «диагностических» мутаций не было обнаружено даже при секвенировании опухолевой ткани тех же пациентов. ...
Introduction . Liquid biopsy is considered as a minimally invasive method of molecular genetic analysis that can be used for early diagnosis, prognosis of disease development, monitoring of residual disease or treatment outcomes, and selection of optimal drug therapy schemes for a patient. Along with the development of tests based on the study of panels of oncologically significant genes or their regions, for various forms of genetically heterogeneous tumors a promising approach could be the use as an object of liquid biopsy of an individual spectrum of somatic mutations of a particular patient that can be detected on the basis of high-throughput sequencing of tumor tissue.
Aim . To determine the applicability of different methods for detecting single-nucleotide somatic mutations detected in tumor tissue of a particular patient in cDNA preparations from blood plasma obtained before surgical removal of the tumor and to evaluate the possibility of quantifying the proportion of the alternative variant in the total pool of cDNA. Materials and methods. We used normal and tumor tissue, as well as blood plasma samples from patients with hepatocellular carcinoma, and various methods for detecting single-nucleotide somatic mutations: real-time polymerase chain reaction (PCR) with intercalating dye or with TaqMan probes, droplet digital PCR and high-throughput sequencing of target amplicons.
Results . Using the example of a somatic mutation in the TLN1 gene detected in tumor tissue of a patient with hepatocellular carcinoma, methods were developed and tested, each of which allows specific detection of the mutant variant in small amounts (2 ng) of cDNA from the blood plasma of the same patient. The use of droplet PCR and target amplicon sequencing methods allowed us to quantify the proportion of the mutant variant in the total cDNA pool, which was 19.7 and 23.5 %, respectively.
Conclusion . Among the methods investigated, droplet digital PCR and targeted amplicon sequencing allow not only reliable detection of mutant variants in small amounts of cDNA, but also adequate quantification, which is particularly important for the development of ways to monitor tumor growth during treatment. The close values of the proportion of mutant variants in cDNA detected by these methods indicate the accuracy of quantitative analysis and the possibility of their use for cross-validation of the results obtained.
... Its importance and promising outlook as a non-invasive marker for cancer has been recognized, utilizing genetic, methylation, and, to a lesser extent, quantitative analyses (Aarthy et al., 2015;Kustanovich et al., 2019). For HCC in particular, methylation analysis of cfDNA has yielded multiple diagnostic biomarkers, including but not limited to p15, p16, GSTP1 and RASSF1A (Ng et al., 2018). ...
Purpose
Hepatocellular carcinoma is the most common primary liver cancer, accounting for 90% of cases, and a major cause of death worldwide. Despite this, alpha-fetoprotein tests are the only blood-based diagnostic tools available, and their use is limited by their low sensitivity. DNA methylation changes, which have been implicated in a majority of cancers, offer an alternative method of diagnosis through measuring such changes in circulating cell-free DNA present in blood plasma.
Method
A genetic programming-based symbolic regression approach was applied to gain the benefits of machine learning while avoiding the opacity drawbacks of “black box” models. The data included plasma samples from 36 patients with hepatocellular carcinoma as well as a control group of 55 that contained patients with and without cirrhosis. A 75-25 train-test splitting was done before training.
Results
The symbolic regression methodology developed an equation utilizing the methylation levels of three biomarkers, with an accuracy of 91.3%, a sensitivity of 100%, and a specificity of 87.5% on the test data. All three biomarkers are differentially methylated in cancerous and non-cancerous samples. The performance matches prior research while providing the added benefits of transparency.
Conclusion
Circulating cell-free DNA presents opportunities for minimally invasive early diagnosis of hepatocellular carcinoma, and utilizing transparent machine learning approaches like symbolic regression can allow accurate diagnosis by combining biological and mathematical principles. Future validation of the model obtained here on a larger and more diverse dataset can reveal the potential for such approaches in cancer diagnosis and open the way for further research.
... Changes in DNA methylation, particularly in the CpG islands of tumor suppressor genes, have been demonstrated to be pivotal in HCC development [66]. Analysis of the methylation pattern of cfDNA may have a value as diagnostic and prognostic biomarker, and might reveal information about tumor size, risk of metastatic spread, and recurrence [67]. Alterations in DNA methylation patterns in HCC tumor tissue after liver resection have been described for many genes. ...
... Alterations in DNA methylation patterns in HCC tumor tissue after liver resection have been described for many genes. In particular, hypermethylation was found in p15, CDKN2A (encoding for p16), glutathione S-transferase (GSTP1), Ras association domain family 1A (RASSF1A), APC, SOCS1, SOCS3, TIMP3, blood vessel epicardial substance (BVES), and Homeobox A9 (HOXA9) genes, while hypomethylation in long interspersed element-1 (LINE-1) repetitive sequence [67][68][69][70][71][72][73]. However, only a proportion of cfDNA carried the same methylation patterns: hypermethylation of GSTP1 and RASSF1A was found in 50% and in 70-93% of cases respectively, while hypomethylation of LINE-1 in approximately 67% of cases [71,72]. ...
Simple Summary
Hepatocellular carcinoma (HCC) is one of the mostly lethal cancers, with a prognosis which is still very poor. Novel reliable biomarkers, useful in early diagnosis and prognosis assessment, are urgently needed in order to improve HCC patient survival. In recent years, several studies focused on liquid biopsy, the molecular analysis of circulating cancer by-products, as a source of novel biomarkers. Extracellular vesicles, circulating tumor cells, cell-free DNA and non-coding RNA provided very interesting results in a large number of studies published recently, but none of them has entered the clinical routine. In this review we will summarize the available evidence on these novel circulating biomarkers as diagnostic, prognostic, and predictive tools. Liquid biopsy proved to be a very useful source of biomarkers, some of which will probably be applied soon in clinical practice.
Abstract
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related death worldwide. Diagnostic, prognostic, and predictive biomarkers are urgently needed in order to improve patient survival. Indeed, the most widely used biomarkers, such as alpha-fetoprotein (AFP), have limited accuracy as both diagnostic and prognostic tests. Liver biopsy provides an insight on the biology of the tumor, but it is an invasive procedure, not routinely used, and not representative of the whole neoplasia due to the demonstrated intra-tumoral heterogeneity. In recent years, liquid biopsy, defined as the molecular analysis of cancer by-products, released by the tumor in the bloodstream, emerged as an appealing source of new biomarkers. Several studies focused on evaluating extracellular vesicles, circulating tumor cells, cell-free DNA and non-coding RNA as novel reliable biomarkers. In this review, we aimed to provide a comprehensive overview on the most relevant available evidence on novel circulating biomarkers for early diagnosis, prognostic stratification, and therapeutic monitoring. Liquid biopsy seems to be a very promising instrument and, in the near future, some of these new non-invasive tools will probably change the clinical management of HCC patients.
... In an animal model study, two potential markers-serum glycoprotein osteopontin and dickkopf-1were determined [130]. Another potential marker is circulating tumor DNA (ctDNA) which can be used to determine if specific genetic mutations have occurred [131]. The ctDNA would be for common pathways involved in HCC oncogenesis such as p53 signaling, the Wnt-β-catenin pathway, chromatin remodeling, responses to oxidative stress (for example, KEAP1 and NFE2L2) or telomere maintenance pathways [96]. ...
Non-alcoholic steatohepatitis (NASH) is a chronic and progressive form of non-alcoholic fatty liver disease (NAFLD). Its global incidence is increasing which makes NASH an epidemic and public health threat. Due to repeated insults to the liver, patients are at risk for developing hepa-tocellular carcinoma (HCC). The progression of NASH to HCC was initially defined according to a two-hit model which involved the development of steatosis, followed by lipid peroxidation and inflammation. However, current research defines a “multi-hit” or “multi-parallel hit” model which synthesizes several contributing pathways involved in progressive fibrosis and oncogene-sis. This perspective considers the effects of cellular, genetic, immunologic, metabolic, and endo-crine pathways leading up to HCC which underscores the complexity of this condition. This arti-cle will provide an updated review of the pathogenic mechanisms leading from NASH to HCC as well as explore the role of biomarkers and screening.
... Flow cytometry is commonly used to search for CTCs through fluorescently labelled cellular tags, many which target stem cell markers such as: Epithelial cell adhesion molecule (EpCAMs), CD133, CD90, CD44, CD13, and cytokeratin 19 [95,96] . A study by Sun et al [97] demonstrated the clinical significance of CTCs in 123 HCC patients by analyzing EpCAM, which showed a high probability of tumour recurrence in individuals with ≥ 2 CTCs in 7.5 mL of blood. Recurrence was found in 26 of 51 patients with ≥ 2 CTCs, whereas only 15 of 72 patients with < 2 CTCs showed recurrence after curative resections. ...
... Recurrence was found in 26 of 51 patients with ≥ 2 CTCs, whereas only 15 of 72 patients with < 2 CTCs showed recurrence after curative resections. The mean follow-up time was reported to be 15.1 ± 2.3 mo [97] . Perioperative analysis of CTCs for individuals with HCC may provide insight on prognosis and can tailor clinical treatment decisions. ...
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. If diagnosed early, curative treatment options such as surgical resection, loco-regional therapies, and liver transplantation are available to patients, increasing their chances of survival and improving their quality of life. Unfortunately, most patients are diagnosed with late stage HCC where only palliative treatment is available. Therefore, biomarkers which could detect HCC early with a high degree of sensitivity and specificity, may play a crucial role in the diagnosis and management of the disease. This review will aim to provide an overview of the different biomarkers of HCC comprising those used in the diagnosis of HCC in at risk populations, as well as others with potential for prognosis, risk predisposition and prediction of response to therapeutic intervention.
... 11 The characterization of cfDNA in HCC patients has been well reviewed in paper. 12 Both methylation analyses and genetic analyses of cfDNA show promising results in previous researches. Especially in a recent report, 5 cfDNA CIN was successfully detected in 87% of hepatocellular carcinoma patients by analyzing chromosome 1 and 8 copy number variations. ...
Purpose
To characterize plasma cell‐free cancer genome chromosomal instabilities (CIN) in patients with liver cancer and to evaluate the potential of CIN as minimally invasive biomarkers for primary liver cancer (PLC) diagnoses.
Experimental Design
We collected 196 plasma samples from 172 individuals in two cohorts, a discovery cohort of surgery ineligible PLC patients and a validation cohort of hepatectomy patients with pathological disease confirmations. All samples were subjected to HiSeq X10 sequencing followed by a customized bioinformatics workflow Ultrasensitive Chromosome Aneuploidy Detection (UCAD).
Results
In the discovery cohort, 29 significant copy number changes were identified in plasma from surgery‐ineligible PLC. Twenty‐two (95.7%) surgery‐ineligible liver cancers were identified as harboring copy number changes in at least 1 of 29 segments. Meanwhile 40/41 (97.6%) noncancers harbored no changes. In the validation cohort, 54 (69.4%) surgery‐eligible liver cancers were identified with positive screening, all of which were subsequently confirmed as cancer by pathological examination. Moreover, 26/27 = 96.3% noncancers were identified with negative screening. UCAD‐positive screening was significantly associated with microvascular invasion (OR > 10, 95% CI:[2.53,]), tumor stages B and C (OR = 8.59, 95% CI [1.07, 400]), and tumor size ≥ 3 cm (OR = 5.68, 95% CI [1.43, 28.1]). Furthermore, we collected 29 followed‐up plasma samples from 19 postsurgery patients. Nine (31.0%) postsurgery samples from 6 (31.5%) patients were identified with positive screening. Among them, 3 patients (50.0%) with positive screening were then confirmed as having disease recurrences.
Conclusions
In addition to AFP, plasma cell‐free DNA sequencing is a useful tool for primary liver cancer diagnoses.
... Since tumor suppressor genes are methylated early during tumorigenesis, analysis of DNA methylation patterns may embody key diagnostic value. With respect to this, and similar to the cfDNA quantification and mutational landscape analysis, cfDNA aberrant methylation allows for diagnostic and prognostic insights in HCC, and might reveal information regarding the tumor size and risk of metastasis or recurrence [114]. A recent study identified a DNA methylation marker panel enriched in HCC tissues, which was replicated in cfDNA of the same patients. ...
Hepatocellular carcinoma (HCC) represents the sixth most common cancer worldwide and the third most common cause of cancer-related death. One of the major problems faced by researchers and clinicians in this area is the lack of reliable disease biomarkers, which would allow for an earlier diagnosis, follow-up or prediction of treatment response, among others. In this regard, the "HCC circulome", defined as the pool of circulating molecules in the bloodstream derived from the primary tumor, represents an appealing target, the so called liquid biopsy. Such molecules encompass circulating tumor proteins, circulating tumor cells (CTCs), extracellular vesicles (EVs), tumor-educated platelets (TEPs), and circulating tumor nucleic acids, namely circulating tumor DNA (ctDNA) and circulating tumor RNA (ctRNA). In this article, we summarize recent findings highlighting the promising role of liquid biopsies as novel potential biomarkers in HCC, emphasizing on its clinical performance.
... www.nature.com/scientificreports/ and therapeutic response in various cancer entities, including HCC 20,22,26,42,43 . Among several important miR-NAs, miR-122 is one of the most crucial microRNAs, which have been reported in liver cancer 40 . ...
Telomerase reverse-transcriptase (TERT) gene promoter mutations in circulating cell-free DNA (cfDNA) as well as the levels of circulating microRNA-122 (miR-122) have been reported as potential noninvasive biomarkers for several. This study evaluates the diagnostic performance of potent biomarker-based panels composing of serological AFP, miR-122 and circulating TERT promoter mutations for screening HBV-related HCC. TERT promoter mutations (C228T and C250T) and miR-122 expression were assessed in the plasma samples from 249 patients with HBV-related liver diseases by nested PCR and qRT-PCR assays, respectively. The diagnostic values of TERT promoter mutations, miR-122 expression and biomarker-based panels were assessed by computation of the area under the curve (AUC). Nested-PCR assays were optimized to detect C228T and C250T mutations in TERT promoter with detection limit of 1%. The common hotspot C228T was observed in 22 HCC cases. The triple combinatory panel (AFP@TERT@miR-122) acquired the best diagnostic value to distinguish HCC from CHB (AUC = 0.98), LC (AUC = 0.88) or non-HCC (LC + CHB, AUC = 0.94) compared to the performance of double combinations or single biomarkers, respectively. Notably, among patients with AFP levels≤20 ng/μl, the double combination panel (TERT@miR-122) retains satisfactory diagnostic performance in discriminating HCC from the others (HCC vs. CHB, AUC = 0.96; HCC vs. LC, AUC = 0.88, HCC vs. non-HCC, AUC = 0.94). The triple combination panel AFP@TERT@miR-122 shows a better diagnostic performance for screening HCC in HBV patients, regardless of AFP levels. The newly established panels can be a potential application in clinical practice in Vietnamese setting.
