Circulating tumor DNA in liquid biopsy: Current diagnostic limitation

With the rapid development of science and technology,cell-free DNA(cfDNA)is rapidly becoming an important biomarker for tumor diagnosis,monitoring and prognosis,and this cfDNA-based liquid biopsy technology has great potential to become an important part of precision medicine.cfDNA is the total amount of free DNA in the systemic circulation,including DNA fragments derived from tumor cells and all other somatic cells.Tumor cells release fragments of DNA into the bloodstream,and this source of cfDNA is called circulating tumor DNA(ctDNA).cfDNA detection has become a major focus in the field of tumor research in recent years,which provides a new opportunity for non-invasive diagnosis and prognosis of cancer.In this paper,we discuss the limitations of the study on the origin and dynamics analysis of ctDNA,and how to solve these problems in the future.Although the future faces major challenges,it also con-tains great potential.

Liquid biopsy in patients with hepatocellular carcinoma: Circulating tumor cells and cell-free nucleic acids

Hepatocellular carcinoma(HCC), with its high incidence and mortality rate, is one of the most common malignant tumors. Despite recent development of a diagnostic and treatment method, the prognosis of HCC remains poor. Therefore, to provide optimal treatment for each patient with HCC, more precise and effective biomarkers are urgently needed which could facilitate a more detailed individualized decision-making during HCC treatment, including the following; risk assessment, early cancer detection, prediction of treatment or prognostic outcome. In the blood of cancer patients, accumulating evidence about circulating tumor cells and cell-free nucleic acids has suggested their potent clinical utilities as novel biomarker. This concept, so-called "liquid biopsy" is widely known as an alternative approach to cancer tissue biopsy. This method might facilitate a more sensitive diagnosis and better decision-making by obtaining genetic and epigenetic aberrations that are closely associated with cancer initiation and progression. In this article, we review recent developments based on the available literature on both circulating tumor cells and cell-free nucleic acids in cancer patients, especially focusing on Hepatocellular carcinoma.

Liquid biopsy in patients with pancreatic cancer: Circulating tumor cells and cell-free nucleic acids

Despite recent advances in surgical techniques and perioperative management, the prognosis of pancreatic cancer(PCa) remains extremely poor. To provide optimal treatment for each patient with Pca, superior biomarkers are urgently needed in all phases of management from early detection to staging, treatment monitoring, and prognosis. In the blood of patients with cancer, circulating tumor cells(CTCs) and cell-free nucleic acids(cf NAs), such as DNA, m RNA, and noncoding RNA have been recognized. In the recent years, their presence in the blood has encouraged researchers to investigate their potential use as novel blood biomarkers, and numerous studies have demonstrated their potential clinical utility as a biomarker for certain types of cancer. This concept, called "liquid biopsy" has been focused on as a less invasive, alternative approach to cancer tissue biopsy for obtaining genetic and epigenetic aberrations that contribute to oncogenesis and cancer progression. In this article, we review the available literature on CTCs and cfN As in patients with cancer, particularly focusing on PCa, and discuss future perspectives in this field.

Liquid biopsy of gastric cancer patients:Circulating tumor cells and cell-free nucleic acids

To improve the clinical outcomes of cancer patients, early detection and accurate monitoring of diseases are necessary. Numerous genetic and epigenetic alterations contribute to oncogenesis and cancer progression, and analyses of these changes have been increasingly utilized for diagnostic, prognostic and therapeutic purposes in malignant diseases including gastric cancer (GC). Surgical and/or biopsy specimens are generally used to understand the tumor-associated alterations; however, those approaches cannot always be performed because of their invasive characteristics and may fail to reflect current tumor dynamics and drug sensitivities, which may change during the therapeutic process. Therefore, the importance of developing a non-invasive biomarker with the ability to monitor real-time tumor dynamics should be emphasized. This concept, so called “liquid biopsy”, would provide an ideal therapeutic strategy for an individual cancer patient and would facilitate the development of “tailor-made” cancer management programs. In the blood of cancer patients, the presence and potent utilities of circulating tumor cells (CTCs) and cell-free nucleic acids (cfNAs) such as DNA, mRNA and microRNA have been recognized, and their clinical relevance is attracting considerable attention. In this review, we discuss recent developments in this research field as well as the relevance and future perspectives of CTCs and cfNAs in cancer patients, especially focusing on GC.

Circulating cell-free nucleic acids as prognostic and therapy predictive tools for metastatic castrate-resistant prostate cancer

Metastatic castrate-resistant prostate cancer remains a disease hard to cure,and for this reason predictive tools to monitor disease progression and therapy response are an urgent need.In this respect,liquid biopsy on circulating cell-free nucleic acids represents an interesting strategy based on robust data.The low invasiveness and the possibility to target circulating cell-free tumor deoxyribonucleic acid underline the high specificity,sensitivity and clinical usability of the technique.Moreover,it has been observed that the cell-free tumor deoxyribonucleic acid of metastatic castrate-resistant prostate cancer patients can be representative of the tumor heterogeneity.Cell-free tumor deoxyribonucleic acids express the same behaviors as mutations:Variation in gene copy number or the methylation rate of the tumor tissue.Recently,circulating cell-free ribonucleic acid molecules have emerged as interesting markers to stratify the disease.Due to high-throughput technologies,liquid biopsy on circulating cell-free nucleic acids will soon be utilized in the clinical management of metastatic castrate-resistant prostate cancer patients.

Clinical Value of Peripheral Blood Circulating Tumor Cells and Cell-Free DNA Combined Detection in Triple-Negative Breast Cancer

Objective:To determine the clinical value of combined detection of circulating tumor cells(CTCs)and cell-free DNA(cfDNA)in peripheral blood of patients with triple-negative breast cancer.Method:41 patients with breast cancer admitted to the First Central Hospital of Baoding from January 2020 to December 2021 were selected and recruited into the experimental group,42 patients with benign breast cancer admitted during the same period were recruited into the conditional control group,and 41 healthy patients admitted during the same period were recruited into the blank control group.The positive rate of peripheral blood CTCs,the level of cfDNA,and the diagnostic efficacy of peripheral blood CTCs,cfDNA alone and the combination thereof for breast cancer were analyzed.Result:The positive rates of peripheral blood CTCs in the experimental group,the conditional control group,and the blank control group were 43.90%,11.90%,and 9.74%,respectively,and there was significant difference among the groups.The levels of cfDNA in peripheral blood of the experimental group,the conditional control group,and the blank control group were 0.26±0.08 bp,0.17±0.03 bp,and 0.15±0.04 bp,respectively,which were statistically significant.The detection levels of 100 bp hTERT/ng mT1 and 241 bp hTERT/ng-mT1 in the experimental group were significantly higher than those in the conditional control group and the blank control group.The accuracy of peripheral blood CTCs detection in the three groups was 66.21%,the accuracy of cfDA241 bp/100 hp hTERT detection was 80.41%,and the accuracy of combined detection of peripheral blood CTCs and cfDNA was 94.03%.Conclusion:The clinical application of peripheral blood CTCs combined with cfDNA level detection can increase detection accuracy,provide data support for clinicians,and improve the clinical diagnostic effect of triple-negative breast cancer.

Circulating micro RNAs and long non-coding RNAs in gastric cancer diagnosis:An update and review

Gastric cancer(GC) is the fourth most common cancer and the third leading cause of cancer mortality worldwide. Micro RNAs(mi RNAs) and long non-coding RNAs(lnc RNAs) are the most popular non-coding RNAs in cancer research. To date,the roles of mi RNAs and lnc RNAs have been extensively studied in GC,suggesting that mi RNAs and lnc RNAs represent a vital component of tumor biology. Furthermore,circulating mi RNAs and lnc RNAs are found to be dysregulated in patients with GC compared with healthy individuals. Circulating mi RNAs and lnc RNAs may function as promising biomarkers to improve the early detection of GC. Multiple possibilities for mi RNA secretion have been elucidated,including active secretion by microvesicles,exosomes,apoptotic bodies,highdensity lipoproteins and protein complexes as well as passive leakage from cells. However,the mechanism underlying lnc RNA secretion and the functions of circulating mi RNAs and lnc RNAs have not been fully illuminated. Concurrently,to standardize results of global investigations of circulating mi RNAs and lnc RNAs biomarker studies,several recommendations for preanalytic considerations are put forward. In this review,we summarize the known circulating mi RNAs and lnc RNAs for GC diagnosis. The possible mechanism of mi RNA and lnc RNA secretion as well as methodologies for identification of circulating mi RNAs and lnc RNAs are also discussed. The topics covered here highlight new insights into GC diagnosis and screening.

Circulating micro RNAs as diagnostic and prognostic tools for hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is an aggressive malignancy and the second leading cause of cancerrelated deaths worldwide. Conventional biomarkers exhibit poor performance in the surveillance,diagnosis,and prognosis of HCC. Micro RNAs(mi RNAs) are a class of evolutionarily conserved small non-coding RNAs that are involved in the regulation of gene expression and protein translation,and they play critical roles in cell growth,differentiation,and the development of various types of cancers,including HCC. Recent evidence revealed the role of mi RNAs as potential novel and ideal biomarkers for HCC. mi RNAs are released to extracellular spaces,and they are extremely stable in bodily fluids,including serum or plasma,where they are packaged into various microparticles or associated with RNA-binding proteins. Numerous studies have demonstrated that circulating mi RNAs have potential applications as minimally invasive biomarkers for HCC diagnosis and prognosis. The present review highlights current understanding of mi RNA biogenesis and the origins and types of circulating mi RNAs. We summarize recent progress in the use of circulating mi RNAs as diagnostic and prognostic biomarkers for HCC. We also discuss the challenges and perspectives of the clinical utility of circulating mi RNAs in HCC.

Diagnostic and prognostic potential of tissue and circulating long non-coding RNAs in colorectal tumors

Long non-coding RNAs(lncRNAs)are members of the non-protein coding RNA family longer than 200 nucleotides.They participate in the regulation of gene and protein expression influencing apoptosis,cell proliferation and immune responses,thereby playing a critical role in the development and progression of various cancers,including colorectal cancer(CRC).As CRC is one of the most frequently diagnosed malignancies worldwide with high mortality,its screening and early detection are crucial,so the identification of disease-specific biomarkers is necessary.LncRNAs are promising candidates as they are involved in carcinogenesis,and certain lncRNAs(e.g.,CCAT1,CRNDE,CRCAL1-4)show altered expression in adenomas,making them potential early diagnostic markers.In addition to being useful as tissue-specific markers,analysis of circulating lncRNAs(e.g.,CCAT1,CCAT2,BLACAT1,CRNDE,NEAT1,UCA1)in peripheral blood offers the possibility to establish minimally invasive,liquid biopsy-based diagnostic tests.This review article aims to describe the origin,structure,and functions of lncRNAs and to discuss their contribution to CRC development.Moreover,our purpose is to summarise lncRNAs showing altered expression levels during tumor formation in both colon tissue and plasma/serum samples and to demonstrate their clinical implications as diagnostic or prognostic biomarkers for CRC.

Non-invasive Prenatal Gene Diagnosis: Progress through Cell-free Fetal DNA and RNA in Maternal Plasma and Urine

Non-invasive prenatal gene diagnosis has been developed rapidly in the recent years, and numerous medical researchers are focusing on it. Such techniques could not only achieve prenatal diagnosis accurately, but also prevent tangential illness in fetuses and thus, reduce the incidence of diseases. Moreover, it is non-invasive prenatal gene diagnosis that prevents potential threaten and danger to both mothers and fetuses. Therefore, it is welcomed by clinical gynecologist and obstetrian, researchers of medical genetics, and especially, pregnancies. This review article touches briefly on the advanced development of using cell-free DNA, RNA in maternal plasma and urine for non-invasive prenatal gene diagnosis.

Circulating MicroRNA-21 is Downregulated in Patients with Metabolic Syndrome

The microRNA-21 （miR-21） is known to play a major role in cancer progression; however, its function in the cardiovascular system appears to be even more complex and conflicting. To characterize miR-21 expression in the plasma of individuals with or without metabolic syndrome （MetS）, 58 MetS cases and 96 non-MetS controls were investigated.

环状RNA ITCH对高糖诱导的心脏成纤维细胞活化的影响与机制研究

目的 探讨环状RNA ITCH(Circ-ITCH)对高糖诱导的心脏成纤维细胞(CF)活化的影响及其机制。方法 体外培养人CF(HCF),按照处理方法的不同分为对照组、高糖组、过表达载体阴性对照(OV-NC)组、Circ-ITCH过表达载体(OV-Circ-ITCH)组;模拟物阴性对照[微小RNA(miR)-NC]组、miR-212-3p模拟物(miR-212-3p mimics)组、抑制物阴性对照(inhibitor-NC)组、miR-212-3p抑制物(miR-212-3p inhibitor)组;Circ-ITCH过表达载体+模拟物阴性对照(OV-Circ-ITCH+miR-NC)组、Circ-ITCH过表达载体+miR-212-3p模拟物(OV-Circ-ITCH+miR-212-3p mimics)组、Circ-ITCH过表达载体+小分子干扰RNA阴性对照(OV-Circ-ITCH+si-NC)组和Circ-ITCH过表达载体+Samd同源物7小分子干扰RNA(OV-Circ-ITCH+si-Smad7)组,除对照组外,其余各组转染12 h后采用30 mmol/L D-葡萄糖处理进行高糖诱导,检测Circ-ITCH、miR-212-3p、Smad7表达、细胞活力及胶原纤维沉积,并验证miR-212-3p与Circ-ITCH、Smad7靶向关系。结果 与对照组比较,高糖组Circ-ITCH、Smad7 mRNA和蛋白表达降低(0.45±0.05 vs 0.99±0.07,0.51±0.06 vs 0.99±0.08,0.68±0.05 vs 1.16±0.07,P<0.05),miR-212-3p表达升高(2.17±0.12 vs 1.01±0.08,P<0.05),Ⅰ型胶原、Ⅲ型胶原、α平滑肌肌动蛋白(α-SMA)表达及细胞活力升高(P<0.05),胶原纤维沉积增多。Circ-ITCH过表达升高高糖诱导的HCF细胞中Smad7表达,降低miR-212-3p、Ⅰ型胶原、Ⅲ型胶原、α-SMA表达及细胞活力,减少胶原纤维沉积。miR-212-3p过表达抑制高糖诱导的HCF细胞中Smad7表达,而抑制miR-212-3p表达则相反。miR-212-3p过表达或抑制Smad7表达均减弱Circ-ITCH过表达对高糖诱导的HCF细胞增殖、胶原纤维沉积的影响。HCF细胞中miR-212-3p与Circ-ITCH、Smad7存在靶向关系。结论 Circ-ITCH靶向抑制miR-212-3p并上调Smad7表达,抑制高糖诱导的HCF细胞活化。

Prognostic and predictive blood biomarkers in gastric cancer and the potential application of circulating tumor cells

Gastric cancer(GC), with its high incidence and mortality rates, is a highly fatal cancer that is common in East Asia particularly in China. Its recurrence and metastasis are the main causes of its poor prognosis. Circulating tumor cells(CTCs) or other blood biomarkers that are released into the circulating blood stream by tumors are thought to play a crucial role in the recurrence and metastasis of gastric cancer. Therefore, the detection of CTCs and other blood biomarkers has an important clinical significance; in fact, they can help predict the prognosis, assess the staging, monitor the therapeutic effects and determine the drug susceptibility. Recent research has identified many blood biomarkers in GC, such as various serum proteins, autoantibodies against tumor associated antigens, and cell-free DNAs. The analysis of CTCs and circulating cell-free tumor DNA(ctDNA) in the peripheral blood of patients with gastric cancer is called as liquid biopsy. These blood biomarkers provide the disease status for individuals and have clinical meaning. In this review, we focus on the recent scientific advances regarding CTCs and other blood biomarkers, and discuss their origins and clinical meaning.

Circulating and stool nucleic acid analysis for colorectal cancer diagnosis

In recent years, the need to identify molecular markers characterized by high sensitivity and specificity in detecting and monitoring early and colorectal cancer lesions has increased. Up to now, none of the markers or panels of markers analyzed have met the rigorous standards required of a screening program. The important discovery of circulating nucleic acids in biological fluids has aroused intense scientific interest because of their usefulness in malignant and non malignant diseases. Over time, their yield and stability have been identified and compared with other &#x0201c;standard&#x0201d; biomarkers. The analysis of circulating DNA from blood and stool is a relatively simple and non-invasive procedure, representing a very attractive marker to detect genetic and epigenetic mutations and to monitor disease progression. A correlation between blood and stool biomarkers could also help to enhance currently available diagnostic approaches. However, various processing and analytic problems need to be resolved before such an approach can be applied in clinical practice.

Circular RNA and its potential as prostate cancer biomarkers

Advancing knowledge of the transcriptome has revealed that circular RNAs(circRNAs)are widely expressed and evolutionarily conserved molecules that may serve relevant biological roles.More interesting is the accumulating evidence which demonstrates the implication of circRNAs in diseases,especially cancers.This revelation has helped to form the rationale for many studies exploring their utility as clinical biomarkers.CircRNAs are highly stable due to their unique structures,exhibit some tissue specificity,and are enriched in exosomes,which facilitate their detection in a range of body fluids.These properties make circRNAs ideal candidates for biomarker development in many diseases.This review will outline the discovery,biogenesis,and proposed functions of circRNAs.

Circulating tumor DNA for diagnosis,prognosis and treatment of gastrointestinal malignancies

Minimally invasive detection of circulating tumor DNA(ctDNA)in peripheral blood or other body fluids of patients with gastrointestinal malignancies via liquid biopsy has emerged as a promising biomarker.This is urgently needed,as conventional imaging and plasma protein-derived biomarkers lack sensitivity and specificity in prognosis,early detection of relapse or treatment monitoring.This review summarizes the potential role of liquid biopsy in diagnosis,prognosis and treatment monitoring of gastrointestinal malignancies,including upper gastrointestinal,liver,bile duct,pancreatic and colorectal cancer.CtDNA can now be part of the clinical routine as a promising,highly sensitive and specific biomarker with a broad range of applicability.Liquid-biopsy based postoperative relapse prediction could lead to improved survival by intensification of adjuvant treatment in patients identified to be at risk of early recurrence.Moreover,ctDNA allows monitoring of antineoplastic treatment success,with identification of potentially developed resistance or therapeutic targets during the course of treatment.It may also assist in early change of chemotherapy in metastatic gastrointestinal malignancies prior to imaging findings of relapse.Nevertheless,clinical utility is dependent on the tumor’s entity and burden.

Cell-free mitochondrial DNA quantification in ischemic stroke patients for non-invasive and real-time monitoring of disease status

BACKGROUND Acute ischemic stroke(AIS)is one of the major causes of the continuous increasing rate of global mortality due to the lack of timely diagnosis,prognosis,and management.This study provides a primitive platform for non-invasive and cost-effective diagnosis and prognosis of patients with AIS using circulating cellfree mitochondrial DNA(cf-mtDNA)quantification and validation.AIM To evaluate the role of cf-mtDNA as s non-invasive,and affordable tool for realtime monitoring and prognosticating AIS patients at disease onset and during treatment.METHODS This study enrolled 88 participants including 44 patients with AIS and 44 healthy controls with almost similar mean age group at stroke onset,and at 24 h and 72 h of treatment.Peripheral blood samples were collected from each study participant and plasma was separated using centrifugation.The cf-mtDNA concentration was quantified using nanodrop reading and validated through real-time quantitative polymerase chain reaction(RT-qPCR)of NADH-ubiquinone oxidoreductase chain 1(ND1)relative transcript expression levels.RESULTS Comparative analysis of cf-mtDNA concentration in patients at disease onset showed significantly increased levels compared to control individuals for both nanodrop reading,as well as ND1 relative expression levels(P<0.0001).Intergroup analysis of cf-mtDNA concentration using nanodrop showed significantly reduced levels in patients at 72 h of treatment compared to onset(P<0.01).However,RT-qPCR analysis showed a significant reduction at 24 h and 72 h of treatment compared to the disease onset(P<0.001).The sensitivity and specificity were relatively higher for RT-qPCR than nanodrop-based cfmtDNA quantification.Correlation analysis of both cf-mtDNA concentration as well as ND1 relative expression with National Institute of Health Stroke Scale score at baseline showed a positive trend.CONCLUSION In summary,quantitative estimation of highly pure cf-mtDNA provides a simple,highly sensitive and specific,non-invasive,and affordable approach for real-time monitoring and prognosticating AIS patients at onset and during treatment.

Role of circulating free DNA in colorectal cancer

The gradual elucidation of the underlying biology of colorectal cancer has provided new insights and therapeutic options for patients with metastatic disease which are selected according to predictive biomarkers. This precision medicine paradigm, however, is incomplete since not all eligible patients respond to these agents and prognostic stratification is largely based on clinicopathologic variants. Importantly, no robust data exist to help properly select patients with localized disease at high risk for recurrence and most likely to benefit from adjuvant chemotherapy. There is a rapidly expanding body of literature regarding the role of the qualitative and quantitative analysis of circulating free DNA in various neoplasms, which consistently outperforms traditional tumor markers both as a predictive and as a prognostic marker. Several lines of evidence suggest that circulating free DNA may exhibit a complementary role to existing modalities for the early diagnosis of colorectal cancer, the selection of patients for adjuvant chemotherapy, for the followup of treated patients, for the selection of treatment for advanced disease and the assessment of response and for determining the prognosis of patients. These data, which are reviewed here, illustrate the important role that circulating biomarkers may soon have at the daily clinical practice.

The Prognostic Value of Cell-Free DNA in Advanced Non-Small-Cell Lung Cancer

Background: Cell-free DNA (cfDNA) holds promise as a tumor marker of clinical importance. We aimed to investigate the prognostic value of baseline cfDNA in non small-cell lung cancer (NSCLC). Material and Methods: During a three-year period, patients with newly diagnosed, previously untreated advanced NSCLC were included in a consecutive, prospective marker-trial. Plasma was isolated from a pre-treatment peripheral blood sample and the level of total cfDNA was measured by an in-house assay qPCR-method. The treatment comprised carboplatin (AUC 5) intravenously day 1), and vinorelbine (30 mg/m2 intravenously day 1 and 60 mg/m2 perorally day 8) q3w for a maximum of six cycles. The primary end-point was overall survival (OS). Secondary end-points were progression free survival (PFS) and overall response rate (ORR). Results: 245 patients were included and received a minimum of 1 cycle of chemotherapy (median 4). The median OS was 8.9 months, the median PFS by intention to treat 5.4 months and the ORR was 25%. The patients were divided into four groups based on quartiles of cfDNA and subsequently dichotomized by the 75th percentile revealing a significantly worse prognosis for patients in the upper 75th percentile (median OS 4.9 months) compared to patients with lower levels (10.0 months) (HR 2.1, 95%CI 1.4 - 3.1, p 0.0001). A multivariate analysis confirmed the independent prognostic value of cfDNA. A subgroup analysis of patients with high cfDNA and poor performance status (PS = 2) identified a group of patients with even worse prognosis (median OS 2.0 versus 9.1 months, HR 3.6, 95%CI 1.4 - 9.2, p 0.0001). Similar and significant results were found when comparing level of cfDNA and PFS. Conclusions: High pre-treatment level of cfDNA seems to have a strong prognostic impact in patients with newly diagnosed advanced NSCLC. Combined with PS it identifies a patient group with minimal or no benefit of chemotherapy.

Role of cell-free DNA for predicting incidence and outcome of patients with ischemic stroke

Early diagnosis and prognosis of ischemic stroke remains a critical challenge in clinical settings.A blood biomarker can be a promising quantitative tool to represent the clinical manifestations in ischemic stroke.Cell-free DNA(cfDNA)has recently turned out to be a popular circulating biomarker due to its potential relevance for diagnostic applications in a variety of disorders.Despite bright outlook of cfDNA in clinical applications,very less is known about its origin,composition,or function.Several recent studies have identified cell-derived mitochondrial components including mitochondrial DNA(mtDNA)in the extracellular spaces including blood and cerebrospinal fluid.However,the time course of alterations in plasma mtDNA concentrations in patients after an ischemic stroke is poorly understood.DNA is thought to be freed into the plasma shortly after the commencement of an ischemic stroke and then gradually decreased.However,the importance of cell-free mtDNA(cf-mtDNA)in ischemic stroke is still unknown.This review summarizes about the utility of biomarkers which has been standardized in clinical settings and role of cfDNA including cfmtDNA as a non-invasive potential biomarker of ischemic stroke.