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.

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 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.

C-TIRADS联合cfDNA鉴别诊断甲状腺结节价值及临床效用评价

目的 探讨中国超声甲状腺结节恶性危险分层指南(C-TIRADS)联合循环游离DNA(cfDNA)鉴别诊断甲状腺结节价值及临床效用。方法 回顾性选取2020年3月至2022年9月于我院甲状腺外科有明确病理结果的395例甲状腺结节患者的临床资料,其中良性结节303例(良性组),恶性结节92例(恶性组)。比较2组一般资料、超声征象、C-TIRADS评分、cfDNA,采用受试者工作特征曲线(ROC)分析C-TIRADS评分、cfDNA及二者联合鉴别诊断甲状腺结节性质的价值,采用OR危险度分析C-TIRADS、cfDNA与甲状腺结节恶性风险的关系,采用临床决策曲线(DCA)分析不同鉴别诊断方案的临床效用。结果 恶性组有甲状腺癌家族史患者多于良性组(P<0.05);恶性组超声征象垂直位、极低回声、微钙化、边缘模糊或不规则或甲状腺外侵犯、实性表现患者多于良性组,彗星尾伪像患者少于良性组(P<0.05);恶性组C-TIRADS评分、cfDNA高于良性组(P<0.05);ROC分析显示,C-TIRADS、cfDNA及二者联合鉴别诊断甲状腺结节的AUC分别为0.843、0.812、0.939,C-TIRADS联合cfDNA的AUC大于C-TIRADS、cfDNA(P<0.05);OR危险度分析显示,C-TIRADS≥4分患者甲状腺结节恶性的风险是<4分患者的12.496倍(P<0.05);cfDNA≥63.70 ng/ml患者甲状腺结节恶性的风险是<63.70 ng/ml患者的9.863倍(P<0.05);绘制DCA曲线显示,C-TIRADS、cfDNA及二者联合鉴别诊断甲状腺结节均具有正向的临床净获益,且采用C-TIRADS联合cfDNA鉴别诊断时,临床效用进一步提高。结论 C-TIRADS联合cfDNA能相互弥补,提高对甲状腺结节的鉴别诊断价值,具有良好的临床效用,为临床鉴别恶性结节提供了一个无创、准确、便捷的方案,有助于提高甲状腺结节的诊疗水平。

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.

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.

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.

系统性红斑狼疮患者外周血不同形式循环游离DNA(cfDNA)的水平和免疫活性比较

目的检测并分析不同形式循环游离DNA(cfDNA)在系统性红斑狼疮(SLE)患者外周血含量和对巨噬细胞和树突状细胞的刺激活性。方法募集58例SLE患者、66例非SLE自身免疫性疾病患者(non-SLE)和60例正常对照,提取血浆中总cfDNA、外泌体cfDNA和免疫复合物cfDNA,采用荧光法检测不同形式cfDNA含量,采用抗体捕获法检测不同形式cfDNA总甲基化水平。体外诱导培养巨噬细胞和树突状细胞,分别与DNA酶1样分子3(DNASE1L3)或IgG特异性降解酶处理前后的SLE来源的外泌体或免疫复合物共培养,采用多重荧光微球法检测巨噬细胞炎性蛋白1a(MIP-1a)、MIP-1b、白细胞介素4(IL-4)、IL-1β、IL-2、白细胞介素1受体拮抗剂(IL-1ra)、单核细胞趋化蛋白1(MCP-1)、IL-6、IL-9、IL-10、IL-12、IL-8、肿瘤坏死因子α(TNF-α)、α干扰素(IFN-α)和IFN-γ分泌以及细胞表面CD40、CD80和CD86。结果SLE组、non-SLE组外泌体cfDNA和免疫复合物cfDNA水平显著增加,SLE患者组也显著高于non-SLE组,单纯cfDNA在三组间无明显变化。除单纯cfDNA以外,SLE患者血浆不同类型cfDNA甲基化水平均显著低于non-SLE组和健康人群。消化酶处理后的外泌体和免疫复合物刺激分泌的MIP-1a、MIP-1b、IL-4、IL-1β、IL-2、IL-1ra、MCP-1、IL-6、IL-9、IL-8、TNF-α和IFN-α水平显著低于未经处理的刺激物,DNASE1L3处理后的外泌体和免疫复合物刺激后分泌的细胞因子量也显著低于IgG酶消化处理后得刺激物。与处理前相比,SLE患者来源的外泌体和免疫复合物经DNASE1L3处理后,对CD80、CD86和CD40在巨噬细胞和树突状细胞的刺激反应性均显著降低(CD86在巨噬细胞上的反应性无明显改变);IgG特异性降解酶降解处理前后的刺激对CD80、CD86和CD40在两种细胞的反应性表现不一,但总体上呈下降趋势。结论SLE患者血浆外泌体和免疫复合物cfDNA水平显著升高,可刺激巨噬细胞和树突状细胞产生较强应答。

DNA methylation signatures in circulating cell-free DNA as biomarkers for the early detection of cancer

Detecting cell-free DNA(cfDNA) or circulating tumor DNA(ctDNA) in plasma or serum could serve as a "liquid biopsy", which would be useful for numerous diagnostic applications. cfDNA methylation detection is one of the most promising approaches for cancer risk assessment. Here, we reviewed the literature related to the use of serum or plasma circulating cell-free DNA for cancer diagnosis in the early stage and their power as future biomarkers.

Liquid biopsies:DNA methylation analyses in circulating cell-free DNA

Analysis of patient＇s materials like cells or nucleic acids obtained in a minimally invasive or noninvasive manner through the sampling of blood or other body fluids serves as liquid biopsies, which has huge potential for numerous diagnostic applications. Circulating cell-free DNA（cfDNA） is explored as a prognostic or predictive marker of liquid biopsies with the improvements in genomic and molecular methods. DNA methylation is an important epigenetic marker known to affect gene expression. cfDNA methylation detection is a very promising approach as abnormal distribution of DNA methylation is one of the hallmarks of many cancers and methylation changes occur early during carcinogenesis. This re？view summarizes the various investigational applications of cfDNA methylation and its oxidized de？rivatives as biomarkers for cancer diagnosis, prenatal diagnosis and organ transplantation monitoring.The review also provides a brief overview of the technologies for cfDNA methylation analysis based on next generation sequencing.

Liquid biopsy for gastric cancer:Techniques,applications,and future directions

After the study of circulating tumor cells in blood through liquid biopsy(LB),this technique has evolved to encompass the analysis of multiple materials originating from the tumor,such as nucleic acids,extracellular vesicles,tumor-educated platelets,and other metabolites.Additionally,research has extended to include the examination of samples other than blood or plasma,such as saliva,gastric juice,urine,or stool.LB techniques are diverse,intricate,and variable.They must be highly sensitive,and pre-analytical,patient,and tumor-related factors significantly influence the detection threshold,diagnostic method selection,and potential results.Consequently,the implementation of LB in clinical practice still faces several challenges.The potential applications of LB range from early cancer detection to guiding targeted therapy or immunotherapy in both early and advanced cancer cases,monitoring treatment response,early identification of relapses,or assessing patient risk.On the other hand,gastric cancer(GC)is a disease often diagnosed at advanced stages.Despite recent advances in molecular understanding,the currently available treatment options have not substantially improved the prognosis for many of these patients.The application of LB in GC could be highly valuable as a non-invasive method for early diagnosis and for enhancing the management and outcomes of these patients.In this comprehensive review,from a pathologist’s perspective,we provide an overview of the main options available in LB,delve into the fundamental principles of the most studied techniques,explore the potential utility of LB application in the context of GC,and address the obstacles that need to be overcome in the future to make this innovative technique a game-changer in cancer diagnosis and treatment within clinical practice.

循环游离DNA在结直肠癌中的临床应用进展

结直肠癌是胃肠道最常见的恶性肿瘤之一。近年来随着高危人群筛查的开展及内镜技术的发展,结直肠癌早期诊断率有所提高,但多数患者出现明显症状时已处于中晚期,预后较差。循环游离DNA(cf DNA)作为一种新型潜在肿瘤标记物近年来得到广泛关注,其在结直肠癌早期诊断、化疗方案的选择、疗效评估及预后判断等方面中具有重要的临床应用前景。本文就cf DNA在结直肠癌中的临床应用进展作一综述。

Circulating Tumor DNA as Biomarkers for CancerDetection

Detection of circulating tumor DNAs(ct DNAs) in cancer patients is an important component of cancer precision medicine ct DNAs. Compared to the traditional physical and biochemical methods, blood-based ct DNA detection offers a non-invasive and easily accessible way for cancer diagnosis, prognostic determination, and guidance for treatment. While studies on this topic are currently underway, clinical translation of ct DNA detection in various types of cancers has been attracting much attention, due to the great potential of ct DNA as blood-based biomarkers for early diagnosis and treatment of cancers. ct DNAs are detected and tracked primarily based on tumorrelated genetic and epigenetic alterations. In this article, we reviewed the available studies on ct DNA detection and described the representative methods. We also discussed the current understanding of ct DNAs in cancer patients and their availability as potential biomarkers for clinical purposes. Considering the progress made and challenges involved in accurate detection of specific cell-free nucleic acids, ct DNAs hold promise to serve as biomarkers for cancer patients, and further validation is needed prior to their broad clinical use.

The size of cell-free mitochondrial DNA in blood is inversely correlated with tumor burden in cancer patients

Circulating cell-free DNAs(cfDNAs)are fragmented DNA molecules released into the blood by cells.Previous studies have suggested that mitochondria-originated cfDNA fragments(mt-cfDNAs)in cancer patients are more fragmented than those from healthy controls.However,it is still unknown where these short mtcfDNAs originate,and whether the length of mt-cfDNAs can be correlated with tumor burden and cancer progression.In this study,we first performed whole-genome sequencing analysis(WGS)of cfDNAs from a human tumor cell line-xenotransplantation mouse model and found that mt-cfDNAs released from transplanted tumor cells were shorter than the mouse counterpart.We next analyzed blood cfDNA samples from hepatocellular carcinoma and prostate cancer patients and found that mt-cfDNA lengths were inversely related to tumor size as well as the concentration of circulating tumor DNA.Our study suggested that monitoring the size of mt-cfDNAs in cancer patients would be a useful way to estimate tumor burden and cancer progression.

Pancreatic ductal adenocarcinoma:the role of circulating tumor DNA

Pancreatic ductal adenocarcinoma(PDAC)is one of the most lethal cancers in humans,and utilized treatments over the past decades have shown little evidence of improvement in survival.This lack of progress in PDAC treatment outcomes has largely been attributed to a variety of limitations in all phases of care.These limitations most notably include late diagnosis leading to limited treatment options and consequently poorer response to treatments and eventual outcomes.Clinical implications regarding the emergence of circulating tumor cells and DNA(ctDNA)have shown promise in augmenting each step in the management of PDAC.This paper will review the emergence of ctDNA and its value in detection of common PDAC DNA alterations,potential clinical implications and utility,followed by the current limitations and the next steps that need to be taken to translate its use into a standard of care.

循环肿瘤细胞在肺癌精准医疗中的研究进展

肺癌是最常见的恶性肿瘤,也是癌症死亡的主要原因。循环肿瘤细胞(circulating tumor cells,CTCs)是从原发病灶或转移病灶脱落到外周血的肿瘤细胞,可在远处播散并引发肿瘤转移。现CTCs的研究已逐渐深入分子层面,CTCs携带的潜在临床信息也进一步被开发,在指导治疗、揭示转移机制、评估程序性细胞凋亡配体1(programmed cell death-ligand 1,PD-L1)表达等方面有巨大的潜在临床价值。本文就CTCs在肺癌精准医疗中的研究进展进行综述,并探讨CTCs与循环游离DNA(circulating free DNA,cfDNA)检测在肺癌中的互补意义,以期为肺癌的临床检测及个体化治疗提供新思路。

The potential for liquid biopsies in the precision medical treatment of breast cancer

Currently the clinical management of breast cancer relies on relatively few prognostic/predictive clinical markers(estrogen receptor, progesterone receptor, HER2), based on primary tumor biology. Circulating biomarkers, such as circulating tumor DNA(ctDNA) or circulating tumor cells(CTCs) may enhance our treatment options by focusing on the very cells that are the direct precursors of distant metastatic disease, and probably inherently different than the primary tumor's biology. To shift the current clinical paradigm, assessing tumor biology in real time by molecularly profiling CTCs or ctDNA may serve to discover therapeutic targets, detect minimal residual disease and predict response to treatment. This review serves to elucidate the detection,characterization, and clinical application of CTCs and ctDNA with the goal of precision treatment of breast cancer.

Recent advances in blood-based and artificial intelligence-enhanced approaches for gastrointestinal cancer diagnosis

Gastrointestinal(GI)cancers are among the most common cancer types and leading causes of cancer-related deaths worldwide.There is a tremendous clinical need for effective early diagnosis for better healthcare of GI cancer patients.In this article,we provide a short overview of the recent advances in GI cancer diagnosis.In the first part,we discuss the applications of blood-based biomarkers,such as plasma circulating cell-free DNA,circulating tumor cells,extracellular vesicles,and circulating cell-free RNA,for cancer liquid biopsies.In the second part,we review the current trends of artificial intelligence(AI)for pathology image and tissue biopsy analysis for GI cancer,as well as deep learning-based approaches for purity assessment of tissue biopsies.We further provide our opinions on the future directions in blood-based and AI-enhanced approaches for GI cancer diagnosis,and we think that these fields will have more intensive integrations with clinical needs in the near future.

Circulating tumor cells and their role in prostate cancer

Circulating tumor cells (CTC)have become an important biomarker in patients with advanced prostate cancer.CTC count has been demonstrated to be a prognostic factor for overall survival in patients with metastatic castration-resistant prostate cancer (mCRPC). In localized prostate cancer,a clear correlation between CTC counts and clinicopathological risk parameters and outcome has not been observed.Currently,the focus of research is shifting from CTC enumeration towards molecular characterization of CTC leading to the discovery of markers predicting treatment response.The role of androgen receptor splice variants expressed by CTC as markers of resistance to abiraterone and enzalutamide has been assessed by various studies.The identification of CTC markers predicting treatment response represents a key step to guide the selection of treatment (e.g.,abiraterone/enzalutamide vs taxanes), particularly in patients with mCRPC.As an alternative to CTC,the analysis of circulating tumor DNA has been shown to enable a noninvasive disease characterization having high potential to promote precision oncology.