A Novel Method for Quantification of Circulating DNA in Serum by Capillary Zone Electrophoresis

In this paper, we first presented a novel method for quantification of circulating DNA in human serum based on capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF). The serum was digested by proteinase to release free DNA, and then CZE-LIF system was used for the quantification of total circulating DNA. This method was successfully used to quantify the circulating DNA levels in sera from healthy individuals and certain cancer patients. We found the significantly elevated circulating DNA levels in certain prostate cancer patients. Our results demonstrated that CZE-LIF system has good linearity, excellent sensitivity (0.5 ng/mL DNA), satisfactory reproducibility (RSDs in one day and between days were both less than 5%) and reliability, and is well suitable to the quantification of the circulating DNA in human serum or plasma.

Circulating tumor DNA and its role in detection, prognosis and therapeutics of hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is considered the fifth most prevalent cancer among all types of cancers and has the third most morbidity value. It has the most frequent duplication time and a high recurrence rate. Recently, the most unique technique used is liquid biopsies, which carry many markers;the most prominent is circulating tumor DNA(ctDNA). Varied methods are used to investigate ctDNA, including various forms of polymerase chain reaction(PCR) [emulsion PCR(ePCR), digital PCR(dPCR), and bead, emulsion, amplification, magnetic(BEAMing) PCR]. Hence ctDNA is being recognized as a potential biomarker that permits early cancer detection,treatment monitoring, and predictive data on tumor burden are subjective to therapy or surgery. Numerous ctDNA biomarkers have been investigated based on their alterations such as 1) single nucleotide variations(either insertion or deletion of a nucleotide) markers including TP53, KRAS, and CCND1;2) copy number variations which include markers such as CDK6, EFGR, MYC and BRAF;3) DNA methylation(RASSF1A, SEPT9, KMT2C and CCNA2);4) homozygous mutation includes ctDNA markers as CDKN2A, AXIN1;and 5) gain or loss of function of the genes, particularly for HCC. Various researchers have conducted many studies and gotten fruitful results.Still, there are some drawbacks to ctDNA namely low quantity, fragment heterogeneity, less stability, limited mutant copies and standards, and differential sensitivity. However, plenty of investigations demonstrate ctDNA's significance as a polyvalent biomarker for cancer and can be viewed as a future diagnostic, prognostic and therapeutic agent. This article overviews many conditions in genetic changes linked to the onset and development of HCC, such as dysregulated signaling pathways, somatic mutations, single-nucleotide polymorphisms, and genomic instability. Additionally, efforts are also made to develop treatments for HCC that are molecularly targeted and to unravel some of the genetic pathways that facilitate its early identification.

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.

Development and validation of a circulating tumor DNA-based optimization-prediction model for short-term postoperative recurrence of endometrial cancer

BACKGROUND Endometrial cancer(EC)is a common gynecological malignancy that typically requires prompt surgical intervention;however,the advantage of surgical management is limited by the high postoperative recurrence rates and adverse outcomes.Previous studies have highlighted the prognostic potential of circulating tumor DNA(ctDNA)monitoring for minimal residual disease in patients with EC.AIM To develop and validate an optimized ctDNA-based model for predicting shortterm postoperative EC recurrence.METHODS We retrospectively analyzed 294 EC patients treated surgically from 2015-2019 to devise a short-term recurrence prediction model,which was validated on 143 EC patients operated between 2020 and 2021.Prognostic factors were identified using univariate Cox,Lasso,and multivariate Cox regressions.A nomogram was created to predict the 1,1.5,and 2-year recurrence-free survival(RFS).Model performance was assessed via receiver operating characteristic(ROC),calibration,and decision curve analyses(DCA),leading to a recurrence risk stratification system.RESULTS Based on the regression analysis and the nomogram created,patients with postoperative ctDNA-negativity,postoperative carcinoembryonic antigen 125(CA125)levels of<19 U/mL,and grade G1 tumors had improved RFS after surgery.The nomogram’s efficacy for recurrence prediction was confirmed through ROC analysis,calibration curves,and DCA methods,highlighting its high accuracy and clinical utility.Furthermore,using the nomogram,the patients were successfully classified into three risk subgroups.CONCLUSION The nomogram accurately predicted RFS after EC surgery at 1,1.5,and 2 years.This model will help clinicians personalize treatments,stratify risks,and enhance clinical outcomes for patients with EC.

Circulating tumor cells and circulating tumor DNA in breast cancer diagnosis and monitoring

Liquid biopsy,including both circulating tumor cells and circulating tumor DNA,is becoming more popular as a diagnostic tool in the clinical management of breast cancer.Elevated concentrations of these biomarkers during cancer treatment may be used as markers for cancer progression as well as to understand the mechanisms underlying metastasis and treatment resistance.Thus,these circulating markers serve as tools for cancer assessing and monitoring through a simple,non-invasive blood draw.However,despite several study results currently noting a potential clinical impact of ctDNA mutation tracking,the method is not used clinically in cancer diagnosis among patients and more studies are required to confirm it.This review focuses on understanding circulating tumor biomarkers,especially in breast cancer.

Significance of postoperative follow-up of patients with metastatic colorectal cancer using circulating tumor DNA

BACKGROUND One of the most notable applications for circulating tumor DNA(ctDNA)detection in peripheral blood of patients with metastatic colorectal cancer(mCRC)is a long-term postoperative follow-up.Sometimes referred to as a“liquid(re)biopsy”it is a minimally invasive procedure and can be performed repeatedly at relatively short intervals(months or even weeks).The presence of the disease and the actual extent of the tumor burden(tumor mass)within the patient’s body can be monitored.This is of particular importance,especially when evaluating radicality of surgical treatment as well as for early detection of disease progression or recurrence.AIM To confirm the radicality of surgery using ctDNA and compare available methods for detection of recurrence in metastatic colorectal cancer.METHODSA total of 47 patients with detected ctDNA and indications for resection of mCRC were enrolled in the multicenter study involving three surgical centers.Standard postoperative follow-ups using imaging techniques and the determination of tumor markers were supplemented by ctDNA sampling.In addition to the baseline ctDNA testing prior to surgery,a postoperative observation was conducted by evaluating ctDNA presence up to a week after surgery and subsequently at approximately three-month intervals.The presence of ctDNA was correlated with radicality of surgical treatment and the actual clinical status of the patient.RESULTS Among the monitored patients,the R0(curative)resection correlated with postoperative ctDNA negativity in 26 out of 28 cases of surgical procedures(26/28,93%).In the remaining cases of R0 surgeries that displayed ctDNA,both patients were diagnosed with a recurrence of the disease after 6 months.In 7 patients who underwent an R1 resection,4 ctDNA positivities(4/7,57%)were detected after surgery and associated with the confirmation of early disease recurrence(after 3 to 7 months).All 15 patients(15/15,100%)undergoing R2 resection remained constantly ctDNA positive during the entire follow-up period.In 22 cases of recurrence,ctDNA positivity was detected 22 times(22/22,100%)compared to 16 positives(16/22,73%)by imaging methods and 15 cases(15/22,68%)of elevated tumor markers.CONCLUSION ctDNA detection in patients with mCRC is a viable tool for early detection of disease recurrence as well as for confirmation of the radicality of surgical treatment.

Circulating tumor DNA dynamics analysis in a xenograft mouse model with esophageal squamous cell carcinoma

BACKGROUND It remains unclear which factors,such as tumor volume and tumor invasion,influence circulating tumor DNA(ctDNA),and the origin of ctDNA in liquid biopsy is always problematic.To use liquid biopsies clinically,it will be very important to address these questions.AIM To assess the origin of ctDNA,clarify the dynamics of ctDNA levels,assess ctDNA levels by using a xenograft mouse after treatment,and to determine whether tumor volume and invasion are related to ctDNA levels.METHODS Tumor xenotransplants were established by inoculating BALB/c-nu/nu mice with the TE11 cell line.Groups of mice were injected with xenografts at two or four sites and sacrificed at the appropriate time point after xenotransplantation for ctDNA analysis.Analysis of ctDNA was performed by droplet digital PCR,using the human telomerase reverse transcriptase(hTERT)gene.RESULTS Mice given two-site xenografts were sacrificed for ctDNA at week 4 and week 8.No hTERT was detected at week 4,but it was detected at week 8.However,in four-site xenograft mice,hTERT was detected both at week 4 and week 6.These experiments revealed that both tumor invasion and tumor volume were asso ciated with the detection of ctDNA.In resection experiments,hTERT was detected at resection,but had decreased by 6 h,and was no longer detected 1 and 3 d after resection.CONCLUSION We clarified the origin and dynamics of ctDNA,showing that tumor volume is an important factor.We also found that when the tumor was completely resected,ctDNA was absent after one or more days.

Early detection of circulating tumor DNA and successful treatment with osimertinib in thr790met-positive leptomeningeal metastatic lung cancer:A case report

BACKGROUND Patients diagnosed with non-small-cell lung cancer with activated epidermal growth factor receptor mutations are more likely to develop leptomeningeal(LM)metastasis than other types of lung cancers and have a poor prognosis.Early diagnosis and effective treatment of leptomeningeal carcinoma can improve the prognosis.CASE SUMMARY A 55-year-old female with a progressive headache and vomiting for one month was admitted to Peking University First Hospital.She was diagnosed with lung adenocarcinoma with osseous metastasis 10 months prior to admittance.epidermal growth factor receptor(EGFR)mutation was detected by genomic examination,so she was first treated with gefitinib for 10 months before acquiring resistance.Cell-free cerebrospinal fluid(CSF)circulating tumor DNA detection by next-generation sequencing was conducted and indicated the EGFR-Thr790Met mutation,while biopsy and cytology from the patient’s CSF and the first enhanced cranial magnetic resonance imaging(MRI)showed no positive findings.A month later,the enhanced MRI showed linear leptomeningeal enhancement,and the cytology and biochemical examination in CSF remained negative.Therefore,osimertinib(80 mg/d)was initiated as a second-line treatment,resulting in a good response within a month.CONCLUSION This report suggests clinical benefit of osimertinib in LM patients with positive detection of the EGFR-Thr790Met mutation in CSF and proposes that the positive findings of CSF circulating tumor DNA as a liquid biopsy technology based on the detection of cancer-associated gene mutations may appear earlier than the imaging and CSF findings and may thus be helpful for therapy.Moreover,the routine screening of chest CT with the novel coronavirus may provide unexpected benefits。

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.

Circulating tumor DNA genomic profiling reveals the complicated olaparib-resistance mechanism in prostate cancer salvage therapy: A case report

BACKGROUND The poly(ADP-ribose)polymerase(PARP)inhibitor olaparib has displayed superior clinical effect in metastatic castration-resistant prostate cancer(mCRPC)patients with the homologous recombination repair(HRR)genes mutations.However,when a patient’s tumor tissue volume is insufficient for genomic profiling of HRR gene mutations,circulating tumor DNA(ctDNA)may be useful in helping to determine and monitor the efficacy of olaparib,as well as in abiraterone-combination treatment,and for understanding any resistance mechanism related to such mutations.CASE SUMMARY A 61-year-old man who was diagnosed with metastatic prostate adenocarcinoma was initially hormone sensitivity,showing high Gleason score(5+5=10)and absolute positive rate(14/14 biopsied specimens).Following failure of several standard therapies,the patient progressed to mCRPC.Surprisingly,the patient showed good response to olaparib-abiraterone-prednisone combination treatment(an androgen-deprivation therapy,provided as the‘final choice’in China).Serum total prostate-specific antigen(TPSA)level reduced and symptoms remitted for 4 months.However,thereafter,serum TPSA levels began slowly increasing,indicating development of olaparib resistance.Subsequent comprehensive genomic profiling of ctDNA, screening 508 cancer-related genes by next-generation sequencing,identified 10 somatic variants as well as 3 copy number alterations. Two identified reversemissense mutations in partner and localizer of BRCA2 (PALB2) may have recovered the readingframe, restoring function of the primary germline PALB2 mutation and causing resistance to thePARP inhibitor olaparib.CONCLUSIONReverse mutations in PALB2, discovered via genomic profiling of ctDNA, may represent apotential resistance mechanism against olaparib in mCRPC.

Circulating tumor DNA:Where are we now?A mini review of the literature

For many years tissue biopsy has been the primary procedure to establish cancer diagnosis and determine further treatment and prognosis.However,this method has multiple drawbacks,including,to mention some,being an invasive procedure carrying significant risk for fragile patients and allowing only for a“snapshot”of the tumor biology in time.The process of liquid biopsy allows for a minimally invasive procedure that provides molecular information about underlying cancer by analyzing circulating tumor DNA(ctDNA)via next-generation sequencing technology and circulating tumor cells.This paper focuses on describing the basis of ctDNA and its current utilities.

Circulating tumour DNA in gastrointestinal cancer in clinical practice:Just a dream or maybe not?

The evaluation of circulating tumor DNA(ctDNA)is increasingly integrated into the management of diagnosis and treatment of gastrointestinal cancer as it represents an innovative and minimally invasive biomarker that could allow us to reach clinical needs not met yet in randomized clinical trials.Recent research provided an interesting overview of the role of circulating tumor DNA in gastric,biliary,liver,pancreatic,and colorectal cancer.Data regarding upper gastrointestinal tumors are currently not practice changing.Tumor detection rates are low in the early stages,while in advanced stages ctDNA is useful for molecular tracking evaluation.Most of the evidence comes from colorectal cancer studies,where ctDNA was evaluated both in the early and advanced stages with the postsurgery minimal residual disease assessment and the response assessment,respectively.ctDNA qualifies as a promising tool in the era of precision medicine,with potential applications in the entire management of gastrointestinal cancer patients.Further evidence is needed to establish which setting may be influenced greatly by liquid biopsy in clinical practice.

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.

Genetic screening of liver cancer:State of the art

Liver cancer,primarily hepatocellular carcinoma,remains a global health challenge with rising incidence and limited therapeutic options.Genetic factors play a pivotal role in the development and progression of liver cancer.This state-of-the-art paper provides a comprehensive review of the current landscape of genetic screening strategies for liver cancer.We discuss the genetic underpinnings of liver cancer,emphasizing the critical role of risk-associated genetic variants,somatic mutations,and epigenetic alterations.We also explore the intricate interplay between environmental factors and genetics,highlighting how genetic screening can aid in risk stratification and early detection via using liquid biopsy,and advancements in high-throughput sequencing technologies.By synthesizing the latest research findings,we aim to provide a comprehensive overview of the state-of-the-art genetic screening methods for liver cancer,shedding light on their potential to revolutionize early detection,risk assessment,and targeted therapies in the fight against this devastating disease.

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.

Research progress on circulating tumor cell detection in brain gliomas

Glioma,the most common primary intracranial tumor,has high morbidity and mortality.The detection of circulating tumor cells(CTCs)is an important part of the liquid biopsy of gliomas.CTCs,carrying the genetic and biological information of tumor tissue,provide a new perspective and dimension for the study of tumor metastasis,progression,chemotherapy sensitivity and drug resistance.Cerebrospinal fluid(CSF)circulates through the ventricle and spinal cord cistern,which can better maintain the original information of tumor cells compared with the complicated environments of tissues and plasma.Study on the dynamic changes of CTCs in the CSF of the central nervous system(CNS)is relatively rare.However,the analysis of CTCs in CSF can be used to guide the treatment of gliomas and reveal the patho-physiological and genetic mechanisms of tumor cell metastasis to the CSF.This paper reviews the progress in the research on CTC detection in gliomas.

Molecular and cellular pathophysiology of circulating cardiomyocyte-specific cell free DNA (cfDNA): Biomarkers of heart failure and potential therapeutic targets

Pathological cardiac damage during heart failure is associated with cell death and damage associated molecular patterns (DAMPs) release which triggers a viscous cycle of sterile inflammation to mediate maladaptive cardiac tissue remodelling during the progression to heart failure. DAMPs like cytokines, chemokines, and nuclear or mitochondrial genomic fragments are released in the pathological myocardium. Interestingly, circulating or cytosolic DNA fragments can play a role in the disease by interaction with nucleic acid sensors expressed in cardiomyocyte and non-myocyte neighbouring cells. The circulating cell free DNA (cfDNA) fragments have been clinically reported as markers for various diseases including cardiovascular pathophysiology. Such cfDNA within the DAMP pool can mediate intra- and inter-cellular signalling cascade to upregulate transcriptional expression of inflammatory mediators and trigger oxidative stress within cells. The cellular role of such genomic equivalents varying with chronic or acute stress might be correlated with the cell death forms encountered in myocardium during disease progression. Thus, cfDNA can be phenotypically correlated as a critical player towards upregulation of pathological processes like interstitial fibrosis, cardiomyocyte contractile dysfunction and cell death. Herein, we review the association of cfDNA with heart failure and analyse their potential usage as novel and effective therapeutic targets towards augmentation of cardiac function.

Clinical outcomes of newly diagnosed primary central nervous system lymphoma treated with zanubrutinib-based combination therapy

BACKGROUND High-dose methotrexate(HD-MTX)combined with other chemotherapeutic agents is an effective treatment for patients with newly diagnosed primary central nervous system lymphoma(PCNSL);however,some patients have adverse reactions.AIM To retrospectively evaluate disease outcomes and mutational profiles in newly diagnosed PCNSL patients treated with a zanubrutinib/HD-MTX combination regimen.METHODS Nineteen newly diagnosed PCNSL patients were treated with zanubrutinib/HDMTX until disease progression,intolerable toxicities,or physician/patientdirected withdrawal.Safety and efficacy were assessed per the CTCAE v5.0 and RECIST v1.1 criteria,respectively.The primary endpoint was the objective response rate(ORR),and the secondary endpoints were progression-free survival,overall survival(OS),and safety.RESULTS The median follow-up duration was 14.7 mo(range,3.9–30 mo).The ORR for all patients was 84.2%,and 2-year progression-free-and OS rates were 75.6%and 94.1%,respectively.All patients completed the induction phase,and nine patients underwent autologous stem cell transplantation as consolidation therapy,resulting in an ORR of 88.9%.Ten patients received zanubrutinib as maintenance therapy and achieved an ORR of 80%.All patients showed an acceptable safety profile.The sequencing results for cerebrospinal fluid(CSF)and tumor tissue showed that PIM1 mutations were the most frequent genetic alterations.Circulating tumor DNA was correlated with disease relapse and response.CONCLUSION Our empirical observations demonstrated that the combination of zanubrutinib with HD-MTX yielded a marked clinical response and tolerability among newly diagnosed PCNSL patients.Non-invasive CSF liquid biopsy profiling may be feasible for evaluating treatment response and tumor burden.

Utility of circulating tumor DNA in patients undergoing hepatectomy for colorectal liver metastases

In this study recently published in Annals of Surgery,Newhook et al.studied the influence of circulating tumor DNA(ctDNA)in the management of patients undergoing hepatic resection for colorectal liver metastases(CRLM)(1).The primary objective was to study the association between the dynamic of ctDNA and somatic mutations with survival after resection of CRLM.The secondary objectives were to evaluate the impact of surgery on perioperative ctDNA dynamics and the impact of its detection on survival.

Circulating tumor DNA(ctDNA)-based minimal residual disease in non-small cell lung cancer

Lung cancer is the second most common cancer worldwide and the leading cause of cancer-related fatalities,with non-small cell lung cancer(NSCLC)accounting for 85%of all lung cancers.Over the past forty years,patients with NSCLC have had a 5-year survival rate of only 16%,despite improvements in chemotherapy,targeted therapy,and immunotherapy.Circulating tumor DNA(ctDNA)in blood can be used to identify minimal residual disease(MRD),and ctDNA-based MRD has been shown to be of significance in prognostic assessment,recurrence monitoring,risk of recurrence assessment,efficacy monitoring,and therapeutic intervention decisions in NSCLC.The level of MRD can be obtained by monitoring ctDNA to provide guidance for more precise and personalized treatment,the scientific feasibility of which could dramatically modify lung cancer treatment paradigm.In this review,we present a comprehensive review of MRD studies in NSCLC and focus on the application of ctDNA-based MRD in different stages of NSCLC in current clinical practice.