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.

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.

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.

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.

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 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.

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: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 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.

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.

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.

Clinical spectrum and prognostic value of TP53 mutations in circulating tumor DNA from breast cancer patients in China

Background:TP53 mutations are common in breast cancer.There is currently no large-scale cohort study to investigate the TP53 landscape in breast cancer patients from China.The predictive value of TP53 mutations for the efficacy of human epidermal growth factor receptor 2(HER2)-targeted therapy in breast cancer remains controversial.In the present study,we aimed to analyze the clinical spectrum and prognostic value of TP53 mutations in circulating tumor DNA(ctDNA)from breast cancer patients in China.Methods:We retrospectively analyzed the clinical data and TP53 mutation features in ctDNA samples from 804 patients withmetastatic breast cancer.TP53 mutations were detected by target region capture-based next-generation sequencing.The relationship between TP53 mutation status and disease-free survival(DFS)was analyzed in 444 patientswithmetastatic breast cancer.Moreover,the relationship between TP53 mutation status and progression-free survival(PFS)was analyzed in 55 HER2-positive patients treated with first-line trastuzumab-based therapy.Kaplan-Meier analysis was performed to estimate the survival curves of the different subgroups,and the log-rank test was used to compare the curves.A Cox regression model was used to estimate multivariable-adjusted hazard ratios and their 95%confidence intervals(CIs)associated with the DFS and PFS.Results:Among the 804 investigated patients,431(53.6%)patients harbored TP53 mutations.TP53 mutations were differentially distributed among different molecular subtypes of breast cancer(P<0.05).Patients with TP53 mutations had a shorter DFS than those with wild-type TP53(hazard ratio=1.32,95%CI=1.09-1.61,P=0.005).TP53 mutations in exons 5-8 were associated with worse outcome(hazard ratio=1.50,95%CI=1.11-2.03,P=0.009).However,TP53 mutation status was not significantly associated with PFS in HER2-positive patients who received firstline trastuzumab-based therapy(P=0.966).Interestingly,in the taxane combination group,patients with TP53 mutations exhibited longer PFS than those without TP53 mutations(hazard ratio=0.08,95%CI=0.02-0.30,P<0.001).However,in the nontaxane combination group,patients with TP53 mutations displayed shorter PFS than those with wild-type TP53(hazard ratio=4.84,95%CI=1.60-14.66,P=0.005).Conclusions:TP53 mutations in exons 5-8 may be an independent prognostic marker for short DFS in patients with metastatic breast cancer.TP53 mutations had opposite effects on trastuzumab-treated patients treated with and without taxanes.

Assessment of circulating tumor DNA in cerebrospinal fluid by whole exome sequencing to detect genomic alterations of glioblastoma

Background:Cerebrospinal fluid(CSF)has been demonstrated as a better source of circulating tumor DNA(ctDNA)than plasma for brain tumors.However,it is unclear whether whole exome sequencing(WES)is qualified for detection of ctDNA in CSF.The aim of this study was to determine if assessment of ctDNA in CSF by WES is a feasible approach to detect genomic alterations of glioblastoma.Methods:CSFs of ten glioblastoma patients were collected pre-operatively at the Department of Neurosurgery,Sun Yat-sen University Cancer Center.ctDNA in CSF and genome DNA in the resected tumor were extracted and subjected to WES.The identified glioblastoma-associated mutations from ctDNA in CSF and genome DNA in the resected tumor were compared.Results:Due to the ctDNA in CSF was unqualified for exome sequencing for one patient,nine patients were included into the final analysis.More glioblastoma-associated mutations tended to be detected in CSF compared with the corresponding tumor tissue samples(3.56±0.75 vs.2.22±0.32,P=0.097),while the statistical significance was limited by the small sample size.The average mutation frequencies were similar in CSF and tumor tissue samples(74.1%±6.0%vs.73.8%±6.0%,P=0.924).The R132H mutation of isocitrate dehydrogenase 1 and the G34V mutation of H3 histone,family 3A(H3F3A)which had been reported in the pathological diagnoses were also detected from ctDNA in CSF by WES.Patients who received temozolomide chemotherapy previously or those whose tumor involved subventricular zone tended to harbor more mutations in their CSF.Conclusion:Assessment of ctDNA in CSF by WES is a feasible approach to detect genomic alterations of glioblastoma,which may provide useful information for the decision of treatment strategy.

Circulating tumor DNA in lung cancer: real-time monitoring of disease evolution and treatment response

Lung cancer is one of the leading causes of all cancer-related deaths. Circulating tumor DNA (ctDNA) is released from apoptotic and necrotic tumor cells. Several sensitive techniques have been invented and adapted to quantify ctDNA genomic alterations. Applications of ctDNA in lung cancer include early diagnosis and detection, prognosis prediction, detecting mutations and structural alterations, minimal residual disease, tumor mutational burden, and tumor evolution tracking. Compared to surgical biopsy and radiographic imaging, the advantages of ctDNA are that it is a non-invasive procedure, allows real-time monitoring, and has relatively high sensitivity and specificity. Given the massive research on non-small cell lung cancer, attention should be paid to small cell lung cancer.

A multiplexed circulating tumor DNA detection platform engineered from 3D-coded interlocked DNA rings

Circulating tumor DNA(ctDNA)is a critical biomarker not only important for the early detection of tumors but also invaluable for personalized treatments.Currently ctDNA detection relies on sequencing.Here,a platform termed three-dimensional-coded interlocked DNA rings(3D-coded ID rings)was created for multiplexed ctDNA identification.The ID rings provide a ctDNA recognition ring that is physically interlocked with a reporter ring.The specific binding of ctDNA to the recognition ring initiates target-responsive cutting via a restriction endonuclease;the cutting then triggers rolling circle amplification on the reporter ring.The signals are further integrated with internal 3D codes for multiplexed readouts.ctDNAs from non-invasive clinical specimens including plasma,feces,and urine were detected and validated at a sensitivity much higher than those obtained through sequencing.This 3D-coded ID ring platform can detect any multiple DNA fragments simultaneously without sequencing.We envision that our platform will facilitate the implementation of future personalized/precision medicine.

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.

Utility of circulating tumor DNA for predicting prognosis in the management of resectable pancreatic cancer

Background:The measurement of circulating tumor DNA(ctDNA)has been studied in several malignancies,including metastatic pancreatic cancer,but less is known about its utility in monitoring treatment response and recurrence in resectable pancreatic cancer.Methods:We conducted a systematic review of the literature examining the association of ctDNA with overall survival(OS)and disease-free survival.Results:Five articles met our exclusion criteria.Baseline and/or postoperative ctDNA was found to be associated with decreased OS and recurrence-free survival.Discussion:ctDNA has the potential to be used as a prognostic biomarker and to guide therapy in resectable pancreatic cancer.

Circulating tumor DNAs and non-coding RNAs as potential biomarkers for hepatocellular carcinoma diagnosis, prognosis and response to therapy

Hepatocellular carcinoma(HCC)is the second leading cause of cancer-related deaths worldwide and despite improvement in therapeutic approaches,prognosis remains poor.This can be partly attributed to the fact that the majority of HCCs are diagnosed at intermediate or advanced stages.Availability of circulating biomarkers able to detect HCC at early stages could improve patients'prognosis.At present,however,alpha fetoprotein or des-g-carboxyprothrombin are unable to reliably detect HCC at early stages and better circulating biomarkers are needed.Circulating tumor DNA(ctDNA)and non-coding RNAs(ncRNAs)are emerging as promising biomarkers to achieve the goal.Genetic and epigenetic alterations in ctDNA allow to pinpoint tumor-specific biomarkers,reveal tumor heterogeneity,help monitor tumor evolution over time and assess therapy efficacy.It remains to be fully evaluated the possibility of detecting these biomarkers at early tumor stages.Circulating ncRNAs are quantitative biomarkers with potential use in diagnostic,prognostic and predictive clinical settings.They may help to reveal HCC at early stages.However,because of heterogeneous and sometimes conflicting reported results,they still require validation and standardization of pre-analytical and analytical approaches before clinical applications could be envisaged.