Circulating DNA level is negatively associated with the long-term survival of hepatocellular carcinoma patients

AIM： To quantify the circulating DNA in plasma from patients with hepatocellular carcinoma （HCC） and to evaluate its prognostic value. METHODS： Blood samples were collected from 79 patients with HCC before operation, 20 patients with liver cirrhosis, and 20 healthy volunteers. Circulating DNA was extracted from plasma and quantified. The association between circulating DNA level and prognosis of HCC patients was evaluated. RESULTS： Compared with the healthy volunteers （17.6 ± 9.5 ng/mL）, a significant higher circulating DNA level was found in the patients with HCC （47.1 ± 43.7 ng/ mL, P = 0.000） or with liver cirrhosis （30.0 ± 13.3 ng/ mL, P = 0.002）. The circulating DNA level was closely associated with tumor size （P = 0.008） and TNM stage （P = 0.040）, negatively associated with the 3-year diseasefree survival （DFS） （P = 0.017） and overall survival （OS） （P = 0.001）. CONCLUSION： Large or invasive tumor may release more circulating DNA, and higher level of circulating DNA may be associated with poor prognosis of HCC patients.

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.

Abnormal DNA methylation as a cell-free circulating DNA biomarker for colorectal cancer detection:A review of literature

Colorectal cancer(CRC) is one of the most prevalent malignancies in the world. CRC-associated morbidity and mortality is continuously increasing, in part due to a lack of early detection. The existing screening tools such as colonoscopy, are invasive and yet high cost, affecting the willingness of patients to participate in screening programs. In recent years, evidence is accumulating that the interaction of aberrant genetic and epigenetic modifications is the cornerstone for the CRC development and progression by alternating the function of tumor suppressor genes, DNA repair genes and oncogenes of colonic cells. Apart from the understanding of the underlying mechanism(s) of carcinogenesis, the aforementioned interaction has also allowed identification of clinical biomarkers, especially epigenetic, for the early detection and prognosis of cancer patients. One of the ways to detect these epigenetic biomarkers is the cell-free circulating DNA(circ DNA), a blood-based cancer diagnostic test, mainly focusing in the molecular alterations found in tumor cells, such as DNA mutations and DNA methylation.In this brief review, we epitomize the current knowledge on the research in circ DNA biomarkers-mainly focusing on DNA methylation-as potential blood-based tests for early detection of colorectal cancer and the challenges for validation and globally implementation of this emergent technology.

Association of the immediate perioperative dynamics of circulating DNA levels and neutrophil extracellular traps formation in cancer patients

Objectives:Elevated circulating DNA(cirDNA)concentrations were found to be associated with trauma or tissue damage which suggests involvement of inflammation or cell death in post-operative cirDNA release.We carried out the first prospective,multicenter study of the dynamics of cirDNA and neutrophil extracellular trap(NETs)markers during the perioperative period from 24 h before surgery up to 72 h after curative surgery in cancer patients.Methods:We examined the plasma levels of two NETs protein markers[myeloperoxidase(MPO)and neutrophil elastase(NE)],as well as levels of cirDNA of nuclear(cir-nDNA)and mitochondrial(cir-mtDNA)origin in 29 colon,prostate,and breast cancer patients and in 114 healthy individuals(HI).Results:The synergistic analytical information provided by these markers revealed that:(i)NETs formation contributes to post-surgery conditions;(i)post-surgery cir-nDNA levels were highly associated with NE and MPO in colon cancer[r=0.60(P<0.001)and r=0.53(P<0.01),respectivelyl,but not in prostate and breast cancer;(i)each tumor type shows a specific pattern of cir-nDNA and NETs marker dynamics,but overall the pre-and post-surgery median values of cir-nDNA,NE,and MPO were significantly higher in cancer patients than in HI.Conclusion:Taken as a whole,our work reveals the association of NETs formation with the elevated cir-nDNA release during a cancer patient's perioperative period,depending on surgical procedure or cancer type.By contrast,cir-mtDNA is poorly associated with NETs formation in the studied perioperative period,which would appear to indicate a different mechanism of release or suggest mitochondrial dysfunction.

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.

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.

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.

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.

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.

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 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.

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.

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.

Potential value of detection of minimal residual disease in colorectal cancer following radical resection

Although there has been significant advancement in the identification and management of colorectal cancer(CRC)in recent years,there is still room for improvement in the current standard treatment regimen.One area of concern is the lack of reliable tumor markers to predict treatment efficacy and guide tailored care.Due to its dynamic,effective,and non-invasive benefits over tissue biopsy,the detection of minimal or molecular residual lesions(MRD)based on circulating tumor DNA(ctDNA)is beneficial to the clinical development of drugs for patients with CRC after radical treatment,as well as for continuous monitoring of tumor recurrence and malignancy molecular gene evolution.The detection of ctDNA can currently be used to guide individual postoperative auxiliary treatment decisions(upgrade or downgrade treatment)in CRC,stratify the risk of clinical recurrence more precisely,and predict the risk of recurrence in advance of imaging examination,according to a large number of observational or prospective clinical studies.With increasing clarity comes the possibility of selecting a regimen of treatment based on postoperative ctDNA,which also improves the accuracy of clinical recurrence risk assessment for CRC.Therefore,it is anticipated that the identification of ctDNA would alter the current framework for dealing with CRC and lead to individualized,stratified precision therapy;however,additional confirmation will require subsequent high-quality,prospective,large-scale randomized controlled studies.This article will provide an overview of the definition and clinical significance of MRD,the primary indications and technological challenges for MRD detection,along with the advancement in clinical research about ctDNA detection following radical resection of the CRC.

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.