In vivo fluorescence flow cytometry reveals that the nanoparticle tumor vaccine OVA@HA-PEI effectively clears circulating tumor cells

Tumor vaccine therapy offers significant advantages over conventional treatments,including reduced toxic side effects.However,it currently functions primarily as an adjuvant treatment modality in clinical oncology due to limitations in tumor antigen selection and delivery methods.Tumor vaccines often fail to elicit a sufficiently robust immune response against progressive tumors,thereby limiting their clinical efficacy.In this study,we developed a nanoparticle-based tumor vaccine,OVA@HA-PEI,utilizing ovalbumin(OVA)as the presenting antigen and hyaluronic acid(HA)and polyethyleneimine(PEI)as adjuvants and carriers.This formulation significantly enhanced the proliferation of immune cells and cytokines,such as CD3,CD8,interferon-,and tumor necrosis factor-,in vivo,effectively activating an immune response against B16–F10 tumors.In vivofluorescenceflow cytometry(IVFC)has already become an effective method for monitoring circulating tumor cells(CTCs)due to its direct,noninvasive,and long-term detection capabilities.Our study utilized a laboratory-constructed IVFC system to monitor the immune processes induced by the OVA@HA-PEI tumor vaccine and an anti-programmed death-1(PD-1)antibody.The results demonstrated that the combined treatment of OVA@HA-PEI and anti-PD-1 antibody significantly improved the survival time of mice compared to anti-PD-1 antibody treatment alone.Additionally,this combination therapy substantially reduced the number of CTCs in vivo,increased the clearance rate of CTCs by the immune system,and slowed tumor progression.Thesefindings greatly enhance the clinical application prospects of IVFC and tumor vaccines.

Prognostic value of circulating tumor cells combined with neutrophil-lymphocyte ratio in patients with hepatocellular carcinoma

BACKGROUND Circulating tumor cell(CTC)count and neutrophil-to-lymphocyte ratio(NLR)are both closely associated with the prognosis of hepatocellular carcinoma(HCC).AIM To investigate the prognostic value of combining these two indicators in HCC.METHODS Clinical data were collected from patients with advanced HCC who received im-mune therapy combined with targeted therapy at the Department of Oncology,the Affiliated Hospital of Southwest Medical University,Sichuan,China,from 2021 to 2023.The optimal cutoff values for CTC programmed death-ligand 1(PD-L1)(+)>1 or CTC PD-L1(+)≤1 and NLR>3.89 or NLR≤3.89 were evaluated using X-Tile software.Patients were categorized into three groups based on CTC PD-L1(+)counts and NLR:CTC-NLR(0),CTC-NLR(1),and CTC-NLR(2).The relationship between CTC-NLR and clinical variables as well as survival rates was assessed.RESULTS Patients with high CTC PD-L1(+)expression or NLR at baseline had shorter median progression-free survival(m-PFS)and median overall survival(mOS)than those with low levels of CTC PD-L1(+)or NLR(P<0.001).Mean-while,patients in the CTC-NLR(2)group showed a significant decrease in mPFS and mOS.Cox regression analysis revealed that alpha-fetoprotein(AFP),CTC PD-L1(+),and CTC-NLR were independent predictors of OS.The time-dependent receiver operating characteristic curve showed that the area under the curve of CTC-NLR at 12 months(0.821)and 18 months(0.821)was superior to that of AFP and CTC PD-L1(+).CONCLUSION HCC patients with high CTC PD-L1(+)or NLR expression tend to exhibit poor prognosis,and a high baseline CTC-NLR score may indicate low survival.CTC-NLR may serve as an effective prognostic indicator for patients with advanced HCC receiving immunotherapy combined with targeted therapy.

Circulating tumor cells as prognostic marker in pancreatic cancer

In this editorial we comment on the article by Zhang et al published in the recent issue of the World Journal of Clinical Oncology.Pancreatic cancer is the fourth most common cause of cancer-related mortality and has the lowest survival rate among all solid cancers.It causes 227000 deaths annually worldwide,and the 5-year survival rate is very low due to early metastasis,which is 4.6%.Cancer survival increases with better knowledge of risk factors and early and accurate diagnosis.Circulating tumor cells(CTCs)are tumor cells that intravasate from the primary tumor or metastasis foci into the peripheral blood circulation system spontan-eously or during surgical operations.Detection of CTC in blood is promising for early diagnosis.In addition,studies have associated high CTC levels with a more advanced stage,and more intensive treatments should be considered in cases with high CTC.In tumors that are considered radiologically resectable,it may be of critical importance in detecting occult metastases and preventing unnecessary surgeries.

Circulating tumor cells in pancreatic cancer:The prognostic impact in surgical patients

Pancreatic cancer is associated with a poor prognosis,even in the early stages,mainly due to metastatic progression.New diagnostic techniques that predict unfavorable outcomes are needed in order to improve treatment strategies.Circulating tumor cells(CTCs)are showing promising results as a predictive biomarker for various tumors.In this editorial we comment on the article by Zhang et al,who published the first systematic review and meta-analysis evaluating the prognostic value of CTCs as biomarkers in early-stage pancreatic cancer patients undergoing surgery.CTCs were detected in peripheral or central venous system blood,before or during surgery.Positive CTCs showed a correlation with decreased overall survival and decreased relapse-free,disease-free and progression-free survival in this meta-analysis.However,the heterogeneity was significant.The authors suggest that this result was related to the separation methods used between studies,but other differences such as the margin status or the neoadjuvant and adjuvant treatments used are also important to consider.CTCs may be a potential prognostic biomarker in pancreatic cancer patients,but it is necessary to compare and standardize the platforms used to isolate CTCs,to compare different biomarkers from liquid biopsy and to determine the impact on prognosis when therapeutic changes are made based on CTCs levels.

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.

Evolutionary model of brain tumor circulating cells: Cellular galaxy

BACKGROUND Although circulating tumor cells(CTCs)have been the focus of consideration for a decade,a categorized cell-based diagnostic strategy is unavailable.The personalized management and complementary/analytical-strategy of data require an alphabetic guide.Therefore,we aimed to determine the behavior of CTCs in tumor and blood in order to provide the hypothetical-based agenda in the brain neoplasms.Exploring the protein expression(PE)using a single cell-based method would clarify the heterogeneity and diversity in tumor and blood,which are key events in the evolution in brain tumors.In fact,heterogeneity,diversity,and evolution are required for cancer initiation and progression.AIM To explore CTCs in brain tumors and blood cells and to assay intensity of PE through personalized insight.METHODS The focal population included 14 patients with meningioma,and four patients with metastatic brain tumors(T).PE was assayed by immunofluorescence in tumors cells and CTCs in 18 patients with brain tumors.Ratio test was applied between the T cells and CTCs in tumor tissue and in vascular system.T/CTC ratio-based classification of PE in macrophage chemoattractant chemokine ligand 2(CCL2),vascular endothelial growth factor(VEGF),epidermal growth factor(EGF),CD133,cyclin E,neurofilament marker,cytokeratin 19,and leukocyte common antigen(CD45)were investigated.RESULTS Total analyzed cells ranged between 10794-92283 for tumor cells and between 117-2870 for CTCs.Characteristics of histopathologic and status of an ataxiatelangiectasia mutated polymorphism(D1853N)in 18 patients affected with brain tumors were also provided.The course of evolution and metastatic event relied on the elevated protein expression in CTCs,which could be considered as a prognostic value.Diverse protein expression of the migrated cells into the blood stream and the tumor was indicative of the occurrence of evolution.Besides,the harmonic co-expression between CCL2/EGF and CCL2/VEGF could facilitate the tumor progression including the metastatic event.Expression of these proteins in the migrated vasculature and into the buccal tissue offered a non-invasive followup detection in neoplastic disorders.PE-exploration of neurofilament marker/CD133/VEGF of the CTCs in meningioma and cytokeratin 19/CD45/cyclin E in the patients with metastatic brain tumor would clarify the tumor biology of the brain neoplastic disorders.CONCLUSION The alphabetical base of the evolutionary mechanisms relies on dual-,triple-,and multi-models with diverse intensity of expression.In fact,cross-talk between initiative and the complementary channels defines the evolutionary insight in cancer.A diverse-model of protein expression,including low,medium,and high intensity,is the key requirement for the completed model.The cluster of cells with diverse expression and remarkable co-expression between CCL2/EGF/VEGF and NM/CD133/VEGF in CTCs may be indicative of probable invasiveness of the tumor.Furthermore,the mode of cytokeratin-19+/CD45-can be traced in the metastatic patients.

Evaluation of epithelial-mesenchymal transitioned circulating tumor cells in patients with resectable gastric cancer: Relevance to therapy response

AIM: To evaluate the epithelial-to-mesenchymal transition(EMT) of circulating tumor cells(CTCs) in gastric cancer patients.METHODS: We detected tumor cells for expression of four epithelial(E^+) transcripts(keratins 8, 18, and 19 and epithelial cell adhesion molecule) and two mesenchymal(M^+) transcripts(Vimentin and Twist) by a quantifiable, dual-colorimetric RNA-in situ hybridization assay. Between July 2014 and October 2014, 44 patients with gastric cancer were recruited for CTC evaluation. Blood samples were obtained from selected patients during the treatment course [before surgery, after surgery and at the 6^(th) cycle of XELOX based chemotherapy(about 6 mo postoperatively)].RESULTS: We found the EMT phenomenon in which there were a few biphenotypic E^+/M^+ cells in primary human gastric cancer specimens. Of the 44 patients, the presence of CTCs was reported in 35(79.5%) patients at baseline. Five types of cells including from exclusively E^+ CTCs to intermediate CTCs and exclusively M^+ CTCs were identified(4 patients with M^+ CTCs and 10 patients with M^+ or M^+ > E^+ CTCs). Further, a chemotherapy patient having progressive disease showed a proportional increase of mesenchymal CTCs in the post-treatment blood specimens. We used NCI-N87 cells to analyze the linearity and sensitivity of Can Patrol^(TM) system and the correlation coefficient(R^2) was 0.999.CONCLUSION: The findings suggest that the EMT phenomenon was both in a few cells of primary tumors and abundantly in CTCs from the blood of gastric cancer patients, which might be used to monitor therapy response.

Circulating endothelial and progenitor cells:Evidence from acute and long-term exercise effects

Circulating bone-marrow-derived cells,named endothelial progenitor cells(EPCs),are capable of maintaining,generating,and replacing terminally differentiated cells within their own specific tissue as a consequence of physiological cell turnover or tissue damage due to injury.Endothelium maintenance and restoration of normal endothelial cell function is guaranteed by a complex physiological procedure in which EPCs play a significant role.Decreased number of peripheral blood EPCs has been associated with endothelial dysfunction and high cardiovascular risk.In this review,we initially report current knowledge with regard to the role of EPCs in healthy subjects and the clinical value of EPCs in different disease populations such as arterial hypertension,obstructive sleep-apnea syndrome,obesity,diabetes mellitus,peripheral arterial disease,coronary artery disease,pulmonary hypertension,and heart failure.Recent studies have introduced the novel concept that physical activity,either performed as a single exercise session or performed as part of an exercise training program,results in a significant increase of circulating EPCs.In the second part of this review we provide preliminary evidence from recent studies investigating the effects of acute and long-term exercise in healthy subjects and athletes as well as in disease populations.

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

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

Circulating tumor cells in pancreatic cancer patients:Enrichment and cultivation

AIM: To investigate the feasibility of separation and cultivation of circulating tumor cells (CTCs) in pancreatic cancer (PaC) using a filtration device.

Molecular detection of epithelial-mesenchymal transition markers in circulating tumor cells from pancreatic cancer patients:Potential role in clinical practice

AIM To evaluate the clinical properties of three subpopulations of circulating tumor cells(CTCs) undergoing epithelial-mesenchymal transition(EMT) in pancreatic ductal adenocarcinoma(PDAC) patients.METHODS We identified CTCs for expression of the epithelial cell marker cytokeratin or epithelial cell adhesion molecule(EpCAM)(E-CTC), the mesenchymal cell markers vimentin and twist(M-CTC), or both(E/M-CTC) using the CanPatrol system. Between July 2014 and July 2016, 107 patients with PDAC were enrolled for CTC evaluation. CTC enumeration and classification were correlated with patient clinicopathological features and outcomes.RESULTS CTCs were detected in 78.5% of PDAC patients. The number of total CTCs ranged from 0 to 26 across all 107 patients, with a median value of six. CTC status correlated with lymph node metastasis, TNM stage, distant metastasis, blood lymphocyte counts, and neutrophil-to-lymphocyte ratio(NLR). Kaplan-Meier survival analysis showed that patients with ≥ 6 total CTCs had significantly decreased overall survival and progression-free survival compared with patients with < 6 total CTCs. The presence of M-CTCs was positively correlated with TNM stage(P < 0.01) and distant metastasis(P < 0.01). Additionally, lymphocyte counts and NLR in patients without CTCs were significantly different from those in patients testing positive for each CTC subpopulation(P < 0.01).CONCLUSION Classifying CTCs by EMT markers helps to identify the more aggressive CTC subpopulations and provides useful evidence for determining a suitable clinical approach.

Monitoring disease progression and treatment efficacy with circulating tumor cells in esophageal squamous cell carcinoma: A case report

This study investigated whether changes in circulating tumor cell(CTC) numbers reflect tumor progression and treatment efficacy in esophageal squamous cell carcinoma(ESCC). A 47-year-old male patient with ESCC is presented in this case study. The patient was evaluated for a series of serum tumor markers and subjected to radiological examinations before and after surgery and during follow-up over the course of five years. In addition, the CTCs in 7.5 m L of peripheral blood were enriched by magneticactivated cell sorting negative selection and identified by immunofluorescence staining. Serum tumor markers remained within normal ranges and were discordant with imaging scans during the follow-up. Initially, one CTC was detected in the peripheral blood sample, and 14 were observed seven days after the operation. After 12 wk, subcutaneous metastases and bone metastases occurred, and the number of CTCs increased to 84. After 48 wk, lung metastases were noted, and the CTC level was 21. At 104 wk, the number of CTCs was 14, and disease recurrence was detected by positron emission tomography-computed tomography. The CTC counts were in accord with the imaging studies at several time points. The additional information provided by CTC enumeration could thus facilitate monitoring of disease status and treatment efficacy and provide support for treatment decisions.

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

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

Targeting of circulating hepatocellular carcinoma cells to prevent postoperative recurrence and metastasis

Currently,the main treatment for hepatocellular carcinoma(HCC)involves the surgical removal of tumors or liver transplantation.However,these treatments are often not completely curative,as they are associated with a risk for postoperative recurrence and metastasis.Circulating tumor cells(CTCs)are increasingly recognized as the main source for recurrence and metastasis after radical hepatectomies are performed.Many studies have demonstrated the association between the presence of either pre-or postoperative CTCs and an increased risk for HCC recurrence.To improve the therapeutic outcome of HCC,a personalized,comprehensive and multidisciplinary approach should be considered,involving the application of appropriate diagnostic and therapeutic measures targeting HCC CTCs in different stages throughout the course of treatment.This article proposes some HCC CTC-based strategies for the treatment of HCC,including the monitoring of HCC CTCs before,during and after radical hepatectomy,therapeutic targeting of HCC CTCs,prevention of the generation and colonization of CTCs,as well as the use of CTC indexes for the selection of indications,prediction of prognoses,and planning of individualized therapeutic regimens.Innovation and technological development of therapies targeting CTCs,as well as their translation into clinical practice,will help to effectively reduce postoperative recurrence and metastasis,and significantly prolong the survival of HCC patients.

Circulating tumor and cancer stem cells in hepatitis C virusassociated liver disease

AIM: To assess the role of circulating tumor cells (CTCs) and cancer stem cells (CSCs) in hepatitis C virus (HCV)-associated liver disease.

Circulating cancer stem cells:the importance to select

It has been demonstrated that even localized tumors withottt chnically apparent metastasis give rise to circulating tumor cells （CTCs）. A growing number of technically diverse platforms are being developed for detecting/isolating CTCs in the circulating blood. Despite the technical challenges of isolating rare CTCs from blood, recent studies have already shown the predictive value of CTCs enumeration. Thus, it is becoming increasingly accepted that CTC numbers are linked to patients＇ outcome and may also be used to monitor treatment response and disease relapse, respectively. Further CTCs provide a non-invasive source for tumor material, ＇liquid biopsy＇, which is particularly important for patients, where no biopsy material can be obtained or where serial biopsies of the tumor, e.g., following treatment, are practically impossible. On the other hand the molecular and biological characterization of CTCs has still remained at a rather experimental stage. Future studies are necessary to define CTC heterogeneity to establish the crucial role of circulating cancer stem cells for driving metastasis, which represent a distinct subpopulation of CTCs that bear metastasis-initiating capabilities based on their sternness properties and invasiveness and thus are critical for the patients＇ clinical outcome. As compared to non-tumorigenic/metastatic bulk CTCs, circulating cancer stem cells may not only be capable of evading from the primary tumor, but also escape from immune surveillance, survive in the circulating blood and subsequently form metastases in distant organs. Thus, circulating cancer stem cells represent a subset of exclusively tumorigenic cancer stem cells characterized by their invasive characteristics and are potential therapeutic targets for preventing disease progression. To date, only a few original reports and reviews have been published focusing on circulating cancer stem cells. This review discusses the potential importance of isolating and characterizing these circulating cancer stem cells, but also highlights current technological limitations.

Aneuploidy of chromosome 8 in circulating tumor cells correlates with prognosis in patients with advanced gastric cancer

Objective： Previous work indicated that aneuploidy of chromosome 8 in circulating tumor cells（CTCs）correlated with therapeutic efficacy for advanced gastric cancer（AGC） patients. In this follow-up study performed on the same population of AGC patients, we investigated whether and how aneuploidy of chromosome 8 in CTCs correlates with patients＇ clinical prognosis.Methods： The prospective study was performed on 31 patients with newly diagnosed AGC. Previously established integrated subtraction enrichment（SE） and immunostaining-fluorescence in situ hybridization（i FISH）platform was applied to identify, enumerate and characterize CTCs. Quantification of CTCs and analysis of their aneuploidy of chromosome 8 were performed on patients before and after therapy.Results： CTCs were measured in 93.5% of AGC patients, and two CTC subtypes with diverse threshold values were identified, multiploid CTCs with the threshold of ≥2 per 7.5 m L and multiploid plus triploid CTCs with the threshold of ≥4, which were found to significantly correlate with poor progression-free survival（PFS） and overall survival（OS）. In particular, patients with ≥10% increased multiploid CTCs after an initial 6 weeks of therapy had poor PFS and OS, whereas improved PFS and OS were observed on those who had ≥10% decreased multiploid CTCs. After adjusting for clinically significant factors, ≥10% increased post-therapy multiploid CTCs was the only independent predictor of PFS and OS.Conclusions： Aneuploidy of CTCs correlates with prognosis of AGC patients. Quantitative comparison monitoring multiploid CTCs before and after therapy may help predict improved or inferior prognosis and chemoresistance.

Advances in isolation and detection of circulating tumor cells based on microfluidics

Circulating tumor cells(CTCs) are the cancer cells that circulate in the peripheral blood after escaping from the original or metastatic tumors. CTCs could be used as non-invasive source of clinical information in early diagnosis of cancer and evaluation of cancer development. In recent years, CTC research has become a hotspot field wherein many novel CTC detection technologies based on microfluidics have been developed. Great advances have been made that exhibit obvious technical advantages, but cannot yet satisfy the current clinical requirements. In this study, we review the main advances in isolation and detection methods of CTC based on microfluidics research over several years, propose five technical indicators for evaluating these methods, and explore the application prospects. We also discuss the concepts, issues, approaches, advantages, limitations, and challenges with an aim of stimulating a broader interest in developing microfluidics-based CTC detection technology.

Cryopreservation for delayed circulating tumor cell isolation is a valid strategy for prognostic association of circulating tumor cells in gastroesophageal cancer

AIM To demonstrate the feasibility of cryopreservation of peripheral blood mononuclear cells(PBMCs) for prognostic circulating tumor cell(CTC) detection in gastroesophageal cancer.METHODS Using 7.5 m L blood samples collected in EDTA tubes from patients with gastroesopheagal adenocarcinoma, CTCs were isolated by epithelial cell adhesion molecule based immunomagnetic capture using the Iso Flux platform. Paired specimens taken during the same blood draw(n = 15) were used to compare number of CTCs isolated from fresh and cryopreserved PBMCs. Blood samples were processed within 24 h to recover the PBMC fraction, with PBMCs used for fresh analysis immediately processed for CTC isolation. Cryopreservation of PBMCs lasted from 2 wk to 25.2 mo(median 14.6 mo). CTCs isolated from pre-treatment cryopreserved PBMCs(n = 43) were examined for associations with clinicopathological variables and survival outcomes.RESULTS While there was a significant trend to a decrease in CTC numbers associated with cryopreserved specimens(mean number of CTCs 34.4 vs 51.5, P = 0.04), this was predominately in samples with a total CTC count of > 50, with low CTC count samples less affected(P = 0.06). There was no significant association between the duration of cryopreservation and number of CTCs. In cryopreserved PBMCs from patient samples prior to treatment, a high CTC count(> 17) was associated with poorer overall survival(OS)(n = 43, HR = 4.4, 95%CI: 1.7-11.7, P = 0.0013). In multivariate analysis, after controlling for sex, age, stage, ECOG performance status, and primary tumor location, a high CTC count remained significantly associated with a poorer OS(HR = 3.7, 95%CI: 1.2-12.4, P = 0.03). CONCLUSION PBMC cryopreservation for delayed CTC isolation is a valid strategy to assist with sample collection, transporting and processing.

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

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