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 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.

Recent progress in aptamer-based microfluidics for the detection of circulating tumor cells and extracellular vesicles

Liquid biopsy is a technology that exhibits potential to detect cancer early,monitor therapies,and predict cancer prognosis due to its unique characteristics,including noninvasive sampling and real-time analysis.Circulating tumor cells(CTCs)and extracellular vesicles(EVs)are two important components of circulating targets,carrying substantial disease-related molecular information and playing a key role in liquid biopsy.Aptamers are single-stranded oligonucleotides with superior affinity and specificity,and they can bind to targets by folding into unique tertiary structures.Aptamer-based microfluidic platforms offer new ways to enhance the purity and capture efficiency of CTCs and EVs by combining the advantages of microfluidic chips as isolation platforms and aptamers as recognition tools.In this review,we first briefly introduce some new strategies for aptamer discovery based on traditional and aptamer-based microfluidic approaches.Then,we subsequently summarize the progress of aptamer-based microfluidics for CTC and EV detection.Finally,we offer an outlook on the future directional challenges of aptamer-based microfluidics for circulating targets in clinical applications.

A pilot study of the relative number of circulating tumor cells and leukocytes containing actin-binding proteins in head and neck cancer patients

Circulating tumor cells(CTCs)play an important role in tumor metastases,which is positively correlated with an increased risk of death.Actin-binding proteins,including cofilin(CFL1),profilin 1(PFN1),and adenylate cyclase-associated protein 1(CAP1),are thought to be involved in tumor cell motility and metastasis,specifically in head and neck squamous cell carcinoma(HNSCC).However,currently,there are no published studies on CFL1,PFN1,and CAP1 in CTCs and leukocytes in HNSCC patients.We assessed serum levels of CFL1,PFN1,and CAP1 and the number of CTCs and leukocytes containing these proteins in blood from 31 HNSCC patients(T1-4N0-2M0).The analysis used flow cytometry and an enzyme-linked immunosorbent assay kit.We found that CAP1+CTCs and CAP1+leukocyte subpopulations were prevalent in these HNSCC patient samples,while the prevalence rates of CFL1+and PFN1+CTCs were relatively low.Patients with stage T2-4N1-2M0 had CFL1+and PFN1+CTCs with an elevated PFN1 serum level,compared with the T1-3N0M0 group.In summary,the PFN1 serum level and the relative number of PFN1+CD326+CTCs could be valuable prognostic markers for HNSCC metastases.The current study is the first to obtain data regarding the contents of actin-binding proteins(ABPs)in CTCs,and leukocytes in blood from HNSCC patients.This is also the first to assess the relationship between the number of CTCs subgroups and disease characteristics.

Circulating tumor cells: Biological features and survival mechanisms

Circulating tumor cells(CTCs)are neoplastic cells that are detached from primary tumors and enter circulation.Enumeration and characterization of CTCs are of significance in cancer diagnosis,prognosis,and treatment monitoring.CTC survival in the bloodstream is a limiting step for the development of metastases in distant organs.Recent technological advances,especially in single-cell molecular analyses have uncovered heterogeneous CTC survival mechanisms.Undergoing epithelial-to-mesenchymal transition(EMT),increasing stem cell-like properties,and forming cell clusters enable CTCs to adapt to the harsh microenvironment of the circulation.Expressing and releasing several immunosuppressive molecules help CTCs escape from anti-cancer immune mechanisms.This review article summarizes the biological characteristics of CTCs and focuses on the recent understanding of the mechanisms by which CTCs survive in circulation.Additionally,the clinical and therapeutic implications of CTCs are discussed.

Clinical implications and perspectives of portal venous circulating tumor cells in pancreatic cancer

Despite recent improvements in the diagnosis and treatment of pancreatic cancer(PC),clinical outcomes remain dismal.Moreover,there are no effective prognostic or predictive biomarkers or options beyond carbohydrate antigen 19-9 for personalized and precise treatment.Circulating tumor cells(CTCs),as a member of the liquid biopsy family,could be a promising biomarker;however,the rarity of CTCs in peripheral venous blood limits their clinical use.Because the first venous drainage of PC is portal circulation,the portal vein can be a more suitable location for the detection of CTCs.Endoscopic ultrasound-guided portal venous sampling of CTCs is both feasible and safe.Several studies have suggested that the detection rate and number of CTCs may be higher in the portal blood than in the peripheral blood.CTC counts in the portal blood are highly associated with hepatic metastasis,recurrence after surgery,and survival.The phenotypic and genotypic properties measured in the captured portal CTCs can help us to understand tumor heterogeneity and predict the prognosis of PC.Small sample sizes and heterogeneous CTC detection methods limit the studies to date.Therefore,a large number of prospective studies are needed to corroborate portal CTCs as a valid biomarker in PC.

Microfluidic platform for circulating tumor cells isolation and detection

Circulating tumor cells(CTCs)are essential biomarkers for liquid biopsies,which are important in the early screening,prognosis,and real-time monitoring of cancer.However,CTCs are less abundant in the peripheral blood of patients,therefore,their isolation is necessary.Recently,the use of microfluidics for CTC sorting has become a research hotspot owing to its low cost,ease of integration,low sample consumption,and unique advantages in the manipulation of micron-sized particles.Herein,we review the latest research on microfluidics-based CTC sorting.Specifically,we consider active sorting using external fields(electric,magnetic,acoustic,and optical tweezers)and passive sorting using the flow effects of cells in specific channel structures(microfiltration sorting,deterministic lateral displacement sorting,and inertial sorting).The advantages and limitations of each method and their recent applications are summarized here.To conclude,a forward-looking perspective is presented on future research on the microfluidic sorting of CTCs.

Case Report: Pazopanib Treatment Response in a Patient with Metastatic Pleomorphic Dermal Sarcoma (Atypical Fibroxanthoma) with Circulating Tumor Cell-Derived Colonies as a Predictive Marker

Atypical fibroxanthomas (AFX) are rare skin tumors. These generally are superficial tumors, usually <3 cm red, fleshy, ulcerated skin lesions, that characteristically occur on sun-damaged skin, sometimes in immunocompromised or previously irradiated patients. These are part of a spectrum of more aggressive fibro-histiocytic neoplasms. In the older literature, these have been termed aggressive or metastatic AFX, but currently these have been reclassified as pleomorphic dermal sarcomas (PDS) and systemic undifferentiated pleomorphic sarcoma (UPS, formerly malignant fibrohistiocytic sarcoma, MFH). We present the case of a 64-year old woman who developed a deeply invasive PDS on the vertex of her scalp invading to the galea, with in-transit scalp metastases. Very little information is available about optimal treatment of metastatic PDS lesions. The patient was initially treated with 2 cycles of epirubicin/ifosfamide chemotherapy, resulting in life-threatening complications. A pretreatment peripheral blood sample was sent for CTC-derived colony assay. This sample grew 8 colonies from 10 ml blood. The tumor failed to respond to epirubicin and ifosfamide, and after several months of hospitalization, a second peripheral blood CTC-derived colony assay grew >376 colonies. The patient could not tolerate additional chemotherapy. She was therefore treated with the oral targeted agent pazopanib. The patient developed a dramatic biopsy-confirmed complete response. After 11 months of pazopanib treatment, a repeat CTC-derived culture sample grew only 8 colonies/10 ml blood. The complete response to pazopanib is still ongoing at over 41 months. To our knowledge, this is the first demonstration of clinical complete response of a PDS tumor following targeted therapy. An additional novel feature was the demonstration that CTC-derived colonies could be grown from the blood of a PDS patient. The number of colonies appeared to correlate with the clinical treatment response and seemed to function as a potential prognostic marker.

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.

Circulating tumor cells(CTCs)in breast cancer:a diagnostic tool for prognosis and molecular analysis

Metastatic breast cancer （MBC） is characterized by a combination of tumor growth, proliferation and metastatic progression and is typically managed with palliative intent. The benefit of standard systemic therapies is relatively limited and the disease is considered incurable suggesting the need to investigate the biological drivers of the various phases of the metastatic process in order to improve the selection of molecularly driven therapies. The detection, enumeration and molecular analysis of circulating tumor cells （CTCs） provide an intriguing opportunity to advance this knowledge. CTCs enumerated by the Food and Drugs Administration-cleared CellSearchTM system are an independent prognostic factor of progression- free survival （PFS） and overall survival （OS） in MBC patients. Several published papers demonstrated the poor prognosis for MBC patients that presented basal CTC count _〉5 in 7.5 mL of blood. Therefore, the enumeration of CTCs during treatment for MBC provides a tool with the ability to predict progression of disease earlier than standard timing of anatomical assessment using conventional radiological tests. During the metastatic process cancer cells exhibit morphological and phenotypic plasticity undergoing epithelial- mesenchymal transition （EMT）. This important phenomenon is associated with down regulation of epithelial marker （e.g., EpCAM） with potential limitations in the applicability of current CTCs enrichment methods. Such observations translated in a number of investigations aimed at improving our capabilities to enumerate and perform molecular characterization of CTCs. Theoretically, the phenotypic analysis of CTCs can represent a ＂liquid＂ biopsy of breast tumor that is able to identify a new potential target against the metastatic disease and advance the development and monitoring of personalized therapies.

Combined peripheral natural killer cell and circulating tumor cell enumeration enhance prognostic efficiency in patients with metastatic triple-negative breast cancer

Objective：Triple-negative breast cancer（TNBC）is a heterogeneous disease with poor prognosis.Circulating tumor cells（CTCs）are a promising predictor for breast cancer prognoses but their reliability regarding progression-free survival（PFS）is controversial.We aim to verify their predictive value in TNBC.Methods：In present prospective cohort study,we used the Pep@MNPs method to enumerate CTCs in baseline blood samples from 75 patients with TNBC（taken at inclusion in this study）and analyzed correlations between CTC numbers and outcomes and other clinical parameters.Results：Median PFS was 6.0（range：1.0–25.0）months for the entire cohort,in whom we found no correlations between baseline CTC status and initial tumor stage（P=0.167）,tumor grade（P=0.783）or histological type（P=0.084）.However,among those getting first-line treatment,baseline CTC status was positively correlated with ratio of peripheral natural killer（NK）cells（P=0.032）,presence of lung metastasis（P=0.034）and number of visceral metastatic site（P=0.037）.Baseline CTC status was predictive for PFS in first-line TNBC（P=0.033）,but not for the cohort as a whole（P=0.118）.This prognostic limitation of CTC could be ameliorated by combining CTC and NK cell enumeration（P=0.049）.Conclusions：Baseline CTC status was predictive of lung metastasis,peripheral NK cell ratio and PFS in TNBC patients undergoing first-line treatment.We have developed a combined CTC-NK enumeration strategy that allows us to predict PFS in TNBC without any preconditions.

Circulating Tumor Cells in Metastatic Breast Cancer:Monitoring Response to Chemotherapy and Predicting Progression-Free Survival

Objective:The purpose of this study is to explore RT-PCR method to set up the examination platform for detecting circulating tumor cells(CTC) in peripheral blood from metastatic breast cancer patients.The primary endpoint is to find out the correlation of existence of CTC with clinical responses and progression-free survival(PFS).Methods:The breast cancer cell line MCF-7 was serially diluted into the peripheral blood from 45 healthy donors to set up the sensitivity of RT-PCR assay.The expression of CK19 mRNA was amplified from both 49 patients and 45 healthy donors respectively.The CK19 protein quantity from plasma was measured by competitive inhibition ELISA assay.Results:The sensitivity of RT-PCR could reach 1/106?107 white blood cells with specificity of 95.6%.The objective response rate(ORR) of patients with CK19 mRNA-negative undertaken one cycle chemotherapy was significantly higher than those with positive(P0.0001).PFS among CK19 mRNA-negative patients was also increased,although there was no significance(P=0.098).The results of ELISA assay showed that CK19 protein was decreased significantly after one cycle chemotherapy,which gave rise to a little higher ORR(P=0.015) and increased PFS(P=0.016).Conclusion:Patients with unamplified CK19 mRNA after one cycle chemotherapy could achieve better radiographic evaluation and increased PFS,which was showed to be of consistency with the CK19 protein assay among the patients treated.

Incorporation of circulating tumor cells and whole-body metabolic tumor volume of 18F-FDG PET/CT improves prediction of outcome inⅢB stage small-cell lung cancer

Objective: We investigated the correlation between the number of circulating tumor cells(CTCs) and wholebody metabolic tumor volume(WBMTV) measured by 18 F-fluorodeoxyglucose(FDG) positron emission tomography/computed tomography(PET/CT).The aim was to evaluate the value of the incorporation of CTC number and WBMTV in the prognostic prediction of stage III small-cell lung cancer(SCLC).Methods: One hundred and twenty-nine patients were enrolled in this study.All patients were treated with four cycles of a platinum-based regimen and concurrent chest irradiation,followed by prophylactic cranial irradiation.Blood samples for CTC analysis were obtained from 112 patients before the initiation of chemotherapy(as a baseline),after cycle 1 and after cycle 4.CTCs were measured using the CELLSEARCH? system.The patients underwent pretreatment FDG PET/CT WBMTV,which included all malignant lesions.The Spearman rank test was used to determine the correlation among CTC counts,WBMTV and disease stage.Overall survival(OS) and progression-free survival(PFS) curves were produced using the Kaplan-Meier method,and survival differences between groups were assessed by the log-rank test.Results: The number of CTCs at baseline did not correlate with WBMTV before the initiation of therapy(P=0.241).The number of CTCs at baseline and the WBMTV before the initiation of therapy were independent relevant factors for PFS and OS.The subgroup analysis(Group A: CTC count >19.5 and a WBMTV >266.5cm~3;Group B: CTC count >19.5 and a WBMTV ≤266.5cm~3; Group C: CTC count ≤19.5 and a WBMTV >266.5cm~3;Group D: CTC count ≤19.5 and a WBMTV ≤266.5cm~3) showed that the differences were statistically significant in the median PFS(Group A vs.D,P<0.001; Group B vs.D,P=0.018; Group C vs.D,P=0.029) and in the median OS(Group A vs.D,P<0.001; Group B vs.D,P=0.012).Conclusions: CTC number and WBMTV are related to progression and death in patients with SCLC.The incorporation of CTC number and WBMTV scans can provide a detailed prognostic prediction for SCLC.

Circulating tumor cells isolation:the "post-EpCAM era"

Circulating tumor cells （CTCs） represent a submicroscopic fraction detached from a primary tumor and in transit to a secondary site. The prognostic significance of CTCs in metastatic cancer patients was demonstrated for the first time more than ten years ago. To date, it seems clear enough that CTCs are highly heterogeneous and dynamically change their shape. Thus, the inadequacy of epithelial cell adhesion molecule （EpCAM） as universal marker for CTCs detection seems unquestionable and alternative methods able to recognize a broader spectrum of phenotypes are definitely needed. In this review the pleiotropic functions of EpCAM are discussed in detail and the role of the molecule in the biology of CTCs is critically dissected.

Epithelial-mesenchymal transition status of circulating tumor cells in breast cancer and its clinical relevance

Objective:Circulating tumor cells(CTCs)play a critical role in cancer metastasis,but their prevalence and significance remain unclear.This study attempted to track the epithelial-mesenchymal transition(EMT)status of CTCs in breast cancer patients and investigate their clinical relevance.Methods:In this study,the established negFACS-IF:E/M platform was applied to isolate rare CTCs and characterize their EMT status in breast cancer.A total of 89 breast cancer patients were recruited,including stage 0–III(n=60)and late stage(n=29)cases.Results:Using the negFACS-IF:E/M platform,it was found that in human epidermal growth factor receptor 2(HER2)+patients,mesenchymal CTCs usually exhibited a high percentage of HER2+cells.Stage IV breast cancer patients had considerably more CTCs than stage 0–III patients.Among stage 0–III breast cancers,the HER2 subtype included a significantly higher percentage of mesenchymal and biphenotypic(epithelial and mesenchymal)CTCs than the luminal A or B subtypes.Among stage IV patients,CTCs were predominantly epithelial in cases with local recurrence and were more mesenchymal in cases with distant metastasis.By applying a support vector machine(SVM)algorithm,the EMT status of CTCs could distinguish between breast cancer cases with metastasis/local recurrence and those without recurrence.Conclusions:The negFACS-IF:E/M platform provides a flexible and generally acceptable method for the highly sensitive and specific detection of CTCs and their EMT traits in breast cancer.This study demonstrated that the EMT status of CTCs had high clinical relevance in breast cancer,especially in predicting the distant metastasis or local recurrence of breast cancer.

Survivin-positive circulating tumor cells as a marker for metastasis of hepatocellular carcinoma

BACKGROUND Circulating tumor cells(CTCs)and survivin are indicators for tumor stage and metastasis,as well as epitheliomesenchymal transition,in various cancers,including hepatocellular cancer(HCC).AIM To explore the potential of survivin-positive CTCs,specifically,as a marker for tumor progression in HCC patients.METHODS We examined the survivin expression pattern in CTCs obtained from 179 HCC patients,and investigated the in vitro effects of survivin silencing and overexpression on the proliferation and invasion of HCC cells.CTC count and survivin expression in patient samples were examined using RNA in situ hybridization.RESULTS All 179 patients were positive for CTC markers,and 94.41%of the CTCs were positive for survivin.The CTC and survivin-positive CTC counts were significantly higher in the HCC patients than in the normal controls,and were significantly associated with tumor stage and degree of differentiation.Further,survivin overexpression was found to induce HepG2 cell proliferation,reduce apoptosis,and improve invasive ability.CONCLUSION Survivin shows upregulated expression(indicative of anti-apoptotic effects)in HCC.Thus,survivin-positive CTCs are promising as a predictor of HCC prognosis and metastasis,and their accurate measurement may be useful for the management of this cancer.

Finding the seed of recurrence:Hepatocellular carcinoma circulating tumor cells and their potential to drive the surgical treatment

The treatment for hepatocellular carcinoma(HCC)relies on liver resection,which is,however,burdened by a high rate of recurrence after surgery,up to 60%at 5 years.No pre-operative tools are currently available to assess the recurrence risk tailored to every single patient.Recently liquid biopsy has shown interesting results in diagnosis,prognosis and treatment allocation strategies in other types of cancers,since its ability to identify circulating tumor cells(CTCs)derived from the primary tumor.Those cells were advocated to be responsible for the majority of cases of recurrence and cancer-related deaths for HCC.In fact,after being modified by the epithelial-mesenchymal transition,CTCs circulate as“seeds”in peripheral blood,then reach the target organ as dormant cells which could be subsequently“awakened”and activated,and then initiate metastasis.Their presence may justify the disagreement registered in terms of efficacy of anatomic vs non-anatomic resections,particularly in the case of microvascular invasion,which has been recently pointed as a histological sign of the spread of those cells.Thus,their presence,also in the early stages,may justify the recurrence event also in the contest of liver transplant.Understanding the mechanism behind the tumor progression may allow improving the treatment selection according to the biological patient-based characteristics.Moreover,it may drive the development of novel biological tailored tests which could address a specific patient to neoadjuvant or adjuvant strategies,and in perspective,it could also become a new method to allocate organs for transplantation,according to the risk of relapse after liver transplant.The present paper will describe the most recent evidence on the role of CTCs in determining the relapse of HCC,highlighting their potential clinical implication as novel tumor behavior biomarkers able to influence the surgical choice.

Impact of chronic exposure to bevacizumab on EpCAM-based detection of circulating tumor cells

Background： Circulating tumor cells （CTCs） are often undetected through the immunomagnetic epithelial cell adhesion molecule （EpCAM）-based CellSearch~ System in breast and colorectal cancer （CRC） patients treated with bevacizumab （BEV）, where low CTC numbers have been reported even in patients with evidence of progression of disease. To date, the reasons for this discrepancy have not been clarified. This study was carried out to investigate the molecular and phenotypic changes in CRC cells after chronic exposure to BEV in vitro. Methods： The human CRC cell line WiDr was exposed to a clinically relevant dose of BEV for 3 months in vitro. The expression of epithelial and mesenchymal markers and EpCAM isoforms was determined by western blotting and immunofluorescence. To evaluate the impact of EpCAM variant isoforms expression on CTC enumeration by CellSearch, untreated and treated colon cancer cells were spiked into 7.5 mL of blood from a healthy donor and enumerated by CellSearch. Results： Chronic exposure of CRC cell line to BEV induced decreased expression of EpCAM 40 kDa isoform and increased expression EpCAM 42 kDa isoform, together with a decreased expression of cytokeratins （CK）, while no evidence of epithelial to mesenchymal transition （EMT） in treated cells was observed. The recovery rate of cells through CellSearch was gradually reduced in course of treatment with BEV, being 84% , 70% and 40% at l, 2 and 3 months, respectively. Conclusions： We hypothesize that BEV may prevent CellSearch from capturing CTCs through altering EpCAM isoforms.

Cultured circulating tumor cells and their derived xenografts for personalized oncology

Recent cancer research has demonstrated the existence of circulating tumor cells(CTCs)in cancer patient’s blood.Once identified,CTC biomarkers will be invaluable tools for clinical diagnosis,prognosis and treatment.In this review,we propose ex vivo culture as a rational strategy for large scale amplification of the limited numbers of CTCs from a patient sample,to derive enough CTCs for accurate and reproducible characterization of the biophysical,biochemical,gene expressional and behavioral properties of the harvested cells.Because of tumor cell heterogeneity,it is important to amplify all the CTCs in a blood sample for a comprehensive understanding of their role in cancer metastasis.By analyzing critical steps and technical issues in ex vivo CTC culture,we developed a cost-effective and reproducible protocol directly culturing whole peripheral blood mononuclear cells,relying on an assumed survival advantage in CTCs and CTC-like cells over the normal cells to amplify this specified cluster of cancer cells.