Epithelial cell adhesion molecule-positive (EpCAM 1 ) hepatocellular carcinoma (HCC) cells may constitute a tumor-initiating subpopulation in tumorigenic cell lines and HCC specimens. In the present study, EpCAM 1 circulating tumor cells (CTCs) were identified prospectively in HCC patients undergoing curative resection, and the prognostic significance and their stem cell-like characteristics were investigated further. Blood samples from 123 HCC patients were tested prior to resection and 1 month thereafter. CTCs were present in 66.67% of patients, and the cell count measured in 7.5 mL of blood (CTC 7.5 ) ranged between 1 and 34. Fifty-one patients had CTC 7.5 of !2 preoperatively, and these patients developed tumor recurrence earlier than those with CTC 7.5 of <2 CTCs (P < 0.001). A preoperative CTC 7.5 of !2 was an independent prognostic factor for tumor recurrence (P < 0.001). Its prognostic significance also applied to patients with alpha-fetoprotein (AFP) levels of 400 ng/mL or subgroups with low recurrence risk (all P < 0.05). A significant decrease of CTC-positive rates (66.67% to 28.15%, P < 0.05) and CTC 7.5 values (2.60 6 0.43 to 1.00 6 0.36, P < 0.05) was observed 1 month after resection. Patients with consistent CTC 7.5 <2 had lower recurrence rates than those with values consistently !2 (15.5% versus 87.50%, P < 0.001). EpCAM 1 CTCs displayed cancer stem cell biomarkers (CD133 and ABCG2), epithelial-mesenchymal transition, Wnt pathway activation, high tumorigenic potential, and low apoptotic propensity. Conclusion: Stem cell-like phenotypes are observed in EpCAM 1 CTCs, and a preoperative CTC 7.5 of !2 is a novel predictor for tumor recurrence in HCC patients after surgery, especially in patient subgroups with AFP levels of 400 ng/mL or low tumor recurrence risk. EpCAM 1 CTCs may serve as a real-time parameter for monitoring treatment response and a therapeutic target in HCC recurrence. (HEPATOLOGY 2013;57:1458-1468 H epatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide, and associated morbidity and mortality rates have escalated in recent years. 1 Despite improvements in surveillance and clinical treatment strategies, the prognosis of HCC remains very poor due to high incidence of recurrence and metastasis. 2 Traditional clinicopathological parameters such as tumor morphology,

EGFR T790M mutation occurs in half of non-small cell lung cancer (NSCLC) patients with acquired EGFR-TKI (TKI) resistance, based on tumor re-biopsies using an invasive clinical procedure. Here, we dynamically monitored T790M mutation in circulating tumor DNA (ctDNA) using serial plasma samples from NSCLC patients receiving TKI through Droplet Digital PCR (ddPCR) method and the associations between overall survival (OS) starting from initial TKI treatment and the T790M ctDNA status detected in plasma were analyzed. Among 318 patients, 117 who acquired TKI resistance were eligible for the analysis. T790M ctDNA was detected in the plasma of 55/117 (47%) patients. Almost half of the T790M ctDNA positive patients were identified at a median time of 2.2 months prior to clinically progressive disease (PD). Furthermore, within the patients receiving TKI treatment at 2nd line or later, the T790M ctDNA positive group had significantly shorter OS than the negative group (median OS: 26.9 months versus NA, P = 0.0489). Our study demonstrates the feasibility of monitoring EGFR mutation dynamics in serial plasma samples from NSCLC patients receiving TKI therapy. T790M ctDNA can be detected in plasma before and after PD as a poor prognostic factor.

Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA from lung cancer patients is an emerging clinical tool. However, compared with tissue testing, the sensitivity of plasma testing is not yet satisfactory because of the highly fragmented nature of plasma cell-free DNA, low fraction of tumor DNA, and limitations of available detection technologies. We therefore developed a highly sensitive and specific droplet digital PCR method for plasma EGFR mutation (exon19 deletions and L858R) testing. Plasma from 86 EGFR-tyrosine kinase inhibitor-naive lung cancer patients was tested and compared with EGFR mutation status of matched tumor tissues tested by amplification refractory mutation system. By using EGFR mutation-positive cell DNA, we optimized the droplet digital PCR assays to reach 0.04% sensitivity. The plasma testing sensitivity and specificity, compared with the matched tumor tissues tested by amplification refractory mutation system, were 81.82% (95% CI, 59.72%-94.81%) and 98.44% (95% CI, 91.60%-99.96%), respectively, for exon19 deletions, with 94.19% concordance rate (κ = 0.840; 95% CI, 0.704-0.976; P < 0.0001), whereas they were 80.00% (95% CI, 51.91%-95.67%) and 95.77% (95% CI, 88.14%-99.12%), respectively, for L858R, with 93.02% concordance rate (κ = 0.758; 95% CI, 0.571-0.945; P < 0.0001). The reported highly sensitive and specific droplet digital PCR assays for EGFR mutation detection have potential in clinical blood testing.

Background Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide because of rapid progression and high incidence of metastasis or recurrence. Accumulating evidence shows that CD73-expressing tumor cell is implicated in development of several types of cancer. However, the role of CD73 in HCC cell has not been systematically investigated and its underlying mechanism remains elusive. Methods CD73 expression in HCC cell was determined by RT-PCR, Western blot, and immunohistochemistry staining. Clinical significance of CD73 was evaluated by Cox regression analysis. Cell counting kit-8 and colony formation assays were used for proliferation evaluation. Transwell assays were used for motility evaluations. Co-immunoprecipitation, cytosolic and plasma membrane fractionation separation, and ELISA were applied for evaluating membrane localization of P110β and its catalytic activity. NOD/SCID/γc(null) (NOG) mice model was used to investigate the in vivo functions of CD73. Results In the present study, we demonstrate that CD73 was crucial for epithelial-mesenchymal transition (EMT), progression and metastasis in HCC. CD73 expression is increased in HCC cells and correlated with aggressive clinicopathological characteristics. Clinically, CD73 is identified as an independent poor prognostic indicator for both time to recurrence and overall survival. CD73 knockdown dramatically inhibits HCC cells proliferation, migration, invasion, and EMT in vitro and hinders tumor growth and metastasis in vivo. Opposite results could be observed when CD73 is overexpressed. Mechanistically, adenosine produced by CD73 binds to adenosine A2A receptor (A2AR) and activates Rap1, which recruits P110β to the plasma membrane and triggers PIP3 production, thereby promoting AKT phosphorylation in HCC cells. Notably, a combination of anti-CD73 and anti-A2AR achieves synergistic depression effects on HCC growth and metastasis than single agent alone. Conclusions CD73 promotes progression and metastasis through activating PI3K/AKT signaling, indicating a novel prognostic biomarker for HCC. Our data demonstrate the importance of CD73 in HCC in addition to its immunosuppressive functions and revealed that co-targeting CD73 and A2AR strategy may be a promising novel therapeutic strategy for future HCC management. Electronic supplementary material The online version of this article (10.1186/s13045-019-0724-7) contains supplementary material, which is available to authorized users.

This review addresses the majority of assays that have been published thus far, including the enrichment and detection steps and the markers used in these assays, accompanied by some biological issues of CTC and the results of clinical application harvested.

Purpose: The spatial heterogeneity of phenotypic and molecular characteristics of CTCs within the circulatory system remains unclear. Herein, we mapped the distribution and characterized biological features of CTCs along the transportation route in hepatocellular carcinoma (HCC).Experimental Design: In 73 localized HCC patients, blood was drawn from peripheral vein (PV), peripheral artery (PA), hepatic veins (HV), infrahepatic inferior vena cava (IHIVC), and portal vein (PoV) before tumor resection. Epithelial and mesenchymal transition (EMT) phenotype in CTCs were analyzed by a 4-channel immunofluorescence CellSearch assay and microfluidic quantitative RT-PCR. The clinical significance of CTCs from different vascular sites was evaluated.Results: The CTC number and size gradient between tumor efferent vessels and postpulmonary peripheral vessels was marked. Tracking the fate of CTC clusters revealed that CTCs displayed an aggregated-singular-aggregated manner of spreading. Single-cell characterization demonstrated that EMT status of CTCs was heterogeneous across different vascular compartments. CTCs were predominantly epithelial at release, but switched to EMT-activated phenotype during hematogeneous transit via Smad2 and b-catenin related signaling pathways. EMT activation in primary tumor correlated with total CTC number at HV, rather than epithelial or EMT-activated subsets of CTCs. Follow-up analysis suggested that CTC and circulating tumor microemboli burden in hepatic veins and peripheral circulation prognosticated postoperative lung metastasis and intrahepatic recurrence, respectively.Conclusions: The current data suggested that a profound spatial heterogeneity in cellular distribution and biological features existed among CTCs during circulation. Multivascular measurement of CTCs could help to reveal novel mechanisms of metastasis and facilitate prediction of postoperative relapse or metastasis pattern in HCC.

Purpose: This study aimed to construct a novel platform for the detection of circulating tumor cells (CTC) in patients with hepatocellular carcinoma (HCC) and to investigate the clinical significance of epithelial cell adhesion molecule mRNA-positive (EpCAM mRNAþ ) CTCs using this platform.Experimental Design: An optimized platform for CTC detection was constructed by evaluating different negative enrichment, mRNA isolation, and cDNA synthesis procedures and compared with the CellSearch system. A total of 299 patients with HCC were recruited into this prospective study; of these, 157 who received curative resection, 76 who received transcatheter arterial chemoembolization (TACE), and 66 who received radiotherapy were tested using our platform. The diagnostic value of EpCAM mRNAþ CTCs was investigated in 122 patients with HCC who underwent resection and 120 control subjects.Results: The optimized negative enrichment and quantitative real-time PCR (qRT-PCR)-based CTC detection platform had high sensitivity, specificity, and reproducibility and a low sample volume requirement. This platform showed a potential diagnostic value in patients with HCC and exhibited 76.7% consistency with the CellSearch system (r ¼ 0.54, P < 0.050). Pretreatment CTC level showed prognostic significance in patients with HCC treated with resection, TACE, and radiotherapy (all P < 0.050). Most of the patients showed a decrease in CTC levels after treatment that reflected tumor response. In contrast, patients with an increased CTC level showed disease progression after treatment.Conclusions: We established an optimized platform based on negative enrichment and qRT-PCR for highly sensitive, specific, and reproducible CTC detection. This platform might be clinically useful in auxiliary diagnosis, treatment response assessment, and early decision-making to tailor the most effective antitumor strategies. Clin Cancer Res; 20(18); 4794-805. Ó2014 AACR.

In the present study, we assessed the clinical value of circulating tumor cells (CTC) with stem-like phenotypes for diagnosis, prognosis, and surveillance in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) by an optimized qPCR-based detection platform. Differing subsets of CTCs were investigated, and a multimarker diagnostic CTC panel was constructed in a multicenter patient study with independent validation (total = 1,006), including healthy individuals and patients with chronic hepatitis B infection (CHB), liver cirrhosis (LC), benign hepatic lesion (BHL), and HBV-related HCC, with area under the receiver operating characteristic curve (AUC-ROC) reflecting diagnostic accuracy. The role of the CTC panel in treatment response surveillance and its prognostic significance were further investigated. The AUC of the CTC panel was 0.88 in the training set [sensitivity = 72.5%, specificity = 95.0%, positive predictive value (PPV) = 92.4, negative predictive value (NPV) = 77.8] and 0.93 in the validation set (sensitivity = 82.1%, specificity = 94.2%, PPV = 89.9, NPV = 89.3). This panel performed equally well in detecting early-stage and α-fetoprotein-negative HCC, as well as differentiating HCC from CHB, LC, and BHL. The CTC load was decreased significantly after tumor resection, and patients with persistently high CTC load showed a propensity of tumor recurrence after surgery. The prognostic significance of the CTC panel in predicting tumor recurrence was further confirmed [training: HR = 2.692; 95% confidence interval (CI), 1.617-4.483; < 0.001; and validation: HR = 3.127; 95% CI, 1.360-7.190; = 0.007]. Our CTC panel showed high sensitivity and specificity in HCC diagnosis and could be a real-time parameter for risk prediction and treatment monitoring, enabling early decision-making to tailor effective antitumor strategies. .