Real-time monitoring of rare circulating hepatocellular carcinoma cells in an orthotopic model by in vivo flow cytometry assesses resection on metastasis

The fate of circulating tumor cells (CTC) is an important determinant of metastasis and recurrence, which leads to most deaths in hepatocellular carcinoma (HCC). Therefore, quantification of CTCs proves to be an emerging tool for diagnosing, stratifying, and monitoring patients with metastatic diseases. In vivo flow cytometry has the capability to monitor the dynamics of fluorescently labeled CTCs continuously and noninvasively. Here, we combine in vivo flow cytometry technique and a GFP-transfected HCC orthotopic metastatic tumor model to monitor CTC dynamics. Our in vivo flow cytometry has approximately 1.8-fold higher sensitivity than whole blood analysis by conventional flow cytometry. We found a significant difference in CTC dynamics between orthotopic and subcutaneous tumor models. We also investigated whether liver resection promotes or restricts hematogenous metastasis in advanced HCC. Our results show that the number of CTCs and early metastases decreases significantly after the resection. The resection prominently restricts hematogenous metastasis and distant metastases. CTC dynamics is correlated with tumor growth in our orthotopic tumor model. The number and size of distant metastases correspond to CTC dynamics. The novel in vivo flow cytometry technique combined with orthotopic tumor models might provide insights to tumor hematogenous metastasis and guidance to cancer therapy.     

Circulating tumor cells in pancreatic cancer patients: Methods of detection and clinical implications

The poor prognosis of pancreatic cancer patients is associated with the frequent and early dissemination of the disease, as well as late detection due to unspecific and late symptoms from the primary tumor. Pancreatic cancers frequently spread to the liver, lung and skeletal system, suggesting that pancreatic tumor cells must be able to intravasate and travel through the circulation to distant organs. Circulating tumor cells (CTCs) are tumor cells that have acquired the ability to enter the circulatory system; this cell population is ultimately responsible for the development of metastases in distant organs. Clinical studies have revealed that the presence of CTCs in blood is correlated with disease progression for other cancers, such as breast, colorectal and prostate cancer. However, as CTCs are extremely rare, both enrichment and sensitive methods of detection are required for their enumeration. This review highlights various enrichment procedures and methods for the detection of CTCs. Furthermore, we systematically review previously reported studies of the clinical relevance of CTC detection in pancreatic cancer patients. There is evidence that the presence of CTCs also correlates with an unfavorable outcome in pancreatic cancer patients. However, technical/methodological issues may explain why some studies only show a trend toward an association between CTC detection and disease progression. Larger studies, as well as characterization of the CTC population, are required to achieve further insight into the clinical implications of CTC detection in pancreatic cancer patients.     

Clinical significance of circulating tumor cells in breast cancer patients

Circulating tumor cells (CTCs) are defined as tumor cells circulating in the peripheral blood of patients, shed from either the primary tumors or its metastases. Many techniques have been developed in the recent years to identify CTCs in breast cancer patients, and trials have proved the prognostic significance of CTCs. In this study, we validated the CTC detection method of combining cell filtration and laser scanning cytometry (LSC), which was highly reproducible with increased sensitivity and accuracy. In 134 non-metastatic breast cancer patients analyzed, HER2 was found to be the only primary tumor characteristics that correlated with the presence of CTCs. 85 patients with definitive stage information were selected for association study between the disease stages and CTC numbers. The detection rate and the number of CTCs were correlated with the disease stages. Moreover, assessment of CTCs in 92 metastatic breast cancer patients was found to be able to predict the efficacy of chemotherapy. Increase in CTC numbers was an independent prognostic factor for treatment outcomes. Our results suggested that CTC assessment could be an indication of the disease progression and analysis of the properties of CTCs is likely to provide new insights into the biology of breast cancer and contribute to defining novel treatments and better prediction of clinical outcomes.     

Microfluidic chip for isolation of viable circulating tumor cells of hepatocellular carcinoma for their culture and drug sensitivity assay

Circulating tumor cells (CTCs) have been proposed to be an active source of metastasis or recurrence of hepatocellular carcinoma (HCC). The enumeration and characterization of CTCs has important clinical significance in recurrence prediction and treatment monitoring in HCC patients. We previously developed a unique method to separate HCC CTCs based on the interaction of the asialoglycoprotein receptor (ASGPR) expressed on their membranes with its ligand. The current study applied the ligand-receptor binding assay to a CTC-chip in a microfluidic device. Efficient capture of HCC CTCs originates from the small dimensions of microfluidic channels and enhanced local topographic interactions between the microfluidic channel and extracellular extensions. With the optimized conditions, a capture yield reached > 85% for artificial CTC blood samples. Clinical utility of the system was further validated. CTCs were detected in all the examined 36 patients with HCC, with an average of 14 ± 10/2 mL. On the contrary, no CTCs were detected in healthy, benign liver disease or non-HCC cancer subjects. The current study also successfully demonstrated that the captured CTCs on our CTC-chip were readily released with ethylene diamine tetraacetic acid (EDTA); released CTCs remained alive and could be expanded to form a spheroid-like structure in a 3-dimensional cell culture assay; furthermore, sensitivity of released CTCs to chemotherapeutic agents (sorafenib or oxaliplatin) could be effectively tested utilizing this culture assay. In conclusion, the methodologies presented here offer great promise for accurate enumeration and easy release of captured CTCs, and released CTCs could be cultured for further functional studies.     

肿瘤循环细胞检测预测肝癌术后复发的临床研究

目的:研究检测肝癌循环细胞对于预测肝癌术后复发的临床价值,以期为临床诊治提供一定的参考。方法:选取2012年1月-2013年1月入院行手术切除的原发性肝癌患者93例,应用CanPatrol 方法分别检测术前、术后外周血中的肝癌循环细胞（CTC）,分析手术前后CTC阳性病例数、CTC计数结果。手术后随访1~2年,观察术后复发情况,并分析术后CTC检测结果与术后复发的相关性。结果:术前CTC阳性39例,阳性率为41.94%,术后CTC阳性59例,阳性率为63.44%。手术前后CTC阳性计数范围为1~29个,术前阳性CTC计数集中分布于1~4个,术后则为1~7个。93例肝癌患者随访期间共复发27例,以CTC手术前后均阴性组的患者复发率最低,为14.29%,CTC手术前后均阳性组的复发率最高,为46.15%;CTC术后阴性组的复发率为17.65%,CTC术后阳性组的复发率为35.59%。经单因素分析,肿瘤数目、肝硬化、微血管侵犯、AFP mRNA与CTC检测是影响肝癌患者术后复发的因素。经多因素Logistic分析,CTC检测阳性与多发肿瘤、合并肝硬化、微血管侵犯、AFP mRNA阳性为肝癌患者术后复发的危险因素。结论:肝癌术后CTC检测阳性的患者复发率显著高于阴性患者,肿瘤数目、肝硬化、微血管侵犯、AFP mRNA与CTC检测是影响肝癌患者术后复发的因素。     

循环肿瘤细胞检测技术的研究进展

循环肿瘤细胞（circulating tumor cells,CTCs）是导致发生肿瘤远处转移和复发的必要前提。随着靶向治疗的不断进步,对于无法取得肿瘤组织的晚期癌症患者,CTCs可作为一种肿瘤组织替代物指导患者治疗方案的选择。精确计数以及分子生物学分析对于肿瘤患者的预后判断、疗效评价以及个体化治疗均有重要的指导作用。随着CTCs检测技术不断发展,CTCs检测的准确性明显提高。本文针对CTCs的检测技术进行综述。      

循环肿瘤细胞在肝癌中的研究及应用进展

肝细胞癌（hepatocellular carcinoma，HCC）是全球范围内最常见的恶性肿瘤之一，发病率及死亡率高，预后差。影响预后的主要原因为早期诊断困难及术后易复发、转移。因此，HCC的早期诊断及术后监测对改善HCC患者的总体预后尤为重要。循环肿瘤细胞（circulating tumor cell，CTC）是存在于血液循环中的恶性肿瘤细胞，在肿瘤的侵袭、转移中发挥重要作用。CTC检测作为“液体活检”技术，能够实时重复监测外周血中的肿瘤细胞，在肿瘤早期诊断、术后动态监测及预后评价等方面具有巨大的临床应用价值。本文回顾了国内外CTC研究进展，对其检测方法及在HCC患者诊断、预后等方面的研究及临床应用等进行系统综述。      

非小细胞肺癌循环肿瘤细胞检测进展

肺癌是世界范围内发病率最高的恶性肿瘤, 约有18%的癌症相关死亡与肺癌有关。循环肿瘤细胞(CTCs)的出现可看做是肿瘤细胞播散入血的直接体现, 而这正是导致非小细胞肺癌患者预后较差的最重要因素之一。检测外周血中循环肿瘤细胞有望成为诊断肺癌的辅助方法, 使肺癌患者得到最佳的个体化治疗。研究者们已尝试多种不同的方法从外周血中分离CTCs。虽然有关CTCs检测的研究众多, 但不同检测方法的敏感度、特异性及重复性限制了其临床意义, 本文就现有的各项技术进行综述。      

Circulating Tumor Cells and Colorectal Cancer

The significance of circulating tumor cells (CTCs) has been discussed for more than a century. The advent of modern technology has allowed for more reliable detection of CTCs, and recent studies have provided compelling evidence that CTCs predict clinical response in metastatic colorectal cancer (mCRC). Combination of CTC analysis with independent prognostic factors has demonstrated powerful synergy in some studies. The ability of CTCs to predict metastasis and therapy-specific response has high potential clinical utility, with early studies showing promising results in colorectal cancer (CRC). Reliable CTC detection has also allowed for examination of tumor cell dissemination during surgery, and there appears to be a heavy dependence on the approach chosen. This review discusses the evidence for CTC significance, with particular focus on detection methods, novel markers, and clinical outcomes in CRC. Numerous opportunities exist for preclinical, clinical, and translational studies to explore molecular determinants within CTCs, as well as the value of CTCs in directing targeted therapeutics.     

Significance of circulating tumor cells in the portal vein regarding metastases and vascular invasion in hepatocellular carcinoma patients

BackgroundVascular invasion is an important risk factor of poor prognosis in hepatocellular carcinoma (HCC) patients. The detection of circulating tumor cells (CTCs) in the blood is direct evidence of tumor presence. There are few reports on CTCs and metastasis and vascular invasion of HCC. The purpose of this study was to analyze the significance of CTCs in the portal vein regarding metastases and vascular invasion in HCC patients.MethodsA total of 104 HCC patients diagnosed and treated in Zhengzhou University People’s Hospital were enrolled. Surgery was performed in 60 individuals. Portal vein blood samples were collected before treatment for CTCs detection. We used the isolation by size of epithelial tumor cells (ISET) and fluorescence in situ hybridization (FISH) to enrich and classify CTCs from blood samples. The patients were divided into metastasis and nonmetastasis groups according to the metastasis status before treatment. Differences in clinical indicators such as alpha-fetoprotein (AFP) levels, tumor size, CTCs count, and macrovascular tumor thrombus between the two groups were analyzed as well as the associations of CTCs count with the above indicators. For individuals with postoperative pathology, the relationship between CTCs counts and microvascular invasion (MVI) was analyzed.ResultsThe amounts of portal vein CTCs were higher in patients with metastases compared with the nonmetastases group (20 vs. 7; z=3.795; P<0.001). Multivariate logistic regression analysis showed that the CTC count was a risk factor for HCC metastasis [odds ratio (OR) =1.044; 95% CI: 1.011–1.079]. The sensitivity and specificity of CTC count in predicting HCC metastasis were 82.93% and 52.38%, respectively. CTC count was significantly correlated with tumor size (r_s=0.308; P=0.001), vascular invasion (z=4.211; P<0.001), and MVI (z=12.763; P=0.002). A threshold CTC count of seven showed the most significant power for predicting metastasis.ConclusionsVascular invasion positivity was closely related to HCC metastasis. Portal vein CTC count before treatment was correlated with vascular invasion and could be considered one of the factors affecting HCC metastasis. However, the ability of CTC count was limited in predicting HCC metastasis due to insufficient specificity.     

Expected clinical applications of circulating tumor cells in breast cancer

Tumor cell invasion and intravascular filtration lead to the presence of circulating tumor cells (CTCs) in peripheral blood. CTCs have, thus, been counted in patients with cancer to analyze metastatic mechanisms or in the hope of developing clinical applications for diagnosis and therapy; various CTC-related studies have been performed. However, the clinical significance of CTCs remains to be established because of the extremely small number of CTCs in peripheral blood as compared with the number of blood cells. Technical problems (e.g. reproducibility and reliability) in the detection of CTCs also remain to be solved. The use of flow cytometric analysis, which can be performed with tumor-cell markers such as anti-epithelial cell adhesion molecule antibodies and anti-cytokeratin antibodies and non-tumor-cell markers such as anti-CD45 antibodies has enhanced specificity for the detection of tumor cells. The CellSearch System^® can detect 1 CTC in 7.5 mL of peripheral blood, with high reproducibility. Its detection rate and accuracy for CTCs have been confirmed. In the United States, clinical trials have used this system to detect CTCs in patients with metastatic breast cancer, metastatic colorectal cancer, and metastatic prostate cancer, and CTCs have been confirmed to be a useful prognostic factor. This system was also suggested to be useful for monitoring treatment response in patients with metastatic breast cancer and was approved by the United States Food and Drug Administration in 2004. Measuring CTC counts can facilitate the early prediction of treatment response and thereby avoid unnecessary therapy. CTCs may also be a useful biomarker for molecular targeted agents, enabling the identification of patients most likely to respond to a given treatment and facilitating treatment selection. However, the widespread use of CTC monitoring as a routine examination requires a further improvement in measurement sensitivity, the establishment of criteria for quantitative and qualitative evaluations, and additional clear-cut evidence supporting the clinical significance of CTCs. We expect that CTCs will be established to be a new diagnostic and therapeutic index for breast cancer.     

Detection and enumeration of circulating tumor cells based on their invasive property

Circulating tumor cells (CTCs) are in limited numbers and heterogeneous, making their detection, isolation, and enumeration a major challenge. To overcome these difficulties, we developed a novel method to detect and enumerate CTCs with invasive property. Our assay consists of three simple steps: enrichment, Matrigel invasion assay, and immunostaining. We have validated this method using mouse xenograft tumor models and confirmed its utility in human cancer patients. Our method does not require special equipment and antigen expression for CTC selection, is less likely to be affected by the heterogeneity of the CTCs, and could be applicable to virtually all cancers. Most important, our method enumerates invasive CTCs, which may allow more accurate correlations with clinical outcome and treatment response compared with other CTC detection methods.     

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 ...     

Clinico-pathological correlation of serial measurement of circulating tumor cells in 24 metastatic colorectal cancer patients receiving chemotherapy reveals interpatient heterogeneity correlated with CEA levels but independent of KRAS and BRAF mutation

Background: The Veridex CellSearch is an FDA-approved technology for enumerating circulating tumor cells in blood samples of metastatic colorectal cancer mCRC) patients and has prognostic value. It is important to understand how counts of circulating tumor cells (CTCs), which are advocated to be tools for “liquid biopsy” of tumors, correlate with clinical and pathologic variables of significance in these patients. In this study, we have attempted to make such correlations along with evaluating how CTC counts change during the course of chemotherapy. Patients and methods: Following an IRB-approved protocol, blood samples were collected from 24 patients with mCRC along with relevant clinico-pathological data. Blood was collected at defined time-points both prior to as well as during the course of treatment with combination chemotherapy, and CTC counts were enumerated from7.5 ml of blood. Results: Seventeen out of 24 patients with mCRC showed a CTC count of 2 or less cells in 7.5 ml of blood at base-line assessment before chemotherapy while 7 patients showed 3 or more cells in 7.5 ml of blood at that point. A correlation was found between high carcino-embryonic antigen (CEA) levels and high CTC counts (P = 0.018) although it was also found that some patients had elevated CTCs without an elevated CEA. No correlation with the time interval between detection of primary tumor and appearance of secondary (metastatic) tumor(s) was found. CTC counts did not correlate with the presence of lung or liver metastases, i.e. a number of mCRC patients with lung or liver metastases had a count of zero CTCs at baseline. We also noted no correlation between CTC number and the status of KRAS or BRAF mutation. CTC counts dropped immediately after the start of chemotherapy in 11 out of 21 patients, and also reduced from the baseline at the end of chemotherapy in 5 out of 10 patients. Six of 7 patients who started with 3 or more CTCs in 7.5 ml at baseline also showed a final CTC reduction at the end of the therapy assessment. Conclusions: Analysis of circulating tumor cells may be of use in monitoring response to therapy in mCRC, either in combination with CEA monitoring or alone when CTCs are elevated but CEA level is not.     

Secondary Circulating Tumor Cells (CTCs) but not Primary CTCs are Associated with the Clinico-Pathological Parameters in Chilean Patients With Colo-Rectal Cancer

Background: The aim of this study was to assess detection of circulating tumor cells (CTC) using anti-CEApre and post surgery in Chilean patients with colo-rectal cancer. Materials and Methods: The presence of CTCswas evaluated in 80 colorectal cancer patients pre and post surgery using standard immunocytochemistry andthe results were compared with findings for standard clinico-pathological parameters. Results: In patients presurgeryCEA (+) CTCs were frequently found, with no relation to tumor size or nodal status. After surgery, thepresence of CTCs was associated with such clinico-pathological parameters. The frequency of CTC detection innode positive patients did not change after surgery. In patients with metastasis there was also no change in thefrequency of CTC detection, and clusters of 3 or more CTCs were evident. Conclusions: Secondary CTCs areassociated with clinico-pathological parameters only after surgical removal of the primary tumor, and mightbe important in identifying patients at high risk of relapse. Primary CTCs detected before surgical removal arefrequently found, are not associated with the clinico-pathological parameters and might have a role in cancerscreening. These findings suggest the need for studies with a larger population of patients.     

Detection of circulating tumor cells is improved by drug-induced antigen up-regulation: preclinical and clinical studies

(51)Cr-prelabelled colon cancer cells (simulating 'circulating tumor cells', CTCs) were added to human peripheral blood and exposed to staurosporine (ST) to increase carcinoembryonic antigen (CEA) expression. CTCs were captured with immunomagnetic beads coated with Ber-EP4 monoclonal antibody, recognizing the common epithelial antigen present in the majority of cancer cells of epithelial origin, with capture efficiency of more than 80%. Moreover, ST treatment increased CEA expression without compromising Ber-EP4 capture efficiency. In a pilot clinical study on 37 patients, CTCs were captured using Ber-EP4 beads, and recognized by RT-PCR set for CEA or cytokeratin-19 (CK) mRNA detection. The results showed that: (a) the percentage of CEA-positive CTCs (CTC(CEA), 54.1%) was lower than that of CK-positive CTCs (CTC(CK), 70.3%); (b) in vitro ST treatment converted a significant number of CTC(CEA)-negative into CTC(CEA)-positive cases. Therefore, immunomagnetic capture combined with exposure to ST provides a feasible and sensitive technique for the detection of functionally-active CTCs responsive to ST-mediated CEA up-regulation.     

循环肿瘤细胞检测在结直肠癌临床中的应用

结直肠癌是常见的恶性肿瘤之一,其复发和转移是导致患者死亡的主要原因。外周血循环肿瘤细胞（circulating tumor cells,CTCs）是导致结直肠癌患者复发转移的关键性因素之一,在结直肠癌患者复发转移的监测、预后、病情评估以及治疗等临床问题中有重要的应用价值,已成为近年来研究的热点。本文从CTCs的分离富集、检测技术及在结直肠癌患者中的临床应用价值等几个方面,全面阐述了CTCs检测在结直肠癌临床应用中的研究进展。     

Early Detection of Recurrence in Patients With Locally Advanced Non–Small-Cell Lung Cancer via Circulating Tumor Cell Analysis

BackgroundAssays to identify circulating tumor cells (CTCs) might allow for noninvasive and sequential monitoring of lung cancer. We investigated whether serial CTC analysis could complement conventional imaging for detecting recurrences after treatment in patients with locally advanced non–small-cell lung cancer (LA-NSCLC).Patients and MethodsPatients with LA-NSCLC (stage II-III) who definitively received concurrent chemoradiation were prospectively enrolled, with CTCs from peripheral blood samples identified using an adenoviral probe that detects elevated telomerase activity present in nearly all lung cancer cells. A “detectable” CTC level was defined as 1.3 green flourescent protein-positive cells per milliliter of collected blood. Samples were obtained before, during (at weeks 2, 4, and 6), and after treatment (post-radiation therapy [RT]; at months 1, 3, 6, 12, 18, and 24).ResultsForty-eight patients were enrolled. At a median follow-up of 10.9 months, 22 (46%) patients had disease recurrence at a median time of 7.6 months post-RT (range, 1.3-32.0 months). Of the 20 of 22 patients for whom post-RT samples were obtained, 15 (75%) had an increase in CTC counts post-RT. In 10 of these 15 patients, CTCs were undetectable on initial post-RT draw but were then detected again before radiographic detection of recurrence, with a median lead time of 6.2 months and mean lead time of 6.1 months (range, 0.1-12.0 months) between CTC count increase and radiographic evidence of recurrence. One patient with an early recurrence (4.7 months) had persistently elevated detectable CTC levels during and after treatment.ConclusionThese results indicate that longitudinal CTC monitoring in patients with LA-NSCLC treated with chemoradiation is feasible, and that detectable CTC levels in many patients meaningfully precede radiologic evidence of disease recurrence.     

Prognostic value of circulating tumor cells in nonmuscle invasive bladder cancer: a CellSearch analysis

BackgroundCirculating tumor cells (CTCs) provide prognostic information in patients with metastatic tumors. Recent studies have shown that CTCs are released in circulation in an early phase of cancer disease so that their presence is under investigation in the adjuvant setting. Few studies investigated the prognostic significance of CTCs enumeration in patients with metastatic and advanced bladder cancer. The current study has analyzed the presence of CTC in patients with nonmuscle-invasive bladder cancer (NMIBC).Patients and methodsForty-four NMIBC patients were enrolled and included in a 24-month follow-up program. Blood drawings were carried out in all patients at the first diagnosis. CellSearch system (Veridex; LLC, Raritan, NJ) was used for CTCs enumeration.ResultsCTC were detectable in 8/44 patients (18%). Presence of CTC was found significantly associated to shorter time to first recurrence (6.5 versus 21.7 months, P < 0.001). Median time to progression was not reached, due to the short follow-up period. CTC presence was found associated to concomitant carcinoma in situ and higher T category.ConclusionThe detection of CTC in this setting of disease may allow to distinguish patients with high risk of recurrence from those with high risk of progression, as well as to early identify patients candidate for adjuvant treatment.