1Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan.
2Department of Gastroenterological Surgery, Esophageal Cancer Division, Cancer Institute Hospital of Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan.
Correspondence Address: Dr. Hideo Baba, Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan. E-mail: firstname.lastname@example.org
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Circulating tumor cells (CTCs) are originated from the primary tumor lesion into the blood stream. CTCs could lead to recurrence of gastrointestinal (GI) cancers, even after a curative resection and colonizing in the distant organs to facilitate tumor distant metastasis; however, it has been challenging in clinic to detect CTCs for a long time, such as detection methodology or molecular markers for identification of CTCs. This review discussed the recent technical advances and biomarkers in the detection of CTCs and the molecular mechanism of CTC in cancer progression and metastasis. Moreover, novel concepts, such as cancer stem cells and epithelial-mesenchymal transition, could lead to CTCs and tumor progression and metastasis. Nevertheless, the involvement of CTCs varies greatly among cancer types in the GI and much remains to be learned. Thus, further study will provide more insightful information from a clinical and translational viewpoint to use CTCs for cancer early diagnosis or prediction of tumor recurrence and investigation of tumor progression and metastasis as well.
Cancer stem cells, circulating tumor cells, epithelial-mesenchymal transition, gastrointestinal cancer, tumor progression and metastasis
Tumor recurrence often occurs in patients with gastrointestinal (GI) cancers, even after a curative resection, which may be because undetectable tumor cells depositor enter into the blood stream at the time of operation. In some cases, tumor recurs despite adjuvant chemotherapy after curative surgery suggesting that chemotherapy failed to eradicate all cancer cells that persist after curative surgery. Thus, tumor cells could be disseminated before surgery. The concept of the circulating tumor cells (CTCs) has been, therefore, established and indicates that tumor cells are in blood stream, which will facilitate tumor progression and metastasis although detection of CTCs in peripheral blood was described more than a century ago. Recent advance on research of CTCs largely contributed to diagnosis and treatment of GI cancers. However, the clinical relevance of CTC detection in GI cancers is still the subject of controversies, and their biology is poorly understood.
Indeed, detection of CTCs becomes a promising means to early diagnosis and prediction of prognosis and tumor recurrence for several types of human cancer.[2-5] Furthermore, the study of CTCs could also elucidate the molecular biological profile of CTC and lead to better understanding of cancer metastasis. To date, standard procedures of CTC detection have to be established, and the clinical relevance should be conﬁrmed by a large-scale clinical study. In this review, we updated and discussed recent progress regarding CTCs in GI cancer. These new data could improve our understanding of the mechanisms of cancer progression and metastasis as well as therapy resistance. This information may also lead to the development of novel clinical targets and improve the clinical management of GI cancer.
In general, methodology in the detection of CTCs consists of two steps, that is, enrichment and detection process. The enrichment process is required because of the rarity of CTCs in peripheral circulation (one CTC per 1 × 106 to 1 × 107 mononuclear cells). To enrich CTCs from blood mononuclear cells, density gradient centrifugation (Ficoll-Hypaque or OncoQuick separation), immunomagnetic or size ﬁltration procedures are used.[6,7] After enrichment, the identiﬁcation of CTCs is then performed. For identiﬁcation techniques, nucleic acid methods and cytometric methods are usually used. Recently, the development of molecular techniques can make molecular and genetic analysis of CTCs after enrichment and identiﬁcation of CTCs, leading to developing CTC characterization.
Isolation of CTCs using the size of epithelial tumors is based on the size of tumor cells without functional modiﬁcation and complex enrichment procedures. It is usual to utilize 5-8 µm probe ﬁlters to enable and to separate small leukocytes from the large epithelial cell and the isolation sensitivity threshold is approximately one tumor cell per milliliter.[8,9] These techniques have a valuable advantage in isolation of CTCs without damaging cells and enable further immunocytochemical or immunoﬂuorescence evaluation of CTCs. Although it is easily handled and cheap, it is considered to be not highly sensitive and poorly speciﬁc.
Furthermore, density gradient separation using Ficoll-Hypaque is an alternative technique to separate CTCs and mononuclear cells from other blood cells and granulocytes. However, Ficoll-Hypaque can be toxic to CTCs. CTCs can also be easily to lose due to the migration of cells to the plasma layer. OnkoQuick was developed to avoid the cross-contamination of different layers, resulting in higher recovery rate of CTCs.[10,11] Recently, RosetteSepTM (Stem Cell Technologies, Vancouver, British Columbia, Canada) developed a novel method based negative selection to improve the speciﬁcity of standard gradient separation.[12,13]
The immunomagnetic CTC enrichment technique is a magnetic bead-based separation method. To date, there have been two methods to identify CTCs expressing targeting-speciﬁc biomarkers. One is using the epithelial cell-speciﬁc marker, e.g. epithelial cell adhesion molecule (EpCAM) or cytokeratin (CK) expressed on the surface of tumor cells from epithelial origin. Another is using the tumor-speciﬁc markers, such as α-fetoprotein, Her2-neu, or carcinoembryonic antigen (CEA) expressed on a particular type of cancer cells. Immunomagnetic isolation technique utilizes monoclonal antibodies that are labeled magnetic microbeads and separates CTCs from the leukocytes background by magnetic force. To separate leukocytes, the negative selection is performed using an anti-CD45 antibody recognizing surface marker of leukocytes. Based on this technique, the Magnetic Activated Cell Sorting System (MACSTM Miltenyi Biotec, Bergisch Gladbach, Nordrhein-Westfalen, Germany) is a useful technology for detecting and analyzing CTCs because it avoids cell lysis and enables cell count by immunocytochemistry and immunoﬂuorescence assay. CellSearch SystemTM (Veridex, Warren, NJ) approved by the US Food and Drug Administration (FDA) is a semi-automated analyzer enriching the CTCs with ferroﬂuid nanoparticles coated with anti-EpCAM antibodies. This system is proved to be useful for detecting and analyzing CTCs with patients with breast, colorectal and prostate cancer in the clinic. However, Alunni-Fabbroni and Sandri argued that this technology has two possible limitations, that is, there is no “universal marker” available for each type of tumor, while epithelial marker (EpCAM) could be down-regulated in epithelial tumor cells after tumor cells undergo epithelial-mesenchymal transition (EMT). Thus, this method could only detect selected CTCs.[17,18]
Reverse transcriptase polymerase chain reaction (RT-PCR) based techniques can increase the speciﬁcity of the molecular methods to discriminate between the higher levels of molecular changes in cancer patients and the low background level in normal cells. Expressions of epithelial or tumor-speciﬁc markers are detected using RT-PCR to evaluate and identify CTCs. Nowadays, multiplex RT-PCR approach has been established to screen at the same time more than one single biomarker. Furthermore, quantitative RT-PCR improves the speciﬁcity of detection for CTCs by deﬁning a cut-off value for biomarker expression. However, there are some limitations of this method: (1) contamination of non-malignant epithelial cells such as skin cells; (2) false positive due to unspeciﬁc markers; and (3) ampliﬁcation of cell-free nucleic acids. Therefore, it is essential to select the appropriate marker that is expressed speciﬁcally by tumor cells to boost the speciﬁcity and reliability of CTC detection.
Cytometric-based technique can isolate and count CTCs using monoclonal antibodies against various antigens. To detect CTCs, CK and EpCAM are most commonly used. It enables to keep CTCs intact during analysis because cell lysis is not necessary. Furthermore, this technique provides information of high statistical precision and subpopulation quantiﬁcation with high speciﬁcity due to simultaneous analysis using multiple parameters. However, in contrast to RT-PCR technique, the disadvantage of this technique has a lower sensitivity.
Fiber-optic Array Scanning Technology, a rapid and accurate CTC location cytometric system, is a scanning technology characterized by a large ﬁeld of view. It allows analyzing large volumes of samples without any puriﬁcation step and minimizing the risk of cell loss. Additional scanning systems such as ACIS (Automated Cellular Imaging System, DAKO, Spatial Technology, USA) and ARIOL (Applied Imaging Corp, Wetzlar, Germany) are available on the market.
As discussed above, the detection of CTCs is involved in two steps of enrichment and identiﬁcation; thus, the development of automated techniques could offer at the same time enrichment, staining and scanning of the samples. The Cell Search System® enriches the CTCs with ferroﬂuid nanoparticles coated with anti-EpCAM antibody. The enriched EpCAM+ population is stained with phycoerythrin-conjugated antibodies against CK-8, -18 and -19 with allophycocyanin-conjugated antibodies speciﬁc for leukocytes (anti-CD45 antibody) and with the nuclear dye 4’, 6-diamino-2-phenylidole (DAPI) for the nucleic acids staining. The CK+/DAPI+/CD45− cells are then counted as CTCs using CellSpotter analyzer (Veridex, Warren, NJ), a four-color semi-automated ﬂuorescent microscope. More recently, CTC-chip based on a microﬂuidic platform has also developed to isolate a high rate of CTCs. CTC-chip consists of an array of 78,000 microspots coated with anti-EpCAM antibodies. Whole blood is pumped through this chip, and EpCAM+ cells are captured and detected by cameras identifying their morphology, viability and the expressions of tumor markers. However, the relevance of this technology in clinical setting remains unclear and clinical validation study is required. Finally, based on enzyme-linked immunospot assay technology, epithelial immunospot (EPISPOT) assay can identify CTCs by detecting speciﬁc CTC-secreted proteins (CK, mucin or prostate speciﬁc antigen).[22,23] EPISPOT makes it possible to detect the only viable CTCs because dying CTCs are unable to secrete an adequate amount of proteins to be detected. Sensitivity of EPISPOT is superior to ELISA assay in a two-order magnitude while detecting the release of CK-19 from tumor cells.
The next desirable step is to elucidate the molecular and genetic characterization of CTCs after enrichment and isolation of CTCs. This step may help us to comprehend the mechanism of cancer metastasis, leading to the development of treatments of tumor metastasis. However, the molecular and genetic characteristics of CTCs are not fully clariﬁed when compared with corresponding tumors in GI cancer. The molecular and genetic characteristics of CTCs are usually analyzed by PCR-based methods, ﬂuorescent in situ hybridization (FISH) or comparative genomic hybridization (CGH). There have been no reports about CTCs characterization analyzed by FISH and CGH in gastric cancer, whereas abnormal copy number alteration was detected in CTCs from patients with metastatic prostate cancer.[25-27] Using PCR-based methods, conventional detection system with epithelial markers such as CEA and CK has been previously performed to show the clinical significance of CTCs in gastric cancer.[28-32] However, Mimori et al. showed in a large-scale study that CTCs circulate even in early stages of the disease indicating that the simultaneous presence of CTC and VEGFR-1 expression is clinically significant for disease progression. It is also well known that there is discordance of expression profile between CTCs and primary tumor, and several markers for regulating metastasis and prognosis have been determined by PCR-based methods.[34-37] Furthermore, a comprehensive molecular profiling using the cDNA microarray was performed to identify novel genes to predict gastric cancer metastasis, recurrence and prognosis, suggesting that expression of MT1-MMP in peripheral blood identified by the cDNA microarray technique in gastric cancer was a powerful indicator of distant metastasis, especially for peritoneal dissemination. van de Stolpe et al. reported that CTCs were heterogeneous and differed among different cancer types. In addition to differences across cancer types, CTCs have heterogeneity within the same patient. Although the heterogeneity of primary tumors has been known, Klein et al. showed that early disseminated cancer cells are genomically very unstable, as well as the primary tumor. In this case, gastric cancer is well known to have histological heterogeneity in primary lesion. Various histological types and differentiation of gastric cancer cells are frequently observed in the same specimens. Therefore, histological heterogeneity may make it difficult to the molecular and genetic characterization of CTCs in GI cancer.
To date, there are a number of methodologies in the detection of CTCs and the clinical relevance of GI cancer have been reported. In clinical setting, the detection of CTC is expected to be useful in early diagnosis of cancer, monitoring of treatment responses and disease progression. In the following, we summarized a comprehensive update of the studies with more than 50 patients or with an outcome analysis and discussed their clinical implications in selected GI cancers.
There are only a few studies of esophageal cancer available as compared to gastric and colorectal cancers [Table 1]. In esophageal cancer, RT-PCR was the main technique to detect CTCs in previous studies.[42-45] As for available molecular markers, CEA and SCC are useful predictive markers for tumor recurrence and survival. Most recently, a large-scale of study using CellSearch System®, morphological technique are also reported.[46,47] Matsushita et al. revealed that CTC detection by CellSearch System® was useful to evaluate the clinical efﬁcacy of chemotherapy and chemoradiation therapy on esophageal cancer patients. Reeh et al. reported that patients with positive CTCs had signiﬁcantly poorer overall survival and progression-free survival rate; therefore, preoperative CTC detection by CellSearch System® was an independent prognostic indicator for patients with esophageal cancer. However, most of previously reported patients had esophageal squamous cell carcinoma. There are some differences of biological behaviors between esophageal squamous cell carcinoma and adenocarcinoma; therefore, further study of esophageal adenocarcinoma is needed.
Clinical relevance of CTC in esophageal cancer
|Author||Year||Case||Method||Molecular marker||Clinical relevance|
|Kaganoi||2004||70||RT-PCR||SCC||Prediction of recurrence|
|Setoyama||2006||106||RT-PCR||CEA||Prediction of recurrence|
|Liu||2007||53||RT-PCR||CEA||Prediction of recurrence|
|Hashimoto||2008||147||RT-PCR||CEA||Prediction of recurrence and prognosis|
|Cao||2009||108||RT-PCR||Survivin||Prediction of haematogenous recurrence and prognosis|
|Tanaka||2010||244||RT-PCR||CEA, SCC||Predictor for hematogenous and local recurrences|
|Yin||2012||72||RT-PCR||CEA, CK-19,||Survivin Clinical efficacy of RT|
|Matsushita||2014||90||CellSearch||EpCAM, CK-8, 18, 19||Clinical efficacy of CT or CRT|
|Reeh||2015||100*||CellSearch||EpCAM, CK-8, 18, 19||Prediction of recurrence and prognosis|
A number of studies of CTC detection in patients with gastric cancer have been reported previously as summarized in Table 2. Although the several methodology of CTC detection including RT-PCR and CellSearch System® [Table 2], it remains unclear which is the best method and molecular marker for detection of CTCs in gastric cancer patients. Recently, various meta-analyses demonstrated that presence of CTCs was associated with poor prognosis and advanced clinicopathological factors.[48-50] It has been reported that detection of CTCs in gastric cancer may be useful in early diagnosis and monitoring of treatment responses and prognosis. However, as for diagnosis, a recent meta-analysis showed that CTC detection alone cannot be recommended as a screening test for gastric cancer because of lower and inconsistent sensitivity estimates for CTC. Furthermore, Mimori et al. showed that CTCs occurred in early stages of the disease, and CTC alone cannot be a predictor of cancer metastasis in a large-scale study. This study also revealed that elevated expression of VEGFR-1 facilitated the establishment of hematogenous metastases of gastric cancer and that the simultaneous presence of CTC and VEGFR-1 expression at premetastatic sites was clinically signiﬁcant in disease progression.
Clinical relevance of CTC in gastric cancer
|Author||Year||Case||Method||Molecuar marker||Clinical relevance|
|Wu||2006||64||MAH||hTERT, CK-19, CEA, MUC1||Associated with recurrence|
|Pituch-Noworolska||2007||57||ICC||CK-8, 18, 19||No prognostic impact|
|Ito||2010||65||ICC||GFP, EpCAM,||Shorter OS|
|Majima||2000||52||RT-PCR||CK-19, 20||Shorter OS|
|Miyazono||2001||57||RT-PCR||CEA||Associated with liver metastaisis, recurrence|
|Sumikura||2003||106||RT-PCR||CEA||Associated with recurrence|
|Ikeguchi||2005||59||RT-PCR||CEA||No association with recurrence|
|Uen||2006||52||RT-PCR||MUC1, c-Met||Shorter OS|
|Koga||2008||101||RT-PCR||CK-18, 19, 20||Shorter OS (CK-19 is better)|
|Yie||2008||55||RT-PCR, ELISA||Survivin||Predictive marker for DFS|
|Mimori||2008||810||RT-PCR||CK-7,19, 20, VEGFR1||Associated with hematogenous metatasis|
|Bertazza||2009||70||RT-PCR||Survivin||Predictive marker for OS|
|Qiu||2010||123||RT-PCR||CEA||Predictive marker for DFS|
|Cao||2011||98||RT-PCR, ELISA||Survivin||Predictive marker for DFS|
|Matsusaka||2010||52||CellSearch||EpCAM, CK-8, 18, 19||Predictive marker for PFS (CTC level after Cx)|
|Uenosono||2013||148||CellSearch||EpCAM, CK-8, 18, 19||Predictive marker for PFS and OS|
To date, there are a large number of studies of CTC detection in colorectal cancer as compared to other GI cancers as summarized in Table 3. RT-PCR and the CellSearch System® have been mainly reported methods to detect CTC in colorectal cancer and data showed that CTCs were associated recurrence and overall survival of patients. For example, Cohen et al. revealed that the number of CTCs detected by the CellSearch System® before and during treatment was an independent predictor of PFS and OS in 430 patients with metastatic colorectal cancer in a prospective multicenter clinical trial. The CellSearch System® using in colorectal cancer was the ﬁrst CTC detection system that was approved by US FDA. Furthermore, a previous meta-analysis reported the aprognostic signiﬁcance of CTC detected by the CellSearch System® has been reported. Eleven studies including 1,847 colorectal cancer patients were analyzed in this study and the presence of CTCs was signiﬁcantly associated with overall and progression-free survival as reported by the previous meta-analysis. In a previous prospective study of non-metastatic colorectal cancer, preoperative CTC detection was a strong and independent prognostic marker. The treatment response rate was signiﬁcantly lower in CTC-positive patients than that of CTC negative patients at base line and during treatment.[23,54-57] Another previous study demonstrated potentially clinical application in detection of KRAS mutational in CTCs for selecting metastatic colorectal cancer patients for cetuximab therapy. In addition, recent development of molecular and genetic characterization of single-CTC demonstrated that there was intra- and inter- heterogeneity of EGFR expression and genetic alterations of EGFR, KRAS and PIK3CA, which possibly explained the variable response rates to EGFR inhibition in patients with colorectal cancer. Therefore, the information on the molecular status of CTCs might be useful for stratiﬁcation of molecular-directed therapy.
Clinical relevance of CTC in colorectal cancer
|Author||Year||Case||Method||Molecuar marker||Clinical relevance|
|Wong||2009||132||ICC||CK-20||Predictive marker for OS|
|Tanigich||2000||53||RT-PCR||CEA||Predictive marker for DFS|
|Yamagichi||2000||52||RT-PCR||CK-20, CEA||Shorter OS|
|Hardingham||2000||94||RT-PCR||CK-19, 20, MUC2||Shorter DFS|
|Bessa||2001||68||RT-PCR||CEA||No prognostic impact|
|Bessa||2003||66*||RT-PCR||CEA||No prognostic impact|
|Sadahiro||2005||93||RT-PCR||CEA||No prognostic impact|
|Douard||2006||121||RT-PCR||CK-20, CGM2||No prognostic impact|
|Iinuma||2006||167||RT-PCR||CK-20, CEA||Shorter DFS and OS|
|Katsumata||2006||57||RT-PCR||CK-20||Strong relation to LN metastais and OS|
|Allen-Mersh||2007||113*||RT-PCR||CK-20, CEA||Shorter DFS|
|Wang||2007||157*||RT-PCR**||CK-19, 20, CEA, hTERT||Shorter DFS|
|Uen||2007||194||RT-PCR**||CK-19, 20, CEA, hTERT||Shorter DFS|
|Uen||2007||438*||RT-PCR||CK-19, 20, CEA, hTERT||Shorter DFS|
|Yie||2008||86||RT-PCR, ELISA||Survivin||Predictive marker for DFS|
|Lu||2011||141*||RT-PCR**||CK-19, 20, CEA, hTERT||Shorter DFS and OS|
|Iinuma||2011||735||RT-PCR||CK-19, 20, CEA, hTERT||Shorter DFS and OS|
|Pilati||2012||50||RT-PCR||CK-19, 20, CEA, CD133, VEGF, EGFR, Survivin||Predictive marker for OS (CD133 CTC)|
|Cohen||2008||430||CellSearch||EpCAM, CK-8, 18, 19||Predictive marker for OS (CTC ≥ 3)|
|Matsusaka||2011||64||CellSearch||EpCAM, CK-8, 18, 19||Predictive marker for PFS and OS (CTC level after Cx)|
|Tol||2010||467||CellSearch||EpCAM, CK-8, 18, 19||Predictive marker for PFS and OS (CTC level before Cx)|
|Sastre||2012||180||CellSearch||EpCAM, CK-8, 18, 19||Predictive marker for PFS and OS (CTC level before Cx)|
|Aggarwal||2013||209||CellSearch||EpCAM, CK-8, 18, 19||Predictive marker for OS (CTC level before Cx)|
|Gazzaniga||2013||119||CellSearch||EpCAM, CK-8, 18, 19||Predictive marker for OS (CTC ≥ 1)|
|Kuboki||2013||63||CellSearch||EpCAM, CK-8, 18, 19||Shorter DFS and OS|
|Sotelo||2015||472||CellSearch||EpCAM, CK-8, 18, 19||No prognostic impact (stage III)|
|Seeberg||2015||194||CellSearch||EpCAM, CK-8, 18, 19||Predict nonresectability and impaired survival|
There are two main approaches in the detection of CTCs, that is, immunological assays using monoclonal antibodies and PCR-based molecular assays, exploiting tissue-speciﬁc transcripts. Immunocytochemical detection of epithelial or tumor-associated antigens is widely accepted. Recent studies have shown that EMT plays a critical role in cancer progression and metastasis in epithelial malignancies including gastric cancer. Our previous study implied that vimentin-positive tumor cells were able to survive in the peripheral circulation and in the bone marrow and that vimentin-positive cancer cells that invade intratumoral vessels must have undergone mesenchymal transition. We assume that not all detected CTCs but rather only a few, which have undergone EMT could give rise to tumor metastasis or recurrence. Most recently, Wu et al. reported that mesenchymal CTCs classiﬁed using EMT markers were more commonly found in patients in metastatic stages of the disease in different types of human cancers. Therefore, it is possible that conventional detection system using epithelial markers fail to detect that population of CTCs.
Furthermore, the concept of rare subpopulations of cancer stem cells (CSCs) has created a novel focus in cancer research but arises a question whether CTCs have CSC property. It is expected that CTC with CSC property may be disseminated from the primary tumor lesion to a distant metastatic site. This hypothesis is supported by the similarities between the properties of CTCs and CSC and suggests that the founder cells of metastases arise from the CTC population. It has been reported that stem cell markers are frequently overexpressed in the CTCs of patients with metastatic breast cancer, and the most CTCs have stem cell phenotypes that are non-proliferating and resistant to chemotherapy. For example, Iinuma et al. revealed that multi genetic markers of CSC, CEA/CK/CD133 in peripheral blood samples could be a useful predictor for recurrence and prognosis. Pilati et al. reported that CD133-positive CTCs might represent a suitable prognostic marker to stratify the risk of patients who undergo liver resection for CRC metastasis.
An increasing number of studies have shown that CTC is associated with GI cancer progression, metastasis and resistance to pharmacotherapy. However, the clinical evidence supporting the role of CTC in cancer progression still remains inconclusive. Therefore, further analysis and clinical trials are required to achieve clinical utility of CTC detection in GI cancers.
This work was supported in part by the following grants and foundations: Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientiﬁc Research; Grant Numbers: 23791550.
This work was supported in part by the following grants and foundations: Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientiﬁc Research; Grant Numbers 23791550.
There are no conﬂicts of interest.
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