Bidard Fc
email: fcbidard@curie.fr

Dr François-Clément C Bidard

Disseminated and circulating tumor cells in gastrointestinal oncology

Keywords: gastrointestinal cancer, circulating tumor cells, disseminated tumor cells, micrometastasis, biomarker

His PhD thesis was about cooperation between heterogeneous cancer cells during the metastatic process in mice models, and his MD thesis concerned circulating tumor cells in breast-cancer patients. He has published more than 20 articles, most of them about disseminated and circulating tumor cells.

Introduction

Onset of metastasis is a complex yet poorly understood process, which is responsible for most of cancer-related deaths. For several years, cellular dissemination from primary to secondary sites has been a "black-box" of research for clinicians. In non-metastatic (M0) patients, clinical studies have isolated strong prognostic factors associated with the risk of metastatic relapse, and these factors are being used to make decisions on adjuvant treatments. Adjuvant chemotherapy targets cancer cells that may have disseminated throughout the body, but is currently given blinded to the real dissemination status of each patient. With continuous technical improvements, detection methods have been recently set up and validated to isolate cancer cells in the blood (circulating tumor cells (CTC)) and in the bone marrow (disseminated tumor cells (DTC)). The opening of these two windows on the metastatic process in patients has raised three main critical issues: Are detection methods accurate? Is quantitative analysis (i.e., counts) of CTCs/DTCs of clinical relevance? Could qualitative analysis (i.e., molecular characterization) of CTCs/DTCs uncover new cancer features/targets? This review focuses on results obtained by different CTC/DTC detection methods in gastro-intestinal cancers.

Technical aspects 2.1 CTC and DTC detection sites

Metastasis is a complex multistep process, which emerged as a biological and clinical research field more than a century ago. In one of the first studies to investigate the metastatic spread of breast cancer, James Paget made the well-known "seed and soil" hypothesis to explain the discrepancy between the blood supply of the different organs and the distribution of breast-cancer metastases [START_REF] Paget | The distribution of secondary growths in cancer of the breast. 1889[END_REF]. Since this seminal report, several steps in the hematogeneous metastatic process have been described: local invasion, intravasation into blood vessels, interaction with bloodformed elements, arrest on the vessel's endothelium, extravasation, invasion of the host-organ microenvironment, cellular dormancy, and establishment of a new growth.

Basically, CTC corresponds to the circulation step of cancer cells, after intravasation, whereas DTC corresponds to the post-extravasation steps. Noteworthy, CTCs are generally detected in the peripheral blood, which is supposed to have a homogeneous cancer-cell concentration, but many gastrointestinal (GI) studies have also looked for CTC in the efferent vein of primary GI tumors, i.e., the portal vein. As looking for DTCs in solid organs is technically difficult, DTCs are almost exclusively detected in bone marrow (by a sternum or iliac-crest puncture). These different detection sites have to be kept in mind, especially in GI cancers, in which the liver may act as a physical blood-filter for CTCs released by the primary tumor. Finally, cancer cells are not definitely set into one of these "compartments": cellular trafficking between the primary tumor site, the blood, the bone-marrow, other target-organs, and metastases are very likely, as has been reported in a few pre-clinical [START_REF] Kim | Tumor self-seeding by circulating cancer cells[END_REF] or clinical [START_REF] Bidard | Disseminated tumor cells and the risk of locoregional recurrence in nonmetastatic breast cancer[END_REF] models.

CTC and DTC detection methods

In a few solid tumors that exhibit specific gene-fusion transcripts (e.g., EWS/FLI1 in Ewing's sarcoma), DTC/CTC detection has already been clinically implemented as a strong prognostic marker for use in everyday clinics, either at diagnosis or after the primary treatment, and is often called a "minimal residual disease". However, since the first CTC description in the late 19 th century [START_REF] Ashworth | A case of cancer in which cells similar to those in the tumours were seen in blood after death[END_REF], as most carcinomas do not have specific transcripts, DTC and CTC have been detected using different techniques, leading to successive results that can appear heterogeneous [START_REF] Pantel | Cancer micrometastases[END_REF][START_REF] Rahbari | Metaanalysis shows that detection of circulating tumor cells indicates poor prognosis in patients with colorectal cancer[END_REF]. These techniques generally consist of isolating cells with epithelial markers out of mesenchymal-derived compartments (blood, bone marrow) [START_REF] Muller | Insights into minimal residual disease in cancer patients: implications for anti-cancer therapies[END_REF]; two steps are generally used: Moreover, high discrepancy rates can be observed using different antibodies against the same membranous marker, as is shown in colorectal cancer [START_REF] Antolovic | Heterogeneous detection of circulating tumor cells in patients with colorectal cancer by immunomagnetic enrichment using different EpCAM-specific antibodies[END_REF]. This preliminary step, which could be followed by either molecular or cytological detection techniques, is highly heterogeneous among techniques, and is critical to understanding discrepancies between published studies in the same setting.

Molecular detection of CTC/DTC relies on the detection of mRNA (by RT-(q)PCR, mRNA hybridization onto cDNA membrane…), which is related either to epithelial function or to specific cancer hallmarks, such as telomerase activity. Target mRNA (CEA, MUC1, cytokeratins…), and primers used for the amplification step are heterogeneous among studies, which are generally monocentric. For example, in the recent meta-analysis by Rahbari et al [START_REF] Rahbari | Metaanalysis shows that detection of circulating tumor cells indicates poor prognosis in patients with colorectal cancer[END_REF], on CTC/DTC detection in colorectal cancer, more than 20 different targets were assessed, alone or in combination, in 31 molecular studies conducted in more than 25 different single centers.

However, the specificity of these molecular techniques is questioned, as nonspecific expression of epithelial markers has been reported in lymphoid cells activated by cancer-related systemic inflammation [START_REF] Chechlinska | Systemic inflammation as a confounding factor in cancer biomarker discovery and validation[END_REF].

Cytological detection of CTC/DTC, after the initial enrichment step, relies on immunostaining of CTC/DTC, using epithelial antigens thought to be expressed by carcinoma cells (e.g., epithelial cytokeratins). Multiple staining can be performed on isolated cells, generally by immunocytofluorescence. The standardized CellSearch® system [START_REF] Allard | Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases[END_REF], which has become the most commonly used CTC detection system over the past few years, enriches the sample (7.5 ml of blood) cells that express the EpCAM molecule, with antibody-coated magnetic beads, and labels the cells with a fluorescent nucleic acid dye (DAPI). Fluorescently labeled monoclonal antibodies specific for leukocytes (CD45-allophycocyan) and epithelial cells (cytokeratin 8, 18, 19phycoerythrin) are used to distinguish epithelial cells from leukocytes. Beyond epithelial-staining, cytological detection methods allow the optical control of stained cells [START_REF] Marrinucci | Cytomorphology of circulating colorectal tumor cells:a small case series[END_REF], in order to distinguish cancer cells from activated lymphoid cells. This final validation step of cell morphology is timeconsuming, hardly automatable, and relies on expert cytologists and/or technicians who should use a standardized classification consensus [START_REF] Fehm | A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation[END_REF] whenever available. Our review is mainly focused on the clinical applications of cytology-based CTC/DTC-detection techniques, which are more homogeneous and have, at least for the CellSearch® system, the advantage of demonstrated reproducibility across centers and studies [START_REF] Riethdorf | Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the CellSearch system[END_REF][START_REF] Kraan | External quality assurance of circulating tumor cell enumeration using the CellSearch((R)) system: A feasibility study[END_REF].

3 Esophageal cancers

DTCs

Although bone and bone marrow are not preferential sites for macrometastasis of esophageal cancer, DTCs have been primarily found in 37% of 90 non-metastatic patients after an iliac-crest puncture using a cytological technique based on cytokeratin detection [START_REF] Thorban | Immunocytochemical detection of disseminated tumor cells in the bone marrow of patients with esophageal carcinoma[END_REF]. DTCs were associated, in this study, with poor survival in patients with completely excised tumors.

Interestingly, bone-marrow DTCs were more frequently detected (79%) when extracted from a rib contiguous to the tumor than from the iliac crest (8%), but rib-derived DTCs had no clear prognostic significance [START_REF] Bonavina | Bone marrow-disseminated tumor cells in patients with carcinoma of the esophagus or cardia[END_REF][START_REF] Ryan | Prevalence of bone marrow micrometastases in esophagogastric cancer patients with and without neoadjuvant chemoradiotherapy[END_REF]. Biologically, whole-genome analysis of single DTCs isolated by micropipetting in 107 patients showed that most genetic aberrations were different in primary tumors and in bone marrow DTCs [START_REF] Stoecklein | Direct genetic analysis of single disseminated cancer cells for prediction of outcome and therapy selection in esophageal cancer[END_REF], supporting the parallel-evolution model, proposed for metastatic processes, by CA Klein [START_REF] Klein | Parallel progression of primary tumours and metastases[END_REF]. In this study, gain of the HER2 gene region on 17q was the most common genomic alteration shared between DTCs, and was associated with shorter overall survival.

CTCs

CTC detection in esophageal cancers has been mainly reported by molecular techniques in patients undergoing surgery with a curative intent. These studies have focused mainly on CEA mRNA expression [START_REF] Noh | Detection of tumor cell contamination in peripheral blood by RT-PCR in gastrointestinal cancer patients[END_REF][START_REF] Nakashima | Clinical significance of circulating tumor cells in blood by molecular detection and tumor markers in esophageal cancer[END_REF][START_REF] Liu | Circulating tumor cells in perioperative esophageal cancer patients: quantitative assay system and potential clinical utility[END_REF][START_REF] Setoyama | Isolated tumour cells in blood and E-cadherin expression in oesophageal squamous cell cancer[END_REF][START_REF] Hashimoto | Circulating micrometastases of esophageal cancer detected by carcinoembryonic antigen mRNA reverse transcriptase-polymerase chain reaction: clinical implications[END_REF], but also on CK19, CK20 [START_REF] Huang | Molecular detection of disseminated tumor cells in the peripheral blood in patients with gastrointestinal cancer[END_REF][START_REF] Ito | Detection and quantification of circulating tumor cells in patients with esophageal cancer by real-time polymerase chain reaction[END_REF], SCC [START_REF] Kaganoi | Detection of circulating oesophageal squamous cancer cells in peripheral blood and its impact on prognosis[END_REF], deltaNp63 [START_REF] Koike | Molecular detection of circulating esophageal squamous cell cancer cells in the peripheral blood[END_REF], and survivin [START_REF] Hoffmann | Preoperative survivin mRNA detection in peripheral blood is an independent predictor of outcome in esophageal carcinoma[END_REF][START_REF] Cao | Detection of survivin-expressing circulating cancer cells in the peripheral blood of patients with esophageal squamous cell carcinoma and its clinical significance[END_REF]. Detection rates are heterogeneous among studies, even when the same mRNA has been quantified in the same clinical setting, e.g., 77% [START_REF] Hoffmann | Preoperative survivin mRNA detection in peripheral blood is an independent predictor of outcome in esophageal carcinoma[END_REF] vs. 47% [START_REF] Cao | Detection of survivin-expressing circulating cancer cells in the peripheral blood of patients with esophageal squamous cell carcinoma and its clinical significance[END_REF] for survivin, 57% [START_REF] Nakashima | Clinical significance of circulating tumor cells in blood by molecular detection and tumor markers in esophageal cancer[END_REF] vs. 28% [START_REF] Liu | Circulating tumor cells in perioperative esophageal cancer patients: quantitative assay system and potential clinical utility[END_REF] for CEA. Almost all of these exploratory studies show a negative prognostic impact of CTC detection on survival, although these reports are on small heterogeneous cohorts.

Reports based on CTC cytological detection are even more limited; a negative prognostic impact and a correlation with pleural dissemination were found in 5 patients out of 23 metastatic esophageal-cancer patients who had ≥2 CTCs/7.5 ml of blood, as assessed with the CellSearch® system [START_REF] Nakamura | Immunocytochemical detection of circulating esophageal carcinoma cells by immunomagnetic separation[END_REF][START_REF] Hiraiwa | Clinical significance of circulating tumor cells in blood from patients with gastrointestinal cancers[END_REF].

Gastric adenocarcinoma 4.1 DTCs

Bone marrow DTCs have been reported by several detection techniques in surgically resected gastric cancers: CK18 [START_REF] Schlimok | Micrometastatic tumour cells in bone marrow of patients with gastric cancer: methodological aspects of detection and prognostic significance[END_REF]; CK2-immunostaining [START_REF] Jauch | Prognostic significance of bone marrow micrometastases in patients with gastric cancer[END_REF]; anti-human epithelial antigen (Ber-EP4) [START_REF] Macadam | Bone marrow micrometastases predict early post-operative recurrence following surgical resection of oesophageal and gastric carcinoma[END_REF]; CK20 RT-PCR [START_REF] Oki | Clinical significance of cytokeratin positive cells in bone marrow of gastric cancer patients[END_REF], CK20/CK19/CEA RT-PCR [START_REF] Fujita | Detection of cancer cells disseminated in bone marrow using real-time quantitative RT-PCR of CEA, CK19, and CK20 mRNA in patients with gastric cancer[END_REF], CK7/CK8 RT-PCR [START_REF] Wang | Detection of disseminated tumor cells in bone marrow of gastric cancer using magnetic activated cell sorting and fluorescent activated cell sorting[END_REF],

CEA/CK20/TFF1/MUC2 RT-PCR [START_REF] Dardaei | The detection of disseminated tumor cells in bone marrow and peripheral blood of gastric cancer patients by multimarker (CEA, CK20, TFF1 and MUC2) quantitative real-time PCR[END_REF]. About half of these studies reported a significant negative prognostic impact on metastases-free and/or overall survival. DTC detection has been associated

with increased tumor-microvessel density [START_REF] Maehara | Tumor angiogenesis and micrometastasis in bone marrow of patients with early gastric cancer[END_REF][START_REF] Matsunami | Detection of bone marrow micrometastasis in gastric cancer patients by immunomagnetic separation[END_REF]; its association with VEGFR expression by the primary tumor appears controversial [START_REF] Kakeji | Clinical significance of micrometastasis in bone marrow of patients with gastric cancer and its relation to angiogenesis[END_REF]. As not every single DTC will later grow into a macrometastase, a German group developed further investigations into DTCs isolated by a cytological technique. They showed that immunostaining of proteins involved in the urokinase plasminogen-activator (uPA) system may distinguish those that have a fully metastatic phenotype and an independent clinical impact [START_REF] Allgayer | Urokinase plasminogen activator receptor (uPA-R): one potential characteristic of metastatic phenotypes in minimal residual tumor disease[END_REF][START_REF] Heiss | Clinical value of extended biologic staging by bone marrow micrometastases and tumorassociated proteases in gastric cancer[END_REF][START_REF] Heiss | Minimal residual disease in gastric cancer: evidence of an independent prognostic relevance of urokinase receptor expression by disseminated tumor cells in the bone marrow[END_REF]. Recently, they also reported that the extracellular matrix-metalloprotease inducer (EMMPRIN) may also play a role in DTCs' evolution to later macrometastases [START_REF] Buergy | Prognostic impact of extracellular matrix metalloprotease inducer: immunohistochemical analyses of colorectal tumors and immunocytochemical screening of disseminated tumor cells in bone marrow from patients with gastrointestinal cancer[END_REF].

CTCs

CTCs in gastric cancer have been studied in several small studies, reported in Table 1. No clear conclusion can be drawn from these heterogeneous studies, which are often underpowered.

Among the several molecular markers tested (CK18, 19, 20, CEA, hTERT (human telomerase reverse transcriptase)), none has demonstrated a clear and confirmed superiority over the others [START_REF] Wu | Molecular detection of disseminated tumor cells in the peripheral blood of patients with gastric cancer: evaluation of their prognostic significance[END_REF][START_REF] Yeh | Detection of circulating cancer cells by nested reverse transcription-polymerase chain reaction of cytokeratin-19 (K19): possible clinical significance in advanced gastric cancer[END_REF][START_REF] Shin | Detection of cancer cells in peripheral blood of stomach cancer patients using RT-PCR amplification of tumour-specific mRNAs[END_REF][START_REF] Shimizu | Usefulness of the real-time reverse transcription-polymerase chain reaction assay targeted to alpha1,4-N-acetylglucosaminyltransferase for the detection of gastric cancer[END_REF][START_REF] Szatanek | Detection of isolated tumour cells in the blood and bone marrow of patients with gastric cancer by combined sorting, isolation and determination of MAGE-1, -2 mRNA expression[END_REF]. CTC-detection rate was related to the surgical maneuvers during the surgical removal of excisable cancers, and was initially correlated with poor survival [START_REF] Miyazono | Surgical maneuvers enhance molecular detection of circulating tumor cells during gastric cancer surgery[END_REF]. This CTC release was confirmed in another study (n=59), though CTC-positivity after surgery was surprisingly associated with an improved prognosis [START_REF] Ikeguchi | Detection of circulating cancer cells after a gastrectomy for gastric cancer[END_REF]. A few reports have also focused on the patient's blood stored for autologous transfusion during surgery, by testing CTSs using various techniques after removal from blood by freezing, filtering, and irradiation [START_REF] Obayashi | Safety and utility of autologous blood transfusion for resection of metastatic liver tumor[END_REF][START_REF] Futamura | The effect of storage on the survival of cancer cells in blood and efficient elimination of contaminating cancer cells by a leukocyte depletion filter[END_REF]. Decreases in CTC have been also reported in about half of patients receiving neoadjuvant chemotherapy [START_REF] Kolodziejczyk | The effects of preoperative chemotherapy on isolated tumour cells in the blood and bone marrow of gastric cancer patients[END_REF]. Biological, preclinical experiments suggest a strong role for CTC arrest in premetastatic niches of target organs, characterized by VEGFR1-positive bone-marrow-derived non-tumoral cells [START_REF] Kaplan | VEGFR1positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche[END_REF]. To date, gastric adenocarcinoma is the only cancer in which the impact of VEGFR1-positive non-tumoral cells has been demonstrated in the presence of CTC, as was reported in 810 Japanese patients [START_REF] Mimori | Hematogenous metastasis in gastric cancer requires isolated tumor cells and expression of vascular endothelial growth factor receptor-1[END_REF].

Using the CellSearch® system, CTC detection rates were 14% and 55% in 14 nonmetastatic and 27 metastatic patients, respectively (≥2 CTCs/7.5ml) [START_REF] Hiraiwa | Clinical significance of circulating tumor cells in blood from patients with gastrointestinal cancers[END_REF]. Recently a Japanese group has reported an association between CTC detection and chemotherapy efficacy in 52 metastatic gastric-cancer patients treated with different chemotherapy regimens in advanced

Pancreatic cancer

In the metastatic setting, the CTC detection rate, using the CellSearch® system, has been recently investigated in four small cohorts (n=16 [START_REF] Allard | Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases[END_REF]; n=23 [START_REF] Ko | Detection and significance of circulating tumor cells (CTC) in patients with metastatic pancreatic cancer (PC) receiving systemic therapy[END_REF]; n=14 [START_REF] Kurihara | Detection of circulating tumor cells in patients with pancreatic cancer: a preliminary result[END_REF]; n=40 [START_REF] Negin | Characterization and prognostic significance of circulating tumor cells in the peripheral blood of patients with metastatic pancreatic cancer[END_REF]): CTCs were detected in ~50% of patients (≥1 CTC/7.5 ml), but the mean count appears lower than in colorectal cancers. Although underpowered, three of these studies addressed the prognostic significance of CTC detection and reported contradictory results (two being rather positive, one rather negative). Using a microfluidic device for CTC sorting (immunoselection and staining being similar to CellSearch®), a group at Massachusetts General Hospital recently reported 100% CTC detection in 15 metastatic pancreatic-cancer patients [START_REF] Nagrath | Isolation of rare circulating tumour cells in cancer patients by microchip technology[END_REF]. However, this detection rate may be overestimated, similar to their prostate cancer results [START_REF] Stott | Isolation and characterization of circulating tumor cells from patients with localized and metastatic prostate cancer[END_REF]. In 2001, a study from the same hospital reported 105 patients with stage I-IV pancreatic cancer with 26% CTC and 28% DTC detection rates (AE1/AE3 immunostaining) [START_REF] Z'graggen | Biological implications of tumor cells in blood and bone marrow of pancreatic cancer patients[END_REF]. Both CTC and DTC detection rates were correlated with disease stage and were not prognostic in multivariate analysis. Similarly, an older cytological study (using a cocktail of six antibodies) [START_REF] Vogel | Disseminated tumor cells in pancreatic cancer patients detected by immunocytology: a new prognostic factor[END_REF] and two molecular-technique-based studies (CK20 and CEA) [START_REF] Uchikura | Intraoperative molecular detection of circulating tumor cells by reverse transcriptionpolymerase chain reaction in patients with biliary-pancreatic cancer is associated with hematogenous metastasis[END_REF][START_REF] Soeth | Detection of tumor cell dissemination in pancreatic ductal carcinoma patients by CK 20 RT-PCR indicates poor survival[END_REF] retrieved an association between DTC and/or CTC detection and disease stage; multivariate analyses of survival were either not reported or were inconclusive

In locally advanced pancreatic cancers, which are unresectable but are still not metastatic, CTC/DTC may indicate how far the dissemination process is advanced, and could guide the physician's choice towards a local (e.g., radiotherapy) or systemic (chemotherapy) treatment. No significant data have been published yet in this setting, but a French companion study of the LAP07 trial (NCT00634725) is currently ongoing, using the CellSearch® system.

In early pancreatic cancer, detection rates are likely to be low with the current cytological techniques. Bone-marrow DTC detection by multiple antibodies has been, however, associated with a shorter time to tumor relapse in 15 patients who underwent complete surgical resection [START_REF] Thorban | Detection of micrometastasis in bone marrow of pancreatic cancer patients[END_REF]. For CTC detection, a single case report has been published using the CellSearch® system for diagnostic purposes in a patient presenting with a pancreatic mass [START_REF] Mudan | Clinical utility of circulating tumor cell measurement in the diagnosis of indeterminate lesions of the pancreas[END_REF], which found 4 CTCs/7.5 ml, whereas the mass turned out to be a pancreatic carcinoma. However, in this diagnostic setting, both sensitivity and specificity of this technique are too low to fulfill the requirements of a diagnostic test. Molecular techniques have been also tested in this setting [START_REF] Hoffmann | Detection of disseminated pancreatic cells by amplification of cytokeratin-19 with quantitative RT-PCR in blood, bone marrow and peritoneal lavage of pancreatic carcinoma patients[END_REF][START_REF] Ishizone | Clinical utility of quantitative RT-PCR targeted to alpha1,4-N-acetylglucosaminyltransferase mRNA for detection of pancreatic cancer[END_REF][START_REF] Zhang | Detection of CK20mRNA in peripheral blood of pancreatic cancer and its clinical significance[END_REF], but their specificity have been discussed when they are used as a single marker, especially for CK20 [START_REF] Zhou | Marker expression in circulating cancer cells of pancreatic cancer patients[END_REF].

Finally, pancreatic and ileal neuroendocrine tumors (NET) strongly express EpCAM, and CTC detection by the CellSearch® system was only reported in one study reported to date: 43% of patients with metastatic ileal NET (n=26) and 21% of patients with metastatic pancreatic NET (n=16) [START_REF] Khan | Circulating tumor cells and EpCAM expression in neuroendocrine tumors[END_REF]. CTC detection was associated with tumor progression in this study.

Colorectal adenocarcinoma: the adjuvant setting 6.1 DTCs

The first large study to show an association between DTC detection and worse prognosis in nonmetastatic colorectal cancer patients (n=88) was reported almost 20 years ago [START_REF] Lindemann | Prognostic significance of micrometastatic tumour cells in bone marrow of colorectal cancer patients[END_REF]. Adjuvant trials that tested the anti-EpCAM monoclonal antibody, edrecolomab, were launched on the bases that most bone-marrow DTCs express EpCAM [START_REF] Braun | Monoclonal antibody therapy with edrecolomab in breast cancer patients: monitoring of elimination of disseminated cytokeratin-positive tumor cells in bone marrow[END_REF] and that tumor response or stabilization has been observed in metastatic patients in Phase-2 studies [START_REF] Skog | Clinical effects of monoclonal antibody 17-1A combined with granulocyte/macrophage-colony-stimulating factor and interleukin-2 for treatment of patients with advanced colorectal carcinoma[END_REF]. However, adjuvant

Phase-3 trials with edrecolomab have shown no clinical benefit [START_REF] Punt | Edrecolomab alone or in combination with fluorouracil and folinic acid in the adjuvant treatment of stage III colon cancer: a randomised study[END_REF][START_REF] Fields | Adjuvant therapy with the monoclonal antibody Edrecolomab plus fluorouracil-based therapy does not improve overall survival of patients with stage III colon cancer[END_REF]. Other reports on DTC detection were included in a large meta-analysis published recently (see after 6.2) [START_REF] Rahbari | Metaanalysis shows that detection of circulating tumor cells indicates poor prognosis in patients with colorectal cancer[END_REF]. Liver DTCs were studied in liver biopsies using molecular techniques, but had no clear clinical impact on resected colorectal cancers [START_REF] Koch | Detection of disseminated tumor cells in liver biopsies of colorectal cancer patients is not associated with a worse prognosis[END_REF].

CTCs

Many studies have been conducted, by different surgical teams, at the time of resecting primary colon tumors. Many have looked for CTCs during surgery in the portal flow, using molecular or cytological [START_REF] Leather | Detection and enumeration of circulating tumour cells in colorectal cancer [see comments[END_REF] techniques, in order to measure circulating tumor load. The focus on portal flow is based on the hypothesis that the portal vein represents a "post-intravasation highway" for CTCs from the primary tumor to the liver, which is the main target organ for the metastatic process in colon cancer. Some studies have also focused on the potential release of CTCs into the portal vein during different surgical procedures using various vascular clamps or "no-touch" techniques [START_REF] Hayashi | No-touch isolation technique reduces intraoperative shedding of tumor cells into the portal vein during resection of colorectal cancer[END_REF][START_REF] Sales | Blood dissemination of colonic epithelial cells during no-touch surgery for rectosigmoid cancer [letter[END_REF][START_REF] Yamaguchi | Significant detection of circulating cancer cells in the blood by reverse transcriptase-polymerase chain reaction during colorectal cancer resection[END_REF]. Also, in patients diagnosed with an occlusive colorectal cancer, a small study (n=58)

suggested that endoscopic insertion of a colonic stent resulted in increased levels of CK20 mRNA, compared to staging colonoscopy [START_REF] Maruthachalam | Tumour cell dissemination following endoscopic stent insertion[END_REF]. Clearance of CTC after surgery has been reported to be a quick event in most patients, possibly associated with the prognosis [START_REF] Patel | Clearance of circulating tumor cells after excision of primary colorectal cancer[END_REF]. A decrease in CTC was also reported after neoadjuvant chemoradiation for rectal cancers [START_REF] Kienle | Decreased detection rate of disseminated tumor cells of rectal cancer patients after preoperative chemoradiation: a first step towards a molecular surrogate marker for neoadjuvant treatment in colorectal cancer[END_REF]. Some other studies have focused on the potential filter role of the liver on CTC dissemination, and compared CTCs in synchronous samples obtained from the portal vein, sus-hepatic veins, and/or the peripheral blood [START_REF] Koch | Comparative analysis of tumor cell dissemination in mesenteric, central, and peripheral venous blood in patients with colorectal cancer[END_REF].

Globally, cytological techniques, including the CellSearch® technique, have reported very low detection rates in stage II and III colon cancers [START_REF] Thorsteinsson | Low number of detectable circulating tumor cells in non-metastatic colon cancer[END_REF]. With molecular techniques, the different published results were reported as heterogeneous, especially for CTC prognostic impact [START_REF] Bessa | Lack of prognostic influence of circulating tumor cells in peripheral blood of patients with colorectal cancer[END_REF],

and no clear conclusion could be drawn. Therefore, a first meta-analysis was published in 2008 based of nine studies that used molecular detection (646 patients) [START_REF] Katsuno | Does the presence of circulating tumor cells in the venous drainage of curative colorectal cancer resections determine prognosis? A meta-analysis[END_REF]. Two interesting conclusions were reported: (i) CTC detection in the portal flow is correlated with nodal invasion (21% of pN0 patients vs. 50% of pN+ patients); and (ii) CTC status at the time of surgery is correlated with disease-free survival and to further liver metastatic relapse, independently of tumor stage (I, II, or III). One of the largest studies in stage II patients (n=194) was published almost synchronously, and reported detection of CTCs in peripheral blood using four mRNAs (CEA, hTERT, CK19, CK20) [START_REF] Uen | Prognostic significance of multiple molecular markers for patients with stage II colorectal cancer undergoing curative resection[END_REF]. In this pilot study, the combination of tumor-invasion depth, vascular invasion, and mRNA markers as predictors of relapse, showed that patients with any one positive predictor had a hazard ratio, of about 27-fold, of developing a postoperative relapse (p<0.001). This result has not been confirmed, but may be of critical importance when selecting stage II cancer patients who may benefit from adjuvant chemotherapy.

A larger meta-analysis has been published recently, which included 36 studies that reported on the detection of DTCs in bone-marrow, on CTCs in the portal vein, and systemic circulation of CTCs, using several molecular and cytological techniques in 3094 patients with nonmetastatic colorectal cancers [START_REF] Rahbari | Metaanalysis shows that detection of circulating tumor cells indicates poor prognosis in patients with colorectal cancer[END_REF]. When pooled together, cancer-cell detection studies were significantly associated with shorter recurrence-free intervals and overall survival rates. When they looked separately at different sampling sites, only peripheral blood CTCs had a prognostic value, whereas bone-marrow DTCs or portal-vein CTCs were not significantly associated with overall survival.

Colorectal adenocarcinoma: the metastatic setting 7.1 DTCs

Bone-marrow DTC detection in the metastatic setting has been reported only in small studies [START_REF] Vlems | Detection of disseminated tumour cells in blood and bone marrow samples of patients undergoing hepatic resection for metastasis of colorectal cancer[END_REF][START_REF] Vogelaar | Clinical impact of different detection methods for disseminated tumor cells in bone marrow of patients undergoing surgical resection of colorectal liver metastases: a prospective follow-up study[END_REF], and has no clear prognostic significance. This is consistent with similar findings for metastatic breast cancer [START_REF] Bidard | Prognosis of women with stage IV breast cancer depends on detection of circulating tumor cells rather than disseminated tumor cells[END_REF].

CTCs

CTCs have been also detected by cytological or molecular techniques in this setting. Several small studies have been published on molecular techniques, using the same markers as already discussed, alone or in combination with CEA, CK 8/18/19/20, hTERT, MUC1/2... Interestingly, detection of K-RAS mutations has been also investigated [START_REF] Nakamori | Genetic detection of colorectal cancer cells in circulation and lymph nodes[END_REF][START_REF] Thebo | K-ras mutational analysis of polyclonal colorectal cancers identifies uniclonal circulating tumor cells[END_REF][START_REF] Iinuma | Detection of tumor cells in blood using CD45 magnetic cell separation followed by nested mutant allele-specific amplification of p53 and K-ras genes in patients with colorectal cancer[END_REF], as have p53 mutations [START_REF] Nakamori | Genetic detection of colorectal cancer cells in circulation and lymph nodes[END_REF][START_REF] Iinuma | Detection of tumor cells in blood using CD45 magnetic cell separation followed by nested mutant allele-specific amplification of p53 and K-ras genes in patients with colorectal cancer[END_REF]100]. Here, again, no molecular marker demonstrated superiority in these small studies.

In metastatic colorectal cancers, surgical teams have focused on patients with resectable liver metastases. In this specific setting, detection of CTCs may reflect an active hematogeneous dissemination process and, therefore, may help to distinguish patients who can experience a short progression-free interval after surgery from those who can have longer complete remission [START_REF] Uchikura | Intraoperative molecular detection of circulating tumor cells by reverse transcriptionpolymerase chain reaction in patients with biliary-pancreatic cancer is associated with hematogenous metastasis[END_REF]101]. Similar concerns for gastric cancers have been raised regarding autologous bloodtransfusion safety during hepatic surgery [START_REF] Obayashi | Safety and utility of autologous blood transfusion for resection of metastatic liver tumor[END_REF]. CTC release during radiofrequency ablation of liver metastases was also reported to be higher than during surgical resection [102]. A randomized clinical trial has been proposed to compare two surgical procedures for CTC detection during surgery as the primary endpoint [103].

For chemotherapy management, some reports with limited patient numbers, have suggested that variations in CTC counts may be associated with treatment response [104]. For this application, the cytological CellSearch® technique has achieved very strong evidence, with several studies published on metastatic colorectal cancers (Table 2). Based on the study reported by Cohen et al.

[105], the CellSearch® system gained, in 2007, FDA approval as an aid to monitor metastatic colorectal-cancer patients. This study reported the strong and independent prognostic impact of CTC-positivity (≥3CTC/7.5ml) at baseline, but also of early CTC changes after 3-5 weeks of treatment (chemotherapy with or without bevacizumab). In a large ancillary study to the CAIRO2 trial (chemotherapy + bevacizumab, with or without cetuximab), Tol et al

[106] reported similar findings at baseline, but a lower CTC-positivity rate after 3-5 weeks of treatment (Table 2).

The correlation with outcome must, therefore, have been inconclusive, and the CTC count after 1-3 weeks of treatment was analyzed instead of its correlation with treatment outcome.

The discrepancy between these studies may be due to the systematic use of bevacizumab in the second trial, which has been reported in breast cancer to lower the CTCdetection rate [107]. Without further research, it is unclear when to perform a CTC count after the start of treatment (i.e., weeks 1-2 vs. weeks 3-5) to detect the best correlation with outcome.

Moreover, these observational studies have focused on patients with good health and have not demonstrated that "managing treatment" according to CTC-count leads to a better outcome.

Interventional randomized trials are therefore needed to compare the standard clinical/radiological vs. quantitative CTC-guided management of metastatic colorectal-cancer patients. Moreover, CTC positivity under treatment corresponds to cancers that are spontaneously resistant to a first-line regimen, and there is no evidence to support the idea that introducing a second-line regimen, according to the CTC count under treatment or to radiological evaluation, which occurs 1-2 months later, will help improve the survival of these refractory patients.

Therefore, it is likely that the main management of chemotherapy according to quantitative CTC changes will be the earlier discontinuation of expensive, harmful, and worthless treatments for some patients, and a gain in the cost/effectiveness ratio, rather than an improvement in survival. CTC may be also of interest to patients with potentially resectable liver metastases, i.e., metastases that may be surgically removed if metastasis shrinkage is obtained.

These patients usually received intensive polychemotherapy combined with a targeted therapy, and CTC might help to select those patients who will most benefit from this treatment strategy. As an example, Figure 1 shows the design of the CTC companion-study in a recently started large multicenter trial.

Hepatocarcinoma (HCC)

Non-metastatic hepatocarcinomas are treated whenever possible by local treatments, which may include liver resection, liver allograft, chemoembolization, and alcoholization. Beyond the issue of a possible surgery-induced CTC release [108], any metastasis-associated biomarker could be clinically relevant, as most patients relapse after treatment of the primary tumor. Following initial studies, which have reported that AFP mRNA detection in blood was associated with disease stage [109,110], several CTC molecular-detection studies have been published (Table 3):

heterogeneous clinical results were reported, whereas AFP mRNA specificity was discussed [111,112]. DTC detection by the same marker has been also reported in small studies [113].

Albumin, human telomerase (hTERT), and melanoma antigen gene-1 (MAGE1) mRNAs detection were also investigated, in blood, in a few small studies [114][115][116][117].

Cytological detection methods have also been used in HCC. The ISET technique, which isolates CTC by size on a filter, has been primarily developed in HCC [118]. In 44 non-metastatic HCC patients, CTC-detection rate was about 50% and was associated with shorter survival [119].

Similar detection rates (45%) were reported more recently with the CellSearch® system in 20 patients (≥1 CTCs/7.5ml), but these patients either had metastatic or locally advanced HCC [120].

Finally, circulating CD45 -/CD90 + cells, described as HCC "stem cells", have been found by flowcytometry in 31 out of 34 (90%) non-metastatic HCC patients, but the correlation with clinical outcome was not reported [121].

Conclusion

Globally, molecular tools used for CTC and DTC detection, based on their epithelial phenotype, are very heterogeneous, and it is hazardous to assess their importance based of them being a small series. Most of the reports in the literature had positive results (i.e., association with outcome), but publication bias may seriously distort any attempts, including those of the published meta-analyses, to estimate the effect of CTC/DTC detection. One may hypothesize that molecular techniques were mostly developed by isolated academic teams, without any strong effort to standardize these techniques. Cytological-detection studies were initially academydriven, and gave interesting insights into the dissemination process and the genetic evolution from the primary tumor, to DTCs, to overt macrometastasis. This technical heterogeneity among these studies has paved the way for the development of the semi-automated and standardized

CellSearch® system, with its development also benefiting large industry-sponsored clinical studies. This system became the 2011 gold standard for CTC detection, and is currently used in exploratory studies for several GI cancer types. The FDA-cleared quantitative approach to manage the chemotherapy of metastatic colorectal cancers is, however, not fully validated, and its clinical interest remains unknown. As recalled in a recent methodological review [122], the validation of CTC changes, as a recognized surrogate end-point for PFS will require data that demonstrate that CTC-changes are prognostic of PFS, but also that the effect of treatment on CTC-changes correlate with that of PFS; this will require the accumulation of data into a large meta-analysis.

The future of DTC and CTC in gastrointestinal cancers could be divided into two main issues. First, the quantitative count obtained by cytological techniques, such as CellSearch®, will be compared to several promising new blood-derived biomarkers, such as circulating tumor DNA or circulating miRNA. However, the future of CTCs as a quantitative biomarker appears somehow compromised by the detection of cancer-specific mutations (e.g., K-RAS mutations) in the blood.

The second issue is that CTC may represent, in the near future, a kind of "liquid biopsy", and provide new insights into tumor biology. The qualitative analysis of CTCs may guide treatment choice at diagnosis, but may also be repeated under treatment to elucidate resistance pathways, without an invasive tumor biopsy. CTCs may become a useful marker to assess many tumor targets by multiple immunostaining, FISH, and/or single-cell RNA/DNA analysis.

Table 1: CTC detection by molecular techniques in gastric adenocarcinoma

Studies with ≤20 patients or without an outcome analysis were not included. Impact on survival was not systematically assessed in multivariate analysis. N/A: not available or not relevant (because M0 and M1 patients were pooled). NS: not significant. DMFS: distant metastasis-free survival;

DFS: disease-free survival; OS: overall survival.

  primary enrichment, followed by DTC/CTC detection. Almost every isolation technique relies on an initial CTC/DTC-enrichment step, generally based on positive immunoselection of cells expressing an epithelial membranous marker, such as epithelial-cell adhesion (EpCAM, MUC1…). Other enrichment techniques have been also reported: size-based differential filtering (ISET), CD45-based leukocyte-negative immunoselection… None of these techniques have a 100% yield for CTC/DTC purification, and the use of multiple-antigen selection (by combining different antibodies) has not yet demonstrated clear superiority over single-antigen selection.

Figure 1 :

 1 Figure 1: CirCe 03, an ancillary study to the Prodige14-METHEPII-ACCORD21 trial

Table 2 : CTC detection in colorectal cancer using the CellSearch® system

 2

	Reference	Patients	CTC detection rate (threshold)	Clinical significance
	Allard, 2004 [10]	N=196 Stage IV	Stage IV: 30% Threshold ≥2CTC/7.5ml	N/A
	Cohen, 2006	N=50	Stage IV: 38%	-CTC positivity was associated with disease
	[134]	Stage IV	Threshold ≥2CTC/7.5ml	status (progression vs. non-progression).
	Sastre, 2008		Stage II : 21%
	[135]	N= 94	Stage III: 24%
	Maestro 2009	Stage I-IV	Stage IV: 61%
	[136]		Threshold ≥2CTC/7.5ml
				-CTC positivity at baseline was independently
	Cohen 2008		Stage IV :	associated with worse PFS (5 vs. 8 months) and
	[105]	N=430	33% before treatment	OS (9 vs. 18 months).
	Cohen 2009	Stage IV	12% at week 3-5	-Among initially CTC positive patients, those
	[137]		Threshold ≥3CTC/7.5ml	who become CTC negative at weeks 3-5 had a
				better PFS (6 vs. 2 months).
	Hiraiwa 2008 [32]	N=40	Stage I-III: 18% Stage IV: 41% Threshold ≥2CTC/7.5ml	-CTC positivity was not associated with outcome in the 29 stage-IV patients
	Jiao 2009 [102]	N=29, with surgically resected liver metastases	N/A 7.5ml of blood analyzed at different sites (peripheral, portal and hepatic veins)	-Surgical resection immediately reduced the number of CTCs compared with radiofrequency ablation, which was associated with an increased number of CTCs -Liver and lungs act as filters for CTCs
				-CTC positivity at baseline was associated with
			Stage IV:	worse PFS (8 vs. 10.5 months) and OS (14 vs.
	Tol 2010 [106]	N=467	29% before treatment 5% at week 3-5	22 months). -Among initially positive patients, those who
			Threshold ≥3CTC/7.5ml	became CTC negative at weeks 1-2 had a better
				PFS (8 vs. 4 months)
	Papavasiliou 2010 [138]	N=20, with surgically resected liver metastases	Preoperative :10% Peroperative: 50% Threshold ≥3CTC/30ml Postoperative:5%	Postoperative CTC detection was associated with DMFS and OS.

Table 3 : Molecular detection of AFP mRNA in hepatocarcinoma (HCC)

 3 Studies with ≤20 patients or without an outcome analysis were not included. Impact on survival was not systematically assessed in multivariate analysis.Periph. Cath: peripheral catheter; Centr. Cath: central catheter N/A: not available DMFS: distant metastasis-free survival; DFS: disease-free survival; OS: overall survival.

	Reference	Number of HCC patients	Blood-puncture location and timing	CTC detection rate	Clinical significance
	Louha M,1997 [139]	84	Periph. Cath. or embolization therapy Before alcoholization??	33%	DMFS
	Matsumura M,1999 [140]	88	Periph. Cath. embolization therapy Before alcoholization or	62%	DMFS and OS
	Wong IH,1999 [115] + 2000 [141]	62	Periph. Cath. surgery Before, during, and after	25% of samples	DMFS
	Kienle P, 2000 [113]	24	Centr. Cath. Before, during, and after surgery	29-45% of patients (time-dependent)	No significance
	Miyamoto A, 2001 [142]	23	N/A surgery Before, during, and after	25% of samples	DFS
			Periph Cath
	Witzigmann H, 2002 [143]	85	Before, during, and after surgical or palliative	28%	No significance for surgery-treated patients
			treatment
	Gross-Goupil M, 2003 [144]	52	Before and after embolization therapy	24%	No significance
	Cillo U, 2004 [112]	40	Periph. Cath. Before surgery	40%	DFS and OS
	Jeng KS, 2004 [145,146]	81	Periph. Cath. 12 weeks after surgery	23%	DFS
	Morimoto O, 2005 [147]	38	Periph. Cath. Before surgery	24%	DMFS and DFS

Conflict of interest statement: none

Funding source: Institut Curie incitative and collaborative programs fund ("PIC CTC"). The funding source had no role in the collection, analyses, and interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication.