Circulating Tumour Microemboli Research Articles

The Impact of Surgery on Circulating Malignant Tumour Cells in Oral Squamous Cell Carcinoma.

The extent to which surgical management of oral squamous cell carcinoma (OSCC) disseminates cancer is currently unknown. To determine changes in numbers of malignant cells released into systemic circulation immediately following tumour removal and over the first seven post-operative days. An observational study from March 2019 to February 2021. This study was undertaken at Queen Mary University Hospital, Hong Kong. Patients with biopsy-proven oral SCC were considered for eligibility. Patients under 18 years of age, pregnant or lactating women and those unable to understand the study details or unable to sign the consent form were excluded. Twenty-two patients were enrolled (12 male and 10 female) with mean age of 65.5 years. Primary tumour management was performed in accord with multi-disciplinary team agreement. Anaesthesia and post-operative care were unaltered and provided in accord with accepted clinical practice. Three types of malignant cells detected in peripheral blood samples were enumerated and sub-typed based on the presence of chromosomal aneuploidy and immunohistochemical characteristics. To test the hypothesis that malignant cells are released by surgery, the numbers of single circulating tumour cells (CTCs), circulating tumour microemboli (CTM) and circulating endothelial cells (CTECs) were recorded pre-operatively, upon tumour removal and the second and seventh post-operative days. Of a potential 88 data collection points, specimens were not obtainable in 12 instances. Tumour removal resulted in a statistically significant increase in CTCs and a non-statistically significant rise in CTMs. CTCs, CTMs and CTECs were detected in the majority of patients up to the seventh post-operative day. Individual patients demonstrated striking increases in post-operative CTCs and CTECs numbers. Surgical management of OSCC has a significant impact on the systemic distribution of cancer cells. Malignant cells persisted post-operatively in a manner independent of recognised staging methods suggesting differences in tumour biology between individuals. Further investigation is warranted to determine whether circulating malignant cell enumeration can be used to refine risk stratification for patients with OSCC.

A comparison of two methods for the detection of circulating tumour cells in patients with oral cavity cancer.

Circulating tumour cells (CTCs) detected in patient blood samples are relevant as diagnostic and prognostic markers offering insights into tumour behaviour and guiding treatment of cancer at an individualised level. The aim of this study was to ascertain the feasibility of detecting CTCs in oral squamous cell carcinoma (OSCC) using two different methods so as to determine the optimal method for the study of this cancer. Comparison of the numbers of CTCs, circulating tumour micro-emboli (CTMs) and circulating tumour endothelial cells (CTECs), was undertaken in forty clinical samples of oral squamous cell carcinoma (OSCC) determined by filtration (ISET® ) and in situ fluorescent immunostaining (i-FISH, Cytelligen® ) immunostaining and in situ hybridisation. i-FISH detected CTCs in 80% of samples compared with 40% of samples analysed by microfiltration. i-FISH detected CTCs in a further 40% of samples in which microfiltration did not detect CTCs. No CTC clusters were detected by microfiltration while i-FISH detected CTM in 12.5% of samples. i-FISH analysis detected CTECs in 20/40 samples. These results highlight significant differences in detection of CTCs, CTM and CTECs between i-FISH and microfiltration when applied to OSCC samples, suggesting that technologies capable of detecting circulating aneuploid cells more accurately detect CTCs. i-FISH also detected CTM and CTEC not detected using ISET® . With proven prognostic relevance in adenocarcinomas, accurate enumeration of CTCs, CTMs and CTECs may be a clinically useful tool in the management of OSCC and may aid in the reduction of false-negative diagnoses.

Clinical significance of circulating tumour cells and Ki-67 in renal cell carcinoma

BackgroundRenal cell carcinoma (RCC) is a common malignant tumour of the genitourinary system. We aimed to analyse the potential value of metastasis-related biomarkers, circulating tumour cells (CTCs) and the proliferative marker Ki-67 in the diagnosis of RCC.MethodsData from 24 laparoscopic radical nephrectomies (RNs) and 17 laparoscopic partial nephrectomies (PNs) were collected in 2018. The numbers and positive rates of CTCs and circulating tumour microemboli (CTM) in the peripheral blood were obtained at three different time points: just before surgery, immediately after surgery and 1 week after surgery. Ki-67 protein expression was evaluated in the RCC tissue by immunohistochemistry.ResultsExcept for the statistically significant association between the preoperative CTC counts and tumour size, no association between the number and positive rate of perioperative CTCs and clinicopathological features was found. The CTC counts gradually decreased during the perioperative period, and at 1 week after surgery, they were significantly lower than those before surgery. High Ki-67 expression was significantly positively correlated with preoperative CTC counts. In addition, Ki-67 expression was higher in the high CTC group (≥ 5 CTCs).ConclusionOur results suggest that surgical nephrectomy is associated with a decrease in CTC counts in RCC patients. CTCs can act as a potential biomarker for the diagnosis and prognosis of RCC. A careful and sufficient long-term follow-up is needed for patients with high preoperative CTC counts.

Capture of Circulating Tumour Cell Clusters Using Straight Microfluidic Chips.

Circulating tumour cells (CTCs) are the metastatic precursors to distant disease in head and neck cancers (HNCs). Whilst the prognostic and predictive value of single CTCs have been well documented, the role of CTC clusters, which potentially have a higher metastatic capacity are limited. In this study, the authors used a novel straight microfluidic chip to focus and capture CTCs. The chip offers high cell recoveries with clinically relevant numbers (10–500 cells/mL) without the need for further purification. Single CTCs were identified in 10/21 patient samples (range 2–24 CTCs/mL), CTC clusters in 9/21 patient samples (range 1–6 CTC clusters/mL) and circulating tumour microemboli (CTM) in 2/21 samples. This study demonstrated that CTC clusters contain EGFR amplified single CTCs within the cluster volume. This novel microfluidic chip demonstrates the efficient sorting and preservation of single CTCs, CTC clusters and CTMs. The authors intend to expand this study to a larger cohort to determine the clinical implication of the CTC subsets in HNC.

Detection of Circulating Tumour Cells in Urothelial Cancers and Clinical Correlations: Comparison of Two Methods

Circulating tumour cells (CTC) are identified exploiting their protein/gene expression patterns or distinct size compared to blood cells. Data on CTC in bladder cancer (BC) are still scarce. We comparatively analyzed CTC enrichment by AdnaTest ProstateCancerSelect (AT) and ScreenCell®Cyto (SC) kits, combined with identification by EPCAM, MUC1, and ERBB2 expression and by cytological criteria, respectively, in 19 nonmetastatic (M0) and 47 metastatic (M+) BC patients, at baseline (T0) and during treatment (T1). At T0, CTC positivity rates by AT were higher in M+ compared to M0 cases (57.4% versus 25%, p = 0.041). EPCAM was detected in 75% of CTC-positive samples by AT, showing increasing expression levels from T0 to T1 (median (interquartile range, IQR): 0.18 (0.07–0.42) versus 0.84 (0.33–1.84), p = 0.005) in M+ cases. Overall, CTC positivity by SC was around 80% regardless of clinical setting and time point of analysis, except for a lower occurrence at T1 in M0 cases. At T0, circulating tumour microemboli were more frequently (25% versus 8%) detected and more numerous in M+ compared to M0 patients. The approach used for CTC detection impacts the outcome of CTC studies. Further investigations are required to clarify the clinical validity of AT and SC in specific BC clinical contexts.

Circulating tumour cells in regionally metastatic cutaneous squamous cell carcinoma: A pilot study.

BackgroundCirculating tumour cells (CTCs) are increasingly being used in the surveillance of cancer, allowing for potential early detection and real-time monitoring of disease progression. The presence of CTCs in patients with metastatic cutaneous head and neck squamous cell carcinoma (cHNSCC) has not been evaluated.ResultsCTCs were detected in eight of ten patients with regional metastatic cHNSCC (80%; range 1–44 cells/9 mL blood). CTMs were detected in three of ten patients (30%, range 1–4 cells/9 mL blood).MethodsPreoperative blood samples from ten patients with nodal metastases from cutaneous squamous cell carcinomas (cSCC) were analyzed using the IsoFluxTM System for the detection and enumeration of CTCs and circulating tumour microemboli (CTMs).ConclusionsFor the first time CTCs have been detected in patients with nodal metastases from cHNSCC. Further work is required to understand their prognostic significance and potential to directly influence clinical practice.

Prevalence and number of circulating tumour cells and microemboli at diagnosis of advanced NSCLC.

Timing and magnitude of blood release of circulating tumour cells (CTC) and circulating tumour microemboli (CTM) from primary solid cancers are uncertain. We investigated prevalence and number of CTC and CTM at diagnosis of advanced non-small cell lung cancer (NSCLC). Twenty-eight consecutive patients with suspected stage III-IV lung cancer gave consent to provide 15 mL of peripheral blood soon before diagnostic CT-guided fine-needle aspiration biopsy (FNAB). CTC and CTM (clusters of ≥3 CTC) were isolated by cell size filtration (ScreenCell), identified and counted by cytopathologists using morphometric criteria and (in 6 cases) immunostained for vimentin. FNAB demonstrated NSCLC in 26 cases. At least one CTC/3 mL blood (mean 6.8 ± 3.7) was detected in 17 (65 %) and one CTM (mean 4.5 ± 3.3) in 15 (58 %) of 26 NSCLC cases. No correlation between number of CTC or CTM and tumour type or stage was observed. Neoplastic cells from both FNA and CTC/CTM were positive for vimentin but heterogeneously. CTC can be detected in two-thirds and CTM in more than half of patients with advanced NSCLC at diagnosis. Reasons underlying lack of CTC and CTM in some advanced lung cancers deserve further investigations.

Prevalence and heterogeneity of circulating tumour cells in metastatic cutaneous melanoma

We previously demonstrated that circulating tumour cells (CTCs) are detectable by the MelCAM and high molecular weight melanoma-associated antigen (HMW-MAA)-dependent CellSearch platform. However, CTCs which do not express these capture and detection markers are not detectable by CellSearch. Consequently, we explored the use of isolation by size of epithelial tumour cells (ISET), a marker independent, filtration-based device to determine the prevalence and heterogeneity of CTCs in metastatic cutaneous melanoma patients. Ninety patients were prospectively recruited and blood samples taken before treatment. Patients' blood was filtered using the ISET platform. CTCs were enumerated using dual immunohistochemistry with positive selection by S100 expression and exclusion of leucocytes and endothelial cells expressing CD45 or CD144, respectively. A panel of markers (Melan-A, MITF, MelCAM, high molecular melanoma-associated antigen, CD271 and MAGEC) was also examined. Fifty-one patients (57%) had CTCs (range 1-44 CTCs/4 ml blood) and 12 patients also had circulating tumour microemboli. Seven patients had S100- CTCs, 11 patients' CTCs were S100+ and 33 patients had S100+ and S100- CTCs. Substantial intrapatient and interpatient heterogeneity was observed for all other melanoma-associated markers. CTCs in metastatic cutaneous melanoma are detectable using the flexible marker-independent ISET platform. CTCs display significant marker expression heterogeneity implying that marker-dependent platforms would not detect all CTCs and multimarker assays are now required to reveal the biological significance of this CTC heterogeneity.

Abstract 1468: Circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) from patients with small cell lung cancer (SCLC) show heterogeneity in epithelial to mesenchymal transition (EMT) phenotype and evidence of vasculogenic mimicry (VM).

Abstract Background: Circulating tumour cells (CTCs) and circulating tumour microemboli (CTM) are prevalent in patients with aggressive small cell lung cancer (SCLC). Heterogeneity in epithelial and mesenchymal phenotypes in CTCs and within CTM was investigated given evidence in preclinical models of epithelial and mesenchymal cell co-operativity advantageous for hematogenous spread and the role of epithelial to mesenchymal transition (EMT) in invasion and metastasis. Methods: Four colour immunofluorescence assays were developed and applied to tumour cells enriched by filtration from peripheral blood of extensive stage SCLC patients prior to treatment. CTCs/CTM were assessed for expression of cytokeratins (CKs, epithelial marker) and vimentin (mesenchymal marker) with DAPI stained nuclei and negative selection of leukocytes and circulating endothelial cells (CECs) by CD45 and/or vascular endothelial-cadherin (VE-cadherin, CD144) expression respectively. Results and Conclusions: CTCs were identified in all patients that expressed epithelial and/or mesenchymal markers. Individual CTM were detected comprised of tumour cells of heterogeneous EMT status and CTM were also observed containing leukocytes and/or CECs. There was substantial heterogeneity in CK and vimentin expression in CTCs and within CTM consistent with EMT plasticity and with co-operativity between epithelial and mesenchymal tumour cells. A subpopulation of CTCs co-expressed CKs and VE-cadherin that intriguingly, raises the possibility that vascular mimicry accompanies and may facilitate hematogenous spread in this aggressive tumor type. Further studies are required to explore this new hypothesis. Citation Format: Robert Metcalf, Jessica Booth, Rajeeb Swain, Steven Bagley, Karen Morris, Matthew Krebs, Andrew Hughes, Gerard Brady, Fiona Blackhall, Caroline Dive. Circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) from patients with small cell lung cancer (SCLC) show heterogeneity in epithelial to mesenchymal transition (EMT) phenotype and evidence of vasculogenic mimicry (VM). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1468. doi:10.1158/1538-7445.AM2013-1468

9 Elucidating the role of the NFkB pathway in cisplatin resistant NSCLC

isolating CTCs are still in their infancy. One such method is the use of the modified invasion assay, VitaAssay. This technique uses CAM (Collagen Adhesion Matrix) coated plates to capture CTCs with an invasive phenotype. Another method of CTC detection is ScreenCell. This technique uses a polycarbonate filtration membrane containing multiple tiny pores. When blood flows across the membrane, tumour cells are captured due to their greater size. Method: Peripheral blood samples were obtained from patients with advanced NSCLC. In addition healthy blood samples spiked with NSCLC cell line cells were also analysed using both VitaAssay and Screencell. VitaAssay: Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density centrifugation. PBMCs were seeded onto VitaAssay plates and cultured for 12 18 hrs. The supernatant is removed and the remaining captured cells are enriched for CTCs. Captured cells can be fixed and stained using immunocytochemistry. ScreenCell: Peripheral blood is diluted with a specified buffer and filtered across the Screencell filter. The filter with captured cells is mounted on a slide for fixation and staining. Fixed CTCs on the filters are then stained using H&E and/or immunocytochemistry. Results: Cells captured from NSCLC patients using VitaAssay were stained for EpCAM/pan-Cytokeratin and CD45. EpCAM/Pan-CK positive, CD45 negative cells were classed as CTCs. In healthy blood samples spiked with A549 and H2228 cells, approximately 20% (range 9 26.4%) of spiked cells were recovered using VitaAssay. In NSCLC patients an average of 30.67 CTCs per ml of blood were identified (range 14 52, n = 6). Using the ScreenCell technique CTCs were identified by size & morphology using H&E staining. In cell spiking experiments, approximately 70% of spiked cells were recovered using Screencell. Also, CTCs were detected in 70% of patient samples (n = 10). Numbers of CTCs detected ranged from 6 82 per ml of blood. In addition, clumps of tumour cells or Circulating Tumour Microemboli were detected in 50% of patient samples. Conclusions: The VitaAssay and ScreenCell techniques both appear to be viable method of isolating CTCs in NSCLC, in both model cell-spiking experiments and in clinical samples as determined by morphology and antigen expression detected with immunocytochemistry.

8 PIK3CA mutations and response to PI3K and MEK targeted therapies in NSCLC

isolating CTCs are still in their infancy. One such method is the use of the modified invasion assay, VitaAssay. This technique uses CAM (Collagen Adhesion Matrix) coated plates to capture CTCs with an invasive phenotype. Another method of CTC detection is ScreenCell. This technique uses a polycarbonate filtration membrane containing multiple tiny pores. When blood flows across the membrane, tumour cells are captured due to their greater size. Method: Peripheral blood samples were obtained from patients with advanced NSCLC. In addition healthy blood samples spiked with NSCLC cell line cells were also analysed using both VitaAssay and Screencell. VitaAssay: Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density centrifugation. PBMCs were seeded onto VitaAssay plates and cultured for 12 18 hrs. The supernatant is removed and the remaining captured cells are enriched for CTCs. Captured cells can be fixed and stained using immunocytochemistry. ScreenCell: Peripheral blood is diluted with a specified buffer and filtered across the Screencell filter. The filter with captured cells is mounted on a slide for fixation and staining. Fixed CTCs on the filters are then stained using H&E and/or immunocytochemistry. Results: Cells captured from NSCLC patients using VitaAssay were stained for EpCAM/pan-Cytokeratin and CD45. EpCAM/Pan-CK positive, CD45 negative cells were classed as CTCs. In healthy blood samples spiked with A549 and H2228 cells, approximately 20% (range 9 26.4%) of spiked cells were recovered using VitaAssay. In NSCLC patients an average of 30.67 CTCs per ml of blood were identified (range 14 52, n = 6). Using the ScreenCell technique CTCs were identified by size & morphology using H&E staining. In cell spiking experiments, approximately 70% of spiked cells were recovered using Screencell. Also, CTCs were detected in 70% of patient samples (n = 10). Numbers of CTCs detected ranged from 6 82 per ml of blood. In addition, clumps of tumour cells or Circulating Tumour Microemboli were detected in 50% of patient samples. Conclusions: The VitaAssay and ScreenCell techniques both appear to be viable method of isolating CTCs in NSCLC, in both model cell-spiking experiments and in clinical samples as determined by morphology and antigen expression detected with immunocytochemistry.

Single-cell profiling of circulating tumour cells: a great leap forward

Single-cell profiling of circulating tumour cells: a great leap forward

227P - Circulating Melanoma Cells (CMCS) in Mucosal and Uveal Melanomas

ABSTRACT Background Melanoma subtypes are increasingly defined by genetic alterations (BRAF mutations in 50% and NRAS in 20% of cutaneous, c-Kit aberrations in acral and mucosal and GNA11 mutations in uveal melanomas. Targeted therapies such as vemurafenib in cutaneous and imatinib in mucosal melanomas have emerged. CMCs are a potential biomarker to assess response to treatment, monitor disease relapse and provide new targets for drug development. Molecular characterisation of CMCs may provide utility as a predictive biomarker to drug treatment. Methods The CellSearch marker dependent platform (Veridex, USA) enriches for CMCs utilising Melcam antibody for CMC capture and High Molecular Weight Melanoma associated Antigen (HMW-MAA) antibody for CMC detection. MART-1 was used as an additional CMC kit detection marker. The ISET marker independent platform (Isolation by Cell Size, Rarecells, France) enriches for CMCs based on cell size. Immunohistochemistry with CD45 and CD144 (leukocyte and endothelial cell markers) and S100 (melanoma marker) was performed to identify CMC characteristic staining and morphology. Results Twenty-three patients with metastatic disease were recruited prospectively (mucosal n = 13, uveal n = 10). CellSearch detected CMCs in 6/11mucosal (range 0-125, mean 21 median 1) and 7/10 uveal (range 0-510, mean 57 median 3) melanomas. MART-1 positive CMCs were detected in both subtypes but circulating tumour microemboli (CTM) were found only in mucosal melanomas. ISET detected CMCs in 10/12 mucosal (range 0-26, mean 4 median 2) and 7/10 uveal (range 0-19, mean 7 median 3) melanomas. CMCs were both S100+ and S100-. No CTM were detected by ISET. Comparison of the platforms in 20 patients (n = 10 of each mucosal and uveal melanomas) revealed variability in CMC detection between platforms (Spearman's correlation p = 0.17). Conclusions This is the first report of CMC detection by CellSearch in mucosal and uveal melanomas. Comparison with ISET revealed no relationship between numbers of CMCs detected in the same patients by each platform thus indicating significant CMC heterogeneity in CMC marker expression and size. Circulating tumour microemboli may indicate collective cell migration thus probing the biology of metastases formation. Disclosure G. Clack: Ownes stocks in Astra-Zeneca pharmaceuticals. A. Hughes: ownes shares in Astra-Zeneca pharmaceuticals. All other authors have declared no conflicts of interest.

Pancreatic circulating tumor cells: Where are they?

e21037 Background: We previously demonstrated that circulating tumor cell (CTC) numbers in advanced pancreatic carcinoma were low in contrast to other epithelial carcinomas. We hypothesized that pancreatic CTCs may sequest in the liver and conceived a study to explore CTC number in the peripheral and portal circulations of patients undergoing Whipple’s surgery. Methods: Seventeen patients undergoing Whipple’s surgery were recruited prospectively after informed written consent. Blood was collected intra-operatively from the portal vein prior to tumor mobilization and from the peripheral circulation. Paired samples were analysed for CTC number (CellSearch system, Veridex, USA), and M30/M65 biomarkers of cell death. Results: Of 17 patients 6 had cholangiocarcinomas and 11 had pancreatic adenocarcinomas (5 inoperable). CTC number in the portal circulation was consistently higher than in the peripheral circulation. All patients with cholangiocarcinoma had no CTCs in the peripheral circulation but two patients had 139 and 21 CTCs in the portal circulation. Five patients with pancreatic carcinoma had no CTCs in the peripheral circulation but 1 CTC in 2 patients, 4 CTCs in 2 patients and 32 and 731 CTCs were seen in 2 other patients in the portal circulation. Five patients had no CTCs in both circulations and the remaining patient had 1 CTC in the peripheral circulation and 4 CTCs in the portal circulation. Where present, circulating tumour microemboli were detected in the portal circulation only. M65 levels were higher in the portal circulation indicating higher levels of cell death. M30 levels did not differ significantly between circulations but appeared to correlate with portal CTC number. Conclusions: This is the first study to enumerate CTCs in peripheral and portal circulations in pancreato-biliary carcinomas. Higher CTC numbers are present in the portal circulation suggesting CTC sequestration in the liver. Five patients with pancreatic cancer (2 of whom were inoperable) had no CTCs in either circulation, a finding consistent with the poor vascularity and dense stromal characteristics of this carcinoma. Nonetheless the portal circulation may be a potential source for CTC characterisation including genomic analysis to further define tumor biology.

10IN NEW TOOLS IN THE MOLECULAR DIAGNOSTIC OF LUNG CANCER

What can CTCs tell us? Are CTCs a surrogate for tissue? What are the practical applications and current limitations of existing technologies for CTC detection and analysis? What is their clinical significance? Can they be molecularly characterised? How do they compare to cell free DNA detectable in blood? Can ‘stem cells’ be identified in the circulation? Answers to these questions are only just beginning to emerge. CellSearch is a semi-automated system that enriches and counts CTCs by EpCam based immunomagnetic capture. Cells are labelled with DAPI (nuclear stain) and antibodies to cytokeratins and CD45. CTCs are identified as EpCAM, DAPI and cytokeratin positive, and CD45 (white blood cell antigen) negative. ISET (Isolation by Size of Epithelial Tumours) detects CTCs by size exclusion and CD45 immunohistochemistry identifies contaminating white blood cells. Both techniques have limitations for practical application and provide complementary information. Cellsearch is more robust for enumerating CTCs whereas ISET is ‘EpCAM independent’ and may detect EpCAM negative cells that have undergone epithelial-mesenchymal transition. Using Cellsearch, the majority of patients with small cell lung cancer (SCLC) have detectable CTCs compared to less than half of patients with stage IV non-small cell lung cancer (NSCLC). CTCs decline following one cycle of chemotherapy, mirroring radiological response, and demonstrating a pharmacodynamic role. CTC number is an independent prognostic factor for survival. Of greatest interest are molecular characteristics. EpCAM negative CTCs can be detected by ISET, in addition to clusters of CTCs, termed circulating tumour microemboli (CTM). Preliminary studies of expression of markers of epithelial (E-Cadherin, Cytokeratin, EpCAM) and mesenchymal (N-cadherin, Vimentin) transition, apoptosis and proliferation, reveal distinct differences in marker expression for cells comprising CTM compared to solitary CTCs from the same patient. For example, apoptotic morphology is observed in solitary CTCs but not in CTM. Our data suggests that collective cell migration occurs in lung cancer and we hypothesise that this mode of cell travel around the circulation may pose the greatest threat. Disclosure: The author has declared no conflicts of interest. 10IN NEW TOOLS IN THE MOLECULAR DIAGNOSTIC OF LUNG CANCER

Circulating tumour cells in colorectal cancer

BackgroundThe detection of circulating tumour cells (CTC) in blood sample in patients with early or advanced colorectal cancer has a potential prognostic value. MethodsThe challenge of CTC detection is related to the requirement of high sensitivity combined with high specificity method. CTCs detection can be distinguished between indirect and direct methods. The former ones are based on the recognition of tissue-, organ- or tumour-specific markers by immuno-histochemistry (indirect immuno-mediated methods) or (real-time) RT-PCR (indirect molecular methods), whilst the latter are related to CTCs selection based on the physical properties of density and sizes. Ongoing and future isolation by size of epithelial tumour cells (ISET) developments concerning automated image analysis on the filter and transmission of high definition images through the web for ‘on line’ cytopathological consultations are aimed to speed up the work of cytopathologists on CTC/ circulating tumour microemboli (CTM) detection. ConclusionsCTC detection in colorectal cancer (CRC) correlates with pathological stage and clinical outcome in particular in those patients with advanced disease. CRC CTC level before and after CT are an independent prognostic factor for progression-free and overall survival. The positive prognostic value of complete clearance CTC after surgery may be useful to select patients for adjuvant chemotherapy.