Circulating tumor cell
Circulating tumor cells (CTCs) are cells that have shed into the vasculature from a primary tumor and circulate in the bloodstream. CTCs thus constitute seeds for subsequent growth of additional tumors (metastasis) in vital distant organs, triggering a mechanism that is responsible for the vast majority of cancer-related deaths.
CTCs were observed for the first time in 1869 in the blood of a man with metastatic cancer by Thomas Ashworth, who postulated that “cells identical with those of the cancer itself being seen in the blood may tend to throw some light upon the mode of origin of multiple tumours existing in the same person”. A thorough comparison of the morphology of the circulating cells to tumor cells from different lesions led Ashworth to conclude that “One thing is certain, that if they [CTC] came from an existing cancer structure, they must have passed through the greater part of the circulatory system to have arrived at the internal saphena vein of the sound leg”. Modern cancer research has demonstrated that CTCs derive from clones in the primary tumor, validating Ashworth's remarks. The significant efforts put into understanding the CTCs biological properties have demonstrated the critical role circulating tumor cells play in the metastatic spread of carcinoma. however, only recently it has been demonstrated that these circulating tumor cells reflect molecular features of cells within tumor masses. CTCs thus could be considered a “liquid biopsy” which reveals metastasis in action, providing live information about the patient’s disease status. Since the accessibility and ease of collection of blood, analysis of CTCs could be an invaluable tool for early stage detection of cancer as well as for neoplastic progression and recurrence monitoring. An important characteristic of a blood test is that it is safe and can be performed at many points during the disease, whereas analysis of solid tumors require invasive procedures that strongly limited the patient compliance. Moreover, the possibility to monitor over the time the disease progress allow to develop appropriate therapy modifications, potentially improving patient’s quality of life. For this purpose technologies with the requisite sensitivity and reproducibility to detect CTCs in patients with metastatic disease have been recently developed.
Frequency of CTCs
The detection of CTCs may have important prognostic and therapeutic implications but because their numbers can be very small, these cells are not easily detected. It is estimated, in fact, that among the cells that have been detached from the primary tumor, only the 0.01% can form metastases.
Circulating tumor cells are found in frequencies on the order of 1-10 CTC per mL of whole blood in patients with metastatic disease. For comparison, a mL of blood contains a few million white blood cells and a billion red blood cells, see figure 1. This low frequency, associated to difficulty of identifying cancerous cells, means that a key component of understanding CTCs biological properties rely on the development of technologies and approaches capable of isolating them in significant numbers (enrichment step) and preserving CTCs for further molecular, morphological and functional analysis. To date CTCs have been detected in several epithelial cancers (breast, prostate, lung, and colon) and clinical evidences indicate that patients with metastatic lesions are more likely to have CTCs isolated.
CTCs are usually captured from the vasculature by using specific antibodies able to recognize specific tumoral marker (usually EpCAM); however this approach is biased by the need for a sufficient expression of the selected protein on the cell surface, event necessary for the enrichment step. Moreover, since EpCAM and other proteins (e.g. cytokeratins) are not expressed in some tumors and can be down regulated during the epithelial to mesenchimal transition (EMT), new enrichment strategies are required.
First evidence indicates that CTC markers applied in human medicine are conserved in other species. Five of the more common markers including CK19 are also useful to detect CTC in the blood of dogs with malignant mammary tumors.
To date, a variety of research methods have been developed to isolate and enumerate CTC. The only U.S. Food and Drug Administration (FDA) cleared methodology for enumeration of CTC in whole blood is the CellSearch system. Extensive clinical testing done using this method shows that presence of CTC is a strong prognostic factor for overall survival in patients with metastatic breast, colorectal or prostate cancer, see figure 2 
This method is based on the use of iron nano-particles coated with a polymer layer carrying biotin analogues and conjugated with antibodies anti EpCAM for capturing CTCs, and on the use of an analyzer to take images of isolated cells upon their staining with specific fluorescent antibody conjugates. Blood is sampled in an EDTA tube with an added preservative. Upon arrival in the lab, 7.5mL of blood is centrifuged and placed in a preparation system. This system first enriches the tumor cells immunomagnetically by means of ferrofluid nano-particles and a magnet. Subsequently recovered cells are permeabilized and stained with a nuclear stain, a fluorescent antibody conjugate against CD45 (leukocyte marker), and cytokeratin 8, 18 and 19 (CKs). The sample is then scanned on an analyzer which takes images of the nuclear, cytokeratin, and CD45 stains. To be considered a CTC a cell must contain a nucleus, be positive for cytoplasmatic expression of cytokeratin as well as negative for the expression of CD45 marker, and have a diameter larger than 5 µm. If the total number of tumor cells found to meet the criteria cited above is 5 or more, a blood sample is positive. In studies done on prostate, breast and colon cancer patients, median survival of metastatic patients with positive samples is about half the median survival of metastatic patients with negative samples. This system is characterized by a recovery capacity of 93% and a detection limit of one CTC per 7.5 mL of whole blood. Despite its sensitivity and reproducibility, the CellSearch Method requires specific equipment to perform the analysis.
CTCs are pivotal to understanding the biology of metastasis and promise potential as a biomarker to noninvasively evaluate tumor progression and response to treatment. However, isolation and characterization of CTCs represent a major technological challenge, since CTCs make up a minute number of the total cells in circulating blood, 1–10 CTCs per mL of whole blood compared to a few million white blood cells and a billion red blood cells. Therefore the major challenge for CTC researchers is the prevailing difficulty of CTC purification that allows the molecular characterization of CTCs. Several methods have been developed to isolate CTCs in the peripheral blood and essentially fall into two categories: biological methods and physical methods.
Biological methods are separation based on antigen-antibody bindings. Antibodies against tumor specific biomarkers including EpCAM, Her2, PSA are used. The most common technique is magnetic nanoparticle-based separation (immunomagnetic assay) as used in CellSearch or MACS. Other techniques under research include microfluidic separation and combination of immunomagnetic assay and microfluidic separation. Oncolytic viruses such as vacinia viruses are developed to detect and identy CTCs.
Physical methods are often filter-based, enabling the capture of CTCs by size. ScreenCell is a filtration based device that allows sensitive and specific isolation of CTCs from human whole blood in a few minutes. Peripheral blood is drawn and processed within 4 hours with a ScreenCell isolation device to capture CTCs. The captured cells are ready for cell culture or for direct characterization using ViewRNA in situ hybridization assay.
Characterization of CTC
Any useful method for isolation of CTCs must allow (i) their identification and enumeration and (ii) their characterization through immunocytochemistry, fluorescence in situ hybridization (FISH) DNA and RNA assays, and all other relevant molecular techniques using DNA and RNA. When circulating tumor cells are captured from blood using filtration devices (such as ScreenCell isolation device), further morphological and molecular characterization is required to reveal important predictive information and report changes in CTC biology, for example during tumor relapse. ViewRNA assay for CTCs characterization is the only in situ hybridization technology that allows multiplex, single-molecule RNA detection of any RNA target. The exceptional sensitivity and specificity is achieved by using proprietary probe design, simultaneous branched DNA (bDNA) signal amplification and background suppression.
The captured CTCs on the filter membrane of a ScreenCell isolation device, are transferred to a 24-well cell culture plate for enumeration/characterization using ViewRNA ISH Cell Assay. A target-specific probe set containing 20 oligonucleotide pairs hybridizes to the target RNA. An oligo pair hybridization event is essential for support of the signal amplification structure, which is assembled by a series of sequential hybridization steps. Each fully assembled amplification structure is contained within 40−50 bp of target RNA with the capacity for 400-fold signal amplification. Therefore, a typical target-specific probe set (containing 20 oligo pairs) can generate 8,000-fold signal amplification at the location of the target RNA.
Further Characterisation of CTC
Some drugs are particularly effective against cancers which fit certain requirements. For example Herceptin is very effective in patients who are Her2 positive, but much less effective in patients who are Her2 negative. Once the primary tumor is removed, biopsy of the current state of the cancer through traditional tissue typing is not possible anymore. Often tissue sections of the primary tumor, removed years prior, are used to do the typing. Further characterisation of CTC may help determining the current tumor phenotype. FISH assays has been performed on CTC to as well as determination of IGF-1R, Her2, Bcl-2, [ERG (gene)|ERG], PTEN, AR status using immunofluorescence.
Morphological appearance is judged by human operators and is therefore subject to large inter operator variation. Several CTC enumeration methods exist which use morphological appearance to identify CTC, which may also apply different morphological criteria. A recent study in prostate cancer showed that many different morphological definitions of circulating tumor cells have similar prognostic value, even though the absolute number of cells found in patients and normal donors varied by more than a decade between different morphological definitions.
- Gupta, GP; Massagué, J (2006 Nov 17). "Cancer metastasis: building a framework.". Cell 127 (4): 679–95. doi:10.1016/j.cell.2006.11.001. PMID 17110329.
- Ashworth, T. R (1869). "A case of cancer in which cells similar to those in the tumours were seen in the blood after death". Australian Medical Journal 14: 146–7.
- Fehm, T; Sagalowsky, A; Clifford, E; Beitsch, P; Saboorian, H; Euhus, D; Meng, S; Morrison, L; Tucker, T; Lane, N; Ghadimi, BM; Heselmeyer-Haddad, K; Ried, T; Rao, C; Uhr, J (2002 Jul). "Cytogenetic evidence that circulating epithelial cells in patients with carcinoma are malignant.". Clinical cancer research : an official journal of the American Association for Cancer Research 8 (7): 2073–84. PMID 12114406.
- Fidler IJ (2003). "Timeline: The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited". Nature Reviews Cancer 3 (6): 453–8. doi:10.1038/nrc1098. PMID 12778135.
- Punnoose, EA; Atwal, SK; Spoerke, JM; Savage, H; Pandita, A; Yeh, RF; Pirzkall, A; Fine, BM; Amler, LC; Chen, DS; Lackner, MR (2010 Sep 8). "Molecular biomarker analyses using circulating tumor cells.". PLoS ONE 5 (9): e12517. doi:10.1371/journal.pone.0012517. PMC 2935889. PMID 20838621.
- Attard, G; Swennenhuis, JF; Olmos, D; Reid, AH; Vickers, E; A'Hern, R; Levink, R; Coumans, F; Moreira, J; Riisnaes, R; Oommen, NB; Hawche, G; Jameson, C; Thompson, E; Sipkema, R; Carden, CP; Parker, C; Dearnaley, D; Kaye, SB; Cooper, CS; Molina, A; Cox, ME; Terstappen, LW; de Bono, JS (2009 Apr 1). "Characterization of ERG, AR and PTEN gene status in circulating tumor cells from patients with castration-resistant prostate cancer.". Cancer Research 69 (7): 2912–8. doi:10.1158/0008-5472.CAN-08-3667. PMID 19339269.
- Cohen, SJ; Punt, CJ; Iannotti, N; Saidman, BH; Sabbath, KD; Gabrail, NY; Picus, J; Morse, M; Mitchell, E; Miller, MC; Doyle, GV; Tissing, H; Terstappen, LW; Meropol, NJ (2008 Jul 1). "Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer.". Journal of clinical oncology : official journal of the American Society of Clinical Oncology 26 (19): 3213–21. doi:10.1200/JCO.2007.15.8923. PMID 18591556.
- Yu M., Haber D. A., et al. (2012). "RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis". Nature 487 (7408): 510–3. doi:10.1038/nature11217.
- Sleijfer S, Gratama JW, Sieuwerts AM, et al. (2007). "Circulating tumour cell detection on its way to routine diagnostic implementation?". Eur J Cancer 43 (18): 2645–50. doi:10.1016/j.ejca.2007.09.016. PMID 17977713.
- Hayes DF, Smerage J. (2008). "Is There a Role for Circulating Tumor Cells in the Management of Breast Cancer?". Clin Cancer Res 14 (12): 3646–50. doi:10.1158/1078-0432.CCR-07-4481. PMID 18559576.
- Pantel K, Alix-Panabières C, Riethdorf (2009). "Cancer micrometastases". Nature Reviews Clinical Oncology 6 (6): 339–51. doi:10.1038/nrclinonc.2009.44. PMID 19399023.
- Pantel K, Riethdorf S. (2009). "Pathology: are circulating tumor cells predictive of overall survival?". Nature Reviews Clinical Oncology 6 (4): 190–1. doi:10.1038/nrclinonc.2009.23. PMID 19333222.
- Panteleakou Z, Lembessis P, Sourla A, et al. (2009). "Detection of circulating tumor cells in prostate cancer patients: methodological pitfalls and clinical relevance". Mol Med 15 (3-4): 101–14. doi:10.2119/molmed.2008.00116. PMC 2600498. PMID 19081770.
- Ghossein RA, Bhattacharya S, Rosai J (1999). "Molecular detection of micrometastases and circulating tumor cells in solid tumors". Clin. Cancer Res. 5 (8): 1950–60. PMID 10473071.
- Zhe, X; Cher M.L., Bonfil R.D. (2011). "Circulating tumor cells: finding the needle in the haystack". Am J Cancer Res 1 (6): 740–751.
- Miller, MC; Doyle, GV; Terstappen, LW (2010). "Significance of Circulating Tumor Cells Detected by the CellSearch System in Patients with Metastatic Breast Colorectal and Prostate Cancer.". Journal of oncology 2010: 617421. doi:10.1155/2010/617421. PMC 2793426. PMID 20016752.
- Swaby, RF; Cristofanilli, M (2011 Apr 21). "Circulating tumor cells in breast cancer: a tool whose time has come of age.". BMC medicine 9: 43. doi:10.1186/1741-7015-9-43. PMC 3107794. PMID 21510857.
- Danila, DC; Fleisher, M; Scher, HI (2011 Jun 15). "Circulating tumor cells as biomarkers in prostate cancer.". Clinical cancer research : an official journal of the American Association for Cancer Research 17 (12): 3903–12. doi:10.1158/1078-0432.CCR-10-2650. PMID 21680546.
- Tanaka, F; Yoneda, K; Kondo, N; Hashimoto, M; Takuwa, T; Matsumoto, S; Okumura, Y; Rahman, S; Tsubota, N; Tsujimura, T; Kuribayashi, K; Fukuoka, K; Nakano, T; Hasegawa, S (2009 Nov 15). "Circulating tumor cell as a diagnostic marker in primary lung cancer.". Clinical cancer research : an official journal of the American Association for Cancer Research 15 (22): 6980–6. doi:10.1158/1078-0432.CCR-09-1095. PMID 19887487.
- Negin, BP; Cohen, SJ (2010 Jun). "Circulating tumor cells in colorectal cancer: past, present, and future challenges.". Current treatment options in oncology 11 (1-2): 1–13. doi:10.1007/s11864-010-0115-3. PMID 20143276.
- Man, Yicun; Wang, Qing; Kemmner, Wolfgang (1 January 2011). "Currently Used Markers for CTC Isolation - Advantages, Limitations and Impact on Cancer Prognosis". Journal of Clinical & Experimental Pathology 01 (01). doi:10.4172/2161-0681.1000102.
- Mikolajczyk, SD; Millar, LS; Tsinberg, P; Coutts, SM; Zomorrodi, M; Pham, T; Bischoff, FZ; Pircher, TJ (2011). "Detection of EpCAM-Negative and Cytokeratin-Negative Circulating Tumor Cells in Peripheral Blood.". Journal of oncology 2011: 252361. doi:10.1155/2011/252361. PMC 3090615. PMID 21577258.
- da Costa A, Oliveira JT, Gärtner F, Kohn B, Gruber AD, Klopfleisch R. (2010). "Potential markers for detection of circulating canine mammary tumor cells in the peripheral blood.". Veterinary Journal. Epub Nov.2 2010. doi:10.1016/j.tvjl.2010.09.027. PMID 21051248.
- MC Miller, GV Doyle, LWMM Terstappen (2010). "Significance of Circulating Tumor Cells detected by the CellSearch System in Patients with Metastatic Breast Colorectal and Prostate Cancer". Journal of Oncology 2010: 1. doi:10.1155/2010/617421. PMC 2793426. PMID 20016752.
- Paterlini-Brechot P, Benali NL. (2007). "Circulating tumor cells (CTC) detection: Clinical impact and future directions". Cancer Lett. 253 (2): 180–204. doi:10.1016/j.canlet.2006.12.014. PMID 17314005.
- "Veridex CellSearch Website". March 2010. Retrieved 2010-03-14.
- "Veridex LLC. CellSearch circulating tumor cell kit premarket notification—expanded indications for use—metastatic prostate cancer". March 2010. Retrieved 2010-03-14.[dead link]
- Cristofanilli M, Budd GT, Ellis MJ, et al. (2004). "Circulating Tumor Cells, Disease Progression and Survival in Metastatic Breast Cancer". NEJM 351 (8): 781–91. doi:10.1056/NEJMoa040766. PMID 15317891.
- Budd G, Cristofanilli M, Ellis M, et al. (2006). "Circulating Tumor Cells versus Imaging - Predicting Overall Survival in Metastatic Breast Cancer". Clin Can Res 12: 6404–09.
- , et al. (2008). "The Relationship of Circulating Tumor Cells to Tumor Response, Progression-Free Survival, and Overall Survival in Patients with Metastatic Colorectal Cancer". JCO 26: 3213–21. doi:10.1200/JCO.2007.15.8923. PMID 18591556.
- JS DeBono, HI Scher, RB Montgomery, et al. (2008). "Circulating Tumor Cells (CTC) predict survival benefit from treatment in metastatic castration resistant prostate cancer (CRPC)". Clin Can Res 14: 6302–9. doi:10.1158/1078-0432.CCR-08-0872.
- Allard W J, Matera J, Miller MC, et al. (2004). "Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with non-malignant diseases". Clin Can Res 10: 6897–6904. doi:10.1158/1078-0432.CCR-04-0378. PMID 15501967.
- Riethdorf et al.; Fritsche, H; Müller, V; Rau, T; Schindlbeck, C; Rack, B; Janni, W; Coith, C et al. (2007). "Detection of Circulating Tumor Cells in Peripheral Blood of Patients with Metastatic Breast Cancer: A Validation Study of the CellSearch System". Clin Cancer Res 13 (3): 920–8. doi:10.1158/1078-0432.CCR-06-1695. PMID 17289886.
- "An Introduction to the CellSearch™".
- Yu M., et al. (2011). "Circulating tumor cells: approaches to isolation and characterization.". The journal of Cell Biology 192 (3): 373–382.
- Nagrath, Sunitha (December 2007). "Isolation of rare circulating tumour cells in cancer patients by microchip technology". Nature 450: 1235–1239. doi:10.1038/nature06385. PMC 3090667. PMID 18097410.
- Hoshino, Kazunori (Oct 2011). "Microchip-based immunomagnetic detection of circulating tumor cells.". Lab on a Chip 11 (20): 3449–3457. doi:10.1039/c1lc20270g. PMID 21863182.
- Wang, Huiqiang (September 2013). "Optical Detection and Virotherapy of Live Metastatic Tumor Cells in Body Fluids with Vaccinia Strains". PLoS ONE 8: e71105. doi:10.1371/journal.pone.0071105. PMID 24019862.
- Zheng, Siyang (Aug 2007). "Membrane microfilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells.". Journal of Chromatography A 1162 (2): 154–161. doi:10.1016/j.chroma.2007.05.064. PMID 17561026.
- Desitter I., et al. (2011). "A New Device for Rapid Isolation by Size and Characterization of Rare Circulating Tumor Cells.". Anticancer research 31 (2): 427–442.
- Meng S, Tripathy D, Shete S et al. (2004). "HER-2 Gene Amplification can be acquired as breast cancer progresses". PNAS 101 (25): 9393–8. doi:10.1073/pnas.0402993101. PMC 438987. PMID 15194824.
- Hayes DF, Walker TM, Singh B, et al. (2002). "Monitoring Expression of HER-2 on Circulating Epithelial Cells in Patients with advanced Breast Cancer". Int J of Oncology 21: 1111–8.
- O'Hara SM, Moreno JG, Zweitzig DR, et al. (2004). "Multigene Reverse Transcription-PCR Profiling of Circulating Tumor Cells in Hormone-Refractory Prostate Cancer". Clin Chem 50 (5): 826–835. doi:10.1373/clinchem.2003.028563. PMID 14988224.
- de Bono JS, Attard G, Adjei A, et al. (2007). "Potential Applications for Circulating Tumor Cells expressing the Insulin Growth Factor-I Receptor". Clin Can Res 13: 3611–6. doi:10.1158/1078-0432.CCR-07-0268.
- Attard G, Swennenhuis JF, Olmos D et al. (2009). "Characterization of ERG, AR and PTEN status in Circulating Tumor Cells from Patients with Castration-Resistant Prostate Cancer". Cancer Research 69 (7): 2912–8. doi:10.1158/0008-5472.CAN-08-3667. PMID 19339269.
- Swennenhuis JF, Tibbe AGJ, Levink R et al. (2009). "Characterization of Circulating Tumor Cells by Fluorescence In-Situ Hybridization". Cytometry Part A 75A: 520–7. doi:10.1002/cyto.a.20718.
- Karp DD, Pollak MN, Cohen RB et al. (2009). "Pharmacokinetics and Pharmacodynamics of the IGF-IR Inhibitor Figitumumab (CP-751,871) in Combination with Paclitaxel and Carboplatin". Journal of Thoracic Oncology 4 (11): 1397–1403. doi:10.1097/JTO.0b013e3181ba2f1d. PMC 2941876. PMID 19745765.
- AGJ Tibbe, MC Miller, LWMM Terstappen (2007). "Statistical Considerations for Enumeration of Circulating Tumor Cells". Cytometry Part A 71A: 132–142.
- F. A. W. Coumans, C. J. M. Doggen, G. Attard, et al. (2010). "All circulating EpCAM1CK1CD452 objects predict overall survival in castration-resistant prostate cancer". Annals of Oncology 21: 1851. doi:10.1093/annonc/mdq030.