Author/Curator: Ritu Saxena, PhD
Word Cloud By Danielle Smolyar
For several decades, research efforts have focused on targeting progression of cancer cells in primary tumors. Primary tumor cell targeting strategies include standard chemotherapy and immunotherapy and modulation of host microenvironment including tumor vasculature. However, cancer progression is comprised of both primary tumor growth and secondary metastasis (Langley RR and Fidler IJ. Tumor cell-organ microenvironment interactions in the pathogenesis of cancer metastasis. Endocr Rev. 2007 May;28(3):297-321; http://www.ncbi.nlm.nih.gov/pubmed/17409287). Owing to the property of unilimited cell division, cells in primary tumor increase rapidly in number and density and are able to favorably influence their microenvironment. Metastasis, on the other hand, depends on the ability of cancer cells to disseminate, circulate, adapt to the harsh environment and seed in different organs to establish secondary tumors. Although tumor cells are shed into the circulation in large numbers since early stages of tumor formation, few tumor cells can survive and proceed to overt metastasis. (Husemann Y et al. Systemic spread is an early step in breast cancer. Cancer Cell. 2008 Jan;13(1):58-68; http://www.ncbi.nlm.nih.gov/pubmed/18167340). Tight vascular wall barriers, unfavorable conditions for survival in distant organs, and a rate-limiting acquisition of organ colonization functions are just some of the impediments to the formation of distant metastasis (Chiang AC and Massagué J. Molecular basis of metastasis. N Engl J Med. 2008 Dec 25;359(26):2814-23; http://www.ncbi.nlm.nih.gov/pubmed/19109576).
It has been hypothesized that metastasis is initiated by a subpopulation of circulating tumor cells (CTC) found in the blood of patients. Therefore, understanding the function of CTC and targeting the CTC is gaining attention as a possible therapeutic avenue in carcinoma treatment.
Figure: Circulating tumor cells in the metastatic cascade
(Image source: Chaffer CL and Weinberg RA. Science 2011,331, pp. 1559-1564; http://www.ncbi.nlm.nih.gov/pubmed/21436443)
Isolation of CTC
Initial methods relied on the difference in physical properties of cells. When spun in a centrifuge, different cells in the blood sample settle in separate layers based on their byoyancy, and CTC are found in the white blood cell fraction. Because CTC are generally larger than white blood cells, a size-based filter could be used to separate the cell types (Vona G, et al, Isolation by size of epithelial tumor cells : a new method for the immunomorphological and molecular characterization of circulating tumor cells. Am J Pathol, 2000 Jan;156(1):57-63; http://www.ncbi.nlm.nih.gov/pubmed/10623654).
Herbert A Fritsche, PhD, Professor and Chief, Clinical Chemistry, Department of Laboratory Medicine, The University of Texas, MD Anderson Cancer Center, demonstrated that the CTC can be captured using antibody labeled magnetic beads, either in positive or negative selection schema. After the circulating tumor cells are isolated, they may be characterized by immunohistochemistry and counted. Alternatively, these cells may be characterized by gene expression analysis using RT-PCR. One of the CTC detection methods, Veridex Inc, Cell Search Assay, has been cleared by the US FDA for use as a prognostic test in patients with metastatic cancers of the breast, prostate and colon. This technology relies on the expression of epithelial cellular adhesion molecular (EpCAM) by epithelial cells and the isolation of these cells by immunomagnetic capture using anti-EpCAM antibodies. Enriched CTC are identified by immunofluorescence. Martin Fleisher, PhD, Chair, Department of Clinical Laboratories, Memorial Sloan-Kettering Cancer Center discussed in a webinar at the biomarker symposia, Cambridge Healthtech Institute, that every new technology has shortcomings, and the reliance on cancer cells to express sufficient EpCAM to enable capture may affect the role of this technology in future clinical use. Heterogeneous downregulation of epithelial surface antigen in invasive tumor cells has been reported. Thus, alternative methods to detect CTC are being developed. These new methods include-
- Flow cytometry that sorts cells by size and surface antigen expression.
- CTC microchips that are designed to capture CTC as whole blood flows past EpCAM-coated mirco-posts.
- Enrichment by filtration using filters with a pore size of 7-8 µm, that permits smaller red blood cell, leukocytes, and platelets to pass, but captures CTC that have diameters of about 12-15 µm.
Better identification of CTC
Baccelli et al (2013) developed a xenograft assay and demonstrated that the primary human luminal breast cancer CTC contain metastasis-initiated cells (MICs) that give rise to bone, lung and liver metastases in mice. These MIC-containing CTC populations expressed EPCAM, CD44, CD47 and MET. It was observed that in a small cohort of patients with metastases, the number of CTC expressing markers EPCAM,CD44, CD47 and MET, but not of bulk EPCAM+ CTC, correlated with lower overall survival and increased number of metastasic sites. These data describe functional circulating MICs and associated markers, which may aid the design of better tools to diagnose and treat metastatic breast cancer. The findings were published in the Nature Biotechnology journal recently (Baccelli I, et al. Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay. Nature Biotechnology 2013 31, 539–544; http://www.ncbi.nlm.nih.gov/pubmed/23609047).
CTC as prognostic and predictive factor for cancer progression
Martin Fleisher, PhD states “detecting CTC in peripheral blood of patients with cancer has become a clinically relevant and important prognostic biomarker and has been shown to be a predictive biomarker post-therapy. But, key to the use of CTC as a biomarker is the technology designed to enrich cancer cells from peripheral blood.”
Since CTC isolation methods started being established, correlation studies between the cells and a patient’s disease emerged. In 2004, investigators at the Department of Breast Medical Oncology, University of Texas MD Anderson Cancer Center (Houston, TX) discovered that the CTC were associated with disease progression and survival in metastatic breast cancer. The clinical trial recruited 177 patients with measurable metastatic breast cancer for levels of CTC both before the patients were to start a new line of treatment and at the first follow-up visit. The progression of the disease or the response to treatment was determined with the use of standard imaging studies at the participating centers. Patients in a training set with levels of CTC equal to or higher than 5 per 7.5 ml of whole blood, as compared with the group with fewer than 5 CTC per 7.5 ml, had a shorter median progression-free survival (2.7 months vs. 7.0 months, P<0.001) and shorter overall survival (10.1 months vs. >18 months, P<0.001). At the first follow-up visit after the initiation of therapy, this difference between the groups persisted (progression-free survival, 2.1 months vs. 7.0 months; P<0.001; overall survival, 8.2 months vs. >18 months; P<0.001), and the reduced proportion of patients (from 49 percent to 30 percent) in the group with an unfavorable prognosis suggested that there was a benefit from therapy. Thus, the number of CTC was found to be an independent predictor of progression-free survival and overall survival in patients with metastatic breast cancer (Cristofanilli M, et al, Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004 Aug 19;351(8):781-91; http://www.ncbi.nlm.nih.gov/pubmed/15317891).
Similar results have been observed in other cancer types, including prostate and colorectal cancer. The Cell Search System developed by Veridex LLC (Huntingdon Valley, PA) enumerated CTC from 7.5 mL of venous blood and was used to compare the outcomes from three prospective multicenter studies investigating the use of CTC to monitor patients undergoing treatment for metastatic breast, colorectal, or prostate cancer. Evaluation of CTC at anytime during the course of disease allowed assessment of patient prognosis and is predictive of overall survival (Miller MC, et al. Significance of Circulating Tumor Cells Detected by the CellSearch System in Patients with Metastatic Breast Colorectal and Prostate Cancer. J Oncol. 2010; http://www.ncbi.nlm.nih.gov/pubmed/20016752). In addition, the CTC test may permit the oncologist to make an early decision to discontinue first line therapy for metastatic breast cancer and pursue more aggressive alternative treatments.
Genetic analysis of CTC
Additional studies have analyzed the genetic mutations that the cells carry, comparing the mutations to those in a primary tumor or correlating the findings to a patient’s disease severity or spread. In one study, lung cancer patients whose CTC carried a mutation known to cause drug resistance had faster disease progression than those whose CTC lacked the mutation. The investigators analyzed the evolutionary aspect of cancer progression and studied the precursor cells of metastases directly for the identification of prognostic and therapeutic markers. Single disseminated cancer cells isolated from lymph nodes and bone marrow of 107 consecutive esophageal cancer patients were analyzed by whole-genome screening which revealed that primary tumors and lymphatically and hematogenously disseminated cancer cells diverged for most genetic aberrations. Chromosome 17q12-21, the region comprising HER2, was identified as the most frequent gain in disseminated tumor cells that were isolated from both ectopic sites. Furthermore, survival analysis demonstrated that HER2 gain in a single disseminated tumor cell but not in primary tumors conferred high risk for early death (Stoecklein NH, et al. Direct genetic analysis of single disseminated cancer cells for prediction of outcome and therapy selection in esophageal cancer. Cancer Cell. 2008 May;13(5):441-53; http://www.ncbi.nlm.nih.gov/pubmed/18455127).
The abovementioned studies indicate that CTC blood tests have been successfully used to track the severity of a cancer or efficacy of a treatment. In conclusion, the evolution of the CTC technology will be critical in the emerging area of targeted therapy. With the development and use of new technologies, the links between the genomic information and CTC could be explored and established for targeted therapy.
Challenges in CTC research
- Potential clinical significance of CTC has been demonstrated as early detection, diagnostic, prognostic, predictive, surrogate, stratification, and pharmacodynamic biomarkers. Hong B and Zu Y (2013) discuss that “the role of CTC as a disease marker may be unique in different clinical conditions and should be carefully interpreted. A good example is the comparison between the prognostic and predictive biomarkers. Both biomarkers employ progression-free survival and overall survival for data interpretation; however, the prognostic biomarker is independent of specific drug treatment or therapy, and used for the determination of outcomes before treatment, while the predictive biomarker is related to a particular treatment to predict the response. Furthermore, inconsistent results are increasingly reported among the various CTC assay methods, specifically pertaining to results for the CTC detection rate, patient positivity rate, and the correlation between the presence of CTC and survival rate (Hong B and Zu Y. Detecting circulating tumor cells: current challenges and new trends. Source. Theranostics. 2013 Apr 23;3(6):377-94; http://www.ncbi.nlm.nih.gov/pubmed/23781285).
- Heterogeneity in CTC along with several other technical factors contribute to discordance, including the changes in methodology, lack of reference standard, spectrum and selection bias, operator variability and bias, sample size, blurred clinical impact with known clinical/pathologic data, use of diverse capture antibodies from different sources, lack of awareness of the pre-analytical phase, oversimplification of the cytopathology process, use of dichotomous decision criteria, etc (Sturgeon C. Limitations of assay techniques for tumor markers. In: (ed.) Diamandis EP, Fritsche HA, Lilja H, Chan DW, Schwartz MK. Tumor markers: physiology, pathobiology, technology, and clinical applications. Washington, DC: AACC Press. 2002:65-82; Gion M and Daidone MG. Circulating biomarkers from tumour bulk to tumour machinery: promises and pitfalls. Eur J Cancer. 2004;40(17):2613-2622; http://www.ncbi.nlm.nih.gov/pubmed/15541962). Therefore, employing a standard protocol is essential in order to minimize a lot of inconsistencies and technical errors.
- CTC in a small amount of blood sample might not represent the actual CTC count in the whole blood. In fact, it has been reported that the Cell Search system might undercount the number of CTC. Nagrath et al (2007) have demonstrated that the average CTC number per mL of whole blood is approximately 79-155 in various cancers (Nagrath S, et al. Isolation of rare circulating tumous cells in cancer patients by microchip technology. Nature. 2007;450(7173):1235-1239; http://www.ncbi.nlm.nih.gov/pubmed/18097410). In addition, an investigative CellSearch Profile approach (for research use only) detected an approximately 30-fold higher number of the median CTC in the same paired blood samples (Flores LM, et al. Improving the yield of circulating tumour cells facilitates molecular characterisation and recognition of discordant HER2 amplification in breast cancer. Br J Cancer. 2010;102(10):1495-502; http://www.ncbi.nlm.nih.gov/pubmed/20461092). Such measurement discrepancies indicate that the actual CTC numbers in the blood of patients could be at least 30-100 fold higher than that currently reported by the only FDA-cleared CellSearch system.
Thus, although promising, the CTC technology faces several challenges both in detection and interpretation, which has resulted in its limited clinical acceptance and use. In order to prepare the CTC technology for future widespread clinical acceptance, a comprehensive guideline for all phases of CTC technology development was published by the Foundation for the National Institutes of Health (FNIH) Biomarkers Consortium. The guidelines describe methods for interactive comparisons of proprietary new technologies, clinical trial designs, a clinical validation qualification strategy, and an approach for effectively carrying out this work through a public-private partnership that includes test developers, drug developers, clinical trialists, the FDA and the National Cancer Institute (NCI) (Parkinson DR, et al. Considerations in the development of circulating tumor cell technology for clinical use. J Transl Med. 2012;10(1):138; http://www.ncbi.nlm.nih.gov/pubmed/22747748).
Reference:
- Langley RR and Fidler IJ. Tumor cell-organ microenvironment interactions in the pathogenesis of cancer metastasis. Endocr Rev. 2007 May;28(3):297-321; http://www.ncbi.nlm.nih.gov/pubmed/17409287
- Husemann Y et al. Systemic spread is an early step in breast cancer. Cancer Cell. 2008 Jan;13(1):58-68; http://www.ncbi.nlm.nih.gov/pubmed/18167340
- Chiang AC and Massagué J. Molecular basis of metastasis. N Engl J Med. 2008 Dec 25;359(26):2814-23; http://www.ncbi.nlm.nih.gov/pubmed/19109576
- Vona G, et al, Isolation by size of epithelial tumor cells : a new method for the immunomorphological and molecular characterization of circulating tumor cells. Am J Pathol, 2000 Jan;156(1):57-63; http://www.ncbi.nlm.nih.gov/pubmed/10623654
- Baccelli I, et al. Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay. Nature Biotechnology 2013 31, 539–544; http://www.ncbi.nlm.nih.gov/pubmed/23609047
- Cristofanilli M, et al, Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004 Aug 19;351(8):781-91; http://www.ncbi.nlm.nih.gov/pubmed/15317891
- Miller MC, et al. Significance of Circulating Tumor Cells Detected by the CellSearch System in Patients with Metastatic Breast Colorectal and Prostate Cancer. J Oncol. 2010; http://www.ncbi.nlm.nih.gov/pubmed/20016752
- Stoecklein NH, et al. Direct genetic analysis of single disseminated cancer cells for prediction of outcome and therapy selection in esophageal cancer. Cancer Cell. 2008 May;13(5):441-53; http://www.ncbi.nlm.nih.gov/pubmed/18455127
- Hong B and Zu Y. Detecting circulating tumor cells: current challenges and new trends. Source. Theranostics. 2013 Apr 23;3(6):377-94; http://www.ncbi.nlm.nih.gov/pubmed/23781285
- 10. Sturgeon C. Limitations of assay techniques for tumor markers. In: (ed.) Diamandis EP, Fritsche HA, Lilja H, Chan DW, Schwartz MK. Tumor markers: physiology, pathobiology, technology, and clinical applications. Washington, DC: AACC Press. 2002:65-82
- Gion M and Daidone MG. Circulating biomarkers from tumour bulk to tumour machinery: promises and pitfalls. Eur J Cancer. 2004;40(17):2613-2622; http://www.ncbi.nlm.nih.gov/pubmed/15541962
- Nagrath S, et al. Isolation of rare circulating tumous cells in cancer patients by microchip technology. Nature. 2007;450(7173):1235-1239; http://www.ncbi.nlm.nih.gov/pubmed/18097410
- Flores LM, et al. Improving the yield of circulating tumour cells facilitates molecular characterisation and recognition of discordant HER2 amplification in breast cancer. Br J Cancer. 2010;102(10):1495-502; http://www.ncbi.nlm.nih.gov/pubmed/20461092
- Chaffer CL and Weinberg RA. Science 2011,331, pp. 1559-1564; http://www.ncbi.nlm.nih.gov/pubmed/21436443
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