Detection and Clinical Significance of Circulating Tumor Cells in Colorectal Cancer—20 Years of Progress
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Circulating tumor cells (CTC) may be defined as tumor- or metastasis-derived cells that are present in the bloodstream. The CTC pool in colorectal cancer (CRC) patients may include not only epithelial tumor cells, but also tumor cells undergoing epithelial-mesenchymal transition (EMT) and tumor stem cells. A significant number of patients diagnosed with early stage CRC subsequently relapse with recurrent or metastatic disease despite undergoing “curative” resection of their primary tumor. This suggests that an occult metastatic disease process was already underway, with viable tumor cells being shed from the primary tumor site, at least some of which have proliferative and metastatic potential and the ability to survive in the bloodstream. Such tumor cells are considered to be responsible for disease relapse in these patients. Their detection in peripheral blood at the time of diagnosis or after resection of the primary tumor may identify those early-stage patients who are at risk of developing recurrent or metastatic disease and who would benefit from adjuvant therapy. CTC may also be a useful adjunct to radiological assessment of tumor response to therapy. Over the last 20 years many approaches have been developed for the isolation and characterization of CTC. However, none of these methods can be considered the gold standard for detection of the entire pool of CTC. Recently our group has developed novel unbiased inertial microfluidics to enrich for CTC, followed by identification of CTC by imaging flow cytometry. Here, we provide a review of progress on CTC detection and clinical significance over the last 20 years.
CRC is the second most common cancer in Australia, with over 15,000 cases diagnosed in 2012, and is the second most common cause of cancer-related deaths (1). In the United States CRC is the third most common cancer and the third leading cause of cancer death in men and women (2). Patients diagnosed with early stage CRC undergo surgical tumor resection with curative intent, yet 20–30% of these patients suffer recurrent or metastatic disease within 5 years of surgery. This suggests that an occult metastatic disease process was already underway (3), or that viable tumor cells with proliferative and metastatic potential had been shed into the bloodstream from the primary tumor site during surgical resection to cause subsequent disease relapse in these patients (4,5). These cells are termed circulating tumor cells (CTC), broadly defined as tumor- or metastasis-derived cells that are present in the bloodstream. In particular, detection of CTC can identify patients with early-stage disease who are at risk of developing recurrent or metastatic disease (4,6) and who would thus more likely benefit from adjuvant therapy after resection of the primary tumor. Although much effort has been directed at the detection of CTCs (reviewed in [7, 8, 9]), large prospective clinical trials have yet to be completed to determine prognostic significance for monitoring minimal residual tumor cells to assess response to therapy in advanced disease as well as in the adjuvant setting.
Presence of CTCs as an Adjunct to Staging
A major determinant of patient prognosis is the stage at which the cancer is diagnosed, because surgery is considered curative in up to 70% of early-stage cases. Screening programs have helped with early diagnosis and intervention, but for those not participating in such programs some 12–25% of CRC patients still present with advanced (stage IV) disease (10). Up to 20% of patients diagnosed with early-stage CRC (stage I or II) and up to 30% with regional spread to lymph nodes or adjacent organs (stage III) have relapsed by 5 years after “curative” surgery (2,11). Furthermore, the introduction of laparoscopic surgery for CRC has not altered the 5-year survival rates after curative surgery compared with open surgery (12). Stage III tumors are a heterogeneous group with respect to outcome: lymph node involvement is of limited reliability in predicting recurrence or the need for adjuvant chemotherapy. Furthermore, a significant proportion of patients still show recurrent disease despite receiving adjuvant chemotherapy. Current tumor staging techniques of histology and radiological imaging are not sensitive enough to detect micrometastases or early tumor cell dissemination, events key to developing metastatic disease. The presence of CTCs may be indicative of a micrometastatic process involving distant organs and may have prognostic implications independent of established staging factors such as the extent of lymph node involvement. We have previously shown, using immunobead reverse-transcription (RT)-PCR, that in stage III patients, detection of epithelial cells in peripheral blood in 13/31 (42%) patients correlated with shorter disease-free survival (DFS) (hazard ratio [HR] 2.8, 95% confidence interval [CI] 1.169–6.716), suggesting that the presence of CTC has the potential to more accurately stratify stage III patients into different prognostic groups (6). Furthermore, our analysis of disseminated tumor cells (DTC) in postresection peritoneal lavage samples showed that stage I and II patients who were positive for DTC showed a significantly poorer outcome (HR for DFS, 6.2; 95% CI, 1.9–19.6), and this was independent of other established risk factors (13). Although adjuvant chemotherapy is offered to stage III patients to eradicate potentially existing micro-metastases, the indication for such treatment in stage II disease is less certain, with only small clinical benefits shown in clinical trials (14). The dilemma is finding a balance between the benefit of therapy, which may be incremental, and risk of harm, and to that end bio-markers, such as CTCs that correlate with impending recurrent or metastatic disease, are much needed.
Enrichment and Detection of CTCs
The isolation and molecular analysis of CTCs in peripheral blood is one of the most promising approaches to identifying disseminated disease but there are challenges in identifying unique tumor physical characteristics and tumor-specific phenotypic or molecular markers. CTCs are detectable at very low numbers relative to white blood cells (in the order of 1 CTC in 107) (15) but may be present at much higher numbers in metastatic cancer patients. The large majority of clinical studies available to date relied solely on the number of CTCs in blood samples determined on the basis of isolation of epithelial tumor cells by monoclonal antibody (Ber-EP4) targeting epithelial cell adhesion molecule (EpCAM), originally selected by our group for the immunomagnetic isolation of carcinoma cells from blood (16). EpCAM, as the target for antibody-labeled magnetic microbeads for this purpose, was later commercialized as Epithelial Enrich™ (Dynal Biotech). EpCAM is also used as the target for CTC capture in the CellSearch™ system (Veridex LLC), approved by the Food and Drug Administration (FDA) for monitoring response to treatment in breast, colon and prostate cancer (17, 18, 19, 20). In the first prospective multicenter study in metastatic CRC using the CellSearch™ System, CTCs were enumerated in a 7.5-mL blood sample from 430 patients. The results showed that patients in whom ≥3 CTC/7.5 mL blood were detected had shorter median and overall survival (OS) compared with patients with <3 CTCs (P < 0.0001), and these differences persisted at follow-up time points after therapy. They concluded that the number of CTCs was an independent predictor of DFS and OS in metastatic CRC and provided prognostic information in addition to that of imaging studies (19). Since these early reports, studies on the detection of CTC using immunoaffinity-based enrichment strategies have abounded and have generally shown that CTC levels of >1 cell/mL in peripheral blood (PB) correlate with poor prognosis. A metaanalysis of 36 studies (3,094 patients) that included PB, mesenteric portal blood (MPB), or bone marrow (BM) sampling showed that detection of CTCs in PB, but not in MPB or BM, correlated with poor prognosis in CRC (HR for DFS 3.06, 95% CI 1.74–5.38). The inclusion criteria were the use of any form of PCR and/or immunologic or flow cytometric detection techniques (21). A more recent metaanalysis of 1,329 patients with metastatic CRC showed that OS (HR, 2.47; 95% CI 1.74–3.51) and DFS (HR, 2.07; 95% CI 1.44–2.98) were shorter in patients with CTCs. Of note, the main inclusion criteria for the studies was that the presence of CTC had to be detected by methods that relied on monoclonal antibodies to EpCAM or cancer-specific antigens (22). Furthermore, most of the studies described in this review and the 12 studies included in the metaanalysis by Sergeant et al. (2008) (7) used expression of cytokeratin 19 (CK19), CK20 or carcinoembryonic antigen (CEA) to identify CTCs, markers that we and others have reported show a high background expression in blood samples from patients with inflammatory bowel disease and result in an unacceptably high rate of false positives (6,7,13,23). Despite these shortcomings, CTC levels enumerated using the CellSearch system showed promise as an early treatment response marker in metastatic breast cancer and could be useful in clinical trials alone or in addition to radiological assessment of response (24). A change in CTC load is also actively being investigated as a surrogate marker for the assessment of therapeutic efficacy: in a study of 90 stage III colon cancer patients receiving adjuvant oxaliplatin with fluorouracil and folinic acid (FOLFOX) chemotherapy the persistent presence of CTCs postchemotherapy was shown to be an effective marker for determining clinical outcome (HR for DFS, 6.27; 95% CI, 2.44–16.12 (25). Interestingly, in a clinical trial of a 4-drug regimen in advanced CRC, patients with high CTC numbers (detected by CellSearch) survived longer than expected compared with historical controls, whereas patients with low CTCs gained no extra benefit. This may identify patients who could be spared high-toxicity regimens (26). In a recent study including 239 patients with nonmetastatic potentially curable CRC, the rate of CTC positivity correlated with increasing stage. In this study multivariate analysis showed that CTC numbers (≥1 CTC/mL blood) were the strongest prognostic factor in nonmetastatic patients (stage I, II, III); HR, 5.5; 95% CI, 2.3–13.6), with similar results for the total study group of 287 patients (HR, 5.6; 95% CI, 2.6–12.0) (27).Although the detection of CTCs as a prognostic marker has been well established, CTC enumeration has yet to be accepted into the clinic in guiding treatment decisions for individual cancer patients. We and others have shown that not all patients positive for CTC suffer disease progression, and conversely disease progression does occur in some patients negative for CTCs (4,13,19,28). Nevertheless, CTCs may be detected much earlier than current standard-of-care imaging (computed tomography [CT] or positron emission tomography [PET]-CT) for metastases (reviewed in ). In a study of advanced CRC (n = 451) the CTC count (by CellSearch) before and during treatment was an independent prognostic marker for PFS and OS (30). There was also a statistically significant correlation between CTC levels and tumor response according to CT imaging; however, there were still issues of sensitivity and specificity, because only 4 of 20 patients (20%) with progressive disease had high CTC levels at 1–2 wks postbaseline (30). Hence further research is needed to improve the sensitivity and specificity of CTC detection. For instance, EpCAM expression was found to be in the order of 10-fold less on CTCs compared with primary and metastatic tissues, suggesting that EpCAM expression is dependent on the local microenvironment and is downregulated in disseminated cells (31). This is quite probably the reason why CTCs were undetected by the CellSearch system in a significant proportion of patients with colorectal (32) and other cancers (reviewed in ). Downregulation of EpCAM has also been reported in DTCs in bone marrow in breast cancer patients following adjuvant therapy (34) and was found to be the reason for failure to detect any CTCs in a murine breast cancer xenograft model (35). Furthermore, tumor cells that have gained entry to the bloodstream interact with platelets, during which platelet-derived transforming growth factor β (TGFβ) contributes to epithelial-mesenchymal transition (EMT) in the tumor cells via activation of the TGFβ/Smad and nuclear factor (NF)-κB pathways (36). EMT+ cells possess a motile, more stemlike phenotype expressing N-cadherin, vimentin and fibronectin (37,38), with concomitant loss of epithelial cell adhesion molecules such as E-cadherin (39) and EpCAM (35). The EMT phenotype of CTC has been linked to resistance to both chemotherapy and radiation therapy (reviewed in ). Methodologies that rely on positive capture of CTC, which have to assume specific criteria such as high expression of EpCAM, are intrinsically flawed, so that the real CTC load is underestimated and stem cell populations of likely high relevance to disease progression are missed (33). On balance, due to the relatively low sensitivity and specificity of the EpCAM-based approach EpCAM-targeted capture strategies cannot still be considered the best approach for CTC isolation (41, 42, 43).
The Emerging Circulating Tumor Stem Cell Hypothesis
In an attempt to address the issues with positive selection, Iinuma et al. (45) used an unbiased approach for CTC isolation and included the stem cell marker CD133 in the identification step. Their multiinstitutional study of 735 CRC patients used mRNA from nonenriched whole blood; they showed that detection of CTCs positive for CEA/CK19/CK20/CD133 expression (RT-PCR) was a significant prognostic factor for poorer DFS and OS in Dukes stage C patients, and in stage B patients with unfavorable pathological features, but not for stage A or stage B patients with otherwise good prognostic features. They concluded that CEA/CK/CD133+ CTC, but not CEA/CK+ CTC, was an independent prognostic factor in patients with Dukes stage B CRC (45). These results also showed a large difference in survival curves for stage C patients, all of whom received adjuvant chemotherapy, suggesting that CEA/CK/CD133+ CTC could be a marker for the presence of treatment-resistant stem cells (41). One caveat is that CD133 is also expressed on endothelial cell progenitors (48), so although not specific for tumor stem cells, CD133 could be a surrogate marker for tumor angiogenesis and tumor progression. This raises the question as to what are the likely markers for detection of CTSCs.
Identification of Stem Cell Markers for CTSC Detection
New Techniques for CTC Enrichment and Identification
The authors declare they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported by grants from the National Health and Medical Research Council Australia (project grant APP1045841), Cancer Council of South Australia, and the Hospital Research Foundation.
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