Detection of circulating tumor cells with CK20 RT-PCR is an independent negative prognostic marker in colon cancer patients – a prospective study
In this study we evaluated the role of CTC and DTC in colon cancer patients who were scheduled for potentially curative colon carcinoma resection. We show that CTC detection by CK20 RT-PCR is a highly sensitive and independent prognostic factor for OS and DFS in colon cancer patients.
In our study we applied two different technological approaches in parallel, i.e. RT-PCR and immunocytochemistry to detect CTC and DTC. Firstly, we employed a highly sensitive and specific nested CK20 RT-PCR to detect CTC and DTC. With this technique we were able to achieve detection rates of 37% in the blood and 35% in the BM. This technique is validly more sensitive than antibody-based detection of either intracellular protein markers (cytokeratins) or the cell surface EpCAM antigen, which yield detection rates of 22.3 and 19.7%, respectively for DTC in the bone marrow. For colorectal cancer patients in particular average detection rates of only 10.5% for CTC with the CellSearch™ system have been reported [19]. In addition to this, it has been demonstrated that the sensitivity of the qRT-PCR method is superior to immunomagnetic-based tools concerning detection of CTC in colorectal cancer patients [20].
Furthermore, we used immunocytochemistry to detect DTC with anti-pan-cytokeratin or anti-EpCAM antibodies. Using this methodological approach, we achieved detection rates of 22.3 and 19.7%, respectively. Recent reports have shown, that additionally incorporating CK20 RT-PCR as a biomarker, the sensitivity of the CellSearchTM system could substantially be enhanced in colorectal cancer patients [21].
Though the major limitation of immunomagnetic enumeration platforms is, that only the subset of EpCAM+ CTC is detected. It has been shown, that a subgroup of CTC may exist, that has undergone epithelial to mesenchymal transition (EMT) and does not express EpCAM [22, 23]. Moreover, the cells that have encountered EMT have undergone dedifferentiation, increased cell mobility and have lost cell adhesions. These attributes make this subset of cells even more likely to have an aggressive metastatic potential and high drug resistance [24, 25].
In our study, we were able to show that disseminated tumor cells in the bone marrow have a different impact on overall survival than circulating tumor cells in the blood. Despite the combined detection rate for DTC in nearly 50% of the patients with either CK20 RT-PCR or immunocytochemistry the prognostic significance of DTC in the bone marrow was negligible compared to CTC in the peripheral blood. In clinical practice BM metastases are rarely seen in colon cancer. Solely in more advanced tumor stages, but what is the biological role of DTC in the bone marrow? This implies, that this organ might have a high ability to clear disseminated colon cancer cells or to prevent their proliferation. During the last years these findings have led to a hypothesis of tumor cell dormancy and tumor stem cells that reside in the bone marrow niche and recirculate after years to form distant metastases [26–28]. Recently, we have been able to show that patients with colorectal liver metastases and detectable DTC in the bone marrow at the time of liver surgery, had an unfavorable prognosis after complete liver metastases resection [29]. Interestingly, in this series of patients with apparent macro-metastases in the liver, CTC in the blood were not an additional negative prognostic marker. These findings support the hypothesis, that detection of DTC in the BM per se is not a negative prognostic factor, but only if under certain circumstances these dormant tumor cells re-circulate and consequently form solid organ metastases.
We included in our study exclusively patients with colon cancer as we have previously reported that in rectal cancer DTC and CTC have no prognostic influence on OS [12]. In accordance to our findings several other groups have also described that in rectal cancer CTC are not a prognostic factor for OS [30–32]. There are several clinical and biological hallmarks indicating that colon and rectal cancer are different with respect to anatomy, function and embryological origin [33, 34]. Furthermore, the treatment of primary non-metastasized colon and rectal cancer is different [35]. Future studies evaluating the role of circulating tumor cells should at least provide subgroup analysis of rectal and colon cancer patients.
The detection of CTC correlates with a higher T-category and the existence of liver metastases. In addition, patients with detectable CTC have a significantly higher risk to develop a recurrent disease. Interestingly, the detection of CTC did not correlate with lymph node metastases, which is in line with previous reports [19, 36]. Furthermore, in our study population we were not able to prove a prognostic influence of detectable CTC or DTC in early stage (UICC stage II) patients. As adjuvant therapy in patients without lymph node metastases remains a controversial issue, further molecular markers or risk factors are urgently needed to identify patients at risk for later metastases.
The biological significance of CTC or DTC is still uncertain. We and other groups can detect CTC in approx. 30% of T1-2 tumor patients [19, 37], but these patients have a very good prognosis. Recently, it has been shown with gene expression profiles of CTCs that there is a strong heterogeneity between the tumor cells. CTC are mostly dormant cells and disguised by the immune system, which may explain the low number of metastases opposing a high number of CTC in the blood flow [38]. It has been shown, that a subset of CTC express functional cancer stem cell characteristics [39]. Furthermore, in breast cancer a subset of metastases-initiating cells (MIC) among CTC was described that have a distinguished phenotype [40]. For the future, not the pure detection of DTC and CTC will be fundamental, but the quantification and phenotypic characterization of molecular markers of CTC that might allow selective targeting of the metastatic cascade of colon cancer.