Abstract
The aim of our study was to describe the frequency of occurrence of circulating tumor cells (CTCs) in patients undergoing surgery for liver metastases from colorectal cancer in relation to treatment (chemotherapy and surgery). We monitored the presence of CTCs before, during and after surgery. Patients and Methods: This prospective study involved 14 patients (9 men and 5 women) undergoing surgical resection or termoablation of liver metastases from colorectal carcinoma. Ten of them received chemotherapy before surgery. Samples of central blood (7.5 ml) were drawn preoperatively, at the time of mobilization of the liver during the surgical procedure, immediately after surgery, and at two and seven days postoperatively. CTCs were detected by ColonCancerSelect and ColonCancerDetect kits (AdnaGen, Langenhagen, Germany). Results: CTCs were detected in three out of 14 patients. For each of the three patients, CTCs were detected via a different gene [tumor-associated antigen GA733-2, carcinoembryonic antigen (CEA) and epidermal growth factor receptor (EGFR)]. This demonstrates the heterogeneity of the CTC population among patients. In one patient, we recorded long-term presence of CTCs, in one patient we detected CTCs only during surgery and in one patient we detected CTCs only before surgery. CTC-positive patients are described in the form of case reports. Conclusion: We detected CTCs only in a minority of patients with liver metastases from colorectal cancer. Observations show that the surgical procedure itself can cause the presence of CTCs in the peripheral blood.
Colorectal carcinoma (CRC) ranks among the most commonly diagnosed cancers in the world (1). In 2008, the incidence in the Czech Republic was 93 men and 63 women in a sample size of 100,000 people (2). The liver is the typical site of metastases of CRC because of its unique blood supply from the portal vein. In patients with recurrence of metastatic liver disease, repeated liver surgery prolongs survival in well-selected patients (3). The 5-year survival rate for surgically-treated patients ranges from 21% to 58% (4-7), which is significantly higher than the rates that are achieved by non-surgical therapies (5-year survival rates less than 5%) (8).
The detachment of malignant cells from the primary tumor into the bloodstream is a principal source of metastases. In various types of solid tumors and their metastases (9-15), including CRC (16, 17), the presence of circulating tumor cells (CTCs) in peripheral blood has been associated with adverse prognosis. There is currently an effort to use molecular characterization of CTCs as a source of information for clinical decisions on therapy (18-20).
In addition to patients with primary CRC, it is not surprising that CTCs have also been identified in patients with liver metastases from CRC (21, 22). The early studies in surgically-treated patients with liver metastases used reverse transcription polymerase chain reaction (RT-PCR) for cytokeratin 20 (CK-20) detection directly from peripheral blood and bone marrow samples. This approach may suffer from both false-negative and false-positive results (23, 24), but it should be mentioned that the other methods used for CTC detection also have the same difficulty to a minor extent (25). At present, it is a priority to capture CTCs themselves and subsequently analyze them, but the number of published results for patients with liver metastases of CRC is still very limited (26).
The aim of our study was to describe the frequency of occurence of CTCs in patients undergoing surgery for liver metastases from CRC, in relation to treatment (chemotherapy and surgery). We monitored the presence of CTCs before, during and after surgery. We detected CTCs by a two-step method. Immunomagnetic enrichment of tumor cells via epithelial and tumor-associated antigens was followed by RT-PCR of tumor-associated antigen GA733-2, carcino-embryonic antigen (CEA) and epidermal growth factor receptor (EGFR) genes.
Patients and Methods
Patients. This prospective study involved 14 patients (9 men and 5 women; median age=64.7 years, range=47-72 years at the time of surgery) undergoing surgical resection or termoablation for liver metastases from CRC at the Department of Surgery, University Hospital Pilsen, in 2011. Ten of them had received chemotherapy before surgery (at various intervals from the date of surgery and with different chemotherapy regimens). Informed consent was obtained from all patients entering the study. The study was approved by the local Ethical Committee (approval no. 110713).
Methods. Central blood samples (7.5 ml) were collected into AdnaCollect Blood Collection Tubes (AdnaGen, Langenhagen, Germany). When possible, samples were obtained at five time points of treatment. Central blood was drawn preoperatively, during the first half of the surgical procedure (at mobilization of the liver during surgery), immediately after surgery, and at two and seven days postoperatively. The assessment for CTCs was performed not later than 24 h after samples were collected and the CTCs were detected by ColonCancerSelect and ColonCancerDetect kits (AdnaGen). The final analysis was performed on an Agilent Bioanalyzer instrument where we obtained electropherograms with densitometric values of bands. The presence of CTCs was detected by expression of genes: GA733-2, CEA and EGFR. The presence of at least one of these genes indicates the presence of CTCs. β-Actin was used as a reference a gene (internal amplification control). Assessment was considered reliable in the case of positive test for a reference gene (Figure 1).
We decided to use a CTC detection method, AdnaGen, targeting epithelial marker on CTC. This method is robust and the relation of the presence of CTCs and prognosis using this method has been published (27-29).
Statistical analysis. Basic statistical description was performed using the Statistica software package (StatSoft, Tulsa, OK, USA).
Results
CTCs were detected in three out of 14 patients; for each of the three patients, CTCs were detected via a different gene GA733-2, CEA or EGFR. The number of CTC-positive and -negative patients and their state of disease are summarised in Table I. Out of the three CTC-positive patients, two were treated by adjuvant chemotherapy after primary surgery. It is necessary to mention that four patients from our group of 14 did not receive adjuvant chemotherapy after primary surgery.
The presence of CTCs in the first of the three patients was detected by the expression of GA733-2 gene (in cells captured by immunomagnetic ColonCancerSelect method). We recorded the presence of CTCs in three out of five blood samples: in samples obtained preoperatively, immediately after surgery and seven days postoperatively. We did not detect CTCs in blood samples obtained during surgery nor in the sample collected two days after surgery. This patient underwent liver resection of segment S7 for metastasis of CRC that appeared three years after surgery for the primary tumor (well-differentiated mucinous adenocarcinoma). No chemotherapy was applied after primary surgery. Metastases in the lungs were diagnosed 22 months after liver surgery; the patient is still alive.
The Agilent electrophoresis record of expression of genes for tumor-associated antigen (GA733-2, 395 bp), carcinoembryonic antigen (CEA, 226 bp), epidermal growth factor receptor (EGFR, 161 bp) and β-actin (114 bp) in lysates of captured circulating tunor cells (CTCs). Lines 1-4 show the patients negative for CTCs. Lines 5, 7 and 9 show the samples positive for CTCs. Line 10 is a positive control.
CTCs were detected in the second patient by expression of the EGFR gene. The only positive sample was taken during the first half of the surgical procedure. In the other four blood samples, we did not detect CTCs. This patient underwent liver resection of segments S4 and S5 according to Habib for metastasis of CRC that appeared 1.1 years after surgery for the primary tumor (well-differentiated adenocarcinoma). The patient was treated by chemotherapy after primary surgery. This patient has been without clinical symptoms of disease for six months.
CTCs were detected by expression of CEA gene in the third patient. We recorded the presence of CTCs in the blood sample taken before surgery but not in any of the other samples. This patient underwent extended right hepatectomy for metastasis of CRC that appeared five months after surgery for the primary tumor (medium-differentiated adenocarcinoma). The patient was treated by chemotherapy after primary surgery. Three months after liver surgery, generalization of disease was diagnosed. Fifteen months after liver surgery, the patient died.
The other patients in this study were negative for CTC detection, although in some of them, a complete set of scheduled blood samples was not available. Data are summarized in Table II.
Overview of circulating tumor cell (CTC) positivity and state of disease of patients.
Discussion
Although liver resection is the most effective treatment, it is not possible in all patients with metastatic liver disease. However, patients undergoing surgery have different outcomes. CTCs are directly involved in disease progression, thus it makes sense that CTCs could be promising biomarkers.
The early studies of surgically-treated patients used RT-PCR for CK-20 detection from peripheral blood and bone marrow samples. CTCs were detected by RT-PCR for CK-20 preoperatively in 23.7% of patients and postoperatively in 28.9% of patients (21). In 2010, Papavasiliou et al. published their work on CTCs in patients undergoing surgery for hepatic metastases from CRC. For detection of CTCs they used the Veridex method (CellSearch; Veridex, LLC, Warren, NJ, USA) for direct detection of CTCs. The study group consisted of 20 patients. Preoperative CTCs were significant in two patients (10%), intraoperative were significant in 10 patients (50%) and postoperative CTCs were significant in one patient (5%). The authors presented a relationship between the presence of postoperative CTCs and clinical outcome (26).
In 2012, Pilati et al. tested the hypothesis of whether the detection of CTCs might identify patients with adverse prognosis after radical liver surgery. The authors did not capture CTCs but they measured transcriptional levels in the peripheral blood of seven selected genes, but it is necessary to point out that they also targeted the stem cell nature of CTCs (expression of CD133): CK-19, CK-20, CEA, prominin-1 (CD133), vascular endothelial growth factor (VEGF), EGFR, and survivin. They concluded that CD133-positive CTCs might represent a suitable prognostic marker to stratify for the risk of patients who undergo liver resection for CRC metastasis (30). Both studies (26, 30) indicate the relationship between the presence of CTCs and poorer clinical outcome, but the authors themselves warn of existing limitations.
In our study, the low number of patients and heterogeneity of surgical procedures applied do not allow for reliable statistical evaluation of the results, therefore we decided to present the results as individual case reports summarized in detail in Table II. We believe that this study provides information on the behavior of CTCs in relation to their heterogeneity and also to the management of blood sampling (samples were obtained at five time points during treatment).
We detected CTCs only in a minority of patients with liver metastases from CRC. The majority of patients received chemotherapy treatment after primary tumor resection, which should prevent survival of residual cells after tumor removal. The presence of CTCs was independent of chemotherapy treatment before surgery. CTC-positive cases were both chemotherapy-treated and without treatment.
From our point of view, a very important finding was that identification of CTCs was achieved using different markers (GA733-2, CEA and EGFR) in the three patients. Each case was only positive for one of these markers. This demonstrates the heterogeneity of CTCs, possibly implicating their different potential in disease dissemination. The patient in whom the CTCs were detected by CEA died 15 months after surgery.
For one of the CTC-positive patients, CTCs were detected only in the blood sample collected during surgery. We interprete this finding as being caused by mobilization of the liver during the surgery. The study of Papavasiliou et al. suggests that CTCs are present in a greater quantity during intraoperative liver manipulation (26).
Result of other studies as well as our data suggest that not only detection but also further molecular characterization of CTCs might provide new insight into the biology of the metastatic process and how it is affected by treatment.
Conclusion
We detected CTCs only in a minority of patients with liver metastases from CRC. The presence of CTCs was detected in patients without chemotherapy but also in patients treated by chemotherapy after primary surgery. Our observations show that the surgical procedure can cause the presence of CTCs in the peripheral blood. Our results also demonstrate the heterogeneity of the CTC population among patients.
Clinicopathological characteristics of patients and circulating tumor cell (CTC) assessments results.
Acknowledgements
This study was supported by the grant NS 9632201 from the Ministry of Health of the Czech Republic, by MH CZ-DRO (Faculty Hospital in Pilsen - FNPl, 00669806) and by the project of LF UK Plzen no. SVV-2013-266804.
Footnotes
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Conflicts of Interest
The Authors declare that they have no conflicts of interest.
- Received February 25, 2013.
- Revision received April 2, 2013.
- Accepted April 4, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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