Impact of circulating tumor cells in colorectal cancer patients undergoing laparoscopic surgery

Abstract

Laparoscopic surgery has recently been widely used for various benign colorectal diseases as well as colorectal cancer. Although laparoscopic surgery has been shown to be with similar prognostic results for certain groups of colorectal cancer patients. The influence of laparoscopic procedures on the oncologist results, especially free tumor cell spreading is still a concern for some surgeons. Tumor cells found in the peripheral blood of patients with cancer are termed circulating tumor cells (CTCs). Presence of CTCs in the peripheral blood of patients with colorectal cancer has been reported to be associated with disease stage, poor prognosis, tumor progression, response to therapy, and drug resistance. Whether laparoscopic procedure enhances tumor spreading during operation remains unknown. Significantly less CTC detected during laparoscopic surgery than open surgery for colorectal cancer has been reported. In our previous experience, no significant elevation in CTC level was found in most patients during laparoscopic resection of colorectal cancer. We have shown that laparoscopic surgery had no significantly deleterious effect on CTCs in colorectal cancer patients. In this review, we aim at the impact of CTCs in patients with colorectal cancer undergoing laparoscopic surgery. The prognostic significance of CTCs in patients with colorectal cancer will also be addressed.

Key Words: Laparoscopic surgery; Circulating tumor cell; Prognosis; Colorectal cancer

Core tip: Although laparoscopic surgery has been widely used for various benign colorectal diseases as well as colorectal cancer, the influence of this procedure on the tumor cell spreading is still unknown. The level of circulating tumor cells (CTCs) in patients with cancer has been reported to be significant prognostic and predictive factors. Whether laparoscopic procedure enhances tumor spreading during operation remains unknown. Significantly less CTC detected during laparoscopic surgery than open surgery for colorectal cancer has been reported. In our previous experience, we have shown that laparoscopic surgery had no significantly deleterious effect on CTCs in colorectal cancer patients.

INTRODUCTION

Laparoscopic surgery has been widely adapted for various benign colorectal conditions[1,2] and now increasingly for colorectal cancer[3] since the first series of laparoscopic colectomy was reported in 1991[4]. Laparoscopic colorectal surgery has the benefits of less postoperative pain, early return of bowel function, improved cosmetic results and equivalent oncologic outcome as compared with open surgery. Therefore, laparoscopic techniques for colorectal cancer surgery are increasingly widespread use and have been regarded as therapeutic option for colorectal cancer. Port site recurrence[5-8], which was considered to be a drawback of laparoscopic cancer surgery, is no more a great concern after the use of protection device; however, the free tumor cell spreading either through the homogenous route or intra-peritoneal cavity during the pneumoperitoneal procedure is still a concern of laparoscopic surgery in cancer patient. Tumor cells found in the peripheral blood of patients diagnosed with cancer are termed circulating tumor cells (CTCs). CTCs were first identified by Ashworth[9] in 1869. Presence of CTCs in the peripheral blood of patients with colorectal cancer has been reported to be associated with poor prognosis, tumor progression and drug resistance[10-12]. This review aims at the prognostic significance of CTCs in patients with colorectal cancer. The impact of CTCs in laparoscopic surgery for patients with colorectal cancer will also be addressed.

LAPAROSCOPIC SURGERY FOR COLORECTAL CANCER

Although the aforementioned clinical benefits of laparoscopic surgery, long-term survival and the disease-free interval after surgery are the main concerns adapting the techniques for colorectal cancer. Equivalent intermediate and long-term outcomes of patient survival and tumor recurrence after laparoscopic surgery as compared to open surgery in colorectal cancer have been reported in the literature[13-16], including randomized controlled clinical trials[3,17-21]. The Clinical Outcomes of Surgical Therapy Group[3,20], the report by Leung et al[19] and MRC CLASSICC[21] showed that overall survival rate and cancer recurrent rate were similar between laparoscopic surgery and open surgery. In the study by Lacy et al[18], cancer-related mortality was lower in patients with stage III disease who underwent laparoscopic surgery. There was no significant difference identified in patients with Stage I and II disease between laparoscopic surgery and open surgery in this trial[18]. Meta-analysis of these randomized controlled trials showed no significant difference in recurrence-free survival and long-term survival between laparoscopic and open surgery[22,23]. For rectal cancer, similar long-term local control and cancer-free survival between laparoscopic surgery and open surgery have been reported in a retrospective comparative study[24]. Long-term outcomes after resection of rectal cancer in CLASICC trail showed that there was no difference in survival, disease-free survival, and local and distant recurrence between laparoscopic surgery and open surgery groups[21,25,26].

CIRCULATING TUMOR CELLS

CTCs are rare in blood and bone marrow. Although CTCs have been identified more than a century[9], CTC-related research did not proceed rapidly until recently due to the lack of sensitive methods to detect these rare cells. CTCs are occurring approximately at the frequency of 1 CTC per 10⁵-10⁷ peripheral blood mononuclear cells[27-30]. Recent advances in technology make the detection of CTCs from a simple blood test possible[31], including quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry, immunomagnetic techniques, assays based on molecular phenotyping, etc.[32-36]. In principle, these methods can be divided into nucleic-acid-based (i.e., mRNA transcripts evaluated by RT-PCR) and cytometric (i.e., immunocytochemistry, immunofluorescence and flow cytometry) approaches[33,37,38]. The nucleic-acid-based approach is highly sensitive, and quantitative RT-PCR permits relative quantification[33,37]. However, the disadvantage is that cells cannot be visualized directly for enumeration or morphology determination[33,37]. The advantage of cytometric approach is that cells can be directly visualized by immunocytochemistry or immunofluorescence[33,37]. Therefore, CTCs enumeration and morphology evaluation are possible. The disadvantage is that the sensitivity is moderate and epithelial-cell adhesion molecule negative cells may be missed[33,37]. The comparison between the two main approaches for detecting CTCs was listed in Table 1. For RT-PCR approach, both single marker and multiple markers have been used. Due to tumor heterogeneity, the use of multiple marker assay may have higher CTC yield as compared with single marker assay[33,37]. Targeting CTCs directly in vivo and the development of leukapheresis and elutriation for subsequent ex vivo CTC analyses are alternative approaches to improve CTC yield[33,37].

Table 1 Comparisons of the two main approaches for detection of circulating tumor cells.

Approaches	Cell viability	Sensitivity	Specificity	Disadvantages
Nucleic-acid-based approach (i.e., RT-PCR)	No	High	Low	Cytological analysis is not possible Cells cannot be visualized
Cytometric approach (i.e., immunocytochemistry, immunofluorescence and flow cytometry)	Yes	Low	High	EpCAM dependent

RT-PCR: Reverse transcriptase-polymerase chain reaction; EpCAM: Epithelial-cell adhesion molecule.

The prognostic significance of CTC enumeration has been reported in breast cancer[39,40], prostate cancer[41], lung cancer[42], and colorectal cancer[43,44]. CTC number has also been reported to be associated with radiological outcome in breast cancer[45] and colorectal cancer[46]. Furthermore, the application of CTC number is now extending beyond prognostication. Due to the progress in characterization of molecular profiles in CTCs, the predictive value of response to therapy and drug resistance has been demonstrated. Expression of HER2 in CTCs may be used as a biomarker for treatment of HER2-receptor antagonists in patients with breast cancer[47]. Epidermal growth factor receptor (EGFR) mutations can also be detected from CTCs in patients with lung cancer[48-50], and can be used as a predictive biomarker for treatment with EGFR tyrosine kinase inhibitors[49]. The convenience of real-time biopsy of CTCs contributes a lot to the development of CTC-guided anticancer therapies and personalized medicine.

PROGNOSTIC IMPACT OF CTC IN COLORECTAL CANCER

Prognostic significance of CTC detection in stage I-III colorectal cancer

Detection of CTCs has been shown to correlate with the stage of colorectal cancer[11,51,52]. Sastre et al[52] showed that stage was the only clinicopathological factor significantly associated with CTC level. The presence of CTCs in the peripheral blood of patients with stage I-IV colorectal cancer has been reported to be associated with poor relapse-free survival and overall survival in a meta-analysis[12]. Several studies have demonstrated the prognostic significance of CTCs in patients with staged I-III colorectal cancer[53-60]. In a review article, Peach et al[58] evaluated the effect of sampling time on prognostic value of CTCs in patients with early stage colorectal cancer. There was no significant difference in detection rate at different sampling time (peri-/early or late postoperative). The presence of CTCs in peripheral blood at least 24 h after resection was a significant prognostic factor for recurrence and poor cancer-specific survival[58]. However, perioperative CTC level was not a significant predictor of recurrence[58].

Prognostic significance of CTC detection in metastatic colorectal cancer

The presence of CTCs in blood has been reported to be associated with worse progression-free survival and overall survival in colorectal cancer patients with metastatic disease[43,44,46,61-64]. Cohen et al[43] first demonstrated the predictive significance of CTCs in 430 patients with metastatic colorectal cancer receiving first, second and third lines of chemotherapy. CTC levels correlated not only with worse progression-free survival and overall survival, but with worse outcome for all treatment types. Their study showed that CTCs level can be used as both prognostic and predictive biomarkers[43,44]. In a multicenter phase III trial (CAIRO2 trial) of patients with metastatic colorectal cancer who received first-line treatment with capecitabine, oxaliplatin, and bevacizumab ± weekly cetuximab, the CTC count before and during treatment independently predicts progression-free survival and overall survival[46]. In a study of 64 patients receiving oxaliplatin-based chemotherapy for metastatic colorectal cancer, patients with ≥ 3 CTC at baseline and at 2 and 8-12 wk had a shorter median progression-free survival and overall survival than those with < 3 CTC[64]. No rise in early CTC level was observed among responders to chemotherapy[64]. In a prospective study of 180 metastatic colorectal cancer patients who received first line XELOX (capecitabine and oxaliplatin) with or without bevacizumab, the CTC count is a strong prognostic factor for progression-free survival and overall survival[63].

IMPACT OF CTC IN LAPAROSCOPIC SURGERY FOR COLORECTAL CANCER

CTC in laparoscopic surgery for primary colorectal cancer

Animal studies have shown that tumor cells shed into the circulation during resection of primary tumor, thus may increase the possibility of metastases[65]. Ito et al[66] demonstrated a significantly higher mean CEA mRNA value in postoperative blood than in preoperative blood by using real-time RT-PCR to detect CTC in peripheral blood of colorectal cancer patients. Their results suggest that tumor cells could be shed into the bloodstream during surgical procedures, and these free tumor cells are accompanied by a poor patient outcome[66]. Weitz et al[67] reported that increased intraoperative levels of CTCs were significantly associated with tumor recurrence in colorectal cancer patients. Clearance of CTCs within 24 h of colorectal cancer resection occurred in tumors with the best prognosis[68,69]. Whether laparoscopic surgery inadvertently facilitates tumor spread in colorectal cancer patients remains unknown. Several animal experiments have shown that pneumoperitoneum may enhance tumor growth or tumor cells dissemination[70-72]. However, Lécuru et al[73] demonstrated that CO₂ laparoscopy had no deleterious effect on circulating tumor DNA in xenograft ovarian cancer model. To evaluate the potential for tumor cell dissemination at time of curative surgery, Wind et al[74] detected and quantified CTCs in peripheral and portal blood of patients who had open or laparoscopic surgery for primary colonic cancer. The detection rate and quantity of CTC were significantly increased intra-operatively and were significantly higher in portal blood compared to peripheral blood[74]. Significantly less CTC were detected during laparoscopic surgery than open surgery[74]. In our previous study[75], we examined the impact of laparoscopic manipulation on circulating CTCs by detecting the guanylyl cyclase C mRNA using real-time RT-PCR in 42 colorectal cancer patients undergoing laparoscopic resections. Quantitation of CTCs was performed preoperatively, intra-operatively, and 14 d after operation. Although there was a trend toward increased CTC level in patients of advanced stage, there was no significant difference in the preoperative CTC level by disease stage. No elevation in CTC level was found during the laparoscopic procedure in most patients, as compared with their preoperative CTC values. We also detected a marginal decrease in CTC level after removing the tumor during laparoscopy, which was sustained at 14 d after surgery. Among the 42 patients, elevated CTC level during the operation was identified in 6 patients. The CTC level concentration returned to undetectable levels or fewer than 100 CTCs/10⁶ nucleated blood cells within 14 d after surgery in all the 6 patients. The return of CTC levels was similar to other studies that clearance of CTCs most commonly occurred within 24 h after operation[68,69,76]. Twelve of the 42 patients with persistently high CTC levels (> 100 CTCs/10⁶ nucleated blood cells) 2 wk after surgery had significantly poor cancer-related survival when compared with those with undetectable or low CTC levels (≤ 100 CTCs/10⁶ nucleated blood cells). Tumor recurrence or metastasis developed in eight patients during follow-up. Persistent high CTC levels at 14 d postoperatively were found in six of these eight patients. The percentage of recurrence in patients with high CTC levels was significantly higher that that in those with low or undetectable CTC levels (P = 0.001) (Table 2). According to our previous published report[75], laparoscopic procedure itself had no significantly deleterious effect on CTCs in colorectal cancer patients.

Table 2 Relationship between postoperative circulating free tumor cells and status of freedom from recurrence in patients of colorectal cancer undergoing laparoscopic surgery (n = 42).

	No recurrence	With recurrence	P value
Post-operative free CTC levels
≤ 100/106 NBCs	28 (82.4%)	2 (25.0%)	0.001
> 100/106 NBCs	6 (17.6%)	6 (75.0%)

CTC: Circulating tumor cell; NBC: Nucleated blood cell.

CTC in laparoscopic surgery for liver metastasis of colorectal cancer

Patients undergoing resection of hepatic metastases of colorectal cancer have a higher risk for extrahepatic tumor recurrence. Weitz et al[67] reported that resection of liver metastasis of colorectal cancer carries an increased risk for intraoperative tumor cell dissemination as compared with resection of primary colorectal cancer by detecting CTCs in blood and bone marrow samples[67]. One possible reason is that extensive manipulation of the liver and liver metastases was usually performed before venous drainage ligation[67]. Koch et al[69] demonstrated the prognostic value of CTCs in pre-, intra-, and postoperative blood and bone marrow samples of patients who underwent curative resection of liver metastasis of colorectal cancer. Multivariate analysis demonstrated that tumor cell detection in intraoperative blood and in bone marrow samples were significant prognostic factors of tumor relapse[69]. Jiao et al[77] determined the CTC level before and immediately after open surgery, laparoscopic resection, open/percutaneous radiofrequency ablation (RFA), in 29 patients with liver metastasis of colorectal cancer. Open liver resection was performed in 11 patients, laparoscopic liver resection in 4, open RFA in 5, and percutaneous RFA in 9. CTCs were localized to the hepatic portosystemic macrocirculation with significantly greater numbers than in the systemic circulation. Surgical procedures led to a statistically significant fall in CTCs at multiple sites measured. Conversely, RFA, either open or percutaneous, was associated with a significant increase in CTCs. Only 4 patients underwent laparoscopic resection in their study. Therefore, the difference between open and laparoscopic surgery was not conclusive. Several other reports also demonstrated that open surgery was superior to RFA for patients of liver metastasis of colorectal cancer[78,79]. Whether CTC was enhanced during laparoscopic surgical procedure for liver metastasis of colorectal cancer and lead to tumor growth and tumor cell dissemination needs further investigation.

CONCLUSION

The level of CTCs in the peripheral blood of patients with colorectal cancer is significantly associated with disease stage, poor prognosis, response to therapy, and drug resistance. It can be used as both prognostic and predictive factors. We have shown that laparoscopic surgery had no significantly deleterious effect on CTCs in colorectal cancer patients. Due to the limited references in the literature, the impact of laparoscopic procedure for colorectal cancer in CTC level and tumor spreading needs further investigation.