Cytokeratin 20 improves the detection of circulating tumor cells in patients with colorectal cancer

Highlights

• CK20 is a biomarker for circulating tumor cells (CTC) in colorectal cancer patients.

• CTCs were detected in 89% of the patients using CK20 as additional biomarker.

• CK20 improves the detection rate of CTCs in colorectal cancer patients.

Abstract

Cytokeratin 20 (CK20) is a well-established marker for colon epithelium. Herein, we suggest that CK20 is a biomarker for detecting circulating tumor cells (CTCs) in patients with metastatic colorectal cancer. Blood specimens (7.5 mL) were collected during surgery after liver mobilization from 25 patients with colorectal cancer. The FDA approved CellSearch™ system and two panels of antibodies against cytokeratins, cytokeratin 8, 18 and 19 (CK8/18/19) and CK8/18/19/20, were used for the detection of CTCs. All the patients' samples were processed using the anti-CK8/18/19 panel. The number of detected CTCs was low, 52% of the patients lacked CTCs and 40% had ≤ 2 CTCs/7.5 mL blood. Nine of the patients' blood samples were processed with both antibody panels. The detection rate of CTCs was significantly higher using the anti-CK8/18/19/20 panel compared with the anti-CK8/18/19 panel, p-value 0.0078. Our data show that inclusion of CK20 as a biomarker efficiently improves the detection of CTCs in colorectal cancer patients. The finding in our study is of clinical importance since a new prognostic biomarker would provide an important tool in individual clinical decision-making for colorectal cancer patients.

Introduction

Colorectal cancer (CRC) is the third most frequent cancer with 6000 new cases per year in Sweden [1] and 1.2 million per year worldwide [2]. Approximately 50% of patients with CRC develop liver metastases (LM) and 15–20% of these patients will be candidates for curative liver resection [3]. Long-term outcome after liver resection for CRC-LM has improved over time and an overall 5-year survival of 45–50% can be expected in combination with modern chemotherapy and targeted drugs [4], [5]. Despite improved overall survival, recurrence rates have remained relatively unchanged and more than 50% of patients will develop recurrence within 2 years after liver resection [6]. For patients with metastatic colorectal cancer the presence of circulating tumor cells (CTCs) has been reported as an independent predictor of overall and progression-free survival [7]. The risks associated with tumor manipulation during surgery and exact mechanisms involved in the development of recurrent metastases are unknown and CTCs can be an important way to monitor tumor cell spread during liver surgery [8].

In year 2004, the CellSearch™ system (Veridex LCC, Raritan, NJ, USA) was cleared by the US Food and Drug Administration as a diagnostic tool for identifying and counting CTCs in blood samples in patients with metastatic breast cancer. Later, 2007 and 2008, FDA also cleared the system for monitoring CTCs in patients with colon cancer and prostate cancer, respectively.

Numerous studies of patients with both advanced colorectal cancer and non-metastatic colorectal cancer have shown that the number of detectable CTCs using the CellSearch™ system is low and even below the clinical threshold of three CTCs, which is in contrast to other epithelial tumor types e.g. breast and prostate cancer [9], [10], [11], [12], [13], [14], [15], [16], [17].

Attempts have been made to improve CellSearch based CTC detection in patients with colorectal cancer using larger sample volumes, 30 mL blood instead of 7.5 mL [14], [18]. The study by Lalmahomed [14], including 15 patients with colorectal liver metastases, showed that the number of CTCs was significantly higher using 30 mL instead of 7.5 mL blood. Before the CellSearch analyses the 30 mL blood was reduced to a volume of 7.5 mL enriched blood using modified Ficoll density gradient separation. Analyzing the enriched blood ≥1 CTC was found in 13 patients (87%) and ≥3 CTCs were found in seven patients (47%) compared with ≥1 CTC in 10 patients (67%) and ≥3 CTCs in two patients (13%) analyzing 7.5 mL blood.

Gervasoni et al. [19] compared three distinct methods for the detection of CTCs in colorectal cancer patients: a multimarker RT-PCR assay, the CellSearch™ system and dHPLC-based gene mutation analysis. The study included 20 patients undergoing elective surgery for colorectal adenocarcinoma and 75% of the patients were positive for the presence of CTCs by the multimarker RT-PCR method. The multimarker RT-PCR assay included expression analysis of four genes, carcinoembryonic antigen (CEA), CK20, CK19 and guanylyl cyclase C. Only 20% were positive by the CellSearch™ system and 14.3% of the patients displayed gene mutations when the dHPLC method was applied.

Cytokeratin 20 expression is a marker for colorectal adenocarcinomas with clinical immunohistochemistry [20]. In this study we wanted to investigate anti-CK20 as an additional marker for CTCs in blood from colorectal cancer patients using the CellSearch™ system, in order to detect those cancer cells that lack the expression of CK 8/18/19.