Specific Detection of Cytokeratin 20-Positive Cells in Blood of Colorectal and Breast Cancer Patients by a High Sensitivity Real-Time Reverse Transcriptase-Polymerase Chain Reaction Method

Abstract

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method for detection of cytokeratin 20-positive cells in blood characterized by two novel features was developed and tested on 99 patients with colorectal cancer, 110 with breast cancer, and 150 healthy subjects. To optimize the specificity and sensitivity of the method, two novel features were used. First, a primer overlapping two adjacent exons was generated to inhibit nonspecific amplification both in healthy donors and cancer patients; second, a non-end-point first-round amplification was used to increase sensitivity. The number of firstround cycles was chosen to reach the highest level of sensitivity while conserving quantitative characteristics. PCR efficiency increased from 88.9% in singleround RT-PCR to 99.0% in nested real-time RT-PCR. To establish sensitivity and specificity of the method, HT29 cells were serially diluted with normal blood. Detection limit improved from 100 HT29 cells (singleround RT-PCR) to 1 to 10 cells (nested real-time RT-PCR) per 3 ml of whole blood. None of the healthy subjects was positive, whereas 22 and 29% of all colorectal and breast cancer patients, respectively, had cytokeratin 20 cell equivalents in blood. The association between cytokeratin 20 cell equivalents and metastasis was statistically significant for breast (P ‫؍‬ 0.026) but not colorectal cancer patients (P ‫؍‬ 0.361). Negativity of all 150 healthy controls examined confers diagnostic potential to the method. Cytokeratin mRNAs are potential markers for detection of epithelial cells in blood. Several reports indicate that cytokeratin 20 (CK20) mRNA in blood acts as a specific cancer cell marker in patients with frequent cancer forms of epithelial origin such as breast 1-5 and colorectal cancer.6 -9 Many breast cancer patients develop metastasis after locoregional and systemic treatment even in the absence of dissemination as assessed by conventional diagnostic tools. Approximately 30 to 50% of colorectal cancer patients who have undergone curative resection have recurrences with fatal outcome.10,11 Most recurrences occur in patients with TNM (tumor, nodes, and metastases) stage II and III cancers, but patients with stage I lesions also have appreciable risk. In these patients, cancer cells were disseminated either before or during surgery of the primary tumor.12-14 Although the relationship between circulating tumor cells and the development of recurrent cancer is not fully understood, it is generally assumed that enhanced dissemination of cancer cells in blood contributes significantly to the development of metastasis. [15][16][17] The detection of circulating metastatic cells would be of great value for the assessment of the metastatic risk.18 Currently, the most powerful prognostic information in cancer patients is obtained from conventional histological assessment of regional lymph nodes. 19 -23 Because 20 to 30% of colorectal cancer patients without metastasis in lymph nodes die from distant metastases or local recurrence within 5 years 24...

Clinical Significance of Cytokeratin 20-Positive Circulating Tumor Cells Detected by a Refined Immunomagnetic Enrichment Assay in Colorectal Cancer Patients

“…Although the clinical significance of CTCs from patients with tumors is still debatable (12,13), the technology platform has improved rapidly. Over the last few years, CTC detection has become more standardized and reliable (14,15).…”

784 Clinical applications of molecular profiling of colorectal cancer

“…Furthermore, the molecular factors involved in prognosis and response to therapy in CRC is poorly understood Detection of disseminated tumour cells in peripheral blood of colorectal cancer patients has been achieved primarily using immunocytological or flow cytometry based techniques [432,433]. The clinical usefulness and high sensitivity of PCR in detecting cancer markers in circulating tumour cells was confirmed before [434,435]. In colorectal cancer, RQ-PCR was used to determine the expression levels of CEA, CK20 and CK19 mRNA in peripheral blood and indicated a valuable tool for cancer staging and disease monitoring [387,436,437].…”

Quantitative RT-PCR Detection of Colorectal Tumor Cells in Peripheral Blood—A Systematic Review

“…In 11 studies, venous blood samples were used to quantify CTCs. A single [32] 2002 No 99 CR Colon: n ϭ 43; rectum: n ϭ 56 TNM stage I-III Miura et al [37] 2003 No 36 CR Dukes A-D Schuster et al [29] 2004 No 129 CR Colon: n ϭ 69; rectum: n ϭ 60 TNM stage I-IV Chen et al [34] 2004 No 42 CR Colon: n ϭ 16; rectum: n ϭ 26 TNM stage I-IV Guo et al [40] 2005 No 40 CR Dukes A-D Dandachi et al [38] 2005 No 82 CR Colon: n ϭ 42; rectum: n ϭ 40 TNM stage I-IV Giribaldi et al [41] 2006 No 99 CR TNM stage I-IV Xu et al [42] 2006 No 168 CR Dukes A-D Iinuma et al [35] 2006 Yes 167 CR Dukes A-D Friederichs et al [39] 2007 NR ϭ not reported; CK ϭ cytokeratin; CEA ϭ carcinoembryonic antigen; ProtM ϭ protease M; GCC ϭ guanylyl cyclase C; GAPDH ϭ glyceraldehyde 3-phosphate dehydrogenase; PBDG ϭ porphobilinogen deaminase; ␤-2-M ϭ ␤-2-microglobulin; RCL ϭ red cell lysis; DC ϭ density centrifugation; Pre ϭ Preoperative blood; I ϭ intraoperative blood; Po ϭ postoperative blood; Fu ϭ at follow-up; NBC ϭ nucleated blood cells; PVB ϭ peripheral venous blood; MVB ϭ mesenteric venous blood. study used blood from the radial artery [32].…”

“…Four different control genes were used for normalization: glyceraldehyde 3-phosphate dehydrogenase [32][33][34][35], ␤-actin [36,37], porphobilinogen deaminase [29,38,39], and ␤2-microglobulin [40]. In two studies, normalization procedure and control genes were not reported [41,42]. When identical marker and control genes were chosen for amplification, primers and probe sequence combinations differed between studies.…”

“…In the study by Miura et al [37], patients with Dukes D disease had significantly higher transcript levels in their peripheral blood versus controls on univariate analysis (P Ͻ 0.0001). In the only multicenter trial CEA and CK20 mRNA positivity rates in both periph- Bustin et al [33] 1999 -----Guller et al [36] 2002 No NR 31 (median) 0.001 (CR,mv) 0.0035 (CR,uv) Ito et al [32] 2002 No 0.16 ( 2 ) -0.03 (BGW,uv) -Miura, et al [37] 2003 Yes Ͻ0.0001 for Dukes D (F) ---Schuster et al [29] 2004 No NR ---Chen, et al [34] 2004 No 0.36 (K-W) 37 (mean) 0.001 (W,uv*) 0.039 (W,uv*) Guo et al [40] 2005 Yes Ͻ0.05 for Dukes C,D ( 2 ) ---Dandachi et al [38] 2005 No NR ---Giribaldi et al [41] 2006 No NR ---Xu et al [42] 2006 eral and tumor drainage blood were stage-dependent on univariate analysis (P ϭ 0.003 and P Ͻ 0.001, respectively) [35]. An increased postoperative detection was found in three of four studies that analyzed postoperative peripheral blood samples [29,32,34].…”