Cytokeratin-20 and Survivin-Expressing Circulating Tumor Cells Predict Survival in Metastatic Colorectal Cancer Patients by a Combined Immunomagnetic qRT-PCR Approach

Abstract

Circulating tumor cells (CTC) express epithelial and stem cell-like genes, though current approved detection methods mainly use epithelial markers. We optimized a CTC isolation method that could capture their molecular heterogeneity and predict overall survival (OS) in metastatic colorectal cancer (mCRC) patients receiving various chemotherapy regimens. We combined immunomagnetic enrichment of CD45-negative, EpCAM-positive circulating cancer cells with qRT-PCR amplification of CK20 and survivin expression in 88 mCRC patients and 20 healthy controls. We then evaluated the prognostic value of baseline CTC CK20 and survivin expression in mCRC patients. The presence of elevated CTC CK20 or survivin expression distinguished mCRC patients from controls with sufficient sensitivity (79.6%) and specificity (85%). In univariate analysis, patients with high CTC-CK20 expression (9 vs. 33.2+ months, log-rank P < 0.001) or high CTC-survivin expression (10 vs. 33.2+ months, log-rank P = 0.032) had a significantly worse median OS than those with low expression of either marker. In multivariable analysis, the high CTC-CK20 group had significantly shortened OS (HR, 3.11; adjusted P = 0.01), and there was a trend toward inferior OS in the high CTC-survivin group (HR, 1.76; adjusted P = 0.099). Patients with either high CTC CK20 or survivin expression had inferior OS compared with those with low expression of both markers (HR, 4.39; 95% confidence interval, 1.56-12.35; adjusted P = 0.005). Colorectal cancer CTCs can be reliably isolated using epithelial and stem cell markers. CTC CK20 and survivin expression may effectively predict OS in mCRC patients receiving chemotherapy.

Figure 1. Cell Spiking Experiments

CK20 expression level detected from serial dilutions of Caco2 cells mRNA. All histogram results are means of triplicate independent experiments (P < 0.05). B. CK20 and survivin gene expression were measured by immunomagnetic qRT-PCR by adding varying numbers of HCT 116 colon cancer cells (5, 10, 50, 100, 1000) into the whole blood of one healthy donor. C. CK20 and survivin gene expression on 2% agarose gel from HT29 colon cancer cells (10, 20, 50, 100) by immunomagnetic RT-PCR. D. The cutoff values for the level of mRNA gene expression were: CK20: 0.14 and survivin: 0.092.

Figure 2. ROC Analysis of CTC CK20 and Survivin Gene Expression

A. ROC curves of CK20 and surviving expression in mCRC patients compared to healthy donors. B. CTC CK20 and survivin gene expression by using immunomagnetic qRT-PCR approach were analyzed following the cutoff values established with sufficient sensitivity and specificity from 20 healthy donors and 88 mCRC patients. Left: An optimal cutoff value of 0.16 for CK20 yields a sensitivity of 76% and specificity of 85%; Right: An optimal cutoff value of 0.15 for survivin yields a sensitivity of 72% and specificity of 100%.

Figure 3. Kaplan-Meier Cumulative Survival Curves Stratified by CTC CK20 and Survivin Expression

Overall survival (OS) curves according to A. CTC CK20 expression (log-rank P < 0.001) B. CTC survivin expression (log-rank P = 0.032) C. CTC elevated CK20 or survivin expression (log-rank P < 0.001).