Discordant quantitative detection of putative biomarkers in nodal micrometastases of colorectal cancer: biological and clinical implications

Abstract

Aims: Nodal expression of the carcinoembryonic antigen (CEA), cytokeratin 20 (CK20), and guanylyl cyclase C (GCC) genes was measured in tandem in patients with colorectal cancer (CRC) to assess whether there would be sufficient agreement between these markers in their ability to detect micrometastasis to qualify one of them as a universal marker, and whether frozen and paraffin wax embedded tissues would yield similar results.

Methods: One hundred and seventy five frozen lymph nodes (FT) and 158 formalin fixed, paraffin wax embedded lymph nodes (PET) from 28 CRC cases were analysed using gene specific quantitative real time polymerase chain reaction, carried out on the LightCycler system with SYBR Green chemistry.

Results: There was significant disparity in positive detection of the three biomarkers in FT versus PET, with notable agreement achieved only for CEA (66.6%) in FT versus PET in Dukes' B disease, and between CK20 and GCC (44.6%) in FT, also in Dukes' B disease. One patient with full concordance in all three tumour markers with both tissue types suffered a relapse and died within two years of follow up.

Conclusions: There was considerable discordance in the positive detection of the three tumour markers in both tissue types (FT versus PET). This brings into question whether using a single tumour marker to detect micrometastasis in one tissue type (FT or PET) is adequately representative, and challenges the concept of universal markers for molecular CRC metastatic detection. Multiple tumour markers would predict more accurately the metastatic potential of Dukes' B CRCs.

Figure 1

Sensitivity of the quantitative real time polymerase chain reaction assay. Amplification runs for (A) CEA, (B) CK20, and (C) GCC were performed with serial dilutions of DLD-1 cells spiked into known concentrations of peripheral mononuclear cells obtained from a healthy donor. Reactions with dilutions greater than the highest dilution depicted in the plots and negative controls yielded no amplified products.

Figure 2

Standard curves for CEA, CK20, and GCC mRNA estimation, constructed by plotting the crossing point cycle numbers against the log concentrations of DLD-1 cells. The expression of (A) CEA, (B) CK20, and (C) GCC mRNA for patient samples was calculated with reference to the appropriate curve. MSE, mean square error; ^θr, regression.

Figure 3

The relative expression of CEA, CK20, and GCC in frozen lymph nodes from patients with Dukes’ B and C colorectal cancer.

Figure 4

The relative expression of CEA, CK20, and GCC in fixed lymph nodes from patients with Dukes’ B and C colorectal cancer.