Differential expression of carcinoembryonic antigen (CEA) splice variants in whole blood of colon cancer patients and healthy volunteers: implication for the detection of circulating colon cancer cells.
Quantification of circulating cancer cells in whole blood samples by real time quantitative RT-PCR might be of clinical value for monitoring therapeutic effectiveness. In colon cancer patients, carcinoembrynic antigen (CEA) and cytokeratin 20 (CK20) have been frequently used for RT-PCR based tumor cell detection, but the specificity in particular for CEA has been questioned. In this study, we compared real-time RT-PCR for CEA and CK20 and analysed patients with metastatic disease (n=32) and healthy volunteers (n=17). CK20 mean values were elevated in cancer patients (P<0.001) and defined a subgroup (38%) who showed CK20 levels at least 100-fold above the highest value of the healthy control group. In contrast, only two cancer patients (6%) showed elevated CEA levels. Samples of the healthy control group showed exclusively a CEA-PCR product of 79 degrees C melting temperature. Thirty per cent of the colon cancer patients showed an additional product of 82 degrees C melting temperature. The 82 degrees C product was identical with the amplification product of CEA-cDNA and cDNA from different colon cancer cell lines. Colon cancer cells were spiked into normal blood in 10-fold dilutions that resulted in a dose dependent shift of the melt curve from 79 degrees C to the 82 degrees C. Sequencing of the PCR products showed that white blood cells express a splice variant of CEA, which hinders detection of tumor cell cDNA in whole blood samples. Our findings have implications for the use of CEA as a diagnostic molecule (e.g. by RT-PCR). The discovery of a physiologically expressed CEA splice variant might lead to a better understanding of the biological function of CEA and its family members.
Hampton
Regina
R
Department of Oncology, Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007, USA.
Walker
Mignon
M
Marshall
John
J
Juhl
Hartmut
H
D000230:Adenocarcinoma / Q000097:blood* / Q000235:genetics / Q000473:pathology; D001483:Base Sequence; D001667:Binding, Competitive; D014408:Biomarkers, Tumor / Q000096:biosynthesis / Q000097:blood* / Q000235:genetics; D002272:Carcinoembryonic Antigen / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D003110:Colonic Neoplasms / Q000097:blood* / Q000235:genetics / Q000473:pathology; D003199:Computer Systems; D018076:DNA, Complementary / Q000097:blood; D005189:False Positive Reactions; D015972:Gene Expression Regulation, Neoplastic; D006358:Hot Temperature; D006801:Humans; D007381:Intermediate Filament Proteins / Q000097:blood; D053554:Keratin-20; D007962:Leukocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000097:blood* / Q000235:genetics; D018365:Neoplasm, Residual; D009360:Neoplastic Cells, Circulating; D011489:Protein Denaturation; D020033:Protein Isoforms / Q000096:biosynthesis / Q000097:blood* / Q000235:genetics; D012326:RNA Splicing; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor; D002272:Carcinoembryonic Antigen; D018076:DNA, Complementary; D007381:Intermediate Filament Proteins; C507499:KRT20 protein, human; D053554:Keratin-20; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D011933:Reagent Kits, Diagnostic