RESUMO
PURPOSE: To examine the utility of guanylyl cyclase C (GC-C)-specific nested reverse transcriptase polymerase chain reaction (RT-PCR) to detect circulating tumor cells in patients with colorectal cancer. PATIENTS AND METHODS: Peripheral-blood mononuclear cells from 24 patients with Dukes' stage D colorectal cancer were analyzed by GC-C-specific nested RT-PCR using 1 microg of total RNA. Peripheral-blood mononuclear cells from 20 healthy volunteers served as controls. Additionally, peripheral-blood CD34+ progenitor cells were assayed for the expression of both GC-C and other epithelial cell-specific markers. RESULTS: GC-C mRNA was detected in blood mononuclear cells from all 24 patients with colorectal cancer and all healthy volunteers. These unexpected positive results reflected low-level ectopic transcription of GC-C in CD34+ progenitor cells. Moreover, CD34+ progenitor cells expressed other epithelial cell-specific markers, including prostate-specific antigen, prostate-specific membrane antigen, carcinoembryonic antigen, CK-19, CK-20, mucin 1, and GA733.2. Limiting the quantity of mononuclear cell total RNA analyzed to < or = 0.8 microg eliminated detection of GC-C and other tissue-specific transcripts in blood of healthy volunteers. However, under the same conditions, GC-C mRNA was detected in mononuclear cells from all 24 patients with metastatic colorectal cancer. Using 0.5 microg of total RNA and GC-C-specific primers, nested RT-PCR detected a single human colon carcinoma cell (approximately 20 to 200 GC-C transcripts/cell) in 10(6) to 10(7) mononuclear blood cells. CONCLUSION: These data suggest that GC-C may be useful for detecting circulating colorectal cancer cells. They also demonstrate that CD34+ cells are a source of ectopically expressed epithelial cell-specific markers and that CD34+ cells may contribute to the high false-positive rate generally observed when those markers are used to detect rare circulating metastatic cancer cells by RT-PCR.
