Rapid quantitative detection of carcinoembryonic antigen-expressing free tumor cells in the peritoneal cavity of gastric-cancer patients with real-time RT-PCR on the lightcycler.
Detection of free cancer cells in the peritoneal cavity by RT-PCR using carcinoembryonic antigen (CEA) as a target gene is a more sensitive predictor of peritoneal dissemination than conventional cytology in gastric-cancer patients. Difficulties with this method are the lack of quantitative assessment of free cancer cells and the length of time before completion. To overcome these problems, we have established a rapid and quantitative detection method using a novel real-time fluorescence PCR system (LightCycler). Using this device with hybridization probes as fluorophores, we detected CEA mRNA in peritoneal washes during surgery (within 3 hr) without any post-PCR procedure. This method could reproducibly quantitate 10 to 10(6) CEA-expressing colon carcinoma cells per 10(7) peripheral blood leukocytes, a comparable sensitivity to conventional RT-PCR with a wide dynamic range. Analysis of peritoneal washes from 109 gastric-cancer patients with this assay revealed relative values of CEA transcripts that correlated well with the depth of tumor invasion (p < 0.01). Average values of CEA transcript in peritoneal washes in patients with cytology (-)/RT-PCR(-), cytology (-)/RT-PCR(+) and cytology (+)/RT-PCR(+) results were 0.64, 1,525 and 6,715, respectively. Moreover, CEA transcripts in peritoneal washes in patients with synchronous peritoneal metastasis were more than 50-fold higher than in those without metastasis. These results suggest a positive correlation between CEA mRNA levels in peritoneal washes and prognosis. We conclude that real-time RT-PCR with hybridization probes is a sensitive, quantitative, specific and rapid method to detect free cancer cells in peritoneal washes. This clinically relevant system is a powerful technique to evaluate the risk of peritoneal recurrence in patients with gastric cancer.
Nakanishi
H
H
Laboratory of Pathology, Aichi Cancer Center Research Institute, Nagoya/Aichi, Japan. hnakanis@aichi-cc.pref.aichi.jp
Kodera
Y
Y
Yamamura
Y
Y
Ito
S
S
Kato
T
T
Ezaki
T
T
Tatematsu
M
M
D002272:Carcinoembryonic Antigen / Q000032:analysis* / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D009367:Neoplasm Staging; D010529:Peritoneal Cavity / Q000473:pathology*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000473:pathology*; D014407:Tumor Cells, Cultured
D002272:Carcinoembryonic Antigen; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases