Significant detection of circulating cancer cells in the blood by reverse transcriptase-polymerase chain reaction during colorectal cancer resection

Abstract

Objective: To analyze the clinical value of reverse transcriptase-polymerase chain reaction (RT-PCR) recognition of mRNA coding for carcinoembryonic antigen (CEA) and cytokeratin 20 in blood obtained from patients with colorectal carcinoma.

Summary background data: RT-PCR has been applied to identify very small numbers of tumor cells. Molecular detection is thought to provide useful information for the clinical management of perioperative prophylaxis of tumor cell implantation or postoperative adjuvant therapy regimens.

Methods: From 52 patients with colorectal cancer, peripheral blood specimens were obtained before and after surgical manipulation; also, a specimen of mesenteric venous blood draining the colorectal tumor was obtained just before tumor resection. Using cDNA primers specific for CEA and cytokeratin 20, RT-PCR was performed to detect tumor cells. Subsequently, the 52 patients were divided into two groups, a group positive for both CEA and cytokeratin 20 and a group negative for CEA, cytokeratin 20, or both.

Results: On the basis of 450 days of follow-up data, the PCR-positive group had a significantly shorter overall survival than the PCR-negative group only with the mesenteric venous blood specimens. Multivariate analysis indicated that detection of the simultaneous presence of CEA and cytokeratin 20 mRNA in mesenteric venous blood is a potent prognostic factor independent of the traditional pathologic parameters. Of the eight peripheral blood specimens found to be PCR-positive, five showed a change of PCR from negative to positive during surgery, and liver metastases developed 11 months later in one of these five patients.

Conclusions: Molecular detection of both CEA and cytokeratin 20 mRNA in mesenteric venous blood may be of prognostic value for patients with colorectal carcinoma. Molecular detection in the peripheral blood at surgery suggests that hematogenic tumor cell dissemination is a common and early event and that surgical manipulation enhances this release of tumor cells into the circulation.

Figure 1. Different numbers of cancer cells were added to 2 mL whole blood. The number of tumor cells in the lanes is 0, 10¹, 10², 10³, 10⁴, and 10⁵ from left to right (lanes 1–6). (M, molecular weight marker; NC, water negative control.)

Figure 2. Representative results of reverse transcriptase–polymerase chain reaction assay using mesenteric venous blood specimens. Lanes 1 and 2 were obtained from the two stage IV patients; lanes 3, 4, 5, and 6 were obtained from the four stage III patients.

Figure 3. Survival curves of the 52 patients with colorectal cancer according to the expression of both carcinoembryonic antigen and cytokeratin 20 mRNA in the mesenteric venous blood specimens. The survival curves were determined by the Kaplan-Meier method. The negative group includes one patient, patient 40, who died of pulmonary vein thrombosis on postoperative day 5 (P value determined using log-rank test).

Figure 4. Representative results of reverse transcriptase–polymerase chain reaction assay using peripheral blood specimens. Lanes 1 and 2 were obtained from patient 19 (stage III) before and after surgery, respectively; lanes 3 and 4 were from patient 9 (stage III); lanes 5 and 6 from patient 35 (stage IV); lanes 7 and 8 from patient 15 (stage I); and lanes 9 and 10 from a patient with benign disease (laparoscopic cholecystectomy).

Figure 5. Survival curves of the 52 patients with colorectal cancer according to the expression of both carcinoembryonic antigen and cytokeratin 20 mRNA in peripheral blood specimens. The negative group includes patient 40 (P value determined using log-rank test.)