Objective : Esophageal cancer is still regarded as a disease with a poor prognosis. In some patients who undergo macroscopically and histopathologically curative resection, metastasis and/or recurrence have been reported. In addition, patients with identical TNM stages do not always show the same prognosis. This suggests that a trace quantity of tumor cells, undetectable by conventional clinicopathological procedures, may be present. While reports describing the detection of trace quantities of tumor cells from esophageal cancer patients have been published, the clinical significance remains unclear at present. In this study, we describe the use of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) of CEA mRNA to identify trace quantities of tumor cells in the bone marrow of patients with esophageal cancer and review the clinical significance of these findings.
Materials : We studied 65 patients who underwent esophageal cancer resection with a 2- or 3-field regional lymph node dissection in our department between March 2003 and February 2004. The subjects consisted of 54 men and 11 women, aged between 39 and 80 (mean age 63.7 yrs). The postoperative follow-up period ranged from 82 to 564 days (316.6 days on average).
Methods : A bone marrow sample was taken from the 4 th or 5 th rib during thoracotomy just after the initiation of surgery. Total RNA was extracted from the sample and purified, then dissolved in RNase-free water. Purity and concentration of the extracted total RNA was determined by UV absorption spectrophotometry.
As a positive control for CEA mRNA, TE-9 cells were used. For the quantitative detection of CEA mRNA, reverse transcription and real-time PCR were performed using total RNA from TE-9 cells and a standard curve was drawn. The amount of CEA mRNA in the bone marrow specimen was corrected by the ratio of GAPDH mRNA, the internal standard, based on the standard curve, and the number of the cells converted to TE-9 cells was calculated.
Quantitative PCR was performed in duplicate to confirm reproducibility. In addition, PCR products of the patients who were positive for CEA mRNA were subjected to 2% agarose gel electrophoresis and the presence of a 131 bp band specific to the amplification of CEA was confirmed to eliminate a false-positive reaction.
Measurement and Results : Fourteen of 65 patients (21.5 %) were positive for CEA mRNA. Although there was no significant correlation between positivity for CEA mRNA and any clinicopathological factors, the positive group showed significantly poorer survival (p=0.0369). Prognosis analysis by multivariate Cox proportional-hazards model suggested that CEA mRNA positivity (p=0.031) and the number of lymph node metastases (p=0.004) were the prognostic factors. On logistic regression analysis, CEA mRNA was not a risk factor.
Conclusions : The detection of a trace quantity tumor cells in bone marrow could be used as a new prognostic factor for esophageal cancer.
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