| ObjectiveIn recent years, the highly sensitive technique of reverse transcriptase-polymerase chain reaction(RT-PCR) has been applied to the detection of micrometastases in blood from patients with malignant solid tumors. A-fetoprotein(AFP) and albumin gene transcripts have been recruited as targets of the RT-PCR assay in blood from HCC patients. However, there is controversy as to whether the detection of these gene transcripts truly reflects the presence of tumor cells, since normal liver cells also abundantly express AFP and albumin messenger RNA(mRNA)s. Another problem related to the use of RT-PCR with limited markers is the evidence that tumors are heterogeneous in gene expression, and down-regulation of marker expression could feasibly occur in metastatic tumors. These observations, which may limit the use of RT-PCR-based assays with a single marker, led us to guess multiple-marker assay may improve the specificity and sensitivity of tumor detection. We examined the expression of hepatocyte-specific AFP gene transcripts, and that of cancer-specific MAGE-1 gene transcripts in blood specimens obtained from patients with HCC and also from HCC-free controls. The purpose of this study was to assess the utility of the multiple marker RT-PCR assay for detecting hepatoma cells and investigated the influence of an alternation of AFPmRNA and MAGE-1mRNA perioperatively detected in peripheral blood in matters of recurrence.Methods Subjects were divided into five groups according to their clinical status: group A, patients with HCC diagnosed by histology(n=59); group B, patients with hepatitisB or liver cirrhosis but no evidence of HCC(n=22); group C, patients with metastatic liver cancer(n=8); group D, patients with hepatic hemangioma(n=8); and group E, healthy volunteers(n=20). A 5 ml quantity of heparinized blood was sampled from peripheral veins of patients before the operation and on 7,28 post-operative days. Then nested RT-PCR was performed to detect AFPmRNA and MAGE-1mRNA in peripheral blood. Results1. Dilution series experimentIn the limited dilution series, 10 HepG2 cells suspended in 5-ml whole blood proved to be sufficient for detection of either AFPmRNA or MAGE-1mRNA. 2. Detection of micrometastases from preoperative blood Samples 2.1 Detection of AFPmRNAWe detected AFPmRNA in 33 of the group A samples(55.9%, 33/59). The frequency of positive cases showed strong correlation with TNM stage. AFPmRNA positivity was significantly more frequent in patients with large(exceeding 5cm) tumors than in those with small(under 5cm) tumors(P<0.05). The frequency of AFPmRNA positivity was also increased in patients with HCC with intrahepatic metastatic nodules, portal cancer thrombosis, and CD44v6 protein expression in cancer tissue (P<0.05). No other parameters, such as serum AFP level, grade of differentiation or extrahepatic metastases correlated with the presence of AFPmRNA in blood. With regard to controls, 3 of 22 samples(13.7%) from group B showed AFPmRNA in their peripheral blood, but none of samples from group C,D,or E showed AFPmRNA .2.2 Detection of MAGE-1mRNAWe also detected MAGE-1mRNA in group A samples(26/59, 44.1%). The frequency of MAGE-1mRNA positivity strongly correlated with HCC with intrahepatic metastatic nodules, portal thrombosis, tumor diameter, and TNM stage(P<0.05). In contrast, the presence of MAGE-1mRNA in blood showed no correlation with serum AFP level, grade of differentiation and extrahepatic metastases(P>0.05). With regard to controls, 3 of 8(37.5%) samples from groups C showed AFPmRNA in their peripheral blood,but groups B, D,or E did not.2.3 Relationship between the presence of AFPmRNA and MAGE-1mRNAMAGE-1mRNA and AFPmRNA were detected in 26(44.1%) and 33(55.9%) of 59 HCC patients repectively, while 40(67.8%) HCC patients were positive for at least one marker. The presense of AFPmRNA in peripheral blood correlated with MAGE-1mRNA (P<0.05), and the coefficient is 0.307. 3. Detection of micrometastases from postoperative blood Sample...
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