| Objectives: Hepatocellular carcinoma (HCC) is the most common malignant tumor in our country with the second death rate on the cancer death list, which has a very aggressive clinical course. Surgical resection and liver transplantation are the main methods of the treatment for HCC patients, but the long-term survive rate was poor. It was reported that cancer cell disseminating from the focus into the blood circulation alreadly before or during surgery resection and liver transplantation, these circulating tumor cells (CTC) might be an important reason of its high recurrence and metastasis rate. The CTC detection of the peripheral blood is important for the judgement of the recurrence and the direction of clinic therapy in HCC patients.To date, CTC in HCC cases were evaluated mainly by detecting alpha- fetoprotein (AFP) mRNA used PT-PCR in peripheral blood that could provide useful information. However, it is controversial whether the detection of the gene transcripts truly reflects the presence of tumor cells, since AFP mRNA is not only specifically detected in HCC cases but also it was significantly found in liver innocuousness disease cases including hepatitis or liver cirrhosis. Therefore, single marker assay may lead to false negative or false positive results. Recently, investigations revealed that telomerase is reactivated in approximately 85-100% of various types of malignant tumors but that it is inactive in most nonneoplastic somatic cells. Human telomerase reverse transcriptase (hTERT) is a catalytic component of reverse transcriptase, and hTERT expression is rate limiting for telomerase, its expression significantly correlated with telomerase activity. The expression rate of hepatocarcinoma is remarkably increasing too, even in the AFP negative HCC tissue, since the sensitivity is 73.9% and the specificity is 100% in HCC, these indicates that hTERT expression is one of more sensitive diagnosis agent than telomerase in HCC. To avoided false positive in hepatitis or liver cirrhosis cases, it is then possible that hTERT mRNA would be a new molecular detection marker gene of hepatocarcinoma. Immunomagnetic bead (IMB) is a new immunologic technique developed recently decade, immmunomagnetic cell seperation is one of main application area, and it has possessed the advantages including easy performance, enhanced separating concentration, higly specificity and maintained cytoactive. Combination with immunocytochemical methods, reverse transcriptase-polymerase chain reaction and flow cytometry etc, IMB could enrich and detect one tumor cell in 106 to 107 peripheral mononuclear blood cells, and could improve the sensitivity and specificity in detection CTC using these technique. Therefore, the present study was to obtain immunomagnetic beads which can be specially and sensitively combined with human hepatocellular carcinoma cells firstly, and secondly expected to assess morphological characteristics of CTCs, and examined the expression of AFPmRNA and hTERT mRNA by nested RT-PCR after immunomagnetic cell separation of peripheral mononuclear blood cells obtained from patients with HCC. Lastly we hope to culture the CTCs from the blood samples and to finding the activities of the cells released from the primary neoplasm after immunomagnetic cell separation. It will be useful to detection and prevention hematogenous metastasis in HCC.Methods: 1. Mediated by 1-ethyl-3, 3-dimethylaminopropyl carbodiimide (EDC), immunomagnetic beads against human hepatocellular carcinoma cells were constructed by binding the monoclonal antibodies HAb18 with magnetic microbeads. Mixing small numbers of cells from the hepatocellular carcinoma line HepG2 with peripheral blood mononuclear cells performed a study. After isolated through immunomagnetic cell separation, the rare cancer cells were observed through microscope and identified by immunocytochemistry, then to evaluate the specificity and sensitivity of the immunomagnetic bead. The recovery rate of tumor cells and detecting efficiency of immunomagnetic cell seperation were caculated. 2. Ten ml of peripheral blood were collected from 56 patients with HCC, 19 with hepatitis and liver cirrhosis, and 11 with metastasis liver cancer. In addition, 18 blood samples were collected from the healthy volunteers as the control. Ficoll density gradient centrifugation and immunomagnetic beads coat HAb18 antibody coated odies were used to isolate and sort circulating carcinoma cells from peripheral blood in HCC patients, then cellar morphological characteristic was observed by immunofluorescence staining technique, and AFP mRNA and hTERT mRNA were tested by nested RT-PCR to detect the circulating tumor cells in peripheral blood and evaluate the clinic implication. 3. There were 8 cases of CTCs cultured and identificated after immunomagnetic beads sorting, cytobiologic characteristic in growth curve, time of cell doubling, anchoring independence, ultrastructre of cell surface, and metastases in nude mouse after transplantated in spleen were observed.Results: 1. The obtained immunomagnetic beads which binding the antibodies HAb18 could specially and sensitively combine HepG2 cells. We detected tumor cell when the number of tumor cell was much more than one in 1×106 peripheral blood mononuclear cells. The 57.2% rare tumor cells were detected by the above methods and no false-positive results were observed. 2. In the preliminary experiments using HepG2 cells, one HepG2 cell could be detected in 5ml blood sample with immunomagnetic beads sorting and nested RT-PCR technique. 3. In the subsequent clinic experiments, there were 42.9%(24/56) HCC patients who were detected CTCs by immunofluorescence staining technique in combination of immunomagnetic beads sorting, Four kinds of morphological characteristic CTCs were observed in peripheral mononuclear blood with HCC patients after immunomagnetic beads sorting, CTCs can be divided into 4 classes: 1) moderately cells; 2) large cells with a large nucleus; 3) nucleate cells debris; 4) enucleate cells debris. 4. The total positive detection rate of AFP mRNA and hTERT mRNA was 55.4% and 48.2% respectively in patients with HCC. The frequency of positive cases showed strong correlation with TNM stage and extrahepatic metastases. Additionally, the positive rate was increasing rapidly in patients with large (exceeding 5cm) tumors (P<0.05). The frequency of AFP mRNA and hTERT mRNA positivity was also increasing in patients with HCC with multi-nodules (P<0.05), portal cancer thrombosis (P<0.05). Other clinical parameters, such as serum AFP level, grade of differentiation, did not correlate with the presence of AFP mRNA and hTERT mRNA in peripheral blood (P>0.05). On the other hand, 67.9% HCC patients were positive for at least one marker. The presense of AFP mRNA in peripheral blood was correlated with hTERT mRNA, and the coefficient is 0.256. The presense of hTERT mRNA in peripheral blood in AFP mRNA negative and positive patients was 64.5% and 28% (P<0.05). Meanwhile, AFP mRNA expressed markedly in HCC patients compared to the hepatitis and liver cirrhosis patients with no-HCC (P<0.05), but none of samples from metastasis liver cancer patients and healthy volunteers showed AFP mRNA. The positive detection rate of hTERT mRNA in metastasis liver cancer patients was 63.6%, and respectively samples demonstrated negative hTERT expression in the hepatitis and liver cirrhosis patients or healthy volunteers. 5. We have got tumor cells in eight blood samples cultured respectively after immunomagnetic beads sorting, but seven were failed. Only one sample's tumor cells emerged endothelial tumor cell proliferation, named HCC-27, and serial cultured for five generation. It was confirmed as hepatocarcinoma cell by morphological analysis and AFP immunocytochemistry analysis, but the growth curve, time of cell doubling, and the ablility of anchoring independence of HCC-27 was significantly lower compared with HepG2 (P>0.05). HCC-27 can survive and metastasize into liver of nude mice (1/2) after transplanted in spleen.Conclusion: 1. The obtained immunomagnetic beads can specially and sensitively separate rare HCC cells from peripheral mononuclear blood cells. 2. After using Ficoll density gradient centrifugation and immunomagnetic beads separation and sorting, there were four kinds of morphological characteristic CTCs in peripheral mononuclear blood with HCC patients such as moderately cells, large cells, nucleate and enucleate cells debris. 3. Combined with immunomagnetic beads sorting and nested RT-PCR technique may be useful to detection CTCs in HCC, detection of AFP mRNA in combination with hTERT mRNA provide useful source to improve sensitivity and specificity of detecting circulating hepatocarcinoma cells. 4. Separation and culture of CTC from peripheral blood samples taken give perfect evidence for hematogenous metastasis of HCC. The ability of growth and clony forming efficiency was low, but it had potency of metastasis.
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