| BachgroundThe incidence of hepatocellular carcinoma (HCC) in our country is very high andthe prognosis of this kind of cancer is very poor. Multiple nodules are usually foundamong those patients at the moment of diagnosis. Two reasons for that includeintrahepatic metastasis (IM) and multiplecentric occurrence (MO). As the treatmentmodalities and prognosis for the two types of HCC are distinct, the discriminationbetween them is of great importance. At present, many literatures referred to the HCVrelated HCC, while few reports showed HBV related multiple HCC in thedistinguishing of MO and IM.ObjectiveTo explore the incident situation of MO and IM in patients with multiple HCCrelated to HBV and to analyze the benefit of some different methods in distinguishingMO and IM.Methods1. The clinicopathologic data of patients with multiple HCC resected in Tianjincancer hospital were collected. Their multiple type were confirmed by analysis ofpathology and immunohistochemistry. Then, the discriminant factors andprognosis between group MO and group IM were appraised.2. The fresh samples of patients with multiple HCC were collected. After theamplification of some microsatellite markers with PCR, the loss of heterozygosity(LOH) and microsatellite instability (MSI) were analyzed by the technique ofmicrosatellite polymorphism. The clonalities of multiple HCC were concludedwith these genetic information.3. The integration of HBV DNA in multiple HCC nodules was confirmed byamplification of HBV-X. In the positive patients, the integrate sites of multiplenodules were compared by Southern Blotting. The clone origins of multiplenodules were then concluded.4. The results from the above methods were analyzed together and the incidence ofMO and IM HCC in our centre was estimated.Results1. (1) By pathology and immunohistochemistry of p53 and osteopotin, 18.0% caseswere diagnosed as MO and 64.0% were concluded to be IM among 89 cases withmultiple HCC. Besides, five cases contain both types of HCC and 11 patientscould not be confirmed. (2) Tumor grade, size, cholinesterase, Child's stage and portal vein invasion were suggested to be discriminant factors by the analysis ofstepwise regression. (3) The prognosis of patients with MO HCCs is better thanthat of patients with IM ones and tumor type (MO or IM)and (4) Child's stageare the independent prognosis elements.2. (1) The LOH rate and MSI rate of 10 markers in the 79 nodules of 35 patientswere 35% and 10% in average, respectively. (2) Through compare these geneticinformation, 14.3% cases were considered as MO and 82.9% patients werediagnosed as IM. Besides, one patient contain both types of HCC.3. (1) Among the 35 cases with multiple HCCs, 97.1% patients were demonstratedbe positive in HBV-X integration, including 3 patients whose serum test ofHBs-Ag were negative. (2) The analysis of integrate sites of HBV-X showed16.1% cases were MO and 71.0% ones were IM. 12.9% cases contain both typesof HCC. (3) Correlation examination demonstrated Child's stage, tumor location,grade and tumor size were correlated with the results of Southern Blotting(P＜0.05).4. (1) With the combined analysis of clinicopathology and molecular genetics,17.1% (5/35) MO and 80% (28/35) IM were found in 35 cases. (2) The analysisof HBV integration and microsatellite polymorphism showed high concordancewith the final results (r_s=0.949, P＜0.001 and r_s=0.829, P＜0.001, respectively).While, the value of immunohistochemistry is limited because of poor concordancewith the final diagnosis (r_s=0.319, P=0.06). (3) The results from the test of HBVintegration is significantly correlated with that of microsatellite polymorphism(r_s=0.787, P＜0.001). (4) Child's stage, tumor grade and satellite nodules areimportant distinguishing factors between MO and IM..Conlusions1. The multiple HCCs is mainly caused by IM (over 70%), while the incidence ofMO is less 20%, which is significantly lower than that of Japan. This may berelated to the different liver cirrhosis and carcinogenesis of HBV and HCV.2. The prognosis of patients with MO HCCs is better than that of cases with IM ones.For former cases, aggressive surgery should be encouraged. Meanwhile, theprotection of liver function is another important treatment.3. the points in the discrimination of two types HCC1) Besides the standards from Japan, the different histological subtypes betweenmultiple nodules in one patient may suggest their different clone origin and shouldbe another discriminant feature. The immunohistochemistry of p53 and osteopotin showed limited value for the discrimination of MO and IM. To find the sensitivemarkers is very important.2) LOH is the main feature of microsatellite polymorphism. Anyway, MSI can alsosupply the valuable genetic information for the diagnosis of MO and IM. The fineneedle biopsy and test of microsatellite polymorphism for those patients withmultiple nodules is essential before operation or liver transplantation, which willbenefit doctors much in choosing treatment modalities.3) The infection of HBV in the development of HCC is persistent and generatedenovo integrate site. The test of HBV DNA for the patients whose serumHBs-Ag is negative is usually essential. The postoperative analysis of clonalitiesof multiple HCCs with Southern Blotting may benefit the appraisal of prognosis.4) The test of HBV DNA integration by Southern Blotting showed highly accuracyin the distinguishing diagnosis. Furthermore, the results of microsatellitepolymorphism can confirm the diagnosis of Southern Blotting. Though the abovemethods can bring us accurate results, they are not suitable for wide application inclinical diagnosis. For clinical doctors, Child's stage, tumor histological grade andmicrosatellite nodules may benefit the discrimination of MO and IM.4. To erect one discriminant only containing the clinicopatholigic data is the future'sobjective with enough cases who are confirmed to be MO or IM by pathology andmolecular genetics.
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