| Background and ObjectiveHepatocellular carcinoma (HCC) is one of the most common human malignancies in our country. According to the statistics of WHO in 2005, liver cancer is the sixth most common cancer worldwide in terms of numbers of cases (626,000 or 5.7% of new cancer cases) because of the very poor prognosis, the number of deaths is almost the same (598,000). Fifty-five percent of these cases (and deaths) are in China, which is a serious threat against our people's life and health. Therefore it has always been the heavy emphasis on the studies of the tumors we laid. The therapeutic effect on HCC is not satisfying, one of the most important reason for which is that we haven't yet understood the molecular biological characteristics of the early small HCC up to now. There is no idea about the molecular mechanisms of the progression from early small well-differentiated HCC to late poorly-differentiated HCC. The condition makes the concept of small HCC confused. The standard set according to the tumor size includes 5cm, 4.5cm, 4cm, 3.5cm, 3cm and 2.5cm, not based upon molecular changes that are hallmarks of HCC development, which affects the improvement of clinical diagnosis and therapeutics of early small HCC and has been the rate-limiting steps of further development of the hepatic surgery. There is an urgent need to establish the new theory and concept of early small HCC for diagnosis and treatment decisions.Studies of modern molecular biology find that tumor is a genic disease. There is accumulating evidence for the multistep nature of HCC. For example, mutations in p53, Rb, and/or the insulin-like growth factor receptor 1 (IGFR1) genes have been reported in HCC, while c-myc and cyclin D1 are frequently overexpressed. Loss of heterozygosity (LOH) involving multiple chromosomes in single tumors also strongly suggests multistep carcinogenesis. Accumulation of genetic alterations or emergence of new alterations in preneoplastic lesions is believed to lead to the development of HCCs. A single gene alteration appears to be insufficient to induce the malignant transformation from normal cells to precancerous lesions, next to early stage tumors and finally to late stage tumors. A set of tumor-associated genes sequential dynamic alterations involves the transformation process. We tried to explore the genetic alterations at the early stages related to the progression of small HCC in the light of genomic instability, which may be the key breakthrough in unvealing the pathological biological evolution nature of small HCC.LOH studies, in certain chromosomal regions, is a frequently employed strategy for the detection of alleles'alteration in the human genome. A LOH indicates a situation where there is loss of a second allele on one chromosome that had already lost the other one by a germinative alteration (two -hits theory by Knudson).In order to identify genetic markers that could serve as a putative predictor of HCC, we analyzed 5 groups lesions including precancerous lesions (low grade dysplastic nodule and high grade dysplastic nodule), hepatocelular adenoma (HCA), micro HCC (d<1cm), small HCC (d<3cm), and large HCC (d>3cm) and detect the alteration characteristics of microsatellites in genomic DNA after carefully preparing the tissue samples by microdissection. Allelotyping was done with 64 markers covering six chromosomes corresponding to tumor suppressor genes (TSGs) that had shown significant alterations in HCCs. We detected both LOH and microsatellite instability (MSI). Finally, comprehensive studies may find clinical application to identify makers useful to early detection of tumors, to predict prognosis or to find new therapeutic targets.MethodsLOH and MSI of 60 microsatellite loci were detected with polymerase chain reaction-based microsatellite polymorphism analyses in tumors or precancerous lesions and corresponding adjacent noncancerous liver tissues of 199 surgically resected HCC using PCR-SSCP based microsatellite polymorphism analysis technique. LOH and MSI on 24 microsatellite loci were detected with polymerase chain reaction-based microsatellite polymorphism analyses in tumors and corresponding adjacent noncancerous liver tissues in 10 surgically resected HCAs, 8 DNs and 49 HCCs using ABI3700 and ABI3730 automatic DNA analysis system. Statistical differences in FAL were analyzed by the t-test (2-tailed); P values less than 0.05 were considered significant. Statistical differences in LOH at a given locus were analyzed by the Chi-square or Fisher exact test; a P value less than 0.05 was considered significant. Chi-square test was used to analyze the correlation between clinicopathological features with fractional allelic loss (FAL) values. The statistical significance was set at P values of less than 0.05.ResultsLOH was found in 185 of 192 informative HCC (96.4%) at one or several loci. As a whole, no significant correlation was observed between the LOH of loci and several clinicopathological features including serum AFP concentration, HBV infection, tumor size, cirrhosis, Edmondson's grade, tumor capsule, tumor thrombosis as well as histopathological type. MSI was found in 123 of 199 HCC (61.8%) at one or several loci. Frequency of LOH on chromosome arms 1p, 4q, 8p, 16q, and 17p is 62.1%(113/182),63.4%(111/175),71.1%(135/190),53.5%(84/157),41.5%(39/94), respectively.The loci with high-frequency of LOH (≥30%) include D1S507 (30.2%) on 1p; D4S415 (51.1%), D4S1538 (42.9%), D4S406 (35.7%) and D4S426 (35.5%) on 4q; D8S520 (36.4%), D8S264 (35.6%), D8S277 (34.7%) and D8S261 (32.3%) on 8p; D16S515 (46.6%) on 16q.The frequencies of LOH on D1S199, D1S234, D1S434, D1S507, D1S2893, RIZ, D4S406, D4S1615, D8S277, D8S1721, D8S1733, D16S408, D16S498, D16S507, D16S512, D17S261, D17S849, D17S921, D17S799, D17S926 and D17S786 were significantly higher in those tumors smaller than 1cm or 2cm in size (P<0.05 or P<0.01). LOH of D1S199 and D4S406 was significantly higher in cases with intact tumor capsule than in those with absent or partially encapsulated tumors (P<0.05). Similarly, LOH frequency on D1S468 and D17S849 was significantly higher in cases with absent or not intact tumor capsule (P<0.05). LOH on D1S243, D1S468, D8S1706 and D16S512 was more frequently detected in tumors with intrahepatic metastasis than in those without (P<0.05). LOH on D1S468 was more frequent in poorly or moderately differentiated HCCs than in those well-differentiated HCCs (P=0.008). In the contrary, LOH on D4S415 was frequently detected in cases with Edmondson grade less thanⅡ(P=0.01). Frequency of LOH on D1S434, D8S277, D16S3091 and D17S926 was frequently detected in the thin trabecular type tumors than those with thick trabecular type (P<0.05) while LOH on D1S468 and D8S1771 was on the contrary. LOH frequency on D8S520 was significantly higher in cases with negative serum HBsAg than in those with positive HBsAg (P=0.043). LOH on D8S1771 was more frequently detected in chronic hepatitis than in cirrhosis (P=0.029), while LOH on D4S415, D8S1733, D17S799, D17S849 and D17S926 was more frequent in those tumors with cirrhosis background. LOH on RIZ, D8S549 and D17S520 was more frequent in female patients than male (P<0.05), while LOH of D4S415 and D16S512 was significantly frequent in male patients (P<0.05).The loci with high-frequency of LOH (≥50%) in HCC include D4S415 (55.6%, 20/36), D17S1791 (66.7%, 16/24), D17S720 (61.8%, 21/34), D17S1796 (60.0%, 9/15), D17S786 (56.7%, 17/30), D17S520 (55.2%, 16/29), D17S960 (55.0%, 11/20), D9S916(62.5%, 10/16), D9S1751 (52.4%, 11/21) and D9S161 (50.0%, 9/18); The loci with LOH in DNs include D17S1796(80.0%, 4/5), D17S720(25%, 2/8), D17S786 (14.3%, 1/7), D17S960 (20%, 1/5), D17S1791 (20%, 1/5), D17S1856 (20%, 1/5), D17S1881 (12.5%, 1/8), D8S1721 (28.6%, 2/7), D9S916(50%, 3/6), D9S1751 (25%, 1/4) and D9S1749 (22.2%, 2/9). The loci with LOH in HCA include D17S720 (12.5%, 1/8), D17S921 (28.6%, 2/7), D17S1791 (12.5%, 1/8), D17S1796 (33.3%, 2/6), D17S1881 (28.6%, 2/7), D4S415 (12.5%, 1/8), D8S1721 (14.3%, 1/7), D1S507 (10.0%, 1/10), D9S916 (33.3%, 1/3), D9S1751 (20.0%, 1/5) and D9S1749 (10.0%. 1/10). Frequency of LOH at D17S520 in HCC is higher significantly than that in DN (P=0.011) and frequency of LOH at 17S1796 in DN higher than that in HCC.In cases 1, 3, and 4, the same microsatellite alterations were observed in both HGDN and HCC at 2, 4, and 6 loci, respectively.Increasing FAL values were seen from DN to Micro HCC, and further to small HCC. FAL value in HCC was higher significantly than that in DN and HCA (PDN<0.001, PHCA<0.001). We observed significant correlation of FAL with Edmondson's classification and liver cirrhosis. FAL value was highter significantly in tumors with Edmondson grade more thanⅢ(P=0.035) and in cases with liver cirrhosis (P=0.031).ConclusionsFrequent microsatellite alterations existed in HCC. The loci with high-frequency of LOH (≥30%) such as D1S507, D4S415, D4S1538, D4S406, D4S426, D8S520, D8S264, D8S277, D8S261, and D16S515 are reported in current study. LOH, which represents tumor suppressor gene pathway, plays a more important role in hepatocarcinogenesis of HCC; MSI, representing mismatch repair gene pathway functions as the next. Furthermore, some clinicopathological features such as serum AFP concentration, HBV infection, tumor size, cirrhosis, Edmondson's grade, tumor capsule, as well as tumor thrombosis, may be significantly correlated with allelic losses on certain chromosome regions.HCA, DN and HCC shared common genetic events on some loci. It indicated that there were some common genetic pathways in the malignant transformation of normal cells. Our results favored a concept of single clone of DN and HCC. Increasing FAL values were seen from DN to Micro HCC, and further to small HCC. There was significant correlation between FAL with Edmondson's classification and liver cirrhosis.
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