Clonal Origins Of Multiple And Recurrent Hepatocellular Carcinoma By Detection Of Microsatellite Loss Of Heterozygosity: Diagnosis And Clinical Significance

BackgroundHepatocellular carcinoma (HCC) is one of the most common cancers in the world and is particularly prevalent in China. Though the survival has been improved greatly compared with the past prognosis, the 5-year survival rate, which is only around 40% after surgical resection. One of main reasons, besides tumor recurrence, is that multiple lesions are detected once HCC is diagnosed. Futher understanding the association of biological features and prognosis of multiple and recurrent HCC may provide more theoretical bases for reasonable treatment of liver cancer.Multiple and recurrent HCCs are mainly caused by intrahepatic metastasis (IM) known as developing from the same tumor or multicentric occurrence (MO) also known as multicentric carcinogenesis, which is independent from the primary tumor. Discriminating them is very important not only for the study of hepatocarcinogenesis, but also for determination of therapeutic strategies. When multiple tumors are MO in HCCs, each nodule has the same biological behavior as primary solitary HCC. Furthermore, the survival in the MO group after surgery was significantly higher than that in the IM group. However, when multiple tumors are monoclonal in HCCs, the nodules regarded as intrahepatic spread fail to be radically resected and consequently result in recurrence rapidly. As a result, aggressive therapy may not be warranted in cases with IM, but in cased with MO, intervention should be taken within the limits of liver functional reserve. HCC with IM recurs earlier and has a poorer prognosis than that with MO. Likewise,recurrent tumor may be monoclonal, that is, it may originate from intrahepatic metastasis (IM) of the remnant foci. In addition, recurrent HCC may originate from multicentric occurrence, which is independent from the primary tumor. There has been no accurate differential diagnosis between IM and MO in multiple and recurrent HCC. Many studies had tried to analyze the clonality of HCC by histopathological examinations and molecular methods. The differential diagnosis of IM and MO is mainly based on histopathological findings as reported by the Liver Cancer Study Group of Japan with modifications, but it is relatively subjective. Previous studies had used DNA ploidy patterns, p53 mutation patterns, the integration pattern of HBV-DNA, and the X chromosome inactivation assay as tumor markers of clonal origins. However, these methods have their limitations. The genesis of liver cancer is a multistep process reflecting cumulative genetic alterations that include activation of oncogenes and inactivation of tumor suppressor genes. Genetic instability and genomic instability can be observed in many human cancers including HCC. Loss of heterozygosity (LOH) in HCC is frequent in the chromosomal arms 1p,4q,5q,8p,8q,9p,10q,11p,13q,14q,16q,17p and 22q, which suggests that tumor suppressor genes may take part in hepatocarcinogenesis. Different LOH patterns in HCC may reflect heterozygosity in origins. The LOH pattern can also be examined by microsatellite markers to clarify the clonality of multiple and recurrent HCC. LOH assay has an outstanding advantage in that only a small amount of DNA is required. Thus, it can be applied to routine, small liver specimens, including liver biopsies and fine-needle aspirates. In this way, tumor clonality can be evaluated in some patients even before surgical resection. Moreover, LOH assay can be applied to formalin-fixed, paraffin-embedded materials. Microdissection under microscopes enables effective DNA extract from paraffin-embedded materials.Objective⑴To distinguish intrahepatic metastasis from multicentric occurrence in multiple and recurrent hepatocellular carcinoma by examining LOH patterns on 15 microsatellite markers with a high incidence of LOH in HCC;⑵To set up a LOH profile consisting of several microsatellite markers with a high percentage of different LOH patterns.⑶To compare clinicopathological features and prognosis between intrahepatic metastasis and multiple occurrence of HCC.Materials and MethodsSeven microsatellite markers were selected for LOH analysis because of their high frequencies of LOH according to the previous study about genomic instability of HCC and microsatellite variations of small HCC in our department. In addition, another 8 microsatellite markers were selected for their high frequencies of LOH reported in the recent literatures.Among the patients with multiple and recurrent HCC receiving radical surgical resection, 30 and 40 cases were selected in each group,respectively. The clonal relations between multiple nodules from individuals were determined in accordance with conventional histological criteria. The tumor and the corresponding non-tumorous liver tissue were harvested by microdissection under direct microscopes separately, then DNA was extracted and examined by PCR-SSCP. LOH of microsatellite markers was determined, and clonality analysis was based on LOH patterns of multiple tumors in 70 patients with multiple HCC or recurrent HCC. LOH assay by PCR-SSCP was confirmed by capillary electrophoresis sequencing. For further analysis between IM group and MO group in 66 cases with multiple or recurrent HCC, the clinicopathological variables and time to progression were investigated.ResultsThe LOH patterns of 15 microsatellite markers for multiple tumor within one individual patient were analyzed according to the criteria for clonality of HCC by as follows: The LOH pattern between the different tumors were regarded as identical when the same marker demonstrated loss of the same allele or retention of heterozygosity. It was regarded as different LOH patterns when the same marker demonstrated loss of one allele in one tumor but no loss or loss of the other allele in the other tumor. The percentage of different LOH patterns equaled the number of different LOH loci divided by the number of informative loci×100%. Tumors with more than 30% of different LOH patterns were designed as having a different origin, multiple occurrence (MO), while those with less than 30% were designed as"undetermined".LOH was found at a frequency ranging from 22.2% (10/45) in D4S415 to 56.9% (33/61) in D16S514 with 36.7% in average in 68 samples from 30 multiple HCC. According to analysis of LOH patterns, 33.3%(10/30), 60.0%(18/30), and 6.7% (2/30) were considered to be MO, IM for intrahepatic metastases, and undetermined group, respectively. The clonality diagnosis by LOH pattern was 23.3% (7/30) different from histopathological diagnosis in the 30 multiple HCC. LOH determined by PCR-SSCP was confirmed by direct DNA sequencing in the samples of 4 randomized multiple HCC.LOH was found at a frequency ranging from 26.8% (19/71) in D4S406 to 46.9% (45/96) in D16S505 with 37.5% in average in 102 samples of 40 recurrent HCC. According to analysis of LOH patterns, 30.0% (12/40), 65% (26/40), and 5% (2/40) were considered to be MO, IM for intrahepatic metastases, and undetermined group, respectively. The clonality diagnosis by LOH pattern was 22.5% (9/40) different from histopathological diagnosis in the 40 recurrent HCC.Among all the 70 cases, multiple HCCs in 22 cases were diagnosed to originate from multicentric occurrence, however, those in 44 cases were diagnosed to originate from intrahepatic metastases by means of PCR-SSCP. In the MO group, 59.1% (13/22) of cases showed different patterns of LOH in D4S402 and D4S406. In addition, more than 30% of the cases showed different patterns of LOH in D17S831, D16S505, D17S938 and D8S277. By contrast, the cases with different patterns of LOH in D8S258 and D8S520 were less than 10%. We also found that all the 22 cases in MO group could be differentiated from the IM group just by analysis of LOH patterns of 14 from the 15 microsatellite markers if D8S258 were ruled out.The following variables were significantly different between group MO and group IM by univariate analysis: tumor size, vascular invasion, histological grade, liver background disease. Time to progression was (33.75±4.45) months and (14.23±2.54) months in group MO and IM of recurrent HCC, respectively. As for their prognosis, Kaplan–Meier and log-rank test demonstrated the disease-free survival in group MO was significantly better than that in group IM (P=0.001).Conclusions1. In our study, loss of heterozygosity of 15 microsatellite markers on multiple chromosomes (1,4, 8, 13, 16, and 17) was examined by PCR-SSCP assay in 70 cases with multiple or recurrent HCC. According to different LOH patterns, the clonality of multiple tumor nodules was determined within individual patients, which could make for inaccuracy of analyzing the clonality by histopathological features alone.2. LOH profiles by 14 microsatellite (except D8S258) markers could yield the same accurate evaluation of clonality in multiple and recurrent HCC as those by all the 15 microsatellite markers. More than 30% of HCC with MO showed different patterns of LOH in D4S402, D4S406, D17S831, D16S505, D17S938 and D8S277 which suggested that these six markers were more sensitive and specific and could be the first choice of microsatellite markers to distinguish multicentric occurrence in multiple and recurrent HCC.3. Microdissection under microscopes ensured effective DNA extract from paraffin-embedded material. In combination with PCR-SSCP assay, LOH could be rapidly, simply and cheap examined. Samples from 8 cases with multiple or recurrent HCC were randomly chosen and LOH was confirmed by amplified DNA sequencing4. Though differentiation between multicentric occurrence and intrahepatic metastasis can not be made completely by histopathological criteria, some multiple HCCs developing on the basis of liver cirrhosis tend to have a higher potential for developing multicentric occurrence if the multiple nodules have smaller sizes with better differentiation, no vascular invasion and longer intervals to tumor progression. Using microsatellite LOH assay combined with clinicopathological features and morphological presentation is a more precise method to assess tumor clonality.5. Time to progression was significantly longer in group MO than that in group IM of recurrent HCC. Recurrent HCC patients could have a better prognosis.