The Value Of Chromosomal Microsatellite Alterations In Molecular Staging And Prognosis Prediction Of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most frequent human cancers worldwide, and it has been ranked the 2nd cancer killer in China since 1990s. Despite clinical advances in surgical resection, the prognosis of HCC still remains not satisfactory. An accurate assessment of postoperative prognosis, is useful information for the triage of patients into groups who may benefit from adjuvant therapy. At present, prognotic predictors primarily include tumor stage and some histopathological parameters. Among the prognostic predictors, histopathological markers, such as tumor size and tumor differentiation, do not always provide enough information upon which to base predictions of patient outcome. Although TNM stage is the most useful prognostic factor, it is not nearly as useful as a predictor of tumor recurrence. And also, we often find that some patients who have undergone curative resection for HCC at the same TNM stage have different prognosis, indicating that the staging contains some inherent deficiencies. Therefore, identification and characterization of molecular biological outcomes are needed to combine into traditional tumor stage, which may provide us with a molecular staging of tumors for more accurate prognosis prediction.It is well known that the stepwise accumulations of genetic alterations, such as the mutation of oncogenes, promoter methylation of tumor suppressor genes, and microsatellite alterations, play an important role in tumor development and progression. Microsatellite alterations, including loss of heterozygosity (LOH) and microsatellite instability (MSI), as two important markers of genomic instability, are considered to be prognostic markers of tumors and have great value in molecular staging and classification. In addition, tumor circulating DNA carrying several of these molecular changes has also been reported by more and more studies, so people prefer genetic markers in peripheral blood to predict prognosis due to their easy accessibility, simple manipulation and clinical utility. In this study, we will detect LOH and MSI in tumor tissue and plasma circulating DNA samples to explore their value in molecular staging and prognosis prediction of HCC.Objective: To explore the value of microsatellite alterations in molecular staging of HCC by survival analyses of these alterations in HCC patients with curative resection.Methods: We screened 225 paraffin-embedded tissue samples of HCC for microsatellite alteraions by tissue microdissection technology using 28 microsatellite markers selected from eight chromosome arms, including 8p, 1p, 17p, 3p, 9p, 4q, 13q, and 16q, all of which are known to contain candidate tumor suppressor genes or to function as prognostic factors, and further tested the associations between the microsatellite alteraions and postoperative 5-year survival of HCC patients.Results: Loss of heterozygosity (LOH) was frequent at chromosomal loci analyzed, whereas microsatellite instability (MSI) seldom occurred. LOH at D8S298 locus was closely associated with intrahepatic metastasis and TNM stage (P=0.010 and P=0.003, respectively). A univariate analysis of patients' outcome showed that LOH at D8S298 was significantly associated with a decreased probability of survival. Patients in whom tumors showed D8S298 LOH in entire cohort had 5-year overall and disease-free survival rates of 39 ± 6 percent and 36 ± 7 percent, respectively, as compared with 54 ± 5 percent (P=0.024) and 60 ± 6 percent (P=0.002), respectively, in patients in whom tumors did not have D8S298 LOH. Likewise, LOH at D1S199 was strongly associated with both decreased overall and decreased disease-free survival. Patients whose tumors showed D1S199 LOH in entire cohort had 5-year overall and disease-free survival rates of 28 ± 7 percent and 29 ± 8 percent, respectively, as compared with 55±5 percent (P=0.003) and 53 ± 5 percent (P=0.02) in the group that did not have D1S199 LOH. Notably, even among the patients with TNM stage I disease, both D8S298 LOH and D1S199 LOH were also highly associated withdecreases in both overall (44 ± 9 vs 68 ± 6, P=0.007 and 30 ± 8 vs 69 ± 6, P<0.001, respectively) and disease-free survival (42 ± 9 vs 66 ± 6, P=0.038 and 39 ± 9 vs 66 ± 6, P=0.015, respectively). Of the patients with TNM stage II or III disease, however, both D8S298 LOH and D1S199 LOH status did not demonstrate the role of risk stratification in survival analysis. Additionally, no statistically significant associations with overall or disease-free survival were observed for any other marker we analyzed in this study. In a multivariate analysis, The Cox proportional hazards model showed that D8S298 LOH was independently associated the decreased DFS (P=0.039), and D1S199 LOH was independently associated with decreased OS (P=0.016).Conclusions: D8S298 LOH and D1S199 LOH are independently associated with a poor prognosis in HCC patients with curative resection, and the both LOH status can divide the patients with TNM I stage disease into two subgroups with different clinical outcome, which indicates that D8S298 and D1S199 LOH have great value in molecular staging of HCC.PART IIThe prognostic value of loss of heterozygosity in plasma circulating DNA of patients with hepatocellular carcinomaObjective: To explore the concordance of LOH in plasma DNA with corresponding tumor tissues and its prognostic value in HCC patients.Methods: D8S298 and D1S199 loci, the screened two microsatellite markers related to prognosis from the results of part I, were used to detect LOH in 126 HCC plasma samples, and their clinicopathological correlations were also analyzed. Because the postoperative time of the plasma samples used in this study is not long, only or so a year, we attempt toexplore the potential prognostic value by their clinicopathological associations. Additionally, three high-polymorphic microsatellite markers (D8S277, D8S298, and D8S1771)located at chromosome 8p were selected to detect LOH in 62 matched HCC tumor tissues and corresponding plasma samples.Results: Of the 39 tissue samples with LOH at the three microsatellite loci,same alteration was detected in 33 (84.6%) of their matched plasma. Among the 126 plasma DNA samples, LOH frequencies of D8S298 and D1S199 loci were 42.0% (37/88)and 29.2%(26/89), respectively. LOH at D8S298 was closely associated with intrahepatic metastasis and TNM stage (P=0.028 and P=0.050, respectively), whereas the associations of D1S199 LOH with clinicopathological features were not observed (P>0.05, both).Conclusions: LOH showed a high concordant pattern between tumor tissue and plasma DNA of HCC. LOH at D8S298 in plasma circulating DNA may serve as a potential marker for assessing metastatic recurrence after HCC resection and play an important role in prognosis prediction.