Diagnostic And Prognostic Value Of Tissue Factor Pathway Inhibitor 2 Gene Methylation In Hepatocellular Carcinoma

PART I Diagnostic Value of Serum Tissue Factor Pathway Inhibitor 2 Gene Methylation in Hepatocellular CarcinomaBackgroundHepatocellular carcinoma (HCC) is ranked as the third leading cause of cancer-related death worldwide and is the main event leading to death in patients with liver cirrhosis. Despite recent advances in the management of HCC, patient prognosis and survival remain frustrating because, by the time of diagnosis, most HCC cases have developed to late stage and missed the best opportunity to receive optimal therapy. Currently used methods to detect and diagnose HCC mainly consist of serum alpha fetoprotein (AFP) and various imaging technologies, including ultrasonography, computed tomography and magnetic resonance imaging, each of which, however, has its own limitations. AFP has been questioned for its relatively low diagnostic sensitivity. There are still 30%-40% patients with HCC whose AFP examinations are negative. The surveillance ability of imaging technologies relies largely on examiner expertise, presence of liver cirrhosis, and size of the tumor, which affects the effective and timely detection of HCC at its initial stage. Therefore, there is an urgent need for development of novel markers to improve early screening and diagnosis of HCC.In the course of the search for new biomarkers, circulating cell-free DNA (cfDNA) has become a popular research focus. Cell-free DNA is defined as extracellular DNA found in cell-free plasma/serum. It has been reported since at least three decades ago that significantly higher cfDNA levels can be found in the sera of cancer patients, as compared to those with non-malignant diseases. Since then, more and more studies have revealed that cfDNA, derived from apoptosis, necrosis, and secretion of tumor cells, can be used for early detection of various cancers, including HCC. CfDNA changes described in blood include oncogene mutations, microsatellite alterations, and epigenetic alterations, as well as mutations in mitochondrial DNA. Current studies indicate that epigenetic alterations can have a vital effect on tumorigenesis and progression. Moreover, epigenetic silencing of tumor-specific genes due to aberrant methylation of gene promoter regions has been proved to play a vital role in hepatocellular carcinogenesis. Several genes such as p16, p15, GSTP1, RASSF1A, APC, and SFRP1 have been reported to be aberrantly methylated in the sera/plasma of HCC patients and potentially used as epigenetic markers in a noninvasive approach to early detection of HCC. The detection of methylated cfDNA represents one of the most promising methods for early diagnosis of HCC.Tissue factor pathway inhibitor 2 (TFPI2) is a member of Kunitz-type serine protease inhibitors that suppress tumor progression, invasion, and metastasis by inhibiting the plasmin- and trypsin-mediated activation of matrix metalloproteinases. Previous studies have proved that TFPI2 is a potential tumor suppressor gene and frequently inactivated through promoter methylation in several kinds of tumors, such as colorectal cancer. Moreover, TFPI2 methylation has been demonstrated to frequently exist in colorectal cancer patients’ sera and has been suggested to be a potential tumor marker in serum for the diagnosis of colorectal cancer. Recently, TFPI2 was found to be a candidate tumor suppressor gene in human HCC, and frequently silenced via epigenetic alterations, including promoter methylation.ObjectiveWe designed this study to detect TFPI2 methylation status in the serum of patients with HCC, and investigated the potential value of serum TFPI2 methylation in the diagnosis of HCC.MethodsThis study included 96 patients who were diagnosed with HCC,51 patients with liver cirrhosis (LC),50 patients with chronic hepatitis B (CHB) and 36 normal controls based on clinical and laboratory tests from July 2011 through June 2015 at Qilu Hospital of Shandong University. Patients with HCC were selected from those diagnosed according to the 2010 update of the American Association for the Study of Liver Diseases (AASLD) Practice Guidelines for Management of Hepatocellular Carcinoma. Patients with LC and CHB were diagnosed according to the 2009 update of AASLD Practice Guidelines for Management of Chronic Hepatitis B. To explore the feasibility of this approach, we compared TFPI2 methylation status in serum samples of HCC, LC, CHB patients and normol control groups using methylation-specific polymerase chain reaction.Results1. Our results revealed that the percentage of serum TFPI2 promoter methylation was significantly higher in HCC group (43.8%,42/96) when compared with LC group (9.8%,5/51; P= 0.000), CHB group (14.0%,7/50; P= 0.000) and normal control group (13.9%,5/36; P= 0.001), respectively, demonstrating that TFPI2 methylation frequently existed in the serum of patients with HCC.2. When used to discriminate HCC from LC patients, aberrant serum TFPI2 methylation showed 43.8% sensitivity and 90.2% specificity, serum AFP (with a cutoff value of 20ng/ml) showed 61.5% sensitivity and 94.1% specificity, and the combination of serum TFPI2 methylation and AFP showed 77.1% sensitivity and 84.3% specificity. When used to discriminate HCC from CHB patients, serum TFPI2 methylation showed 43.8% sensitivity and 86.0% specificity, serum AFP showed 61.5% sensitivity and 88.0% specificity, and the combination of serum TFPI2 methylation and AFP showed 77.1% sensitivity and 76.0% specificity. When used to discriminate HCC patients from normal controls, serum TFPI2 methylation showed 43.8% sensitivity and 86.1% specificity, serum AFP showed 61.5% sensitivity and 100% specificity, and the combination of serum TFPI2 methylation and AFP showed 77.1% sensitivity and 86.1% specificity. Although the diagnostic sensitivity and specificity of serum TFPI2 methylation in HCC was lower than those of serum AFP in this study, both markers could be combined to improve the detection ability of serum AFP in HCC. Our data indicated that serum TFPI2 methylation could be used as a potential marker for noninvasive detection and diagnosis of HCC.3. Patients with advanced TNM stage (Ⅲ-Ⅳ) showed significantly elevated serum methylation percentage of TFPI2 in comparison with those with early TNM stage (Ⅰ-Ⅱ) (63.2% vs 31.0%; P= 0.002). Moreover, TFPI2 methylation was observed more frequently according to the progression of TNM stage. No significant correlation was observed between the methylation status of TFPI2 and other clinicopathological parameters, such as patient gender, age, tumor size, tumor number, hepatitis B virus infection or serum AFP levels (P> 0.05, respectively).Conclusions1. TFPI2 methylation frequently existed in the serum of patients with HCC, indicating that serum TFPI2 methylation can be used as a potential marker for noninvasive detection and diagnosis of HCC.2. TFPI2 methylation in serum tended to be detected more easily in patients with advanced HCC and might be used as a predictor of HCC progression.PART Ⅱ Prognostic Value of Tissue Factor Pathway Inhibitor 2 Gene Methylation in Hepatocellular Carcinoma after HepatectomyBackgroundHepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the third most frequent cause of cancer-related mortality worldwide. Hepatocarcinogenesis is an extremely complex multistep process in which many signaling cascades are involved, resulting in a heterogeneous molecular profile. A number of genetic alterations associated with the carcinogenesis, development, progression and metastasis of HCC have been described. In addition to genetic abnormalities, epigenetic alterations have been demonstrated to play a crucial role in the initiation, progression and invasion of HCC. As an important and common epigenetic mechanism, DNA methylation leads to the silencing of a broad range of tumor suppressor genes which closely associate with HCC. Interestingly, methylation of these genes in the peripheral blood or tissue specimen could serve as biomarkers for early diagnosis or prediction of prognosis in HCC.Tissue factor pathway inhibitor 2 (TFPI2), a Kunitz-type serine protease inhibitor, can suppress tumor invasion and metastasis by negatively regulating the enzymatic activity of plasmin and trypsin, both of which mediate activation of matrix metalloproteinases and regulation of extracellular matrix degradation. It has been demonstrated that TFPI2 is a candidate tumor suppressor gene. Ectopic overexpression of TFPI2 could significantly inhibit the proliferation and invasiveness of HCC cells, while TFPI2 downregulation can contribute to tumor invasion of HCC cells through alteration in the expression of metastasis-related genes. TFPI2 has been reported to be frequently silenced via promoter methylation in various human cancer types, including HCC. In our previous study, we detected TFPI2 promoter methylation status in the serum of HCC patients and found that the degree of TFPI2 methylation in circulating cell-free tumor DNA was positively related to the tumor, node, metastasis (TNM) stage of HCC. TFPI2 methylation was observed more frequently according to the progression of TNM stage. Based on the above knowledge, we speculate that the aberrant TFPI2 promoter methylation may be closely associated with HCC progression and serve as a potential prognostic marker for HCC.ObjectiveThis study aimed to evaluate the prognostic value of TFPI2 methylation in HCC after hepatectomy, and to investigate its correlation with various clinicopathologcial factors by examining TFPI2 methylation status in 178 surgical specimens of HCC.MethodsWe obtained specimens of hepatocellular carcinoma and corresponding non-tumorous liver samples from 178 patients at the Qilu Hospital of Shandong University from December 2008 through September 2012. All patients with HCC were histologically confirmed and selected from those diagnosed according to the 2010 update of the American Association for the Study of Liver Diseases (AASLD) Practice Guidelines for Management of Hepatocellular Carcinoma. A total of 178 pairs of HCC samples and adjacent non-tumorous liver tissues were used for methylation-specific polymerase chain reaction. Twenty pairs of HCC tumor and matched non-tumorous tissues randomly selected from 178 tissue samples and twenty cases of normal liver tissues without cirrhosis from non-tumor liver tissues were used for real-time quantitative PCR. Survival time between groups was determined by Kaplan-Meier method and compared by log-rank test. Multivariate analyses were performed by Cox proportional hazards models to investigate those prognostic factors that predicted overall survival (OS), disease-free survival (DFS) and early tumor recurrence (ETR).Results1. Methylation of TFPI2 gene was detected in 44.9%(80/178) of primary HCC samples. In contrast, only 10.7%(19/178) of the corresponding non-tumorous liver samples and 5.0%(1/20) of the normal liver samples had TFPI2 methylation detected. The frequency of TFPI2 methylation was significantly higher in primary HCC samples than that in corresponding non-tumorous liver samples (χ2=52.064, P< 0.001) and that in normal liver samples (χ2=11.867, P=0.001).2. The mRNA levels of TFPI2 were detected in the 20 pairs of 178 HCC tissue and the 20 normal liver samples by real-time quantitative PCR. The relative mRNA level of TFPI2 in primary HCC tissue was significantly lower than that in the corresponding non-tumorous liver tissue (Z= 4.626, P< 0.001) and that in the normal liver samples (Z= 5.416, P< 0.001).3. The clinicopathological significance of TFPI2 methylation was evaluated by comparing the TFPI2 methylation group (n= 80) with the unmethylation group (n= 98) of patients with HCC. The results revealed that TFPI2 methylation was significantly correlated with the TNM stage (P= 0.004). TFPI2 methylation was observed in 35 out of 58 cases (60.3%) with higher TNM stage (III+IV) but only in 45 out of 120 cases (37.5%) with lower stage (I+II). No significant correlation was observed between the methylation status of TFPI2 and other clinicopathological variables, such as patients’gender, age, hepatitis B virus (HBV) infection, serum alpha fetoprotein level, number and size of tumor, liver cirrhosis, tumor differentiation, or severe portal vein invasion (P> 0.05, respectively).4. Patients with TFPI2 methylation demonstrated a significantly poorer prognosis than those without TFPI2 methylation for both overall survival and disease-free survival (P < 0.001, respectively). Univariate analyses revealed that tumor size, severe portal vein invasion, early tumor recurrence, TNM stage, and TFPI2 methylation status significantly affected postoperative survival of patients with HCC (P< 0.05, respectively). Multivariate analyses were used to investigate the correlation between TFPI2 methylation and postoperative survival in the presence of other prognostic factors for OS and DFS. Multivariate analysis with respect to OS indicated that TFPI2 methylation (P= 0.002), TNM stage (P= 0.000), and ETR (P= 0.000) were independent prognostic factors for OS. Multivariate analysis with respect to DFS revealed that TFPI2 methylation (P= 0.000) and TNM stage (P= 0.015) were independent prognostic factors for DFS. If we only selected the 158 patients infected with HBV as the study population and included HBV viral load as the variable, we found that HBV viral load, tumor size, severe portal vein invasion, early tumor recurrence (ETR), TNM stage and TFPI2 methylation status significantly affected overall survival of patients with HBV-related HCC by univariate analyses (P< 0.05, respectively). Multivariate analysis with respect to OS indicated that TFPI2 methylation was still one of the independent prognostic factors for OS (P= 0.002). Besides, HBV viral load (P= 0.015), TNM stage (P= 0.000) and ETR (P= 0.000) were also found to be prognostic factors for OS of patients with HBV-related HCC.5. Sixty one patients (34.3%) had early tumor recurrence. ETR was significantly correlated with tumor number, TNM stage, and TFPI2 methylation (P< 0.05, respectively). ETR after hepatectomy was observed in 38 out of 80 cases (47.5%) with TFPI2 methylation HCC, but in only 23 out of 98 cases (23.5%) without TFPI2 methylation HCC (P= 0.001). Finally, the multivariate analysis on ETR was carried out with these aforementioned factors, and we found that the methylation of TFPI2 was the only independent predictor for ETR of HCC after resection (Hazard ratio= 2.295, P= 0.002). If we only selected the 158 patients infected with HBV as the study subjects and included HBV viral load as the variable, we found that tumor number and TFPI2 methylation were closely associated with ETR in patients with HBV-related HCC by univariate analyses (P< 0.05, respectively). Multivariate analysis with respect to ETR indicated that TFPI2 methylation was still the only independent prognostic factors for ETR of HBV-related HCC after resection (Hazard ratio= 2.448, P= 0.001).Conclusions1. Our results indicated that TFPI2 methylation frequently existed in the tumor tissue of patients with HCC, which contributed to the downregulation of TFPI2 mRNA levels in HCC.2. Methylation of TFPI2 gene predicts high risk of advanced tumor stage, early tumor recurrence and poor prognosis, and it could be a potential prognostic biomarker in patients with HCC after hepatectomy.