Clinical Cancer Research: Gene Expression Profiling Reveals Potential Biomarkers of Human Hepatocellular Carcinoma

Hu-Liang JiaQing-Hai YeLun-Xiu QinAnuradha BudhuMarshonna ForguesYidong ChenYin-Kun LiuHui-Chuan SunLu WangHong-Zhou LuFang ShenZhao-You Tang and Xin Wei Wang


Purpose: Hepatocellular carcinoma (HCC), common cancer worldwide, has a dismal outcome partly due to the poor identification of early-stage HCC. Currently, one-third of HCC patients present with low serum α-fetoprotein (AFP) levels, the only clinically available diagnostic marker for HCC. The aim of this study was to identify new diagnostic molecular markers for HCC, especially for individuals with low serum AFP.

Experimental Design: We used the microarray technique to determine the expression profiles of 218 HCC specimens from patients with either high or low serum AFP. From the microarray study, we selected five candidate genes (i.e., GPC3, PEG10, MDK, SERPINI1, and QP-C), which were overexpressed in HCCs. Using quantitative real-time PCR analyses, we validated the expression of these five genes in 50 AFP-normal and 8 AFP-positive HCC specimens and 36 cirrhotic noncancerous hepatic specimens, which include 52 independent specimens not used in microarray analysis.

Results: A significant increase in the expression of the five candidate genes could be detected in most of the HCC samples, including those with normal serum AFP and small tumors. GPC3, MDK, and SERPINI1 encode known serum proteins. Consistently, a significant increase in serum midkine, encoded by MDK, was associated with HCC patients, including those with normal serum AFP. Using a prediction analysis of microarray, we showed that a combined score of these five genes can accurately classify noncancerous hepatic tissues (100%) and HCC (71%).

Conclusions: We suggest that a diagnostic signature approach using a combined score of these five biomarkers rather than a single marker may improve the prediction accuracy of HCC patients, including those with normal serum AFP and smaller-sized tumors.

Hepatocellular carcinoma (HCC), endemic to Asia and Africa with a rising incidence in Western countries (13), is one of the most common and aggressive cancers worldwide. It has been the third cancer killer worldwide and the second cancer killer in China since the 1990s (45). Globally, the 5-year survival rate of HCC is <5% and ∼598,000 HCC patients die each year (6). The high mortality associated with this disease is mainly attributed to the inability to diagnose HCC patients at an early stage. In fact, most symptomatic HCC patients are diagnosed at an advanced stage, thus precluding their chance for surgical intervention (7). In contrast, HCC patients who were diagnosed at an early stage and received curative resection had a significantly improved survival time (810). Thus, early detection and resection have been generally recognized as the most important factors to achieve long-term survival for HCC patients.

Since its detection in the serum of HCC patients in the 1970s, α-fetoprotein (AFP) has been the only serologic marker widely used for diagnosing HCC patients. This marker allows the identification of a small set of HCC patients with smaller tumors, and these patients have a relatively long-term survival rate following curative treatment (810). Presently, the only approach to screen for the presence of HCC in high-risk populations is the combination of serum AFP and ultrasonography (1113). However, elevated serum AFP is only observed in about 60% to 70% of HCC patients and, to a lesser extent (33-65%), in patients with smaller HCCs (14). Moreover, the nonspecific elevation of serum AFP has been found in 15% to 58% of patients with chronic hepatitis and 11% to 47% of patients with liver cirrhosis (14). Therefore, it is necessary to identify new serologic HCC biomarkers that have sufficient sensitivity and specificity for the diagnosis of HCC patients, especially in AFP-normal and/or smaller HCC cases.

This study was designed to identify new biomarkers for HCC. In this study, we first conducted microarray analyses to identify genes that were differentially expressed in HCC samples among patients with high serum AFP to those with low serum AFP. We then selected five candidate genes that were highly expressed in HCC samples, including those with low serum AFP and whose protein products could potentially be identified in sera. We did validation analysis by quantitative real-time PCR analyses (qRT-PCR). We show that these five genes separate HCC specimens from cirrhotic and noncancerous liver specimens. A significantly increased expression can be found in most of the HCC specimens, including cases with normal serum AFP and small tumor size, suggesting that the products of these five genes may serve as biomarkers to aid HCC diagnosis. Serum midkine was significantly elevated in HCC patients, including those who had normal serum AFP and smaller tumors. These results warrant further efforts in the development of serum-based detection assays for GPC3 and SERPINI1, and possibly PEG10 and QP-C, and an algorithm by combining these five markers as a potential early indicator for HCC.

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