Lymphatic vessel endothelial hyaluronan receptor‑1 is a novel prognostic indicator for human hepatocellular carcinoma
- Authors:
- Published online on: August 6, 2013 https://doi.org/10.3892/mco.2013.167
- Pages: 1039-1048
Abstract
Introduction
Hepatocellular carcinoma (HCC) is the sixth most common type of cancer worldwide (1). The median survival time of patients with unresectable tumors and untreated patients with less advanced disease is <4 months and <1 year, respectively (2–6). The total survival rate of HCC patients is 3–5% (7), due to the high rate of recurrence following resection and the resistance to chemotherapy.
This type of cancer is particularly aggressive as a result of its high degree of vascularization. Multiple angiogenic and anti-angiogenic factors released by the tumor and host cells are involved in this process (8). The microvascular density of HCCs correlates with disease prognosis and postoperative disease recurrence (9–12). Angiogenesis, the formation of new blood vessels from preexisting vasculature, is crucial in the development, growth and metastasis of various neoplasms, including HCCs (13,14). Although angiogenesis constitutes a promising avenue for the identification of markers and novel therapeutic approaches, the ramifications of the signaling pathways are complex and have not yet been fully elucidated, particularly with respect to vascularization.
This study aimed to identify angiogenic genes that are deregulated by HCC and determine their potential as predictors of postoperative survival. Liver tissue samples and nodules from three groups of HCC patients were used to perform TaqMan gene array analysis and to identify the most promising biomarker of HCC in terms of patient characteristics, survival rates and tissue histology.
Materials and methods
Paired analysis of angiogenic gene expression in HCC nodules and non-HCC liver tissue
A preliminary experiment was conducted, using tissue samples from 12 HCC patients to identify the affected angiogenesis-related target genes to be investigated in this study. All the patients were Japanese and they had undergone surgical HCC resection between October, 2008 and October, 2009 at the Department of Surgery, Institute of Gastroenterology, Tokyo Women’s Medical University, Japan. The majority of the patients were male, with moderately differentiated HCC histology and negative for intrahepatic metastases (IM), portal vein invasion (Vp) or venous invasion (Vv). Half of the patients had liver cirrhosis or chronic hepatitis resulting from viral infection (Table I). The patients provided written informed consent according to the institutional regulations. This study was approved by the Ethics Committee and Institutional Review Board of the Tokyo Women’s Medical University.
 | Table I.Characteristics of the 12 HCC patients who provided liver samples for the identification of angiogenic genes deregulated by HCC. |
The tissue samples collected from primary HCC nodules and non-HCC liver tissue of each patient were immediately snap-frozen and stored at −80°C until further use. The samples were then homogenized and total RNA was isolated using the RNeasy® Mini kit (Qiagen, Valencia, CA, USA). Subsequently, complementary DNA (cDNA) was synthesized using 2 μg of total RNA and High Capacity RNA-to-cDNA Master Mix (Applied Biosystems Inc., Foster City, CA, USA) according to the manufacturer’s protocol. We used the TaqMan® Array Gene Expression 96-well Human Angiogenesis Plate (Applied Biosystems Inc.) to determine the angiogenic gene profiles of the specimens in each sample set. A total of 92 angiogenesis- or lymphangiogenesis-associated gene assays and 4 control endogenous gene assays were performed in each plate. The target genes investigated in this study are listed in Table II. The gene expression level was analyzed using a 7500 Real-Time PCR system (Applied Biosystems Inc.). Polymerase chain reaction (PCR) using TaqMan® Gene Expression Master Mix (Applied Biosystems Inc.) was performed under the following conditions: 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 30 sec at 95°C and 1 min at 60°C. Data were analyzed using SDS software, version 1.4 (Applied Biosystems Inc.) and gene expression levels were compared using the ΔΔCt method (15). Significantly upregulated or downregulated genes were screened using a cut-off P-value of <0.01.
Paired analysis of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) expression in HCC nodules and non-HCC liver tissue
Archived liver tissue samples (primary HCC tumors; >95% HCC cells and non-HCC tissue from the same patient) from HCC patients were tested for LYVE-1 expression. The 58 complete sets were obtained from Japanese patients who had undergone surgical HCC resection between December, 1993 and May, 2007 at the Department of Surgery, Institute of Gastroenterology, Tokyo Women’s Medical University, Japan. Similar to the 12-patient group, the archived samples were collected primarily from males with moderately differentiated HCC histology and cirrhosis or chronic hepatitis resulting from viral infection (Table III). The patients provided written informed consent in accordance with institutional regulations.
| Table III.Characteristics of the 58 HCC patients investigated for the histology of HCC nodules and non-HCC liver tissue and survival curves. |
The formalin-fixed paraffin-embedded (FFPE) samples were preserved using the general protocol of the Institute of Pathology, Tokyo Women’s Medical University, Japan. Each FFPE specimen was cut into 10-μm sections, deparaffinized in xylene and rehydrated in graded ethanols. The tissues were dissected and total RNA was isolated using the RNeasy® FFPE kit (Qiagen). Subsequently, cDNA was synthesized using High-Capacity cDNA Reverse Transcription kits (Applied Biosystems Inc.) with 1 μg of total RNA, according to the manufacturer’s protocol. The expression of LYVE-1 and β-2 microglobulin (B2M), which was used as endogenous control, were measured using a StepOne™ Real-Time PCR system (Applied Biosystems Inc.). The TaqMan® primers/probe for LYVE-1 (Assay ID: Hs00272659_m1) and B2M (Assay ID: Hs99999907_m1) were purchased from TaqMan® Gene Expression Assays (Applied Biosystems Inc.). PCR was performed using TaqMan® Fast Master Mix under the following conditions: 20 sec at 95°C, followed by 40 cycles of 1 sec at 95°C and 20 sec at 60°C. Data were analyzed using StepOne™ software, version 2.1 and the gene expression level was quantified by the ΔΔCt method.
Histological analysis of the nodules
All the HCC specimens, including the fresh specimens from the 12 patients, were histologically evaluated according to the general rules for the clinical and pathological study of primary liver cancer (16). The clinicopathological parameters of the specimens, including tumor diameter, liver status, IM, Vp, Vv and histopathological classification were obtained.
Correlations between LYVE-1 expression, HCC differentiation and patient survival
We analyzed archived HCC samples from 103 HCC patients. Those archived samples had been primarily collected from males with moderately differentiated HCCs and cirrhosis or chronic hepatitis resulting from viral infection (Table IV). The patients were Japanese and had undergone surgical HCC resection between December, 1993 and May, 2007 at the Department of Surgery, Institute of Gastroenterology, Tokyo Women’s Medical University, Japan. The patients provided written informed consent in accordance with institutional regulations.
Statistical analysis
We used Wilcoxon signed-rank tests to compare gene expression levels between HCC nodules and non-HCC liver tissue. The correlation between LYVE-1 expression levels in HCC nodules and the degree of nodule differentiation was assessed using Steel-Dwass tests. Disease-free survival (DFS) and overall survival (OS) were calculated by the Kaplan-Meier method and differences in survival curves were analyzed using log-rank tests. The follow-up time was defined as the time from the date of surgery to the date of death or the last known follow-up. The correlation of LYVE-1 expression to the clinicopathological parameters was evaluated using Fisher’s exact probability tests or Chi-square tests. Independent prognostic factors were analyzed using the Cox proportional hazards regression model. P<0.05 was considered to indicate a statistically significant difference. All tests were two-sided. We used JMP® software, version 9.0.1 (SAS Institute Inc., Cary, NC, USA) to compute all the statistics.
Results
Identification of angiogenic genes deregulated by HCC
The gene array analysis of liver tissue samples collected from the initial 12-patient group identified 14 genes differentially expressed in HCC and non-HCC tissues (Table V). Among these, the genes encoding collagen type XVα1, IVα1 and IVα2, as well as two growth factor-related genes [EGF-like repeats and discoidin I-like domains 3 (EDIL3) and platelet-derived growth factor β polypeptide (PDGFB)] were upregulated by HCC. HCC was also associated with upregulation of the gene encoding neurite growth-promoting factor 2 (midkine, MDK), which is involved in embryonic development and inflammation. By contrast, HCC caused downregulation of genes encoding inflammatory chemokines (CXCL2 and CXCL12) and genes associated with vessel growth, namely neuropilin 2 (NRP2) and LYVE-1 (Table V).
Interpatient variability in LYVE-1 downregulation by HCC
The effect of HCC on LYVE-1 expression was verified using a larger cohort of 58 patients. LYVE-1 expression was significantly lower in HCC nodules compared to the corresponding non-HCC liver tissue (P<0.0001). Paired analysis of HCC nodule and non-HCC liver tissue samples from each patient revealed a large variability in LYVE-1 expression between the patients (Fig. 1A).
Correlation between LYVE-1 downregulation and HCC nodule differentiation
Since the only parameter affected by LYVE-1 expression was the histology of the nodules, this association was further investigated by analysis of HCC nodule samples. The possible contribution of disease severity to interpatient variability in LYVE-1 expression was assessed using a large number of patients for whom nodule histology reports and archived tissue samples were available for correlation analysis. The loss of nodule differentiation was associated with a decrease in LYVE-1 expression, which would occur early in the evolution of the disease (P=0.0006). The LYVE-1 expression level was decreased >5-fold between the first two stages (P<0.0001) and remained comparable in poorly differentiated HCC nodules (P=0.91). These data support an association between LYVE-1 expression and HCC progression (Fig. 1B).
Correlation between LYVE-1 expression and patient survival
The detrimental effect of LYVE-1 downregulation on the survival of HCC patients was confirmed in the cohort of the 103 HCCs based on a similar analysis of HCC nodules. Based on a median observation frequency of 2,752 days, this group was characterized by a 5-year DFS rate of 34.1% and a 5-year OS rate of 66.6% and was used to assess the effect of LYVE-1 expression on survival by dividing the patients into groups with high expression (>7-fold relative to the lowest value) and low expression (<7-fold relative to the lowest value) in HCC nodules. Fig. 2A shows that DFS was not significantly affected by the LYVE-1 expression level in HCC nodules. By contrast, the OS curve decayed less rapidly for the high-expression group compared to that for the low-expression group, resulting in 5-year OS rates of 81 and 45%, respectively (P=0.004; Fig. 2B). In fact, all the patients with low LYVE-1 expression reached the 45% OS plateau phase within 4 years after surgery. Accordingly, these data were confirmed by univariate Cox regression analyses for DFS [hazard ratio (HR)=1.394; 95% confidence interval (CI): 0.864–2.203; P=0.1694] and OS (HR=2.458; 95% CI: 1.298–4.625; P=0.0063). Multivariate Cox regression analyses identified LYVE-1 expression as a significant independent prognostic parameter of OS (HR=3.067; 95% CI: 1.507–6.273; P=0.0021) (Tables VI and VII).
Specificity of factors affected by LYVE-1 expression in HCC patients
Analyses were performed to determine whether other aspects of the disease were associated with the downregulation of LYVE-1 expression. The patients were re-examined by comparing the low- and high-expression groups with respect to the general characteristics and the histology of the HCC nodules (Table VIII). The expression of LYVE-1 did not appear to exert any effect on basic characteristics, such as age, gender ratio, liver status or viral infection and IM, Vp and Vv in neither one of the two groups. With respect to tissue histology, the HCC nodules were significantly less differentiated in the low-expression group (P<0.0064; Table VIII). These data suggest that LYVE-1 downregulation may be a marker of nodule dedifferentiation in HCC tissues.
| Table VIII.Association between LYVE-1 expression in HCC liver nodules and clinicopathological characteristics of the 103 patients. |
Discussion
The field of cancer research has benefited significantly from genetic and functional analyses of oncogenes and tumor suppressor genes (17). Among the 92 angiogenic genes investigated, 14 genes were shown to be significantly deregulated in HCC. Some of these genes (COL15A1, COL4A1, COL4A2, PDGFB, MDK and EDIL3) were upregulated, whereas others (ANGPTL1, CXCL12, CXCL2, NRP, HGF, LYVE-1, PDGFRA and PLG) were downregulated, suggesting that they may be involved in the mechanism of carcinogenesis or tumor growth. Among these genes, LYVE-1 was one of the most strongly downregulated genes in HCC nodules, compared to adjacent non-HCC tissue. This gene is of particular interest, as the triad of glypican-3, LYVE-1 and survivin was previously demonstrated to provide a reliable diagnosis of early HCC (18). The present study demonstrates the potential of LYVE-1 deregulation as an independent biomarker of postsurgical outcome in HCC patients.
In the present study, the clinicopathological findings revealed a significant correlation between LYVE-1 expression and the histology of HCC nodules. From a dynamic perspective, the gradual loss of differentiation may be associated with LYVE-1 downregulation occurring early during this process. LYVE-1 expression levels in poorly or moderately differentiated nodules were comparable and were decreased by >5-fold compared to the levels in well-differentiated nodules. These data are consistent with those of a previous study, demonstrating that LYVE-1 expression decreases progressively in HCC nodules transitioning from a polyclonal cirrhotic to a monoclonal cirrhotic phenotype (19). In addition, our study suggests that LYVE-1 may be an early marker of HCC tumorigenesis.
The potential of LYVE-1 as a predictor of postsurgical outcome in HCC patients was clearly demonstrated in terms of the 5-year OS. Logistic regression analyses revealed that low LYVE-1 expression in HCC nodules was significantly predictive of shorter OS. Since the decrease in LYVE-1 expression occurs early during the nodule transformation phase, these data suggested that close monitoring of LYVE-1 expression after surgery may considerably improve survival in HCC patients.
Our understanding of the role of LYVE-1 in tumorigenesis is evolving rapidly as the dogma is challenged by thorough immunohistochemical examination (20). This marker of lymphatic endothelial cells has been detected in the endothelial cells of the hepatic blood sinusoids of healthy subjects and patients diagnosed with liver cancer and cirrhosis. Notably, this protein is not detected in angiogenic blood vessels of liver tumors and is weakly detected in the microcirculation of regenerative hepatic nodules in cirrhosis, despite the fact that both types of vessels are derived from liver sinusoids. Furthermore, the lymphatics are restricted to the margins of HCCs and the surrounding tissues. This distribution is consistent with the LYVE-1 downregulation observed in the highly vascularized HCC nodules compared to non-HCC tissues. Accordingly, the restriction of LYVE-1 to the periphery of the tumor may translate into progressive decrease, in relative expression with an increase in tumor size, as supported by a previous study demonstrating that LYVE-1 attenuation in the sinusoidal endothelium was associated with hepatic disease progression (21).
The most common cause of mortality in HCC patients is tumor recurrence following surgery, which may be caused by small metastatic lesions or metachronous multicentric lesions in the case of liver inflammation or cirrhosis. Chronic aggressive hepatitis is a significant risk factor of HCC recurrence following hepatectomy (22). Notably, the expression of LYVE-1 in the lymphatic endothelium is downregulated by the pro-inflammatory cytokine tumor necrosis factor-α in vitro and in vivo(23–25), suggesting that LYVE-1 expression may be suppressed by hepatitis. The fact that inflammation is initiated early during the course of liver disease is consistent with our hypothesis that LYVE-1 may be an early marker of HCC tumorigenesis.
LYVE-1 is a member of the Link protein superfamily and is similar to the leukocyte hyaluronan receptor CD44, which is known to facilitate tumor cell invasion. Hyaluronan is a key substrate for cell migration among tissues during inflammation, wound healing and neoplasia (26). Recent studies suggested that the ligands of LYVE-1 receptors may enhance tumor cell adhesion to the vessel wall (27) and open lymphatic intercellular junctions (28), allowing tumor cells to invade the surrounding tissue (29). Therefore, although the overall LYVE-1 expression is decreased in HCC nodules, the strategic positioning of its receptor at the periphery of the tumor may favor tumorigenesis and metastasis through the facilitation of tumor cell passage in and out of the tumor. This hypothesis is consistent with the recent finding that LYVE-1 expression may be associated with chemoresistance (30). Therefore, the progressive loss of LYVE-1 expression during the transformation of HCC nodules may correlate with the severity of inflammation and tumor growth.
To the best of our knowledge, this study is the first to demonstrate a direct correlation between LYVE-1 expression and tumor dedifferentiation, which strengthens the hypothesis that LYVE-1 may be a potent independent marker for the clinical prognosis of HCC.
Acknowledgements
We would like to thank Mrs. Mieko Hirokawa, Mr. Kanta Ohsuga and Mrs. Saki Okamoto for their technical support. This study was supported by Health and Labour Sciences Research Grants from the Ministry of Health, Labour and Welfare of Japan - development of early detection systems for liver cancer using molecular markers and diagnostic imaging in research on hepatitis.
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