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10.1245/s10434-006-9277-4
Annals of Surgical Oncology 14:1191-1199 (2007)
© 2007 Society of Surgical Oncology
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Original Article

Receptor Activator of Nuclear Factor-{kappa}B Ligand (RANKL) Expression in Hepatocellular Carcinoma With Bone Metastasis

Atsushi Sasaki, MD1,2, Kenji Ishikawa, MD1, Naotsugu Haraguchi, MD1, Hiroshi Inoue, MD1, Tetsuya Ishio, MD2, Kohei Shibata, MD2, Masayuki Ohta, MD2, Seigo Kitano, MD2 and Masaki Mori, MD1

1 Department of Surgical Oncology, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara, Beppu 874-0838, Japan
2 Department of Surgery I, Oita University Faculty of Medicine, 1-1 Hasama-machi, 879-5593, Oita, Japan

Correspondence: Address correspondence and reprint requests to: Masaki Mori, MD; E-mail: mmori{at}beppu.kyushu-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Although receptor activator of nuclear factor-{kappa}B ligand (RANKL) seems to be involved in the development of bone metastases in several malignant tumors, its role in hepatocellular carcinoma (HCC) has not been investigated.

Methods: We retrospectively examined the immunohistochemical expression of RANKL in formalin-fixed, paraffin-embedded resected specimens obtained from 96 patients with HCC with (n = 16) and without (n = 80) bone metastases. In addition, tumor RANKL mRNA expression was evaluated by reverse transcriptase–polymerase chain reaction (RT-PCR) in five selected patients. We analyzed the relationship between RANKL expression level, bone metastasis development, and survival rate of patients with HCC after hepatic resection.

Results: Of the 96 patients with HCC, serum hepatitis C virus antibody was detected in 43.5% of patients and hepatitis B surface antigen in 29.5% of patients. Thirty-three patients (36.5%) also had liver cirrhosis. Immunohistochemical analysis showed that RANKL protein was present in 10 (62.5%) of 16 patients with HCC with bone metastasis compared with 21 (26.3%) of 80 patients with HCC without bone metastasis; we found that RANKL expression was statistically significantly correlated to bone metastasis development (P < .01). RANKL mRNA expression was confirmed by RT-PCR in patients positive for RANKL protein expression by immunohistochemistry. The 5-year cancer-related (P < .01) and disease-free survival (P < .01) rates after hepatic resection were statistically significantly worse in patients positive for RANKL expression compared with RANKL-negative patients.

Conclusions: Some HCC cells produced the crucial bone resorption regulator RANKL. Because RANKL modulates bone turnover, its presence would have profound implications for the establishment and development of bone metastases.

Key Words: Hepatocellular carcinoma • Receptor activator of nuclear factor-{kappa}B ligand • Bone metastasis • Prognosis • Recurrence


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. Although hepatic resection is regarded as the most effective therapy for advanced HCC,1 the survival rate after hepatic resection is unsatisfactory as a result of the high recurrence rate of the tumor and the relatively low disease-free survival after hepatic resection (less than 30%) in areas with endemic hepatitis C virus (HCV) infection.2–4 Also, more than 80% of patients with tumor recurrence after hepatic resection have recurrent tumors in the remnant liver as a result of portal vein invasion of the tumor cells or multicentric cancer development.4,5 Because of recent advances in locoregional therapies, including thermal or chemical ablation therapies and transarterial chemoembolization, the survival periods after disease recurrence have been improved in patients with intrahepatic recurrence only.6 However, patients with HCC can also develop hematogenous metastases in extrahepatic organs after hepatic resection, with the most frequent organs of extrahepatic metastases being the lung, followed by bone, adrenal glands, and brain.4 Compared with intrahepatic metastasis, effective therapies for recurrent extrahepatic metastasis have yet to be developed.

The frequency of bone metastasis in patients with HCC has been reported to be approximately 10% in patients who could not undergo hepatic resection7,8 and 1.6% to 7.0% in patients who underwent hepatic resection.4,9,10 HCC cells can develop as osteolytic bone metastasis, which frequently leads to pain, pathologic fractures, nerve compression syndromes, and sometimes hypercalcemia.9,11,12 However, the diagnosis of bone metastases is difficult when patients do not yet feel pain, and effective therapies for bone metastases have not yet been established. Current treatments are mainly palliative, and patients with bone metastases have a poor survival rate.9,11,12 Therefore, it would be extremely useful to predict bone metastasis after hepatic resection for the postoperative treatment of patients with HCC. However, the risk factors for development of bone metastases in patients with HCC have not yet been fully investigated.

In 1889, Stephen Paget13 set forth the "seed and soil" hypothesis, which proposed an interplay between cancer cell properties and particular organ microenvironments that confers a selective advantage to growing cancer cells. Therefore, an important consideration in the understanding of bone metastasis development is the unique composition of the "soil," or the bone microenvironment. An important factor in the regulation of bone remodeling is the direct interaction between osteoblasts and osteoclasts. Many cytokines and hormones can affect osteoclast formation. A recently characterized novel cytokine system was found to be capable of regulating the proliferation, differentiation, fusion activation, and apoptosis of osteoclasts.14–17 This system comprised a ligand (receptor activator of nuclear factor-{kappa}B ligand [RANKL]), a specific receptor (receptor activator of nuclear factor-{kappa}B [RANK]), and a decoy receptor (osteoprotegerin [OPG]).18–20 RANKL, a cell membrane-bound tumor necrosis factor superfamily member, binds to RANK expressed on osteoclast precursors, which then leads to osteoclast precursor maturation. RANKL is produced by various tissues and cell types but is most abundant in bone and lymphoid tissues.18–22 Several malignant tumors, including prostate cancer,23 neuroblastoma,24 chondroblastoma,25 and giant cell tumors,26 produce RANKL and promote osteolytic bone metastasis and/or invasion by maturating and activating bone osteoclasts. However, expression of RANKL by HCC cells and the relationship between RANKL expression and bone metastasis development in patients with HCC have not yet been elucidated.

To clarify the role of RANKL in bone metastasis development and survival rate after hepatic resection in patients with HCC, we retrospectively investigated RANKL expression in HCC tumors and examined the relationship between RANKL expression, bone metastasis development, and survival rates in patients who underwent hepatic resection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Clinicopathologic Factors
Between July 1982 and December 2004, excluding patients who died of postoperative complications, 230 patients with HCC underwent initial hepatic resection with curative intent at the Department of Surgery I, Oita University Faculty of Medicine, Japan. Of these, 17 patients (7.4%) experienced bone metastasis after hepatic resection. The patients with HCC who were identified as having bone metastasis before hepatic resection were excluded from the present study. Of the bone metastasis group, one patient was excluded from the protein and mRNA expression studies because of the large amount of necrotic liver tumor tissue caused by preoperative thermal ablation therapy and transarterial chemoembolization. Therefore, we retrospectively selected specimens from 16 patients with bone metastasis for immunohistochemical analysis. Of these 16 patients, 2 received a second hepatic resection for multicentric cancer development (71 months and 95 months after the initial hepatic resection), with HCC tumors obtained at second hepatic resection used for analysis. Of the remaining 214 patients without bone metastases, a total of 80, who were followed up more than 12 months after surgery, were matched to the bone metastasis patients according to sex and tumor diameter, and specimens from these 80 were used for immunohistochemical and polymerase chain reaction (PCR) studies.

Pathologic factors examined included tumor differentiation, presence or absence of tumor invasion into the portal or hepatic veins, and background liver tissue diagnosis by routine light microscopic examination. Histologic tumor grade and curability of the surgery were defined according to definitions of the Liver Cancer Study Group of Japan.27 All patients were regularly followed up at the outpatient clinic at Oita University Hospital and prospectively monitored for tumor recurrence by monthly assessment of serum tumor markers and by ultrasound or contrast computed tomography studies every 2 to 4 months. When bone metastases were suspected, bone scintigraphy with 99mTc-hydroxymethyl diphosphonate and/or magnetic resonance imaging were performed. None of the patients received bone scintigraphy before surgery. This work was approved by the ethical committee of the Oita University Faculty of Medicine, and all patients provided written informed consent.

Immunohistochemistry
Resected liver specimens were immediately fixed in 10% formalin for 7 days and routinely processed for light microscopy. Paraffin-embedded blocks were sectioned, and the sections were stained with hematoxylin and eosin. A single slide that included the HCC tumor with representative histologic features was selected from each case. When a patient had two or more HCC tumors consistent with multicentric cancer development, we selected the HCC tumor with the largest tumor diameter and/or poorest tumor differentiation. The selected paraffin-embedded blocks were sectioned at 4 µm and treated for 10 minutes with 3% H2O2/methanol at room temperature. Sections were then incubated with the primary antibody or control anti-goat immunoglobulin G horseradish peroxidase conjugate (Zymed Laboratories, San Francisco, CA) for 1 hour at room temperature. The antibody used was a goat polyclonal antibody against an N-terminal peptide of murine RANKL (N-19; Santa Cruz Biotechnology, Santa Cruz, CA; diluted 1:50). After rinsing, sections were blocked for 20 minutes at room temperature with 1% bovine serum albumin, then incubated for 30 minutes with amino acid polymers conjugated with peroxidase and rabbit anti-goat antibody (Histofine Simple Stain Max-PO (G), Nichirei, Tokyo, Japan). Reaction products were visualized with .02% 3,3'-diam-inobenzidine tetrahydrochloride. Sections were counterstained with Mayer hematoxylin.

When more than 10% of HCC cells in the sample showed a positive immunoreaction with the specific antibody, the tumor was defined as positive for the corresponding antigen, such that immunohistologic reactions were defined as either negative (<10% of neoplastic cells) or positive (≥ 10% of neoplastic cells).

Reverse Transcriptase–PCR Analysis of RANKL mRNA Expression in HCC Cells
To investigate RANKL mRNA expression by HCC tumor cells, we selected five patients (one patient with bone metastases and four without bone metastases) who underwent hepatic resection between 1999 and 2004, assessed RANKL mRNA expression in HCC tumor cells by reverse transcriptase (RT)-PCR, and compared results with those obtained for RANKL protein expression immunohistochemistry. RT-PCR was performed according to previously described methods.28 Briefly, total RNA was prepared from resected HCC tissues with Bio Robot EZ1 (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Total RNA (2.5 mg) was reverse transcribed at 37°C for 60 minutes in the presence of random primers (Takara Bio, Shiga, Japan), and the resultant cDNA was amplified with 43 cycles of denaturation at 95°C for 5 seconds (10 minutes for the first cycle), annealing at 62°C for 8 seconds, and extension at 72°C for 8 seconds. RT-PCR products (1 µL each) were electrophoresed on 2% agarose gels and stained with ethidium bromide. PCR primers used to amplify RANKL cDNA were 5'-CTCACTATTAATGCCA-CCGACA-3' (upstream) and: 5'-CATGATGTCG-AAAGCAAATGTT-3' (downstream). The length of the amplified product was 175 bp. Human Universal Reference Total RNA (BD Clontech, Palo Alto, CA) was used as a positive control.

Survival and Statistical Analyses
Cancer-related and disease-free survival after hepatic resection was examined by univariate analysis. For cancer-related survival analysis, data were censored when a patient remained alive or had died of an unrelated disease or cirrhosis-related disease (hepatic failure or ruptured esophagogastric varices). Relationships between RANKL immunoreactivity, presence or absence of bone metastases, and clinicopathologic factors were evaluated by {chi}2 or Fisher’s exact tests for nominal variables, and the Kruskal-Wallis test for continuous variables. Survival rates were calculated by the Kaplan-Meier method. Cancer-related and disease-free survival curves were drawn according to immunoreactivity status (positive or negative) of anti-RANKL antibody and checked by the log rank test. For all analyses, P < .05 was considered statistically significant. Statistical analyses were performed by JMP software (JMP, SAS Institute, Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Characteristics
The 96 patient HCC group consisted of 76 men and 20 women, with a mean age at the time of last hepatic resection of 64.0 years (range, 37 to 84 years). Of 85 patients tested, 37 (43.5%) were positive for serum hepatitis C virus (HCV) antibody. Mean greatest tumor dimension was 58.3 mm (median, 55 mm; range, 21 mm to 175 mm). Preoperatively, mean and median values for indocyanine green retention rate at 15 minutes were 15.4% and 13.8%, respectively, and for preoperative serum alfa-fetoprotein, 2527.0 and 55 ng/mL, respectively. In terms of tumor differentiation, 10 patients had well-differentiated, 75 moderately differentiated, and 11 poorly differentiated tumors. Microscopic invasion into the portal vein was observed in 54 patients (56.3%) and into the hepatic vein in 20 patients (20.8%). Histologic diagnosis of liver cirrhosis as the underlying cause of liver disease was made in 35 patients (36.5%).

Expression of RANKL in HCC Cells
Of the 96 resected HCC tumor samples, 58 (60.4%) showed an immunohistochemical reaction to anti-RANKL antibody in the cytoplasm of HCC cells to various degrees and intensities (Fig. 1a, bGo). In some HCC tumors, scattered giant cells with bizarre nuclei (nonosteoclastic type) strongly stained with RANKL antibody (Fig. 1cGo). However, 38 samples (39.6%) showed no immunohistochemically detectable expression of RANKL. According to our criteria for immunohistochemical expression, 31 patients (32.3%) were classified as positive and 65 (67.7%) as negative for RANKL immunohistochemical staining. Of five HCC tumors investigated by RT-PCR, three HCC tumors positive by immunohistochemistry were found to express RANKL mRNA, and two tumors immunohistochemically negative for RANKL expression were found not to express RANKL mRNA. A tumor sample from a patient with HCC with bone metastasis expressed both RANKL protein and mRNA (Fig. 2Go).


Figure 1
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  		FIG. 1. Representative microphotographs (original magnification, x 200) showing positive immunohistochemical expression of receptor activator of nuclear activator-{kappa}B ligand (RANKL) in hepatocellular carcinoma (HCC) cells. Immunohistochemical reactions are observed in the cytoplasm of tumor cells (a). In HCC tumors, nonosteoclastic multinucleated giant cells frequently show strong RANKL protein expression (b).

 

Figure 2
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  		FIG. 2. Gel analysis of reverse transcriptase–polymerase chain reaction (RT-PCR) products derived from receptor activator of nuclear activator-{kappa}B ligand (RANKL) mRNA. The RT-PCR results are consistent with those of the immunohistochemical study. Of five hepatocellular carcinoma (HCC) tumors, three positive for immunohistochemical expression show RANKL mRNA expression, and two negative for immunohistochemical expression show no mRNA expression. HCC tumor with bone metastasis expresses both protein and mRNA for RANKL (sample 61).

 
Relationship Between Bone Metastases and Clinicopathologic Factors
Patient characteristics according to the presence or absence of bone metastases are listed in Table 1Go. Patients with bone metastases (n = 16) had no well-differentiated tumors and higher preoperative serum alfa-fetoprotein values (P < .01) compared with patients without bone metastasis. The frequencies of serum hepatitis B surface antigen (P = .12), tumor invasion into the portal (P = 1.00) or hepatic (P = .09) veins, and accompanying liver cirrhosis (P = .92) were not correlated to development of bone metastasis in our patient group.


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  		TABLE 1. Relationship between bone metastases and clinicopathologic factors in patients with hepatocellular carcinoma
 
Relationship Between Immunohistochemical Expression of RANKL in HCC Tumor Cells and Clinicopathologic Factors
Correlation between RANKL expression in HCC cells and clinicopathologic factors is listed in Table 2Go. No correlations between HCC tumor RANKL expression and any of the clinicopathologic factors were observed. Although RANKL-positive HCC tumors tended to have smaller tumor diameters than RANKL-negative HCC tumors, this difference did not reach statistical significance (P = .10). RANKL expression in HCC tumors was significantly related to the development of bone metastases, with RANKL-positive HCC tumors observed in 10 (62.5%) of 16 patients with bone metastases, compared with 21 (26.3%) of 80 patients without bone metastases (P < .01).


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  		TABLE 2. Relationship between immunohistochemical expression of RANKL in hepatocellular carcinoma tumors and clinicopathologic factors
 
Patient Outcomes and Survival Analysis
At the end of the follow-up period, of the 96 patients with HCC who underwent curative hepatic resection, 21 were alive without disease recurrence, 29 were alive with disease recurrence, 43 had died of the disease, 2 had died of liver failure, and 1 had died of pneumonia. The mean and median follow-up periods after hepatic resection in surviving patients were 59.5 and 55.0 months, respectively. Of the 16 patients with bone metastases, 10 also had intrahepatic metastases and/or multicentric cancer development, and three also had lung metastases at the time of bone metastasis diagnosis. The mean and median intervals between last hepatic resection and bone metastasis diagnosis were 17.8 months and 9 months, respectively.

The 5-year cancer-related and disease-free survival rates after the last hepatic resection were 57.7% and 16.9%, respectively, for all 96 patients with HCC. Cancer-related survival and disease-free survival curves according to RANKL expression are shown in Fig. 3Go. The 1-, 3-, and 5-year cancer-related survival rates after last hepatic resection were 96.9%, 78.2%, and 68.8%, respectively, in RANKL-negative patients and 83.9%, and 44.4%, and 35.6%, respectively, in RANKL-positive patients. The 1-, 3-, and 5- disease-free survival rates were 59.4%, 36.2%, and 20.9%, respectively, in RANKL-negative patients and 32.3%, and 9.7%, and 9.7%, respectively, in RANKL-positive patients. Thus, after the last hepatic resection, cancer-related survival and disease-free survival rates (P < .01) were significantly decreased in RANKL-positive compared with RANKL-negative patients.


Figure 3
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  		FIG. 3. Cancer-related and disease-free survival curves after hepatic resection according to receptor activator of nuclear activator-{kappa}B ligand (RANKL) expression in tumor cells. Cancer-related and disease-free survival rates are statistically significantly worse in RANKL-positive patients than in RANKL-negative patients (P < .01 each for cancer-related survival and disease-free survival).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although it is known that HCC tumors can develop osteolytic bone metastases, the diagnosis of bone metastases in HCC cases remains difficult when conventional diagnostic modalities such as computed tomography and magnetic resonance imaging are used.29 Although several therapies, including irradiation, systemic chemotherapy, and transarterial infusion therapy, are used to treat bone metastases in HCC, most of these therapies are regarded as palliative only, and the survival rate of patients with bone metastasis is poor.8 Therefore, the ability to predict bone metastasis would be an important advance in the treatment of patients with HCC. However, the risk factors for bone metastasis have not yet been fully investigated in patients with HCC.

RANKL, also known as osteoclast differentiating factor, OPG ligand, or tumor necrosis factor {alpha}-converting enzyme, is a newly identified membrane-bound member of the tumor necrosis factor ligand superfamily.18,19 Two receptors have been identified for RANKL: RANK, a membrane-bound signaling receptor for RANKL; and OPG, a secreted receptor that is thought to act as a natural decoy receptor to limit the biological actions of RANKL. It is thought that the balance between RANKL and OPG levels regulates the development and activation of osteoclasts and bone metabolism.18 RANKL expression has been observed in some malignant tumors from several organs, including the prostate, and osseous or cartilaginous tissues. Most studies of RANKL expression in malignant cells have been performed on metastatic bone lesions.

Good et al.30 studied metastatic osteolytic bone tumors and showed that RANKL protein was expressed in renal and pulmonary carcinomas, but not in breast cancer or myeloma cells. Huang et al.31 demonstrated that osteolytic bone metastases, including breast, lung, prostate, and thyroid carcinomas, expressed RANKL protein. However, it remains unclear whether RANKL expression in the primary tumors is predictive of an increased propensity toward skeletal metastases. Brown et al.23 investigated primary and metastatic prostate cancer and showed that RANKL was heterogeneously expressed in 10 of 11 primary prostate cancer specimens, and that the proportion of tumor cells expressing RANKL was greatly increased in bone metastases compared with nonosseous metastases or the primary prostate cancer. Breast cancer cells are known to secrete high levels of parathyroid hormone-related protein (PTHrP), which can enhance RANKL mRNA and inhibit OPG production by osteoblastic lineage cells.32–34 We did not analyze the expression of PTHrP in HCC tumors in the present study because ordinary-type HCC tumors are known not to produce PTHrP.35 Our study investigated cases of primary HCC and demonstrated that RANKL expression in HCC cells correlated with the development of bone metastasis after hepatic resection. Our findings suggested that RANKL, rather than PTHrP, produced by HCC cells activated and differentiated osteoclast precursors, which then mediated osteolysis and promoted the development of bone metastases.

Osteoporosis is a common complication of chronic liver disease. The pathogenesis of hepatic osteodystrophy is thought to consist of multiple factors, including genetic background, nutritional deficiency, low vitamin D levels, Ca2+ deficiency, low insulin-like growth factor 1 levels, and excessive alcohol intake.36 Recently, serum levels of soluble RANKL were measured in patients with chronic liver disease. Szalay et al.37 investigated the serum levels of OPG and RANKL in patients with primary biliary cirrhosis, chronic hepatitis related to HCV, and post-menopausal osteoporosis and demonstrated that patients with primary biliary cirrhosis tended to have high OPG and low RANKL levels, whereas patients with chronic hepatitis related to HCV exhibited high OPG and RANKL levels. Fábrega et al.38 observed high OPG and RANKL levels in patients with alcoholic liver cirrhosis, and Moschen et al.39 demonstrated high OPG and RANKL levels in patients with liver disease but without cirrhosis, including those with alcoholic hepatitis, primary biliary cirrhosis, and viral hepatitis.

However, to our knowledge, there have been no previous reports of RANKL expression in HCC cells correlating with bone metastasis development. Several previous reports have demonstrated the use of RANKL and/or OPG serum levels in predicting bone metastases in patients with multiple myeloma.40 However, RANKL and/or OPG serum levels cannot be used to predict bone metastasis in patients with HCC because most cases of HCC seem to develop from chronic liver disease, especially hepatitis B virus or HCV infection.

Therapeutic strategies based on the OPG/RANK/ RANKL triad have been proposed for myeloma and prostate cancer. Zhang et al.41 demonstrated that soluble RANK-Fc diminished prostate cancer progression in murine bone through inhibition of bone remodeling. Also, Honore et al.42 demonstrated that OPG administration blocked behaviors indicative of pain in mice through inhibition of tumor-induced bone destruction. For patients with bone metastases from myeloma, breast cancer, prostate cancer, or lung cancer, bisphosphonates have been used for pain reduction. Ohnishi et al.43 reported the efficacy of alendronate against hypercalcemia due to HCC bone metastases. Our results support the possible use of soluble RANKL-Fc, OPG, and bisphosphonates in human patients with HCC with bone metastases.

In conclusion, our findings indicated that HCC cells can produce the crucial bone resorption regulator RANKL. RANKL may modulate bone turnover and may have profound implications for the establishment and development of bone metastases.


   ACKNOWLEDGMENTS
 
Presented in part at The Society of Surgical Oncology 59th Annual Cancer Symposium, March 23–26, 2006, San Diego, CA. This work was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (grant 16591329), Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, and Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (grants 17109013, 17591411, and 17591413). The authors thank MS. Yuko Nakagawa and Fusayo Kawamura for their technical assistance with the immunohistochemistry, and Yuichi Endo M.D. and MS. Kazue Ogata for assistance with the reverse transcriptase–polymerase chain reaction analysis.

Received for publication March 30, 2006. Accepted for publication October 4, 2006.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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