Abstract
The clinical relevance of frizzled-related protein (FRZB) in hepatocellular carcinoma (HCC) bone metastasis remains uncertain. The aim of this study was to assess the clinical relationship of FRZB in patients with HCC bone metastasis after surgical resection. FRZB expression was evaluated by immunohistochemistry in formalin-fixed paraffin embedded (FFPE) HCC and paired bone metastasis tissues from 13 patients that underwent surgical resection. The clinical characteristics of 13 HCC patients with synchronous or metachronous bone metastasis received surgery were retrospectively reviewed. We found that FRZB was positive in 9 HCC tissues (69.2%) and in 11 paired bone metastatic tissues (84.6%) among these 13 paired samples. The expression of FRZB in the bone metastases was noticeably higher than that in the paired HCC tissues. The expression of FRZB was up-regulated in 10 (76.9%) paired bone metastases tissues. FRZB expression was up-regulated in HCC bone metastasis tissue, which suggested that FRZB might play a key role in the HCC bone metastasis.
Keywords: Hepatocellular carcinoma, bone metastasis, frizzled-related protein (FRZB)
Introduction
Liver cancer in men is the fifth most frequently diagnosed cancer worldwide but the second most frequent cause of cancer death. In women, it is the seventh most commonly diagnosed cancer and the sixth leading cause of cancer death. Half of these cases and deaths were estimated to occur in China [1]. Among primary liver cancers, hepatocellular carcinoma (HCC) represents the major histological subtype, accounting for 70% to 85% of the total liver cancer burden worldwide [1]. The primary risk factor for HCC is liver injury from diverse causes that leads to hepatic cirrhosis in most patients. An estimated 78% of HCC cases and 57% of cases of liver cirrhosis are caused by chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) [2,3]. HCC is the sixth most prevalent cancer worldwide and the third leading cause of cancer-related death, although its geographical distribution is heterogeneous with the highest incidence in sub-Saharan Africa and Eastern Asia [4]. Bone is an uncommon site of metastasis in HCC, with the incidence ranging from 3% to 20% [5]. Although bone involvement is reported as uncommon in HCC, its incidence has significantly increased in the last decade due to the longer survival of HCC patients related to recent progresses made both in the diagnosis and treatment of the disease [5-7]. A better understanding of the pathogenic mechanisms underlying the spread of bone metastases in HCC is important. Some retrospective studies have described the characteristics of bone metastasis from HCC [5,8-10]. However, few data are yet available about bone involvement in patients with HCC, and no agreement has yet been reached about the treatment strategy for extrahepatic HCC metastases. The nature and the characteristics of bone metastases in HCC have not been fully explored in literature, presumably because HCC skeletal involvement was rarely diagnosed.
Frizled-related protein (FRZB), a member of the secreted frizzled related protein (SFRP) family, also known as sFRP3, plays an important role in embryonic development. FRZB is one of the Wnt signaling pathway regulators. FRZB contains a 24-amino acid putative transmembrane segment [11], a cysteine-rich domain (CRD) which is similar to the putative Wnt-binding region of the frizzled family of transmembrane receptors, a netrin-like domain (NTR) which is homologous with tissue inhibitors of metalloproteases (TIMPs) [12]. Polymorphisms in the FRZB gene have been associated with osteoarthritis [13] and are considered one of the osteoblast regulatory genes. FRZB affects the cartilage integrity as well as cortical bone thickness and density. The mechanism of this protection can be partly attributed to FRZB suppression of the expression of WNT/β-catenin target genes, including genes for MMP3 and cyclooxygenase 2 (COX2) [14]. Further study demonstrated that FRZB may bind and inhibit MMP3 proteinase activity through its NTR domain [14]. FRZB was seen to be acting as an oncogene in metastatic renal cancer [15]. Tissue microarray analysis showed that the level of FRZB protein was low in primary renal cancer tissues but high in metastatic renal cancer tissues. Functional analysis showed increased cell growth, invasion, and tube formation, and decreased numbers of apoptotic cells in the FRZB-transfected renal cancer cell line A498. The reverse trend was seen in metastatic cells (ACHN and Hs891.T) when FRZB expression was silenced by using a knock-down approach [15].
In this study, we evaluated the potential clinical relationship of FRZB in patients with HCC bone metastasis after surgical resection.
Patients and methods
Patients and tumor tissue samples
The institutional ethical committee approved the current retrospective study. A written informed consent was obtained for all patients. We reviewed the electronic medical records of consecutive patients in whom HCC and synchronous or metachronous bone metastasis was newly diagnosed from January 2009 to October 2014 at the Department of Orthopedics, the First Affiliated Hospital, Zhejiang University School of Medicine (Table 1). The diagnosis of HCC was mainly based on recommendations of the American Association for the Study of Liver Diseases [16]. All patients underwent blood investigations, which included complete blood count, liver function tests, and tests for viral markers of hepatitis B and C infection. Serum alpha-fetoprotein (AFP) was estimated using a particle enzyme immunoassay (Axsym System; Abbott Laboratories, Abbot Park, Illinois, USA; normal value <20 ng/ml). Upper gastrointestinal endoscopy was done in each case to detect the presence of esophageal varices. Patients with underlying cirrhosis were classified into Child’s A, B or C based on the Child-Pugh classification [17]. Staging of HCC was done based on the Barcelona Clinic Liver Cancer (BCLC) staging protocol [18]. All patients were evaluated pre- and post-operatively using abdominal computed tomographic (CT) scan, magnetic resonance imaging (MRI), or fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomographic scan (18F-FDG PET/CT).
Table 1.
Clinical characteristics of 13 hepatocellular carcinoma patients with synchronous or metachronous bone metastasis received surgery
| Gender (Male/Female) | Age (Years) | AFP level (ng/ml) | ALT (IU/L) | AST (IU/L) | HBV (Po/Ne) | HCV (Po/Ne) | BCLC staging (A/B/C) | Child’s score (A/B/C) | HCC NM | BM (S/M) | OS | Chem (Y/N) | Rad (Y/N) | MTT (Y/N) | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Case 1 | Female | 67 | NA | NA | NA | Po | Ne | NA | NA | 2 | M | 84.0 | Y | N | N |
| Case2 | Male | 39 | 8.4 | NA | NA | Po | Ne | NA | NA | 2 | M | 57.5 | Y | Y | Y |
| Case 3 | Male | 49 | 24.2 | 349 | 229 | Po | Ne | C | B | >3 | M | NA | Y | N | N |
| Case 4 | Male | 58 | 27.1 | NA | NA | Po | Ne | A | A | 2 | M | 42.3 | NA | N | N |
| Case 5 | Male | 74 | 5.7 | 41 | 32 | Po | Ne | C | A | 1 | M | NA | Y | N | N |
| Case 6 | Male | 43 | 2335.3 | 48 | 83 | Po | Ne | B | A | >3 | M | 3.0 | Y | Y | Y |
| Case 7 | Male | 62 | 8.3 | 33 | 34 | Po | Ne | A | A | 1 | M | NA | Y | N | N |
| Case 8 | Male | 50 | 14.7 | 27 | 117 | Po | Ne | C | A | >3 | M | 25.0 | Y | N | Y |
| Case 9 | Male | 67 | 89.6 | NA | NA | Po | Ne | A | NA | >3 | M | NA | Y | N | N |
| Case 10 | Male | 55 | 4.4 | 28 | 30 | Po | Ne | NA | A | >3 | S | 9.3 | Y | N | N |
| Case 11 | Male | 52 | 27.5 | 28 | 21 | Po | Ne | B | A | >3 | M | 36.0 | Y | N | N |
| Case 12 | Male | 26 | 2175.2 | 16 | 21 | Po | Ne | C | A | 3 | M | 21.6 | Y | N | N |
| Case 13 | Male | 42 | 228.9 | 23 | 21 | Po | Ne | B | A | >3 | M | 27.5 | Y | N | Y |
Abbreviations: HBV, hepatitis B virus infection. HCV, hepatitis C virus infection. Po, positive. Ne, negative. AST, aspartate aminotransferase. ALT, aspartate aminotransferase. AFP, alpha-fetoprotein. BCLC staging, Barcelona clinic liver cancer staging. NA, not available. OS, overall survival (months), Chem, chemotherapy. MTT, Molecularly targeted therapy. Rad, radiotherapy. HCC NM, hepatocellular carcinoma number of masses. BM (S/M), bone metastases (S, synchronous; M, metachronous). Y, yes. N, No.
Immunohistochemistry
Five micromolar FFPE sections were cut, dewaxed, rehydrated, and subjected to antigen retrieval. After blocking endogenous peroxidase activity, the sections were incubated with the primary antibody against FRZB (Abcam, Cambridge, MA) (1:200) (overnight at 4 at 4°C). Immunohistochemistry was performed using the streptavidin-biotin peroxidase complex method (Lab Vision, Fremont, CA). The slides were examined and pictures were taken using an Olympus BX60 (Olympus, Japan). Sections known to stain positively were incubated in each batch and negative controls were also prepared by replacing the primary antibody with preimmune sera.
Expression analysis of FRZB in tumor tissue was performed by comparing staining intensity and the percentage of immunoreactive cells. Staining intensity was arbitrarily scored on a scale of four grades: 0 (no staining of cancer cells), 1 (weak staining), 2 (moderate staining), and 3 (strong staining), and the percentage of positive cells was scored as follows: 0 (0%), 1 (1% to 25%), 2 (26% to 50%), and 3 (>50%). FRZB staining positivity was determined using the following formula: overall score = positive percentage score × intensity score. A score of 0 was defined as “0”, >0 to ≤2 as “1”, >2 to ≤6 as “2”, and >6 to ≤9 as “3”. In the end, tumor samples rated as level 0 or 1 were defined as negative for expression, whereas samples rated as level 2 or 3 were defined as positive.
Data collection and follow-up
The clinical, laboratory, and radiologic records of all patients were retrospectively reviewed (Table 1). Liver function tests were checked in all patients every three months in order to evaluate hepatic functional reserve. The results of all 13 HCC patients with synchronous or metachronous bone metastasis were analyzed. Follow-up cross-sectional imaging (contrast-enhanced CT or MRI) was performed one month after treatment. Further treatments were based on clinical evaluation, laboratory values and imaging response. Patients were followed-up every 3 months. The patients were followed up until death or until the date of last follow-up. Follow-up was finished on February 28, 2015. Overall survival was calculated from the date after hepatic resection to the date of death for any cause or last follow-up.
Results
Clinicopathological characteristics of HCC patients with bone metastasis
The clinicopathological characteristics of the cohort are summarized in Table 1. The 13 patients (12 males, 1 female) had an age range from 26 to 74 years. Hepatitis B virus (HBV) infection was the most common etiological factor of HCC, seen in 13 (100%) patients. No patient with hepatitis C virus (HCV) infection was found. BCLC B patients were 3 (23.1%), and BCLC C patients were 4 (30.8%). Child’s score a patients were 9 (69.2%), and Child’s score B patients were 1 (7.7%). All the patients received synchronous or metachronous resections of the HCC and bone metastases. Of the patients, only 1 patient (7.7%) was treated with surgery alone, 12 (92.3%) patients received adjuvant chemotherapy, 2 (15.4%) patients received adjuvant radiotherapy, 4 (30.8%) patients received molecularly targeted therapy preoperatively or postoperatively.
FRZB up-regulated in HCC bone metastasis tissue
We evaluated the expression of FRZB in 13 HCC samples and paired bone metastatic samples using the method of immunohistochemical staining. We found that FRZB was positive in 9 HCC tissues (69.2%) and in 11 paired bone metastatic tissues (84.6%). The expression of FRZB in the bone metastases was noticeably higher than that in the paired HCC tissues (Figure 1). The expression of FRZB was up-regulated in 10 (76.9%) paired bone metastases tissues. The potential clinical relevance of FRZB expression in HCC patients with synchronous or metachronous bone metastasis received surgery was shown in Table 2. These results suggested that FRZB might play a key role in the HCC bone metastasis.
Figure 1.
Expression of FRZB in hepatocellular carcinoma (HCC) and paired bone metastatic tissues. A. Negative and positive staining of FRZB in HCC and paired bone metastatic tissues (original magnification × 200). B. Representative immunohistochemical staining of FRZB in HCCr tissues and paired bone metastatic tissues. The expression of FRZB is significantly greater in the HCC bone metastatic tissues than in HCC tissues (original magnification × 200).
Table 2.
The clinical relevance of FRZB expression in HCC patients with synchronous or metachronous bone metastasis received surgery
| FRZB expression (Positive/Negative) | FRZB up-regulation in bone metastasis (Yes/No) | OS | ||
|---|---|---|---|---|
|
|
||||
| HCC | Paired bone metastasis | |||
| Case 1 | Negative | Negative | No | 84.0 |
| Case2 | Negative | Positive | Yes | 57.5 |
| Case 3 | Positive | Positive | Yes | NA |
| Case 4 | Positive | Positive | No | 42.3 |
| Case 5 | Positive | Positive | Yes | NA |
| Case 6 | Positive | Positive | Yes | 3.0 |
| Case 7 | Negative | Negative | No | NA |
| Case 8 | Positive | Positive | Yes | 25.0 |
| Case 9 | Negative | Positive | Yes | NA |
| Case 10 | Positive | Positive | Yes | 9.3 |
| Case 11 | Positive | Positive | Yes | 36.0 |
| Case 12 | Positive | Positive | Yes | 21.6 |
| Case 13 | Positive | Positive | Yes | 27.5 |
Abbreviations: HCC, hepatocellular carcinoma. NA, not available. OS, overall survival (months).
Discussion
HCC is the fifth most common cancer in men worldwide [4]. The bone is well known to be the third most frequent site of metastases by all tumors, after the lungs and lymph nodes, and HCC bone colonization has been reported in approximately 20 % of patients affected by this tumor [5,19,20]. Recently, the progress in both diagnostic modalities and therapeutic procedures, such as surgical resection, radiofrequency ablation, and transcatheter arterial chemoembolization in association with treatments using small molecules as multikinase inhibitors, has prolonged the survival in HCC patients which led to a concurrent worsening of the tumor progression within the skeleton and the formation of bone metastases [5-7,19]. To date, few data are yet available about bone involvement in patients with HCC, and the nature and the characteristics of bone metastases in HCC have not been fully explored.
FRZB, the first member of the sFRP family, was isolated as a chondrogenic factor in developing cartilage [11]. FRZB binds to both Wnt-8 and Wnt-1 and acts as a functional inhibitor of Wnt-8 activity [21,22]. It was demonstrated that FRZB was seen to be acting as an oncogene in metastatic renal cancer [15]. Hirata, et al. found that FRZB protein was up-regulated in metastatic renal cancer tissues compared with primary renal cancer tissues [15]. However, FRZB is also involved in malignant tumor generation and progression. Numerous studies strongly suggest a tumor suppressor role of FRZB. Deregulation of FRZB is found in bone-originated malignant diseases. Expression of FRZB was also found to be related to bone involvement at diagnosis in myeloma plasma cells [23]. Loss of FRZB expression was commonly found in osteogenic sarcoma tissues [24]. Expression of FRZB suppresses epithelial original prostate cancer cell in vivo growth and progression [25]. FRZB can function as a melanoma migration and invasion suppressor by interfering with Wnt5a signaling [26]. FRZB decreases growth and invasiveness of fibrosarcoma cells and this inhibition is associated with downregulation of c-Met expression and inhibited Met-mediated signaling [27]. FRZB suppressed gastric cancer cell proliferation and modulated the balance between proliferation and differentiation in gastric cancer [28]. Knockdown of FRZB in gastric cancer cells increased cell growth and migration/invasion [29]. FRZB knockdown may upregulate the Wnt/βcatenin pathway and promote aggressiveness in gastric cancer [29].
The aim of this study was to assess the clinical relationship of FRZB in patients with HCC bone metastasis after surgical resection. Herein, we evaluated the FRZB expression in HCC and paired bone metastatic tissues from 13 patients, which had clinical follow-up records. Our results indicated that the positive expression of FRZB in HCC bone metastasis might have a shorter overall survival and that FRZB might play a key role in the HCC bone metastasis. These data, for the first time, imply that FRZB has distinct roles in HCC bone metastasis and is worthy of further investigation.
In summary, this is the first study showing the expression of FRZB in HCC bone metastasis. Our results found that FRZB expression was up-regulated in HCC bone metastasis tissue, which suggested that FRZB might play a key role in the HCC bone metastasis. Our results indicated FRZB might be a novel predictor for poor prognosis of HCC patients with bone metastasis after surgical resection and FRZB might be a promising candidate for targeted therapy of HCC bone metastasis.
This work was supported by National Natural Science Foundation of China (Grants No. 81374014 and No. 81472210), Zhejiang Provincial Medical and Healthy Science and Technology Projects (Grant No. 2013KYA228), Zhejiang Provincial Science and Technology Project (Grant No. 2013C33112), Science Research Fund of Taizhou (Grants No. A121KY08, A131KY13-3 and A131KY13-12), and Enze Medical Research Fund (Grants No. 12EZA1, 13EZA2 and 13EZB6).
Acknowledgements
This work was supported by National Natural Science Foundation of China (Grants No. 81374014 and No. 81472210), Zhejiang Provincial Medical and Healthy Science and Technology Projects (Grant No. 2013KYA228), Zhejiang Provincial Science and Technology Project (Grant No. 2013C33112), Science Research Fund of Taizhou (Grants No. A121KY08, A131KY13-3 and A131KY13-12), and Enze Medical Research Fund (Grants No. 12EZA1, 13EZA2 and 13EZB6).
Disclosure of conflict of interest
None.
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