Author Affiliations: Department of Radiology (Pan J) and Department of Liver Surgery (Zhang YF, Yang HY, Xu HF, Lu X, Sang XT, Zhong SX and Mao YL), Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China; Department of Abdominal Surgery, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China (Zhang YF)

Corresponding Author: Yi-Lei Mao, MD, PhD, Professor of Surgery, Department of Liver Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 1# Shuaifuyuan, Wangfujing, Beijing 100730, China (Tel: +86-10-69156042; Fax: +86-10-69156043; Email: yileimao@126.com)

 

© 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60403-9

Published online July 17, 2015.

 

Acknowledgement: We thank Xiang-Yang Liu who provided medical writing services.

Contributors: YHY and MYL proposed the study. PJ, ZYF, YHY, XHF and LX performed research. SXT, ZSX and MYL wrote the first draft. ZYF and YHY collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. PJ, ZYF and YHY contributed equally to this article. MYL is the guarantor.

Funding: This study was supported by grants from the National Key Technology Research and Development Program of China 2012 (BAI06B01), the National Natural Science Foundation of China (81201566), the Specialized Research Fund for the Doctoral Program of Higher Education (20121106110002) and Wu Jieping Medical Foundation (LDWMF-SY-2011B002).

Ethical approval: The study protocol was approved by the Independent Ethics Committee of Peking Union Medical College Hospital and Cancer Hospital, Chinese Academy of Medical Sciences.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

BACKGROUND: Golgi protein 73 (GP73) is a promising biomarker of hepatocellular carcinoma (HCC). It decreases after surgical resection, and resumes upon recurrence, indicating a potential indicator for the effectiveness of the treatment. But changes of GP73 after transcatheter arterial chemoembolization (TACE) have not been reported so far. This study was to investigate the dynamic changes of GP73 in HCC patients after TACE treatment, and the possible underlying mechanisms in the cell cultures.

 

METHODS: Blood samples were collected from 72 HCC patients, before TACE, at day 1 and day 30 after TACE. GP73 levels were measured by Western blotting. The dynamic changes of GP73 were analyzed and compared with image changes and clinical data. The effects of chemotherapeutic agents (5-FU and pirarubicin) on GP73 expression were tested in three HCC cell lines (HepG2, HCCLM3 and MHCC97H).

 

RESULTS: The GP73 level was significantly elevated at day 1 and day 30 after TACE in HCC patients compared with that before the procedure (P<0.05). There was no statistical difference between the two time points after TACE, nor correlation between GP73 levels and clinicopathological features, tumor metastasis, and patient survival. Pirarubicin, not 5-FU, significantly increased GP73 expression in three cell lines.

 

CONCLUSIONS: Unlike surgical resection which decreases the GP73 level, TACE significantly increased GP73 expression in patients with HCC. No correlations were observed among GP73 levels, tumor characteristics and prognosis of patients with HCC.

 

(Hepatobiliary Pancreat Dis Int 2015;14:406-412)

 

KEY WORDS: hepatocellular carcinoma; transcatheter arterial chemoembolization; Golgi protein 73; chemotherapeutics

Hepatocellular carcinoma (HCC) is one of the most malignant cancers in the world, with morbidity ranks the 5th and mortality the 3rd among various cancers.^{[1, 2]} Earlier diagnosis of HCC is essential for therapeutic selection. However, there is no accurate biomarker to diagnose and monitor the patients with HCC; traditional alpha-fetoprotein (AFP) has a limited role in HCC diagnosis and monitoring due to the lack of sensitivity and specificity. Clinicians therefore are trying to find a better biomarker to be added to or replace AFP. Golgi protein 73 (GP73), a Golgi type II transmembrane protein, has been indicated as one of the most promising HCC serum markers that might be applicable in clinical practice. This protein is normally expressed in epithelial cells of tissues including bile ducts, intestinal tracts, trachea and prostate, and increased significantly in HCC patients.^[3-6] A large sample, multi-center and multi-nation clinical study^[5] showed that the sensitivity and specificity are 74.6% and 97.4% respectively for the diagnosis of HCC, which are significantly higher than those of AFP in the same cohort. More interestingly, serum GP73 value in HCC patients is correlated with tumor mass: surgical resection almost normalized serum GP73 and its value was rebounded upon tumor recurrence.^[5] Our data implied its potential role in evaluating or monitoring the effectiveness of treatments for HCC. These treatments might include surgery, radiofrequency ablation, sorafinib, and transcatheter arterial chemoembolization (TACE) as recommended by the guidelines elsewhere. However, sporadic data up to date showed the changes of GP73 level after treatments of HCC other than surgical resection.

 

TACE is widely employed in patients with HCC, and has been recommended as the first choice for the middle-advanced stage HCC patients who cannot accept surgery.^{[7, 8]} However, there is still lack of an effective way to evaluate the efficacy of TACE, whereas the changes of tumor size may not be reliable enough, and AFP is not sensitive.^[9-11]

 

The present prospective clinical study was to observe the changes of serum GP73 after TACE at different time points, to investigate the correlations between the changes and the clinical prognosis, to see if GP73 could be a potential indicator to evaluate the effectiveness of TACE, and to study the underlying mechanisms of GP73 fluctuation in HCC.

 

(1) Patients diagnosed with HCC based on either pathologic results or AFP and images as indicated by the guidelines;

 

(2) Patients with Child-Pugh A and large or multifocal HCC which were not suitable for surgical resection or radiofrequency ablation;

 

(3) Patients who underwent TACE as the initial treatment and had no history of surgery, ablation or chemical therapy;

 

(4) Patients included on a voluntary basis were able to actively have related test.

 

(1) Liver function ranked Child-Pugh B and C;

 

(2) Active gastrointestinal bleeding;

 

(3) Impaired coagulation functions (platelet count below 50×10⁹/L or prothrombin activity below 50%) and renal failure;

 

(4) Regarded as unsuitable for this study by the researchers.

 

Seventy-two HCC patients treated with TACE from December 2010 to April 2014 were enrolled. They were from Peking Union Medical College Hospital and Cancer Hospital, Chinese Academy of Medical Sciences. The diagnosis of the patients was based on the American Association for the Study of Liver Diseases criteria (2010) and China Ministry of Health Guidelines for Primary Liver Cancer (2011). The study protocol was approved by the Independent Ethics Committee of Peking Union Medical College Hospital and Cancer Hospital, Chinese Academy of Medical Sciences. A written informed consent was obtained from each patient.

 

After local anesthesia, the right femoral artery was punctured with the Seldinger technique. With the guidance of a wire, the catheter was placed into the celiac trunk and common hepatic artery, and angiography was performed. According to the findings of angiography, a 3F micro-catheter was super-selectively catheterized into the tumor-feeding artery, and then, the chemoembolization was conducted through this micro-catheter. The dose of chemical drugs was individualized according to the tumor condition and liver function. The drugs included doxorubicin (epirubicin or pirarubicin), mitomycin, calcium folinate and 5-FU, and the patients were given 3 or 4 drugs combined therapy according to individual situation. After drug administrations, lipiodol-doxorubicin was used to block the vessels. Gelatin sponge strips were applied to block the tumor-feeding artery when necessary.

 

Three milliliters of venous blood were collected on fast in the morning before TACE, at day 1 and day 30 after TACE. The blood was centrifuged (6000 rpm, 5 minutes), serum was harvested and stored at -80 �� until use.

 

All patients were examined every 1-2 months after TACE. Imaging test (computed tomography or magnetic resonance imaging), AFP and serum liver function test were evaluated to assess the patients' response to the treatment, and to detect the remaining or recurrent lesions of HCC. The follow-up started in December 2010 and ended in June 2014. The median follow-up time was 11.2 months (range 2.0-17.2). Thirty-nine patients died during the follow-up and their deaths were regarded as endpoints for overall survival.

 

Serum GP73 concentration was evaluated by Western blotting as described in our previous study.^[5] The Novex Protein Gels and iBlot Dry Blotting System (Invitrogen, Carlsbad, CA, USA) and the ECL Plus chemiluminescent detection system (Thermo, Rockford, IL, USA) were used; goat anti-GP73 polyclonal antibody and horseradish peroxidase-conjugated mouse anti-goat secondary antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The brief procedure was described in the following paragraph.

 

HepG2, HCCLM3 and MHCC97H cell lines at logarithmic growth phase were collected. After centrifugation, the cells were diluted to 1×10⁸ cells/L and planted in 6 orifice plate with 1 mL per orifice. After the cells were cultured at 37 �� with 5% CO₂ for 24 hours, two chemotherapeutics, that were actually used in the TACE procedure in the HCC patients, were added (0, 20, 40, 60 µmol/L 5-FU and 0, 2.5, 5, 10, 20 µmol/L pirarubicin). After 24-hour incubation, the proteins were isolated from the cells according to the RIPA instructions and the protein concentrations were measured with the BCA kit. Thirty micrograms of protein were loaded and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferred onto nitrocellulose membranes. The membranes were incubated with primary antibodies (anti GP73 1:1000, anti β-actin 1:4000) which were diluted with 3% bovine serum albumin in 0.1% Tween Tris-buffered saline buffer (TBST) overnight at 4 ��. The blots were washed by TBST and incubated with secondary antibody (1:4000) for 60 minutes. After washing with TBST, the blots were then developed using the ECL system and densitometry analysis was performed.

 

Statistical analyses were performed using SPSS 12.0 (SPSS Inc., Chicago, IL, USA). Quantitative values were presented as mean±SD or median (25th-75th percentiles). The Chi-square test was used to compare general characteristics of the patients and the Mann-Whitney U test to compare GP73 between different subgroups according to the clinicopathological features. Spearman's rank-order correlation coefficient was used to analyze relations between GP73 and biochemical indexes before and after TACE. The dynamic change of GP73 before and after TACE was analyzed by one-way analysis of variance (ANOVA) with repeated measures and Bonferroni's correction between groups. The influence of clinicopathological features on the change of GP73 was assessed by multivariate analysis of variance with repeated measures. The relation between recurrence or metastasis and the change of GP73 was evaluated by the corrected Chi-square test. Overall survival (OS) and progression-free survival (PFS) were analyzed by the Kaplan-Meier method. A two-tailed P value of <0.05 was regarded statistically significant.

 

 

In the 72 patients with HCC, 59 were male and 13 female with an average age of 55.0±13.1 years. HBV was the main etiological factor of the tumor (80.6%). Forty-one (56.9%) patients had single tumor and 31 (43.1%) had multiple tumors. Twenty-eight (38.9%) patients had tumors larger than 10 cm, and 44 (61.1%) smaller than 10 cm. Vascular invasion occurred in 29 (40.3%) patients and lymph nodes metastases in 15 (20.8%) patients. Of 13 patients with distant metastases, 10 had the involvement in the lung, 2 in the adrenal grand and 1 in the left cervical lymph node. When more than 200 ng/mL of AFP level was defined as positive, 44 (61.1%) of our patients were AFP positive and 28 (38.9%) negative. Liver function of all patients was Child-Pugh A, which was suitable for TACE. Clinical data of the patients are shown in Table 1.

 

The Mann-Whitney U test showed that there was no significant difference of basic serum GP73 level in different subgroups (including gender, age, etiology, tumor number, tumor size, vascular invasion, lymph node metastasis, distant metastasis and AFP level) (Table 1).

 

Serum GP73 was negatively correlated with albumin and positively correlated with aspartate aminotransferase, gamma-glutamyl transpeptidase, alkaline phosphatase, total bilirubin, direct bilirubin, and lactate dehydrogenase (P<0.05, Table 2).

 

GP73 level was significantly elevated at day 1 [5.0 (3.4-7.5)] and day 30 [5.5 (4.1-9.0)] after TACE in contrast with that before the treatment [4.6 (3.1-7.4)] (P<0.05). However, the GP73 level was not significantly different between day 1 and day 30 after TACE.

 

Multivariate analysis showed that basic serum GP73 was not correlated with gender, age, etiology, tumor number, tumor size, vascular invasion, lymph node metastasis, distant metastasis and AFP level.

 

Forty-four patients had new metastases of HCC within 17.2 months including 32 intrahepatic and 13 distant metastases and one of these patients had both intrahepatic and lung metastases. The Chi-square test indicated no correlation between the change of GP73 and new metastasis (Table 3).

 

We divided the patients into two groups: those with increased and those with decreased GP73 at day 1 after TACE. Survival analysis showed no significant difference of OS or PFS (P>0.05) (Fig. 1A and C) in the two groups. This was also true for the GP73 changes at day 30 after TACE (P>0.05) (Fig. 1B and D).

 

To investigate the mechanism of the increases of GP73 values in HCC patients in response to TACE treatment, we incubated three HCC cell lines with 5-FU and/or pirarubicin. We found that the GP73 levels in all the three cells were not significantly changed after 24-hour incubation with 5-FU at various concentrations. However, pirarubicin (20 µmol/L) significantly increased GP73 expression in three cell lines (Fig. 2). These results implied that pirarubicin enhanced GP73 expression after TACE.

 

 

GP73 has been recognized as a promising biomarker for HCC. This Golgi type II transmembrane protein consisting of 400 amino acids can be found in biliary epithelial cells but rarely in hepatocytes in normal human liver.^[12] However, GP73 level is significantly elevated in serum and tumor tissues from HCC patients.^{[6, 13, 14]} Our recent multi-center, multi-nation, large sample cohort study demonstrated that GP73 decreased to the normal level in HCC patients after surgical resection, and rebounded in most of the cases after recurrence of the tumor,^[5] indicating that GP73 change is an indicator of the therapeutic effectiveness of patients with HCC.

 

The proposal of this study was actually triggered by a question raised from the readers of our previous publication,^[5] that if GP73 level could also be a potential indicator of the routine treatments of HCC other than surgery, namely TACE or sorafinib, etc. TACE is currently the first-line therapy for unresectable HCC patients and the assessments of therapeutic effects rely on imaging and HCC biomarkers. However, both of which are neither satisfactory nor reliable. Our study aimed to observe the dynamic changes of GP73 before and after TACE.

 

To our surprise, the results of this study revealed that the GP73 level was significantly elevated, not dropped, at day 1 after TACE, and this elevation maintained up to day 30, showing that this was a real increase, not an acute reaction, by the strike of TACE manipulation. The relationship between GP73 and HCC is not clear yet. Our previous study^[5] showed that surgical resection significantly decreased GP73 levels in HCC patients. Yamamoto and colleagues^[15] showed that hepatectomy did not significantly change the serum GP73 levels. How to explain this inconsistency? It was reported that a majority of HCC patients in Japan were HCV-related, whereas our HCC patients were HBV-related. Another difference was found in the GP73 measurement. We used Western blotting, and Yamamoto et al used ELISA methods. Since TACE has a therapeutic effect on HCC, the elevation of the GP73 levels after TACE needs to be investigated.

 

We have thus performed cell culture to see if certain chemotherapeutics, particularly 5-FU and pirarubicin that we employed in our patients, contribute to the increased GP73 levels after TACE. The results showed that 20 µmol/L pirarubicin, but not 5-FU, significantly elevated GP73 expression in three HCC cell lines. HCC cells treated with 20 µmol/L pirarubicin were equivalent to the clinical dosage of TACE in patients. Thus we speculated that TACE might have limited effects on suppressing the GP73 level and that the increased GP73 was from the HCC reaction to pirarubicin. We also observed the changes of GP73 in 11 patients who received only transarterial embolization treatment without chemotherapy (data not show). The results showed that transarterial embolization did not change the GP73 levels of the patients. The patients' serum GP73 levels at day 1 and day 30 after TACE were significantly higher than those treated with transarterial embolization. These findings indicated that chemotherapeutics (pirarubicin) increased the GP73 values.

 

In our study, there was no correlation between GP73 and clinicopathological features, such as gender, age, etiology, tumor number, tumor size, vascular invasion, lymph node metastasis, distant metastasis and AFP level. However, the GP73 levels were positively correlated with most of the factors of liver damage, such as aspartate aminotransferase, gamma-glutamyl transpeptidase, alkaline phosphatase, total bilirubin, direct bilirubin and lactate dehydrogenase, which indicate acute liver injuries caused by inflammatory reaction following TACE treatment.

 

Survival analysis suggested that the change of GP73 was not related to OS or PFS, which was consistent with a previous study.^[6] We believed that the follow-up time for our patients was relatively short. However, for patients with middle-advanced HCC that could not be surgically removed, it was hard to prolong the follow-up time. Therefore, GP73 may not be a suitable biomarker for the therapeutic effectiveness of TACE in HCC patients.

 

In summary, we first observed the dynamic changes of GP73 in HCC patients before and after TACE, and found a progressive increase of GP73 following that treatment. Thus it might not be suitable to serve as an indicator for the effectiveness of TACE treatment. The use of certain chemotherapeutics such as pirarubicin during TACE and acute inflammation and injury of the liver may cause the elevated levels of GP73.

 

 

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