Predictive value of tumor markers in patients with recurrent hepatocellular carcinoma in different vascular invasion pattern

Feng Gao， Heng-Kai Zhu， Yang-Bo Zhu， Qiao-Nan Shan， Qi Ling， Xu-Yong Wei，Hai-Yang Xie， Lin Zhou， Xiao Xu and Shu-Sen ZhengHangzhou， China

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Predictive value of tumor markers in patients with recurrent hepatocellular carcinoma in different vascular invasion pattern

Feng Gao， Heng-Kai Zhu， Yang-Bo Zhu， Qiao-Nan Shan， Qi Ling， Xu-Yong Wei，Hai-Yang Xie， Lin Zhou， Xiao Xu and Shu-Sen Zheng
Hangzhou， China

BACKGROUND： Four tumor markers for hepatocellular carcinoma （HCC）， alpha-fetoprotein （AFP）， glypican-3 （GPC3），vascular endothelial growth factor （VEGF） and des-gammacarboxy prothrombin （DCP）， are closely associated with tumor invasion and patient's survival. This study estimated the predictability of preoperative tumor marker levels along with pathological parameters on HCC recurrence after hepatectomy.

METHODS： A total of 140 patients with HCC who underwent hepatectomy between January 2012 and August 2012 were enrolled. The demographics， clinical and follow-up data were collected and analyzed. The patients were divided into two groups： patients with macroscopic vascular invasion （MaVI+）and those without MaVI （MaVI-）. The predictive value of tumor markers and clinical parameters were evaluated by univariate and multivariate analysis.RESULTS： In all patients， tumor size （＞8 cm） and MaVI were closely related to HCC recurrence after hepatectomy. For MaVI+ patients， VEGF （＞900 pg/mL） was a significant predictor for recurrence （RR=2.421； 95% CI： 1.272-4.606； P=0.007）. The 1- and 2-year tumor-free survival rates for MaVI+ patients with VEGF ≤900 pg/mL versus for those with VEGF ＞900 pg/mL were 51.5% and 17.6% versus 19.0% and 4.8% （P＜0.001）. For MaVI- patients， DCP ＞445 mAu/mL and tumor size ＞8 cm were two independent risk factors for tumor recurrence （RR=2.307， 95% CI： 1.132-4.703， P=0.021； RR=3.150，95% CI： 1.392-7.127， P=0.006； respectively）. The 1- and 2-year tumor-free survival rates for the patients with DCP ≤445 mAu/mL and those with DCP ＞445 mAu/mL were 90.4% and 70.7% versus 73.2% and 50.5% respectively （P=0.048）. The 1-and 2-year tumor-free survival rates for the patients with tumor size ≤8 cm and ＞8 cm were 83.2% and 62.1% versus 50.0% and 30.0%， respectively （P=0.003）.

CONCLUSIONS： The MaVI+ patients with VEGF ≤900 pg/mL had a relatively high tumor-free survival than those with VEGF ＞900 pg/mL. In the MaVI- patients， DCP ＞445 mAu/mL and tumor size ＞8 cm were predictive factors for postoperative recurrence.

（Hepatobiliary Pancreat Dis Int 2016；15：371-377）

tumor markers；

hepatocellular carcinoma；

recurrence；

vascular invasion；

prediction

Introduction

Hepatocellular carcinoma （HCC） is the sixth most prevalent malignancy and the third most frequent cause of tumor-related death throughout the world.［1］Though current options for treatment of HCC consist of surgical resection， liver transplantation，transcatheter arterial embolization and radiofrequencyablation， hepatectomy is still the first choice.［2-4］However，the high incidence of HCC recurrence following resection reduces the survival rate.

Many studies［5-8］have developed various serum and tissue tumor markers to predict the prognosis of patients with HCC. Alpha-fetoprotein （AFP） is the first introduced and most extensively utilized marker for diagnosis，prognosis and monitoring of HCC. Glypican-3 （GPC3），vascular endothelial growth factor （VEGF） and desgamma-carboxy prothrombin （DCP） were found to be associated with carcinogenesis and vascular invasion that enabled them to be potential and reliable biomarkers for predicting tumor recurrence and survival in HCC patients after resection.

Nevertheless， the studies investigating predictive ability focus on the universality of multiple tumor markers in spite of varied tumor features， such as tumor size，number and vascular invasion. Macroscopic vascular invasion （MaVI） is usually evaluated before treatment and regarded as a critical feature reflecting biological behavior of tumor and influencing outcome of patients.［9， 10］

Therefore， the aim of the present study was to estimate the prognostic role of preoperative levels of serum tumor markers and pathological parameters in the recurrence of HCC in different vascular invasion patterns after hepatectomy. Early prediction of tumor recurrence after treatments is essential for precise selection of personalized therapeutic strategies， thus improving the survival of HCC patients.

Methods

Patients

A cohort of 140 adult patients who had been diagnosed with HCC and received liver resection at the First Affiliated Hospital， Zhejiang University School of Medicine，Hangzhou， China from January 2012 to August 2012 were enrolled while excluding those who had received preoperative adjuvant therapy or died of noncancerous causes or took oral warfarin. Demographics and clinical information were analyzed from original medical records after approval from the institutional review board. Tumor invasiveness was divided into 3 types： type 1， single nodular type with a clear demarcation； type 2， single nodular type with extranodular growth； type 3， contiguous multi-nodular type formed by a cluster of small and contiguous nodules.［11， 12］And tumor differentiation was determined by biopsy. Serum samples were taken from each patient for the measurement of the levels of four tumor markers within 3 days before operation. The diagnosis of HCC was confirmed by pathologic examination of resected specimens. MaVI was defined as portal vein invasion， and venous invasion was evaluated by imagings including computed tomography （CT）， magnetic resonance imaging （MRI）， or positron emission tomographycomputed tomography （PET-CT）.

The patients were followed up with contrast enhanced CT scan of the chest and abdomen or contrast enhanced MRI of the abdomen and non-contrast CT of the chest every 3 months for the first year， and subsequently every 6 months. The end of follow-up was as follows： the last follow-up （May 2015）， tumor recurrence or death.

Assays

A 5-mL blood sample was drawn into a serum separator tube and centrifuged at 1500 ×g for 10 minutes to obtain serum that was subsequently stored at -80 ℃. The serum was diluted to fit the standard curve of each measurement. Serum concentrations of VEGF were measured in duplicate with an enzyme-linked immunosorbent assay （ELISA） kit （Quantikine Human VEGF Immunoassay； R&D Systems， Minneapolis， MN， USA），by an investigator who was blinded to the clinical information of the patients. AFP， GPC3， and DCP were measured with an ELISA kit （Uscn Life Science Inc.， Wuhan，China）. The detection limits and the quantitation limits are 0.27 ng/mL and 0.78 ng/mL for AFP， 57 ng/L and 156 ng/L for GPC3， 9.00 pg/mL and 31.20 pg/mL for VEGF，and 5 mAu/mL and 100 mAu/mL for DCP.

Statistical analysis

Statistical analysis was performed with the software SPSS， version 16.0 （Chicago， IL， USA）. Variables were expressed as means and standard deviations or as medians and interquartile ranges. Pearson's correlation test， the Chi-square test， the Mann-Whitney U test or Student's t test was used appropriately to calculate the statistical significance of variables. Tumor-free survival rates were calculated using the Kaplan-Meier method. Univariate analysis was performed to identify the risk factors for tumor recurrence. After the univariate analysis， only variables with P value ＜0.1 were used in the multivariate analysis， which used the Cox proportional hazard regression analysis to identify independent survival predictors. Pearson's correlation test was used to assess the correlation between serum levels of tumor markers and pathological features of the resected HCC. A P value ＜0.05 was considered statistically significant.

Results

Table 1 presents baseline demographics and clinical parameters. The study population is composed of 124males and 16 females with a mean age of 54.6 years. The etiologies of HCC were as follows： HBV， 112 （80.0%）； alcoholic， 22 （15.7%）； and others， 6 （4.3%）. The follow-up ranged from 3 to 37 months （mean 15 months）.

Prognosis of HCC patients

During the follow-up， 81 （57.9%） patients were documented as experiencing HCC recurrence. The 1-year disease-free survival rate was 64.4%. The HCC recurrent rate was 27/34 for the patients whose tumor size was ＞8 cm and this ratio was 54/106 for those whose tumor size was ≤8 cm （P=0.003）； the recurrent ratio for patients with positive MaVI was 42/50 and this ratio was 39/90 for those with negative MaVI （P＜0.001， Table 1）.

MaVI was observed in 50 （35.7%） of the 140 patients under preoperative imaging modalities. As shown in Fig. 1， the prognosis of the patients with MaVI+ was significantly poorer than that of those with MaVI-（P＜0.001）. The 1-year disease-free survival rate of MaVI+ and MaVI- patients were 37.9% and 89.9%， respectively.

Table 1. Characteristics of 140 patients with hepatocellular carcinoma

Risk factors of HCC recurrence for patients with MaVI

Of the 50 MaVI+ patients， 42 （84.0%） experienced tumor recurrence. Multivariate analysis revealed that the level of VEGF was an independent risk factor affecting cumulative incidence of HCC recurrence following liver resection （RR=2.421； 95% CI： 1.272-4.606； P=0.007，Table 2）. The 1- and 2-year tumor-free survival rates for the patients with VEGF ≤900 pg/mL and for those with VEGF ＞900 pg/mL were 51.5% and 17.6% versus 19.0% and 4.8% （P＜0.001， Fig. 2）. There was no significant correlation between VEGF and microscopic vascular invasion，lymph node metastasis or tumor invasiveness （r=0.140，P=0.384； r=-0.054， P=0.739； r=0.078， P=0.279； respectively）.

Fig. 1. Tumor-free survival rates of patients with and without macroscopic vascular invasion. MaVI+ patients： patients with macroscopic vascular invasion； MaVI- patients： patients without macroscopic vascular invasion.

Risk factors of HCC recurrence for patients without MaVI

Of the 90 MaVI- patients， 58 （64.4%） had a level of DCP higher than 445 mAu/mL and tumor size was larger than 8 cm in 10 （11.1%） （Table 3）. Multivariate analysis indicated that DCP ＞445 mAu/mL and tumor size ＞8 cm were the independent risk factors for tumor recurrence （RR=2.307， 95% CI： 1.132-4.703， P=0.021； RR=3.150，95% CI： 1.392-7.127， P=0.006， respectively； Table 3）. The 1- and 2-year tumor-free survival rates for the patients with DCP ≤445 mAu/mL （n=58） and those with DCP ＞445 mAu/mL （n=32） were 90.4% and 70.7% versus 73.2% and 50.5% respectively （P=0.048， Fig. 3）. The 1-and 2-year tumor-free survival rates for the patients with tumor size ≤8 cm and for those with tumor size ＞8 cm were 83.2% and 62.1% versus 50.0% and 30.0%， respectively （P=0.003， Fig. 4）. There was no significant correlation between the concentration of DCP and microscopic vascular invasion or tumor invasiveness （r=0.029，P=0.786； r=0.022， P=0.835； respectively）.

Table 2. Univariate and multivariate analysis of variables associated with tumor-free survival in MaVI+ patients

Table 3. Univariate and multivariate analysis of variables associated with tumor-free survival in MaVI- patients

Fig. 2. Tumor-free survival rates of MaVI+ patients with VEGF≤?900 pg/mL and with VEGF ＞900 pg/mL. MaVI+ patients： patients with macroscopic vascular invasion.

Fig. 3. Tumor-free survival rates of MaVI- patients with DCP≤?445 mAu/mL and with DCP ＞445 mAu/mL. MaVI- patients： patients without macroscopic vascular invasion.

Fig. 4. Tumor-free survival rates of MaVI- patients with tumor size （≤8 cm or ＞8 cm）. MaVI- patients： patients without macroscopic vascular invasion.

Discussion

In the present study， we investigated the prediction of HCC recurrence in MaVI+ and MaVI- patients undergoing hepatectomy with preoperative tumor markers （AFP，GPC3， VEGF and DCP） and pathological parameters. There was a significant difference between the MaVI+ and MaVI- patients in parameters selection for recurrence prediction before surgery. In the MaVI+ patients，low VEGF levels were correlated with a relatively good prognosis. In the MaVI- patients， high DCP level and bigger tumor size were poor prognostic factors.

Tumor recurrence remains one of the most dominant morbidities and a major cause of poor prognosis in HCC patients after resection.［13， 14］The researchers have spent decades looking for valuable tumor markers of HCC diagnosis， monitoring and prognosis， which may establish framework for the precision taxonomy of HCC by molecular classification and assist in personalized therapeutic strategy.［14， 15］Nevertheless， the studies investigating prognosis prediction focus on the universal applicability of multiple tumor markers besides varied gross morphological features， such as tumor size， number and MaVI.

MaVI is usually evaluated before treatment and has been enrolled into scoring systems as a critical risk factor for poor survival.［9， 10］Differentiation is not an independent risk factor for tumor recurrence， which means that MaVI is a critical characteristic representing an advanced status of HCC （Table 2）. A reliable early prediction of tumor recurrence would benefit patients in three ways. The tumor markers may act as potential molecular treatment targets in personalized therapy. Moreover， more aggressive therapies such as transarterial chemoembolization， radiation therapy and systemic therapy would be performed. The risk of recurrence prompts a closer follow-up for HCC patients. A more precise and integrated therapy thus improves outcomes especially for the MaVI+ patients.

It has been well accepted that tumor cells secrete VEGF and induce angiogenesis to enhance tumor growth.［16］Peng et al［17］demonstrated that despite the well-known effects on angiogenesis， autocrine VEGF signaling promotes tumor cell proliferation and thus， VEGF inhibitor，such as sorafenib， inhibits angiogenesis and tumor cell proliferation. VEGF was shown to contribute to HCC recurrence after resection. The present study indicated that lower level of VEGF means a relatively good tumor-free survival among MaVI+ patients. As VEGF is a functional angiogenesis inhibiting target of sorafenib， our data may offer a new strategy of treatment.

Previous studies［18， 19］reported that preoperative elevation of DCP level implied advanced stage of HCC and poor prognosis. And it might play a vital role in predicting tumor behavior in HCC patients.［19］DCP showed high predictability of poor recurrence after hepatectomy for MaVI- patients rather than MaVI+ patients who already possessed an advanced tumor.

The mechanisms of the elevated tumor markers influencing the tumor-free survival rates are not clear because none of the mentioned pathological parameters were significantly correlated to VEGF and DCP. There is only a trend that tumor invasiveness may play a role in affecting tumor-free survival rates in MaVI+ patients. It was reported that VEGF modulates angiogenesis and promotes HCC cell proliferation.［17， 20］Accelerated growth of cancer cells and increased probability of tumor metastasis would worsen the prognosis and therefore， higher level of serum VEGF implies lower tumorfree survival in MaVI+ patients. Similarly， high level of serum DCP means poor prognosis in MaVI- patients.

Tumor size is a risk factor of recurrence in HCC patients.［21］Ishii et al［22］suggested that the patients with large HCC tumors more than 7 cm in diameter were at high risk for a poor prognosis due to a high percentage of lung metastasis. Our data showed that tumor size ＞8 cm was an independent risk factor of HCC recurrence for patients without MaVI.

Discovered by Abelev et al［23］in 1963， AFP has been a well-established biomarker for the diagnosis and prognosis of HCC for the past 5 decades.［24-26］Cucchetti et al［27］concluded that there was a significant association between preoperative AFP level and early recurrence in HCC patients. In contrast， Chon et al［28］did not find that the correlation between the level of AFP and the outcome of patients with HCC. Our data did not show the correlation between the level of AFP and HCC recurrence neither in MaVI+ nor in MaVI- patients.

GPC3， a member of the glypican family， is recentlyidentified as a sensitive and specific serological marker for HCC. A meta-analysis conducted by Li et al［29］indicated that GPC3 expression in tumor samples tends to be associated with cirrhosis， poor differentiation， advanced staging and vascular invasion. And Liu et al［8］reported that high level of GPC3 in the samples of cirrhotic livers with or without HCC enhances expression of HCC-related oncogenic mRNA and thus serves as a useful biomarker. However， the expression of GPC3 is detected in the tumor samples by immunohistochemistry which is not absolutely comparable with the serum level in HCC patients. The prognostic value of serum GPC3 is unclear. Our study chose HCC patients and detected serum GPC3 but did not find out significant correlation between GPC3 and recurrence of HCC after hepatectomy.

The limitations of the present were： patients were from a single center which may not be universally validated； and the relatively short follow-up period after hepatectomy. Further study might need to be conducted in a large population in multicenter prospective studies with an increased follow-up time span.

In conclusion， the MaVI+ patients with VEGF ≤?900 pg/mL could achieve a relatively high tumor-free survival compared with those with VEGF ＞900 pg/mL； DCP ＞445 mAu/mL and tumor size ＞8 cm have good predictive ability for postoperative recurrence of HCC in MaVI-patients.

Contributors： ZSS and XX conceived and designed the research；GF， ZHK and ZYB collected the data； GF and ZHK wrote the manuscript； GF， ZHK， SQN， LQ， WXY， XHY and ZL analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. GF and ZHK contributed equally to this work. ZSS is the guarantor.

Funding： This study was supported by grants from the National High Technology Research and Development Program of China （863 Program 2012AA020204）， the “New-Century 151 Talent Program” of Zhejiang Province （the 1st level）， Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents， and Public Technology Research Projects of Science and Technology Department of Zhejiang， China （2014C37061）.

Ethical approval： This study was approved by the Institutional Review Board and informed consent in writing for each individual was obtained.

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.

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Accepted after revision March 3， 2016

Author Affiliations： Division of Hepatobiliary and Pancreatic Surgery，Department of Surgery， First Affiliated Hospital， Zhejiang University School of Medicine （Gao F， Zhu HK， Zhu YB， Shan QN， Ling Q， Wei XY，Xu X and Zheng SS）； Key Lab of Combined Multi-Organ Transplantation，Ministry of Public Health， First Affiliated Hospital， Zhejiang University School of Medicine （Zhu HK， Wei XY， Xie HY， Zhou L， Xu X and Zheng SS）； and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases， Zhejiang University （Gao F， Ling Q， Xu X and Zheng SS）， 79 Qingchun Road， Hangzhou 310003， China

Prof. Shu-Sen Zheng， MD， PhD， FACS， Division of Hepatobiliary and Pancreatic Surgery， Department of Surgery， First Affiliated Hospital， Zhejiang University School of Medicine； Key Lab of Combined Multi-Organ Transplantation， Ministry of Public Health， First Affiliated Hospital， Zhejiang University School of Medicine； Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases，Zhejiang University， 79 Qingchun Road， Hangzhou 310003， China （Tel/ Fax： +86-571-87236567； Email： zyzss@zju.edu.cn）
? 2016， Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872（16）60095-4
Published online May 9， 2016.

August 26， 2015