Introduction

Oncology Letters

1792-1074 1792-1082

D.A. Spandidos

10.3892/ol.2017.5890

OL-0-0-5890

Articles

Assessment of high-sensitivity C-reactive protein tests for the diagnosis of hepatocellular carcinoma in patients with hepatitis B-associated liver cirrhosis

Li-Na

1 * Liu

Xiao-Yan

1 * Lu

Zhen-Hui

2 * Wu

Li-Gang

3 * Tang

Yuan-Yuan

1 * Luo

Xia

1 * Hu

Yan-Chao

1 * Yan

Ting-Ting

4 * Wang

2 Ding

Xiang-Chun

1 Xie

Yan

5 6

1Department of Infectious Disease, General Hospital of Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China 2Department of Hepatobiliary Surgery, General Hospital of Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China 3Department of Oncological Surgery, General Hospital of Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China 4Clinical Medical College, Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China 5Tissue Organ Bank & Tissue Engineering Centre, General Hospital of Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China 6Tissue Repair and Regeneration Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Queensland QLD 4059, Australia

Correspondence to: Dr Xiang-Chun Ding, Department of Infectious Disease, General Hospital of Ningxia Medical University, 804 Shengli Street, Yinchuan, Ningxia 750004, P.R. China, E-mail: 13619511768@163.com *

Contributed equally

05 2017

22 03 2017

13 5 3457 3464 05062015 27022017

2017

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide, with high morbidity and mortality. Chronic infection with hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma and the majority (~80%) of hepatocellular carcinoma patients in China exhibit co-morbidity with HBV-associated liver cirrhosis. The goal of reliable early diagnostic and prognostic techniques for HBV-associated HCC remains unrealized. The aim of the present study was to explore the efficacy of serum high-sensitivity C-reactive protein (hs-CRP) tests in the early diagnosis of HCC in patients with HBV-associated liver cirrhosis. A cohort of 493 patients with HBV-associated liver disease was divided into three groups: Chronic HBV (CHB) group; liver cirrhosis without HCC (LC) group; and liver cirrhosis with HCC (HCC) group. A further 47 healthy individuals comprised the healthy control (CN) group. Comparative analyses of clinical symptoms, histopathology, ultrasound imagery, computed tomography, magnetic resonance imaging, biochemistry [α-fetoprotein (AFP) and liver function enzymes], and hs-CRP tests were conducted across these four groups. Immunohistochemical analysis showed that CRP is strongly expressed in HCC tumor tissue, but is not expressed elsewhere. Analyses of the correlations between serum hs-CRP levels and HCC clinical parameters indicated that there was no correlation between serum hs-CRP levels, tumor Edmondson grade, tumor-node-metastasis stage and AFP status. Serum hs-CRP and AFP levels were found to be significantly elevated in the HCC group compared to those in the LC, CHB and CN groups (P<0.01). Receiver operator characteristic analysis showed that measurement of serum hs-CRP could differentiate HCC from HBV-associated liver cirrhosis, as well as increase the accuracy of HCC diagnoses. Additionally, measurement of hs-CRP and AFP together improved diagnostic accuracy for HCC compared with either test alone. Serum hs-CRP could have potential as an effective diagnostic tool to complement AFP in diagnosing HCC and improving the identification of AFP-negative HCC in patients with HBV-associated liver cirrhosis. The present findings may facilitate the earlier diagnosis of hepatocellular carcinoma, permitting more effective treatment and a broader spectrum of treatment modalities for patients with advanced hepatic disease.

chronic hepatitis B virus liver cirrhosis hepatocellular carcinoma α-fetoprotein high-sensitivity C-reactive protein

Introduction

Hepatocellular carcinoma (HCC) is a common malignant tumor, with high morbidity and mortality, which affects patients worldwide. Approximately 564,000 novel cases of HCC are diagnosed each year (1,2). HCC is particularly common in Asia due to a high prevalence of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. In China, HCC has been the second-leading cause of cancer-associated mortalities since the 1990s (3). The majority of HCC patients in China (~80%) have co-morbid cirrhosis associated with chronic HBV infection (4,5). The paucity of methods for the early diagnosis of HCC results in a high mortality rate (6–9). The mean 5-year survival rate of advanced liver cancer is 3–5% (10,11). Although a combination of α-fetoprotein (AFP) assays with ultrasound examination is used for early diagnosis of liver cancer, only one-third of patients are accurately diagnosed and treated at an early stage of the disease (7). Accordingly, identifying more effective alternative methods for the early diagnosis of hepatocellular carcinoma in patients with HBV-associated liver cirrhosis is imperative.

HCC with HBV-associated liver cirrhosis is usually diagnosed through a combination of patient medical history, physical examination, imaging (ultrasound, MRI or CT scans showing a liver mass consistent with HCC), and optionally, elevated serum AFP level (>400 ng/ml). A suspicious lesion on the sonogram generally requires additional imaging studies to confirm the stage of the tumor, while their sensitivity in detecting small nodules may be low. The addition of arterial phase imaging to conventional CT scanning increases the number of tumor nodules detected, but in nodular cirrhotic livers, detecting HCC is more challenging. While the overall sensitivity of MRI is similar to that of triphasic CT scans, in patients with nodular cirrhotic livers MRI has better sensitivity and specificity. Confirmation of the diagnosis is made by fine needle aspiration or biopsy. Elevation of AFP >400 ng/ml can be used instead of fine needle cytology for diagnosis of HCC in patients with liver cirrhosis and a focal hypervascular liver lesion (>2 cm) in at least one imaging technique. Patients with potentially resectable liver mass and AFP >400 ng/ml should undergo surgery without pre-operative fine needle aspiration cytology or biopsy. Any deterioration in liver function in a patient with known liver cirrhosis of any etiology should raise a suspicion of HCC. As an increasing number of cirrhotic patients and/or HBV/HCV carriers undergo diagnostic procedures, it is likely that, in a substantial number of patients, classical echosonography will be the initial imaging technique that raises a suspicion of HCC. Subsequently, the size of lesions and the presence or otherwise of cirrhosis may affect the sequencing of tests used to diagnose HCC. Suspect nodules <1 cm should be examined with ultrasound at intervals of 3–6 months; nodules between 1 and 2 cm in a cirrhotic liver should be investigated with at least two dynamic studies (triphasic CT scan, ultrasound, or MRI with contrast). If two techniques show a typical appearance of HCC, the nodule should be interpreted as such; if that is not the case, the lesion should either be biopsied when possible or be extirpated, at the discretion of the physician. Nodules >2 cm with a typical feature of HCC found through a dynamic imaging technique, as well as any nodule associated with AFP concentration >400 ng/ml or rising AFP on sequential determinations, does not require biopsy, but should be considered as proven HCC, and appropriate treatment should be started (12). The Barcelona Clinic Liver Cancer (BCLC) staging system was used to define tumor stage (12,13).

C-reactive protein (CRP) is a non-specific acute-phase protein synthesized by the liver in response to acute and chronic inflammation, and thus represents a molecular indicator of inflammation, infection, trauma and tissue necrosis (14–18). Previous studies have found that the serum CRP measurement can be applied not only to assess the severity of liver damage and fibrosis in cases of fatty liver and chronic HCV, but also serves as an independent marker of poor prognosis in patients with hepatocellular carcinoma (19–25). Elevated CRP expression has also been detected in cancers of the lung, ovaries, colon and prostate, as well as colorectal cancer and HCC (26–31). Therefore, the present study proposed that serum CRP has potential as a diagnostic and prognostic biomarker for multiple human cancers, including HCC. However, the usefulness of serum CRP tests in HCC and other cancers remains unknown.

A high-sensitivity CRP (hs-CRP) assay, which gives results in 25 min, quantifies low levels of CRP (as low as 0.04 mg/l) using laser nephelometry. This test has been applied to detect CRP. It is not yet known, however, whether the hs-CRP assay can improve diagnostic accuracy for HCC in patients with HBV-associated liver cirrhosis. The present study evaluated whether the hs-CRP assay can improve the diagnostic accuracy of HCC in patients with cirrhosis associated with HBV. A total of 491 candidates were recruited and analyzed. The present findings may assist clinicians in the earlier diagnosis of hepatocellular carcinoma, in turn resulting in more effective and alternative treatment modalities for patients with advanced hepatic disease.

Materials and methods <sec> <title>Patients

Ethical approval for the present study was obtained from the Ethics Committee of the General Hospital of Ningxia Medical University (Yinchuan, China) and written informed consent was acquired from the candidates prior to any treatment. A total of 493 cases of patients diagnosed with HBV-associated liver disease between July 2012 and October 2014 were recruited from the Department of Infectious Disease, General Hospital of Ningxia Medical University, (Yinchuan, China). Patients with liver damage due to autoimmune status, metabolism, medication or alcohol intake, and those with acute infection, rheumatoid arthritis, gout, asthma, chronic lung disease, myocardial infarction or stroke, any of which could alter serum CRP levels, were excluded. Patients with portal vein thrombosis were also excluded from the present study. None of the included patients had been treated with any hepatoprotective, anti-fibrotic or anti-viral drugs prior to the serum samples being collected. All the included patients had a complete data set, including a full medical history, HBV markers, serum HBV DNA level, liver function, AFP level, hs-CRP assay, and magnetic resonance imaging (MRI), computed tomography (CT) and ultrasound imagery. Patients, including 47 healthy individuals, were classified into four groups: Chronic HBV (CHB) group; liver cirrhosis without HCC (LC) group; liver cirrhosis with HCC (HCC) group; and healthy control (CN) group. The demographic and clinical/pathological data are summarized in Table I.

CHB was diagnosed according to the clinical practice guidelines criteria laid down by Asian Pacific Association for the Study of the Liver, European Association for the Study of the Liver and American Association for the Study of Liver Diseases (32). LC diagnosis was confirmed by clinical symptoms, diagnostic ultrasound imaging, histopathology of liver biopsy samples, and laboratory diagnosis, including the presence of ascites, enlargement of the caudate lobe, collateral portal-venous anastomoses, portal hypertension, nodular liver contour, splenomegaly and varices (11).

In total, 47 healthy volunteers, who had no history of alcoholism, taking medications (including traditional Chinese medicines) that could damage the liver, or autoimmune or metabolic disorders, and whose blood chemistry was normal, acted as a control group.

Patients currently undergoing, or with any history of, treatment with any hepatoprotective, anti-fibrotic, or anti-viral drugs were excluded from the present study. Additionally, patients and healthy volunteers with conditions such as infection, arthritis, gout, asthma, chronic lung disease, myocardial infarction or stroke were excluded.

Measurements

Serum hs-CRP levels were assayed by Ultra ion immuno-turbidmetric detection kit (Shanghai Changzheng Biological Company, Shanghai, China). Serum AFP levels were analyzed with a chemiluminescence immunoassay analyzer detection kit (Shanghai Abbott Pharmaceutical Co., Ltd., Shanghai, China).

Immunohistochemistry (IHC)

Liver tissue samples were stained with Masson Trichrome (ab150686; Abcam, Cambridge, UK) and examined by two independent liver pathologists. For CRP staining, samples were cut into 3–5 mm sections, formalin-fixed and embedded in paraffin. The sections were then dewaxed, rehydrated, and incubated in 3% hydrogen peroxide to quench endogenous peroxidase activity. The samples were incubated with anti-CRP primary antibody (dilution, 1:1,000; bs-0115R; Hotgen Biotech, Beijing, China) and secondary antibody (HRP-labeled anti-rabbit; dilution, 1:5,000; BA1003; BosterBio, Wuhan, China) overnight at 4°C. Subsequently, 3,3′-Diaminobenzidine Substrate Chromogen System (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) was used to detect positive staining. Images were captured using an Olympus E520 microscope (Olympus, Tokyo, Japan). Protein expression was semi-quantified as follows: The positive staining intensity was scored according to the number of cells exhibiting cytoplasmic and nuclear staining, using the following classification system: 0, no staining; 1, nuclear staining in <10% of cells and/or weak cytoplasmic staining; 2, nuclear staining in 10–50% of cells and/or distinct cytoplasmic staining; and 3, nuclear staining in >50% of cells and/or strong cytoplasmic staining. The positive staining intensity (scale 0–3) and the percentage of positive cells (0–100%) were determined and multiplied to produce a final score (0–300). The same experienced pathologists analyzed the IHC data to maintain consistency.

Statistical analysis

Statistical analyses were performed using the SPSS v17.0 software package (SPSS Inc., Chicago, IL, USA). Quantitative data is expressed as the mean ± standard deviation. The differences between abnormally distributed data were compared using a Wilcoxon rank-sum test, while means from normally distributed data were compared using Student's t-test. A χ² test was used to analyze the count data, while Spearman's rank correlation coefficient was applied to analyze correlation. The area under the curve (AUC) was calculated using receiver operator characteristic (ROC) curve analysis. P<0.05 was considered to indicate a statistically significant difference.

Results <sec> <title>Baseline demographic and clinical characteristics

A total of 493 cases of previously untreated patients with HBV-associated cirrhosis were recruited into the present study. The patients were classified into CHB (137 cases, 27.79%), LC (169 cases, 34.28%) and HCC (187 cases, 37.93%) groups. An additional 47 healthy volunteers were recruited as an age-matched control group (Table I).

CRP expression in human tissues

To further validate CRP expression in HCC and non-tumor liver tissues in patients, immunohistochemical analysis was employed to detect the presence of CRP in 126 cases of non-tumor tissue samples and 134 cases of HCC tissue samples. IHC analysis showed that CRP is abundantly expressed in HCC tissues, but not in non-tumor tissues. CRP-positive staining was mainly found in parenchymal cells, but several non-parenchymal cells also showed positive staining of CRP (Fig. 1). Staining defined as ‘negative’, ‘weak positive’, ‘moderate positive’ and ‘strong positive’ was found in 21.43, 33.33, 25.40 and 19.18%, and 7.46, 4.48, 41.79 and 46.27% of non-tumor and HCC tissue samples, respectively. In addition, the total positive rates of CRP expression in non-tumor and HCC tissues samples were 78.57 and 92.54%, respectively (Table II). Additionally, IHC scores for CRP were significantly increased in tumor tissue samples (64.04±91.52) compared with non-tumor tissue samples (21.40±38.72) (P<0.05; Table II).

Serum hs-CRP and AFP levels

Serum hs-CRP and AFP levels were found to be significantly elevated in the HCC group compared to those in the LC, CHB and CN groups (P<0.01). The mean levels of hs-CRP and AFP in the HCC group (17.34±22.61 and 492.61±544.15 ng/ml) were significantly increased with the levels in the LC (2.83±3.05 and 104.08±186.3 nm/l), CHB (1.53±2.7 and 16.15±43.35 ng/ml) and CN (0.59±0.53 and 4.48±1.29 ng/ml) groups (P<0.05; Table I).

Correlation between serum hs-CRP level and HCC clinical parameters

The clinical parameters of patients with HCC were AFP, tumor Edmondson grade and tumor-node-metastasis (TNM) stage. The differences among the groups were analyzed by χ² test. To evaluate clinical relevance, the association between serum hs-CRP concentration and the pathological characteristics of HCC patients were examined. Analysis of the correlation between serum hs-CRP level and HCC clinical parameters indicated that there was no correlation between hs-CRP serum levels and tumor Edmondson grade (r=0.238, P=0.229), TNM stage (r=0.159, P=0.498) or AFP status (r=0.336, P=0.737) (Table III).

ROC curve analyses of serum hs-CRP and AFP levels for the diagnosis of HCC

ROC curve analysis was used to clarify the optimal cut-off value of CRP expression in the diagnosis of HCC. The ROC curve showed that the sensitivity and specificity of serum hs-CRP levels for the diagnosis of HCC were 84.16 and 61.59%, respectively [95% confidence interval (CI), 0.867–0.933], while those of serum AFP for HCC diagnosis were 74.42 and 55.63%, respectively (95% CI, 0.778–0.864). The AUC of CRP and AFP levels for HCC diagnosis were 0.903 and 0.824, respectively. The optimal cut-off levels for serum CRP and AFP in the diagnosis of HCC were 2.17 mg/l and 20 ng/ml, respectively (Fig. 2). ROC analysis showed that measurement of serum hs-CRP could differentiate between HCC and HBV-associated liver cirrhosis and increase the diagnostic accuracy for HCC. Additionally, measurement of hs-CRP and AFP together improved the diagnostic accuracy for HCC compared with all controls and either test alone (Table IV).

Comparison of positive rates of serum CRP and AFP expression

In the present study, 20 ng/ml AFP and 2.17 mg/l hs-CRP were regarded as the optimal cut-off levels. The results showed that 161 patients (86.1%) were positive for CRP, whereas 130 patients (69.52%) were positive for AFP (187 patients; χ²=14.881; P<0.001) (Table V). The CRP positive rate in HCC patients was increased compared with liver cirrhosis patients (χ²=93.761; P<0.001) (Table IV), while the positive rate of AFP in patients with HCC was similar to that in patients with liver cirrhosis (χ²=2.16; P>0.01) (Table V). The positive rate of CRP in AFP-positive patients with liver cirrhosis was increased compared with that in AFP-negative patients with liver cirrhosis, while the positive rate of CRP in AFP-positive patients with HCC was similar to that of AFP-negative patients with HCC (Table VI). Overall, these data indicate that serum hs-CRP measurement can raise the diagnostic accuracy for AFP-negative HCC in patients with HBV-associated liver cirrhosis.

Discussion

Chronic inflammation has been reported to be involved in tumor initiation, promotion, progression, invasion, and metastasis (33–41). HBV-associated HCC is a typical inflammation-associated malignancy (42–44). CRP is a sensitive acute-phase reactant and a particularly sensitive marker of inflammation and tissue damage in the liver (37–40). At present, several studies have described elevated CRP expression in cancer cells and tissues, including HCC (25–31), but whether CRP expression plays a role in the development or progression of HBV-associated HCC remains unknown. The present study aimed to determine whether CRP is expressed in the tissue of HBV-associated HCC with liver cirrhosis, and whether there is any difference in serum CRP levels between HBV-associated HCC with liver cirrhosis, HBV-associated liver cirrhosis without HCC, CHB patients and healthy control subjects. The present study found that CRP is strongly expressed in HCC tissues [including AFP (−) and (+) HCC patients], but not in non-tumor tissues. Additionally, IHC scores for CRP were significantly increased in tumor tissue samples compared with non-tumor tissue samples (P<0.05). It was also observed that serum CRP levels were significantly increased in patients with HBV-associated HCC compared with patients with HBV-associated liver cirrhosis without HCC, CHB patients, and the healthy control group (P<0.05). On the basis of the aforementioned data, the present study hypothesizes that CRP plays an important role in the development and progression of HCC, and serum CRP level has potential as a diagnostic and prognostic biomarker for HCC. However, earlier studies have indicated that serum CRP measurement is not a good diagnostic marker for HCC (21–23). It was found that serum CRP levels in earlier studies were measured with a commercial kit by a nephelometric method, whose lower accuracy limits its diagnostic efficacy.

hs-CRP, an ultra-sensitive detection technology, can accurately detect low concentrations of CRP; however, whether hs-CRP measurement can improve diagnostic accuracy for HCC in patients with HBV-associated liver cirrhosis remains unknown. To further clarify the optimal cut-off value of CRP for diagnosing HCC in patients with HBV-associated liver cirrhosis, ROC curve analysis was performed. The results showed that the sensitivity and specificity of serum hs-CRP for HCC diagnosis were 84.16 and 61.59%; those of serum AFP for HCC diagnosis were 74.42 and 55.63% respectively. The areas under ROC curve of CRP and AFP were 0.903 and 0.824, respectively. The optimal cut-off levels for serum CRP and AFP in the diagnosis of HCC were 2.17 mg/l and 20 ng/ml, respectively. ROC analysis indicated that measurement of serum hs-CRP could differentiate HCC from HBV-associated liver cirrhosis and increase the diagnostic accuracy for HCC. Additionally, measurement of hs-CRP and AFP together improved the diagnostic accuracy for HCC compared with all controls compared with either test alone.

Since the identification of AFP in the 1970s, AFP has been the only serologic marker widely used in HCC diagnosis. However, its diagnostic power has been continuously questioned and debated. For example, elevated serum AFP was only observed in 60–70% of HCC patients, while the proportion was just 33–65% in patients harboring HCCs <3 cm in diameter (7,8). In addition, the non-specific elevation of serum concentrations of AFP may be present in patients with non-malignant chronic liver disease, such as patients with chronic HBV (15–58%) and patients with liver cirrhosis (11–47%) (7,8,11,28,45). Therefore, the efficacy of AFP as a biomarker for HCC diagnosis has been questioned. The development of more effective alternative diagnostic methods to complement AFP is urgently required to improve clinical outcomes. In the present study, 20 ng/ml AFP and 2.17 mg/l hs-CRP were regarded as the optimal cut-off levels for the diagnosis of HCC. The results showed that 86.1% of HCC patients were positive for CRP, whereas 69.52% of HCC patients were positive for AFP. The CRP positive rate in HCC patients was increased compared with liver cirrhosis patients, while the positive rate of AFP in patients with HCC was similar to patients with liver cirrhosis. The positive rate of CRP in AFP-positive HCC patients was similar to the rate in AFP-negative HCC patients. These results indicated that serum hs-CRP measurement is able to differentiate between HCC and HBV-associated liver cirrhosis, and the diagnostic accuracy for AFP-negative HCC with HBV-associated liver cirrhosis was increased. However, the association between CRP and HBV-associated HCC is more complicated than expected. Serum AFP and CRP are also frequently elevated in patients with chronic HBV and HBV-associated liver cirrhosis who do not have HCC. Yang et al (46) recently reported that in cases where there is a delayed response in AFP levels to entecavir in patients with elevated AFP levels, this delay acts as an indicator of high HCC risk (46,47). However, the clinical significance of CRP response to antiviral treatments remains to be fully studied. Thus, the diagnostic value of serum CRP level for HCC diagnosis requires additional investigation.

Serum hs-CRP level may be an effective diagnostic metric to complement AFP level in diagnosing HCC and improving the identification of patients with AFP-negative HCC in cases of HBV-associated liver cirrhosis. The present findings may improve the early diagnosis of hepatocellular carcinoma, permitting more effective and alternative treatment modalities for patients with advanced hepatic disease.

Acknowledgements

The present study was supported by the National Natural Science Foundation of China (grant no. 81460301), the Natural Science Foundation of Ningxia (grant nos. NZ09115 and NZ11199), the Scientific and Technological Foundation of Ningxia (grant no. NZI5134), and Chinese Foundation for Hepatitis Wang Bao-en Liver Fibrosis Fund (grant no. xjs).

Abbreviations

HCC

hepatocellular carcinoma

HBV

hepatitis B virus

CRP

C-reactive protein

CHB

chronic hepatitis B virus

References 1

El-Serag

Epidemiology of viral hepatitis and hepatocellular carcinoma

Gastroenterology14212641273.e12012

10.1053/j.gastro.2011.12.061

22537432

Luk

Liu

Proteomics of hepatocellular carcinoma in Chinese patients

OMICS152612662011

10.1089/omi.2010.0099

21348761

Tang

Hepatocellular carcinoma-cause, treatment and metastasis

World J Gastroenterol74454542001

10.3748/wjg.v7.i4.445

11819809

Lin

Kao

Risk stratification for hepatitis B virus related hepatocellular carcinoma

J Gastroenterol Hepatol2810172013

10.1111/jgh.12010

23094699

Gao

Jiang

Zhou

Ding

Wang

Zhao

Chen

HBV infection and familial aggregation of liver cancer: An analysis of case-control family study

Cancer Causes Control158458502004

10.1023/B:CACO.0000043435.59195.3c

15456998

Zhang

Wang

Zhuang

Zhu

Chen

Chan

Leung

Heat shock protein 27 mediates the effect of 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b] xanthone on mitochondrial apoptosis in hepatocellular carcinoma

J Proteomics75483348432012

10.1016/j.jprot.2012.05.032

22677112

Patel

Shariff

Ladep

Thillainayagam

Thomas

Khan

Taylor-Robinson

Hepatocellular carcinoma: Diagnostics and screening

J Eval Clin Pract183353422012

10.1111/j.1365-2753.2010.01599.x

21114800

Shariff

Cox

Gomaa

Khan

Gedroyc

Taylor-Robinson

Hepatocellular carcinoma: Current trends in worldwide epidemiology, risk factors, diagnosis and therapeutics

Expert Rev Gastroenterol Hepatol33533672009

10.1586/egh.09.35

19673623

Lee

Chen

RNA interference as therapeutics for hepatocellular carcinoma

Recent Pat Anticancer Drug Discov61061152011

10.2174/157489211793980097

21110827

Siegel

Naishadham

Jemal

Cancer Statistics, 2013

CA Cancer J Clin6311302013

10.3322/caac.21166

23335087

Shen

Fan

Yang

Tan

Zhao

Wang

Niu

Zhou

Serum DKK1 as a protein biomarker for the diagnosis of hepatocellular carcinoma: A large-scale, multicentre study

Lancet Oncol138178262012

10.1016/S1470-2045(12)70233-4

22738799

Jelic

Sotiropoulos

ESMO Guidelines Working Group: Hepatocellular carcinoma: ESMO clinical practice guidelines for diagnosis, treatment and follow-up

Ann Oncol21(Suppl 5)v59v642010

10.1093/annonc/mdq166

20555104

Llovet

Di Bisceglie

Bruix

Kramer

Lencioni

Zhu

Sherman

Schwartz

Lotze

Talwalkar

Design and endpoints of clinical trials in hepatocellular carcinoma

J Natl Cancer Inst1006987112008

10.1093/jnci/djn134

18477802

Shameem

Bhargava

Ahmad

Saad

Fatima

Malik

Association between serum C-reactive protein levels and other important predictive markers of outcome in COPD

Acta Med Iran4918202011

21425065

Tanrıverdi

Tor

Kart

Altın

Atalay

SumbSümbüloğlu

Prognostic value of serum procalcitonin and C-reactive protein levels in critically ill patients who developed ventilator-associated pneumonia

Ann Thorac Med101371422015

10.4103/1817-1737.151442

25829966

Garcia-Rio

Miravitlles

Soriano

Muñoz

Duran-Tauleria

Sánchez

Sobradillo

Ancochea

EPI-SCAN Steering Committee: Systemic inflammation in chronic obstructive pulmonary disease: A population-based study

Respir Res11632010

10.1186/1465-9921-11-63

20500811

Abd

Eapen

Bajpai

Goyal

Dollar

Sperling

The role of C-reactive protein as a risk predictor of coronary atherosclerosis: Implications from the JUPITER trial

Curr Atheroscler Rep131541612011

10.1007/s11883-011-0164-5

21274757

Peisajovich

Marnell

Mold

Du Clos

C reactive protein at the interface between innate immunity and inflammation

Expert Rev Clin Immunol43793902008

10.1586/1744666X.4.3.379

20476927

Lin

Wang

Chen

Chuang

Hsieh

Tsai

Chang

Role of serum C-reactive protein as a marker of hepatocellular carcinoma in patients with cirrhosis

J Gastroenterol Hepatol154174212000

10.1046/j.1440-1746.2000.02149.x

10824887

Fabris

Pirisi

Soardo

Falleti

Pezzetta

Vitulli

Toniutto

Bortolotti

Gonano

Bartoli

Value of serum C-reactive protein measurement in the detection of hepatocellular carcinoma superimposed on liver cirrhosis

J Cancer Res Clin Oncol1202292321994

10.1007/BF01372561

7507117

Lee

Tsai

Lai

Serum C-reactive protein as a serum marker for the diagnosis of hepatocellular carcinoma

Cancer63156715711989

10.1002/1097-0142(19890415)63:8<1567::AID-CNCR2820630820>3.0.CO;2-J

2466549

Andreozzi

Viscogliosi

Colella

Subic

Cipriani

Marigliano

Verrusio

Servello

Ettorre

Marigliano

Predictors of liver fibrosis in patients with non-alcoholic fatty liver disease. The role of metabolic syndrome, insulin-resistance and inflammation

Recenti Prog Med1035705742012(In Italian)

23258240

Komoriya

Inoue

Yoshimune

Ogawa

Moriyama

Kohno

Use of a highly sensitive latex reagent with amino acid spacer for determination of C-reactive protein concentration in a variety of liver diseases

J Biosci Bioeng1145605632012

10.1016/j.jbiosc.2012.06.005

22770697

Atta

Cabral

Santos

Paraná

Atta

Inflammation biomarkers in chronic hepatitis C: Association with liver histopathology, HCV genotype and cryoglobulinemia

Inflamm Res61110111062012

10.1007/s00011-012-0502-2

22718074

Sjöwall

Cardell

Boström

Bokarewa

Enocsson

Ekstedt

Lindvall

Frydén

Almer

High prevalence of autoantibodies to C-reactive protein in patients with chronic hepatitis C infection: Association with liver fibrosis and portal inflammation

Hum Immunol733823882012

10.1016/j.humimm.2012.01.009

22333691

Rocha

Morgan

Templeton

Pond

Naik

Sonpavde

Prognostic impact of C-reactive protein in metastatic prostate cancer: A systematic review and meta-analysis

Oncol Res Treat377727762014

10.1159/000369545

25531724

Shibutani

Maeda

Nagahara

Ohtani

Sugano

Ikeya

Kimura

Amano

Kubo

Tanaka

Elevated preoperative serum C-reactive protein levels are associated with poor survival in patients with colorectal cancer

Hepatogastroenterology61223622402014

25699359

Chen

Wang

Abnet

Dawsey

Fan

Yin

Taylor

Qiao

Freedman

Association between C-reactive protein, incident liver cancer, and chronic liver disease mortality in the Linxian nutrition intervention trials: A nested case-control study

Cancer Epidemiol Biomarkers Prev243863922015

10.1158/1055-9965.EPI-14-1038

25613115

Hefler

Concin

Hofstetter

Marth

Mustea

Sehouli

Zeillinger

Leipold

Lass

Grimm

Serum C-reactive protein as independent prognostic variable in patients with ovarian cancer

Clin Cancer Res147107142008

10.1158/1078-0432.CCR-07-1044

18245530

Zhu

Yan

Wang

Zhao

Serum C-reactive protein and risk of lung cancer: A case-control study

Med Oncol303192013

10.1007/s12032-012-0319-4

23254959

Nozoe

Matsumata

Kitamura

Sugimachi

Significance of preoperative elevation of serum C-reactive protein as an indicator for prognosis in colorectal cancer

Am J Surg1763353381998

10.1016/S0002-9610(98)00204-9

9817250

Alberti

Caporaso

HBV therapy: Guidelines and open issues

Dig Liv Dis43(Suppl 1)S57S632011

10.1016/S1590-8658(10)60693-7

Balkwill

Mantovani

Inflammation and cancer: Back to Virchow?

Lancet3575395452001

10.1016/S0140-6736(00)04046-0

11229684

Okada

Inflammation-related carcinogenesis: Current findings in epidemiological trends, causes and mechanisms

Yonago Acta Med5765722014

25324587

Feng

Huang

Chen

A new inflammation index is useful for patients with esophageal squamous cell carcinoma

Onco Targets Ther7181118152014

10.2147/OTT.S68084

25336972

Nishikawa

Arimoto

Wakasa

Kita

Kimura

Osaki

Pre-treatment C-reactive protein as a prognostic factor for recurrence after surgical resection of hepatocellular carcinoma

Anticancer Res33118111882013

23482800

Zhao

Luo

Liu

The association between preoperative serum C-reactive protein and hepatocellular carcinoma recurrence in patients with chronic hepatitis B virus HBV infection-a retrospective study

PLoS One10e01169092015

10.1371/journal.pone.0116909

25602444

Wang

Zhang

Luo

Zhou

Liao

Huang

Xie

Screening differential expression of serum proteins in AFP-negative HBV-related hepatocellular carcinoma using iTRAQ-MALDI-MS/MS

Neoplasma6117262014

10.4149/neo_2014_001

24195504

Imai

Kinoshita

Onoda

Iwaku

Oishi

Tanaka

Fushiya

Koike

Nishino

Tajiri

Persistent elevated C-reactive protein after treatment is an independent marker of a poor prognosis in patients with hepatocellular carcinoma

Clin Transl Oncol155755812013

10.1007/s12094-012-0976-y

23263911

Sieghart

Pinter

Hucke

Graziadei

Schöniger-Hekele

Müller

Vogel

Trauner

Peck-Radosavljevic

Single determination of C-reactive protein at the time of diagnosis predicts long-term outcome of patients with hepatocellular carcinoma

Hepatology57222422342013

10.1002/hep.26057

22961713

Liu

Zhang

Zhan

Zhao

Wan

Peng

Zhu

Interleukin-23A is associated with tumor growth in Helicobacter-pylori-related human gastric cancer

Cancer Cell Int141042014

10.1186/s12935-014-0104-x

25349535

Szabo

Lippai

Molecular hepatic carcinogenesis: Impact of inflammation

Dig Dis302432482012

10.1159/000336913

22722548

Morales-Sánchez

Fuentes-Pananá

Human Viruses and Cancer

Viruses6404740792014

10.3390/v6104047

25341666

Chen

Williams

Filippova

Filippov

Duerksen-Hughes

Viral carcinogenesis: Factors inducing DNA damage and virus integration

Cancers (Basel)6215521862014

10.3390/cancers6042155

25340830

Sherman

Colombo

Hepatocellular carcinoma screening and diagnosis

Semin Liver Dis343893972014

10.1055/s-0034-1394139

25369301

Yang

Kim

Chung

Sohn

Lee

Hong

Chung

Jang

Jeong

Kim

Prediction of risk for hepatocellular carcinoma by response of serum α-fetoprotein to entecavir therapy

J Gastroenterol Hepatol30117511822015

10.1111/jgh.12921

25707935

Triolo

Corte

C Della

Colombo

Impact of HBV therapy on the incidence of hepatocellular carcinoma

Liver Int34(Suppl 1)S139S1452014

10.1111/liv.12394

Figure 1.

Representative immunohistochemistry images of C-reactive protein in non-tumor and HCC (magnification, ×100) tissues, consisting of (A) chronic HBV, (B) HBV-associated liver cirrhosis, (C) AFP-positive HBV-associated HCC and (D) AFP-negative HBV-associated HCC. HCC, hepatocellular carcinoma; HBV, hepatits B virus; AFP, α-fetoprotein.

Figure 2.

Receiver operating characteristic curve for CRP and AFP in patients with hepatocellular carcinoma compared with the controls. AFP, α-fetoprotein; CRP, C-reactive protein.

Table I.

Patient demographic and clinical characteristics.

	Group

Characteristic	HC	CHB	LC	HCC
Total, n	47	137	169	187
Age, mean years ± SD	39±12.1	34±10.6	46±10.2	48±10.6
Gender ratio, M:F	39:8	110:27	131:38	144:43
HBeAg status, n
HBeAg positive	NA	87	45	42
HBeAg negative	NA	50	124	145
HBV DNA, mean logIU/ml ± SD	NA	3.26±2.25	2.90±2.72	3.35±2.71
ALT, mean IU/l ± SD	19.80±13.17	131.33±181.22	101.78±177.24	53.64±66.04
AFP, mean ng/ml ± SD	4.48±1.29	16.15±43.35	104.08±186.30	492.61±544.00
AFP status, n
AFP negative	NA	NA	64	65
AFP positive			105	122
hs-CRP, mean mg/l ± SD	0.59±0.53	1.53±2.70	2.83±3.05	17.34±22.61
Child class (A:B:C), %	NA	NA	47:53:69	52:75:60
MELD score, mean ± SD	NA	NA	9.0±6	11.5±4
TNM stage, n
I–II	NA	NA	NA	109
III–IV				78
Metastasis, n
Yes	NA	NA	NA	123
No				64

The diagnostic cutoff values of AFP was 20 ng/l. HCC, hepatocellular carcinoma; CHB, chronic hepatitis B virus; LC, liver cirrhosis; HC, healthy control; AFP, α-fetoprotein; MELD, model for end-stage liver disease; ALT, alanine aminotransferase; hs-CRP, high-sensitivity C-reactive protein; AFP, α-fetoprotein; HBV DNA, hepatitis B virus deoxyribose nucleic acid; HbeAg, hepatitis B eantigen; NA, not applicable; SD, standard deviation; TNM, tumor-node-metastasis.

Table II.

CRP expression in non-tumor and HCC tissue samples.

		CRP staining intensity, n (%)

Tissue type	n	−	+	++	+++	Total positive rate, %	CRP staining score
Non-tumor	126	27 (21.43)	42 (33.33)	32 (25.40)	25 (19.84)	78.57	21.40±38.72
HCC	134	10 (7.46)	6 (4.48)	56 (41.79)	62 (46.27)	92.54	64.04±91.52

−, negative; +, weakly positive; ++, moderate positive; +++, strongly positive; CRP, C-reactive protein; HCC, hepatocellular carcinoma.

Table III.

Correlation between serum hs-CRP concentration and AFP status, TNM stage and tumor Edmondson grading in hepatocellular carcinoma patients.

Parameter	n	hs-CRP concentration, mg/l	r	P-value
AFP status			0.336	0.737
AFP negative	65	15.51±22.016
AFP positive	122	17.24±22.044
TNM stage			0.238	0.229
I	40	18.24±19.39
II	69	19.37±21.06
III	45	20.40±26.37
IV	33	32.71±32.41
Edmondson grading			0.159	0.498
Early stages (I–II)	109	19.02±19.81
Advanced stages (III–IV)	78	24.02±27.82

The diagnostic cutoff values of AFP was 20 ng/l. Data are presented as the mean ± standard deviation. hs-CRP, high-sensitivity C-reactive protein; AFP, α-fetoprotein; HCC, hepatocellular carcinoma; TNM, tumor-node-metastasis.

Table IV.

Assessment of serum hs-CRP, AFP and CRP+AFP in the diagnosis of hepatocellular carcinoma.

Parameter	AUC	Sensitivity, %	Specificity, %	PPV, %	NPV, %
AFP	0.824	74.42	55.63	51.60	89.00
CRP	0.903	84.16	61.59	59.44	85.32
CRP+AFP	0.998	94.06	37.75	81.36	90.00

The diagnostic cutoff values of serum CRP and AFP were 2.17 mg/l and 20 ng/l, respectively. hs-CRP, high-sensitivity C-reactive protein; AFP, α-fetoprotein; AUC, area under the curve; PPV, positive predictive value; NPV, negative predictive value.

Table V.

Comparison between the positive rate of serum CRP and AFP expression.

Group	n	CRP positive, n (%)	AFP positive, n (%)
HC	47	0	0
CHB	137	16 (11.68)	17 (12.41)
LC	169	71 (42.01)	105 (62.13)
HCC	187	161 (86.10)	130 (69.52)

The diagnostic cutoff values of serum CRP and AFP were 2.17 mg/l and 20 ng/ml, respectively. CRP, C-reactive protein; AFP, α-fetoprotein; HCC, hepatocellular carcinoma; CHB, chronic hepatitis B virus; LC, liver cirrhosis; HC, healthy control.

Table VI.

Serum hs-CRP positive rate in AFP-negative or AFP-positive patients with liver cirrhosis and HCC.

Patient group	Total, n	CRP positive, n	CRP positive rate, %	χ²	P-value
LC	169			6.422	0.011
AFP negative	64	19	29.69
AFP positive	105	52	49.52
HCC	187			0.183	0.669
AFP negative	65	55	84.62
AFP positive	122	106	86.89

The diagnostic cutoff values of serum CRP and AFP were 2.17 mg/l and 20 ng/l, respectively. CRP, C-reactive protein; AFP, α-fetoprotein; HCC, hepatocellular carcinoma; LC, liver cirrhosis.