Diagnostic specificity and sensitivity of PIVKAII, GP3

DOI: 10.1177/0004563217726808

Diagnostic specificity and sensitivity of PIVKAII, GP3, CSTB, SCCA1 and HGF for diagnosis of hepatocellular carcinoma in patients with alcoholic liver cirrhosis

Journal:

Annals of Clinical Biochemistry

Manuscript ID

ACB-17-045.R2

Manuscript Type:

Research Article

Date Submitted by the Author: Complete List of Authors:

Keywords:

04-Jul-2017 Unic, Adriana; Medical School University Hospital Sestre Milosrdnice, University Department of Chemistry Derek, Lovorka; Medical School University Hospital Sestre Milosrdnice, University Department of Chemistry Duvnjak, Marko; Medical School University Hospital Sestre Milosrdnice, Division of Gastroenterology and Hepatology, Department of Medicine; Sveuciliste u Zagrebu Medicinski fakultet Patrlj, Leonardo; University Hospital Dubrava, Department of Surgery; Sveuciliste u Zagrebu Medicinski fakultet Rakic, Mislav; University Hospital Dubrava, Department of Surgery; Sveuciliste u Zagrebu Medicinski fakultet Kujundzic, Milan; University Hospital Dubrava, Department of Gastroenterology, Hepatology and Clinical Nutrition; Sveuciliste u Zagrebu Medicinski fakultet; Sveuciliste u Zagrebu Farmaceutsko-biokemijski fakultet Renjic, Vesna; University Hospital Dubrava, Department of Gastroenterology, Hepatology and Clinical Nutrition Stokovic, Nikola; Sveuciliste u Zagrebu Medicinski fakultet Dinjar, Petra; University Hospital Merkur, Department of Gastroenterology and Hepatology Jukic, Anita; University Hospital Centre Split, Department of Internal Medicine Grgurevic, Ivica; University Hospital Dubrava, Department of Gastroenterology, Hepatology and Clinical Nutrition; Sveuciliste u Zagrebu Medicinski fakultet; Sveuciliste u Zagrebu Farmaceutsko-biokemijski fakultet Tumour markers < Analytes, Cancer < Clinical studies, Liver disease < Clinical studies


Page 1 of 31 DOI: 10.1177/0004563217726808 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Annals of Clinical Biochemistry

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Diagnostic specificity and sensitivity of PIVKAII, GP3, CSTB, SCCA1 and HGF for diagnosis of hepatocellular carcinoma in patients with alcoholic liver cirrhosis Adriana Unić1, Lovorka Derek1, Marko Duvnjak2,3 , Leonardo Patrlj3,4, Mislav Rakić3,4, Milan Kujundžić3,5,6, Vesna Renjić5, Nikola Štoković3, Petra Dinjar7, Anita Jukic8 and Ivica Grgurević3,5,6 1

University Department of Chemistry, Medical School University Hospital Sestre

Milosrdnice, Zagreb, Croatia 2

Division of Gastroenterology and Hepatology, Department of Medicine, Sestre

milosrdnice University Hospital, Zagreb, Croatia 3

University of Zagreb School of Medicine, Zagreb, Croatia

4

Department of Surgery, University Hospital Dubrava, Zagreb, Croatia

5

Department of Gastroenterology, Hepatology and Clinical Nutrition, University

Hospital Dubrava, Zagreb, Croatia 6

University of Zagreb Faculty of Pharmacy and Biochemistry, Zagreb, Croatia

7

Department of Gastroenterology and Hepatology, University Hospital Merkur,

Zagreb, Croatia 8

Department of Internal Medicine, University Hospital Centre Split, Split, Croatia

Corresponding author: Ivica Grgurevic, MD, PhD, Assist. Prof.


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Department of Gastroenterology, University Hospital Dubrava, Avenija Gojka Suska 6, 10 000 Zagreb, Croatia Tel. +385 1 290 2554 Fax.+385 1 290 2550 E-mail: [email protected]

Abstract word count: 228 Article word count: 3408

DECLARATIONS Conflict of interest: None declared. Funding: None Ethical approval: University Hospital Dubrava Ethical approval issued 23.12.2008. Guarantor: IG Contributorship: AU and IG were involved with study conception, design and drafting of manuscript. All authors were involved in acquisition, analysis and interpretation of data. All authors reviewed and edited the manuscript and approved the final version of the manuscript.


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ABSTRACT Introduction: Despite of some new treatment possibilities the improvement in survival rate for hepatocellular carcinoma (HCC) patients is still poor due to late diagnosis. The aim of this study was to investigate diagnostic sensitivity and specificity of Protein induced by vitamin K absence or antagonist-II (PIVKAII), Glypican-3 (GP3), Cystatin B (CSTB), Squamous cell carcinoma antigen 1 (SCCA1) and Hepatocyte growth factor (HGF) as potential tumour markers for HCC in patients with alcoholic liver cirrhosis (ALC) using imaging techniques (MSCT and MRI) as reference standards. Patients and methods: Eighty three participants were included: 20 healthy volunteers, 31 patients with ALC and 32 patients with HCC. Peripheral blood sampling was performed for each participant and serum concentrations of PIVKAII, GP3, CSTB, SCCA1 and HGF were determined using commercial ELISA kits. Results: Only serum concentrations of PIVKAII were significantly higher in HCC patients as compared to ALC and healthy controls (cut-off 2.06 µg/L; AUC 0.903),

whereas

individual

diagnostic

performance of

other

individual

compounds was inadequate. The “best” combination of tumour markers in our study includes all tested markers with AUC of 0.967. Conclusion: While novel diagnostic tumour markers are urgently needed, the examined potential tumour markers, with the exception of PIVKAII seem to be inadequate for diagnosing HCC in ALC. Furthermore, probably the future is in the finding the best optimal combination of tumour markers for diagnosing HCC based on cost-effectivenes


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INTRODUCTION Hepatocellular carcinoma (HCC) is the third most common cause of cancer related death worldwide, and accounts for 90% of all primary liver malignancies1. Incidence of HCC in developed countries is not high when compared to other malignancies, but has high mortality rate, despite new treatment possibilities, indicating that early diagnosis is crucial in order to improve survival1-4. Between 90-95 % of HCC cases are diagnosed in patients with pre-existing cirrhotic livers1. Among risk factors for both cirrhosis and HCC the most prevalent are hepatitis B (HBV) and hepatitis C (HCV) infections, alcoholism, fatty liver, haemochromatosis and primary biliary cirrhosis1, 2. Since the treatment is the most effective when administered in the early stage of HCC there is an obvious need for accurate diagnostic tool (-s) for patients at risk. Although HCC meets the criteria for a surveillance program, poor diagnostic performance of available tools has prevented its widespread use3,

5-7

. Up to

now, HCC screening has been based on alpha-fetoprotein (AFP) and liver ultrasound (US) both of which have a limited diagnostic performance3,

4, 8

.

According to many international guidelines and reports US is the most appropriate tool for HCC screening6, 7. Recent research on 2293 high risk patients has shown that US could detect almost all HCC lesions with a sensitivity of 94% and specificity of 82% and more efficiently than using serum AFP6. The combined use of AFP and US seems to be useless because of increased false positive results which lead to increased costs6. Therefore, implementation of more sensitive and specific tumour markers for HCC detection is the focus of many researches in order to improve sensitivity and objectifiy diagnosis5-7, 9.

Besides AFP, some of the potential serum tumour

markers for HCC detection include Protein induced by vitamin K absence or


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antagonist-II (PIVKAII)7,

10, 11

, Glypican-3 (GP3)7,

10, 12, 13

, Squamous cell

carcinoma antigen 1 (SCCA1)14-16 while data on the performance of Cystatin B and Hepatocyte growth factor (HGF) are still unclear17,18. PIVKAII is with AFP the most investigated marker for HCC detection. It has been shown that PIVKA II increased concentrations may be independent of AFP increase in HCC patients11. Furthermore, the Japanese Evidence-based Clinical Practice Guidelines and Consensus-based Clinical Practice Manual recommend measurement of PIVKA and AFP (or AFP-L3/AFP) for HCC screening in high-risk patients19. Recently, research on AFP, AFP-L3 and PIVKAII diagnostic and prognostic performance using two statistical models (GALAD and BALAD-2) for estimating the likelihood of the presence of HCC in individual patients with chronic liver disease and the survival of patients with HCC, were published. The authors concluded that these models might be used in HCC surveillance and determination of patient prognosis20. Glypican-3 (GP3) is an oncofetal protein, and is one of the members of heparan sulfate proteoglycans anchored to the plasma membrane. Since GP3 has been exclusively detected in HCC cells and not in benign liver tissues, it has been investigated as another potential tumour marker for HCC detection10,

12, 13

.

Results of meta-analysis conducted in 2014 indicate that serum GP3 is elevated in HCC patients when compared with healthy individuals, but it is still unclear whether it is useful in differentiation of patients with cirrhosis or HCC12. SCCA is a member of serine protease inhibitor family, and recent studies have shown that SCCA consists of two homologous proteins SCCA1 and SCCA216.


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Although SCCA1 is found to be elevated in patients with HCC, its usefulness in diagnosing HCC is still doubtful14-16. Cystatins are endogenous inhibitors of lysosomal cysteine proteinases (cathepsin L, H, and S)17. Overexpression of Cystatin B (CSTB), a member of the cystatin superfamily, has been reported in patients with HCC17. HGF is produced by stromal cells, and stimulates proliferation of epithelial cells, motility, morphogenesis and angiogenesis in various organs21. Increased HGF serum concentrations have been reported in HCC patients as compared to patients with cirrhosis or chronic hepatitis18. The diagnostic performance of the mentioned HCC tumour markers has mostly been investigated in patients with cirrhosis of viral etiology. Considering the possible differences in pathogenic pathways leading to cirrhosis and HCC development22-24, the choice of marker(s) for screening might differ in relation to the etiology of liver disease. Since alcohol has become an important cause of liver cirrhosis in Europe23, an increasing number of HCC patients of this etiological background should be expected. Keeping

in mind these

epidemiological trends and unmet need for an accurate diagnostic test, we aimed to investigate both an individual and combined diagnostic performance of PIVKAII, GP3, SCCA1, CSTB and HGF as potential tumour markers for HCC detection in patients with alcoholic liver cirrhosis (ALC) when compared to imaging techniques as a diagnostic standard for HCC screening in a high risk population.


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SUBJECTS AND METHODS Eighty three participants were included in the study: 20 healthy volunteers (HV), 31 patients with ALC and 32 patients with ALC and HCC. The study was approved by the Institutional Ethics Committee, and all participants signed informed consent. Patients, hospitalized at the Department of Gastroenterology and Hepatobiliary surgery of a tertiary care University hospital, were consecutively recruited in the study (during a three-year period (2009–2011).), when alcohol cirrhosis with HCC was suspected according to the physical exam, anamnesis (where history of alcohol exposure was recorded) and results of laboratory tests (alanine aminotransferase

(ALT),

aspartate

aminotransferase

(AST),

alkaline

phosphatase (ALP), gamma-glutamyl transferase (GGT), total bilirubin, albumin, creatinine, complete blood count (CBC) and prothrombin time (PT/INR)). Cirrhosis was confirmed by US, and all patients underwent multislice computed tomography (MSCT) and magnetic resonance imaging (MRI) in order to confirm or to reject presence of HCC in cirrhotic liver. After diagnosis patients were divided into subgroups. Healthy participants were included in order to confirm the reference range of HCC tumour markers. The absence of cirrhosis, if a liver abnormality was suspected, was defined according to the common clinical ultrasonographic and contrast-enhanced imaging methods such as MSCT and MRI. The following data were obtained from all participants: demographics (age, gender), history of alcohol exposure, laboratory data (including ALT, AST, ALP, GGT, total bilirubin, albumin, creatinine, CBC and PT/INR) and imaging results if needed (ultrasound, CT and MRI) (Table 1).


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For the patients included in the study, additional laboratory tests were performed: hepatitis B surface antigen, anti-hepatitis B core IgG+IgM, hepatitis C antibody test and serum iron. Exclusion

criteria

were:

presence

of

HBV

and/or

HCV,

suspected

haemochromatosis, presence of metastases, prior treatment of HCC and history of other solid tumour (-s). Sample size estimation using comparison of two means was performed in order to assure an adequate power to detect statistical significance in the study. We have estimated that in order to achieve a power of 80% (to detect a significant difference (P