Assessment of Plasma Vitronectin as Diagnostic and Prognostic Marker of Hepatocellular Carcinoma in Patients with Hepatitis C Virus Cirrhosis

Abstract

Background: hepatitis C is an inflammatory liver disease caused by the hepatitis C infection (HCV), and without treatment, almost 50% will progress to liver cirrhosis. Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and the fourth leading cause of cancer-related mortality. Aim of the study: the objective of this study was to evaluate the serum level of vitronectin (VTN) compared to AFP and determine their role as diagnostic and prognostic markers of HCV-related liver diseases. Subject and Methods: this study involved 52 HCV patients from which 26 patients were cirrhotic, and 26 patients had HCC (on top of hepatitis C virus-related cirrhosis) plus 10 healthy people as a control group. It was carried out in Gastroenterology and Hepatology Unit, Internal Medicine Department, Zagazig University Hospitals, Egypt. All individuals in this study were subjected to physical examination, full history taking, liver function tests, assessment of serum levels of Vitronectin (VTN) and alpha-fetoprotein (AFP) before and after the intervention within three months. Results: serum level of vitronectin increased significantly in cirrhosis patients and HCC patients than controls (p = 0.0041), (p < 0.001), respectively, and in HCC than cirrhosis patients (p < 0.001). Significant positive correlations were observed between levels of serum VTN and AFP in all HCV patients as well as cirrhotic patients (p < 0.001, p = 0.011, respectively). On the contrary, VTN and AFP didn’t show a significant correlation in HCC patients’ group. Moreover, the median serum level of VTN decreased significantly after treatment in patients with HCC (p < 0.001). At cut-off 38.5 ng/mL for AFP it shows sensitivity 80.8%, specificity 76.9% to differentiate HCC from cirrhosis cases. While VTN shows 84.6% sensitivity, 96.2% specificity at cut-off 26.5 μg/mL. Regarding clinicopathological characteristics and VTN levels, half of patients were stage B, 63.9% had tumor size >3 cm, 84.6% had more than one focal lesion. Conclusions: these results may allow one to speculate a potential role of Vitronectin in diagnosis and prognosis of HCC on top of cirrhosis related to HCV infection in addition to AFP and US and CT.

1. Introduction

Hepatitis C virus (HCV), an RNA virus from the Flaviviridae family, causes hepatitis C, inflammatory liver disease, which can be transmitted through blood and without treatment almost 50% will progress to liver cirrhosis which may develop HCC [1].

Hepatocellular carcinoma (HCC) is the most frequent form of primary liver cancer and the fourth most prevalent cause of cancer-related mortality [2].

Most cases of HCC are combined with cirrhosis caused by chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), so they should be enrolled into surveillance programs using ultrasound and serum α-fetoprotein (AFP) [3].

Direct-acting antivirals (DAA) are highly effective for the treatment of chronic hepatitis C virus (HCV) infection. Rates of sustained virologic response (SVR) in clinical trials exceed 95% and treatment is well tolerated [4].

The goal of antiviral therapy is to reduce transmission and prevent the consequences of chronic HCV infection including cirrhosis, hepatocellular carcinoma (HCC), and death [5].

HCC can be diagnosed with laboratory tests and radiology methods such as ultrasonography, triphasic computerized tomography (triphasic CT-scan), and dynamic magnetic resonance imaging (dynamic MRI) [6].

Alpha feto-protein (AFP) has a limited sensitivity because it can be normal in up to 40 percent of HCC cases, particularly in the initial stages of the tumor, and a low specificity because its levels can be raised in cases other than HCC, such as cirrhosis, chronic hepatitis exacerbation, and even cholangiocarcinoma in some cases. In clinical practice, AFP is beneficial in HCC screening and diagnosis in conjunction with US, HCC staging in the Cancer of the Liver Italian Program (CLIP) staging method, and tumor progression assessment [7].

In chronic liver diseases, investigations for HCC are recommended if AFP was above 20 ng/mL [8].

Vitronectin (VTN) has been identified as a ‘serum spreading factor’ that attaches to glass. It was also known as ‘epibolin’ and ‘S protein,’ and it was discovered to be a complement membrane assault complex inhibitor [9].

Vitronectin concentrations in plasma range from 200 to 400 mg/mL. Plasma levels of VTN have been shown to be lower in individuals with severe liver impairment, suggesting that the liver is the primary source of plasma vitronectin [10].

Although the liver has a high quantity of VTN mRNA in rats, VTN mRNA has also been detected in other organs such as the brain, heart, skeletal muscle, lung, uterus, testis, and thymus. VTN mRNA has only been found in the male genital tract in normal tissues, but it is expressed at high quantities in tumors, proposing that it may has a role in cancer [11].

Patients with chronic hepatitis, compensated cirrhosis, and decompensated cirrhosis had lower plasma VTN levels than healthy people. The amount of plasma VTN reduced as hepatic dysfunction worsened in chronic liver disorders [12].

The data above indicates that the exact role of VTN in liver disease isn’t clear. So, in this study, the aim was to evaluate the level of serum vitronectin (VTN), compared to AFP and determine their role in the diagnosis and prognosis of HCV-related liver disease.

2. Study Design and Participants

This case control research involved 52 patients with HCV, 26 of whom were cirrhotic and 26 others developed HCC (on top of hepatitis C virus related cirrhosis) before and after intervention within three months. Patients were admitted to Gastroenterology and Hepatology unit (ICU, inpatient wards and outpatient clinic), Internal Medicine Department, Zagazig University Hospitals), Egypt, in addition to 10 apparently healthy individuals were selected to act as a control reference group.

All participants underwent complete history taking and thorough clinical examination. All patients were subjected to abdominal ultrasonography using probe 3.5 MHZ of TDI Philips machine. Cirrhotic patients were diagnosed by clinical, biochemical, and abdominal ultrasonographic findings. HCC was diagnosed according to the American Association for the Study of Liver Diseases practice guidelines. Clinical staging of HCC was evaluated cording to the Barcelona Clinic Liver Cancer staging classification [13] and Child-Pugh classification [14]. Clinicopathological features of HCC cases including tumor number and size were obtained at the time of blood collection.

Patients with hepatitis B virus chronic infection, non-viral hepatitis (alcoholic, Wilson’s disease, hemochromatosis, autoimmune hepatitis), HCC with vascular invasion, LN infiltration, distant metastasis and malignancies other than HCC, or severe co-morbidity were excluded from this study.

The sample size was calculated using open Epi to the following mean vitronectin among Hcc patients was 80.2 $\pm$ 80 and among control 33 $\pm$ 30 (Yang XP; et al., 2016). Power of the study 80% and C.I 95%.

Laboratory tests:

Blood samples were drawn from all subjects after an overnight fast. We divided the blood sample into:

Two ml of whole blood was collected into plain tubes BD Vacutainer^® for determination of serum VTN and AFP levels, plain tubes were permitted to clot for 30 min then were centrifuged at 1200× g for 10 min. Sera were separated immediately stored at −20 °C until analysis. For routine investigation we used BD Vacutainer^® plastic Citrate Tubes and plain tubes then centrifuged promptly at 1500× g for 15 min, Plasma was tested on the Sysmex^® CA-1500 System (Siemens, Kobe, Japan) for an international normalized ratio of prothrombin time (INR) estimation. Two ml whole blood in the EDTA tube was utilized for complete blood counts using an XS500i Hematology analyzer (Sysmex, Kobe, Japan). 2 mL of whole blood was collected into plain tubes BD Vacutainer^® for determination of for bilirubin, ALT, AST and creatinine using full automated Cobas 8000 c702 (Roche diagnostic, Mannheim, Germany).

Measurement of serum Vitronectin and AFP levels

Enzyme-linked immunosorbent assay (ELISA) technique was used for determination of serum VTN and AFP levels by (Takara Bio Inc., Shiga, Japan) (Enzymun-Test, Boehringer, Mannheim, Germany) according to the manufacturer’s instructions. The VTN and AFP levels were calculated by a standard curve. The results were interpreted as μg/mL for VTN and ng/mL for AFP. The intra assay precision of VTN at concentration 148.4, 33.5, 12.4 the CV is 4.7%, 4.3%, 0.43%, respectively. Inter assay precision of VTN at concentration 155.4, 35.3, 12.7 the CV is 5.9%, 4.6%, 7.5%, respectively. Regarding AFP intra assay precision at concentration of 66.9, 31.5, 12.8 the CV is 9.7%, 04.4%, 5.5%, respectively. In addition, inter assay precision of AFP at concentration 64.2, 29.8, 11.6 the CV is 3%, 6%, 8.8%, respectively. The results were interpreted as μg/mL for VTN and ng/mL for AFP.

Statistical Analysis of Data

SPSS, statistical package for social science, program (SPSS Inc., version 20, Chicago, IL, USA), and the GraphPad Prism 3.0 software (GraphPad Software, Inc., San Diego, CA, USA) were used to conduct statistical analysis of the data. Chi square analyzed comparison between groups. Quantitative data were presented by median (minimum and maximum). Comparisons between the mean value of two groups were analyzed by using Mann-Whitney (for non-parametric). Comparisons between multiple groups were carried out by one-way analysis of variance (ANOVA) test for parametric comparisons and by Kruskal Wallis test for non-parametric analysis. Spearman’s correlation was used for non-parametric correlations. At a confidence interval of 95 percent, a probability value of p < 0.05 was regarded statistically significant.

All numerical variables, including vitronectin, were tested for normality to determine if they were normally distributed or not before performing any statistical analysis of the data. Tests of normality we used were Kolmogorov-Smirnov test, Shapiro–Wilk test, in addition to normal Q–Q plot, and histograms. For vitronectin, p value of Kolmogorov-Smirnov and Shapiro–Wilk tests was less than 0.05 indicating that it wasn’t normally distributed.

Receiver Operator Characteristic (ROC) was used to analyze the serum levels of VTN and AFP to differentiation between patients of HCC and cirrhosis

3. Results

During the study period, we observed 62 individuals, 10 normal persons as control group and 52 HCV patients; Among them, 26 cirrhotic patients with HCV and 26 HCC patients on top of HCV (Figure 1).

3.1. Clinical and Biochemical Characteristics of the Studied Groups

According to HCV patients in the study; 26 cirrhotic patients with HCV all of them received direct acting antiviral drugs according to the followed protocols, HCC patients who had HCV infection, 20 patients received direct acting antiviral drugs, 4 patients did not take medications and two patients took medication but did not continue treatment.

Concerning the relationship between the serum levels of VTN and clinicopathological characteristics in HCC patients, our results showed that about 50% of HCC patients were in stage B, and 63.9% of HCC patients had tumor size >3 cm, and 84.6% of HCC patients had more than single focal lesion (

Table 1. The relationship between plasma level of VTN before and after treatment and clinicopathological characteristics and child-pugh classification in HCC patients.

Parameters	HCC, (n = 26) n (%)	Vtn before Treatment (µg/mL)	Vtn after Treatment (µg/mL)
Stage
Stage 0	2 (7.7%)	25 ± 4.246	11 ± 2.828
Stage A	11(42.3%)	43 ± 7.603	15 ± 3.526
Stage B	13 (50%)	52 ± 5.236	20 ± 3.957
p value		0.108	0.201
Tumor size
<3 cm	12 (46.1%)	40.46 ± 3.563	16.356 ± 2.523
˃3 cm	14(63.9%)	50.93 ± 3.112	18 ± 1.362
p value		0.837	0.536
Number of tumor lesions
Single	4 (15.4%)	25.3 ± 5.623	14 ± 3.365
Multiple	22(84.6%)	52 ± 1.3	17 ± 2.748
p value		0.277	0.997
Child-Pugh classification
Child A	17(65.4%)	35.658	14.73
Child B	9(34.6%)	36.988	15.38
p value		0.36	0.309

).

Serum levels of VTN and AFP in cirrhosis, HCC patients and controls were analyzed as illustrated in

Table 2. Serum levels of Vitronectin and AFP in cirrhosis, HCC patients, and controls.

Parameters	Control	Cirrhosis	HCC	Test of Sig. between All Groups
VTN before treatment	6.5 (4–9)	18.15 (11–27)	34 (26–56)	KW = 50.8 p < 0.001
Test of sig. between each two groups	P1 < 0.001	P2 < 0.001	P3 < 0.001
AFP before treatment	3.5 (1.2–27.2)	23.5 (1.1–63)	89.5 (5–970)	KW = 34.638 p < 0.001
Test of sig. between each two groups	P1 = 0.004	P2 < 0.001	P3 < 0.001

KW = Kruskal Wallis test was used for comparison between the three groups, Mann-Whitney test was used for comparison between each two groups, P1 = test of significance between control and cirrhosis groups, P2 = test of significance between cirrhosis and HCC groups, P3 = test of significance between control and HCC groups.

, Figure 2 and Figure 3. The median serum level of VTN and AFP differed significantly among the three groups (p < 0.001). The median serum level of VTN and AFP increased significantly in cirrhosis patient than controls (p < 0.001, p = 0.004, respectively). The median serum level of VTN and AFP increased significantly in HCC patient than controls (p < 0.001, p < 0.001, respectively). The median serum level of VTN and AFP increased significantly in HCC than cirrhosis patient (p < 0.001, p < 0.001, respectively).

3.2. Serum Levels of Vitronectin (VTN) in HCC Patients before and after Treatment

Serum levels of VTN in HCC patients’ group before and after treatment were analyzed as illustrated in

Table 3. Serum levels of Vitronectin in HCC patients’ group before and after treatment.

Parameter	Before Treatment	After Treatment	Test of Sig.
Vitronectin	34 (26–56)	15.25 (8–21)	Z = −4.460 p < 0.001

and Figure 4.

The median serum level of VTN decreased significantly after treatment in HCC patients (p < 0.001).

3.3. Comparison of Vitronectin (VTN) and AFP Levels in HCV-Infected Patients According to Child-Pugh Classification

No significant statistical difference of Vitronectin (VTN) and AFP was observed in HCV-infected patients with different Child-Pugh classifications (

Table 4. Serum levels of Vitronectin and AFP in patients according to Child-Pugh classification.

Child-Pugh Classification	Child A n = 32	Child B n = 16	Child C n = 4	Test of Sig.
VTN before treatment	26 (11–53)	26 (11.6–56)	18.5 (17–19)	KW = 2.827 p = 0.243
AFP	37.3 (1.1–636)	57 (3.5–970)	31.5 (25–43)	KW = 1.877 p = 0.391

KW = Kruskal Wallis test.

).

3.4. Correlations between Vitronectin (VTN) and AFP Levels in HCV-Infected Patients

Significant positive correlations were observed between serum levels of VTN and AFP in all HCV patients as well as cirrhotic patients (p < 0.001, p = 0.011, respectively), On the other hand, VTN and AFP didn’t show a significant correlation in the HCC patients’ group (

Table 5. Correlations between Vitronectin (VTN) and AFP levels in HCV-infected patients.

	All HCV Infected Patient n = 52		Cirrhosis Patients n = 26		HCC Patients n = 26
	r	p	r	p	r	p
VTN versus AFP	0.689	<0.001	0.492	0.011	0.261	0.198

, Figure 5 and Figure 6).

3.5. Area under ROC Curves (AUC) of Serum Levels of VTN and AFP before Treatment

AUC of serum levels of VTN and AFP were conducted for determination of cutoff values and differentiation between patients of HCC and cirrhosis (

Table 6. AUC, and performance characteristics of serum levels of VTN and AFP for discrimination between HCC and cirrhosis cases.

Marker	AUC (95% CI)	p-Value	Cutoff	Sensitivity (%)	Specificity (%)	PPV (%)	NPV (%)	Accuracy (%)
AFP	0.890 (0.799–0.981)	<0.0001 *	≥38.5	80.8	76.9	77.8	80	78.9
Vitronectin	0.990 (0.973–1.008)	<0.0001 *	≥26.5	84.6	96.2	95.7	86.2	90.4

* sig p < 0.05.

and Figure 7). Serum VTN and AFP showed significant AUCs (p < 0.0001, p < 0.0001, respectively) that could differentiate between HCC and cirrhosis patients. The cut–off level of serum VTN 26.5 μg/mL (AUC = 0.990, 95% CI = 0.973–1.008, p < 0.0001, sensitivity = 84.6%, specificity = 96.2%), AFP 38.5 ng/mL (AUC = 0.890, 95% CI = 0.799–0.981, p < 0.0001, sensitivity = 80.8%, specificity = 76.9%) was decided by using ROC Curve analysis.

4. Discussion

Vitronectin is a secreted acute-phase glycoprotein present in serum and extracellular matrix that stimulates cell adhesion and spreading through interactions with integrins. It is primarily generated by hepatocytes [15].

It is increased in hepatocellular (HCC) patients especially in those with adverse prognostic factors [16].

In this research, the median value of AFP level in HCC patients’ group was 89.5 IU/mL which was higher than its median value in the other study groups with high level of significance between the study groups (p < 0.001). This agreed with Yang et al. [17] who revealed that serum AFP level in patients with HCC was significantly higher than those of liver cirrhosis and chronic hepatitis patients and apparently healthy controls.

Patel et al. [18] found that AFP elevated in some patients with HCC and may directly correlate with tumor size. Another study reported that the median value of AFP levels in patients with HCC was 69.1 IU/mL that was statistically higher as compared to the patients with the cirrhotic and fibrotic group [19]. AFP increased in HCC patients compared to normal patients [20]. This also came in accordance with another study where the AFP serum level showed a highly significant elevation in HCC patients [21]. This was in line with Hussein et al. [22] who showed a substantial increase in serum AFP levels in HCC patients. Durazo et al. [23]. observed that the serum level of AFP was considerably greater in HCC patients than in non-HCC patients (p < 0.0001)), which was similarly found by Yasmin-Anum et al. [24] and El-Tayeh et al. [25]. Battaglia et al. [26]. also found that the mean plasma concentration of AFP was significantly higher in untreated HCC patients as compared with chronic liver disease.

The median level of vitronectin in the different study groups showed a statistically significant difference between the different study groups. Serum level of vitronectin increased significantly in cirrhosis patients than controls (p = 0.0041), in HCC patients than control (p < 0.001), and in HCC than cirrhosis patients (p < 0.001). In contrast to a study performed by Peng et al. [27]. to analyze the changes in serum protein levels including VTN in the progression of hepatitis B which revealed that VTN levels decreased in the progression of hepatitis B from chronic hepatitis B to HBV-induced acute-on-chronic liver failure. Tomihira et al. [28]. demonstrated chronic liver disease patients had reduced plasma VTN levels, which might be attributable to decreased generation, deposition in damaged tissues, or a combination of the two. Reduced plasma VTN levels in patients with chronic liver diseases were linked to hepatic dysfunction, and changes in the levels of glycoproteins involved in cell attachment were linked to the development of hepatic fibrosis in patients with chronic liver diseases, according to the same group of authors. This was in agreement to another study where serum VTN levels were significantly higher in HCC patients than in the other groups [17]. Vitronectin is also a glycol-biomarker candidate for HCC. At three N-glycosites in VTN, 14 distinct N-linked glycans corresponding to 27 unique N-linked glycol peptides were identified. The abundance ratio of two typical glycoforms (fucosyl vs. non-fucosyl) in HCC plasma was dramatically elevated [29]. Vitronectin was up-regulated in liver disease and/or in hepatic fibrosis through its collagen-binding domain [30]. Current study also agreed with the results of Ferrin et al. [16], who revealed that serum level of VTN was significantly higher in HCC patients with or without chronic HCV infection. According to Kim KH et al. [31], the serum level of VTN was very high in cirrhotic patients and advanced HCC patients. The same results were also reported by Hwang et al. [32], who found that serum VTN level was higher in HCC patients compared to healthy controls. These results agreed with Yang et al. [17], who reported that VTN levels had significant diagnostic values for differentiating HCC from control, chronic hepatitis, and liver cirrhosis with an accuracy of 88.7%, 84.6%, and 80%, respectively. In addition, this study reported a significantly low serum level of VTN in HCC patients after treatment (p < 0.001), so it can be used as a prognostic marker and follow-up treatment response.

According to Amin et al. [33] found that cirrhotic patients have significantly lower levels of serum Cholinesterase than the control group and the lower levels were correlated with Child Score of such patients. Therefore, we can depend on new serological markers related to the severity of liver disease.

Limitation of the Study

This study had some limitations: It was designed to enroll only 62 subjects, included cirrhotic patients and patients with HCC on top of HCV infection. The analysis was performed in a limited geographical setting (Zagazig) and short time follow up, so, future research will include larger number of patients in multiple centers with longer time for follow up.

5. Conclusions

In conclusion, it can be depended on Vitronectin in diagnosis and prognosis of HCC on top of cirrhosis related to HCV positive infection in addition to AFP and US and CT. It revealed a statistically significant difference between the different study groups.