Significance of Serum Hepatocyte Growth Factor Level in Diagnosis of Hepatocellular Carcinoma

Background and study aim :


INTRODUCTION
Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver [1].Over a decade (1993)(1994)(1995)(1996)(1997)(1998)(1999)(2000)(2001)(2002), there was nearly a two fold increase of the proportion of HCC among chronic liver disease (CLD) patients in Egypt with a significant decline of hepatitis B virus (HBV) and slight increase of hepatitis C virus (HCV) as risk factors [2].The incidence and mortality rates for HCC are nearly identical, indicating the overall poor survival of patients with this tumor.Therefore, the most effective treatment relies on the early diagnosis of HCC [3].Alpha-fetoprotein (AFP) is a serological marker currently available for the detection of hepatocellular carcinoma.Its poor sensitivity renders it unsatisfactory for this purpose and suggests an urgent need for novel biomarkers for early stage HCC detection [4].HGF is a pleiotropic growth factor originally isolated from rat platelets.HGF-like factor known as macrophage-stimulating protein (MSP) is an 82 kDa, 674 amino acid residue hetero dimeric glucoprotein [5].It has a wide range of effects from embryonic development and liver regeneration to protection and/or repair of various organs including kidneys, lungs, and cardiovascular system [6].HGF (scatter factor) is the most potent growth factor for hepatocytes and its receptor, the Nmethyl-N′-nitroso-guanidine human osteosarcoma transforming gene transmembrane tyrosine kinase (c-Met) is implicated in HCC carcinogenesis and progression through activation of multiple signaling pathways that direct cell growth, proliferation, survival and motility [7].Aberrant Met/HGF activation has been observed in many tumor types [8].Met/HGF inhibition has emerged as targeted anticancer therapies [9].Ongoing clinical development with tivantinib, cabozantinib, onartuzumab, crizotinib, rilotumumab and ficlatuzumab has shown encouraging results [8].The aim of this work was to investigate the diagnostic significance of hepatocyte growth factor in HCV related HCC cirrhotic patients and to assess its sensitivity and specificity as compared to AFP.

Principle:
An anti-human HGF coating antibody is adsorbed onto micro wells.Human HGF present in the sample or standard binds to antibodies adsorbed to the micro wells; a biotin-conjugated antihuman HGF antibody binds to human HGF captured by the first antibody.Streptavidin-HRP binds to the biotin conjugated anti-human HGF.
Assay protocol : -The following steps were done as recommended by (Sunred Bio Biomedical Company): All reagents were brought to room temperature before use and each sample, standard, blank and optional control sample was assayed .First each test sample was diluted before use (50 μL of diluted sample was added into pre-designated wells).Then calibrator solutions were prepared as recommended by manufacture.The Micro titer Plate was incubated at 22°Croom temperature for thirty (30 ± 2) minutes (Plate was kept covered during incubation).The contents of the wells were aspirated and each well was filled completely with appropriately diluted wash solution and aspirate.This step was repeated three times, Then 50 μL of appropriately diluted Enzyme-Antibody was conjugated to each well and incubated at 22°C room temperature for thirty (30± 2) minutes( Plate was kept covered in the dark during incubation).The wells were washed then 50 μL of tetra methylbenzidine (TMB) Substrate Solution was added to each well.(Theplate was incubated in the dark at RT for precisely 10 minutes).After ten minutes, 50 μL of Stop Solution was added to each well.and the absorbance was determined at 450 nm of the contents of each well.

RESULTS
Demographic criteria of studied groups were shown in table (1).Mean age of patients with HCC was highly significantly elevated than other groups (P<0.001).The number of males within HCC, cirrhosis and control groups were higher than females.Three patients (10%) in cirrhotic group were Child class A compared to one patient (3.3%) in HCC group.Seventeen patients (56.7%) in cirrhosis group and 10 patients (33.3%) of the HCC group were Child class B, while 10 patients (33.3%) in the cirrhosis group were Child class C compared to 19 patients (63.3%) in the HCC group with no statistically significant difference between both groups (P value = 0.25).Table (2) showed highly statistically significant difference between studied groups as regard all laboratory parameters with higher values of liver profile tests and low serum albumin level in HCC group when compared to other groups.The mean value of serum AFP and HGF was highly significantly elevated in HCC and cirrhotic groups than control groups, and the mean level of HGF was higher in HCC group (795.8 ± 312.04 pg/ml) than cirrhotic group (322.7 ± 45.2 pg/ml) with highly significant difference (P<0.001)(Table 3).Correlation studies revealed significant correlation between serum level of HGF and ALT (P value = 0.005) (Table 4 and Fig. 1) and MELD score (P value = 0.02) (Table 5 and Fig. 2) in HCC group.Table (6) showed no statistically significant correlation between HGF level and tumor number, size, site and shape.Table (7)  As regard CT pattern of HCC in triphasic CT scan confirmed HCC diagnosis as all lesions showed typical enhancement in arterial phase followed by venous washout in the delayed portal/venous phase.Currently, early diagnosis of HCC is the most important step in HCC management.Most imaging techniques help to discover HCC after considerable time of onset of tumor.In most instances, oncologists depend on AFP as the commonest and feasible marker for assessing HCC in addition to imaging.However, AFP is not completely reliable marker in early HCC diagnosis, prevention or therapy due to its low specifiity and sensitivity.Liver biopsy is always considered as an invasive procedure, so chemical findings are still greatly appreciated [20].Despite accumulating data regarding the risk factors for HCC, the mechanisms that contribute to HCC tumorigenesis remain poorly understood.There is vast evidence for protumorigenic growth factor signaling dysregulation in human HCCs affecting different signaling systems such as insulin growth factor, HGF, wingless, TGF-α, epidermal growth factor, and TGF-β signaling.There are many studies about HGF with tissue, cell line, and serum in HCC patients [21].
The mitogenic, motogenic, morphogenic, and pro neoangiogenic HGF is produced by nonparenchymal cells such as hepatic stellate cells [22], sinusoidal endothelial cells, and kupffer cells [23,24].Increased HGF serum levels have been reported in patients with squamous cell carcinoma of the esophagus [25].There is a correlation between HGF serum values and a worsening of hepatic chronic disease [26].In the presence of liver cirrhosis, the functions of all these cells deteriorate, as do those of parenchymal cells.The mechanism of the high serum HGF level in liver disease could be a disorder of either production or elimination of HGF itself, but the role of HGF in liver disease is complicated and is not yet completely understood [8].
In the present study, we evaluated the diagnostic value of HGF in patients with HCV related HCC.In the present study, it was found that the mean age of patients with HCC was higher than other groups with highly significant difference between them (P<0.001).Also, Liver profile tests and AFP were significantly higher and s.albumin was significantly lower in HCC group than the other groups.In agreement with our findings, the study done by Breuhan et al. [27] which concluded that the mean age of patients with HCC was significantly higher than that of the other groups (P<0.001) and they had the highest values for various concurrently measured liver profile tests.The present study revealed that the mean of HGF level was higher in HCC group than the other groups with highly statistically significant difference between studied groups (P<0.001) that in agreement with Funakoshi et al. [28] who reported that levels of HGF in serum tend to be higher in patients with liver cirrhosis and HCC than with chronic hepatitis.Also the results of the present work supported by study of Yamagamim et al. [29] in which plasma samples were taken from 99 patients with chronic viral hepatitis C, cirrhosis, and HCC.The mean HGF level in HCC group (0.533 ± 0.167 ng/ml) was higher than in either chronic hepatitis group (0.383 ± 0.076 ng/ml) with P value = 0.008 or liver cirrhosis group (0.377 ± 0.054 ng/ml) with P value = 0.0016 and concluded that the serum levels of HGF represents the degree of the carcinogenic state (≥0.3 ng/ml, a high risk of developing cancer) in the liver of patients with viral chronic hepatitis C and cirrhosis.
The increase of HGF serum levels (≥0.6 ng/ml for the diagnosis) in cirrhotic patients is an indicator of HCC development.Furthermore, serum HGF levels reveal high carcinogenic states in liver cirrhosis type C [30].Induction is due to the increased production of HGF, not only in the liver, but also in distant organs, such as the lung.With the progression of liver damage, clearance of HGF in the liver diminishes.In addition, although patients with liver cirrhosis show a marked increase in serum HGF levels as the molecule is processed from a biologically inactive single-chain precursor from of HGF into the two-chain active form.Levels may be significantly disturbed in the damaged liver, and a single-chain precursor form can become a major form in the serum [28].
In the present work, correlation study revealed that there was a highly significant positive correlation between HGF level and ALT level (P = 0.005) and significant positive correlation with MELD score (P=0.02) in HCC group but there was insignificant correlation between HGF level and other studied parameters in both cirrhotic and HCC group such as age, CBC, other liver profile tests, AFP and Child score.This results in agreement with that reported by Karabulut et el.
[8] who concluded that serum HGF levels were significant higher in patients with elevated serum ALT levels than others with normal ALT levels (P = 0.05) but in the other hand they found that no correlation between serum levels of HGF and MELD score (P = 0.09), which was in contrast to our results.This difference may be related to difference in patients criteria as the last study included HCV, HBV, alcoholic patients.The present study demonstrated that no correlation between serum HGF level and tumor number, site, size and echogenicity in HCC group.This result were in a concordance with that reported by Karabulut et el. [8] as they found no correlation between HGF level and tumor number but there was positive correlation with tumor size (P = 0.01).That can be explained by small number of studied patients in the present work (30 patients)

Fig. ( 1 )
Fig. (1): Scatter Plot curve is showing correlation between serum level of HGF and ALT in HCC Group

Fig. ( 3 )
Fig. (3): Receiver Operating Characteristics (ROC) curve which show AFP and HGF sensitivity and specificity in diagnosis of HCC

liver cirrhosis group): included
30 patients with post hepatitic liver cirrhosis without HCC, the majority of them were males [17 patients (56.7%)].Group II (HCC group): included 30 cirrhotic patients with HCC, the majority of them were males [23 patients (76.7%)].Group III (control group): included 25 apparently healthy subjects, 13 of them were males [13 patients (52%)].Patients less than 18 years old, patients with liver cirrhosis due to other causes than HCV infection (HBV, Autoimmune hepatitis, Alcoholic,… etc), patients with hepatic focal lesions other than HCC (Hemangiona, Hepatoblastoma,…etc), and patients with metastatic focal lesions or with vascular invasion were excluded from this study.

:
Median, range, mean, and standard deviation were used for descriptive statistics, as appropriate.ROC) analysis was used to evaluate the diagnostic value of HGF and AFP and to identify the optimal threshold values.Sensitivity and specificity, positive and negative predictive values of HGF and AFP were profiled by curves.Calculations were done with the Statistical Package for the Social Sciences version 22 (IBM, SPSS, Statistics, V.22, 2012, IBM crop., New York.USA).

Table ( 1
): Demographic criteria of studied groups

Table ( 3
): Serum levels of AFP and HGF among studied groups

Table ( 5
): Spearman's correlation between serum level of HGF and severity of liver disease assessed by (Child-Pugh score, MELD scores)

Table ( 7
): Diagnostic performance of serum level of AFP and serum level of HGF in HCC group

:Non. Ethical approval: Written
in comparison to the last study (54 patients).Analysis of serum HGF level by ROC curve in the present work revealed satisfactory values regarding sensitivity and specificity at a cut off value (≥ 426.1 pg/ml).Serum HGF had higher sensitivity, specificity, PPV and NPV than AFP for diagnosis of HCC (96.7%, 98.2%, 96.7, 98.2 Vs 83.3%, 92.7%, 86.2%, 91.1% respectively) there for, assessment of serum HGF in HCV cirrhotic patients would be beneficial for diagnosis of HCC.informed consent was taken from all patients before participation in this study.The study protocol was approved by the ethical committee of Benha Faculty of Medicine and its University Hospitals.