|Year : 2019 | Volume
| Issue : 4 | Page : 451-457
Noninvasive prediction of hepatitis C-associated hepatocellular carcinoma using circulating apolipoproteins
Hasan El Garem1, Gamal Esmat1, Rabab Salama1, Hanan Fouad2, Dina Sabry2, Yomna Mostafa3, Dalia Omran1, Heba Omar1
1 Department of Endemic Medicine and Hepatogastroenterology, Faculty of Medicine, Cairo University, Cairo, Egypt
2 Department of Medical Biochemistry, Faculty of Medicine, Cairo University, Cairo, Egypt
3 Department of Hepatogastroenterology, Egypt Air Hospital, Cairo, Egypt
|Date of Submission||13-Feb-2019|
|Date of Acceptance||30-May-2019|
|Date of Web Publication||18-Aug-2020|
MD, GCSRT Heba Omar
Faculty of Medicine, Cairo University, Cairo, 11652
Source of Support: None, Conflict of Interest: None
Background and aims Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. We investigated the potential usefulness of circulating apolipoproteins (Apo-A1 and Apo-A4) in HCC screening and diagnosis.
Patients and methods We included 60 adult patients with hepatitis C virus-related chronic liver disease including HCC, in addition to 20 healthy controls. Patients were stratified into three equal groups, with 20 patients each: chronic hepatitis C, posthepatitis C cirrhosis (liver cirrhosis), and HCC. All patients and controls underwent full clinical assessment, laboratory investigations, and evaluation of candidate apolipoproteins by enzyme-linked immunoassay.
Results Significantly higher Apo-A1 and Apo-A4 levels were detected in patients with HCC than in those with liver cirrhosis (P<0.001). Receiver operator characteristic curve showed that for HCC diagnosis, a cutoff of 78.6 mg/dl for Apo-A1 yielded 90% sensitivity and 100% specificity and a cutoff of 16.5 mg/dl for Apo-A4 yielded 85% sensitivity and 80% specificity. Furthermore, within HCC group, Apo-A1 was significantly higher in patients with small HCC (>2 cm) than those with large tumors (P=0.01). Lower Apo-A1 level correlated significantly with pylethrombosis (P=0.007).
Conclusion Apo-A1 and Apo-A4 are novel biomarkers for HCC screening and diagnosis, with a special discriminative ability for Apo-A1 for those with small tumors and those with pylethrombosis.
Keywords: apolipoprotein-A1, apolipoprotein-A4, diagnosis, pylethrombosis, screening, small hepatocellular carcinoma
|How to cite this article:|
El Garem H, Esmat G, Salama R, Fouad H, Sabry D, Mostafa Y, Omran D, Omar H. Noninvasive prediction of hepatitis C-associated hepatocellular carcinoma using circulating apolipoproteins. Egypt J Intern Med 2019;31:451-7
|How to cite this URL:|
El Garem H, Esmat G, Salama R, Fouad H, Sabry D, Mostafa Y, Omran D, Omar H. Noninvasive prediction of hepatitis C-associated hepatocellular carcinoma using circulating apolipoproteins. Egypt J Intern Med [serial online] 2019 [cited 2020 Oct 1];31:451-7. Available from: http://www.esim.eg.net/text.asp?2019/31/4/451/292220
| Introduction|| |
Chronic hepatitis C (CHC) infection is a global health problem that infects between 130 and 150 million people worldwide. The link between CHC infection and the development of hepatocellular carcinoma (HCC), which is the most common type of primary hepatic tumors (90% of all primary hepatic malignancies), is well known , with an estimated 17-fold increase in the risk of HCC development among hepatitis C virus (HCV)-infected patients . Annually, there are more than 620 000 newly diagnosed HCC cases , of them 22% (>100 000) were attributed to CHC infection .
In Egypt, the incidence rate of HCC has doubled in the past decade. This could be attributed to high prevalence of HCV in Egyptian population, with an estimated prevalence of HCC among patients with liver cirrhosis (LC) of 21% .
Early detection of HCC by the screening of at-risk population is considered one of the best strategies to reduce disease-related mortality and mortality ,,. Although serum biomarkers are minimally invasive and acceptable, the sensitivity and specificity of biomarkers like alpha-fetoprotein (AFP) for early detection of HCC are marginal .
Proteins perform and regulate most biological cell functions. Several recent proteomic analyses had pointed to the potential role of apolipoproteins especially ApoA-1 and Apo-A4 in the diagnosis of HCC .
The current study aimed at determination of the diagnostic accuracy of apolipoproteins (Apo-A1 and Apo-A4) as noninvasive biomarkers for HCC screening and diagnosis and to set a cutoff value of those apolipoproteins at which further investigations for HCC become of high importance.
Patient and methods
Research design and study population
This was a case–control study using prospectively collected data from 60 treatment-naïve HCV patients within the spectrum of HCV-related chronic liver disease, including HCC. All included patients had confirmed positivity of their HCV antibody and HCV RNA by PCR. The presence or absence of LC was judged base on their clinical, laboratory, and ultrasound findings. Patients with HCC were diagnosed by typical enhancement (concurrent wash-in in the arterial phase and wash-out in delayed/venous phase) of hepatic focal lesion more than 1 cm on four-phase multidetector computed tomography or dynamic contrast-enhanced MRI examination of cirrhotic liver according to EASL guidelines .
Patients were categorized into three equal groups of 20 patients each (CHC without LC group, LC group, and posthepatitis C HCC group). In addition, 20 healthy volunteers were included as a control group. Participants were consecutively recruited from the outpatient clinic of Endemic Medicine and Hepatology Department, Faculty of Medicine, Cairo University, during the period from May 2015 to March 2016. Patients with concomitant chronic HBV or HIV infection, patients with any other diagnosed malignancy or metastasis, and those who received any treatment for their diagnosed HCC were excluded.
The study was conducted according to guidelines laid down in Declaration of Helsinki 1975 after its approval by the clinical research ethics committee of the Endemic Medicine and Hepatology Department, Faculty of Medicine, Cairo University. All study participants agreed to give written informed consent before participation in the study.
The primary outcome of interest for the current work was to determine the diagnostic accuracy of serum expression levels of apolipoproteins (Apo-A1 and Apo-A4) as noninvasive diagnostic biomarkers for posthepatitis C HCC and to set the best cutoff value for those biomarkers at which HCC diagnosis is highly suspected. Peripheral blood levels of Apo-A1 and Apo-A4 were evaluated using enzyme-linked immunoassay (ELISA) kits Apo-A1 ELISA kit E0604h and Apo-AIV ELISA kit E1967h, respectively, according to the manufacturer’s instructions (EIAab Reference Database online at www.eiaab.com).
Data regarding the following variables were also obtained from all patients included in our study.
Demographics and clinical data
Age, sex, comorbidities (diabetes), smoking practice, risk factors for acquiring HCV infection (previous parenteral antischistosomal therapy, operations, or blood transfusion) were noted. For patients with LC, their Child–Pugh classification  was calculated.
Laboratory data were collected at the time of enrollment and included complete blood count, hepatic necroinflammatory markers (aspartate and alanine aminotransferases), indicators of liver synthetic functions (total bilirubin, albumin, and international normalized ration), and AFP.
Data were entered, validated, and analyzed using STATA 14 (College Station, Texas, USA) software. Patients’ demographic and routine laboratory values data were expressed as number (%) for categorical variables and as mean±SD or median (interquartile range) for continuous variables. Continuous variables were tested for normality using histograms and sktest. Those variables with a small P value less than 0.05 on sktest were considered not normally distributed. All quantitative parametric and nonparametric variables were analyzed using either Student t test or Mann–Whitney test for comparison of two groups whenever appropriate. A comparison of three groups was done using Kruskal–Wallis for nonparametric or analysis of variance test for parametric variables whenever appropriate . χ2 test was used for comparison of categorical data and Fisher’s exact test for categorical data with lower number of samples . Associations between HCC diagnosis and candidate apolipoproteins were investigated using univariate logistic regression models. Data were presented as odds ratios (OR) with 95% confidence intervals (95% CI). The diagnostic performance of Apo-A1and Apo-A4 in the prediction of HCC was evaluated using receiver operator characteristic (ROC) curves. The area under the ROC curves and the 95% CI were used as indexes of accuracy ROC curve. All statistical analyses were based on two-sided hypothesis tests with a significance level of P value less than 0.05.
| Results|| |
Characteristics of the included participants
[Table 1] shows demographics, clinical, and laboratory characteristics of enrolled participants. A total of 60 patients met the study inclusion criteria: 20 had chronic hepatitis, 20 had cirrhosis of the liver, and 20 had HCC. In addition, 20 normal participants were analyzed. There was significant male predominance across all patient groups; males represented 35, 75, and 85% of CHC, LC, and HCC groups, respectively (P<0.001). The mean±SD age of all patients was in the seventh decade of life (61.11±11.1), with the youngest mean age in the LC group at 58.90±10.72 years and oldest age in HCC at 64.85±7.16 years, with significant difference in age among the studied groups (P<0.001).
|Table 1 Demographic, clinical, and laboratory data of studied participants (N=80)|
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Circulating Apo-A1 and Apo-A4 levels in patients with liver diseases and healthy controls
Circulating Apo-A1 and Apo-A4 were measured in the blood of patients with HCV-related chronic liver disease as well as controls using ELISA. Mean serum expression level of Apo-A1 was significantly higher (P<0.001) in HCC group (212.12±101.24 mg/dl) compared with LC (30.25±12.71 mg/dl) and control (32.65±2.70 mg/dl) groups. Apo-A4 expression was significantly higher in patients with HCC (19.00±4.13 mg/dl) compared with patients with LC (11.22±6.63 mg/dl) and significantly lower in patients with HCC than controls (23.17±1.56 mg/dl) (P<0.001). In relation to LC group, CHC group had significant higher level of Apo-A1 (30.25±12.71 vs. 100.50±31.31 mg/dl, respectively, P≤0.001) and significant lower levels of Apo-A4 (11.22±6.63 vs. 38.39±17.05 mg/dl, respectively, P≤0.001), as shown in [Table 2].
|Table 2 Apolipoprotein-A1 and apolipoprotein-A4 levels in different patient groups|
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Features of patients with hepatocellular carcinoma
Clinical characteristics of the 20 patients with HCC (17 male and three female) are shown in [Table 3]. Overall, 70% (14) of patients with HCC were older than 60 years, a similar percent (14) had single focal lesion, 20% had small focal lesion (<2 cm in largest diameter), 85% had no vascular invasion, 85% had no lymphadenopathy, and 60% had Barcelona clinic liver cancer stage 0-B. Apo-A1 level was significantly higher in patients with small HCC than those with large tumors (319.23±108.58 vs. 185.35±82.47 mg/dl, respectively, P=0.01). On the contrary, Apo-A4 showed nonsignificant higher values in patients with small HCC compared with those with large HCC (21.18±4.41 vs. 18.46±4.01 mg/dl, respectively, P=0.25). Apo-A1 had significantly lower expression level in patients with portal vascular thrombosis (n=3) than patients without pylethrombosis (188.18±86.45 vs. 347.83±71.33 mg/dl, respectively, P=0.007). On the contrary, Apo-A4 had nonsignificant lower levels in patients with pylethrombosis compared with those without pylethrombosis (18.64± 4.35 vs. 21.07±1.68 mg/dl, P=0.36).
|Table 3 Clinical characteristics of patients with hepatocellular carcinoma (N=20)|
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Diagnostic value of Apo-A1 and Apo-A4
We analyzed the properties of Apo-A1 and Apo-A4 for HCC diagnosis using univariate logistic regression model, with ‘0’ representing cirrhosis group and ‘1’ representing HCC group. Logistic regression analysis revealed that Apo-A1 (OR, 1.10; 95% CI, 1.02–1.20; P=0.03) and Apo-A4 (OR, 1.28; 95% CI, 1.09–1.50; P<0.001) were significantly associated with increased risk of HCC. However, AFP was not significantly associated with risk of HCC (OR, 1.02; 95% CI, 0.99–1.06; P=0.22) ([Table 4]).
|Table 4 Univariate logistic regression analysis for candidate biomarkers|
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ROC curve showed that for HCC diagnosis, a cutoff of 78.6 mg/dl for Apo-A1 yielded 90% sensitivity and 100% specificity with 100% positive predictive value and 86.96% negative predictive value (C statistics=0.99) and at a cutoff of 16.5 mg/dl for Apo-A4 yielded 85% sensitivity and 80% specificity with 80% positive predictive value and 80% negative predictive value (C statistics=0.84), as depicted in [Figure 1].
|Figure 1 ROC curve for the diagnostic performance of apolipoproteins (Apo-A1 and Apo-A4) in HCC. HCC, hepatocellular carcinoma; ROC, receiver operator characteristic.|
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| Discussion|| |
In the current case–control study, we investigated the diagnostic utility of circulating Apo-A1 and Apo-A4 for prediction of HCV-related HCC. Apo-A1 and Apo-A4 showed superiority to AFP in the diagnosis of HCV-related HCC (C statistics, 0.99 and 0.84, respectively). Furthermore, Apo-A1 could significantly identify patients with small HCC (<2 cm in its largest diameter) as well as those with pylethrombosis.
HCC is a life-threating tumor that accounts for more than 90% of primary liver malignancies. It ranks the fifth most common cancer in males and the seventh in females worldwide. Owing to its very poor prognosis, it has been regarded as the third most common leading cause of death owing to malignancies worldwide . The prognosis of HCC remains unsatisfying with an overall 5-year survival rate of less than 10% owing to a lack of early detecting methods . Progression of HCV-related liver disease from chronic infection to HCC through LC is associated with alterations in circulating proteins as well as proteins present in hepatic tissues .
Apo-A1 is a major constituent of high-density lipoproteins encoded on chromosome 11q23-q24 , and has a well-known anti-atherogenic function that protects against cardiovascular diseases ,,,,. The role of Apo-A1 in HCC is still unclear, but few recent reports have discussed the potential diagnostic role of Apo-A1 for early detection of HCV-related HCC ,.
Most apolipoproteins, lipids, and lipoproteins are formed in hepatocytes. Thus, hepatic injury or chronic liver diseases including HCC may result in abnormal circulating patterns of those molecules secondary to alterations of many cytokines and/ or metabolic cellular substances, or tumor factors , but the exact mechanisms are not fully understood . In the present work, high levels of circulating Apo-A1 and Apo-A4 were detected in patients with HCC compared with patients with LC (212.12±101.24 vs. 30.25±12.71 mg/dl, respectively, and 19.00±4.13 vs. 11.22±6.63 mg/dl, respectively). Both were associated with increased risk of HCC (OR, 1.10 and 1.28, respectively). Pleguezuelo et al.  reported an increase in risk for HCC with increased Apo-A1 and decreased Apo-A4 levels. Nonmatching Apo-A4 results between Pleguezuelo study and ours could be explained to the different HCC etiology in both studies. Liver is the main site for the formation, storage, transportation, and breakdown of some Apo . Each Apo might be affected by liver disease in a different way.
Furthermore, within HCC group, we found that Apo-A1 level was significantly higher in patients with small HCC than those with large tumors (319.23±108.58 vs. 185.35±82.47 mg/dl, respectively, P=0.01). The prognosis of HCC could be markedly improved if it is detected at an early potentially curable stage. However, precise identification of early-stage HCC is still clinically difficult, even with the help of advanced radiology technology. These results may suggest that Apo-A1 might be a useful marker for diagnosis of small HCC .Interestingly, we found that within HCC group, lower Apo-A1 level correlated significantly with the occurrence of pylethrombosis (188.18±86.45 vs. 347.83±71.33 mg/dl, respectively, P=0.007). High-density lipoprotein had multiple direct and indirect antithrombotic effects, directly through enhancement of endothelial nitric oxide synthase activity and decrease leukocytes endothelial adhesiveness  and indirectly through induction of anticoagulant action of the protein C pathway with subsequent to downregulation of thrombin production . Matching our results, Xu et al.  reported lower levels of Apo A-I expressed in most HCC tumors with pylethrombosis (n=20) compared with HCC tumors without pylethrombosis (n=20). Eichinger et al.  found that high levels of Apo A-I meant a decreased risk of recurrent venous thromboembolism. Dissimilar profiles of up- and down-regulated apolipoprotein have been identified; such discrepant results could be attributed to different disease etiology and pathological differentiation of the analyzed HCC. However, it could be also owing to molecular heterogeneity of HCC .
The major strength of the current study is being among very few Egyptian reports on the performance of apolipoproteins in the diagnosis of HCV-related HCC patients. A limitation of the present study was the relatively small sample.
| Conclusion|| |
Apo-A1 and Apo-A4 are novel biomarkers for HCC screening and diagnosis with special importance for Apo-A1, which could be a valuable biomarker for identification of patients with HCC with small tumors as well as those with pylethrombosis.
Author contributions: Hassan El Garem and Gamal Esmat set the research design and drafted the protocol with support from Rabab Salama and Dalia Omran. Yomna Mostafa and Heba Omar did the data collection and literature review. Hanan Foud and Dina Sabry did the laboratory work. Heba Omar did the statistical work. All authors were involved in the development of the search strategy, the eligibility criteria, and manuscript drafting and revision.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]