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Hepatitis B in hepatocellular carcinoma patients and its correlation with alpha-fetoprotein and liver enzymes

1 Department of Biochemistry, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India
2 Department of Radiation Oncology, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India
3 Department of Pathology, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India
4 Department of Microbiology, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India

Date of Submission08-Apr-2019
Date of Decision22-Nov-2019
Date of Acceptance19-Dec-2019
Date of Web Publication22-Jul-2020

Correspondence Address:
Dinesh Kumar Sinha,
Department of Radiation Oncology, Regional Cancer Centre, Indira Gandhi Institute of Medical Sciences, Sheikhpura, Patna - 800 014, Bihar
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_239_19

 > Abstract 

Background: Hepatocellular carcinoma (HCC) in India ranges from 0.7 to 7.5 for men and 0.2 to 2.2 for women, per 100,000 population per year. The major risk factors for the development of HCC are infection with hepatitis B virus (HBV), hepatitis C virus, and cirrhosis of liver. Alpha-fetoprotein (AFP) and liver enzymes are widely used by clinicians for diagnostic purpose in HCC.
Aims and Objective: This study was conducted in HCC patients related to HBV infection and to assess the significance of AFP and liver enzymes in it.
Materials and Methods: Blood samples of 68 patients were taken. The samples were analyzed for AFP and liver enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], and alkaline phosphatase [ALP]). Liver enzymes were estimated by auto analyzer OLYMPUS AU400. AFP was analyzed by chemiluminescence immunoassay.
Results: The mean values of AFP in serum hepatitis B surface antigen (HBsAg) negative and positive patients ranges from 22745.4 to 23269.3 ng/ml with P = 0.921. The mean value of ALP in HbsAg-negative patients was 418 U/ml, whereas in positive patients, it was 310 U/ml. Both the groups did not show any significant changes in AFP levels. The ALP showed slight rise in negative group. The other parameters did not show significant rise in all patients.
Conclusion: These values suggest that there was no significant influence of viral etiology on AFP and liver enzymes level in HCC patients.

Keywords: Alpha-fetoprotein, hepatocellular carcinoma, liver enzymes, serum hepatitis B surface antigen

How to cite this URL:
Surit R, Shekhar R, Sinha DK, Singh SK, Kumar U, Prasad N. Hepatitis B in hepatocellular carcinoma patients and its correlation with alpha-fetoprotein and liver enzymes. J Can Res Ther [Epub ahead of print] [cited 2022 Jul 1]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=290467

 > Introduction Top

Hepatocellular carcinoma (HCC) is one of the most common cancers and also a leading cause of cancer-related deaths in world population. HCC has an increasing trend as documented, and it has a poor prognosis in spite of recent advancements in therapeutic intervention because of its diagnosis at advanced stages.[1]

The age-adjusted incidence rate of HCC in various cancer registries including most in India as depicted by World Health Organization (WHO), provided the demographics [Table 1], which was updated in 2011.[2],[3] The majority of HCC patients have background of chronic liver disease and cirrhosis. The major risk factors for developing cirrhosis include chronic infection with hepatitis B virus (HBV), hepatitis C virus (HCV), alcoholic liver disease, and nonalcoholic steatohepatitis.[4] HBV was categorized as a Group 1 human carcinogen and one of the most important oncogenic agents, as said by the WHO.
Table 1: Demographic data for hepatitis B virus and hepatocellular carcinoma in world and India

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HBV promotes carcinogenesis by involvement in many signaling pathways in hepatocytes, thereby affecting the expression and functions of specific genes associated with HCC. Repeated cycles of inflammation-induced apoptosis and hepatocyte regeneration increase the risk of carcinogenesis.[5]

Alpha-fetoprotein (AFP) has been used as a tumor marker for the diagnosis and surveillance of HCC. The sensitivity and specificity of AFP, however, have been reported to vary from 39% to 64% and from 76% to 91%, respectively.[6],[7],[8],[9] According to some studies, patients with AFP level >400 ng/ml should be investigated to confirm the presence of HCC.[10] Physicians generally use significant elevations of liver enzyme levels as complementary markers to aid the diagnosis of various diseases. In some studies, the association between common liver enzymes (ALT, aspartate aminotransferase [AST], alkaline phosphatase [ALP], and gamma-glutamyl transpeptidase [GGT]) and HCC was done, but results did not show any significant association. Hence, it suggests the inability of liver enzymes as a prospective predictor of HCC risk in HBV patients.[1] The etiological difference of serum AFP level between HBV and non-HBV infection-related HCC has been noticed by Liu et al.[11] Many reports have shown that serum AFP levels in HCC patients are not influenced by hepatitis B or C virus pattern and that AFP levels might not be used for HCC diagnosis in noncirrhotic patients.[12] In addition, others have postulated that the usefulness of AFP in the diagnosis of HCC of viral etiology is limited and is more useful in HCC of nonviral etiology.

Hence, we designed a study to analyze the influence of HBV infection on liver enzymes and AFP in HCC patients.

 > Materials and Methods Top

Patients and study design

A total of 68 newly diagnosed patients of HCC were enrolled in this prospective study. The diagnosis was based on liver mass cytology or biopsy with appropriate imaging characteristics as defined by approved guidelines.[13] After the patient's consent, 5 ml of blood sample was taken in each patients. A study pro forma was recorded for each patient's detail about age, sex, personal history, clinical history, and general examination. All patients' samples were analyzed for

  • AFP
  • ALT
  • AST
  • ALP
  • Total bilirubin
  • Viral serology test for antihepatitis B surface antigen (HbsAg), anti-HCV, and HIV.

They all were divided into two groups: Group 1 comprised patients with HCC with HbsAg positivity, and the Group 2 comprised patients with HCC with HbsAg negativity.

AFP was analyzed in ACCESS2 (chemiluminescence immunoassay). Liver function test (LFT) was estimated by the OLYMPUS AU400 (chemistry analyzer). Viral serology test was done with DYNEX DSX4 (automated ELISA machine) using AVANTOR BeneSphera kit (microwell ELISA). The quality control and calibration was done before analyzing the samples.

This study was approved by the Institutional Ethics Committee for Human Research, Indira Gandhi Institute of Medical Sciences, Patna. The study was conducted for a total period of 1 year.

The inclusion criteria were as follows: (1) age >18 years, (2) patient diagnosed of HCC by cytology or histopathology, and (3) patient willingness for participation in the study.

The exclusion criteria were as follows: (1) patient having suspicion of metastatic adenocarcinoma in liver, secondary metastasis in liver or other malignancy of liver with or without raised AFP. (2) patients having history of drug addiction, alcoholism, and/or smoking, and (3) Patients having HIV infection.

Statistical analysis

The values of different parameters were expressed as mean with standard deviation and compared using the Student's t-test. P < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS, version 16.0.

 > Results Top

A total of 68 cases of diagnosed HCC were included in this study. On the basis of viral serology test reports, there were 40 HBV-positive and 28 HBV-negative patients. The mean age for HBV positive and negative patients was 57.5 ± 12.18 years and 53.30 ± 15.99 years, respectively. [Figure 1] shows the clinical characteristics of the analyzed patients.
Figure 1: Clinical characteristics of the analyzed patients

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The AFP > 400 ng/ml was seen in 96.4% of HBV-positive patients, whereas 92.5% patients in negative group. The AFP ranges from 352 to 54,000 ng/ml in positive group and 128 to 77,035 ng/ml in negative group. The distribution of AFP is shown in [Figure 2]. The mean value of raised AFP in HBV-positive patients was 22745.4 ± 20678.6 ng/ml, whereas in negative group, the range was 23269.3 ± 22204.4 ng/dl and P = 0.921. The patients with raised AFP >400 ng/ml did not show any significant changes in both the groups [Table 2].
Figure 2: Boxplot showing distribution of AFP, AST, ALP and ALT. The median of AFP, AST, ALT and ALP were 15391.5 ng/ml, 132, 59 and 315 U/ml, respectively, in positive group and 13224.5 ng/ml, 108.5, 56 and 337 U/ml, respectively, in negative group. AFP = Alphafetoprotein, ALP = Alkaline phosphatase, AST = Aspartate transaminases, ALT = Alanine transaminases, HBsAg = Serum hepatitis B surface antigen

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Table 2: Mean with standard deviation and P values in both groups

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We also analyzed three liver enzymes (AST, ALT, and ALP) and total bilirubin. Abnormalities in LFTs, i.e., raised ALP, AST, and ALT were observed in 85.18%, 92.59%, and 61.85%, respectively, in HBV-positive patients. The distribution of three enzymes (AST, ALT, and ALP) in both groups is shown in [Figure 2]. The mean value of abnormal level of ALP in HBV-negative patients was 429.6 ± 218.27 U/ml, whereas in positive patients, it was 353.96 ± 130.17 U/ml, i.e., more raised in negative group with P = 0.104 [Table 2]. Thus, it seems to be no difference in mean values. The mean value of abnormal AST and ALT in HBV-positive patients was 195.96 ± 139.06 and 108.86 ± 65.13, respectively, and for negative group, it was 164.32 ± 109.55 and 86.28 ± 43.74, respectively. P = 0.332 and 0.250, respectively, for HBV-positive and negative groups also were not significant for AST and ALT [Table 2].

The total bilirubin did not show any significant changes in both groups of patients.

 > Discussion Top

AFP is a 70 kDa glycoprotein and homologous to albumin. AFP produced by malignancies appears to be more highly fucosylated than that formed by normal tissues. Increased levels are mainly confined to malignancies such as nonseminomatous germ cell tumors of testis, ovary and other sites, HCC, and hepatoblastoma (in children, extremely rare in adults). Benign conditions, which may have elevated AFP levels are hepatitis, cirrhosis, biliary tract obstruction, alcoholic liver disease, ataxia telangiectasia, and hereditary tyrosinemia, and its reference range is 0–10 ng/ml.[14]

HBV-related HCC typically presents more frequently as an aggressive tumor compared with HCV-related HCC, and the levels of AFP are higher in HBV-related HCC than in HCV-related HCC.[15] To our knowledge, very few studies have been done to enlighten the effect of HBV on AFP and liver enzymes in HCC. Ming Jie Yao et al. mentioned in his study that a majority (79.55%) of HCC patients with serum HBsAg-positive status had serum AFP levels >11.62 ng/ml, which dropped to 56.49% in the HCC patients irrelevant to HBV infection. According to him, in HBV-infected patients, the median serum AFP level in HCC patient group was significantly higher than that in chronic hepatitis B or cirrhosis patients (423.89 ng/ml vs. 40.82 ng/ml, P < 0.000). These results suggested that serum AFP levels are of diagnostic value for HCC patients with chronic HBV infection,[16] whereas, few studies from the West world have indicated that the levels of serum AFP in HCC patients with and without HBV infection did not show significant results.[12]

We analyzed the AFP level in two defined groups of HCC, but there was no significant difference. It seems that significant elevations of AFP in HBV-infected HCC occur due to viral effect, but we did not find this elevation in our study. Therefore, this clinical hypothesis comes under the questionable debate and needs more study about elevated AFP significance. We also extensively evaluated the association between three common liver enzymes (ALT, AST, and ALP) that are routinely tested in the clinical setting in HCC patients. There are two major types of serum liver enzymes which are commonly seen in clinical practice: hepatocellular predominance with elevated ALT and AST and cholestatic predominance with elevated ALP and GGT. Serum ALT and AST are released from damaged hepatocytes into blood.[17] Elevated ALT and AST have been associated with the biochemical changes of various liver diseases and frequently used by clinicians. Lopez et al. reported that abnormalities in GGT, ALP, and other liver enzymes were related to more advanced stages in patients with HCC.[18] Jüngst et al. reported that the occurrence of intrahepatic cholestasis in patients with chronic hepatitis B indicates severe progressive liver disease or an acute exacerbation of HBV infection.[19]

 > Conclusion Top

In our study, we did not observe a significant association between ALP, ALT, and AST abnormalities in both groups of HCC. All enzymes were raised in both groups. However, ALP shows a slight decrease in the levels in positive group which is also area to look at. Conclusively, it can be said that the treatment protocol should not be based on etiological factor; rather it should be much more influenced by the condition of patient.

Study endpoints

The primary endpoint was the proportion of patients with HCC who had undergone investigations. Secondary endpoint was the association between elevated liver enzymes and AFP to both defined groups of HCC.

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Conflicts of interest

There are no conflicts of interest.

 > References Top

Seo SI, Kim HS, Kim WJ, Shin WG, Kim DJ, Kim KH, et al. Diagnostic value of PIVKA-II and alpha-fetoprotein in hepatitis B virus-associated hepatocellular carcinoma. World J Gastroenterol 2015;21:3928-35.  Back to cited text no. 1
Acharya SK. Epidemiology of hepatocellular carcinoma in India. J Clin Exp Hepatol 2014;4:S27-33.  Back to cited text no. 2
International Agency for Research on Cancer (IARC–WHO). Available from: http://Ci5.iarc.fr. [Last accessed on 2018 Sep 09].  Back to cited text no. 3
Sanyal AJ, Yoon SK, Lencioni R. The etiology of hepatocellular carcinoma and consequences for treatment. Oncologist 2010;15 Suppl 4:14-22.  Back to cited text no. 4
Pollicino T, Saitta C, Raimondo G. Hepatocellular carcinoma: The point of view of the hepatitis B virus. Carcinogenesis 2011;32:1122-32.  Back to cited text no. 5
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Colombo M, de Franchis R, Del Ninno E, Sangiovanni A, De Fazio C, Tommasini M, et al. Hepatocellular carcinoma in Italian patients with cirrhosis. N Engl J Med 1991;325:675-80.  Back to cited text no. 7
Pateron D, Ganne N, Trinchet JC, Aurousseau MH, Mal F, Meicler C, et al. Prospective study of screening for hepatocellular carcinoma in Caucasian patients with cirrhosis. J Hepatol 1994;20:65-71.  Back to cited text no. 8
Di Bisceglie AM, Hoofnagle JH. Elevations in serum alpha-fetoprotein levels in patients with chronic hepatitis B. Cancer 1989;64:2117-20.  Back to cited text no. 9
Chen DS, Sung JL. Hepatitis B virus infection on Taiwan. N Engl J Med 1977;297:668-9.  Back to cited text no. 10
Bruix J, Sherman M; American Association for the Study of Liver Diseases. Management of hepatocellular carcinoma: An update. Hepatology 2011;53:1020-2.  Back to cited text no. 11
Cedrone A, Covino M, Caturelli E, Pompili M, Lorenzelli G, Villani MR, et al. Utility of alpha-fetoprotein (AFP) in the screening of patients with virus-related chronic liver disease: Does different viral etiology influence AFP levels in HCC? A study in 350 western patients. Hepatogastroenterology 2000;47:1654-8.  Back to cited text no. 12
Liu C, Xiao GQ, Yan LN, Li B, Jiang L, Wen TF, et al. Value of α-fetoprotein in association with clinicopathological features of hepatocellular carcinoma. World J Gastroenterol 2013;19:1811-9.  Back to cited text no. 13
Giannini EG, Testa R, Savarino V. Liver enzyme alteration: A guide for clinicians. CMAJ 2005;172:367-79.  Back to cited text no. 14
Duffy MJ, McGing P. The Association of Biochemists in Ireland: Guidelines for the use of Tumour Markers. 5th ed. November 2018.  Back to cited text no. 15
Wroblewski F. The clinical significance of transaminase activities of serum. Am J Med 1959;27:911-23.  Back to cited text no. 16
Amaddeo G, Cao Q, Ladeiro Y, Imbeaud S, Nault JC, Jaoui D, et al. Integration of tumour and viral genomic characterizations in HBV-related hepatocellular carcinomas. Gut 2015;64:820-9.  Back to cited text no. 17
Lopez JB, Balasegaram M, Thambyrajah V, Timor J. The value of liver function tests in hepatocellular carcinoma. Malays J Pathol 1996;18:95-9.  Back to cited text no. 18
Jüngst C, Berg T, Cheng J, Green RM, Jia J, Mason AL, et al. Intrahepatic cholestasis in common chronic liver diseases. Eur J Clin Invest 2013;43:1069-83.  Back to cited text no. 19


  [Figure 1], [Figure 2]

  [Table 1], [Table 2]


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