Alpha-fetoprotein as a tumor marker in hepatocellular carcinoma: investigations in south Indian subjects with hepatotropic virus and aflatoxin etiologies

Article Outline

• Summary
• Introduction
• Materials and methods
  • Study design
  • Patients and specimens
  • Serological assays
• Results
• Discussion
• Acknowledgements
• References
• Copyright

Summary 

Objectives

The prevalence of hepatitis B virus (HBV) is reportedly the main cause of hepatocellular carcinoma (HCC) in India, where hepatitis C virus (HCV)-associated HCC is believed to be relatively less prevalent. We verified the usefulness of alpha-fetoprotein (AFP) as a tumor marker and analyzed the influence of viral etiology on AFP levels in HCC.

Methods

Of a total of 1012 cases with liver disease, 202 were investigated for the presence of AFP (142 HCC cases, 30 cirrhosis cases, and 30 chronic liver disease (CLD) cases). In addition, serum samples from 30 healthy patients, 30 hepatitis B surface antigen (HBsAg) carriers, and 30 acute viral hepatitis cases were included as controls. AFP was quantitatively determined using a commercial ELISA (Quorum Diagnostics, Canada). Out of the 142 HCC cases screened for AFP, aflatoxin B1 (AFB1) detection was carried out in 38 HCC cases using an in-house immunoperoxidase test.

Results

In HBV and HCV co-infected HCC cases, the AFP positivity was 85.7%. In HBV alone-associated HCC, the positivity was 62.9%, and 54.5% of AFB1 positive HCC cases showed AFP positivity. In HBV and HCV negative HCC cases, the positivity was 20.5%, and in HCV-associated HCC it was 17.6%. The HBV/HCV co-infected group and HBV alone positive HCC cases had significantly elevated levels of AFP. When AFP positivity was analyzed based on the marker profile of HBV, 89.7% of AFP positive cases were HBV-DNA positive.

Conclusions

The overall positivity pattern of AFP in HCC does indicate that higher levels of AFP are observed with hepatitis virus positivity, especially with HBV. Further studies must be carried out to correlate the serum levels of AFP with the size, number, and degree of differentiation of HCC nodules.

Keywords: Aflatoxin B1, Alpha-fetoprotein, Cirrhosis, Hepatocellular carcinoma, Hepatitis

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Introduction 

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death worldwide.¹, ², ³, ⁴, ⁵ The prevalence of HCC in autopsied Indians is low, and varies between 0.2% and 1.9%.² Alpha-fetoprotein (AFP), a 70-KDa glycoprotein tumor marker, is increased in the majority of patients with HCC and other gastrointestinal tumors. AFP is normally produced during fetal development by the liver and yolk sac. After birth, the levels drop off rapidly, and by the second year only trace amounts are detectable in serum. AFP may be useful in the diagnosis and follow-up of cases of HCC, although increased levels are associated with malignancies other than primary HCC. Studies suggest that in patients thought to have HCC on clinical grounds, AFP levels >400ng/ml should strongly confirm the presence of HCC by a tissue diagnosis.⁶ Some patients with primary hepatic cancer will have normal AFP levels, and normal or moderately elevated levels should not exclude a diagnosis of HCC, although the usefulness in follow-up of patients under treatment for hepatoma has not been examined systematically. Thus, this marker is believed to be useful in following the clinical course of persons with HCC. It has been suggested that the AFP values are more likely to be elevated with the stage of such cancers and are of prognostic value to check the efficacy of treatment of HCC. Levels of serum AFP are widely used for HCC screening in patients with chronic liver disease (CLD). The relationship between viral etiology and AFP levels in HCC is still unclear. The aim of this study was to verify, in Indian patients, the usefulness of AFP as a tumor marker, and to analyze the influence of viral etiology on AFP levels in HCC.

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Materials and methods 

Study design 

This study was conducted between January 1994 and December 1998, after prior approval from the Institutional Review Board (IRB) of the University of Madras. Written, informed consent was obtained from all the patients, or their legal representatives, before study enrolment. The study was carried out in compliance with good clinical practice, including the International Conference on Harmonization Guidelines and the Declaration of Helsinki.

Patients and specimens 

We analyzed 1012 liver disease cases admitted to the medical and surgical gastroenterology units of five major hospitals in the city of Chennai. Inclusion into the study was based on the clinical diagnosis of HCC, i.e., patients who showed mass abdomen, mass lesion on ultrasound, and elevated AFP levels were enrolled. Each subject provided written, informed consent to conduct an interview, collect a blood specimen, undergo a liver biopsy, and perform serological assays. All the cases were interviewed in person with the use of a structured questionnaire. The subjects were questioned on their socio-demographic characteristics, diet, cigarette smoking habits, consumption of alcohol, betel nut chewing, their medical and surgical history, and any family history of HCC or liver cirrhosis. Of the 1012 liver disease cases, 202 were analyzed for the presence of the tumor marker AFP (142 HCC cases, 30 cirrhosis cases, and 30 CLD cases). In addition, serum samples from healthy patients (n=30), hepatitis B surface antigen (HBsAg) carriers (n=30), and acute viral hepatitis cases (n=30) were used as controls for AFP ELISA.

Serological assays 

A 10-ml specimen of blood was collected with the use of a disposable vacuum syringe and the separated serum kept in a freezer (−70°C) until assay. The serum specimens were tested for HBsAg, hepatitis B ‘e’ antigen (HBeAg), anti-hepatitis B ‘e’ antigen antibodies (anti-HBe), anti-hepatitis B core antigen IgM antibodies (anti-HBc IgM), hepatitis delta antigen (HDAg), and anti-hepatitis delta antigen antibodies (anti-HD) (Wellcozyme, Murex Diagnostics, UK) by ELISA, with the use of commercially available kits according to the manufacturer's instructions. Anti-hepatitis C virus (anti-HCV) status was tested using RIBA 3.0, SIA in vitro qualitative enzyme immunoassay (EIA). Aflatoxin B1 (AFB1) detection in liver biopsies was carried out using an in-house immunoperoxidase test on 38 HCC cases.⁷ The test was sensitive enough to detect up to approximately 1ng/ml of AFB1 in serum.⁷ AFP in the sera was quantitatively determined using a commercial ELISA kit procured from Quorum Diagnostics, Canada. This was a sandwich ELISA; the solid phase was coated with specific rabbit anti-AFP antibodies. AFP present in the specimen will bind to the antibodies in the wells. Then mouse monoclonal anti-AFP antibody labeled with horseradish peroxidase enzyme was added to detect the AFP bound to the solid phase antibody. A reference standard set containing 0, 5, 20, 50, 150, and 300ng/ml of AFP in the lyophilized form was reconstituted before starting the test.

Briefly, 20μl of standards, specimens, and controls were added to the appropriate wells. A hundred microliters of zero buffer was added to each well and mixed for 10s. Wells were covered and incubated at room temperature for 30min. After aspirating the contents, the wells were washed five times with distilled water. The remaining fluid was removed by tapping the strips on a tissue paper. Enzyme conjugate (150μl) was added to each well and mixed for 5s. Wells were incubated for 30min and the strips were washed. TMB (3,3′,5,5′-tetramethylbenzidine) substrate solution (200μl) was added to all the wells and the plate was incubated in the dark at room temperature. Enzyme was stopped by the addition of 50μl per well of 2M H₂SO₄. The ELISA reader was blanked in the air and the absorbance was measured at 450nm (ELx800 ELISA Reader, Biotech Inc., USA). The absorbance was measured for each set of reference standards, specimens, and controls. A standard curve was constructed by plotting the absorbance obtained from each reference standard against its concentration (ng/ml). The amount of AFP (ng/ml) in each specimen was quantitated against the standard curve obtained by plotting the absorbance of the reference standards against its concentration.

Statistical analysis was done using Epi Info statistical software (http://www.cdc.gov/epiinfo/epiinfo.htm). The positivity profiles of HCC cases in the study groups are given in frequencies with their percentage. Mean titer values are given as mean±standard deviation (SD) and the AFP positivity profiles are given using proportions with 95% confidence intervals (CI). The positivity profiles of AFP in HCC cases were analyzed using Chi-square trend and unadjusted odds ratios (OR). The hepatitis B virus (HBV) marker pattern observed among the AFP positive and negative HBsAg positive HCC cases were analyzed using the two sample binomial proportion test, and proportions of percentage with 95% CI are given. In addition, the AFP levels in AFB1 tissue positive vs. negative HBV positive and negative cases were analyzed using Fisher's exact test. A p-value of <0.05 was considered statistically significant.

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Results 

A total of 202 liver disease cases were investigated for the presence of the tumor marker AFP (142 HCC cases, 30 cirrhosis cases, and 30 CLD cases). All these cases were screened for HBsAg and anti-HCV to establish HBV and hepatitis C virus (HCV) status in addition to certain serological HBV and HCV markers.⁸ In addition, serum samples from healthy controls (n=30), HBsAg carriers (n=30), and acute viral hepatitis cases (n=30) were used as controls for AFP ELISA. The male:female ratio of HCC cases in the present study was 6.4:1. The mean age of male HCC cases was 62.7 years as against 46 years in females. The clinical symptomatology revealed mass abdomen in 64.1% of cases, while anorexia and abdomen pain were seen in 43.5% and 35.9%, respectively. Other common symptoms were pedal edema (27.6%), ascites (24.7%), weight loss (21.2%), fever (12.9%), and hematemesis (8.8%). Along with the 142 HCC cases, 30 cases each of cirrhosis, CLD, HBsAg carriers, and healthy controls were analyzed for their serum AFP. Among the 142 cases with HCC screened, 65 (45.8% with CI 38–54%) revealed positive AFP, while the cirrhosis, CLD, and control groups did not have detectable AFP levels. Of the total 170 HCC cases, HBV and HCV co-infection was seen in seven cases (4.1%).

A correlation of AFP positivity with viral marker positivity is shown in Table 1. In HBV and HCV co-infected HCC cases, the AFP positivity was 85.7% (n=6). In HBV alone-associated HCC, AFP positivity was 62.9% (n=39), and 54.5% (n=6) of AFB1 positive HCC cases showed AFP positivity. In HBV and HCV negative HCC cases, the AFP positivity was 20.5% (n=8), and in HCV-associated HCC it was 17.6% (n=3). When quantitative levels of AFP were analyzed, the HBV/HCV co-infected group and HBV alone positive HCC cases had significantly elevated levels of AFP.

Table 1. Alpha-fetoprotein positivity profile in hepatocellular carcinoma cases (N=142)

Study groups	AFP positivity			Mean titer
	Number	AFP positivity	Chi-square trend OR	Mean titer±2 SD (ng/ml)	One-way ANOVA F-test	Multiple comparison by Bonferroni t-test
HBV positive	62	39 (62.9%)	7.91	502±390	F=4.09; p=0.02	1 and 6; p<0.05
HCV positive	17	3 (17.6%)	1.00	492±215		1 and 6; p<0.05
HBV and HCV positive	7	6 (85.7%)	28.01	567.7±199.4		1 and 6; p<0.05
HBV and HCV negative	39	8 (20.5%)	1.2	463.9±390.9		-
AFB1 positive	11	6 (54.5%)	5.6	483±497.7		-
HBV and AFB1 positive	6	3 (50%)	5.67	233.3±194.2		-

AFP, alpha-fetoprotein; OR, odds ratio; SD, standard deviation; ANOVA, analysis of variance; HBV, hepatitis B virus; HCV, hepatitis C virus; AFB1, aflatoxin B1.

Compared to HCV positive AFP positive subjects, HBV positive AFP positive cases had a 7.91-times greater chance of developing HCC, and both HBV and HCV positivity revealed a 28.01-times greater chance of developing HCC than HBV alone. One-way ANOVA showed the existence of differences between the five groups analyzed. The Bonferroni t-test also showed that groups 1 and 6 differed significantly.

When AFP positivity was analyzed based on the marker profile of HBV, 89.7% of AFP positive cases were HBV-DNA positive indicating the close association of replicating HBV in inducing AFP (Z=2.02; p=0.04 and 24.5% with CI 1–49%; Table 2). Similarly analysis was made with regard to AFB1 positivity with or without viral positivity. Even though no significant correlation could be made, 71.4% of HBV/AFB1 positive cases were also positive for AFP (Table 3).

Table 2. Hepatitis B virus marker pattern observed in alpha-fetoprotein positive and negative, hepatitis B surface antigen (HBsAg) positive hepatocellular carcinoma cases

Viral marker	AFP positive		AFP negative		Two sample binomial proportion test	Proportion with 95% CI
	Number	Positive	Number	Positive
HBeAg	39	10 (25.6%)	23	7 (30.4%)	Z=0.11; p=0.91	-
Anti-HBe	39	29 (74.4%)	23	16 (69.6%)	Z=0.11; p=0.91	-
Anti-HBc IgM	12	9 (75%)	9	5 (55.6%)	Z=0.47; p=0.63	-
HBV-DNA	39	35 (89.7%)	23	15 (65.2%)	Z=2.02; p=0.04	24.5% (1–49%)
HDV	27	4 (14.8%)	16	6 (37.5%)	Z=1.33; p=0.18	-

AFP, alpha-fetoprotein; CI, confidence interval; HBeAg, hepatitis B ‘e’ antigen; anti-HBe, anti-hepatitis B ‘e’ antigen antibodies; anti-HBc IgM, anti-hepatitis B core antigen IgM antibodies; HDV, hepatitis D virus.

Table 3. Alpha-fetoprotein levels in aflatoxin B1 tissue positive vs. negative hepatitis B virus positive and negative cases

	AFB1	Numbers screened	AFP positivity	Fisher's exact test	Mean (ng/ml)
HBsAg positive group (n=15)	Positive	7	5 (71.4%)	p=0.60	346
	Negative	8	4 (50%)		557
HBsAg negative group (n=16)	Positive	11	6 (54.5%)	p=0.60	549
	Negative	5	1 (20%)		568

HBsAg, hepatitis B surface antigen; AFB1, aflatoxin B1; AFP, alpha-fetoprotein.

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Discussion 

AFP is often elevated in HCC.⁸ Our study revealed 85.7% AFP positivity among HBV/HCV co-infected HCC cases. In addition, the HBV alone-associated HCC cases showed a positivity of 62.9%. The study is in agreement with studies by others.⁸, ⁹ In HBV and HCV negative HCC cases, the AFP positivity was 20.5%, and in HCV-associated HCC it was 17.6%, which seems to show little or almost no influence from HCV. Further, when quantitative levels of AFP were analyzed, the HBV/HCV co-infected group and HBV alone positive HCC cases had significantly elevated levels of AFP. Again, when AFP positivity was analyzed based on the marker profile of HBV, 89.7% of AFP positive cases were HBV-DNA positive, indicating the close association of replicating HBV in inducing AFP. The frequency and the extent of AFP elevation are greater in HBsAg positive than HBsAg negative patients.¹⁰, ¹¹ We recently reported that 58.1% of HCC cases showed aflatoxin B1 (AFB1) in liver biopsies, but none was found from the cirrhosis cases using an in-house immunoperoxidase staining test.⁷ Further, we also reported that 68.8% of HBsAg negative HCC cases and 46.1% of the HBsAg positive subjects were concurrently positive for AFB1 in liver biopsy, which proves that aflatoxins have a significant association with HCC in this part of the tropical subcontinent using an in-house immunohistochemical method.⁷

The strong association between HBsAg and primary liver cancer in countries where liver tumors are often AFP secretors, suggests a role for HBV, not only in the etiology of the cancer, but also in the reactivation of the gene encoding this fetal protein. A report from the south of India has shown AFP positivity to be higher in HBsAg positive HCC cases (69.9%) and lower in HBsAg negative HCC cases (38.1%).¹² The quantitative levels of AFP were also significantly higher in the HBV positive (502ng/ml) and HBV/HCV co-infected groups (567.7ng/ml) than in the other groups in our study. Similar higher levels of AFP in HBV positive HCC cases (3200ng/ml in 95%) have been reported by others.¹³ In contrast, other reports from the West have shown that serum AFP levels in HCC patients are not influenced by hepatitis B or C virus pattern and that AFP dosage might not be advocated for HCC diagnosis in non-cirrhotic patients.¹⁴ 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 non-viral etiology.¹⁵

Studies on AFP levels in patients with HCC and other benign and malignant liver diseases have shown that serum AFP is greater than 400ng/ml in 69% of patients with hepatoma.⁶ In none of 66 cases of cancer other than HCC was the AFP level greater than 400ng/ml. In 211 patients with non-malignant liver disease, an AFP level greater than 400ng/ml was seen in only one patient, a person with chronic aggressive hepatitis. These data suggest that in patients thought to have HCC on clinical grounds, AFP levels about 400ng/ml should strongly confirm the presence of HCC by a tissue diagnosis. However, clinicians should remember that some patients with primary hepatic cancer will have normal AFP levels, and normal or moderately elevated levels should not be used to exclude the diagnosis of HCC.

In the present study AFP positivity was analyzed to check the utility of this marker in our HCC, cirrhosis, and CLD cases, and to define the normal AFP levels in matched control groups. It is generally accepted that a considerable number of patients with acute and chronic hepatitis and cirrhosis without malignancy might also have slight but significant elevation of serum AFP.¹⁶, ¹⁷ Frequent rises in adult serum level of AFP are seen in patients with hepatitis and cirrhosis. Positive rates (>10 or 20ng/ml without detectable differences in the results) reported are 31–52% for acute hepatitis, 15–58% for chronic hepatitis, and 11–47% for cirrhosis.⁶, ¹⁶, ¹⁸ Though there are reports on AFP positivity in CLD and cirrhosis cases, in the present study none of our CLD, cirrhosis, HBsAg carriers, and healthy control samples showed AFP positivity. A major limitation is the smaller sample size to assess the AFP levels in this group. Others have analyzed histopathologically confirmed HCC cases and have found that serum AFP is normal (<8.5ng/ml) in 20%, moderately elevated (8.5–300ng/ml) in 48%, and considerably elevated (>300ng/ml) in 32% of cases.¹⁹ In a north Indian study, the AFP levels were raised in 65% of the HCC cases, the highest level recorded being 580ng/ml.²⁰ In another south Indian study, elevated AFP levels were observed in 47.4% of the cases.¹² Our results corroborate those of the south Indian study revealing a positivity of 45.4% observed among HCC cases. The highest AFP level detected in our HCC cases was 739ng/ml. In acute hepatitis, transient and simultaneous rises of serum AFP and transaminase or an AFP rise shortly after the transaminase peak, have been observed.¹⁸, ²¹ In the present study AFB1 positive non-viral HCC cases also showed higher levels of AFP (483ng/ml). It may be postulated that the levels of AFP might be indicative of the severity of hepatocarcinogenesis, irrespective of the hepatocarcinogen. This hypothesis is in line with the other assumptions that viruses and certain other hepatotoxins may induce a distinct type of cell injury, leading to AFP synthesis and release into the serum. Studies have shown that AFP, AFP-L3, and des-γ-carboxyprothrombin (DCP) are the most useful serum tumor markers for the detection of HCC, and their simultaneous determination could improve the accuracy, especially in differentiating HCC from non-malignant hepatopathy.²², ²³, ²⁴, ²⁵, ²⁶ Some clinical researchers have indicated that the simultaneous determination of supplementary markers especially glypican-3 (GPC3) along with AFP could significantly increase the sensitivity in the diagnosis of HCC,²³ which was not attempted in our study. Further, the major limitation of this study was that the study failed to evaluate the factors closely associated with tumors. It should be remembered that serum levels of AFP reflect the size, number, and degree of differentiation of HCC nodules.²⁴, ²⁵

In conclusion, there has been no report on the AFP levels of aflatoxin B1 positive HCC cases to the best of our knowledge from the south Indian context. Based on the data analyzed and investigations performed, we postulate that serum AFP levels in HCC patients are influenced by HBV pattern, in contrast to reports from the West, and could be used in HCC diagnosis. We conclude that the overall positivity pattern of AFP in HCC does indicate that higher levels of AFP are observed with hepatitis virus positivity, especially with HBV. Further studies are required to correlate the serum levels of AFP with the size, number, and degree of differentiation of HCC nodules.

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Acknowledgements 

The authors are grateful for the financial assistance provided by the Indian Council of Medical Research (ICMR), New Delhi to K.G. Murugavel. Thanks are due to all the study participants for their active role in conducting the study.

Conflict of interest: No conflict of interest to declare.

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