Hepatocellular carcinoma (HCC) is the third leading cause of death from cancer worldwide. The rising trend of HCC has been associated with increased prevalence of hepatitis C virus (HCV) infection. In Egypt, the prevalence of HCV infection among the general population has been estimated to be around 14% 1. HCC, when diagnosed at an early stage, can be treated by liver transplantation or surgical resection. However, despite effective therapies for early-stage HCCs and efforts at early diagnosis through screening of patients at risk for this cancer, most HCCs in Egypt are diagnosed at an advanced stage.
In Egypt, less than 10.0% of patients diagnosed with HCC are eligible for liver transplantation or surgical resection with curative intent. Therefore, there is an urgent need for improved methods of screening and surveillance of individuals at risk for HCC 2,3.
A major problem with HCC surveillance is the lack of reliable biomarkers. α-Fetoprotein (AFP) is the most widely used biomarker for surveillance of HCC; however, not all HCCs secrete AFP 4 and its levels do not increase in 35–45% of HCC patients 5,6. In addition, only one-third of patients with HCC have AFP levels higher than 100 ng/ml 7. Further, AFP levels may be elevated in patients with chronic liver disease, especially in those with a high degree of hepatocyte regeneration (e.g. on HCV infection), and they can express these elevated levels in the absence of malignancy 8,9.
The lens culinaris agglutinin-reactive AFP percentage of total AFP concentration [(AFP-L3/total AFP)×100 or AFP-L3%] has been used as a marker for early diagnosis, assessment of therapeutic effects, and predicting the prognosis of HCC 10,11. The AFP-L3 isoform has been reported to be more specific in the diagnosis of HCC than the total AFP level. It has also been shown to be associated with more aggressive HCCs and predicting a worse outcome 12.
With recent progress in the identification and characterization of new serum markers for HCC, such as GP73 and glypican-3, and also with recent improvements in the assays for determination of AFP-L3% fraction and des-γ-carboxy prothrombin levels, it is probable that future efforts at surveillance of patients with cirrhosis who are at risk for HCC will use a panel of serum markers. To achieve optimal results with a combination of markers it is important that each marker be carefully evaluated to establish its potential place in the panel 13.
Aim of the study
The aim of this study was to evaluate the role of the protein induced by the absence of vitamin K or antagonist II (PIVKA-II) and AFP-L3% in diagnosis of patients with HCC having normal and abnormal AFP levels.
Patients and methods
This study was conducted on 441 patients. A total of 148 patients had advanced liver cirrhosis with no evidence of HCC, 137 had HCC with AFP levels below 400 ng/dl, and 76 had HCC with AFP levels above 400 ng/dl; the study also included 80 healthy controls. Venous blood samples (10 ml) were drawn from all included patients and tested for liver function and viral markers (HBsAg, HCV antibodies); they were also serodiagnosed for schistosomiasis. The levels of AFP, AFP-L3%, and PIVKA-II were also determined. Levels of AFP-L3% and PIVKA-II (des-γ-carboxy prothrombin) in the serum were determined using a liquid-phase binding assay performed on a Wako LiBASys clinical auto analyzer (Wako Pure Chemical Industries Ltd, Osaka, Japan). HCV antibodies were assayed using the Innotest kit (Innogenetics Biotechnology, USA). Antibilharzial antibodies were assayed using Amibiase H.A.I. fumouze (Wako Pure Chemical Industries Ltd., Osaka, Japan).
The results were analyzed using SPSS version 16 (SPSS Inc., Chicago, Illinois, USA). Data are presented as mean±SD (range). The Kruskal–Wallis test was used for independent sample analysis, and the Tamhane post-hoc test was used to test differences within the same group. Receiver operator characteristic (ROC) curve analysis was used to detect the sensitivity and accuracy of levels of AFP-L3% and PIVKA-II in the diagnosis of HCC. P-values less than 0.05% were considered significant.
This study was conducted on 441 patients. A total of 148 patients had advanced liver cirrhosis with no evidence of HCC, 137 had HCC with AFP levels below 400 ng/dl, and 76 had HCC with AFP levels above 400 ng/dl; the study also included 80 healthy controls. Males represented 72.9% of individuals in the cirrhotic group, 57.5% in the control group and 72.8% of the HCC group. HCV infection was the most common etiology in the cirrhotic group (64.9%) and HCC group (68.1%). Antibilharzial Abs accounted for 31.1% of patients in the cirrhotic group and 55.4% in the HCC group. In the cirrhotic group, 32.4% of patients were classified as Child class C versus 44.6% in the HCC group. See (Tables 1–6).
HCC accounts for 85–90% of all primary liver cancers. Every year, approximately half a million people worldwide are newly diagnosed with HCC and a similar number of people die from HCC, thus showing the dismal prognosis of this cancer 14.
Worldwide, HCC is one of the most common malignancies associated with poor prognosis 15. According to recent reports, the incidence of HCC has increased markedly in the last 5–10 years 16. Not enough attention has been paid to exploring the magnitude of the problem of HCC in Egypt, although there has been a remarkable increase in the incidence of HCC among CLD patients (from 4.0 to 7.2%) in over a decade. This rise may be explained by the increasing risk factors such as the emergence of HCV infection during the same period of time, the contribution of HBV infection, improvements in the screening programs and diagnostic tools for HCC 7, and increased survival rate among patients with cirrhosis, which sometimes leads to the development of HCC.
The mean age of HCC patients in this study was ∼54 years, and HCC was more common in men in all the three groups. This is in concordance with the findings of Sherman 17, who showed that the incidence of HCC started to increase above 45 years of age, whereas Leerapum et al. 18 and Sharma et al. 19 demonstrated a higher mean age of ∼70 years. El-Serag emphasized the role of the duration of HCV infection in the development of cirrhosis and HCC as he reported that 3–35% of patients progress to cirrhosis 25 years after infection and 1–3% progress to HCC 30 years after infection 20. The sex frequency in this study agrees with the fact that HCC is the most common cancer in men and the eighth most common cancer in women worldwide 21.
In most of the patients investigated in the present study, HCV-related cirrhosis was the underlying cause for HCC. Murugavel and colleagues analyzed the influence of viral etiology on AFP levels in HCC patients. In Egypt, HCV-associated HCC is believed to be more prevalent.
With regard to tumor markers, the present study shows a statistically significant elevation in the mean serum level of AFP in the HCC group compared with the cirrhotic and control groups (P<0.01); it also shows statistical significance in the cirrhotic group compared with the control group (P<0.01). These results are in agreement with the results of many other studies, in which Sharma and colleagues 19,22 observed that the mean serum level of AFP was statistically higher in HCC patients than in liver cirrhosis patients. It has been reported that AFP concentrations greater than 200 ng/ml strongly correlate with the development of HCC 23.
The aforementioned results demonstrate that increased serum AFP concentration may be found in HCC patients and in those with other benign liver diseases such as liver cirrhosis. This increase may be attributed to the progressive liver pathology or cell injury rather than HCC or both factors together 24.
On testing the level of AFP-L3% in the present study (Tables 2 and 4), the mean value was found to be statistically higher in the HCC group than in the cirrhotic and control groups (P<0.01). Moreover, there was also a highly statistically significant elevation in AFP-L3% levels in the cirrhotic group compared with controls (P<0.01). These results are in agreement with the findings of Durazo et al.22, who found that levels of AFP-L3% were statistically higher in patients with HCC than in those without HCC. Similarly, Xu et al.25 found that the levels of AFP-L3% in the serum of patients with HCC were significantly higher than those in patients with other liver diseases (P<0.01).
With regard to the levels of PIVKA-II (Tables 2 and 4), the present study shows that there is a highly statistically significant increase in the mean serum level of PIVKA-II (P<0.01) in the HCC group compared with the cirrhotic and control groups. Meanwhile, there was no significant difference between the cirrhotic and control groups (P>0.05). In accordance with these results, Sharma et al.19 and Tada et al.26 reported that the mean serum level of PIVKA-II was statistically higher in HCC patients than in cirrhosis patients. Similarly, in another study carried out by Wang et al.9, the mean level of PIVKA-II was significantly higher in patients with HCC than in those with chronic hepatitis and cirrhosis, and the authors reported that PIVKA-II has a better diagnostic value than AFP in differentiating HCC from nonmalignant chronic liver disease.
On evaluating the levels of AFP-L3% and PIVKA-II in the HCC group with abnormal AFP levels (<400 ng/ml and ≥400 ng/ml; Tables 2 and 4), the mean value of AFP-L3% was found to be statistically significantly higher in patients with abnormal (diagnostic) AFP levels than in those with normal levels. This result is in agreement with the findings of Tada et al.26, who showed that total AFP was significantly higher in the AFP-L3%-positive group than in the AFP-L3%-negative group. Yamamoto et al.27 found that the significance of the level of AFP-L3% depends on that of AFP, and it is thought to play a role in patients with intermediately elevated AFP levels because of its high specificity. In contrast, Kumada et al.28 reported that the elevation in the level of AFP-L3% was independent of the elevation in the total AFP level in patients with HCC, and AFP-L3% has been shown to be a better marker for HCC than total AFP.
The optimal cutoff value for the level of AFP-L3% obtained from ROC curves for the HCC group with abnormal AFP levels (<400 ng/ml) was 10.05 ng/ml, with a sensitivity, specificity, and accuracy of 46.7, 97.6, and 61.7%, respectively (Table 3). Moreover, the optimal cutoff value of AFP-L3% obtained from ROC curves for the HCC group with abnormal AFP levels (≥400 ng/ml) was 10.05 ng/ml, with a sensitivity, specificity, and accuracy of 79.0, 97.8, and 99.2%, respectively (Table 5). Although these results are in agreement with the findings of Xua et al.25, they considered AFP-L3% levels of 10% or more to be a diagnostic criterion and found that the sensitivity for diagnosis of HCC was 90.9% and concluded that detection of AFP-L3% seemed to be of clinical value in the diagnosis and differential diagnosis of HCC. Moreover, Sun et al.29 stated that for AFP-L3% levels of 10% or more, which was taken as a diagnostic criterion, the sensitivity of AFP-L3% in HCC diagnosis was 84.4% and specificity was 92.5% with a total confirmatory rate of 87.9%. Xua et al. 25 and Sun et al. 29 stated that the AFP-L3% level is a valuable biomarker in the diagnosis and prediction of the prognosis of HCC. In contrast, Durazo et al.22 stated that AFP-L3% shows good specificity but a lower sensitivity that is not enough for it to be considered as a surveillance tool for HCC. Li and colleagues and Wang et al.9 stated that AFP-L3% has been related to tumors with rapid growth, portal vein invasion, and large-sized tumors with metastasis. Durazo et al.22 also state that the AFP-L3% may be a better prognostic marker for HCC than for a screening test.
The optimal cutoff value for PIVKA-II obtained from ROC curves in the HCC group with abnormal AFP levels (<400 ng/ml) was 17.95 ng/ml, with a sensitivity, specificity, and accuracy of 41.6, 88.2, and 66.9%, respectively (Table 3). Moreover, the optimal cutoff value for PIVKA-II obtained from ROC curves in the HCC group with abnormal AFP (≥400 ng/ml) was 3.55 ng/ml, with a sensitivity, specificity, and accuracy of 71.1, 67.5, and 76.3%, respectively (Table 5). Data from this study show that PIVKA-II is more sensitive and accurate in diagnosing HCC in patients with normal or abnormal AFP levels. These results are in accordance with the findings of Durazo et al.22, who had found that PIVKA-II appears to be the most accurate tumor marker and should be used for HCC surveillance. Moreover, Yamamoto et al.27 concluded that PIVKA-II is a more effective tumor marker than AFP and AFP-L3%.
The above mentioned results on tumor markers reveal that levels of PIVKA-II are elevated in most patients with HCC and do not correlate with AFP elevation. In fact, AFP-L3% appears to be more specific and more closely associated with the presence of HCC as it is less often elevated in cirrhotic patients without HCC, in contrast to AFP and PIVKA-II.
From the above findings we conclude that in the HCC group with abnormal AFP levels (≥400 ng/ml), serum AFP remains the most useful tumor marker in diagnosis of HCC. The mean values of AFP-L3% appear to be more specific than mean AFP values in the diagnosis of HCC group with normal AFP levels (<400 ng/ml) as it is less often elevated in patients having cirrhosis without HCC, which does not hold true for AFP or PIVKA-II, and this implies that AFP-L3% is closely associated with the presence of HCC.
This research was supported by Wako Pure Chemical Industries Ltd.
Conflicts of interest
There are no conflicts of interest.
1. El-Serag HB. Hepatocellular carcinoma
: an epidemiologic view. J Clin Gastroenterol. 2002;35(Suppl 2):572–578
2. El-Serag HB. Epidemiology of hepatocellular carcinoma
. Clin Liver Dis. 2001;5:87–107
3. El-Serag HB, Siegel AB, Davila JA, Shaib YH, Cayton-Woody M, McBride R, et al. Treatment and outcomes of treating of hepatocellular carcinoma
among Medicare recipients in the United States: a population-based study. J Hepatol. 2006;44:158–166
4. Debruyne EH, Delanghe JR. Diagnosing and monitoring hepatocellular carcinoma
: new aspects and applications. Clin Chim Acta. 2008;395:19–26
5. Nguyen MH, Garcia RT, Simpson PW, Wright TL, Keeffe EB. Racial differences in effectiveness of alpha-fetoprotein
for diagnosis of hepatocellular carcinoma
in hepatitis C virus cirrhosis. Hepatology. 2002;3:410–417
6. Yamaguchi I, Nakamura K, Kitano H, Masuda Y, Kanke F, Kobatake S, et al. Development of des-gamma carboxy prothrombin (DCP) measuring reagent using the LiBASys clinical analyzer. Clin Chem Lab Med. 2008;46:411–416
7. El-Serag HB, Mason AC, Key C. Trends in survival of patients with hepatocellular carcinoma
between 1977 and 1996 in the United States. Hepatology. 2001;33:62–65
8. Bae JS, Park SJ, Park KB, Paik SY, Ryu JK, Choi CK. Acute exacerbation of hepatitis in liver cirrhosis
with very high levels of alpha-fetoprotein
but no occurrence of hepatocellular carcinoma
. Korean J Intern Med. 2005;20:80–85
9. Wang CS, Lin CL, Lee HC, Chen KY, Chiang MF, Chen HS, et al. Usefulness of serum des-gamma-carboxy prothrombin in detection of hepatocellular carcinoma
. World J Gastroenterol. 2005;11:6115–6119
10. Hayashi K, Kumada T, Nakano S, Takeda I, Sugiyama K, Kiriyama S, et al. Usefulness of measurement of Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein
as a marker of prognosis and recurrence of small hepatocellular carcinoma
. Am J Gastroenterol. 1999;94:3028–3033
11. Okuda K, Tanaka M, Kanazawa N, Nagashima J, Satomura S, Kinoshita H, et al. Evaluation of curability and prediction of prognosis after surgical treatment for hepatocellular carcinoma
by lens culinaris agglutinin-reactive alpha-fetoprotein
. Int J Oncol. 1999;14:265–271
12. Hirai Y, Waki II, Momose A, Fukazawa T, Aida T, Takagi K, et al. Increase of O 2p unoccupied electronic states within the ab plane of YBa2Cu3O6.8 due to a superconducting transition. Phys Rev B Condens Matter. 1992;45:2573–2576
13. Song BC, Suh DJ, Yang SH, Lee HC, Chung YH, Sung KB, et al. Lens culinaris agglutinin-reactive alpha-fetoprotein
as a prognostic marker in patients with hepatocellular carcinoma
undergoing transcatheter arterial chemoembolization. J Clin Gastroenterol. 2002;35:398–402
14. El-Serag HB, Davila JA. Surveillance for hepatocellular carcinoma
: in whom and how? Therap Adv. Gastroenterol. 2011;4:5–10
15. Okano H, Shiraki K, Inoue H, Deguchi M, Sugimoto K, Sakai T, et al. Treatment of hepatocellular carcinoma
and the exacerbation of liver function. Int J Oncol. 2001;19:1279–1282
16. Velazquez RE, Rodriguez M, Navascues CA, Linares A, Perez R, Sotorrios NG, et al. Prospective analysis of risk factors for hepatocellular carcinoma
in patients with liver cirrhosis
. Hepatology. 2003;37:520–527
17. Sherman M. Hepatocellular carcinoma
: epidemiology, risk factors, and screening. Semin Liver Dis. 2005;25:143–154
18. Leerapum A, Suravarapu SV, Bida JP, Clark RJ, Sand EL, Mettler TA, et al. The utility of lens culinaris agglutinin-reactive alpha-fetoprotein
in the diagnosis of carcinoma: evaluation in a United States referral population. Gastroenterol Hepatol. 2007;5:394–402
19. Sharma B, Srinivasan R, Chawla YK, Kapil S, Saini N, Singla B, et al. Clinical utility of prothrombin induced by vitamin K absence in the detection of hepatocellular carcinoma
in Indian population. Hepatol Int. 2010;4:569–576
20. El-Serag HB. Hepatocellular carcinoma
and hepatitis C in the United States. Hepatology. 2002;36:S74–S83
21. El-Serag HB, Rudolph KL. Hepatocellular carcinoma
: epidemiology and molecular carcinogenesis. Gastroenterology. 2007;132:2557–2576
22. Durazo FA, Blatt LM, Crey WG, Lin JH, Han S, Saab S, et al. Des-Gamma-carboxyprothrombin, alpha-fetoprotein
and AFP-L3% in patients with chronic hepatitis, cirrhosis and hepatocellular carcinoma
. J Gastroenterol Hepatol. 2008;23:1541–1548
23. Farinati F, Marino D, De Giorgio M. Diagnostic and prognostic role of alpha-fetoprotein
in hepatocellular carcinoma
: both or neither? Am J Gastroenterol. 2006;101:524–532
24. Choi J, Ou JH. Mechanisms of Liver Injury III. Oxidative stress in the pathogenesis of hepatitis C virus. Am J Physiol Gastrointest Liver Physiol. 2006;290:G847–G851
25. Xu AF, Wang MC, Sui DM, Yuan YH, Chen G, Lou GO. Subject diagnostic value of detecting alpha-fetoprotein
variants with a new microspincolumn method in hepatocellular carcinoma
. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2007;21:67–69
26. Tada T, Kumada T, Toyoda H, Kiriyama S, Sone Y, Tanikawa M, et al. Relationship between Lens culinaris agglutinin-reactive alpha-fetoprotein
and pathologic features of hepatocellular carcinoma
. Liver Int. 2005;25:848–853
27. Yamamoto K, Immura H, Matsuyama Y, Kume Y, Ikeda H, Norman GL, et al. AFP, AFP-L3%, DCP and GP73 as markers for monitoring treatment response and recurrence and as surrogate markers of clinicopathological variables of HCC. J Gastroenterol. 2010;45:1272–1782
28. Kumada T, Nakano S, Takeda I, Kiriyama S, Sone Y, Hayashi K, et al. Clinical utility of lens culinaris agglutinin-reactive alpha-fetoprotein
in small hepatocellular carcinoma
: special reference to imaging diagnosis. J Hepatol. 1999;30:125–130
29. Sun GZ, Zhao LiJH, Zhao GQ, Wang SX, Kong SL. Detection of AFP-L3% using agglutinin-coupled spin column to be used in diagnosis of hepatocellular carcinoma
. Zhonqhua Yi Xue Za Zhi. 2008;88:1986–1988