AFP was discovered in 1956 by Bergstrand and Czar[15], who used paper for electrophoretic separation of human fetoprotein in serum. The first reports on the usefulness of AFP as a diagnostic marker for HCC were presented in 1964 by Tatarinov and in 1968 by Abelev[16]. AFP is a glycoprotein with molecular weight of around 70 kDa, synthesized in the endodermal cells of the yolk sac during early fetal development, and then in embryonic hepatocytes[17]. It reaches a maximum serum concentration of 3 g/L in the 12-16th wk of fetal life and during the next 18 mo, AFP values decrease and normalize[18]. Its synthesis in adult life is repressed. Pathological elevation is seen in hepatocyte regeneration and hepatocarcinogenesis. Numerous data have proved that significantly higher AFP serum levels accompany various liver diseases (viral hepatitis, liver cirrhosis, liver tumors: primarily HCC and hepatoblastoma, also metastasis in 5%-10% cases), other neoplasms (mainly cancers of the digestive tract: pancreas -24%, stomach -15%, large intestine -3%, and gallbladder). Positive predictive values (PPV) for AFP are paradoxically significantly lower among patients with HCC viral etiology than non-viral (PPV: 70% vs 94%, P < 0.05)[19]. It has been confirmed on numerous occasions that AFP serum concentration increases in parallel with HCC tumor size. For this reason AFP has to be considered ‘the golden standard’ for HCC serum markers. However, the usefulness of AFP testing for the population at risk should be seriously questioned. AFP diagnostic values for this assay are undoubtedly poor. AFP specificity varies from about 76% to 96% and increases with elevated cut-off value. Simultaneous sensitivity decreases much more from about 25% for potentially resectable tumors of less than 3 cm in diameter to about 50% for lesions of > 3 cm in diameter (Table 3)[20,21]. 20%-30% AFP sensitivity coincides with cut-off values > 100 μg/L, which means that 70%-80% of the results - of a conventional test used as “a gold standard” for HCC diagnosis - are falsely negative. Based on these data 70%-80% of liver tumors, normally resectable are non-detectable. For this reason only one patient out of 5 receives potentially curative treatment. Unfortunately, the remaining majority unfortunately do not undergo treatment or are subjected to it too late.

Nowadays it is believed that early HCC diagnosis is presently considered feasible in 30%-60% of the cases in developed countries. Tumors smaller than 2 cm in diameter represented < 5% of cases in the 1990s in Europe, whereas now they represent up to 30% of cases in Japan[6]. Significantly more effective surveillance strategies in Japan lead to earlier HCC detection and earlier qualification for effectively curative radical surgery, with very good postoperative survival rates[22]. According to these assumptions, European and American experts have defined trends and expected aims of surveillance policies in Western countries in 1980-2020. The applicability of potentially curative treatments have been divided into 3 periods: until 1990: 5%-10% of cases; 1990-2010: 30%-40% of cases; and 2010-2020: 40%-60% of cases[6]. Limitations of available therapies constitute a major challenge for diagnostic techniques, which are, most of all, modern visual methods and novel HCC specific biomarkers.

Numerous studies analyzing the chemical structure of AFP have shown that different sugar moieties of the bonds determine their binding capacity to lectin lens culinaris agglutin (LCA)[23]. Taking those facts into consideration, Polish scientists, Breborowicz et al[24] identified in 1981 3 main glycoforms, namely AFP-L1, AFP-L2, AFP-L3. AFP-L1, the non-LCA-bound fraction, is the major AFP isoform in the serum of nonmalignant hepatopathy patients (chronic hepatitis, cirrhosis). AFP-L2 presents intermediate binding capability with its serum concentration increasing during pregnancy, and it is also present in cases of yolk sac tumors. AFP-L3, as the LCA-bound fraction, is the major glycoform in the serum of HCC patients[24-26]. It can be detected in 35% of patients with small HCC (< 3 cm). Some clinical studies have indicated that AFP-L3 can be detected 9-12 mo ahead of changes using visual techniques[27,28]. Sensitivities of AFP-L3 in detecting HCC range from 45% for lesions < 2 cm to > 90% for changes > 5 cm in diameter[20]. The specificity is more than 95%[26,29]. Fucosylation rate can be used in clinical practice (AFP-L3/AFP total). It has been confirmed that the ratio of more than 10% is closely associated with worse liver function and poorer tumor histology with implications such as larger tumor mass, a more invasive/malignant character, and earlier metastatic tendency[27,28]. Therefore AFP-L3 could be used as a reliable early HCC biomarker and a valuable indicator of poor prognosis. It is possible to achieve particularly accurate results for HCC screening with the use of AFP-L3 in combination with one of the 3 newly-discovered AFP glycoforms which can also be used as single tests, that is AFP-P4, AFP-P5 (E-PHA), and monosialylated AFP (IEF)[19].

DCP, also known as PIVKA-II (protein induced by vitamin K absence or antagonist-II), is an abnormal, inactive prothrombin, lacking carboxylation of the 10 glutamic-acid residues in the N-terminus, which is the result of an acquired post-translational defect of the prothrombin precursor in HCC cell lines. DCP was discovered in serum of patients during their anticoagulant therapy with a vitamin K antagonist. In 1984 Liebman et al[27] first described a higher DCP level both in patients with HCC and in cases of HCC recurrence after surgical resection, suggesting the usefulness of DCP as an HCC biomarker. It has been proved that significant concentrations of serum DCP are present in 50%-60% of all HCC patients, but in only 15%-30% of early HCC cases[31]. In the analyses of Nakagawa et al[32] the sensitivity of this test is 48%-62% and the specificity is 81%-98%. The diagnostic value of DCP as a biomarker is approximately comparable with AFP. Grazi et al[33] proved that AFP and DCP are not correlated, so the combination of those markers significantly improves HCC detection: sensitivity 74.2%, specificity 87.2%. Carr et al[34] reported in 2007 interesting data based on prospective analyses of 99 patients with non-resectable HCC verified using liver biopsy (Table 4)[34-36]. Nowadays the best way to diagnose HCC is the use of AFP-L3 with DCP analyzed by immunoenzymatic higher sensitivity methodology[33,37].

AFU is a normal lysosomal enzyme which hydrolyzes sugars containing L-fucose. In 1984 Deugnier et al[38] first reported that AFU is overexpressed in patients with HCC liver changes. It has been proved that the values of AFU serum concentration were not correlated with the tumor size and were frequent in early HCC cases[23]. Tangkijvanich et al[39] indicated that the sensitivity and specificity of AFU were about 80% and 70% respectively, in contrast with 40% and almost 100% for AFP. A simultaneous determination of both markers can improve the sensitivity to 82%[39]. This conclusion suggested that AFU could serve as a valuable supplement to AFP in early detection of HCC, similar to another popular serum enzyme - γ-glutamyl transferase.

GGT is a glycosylated membrane enzyme which activity is modulated in many physiological and pathological conditions, including differentiation and carcinogenesis[40]. It is mainly secreted by the hepatic Kupffer cell and endothelium of the bile duct. GGT is also overexpressed, similar to AFP, by fetal hepatoblasts and HCC cell lines[23]. The total serum GGT, a generally accepted cholestatic marker, has poor HCC specificity so can be useful only supplementary to AFP and other newer biomarkers for more effective HCC screening. In 1965, Polish scientists Kokot et al[41] separated the serum γ-glutamyl transferase into 3 to 4 bands by means of paper electrophoresis[38]. Since then, other methods have been used, that is, separation of GGT bands by means of starch gel (Orlowski et al[42]), cellulose acetate (Hitoi et al[43]), agarose gel (Hetland et al[44]), polyacrylamide gel electrophoresis (Kojima et al[45]; Suzuki et al[46]; Sawabu et al[47]; Kew et al[48]), and polyacrylamide stage gel plate (Xu et al[49]). Xu reported that they had fractionated 9 to 11 activity bands of GGT, in which GGTII was found in the sera of all patients with hepatoma. The positive rate of GGT was 90% and no correlation was observed with AFP[49] and DCP[50]. After 10 years of follow-up they reported that GGTII was positive in 90% of cases with HCC and negative in most patients with acute and chronic viral hepatitis, extrahepatic tumors, in pregnant women, and in healthy controls[51].

GPC-3 is an oncofetal protein being one of the members of heparan sulfate proteoglycans anchored to the plasma membrane through glycosylphosphatidyinositol[52]. GPC-3 is normally involved in the regulation of cell proliferation and survival during embryonic development and functions as a tumor suppressor. It has been reported to be downregulated in breast cancer, ovarian cancer and lung adenocarcinoma[53] but upregulated in HCC[54]. GPC-3 is absent in hepatocytes of healthy subjects and patients with nonmalignant hepatopathy, and can be detected in about 50% of HCC patients and 33% of HCC patients seronegative for both AFP and DCP. The specificity of GPC-3 is 100%[55]. Some clinical studies have indicated that the simultaneous determination of GPC-3 and AFP could significantly increase the sensitivity in HCC detection, without a reduction in the specificity[56]. More trials have confirmed the diagnostic value of 2 other, newly-discovered membranous proteins: Golgi protein 73 (GP73) and mucin 1 (MUC-1).

GP73 is a resident Golgi protein, shown to be upregulated in hepatocytes of patients with acute hepatitis[57] and cirrhosis[58] and in the sera of patients with HBV- and HCV-related HCC[59,60]. Marrero et al[60] reported a sensitivity of 69% and a specificity of 75% in HCC versus cirrhotic patients, indicating its superiority in comparison with AFP: sensitivity 30%, specificity 96%.

MUC-1 is a membrane protein expressed in many epithelial cells, but overexpressed in patients with breast cancer[61], inflammatory lung diseases[62], and HCC[63,64]. Moriyama et al[63] demonstrated expression of MUC-1 in HCC cells and in serum of patients with HCV-related HCC. Gad et al[64] reported specificity of 99%, sensitivity of 87% for combined MUC-1, DCP and AFP in Japanese and Egyptian patients with HCC.

SCCA represents a family of serine proteases of high molecular weight, also known as serpins. There are 2 homologous genes: SCCA1 and SCCA2, encoding 2 different SCCA isoforms, both expressed in many normal squamous epithelial cells. Increased SCCA levels have been detected in head and neck cancers and other epithelial malignancies, including cervix and lung. Recently Pontisso et al[65] first reported a high SCCA expression in HCC tissues, which seems very interesting, as liver does not possess squamous epithelial cells. Hepatocytes, however, share a common embryogenic origin. The sensitivity and specificity for SCCA in HCC diagnosis are 84% and 46% respectively. The complementary strengths (high sensitivity/low specificity) and total AFP (low sensitivity/high specificity) suggest that the combination of the 2 markers should be of more value for screening, and in fact it leads to a diagnostic accuracy of 90%[66].

A new step for HCC testing is represented by forming known antigens (AFP, SCCA, DCP) into immunocomplexes (IC) with immunoglobulins of the IgM class. Monitoring of SCCA-IgM IC, AFP-IgM IC and DCP-IgM IC appears to be a much more advantageous approach for detecting patients with small HCC changes[67-70]. Giannelli et al[67] confirmed that the combined use of AFP IgM IC, SCCA and SCCA IgM IC in patients displaying low levels of AFP (< 20 IU/mL) identified 25.6% HCC. This study suggests that the use of a combination of all these markers in clinical practice provides a non-invasive and simple test that could increase the accuracy of HCC diagnosis. According to the results of Beneduce et al[68]: SCCA IgM IC significantly improves accuracy of HCC testing with sensitivity of 100%, specificity of 70%, PPV of 100%, and negative predictive value of 83%; AFP-IgM IC is a complementary serological marker to free AFP and the combination of these biomarkers may be useful in the diagnosis of liver cancer[69]; DCP-IgM IC in HCC patients was not associated with an increase in IgM concentration and was more frequently detected in HCC patients than DCP and AFP, strengthening the diagnostic role of IgM immune complexes in liver cancer[70]. The novel generation of HCC biomarkers seems very promising as it introduces new hope in supporting US for more accurate HCC screening.