AFP, PIVKAII, GP3, SCCA-1 and follisatin as surveillance biomarkers for hepatocellular cancer in non-alcoholic and alcoholic fatty liver disease

Hepatocellular carcinoma (HCC) is a major health problem worldwide, with more than 500,000 cases diagnosed annually [1]. While the incidence of HCC has reportedly risen over the last 5–8 years, the survival of those affected has not changed significantly in the last two decades [1‐3]. This is related to both its late detection the lack of effective therapies for advanced stage disease [4]. Up to 80% of HCCs develop against a background of cirrhosis of the liver and while we believe that surveillance of the at risk cirrhotic population could aid earlier detection of the disease and decrease the cancer related mortality rate, our present success is limited by the lack of sensitive biomarkers. Currently, standard surveillance includes a combination of 6 monthly abdominal ultrasound scan (USS) and serum alphafetoprotein (AFP) measurement, but this strategy does not reliably detect early disease. The diagnostic performance of AFP is inadequate[5] as it is only elevated in 40–60% of cases, while abdominal USS is difficult in cirrhotic nodular livers and notoriously user dependent[6]. Alternative serum biomarkers are being actively sought and proposed candidates include Prothrombin Induced by Vitamin K Absence (PIVKA-II), glypican-3 (GP3), and more recently, Squamous Cell Carcinoma Antigen -1 (SCCA-1).

PIVKA-II is an abnormal prothrombin identified as an HCC biomarker in 1984 [7] and since reported elevated most notably in advanced cases with portal vein invasion [8, 9]. It is proposed that PIVKA-II may be useful primarily as a prognostic biomarker, predicting rapid tumour progression and a poorer prognosis [10]. The oncofetal antigen glypican3 (GP3) is a heparan sulfate proteoglycan that is expressed in more than 70% of HCC[11]. When combined with AFP it has a sensitivity of up to 82% for HCC detection on a background of viral hepatitis [12]. SCCA-1 is a member of the high molecular weight serine protease family called serpins [13] initially reported elevated in epithelial tumours such as the cancer of the head [14] and more recently in the serum of individuals with HCC and cirrhosis. [15]

On a global scale, viral causes of chronic liver disease are the commonest predecessors of HCC and these proposed biomarkers [16] have largely been studied in this disease group. Our own HCC patients have tumours arising predominantly on a background of alcoholic (ALD) and non-alcoholic fatty liver diseases (NAFLD). Here we present the data on a cross-sectional study comparing the efficacy of these markers, as well as a novel candidate biomarker, Follistatin, for the diagnosis of HCC arising on a background of steatohepatitis related cirrhosis. Follistatin is a secreted monomeric protein overexpressed in rat and human liver tumours and reportedly contributing to hepatocarcinogenesis by the inhibition of activins [17]. Follistatin mRNA was markedly overexpressed in HCC cell line microarray studies performed in our own laboratory (unpublished data).

Our data indicate differences in biomarker performance in NAFLD and ALD patients compared to performances reported in viral hepatitis. Neither PIVKAII, GP3, SCCA-1, nor the novel candidate Follistatin, has a role independent of AFP in HCC surveillance in steatohepatitis related cirrhosis. We show that the combination of AFP and PIVKAII is more valuable than AFP alone and suggest this approach be adopted as standard surveillance in this disease group.

Serum AFP, PIVKA-II, GP3 and SCCA-1 levels were determined in 50 patients with HCC arising in a background of ALD or NAFLD cirrhosis. A control group of 41 patients with cirrhosis from ALD/NAFLD was used for comparison. The clinical characteristics of the patients in these groups are shown in Table 1.

The increasing incidence of HCC[3], compounded by the fact that the majority of these tumours are diagnosed at a late stage when curative treatments are not possible[19], has prompted the international community into performing regular surveillance of high risk individuals. Unfortunately, surveillance programmes are hindered by the poor performance of the commonly used serum marker, namely AFP[6], even in combination with abdominal USS. A tremendous amount of effort has been and continues to be applied to the search for improved HCC biomarkers. As yet, none has proved superior to AFP in performance, but in combination some may have complimentary roles in HCC arising on a background of viral hepatitis [20]. Our own particular concern relates to the marked increase in the prevalence of ALD and NAFLD related HCC on our own unit.

In our study, serum AFP performs moderately well as a biomarker of HCC in ALD/NAFLD patients, with a sensitivity of 58% (15 ng/ml) in combination with a specificity of 100%. The AASLD recommended cut off level for diagnosis of HCC is 200 ng/ml [21], although lower levels, particularly if rising, should be deemed suspicious and followed very carefully. In ALD/NAFLD patients, where a mild to moderately elevated but stable AFP level similar to that occasionally observed in individuals with viral hepatitis is rare, it may be possible to attach a more sinister connotation to much lower levels of expression. While this data is encouraging, the sensitivity of AFP is not good enough for it to be used in isolation, as over a third of cancers will be missed.

Both the sensitivity and specificity for PIVKAII as an HCC biomarker were in the order of 80% at a level of 20 ng/ml. The addition of PIVKAII serum analysis to that of AFP increases the combined sensitivity to 94%. While this is at the modest expense of the specificity (reduced to 80.5%), the combination of both AFP and PIVKAII analyses may well be justified in our patients. It should be noted, however, that the added benefit is only in the detection of more advanced disease – as indicated in previous viral hepatitis studies and confirmed in our own NAFLD/ALD patients -the encouraging performance of PIVKA-II is predominantly a result of detection of larger, more advanced cancers.

Assessment of the other candidate biomarkers was disappointing. Both the sensitivity and specificity of GP3 were poor in our patient set, indicating that it has no role at all in the surveillance of HCC in individuals with steatohepatitis related cirrhosis. Follistatin is an expressed transcript in fetal liver and has previously been identified by microarray as an up-regulated gene in HCC relative to dysplastic nodules [22]. Although we had high hopes for this activin antagonising protein, [23] based on both our preliminary microarray data and a pilot study in immune depleted sera, the ELISA data assessing its discriminatory function between cirrhotic individuals with and without HCC was poor. The discrepancy between the western and ELISA data is most likely a result simply of assessing a greater number of patients using the latter method, but it is also that follistatin, or an isoform of it, was enriched during the column preparation phase of the serum of HCC patients assessed by western blotting. Perhaps the most surprising of results, however, in this homogenous group of patients with steatohepatitis related HCC, was the disappointing performance of SCCA-1. SCCA-1 has previously shown promise, particularly as an AFP complementary biomarker, in viral hepatitis related HCC [24, 25]. In our study in NAFLD/ALD, however, there was no significant difference between levels in patients with and without HCC and while the combination with AFP does modestly improve its sensitivity (78% from 56%), this is at an unacceptable cost to specificity (73% from 100%). Why this serum protein should be significantly elevated in the serum of HCV related HCC patients relative to HCV cirrhosis alone, and not similarly elevated in steatohepatitis related HCC patients relative to steatohepatitis cirrhosis alone is unclear. It is possible that the study of these novel candidate biomarkers complexed to immunoglobulins, rather than the study of their free forms, may yet improve their performance, as has been shown for other biomarkers [26]. Whether or not there is room for improvement, however, our SCCA-1 data clearly indicate that as we come to consider further candidate biomarkers it is important to assess HCC arising in different disease backgrounds independently when performing validation studies.

In summary, while we propose the combination of AFP and PIVKAII for HCC surveillance in NAFLD/ALD patients, the search for novel biomarkers of early HCC disease should continue.