The prevalence of chronic liver disease is increasing steadily in the UK, as is the population at risk of developing and dying from HCC. An increasing incidence of HCC has already been documented  and this is unfortunately compounded by the majority of tumours being diagnosed at a late stage when curative treatments are not possible.  Regular surveillance of high risk individuals is recommended but is presently hindered by the poor performance of the commonly used serum marker, AFP,  even in combination with abdominal USS. A significant 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. [15, 16] Our particular concern relates to the marked increase in the prevalence of ALD and NAFLD related HCC on our own unit. These individuals are often over 65 years of age and have cardiovascular co-morbidities precluding curative resection or transplantation. Much attention has been focused on validating quantitative fibrosis or cirrhosis markers in these individuals, with combinations of serum markers  showing encouraging if still suboptimal improvements in performance. Imaging methods such as the fibroscan may improve the efficiency of cirrhosis detection [17–19], but presently this technique needs to be interpreted with caution in obese individuals. [19–23] The need for further improvements in serum biomarkers for early detection of both advanced fibrosis and HCC grows ever more pressing.
We have applied a proteomic strategy using an optimised method of patient serum preparation in order to identify candidate biomarkers of either cirrhosis or HCC. This includes immune depletion prior to separation of serum by 2D gel electrophoresis. While this might remove some key biomarkers, we believe that this strategy improves the sensitivity of the technique applied to the remaining serum proteins. Having identified a number of spots able to discriminate between patients in at least one of our pre-defined disease groups, namely pre-cirrhotic NAFLD, cirrhotic NAFLD, and cirrhotic NAFLD with cancer, we selected candidates for identification by peptide mass spectroscopy.
The novel serum protein CD5L was identified in the serum of cirrhotic individuals with and without HCC by our proteomic strategy. CD5L, also known as Sp alpha, is a soluble protein belonging to group B of the scavenger receptor cysteine-rich (SRCR) superfamily for which little functional information is available. It is expressed by macrophages present in lymphoid tissues (spleen, lymph node, thymus, and bone marrow), and it binds to myelomonocytic and lymphoid cells, suggesting that it may play an important role in the regulation of the innate and adaptive immune systems. It was recently identified in the sera of individuals with liver fibrosis related to HCV infection and, based on its proposed role in immune system regulation, was thought most likely associated with viral infection rather than cirrhosis. While this may yet be true, as a mRNA transcript, it has been previously reported upregulated in HCC relative to dysplastic nodules. We have investigated the potential of CD5L as a candidate biomarker for either advanced liver disease, or advanced liver disease and cancer. Our ELISA assay indicated a poor performance for CD5L as a surveillance tool for HCC, but again suggested value for cirrhosis detection. Our data clearly indicate, however, an association with cirrhosis in individuals without viral infection, which is independent of the presence or absence of histologically assessed inflammation. Our CD5L mRNA expression data from pre-cirrhotic NAFLD liver biopsies indicate an increase in association with fatty liver disease which, again, is not altered by the grade of either fat or inflammation. As CD5L does not increase incrementally with the level or stage of fibrosis, we propose that its dramatic increase in the serum of individuals with a background cirrhosis reflects hepatocyte regeneration, rather than the advanced fibrosis per se. The recent identification of CD5L by microarray as one of 30 mRNA transcripts expressed in patients with cirrhosis with a 'high risk' of HCC development  was one reason for our focus on this serum protein. Certainly some of our HCC patients had markedly high CD5L levels, while those with cirrhosis who had particularly high levels may be at high risk and may yet develop HCC. As a cirrhosis biomarker, the ROC analysis for CD5L indicates an accuracy of 72%. While this falls short of the accuracy of the European Liver Fibrosis (ELF) panel, recently validated in NAFLD patients and having an AUC of 0.9 for advanced fibrosis, CD5L is a single biomarker whose performance in conjunction with others may yet improve. In addition, it may have a value complementing that of AFP, which is predominantly elevated in patients with advanced HCC, in highlighting individuals with cirrhosis who are at greater risk of HCC development. At a level > 200 ng/ml, CD5L had a specificity for cirrhosis of 96% and a specificity for cancer of 60%.
Particularly prominent on our gel images were three spots identified as apolipoprotein A1 (ApoA1). ApoA1 is the major protein component of high density lipoprotein in plasma. It promotes cholesterol efflux from tissues to the liver for excretion and it is a cofactor for lecithin cholesterolacyltransferase which is responsible for the formation of most plasma cholesterol esters. It is not that surprising that this liver function associated protein alters in the serum of individuals with chronic liver disease and this has been previously reported in serum proteomic studies. The mass of spots 1 and 2 (Figure 1) varied by approximately 30 daltons using MALDI analysis (Additional files 1 and 3) and are most likely attributable to post translational modification by oxidation. Notably, these two spots were reduced in cirrhotic individuals relative to those with pre-cirrhotic NAFLD, which is in keeping with previous reports and validates the clinical relevance of our methodology. In fact, a reduction of ApoA1 in the serum of patients with cirrhosis has remained a constitutive part of combined peptide panels used to predict fibrosis for a number of years. [6, 29] In addition, however, we identified a novel isoform - spot 3. The difference in mass between spots 1 and 2 and spot 3 was much greater and in the order of 900 daltons (Additional files 1, 3 and 5). MALDI MS analysis of this spot has identified it as pro-Apolipoprotein A1 (Additional files 6 and 11), similarly detected and reported by an independent group of researchers studying patients with lung cancer.  Pro-Apolipoprotein A1 is proposed as a novel serum marker of brain metastases in lung cancer patients  and there may well be a rationale for its upregulation also in HCC patients. ApoA1 is secreted as the proprotein (pro-Apolipoprotein A1/spot 3) and is then cleaved, regulating its activation for lipid binding, by a metalloproteinase. One candidate metalloproteinase responsible is the c-terminal procollagen endoproteinase, Bone morphogenic protein 1 (BMP-1).  BMP-1 is secreted by the liver, but protease inhibitors, such as alpha-2-macroglobulin (A2M), are also secreted by the liver, often at elevated levels in inflammation or chronic disease. Either a reduction in BMP-1, or an increase in inhibitors such as A2M - as reportedly occurs in HCC , could block the maturation of pro-Apolipoprotein A1, hence contributing to relative increase in this isoform (spot 3). In fact, the relative decrease in mature apoA1 in cirrhotic patients (spot 2) may also reflect increases in protease inhibition and A2M has similarly contributed significantly to serum diagnostic tests for fibrosis. [32, 34] Further investigation has yet to determine the sensitivity and specificity of pro-Apolipoprotein A1 as a novel candidate biomarker in patients with chronic liver disease and HCC, but our pilot study suggests that its up-regulation is specifically a feature in the serum of patients with HCC.