GlycoFibroTest Is a Highly Performant Liver Fibrosis Biomarker Derived from DNA Sequencer-based Serum Protein Glycomics

Liver fibrosis in chronic hepatitis patients is currently assessed by liver biopsy. However, this expensive technique can be accompanied by severe complications (1), sampling error (2), and up to 20% interlaboratory variance (3). These shortcomings make it unsuitable for regular monitoring of the patient's condition. Nevertheless monitoring the progression of fibrosis would allow determination of the long term responses to existing therapies (e.g. interferon/ribavirin) and emerging candidates for therapy (e.g. small interfering RNA against heat shock protein 47 (4)). Moreover it is important to follow up patients who have clinically significant fibrosis (METAVIR F2–F4 (5)) and to detect cirrhosis in an early stage because cirrhosis substantially increases the risk of developing hepatocellular carcinoma (HCC)1 (6). Therefore, non-invasive markers are needed as alternatives or at least complements to the liver biopsy golden standard for monitoring fibrosis evolution over time. Increased levels of extracellular matrix components are useful for monitoring evolution of liver fibrosis, and in this context serum hyaluronate has been thoroughly studied. This linear polysaccharide is a constituent of the extracellular matrix of peripheral tissues (7). Its concentration in the serum increases as liver fibrosis progresses because of an increase in its synthesis by activated hepatic stellate cells and in later fibrosis stages also because of reduced clearance by the liver sinusoidal endothelial cells (8, 9). In patients with clinically significant fibrosis, α2-macroglobulin concentrations also increase (10). This acute phase protein inhibits matrix metalloproteinases, and its production by hepatocytes and activated stellate cells is up-regulated during fibrosis. The aspartate transaminase to platelets ratio index (APRI) (11) measures two routinely assessed parameters: aspartate transaminase concentration and platelet count. Thrombocytopenia during fibrogenesis in patients infected with hepatitis C virus (HCV) can be attributed to hypersplenism (12) as well as to reduced production of thrombopoietin by hepatocytes (13). The increase of serum aspartate transaminase concentration during fibrosis progression may be due to reduced clearance by the liver (14). APRI can predict significant fibrosis and cirrhosis (11). In an attempt to improve upon these biochemical parameters, several complex regression and classification algorithms have been designed. Tissue inhibitor of metalloproteinases-1, α2-macroglobulin, and hyaluronate are the three components of the FibroSpect test (15) (Prometheus Laboratories, San Diego, CA). Tissue inhibitor of metalloproteinases-1 is produced in the liver mainly by stellate cells and acts as a specific inhibitor of matrix metalloproteinases. Its concentration is increased in advanced liver fibrosis. FibroSpect components are measured in laboratories of the commercial supplier, which should ensure appropriate quality control and reliability. FibroSpect has been validated in HCV patients by some groups (16, 17). FibroTest (18) (Biopredictive, Paris, France) is a binary logistic regression model designed to distinguish between chronic HCV patients who have clinically significant fibrosis (F2–F4) and those who do not (F0–F1). It consists of five serum biochemical markers (α2-macroglobulin, apolipoprotein A-I, γ-glutamyl transpeptidase, haptoglobin, and total bilirubin) as well as the patient's age and gender. The investigators who developed and commercialized the FibroTest algorithm have published several studies to validate it. However, only a few other groups have independently assessed the performance of FibroTest, and few studies compared the performance of the algorithm with the performance of the individual parameters constituting the model and with other fibrosis correlates. Moreover as FibroTest is in principle accessible only via a Web interface in which one enters the clinical chemistry values measured in their own laboratory, it may be quite difficult to assure the quality of output. Indeed measured α2-macroglobulin and apolipoprotein A-I values can be different when measured on two different analyzers even when they have been calibrated against the same standard (Beckman-Coulter, Krefeld, Germany versus Dade Behring, Eschborn, Germany) (19). This is an important issue given the strong dependence of FibroTest on its α2-macroglobulin component (see below). FibroTest results have to be interpreted cautiously because measuring five components not only increases overall variance but also broadens the range of possible interferences (2), such as inflammation (increase in either haptoglobin or α2-macroglobulin), Gilbert syndrome (increase in unconjugated bilirubin), and a decrease in haptoglobin and/or elevation of unconjugated bilirubin because of hemolysis. Several new approaches have recently been developed to assess liver fibrosis. FibroScan (20) (Echosens, Paris, France) uses transient elastography to measure the stiffness of the liver, which correlates with the amount of scar tissue formed. Briefly when an elastic shear wave is introduced in the liver by low frequency vibrations, its velocity will depend on the stiffness of the tissue. Although the test is rapid, reproducible, and useful in diagnosing advanced fibrosis (F3–F4), it is rather expensive, and measurements are difficult to perform in obese patients. Increased values are also seen in patients with acute liver inflammation. In 2001 (21) we introduced an ultrasensitive method to profile protein-linked N-glycans using a slab gel-based DNA sequencer. We used this method to identify GlycoCirrhoTest as a serum N-glycome-derived biomarker for advanced liver disease (cirrhosis) (22). However, the method was still rather cumbersome and hard to use. Here we introduce a more simple assay (Fig. 1) that is optimized to prepare 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled N-glycans from total serum protein. All reaction steps can be done in a 96-well plate format only requiring a thermocycler and reagent transfer steps making it suitable for clinical implementation. These samples can then be analyzed on state-of-the-art and high throughput DNA sequencers without changing the settings needed for genomics (23). From a simple serum N-glycome analysis on standard DNA analyzers, three biomarkers can be derived (Fig. 2). GlycoCirrhoTest (22) can distinguish compensated (early stage) cirrhosis from non-cirrhotic chronic liver disease with 79% sensitivity and 86% specificity (both 100% in decompensated cirrhosis). GlycoHCCTest helps in distinguishing cirrhosis patients with HCC from those without (24). Moreover we found indications that GlycoFibroTest, which is the ratio between the agalacto glycans shown in Fig. 2 (NGA2FB) and the fully galactosylated triantennary glycans (NA3; Fig. 2), appeared to rise gradually with increasing fibrosis stage. Consequently this ratio might be a promising marker for the follow-up of liver fibrosis progression. To validate this GlycoFibroTest, we undertook the current study. Fig. 1. Serum N-glycomics sample preparation protocol. APTS-labeled N-glycans are prepared with consecutive reactions (1–6) that are performed in a 96-well plate incubated in a thermocycler (see “Experimental Procedures”). Reagent A, 5% ... Fig. 2. Structure and nomenclature of the N-glycans mentioned in this study (upper panel) and N-glycome-derived biomarkers for chronic liver disease (lower panel). From the N-glycan profile derived from total serum protein (N-glycome), one can derive three glycomics-based ...