The Mehta laboratory has identified a number of potential biomarkers of HCC. These biomarkers, which are fucosylated serum glycoproteins, can be used alone or in combination with other biomarkers and clinical factors in a diagnostic algorithm. The lead biomarker is fucosylated kininogen, which combines with alpha feto protein (AFP), age, gender, alkaline phosphatase (ALK) and Alanine transaminase (ALT) to greatly increase the detection of HCC as compared to the currently used markers. In collaboration with Dr. Goldman we have recently performed glycan analysis of human kininogen from patients with cirrhosis or patients with HCC in the background of cirrhosis. Increased levels of fucosylation were observed in patients with HCC as compared to patients with just cirrhosis. Through glycoproteomics we identified the specific sites of glycan modification in HCC as well. Surprisingly, the level of fucosylation on one specific site had substantially better discriminatory ability than lectin analysis of the whole protein or the application of this fucosylated kininogen algorithm. When comparing the methods on the exact same samples, the lectin-ELISA for fucosylated kininogen resulted in an AUROC of 0.65, and the diagnostic algorithm containing AFP, ALT, AST, age and gender increased this to 0.91. Amazingly, the fucosylation from one specific glycan site resulted in an AUROC of 0.99 on these exact sample samples. Clearly, our results highlight the benefit of looking at one specific site of glycan modification as opposed to the whole protein and suggest a new avenue for the development of better biomarkers of HCC. Currently the mass spectrometry based glycopeptide method is the only way to examine the glycosylation of one specific site of N-linked glycan. However, it is not a clinically or commercially viable method for use in large scale studies. Thus, we propose to create a new method for the analysis of glycopeptides. We refer to this method as a Lectin-Tryptic-ELISA. In this method, an antibody is made to a specific peptide (non glycosylated) that is formed from the tryptic cleavage of kininogen. This peptide is synthesized and used as an standard immunogen. The key point is that in human kininogen, the selected peptide contains the altered N-linked glycan of interest. For the Lectin-Tryptic-ELISA, the serum is treated with trypsin and allowed to go to completion. Next, the tryptic peptide (in serum this would be a glycopeptide) of interest is captured through a simple antibody-antigen reaction and the level of fucosylation detected using a lectin. A key requirement is knowledge of the site of fucosylation and the development of the antibody. In principle, the assay is very simple and will depend upon the ability to develop peptide specific antibodies. Currently, commercial antibody development utilizes peptides for antibody generation and thus, this aspect of the proposal can be achieved easily through outside vendors. The key thing will be to identify the specific peptides to target and refinement of the conditions that allow for peptide generation and assay performance. ! !
