Serum Glyco-Markers of Early Hepatocellular Carcinoma Using a Mass Spec Approach

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Abstract: Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death worldwide and is rising in incidence in the US. Over 80% of patients in the US with HCC have underlying cirrhosis, and professional societies guidelines recommend surveillance in all patients with cirrhosis to facilitate early detection. Unfortunately, only 20-30% of patients are detected at an early stage when potentially curative treatments are possible. Our proposal, based on our strong preliminary data evaluating glycoproteomic profiles, addresses the clear unmet need for accurate early HCC detection biomarkers. Unique changes in glycosylation in proteins, which involve structural changes in glycan groups, have been shown to be important serum biomarkers for early HCC detection. Importantly, even subtle changes involving minor structures can be very specific in differentiating cirrhosis versus early stage HCC. In this study, we will use a mass spectrometry approach, which has proven to be a highly sensitive and accurate way to detect and quantitatively monitor glycan structural changes. Our published mass analysis discovery work, tandem mass spectrometry measurements, and novel bioinformatic tools for glycan and glycopeptide analysis demonstrate these minor changes in structure can act as promising early HCC detection biomarkers. In aim 1 these methods will be demonstrated for glycopeptide screening from serum using novel mass spec assays using parallel reaction monitoring (PRM) analysis based on a stepped HCD fragmentation method which can ultimately be used to distinguish early stage HCC from cirrhosis. The PRM method is a targeted assay which can distinguish small changes in glycan structure and can be multiplexed to monitor large numbers of markers simultaneously. In addition, using separations coupled to the PRM strategy, we will be able to distinguish different isomeric forms of fucosylation and sialylation in glycan structures which may contain important disease related markers. In aim 2 the methods developed herein will be optimized and then applied to a large confirmation set of 300 early stage HCC and cirrhosis samples for 20 target glycopeptides from Haptoglobin and to several other potential glycopeptide markers discovered and evaluated in our prior work. In aim 3 we will then validate the biomarker performance in a blinded phase II biomarker study using a large set of 800-1000 early stage HCC and cirrhosis patients, diverse with regard to sex, race/ethnicity, and liver disease etiology. Through these aims, we will identify a panel of glycopeptides that can serve as highly accurate biomarkers for early HCC detection.