MULTIPLEXED PROTEOMIC APPROACH FOR IDENTIFICATION OF POTENTIAL SERUM BIOMARKERS IN HEPATOCELLULAR CARCINOMA PATIENTS WITH NORMAL AFP

초록

Background: The high mortality associated with Hepatocellular carcinoma (HCC) is partly due to diagnosis of HCC at advanced stage where effective therapies are lacking. AFP, as a serologic indicator of HCC, often elevated in non-cancerous condition and not elevated in as many as 40% of HCC. The sensitivity and specificity of ultrasonography have been reported to be 71%, 78%, respectively. Other serum markers such as DCP, GP73 levels are known to be elevated in late stage of HCC or advanced liver disease. In recent advance in mass spectrometry based multiplexed targeted protein quantification facilitates the discovery and validation of potential biomarkers from large number of clinical samples in a high-throughput manner. Aims: Aims of our study were to analyze the serum proteins in the processes of hepatocarcinogensis and to identify and validate the HCC specific potential serum biomarkers for the early detection of HCC using multiplexed proteomic techniques such as nano-LC/MS/MS and MRM (multiple reaction monitoring) in HBV infected HCC patients. Methods: Pooled sera from liver cirrhosis and HCC with normal or elevated AFP were used for analysis. Differentially expressed proteins in HCC patients were identified through the intensive protein profiling of serum proteomes using nano-LC/MS/MS and the selected multiple biomarkers were validated quantitatively and simultaneously by using triple quadruple mass spectrometer. Results: A total of 267 proteins were identified and quantified in the sera of HCC patients, of which 28 proteins and 86 proteins showed abundance changes of at least two fold elevations or reduction, respectively in the pooled sera of HCC with normal AFP. Through the gene ontology analysis of differentially expressed proteins in HCC patients, many of the identified proteins were categorized into the inflammatory response, wound healing, acute phase response, defense response, response to stress, localization, immune process, cellular developmental process, complement activation and so on. Selected target proteins among the differentially expressed proteins were validated with extended cases of patients’ sera by MRM method. Conclusion: With the advantage of multiplexed proteomic approach and MRM, we discovered several serum biomarker candidates associated with HCC, which could be developed as potential biomarkers for early diagnosis of HCC.