Molecular characterization of blood-circulating protein biomarkers for non-invasive and rapid diagnosis of Hepatocellular Carcinoma (HCC)

Abstract

Major burden of cancer is attributed to Hepatocellular carcinoma. Third world countries are more prone to HCC because they possess poor healthcare systems. Annually around 7.6 persons per 100,000 suffer from HCC in Pakistan. Hepatitis C is a major contributor towards HCC in Pakistan contributing 60-70% of HCC incidences. Lack of proper healthcare systems in the poor countries lead to late diagnosis of HCC because the imaging techniques employed to diagnose HCC i-e MRI, Ultrasound and histopathology require expensive healthcare systems. Various studies have been focused on developing biomarkers for HCC to detect HCC at an early stage. Circulating proteins can easily be detected in the body fluids, therefore offer excellent potential for the development of biomarker for the disease. For easy, and early detection of HCC, the need of the hour is to develop blood-based serum biomarkers. Alpha Fetoprotein (AFP) possess less clinical efficacy because it is used in conjunction with imaging techniques. Multiple circulating proteins are used in various research to check their potential to act as efficient biomarker candidates for HCC detection previously in our lab. These findings are in continuation of our previous studies by (Awan et al., 2015) in which bioinformatics pipeline was established. Subsequently, the blood samples were collected from HCC patients and ELISA was performed on blood serum of C8A, SERPINC1, HSD11B1 and MBL2 along with Western blot of C8A (Abdullah, MS Thesis 2021). Mr. Abdullah Ahmed performed ELISA of the first four proteins namely C8A, SERPINC1, HSD11B1 and MBL2. Among them C8A showed excellent ability to act as a potential biomarker for HCC having sensitivity and specificity values far better than AFP while other proteins did not show significant potential to act as biomarker for HCC. He then qualitatively validated the positive results of C8A through Western Blot. In the current study, ELISA of the remaining three proteins along with Western Blot analysis and Multiplex ELISA is performed. Proteins namely Cyp2a6, UPB1 and ADH6 were checked in the serum of 150 HCC patients and 50 control samples via quantitative ELISA. Among these, ADH6 poses decent biomarker potential with 70.67% sensitivity and 64% specificity values and Cyp2a6 showed 64% sensitivity and 54% specificity whereas UPB1 did not show significant biomarker potential. These results were then confirmed via quantitatively and qualitatively through Western blot of selected serum samples of all three proteins i-e Cyp2a6, UPB1 and ADH6. Qualitative validation was performed to determine the antibody‘s specificity to that particular protein while Abstract 2 quantitative validation was performed by setting the first band in the gel as a reference band and then finding the relative intensity of all the remaining bands in comparison to the reference band by using ImageLab software. Multiplex ELISA was performed to check whether the combination of two proteins yielded better results. The best performing biomarker ADH6 was combined with AFP (the current gold standard biomarker for diagnosing HCC). The combination yielded better sensitivity (88.68%) and specificity (80%). Conclusively, the combination of two biomarkers ADH6 and AFP showed excellent potential to serve as a circulating blood-based protein biomarker panels for non-invasive and rapid detection of HCC