Development and application of the antigen microarray for the detection of autoantibodies in hepatocellular carcinoma patients

Hepatocellular carcinoma (HCC) is a primary liver cancer characterised by mutations in the cellular machinery, which induces the liver cells to replicate at a higher rate. It is the fifth most common cancer and the third most common cause of cancer-related deaths worldwide. The extensive prevalence of this malignancy carries a significant economic burden on a country. HCC occurs within the background of cirrhosis and chronic liver diseases, where hepatitis B virus (HBV), hepatitis C virus (HCV) infections and alcoholism are regarded as the most frequent risk factors of the disease. However, current diagnostic tools are sensitive only at the advanced stages, which hugely contribute to the grave prognosis and compromised effective curative treatment, resulting in poor 5-year patient survival. With the continuous devastating effect of this malignancy, there is an urgent need for an efficient tool, which could detect the malignancy at both the early and late stages. Recently, an increasing number of studies have shown that cancer patients produce detectable serological biomarkers in the form of IgG autoantibodies, which appear five years even before clinical manifestation of the disease. This evidence has recently generated opportunities for immune system exploitation as source of cancer biomarkers. Autoantibodies are elicited by the release of tumour-associated antigens (TAAs) during tumour development, making the autoantibody signature an effective approach for the early diagnosis of HCC. They could also represent as novel tools for prognosis, monitoring and prediction of response for chemotherapy. It is therefore crucial to design a non-invasive, high-throughput and robust screening system that would allow autoantibodies to be detected early in HCC and high-risk patients. In recent times, the antigen microarray, which is a new generation probing technique, has emerged as a promising tool for the examination of humoral immune responses in cancer in a multiplexed and high-throughput manner. This technology is adapted as an alternative approach to the already existing Enzyme Linked Immunosorbent Assay (ELISA). In Antigen Microarrays, the recombinant TAAs are spotted robotically on a glass slide and probed with sera after which signals are amplified and quantified through fluorescence. The technology has the advantage of quantifying serum autoantibodies to hundreds of TAAs simultaneously and further providing a form of protein analysis at a scale beyond that which is achievable by traditional immunological techniques. This thesis documents the development, validation and application of the in-house Antigen Microarray platform to detect and characterise specific anti-TAA IgG autoantibodies to a panel of TAAs in the sera of HCC and underlying liver disease patients. Important parameters associated with the antigen microarray such as slide surface chemistry, target humidity, printing buffer, blocking buffer and antigen concentration were optimised to improve the accuracy and precision of the technology for this early biomarker detection. Secondly, the validity of the developed technology was further assessed using guidelines drawn by the Food and Drug Administration for pharmacokinetic assay validation. Additionally, we opted to produce a panel of eight recombinant TAAs in a mammalian expression system, to be sure of the quality and purity of antigens generated. The suitability of in-house TAAs were also tested on the microarray assay to reveal precision of the assay. Lastly, serum anti-TAA autoantibody levels were measured in HCC (n=183), liver cirrhosis (n=90), chronic hepatitis patients (n=133) as well as healthy groups (n=46), over a wide range of in-house (KRAS, HRAS, HDGF, CDKN2A, RALA, P62, P53, Gankyrin) and commercial TAAs produced both in mammalian (KRAS, HRAS, HDGF, CDKN2A, RALA, P62, P53, Gankyrin) and E. coli expression systems (KRAS, HRAS, HDGF, RALA, P62, APCN, AFP, IMP1, GRP78, Gankyrin). Validation studies showed high reproducibility as precision values were within the acceptable limits, namely, intra (within) and inter (across days) coefficient of variation were less than 15% and 20% respectively. Eight highly expressed Flag-tagged recombinant TAAs (KRAS, HRAS, HDGF, CDKN2A, RALA, P62, P53, Gankyrin) in lysates were produced and evaluated by western blotting. Preliminary studies conducted to investigate whether heat treatment reduces or elevates autoantibody levels in sera revealed no significant effect (P>0.05) of heat inactivation on the detectability of autoantibodies to TAAs. There was a significant difference (p<0.05) in the distribution of specific anti-TAA autoantibody responses across the four cohorts with highest responses detected in HCC patients followed by chronic hepatitis and liver cirrhosis patients. This suggests that high anti-TAA autoantibody levels are strongly associated with the cancer. Poor correlation (r