Advanced Liquid Biopsy Technologies for Circulating Cancer Biomarker Detection

Advanced Liquid Biopsy Technologies for Circulating Cancer Biomarker Detection File version Author(s) Primary Supervisor Shiddiky, Muhammad J Other Supervisors Nguyen, Nam-Trung Rehm, Bernd Editor(s) Date Size File type(s) Location License Abstract Epithelial ovarian cancer is one of the most prevalent gynaecological cancers in women and is often diagnosed in the late stage due to the mild symptoms. Currently, ovarian cancer screening is reliant on the prevailing usage of blood based CA125 protein biomarker and transvaginal ultrasound which can detect ovarian cancer in the preclinical phase in a substantial portion of cases. However, there are also other elements that can result in elevated CA125 levels such as menstruation, endometriosis or ovarian cysts. As such, the lack of accurate disease risk classification during ovarian cancer screening has led to several health burdens associated with unnecessary biopsies and overtreatment of patients. Thus, new diagnostic methods with improved sensitivity and specificity for ovarian cancer are a clinical priority. To address the enigma associated with ovarian cancer screening, liquid biopsy technologies have been developed. Molecular profiling of liquid biopsies has the potential to detect changes associated with the tumuor in collected, non-invasively body fluid samples. Detection of tumour origin biomolecules such as; circulating tumour cells (CTCs), circulating tumour specific nucleic acids (ctDNA, ctRNA, miRNAs, lnRNAs), exosomes, autoantibodies in blood, saliva, stool, urine etc. has brought about a paradigm shift in the management and diagnosis of cancer. From reliance on painful and hazardous tissue biopsies or sophisticated equipment dependent imaging, cancer management schemes are witnessing rapid evolution towards minimally invasive yet highly sensitive liquid biopsy-based tools. Clinical application of liquid biopsy is already paving the way for precision theranostics and personalised medicine, especially by enabling repeated sampling, which in turn provides a more comprehensive molecular profile of tumours. On the other hand, integration with novel miniaturised platforms, engineered nanomaterials, as well as electrochemical detection has helped in the development of low cost and simple platforms suited for point-of-care application. Despite excellent analytical performances of the existing detection methodologies, electrochemical approaches offer a promising alternative for simple, sensitive, specific, rapid, and cost effective analysis of genetic and epigenetic biomarkers in cancer samples. Therefore, innovative technology using electrochemical approach would be an effective method for the detection of biomarkers in patients with cancer. This thesis focuses on the use of nucleic acids (i.e., genetic and epigenetic) biomarkers, specifically HOX antisense intergenic RNA (HOTAIR) lncRNA and DNA methylation to identify tumour specific changes and their performance as diagnostic biomarkers in non-invasively collected biofluid samples. Novel electrochemical and colourimetric approaches have been demonstrated for the construction of a sensitive, and specific biosensor platform for the complex task of detecting and quantifying circulating ovarian cancer biomarkers. To achieve this goal, first a comprehensive literature review on the biogenesis, significance, and potential role of four widely known biomarkers (CTCs, ctDNA, miRNA and exosomes) in cancer diagnostics and therapeutics has been provided. A detailed discussion of the inherent biological and technical challenges associated with currently available methods and the possible pathways to overcome these challenges is also provided. The recent advances in the application of a wide range of nanomaterials in detecting these biomarkers are also highlighted. Next, an amplification-free electrochemical method for the detection of HOTAIR lncRNA was developed. In this method, HOTAIR sequences were magnetically isolated, purified and detected by a sandwich hybridisation method at a screen-printed gold electrode (SPE-Au). This event was monitored by amperometry using the hydrogen peroxide/horseradish peroxidase/hydroquinone (H2O2/HRP/HQ) system which enabled a catalytic enhancement of the signal. In the following chapter, a more sensitive assay was discussed which utilised colourimetric and electrochemical readout for HOTAIR detection. In this approach, subsequent detection of magnetically purified and isolated sequences was performed using the sandwich hybridisation event coupled with HRP-catalysed reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2 which facilitated the naked eye observation and enabled an alternative amperometric quantification of HOTAIR. We then explored the bioengineering and characterisation of self-assembled superparamagnetic polyhydroxybutyrate (PHB) nanobeads for the development of a platform method for the analysis of circulating biomarkers, where these nanobeads were modified with specific bio-recognition antibodies, dispersed in analyte fluids where they worked as “dispersible capture agents” to bind specific targets. The enormous active sites of PHB nanobeads allow the direct attachment of a larger number of antibodies which can significantly enhance the capture efficiency. Their magnetic property allows magnetic nanoparticle-based mixing, separation and purification which can improve assay performance by reducing the matrix effects of the biological samples, as non-target species can be removed via magnetic isolation and purification steps. Two common circulating biomarkers namely global DNA methylation and exosomes were chosen for this method. After purification and magnetic collection, the isolated targets were directly adsorbed onto a screen-printed gold electrode (SPE-Au) and electrochemically quantified using a catalytic redox cycling system of hydrogen peroxide/horseradish peroxidase/hydroquinone (H2O2/HRP/HQ). In another approach, to simplify the assay protocol, the PHB nanobeads were directly adsorbed onto the SPE-Au electrode via PHB-gold affinity interaction followed by the immune attachment of the methylated DNA targets onto the surface-bound PHB nanobeads/anti 5mC-HRP conjugates. The targets were then quantified using the similar catalytic redox cycling of H2O2/HRP/HQ. Lastly, the clinical utility of these novel technologies was demonstrated using ovarian cancer cell lines and a cohort of well-annotated patient samples. This illustrates an attempt to translate the developed technologies from an academic research phase to patient usage by assessing the clinical performance metrics. To date, there are various ovarian cancer treatment options such as surgery, chemotherapy, targeted therapy, radiation therapy and palliative treatment. Each treatment method is dependent on various factors such as the cancer stage, gene type, overall health and fitness, as well as the desire to bear children. Thus, it is envisioned that the research that integrates new cutting-edge biomarkers and innovative detection strategies (as showcased in this thesis) could advance ovarian cancer diagnosis and risk stratification in clinical settings. This will enable a more personalised treatment approach accustomed to the needs of individual patients. Journal Title Conference Title Book Title Edition Volume Issue Thesis Type Thesis (PhD Doctorate) Degree Program Doctor of Philosophy (PhD) School School of Environment and Sc Publisher link Patent number Funder(s) Grant identifier(s) Rights Statement Rights Statement The author owns the copyright in this thesis, unless stated otherwise. Item Access Status Note Access the data Related item(s) Subject Epithelial ovarian cancer blood based protein CA125 diagnostic methods sensitivity specificity