Advancing Gene Fusion Detection towards Personalized Cancer Nanodiagnostics Kevin Maisheng Koo Bachelor of Science (Hons I) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017 Australian Institute for Bioengineering and Nanotechnology Abstract Prostate cancer (PCa) is one of the most prevalent non-cutaneous cancers in men, and is also one of the most lethal oncogenic diseases that accounts for a vast majority of male cancer-related deaths. Currently, widespread PCa screening is reliant on the prevailing usage of the FDA-approved blood- based prostate specific antigen (PSA) biomarker. Yet, landmark clinical trials in recent years have indicated that serum PSA screening holds a substantial risk of over-diagnosing low grade indolent PCa which are unlikely to result in mortality. Consequently, this paucity of accurate disease risk stratification during PCa screening has led to a variety of health burden associated with unnecessary biopsies, and over-treatment in a considerable fraction of patient population. Given that the screening shortcoming of the PSA test is outweighing its benefit, there is a clear need for better strategies to improve PCa risk stratification and accurately detect high-grade aggressive PCa molecular subtypes at an early stage for timely personalized treatment. To address this PCa screening conundrum, the research work described in this thesis primarily embodies a bipartite strategy which pairs together the use of next-generation PCa-specific molecular biomarkers, and the development of innovative nanodiagnostic technologies to target these superior biomarkers. In recent years, massive advances in next-generation sequencing techniques have led to the discoveries of novel PCa molecular targets which possess excellent PCa- specificity (i.e. next-generation PCa biomarkers); and have been shown to improve PCa diagnosis, prognosis, and prediction of therapeutic resistance in a multitude of seminal clinical studies. The most tantalizing prospect among such next-generation PCa biomarkers is the chromosomal gene fusion mutation between the TMPRSS2 and ERG genes (TMPRSS2:ERG). TMPRSS2:ERG is a highly-recurrent biomarker in the majority of clinically-detected PCa tumor foci, and its exclusive presence in cancer cells facilitates the accurate identification of a major PCa subtype. Through combinatorial detection of TMPRSS2:ERG with other next-generation PCa biomarkers, it is anticipated that PCa molecular subtyping and better disease risk stratification could be achieved. Furthermore, these next-generation biomarkers are detectable in patient urine samples as a form of liquid biopsy, thus opening up new avenues of truly non-invasive PCa diagnostics. Presently, urinary biomarker detection is achieved through conventional laboratory-based methods which are not ideal for use in a clinical setting. Hence, to realize the use of PCa diagnostics for clinical screening, this thesis also details the use of nanotechnology to design various cutting-edge diagnostic platforms for next-generation PCa urinary biomarker screening. It is envisioned that such PCa nanodiagnostics which exhibits unique physical properties at the nanoscale; could result in accurate early PCa detection with unprecedented detection sensitivity, speed, and reliability. This thesis first describes the design of miniaturized solution-based biosensing systems which coupled rapid isothermal RNA target amplification with innovative detection readouts that are enabled by nanoscaled mechanisms. These lab-in-a-drop systems integrated the detection of urinary TMPRSS2:ERG transcripts within a fluidic microdroplet, and exhibited single-cell detection sensitivity through visual and/or quantitative colorimetric readouts. Next, this thesis reports the pioneering use of label-free surface-enhanced Raman scattering (SERS) for RNA detection in clinical samples. Label-free SERS is a spectroscopic technique for direct detection of adsorbed nucleic acid on metallic nanoparticle surfaces but has prior been limited to detection of short-length oligonucleotides. Herein, label-free SERS detection of TMPRSS2:ERG RNA in patient urinary specimens was shown using a novel combination of isothermal RNA amplification and/or chemometrics. In addition to the use of isothermal amplification, this thesis also investigated the development of enzymatic amplification-free nanobiosensors for detection of various RNA biomarker species. By exploiting adenine-bare gold affinity interactions, the detection of different species of next- generation PCa RNA biomarkers was shown. Further, to attain a more comprehensive screening outcome to aid in clinical decision-making, this thesis has also devised new nanobioassays for concurrent analysis of multiple next-generation PCa biomarkers. Lastly, to facilitate clinical translation, clinical evaluation of both biomarkers and nanodiagnostic technologies (developed through this thesis) was carried out using a cohort of well-annotated patient samples. This represents an effort to progress the developed nanotechnologies from an academic research phase to patient usage by assessment of clinical performance metrics. Today, there is an assortment of PCa treatment options such as active surveillance, radical prostatectomy, radiation or hormonal therapies; and each treatment has differing molecular subtype- dependent effectiveness and necessity. It is envisioned that research in the fusion of powerful new- age biomarkers and avant-garde detection strategies (as showcased in this thesis) could progress the field of PCa subtyping and risk stratification in the clinic, therefore granting a more personalized treatment approach that is tailored to the needs of individual patients. Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. Publications during candidature Peer-Reviewed Articles Koo, K. M.; Wang, J.; Richards, R. S.; Farrell, A.; Yaxley, J. W.; Samaratunga, H.; Teloken, P. E.; Roberts, M. J.; Coughlin, G. D.; Lavin, M. F.; Mainwaring, P. N.; Wang, Y.; Gardiner, R. A. & Trau, M. (2018) Design and clinical verification of surface-enhanced Raman spectroscopy diagnostic technology for individual cancer risk prediction. ACS Nano 12: 8362-8371. Koo, K. M.; Dey, S. & Trau, M. (2018) Amplification-free multi-RNA type profiling for cancer risk stratification via alternating current electrohydrodynamic nano-mixing. Small 14: 1704025. Koo, K. M.; Carrascosa, L. G. & Trau, M. (2018) DNA-directed assembly of copper nanoblocks with inbuilt fluorescent and electrochemical properties: Application in simultaneous amplification-free analysis of multiple RNA species. Nano Res. 11: 940-952. Koo, K. M.; Wee, E. J. H.; Wang, Y. & Trau, M. (2017) Enabling miniaturised personalised diagnostics: From lab-on-a-chip to lab-in-a-drop. Lab Chip 17: 3193-3340. *Featured as Issue Front Cover Image Wang, J.; Koo, K.M.; Wee, E. J. H.; Wang, Y. & Trau, M. (2017) Nanoplasmonic label-free surface-enhanced Raman scattering strategy for non-invasive cancer genetic subtyping in patient samples. Nanoscale 9: 3496-3503. (co-first author) Koo, K. M.; Wee, E. J. H. & Trau, M. (2017) High-speed biosensing strategy for non-invasive profiling of multiple cancer fusion genes in urine. Biosens. Bioelectron. 89: 715-720. Koo, K. M.; Wee, E. J. H.; Mainwaring, P. N.; Wang, Y. & Trau, M. (2016) Toward precision medicine: A cancer molecular subtyping nano-strategy for RNA biomarkers in tumor and urine. Small 12: 6233-6242. *Featured as Issue Cover Image Koo, K. M.; McNamara, B.; Wee, E. J. H.; Wang, Y. & Trau, M. (2016) Rapid and sensitive fusion gene detection in prostate cancer urinary specimens by label-free SERS. J. Biomed. Nanotech. 12: 1798-1805. Koo, K. M.; Wee, E. J. H.; Mainwaring, P. N. & Trau, M. (2016) A simple, rapid, low-cost technique for naked-eye detection of urine-isolated TMPRSS2:ERG gene fusion RNA. Sci. Rep. 6: 30722. Koo,