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Hassall, James (2021) Investigating radiation responsiveness in rectal cancer. PhD thesis, University of Nottingham.

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Abstract

Currently rectal cancer patients undergoing radiotherapy are subject to a ‘watch-and-wait’ scenario whereby the clinician and patient are not certain if the tumour is responding to the radiotherapy until completion. Real-time monitoring of response to radiotherapy may be possible via liquid biopsy testing and it is hoped that this will inevitably improve outcome by streamlined patient stratification. Liquid biopsies are a promising method to interrogate the molecular characteristics of the tumour and determine presence of the tumour in real-time by blood test. This is possible when dying cells release DNA into the blood stream (cell-free DNA). This also occurs in tumour cells (circulating tumour DNA) at a higher rate due to their instability, producing regular injections of DNA into the blood stream. Each tumour contains a plethora of mutations which can be identified in cell free DNA by an appropriate mutation detection methodology. Cell-free DNA is highly fragmented (130-170bp), low in mutant allele frequency (as low as 0.01%) and low in concentration (1-100ng/ml of blood). Therefore, these characteristics present a major hurdle for mutation detection methods to determine presence of circulating tumour DNA.

This study has developed two novel methods that are suitable for cell-free DNA mutation detection as other available methods are currently flawed in a number of ways: either they are too expensive for serial analysis of liquid biopsy (which is required for treatment-response monitoring), not sensitive enough, not scalable enough or too complicated for clinical integration. The two methods ‘Highly Optimised annealing Temperature_Amlification Refractory Mutation System PCR (HOT_ARMS PCR)’ and Highly Optimised annealing Temperature_Probe Inhibited PCR (HOT_PI PCR)’ are a combination of methods for liquid biopsy interrogation. HOT_ARMS is an extremely simple, robust, inexpensive, and exquisitely sensitive test that uses mutation-specific primers for detection of any kind of mutation which results in a sequence change. HOT_ARMS improves the previously published ARMS system by increasing the specificity of the PCR via primer modification to raise the annealing temperature (Ta) to >65°C. The increased kinetic energy of the primers reduces non-specific 3’ base-pairing and the modification increases selectivity for the mutant amplicon. This simple adaptation of ARMS PCR with a working annealing of 71°C generates an unrivalled all-round PCR-based method of detecting low frequency mutant alleles. It is a single stage closed-tube test which does not require expensive equipment and, because it is not reliant on probes, it is easy to set up and requires little optimisation for most mutations. The speed of the test means it could theoretically be established in the hospital out-patient and even the primary care setting.

HOT_PI PCR is an extremely simple, robust, inexpensive, and efficient method of mutation enrichment on up to 6 nucleotides for any sequence change. This can result in 60-fold enrichment of mutant alleles, allowing methods with insufficient limits of detection the ability to test liquid biopsies. High-resolution melting analysis was deployed and could detect 0.1% MAF (a major improvement from 6% MAF). HOT_PI also uses a working annealing temperature of 71°C meaning that it can be combined with HOT_ARMS PCR for a streamlined approach to screen for the presence of mutations in pre-treatment liquid biopsies. This will determine the mutational profile of the tumour for treatment-response monitoring; allowing for quantitative singleplex HOT_ARMS assays to be deployed and ultimately save precious FFPE tissue for histology testing. HOT_PI is a single stage closed-tube test which is easy to set up and does not require expensive equipment. The speed of the test means it could work alongside HOT_ARMS PCR and reduce the number of required tests.

Rectal cancer patients undergoing radiotherapy currently vary in response. Twenty percent of patients will completely respond to radiotherapy and up to 60% will achieve tumour downstaging. This leaves a large proportion of patients receiving treatment unnecessarily. It may be possible to create a predictive test through biomarker analysis by comparing biopsy specimens from patients with known response status. This study interrogated redox-homeostasis genes in 10 biopsy samples known to respond to radiotherapy (Mandard 1-2) and 10 samples known to not respond to radiotherapy (Mandard 3-5) by reverse-transcription real-time PCR using custom designed assays. Redox-homeostasis has previously been found to be predictive of radiotherapy response in breast and lung cancer.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Ilyas, Mohammad
Keywords:	Circulating tumour DNA, liquid biopsy, cell-free DNA, HOT_ARMS PCR, HOT_PI PCR, HOT_PCR, N_CaRT, ARMS-PCR, wild-type blocking PCR, redox-homeostasis, glutathione, thioredoxin, rectal cancer, cancer.
Subjects:	W Medicine and related subjects (NLM Classification) > WI Digestive system
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Related URLs:	URL URL Type https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019145734 Dataset
Item ID:	64466
Depositing User:	Hassall, James
Date Deposited:	12 May 2022 08:22
Last Modified:	29 Feb 2024 15:59
URI:	https://eprints.nottingham.ac.uk/id/eprint/64466

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