The Development of Circulating Tumour DNA as a Clinical Surveillance Tool for New Zealand Breast Cancer Patients
Parackal, Sarah Grace

View/ Open
Cite this item:
Parackal, S. G. (2018). The Development of Circulating Tumour DNA as a Clinical Surveillance Tool for New Zealand Breast Cancer Patients (Thesis, Master of Science). University of Otago. Retrieved from http://hdl.handle.net/10523/8071
Permanent link to OUR Archive version:
http://hdl.handle.net/10523/8071
Abstract:
Breast cancer is a heterogeneous group of diseases where metastatic spread is the major cause of death. Surveillance of treatment response and tumour burden is currently achieved with periodic medical imaging over the course of the disease. Circulating tumour DNA (ctDNA) is a blood-based biomarker where short fragments of DNA are released from the tumour into the blood circulation. These fragments contain identical somatic mutations from the tumour and can be acquired through a routine blood draw for further analysis such as variant detection (Next Generation Sequencing) and quantification (Droplet Digital PCR). Routine analysis of ctDNA provides a means of monitoring tumour burden, measuring treatment response and surveillance of metastatic spread. Despite previously successful ctDNA analytical studies, its adoption into clinical diagnostics is hindered by a lack of standardised operating protocols. This thesis aimed to 1) contribute to the development of a ctDNA analytical pipeline for New Zealand and, 2) demonstrate ctDNA sensitivity for the surveillance of metastatic breast cancer.
The first objective was accomplished by comparing two commercially available cfDNA blood collection tubes. Differences in sample stabilisation by the prevention of gDNA contamination from WBC lysis was measured for both tubes over a period of 14 days. No statistically significant results were observed between the two tubes, hence consideration of price and product availability were considered for choosing a tube for clinical sample collection. Optimisation of mutation identification and quantification techniques were carried out using a commercial cfDNA reference standard set. The standards were used in targeted NGS to determine mean target coverage and number of variants detected for various DNA input amounts (20 ng, 5 ng and 1ng) at a low mutant allele frequency (1%). It was observed that the 20 ng sample achieved the highest mean target coverage and thus the highest number of somatic variants detected. The standards were also used to optimise in-house ddPCR assay design for two variants (NRAS Q61K and PIK3CA E545K). The detection sensitivity of each assay was determined to identify the mutant variants at three allele frequencies within a background of 10, 000 wild type copies. Detection sensitivities of 0.03% and 0.49% were observed for the NRAS and PIK3CA assays, respectively.
To achieve the second objective, the ctDNA methodology was applied to a small pilot study of six metastatic breast cancer patients to determine response to treatment and monitor tumour burden in comparison to current clinical practices. A significant reduction in ctDNA levels was observed in three patients within two chemotherapy cycles, potentially indicating a positive response to treatment. However, ctDNA analysis for the remaining three patients was inconclusive.
The findings of this study contribute to the development of a standardised ctDNA analytical protocol and demonstrate its utility for surveillance of metastatic breast cancer patients. Although further research in this area is yet to be done, the adoption of ctDNA analysis for the surveillance of cancer within New Zealand will contribute to a shift in cancer care towards primary services and help achieve the New Zealand Primary Health Care strategy “Better, Sooner, More Convenient”.
Date:
2018
Advisor:
Guilford, Parry
Degree Name:
Master of Science
Degree Discipline:
Biochemistry
Publisher:
University of Otago
Keywords:
ctDNA; New Zealand; Breast Cancer; Clinical Tool
Research Type:
Thesis
Languages:
English
Collections
- Biochemistry collection [228]
- Thesis - Masters [3378]