Date of Award

2017

Degree Type

Thesis

Degree Name

Master of Science (Human Biology)

School

School of Medical and Health Sciences

First Advisor

Dr Elin Gray

Second Advisor

Professor Mel Ziman

Field of Research Code

1 1 1 2 0 1, 1 1 1 2 0 2, 1 1 1 2 0 3, 1 1 1 3 0 1

Abstract

Background: Uveal melanoma (UM) is an extremely aggressive disease with approximately 50% of patients developing incurable metastatic disease. Therefore, accurate prognosis of a patient is necessary for closer follow up and the earlier implementation of systemic adjuvant therapies in those most likely to develop metastatic disease. Fortunately, UM can be classified into two distinct molecular classes based on clinically validated gene expression profiling, chromosomal aberrations and specific driver mutations, which accurately predict the metastatic propensity of the primary tumour. However, genetic testing currently requires biopsy of the eye which can lead to serious complications including permanent blindness. Therefore, an alternative source of primary tumour genetic material is needed to avoid these complications.

Aims: We proposed that circulating tumour cells (CTCs) are a viable source of tumour genetic material in which patient prognosis could be analysed. Firstly, we aimed to increase the sensitivity of an immunomagnetic enrichment protocol to capture CTCs. Secondly, we aimed to evaluate whole genome amplification methods for accurate single cells analysis to determine the genomic profile of UM cells. The combination of both aims would allow the use of UM CTCs for determining disease prognosis from an easily accessible blood sample.

Methodologies:

Aim 1 - To refine and evaluate methods for multi-marker immunomagnetic capture of UM CTCs. A tissue microarray (TMA) was created from 1mm cores taken from archived primary UM tissue. Normal tissue and cutaneous melanoma were added as controls. The TMA was stained by immunohistochemistry (IHC) for melanoma, melanocyte, and stem cell markers. Stained tissue was assessed to determine intensity and coverage of staining. In addition to primary UM tissue, five UM cell lines were assessed for the same markers using flow cytometry and immunocytochemistry. Given their high level of staining of UM, 5HT2B, ABCB5, surface gp100 (BETEB), MCAM, and MCSP were coated to immunomagnetic beads and used to determine the retrieval rate of UM cell lines cells spiked into peripheral blood mononuclear cells at a known quantity. CTCs could be detected by immunofluorescent staining of MART1, gp100, and S100β.

Aim 2 - Aim 2: To develop methodologies for the detection of genetic markers of metastatic propensity using single UM cells. Single UM cell line cells plus respective bulk genomic DNA whole genome amplified and bulk genomic DNA were amplified using PicoPlex and Repli-G WGA kits to determine each kits’ respective viability of detecting CNVs using low-pass (0.01-0.1x) whole genome sequencing (WGS) on the IonPGM platform. Peripheral blood mononuclear cells (PBMCs) were used as negative controls. In addition, we tested if these methods allowed accurate CNV data after fixation, permeabilisation, and immunostaining. After ensuring cell processing had no significant effects on genomic profile of single cells, blood samples from patients were processed to isolate CTCs from PBMCs. Isolated CTCs were then whole genome amplified using PicoPlex and shallow sequenced using the IonPGM system.

Results: We validated several melanoma, melanocyte, and stem cell markers which have been previously shown to be expressed in cancer, cutaneous melanoma, or UM. We found that 5HT2B, and ABCB5, surface gp100 (BETEB), MCAM, and MCSP were highly expressed in primary UM tissue or UM cell lines and were able to immunomagnetically capture UM cell line cells. Concurrently, we validated the use of shallow (0.01x-0.1x depth) whole genome sequencing of single UM cells amplified using the PicoPlex WGA Kit and found that PicoPlex offered a robust method of amplifying single cells that have undergone immunomagnetic isolation, fixation, staining, and capture whilst retaining the original genetic profile of the parent cell line. Upon testing this in a patient, we found a gain of chromosome 8 which is an early event in UM tumourigenesis; aneuploidy of chromosome 8 is a genetic feature that may, with the aid of future studies, delineate patient metastatic risk.

Available for download on Tuesday, May 29, 2018

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