Title

The identification and validation of a diagnostic signature of tumour-associated autoantibodies for the early diagnosis of cutaneous melanoma

Author Identifiers

Pauline Zaenker
ORCID ID: 0000-0002-0807-1968

Date of Award

2018

Degree Type

Thesis

Degree Name

Doctor of Philosophy

School

School of Medical and Health Sciences

First Advisor

Professor Mel Ziman

Second Advisor

Dr Elin Gray

Field of Research Code

1107, 1112

Abstract

Background

Cutaneous melanoma is an aggressive cancer with a high propensity to metastasise. Melanomarelated fatalities are however preventable through early detection and complete surgical excision of the primary tumour. Current screening methods for melanoma are effective but limited to the visual examination of the skin, potentially leading to a delay in diagnosis of less obvious lesions.

Circulating biomarkers have been proposed to aid with early melanoma diagnosis and are easily accessible by routine blood collection. However, despite intensive scientific efforts, there are currently no blood-based biomarkers that are sufficiently sensitive for detecting melanoma at its earliest stages. Autoantibodies represent an amplified signal of tumour immunosurveillance that is measurable in patients with various cancers and detectable prior to the clinical manifestation of a tumour. Single and combinations of autoantibody biomarkers have shown potential as diagnostic biomarkers in numerous malignancies. However, the diagnostic utility of autoantibodies in melanoma is yet to be comprehensively investigated. The purpose of this thesis was therefore to identify a distinctive autoantibody signature in primary melanoma patient blood indicative of a positive disease status.

Aims

The initial aim of the thesis was to perform a comprehensive literature review (Chapter 2) identifying the causes of autoantibody production in cancer, indicating their biological relevance in the initial stages of carcinogenesis. Subsequently, we aimed to identify individual and combinations of suitable autoantibody biomarkers for the diagnosis of primary melanoma (Chapter 3). Finally, this thesis aimed to develop clinically applicable assays for a selection of identified autoantibodies to ascertain their diagnostic utility in primary melanoma detection (Chapter 4).

Methods

A total of 245 sera from primary melanoma patients and healthy volunteers were screened against a high-throughput microarray platform containing 1627 functional proteins to measure IgG autoantibody levels (Chapter 3). Data was analysed using stringent published and machinelearning methods. Pathway analysis of identified biomarkers was also undertaken. In-house Bio-Plex assays were developed and optimised for the detection of specific IgG autoantibody levels in a cohort of 104 primary melanoma patients and 104 healthy volunteers (Chapter 4).

Results

The literature search (Chapter 2) revealed that autoantibody production in cancer may be due to defects in tolerance and inflammation, changes in protein expression levels, altered protein structure and cellular death mechanisms. Following the high-throughput screening of primary melanoma patient and healthy control sera, a list of 139 autoantibody biomarkers with potential for melanoma diagnosis were identified (Chapter 3). The identified biomarkers were associated with major cancer-related pathways, indicating their relevance for cancer detection. To increase the overall sensitivity of melanoma detection whilst maintaining high specificity, we identified a combination of 10 autoantibody biomarkers that, as a panel, displays a sensitivity of 79%, specificity of 84% and an AUC of 0.828 for primary melanoma detection. Finally using inhouse developed bead assays for three of the biomarkers, individual biomarker sensitivities ranged from 10.6% to 18.3% at greater than 90% specificity. As a combination, the panel was able to detect melanoma with 28% sensitivity at 85% specificity.

Conclusions

The work presented here shows that autoantibodies may have potential as diagnostic biomarkers for melanoma. Although the causes of their production in cancer are not well understood, the literature indicates (Chapter 2) that it is the abnormal expression and/or alteration in the structure of the corresponding antigen that is most accountable for an immune response. However, failure of tolerance mechanisms, inflammation and cell death affect the context in which the antigens are presented to the immune system, initiating the production of autoantibodies. The identified biomarkers in Chapters 3 and 4 show upregulated levels in the patient cohort relative to healthy controls. As a single biomarker, autoantibodies do not appear sufficiently sensitive for melanoma detection, but in combination their overall increased sensitivity and specificity warrants further investigation in larger cohorts using clinically applicable screening platforms and assay formats that require extensive independent validation.

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