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Characterisation of the Expression of Tumour Antigens and Biomarkers in Myeloid Leukaemia and Ovarian Cancer

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Characterisation of the Expression of Tumour Antigens and Biomarkers in Myeloid Leukaemia and Ovarian Cancer Title : Characterisation of the expression of tumour antigens and biomarkers in myeloid leukaemia and ovarian cancer Name : Ghazala Naz Khan This is a digitised version of a dissertation submitted to the University of Bedfordshire. It is available to view only. This item is subject to copyright. Characterisation of the Expression of Tumour Antigens and Biomarkers in Myeloid Leukaemia and Ovarian Cancer By Ghazala Naz Khan PhD A thesis submitted to the Faculty of Creative Arts, Technologies and Science, University of Bedfordshire in fulfilment of the requirements for the degree of Doctor of Philosophy December 2016 Abstract Acute myeloid leukaemia (AML) and ovarian cancer (OVC) are two difficult to treat cancers. AML is often treatable however minimal residual disease (MRD) endures such that many patients who achieve remission eventually relapse and succumb to the disease. OVC affects approximately 7000 women in the U.K. every year. It can occur at any age but is most common after menopause. Diagnosis at an early stage of disease greatly improves the chances of survival however, patients tend to be diagnosed in the later stages of disease when treatment is often less effective. Immunotherapy has the potential to reduce MRD and delay or prevent relapse. In order for immunotherapy to work, tumour antigens need to be identified and characterised so they can be effectively targeted. Personalised treatments require the identification of biomarkers, for disease detection and confirmation, as well as to provide an indication of best treatment and the prediction of survival. PASD1 has been found to be frequently expressed in haematological malignancies and I wanted to determine if there was a correlation between the presence of antigen-specific T cells in the periphery of patients with AML and PASD1 protein expression in the leukaemic cells. The expression of other leukaemia antigens were concurrently examined as comparators. I performed RT-PCR on nine antigens and immunocytochemistry on PASD1 in 18 samples from AML patients. I found a correlation between PASD1 expression in AML samples and the presence of PASD1-specific T cells as detected on the pMHC array. OVC lacks suitable targets for immunotherapy with few CTAs having been identified. I examined the expression of SSX2IP and the CTAs PASD1 and SSX2 in OVC. I compared the protein expression of these known tumour antigens to the “gold standard” biomarker for the diagnosis of OVC, CA125 and two other proteins known to be promising in the diagnosis of OVC, HE4 and WT1. I analysed commercially available paraffin-embedded OVC multiple i tissue arrays (MTAs) containing 191 samples, predominantly stage I (n= 166), II (n= 15) and III (n= 6) OVC as well as healthy donor (n= 8) and normal adjacent tissues (n= 8). Scoring was performed in a single blinded fashion. I found SSX2A to be expressed at a score level of 3 with a frequency (37/191) that exceeded that of CA125 (14/191), HE4 (14/191), WT1 (1//191) or PASD1 (0/191). To confirm this expression I used two additional commercially-available antibodies that recognise the region common to SSX2A and B, and an antibody specific for SSX2A. Using SSX2 peptides, I blocked the immunolabelling of SSX2 in SSX2-positive cell lines showing that the immunolabelling of SSX2 and SSX2A was specific. I demonstrated that the expression of SSX2 and specifically SSX2A was reproducible and restricted to ovarian cancer with little or no expression in endometrial tissues, or diseased or inflamed endometrial tissue. In summary, these studies demonstrated that PASD1 expression in leukaemia cells correlated with the presence of PASD1-specific T cells in the periphery of presentation AML patients. I have shown that PASD1 specific-T cells are present in AML patients at diagnosis and that immunotherapy targeting PASD1 could be used to break tolerance and clear residual leukaemia cells during first remission. Analysis of the expression of three antigens in OVC, identified the specific expression of SSX2, in particular SSX2A in OVC but not healthy or diseased endometrial tissues. The expression of SSX2A was more frequent and more specific to OVC, than HE4 and WT1, and more frequent at higher intensity, especially in early stage OVC, than CA125. SSX2 and explicitly SSX2A requires further investigation to determine whether the high level of background at score 2 can be reduced with better blocking of non-specific sites. This may require the use of different SSX2 antibodies or an improved staining protocol. ii Author’s Declaration I declare that this thesis is my own unaided work. It is being submitted for the degree of Doctor of Philosophy at the University of Bedfordshire. It has not been submitted before for any degree or examination in any other University. Name of candidate: Ghazala Khan Signature: Date: 20 December 2016 iii Dedications For my Dad. For my parents who gave me the world. They allowed me to follow my dreams and encouraged me to be the best. I am who I am because of them. iv Acknowledgements To my supervisor, Dr Barbara Guinn who made the impossible possible with unwavering support, friendship and mentoring. What I have learned professionally and personally will stay with me forever. Thank you to Dr Kim Orchard, Department of Haematology, Southampton University Hospital Trust for kindly providing the haematological samples used in this study. I am indebted to Linden Lyne and Professor Alison Banham for kindly teaching me how to perform immunohistochemistry on paraffin-embedded samples. To a true friend and colleague, Khadar, for always being there through the good and bad times. Our group has been supported by Leukaemia and Lymphoma Research, Wessex Cancer Trust and a Wessex Medical Research Innovations grant. v Peer-reviewed publications Manuscripts 1. Brooks, S.E., Bonney, S.A., Lee, C., Khan, G., Smits, E., Publicover, A., Sigurdardottir, D., Li, D., Pulford, K.P. Banham, A.H., Tendeloo, V., Mufti, G.J., Rammensee, H.-G., Elliott, T.J., Orchard, K.H. & Guinn, B.A. (2015) PASD1 epitopes are frequently recognised by “untouched” CD8+ T cells from presentation myeloid leukaemia patients. Public Library of Science One, 10: e0140483. 2. Khan, G., Brooks, S.E., Banham, A.H. & Guinn, B.A. (2015) Infrequent Expression of the Cancer-Testis Antigen, PASD1, in Ovarian Cancer. Biomarkers in Cancer, 7: 31-8. 3. Khan, G., Denniss, F., Mills, K.I., Pulford, K. & Guinn, B.A. (2014) PASD1: a promising target for the immunotherapy of haematological malignancies. Journal of Genetic Syndromes & Gene Therapy, 4, 186. Invited review for a special issue on "Cancer Genetics". 4. Hardwick, N.R., Buchan, S., Ingram, W., Khan, G., Vittes, G., Rice, J., Pulford, K., Mufti, G.J., Stevenson, F.K. & Guinn, B.A. (2013) An analogue peptide from the cancer testis antigen, PASD1, induces CD8+ T cell-responses against naturally processed peptide. Cancer Immunity, 13, 16-26. 5. Lam, P., Khan, G., Stripecke, R., Hui, K.M., Kasahara, N., Peng, K.-W. & Guinn, B.A. (2013) The innovative evolution of cancer gene and cellular therapies. Cancer Gene Therapy, 20, 141-149. Book Chapters 1. Hofmann, S., Khan, G., Boncheva, V., Greiner, J. & Guinn, B.A. (2014) Vaccination against myeloid leukaemias using newly defined antigens. In Cancer Immunology Immunotherapy. (Ed. by Rees, R.) Oxford University Press. ISBN 978-0-19-967686-6. 2. Khan, G., Brooks, S.E., Denniss, F.K., Sigurdardottir, D. & Guinn, B.A. (2013) Identification and validation of targets for cancer immunotherapy: from the bench-to- bedside. In Gene Therapy (Ed. by Wei, M. & Good, D.). IntechOpen. ISBN 980-953-307- 743-2. Conference presentations 1. Khan, G., Brooks, S. & Guinn, B.A. (2016) Can cancer-testis antigens act as biomarkers for stage I and II ovarian cancer? In Press, 2016 National Cancer Research Institute Cancer Conference, Liverpool, England. 2. Khan, G., Mead, A., Brooks, S.E. & Guinn, B.A. (2016) Detection of tumour antigens as targets for immunotherapy and biomarkers of early disease. Poster Presentation at the Department of Life, Health & Chemical Away Day, Open University. 3. Khan, G.,* Mead, A., Brooks, S.E. & Guinn, B.A. (2014) Characterising a novel biomarker for ovarian cancer. Poster Presentation at the National Cancer Research Institute annual vi meeting, Liverpool, U.K. Short-listed for a British Association of Cancer Research (BACR)/Gordon Hamilton-Fairley Young Investigator Award. 4. Khan, G.,* Brooks, S.E. & Guinn, B.A. (2014) A cancer-testis antigen which acts as a novel biomarker for stage I and II ovarian cancer. Poster Presentation at the British Gynaecological Cancer Society Annual Conference, London, U.K. 5. Khan, G.,* Brooks, S.E. & Guinn, B.A. (2014) Identification of a new biomarker for ovarian cancer? Poster Presentation at the Association for Cancer Immunotherapy Annual meeting, Mainz, Germany. 6. Khan, G., Brooks, S.E. & Guinn, B.A. (2014) Identification of a new biomarker for ovarian cancer: a new target for site specific therapies? Oncolytic Virus Therapeutics Annual Meeting, Oxford, U.K. Human Gene Therapy, 25, A68. 7. Khan, G.,* Boncheva, V., Mirnezami, A. & Guinn, B.A. (2013) Characterisation of SSX2 and SSX2IP expression in colon cancer. National Cancer Research Institute Annual Meeting, Liverpool, U.K. 8. Khan, G., Denniss, F., Mills, K.I. & Guinn, B.A. (2012) SSX2 and SSX2IP expression in cancer. International Society for the Cell and Gene Therapy of Cancer, Singapore. *Presenting author Non-peer reviewed publications 1. Khan, G. & Guinn, B.A. (2013) PASD1 (Xq28). Atlas of Genetics
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