Studies on Biomarker Development In Breast Cancer Ewan K A Millar Doctor of Medicine The University of New South Wales 2011 1 Studies on Biomarker Development In Breast Cancer By Ewan K A Millar BSc (Hons) MB ChB (Glasg) FRCPath (UK) FRCPA A report submitted for the degree of Doctor of Medicine (MD) by Published Works at The University of New South Wales August 2011 2 Originality Statement ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Ewan KA Millar Date 3 Acknowledgements I would like to thank several individuals, without whom the completion of the work in this thesis would not have been possible. Firstly, I would like to thank my beautiful wife Julia, for all her love and support and who has always been so understanding and encouraging of my career and research work, which has at times, kept me from being quite as involved in family life as I should have been. To my rapidly growing and wonderful children David, William and Georgia, thanks for all your love and support. I would not be in the position of submitting this Thesis without the generous support and guidance offered to me by Professor Rob Sutherland. His mentorship, intellectual input and friendship over the past 6 years have been immense and have helped develop my research career immeasurably. His skills and expertise as a researcher have given me much to aspire to in the years ahead. Other members of the Translational Breast Cancer Research Group also stand out for a special mention: A/Professor Sandra O’Toole and Dr Catriona McNeil have always provided invaluable knowledge and support in all aspects of our joint research goals. To Alice Boulghourjian and her predecessor Sarah Eggleton for tolerating, at times exhausting and demanding, requests for scientific support with their skills in immunohistochemistry. Associate Professor Peter Graham for his generous support and expert clinical input to our collaborative studies which will no doubt continue into the future. To Professor Soon Lee for long-standing support and encouragement, over the years, to pursue an academic career, with much useful advice. 4 To other colleagues at the Garvan Institute, Professor Liz Musgrove and Dr Alison Butt who have continued to generously support my work and research career. 5 Dedication For Julia, David, William and Georgia. 6 Contents Page Publications.......................................................................................8 Conference Abstracts......................................................................14 Abstract...........................................................................................17 Chapter 1. Background..................................................................18 Chapter 2. Immunophenotyping breast cancer using surrogate biomarker panels for molecular subtype ........................................35 Chapter 3. Endocrine Resistance in ER+ Breast Cancer..............41 3.1 Apoptotic Pathways.....................................................43 3.2 Proliferative Pathways.................................................46 Chapter 4. Signalling Pathways.....................................................51 Chapter 5. Tumour Hypoxia & HIF Pathway...................................63 Chapter 6. DNA Repair Pathways.................................................68 Chapter 7. Ductal Carcinoma in-Situ..............................................70 Chapter 8. Rare types of breast tumours: phyllodes tumours & non-Hodgkin’s lymphoma...............................................................72 Chapter 9. Authorship of papers for Thesis....................................75 Chapter 10. Significance of findings from published work..............83 References.....................................................................................85 Publications arising from this work................................................102 7 Publications in peer reviewed journals EM1. Murphy N, Millar E, Lee CS. Gene expression profiling in breast cancer: towards individualising patient management. Pathology. 2005; 37: 271-277.(IF 2.17). EM2. O’Toole SA, Selinger T, Millar EKA , Lum T, Beith JM. Molecular assays in breast cancer pathology. Pathology. 2011; 43: 116-127. (IF 2.17). EM3. Millar EKA, Graham PH, O’Toole SA, McNeil CM, Browne L, Morey AL, Eggleton S, Beretov J, Theocharous C, Capp A, Nasser E, Kearsley JH, Papadatos G, Delaney G, Fox C, Sutherland RL. Prediction of local recurrence, distant metastases and death following breast-conserving therapy in early-stage invasive breast cancer using a five biomarker panel. Journal of Clinical Oncology. 2009; 27: 4701-8. (IF 18.97) EM4. Millar EKA, Graham PH, McNeil CM, Browne L, O’Toole SA, Boulghourjian A, Papadatos G, Delaney G Nasser E, Kearsley JH, Fox C, Capp A, Sutherland RL. Prediction of outcome in early ER+ breast cancer is improved using a biomarker panel which includes Ki-67 and p53. British Journal of Cancer. 2011. 105:272-80. (IF 4.83) EM5. Graham P, Jagavkar R, Brown L, Millar E. Supraclavicular radiotherapy must be limited laterally by the coracoid to avoid significant adjuvant breast nodal radiotherapy lymphoedema risk. Australasian Radiology (Journal of Medical Imaging and Radiation Oncology). 2006. 50: 578-582. (IF 0.947) 8 EM6. Millar EKA, Anderson LR, McNeil CM, O’Toole SA, Pinese M, Crea P, Morey AL, Biankin AV, Henshall SM, Musgrove EA, Sutherland RL, Butt AJ. BAG-1 predicts patient outcome and tamoxifen responsiveness in ER positive invasive ductal carcinoma of the breast. Br J Cancer.100; 122-133, 2009. (IF 4.83). EM7. Roberts CG*, Millar EKA*, O’Toole SA, McNeil CM, Lehrbach GM, Pinese M, Tobelmann P, McCloy RA, Musgrove EA, Sutherland RL and Butt AJ. Identification of PUMA as an estrogen target gene that mediates the apoptotic response to tamoxifen in human breast cancer cells and predicts patient outcome and tamoxifen responsiveness in breast cancer. Oncogene. 2011.30:3186-97. (IF 7.41) *Both authors contributed equally to this work. EM8. McNeil CM, Sergio CM, Anderson LR, Inman CK, Murphy NC, Millar EKA, Crea P, Kench JG, Alles MC, Gardiner-Garden M, Ormandy CJ, Butt AJ, Henshall SM, Musgrove EA, Sutherland RL. C-Myc overexpression and endocrine resistance in breast cancer. Journal of Steroid Biochemistry and Molecular Biology. 2006; 102:147-55. (IF 2.89). EM9. Wang Y, Dean JL, Millar EKA, Tran TH, McNeil CM, Burd CJ, Henshall SM, Utama FE, Witkiewicz A, Rui H, Sutherland RL, Knudsen KE, Knudsen ES. Cyclin D1b is aberrantly regulated in response to therapeutic challenge and promotes resistance to estrogen antagonists. Cancer Research. 2008; 68:5628- 5638. (IF 8.23) 9 EM10. Millar EKA, Dean JL, McNeil CM, O’Toole SA, Henshall SM, Tran T, Lin J, Quong A, Comstock CES, Witkiewicz A, Musgrove EA, Rui H, Le Marchand L, Setiawan VW, Haiman CA, Knudsen KE, Sutherland RL, Knudsen ES. Cyclin D1b Protein expression in breast cancer is independent of cyclin D1a and associated with poor disease outcome. Oncogene. 2009; 28:1812-20. (IF 7.41). EM11. López-Knowles E, O’Toole SA, McNeil CM, Millar EKA, Qiu MR, Crea P, Musgrove EA, Sutherland RL. PI3K pathway activation in breast cancer is associated with the basal-like phenotype and cancer-specific mortality. International Journal of Cancer. 2010. 126:1121-31. (IF 4.93). EM12. Fedele CG, Ooms LM, Ho M, Vieusseux J, O’Toole SA, Millar EKA, Lopez- Knowles E, Sriratana A, Gurung R, Baglietto L, Giles GG, Bailey CG, Rasko JEJ, Shields BJ, Price JT, Majerus PW, Sutherland RL, Tiganis T, McLean CA, Mitchell CA. The PtdIns(3,4)P2 4-phosphatase, INPP4B, regulates ER-positive mammary cell proliferation and is lost in human basal-like breast carcinomas. Proceedings of the National Academy of Science (USA). 2010.107:22231-6. (IF 9.77). EM13. O’Toole SA, Machalek D, Shearer R , Millar EKA, Nair R, McLeod D, Cooper C, McFarland A, Ru Qiu M, McNeil CM, Rabinovich B, Martelotto L, Vu D, Musgrove E, Sutherland RL, Watkins N, Swarbrick A. Hedgehog overexpression is associated with stromal interactions and predicts for poor outcome in breast cancer. Cancer Research. 2011. 71:4002-4014. (IF 8.23). 10 EM14. Zardawi SJ, Zardawi I, McNeil CM, Millar EKA, McLeod D, Morey AL, Crea P, Murphy NM, Lopez-Knowles E, Oakes SR, Ormandy CJ, Qiu MR, Hamilton A, Spillane A, Lee CS, Sutherland RL, Musgrove EA, O’Toole SA. Notch1 expression is an early event in breast cancer development and is associated with the HER-2 molecular subtype. Histopathology. 2010; 56, 286–296. (IF 3.57). EM15. López-Knowles E, Zardawi SJ, McNeil CM, Millar EKA, Crea P, Musgrove EA, Sutherland RL,. O’Toole SA. Cytoplasmic localization of ß catenin is a marker of poor outcome in breast cancer patients. Cancer Epidemiology Biomarkers Prevention. 2010;19:301-9. (IF 4.19). EM16. Murphy NC*, Biankin AV*, Millar EKA*, McNeil CM, O’Toole SA, Segara D, Crea P, Olayioye MA, Lee CS,