Activating Kinase Mutations in Melanoma Ricardo Enrique Vilain BSc (Hons) MBBS (Hons) Doctor of Philosophy (Medical Genetics) University of Newcastle Newcastle, September 2012 ii Declaration The thesis contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to this copy of my thesis, when deposited in the University Library, being made available for loan and photocopying subject to the provisions of the Copyright Act 1968. I hereby certify that the work embodied in this thesis has been done in collaboration with other researchers. I have included as part of the thesis a statement clearly outlining the extent of collaboration, with whom and under what auspices. I hereby certify that the work embodied in this thesis contains a published paper work of which I am a joint author. I have included as part of the thesis a written statement, endorsed by my supervisor, attesting to my contribution to the joint publication work. _____________ Ricardo Vilain BSc (Hons) MBBS (Hons) i ii Dedication To my four beautiful girls: Anne-Marie, Vivian, Connie and Ingrid for a lifetime of love and support. iii iv Publications from Thesis Papers 1. Vilain RE, Dudding T, Braye SG, Groombridge C, Meldrum C, Spigelman AC, Ackland S, Ashman L, Scott RJ. Can a familial gastrointestinal tumour syndrome be allelic with Waardenburg syndrome? Clin Genet 2011;79:554-60. 2. Vilain RE, Braye SG, Ashman L, Scott RJ. (2011) Characterisation of KIT and other common oncogene mutations in malignant melanomas. In preparation. 3. Vilain RE, Braye SG, Ashman L, Scott RJ. (2011) BRAF and NRAS mutational status are prognostically important in thick and locally advanced cutaneous melanoma. In preparation. 4. Vilain RE, Braye SG, Ashman L, Scott RJ. (2011) MET juxtamembranous SNPs R988C and T1010I are over-represented in patients with Melanoma. In preparation. v vi Conferences Proceedings 1. Vilain RE, Braye SG, Ashman L, Scott RJ. The Importance of NRAS and BRAF mutations in thick and advanced melanomas (Poster). 8th Internal Melanoma Congress, Tampa FL, USA 2011. 2. Vilain RE, Ashton KA, Bowden NA, Braye SG, Ashman L, Scott RJ. Clinicopathological and gene expression profiling of advanced melanomas harboring KIT and other common kinase mutations (Poster). 7th Internal Melanoma Congress. Sydney NSW 2010. 3. Vilain RE, Braye SG, Ashman L, Scott RJ. Characterisation of KIT and other common kinase mutations in melanomas: A step in the development of patient-tailored treatment for melanoma (Speaker). Hunter Medical Research Conference on Translational Research, Newcastle NSW 2010. 4. Vilain R, Braye S, Ashman L, et al. Characterisation of KIT mutations in thick/metastatic melanomas: An Australian Perspective (Poster). 6th Internal Melanoma Congress, Boston MA, USA 2009. 5. Vilain R, Dudding T, Braye S, et al. Familial Multifocal Gastrointestinal Stromal Tumours and an associated with a White Spotting disorder (Poster). Hunter Medical Research Conference on Translational Research, Newcastle NSW 2008. vii viii Acknowledgments It takes a whole village to raise a child, and it has taken three supervisors, four laboratories and countless friends to make this PhD come to fruition. The lion’s share of my gratitude goes to my principal supervisor Prof. Rodney Scott, for his encouragement of my ideas and belief in my ability to see them through. I hope this is start of a long and productive research partnership. Those important early days of this research were conducted under the tutelage of Prof. Leonie Ashman, whose encouragement was instrumental in my decision to take on this work. To my mentor Dr. Stephen Braye, my eternal thanks for his unwavering support and enthusiasm for this project. I’m fortunate to have Dr. Braye as source of wise counsel in matters of research, pathology and life. The benefits of postgraduate studies are only realized when the students design, implement and, inevitably, troubleshoot their research. The best supervisors ensure their students feel this independence, while always making it clear they have their backs if things ever truly become dire. Amongst all colleagues in the department of Genetics at the Hunter Area Pathology Service, I’d like to particularly thank Ms. Gordana Pecenpetelovska, Dr Cliff Meldrum and Mr Michael Hipwell for bringing me up to speed with the techniques required to make this work possible. To all my friends in the Information Based Medicine laboratory at the Hunter Medical Research, thank you for welcoming a complete stranger with such warmth and kindness. A big thanks to Dr Katie Ashton for her advice, support and amazing technical abilities. Dr Ashton provided the gene expression data on the formalin fixed paraffin embedded tissues in this thesis. To Dr Nikola Bowden, my adoptive supervisor, whose passion for science, keen intellect and masterful use of social media were instrumental not only in the nature and direction of my work, but also in ensuring it was completed in a timely fashion. I hope to remain Dr Bowden’s pathologist-of-choice for years to come. Despite all these collaborations, my home always remained the department of Anatomical Pathology at Hunter Area Pathology Service. I’m in the debt of all the consultants, the secretarial and technical staff but I need to highlight key individuals: Mr Robert Borwell made his considerable technical expertise and all the anatomical pathology department’s resources available to me; Ms Zenobia Haffajee and Ms Megan Clarke provided technical assistance with the automated immunohistochemical staining runs; Ms Tina Hope assisted in the construction of the melanoma tissue microarrays; Mr Michael Hadlow, whose knowledge of the NSW Human Tissue Act (1983) underpinned this entire project, I could not have got this PhD up off the ground without his advice; To my departmental director A/Prof. Barbara Young, I thank her for taking a risk in employing a half-baked PhD as one of the senior registrars. I also would like to acknowledge Pfizer Australia, the National Health and Medical Research Council and the Royal College of Pathologists of Australasia for their financial support. Dr Darryl Irwin is thanked for his technical assistance with the generation of SequenomTM MassARRAY multiplex genotype assays. Dr Irwin also provided an independent assessment of the genotyping results. ix Despite the most supportive supervision and closest of collaborative partnerships, a PhD by its very nature can feel like a very lonely route to take. I believe the support only a family can provide gives the best chance of remaining sufficiently optimistic to see this massive undertaking through. I thank my mother for doing everything in her power to give her children the opportunity to achieve their full potential. She is the only role model I have ever needed. To my wife, who has borne the burden of my PhD, I thank her for the love and support which made it possible to see the successful completion of this project, my specialty exams and the birth of our daughter. x Table of Contents Declaration .................................................................................................................................... i! Dedication ................................................................................................................................... iii! Publications from Thesis ........................................................................................................... v! Conferences Proceedings ....................................................................................................... vii! Acknowledgments ..................................................................................................................... ix! Table of Contents ....................................................................................................................... xi! Figures and Tables ................................................................................................................. xvii! Abstract .................................................................................................................................. xxvii! Chapter 1 Introduction ................................................................................................................ 1! 1.1! Epidemiology of Melanoma in Australia .......................................................................... 3! 1.2! Management of Melanoma .............................................................................................. 6! 1.2.1! Clinical Detection ...................................................................................................... 6! 1.2.2! Melanoma Diagnosis ................................................................................................ 6! 1.2.3! Melanoma Subtype .................................................................................................. 7! 1.2.3.1! Superficial Spreading Melanoma ...................................................................... 7! 1.2.3.2! Nodular Melanoma ............................................................................................ 7! 1.2.3.3! Lentigo Maligna Melanoma ............................................................................... 7! 1.2.3.4! Acral Lentiginous Melanoma ............................................................................