Overcoming primary and acquired erlotinib resistance with epidermal growth factor receptor (EGFR ...

390 Pages·2015·24.24 MB·English

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OVERCOMING PRIMARY AND ACQUIRED
ERLOTINIB RESISTANCE WITH
EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR)
AND PHOSPHOINOSITIDE 3-KINASE (PI3K)
CO-INHIBITION IN PANCREATIC CANCER

SUBMITTED IN FULFILLMENT OF THE REQUIREMENTS
FOR ADMISSION TO THE DEGREE
DOCTOR OF PHILOPSOPHY
UNIVERSITY OF SYDNEY

MATTHEW H WONG (311260888)
SYDNEY MEDICAL SCHOOL
Sep 2015
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DEDICATION

This thesis is dedicated to my wife Wendy Ho; my two beautiful daughters,
Tiffany and Nadia Wong; and to my parents, Andy and Tammy Wong for
their love, faith and patience these three years as I pursue the challenges
of post-graduate research.

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DECLARATION AND CONTRIBUTION
The work in this thesis was performed by the candidate, except where due
acknowledge has been made.
This work was undertaken at the Department of Cancer Surgery, Kolling Institute of
Medical Research, The University of Sydney, Royal North Shore Hospital. I declare
that no part of this work has been submitted previously for the purpose of obtaining a
degree or diploma in any other universities.
The in-vitro work was independently designed and performed by myself (MW), with
advice and assistance to methods development by supervisors and lab technicians.
The in-vivo work was a group project undertaken by the whole of Cancer Surgery
group. Planning of experiments, preparation of drugs and statistical analyses were
independently performed by myself, the surgical and drug administration procedures
were shared (AX, SJ, MW, RM); H&E was primarily undertaken by SJ and IHC
undertaken by AX to maintain quality control, with some assistance provided by
myself.

Matthew Wong
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ACKNOWLEDGMENT
I would like to thank my Primary supervisor, Professor Ross Smith, for his guidance,
supervision, support and funding for this research work to be completed. He is not only
an outstanding researcher, but also a caring surgeon and a great friend. He always
has time for his students, and he will strive his very best to make our dreams happen.
I also like to thank my co-supervisor Professor Rob Baxter for his guidance and
advice throughout the planning and execution of this research project. His immense
knowledge in the IGF1R and PI3K pathways and his many years of experience in
research has helped me solidify my concepts and translate them into practice. I
especially like to thank him for the many hours he put in to edit my research to
publication qualities for conference and the Clinical Cancer Research paper.
I very much thank my co-supervisor A/Professor Nick Pavlakis for his many years of
support of me as his advanced trainee, research fellow and now staff specialist in
Medical Oncology. His expertise and experience in Medical Oncology, Clinical Trials
and his associations with Core research groups and pharmaceutical companies have
been instrumental to the progress of this research work, and I thank him for the way I
have become as a researcher, as a doctor and as a person.
A special thanks extends to my co-supervisor Dr. Aiqun Xue, for the countless hours
she has put in to develop my research skills and laboratory skills, and help me design
the appropriate experiments for this project. It must be tiring teaching a doctor with
little previous research experience the basics of in vitro and in vivo experiments. I
certainly could not achieve this research project without Aiqun’s help.
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I also like to thank Dr. Sohel Julovi, post-doc scientist in our CanSur Research Group.
His help and assistance to the directions of my project is much appreciated. I
particularly like to thank his help in the designing, planning and teaching me in regards
to the animal experiments.
I would like to give a special thanks to A/Prof Jas Samra, a great teacher, pancreatic
surgeon and partner in pancreatic cancer research. His help with the animal studies
and providing the surgical specimen are most crucial to the success of this large scale
in vivo study. I thank him for the constant support and advice he gives me for the
pancreatic cancer patients whom I share care.
I also need to thank Dr Tom Hugh, our liver surgeon and great teacher in upper GI
research. I thank him for his constant support of me and my project, and the great
lengths of time he put in for assisting with grant applications.
Here I should express my appreciation to Mr. Lyndsay Peters, the manager for the
flow cytometer in Kolling Institute. I thank him for his expert advice and assistance for
the operation of the flow cytometer, which has occupied much of my research and time
in the first 2 years of my study.
I also thank Dr. Sergey Kurdyukov, the manager for confocal microscopy and
genomic facility. His assistance is vital for ensuring the quality of my research with
immunofluorescence and PCR.
I also like to thank A/Professor Anthony Gill, head of Cancer diagnosis and
pathology, for his valuable assistance and advice on surgical pathology,
histopathology and immunohistochemistry, which is crucial to this project.
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I like to thank Dr. Mike Lin very much for his support and advice in many of my in vitro
experiments, particularly the confocal microscopy and western blotting techniques.
I would like to thank Joy McPherson, secretary of Professor Ross Smith, who has
looked after me for many years before me undertaking research. I thank her for her
for organising meetings and her involvement in the many funding functions for CanSur.
I greatly appreciate my funding partners: Amgen Research Scholarship, Royal
North Shore Staff Specialist Scholarship, Ramsey Scholarship, Pink Ladies and
Cancer Surgery Foundation, and CanSur. Without them I would not be able to
complete this project within the 3 year time frame.
I also like to thank Novartis Pharmaceuticals for providing BYL-719 and BEZ-235
targeted therapies, and their assistance for my clinical trial proposal.

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TABLE OF CONTENTS
DEDICATION………………………………………………………….……...……. 2
DECALARATON AND CONTRIBUTIONS……………….….……...……. 3
ACKNOLWEDGMENTS…………………………..……………………...……. 4
TABLE OF CONTENTS………………………………...………………...……. 7
SUMMARY……………………………………………………….…………...……. 14
LIST OF PUBLICATIONS…………………...…………………………...……. 16
ABBREVIATIONS………………………………………………………………... 18
LISTS OF TABLES……………………………………………………………..... 20
LISTS OF FIGURES……………………………………………………………... 23
CHAPTER 1: LITERATURES REVIEW
1.1 Epidemiology of Pancreatic Cancer…………………………………….. 32
1.1.1 Incidence and Survival
1.1.2 Causes of pancreatic cancer
1.2 Molecular pathways in Pancreatic Cancer………………………………38
1.2.1 Multiple genetic alterations in Pancreatic Cancer
1.2.2 Clonal Heterogeneity, Driver mutations and Actionable Targets
1.2.3 Key molecular pathways in Pancreatic Cancer
7
1.3 Current treatment strategies in Advanced and Metastatic Pancreatic
Cancer……………………………………………………………………………..….. 51
1.3.1 Chemotherapy and Targeted therapies
1.3.2 Comparison of EGFR Drug Clinical Trials in PDAC, NSCLC and CRC
1.3.3 Resistance in EGFR targeted therapies in cancer
1.4 Strategies to improve targeted therapies efficacy ……….………….. 63
1.4.1 Novel targeted therapies
1.4.2 Combined blockade targeted therapies
1.4.3 Biomarkers in EGFR inhibitor therapies
1.5 Study Hypotheses………………………………………………………….. 78

CHAPTER 2: MATERIALS AND METHODS
2.1 Materials…………………………………………….……………………….. 81
2.1.1 Cell Lines
2.1.2 Media for Cell Culture
2.1.3 Novel Tyrosine Kinase Inhibitors (inhibitor)
2.1.4 Chemotherapeutic Agents
2.1.5 Antibodies
2.1.6 Primers
2.2 Methods……………………………………………………………………... 93
2.2.1 Cell Culture Protocols
2.2.2 Cryopreservation and Thawing Frozen Cells Protocol
2.2.3 Cell Proliferation (MTT Assay)
8
2.2.4 Cell Proliferation (Incucyte)
2.2.5 Clonogenic Assay
2.2.6 Phospho- RTK Array
2.2.7 Western Blotting
2.2.8 Immunofluorescence (Confocal Microscopy)
2.2.9 PCR Array
2.2.10 Quantitative Real-time PCR (qRT-PCR)
2.2.11 Cell Cycle (Flow Cytometry)
2.2.12 Apoptotic Assay (Flow Cytometry)
2.2.13 Migration Assay (Incucyte)
2.2.14 General Methods of Animal Experiment
2.2.15 Haematoxylin and Eosin Staining (H&E) and Immunohistochemistry
(IHC)
2.2.16 General Statistical Analysis

CHAPTER 3: GENETIC AND MOLECULAR CHARACTERISATION OF
PRIMARY AND ACQUIRED ERLOTINIB RESISTANCE
3.1 Introduction………………………………………………………..……….. 117
3.1.1 Chapter Background
3.1.2 Chapter Aims
3.1.3 Chapter Methods
3.2 Measuring Erlotinib Sensitivity in 5 PDAC Cell Lines…..………….. 120
3.2.1 Cell Proliferation Results to Erlotinib and Gefitinib Single Agent
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