THE ROLE OF INSULIN IN ADVANCED PROSTATE CANCER · ii The Role of Insulin in Advanced Prostate...

THE ROLE OF INSULIN IN ADVANCED PROSTATE CANCER

Dr Ian McKenzie BMedSci, MBBS (Hons)

Submitted in fulfilment of the requirements for the degree of

Masters of Applied Science (Research)

Faculty of Science and Technology

Queensland University of Technology

Australian Prostate Cancer Research Centre - Queensland

February 2011

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The Role of Insulin in Advanced Prostate Cancer i

Keywords

Advanced Prostate Cancer

Androgen Deprivation Therapy

Castrate Resistant Prostate Cancer

Hormone Therapy

Insulin

Insulin-like Growth Factor (IGF)

Metabolic Syndrome

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ii The Role of Insulin in Advanced Prostate Cancer

Abstract

Advanced prostate cancer is a common and generally incurable disease.

Androgen deprivation therapy is used to treat advanced prostate cancer with good

benefits to quality of life and regression of disease. Prostate cancer invariably

progresses however despite ongoing treatment, to a castrate resistant state. Androgen

deprivation is associated with a form of metabolic syndrome, which includes insulin

resistance and hyperinsulinaemia. The mitogenic and anti-apoptotic properties of

insulin acting through the insulin and hybrid insulin/IGF-1 receptors seem to have

positive effects on prostate tumour growth. This pilot study was designed to assess

any correlation between elevated insulin levels and progression to castrate resistant

prostate cancer.

Methods: 36 men receiving ADT for advanced prostate cancer were recruited,

at various stages of their treatment, along with 47 controls, men with localised

prostate cancer pre-treatment. Serum measurements of C-peptide (used as a surrogate

marker for insulin production) were performed and compared between groups.

Correlation between serum C-peptide level and time to progression to castrate

resistant disease was assessed.

Results: There was a significant elevation of C-peptide levels in the ADT

group (mean = 1639pmol/L)) compared to the control group (mean = 1169pmol/L),

with a p-value of 0.025. In 17 men with good initial response to androgen

deprivation, a small negative trend towards earlier progression to castrate resistance

with increasing C-peptide level was seen in the ADT group (r = -0.050), however

this did not reach statistical significance (p>0.1).

Conclusions: This pilot study confirms an increase in serum C-peptide levels in

men receiving ADT for advance prostate cancer. A non-significant, but negative

trend towards earlier progression to castrate resistance with increasing C-peptide

suggests the need for a formal prospective study assessing this hypothesis.

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The Role of Insulin in Advanced Prostate Cancer iii

Table of Contents

Keywords.................................................................................................................................................. iAbstract ................................................................................................................................................... iiTable of Contents ................................................................................................................................... iiiList of Figures ......................................................................................................................................... vList of Tables.......................................................................................................................................... viList of Abbreviations............................................................................................................................. viiCHAPTER 1: INTRODUCTION........................................................................................................ 11.1 Background.................................................................................................................................... 11.2 Context........................................................................................................................................... 31.4 Hypothesis ..................................................................................................................................... 41.5 Purposes......................................................................................................................................... 51.6 Thesis Outline................................................................................................................................ 6CHAPTER 2:SCIENTIFIC BACKGROUND.................................................................................. 92.1 The Prostate ................................................................................................................................... 92.2 Prostate Cancer ............................................................................................................................ 10

2.2.1 Localised Prostate Cancer............................................................................................... 112.2.2 Advanced Prostate Cancer .............................................................................................. 14

2.3 Androgen Deprivation Therapy................................................................................................... 152.4 The Metabolic Syndrome ............................................................................................................ 192.5 Insulin and Insulin-Like Growth Factor ...................................................................................... 20CHAPTER 3:LITERATURE REVIEW ......................................................................................... 253.1 Historical Background ................................................................................................................. 253.2 Metabolic Syndrome and Androgen Deprivation Therapy ......................................................... 263.3 Insulin and Cancer ....................................................................................................................... 313.4 Insulin and Prostate cancer .......................................................................................................... 353.5 The Role of Androgen in Castrate Resistant Prostate Cancer ..................................................... 413.6 Discussion and Future ................................................................................................................. 44CHAPTER 4:RESEARCH DESIGN ............................................................................................... 474.1 Methodology and Research Design ............................................................................................. 47

4.1.1 Methodology................................................................................................................... 474.1.2 Research Design ............................................................................................................. 47

4.2 Participants .................................................................................................................................. 484.3 C-Peptide ..................................................................................................................................... 494.4 Instruments .................................................................................................................................. 504.5 Procedure and Timeline............................................................................................................... 534.6 Analysis ....................................................................................................................................... 534.7 Ethics and Limitations ................................................................................................................. 53

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iv The Role of Insulin in Advanced Prostate Cancer

CHAPTER 5:RESULTS ................................................................................................................... 555.1 Androgen Deprivation Therapy and C-Peptide ........................................................................... 575.2 C-Peptide and Progression of Prostate Cancer ............................................................................ 60CHAPTER 6:ANALYSIS ................................................................................................................. 676.1 Androgen Deprivation Therapy and C-Peptide ........................................................................... 676.2 C-Peptide and Progression of Prostate Cancer ............................................................................ 72CHAPTER 7:CONCLUSIONS ........................................................................................................ 777.1 Summary of Findings and Future Plans ...................................................................................... 77BIBLIOGRAPHY ............................................................................................................................... 81 ACKNOWLEDGEMENTS …………………………………………………………………………86

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The Role of Insulin in Advanced Prostate Cancer v

List of Figures

Figure 1 Insulin, IGF-I and IGF-II signalling through IGF-I receptor5 .................................................. 4Figure 2 Insulin Receptor and its downstream pathways17 ................................................................... 21Figure 3 Schematic diagram of different ligand and receptor subtypes of the Insulin/IGF

system in physiological and pathological conditions (HR – Hybrid Receptor). IR-A and HR-A present in tumour and poorly differentiated tissues19 ......................................... 23

Figure 4 Representative staining of benign and malignant prostate tissue showing expression of IGF-1 and insulin receptors49 ............................................................................................... 41

Figure 5 Box Plot demonstrating Age (in years) range between control and ADT groups .................. 56Figure 6 Box Plot comparing age at time of prostate cancer diagnosis between control and

ADT groups .......................................................................................................................... 56Figure 7 Box Plot comparing age at time of onset of ADT with control group.................................... 57Figure 8 Box plot comparing serum C-peptide levels between control and ADT groups .................... 58Figure 9 Scatter plot comparing age to C-peptide level for the total study population......................... 59Figure 10 Scatter plot comparing age to C-peptide level for the control group only............................ 59Figure 11 Scatter plot comparing duration of ADT to serum C-peptide level...................................... 60Figure 12 Scatter plot comparing time to castrate resistant prostate cancer with serum C-

peptide .................................................................................................................................. 61Figure 13 Scatter plot comparing time to castrate resistance excluding PSA non-responders ............. 62Figure 14 Scatter plot of time to castrate resistance excluding diabetics and PSA non-

responders............................................................................................................................. 63Figure 15 PSA kinetics of ADT group patients with PSA<100 at time of onset ADT grouped

by serum C-peptide .............................................................................................................. 65

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vi The Role of Insulin in Advanced Prostate Cancer

List of Tables

Table 1 Adaptation of risk stratification for localised prostate cancer8 ................................................ 11Table 2 Summary of characteristics of two study groups ..................................................................... 55

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The Role of Insulin in Advanced Prostate Cancer vii

List of Abbreviations

ADT – Androgen Deprivation Therapy BMI – Body Mass Index – equal to mass/ (height in metres) 2 CRPC - Castrate Resistant Prostate Cancer DES - Diethylstilboestrol DHT – Dihydrotestosterone GnRH – Gonadotrophin Releasing Hormone HDL – High Density Lipoprotein IGF-1 and IGF-2 – Insulin-like Growth Factor types 1 and 2 IGF-1R – Insulin-like Growth Factor type 1 Receptor IR – Insulin Receptor IRS – Insulin Receptor Substrate LDL – Low Density Lipoprotein LH – Luteinizing Hormone LHRH – Luteinizing Hormone Releasing Hormone MAPK – Mitogen-associated Protein Kinase PI3K – Phosphatidylinositol 3 Kinase PSA – Prostate Specific Antigen SHBG – Sex-hormone Binding Globulin SREBP – Sterol Response Element Binding Protein

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viii The Role of Insulin in Advanced Prostate Cancer

Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet

requirements for an award at this or any other higher education institution. To the

best of my knowledge and belief, the thesis contains no material previously

published or written by another person except where due reference is made.

Signature: _________________________

Date: _________________________

Chapter 1: Introduction 1

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Chapter 1: Introduction

This chapter outlines the background (section 1.1) and context (section 1.2) of

this research. It describes the purposes (section 1.3) of the research project as well as

the scope and significance (section 1.4) of the issues identified. Section 1.5 will

outline the remaining chapters of this thesis.

1.1 BACKGROUND

Prostate cancer is the most commonly diagnosed cancer in Australia. The most

recently published data by the Australian Institute of Health and Welfare showed that

in 2006, prostate cancer constituted 29.5% of all cancers diagnosed in Australian

men. It was the second leading cause of cancer-related mortality in Australian males

behind lung cancer1.

Prostate cancer is a disease unique to men (the prostate gland being present

only in men) and increases in incidence with increasing age. Peak incidence occurs

between the ages of 70-74 years of age with the disease exceedingly rare below the

age of 40 years. In prostate cancer cases, greater than 85% are diagnosed in men over

the age of 65 years. The incidence of prostate cancer is increasing. Each year of the

decade from 1997 to 2006, the number of cases of prostate cancer diagnosed per year

increased steadily. The Age Standardised incidence for prostate cancer in men for

2006 was 170 cases per 100,000 people. An Australian man had a 1 in 7 risk of being

diagnosed with prostate cancer to the age of 75 years old, with a 1 in 89 chance of

dying of prostate cancer from the latest available data in 20061.

There are two possible explanations for this increase. The first is that more

people are developing prostate cancer. The alternative to this is that diagnostic

practices are changing, leading to an increase in detection of the unchanged,

previously undiagnosed volume of prostate cancer in the community. Prostate

Specific Antigen (PSA), a protein secreted uniquely by the prostate has been used to

varying degrees to screen for prostate cancer. Whilst the test is not specific for

prostate cancer, it has been promoted, and its use significantly increased, as a means

of early detection of prostate cancer. Testing for PSA became widely available in

Australia during the late 1980s. Since that time the rates of prostate cancer diagnosis

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2 Chapter 1: Introduction

have steadily increased1. It is likely that with the introduction of PSA testing,

prostate cancers are now being diagnosed, that prior to PSA testing in the community

would have remained clinically asymptomatic and therefore undiagnosed. There is

significant controversy surrounding this issue with debate over the appropriateness of

widespread PSA screening. The risk of early detection of prostate cancer that would

otherwise have remained indolent is that of overtreatment. Treatment modalities for

localised prostate cancer carry significant morbidity and low but defined risk of

mortality. The early detection of low-grade prostate cancers via PSA screening has

likely subjected a number of patients to unnecessary intervention and morbidity for

otherwise indolent disease. PSA testing is more widely used as a means of

monitoring for prostate cancer progression after diagnosis whether or not the patient

receives treatment for their prostate cancer, a use for which it is proven to be of great

value.

Regardless of the reason for the increasing incidence of prostate cancer, it

remains a significant public health problem with a significant associated morbidity

and mortality.

The demographics of the Australian population are changing also. Australia's

population, like that of most developed countries, is ageing as a result of sustained

low fertility and increasing life expectancy. During the period 30 June 1990 to 30

June 2010, the median age (the age at which fifty percent of the population is older

and fifty percent younger) of the Australian population has increased by 4.8 years,

from 32.1 to 36.9 years. Over the same period, the proportion of the Australian

population aged over 65 years increased from 11.1% to 13.5%. The total number of

elderly people increased by 170.6% compared with a total population growth of

30.9% over the same period2. Over the next several decades, population ageing is

expected to have significant implications for Australia in many areas including

health, labour force participation, housing and demand for skilled labour3. Given that

prostate cancer is a disease of the older population, one would therefore expect its

incidence to increase, making its management an increasing health burden for

society.

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1.2 CONTEXT

Prostate cancer that is localized can be successfully treated with either surgery

or radiotherapy with each giving a good chance of cure. Once prostate cancer has

spread outside the prostate, either locally into adjacent structures or to distant sites,

most commonly lymph nodes or bone, the management is less successful. In these

cases, the most common treatment is androgen deprivation therapy (ADT). This

entails use of medication or surgical removal of the testes to lower serum androgen

levels and therefore deprive prostate cancer cells of testosterone. This causes

apoptosis in androgen-dependent prostate cancer cells and in most cases, clinical

regression of disease. This is clinically best measured as a decline in serum PSA.

Advanced prostate cancer is defined to include the following patient groups:

1. Clinically locally advanced prostate cancer. This is clinical stage T3

(extending outside the prostatic capsule) or T4 disease (invading

outside the prostate into adjacent structures e.g. the bladder).

2. Prostate cancer that has metastasized to any site distant from the

prostate. This most commonly involves the lymph nodes or bones,

though it can spread to other organs as well.

3. Patients with a biochemical recurrence of prostate cancer. This is

defined as a rising PSA after previous definitive therapy for prostate

cancer (usually in the form of surgery or radiotherapy).

In these patient groups, when ADT is used it achieves a good response in the

majority of cases with a decline in PSA, clinical improvement in extent of disease,

and often symptomatic relief.

Unfortunately, the response to ADT is not a durable one. Almost universally,

men on ADT will eventually progress, often initially signalled by a rise in PSA

despite castrate serum testosterone levels. The median time to progression for men

with metastatic disease is 14-20 months post initiation of ADT4. This phase of

disease is known as Castrate Resistant Prostate Cancer (CRPC) and is almost

universally fatal. To date, despite many theories as to why and how CRPC develops,

there is no clear consensus. Clearly, understanding the mechanism behind CRPC

development is the key to developing new therapies and improving the outlook for

men with advanced prostate cancer. It is with this cohort of patients in mind that this

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research project has been developed, attempting to assist in the understanding of the

development of CRPC to help direct future development of possible therapeutic

agents for this poorly understood phase of prostate cancer.

1.4 HYPOTHESIS

There is a large amount of published and ongoing research, examining the

effects of insulin and the insulin-like growth factors 1 and 2 (IGF-1 and IGF-II) on

cancer, including prostate cancer as will be outlined later in the literature review

(Chapter 3). The insulin and IGF-1 receptors are tyrosine kinase receptors, sharing

common downstream pathways; exhibiting both mitogenic and anti-apoptotic

properties (see Figure 1). Because these effects are ideal for cancer cell growth, it has

sparked significant interest in what role, if any, they may play in cancer, and

specifically prostate cancer progression.

Figure 1 Insulin, IGF-I and IGF-II signalling through IGF-I receptor5

There is significant epidemiological evidence suggesting a link between

elevated serum insulin levels and development and progression of multiple types of

cancers, including prostate cancer. The association of elevated serum insulin levels

and faster progression of prostate cancer have been demonstrated by in vitro studies

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of prostate cancer cell lines and in vivo mouse models of prostate cancer. This

evidence is described in detail in Chapter 3.

ADT has numerous side effects. The most common of these include hot

flashes, anaemia, loss of bone mineral density and osteoporosis, loss of libido and

erectile dysfunction. ADT also induces weight gain with increases in percentage

body fat mass and loss of lean body mass. More recently, the extent of the metabolic

side effects associated with ADT has become apparent. ADT induces insulin

resistance, with associated elevation of serum insulin levels, altered serum lipid

levels, and an increased risk of developing both diabetes mellitus and cardiovascular

disease. The details of these changes are further discussed in chapter 3.

Combining this information, we identified four key points:

ADT whilst initially causing regression of prostate cancer invariably

progresses despite ongoing therapy with castrate serum testosterone levels

(castrate resistant prostate cancer)

Serum insulin increases with androgen deprivation therapy in advanced

prostate cancer

Elevated serum insulin is associated with the development and progression of

cancers including prostate cancer

In vitro experiments demonstrate more rapid prostate cancer progression with

elevated serum insulin levels

This led to the development of the following hypothesis upon which this

project is based:

Serum insulin increases due to Androgen Deprivation Therapy for

prostate cancer, and this hyperinsulinaemia is directly correlated to prostate

cancer progression to Castrate Resistant Prostate Cancer.

1.5 PURPOSES

New studies are being developed looking at altering the insulin axis and

assessing how this affects prostate cancer growth and progression. Areas of

particular interest focus on affecting the availability of ligand available to bind to the

insulin receptor, or altering the insulin receptor and its actions with either anti-insulin

receptor antibodies or tyrosine kinase inhibitors. This work is based on the

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assumption that insulin is positively affecting prostate cancer growth and

progression. In the available literature however, there are no investigations in the

human prostate cancer patient population correlating elevated serum insulin levels

with progression of disease in men with advanced prostate cancer receiving androgen

deprivation therapy. This significant gap in the literature is what this project is

aiming to address.

This project was designed as a pilot study to assess serum insulin levels,

using serum C-peptide, a surrogate marker for endogenous insulin production, in

men receiving ADT for their advanced prostate cancer. The main objectives of the

study were as follows:

To confirm the literature findings that men who are receiving ADT have

elevated serum C-peptide levels compared to men not receiving ADT.

To correlate elevated C-peptide levels with ADT with the progression of

advanced prostate cancer to the castrate resistant phase. In accordance with

our hypothesis, we believe that men with a higher serum C-peptide level, will

progress to castrate resistance at a more rapid rate than those with lower

serum C-peptide level. This pilot study aims to identify a correlation to justify

the design of a formal trial to further investigate this correlation.

1.6 THESIS OUTLINE

This thesis takes the following structure. Chapter 2 describes the scientific

background to the problem under consideration. It describes prostate cancer

pathology, pathogenesis, diagnosis and treatment. It describes the physiology of

androgen deprivation therapy as well as its side effects. It discusses the metabolic

syndrome, its definition and its consequences. Finally, chapter 2 outlines the

structure and function of insulin, the insulin receptor and its signalling.

Chapter 3 encompasses the literature review. Section 3.1 outlines the

evidence linking the metabolic syndrome and androgen deprivation therapy. Section

3.2 describes the association between insulin and cancer and section 3.3 between

insulin and prostate cancer. The combined literature forms the grounding for the

hypothesis under investigation in this thesis.

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Chapter 4 describes the materials and methods of this study. It explains

participant features and methods of patient recruitment, techniques for measurement

of serum C-peptide and the methods of statistical analysis performed.

Chapter 5 presents the results of this study.

Chapter 6 synthesises the relevant results and discusses them in relation to the

study hypothesis.

Chapter 7 summarises our findings and presents a plan for future investigation

of this topic.

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Chapter 2: Scientific Background 9

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Chapter 2: Scientific Background

This chapter outlines the basic scientific context of this project. Section 2.1

outlines the anatomy and physiology of the prostate gland. Section 2.2 discusses the

pathophysiology of prostate cancer, its diagnosis and staging, and its management.

Section 2.3 discusses androgen deprivation therapy, its uses and mechanisms, and

discusses its side effects. Section 2.4 discusses the metabolic syndrome, its definition

and its consequences.

2.1 THE PROSTATE

The prostate is a male sex accessory organ. It is retroperitoneal in location

lying beneath the bladder in the pelvis, above the urogenital diaphragm, and

immediately anterior to the rectum. It is penetrated throughout its length by the

proximal urethra. It is a glandular organ and provides approximately 30% of the

volume of the seminal fluid. It is made up of glandular acini embedded in a fibro-

muscular stroma. It can be divided into 4 zones, differing on histology. The anterior

or fibro-muscular zone consists of smooth muscle, with little to no glandular

elements. The central and transitional zones surround the urethra and contain

approximately 25% of the prostate glandular elements. Finally the peripheral zone

makes up 75% of the glandular component of the prostate6.

The prostate is supported in its growth, maintenance and secretion by various

hormones, the foremost being testosterone. This hormone is converted in the prostate

to its more active constituent dihydrotestosterone (DHT). The primary source of

testosterone is from the testes. Testosterone is produced de novo in the Leydig cells

of the testes under the influence of luteinizing hormone (LH), and is secreted into the

circulation where it is mostly bound to sex-hormone binding globulin (SHBG). LH is

produced by the pituitary gland in response to Luteinizing Hormone Releasing

Hormone (LHRH) from the hypothalamus. These three hormones work in a feedback

loop such that when testosterone is low, it stimulates the hypothalamus to produce

more LHRH, which in turn stimulates the pituitary to produce LH, leading to

increased testosterone production from the testes. Alternatively, when serum

testosterone is elevated, LHRH production by the hypothalamus decreases, leading to

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10 Chapter 2: Scientific Background

decreased LH secretion from the pituitary gland and subsequently decreased

testosterone production from the testes. This is known as the hypothalamic-pituitary-

testicular axis.

In addition to testosterone, androgen is produced from the adrenal gland in the

form of androstenedione. This is a weak androgen, which can be converted

peripherally to testosterone. This likely only contributes approximately 5% of serum

testosterone concentration. The contribution of adrenal androgen to prostatic growth

and function appears to be minimal7.

2.2 PROSTATE CANCER

Prostate cancer, in the majority of cases is an adenocarcinoma with well-

defined, readily demonstrable glandular patterns. Approximately 70% of cases

involve the lateral or peripheral zone of the prostate. Adenocarcinoma of the prostate

is multifocal in more than 85% of cases8. Because of this classically posterior

location for cancer, tumours may be palpable by clinical digital rectal examination.

Prostate cancer is graded according to the histological appearance of the

prostate tissue. The tissue is allocated a numerical grade, from 1 to 5, based on

glandular patterns and the degree of differentiation. Grade 1 is well differentiated,

down to grade 5, which is very poorly or undifferentiated. Tissue usually contains

more than one pattern of grade so the predominant pattern is assigned a primary

grade, and the subdominant pattern assigned a secondary grade. The two grades are

added to give an overall score known as the Gleason score. This is known as the

Gleason system of grading. Tissue grading is important in prostate cancer, as it has

been shown, along with the stage of disease and PSA to accurately predict

prognosis9.

Like the benign prostate, prostate cancer is heavily dependent on testosterone

for both its development and progression. The anabolic effects resulting from

activation of the androgen receptor play a critical role in the growth of prostate

cancer cells. This becomes extremely important when considering possible treatment

options for prostate cancer patients. It is this dependence that forms the underlying

basis for androgen deprivation therapy.

Staging of prostate cancer is routinely via the TNM system. This is the

Tumour, Node and Metastasis system of staging. T1 represents cancer found

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incidentally on trans-urethral resection for benign hypertrophy symptoms (T1a or

T1b depending on the volume of disease found) or T1c for disease, found on needle

biopsy performed for investigation of an elevated PSA, which is not clinically

palpable on digital rectal examination. T2 is cancer palpable on digital rectal

examination that is still organ confined. T3a and T3b tumours extend outside the

prostatic capsule without and with seminal vesicle involvement respectively. T4

disease directly invades adjacent organs. Any spread to lymph nodes is recorded as

either present or absent, regardless of extent (N1 or N0 respectively). Any metastatic

spread, whether to bones or soft tissue is denoted as M1 disease (M0 allocated if no

metastases). Each individual tumour is allocated a T, N and M rating based on its

individual characteristics, and this is the clinical stage of disease10.

D’Amico and colleagues designed a category description (low, intermediate or

high) based on clinical prostate cancer stage, prostate biopsy Gleason grade, and

PSA level at the time of diagnosis of prostate cancer. This category was designed to

reflect the risk of prostate cancer recurrence following attempted curative therapy for

localized prostate cancer. This classification has been shown to reflect clinical

behaviour and prognosis for prostate cancer11.

Level of Risk Low Intermediate High

PSA level (ng/mL) <10 10-20 >20

Biopsy Gleason Score <6 7 8-10

Clinical T stage T1, T2a T2b T2c

Table 1 Adaptation of risk stratification for localised prostate cancer11

2.2.1 LOCALISED PROSTATE CANCER

Localized prostate cancer is defined as tumour that remains localized to the

prostate gland (i.e. clinical T1 or T2 disease). Within this group of tumours, there is

wide variation in natural history of the disease based on the level of differentiation

measured via Gleason grade, and serum PSA, both of which help to determine

prognosis for an individual’s disease. Localized prostate cancer can be managed in

multiple ways. These include active surveillance, surgery and radiotherapy. The

choice of appropriate treatment depends on both tumour and patient related factors.

These include the individual tumour characteristics such as Gleason grade, serum

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PSA and clinical stage. The patient’s age, other medical co-morbidities and projected

survival from non-cancer causes, as well as the personal choice of the patient and the

treating physician also play a significant role in the decision for any treatment.

Active surveillance is a method of closely monitoring a patient with low risk,

localized prostate cancer with the aim of instituting active management should there

be concerning changes in PSA or on a repeat prostate biopsy. One rationale for this

management is the belief that PSA screening is detecting a population of prostate

cancers that, without screening would never have been detected in the patient’s

lifetime and wouldn’t have caused significant disability or death. Because of the

possible significant side effects of treatment for prostate cancer, there may be a

population of patients in whom the risks of side effects of curative treatment are

deemed to be more significant than the risk of progression of cancer. To date

however there is no consensus as to which patients are suitable for this form of

management and at what point active treatment should be instigated. Delayed

treatment has usually been reserved for men with less than 10 years life expectancy

and low grade localized disease. There is however a push to define a group of

younger men with low risk, small volume, low grade disease who may be suitable for

active surveillance7. There is however more research required to properly define this

group and to validate outcomes over a prolonged period before this can be routinely

recommended for younger men12.

Clinical T1 or T2 disease, and in a small number of cases, favourable T3

disease, can be treated with surgery in the form of a radical prostatectomy. This

operation removes the entire prostate gland including the seminal vesicles, but

sparing the external urinary sphincter mechanism. The bladder neck is reconstituted,

and anastomosed to the proximal urethra. The operation may be accompanied by a

pelvic lymph node dissection, which gives additional pathological staging

information. There is ongoing debate as to whether lymph node dissection provides

any additional benefit with regards to local control of prostate cancer7. Currently in

Australia, pelvic lymph node dissection is not routinely performed during a radical

prostatectomy. It is considered in high-risk localized disease for its pathological

staging information. This operation gives a good chance of cure of localized disease,

although progression rates, either biochemical recurrence or metastases, as well as

survival rates vary, depending on the individual pathological characteristics of the

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tumour13. Adverse prognostic features following radical prostatectomy include non–

organ-confined disease, perineural, lymphatic or vascular space invasion, extra

capsular tumour extension, positive surgical margins, seminal vesicle invasion, and

lymph node metastases. In men with pathologically organ-confined disease, the 10-

year cancer progression-free survival approaches 90% following radical

prostatectomy13.

External beam radiotherapy uses beams of gamma radiation directed at the

prostate and surrounding tissues. Modern 3-dimensional conformal therapy allows

more accurate focusing of the radiation dose to the prostate. This allows higher doses

to be given which has been shown to improve cancer outcomes, also minimizing

radiation damage to adjacent structures such as the bladder and rectum, which were

significant limitations with older forms of radiation therapy to the prostate. In spite

of these improvements the main side effects of radiation therapy remain bladder and

rectal toxicity, as well as damage to the sphincter muscle and urethra, with associated

urinary incontinence. There is also risk of erectile dysfunction due to damage to the

vasculature of the cavernous nerves and corpora cavernosa involved in penile

erection. Radical prostatectomy is still considered the gold standard treatment for

men with localized prostate cancer. Therefore, the majority of radiotherapy is given

to older men, or men who would not tolerate surgery with localized disease, or men

with high risk localized disease or locally advanced disease7. Research has shown

that better outcomes in all parameters are achieved from radiotherapy for prostate

cancer, if adjuvant long-term androgen deprivation therapy is given to men with

locally advanced or high-risk localized disease, or short-term (6 months) ADT for

men with intermediate risk localized disease14. 10-year cancer cure rates for localized

disease with standard external beam radiotherapy are about 50%. With newer 3D

conformal radiotherapy allowing radiation dose escalation and adjuvant androgen

deprivation, better outcomes are reported with 5-year progression-free probabilities

for localized tumours reported between 70-85%. At this time there is little data on

long-term durability of response in younger patients with these newer treatment

regimens. There are no randomized controlled trials comparing results of

radiotherapy with radical prostatectomy for low risk disease7.

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2.2.2 ADVANCED PROSTATE CANCER

Advanced prostate cancer has been defined in multiple ways. Current consensus

includes any of the following groups of patients:

Locally advanced prostate cancer. This includes clinical stage T3 or T4

disease.

Biochemical recurrence following previous curative therapy. This can be due

to residual local tumour, metastatic tumour deposits, or both. The rising PSA

usually occurs well before any clinical parameters or imaging modalities can

detect recurrence of disease. Biochemical recurrence is defined as a rise in

PSA after reaching a post-treatment nadir (the lowest point PSA reaches after

treatment).

Metastatic prostate cancer, whether to lymph nodes, bone or soft tissue. The

most common sites for metastases from prostate cancer are lymph nodes and

bone15.

There are different definitions for what should be considered a recurrence

following either radical prostatectomy or radiotherapy. Following radical

prostatectomy, PSA should become undetectable in blood, due to the removal of all

viable prostate tissue. Multiple definitions have been used however a consensus

definition of biochemical failure post radical prostatectomy published by the Prostate

Cancer Working Group, defined it as a PSA of 0.4ng/mL or higher, greater than 8

weeks post surgery, and that continues to rise on a subsequent measurement16.

Following radiotherapy, the prostate is still in situ, therefore benign prostate tissue

may still survive, producing PSA without there being any viable tumour remaining.

Therefore the definition of biochemical recurrence following radiotherapy is not so

clear. The American Society for Therapeutic Radiology and Oncology (ASTRO)

produced guidelines on this issue, defining recurrence as three consecutive PSA rises

after reaching nadir PSA level, measured at least 3 months apart, with the date of

failure taken as the mid-point between nadir PSA and the date of the first rise in

PSA17. This definition however has been widely criticized for underestimating the

incidence of true cancer recurrence and perhaps missing the opportunity for further

curative treatment in some patients. Other definitions have been proposed based on

various absolute PSA values however these are undergoing validation7.

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In advanced cases, while a proportion will be considered for radiotherapy,

and a smaller proportion of favourable cases, for radical prostatectomy, the mainstay

of treatment, particularly for metastatic disease and biochemical recurrence, will be

androgen deprivation therapy. By blocking the production of androgen, or inhibiting

androgen receptor binding, the androgen-dependent prostate cells are deprived of

androgen, which induces apoptosis in these cells, with shrinking of the prostate (if

still in situ) and extra-prostatic tumour deposits18. This is clinically seen as a decline

in serum PSA (due to apoptosis of PSA-producing prostate cells), and symptomatic

improvement such as decreased bone pain and improvement in urinary symptoms7.

2.3 ANDROGEN DEPRIVATION THERAPY

It has been known for almost 150 years that prostatic epithelium is

exquisitely sensitive to androgens and will atrophy with castration. In their series of

papers published in the 1940s, Huggins and Hodges presented a small study of 21

men with metastatic prostate cancer who were treated with surgical castration. They

documented a decrease in serum acid phosphatase levels, improvements in X-Ray

appearance of bony metastases as well as an improvement in symptoms in all but 3

cases19. This work was based on the initial findings that acid phosphatase levels were

elevated in both benign and malignant prostate tissue (both primary and secondary)

and increased in response to androgen with the realization that they were

biochemically analogous.

Since those original findings, multiple methods of altering the androgen axis

have been developed. All forms of Androgen Deprivation Therapy function by

decreasing the ability of androgen to activate the Androgen Receptor (AR). This can

be achieved by either decreasing the amount of available androgen, or via blocking

androgen binding to the androgen receptor.

The earliest forms of ADT were via bilateral orchidectomy (surgical removal

of the testes). By removing the testosterone producing Leydig cells of the testes, the

major contributor to serum androgen levels is removed. This procedure leads to a

greater than 90% decrease in serum androgen levels within 24 hours. A small amount

of androgen is produced by the adrenal glands, in the form of androstenedione, and

16


from peripheral conversion of oestrogen, but these sources provide insignificant

amounts to the overall serum levels of available androgen7.

Androgen supply is controlled centrally via the Hypothalamic-pituitary-

testicular axis. Luteinizing Hormone Releasing Hormone (LHRH) is released from

the hypothalamus to activate the anterior pituitary to produce Luteinizing Hormone

(LH), which acts on the Leydig cells of the testes to stimulate testosterone

production. Serum androgen levels provide a negative feedback loop to the

hypothalamus to regulate LHRH release. The first drugs used to manipulate this

central control of androgen production, was the administration of exogenous

oestrogens, mostly in the form of Diethylstilboestrol (DES). Oestrogens are very

potent inhibitors of LH secretion, therefore causing dramatic decreases in serum

testosterone. DES has been widely studied in advanced prostate cancer, but its use is

limited by an unacceptable cardiovascular toxicity.

The more modern manipulator of central control of androgen production is

the class of LHRH agonists such as Goserelin acetate. After causing an initial surge

in LH secretion, the loss of physiological phasic pituitary stimulation causes

dramatic decreases in LH production, and subsequent castrate testosterone levels.

These are the most common current form of ADT, administered via subcutaneous

depot injection in 1, 3 or 6 monthly preparations. The initial surge in LH, and

therefore testosterone can cause an initial worsening of symptoms, so they are often

administered initially in combination with androgen receptor antagonists in men with

metastatic disease. These preparations are the most common form of ADT currently

in use, with or without androgen receptor antagonists. LHRH antagonists are also

available and do not cause the initial LH and testosterone surge, though their use is

much less common than the agonist preparation7.

Moving from central androgen regulation, there are a wide variety of

androgen receptor antagonists available. All these preparations inhibit androgen

action by competitive binding to the androgen receptor. These come in two classes,

steroidal and non-steroidal forms. The steroidal anti-androgens (e.g. cyproterone

acetate) block androgen receptor binding, but also have progestational effects

causing suppression of central LH release. These drugs therefore work at the cellular

level, as well as decreasing circulating testosterone levels. The non-steroidal anti-

androgens have no anti-gonadotrophic effects and simply block androgen receptor

17


binding, both at the prostate, and centrally. This has the effect of blocking serum

testosterone’s inhibitory feedback on the hypothalamus, causing a paradoxical

increase in LH and testosterone levels. The advantage of this is that it can minimize

the effect on erection function, one of the major side effects of any androgen

deprivation therapy.

Less commonly used now, some drugs act to inhibit non-testicular androgen

supply. Ketoconazole acts to inhibit adrenal steroid synthesis and also inhibits

testicular Leydig cell testosterone synthesis. Whilst it rapidly induces castrate

testosterone levels, its response is not durable long-term, with testosterone levels

returning to near normal within 5 weeks of continuous therapy. It is now more

commonly used as second line therapy in castrate resistant prostate cancer.

Aminoglutethimide inhibits peripheral conversion of cholesterol to pregnenolone, an

early step in steroid synthesis. Whilst it inhibits testosterone synthesis, it also inhibits

cortisol and aldosterone synthesis. Because of these effects, patients must take long-

term steroid replacement and the medication is associated with significant side

effects. It is now rarely used in practice7.

Androgen deprivation therapy gives a very good, reproducible and durable

response in advanced prostate cancer. The significant majority of patients will show

evidence of clinical response, most commonly in the form of a decline in serum PSA

level. For patients with significant symptoms from their prostate cancer, the initiation

of ADT is very effective in providing relief, as left untreated, these symptoms can

cause significant decline in quality of life. Sharifi et al published a systematic review

of Randomised Controlled Trials examining the efficacy of ADT in 2005. This

demonstrated clear benefit from ADT in terms of quality of life in the setting of

advanced prostate cancer. There were clear reductions in bone pain, pathological

fractures, spinal cord compression and ureteric obstruction. They concluded that any

benefit in terms of survival is as yet unclear4.

Unfortunately, the effect of ADT on prostate cancer almost invariably

diminishes over time. This is most commonly signaled by a rise in PSA after

reaching a nadir level. This phenomenon is known as Castrate Resistant Prostate

Cancer. It is believed that this occurs due to the growth of a subset of cells requiring

minimal androgen to support growth. The exact mechanisms causing this are as yet

unclear and it is the topic of extensive research, including this study. The average

18


time to development of CRPC in men with metastatic disease is 14-20 months post

onset of androgen deprivation therapy4. CRPC onset signals a slow decline with

eventual radiographic and clinical progression of disease, and is the fatal form of

prostate cancer.

Unfortunately androgen deprivation, while a very effective treatment for

advanced prostate cancer, does come with significant side effects. These common

side effects have been well documented and much published.

The most common side effects are documented below:

Hot flashes - a subjective perception of sudden increase in temperature,

specifically a feeling of warmth in the face, neck, upper chest and back.

These symptoms may be accompanied by a reddening of the skin, as well as

perspiration. This troublesome effect has been documented to occur in up to

80% of men undergoing treatment with an LHRH agonist20.

Loss of libido and sexual function – Testosterone is very important in the

male sexual response. Decreasing serum testosterone levels with ADT have

been shown to negatively impact upon both of sexual desire and libido, as

well as erectile function. Assessment of the SEER database of Prostate

Cancer Outcomes, examining quality of life outcomes in 431 men treated

with ADT, showed complete loss of erectile function in up to 78% of men,

and loss of sexual desire in up to 63%. Both effects were significant after

adjusting for baseline function20.

Anaemia – Up to 90% of men receiving ADT will have at least a 10% decline

in haemoglobin level. It is believed to be in part due to the effects of

testosterone on erythropoietin levels, a hormone required for new

haemoglobin synthesis. A recent prospective study of 83 men receiving ADT

however demonstrated a decline in haemoglobin but maintenance of

erythropoietin levels. It seems that the relation between haematopoiesis and

androgens needs to be defined further21.

Osteoporosis – Studies have shown that bone mineral density decreases

significantly with ADT. It is normal for men to lose bone mineral density

with age; however ADT seems to accelerate this process. The rate of

development of osteoporosis is between 1.4 and 2.6% per year. Associated

19


with this accelerated loss of bone mineral density is an increase in the risk of

fractures. Most significantly is fracture of the neck of femur, which is

associated with significant morbidity and mortality in the elderly population.

The SEER database revealed an increased risk of fracture over the normal

population after 1year of ADT and this risk increased with increased number

of doses of GnRH agonist20.

Cognitive function – Whilst there is some conflict in the literature, a decline

in attention and memory has been reported for men undergoing ADT. In

addition, impairment in concentration and verbal skills has been reported.

Likewise, reports of increased incidence of depression have been linked to

ADT.

These side effects range from being bothersome, to those impacting significantly on

the physical and mental health of patients.

More recent research has focused on the metabolic side effects of ADT.

These metabolic changes include decreased insulin sensitivity and associated

increased serum insulin, sarcopenic obesity and serum lipid profile changes. These

side effects are similar to the classic metabolic syndrome. These are gaining more

interest as the increased risk of diabetes mellitus and cardiovascular disease

associated with ADT becomes apparent. A large population based cohort study

published in 2004 demonstrated that cardiovascular disease was the leading non-

cancer cause of mortality in prostate cancer patients22. This has focused attention on

what effect ADT has on cardiovascular risk, and how these effects can be modified.

These significant metabolic side effects will be discussed in more detail in chapter 3.

2.4 THE METABOLIC SYNDROME

The Metabolic Syndrome is a name given to a collection of metabolic

disturbances associated with an increased risk of developing cardiovascular disease

and diabetes mellitus. Unfortunately this cluster of changes is becoming increasingly

common in modern society as the Western lifestyle of high calorie and fat intake, and

increased sedentary lifestyle becomes more common, with increasing weight gain

and obesity. The most recent health statistics from the Australian Bureau of Statistics

shows that in the 2007/08 Australian National Health Survey, 25% of adults over 18

years old were obese (BMI>30) and 37% overweight (BMI 25-30) based on body

20


mass index (BMI). This consisted of 68% of adult males and 55% of adult females.

This had increased from the 1995 National Nutrition Survey with 19% of adults were

obese and 38% overweight23.

There are numerous definitions of the metabolic syndrome, mostly differing

in their emphasis on components, and how to define each criterion. The most

commonly accepted definitions all include a combination of:

Central obesity - measured in various ways depending on classification, either

as elevated BMI >30kg/m2, elevated waist circumference, or elevated waist:

hip ratio

Dyslipidaemia – elevated triglycerides and decreased High Density

Lipoprotein (HDL)

Elevated blood pressure

Impaired glucose tolerance – depending on definition, this includes measured

insulin resistance, elevated fasting glucose, or previously diagnosed diabetes

mellitus

The most accepted criteria in recent times are those defined by the World Health

Organization, the National Cholesterol Education Program and the International

Diabetes Federation. The WHO definition places particular focus on insulin

resistance as a necessary component, whereas that of NCEP places equal emphasis

on each factor with at least 3 required for the diagnosis. The IDF definition places

emphasis on central obesity as defined by waist circumference24. Regardless of the

definition, the metabolic alterations of this syndrome are all individually risk factors

for the development of atherosclerotic vascular disease. There is now debate in the

literature as to whether combining these components has any added diagnostic or

prognostic value over each in isolation, however the term persists25.

2.5 INSULIN AND INSULIN-LIKE GROWTH FACTOR

Insulin is a hormone secreted by the beta cells of the pancreas, primarily in

response to an increase in circulating nutrient levels. It interacts with its receptor, the

insulin receptor on insulin sensitive cells. In general, the insulin receptor is

expressed at high levels only in adipose tissue, muscle and in the liver. These tissues

21


express one of the two IR isoforms, IR-B. The second isoform, IR-A, primarily

considered a foetal isoform, is present in these tissues at low levels.

The insulin receptor is a tyrosine kinase receptor. Activation by insulin

binding increases transcription of Sterol Response Element Binding Protein

(SREBP), and phosphorylation of tyrosine residues on the Insulin Receptor Substrate

(IRS) family of proteins. Phosphorylation triggers interaction with key signalling

molecules, activating a variety of signalling pathways, most importantly

Phosphatidylinositol 3 Kinase (PI3K)/Akt, Mitogen-activated Protein Kinase

(MAPK), and the Cbl/CAP complex. Insulin’s primary role is in glucose

homeostasis, stimulating cells to transport glucose from the bloodstream into the

cells via glucose transporters. Insulin functions to promote storage of energy in the

body. Activation of these pathways acts in a co-ordinated manner to stimulate uptake

of glucose, fatty acids and amino acids into liver, adipose tissue and muscle and

promote the storage of these nutrients in the form of glycogen, lipids and protein

respectively. At the same time, it inhibits glycogenolysis and gluconeogenesis,

protein degradation and lipolysis (See Figure 2). As well as these metabolic

functions, activation of the insulin receptor also has effects on cell survival with

activation of the MAPK cascade leading to mitogenic responses26.

Figure 2 Insulin Receptor and its downstream pathways26

22


The IGF-1 Receptor is structurally highly homologous to the insulin receptor,

and is a tyrosine kinase receptor, activating similar downstream pathways to the

insulin receptor. It is activated by either of its ligands, IGF-1 and IGF-2. Liver

production of IGF-1 accounts for the majority of IGF-1 in circulation. Regulation of

IGF-1 production is complex, but is primarily regulated by Growth Hormone.

Circulating IGF-1 acts on target tissues in an endocrine fashion, however IGF-1 is

also synthesised by other organs having both autocrine and paracrine effects. IGF-2

is also produced by the liver and other tissues but is not closely regulated by growth

hormone27. The bioavailability of IGF-1 and -2 is tightly regulated by binding to a

family of IGF Binding Proteins (IGFBPs). IGF-2 is also bound by the IGF-2

receptor, however this receptor has no known signalling activity and may act only to

sequester IGF-2 preventing its binding to IGF-1 or insulin receptors. While the

insulin receptor is primarily involved in metabolism and energy storage, the IGF-1

receptor is expressed on virtually all tissues of the body and plays more of a role in

growth regulation of the body stimulating cell proliferation and inhibiting

apoptosis27,28 (see Figure 1).

Both the IGF-1 and insulin receptors are tetramer structures comprising two

hemi-dimers, each containing an intra and an extracellular subunit. As mentioned

above there are two types of insulin receptor, subtypes IR-A and IR-B. The IR-A

receptor subtype is primarily expressed in foetal tissues. It is also expressed in tissues

of different malignancies. This subtype primarily stimulates mitogenic responses,

while the IR-B subtype is primarily involved in metabolic effects. Cells that express

both IGF-1R and IR, because of their high degree of homology, can form hybrid

IGF-1/insulin receptors. These can be formed from a hemi-dimer of each of IGF-1

and IR29. Whilst IGF-2 has minimal affinity for the IR-B subtype, it can bind and

activate the IR-A subtype and therefore the IGF-1/IR-A hybrid receptor. Therefore,

in the presence of each of IGF-1, insulin and hybrid receptors, IGF-1 can bind to the

IGF-1R or either of the hybrid receptors, IGF-2 can bind the IGF-1R or the IGF1/IR-

A hybrid receptor, and insulin can activate any of the IR subtypes and to a lesser

extent the IGF-1R, but has minimal affinity for hybrid receptors28. This is outlined in

Figure 3 below. The net result is that each of the three ligands, IGF-1, IGF-2 and

23


insulin are able to activate the growth promoting and anti-apoptotic effects on a wide

range of tissues, depending on the individual receptor expression within each tissue.

Figure 3 Schematic diagram of different ligand and receptor subtypes of the Insulin/IGF system in physiological and pathological conditions (HR – Hybrid Receptor). IR-A and HR-A present in tumour and poorly differentiated tissues28

24


Chapter 3: Literature Review 25

25

Chapter 3: Literature Review

This chapter gives a historical background (section 3.1) and reviews literature

on the following topics: Androgen Deprivation and the Metabolic Syndrome (section

3.2); the role of insulin in cancer in general (section 3.3); the role of insulin in

prostate cancer (section 3.4); and the role of androgen in castrate resistant prostate

cancer (Section 3.5). Section 3.6 will synthesise, summarise the conclusions from the

literature, and establish the gap in knowledge that this study is investigating.

3.1 HISTORICAL BACKGROUND

The common side effects of Androgen Deprivation Therapy have been much

studied and are well documented as discussed in Chapter 2. The first papers

published by Huggins and Hodges in the 1940’s on the use of castration as a form of

treatment for prostate cancer noted a marked increase in appetite and subsequent

weight gain in the vast majority of their patient cohort post castration30.

In 1990, Tayek et al published a small study of 10 patients with advanced

prostate cancer undergoing 12 months of GnRH agonist therapy. This study

confirmed a significant increase in body weight and body fat percentage after 12

months of treatment. They also documented an increase in total cholesterol up to

17% by 2 months of treatment, which was maintained throughout the study.

Interestingly 60% of their patients were malnourished prior to commencing this

study, and with no control group it was uncertain what effect this may have had on

the results31.

After a period of little literature published on the metabolic side effects of

ADT, the new millennium saw a renewed interest in the topic. The last decade has

seen a renewed interest in the changes in body mass and associated metabolic

changes of androgen deprivation and these initial findings have been confirmed and

clarified in multiple studies.

26

26 Chapter 3: Literature Review

3.2 METABOLIC SYNDROME AND ANDROGEN DEPRIVATION THERAPY

A metabolic syndrome occurs with androgen deprivation therapy. This is the

conclusion of multiple studies investigating the metabolic side effects of ADT. Initial

findings on the role of androgens in glucose and lipid metabolism were made during

observation of elderly men, in which the prevalence of hypogonadism is

significant32. One study found the prevalence of hypogonadism in men over the age

of 45 years was 36.3%. This prevalence increased with increasing age33. Multiple

studies on the correlation between testosterone and control of metabolism have been

published assessing the effect of the natural decline of serum testosterone with aging.

In the published literature, two main author groups have dominated this topic.

Basaria et al, based at Johns Hopkins University in Baltimore, Maryland, USA, have

published multiple papers examining the metabolic side effects of ADT. These

papers were all based on cross-sectional studies of similar design. They matched

patients receiving ADT for more than 12 months against two control groups, men

with prostate cancer of similar stage and grade not receiving ADT, and age-matched

eugonadal men without prostate cancer. The first control group was designed to

account for changes associated with prostate cancer alone, and the second control

group to account for age-related changes. These studies were unanimous in their

findings of an increase in body mass index (BMI), and an increase in percentage fat

mass in the ADT group compared to both control groups. They also assessed lipid

changes, finding an increase in total cholesterol and LDL cholesterol levels,

remaining after accounting for BMI. The main issue with these studies were their

cross-sectional design and their very small numbers (n=20 for ADT group in the

largest study34). Despite this they, chose good control groups and their findings are

consistent with those of the prospective studies in the published literature.

Importantly also, their study group were men who had been on ADT for at least 12

months with an average of 45 months duration, putting them in the significant

minority of studies that examine longer term effects of ADT35,36.

The second author group responsible for a large amount of the published

literature on this topic is that of Smith et al, based in Boston, Massachusetts, USA.

Their studies were prospective assessments of the changes in body composition,

insulin sensitivity and lipid profile during ADT. Whilst these studies benefitted from

27


prospective assessment, they had small numbers of patients (n=25 and 26

respectively) and lacked any control groups. Their initial work suffered also from a

very short follow-up of only 3 months37,38, however subsequent prospective data

gathered over 12 months was published in 200839. Given the long natural history of

advanced prostate cancer however, and the generally long period of time that patients

remain on ADT, this would still seem to be a short duration over which to assess

outcomes from this type of therapy.

These studies demonstrated that after 3 months of ADT, insulin sensitivity had

decreased by 12.9% and serum insulin increased by 25.9%. Patients had increased

percentage fat mass and decreased lean body mass by 4.3% and 1.4% respectively by

3 months of treatment, and by 12 months this had increased to 11.2% and 3.6%

respectively38,39. These figures are very similar to another prospective study of 79

patients on ADT trialling Zoledronic acid for management of bone metastases

published by the same group. This likewise showed increased fat mass of 11% and

decreased lean body mass of 3.8% after 12 months of treatment40. They have also

reported serum lipid changes with a statistically significant increase in each of total

cholesterol, LDL and HDL cholesterol and triglycerides by 3 months37.

Outside of these two research groups, multiple other papers have been

published on this topic. These have tended to each focus on different aspects of the

metabolic syndrome and which, if any of these occur with ADT. These will be

discussed by type of metabolic alteration.

Insulin

The most consistent changes associated with ADT reported in the literature are

changes to serum insulin and insulin sensitivity. Insulin resistance is a state of

reduced sensitivity of normally insulin-responsive tissues to insulin. This results in

an impaired ability of insulin to suppress hepatic glucose production and stimulate

peripheral glucose uptake. Insulin production is the prime regulator of circulating

glucose levels. Insulin resistance would therefore be expected to result in an increase

in circulating glucose levels, however, if pancreatic beta cell (insulin secreting cells)

function is normal, increased amounts of insulin are secreted to overcome insulin

resistance and thereby normalize glucose levels. This hyperinsulinaemia is a

hallmark of insulin resistance41.

28


Consistent with the findings reported above, Smith et al performed a

prospective study of 22 patients receiving ADT for three months. The patients were

assessed pre-treatment, at one, three and six months. At three months, half the

patients ceased treatment to assess reversibility of changes. They confirmed an

increase in fat mass and a decreased lean body mass, and found that serum insulin

levels were significantly increased at all 3 time points of treatment, although fasting

glucose remained normal. Whilst this was a small study, it was particularly

significant in its finding that metabolic changes were occurring at such an early point

in treatment (within 4 weeks) 42. The elevated blood insulin but normal glucose

implies impaired insulin sensitivity, but increased insulin production sufficient to

maintain normal glucose levels.

Persistent elevation of serum insulin and insulin resistance was confirmed in a

larger prospective study of 139 patients. This study divided patients into either

continuous or intermittent androgen deprivation, then further divided each treatment

group according to BMI into two groups, those with BMI<24 and BMI>24. The

average duration of ADT in the intermittent group was 7.2 months such that patients

received ADT for the majority of the twelve months duration of the study. They

found that insulin and insulin resistance were significantly elevated in all four groups

at six months. Of interest, those receiving intermittent therapy, (those that ceased

ADT part way through the study), showed insulin levels returning almost to baseline

by 12 months. This demonstrated reversibility with cessation of treatment. This is

important as it strengthens the conclusion that the metabolic changes are a true

treatment effect. This study is one of the largest prospective studies published on this

topic. One issue identified however is that it was performed on a Chinese population,

such that extrapolating results to a Western population may be unreliable43.

Insulin resistance is a key feature of type 2 diabetes mellitus. Increased insulin

is produced to maintain serum glucose levels, but eventually is insufficient and frank

diabetes occurs with elevated fasting serum glucose levels. Three retrospective

studies have assessed the incidence of new onset diabetes mellitus in prostate cancer

patients receiving ADT. The prominent feature of these studies is the significant

sample size of each. This has been possible by the use of patient databases with

retrospective assessment of their prospectively collected data. This is an important

difference as the prospective studies have all suffered from small patient numbers

29


limiting the power of their findings. The largest study was of 73,196 men with loco-

regional prostate cancer from the SEER (Surveillance, Epidemiology and End

Results) American Medicare database. They assessed the incidence of new onset

diabetes, cardiovascular disease, myocardial infarction and sudden cardiac death in

prostate cancer patients receiving ADT (both GnRH agonist and surgical

orchidectomy were included), with prostate cancer patients not on ADT. The

incidence of all outcomes was significantly higher in the ADT group versus the no

androgen deprivation group. The adjusted risk for developing diabetes mellitus was

significantly increased with any form of ADT (either medical or surgical castration)

however for all other outcomes the adjusted risk was only significantly elevated for

GnRH agonist therapy. Most notably in this study, this increased risk of diabetes was

present from as early as 1 to 4 months of treatment and remained high with ongoing

therapy44.

Two smaller retrospective database reviews had similar findings of increased

risk of developing diabetes with ADT45,46. The review of Derweesh et al showed a

worsening of fasting blood glucose in patients who were known to have diabetes

prior to onset of ADT. Data analysed retrospectively from a database is not as

reliable as that obtained prospectively, particularly as database information may miss

significant details, particularly those that rely solely on diagnosis codes from patient

records, which can be extremely unreliable.

Lipids

There is some debate in the literature regarding the extent of lipid alterations

associated with ADT. The classical metabolic syndrome is characterised by an

increase in triglycerides and a decrease in HDL cholesterol. Along with these

changes, increased LDL cholesterol has been shown to increase a patient’s risk of

developing cardiovascular disease. The majority of the literature published on this

subject, are small prospective studies. The findings from all of these studies are

consistent, finding a significant increase in total cholesterol within the first 12

months of ADT. The data however is mixed on changes to triglycerides, LDL and

HDL cholesterol. Three separate prospective studies by Smith et al demonstrated

significant increases in each of total cholesterol (range 9-9.4%), LDL cholesterol

(7.3-9.9%), HDL cholesterol (8.7-11.3%) and triglycerides (23-26.5%). Whilst these

studies each used small cohorts, diminishing their statistical power, the findings

30


between studies were very consistent37,39,47. These studies also suffer from the lack of

a control group for comparison. Interestingly, one of these studies was of short

duration at 12 weeks (the other studies lasted 12 months) yet still demonstrated

significant changes, indicating an early effect of therapy37.

A cross-sectional, small study of 16 men on long-term ADT agreed with the

significant increase in total cholesterol found by Smith et al. They only found

significant increases in other lipid parameters however, compared against age-

matched controls, after adjusting for BMI. There was no significant difference

comparing these lipid parameters between ADT and non-ADT prostate cancer

groups48.

Pooled data from three prospective phase 3 trials comparing LHRH agonists

against LHRH antagonists, assessed lipid changes over six months of therapy, but

most significantly assessed changes in men receiving cholesterol lowering agents in

the form of the statin class of medications. This study demonstrated a significant

increase in both total cholesterol and HDL cholesterol regardless of statin therapy,

occurring within twelve weeks of therapy. Importantly it must be noted however that

a therapeutic effect of statin drugs is an elevation of HDL cholesterol. The findings

for LDL and triglycerides was mixed however with an increase in LDL cholesterol in

the non-statin group only and both increased and decreased triglycerides depending

on which type of ADT was received49. This does confuse the overall conclusions

somewhat; however the strength of an elevation in total cholesterol despite lipid-

lowering therapy suggests that this effect of ADT is significant.

Summary

The available literature clearly demonstrates that ADT induces a form of

metabolic syndrome. The classical metabolic syndrome includes a combination of

central obesity, dyslipidaemia, elevated blood pressure and impaired glucose

tolerance, although there are multiple definitions with slightly different requirements

within each group of changes. The available literature agrees that ADT causes

sarcopenic obesity that is a gain in percentage fat mass with a loss of lean muscle

mass, regardless of change to overall weight. Insulin sensitivity decreases, with an

associated increase in serum insulin, also a worsening of diabetic control in diabetic

patients that are commenced on ADT. There is an increase in total cholesterol levels,

and perhaps an increase in LDL cholesterol and triglycerides, though these changes

31


need further study to draw a firm conclusion. The data on changes to HDL

cholesterol are inconsistent, but there appears to be a trend towards increased serum

levels. ADT does not cause any change in blood pressure. From this evidence, the

metabolic syndrome induced by ADT is different to the classical type39.

The result of the metabolic changes caused by ADT is a trend towards an

increased risk of developing diabetes mellitus and cardiovascular disease. Prostate

cancer has its highest incidence between 60-70 years of age. This coincides with the

peak incidence of cardiovascular disease, mostly due to an increase in the incidence

of risk factors for cardiovascular disease such as hypertension,

hypercholesterolaemia and type 2 diabetes mellitus occurring at a similar age. It is

clear that androgen deprivation therapy induces a form of metabolic syndrome, and

given the age of patients in which it is commonly used, this will likely increase the

risk of developing cardiovascular disease with its resulting morbidity and mortality.

Lu Yao et al demonstrated that indeed cardiovascular disease is the leading cause of

mortality in prostate cancer patients other than prostate cancer specific mortality22.

This is believed to be because of the age group of prostate cancer patients and the

incidence of cardiovascular disease in this age group in the general population. The

findings of the metabolic syndrome shown to occur with ADT may well be

contributing to this statistic.

3.3 INSULIN AND CANCER

The role of insulin in neoplasia is a topic that continues to receive significant

interest. Insulin and the closely related Insulin-like Growth Factors 1 and 2 are able

to interact with multiple receptors. These are either of the insulin or IGF-1 receptors,

and the insulin/IGF receptor hybrid. The insulin and IGF-1 receptors are both

tyrosine kinase receptors with mitogenic and anti-apoptotic effects, to varying

degrees as outlined in Chapter 2. There is significant homology between the two

types of receptor, with both activating similar downstream pathways, in particular

those of PI3K and Ras/MAPK (see Figure 1). Their roles in neoplasia seem to be

intrinsically linked, with evidence implicating all three in the development and

progression of various cancers. Whilst their actions in vivo differ, blood levels of

each are linked, varying with nutritional state, and high levels of insulin directly

increasing IGF-1 production by the liver50. Their role in cancer has been

hypothesised to occur via insulin, IGF-1, or IGF-2 binding to and activating any of

32


the insulin, IGF-1 or hybrid insulin/IGF-1 receptors51. The general underlying

principle is that deregulation of insulin and IGF-I signaling, associated with

increased activation of IR and IGF-IR, might occur in tumors, with subsequent

increases in signaling through the PI3K and MAPK pathways, leading to unregulated

protein synthesis, cell cycle progression and cell growth, as well as prevention of

apoptosis5.

The evidence, whilst circumstantial, suggests an association between serum

insulin and IGF-1 levels, and the development and progression of malignancy. Two

well-designed meta-analyses have been published on this topic.

The first, by Pisani et al focussed on epidemiological studies of circulating

insulin, and its relation to the risk of developing cancer. They pooled data from case-

control and prospective studies, looking particularly at colorectal, breast, endometrial

and pancreatic cancer. With respect to colorectal cancer, they demonstrated a pooled

relative risk of cancer development of 1.35 (95% CI 1.13-1.61) for patients with the

upper categories of blood insulin or C-peptide levels. This analysis included data

from 10 prospective studies and demonstrated the strongest association, with the

least heterogeneity between studies. In breast cancer, a pooled relative risk for cancer

development of 1.26 (95% CI 1.06-1.48) likewise suggested an association with

elevated insulin levels. In this case, however, the positive associations were only

seen with retrospective studies, while the four prospective studies didn’t reach

statistical significance individually. The data was mixed on both endometrial and

pancreatic cancer; however an association was suggested, though not statistically

significant. The main issue identified in this area of research is the significant

heterogeneity between studies in terms of design of the studies, the type of assays

performed, and whether patients were fasted or non-fasted which can significantly

alter the results. In addition, adjusting for confounding variables such as sex

hormone and other growth factor exposure was rarely done making a true

quantitative analysis almost impossible52. This review also suffers from a relatively

narrow literature search only using articles available through Pub med, and excluding

those not written in the English language. This may have resulted in significant

studies being missed. However, the overall qualitative indication from this review,

bearing these factors in mind, is that there is an increased risk of developing certain

types of malignancy with elevated levels of insulin.

33


The second review, a systematic review and meta-analysis focussed on

patient exposure to elevated levels of IGF-1 and its binding protein IGFBP-3. This

review was of better design than that of Pisani et al, with a much broader search of

the literature, and a clearly described method of analysis. They concluded that

elevated circulating IGF-1 levels were associated with an increased risk of colorectal,

prostate and pre-menopausal breast cancer. This association remained after

multivariate analysis. For prostate and pre-menopausal breast cancer, this was

demonstrated to have a direct dose-response relationship53. Again, this review

suffered from heterogeneity between study design, but with its more rigorous

approach to systematic review, it concludes that the associations found, while

modest, are significant. The findings of these meta-analyses have been supported by

multiple rigorous reviews published over the last few years affirming the positive

correlation between elevated blood insulin and IGF-1 levels, and development of

cancer41,54.

A large proportion of the work in this area has been performed on patients

with type II diabetes mellitus. This disease is characterised by insulin resistance,

elevated serum insulin levels and hyperglycaemia. Being so prevalent in modern

society, it provides a large cohort of patients from which to gather epidemiological

data. This evidence suggests that elevated serum insulin levels increase the risk of

developing various malignancies. The strongest evidence relates to colorectal, breast,

and pancreatic cancer, and to a lesser extent endometrial cancer.

The various treatments for type II diabetes have generated considerable

interest. The biguanide drug metformin acts to decrease circulating glucose and

insulin levels. This occurs by activation of the AMP-activated Protein Kinase

(AMPK)/LKB1 pathway, situated downstream of both the insulin and IGF-1 receptor

pathways, acting to inhibit mammalian Target of Rapamycin (mTOR) and therefore

the downstream effects of IR/IGF-1R activation (see Figure 1). The result of this in

the liver is decreased gluconeogenesis and hepatic glucose output. This consequently

leads to a decrease in circulating insulin levels54. The sulfonylurea class of drugs act

to increase pancreatic insulin production and therefore increase circulating insulin

levels. These drugs are also commonly used in type 2 diabetes. Exogenous insulin is

also administered in more advanced cases.

34


A retrospective cohort study by Libby et al, assessed diabetic patients

commencing on metformin, and compared them against matched diabetic patients

not receiving metformin, with the primary outcome measure being new cancer

diagnosis. They showed that the metformin group had a significantly decreased risk

of cancer diagnosis with an adjusted hazard ratio of 0.63 (95% CI 0.53-0.75)

compared to non-metformin control. The metformin group also demonstrated a

decreased cancer-related mortality, as well as a prolonged median time to cancer

diagnosis55. These results were supported by a further observational study of 11,876

diabetic patients demonstrating a 33% decreased risk of developing cancer with

metformin treatment compared to other treatments56.

Similar results were also obtained in a retrospective cohort study by Bowker

et al. Prospectively obtained data on 10,309 newly treated diabetic patients over 5

years of treatment was analysed. This demonstrated that those receiving exogenous

insulin therapy had an increased rate of cancer-related mortality compared to non-

insulin treated diabetic patients, with an adjusted hazard ratio of 1.9. An increased

risk was also seen with patients receiving the sulfonylurea class of drug (a

medication that increases serum insulin) compared with those receiving metformin, a

biguanide drug that lowers serum insulin levels. This was interpreted as a negative

effect of circulating insulin on cancer behaviour57. A caveat with this study however

is the relatively small number of deaths (407 in total) in a large population, so the

power of these findings can be questioned.

The main issue with all these studies is that, being retrospective in design, it

is difficult to control for confounders such as body weight and glycaemic control,

which were not recorded in the data. In addition, circulating insulin levels were not

measured, only inferred by treatment type. These factors may all be adversely

affecting the results. With no diabetic, non-medicated control group, one is unable to

comment on whether this effect was due to deleterious effects of insulin or

sulfonylurea’s, or perhaps a protective effect of metformin, as opposed to any direct

effect of insulin levels on cancer development and behaviour.

Over the last few years however, several good quality, rigorous reviews have

been published. They have examined the evidence around the association between

blood insulin levels and cancer risk. They found that as well as the population based,

35


epidemiological evidence there is also both experimental and clinical evidence

supporting this association.

Experimental evidence is from a combination of human cell and animal

studies. Early studies showed that physiological concentrations of insulin, applied to

breast cancer cells in vitro, was able to stimulate cell DNA synthesis54. In animal

models of colorectal cancer, development of mouse aberrant colonic crypt foci, a

precancerous change, was positively correlated with insulin administration, while

caloric restriction, known to improve insulin sensitivity and decrease circulating

insulin levels showed a negative correlation with development. Similarly, an

increased non-fasting insulin level correlated with increased size and number of

aberrant foci41. As well as elevated circulating ligand, examination of human tumour

tissue has demonstrated over-expression of the insulin receptor in both breast28 and

prostate58 cancer tissue when compared to their benign counterparts (see Figure 4).

Summary

The combined evidence demonstrates a positive association between elevated

blood insulin and IGF-1 levels and an increased risk of the development of various

cancers in humans. The strongest correlation seems to be with colorectal, post-

menopausal breast and pancreatic cancers. Whilst the majority of evidence is

circumstantial, derived from retrospective, observational, population based studies

with their inherent drawbacks, the overall weight of evidence lies in support of this

assertion.

3.4 INSULIN AND PROSTATE CANCER

Similar to the relationship between insulin, IGF and cancer in general,

significant interest surrounds their role in the development and progression of

prostate cancer. This association again derives from a combination of experimental,

population based data and clinical evidence.

There is mixed evidence regarding the overall risk of prostate cancer in

patients with hyperinsulinaemia. A well-designed systematic review performed by

Hsing et al concluded that the currently available evidence was insufficient to prove

any increased risk59. They also found that long-term diabetes actually decreased the

risk of development of prostate cancer. While type II diabetes demonstrates insulin

resistance and hyperinsulinaemia in its early stages, in long-term diabetes, insulin

36


levels eventually decline despite ongoing hyperglycaemia. Testosterone and IGF-1

levels also decline to low levels. Given prostate cancer’s reliance on androgen for

growth, it is possible that this, and possibly eventual low insulin and IGF levels

which can explain this finding59,60.

Epidemiological studies have however suggested a direct correlation between

insulin levels and prostate cancer mortality, and the risk of developing high-grade

prostate cancer61-63. A paper by Ma et al assessed the effect of pre-diagnosis body

mass index (BMI) and C-peptide (a surrogate marker for endogenous insulin

production) on prostate cancer specific mortality. They found that as baseline BMI

increased so too did the risk of prostate cancer specific mortality. In keeping with

this finding, obese men (BMI>30) were more likely to have extra prostatic or

metastatic prostate cancer or a higher Gleason grade of cancer at the time of

diagnosis than men with BMI <30 Obesity is associated with insulin resistance and

an increased risk of type II diabetes. They also demonstrated that patients with C-

peptide in the highest quartile had an increased prostate-cancer specific mortality

compared to those in the lowest quartile. They concluded from this that at least part

of the effect of increased BMI on mortality was related to insulin levels61. This study

was a retrospective assessment of prospectively accrued data from the Physicians

health study, a large study assessing the effects of aspirin and beta-carotene on men

without a history of heart disease, cancer or chronic illness. The best aspect of this

study was the numbers of patients accrued (2546 men diagnosed with prostate

cancer) and followed over a long period (24 years with median follow up of 7 years

between diagnosis and death or end of follow-up), which is important when assessing

prostate cancer with a long time course of progression.

Similarly, a nested case-control trial within the Prostate Cancer Prevention

Trial found similar results. While increasing C-peptide level was only weakly

associated with cancer risk, there was a strong association with the development of

high-grade prostate cancer of Gleason grade 7 or greater. For every unit increase in

log (C-peptide), there was a 39% increased risk of high-grade prostate cancer. This

association remained after adjusting for BMI and adiposity62.

Hammarsten et al prospectively assessed baseline insulin levels at time of

prostate cancer diagnosis and compared them between men who died from prostate

cancer during 5 years of follow-up, and men who survived. Statistically significant

37


risk factors identified for lethal prostate cancer included both Type 2 diabetes and

hyperinsulinaemia. Hyperinsulinaemia remained significant even after adjusting for

stage and grade of prostate cancer, factors known to affect prognosis. They

concluded that hyperinsulinaemia is a promoter of clinical prostate cancer63.

The hyperinsulinaemia of ADT has been shown to begin early in the course

of treatment. Some studies have documented a significant rise in serum insulin

within 1 month of onset of therapy with increased insulin resistance persisting

through to at least 12 months of therapy42. This shows that patients receiving ADT

undergo chronic exposure to elevated serum insulin from very early in their

treatment, and this is sustained as treatment progresses. Therefore, it is important to

consider what effects long-term exposure to elevated insulin levels have on prostate

cancer growth.

Experimental examination of the effects of insulin and IGF-1 on prostate

cancer is from both in vitro and in vivo experiments. Multiple prostate cancer cell

lines have been derived from both primary prostate cancer tissue and metastatic

tissue and have been used for many years to assess how prostate cancer cells respond

with manipulations of their growth environment. The effects of many important

treatments currently in use for prostate cancer have originally derived from work on

tissue cell lines. Each cell line has slightly different features in terms of receptor

expression, among others, providing very valuable first line indications of factors

affecting prostate cancer growth.

Cancer cell lines can be examined either growing in medium in a laboratory

dish or they can be implanted into animals, most commonly mice, to attempt to

mimic tumour growth in humans. Cell line experiments using multiple different

prostate cancer cell lines have shown a positive correlation between both insulin and

IGF-1 levels and prostate cancer cell growth. Mouse models containing either

LAPC-4 or LNCaP cell line xenografts have been assessed for their response to

manipulation of the insulin axis by dietary modification64-66. Xenograft mice were

grown and then separated into groups receiving different combinations of low and

high fat, and low or high carbohydrate diets. Freedland et al assessed LAPC-4

xenograft mice and found that those receiving the high fat, high carbohydrate diet

(termed the “Western” diet in this study) developed a 3 fold higher insulin level, and

a 26% higher IGF-1 level than their no carbohydrate, and low carbohydrate high fat

38


diet counterparts. The non-Western diet mice had smaller tumour size and longer

survival than the Western diet mice64. Ngo et al showed similar results, again

investigating LAPC4 xenograft mice. Mice on a low fat diet had significantly lower

insulin levels. Analysis revealed that serum insulin levels positively correlated with

each of PSA level, tumour volume and rate of tumour growth65. Mice xenografted

with LNCaP cells by Venkateswaran et al, and fed a high fat, high carbohydrate diet

developed elevated serum insulin and IGF-1 levels. Tumour cells displayed increased

insulin receptor expression and increased activation of Akt, situated downstream

from the insulin receptor. Elevated serum insulin was associated with significantly

increased size of the prostate tumour66. Each of these studies, though slightly

different in design, gives strong evidence for an in vivo relationship between serum

insulin levels and prostate cancer growth. The strength of these studies in

combination is that this association is demonstrated in two separate prostate cancer

cell lines.

Moving from laboratory studies, various studies have attempted to assess this

relationship in human patients. Snyder et al assessed the effect of pre-diagnosis

diabetes mellitus on prostate cancer prognosis. They performed a systematic review

and meta-analysis and found that the overall mortality rate for diabetic patients was

higher than that of non-diabetics with a pooled HR of 1.57 (95% CI 1.12-2.2).

However there was insufficient evidence to assess the effect of diabetes on prostate

cancer specific mortality67. Given the known increased risk of cardiovascular disease

with diabetes, it may be that the overall mortality increase was unrelated to prostate

cancer effects. This systematic review, whilst well designed does suffer from the lack

of available evidence on this topic and significant heterogeneity between included

studies. In addition, because of the lack of studies considered adequate for inclusion,

there is no distinction between type 1 (typified by low insulin levels) and type II

diabetes; therefore, it is impossible to extrapolate what effect serum insulin levels

may be having on prostate cancer growth.

Patel et al performed a retrospective analysis of prospectively collected data

on patients undergoing radical prostatectomy at their institution. They compared

outcome between diabetic patients, and non-diabetic control group matched for

prognosis based on age and prostate cancer pathology. They also divided the diabetic

group into those receiving, and those not receiving metformin. They found that

39


diabetes was associated with a statistically significant increased risk of biochemical

recurrence of prostate cancer with an overall hazard ratio of 1.55 (95% CI 1.03-2.33).

The 5-year biochemical recurrence free survival was 75% for non-diabetics, but only

59% for diabetic patients not receiving metformin. Interestingly metformin users

biochemical recurrence free survival was 66%, suggesting a protective effect of

metformin on univariate analysis, but this was not borne out on multivariate

analysis68. Unfortunately, serum insulin was not measured in these patients, and

again there was no differentiation between type1 and type 2 diabetics. Therefore

extrapolating these results to suggest a detrimental effect of exposure to elevated

insulin levels could only be done with extreme caution.

Attempting to clarify this issue, Lehrer et al actually assessed serum insulin

levels in patients of 3 different stages of prostate cancer undergoing either

brachytherapy or external beam radiotherapy in their institution. These patient groups

were divided into low, intermediate and high risk groups in line with the D’Amico

classification discussed in chapter 2 (see Table 1) with slight modification in PSA

levels between intermediate and high risk groups. They found that serum insulin

increased with increasing risk group. The high-risk group had significantly higher

serum insulin compared to the intermediate or low risk groups. There was no

significant difference between intermediate and low risk groups although the overall

numbers in the intermediate risk group were small such that insufficient numbers

may have been present to display any true difference. There is a major criticism of

this study, which may have significant bearing on our ability to interpret these

results. As part of the protocol, both the intermediate and high-risk groups received 3

months of neo-adjuvant androgen deprivation therapy. There is no statement

describing at what time serum insulin levels were measured. With the knowledge that

androgen deprivation therapy significantly elevates serum insulin within a short

duration of therapy, if serum insulin was measured post-treatment than the results of

this study bear no reflection on prostate cancer association with insulin, instead likely

only reflecting the effect of ADT used on their patient groups. This is a significant

flaw and one that would need to be clarified before putting any weight on the

findings of this study. The implication of the study is that measurements were

performed pre-treatment. If this is the case, this study demonstrates a significant

40


direct correlation between serum insulin and grade and stage of localized prostate

cancer69.

Whilst not specifically assessing insulin level, a recently published paper by

Flanagan et al, assessed men receiving ADT, and divided the group into men who fit

criteria for diagnosis of metabolic syndrome at the time of onset of ADT versus men

who did not. They used a modification of the ATP-III criteria for metabolic

syndrome. These required 3 or more of elevated triglycerides, obesity

(BMI>30Kg/m2), hypertension, low High Density Lipoprotein level and fasting

hyperglycaemia. They demonstrated a statistically significant decrease in time to

development of castrate resistance for men with metabolic syndrome. They also

performed a multivariate analysis demonstrating that each of the five criteria for

diagnosis of metabolic syndrome was individually associated with a decreased time

to castrate resistance, except for elevated triglyceride level70. This study was a

retrospective chart review; however they appear to have had good data medical data

available for their analysis. The study also suffers in its definition of castrate

resistance, using a non-consensus definition of time to reach 50% elevation of PSA

above nadir level. This may have inaccurately skewed their results. However, the

results of this study do loosely support our hypothesis in that the features of

metabolic syndrome, which have shown to be aggravated by ADT, negatively

affected time to castrate resistance. This support is tempered by the fact that insulin

was not analysed specifically in this study, nor was an elevated insulin level a

requirement for diagnosis of metabolic syndrome.

While all these studies give circumstantial evidence of insulin positively

affecting prostate cancer growth, the ability of prostate cancer to respond to insulin

via an appropriate receptor was further assessed in a key study by Cox et al. They

examined prostate cancer tissue expression of insulin receptors and insulin and IGF-1

receptor mRNA expression. Immunohistochemical staining of both benign and

malignant prostate tissue demonstrated significant increased expression of insulin

receptor, by two fold in malignant prostate tissue compared to benign prostate tissue.

Insulin receptor expression increased with increasing Gleason grade of cancer,

except for grade 5, which was not significantly different to grade 3. They

demonstrated that there was no difference in IGF-1 receptor expression between

benign and malignant prostate tissue (See Figure 4). Expression of insulin and IGF-1

41


receptor mRNA was significantly increased in prostate cancer specimens. The other

major conclusion was that prostate cancer tissue expresses a mixture of insulin, IGF-

1 and hybrid receptors. Whilst this study does not demonstrate insulin signalling, the

increased expression of insulin receptors on cancer tissue suggests an increased

sensitivity to insulin58.

Figure 4 Representative staining of benign and malignant prostate tissue showing expression of IGF-1 and insulin receptors58

3.5 THE ROLE OF ANDROGEN IN CASTRATE RESISTANT PROSTATE CANCER

There is growing evidence for an ongoing role of androgens in stimulating

prostate cancer growth despite androgen deprivation therapy, and in the presence of

castrate serum testosterone levels. The exact mechanism behind the development of

castrate resistant prostate cancer, at this time is not entirely clear. There is ongoing

debate regarding the exact definition of castrate resistant disease, however it is

generally accepted as a consistent rise in PSA after levels have reached a nadir or

lowest point following the institution of androgen deprivation therapy (medical or

surgical castration), occurring in the presence of castrate serum testosterone levels

with or without metastatic disease71. PSA (properly known as human kallikrein 3) is

42


an androgen-regulated gene. The recurrent expression of this gene following the

initial switching off with androgen deprivation therapy implies that the androgen

receptor resumes activation during progression to castrate resistance. Similarly the

fact that up to 30% of patients respond to secondary androgen axis manipulation

during castrate resistance implies an ongoing dependence on the androgen receptor

pathway in these patients72.

There are five main hypotheses surrounding the ongoing activation of the

androgen receptor after castration.

1. Mutation or amplification of the androgen receptor – Either mutations in the

androgen receptor, or increased expression of the androgen receptor can

render the prostate cells more sensitive to androgen such that they may be

activated by much lower concentrations of androgen73-76. Titus et al assayed

both castrate resistant prostate cancer and benign prostate tissue for

testosterone and dihydrotestosterone (DHT) levels. They found that castrate

resistant prostate cancer tissue had median DHT levels 90% lower than that

of benign tissue; however these levels were still sufficient to activate the

androgen receptor. Tissue testosterone levels were similar in both groups77.

These findings were further analysed by Locke et al. They demonstrated a

dramatic decline in tissue testosterone and DHT levels from castration to the

time of nadir PSA level; however levels increased again to reach

approximately 20% of pre-castrate levels by the time of castrate resistance78.

Leon et al demonstrated an increase in androgen receptor levels in an LNCaP

xenograft mouse model during progression from nadir PSA to castrate

resistance79. Visakorpi et al demonstrated this same phenomenon in humans

via in situ hybridisation, with up to 30% of prostate cancers examined having

amplification of the androgen receptor gene with increased expression of the

androgen receptor in the castrate resistant phase80.

2. Promiscuous Androgen Receptor – Missense mutations in the androgen

receptor gene can lead to decreased specificity of ligand binding, leading to

inappropriate activation of the receptor by non-androgen steroids and

androgen antagonists. Examples include adrenal androgens and some of the

androgen receptor antagonists. This mechanism is proposed to explain the

phenomenon of anti-androgen withdrawal effect. This is a phenomenon

43


occasionally seen in men with advanced prostate cancer where addition of an

androgen receptor antagonist, most commonly flutamide, results in worsening

of their disease, then cessation of the drug leads to a counter-intuitive decline

in PSA level74-76.

3. Outlaw Receptor – a term to describe steroid hormone receptors that can be

activated to a lesser, but still significant extent by ligand-independent

mechanisms81. The androgen receptor can be activated by certain growth

factors in the absence of androgen, including IGF-1, Epidermal Growth

Factor (EGF) and Keratinocyte Growth Factor (KGF). IGF-1 is the most

potent of these and has been shown to induce a five increase in PSA

expression in LNCaP cells82.

4. Bypass Pathway – describes alternative pathways that can be invoked that

enable bypassing of the androgen receptor entirely. With treatment targeting

critical survival pathways such as the androgen-dependent growth of prostate

cancer, there may be mutations that are selected out for a pathway allowing

growth stimulation and suppression of apoptosis. One example is expression

of the anti-apoptotic gene Bcl-2, not normally expressed in prostate tissue,

whose presence has been documented in castrate resistant prostate cancer in

both xenograft mouse models and human tissue73,74.

5. Lurker Cell Pathway – Describes the stimulated growth of cells not

dependent on androgen for survival, such as prostate epithelial basal cells,

caused by selection pressure after the androgen deprivation induced ablation

of androgen-sensitive prostate cancer cells.

All of these mechanisms have been proposed as a means of describing the

phenomenon of castrate resistant growth of prostate cancer. Each mechanism, which

can be supported by scattered evidence, are also contradicted by other findings such

that, to date, none have been shown to be able to adequately explain this

phenomenon and a combination, or perhaps another mechanism altogether may be

occurring83.

Another hypothesis to attempt to explain castrate resistant prostate growth is

the local production of androgens within the prostate sufficient to activate the

androgen receptor. This mechanism would explain the re-activation of the androgen-

dependent PSA gene despite castrate serum levels of testosterone. Locke et al

44


published a paper in Cancer Research assessing this topic. They assessed prostate

cancer tissue samples from an LNCaP mouse model pre-castration, at nadir PSA and

then at the time of castrate resistance. They demonstrated that tissue testosterone and

dihydrotestosterone levels dramatically decrease with castration to the time of nadir

PSA. However, levels increase again as the tumour progresses to castrate resistance,

reaching up to 20% of pre-castrate levels78. Similar to the findings of Titus et al in

human prostate tissue, these levels are indeed sufficient to activate the androgen

receptor77. Equally significant was the finding that the mRNA and protein of

enzymes required for synthesis of dihydrotestosterone from cholesterol were

expressed at all stages of disease and increased with progression to castrate

resistance. The other conclusions from this study was that castrate resistant tumours

are capable of conversion of acetic acid to dihydrotestosterone, and that they use

progesterone to synthesise DHT via both the classical pathway and a backdoor

pathway of steroid synthesis78(See Fig 3 – steroid pathways).

Following on from this work, our laboratory has shown, in soon to be

published data, that insulin treatment of LNCaP cells up-regulates key enzymes in

this steroid genesis pathway. There is also evidence that insulin treatment of LNCaP

cells causes increased dihydrotestosterone production.

3.6 DISCUSSION AND FUTURE

This study is investigating the role of hyperinsulinaemia in the progression of

advanced prostate cancer. The available evidence strongly suggests an association

between insulin levels and a variety of cancers, both development, and progression

of disease.

The association between insulin and prostate cancer is probably not as strong

with mixed quality evidence assessing this relationship. The current evidence

suggests that insulin levels bear no relationship to the risk of developing prostate

cancer. In fact, some studies suggest a possible decreased risk although these results

are confounded somewhat by alterations in androgen levels in diabetic patients from

which this data is extracted. Contrary to this however, emerging evidence suggests

that elevated insulin levels predict for a more aggressive and advanced prostate

cancer phenotype. Evidence also exists, though certainly requires further

45


investigation, that in patients already diagnosed with prostate cancer, elevated insulin

levels increase the risk of prostate cancer specific mortality. Certainly, the increased

expression of insulin receptors with increasingly aggressive Gleason grade of

prostate cancer would seem concordant with these findings.

In animal studies using xenografted prostate cancer cells, the evidence seems

far stronger for a positive effect of serum insulin on prostate cancer growth and

progression. It has been well demonstrated that insulin, IGF-1 and hybrid insulin/IGF

receptors are over-expressed in prostate cancer tissue compared with the benign

prostate. While these results taken individually are of varied significance, assessing

the body of evidence in its entirety suggests that indeed insulin does play a role in

prostate cancer, seemingly positively affecting prostate cancer growth and

progression.

It has been clearly shown that Androgen Deprivation Therapy (ADT), used as

treatment in advanced prostate cancer, induces a variety of changes to body

composition and metabolism from very early in the course of treatment. In particular,

patients develop increased body fat mass and decreased lean body mass, serum lipid

alterations, as well as target tissue insulin resistance, with associated

hyperinsulinaemia. Insulin is a known mitogen, also with anti-apoptotic properties

when signalling through both the insulin receptor and its closely related insulin/IGF-

1 receptor hybrid.

Recent evidence has demonstrated a more rapid development of castrate

resistant disease in the presence of metabolic syndrome in men receiving ADT for

advanced prostate cancer. This supports a role for the metabolic effects of ADT

negatively impacting upon the progression of prostate cancer.

Prostate cancer testosterone and dihydrotestosterone levels following

androgen deprivation, while low are sufficient to activate the androgen receptor, with

up-regulation of the androgen receptor occurring in a proportion of castrate resistant

prostate cancer cells. These residual intra-prostatic androgens may be derived from

prostatic de novo steroid genesis with the enzymes required for this process present

in castrate resistant prostate cancer cells. Recently completed data from our group

shows that these enzymes can be up regulated by treatment with insulin in vitro.

46


From these findings, we believe that the elevated insulin of androgen

deprivation therapy eventually adversely affects prostate cancer behaviour. Indeed,

studies are already being designed to examine different methods for manipulating the

insulin axis in this patient group and assessing its effect on prostate cancer growth

and progression. The gap in knowledge that we have identified in this area is a

demonstration of how serum insulin levels correlate with progression of prostate

cancer in men receiving ADT for their advanced disease. This study has been

designed to analyse this relationship. Our hypothesis is that the elevated serum

insulin caused by androgen deprivation therapy, promotes de novo prostate synthesis

of androgens leading to progression to castrate resistant prostate cancer.

A prospective study, with sufficient patient numbers and follow-up to

properly address this issue is an undertaking with significant time and monetary

costs. Therefore, we designed this study as a pilot study to assess whether any trend

could be found between insulin levels and prostate cancer progression to castrate

resistance sufficient to warrant a larger, prospectively designed study to be

performed to further clarify this issue. We assessed men receiving ADT for their

advanced prostate cancer in a cross-sectional manner. Serum samples were obtained

and tested for C-peptide. PSA data was obtained as well as information on prostate

cancer stage and grade, and other medical co-morbidities and medications, which

may affect our results. The study design and our subsequent findings are set out in

the following chapter.

Chapter 4: Research Design 47

47

Chapter 4: Research Design

This chapter outlines the design of this project used to achieve the aims and

objectives outlined in section 1.3. Section 3.1 discusses the methodology used in the

study, the study design and how it was implemented. Section 3.2 describes the

characteristics of the participants in the study. Section 3.3 lists all the equipment used

for sample analysis. Section 3.4 outlines how the data was analysed. Section 3.5

describes all ethical implications and approvals involved in the completion of this

research.

4.1 METHODOLOGY AND RESEARCH DESIGN

4.1.1 METHODOLOGY

This project was designed as an observational cross-sectional study design.

Cases were selected based on their diagnosis of advanced prostate cancer and

treatment with Androgen Deprivation Therapy as outlined in Section 4.2. Controls

were selected from patients with localised prostate cancer due to undergo radical

prostatectomy. This group was selected to account for any metabolic changes that

may be attributable to prostate cancer itself.

4.1.2 RESEARCH DESIGN

The primary hypothesis of this study is that serum insulin increases due to

Androgen Deprivation Therapy for prostate cancer, and that this hyperinsulinaemia is

directly correlated to prostate cancer progression to Castrate Resistant Prostate

Cancer. This study was designed as a pilot study to assess whether there is any

correlation between serum insulin level and the rate of progression to castrate

resistance.

The study was designed in a cross-sectional manner, consecutively recruiting

men over a 9-month period with advanced prostate cancer receiving androgen

deprivation therapy. Patients could be at any stage of their treatment. Retrospective

data was collected regarding their prostate cancer diagnosis and treatment to date,

including their PSA history, their general demographics, personal medical and

medication history. A control group of men with localised prostate cancer, due to

48

48 Chapter 4: Research Design

undergo radical prostatectomy as attempted curative treatment, was recruited and the

same information collected. This control group was selected to enable comparison to

account for any changes to serum insulin level caused by prostate cancer itself.

4.2 PARTICIPANTS

This project was designed to examine men undergoing androgen deprivation

therapy as treatment for advanced prostate cancer. Androgen deprivation therapy

was defined as any of three types:

1. Androgen receptor antagonist (most commonly cyproterone acetate)

2. Gonadotrophin Releasing Hormone agonist (eg Leuprorelin acetate or

Goserelin acetate)

3. Bilateral orchidectomy (surgical removal of both testes)

Commonly a combination of therapies was in use in individual patients. All these

treatments act to deprive the prostate tissue of androgen or block androgen action at

the prostate.

Advanced prostate cancer was defined as any of:

‐ Biochemical recurrence after previous attempted curative therapy (surgery or

radiotherapy) – measured as a rise in PSA after reaching post-treatment nadir

‐ Locally advanced prostate cancer – clinical T3 (spread outside the prostate

capsule) or T4 (invading into local structures) disease. This was either not

deemed suitable for curative therapy, or was receiving androgen deprivation

therapy as a neo-adjuvant or adjuvant therapy to attempted curative radiation

therapy.

‐ Metastatic prostate cancer to any distant site (either bones or soft tissue) or

lymph nodes

Patients were recruited through either Urology or Radiation Oncology outpatient

clinics at either of two hospitals in Brisbane, Australia. These were the Mater

Hospital, South Brisbane and the Princess Alexandra Hospital, Woolloongabba.

Patients were also recruited whilst inpatients under the Urology or Radiation

Oncology teams receiving treatment at the Mater Hospital, South Brisbane.

49


To be considered for recruitment patients had to be diagnosed with advanced

prostate cancer according to the above definition, and be receiving Androgen

Deprivation Therapy as treatment. This group therefore included patients receiving

androgen deprivation as sole therapy, those receiving it as neoadjuvant or adjuvant

therapy to a course of radiotherapy, or receiving supplemental chemotherapy.

Patients receiving short course (6 month) Androgen Deprivation Therapy prior to

radiotherapy for localised prostate cancer not meeting the above definition of

advanced disease were excluded from the study.

The control group in this study consisted of men diagnosed with localised

prostate cancer due to undergo radical prostatectomy as treatment. This group of men

were recruited from multiple hospitals within Brisbane, Queensland, Australia. This

recruitment was performed under the protocols and practices of the Australian

Prostate Cancer Bioresource.

The end-point under examination in this study was the diagnosis of castrate

resistant prostate cancer. Castrate resistance was defined as 2 consecutive elevations

of PSA, post attaining a nadir level after onset of ADT, in the setting of a castrate

testosterone level of less than or equal to 0.7nmol/L. The elevated PSA value had to

be greater than 2ng/mL. Castrate resistance was also deemed to have occurred if any

form of salvage therapy was instituted, or if there was radiological or clinical

evidence of disease progression. This definition is taken from the Prostate Cancer

Working Group consensus criteria 200884.

4.3 C-PEPTIDE

In experimental and clinical medicine, serum C-peptide is often measured as a

surrogate for serum insulin. Insulin originates in the beta cells of the pancreas in the

form of pro-insulin. This polypeptide is cleaved into the active insulin and C-peptide

and the two fragments are secreted into the circulation in equimolar concentrations.

Measurements of serum insulin or C-peptide give a surrogate marker of pancreatic

beta cell function. Unlike insulin, C-peptide has no known physiological function.

Anatomically, insulin and C-peptide are secreted into the portal circulation meaning

they reach the liver prior to reaching the systemic circulation where they can be

measured via venous blood samples. Insulin undergoes significant first pass

metabolism, meaning it is removed from the portal circulation by the liver to varying

50


extents depending on requirements. This means that the systemic levels of insulin are

not always an accurate reflection of pancreatic insulin production. C-peptide

however, is not metabolised by the liver and circulates in the peripheral bloodstream

before eventual excretion by the kidneys. C-peptide has a half-life up to 5 times

longer than does insulin such that high concentrations of C-peptide persist in the

peripheral circulation for a longer period than insulin. Whilst blood levels of both

insulin and C-peptide fluctuate with food intake, the longer time to elimination of C-

peptide means its measurement may give a more accurate measure of overall insulin

production with less fluctuation than measurement of insulin itself41,85,86.

For a reliable measurement of serum insulin levels, samples should be acquired

from the patient in the fasted state. Due to the recruitment of patients in an outpatient

setting, assessing patients in the fasted state was not possible. As C-peptide levels

fluctuate less with time, C-peptide was chosen for analysis in this study rather than

insulin to give a more accurate measure of endogenous insulin production in our

patient group. The normal range for fasting C-peptide is between 300-1000pmol/L. It

has been shown that the non-fasting reference range is higher, but only slightly stated

to be between 300-1500pmol/L.

4.4 INSTRUMENTS

Ethics Approvals

The protocol for this study was assessed and approved by the Queensland

University of Technology Human Research Ethics Committee. Patient recruitment

and sample collection was under the direction of the Australian Prostate Cancer

Bioresource, Queensland node managed by the Institute of Health and Biomedical

Innovation at the Queensland University of Technology, Brisbane. The protocol for

recruitment and sample collection was approved by both of the Mater Health

Services and the Metro South Health Service District Human Research Ethics

Committees, Brisbane Australia.

Recruitment of Cases

Participants were recruited from the public urology and radiation oncology

outpatient departments at the Mater and Princess Alexandra Hospitals, or whilst an

inpatient under the care of Urology or Radiation Oncology teams at the Mater

51


Hospital, Brisbane, Australia. These patients were all diagnosed by their treating

Urologist as having Advanced Prostate Cancer as defined above and were all

receiving Androgen Deprivation Therapy under the direction of their treating

physician. During the recruitment period from April 2010 to December 2010, all

patients meeting selection criteria were invited to participate in the study. Only one

patient declined to participate after project information was provided. The remainder

of patients were recruited at the time of consultation. The treating Urologist,

Radiation Oncologist or Urology Registrar identified appropriate potential

participants for involvement in this study, and the design of the project was

explained to them. Appropriate informed written consent for participation was

obtained from all participants in accordance with Ethics Committee guidelines prior

to sample and data collection.

Thirty-six patients with advanced prostate cancer receiving ADT were

recruited and venous blood samples were obtained. These samples were collected

from the patient in a non-fasting state due to the nature of their recruitment on an

opportunistic basis in an outpatient clinic setting.

Recruitment of Controls

The Australian Prostate Cancer (APC) Bioresource is a storage bank of

prostate tissue and blood products collected from men with prostate cancer at various

stages in their disease. This is a national bank of samples and patient data with

separate nodes of the tissue bank within each state, each node operating under the

approval of human research ethics committees at each hospital from which they

recruit. The Queensland node is based at the Queensland University of Technology.

Approval was obtained from the Queensland University of Technology Human

Research Ethics Committee to obtain samples from the APC Bioresource for use in

this research project. Access to samples form APC Bioresource was via a process of

application and approval by the APC Bioresource National Committee.

Forty-seven samples of previously collected and stored human serum were

obtained from the APC Bioresource for use in this project. These samples had been

collected from men with localised prostate cancer prior to undergoing Radical

Prostatectomy for treatment of their prostate cancer. All samples were collected in

the non-fasted state. These samples were originally processed from venous blood

samples collected and processed under the same protocol used for case samples.

52


After snap freezing in liquid nitrogen, the serum was stored at -80ºC at the

Queensland University of Technology Brisbane.

Sample Preparation

The protocol for collection and processing of samples was taken from the

protocols of the APC Bioresource. The same protocol was used for both the case and

control patient samples. An 8mL venous blood sample was extracted from the

patient, in a non-fasted state, into a standard SST tube. The SST sample was allowed

to stand at room temperature for 1 hour to allow blood clotting. The sample was then

centrifuged at 4000rpm, at 22°C for 10 minutes. Serum was removed and aliquotted

into 500µL vials. Samples were snap frozen in liquid nitrogen and stored at -80°C

until required for use.

Sample Testing

Patient serum samples were analysed for C-peptide content using a

commercially available, enzyme linked immunosorbent assay kit specific for human

C-peptide, purchased from ALPCO diagnostics (catalogue number 80-CPTHU-

E01.1). All samples were removed from -80ºC freezer and thawed to room

temperature immediately prior to testing. The ELISA was a standard sandwich-type

immunoassay with results read by spectrophotometer at 450nm with a reference

wavelength of 620nm. This assay has an intra-assay co-efficient of variation (CV%)

of 3.87% and an inter-assay co-efficient of variation (CV%) of 7.9%. Storage and

thawing of serum samples were in line with manufacturer’s recommendations for this

assay kit for c-peptide. Assays were performed according to the manufacturer’s

protocol by Dr McKenzie in the laboratory of the Australian Prostate Cancer

Research Centre – QLD, Princess Alexandra Hospital, Brisbane, QLD, Australia. All

samples were tested in duplicate and the average value calculated for the result.

The initial approach to patients was by the treating Urologist, Radiation

Oncologist or Urology Registrar. The participant was then met and interviewed by

the lead investigator, Dr Ian McKenzie. This interview comprised the description of

the project and provision of approved information and consent forms, which were

read through with the participant and all questions answered by the investigator.

After written, informed consent for participation was obtained; information on the

participant’s history was obtained and recorded. This information comprised general

53


demographics, history of prostate cancer diagnosis and treatment, including PSA

history, past medical and surgical history and medication history. All information

obtained was confirmed and clarified via the patient’s hospital medical record with

the patient’s written permission. Following this, a venous blood sample was collected

by the investigator and then processed as outlined above.

All information obtained, and blood samples acquired were labelled with a

participant identification number allocated by the APC Bioresource. Only the

participant’s identification number was recorded and the patients name and personal

details were removed from the record for storage to protect patient privacy.

Information on the prostate cancer, medical and surgical history and

medication history of the participant samples obtained from APC Bioresource for the

control group were received in de-identified form by the researcher, identified only

by the APC Bioresource patient identification number.

4.5 PROCEDURE AND TIMELINE

Recruitment for this study ran from April 2010 to the 31st of December 2010.

All laboratory testing was performed in three batches, with each assay using internal

controls and standards to ensure inter-assay concordance.

Data was recorded, in de-identified form as a spreadsheet using Microsoft

Excel 2008. All data collection, verification and transcription performed by Dr

McKenzie.

4.6 ANALYSIS

Sorting and refining of data was performed using Microsoft Excel 2008.

Statistical comparisons between case and control groups were performed using

unpaired, two-tail t-test. Box-plot and scatter plot graphs were constructed through

Microsoft Excel. All calculations of correlation were performed using LaTeX

mathematical and statistical software.

4.7 ETHICS AND LIMITATIONS

This project and its protocol were performed under the guidance and approval

of each of the Mater Health Services, Metro South Health Service District, and

Queensland University of Technology Human Research Ethics Committees,

54


Brisbane, Australia. The project provided minimal risk to participants and involved

no alteration to their management, which remained unchanged as directed by the

treating physician.

The major limitation to this study is its small sample size. The project was

designed as a pilot study to guide future research direction. Because of the long time

course of prostate cancer development and progression, a large prospective study

would need to be performed over a long period to achieve significant results. Positive

results from a pilot study would give stronger grounding to justify the significant

time and cost that such a study would require. This study was designed to attempt to

provide such grounding. However, because of the small numbers recruited, it is

difficult to achieve significant statistical power.

The best type of study to prove causality of elevated C-peptide and progression

of advanced prostate cancer would be a randomised controlled trial. This type of

study suffers from the ethical flaw of denying patients treatment, which has been

shown via level-1 evidence to be beneficial. An ethically appropriate study would be

a prospective study analysing patients from the time of onset of androgen deprivation

therapy. Due to the long time course of prostate cancer progression, this type of

study would have to be run over a long period, likely 5-10 years to achieve sufficient

results for statistical analysis. A proposed protocol for this type of study is outlined

in Chapter 6. For the purposes of a pilot study however, we believe a cross-sectional

study such as this project provides adequate data to enable comment on correlation to

direct future, prospective research in this topic. This will be further discussed in

Chapter 6.

Chapter 5: Results 55

55

Chapter 5: Results

Eighty-three participants were recruited for this study in total. Thirty-six

patients receiving androgen deprivation therapy for advanced prostate cancer formed

the study ADT group, and forty-seven patients assessed pre-radical prostatectomy for

localised prostate cancer formed the control group. Table 2 compares the two groups.

Variable Control Group ADT Group Number Patients 47 36 Age (Years) 61.9 (61, 50-74) 72.1 (70.5, 52-91) Mean (Median,

Range) C-Peptide (pmol/L)

1169 (833, 143-4117)

1639 (1400, 232-4855)

Castration Type - Medical - Surgical

N/A 35 (97.2) 1 (2.8)

ADT Type Primary Salvage - Radiotherapy - Surgery - Surveillance

N/A

27 (75) 3 (0.07) 1 (0.03) 5 (14)

Clinical Stage - T1a/b - T1c - T2a/b - T2c - T3a/b/c - T4 - N+ - M+ - N+/M+

1 (2.1) 5 (10.6) 8 (17) 26 (55.3) 7 (15) 0 1 (2.1) 0 0

0 3 (8.3) 7 (19.4) 1 (2.8) 10 (27.8) 15 (41.7) 5 (13.9) 17 (47.2) 7 (19.4)

Number (%)

Progressing Disease 0 20 (55.6)

Table 2 Summary of characteristics of two study groups

The mean age of the ADT and control groups at the time of study analysis was

72.1 and 61.9 years respectively. There was a statistically significant difference

between the two groups of 10.24 years (P<0.0001, 95%CI 6.65 - 13.83). The median

ages of the two groups were 70.5 and 61 years respectively as displayed in Figure 5.

56

56 Chapter 5: Results

Figure 5 Box Plot demonstrating Age (in years) range between control and ADT groups

Comparison was made between the age at the time of prostate cancer

diagnosis between the two groups. We also compared age at time of onset of

androgen deprivation therapy in the ADT group, with age at time of treatment in the

control group. The difference between the mean ages for these comparisons were 6.1

and 7.3 years respectively. Both these differences retained statistical significance.

The results of these comparisons are shown in Figures 6 and 7.

Figure 6 Box Plot comparing age at time of prostate cancer diagnosis between control and ADT groups

57


Figure 7 Box Plot comparing age at time of onset of ADT with control group As would be expected, the ADT group had more advanced stage disease than the control group. In the ADT group, the majority were receiving ADT as a primary therapy (75%), with only a small number receiving ADT for progression or relapse of their disease. The vast majority of patients were receiving medical castration in the form of an LHRH agonist (97.2%). Follow-up PSA data was available for 15 of the 47 control group patients. Of these 15 patients, none had evidence of disease relapse or progression following their radical prostatectomy at the time of analysis, with a median follow-up of 12 months. In the ADT group, 20 of the 36 patients had reached castrate resistant phase according to our definition.

5.1 ANDROGEN DEPRIVATION THERAPY AND C-PEPTIDE

The first aim of this project was to confirm the literature findings of elevated

serum insulin occurring with ADT treatment. Serum samples were analysed for C-

peptide levels (a surrogate marker for insulin production). Analysis of serum C-

peptide revealed a significantly elevated level in the study ADT group compared to

the control group. The mean C-peptide was 1639pM for the ADT group compared to

1169pM for the control group. The difference between the groups was 470.6pM.

This difference reached statistical significance with a P-value of 0.025 (95% CI 61.4

– 879.8). The distribution in each group was not symmetrical however, and a clearer

indication of the difference between groups appears when assessing the median

values of the two groups. The median value in the control group was 833pM,

compared to an ADT group median of 1399pM. The data in each group is positively

58


skewed with the mean value being higher than the median value due to high outlier

values. This effect is more marked in the control group. This is well demonstrated in

the box plot below (Figure 8).

Figure 8 Box plot comparing serum C-peptide levels between control and ADT groups

These results seem to concur with the literature findings of increased insulin

resistance, and therefore serum insulin levels occurring with ADT.

We also examined other factors to assess for any other possible correlation to

C-peptide levels. Firstly we compared age with C-peptide amongst the two groups,

separately and then combined. Comparing both the ADT group and the total study

population with C-peptide level produced a statistically significant correlation (r(34)

= 0.422, P<0.01 for the ADT group, and r(81) = 0.400, P<0.01 for the total study

group (Figure 9)). When the control group was analysed alone however, the

correlation was much weaker and no longer reached statistical significance (r(45) =

0.208, P>0.1 (Figure 10)). This is important as it excludes the patients receiving

ADT, which the literature, and our preliminary findings above suggest positively

alters C-peptide level. Therefore including these patients in the analysis of age and

C-peptide may be suggesting a correlation that does not truly exist, which would be

in keeping with our findings of the analysis of the control group alone. Alternatively,

the ADT group is significantly older than the control group, therefore this may only

strengthen the correlation between increasing age and C-peptide level.

59


Figure 9 Scatter plot comparing age to C-peptide level for the total study population

Figure 10 Scatter plot comparing age to C-peptide level for the control group only Comparing PSA level to C-peptide for the entire group, no significant

correlation was found (r(81) = 0.157, P>0.1). Again, ADT alters C-peptide levels,

but it also alters PSA levels by virtue of its apoptotic effect on PSA producing,

androgen dependent prostate cancer cells. Therefore, we analysed the control group

alone, patients with as yet untreated, but diagnosed prostate cancer. Again, no

significant correlation was found (r(45) = -0.164 (P>0.1)) between the two variables.

60


Finally, we compared duration of ADT to C-peptide level. No significant

correlation could be drawn from this comparison (r(34) = -0.091 (P>0.1)). Indeed,

the trend, while small was toward a decreasing C-peptide level with longer duration

of ADT (Figure 11).

Figure 11: Scatter plot comparing duration of ADT to serum C-peptide level. C-peptide values for the control group are shown at time 0 (have not received ADT) in red for comparison. The normal range for serum C-peptide is shown between the blue lines (range 300-150pmol/L) An important determinant of body insulin status is adiposity. Increased body fat mass is a known contributor to insulin resistance and serum insulin levels. This can be measured in the form of body weight or body mass index (BMI). Unfortunately, this data was not available for the majority of patients. Of particular importance is the change in body weight or BMI from the time of onset of ADT through to the time of analysis. Due to the cross-sectional nature of this study, data on body weight from the time of onset of treatment was not available. Likewise for the control group, this data was not recorded as part of the recruitment questionnaire. This is a confounding factor that will be discussed further in Chapter 6.

5.2 C-PEPTIDE AND PROGRESSION OF PROSTATE CANCER

The second aim of this study was to assess how serum C-peptide levels

correlated, if at all, with progression of advanced prostate cancer to the castrate

resistant phase. Serial patient PSA data was obtained from time of onset of ADT

through to time of recruitment for this study, and beyond where available. For the

61


purposes of this study, castrate resistance was defined as 2 consecutive elevations of

PSA, post attaining a nadir level after onset of ADT, in the setting of a castrate

testosterone level of less than or equal to 0.7nmol/L. The elevated PSA value has to

be greater than 2ng/mL. Castrate resistance was also deemed to have occurred if any

form of salvage therapy was instituted, or if there was radiological or clinical

evidence of disease progression. This definition is taken from the Prostate Cancer

Working Group consensus criteria 200884. Of the 36 cases in the ADT group, 20

patients had reached castrate resistance by this definition.

Time to development of castrate resistance was measured in months from the

date of onset of ADT. Figure 12 shows a scatter plot of time to castrate resistance in

months against serum C-peptide in pmol/L. A linear trend line shows no change with

increasing C-peptide level (Figure 12). The correlation co-efficient, as expected, was

not statistically significant (R (18) = 0.023 P>0.1).

Figure 12 Scatter plot comparing time to castrate resistant prostate cancer with serum C-peptide

Of this group of patients, 3 patients reached castrate resistance in less than 10

months. Analysis of these patients showed that none of them had a PSA response to

ADT, that is, their PSA continued to rise despite commencing ADT. This lack of

response suggests a prostate cancer phenotype that is not androgen sensitive, for

example a neuroendocrine tumour. These 3 patients were removed from the analysis

as their data is likely to skew the results, particularly in such a small population. This

62


produced a correlation co-efficient of r = -0.050. Whilst this was not statistically

significant (P>0.1), it suggests a trend towards earlier progression to castrate

resistance with increasing C-peptide as shown in Figure 13.

Figure 13 Scatter plot comparing time to castrate resistance excluding PSA non-responders

Type 2 diabetes mellitus is associated with insulin resistance. Typically, in the

early stages of the disease, insulin resistance, and therefore serum insulin levels are

elevated in an attempt to maintain blood glucose levels. Many of the treatments used

for type 2 diabetes alter insulin levels and insulin sensitivity. As diabetes progresses

however, the ability of the pancreas to produce insulin fails and these patients require

administration of exogenous insulin to maintain blood glucose levels. These

fluctuations in serum insulin throughout the course of type 2 diabetes, as well as the

manipulations of diabetic medications may have affected our results. Therefore, we

assessed the effect of serum C-peptide on time to progression to castrate resistance

excluding those patients with diagnosed diabetes mellitus (whether they were

receiving treatment for diabetes or not), as well as excluding the PSA non-

responders. 5 diabetic patients in total were identified, 2 of whom had already been

excluded as PSA non-responders, therefore bringing the total number of patients for

analysis to 14. The correlation between C-peptide and time to castrate resistance

remained negative but also remained non-significant (r(12) = 0.049, P>0.1).

63


Figure 14 Scatter plot of time to castrate resistance excluding diabetics and PSA non-responders

A second way of assessing the effect of C-peptide on progression of prostate

cancer is by monitoring the kinetics of PSA progression (change of PSA value with

time). Fig 15 shows this in graphical form. The ADT group was divided into two by

level of C-peptide, either less than (19 patients) or greater than 1500pmol/L (17

patients). Plotting time since diagnosis against PSA level we noted a large proportion

of patients commencing ADT with a very high PSA level (greater than 100ng/mL).

Such a high PSA suggests very active and/or extensive disease so these people were

removed from the analysis as they would be expected to recur more quickly given

their pre-treatment disease burden. This left two groups of 11 patients for analysis.

We plotted these patients in their respective groups based on level of C-peptide and

performed subgroup analysis on patients with diabetes (Fig 15a), patient age at time

of recruitment greater or less than 70 years (Fig 15b), and patients with significant

medical co-morbidities including any of ischaemic heart disease, cerebrovascular

disease or other malignancy (Fig 15c). Each line represents the PSA kinetics of an

individual patient, the solid, heavy line indicating patients with the feature under

study.

64


A i

Aii

B i

Bii

65


C i

Cii

Figure 15 PSA kinetics of the ADT patient group, with PSA<100, at time of onset of ADT. Figures grouped by serum C-peptide <1500pmol/L (i) or >1500pmol/L (ii). Patient kinetics in heavy black include: patients with diabetes (Fig 15A); age greater than 70 years (Fig 15B i) or less than 70 years (Fig 15B ii); and patients with significant medical co-morbidities (Fig 15C).


66

67

Chapter 6: Analysis 67

Chapter 6: Analysis

This chapter will analyse the results of this study in the context of current

evidence in the literature. It will assess the gap in knowledge that we identified and

discuss to what extent this gap has been addressed. This will be done using the two

aims of this research project. Chapter 6.1 will discuss the effect of androgen

deprivation therapy on insulin levels. Chapter 6.2 will discuss the effect of insulin

levels on advanced prostate cancer according to our results.

6.1 ANDROGEN DEPRIVATION THERAPY AND C-PEPTIDE

There is strong evidence, consistent throughout the literature that insulin

resistance and serum insulin increase with ADT. It has been shown to occur from

early in the course of treatment and is maintained long-term. This leads to an overall

increased risk in this population group of developing clinical type 2 Diabetes

Mellitus87.

The first aim of this pilot study was to confirm these findings by comparing

two cohorts of patients, 36 men receiving ADT for advanced prostate cancer (ADT

Group), and 47 men with localised prostate cancer who were not receiving ADT

(Control Group). Primary analysis of the two groups showed a significant elevation

of mean insulin level (measured in this study as serum C-peptide) in the ADT group

compared to the control group. This was a pilot study conducted with small numbers

of patients (n=36 in ADT group) yet the difference between the groups still attained

statistical significance (P=0.025). Comparing the median values of each group gives

a more pronounced picture of the difference between the two groups. While both

groups are positively skewed (the mean value is higher than the median value,

indicating a non-symmetrical distribution of values within the groups) by high outlier

values for C-peptide, this is more pronounced in the control group. It is possible that

a more significant difference actually exists between the groups, which are being

masked by these outlier results. Closer analysis of the two groups shows that, in the

control group, of the thirteen men with an elevated C-peptide over 1500pmol/L, nine

of these men are being treated for either of hypertension, hypercholesterolaemia, or

both. It may be that these men have, or are developing, an undiagnosed metabolic

68

68 Chapter 6: Analysis

syndrome and insulin resistance, which may account for these high C-peptide values.

This may be positively skewing the control C-peptide data and masking a more

significant difference between the two groups.

Type 2 diabetes mellitus, and the treatment of diabetes, alters the serum insulin

profiles of patients depending on how advanced their diabetes is. In early disease, the

pancreas increases insulin production to overcome target cell insulin resistance. As

the disease progresses over time however, the pancreas is unable to maintain insulin

production and serum levels decline with worsening blood glucose control. It is at

this time that patients require administration of exogenous insulin to maintain blood

glucose levels. It is uncertain at what stage in their disease the diabetic patients in

this study are at, however their serum insulin levels are likely to be altered due to

diabetes, and the drugs used to treat it. After removing all diabetic patients from the

analysis, the mean difference between the two groups decreased but remained

significant (P=0.043). The difference between median values for each group however

became more pronounced, with the median control value decreasing to 740pM, and

the median value for the ADT group increasing to 1424pM (original values were

833pM and 1400pM respectively). Taken alone, these findings would seem to concur

with the published literature that ADT does indeed induce insulin resistance and

hyperinsulinaemia.

One deficiency in this study is the lack if data regarding patient body weight

and BMI. Increased body weight, mostly reflecting increased adiposity is a well-

documented risk factor for development of insulin resistance and hyperinsulinaemia.

Whether there was any difference in BMI between the study and control groups prior

to the study group patients commencing ADT is unknown. It is possible that the

study group had increased body weight, adiposity and possibly insulin resistance

prior to commencing ADT. Clearly if this were the case, it would significantly alter

our interpretation of the studies findings that the ADT group have significantly

higher serum C-peptide levels than the control group. Caution must therefore be

taken in interpreting our data as significant given this confounding variable that

cannot be accounted for. Any future studies should acquire this data, something that

would be possible with a prospectively designed study.

An important finding from this study is that there were significantly elevated

C-peptide levels in both groups, though more significant in the ADT group. Using a

69


normal range for non-fasting serum C-peptide of 300-1500pmol/L, in the ADT group

17 of the 36 patients (47%) had C-peptide values greater than the upper limit of

normal; while in the control group this number was 13 of 47 patients (28%). The rate

of obesity, diabetes and the metabolic syndrome are all increasing in modern western

society88. Certainly, of the patients in this study, a significant number were found to

have elevated C-peptide levels, perhaps suggesting an underlying insulin resistance

syndrome. Left unmonitored, and without appropriate intervention, this proportion of

patients may progress to frank diabetes. This may be an important public health

finding given the significant impact that diabetes can have on morbidity and

mortality, and its associated cost to the community. This perhaps suggests a need for

more widespread screening of the general population for insulin and glucose levels to

identify these patients at an early stage. Certainly, in men, receiving ADT, with a

documented increase in insulin resistance, screening for its development, and

intervention with exercise and medication at an early stage may have significant

health benefits.

We further analysed these groups in terms of their age. There was a significant

difference between the two groups with the ADT group being older than the control

group, even after accounting for age at the time of diagnosis of prostate cancer, and

the time of onset of treatment with ADT. In prostate cancer, because of its generally

long time course of progression, the patient’s age at diagnosis, as well as medical co-

morbidities plays a considerable role in the choice of treatment. For localised disease

(disease within the prostate and no evidence of metastases), the vast majority of men

over the age of 70 years, with disease that may be suitable for surgical treatment via

radical prostatectomy, are much more likely to receive radiotherapy or androgen

deprivation therapy to avoid the risks associated with surgical treatment in older

patients. This is also due to the long-term good outcomes that can be achieved with

radiation therapy. As a broad generalisation, with advancing age, men are more

likely to die of other causes than prostate cancer, making the risk-benefit analysis of

radical surgery that is discussed with patients by their urologist, favour treatments

other than surgery. This is particularly the case with significant medical co-

morbidities at the time of diagnosis of prostate cancer, which is more common with

increasing age. As a result, there is a natural bias in treatment choice for localised

70


prostate cancer, meaning that men receiving surgery are generally younger than men

receiving ADT.

In our study however, to be included in the ADT group for analysis, men had

to be diagnosed with advanced prostate cancer. Indeed 29 of the 36 men in the ADT

group had metastatic disease (either lymph node or distant bone or solid organ

metastases), which would largely preclude them from surgical treatment as the

chance of cure would be low. The remaining seven men all had locally advanced,

either clinical stage T3 or T4 disease, usually considered not suitable for surgical

resection (See Table 2 for comparison of stage between groups). Indeed, in this

study, 75% of patients in the ADT group were commenced on ADT as a primary

therapy, rather than as salvage following recurrence after previous curative therapy,

reflecting the advanced stage of cancer in this group. Therefore, whilst the ADT

group were indeed significantly older, this would not appear to be due to treatment

choice based on age criteria. These men are receiving ADT because of their

advanced prostate cancer. This means that the significant difference in age between

our two study groups is not due to treatment bias.

We analysed the effect of age on serum C-peptide level. This demonstrated a

statistically significant correlation between increasing age and increasing C-peptide

levels across the entire study population, as well as the ADT group when analysed

alone. This comparison only lost statistical significance when the control group was

analysed alone. The ADT group was receiving ADT, known to elevate serum insulin.

The loss of significant correlation in the non-ADT control group suggests that a

correlation with age may have been coincidental, given the older age of the ADT

group as well as their use of ADT. This explanation is reassuring however the result

makes the attribution of elevated C-peptide levels to ADT alone more difficult.

Direct correlations with serum insulin are difficult as levels can be influenced

by many factors including body weight and fat mass, physical activity and

medications such as thiazide diuretics and beta-adrenergic antagonists89. Therefore,

any interpretation of correlation must be taken with caution. A further possible

explanation for this result may lie in the fact that the incidence of hypertension,

hypercholesterolaemia, coronary heart disease and diabetes mellitus all increase with

increasing age88. It may be that an increase in serum insulin and insulin resistance in

older patients may reflect an underlying undiagnosed metabolic syndrome with

71


increasing age, regardless of use of ADT. This is supported by a suggestion in the

literature that indeed serum insulin does increase with advancing age89.

This result is likely to be a problem that would recur in any future study as; in

general, patient groups receiving ADT will always tend to be of more advanced age.

We would suggest that a method of accounting for this possible confounding factor

in future studies would be to compare serum C-peptide between men receiving ADT,

and a cohort of age-matched, eugonadal control patients. Indeed, the studies that

have used age-matched controls to compare serum insulin against men receiving

ADT have found that there still remains a significant elevation of insulin in patients

on ADT that is not seen in the age-matched control group35.

PSA level and duration of ADT may have also contributed to elevation of C-

peptide in our study. Calculation of correlation co-efficient for each of these

variables demonstrated no statistically significant correlation with C-peptide level.

Interestingly the analysis of duration of ADT with C-peptide level suggested a

negative correlation with serum C-peptide decreasing with longer duration of ADT.

It is possible that men on long-term ADT with its induced insulin resistance and

hyperinsulinaemia may, like long-term diabetic patients, begin to lose pancreatic

production of insulin producing decreasing serum levels that have been observed in

this study. This phenomenon should be further investigated.

We also analysed, via PSA kinetics, the effect of diabetes, age, and medical co-

morbidities on PSA progression. This was achieved dividing the ADT group into two

according to C-peptide level. The first group was patients with C-peptide values

below the upper limit of the normal range of values (<1500pmol/L); the second

group those with values above the normal range. No correlations were obvious after

analysing these different trends.

Serum C-peptide is a surrogate marker for insulin production. It should ideally

be collected in the fasting state, which due to the outpatient nature of this study was

not possible. The non-fasting nature of collection in this study has been adjusted for

in the normal reference range. In any future studies serum samples should be

collected in the fasting state. Another possible confounder in this study is that of a

normal range for C-peptide. Normal ranges are usually acquired from analysis of a

young, healthy group of patients. Whether C-peptide levels in the young population

are an accurate reflection of what is ‘normal’ in the elderly population of this study

72


has not been assessed in the literature. This is unlikely to be answered with future

studies and the literature documented normal ranges should be used.

6.2 C-PEPTIDE AND PROGRESSION OF PROSTATE CANCER

The hypothesis of this study was that the hyperinsulinaemia of ADT was

correlated to the progression of advanced prostate cancer to castrate resistance.

Published literature has identified four key points regarding the role of insulin in

prostate cancer:

ADT, whilst initially causing regression of prostate cancer, invariably

progresses despite ongoing therapy with castrate serum testosterone levels

(castrate resistant prostate cancer)

Serum insulin increases with Androgen Deprivation Therapy in advanced

prostate cancer

Elevated serum insulin is associated with the development and progression of

cancers including prostate cancer

In vitro experiments demonstrate more rapid prostate cancer progression with

elevated serum insulin levels

Clinical research has now moved towards trials manipulating the insulin/IGF

axis in an attempt to slow the progression of advanced prostate cancer while

receiving ADT. For example, a trial is currently in the recruitment phase, based at

Massachusetts General Hospital in USA, to assess the response to treatment of

castrate resistant prostate cancer with metformin; a biguanide drug used commonly

in type 2 diabetes to improve insulin resistance. (www.clinicaltrials.gov)

The gap in literature that we identified was a correlation between serum insulin

levels and progression of advanced prostate cancer in human patients while

undergoing ADT. A recently published paper in the Annals of Oncology,

demonstrated that in men receiving ADT, the presence of metabolic syndrome at the

time of onset of treatment, decreased the time to progression of prostate cancer to the

castrate resistant phase. This paper did not however specifically assess insulin levels,

nor was hyperinsulinaemia required for the diagnosis of metabolic syndrome70.

Therefore, while their findings somewhat support our hypothesis, the question

remains unaddressed.

73


We assessed men at various stages of advanced disease, receiving ADT for

varying durations. The time between onset of ADT and the progression to castrate

resistance was calculated and compared to serum C-peptide levels (as a surrogate

marker for insulin production). The overall findings of this comparison, was that

there was no significant correlation between the two variables. Analysis was repeated

after removing men who had no PSA response to ADT. This correlation remained

statistically non-significant however a trend towards more rapid progression to

castrate resistance with increasing level of C-peptide was seen. A similar, non-

significant trend was seen after removing diabetic patients from this analysis. This

result was not sufficient to reject the null hypothesis of this study, that there is no

correlation between C-peptide and time to castrate resistance. Given the small

numbers recruited for this pilot study, it was unlikely that significance would be

reached for any correlation found. The trend that was identified however suggests

that this question certainly warrants further investigation, ideally in the form of a

prospective analysis. This achieves one of the main aims of this study.

One confounding variable identified in our study was the frequency of

measurement of PSA. Commonly men, after onset of ADT, received PSA testing at

sporadic, seemingly random periods, often with periods of over 12 months between

measurements. This inconsistency in measurement, both within individual kinetics,

and between different individuals, made an accurate assessment of the time of

progression of disease very difficult. The benefit of a prospectively designed trial is

that this inconsistency can be removed with a regular testing interval scheduled in the

study protocol. This would make for a much more accurate assessment of effect.

A further confounding variable is the patient’s body weight and BMI

measurements. This data was not available for the majority of study group patients,

and for any of the control group. As was discussed above, there is an increased risk

of insulin resistance with increased adiposity. As this data was not available at the

time of onset of ADT, any possible difference between the groups at that stage

cannot be analysed. The effect of ADT on body weight from the time of onset of

ADT to time of castrate resistance is also not known. This confounding variable

could be eliminated by a prospective study accruing this data from the time of onset

of treatment.

74


An interesting fact from the results of this trial was the significantly elevated

PSA levels at which patients were being diagnosed with advanced prostate cancer. In

many cases, the PSA at the time of onset of therapy was well in excess of 100ng/mL.

In some cases, this was after serial PSA monitoring of significantly elevated PSA

levels. This suggests that a significant proportion of men are presenting with very

advanced disease, and that in some cases, this is despite PSA monitoring by their

physician, which is concerning. We analysed this subgroup to assess for reasons that

may explain this phenomenon.

This study was performed in Queensland, Australia. Queensland is an

extremely large state with a land area of 1,730,648 sq km. Of this, 66% of the

population lives in the South-East corner around the capital, Brisbane, as at June 30,

200990. Parts of the state are extremely isolated, situated very large distances from

tertiary medical, and more specifically Urological care. The Mater and Princess

Alexandra Hospitals, Brisbane, from where patients were recruited for this study,

provide Urology care for a large proportion of the state of Queensland via an

outreach service. We therefore assessed the geographical location of the patients with

PSA values greater than 100ng/mL at time of onset of treatment. Of the 12 patients

who commenced ADT at a PSA greater than 100ng/mL, 7 lived outside the Greater

Brisbane regional area. 3 of the 12 patients either declined initial investigation of an

elevated PSA or declined treatment after diagnosis, 2 of these patients living within

the Brisbane area. This leaves 6 of 9 patients who were diagnosed at a very advanced

stage of their disease deriving from a regional area of Queensland. This is a

concerning statistic from a public health point of view. It highlights the need for

more education in these regional areas about prostate cancer and its diagnosis,

screening and treatment. This is something that should be further examined.

A significant factor in the design and analysis of this study was the definition

of castrate resistance. Castrate resistant prostate cancer presents a spectrum of

disease ranging from patients without metastases or symptoms, with rising PSA

levels despite ADT, to patients with metastases and significant debilitation due to

cancer symptoms91. The literature contains numerous different definitions of this

disease state, and in clinical practice, significant differences in definition exist. There

appears to be consensus that radiological or clinical progression of disease, in the

setting of castrate testosterone levels defines a subset of these patients. There are a

75


large number of patients however, for whom the only sign of disease progression is a

rising PSA despite ongoing ADT. Multiple definitions exist for this disease state.

After reaching a nadir level of PSA with ADT, the time of progression has

previously been defined in varying studies. This can be any of: the time of the first

confirmed rise in PSA, the time of two consecutive increases in PSA, the midpoint

between the time of first confirmed rise and nadir level, the time at which PSA

reaches 50% of baseline (pre-treatment) value, or the time at which PSA reaches a

level 50% above nadir. Each of these different definitions gives a different duration

of response to treatment, and in a clinical trial setting, can significantly alter the

results, either positively or negatively depending on the desired outcome for the

study. For this study, we chose a definition published by the Prostate Cancer

Working Group. This group of US specialist prostate cancer clinicians combined to

produce a consensus paper to guide future research outcomes reporting in prostate

cancer research. Two consensus papers have been published on this topic by this

group, and our definition derives from the most recent paper, published in 200884.

Whilst this definition does not give our results the most favourable outcome in

comparison to other definitions, it is the closest to an international consensus

definition available at this time, which we feel is an appropriate way to present our

results.

A good study to investigate any correlation between insulin levels and

progression of advanced prostate cancer in a formal trial setting would be a

prospective cohort study. A possible design for such a study could be as follows.

Patients are recruited with informed consent at the time of decision to commence

androgen deprivation therapy for their advanced prostate cancer, as was defined in

this study. The decision on when to commence treatment would be made by the

patient’s usual treating physician. Prior to onset of treatment with ADT, a fasting

blood sample should be collected and analysed for C-peptide and glucose. Data on

medical co-morbidities, medications and body weight and BMI should also be

collected. This information should then be updated at each 3 month interval. After 3

months of treatment, a repeat fasting blood sample should be collected and analysed

again for C-peptide, glucose and PSA. These men can then be followed out with

serial PSA measurements every 3 months up to the time of diagnosis of castrate

resistant prostate cancer. If our hypothesis is correct, and there is a positive

76


correlation between serum C-peptide level and time to progression to castrate

resistant disease, we would expect to see the men with the higher levels of C-peptide

progressing more rapidly. Patient samples should be compared with those of age-

matched, eugonadal control subjects to account for any possible association of age

with serum C-peptide level. Weight and BMI can also be compared to exclude this

possible confounding factor as affecting insulin sensitivity. This study would take a

period of some years to properly recruit and follow-up adequately, to achieve

appropriate power for statistical significance of any correlation.

Chapter 7: Conclusions 77

77

Chapter 7: Conclusions

This chapter discusses our conclusions from this project. It addresses the

limitations of our findings. Finally, it provides a plan for future research in this area

that may address the limitations identified from this project.

7.1 SUMMARY OF FINDINGS AND FUTURE PLANS

This study had two main aims:

To confirm the literature findings that men who are receiving ADT have

elevated serum C-peptide levels compared to men not receiving ADT. This

will be achieved by comparing serum C-peptide levels in the ADT group to

those in the control group of men with localised prostate cancer who are not

receiving ADT.

To correlate elevated C-peptide levels with ADT with the progression of

advanced prostate cancer to the castrate resistant phase. In accordance with

our hypothesis, we believe that men with a higher serum C-peptide level, will

progress to castrate resistance at a more rapid rate than those with lower

serum C-peptide level. This pilot study aims to identify a correlation to justify

the design of a formal trial to further investigate this correlation.

In addressing the first aim, we did indeed demonstrate that the group of men

receiving ADT had a statistically significantly higher serum C-peptide level than

those not receiving ADT. The range of values between groups did however have

significant overlap. With such small numbers in this study caution must be used in

interpreting the significance of this finding. These findings were also tempered

somewhat by the confounding increased age of our ADT group compared to control

which may play a part in the insulin resistance seen with ADT as; in general, men

receiving ADT for advanced prostate cancer will tend to be of more advanced age. In

future studies on this topic, this confounder can be removed by comparison with an

age-matched, eugonadal, non-prostate cancer control group.

78

78 Chapter 7: Conclusions

Our second aim was partly achieved. In our final analysis, there was a slight,

though non-significant trend towards more rapid development of castrate resistance

with elevated C-peptide level. This result was not sufficient to reject the null

hypothesis that there is no correlation between the two variables. However, this study

was designed as a pilot study, with very small numbers of patients and varying

lengths of follow-up of our patients with only 20 of the 36 ADT group patients

having reached castrate resistance at the time of analysis. The slight trend that we

identified, in our opinion, justifies further investigation in the form of a prospective

pilot study, the design of which we have suggested at the end of chapter 6 aiming to

account for the confounding factors that limit the interpretation of this study’s

findings.

There were some difficulties encountered in this project. There are numerous

definitions for castrate resistant prostate cancer used in the literature. Of these, we

selected the Prostate Cancer Working Group definition. This is as close to a

consensus definition as was available and we feel this definition should be used in

future studies to enable accurate interpretation of findings between studies.

Being largely retrospective in nature, our results were confounded by the

inconsistencies in PSA data available on a number of our patient cohort. In many of

the patients, there were long periods without PSA monitoring once patients

commenced on ADT. This makes definitions of disease based purely on PSA criteria

very difficult to interpret accurately. This problem can be removed in a prospectively

designed study with uniform timing of serial PSA measurements between patients.

Similarly, the retrospective, cross-sectional nature of this study meant that

important data such as body weight and BMI of all patients, both at time of onset of

treatment and at time of analysis was not known. This adds confounding variables

that limit the interpretation of our results in such a small study. A prospectively

accrued data set in future studies would address this issue.

Importantly, despite our small data set, we identified some features within our

population group that we feel deserve further investigation. Firstly, the large number

of patients, in both the ADT and the control group with significantly elevated C-

peptide levels suggesting a possible underlying insulin resistance. This may reflect a

significant distribution of early metabolic syndrome within not only those patients on

ADT, but also the wider community. We feel that the results of this study, suggesting

79

Chapter 7: Conclusions 79

a higher incidence of hyperinsulinaemia in those patients on ADT than in the control

group, in accordance with the published literature, are important. Regardless of the

effect of hyperinsulinaemia on prostate cancer progression, a hypothesis, which we

feel warrants further investigation as outlined above, long-term hyperinsulinaemia

has significant effects on the risk of long-term diabetes mellitus and its associated

morbidities, as well as long-term cardiovascular risk. We feel it is therefore very

important for medical practitioners to monitor their patients receiving ADT, as early

detection and management of developing insulin resistance could have potentially

great benefits for the patient’s long-term health, and may have yet to be determined

effects on their long-term prostate cancer progression.

With this in mind, there is currently a randomised trial underway in Australia,

investigating the use of metformin, used to manage insulin resistance, and its effects

on serum insulin in patients receiving ADT for advanced prostate cancer. We eagerly

await the results of this trial with hopes that it will strengthen and support some of

the findings of this study and perhaps offer evidence for early intervention and

management of insulin resistance in this patient population.

Secondly, a significant number of patients in our study were diagnosed with

very advanced prostate cancer and extremely high PSA values. More importantly in

this is the fact that a number were monitored for significant periods of time with no

investigation or treatment at these high PSA levels. Of these patients a large

proportion derive from rural and remote areas of our state, Queensland, Australia.

This may suggest a need for greater education on prostate cancer detection and

management for both the community and medical staff in these regions.

The issues that we have identified may have significant public health

ramifications in terms of morbidity, mortality and financial cost to the community.

We anticipate these important findings stimulating further study and improvement to

the general healthcare of the Queensland, and Australian population in future.

80

80 Chapter 7: Conclusions

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86 Bibliography

Acknowledgements

Principal Supervisors

Prof Colleen Nelson BSc, PhD

Director of Australian Prostate Cancer Research Centre – QLD, Australia

Professor and Chair of Prostate Cancer Research, Institute of Health and Biomedical

Innovation, Queensland University of Technology, Australia

Dr Peter Swindle, MBBS, FRACS, MS

Consultant Urologist, Mater Adult Hospital, Brisbane, QLD, Australia

Senior Lecturer, University of Queensland School of Medicine, Australia

Associate Supervisors

Prof Adrian Herington, BSc (Hons), PhD

Deputy Executive Dean, Faculty of Science and Technology, Queensland University

of Technology, Australia

Assistance From

Dr Jennifer Gunter, BAppSc (Hons), PhD

Post-doctoral research fellow, Australian Prostate Cancer Research Centre – QLD,

Australia

Ms Amy Lubik, BSc (Hons)

PhD student, Australian Prostate Cancer Research Centre – QLD, Australia

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THE ROLE OF INSULIN IN ADVANCED PROSTATE CANCER · ii The Role of Insulin in Advanced Prostate...

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