An Innovative Method of Increasing Early Detection for Skin Cancer in Australia

Abstract Objective: To evaluate a family practice intervention to encourage patients to request a skin examination during their consultation. Methods: Family physicians in Queensland, Australia, were randomized to intervention or control groups. In the intervention group, materials were provided by the office receptionist and supported by the family physician. Results: The rate of full-body skin examination was 99.3/ 1000 consultations in intervention-group practices compared to 22.4/ 1000 in control-group practices (p

A measure of skin familiarity and its role in the early detection of skin cancer

Skin self-examination (SSE) is promoted widely so that individuals will become familiar with their skin and be better able to identify suspicious changes earlier. However, individuals can also become familiar with their skin other than through purposeful SSE. In this article, we develop a measure of skin familiarity based on the density of spots on 14 different areas of the body. A factor analysis of the 14 body-area scores revealed that they could be grouped into four broad body regions (shoulders and back, front of legs, back of legs, and feet). Each total body score and body-region score has high internal consistency (Cronbach's alpha coefficients ranging from 0.79 to 0.93). Moreover, the scores correlate as expected with skin self-examination behaviors and other personal characteristics, indicating high construct validity. We consider the advantages that skin familiarity measures offer over the exclusive use of SSE measures in the assessment of early detection activities and discuss the direction of future research in this area

Optical redox ratio and endogenous porphyrins in the detection of urinary bladder cancer:a patient biopsy analysis

Bladder cancer is among the most common cancers in the UK and conventional detection techniques suffer from low sensitivity, low specificity, or both. Recent attempts to address the disparity have led to progress in the field of autofluorescence as a means to diagnose the disease with high efficiency, however there is still a lot not known about autofluorescence profiles in the disease. The multi-functional diagnostic system "LAKK-M" was used to assess autofluorescence profiles of healthy and cancerous bladder tissue to identify novel biomarkers of the disease. Statistically significant differences were observed in the optical redox ratio (a measure of tissue metabolic activity), the amplitude of endogenous porphyrins and the NADH/porphyrin ratio between tissue types. These findings could advance understanding of bladder cancer and aid in the development of new techniques for detection and surveillance.

Early detection : a strategy to reduce risk and severity?

Despite changes in surgical techniques, radiotherapy targeting and the apparent earlier detection of cancers, secondary lymphoedema is still a significant problem for about 20–30% of those who receive treatment for cancer, although the incidence and prevalence does seem to be falling. The figures above generally relate to detection of an enlarged limb or other area, but it seems that about 60% of all patients also suffer other problems with how the limb feels, what can or cannot be done with it and a range of social or psychological issues. Often these ‘subjective’ changes occur before the objective ones, such as a change in arm volume or circumference. For most of those treated for cancer lymphoedema does not develop immediately, and, while about 60–70% develop it in the first few years, some do not develop lymphoedema for up to 15 or 20 years. Those who will develop clinically manifest lymphoedema in the future are, for some time, in a latent or hidden phase of lymphoedema. There also seems to be some risk factors which are indicators for a higher likelihood of lymphoedema post treatment, including oedema at the surgical site, arm dominance, age, skin conditions, and body mass index (BMI).

Factors related to the presentation of thin and thick nodular melanoma from a population-based cancer registry in Queensland Australia

Purpose: Worldwide, the incidence of thick melanoma has not declined, and the nodular melanoma (NM) subtype accounts for nearly 40% of newly-diagnosed thick melanoma. To assess differences between patients with thin (≤2.00 mm) and thick (≥2.01 mm) nodular melanoma, we evaluated factors such as demographics, melanoma detection patterns, tumor visibility, and physician screening for NM alone and compared clinical presentation and anatomic location of NM with superficial spreading melanoma (SSM). Methods We utilized data from a large population-based study of Queensland (Australia) residents diagnosed with melanoma. Queensland residents aged 20 to 75 years with histologically confirmed first primary invasive cutaneous melanoma were eligible for the study, and all questionnaires were conducted by telephone (response rate 77.9%). Results During this four-year period, 369 patients with nodular melanoma were interviewed, of whom 56.7% were diagnosed with tumors ≤ 2.00 mm. Men, older individuals, and those who had not been screened by a physician in the past three years were more likely to have nodular tumors of greater thickness. Thickest nodular melanoma (4 mm+) was also most common in persons who had not been screened by a doctor within the past three years (OR 3.75; 95% CI 1.47-9.59). Forty-six percent of patients with thin nodular melanoma (≤ 2.00 mm) reported a change in color, compared with 64% of patients with thin SSM and 26% of patients with thick nodular melanoma (>2.00 mm). Conclusion Awareness of factors related to earlier detection of potentially fatal nodular melanomas, including the benefits of a physician examination, should be useful in enhancing public and professional education strategies. Particular awareness of clinical warning signs associated with thin nodular melanoma should allow for more prompt diagnosis and treatment of this subtype.

Androgen levels increase by intratumoral de novo steroidogenesis during progression of castration-resistant prostate cancer

Although systemic androgen deprivation prolongs life in advanced prostate cancer, remissions are temporary because patients almost uniformly progress to a state of a castration-resistant prostate cancer (CRPC) as indicated by recurring PSA. This complex process of progression does not seem to be stochastic as the timing and phenotype are highly predictable, including the observation that most androgen-regulated genes are reactivated despite castrate levels of serum androgens. Recent evidence indicates that intraprostatic levels of androgens remain moderately high following systemic androgen deprivation therapy, whereas the androgen receptor (AR) remains functional, and silencing the AR expression following castration suppresses tumor growth and blocks the expression of genes known to be regulated by androgens. From these observations, we hypothesized that CRPC progression is not independent of androgen-driven activity and that androgens may be synthesized de novo in CRPC tumors leading to AR activation. Using the LNCaP xenograft model, we showed that tumor androgens increase during CRPC progression in correlation to PSA up-regulation. We show here that all enzymes necessary for androgen synthesis are expressed in prostate cancer tumors and some seem to be up-regulated during CRPC progression. Using an ex vivo radiotracing assays coupled to high-performance liquid chromatography-radiometric/mass spectrometry detection, we show that tumor explants isolated from CRPC progression are capable of de novo conversion of [(14)C]acetic acid to dihydrotestosterone and uptake of [(3)H]progesterone allows detection of the production of six other steroids upstream of dihydrotestosterone. This evidence suggests that de novo androgen synthesis may be a driving mechanism leading to CRPC progression following castration.

The skin awareness study : promoting thorough skin self-examination for skin cancer among men 50 years or older

Background: Incidence and mortality from skin cancers including melanoma are highest among men 50 years or older. Thorough skin self-examination may be beneficial to improve skin cancers outcomes.--------- Objectives: To develop and conduct a randomized-controlled trial of a video-based intervention to improve skin self-examination behavior among men 50 years or older.--------- Methods: Pilot work ascertained appropriate targeting of the 12-minute intervention video towards men 50 years or older. Overall, 968 men were recruited and 929 completed baseline telephone assessment. Baseline analysis assessed randomization balance and demographic, skin cancer risk and attitudinal factors associated with conducting a whole-body skin self-examination or receiving a whole-body clinical skin examination by a doctor during the past 12 months.--------- Results: Randomization resulted in well-balanced intervention and control groups. Overall 13% of men reported conducting a thorough skin self-examination using a mirror or the help of another person to check difficult to see areas, while 39% reported having received a whole-body skin examination by a doctor within the past 12 months. Confidence in finding time for and receiving advice or instructions by a doctor to perform a skin self-examination were among the factors associated with thorough skin self-examination at baseline.---------- Conclusions: Men 50 years or older can successfully be recruited to a video-based intervention trial with the aim reduce their burden through skin cancer. Randomization by computer generated randomization list resulted in good balance between control and intervention group and baseline analysis determined factors associated with skin cancer early detection behavior at baseline.

Quality issues for the National Bowel Cancer Screening Program (NBCSP)

Purpose: This two-part research project was undertaken as part of the planning process by Queensland Health (QH), Cancer Screening Services Unit (CSSU), Queensland Bowel Cancer Screening Program (QBCSP), in partnership with the National Bowel Cancer Screening Program (NBCSP), to prepare for the implementation of the NBCSP in public sector colonoscopy services in QLD in late 2006. There was no prior information available on the quality of colonoscopy services in Queensland (QLD) and no prior studies that assessed the quality of colonoscopy training in Australia. Furthermore, the NBCSP was introduced without extra funding for colonoscopy service improvement or provision for increases in colonoscopic capacity resulting from the introduction of the NBCSP. The main purpose of the research was to record baseline data on colonoscopy referral and practice in QLD and current training in colonoscopy Australia-wide. It was undertaken from a quality improvement perspective. Implementation of the NBCSP requires that all aspects of the screening pathway, in particular colonoscopy services for the assessment of positive Faecal Occult Blood Tests (FOBTs), will be effective, efficient, equitable and evidence-based. This study examined two important aspects of the continuous quality improvement framework for the NBCSP as they relate to colonoscopy services: (1) evidence-based practice, and (2) quality of colonoscopy training. The Principal Investigator was employed as Senior Project Officer (Training) in the QBCSP during the conduct of this research project. Recommendations from this research have been used to inform the development and implementation of quality improvement initiatives for provision of colonoscopy in the NBCSP, its QLD counterpart the QBCSP and colonoscopy services in QLD, in general. Methods – Part 1 Chart audit of evidence-based practice: The research was undertaken in two parts from 2005-2007. The first part of this research comprised a retrospective chart audit of 1484 colonoscopy records (some 13% of all colonoscopies conducted in public sector facilities in the year 2005) in three QLD colonoscopy services. Whilst some 70% of colonoscopies are currently conducted in the private sector, only public sector colonoscopy facilities provided colonoscopies under the NBCSP. The aim of this study was to compare colonoscopy referral and practice with explicit criteria derived from the National Health & Medical Research Council (NHMRC) (1999) Clinical Practice Guidelines for the Prevention, Early Detection and Management of Colorectal Cancer, and describe the nature of variance with the guidelines. Symptomatic presentations were the most common indication for colonoscopy (60.9%). These comprised per rectal bleeding (31.0%), change of bowel habit (22.1%), abdominal pain (19.6%), iron deficiency anaemia (16.2%), inflammatory bowel disease (8.9%) and other symptoms (11.4%). Surveillance and follow-up colonoscopies accounted for approximately one-third of the remaining colonoscopy workload across sites. Gastroenterologists (GEs) performed relatively more colonoscopies per annum (59.9%) compared to general surgeons (GS) (24.1%), colorectal surgeons (CRS) (9.4%) and general physicians (GPs) (6.5%). Guideline compliance varied with the designation of the colonoscopist. Compliance was lower for CRS (62.9%) compared to GPs (76.0%), GEs (75.0%), GSs (70.9%, p<0.05). Compliance with guideline recommendations for colonoscopic surveillance for family history of colorectal cancer (23.9%), polyps (37.0%) and a past history of bowel cancer (42.7%), was by comparison significantly lower than for symptomatic presentations (94.4%), (p<0.001). Variation with guideline recommendations occurred more frequently for polyp surveillance (earlier than guidelines recommend, 47.9%) and follow-up for past history of bowel cancer (later than recommended, 61.7%, p<0.001). Bowel cancer cases detected at colonoscopy comprised 3.6% of all audited colonoscopies. Incomplete colonoscopies occurred in 4.3% of audited colonoscopies and were more common among women (76.6%). For all colonoscopies audited, the rate of incomplete colonoscopies for GEs was 1.6% (CI 0.9-2.6), GPs 2.0% (CI 0.6-7.2), GS 7.0% (CI 4.8-10.1) and CRS 16.4% (CI 11.2-23.5). 18.6% (n=55) of patients with a documented family history of bowel cancer had colonoscopy performed against guidelines recommendations (for general (category 1) population risk, for reasons of patient request or family history of polyps, rather than for high risk status for colorectal cancer). In general, family history was inadequately documented and subsequently applied to colonoscopy referral and practice. Methods - Part 2 Surveys of quality of colonoscopy training: The second part of the research consisted of Australia-wide anonymous, self-completed surveys of colonoscopy trainers and their trainees to ascertain their opinions on the current apprenticeship model of colonoscopy in Australia and to identify any training needs. Overall, 127 surveys were received from colonoscopy trainers (estimated response rate 30.2%). Approximately 50% of trainers agreed and 27% disagreed that current numbers of training places were adequate to maintain a skilled colonoscopy workforce in preparation for the NBCSP. Approximately 70% of trainers also supported UK-style colonoscopy training within dedicated accredited training centres using a variety of training approaches including simulation. A collaborative approach with the private sector was seen as beneficial by 65% of trainers. Non-gastroenterologists (non-GEs) were more likely than GEs to be of the opinion that simulators are beneficial for colonoscopy training (χ2-test = 5.55, P = 0.026). Approximately 60% of trainers considered that the current requirements for recognition of training in colonoscopy could be insufficient for trainees to gain competence and 80% of those indicated that ≥ 200 colonoscopies were needed. GEs (73.4%) were more likely than non-GEs (36.2%) to be of the opinion that the Conjoint Committee standard is insufficient to gain competence in colonoscopy (χ2-test = 16.97, P = 0.0001). The majority of trainers did not support training either nurses (73%) or GPs in colonoscopy (71%). Only 81 (estimated response rate 17.9%) surveys were received from GS trainees (72.1%), GE trainees (26.3%) and GP trainees (1.2%). The majority were males (75.9%), with a median age 32 years and who had trained in New South Wales (41.0%) or Victoria (30%). Overall, two-thirds (60.8%) of trainees indicated that they deemed the Conjoint Committee standard sufficient to gain competency in colonoscopy. Between specialties, 75.4% of GS trainees indicated that the Conjoint Committee standard for recognition of colonoscopy was sufficient to gain competence in colonoscopy compared to only 38.5% of GE trainees. Measures of competency assessed and recorded by trainees in logbooks centred mainly on caecal intubation (94.7-100%), complications (78.9-100%) and withdrawal time (51-76.2%). Trainees described limited access to colonoscopy training lists due to the time inefficiency of the apprenticeship model and perceived monopolisation of these by GEs and their trainees. Improvements to the current training model suggested by trainees included: more use of simulation, training tools, a United Kingdom (UK)-style training course, concentration on quality indicators, increased access to training lists, accreditation of trainers and interdisciplinary colonoscopy training. Implications for the NBCSP/QBCSP: The introduction of the NBCSP/QBCSP necessitates higher quality colonoscopy services if it is to achieve its ultimate goal of decreasing the incidence of morbidity and mortality associated with bowel cancer in Australia. This will be achieved under a new paradigm for colonoscopy training and implementation of evidence-based practice across the screening pathway and specifically targeting areas highlighted in this thesis. Recommendations for improvement of NBCSP/QBCSP effectiveness and efficiency include the following: 1. Implementation of NBCSP and QBCSP health promotion activities that target men, in particular, to increase FOBT screening uptake. 2. Improved colonoscopy training for trainees and refresher courses or retraining for existing proceduralists to improve completion rates (especially for female NBCSP/QBCSP participants), and polyp and adenoma detection and removal, including newer techniques to detect flat and depressed lesions. 3. Introduction of colonoscopy training initiatives for trainees that are aligned with NBCSP/QBCSP colonoscopy quality indicators, including measurement of training outcomes using objective quality indicators such as caecal intubation, withdrawal time, and adenoma detection rate. 4. Introduction of standardised, interdisciplinary colonoscopy training to reduce apparent differences between specialties with regard to compliance with guideline recommendations, completion rates, and quality of polypectomy. 5. Improved quality of colonoscopy training by adoption of a UK-style training program with centres of excellence, incorporating newer, more objective assessment methods, use of a variety of training tools such as simulation and rotations of trainees between metropolitan, rural, and public and private sector training facilities. 6. Incorporation of NHMRC guidelines into colonoscopy information systems to improve documentation, provide guideline recommendations at the point of care, use of gastroenterology nurse coordinators to facilitate compliance with guidelines and provision of guideline-based colonoscopy referral letters for GPs. 7. Provision of information and education about the NBCSP/QBCSP, bowel cancer risk factors, including family history and polyp surveillance guidelines, for participants, GPs and proceduralists. 8. Improved referral of NBCSP/QBCSP participants found to have a high-risk family history of bowel cancer to appropriate genetics services.

Long term survival following the detection of circulating tumour cells in head and neck squamous cell carcinoma

Background Techniques for detecting circulating tumor cells in the peripheral blood of patients with head and neck cancers may identify individuals likely to benefit from early systemic treatment. Methods Reconstruction experiments were used to optimise immunomagnetic enrichment and RT-PCR detection of circulating tumor cells using four markers (ELF3, CK19, EGFR and EphB4). This method was then tested in a pilot study using samples from 16 patients with advanced head and neck carcinomas. Results Seven patients were positive for circulating tumour cells both prior to and after surgery, 4 patients were positive prior to but not after surgery, 3 patients were positive after but not prior to surgery and 2 patients were negative. Two patients tested positive for circulating cells but there was no other evidence of tumor spread. Given this patient cohort had mostly advanced disease, as expected the detection of circulating tumour cells was not associated with significant differences in overall or disease free survival. Conclusion For the first time, we show that almost all patients with advanced head and neck cancers have circulating cells at the time of surgery. The clinical application of techniques for detection of spreading disease, such as the immunomagnetic enrichment RT-PCR analysis used in this study, should be explored further.

Identification of early-stage colorectal cancer patients at risk of relapse post-resection by immunobead reverse transcription-PCR analysis of peritoneal lavage fluid for malignant cells

Purpose: Colorectal cancer patients diagnosed with stage I or II disease are not routinely offered adjuvant chemotherapy following resection of the primary tumor. However, up to 10% of stage I and 30% of stage II patients relapse within 5 years of surgery from recurrent or metastatic disease. The aim of this study was to determine if tumor-associated markers could detect disseminated malignant cells and so identify a subgroup of patients with early-stage colorectal cancer that were at risk of relapse. Experimental Design: We recruited consecutive patients undergoing curative resection for early-stage colorectal cancer. Immunobead reverse transcription-PCR of five tumor-associated markers (carcinoembryonic antigen, laminin γ2, ephrin B4, matrilysin, and cytokeratin 20) was used to detect the presence of colon tumor cells in peripheral blood and within the peritoneal cavity of colon cancer patients perioperatively. Clinicopathologic variables were tested for their effect on survival outcomes in univariate analyses using the Kaplan-Meier method. A multivariate Cox proportional hazards regression analysis was done to determine whether detection of tumor cells was an independent prognostic marker for disease relapse. Results: Overall, 41 of 125 (32.8%) early-stage patients were positive for disseminated tumor cells. Patients who were marker positive for disseminated cells in post-resection lavage samples showed a significantly poorer prognosis (hazard ratio, 6.2; 95% confidence interval, 1.9-19.6; P = 0.002), and this was independent of other risk factors. Conclusion: The markers used in this study identified a subgroup of early-stage patients at increased risk of relapse post-resection for primary colorectal cancer. This method may be considered as a new diagnostic tool to improve the staging and management of colorectal cancer. © 2006 American Association for Cancer Research.

The expression, regulation and function of kallikrein 4 in prostate cancer

Prostate cancer is a significant health problem faced by aging men. Currently, diagnostic strategies for the detection of prostate cancer are either unreliable, yielding high numbers of false positive results, or too invasive to be used widely as screening tests. Furthermore, the current therapeutic strategies for the treatment of the disease carry considerable side effects. Although organ confined prostate cancer can be curable, most detectable clinical symptoms occur in advanced disease when primary tumour cells have metastasised to distant sites - usually lymph nodes and bone. Many growth factors and steroids assist the continued growth and maintenance of prostatic tumour cells. Of these mitogens, androgens are important in the development of the normal prostate but are also required to sustain the growth of prostate cancer cells in the early stage of the disease. Not only are androgens required in the early stage of disease, but also many other growth factors and hormones interact to cause uncontrolled proliferation of malignant cells. The early, androgen sensitive phase of disease is followed by an androgen insensitive phase, whereby androgens are no longer required to stimulate the growth of the tumour cells. Growth factors such as transforming growth factor  and  (TGF/), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factors (IGFs), Vitamin D and thyroid hormone have been suggested to be important at this stage of disease. Interestingly, some of the kallikrein family of genes, including prostate specific antigen (PSA), the current serum diagnostic marker for prostate cancer, are regulated by androgens and many of the aforementioned growth factors. The kallikrein gene family is a group of serine proteases that are involved in a diverse range of physiological processes: regulation of local blood flow, angiogenesis, tissue invasion and mitogenesis. The earliest members of the kallikrein gene family (KLK1-KLK3) have been strongly associated with general disease states, such as hypertension, inflammation, pancreatitis and renal disease, but are also linked to various cancers. Recently, this family was extended to include 15 genes (KLK1-15). Several newer members of the kallikrein family have been implicated in the carcinogenesis and tumour metastasis of hormone-dependent cancers such as prostate, breast, endometrial and ovarian cancer. The aims of this project were to investigate the expression of the newly identified kallikrein, KLK4, in benign and malignant prostate tissues, and prostate cancer cell lines. This thesis has demonstrated the elevated expression of KLK4 mRNA transcripts in malignant prostate tissue compared to benign prostates. Additionally, expression of the full length KLK4 transcript was detected in the androgen dependent prostate cancer cell line, LNCaP. Based on the above finding, the LNCaP cell line was chosen to assess the potential regulation of full length KLK4 by androgen, thyroid hormone and epidermal growth factor. KLK4 mRNA and protein was found to be up-regulated by androgen and a combination of androgen and thyroid hormone. Thyroid hormone alone produced no significant change in KLK4 mRNA or protein over the control. Epidermal growth factor treatment also resulted in elevated expression levels of KLK4 mRNA and protein. To assess the potential functional role(s) of KLK4/hK4 in processes associated with tumour progression, full length KLK4 was transfected into PC-3 cells - a prostate cancer cell line originally derived from a secondary bone lesion. The KLK4/hK4 over-expressing cells were assessed for their proliferation, migration, invasion and attachment properties. The KLK4 over-expressing clones exhibited a marked change in morphology, indicative of a more aggressive phenotype. The KLK4 clones were irregularly shaped with compromised adhesion to the growth surface. In contrast, the control cell lines (parent PC-3 and empty vector clones) retained a rounded morphology with obvious cell to cell adhesion, as well as significant adhesion to their growth surface. The KLK4 clones exhibited significantly greater attachment to Collagen I and IV than native PC-3s and empty vector controls. Over a 12 hour period, in comparison to the control cells, the KLK4 clones displayed an increase in migration towards PC-3 native conditioned media, a 3 fold increase towards conditioned media from an osteoblastic cell line (Saos-2) and no change in migration towards conditioned media from neonatal foreskin fibroblast cells or 20% foetal bovine serum. Furthermore, the increase in migration exhibited by the KLK4 clones was partially blocked by the serine protease inhibitor, aprotinin. The data presented in this thesis suggests that KLK4/hK4 is important in prostate carcinogenesis due to its over-expression in malignant prostate tissues, its regulation by hormones and growth factors associated with prostate disease and the functional consequences of over-expression of KLK4/hK4 in the PC-3 cell line. These results indicate that KLK4/hK4 may play an important role in tumour invasion and bone metastasis via increased attachment to the bone matrix protein, Collagen I, and enhanced migration due to soluble factors produced by osteoblast cells. This suggestion is further supported by the morphological changes displayed by the KLK4 over-expressing cells. Overall, this data suggests that KLK4/hK4 should be further studied to more fully investigate the potential value of KLK4/hK4 as a diagnostic/prognostic biomarker or in therapeutic applications.

Purine Nucleoside Phosphorylase mediated molecular chemotherapy and conventional chemotherapy: A tangible union against chemoresistant cancer

Background Late stage Ovarian Cancer is essentially incurable primarily due to late diagnosis and its inherent heterogeneity. Single agent treatments are inadequate and generally lead to severe side effects at therapeutic doses. It is crucial to develop clinically relevant novel combination regimens involving synergistic modalities that target a wider repertoire of cells and lead to lowered individual doses. Stemming from this premise, this is the first report of two- and three-way synergies between Adenovirus-mediated Purine Nucleoside Phosphorylase based gene directed enzyme prodrug therapy (PNP-GDEPT), docetaxel and/or carboplatin in multidrug-resistant ovarian cancer cells. Methods The effects of PNP-GDEPT on different cellular processes were determined using Shotgun Proteomics analyses. The in vitro cell growth inhibition in differentially treated drug resistant human ovarian cancer cell lines was established using a cell-viability assay. The extent of synergy, additivity, or antagonism between treatments was evaluated using CalcuSyn statistical analyses. The involvement of apoptosis and implicated proteins in effects of different treatments was established using flow cytometry based detection of M30 (an early marker of apoptosis), cell cycle analyses and finally western blot based analyses. Results Efficacy of the trimodal treatment was significantly greater than that achieved with bimodal- or individual treatments with potential for 10-50 fold dose reduction compared to that required for individual treatments. Of note was the marked enhancement in apoptosis that specifically accompanied the combinations that included PNP-GDEPT and accordingly correlated with a shift in the expression of anti- and pro-apoptotic proteins. PNP-GDEPT mediated enhancement of apoptosis was reinforced by cell cycle analyses. Proteomic analyses of PNP-GDEPT treated cells indicated a dowregulation of proteins involved in oncogenesis or cancer drug resistance in treated cells with accompanying upregulation of apoptotic- and tumour- suppressor proteins. Conclusion Inclusion of PNP-GDEPT in regular chemotherapy regimens can lead to significant enhancement of the cancer cell susceptibility to the combined treatment. Overall, these data will underpin the development of regimens that can benefit patients with late stage ovarian cancer leading to significantly improved efficacy and increased quality of life.

Identification of a novel prostate cancer susceptibility variant in the KLK3 gene transcript

Abstract Genome-wide association studies (GWAS) have identified more than 30 prostate cancer (PrCa) susceptibility loci. One of these (rs2735839) is located close to a plausible candidate susceptibility gene, KLK3, which encodes prostate-specific antigen (PSA). PSA is widely used as a biomarker for PrCa detection and disease monitoring. To refine the association between PrCa and variants in this region, we used genotyping data from a two-stage GWAS using samples from the UK and Australia, and the Cancer Genetic Markers of Susceptibility (CGEMS) study. Genotypes were imputed for 197 and 312 single nucleotide polymorphisms (SNPs) from HapMap2 and the 1000 Genome Project, respectively. The most significant association with PrCa was with a previously unidentified SNP, rs17632542 (combined P = 3.9 × 10−22). This association was confirmed by direct genotyping in three stages of the UK/Australian GWAS, involving 10,405 cases and 10,681 controls (combined P = 1.9 × 10−34). rs17632542 is also shown to be associated with PSA levels and it is a non-synonymous coding SNP (Ile179Thr) in KLK3. Using molecular dynamic simulation, we showed evidence that this variant has the potential to introduce alterations in the protein or affect RNA splicing. We propose that rs17632542 may directly influence PrCa risk.

The roles of the preproghrelin-derived peptides - ghrelin, desacyl ghrelin and obestatin - in prostate cancer

Prostate cancer is the second most common cause of cancer related deaths in Western men. Despite the significant improvements in current treatment techniques, there is no cure for advanced metastatic, castrate-resistant disease. Early detection and prevention of progression to a castrate-resistant state may provide new strategies to improve survival. A number of growth factors have been shown to act in an autocrine/paracrine manner to modulate prostate cancer tumour growth. Our laboratory has previously shown that ghrelin and its receptors (the functional GHS-R1a and the non-functional GHS-R1b) are expressed in prostate cancer specimens and cell lines. We have shown that ghrelin increases cell proliferation in the PC3 and LNCaP prostate cancer cell lines through activation of ERK1/2, suggesting that ghrelin could regulate prostate cancer cell growth and play a role in the progression of the disease. Ghrelin is a 28 amino-acid peptide hormone, identified to be the natural ligand of the growth hormone secretagogue receptor (GHS-R1a). It is well characterised as a growth hormone releasing and as an orexigenic peptide that stimulates appetite and feeding and regulates energy expenditure and bodyweight. In addition to its orexigenic properties, ghrelin has been shown to play a regulatory role in a number of systems, including the reproductive, immune and cardiovascular systems and may play a role in a number of pathological conditions such as chronic heart failure, anorexia, cachexia, obesity, diabetes and cancer. In cancer, ghrelin and its receptor are expressed in a range of tumours and cancer cell lines and ghrelin has been demonstrated to modulate cell proliferation, apoptosis, migration and invasion in some cell types. The ghrelin gene (GHRL) encodes preproghrelin peptide, which is processed to produce three currently known functional peptides - ghrelin, desacyl ghrelin and obestatin. Prohormone convertases (PCs) have been shown to cleave the preproghrelin peptide into two primary products - the 28 amino acid peptide, ghrelin, and the remaining 117 amino acid C-terminal peptide, C-ghrelin. C-ghrelin can then be further processed to produce the 23 amino acid peptide, obestatin. Ghrelin circulates in two different forms - an octanoylated form (known as ghrelin) and a non-octanoylated form, desacyl ghrelin. The unique post-translational addition of octanoic acid to the serine 3 residue of the propeptide chain to form acylated ghrelin is catalysed by ghrelin O-acyltransferase (GOAT). This modification is necessary for binding of ghrelin to its only known functional receptor, the GHS-R1a. As desacyl ghrelin cannot bind and activate the GHS-R1a, it was initially thought to be an inactive peptide, despite the fact that it circulates at much higher levels than ghrelin. Further research has demonstrated that desacyl ghrelin is biologically active and shares some of the actions of ghrelin, as well as having some opposing and distinct roles. Interestingly, both ghrelin and desacyl ghrelin have been shown to modulate apoptosis, cell differentiation and proliferation in some cell types, and to stimulate cell proliferation through activation of ERK1/2 and PI3K/Akt pathways. The third known peptide product of the ghrelin preprohormone, obestatin, was initially thought to oppose the actions of ghrelin in appetite regulation and food intake and to mediate its effects through the G protein-coupled receptor 39 (GPR39). Subsequent research failed to reproduce the initial findings, however, and the possible anorexigenic effects of obestatin, as well as the identity of its receptor, remain unclear. Obestatin plays some important physiological roles, including roles in improving memory, the inhibition of thirst and anxiety, increased secretion of pancreatic juice, and regulation of cell proliferation, survival, apoptosis and differentiation. Preliminary studies have also shown that obestatin stimulates cell proliferation in some cell types through activation of ERK1/2, Akt and PKC pathways. Overall, however, at the commencement of this PhD project, relatively little was known regarding the functions and mechanisms of action of the preproghrelin-derived functional peptides in modulating prostate cancer cell proliferation. The roles of obestatin, and desacyl ghrelin as potential growth factors had not previously been investigated, and the potential expression and regulation of the preproghrelin processing enzymes, GOAT and prohormone convertases was unknown in prostate cancer cell lines. Therefore, the overall objectives of this study were to: 1. investigate the effects of obestatin on cell proliferation and signaling in prostate cancer cell lines 2. compare the effects of desacyl ghrelin and ghrelin on cell proliferation and signaling in prostate cancer cell lines 3. investigate whether prostate cancer cell lines possess the necessary enzymatic machinery to produce ghrelin and desacyl ghrelin and if these peptides can regulate GOAT expression Our laboratory has previously shown that ghrelin stimulates cell proliferation in the PC3 and LNCaP prostate cancer cell line through activation of the ERK1/2 pathway. In this study it has been demonstrated that treatments with either ghrelin, desacyl ghrelin or obestatin over 72 hours significantly increased cell proliferation in the PC3 prostate cancer cell line but had no significant effect in the RWPE-1 transformed normal prostate cell line. Ghrelin (1000nM) stimulated cell proliferation in the PC3 prostate cancer cell line by 31.66 6.68% (p<0.01) with the WST-1 method, and 13.55 5.68% (p<0.05) with the CyQUANT assay. Desacyl ghrelin (1000nM) increased cell proliferation in PC3 cells by 21.73 2.62% (p<0.01) (WST-1), and 15.46 7.05% (p<0.05) (CyQUANT) above untreated control. Obestatin (1000nM) induced a 28.37 7.47% (p<0.01) (WST-1) and 12.14 7.47% (p<0.05) (CyQUANT) significant increase in cell proliferation in the PC3 prostate cancer cell line. Ghrelin and desacyl ghrelin treatments stimulated Akt and ERK phosphorylation across a range of concentrations (p<0.01). Obestatin treatment significantly stimulated Akt, ERK and PKC phosphorylation (p<0.05). Through the use of specific inhibitors, the MAPK inhibitor U0126 and the Akt1/2 kinase inhibitor, it was demonstrated that ghrelin- and obestatin-induced cell proliferation in the PC3 prostate cancer cell line is mediated through activation of ERK1/2 and Akt pathways. Although desacyl ghrelin significantly stimulated Akt and ERK phosphorylation, U0126 failed to prevent desacyl ghrelin-induced cell proliferation suggesting ghrelin and desacyl ghrelin might act through different mechanisms to increase cell proliferation. Ghrelin and desacyl ghrelin have shown a proliferative effect in osteoblasts, pancreatic -cells and cardiomyocytes through activation of ERK1/2 and PI3K/Akt pathways. Here it has been shown that ghrelin and its non-acylated form exert the same function and stimulate cell proliferation in the PC3 prostate cancer cell line through activation of the Akt pathway. Ghrelin-induced proliferation was also mediated through activation of the ERK1/2 pathway, however, desacyl ghrelin seems to stimulate cell proliferation in an ERK1/2-independent manner. As desacyl ghrelin does not bind and activate GHSR1a, the only known functional ghrelin receptor, the finding that both ghrelin and desacyl ghrelin stimulate cell proliferation in the PC3 cell line suggests that these peptides could be acting through the yet unidentified alternative ghrelin receptor in this cell type. Obestatin treatment also stimulated PKC phosphorylation, however, a direct role for this pathway in stimulating cell proliferation could not be proven using available PKC pathway inhibitors, as they caused significant cell death over the extended timeframe of the cell proliferation assays. Obestatin has been shown to stimulate cell proliferation through activation of PKC isoforms in human retinal epithelial cells and in the human gastric cancer cell line KATO-III. We have demonstrated that all of the prostate-derived cell lines examined (PC3, LNCaP, DU145, 22Rv1, RWPE-1 and RWPE-2) expressed GOAT and at least one of the prohormone convertases, which are known to cleave the proghrelin peptide, PC1/3, PC2 and furin, at the mRNA level. These cells, therefore, are likely to possess the necessary machinery to cleave the preproghrelin protein and to produce the mature ghrelin and desacyl ghrelin peptides. In addition to prohormone convertases, the presence of octanoic acid is essential for acylated ghrelin production. In this study octanoic acid supplementation significantly increased cell proliferation in the PC3 prostate cancer cell line by over 20% compared to untreated controls (p<0.01), but surprisingly, not in the DU145, LNCaP or 22Rv1 prostate cancer cell lines or in the RWPE-1 and RWPE-2 prostate-derived cell lines. In addition, we demonstrated that exogenous ghrelin induced a statistically significant two-fold decrease in GOAT mRNA expression in the PC3 cell line (p<0.05), suggesting that ghrelin could pontentially downregulate its own acylation and, therefore, regulate the balance between ghrelin and desacyl ghrelin. This was not observed, however, in the DU145 and LNCaP prostate cancer cell lines. The GOAT-ghrelin system represents a direct link between ingested nutrients and regulation of ghrelin production and the ghrelin/desacyl ghrelin ratio. Regulation of ghrelin acylation is a potentially attractive and desirable tool for the development of better therapies for a number of pathological conditions where ghrelin has been shown to play a key role. The finding that desacyl ghrelin stimulates cell proliferation in the PC3 prostate cancer cell line, and responds to ghrelin in the same way, suggests that this cell line expresses an alternative ghrelin receptor. Although all the cell lines examined expressed both GHS-R1a and GHS-R1b mRNA, it remains uncertain whether these cell lines express the unidentified alternative ghrelin receptor. It is possible that the varied responses seen could be due to the expression of different ghrelin receptors in different cell lines. In addition to GOAT, prohormone convertases and octanoic acid availability may regulate the production of different peptides from the ghrelin preprohormone. The studies presented in this thesis provide significant new information regarding the roles and mechanisms of action of the preproghrelin-derived peptides, ghrelin, desacyl ghrelin and obestatin, in modulating prostate cancer cell line proliferation. A number of key questions remain to be resolved, however, including the identification of the alternative ghrelin/desacyl ghrelin receptor, the identification of the obestatin receptor, a clarification of the signaling mechanisms which mediate cell proliferation in response to obestatin treatment and a better understanding of the regulation at both the gene and post-translational levels of functional peptide generation. Further studies investigating the role of the ghrelin axis using in vivo prostate cancer models may be warranted. Until these issues are determined, the potential for the ghrelin axis, to be recognised as a novel useful target for therapy for cancer or other pathologies will be uncertain.

Prostate cancer biomarkers and the emerging proteomic techniques used in biomarker research

Prostate cancer (CaP) is the second leading cause of cancer-related deaths in North American males and the most common newly diagnosed cancer in men world wide. Biomarkers are widely used for both early detection and prognostic tests for cancer. The current, commonly used biomarker for CaP is serum prostate specific antigen (PSA). However, the specificity of this biomarker is low as its serum level is not only increased in CaP but also in various other diseases, with age and even body mass index. Human body fluids provide an excellent resource for the discovery of biomarkers, with the advantage over tissue/biopsy samples of their ease of access, due to the less invasive nature of collection. However, their analysis presents challenges in terms of variability and validation. Blood and urine are two human body fluids commonly used for CaP research, but their proteomic analyses are limited both by the large dynamic range of protein abundance making detection of low abundance proteins difficult and in the case of urine, by the high salt concentration. To overcome these challenges, different techniques for removal of high abundance proteins and enrichment of low abundance proteins are used. Their applications and limitations are discussed in this review. A number of innovative proteomic techniques have improved detection of biomarkers. They include two dimensional differential gel electrophoresis (2D-DIGE), quantitative mass spectrometry (MS) and functional proteomic studies, i.e., investigating the association of post translational modifications (PTMs) such as phosphorylation, glycosylation and protein degradation. The recent development of quantitative MS techniques such as stable isotope labeling with amino acids in cell culture (SILAC), isobaric tags for relative and absolute quantitation (iTRAQ) and multiple reaction monitoring (MRM) have allowed proteomic researchers to quantitatively compare data from different samples. 2D-DIGE has greatly improved the statistical power of classical 2D gel analysis by introducing an internal control. This chapter aims to review novel CaP biomarkers as well as to discuss current trends in biomarker research from two angles: the source of biomarkers (particularly human body fluids such as blood and urine), and emerging proteomic approaches for biomarker research.