Ovarian Cancer Research Foundation March 2009
The OCRF has traditionally had a broad based funding objective to prosper basic research within Australia, since its inception in 2000. It is not generally aiming its research agenda at clinically based research studies as these are felt to be dealt with by the Australian Society for Gynaecological Oncology and the National Health and Medical Research Council.
So far projects which have been supported include proteomics research based in South Australia, as well as supporting a new study in Adelaide on “Autoantibodies in ovarian cancer: their potential use as diagnostic markers”, under the direction of Associate Professor Martin Oehler, Dr Peter Hoffmann and Dr Carmela Ricciardelli, as well as a clinical study “Does Palliative Chemotherapy improve symptoms in women with recurrent ovarian cancer”, under the auspices of the Australian and New Zealand Gynaecological Oncology Group.
Further research endeavours in New South Wales have been supported by employing a research nurse based at Royal Prince Alfred Hospital under the direction of Dr Chris Dalrymple and it is hoped that a similar arrangement can be progressed in Queensland at the Royal Women’s Hospital.
Applications for basic science research are invited and all applications will be independently reviewed by non-OCRF reviewers. It is hoped that applications for research will be consistent with the stated aims of the OCRF and thus in broad terms be restricted to basic science research into the development of early detection systems as well as understanding the basic biology of ovarian cancer.
Proposed screening tests for ovarian cancer have featured in the lay-press, both electronic and print, in recent months. The view of OCRF, consistent with the draft National Statement prepared by NBOCC, is that no test currently exists which meets acceptable standards of accuracy for population-based screening for ovarian cancer.
Australia has no mechanism in place as yet to evaluate or licence tests which claim to detect hidden diseases. Nevertheless, there has to be good evidence that a test is sensitive (ie, can reliably detect the disease whenever it is present) AND specific (ie, can rule out the disease in healthy persons). No test for ovarian cancer has been shown to meet acceptable levels for these criteria. In the United States, the Food and Drug Administration examines evidence that tests meet suitable standards, and has recently forced the withdrawal of one heavily marketed test which failed to meet minimum standards.
Professor Tom Jobling
Progress Update - 2009 First Quarter
During the first quarter of 2009, we have made excellent progress and our efforts towards the early detection of ovarian cancers are now focussed in two key areas - “discovery” and “development”. This reflects the substantial progress that has been by the research team made over the past 4 years, as we continue to focus our efforts on developing a diagnostic test for early cancer detection.
Discovery
The OCRF research team has successfully developed a new technology for identifying cancer-specific changes in very small proteins from patient blood samples. Using nanoparticles we are now able to specifically capture these small proteins, which are otherwise not detected by existing techniques. These proteins are then analysed using our SELDI TOF mass spectrometer, acquired through the generosity of the National Australia Bank Silver Ribbon campaign in 2004, to locate cancer-specific changes. This combination of new technologies is proving very promising, and based on current findings we already have strong evidence for the presence of additional highly specific and sensitive markers of ovarian cancer.
We have also developed a novel approach that works with a patient’s immune system to identify cancer-specific changes. Using this platform we have been able to specifically identify proteins that are believed to elicit an immune response in cancer patients; this is a very promising area of research, and opens up potential therapeutic opportunities in addition to providing new leads for early cancer detection.
Each of these technologies is now being applied to the discovery of new cancer markers, to increase the effective sensitivity and specificity of the test being developed. We are now in the initial stages of profiling individual patient samples using these new approaches. It is expected that these new technologies will be important in our efforts to establish routine screening for early ovarian tumour detection.
Development
The technology required for laboratory-based discovery is different to that required for routine clinical testing. The panel of tumour markers developed over the course of the previous two years is now being assessed for development in single and combination tests, in a format more suitable for routine clinical use. We are working closely with expert assay specialist, Associate Professor David Robertson, to develop the approach required to move our research findings into a clinical context. This new development program reflects a move for the research team into the next phase of development. Once testing for individual proteins is established, it will be necessary to determine whether they are able to function correctly in combination. This represents a future direction for the development team.
Dr Andrew Stevens
NAB Ovarian Cancer Research Foundation Research Fellow Group Leader
Background
Ovarian cancer remains as the most lethal form of gynaecological cancer, with an overall 5-year survival rate of less than 30% for the majority of women diagnosed. Early detection leads to survival rates exceeding 90% - hence, early detection of these cancers is the key to improving patient prognosis. Around 1500 new diagnoses are made in Australia each year, with this number expected to increase as our population ages; our work therefore continues to focus on developing a routine screening test for the early detection of ovarian cancers.
Progress to Date
By identifying proteins that are specifically produced by cancer patients, we can test for their presence in blood as a way to identify ovarian cancers early. To be useful for cancer diagnosis, a test must achieve two specific goals. First, it must be sensitive – that is, able to correctly identify 100% of the cancer patients in any group. Second, it must also be specific - able to identify 100% of the healthy women correctly. By combining these two parameters, the accuracy of the test can be assessed.
In our initial pilot program using proteomics technologies, we identified 146 cancer-specific changes in patient blood samples. Over the past 12 months, we have completed this initial phase with the final identification of over 100 of these proteins. The proteins identified include a number that are involved in causing cells to grow inappropriately – the hallmark of a developing tumour.
We are now nearing completion of phase two of our marker discovery program, which involves testing for the presence of these proteins in a set of 50 patient samples. Preliminary evidence suggests that 18 of the proteins that have been identified can, in combination, correctly identify 100% of cancer patients based on their levels in a patient’s blood. The same set of proteins can also correctly identify over 90% of healthy women – a very promising beginning for an early detection test.
The next phase of this research is to develop a specific combination test for each of these proteins, and determine its effectiveness for identifying cancer-specific changes in a much larger group of patients. To this end, we have begun recruiting healthy women to participate in the study. By donating a small amount of blood, participants are helping us to generate information that will ultimately be used to benefit ovarian cancer patients.
We are also continuing our research to identify new and more specific markers, to increase the specificity of the test – that is, its ability to correctly identify healthy women. This is very important to prevent “false positives”, or an incorrect diagnosis of cancer when it is not present. We are currently working on the development of a new technology aimed at examining proteins that are too small to be assessed using our current systems. This represents a future direction for our research team, and is expected to contribute further to developing a clinically useful early detection test.
Dr Andrew Stevens
NAB Ovarian Cancer Research Foundation Research Fellow Group Leader
PROJECT: PROTEOMICS OF OVARIAN CANCER IMPLANTATION
DECEMBER 2008
Ovarian cancer has a distinct predisposition to metastasize by the implantation of cells onto the mesothelium that lines the peritoneal cavity. Once ovarian cancer cells adhere to those cells, they may migrate through the mesothelial layer and invade local organs. The local invasion of organs like the bowel eventually results in the death of the patient. It remains unclear which factors promote ovarian cancer metastasis and implantation. This project funded by the OCRF at Adelaide University (Researchers: A/Prof Martin Oehler (Dept. of Gynaecological Oncology), Dr. Carmela Ricciardelli, Miranda Ween (Discipline of Obstetrics & Gynaecology) and Dr Peter Hoffmann (Proteomics Centre)) investigates the interaction between ovarian cancer cells and peritoneal cells using a proteomics approach. This novel strategy aims to identify important proteins likely to be mechanistically involved in implantation onto the peritoneum. Several proteins which are differentially expressed in co-culture experiments with ovarian and mesothelial cells have been identified using MALDI-TOF/TOF mass spectrometry. Ongoing functional studies are investigating whether these candidate proteins play an important role in ovarian cancer implantation and invasion. If this is the case these proteins may serve as targets for the development of therapeutics inhibiting ovarian cancer metastasis and decreasing mortality.
Assoc Prof Martin K. Oehler
Report For Ovarian Cancer Research Foundation, Oct 2008
ANZGOG is pleased to provide this report on the SYMPTOM BENEFIT study, Stage 1 of which received $25,000 in funding from the Ovarian Cancer Research Foundation in 2008. This funding is being used to cover the costs of central trial coordination. A trial coordinator has been employed to facilitate starting up this study including meeting all ethical and regulatory requirements, site management, working with the Data Management group at the ANZGOG Coordinating Centre, NHMRC Clinical Trials Centre, to establish the trial database, through to patient registration and data entry.
STUDY TITLE:Evaluation of Symptom Benefit during Palliative Chemotherapy in Women with Recurrent Ovarian Cancer: Measuring subjective improvement as well as objective response to estimate the benefit of palliative chemotherapy in women with platinum resistant or refractory ovarian cancer (ANZGOG-0701).
PRINCIPAL INVESTIGATOR: Prof Michael Friedlander, Prince of Wales Hospital, Randwick NSW.
TRIAL STATUS:
Background
Ovarian cancer persists as the most lethal of gynaecological cancers, with late stage diagnoses making up the majority of patients and an overall 5-year survival rate of less than 30%. Current statistics show that if tumours are detected whilst still confined to the ovary, over 90% of patients can expect a good outcome. Our work therefore continues to develop a routine screening test for the early detection of ovarian cancers.
Progress to date
Proteins that are inappropriately produced by cancer cells and then released into the bloodstream remain the ideal markers for detecting ovarian cancers at an early stage. Detecting these markers early will allow for the diagnosis of ovarian cancers in the pre-cancerous or beginning stages of tumour development, when treatment is the most successful. The major challenge is to identify these cancer-specific markers amongst the tens of thousands of normal proteins already present in blood.
Over the past year we have made substantial progress in the development of proteomic technologies capable of identifying cancer-specific changes in patient blood. A suitable procedure has now been developed that enables us to detect and analyse cancer-specific alterations in proteins at the picogram level (one trillionth of a gram) from a small amount patient’s blood.
A pilot research program has been operating for the past 12 months, aimed at identifying protein changes that are specific to cancer patients using a small group of patient samples. Analysis of these samples have thus far identified 156 specific changes that are found in ovarian cancer patients, but not in healthy women. Of these, 47 proteins have been identified by mass spectrometry, with the remainder currently under investigation. The identification of these proteins is extremely promising and our focus is now to identify the remaining proteins.
The next stage of our research program will examine the prevalence of these cancer-specific changes in a group of 50 patients, to determine which markers may hold promise as diagnostic predictors. The ideal marker will be one or more proteins that can identify these changes in all cancer patients, and also correctly identify their absence in healthy women. Once the efficiency of these proteins at detecting ovarian cancers is known, the proteins can proceed to the next stage of test development. We are confident that we are now one step closer to the routine application of an early stage test for ovarian cancers.
Dr Andrew Stevens
NAB Ovarian Cancer Research Foundation Research Fellow Group Leader
RESEARCH ACTIVITIES:
Background
Ovarian cancer presents at a late clinical stage in more than 80% of patients, and is associated with a five year survival of only 35% in this group. By contrast, the five year survival rate for patients with ovarian cancer that has not spread beyond the ovaries (stage I) exceeds 90%, with most patients cured. Thus, the detection of early-stage ovarian cancer is the best way to improve survival. A highly sensitive and specific diagnostic test or biomarker for early-detection of ovarian cancer is therefore vital.
Ovarian Cancer Proteomics
Proteomics is the study of protein shape, function and patterns of expression. A major application of proteomics is in the field of cancer research. The OCRF is using proteomics technology with the aim of identifying early detection markers in ovarian cancer. Our strategy involves the use of two key technologies – SELDI-TOF-MS (surface-enhanced laser desorption/ionization time of flight mass spectrometer) and 2D PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis).
With the very generous support of the National Australia Bank Silver Ribbon Campaign 2004 the OCRF was able to purchase the ‘Ciphergen ProteinChip SELDI-TOF-MS proteomics system, Series 4000’ - some of the latest technology for proteomics research. The SELDI system is a very sensitive tool for identifying new proteins and unique expression patterns of proteins ('protein signatures') in patients with ovarian cancer.
2D PAGE is an alternative technique used to separate and isolate thousands of proteins in a single experiment. Unlike SELDI, which compares patterns of proteins, the focus of 2D PAGE is on the identification of individual proteins, facilitating their use in the development of stand-alone diagnostic tools. Using some of the latest fluorescent detection technologies, we are able to examine thousands of proteins at a time for cancer-specific changes in protein expression patterns.
Current research employing both SELDI and 2D PAGE has delivered very promising results. We are confident that by using these technologies we are moving an important step closer to the development of an early detection test for ovarian cancer.
Ovarian Cancer Proteomics
Proteins that are inappropriately produced by cancer cells and then released into the bloodstream remain the ideal markers for early detection of ovarian cancer. The major challenge is to identify these cancer-specific markers amongst the tens of thousands of normal proteins already present in blood. Our focus is the detection of proteins that are produced very early in the progression of cancer. Detecting these markers early will allow diagnosis of ovarian cancers in the pre-cancerous or beginning stages of tumour development, when treatment is the most successful.
Our current SELDI work is focussed on comparison of protein signatures between healthy women and cancer patients. We have developed a comprehensive strategy for the removal of non-cancer-related proteins from patient samples, leaving only those proteins that are important in disease. This has resulted in the detection of several promising candidate proteins that can discriminate between cancer patients and healthy women. We are continuing to work towards an increased sensitivity for detection of these differences, and also towards the biochemical identification and analysis of the proteins flagged as changing in a cancer-specific manner.
Our 2D PAGE strategy has also been highly successful to date. We have developed a series of protocols used to sequentially fractionate patient blood samples into more simple components for analysis. This has allowed detection of proteins present in blood at concentrations of picograms (one trillionth of a gram) per ml.
RESEARCH ACTIVITIES:
Background
Ovarian cancer presents at a late clinical stage in more than 80% of patients, and is associated with a five year survival of only 35% in this group. In contrast, the five year survival for patients with organ-confined (stage I) ovarian cancer exceeds 90%, and most patients are cured. Thus, the detection of early-stage ovarian cancer is the best way to improve survival. A highly sensitive and specific diagnostic test or biomarker for early-detection of ovarian cancer is therefore warranted.
Ovarian Cancer Proteomics
Proteomics is the study of protein shape, function and patterns of expression. The field of proteomics has experienced substantial growth in the past decade. A major application of proteomics is in the field of cancer research. The OCRF is using proteomics technology with the aim to identify early detection markers in ovarian cancer.
With the very generous support of the National Australia Bank Silver Ribbon Campaign 2004 the OCRF was able to purchase latest proteomics technology - the Ciphergen ProteinChip SELDI-TOF-MS (surface-enhanced laser desorption/ionization time of flight mass spectrometer) proteomics system, Series 4000.
The SELDI system is a very sensitive tool for identifying new proteins and unique expression patterns of proteins (called 'protein signatures') in patients with ovarian cancer. It consists of two components:
Protein capturing tool: Biological samples such as serum, cell lysates or other protein preparations are applied directly to ProteinChip Arrays which exhibit different chromatographic properties. They are utilized to enrich for subsets of different proteins.
Protein detection tool: After a short incubation period, unbound proteins are washed off the surface of the array; only proteins interacting with the chemistry of the array surface are retained for analysis. The arrays are finally analyzed in the ProteinChip Reader, a time-of-flight mass spectrometer, which records the mass values and signal intensities of the detected proteins and peptides. The raw data is then analyzed in-depth with complex statistical software.
The acquired most advanced SELDI model (Series 4000) which is based at Prince Henry's Institute of Medical Research is currently the only one in Australia.
First experiments with the SELDI system have resulted in very promising results. We are confident that by the use of this technology we will be able to move an important step closer to the development of an early detection test for ovarian cancer.
Cell death receptor P2X7 in ovarian cancer
Apoptosis (programmed cell death) is characterized by an organized and controlled sequence of molecular and cellular events leading to the removal of a cell from its potential or current physiological or pathological function. The ability to resist apoptosis is a hallmark of most, and perhaps all types of cancer.
The P2X7 receptor mediates apoptosis in various cell types. In cancer, however, the P2X7 receptor is believed to be non-functional and unable to initiate programmed cell death and to control cell proliferation. An antibody raised against non-functional receptor P2X7 (Biosceptre International Inc., Sydney) was found to bind to a wide range of prostate, breast and skin cancer cells. No confirmed cancer cells from these tissues were found to be free of the aberrant receptor
The OCRF has conducted a collaborative study with Biosceptre International Inc to examine the potential use of the non-functional P2X7 receptor as a diagnostic tool in ovarian cancer.
RESEARCH ACTIVITIES:
Background
Ovarian cancer presents at a late clinical stage in more than 80% of patients, and is associated with a 5-year survival of only 35% in this group. In contrast, the 5-year survival for patients with organ-confined stage I ovarian cancer exceeds 90%, and most patients are cured of their disease. Thus, the detection of early-stage ovarian cancer is the best way to improve survival. A highly sensitive and specific diagnostic test or biomarker for early-detection of ovarian cancer is therefore warranted.
Identification of Genes Involved in Ovarian Cancer Development
The molecular and genetic events associated with the development of ovarian cancer are still largely unknown, thus contributing to the lack of reliable biomarkers for disease detection. Contrary to colorectal cancer which develops stepwise in a distinct series of well-defined lesions, the ovarian cancer research community disagrees on which are the precursors of epithelial ovarian cancer.
To identify early genetic events involved in the development of ovarian cancer, we are analyzing gene expression profiles of ovarian malignancies (cancers) by means of complementary DNA (cDNA) microarrays. To obtain precise expression profiles, laser microdissection is being employed to isolate the cells from tissue samples. The identification of molecular markers, whose expression is elevated at an early stage and remain elevated during cancer progression, would be especially useful for early cancer detection. If those molecules would be functionally involved in cancer growth, they would also be a therapeutic target. A better understanding of the molecular basis of ovarian cancer development would lead to new paradigms in early ovarian cancer detection and new therapeutic strategies.
Ovarian Cancer Proteomics