Research Reports

 

RESEARCH REPORT - MARCH 2007
by Andrew N. Stephens, PhD
National Australia Bank Ovarian Cancer Research Fellow

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.

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RESEARCH REPORT - HEALTH STUDIES TO DATE, SEPTEMBER 2006

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RESEARCH REPORT - SEPTEMBER 2005
by Dr Martin K. Oehler,
National Australia Bank Ovarian Cancer Research Fellow

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.

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RESEARCH REPORT - JULY 2004
by Dr Martin K. Oehler,
National Australia Bank Ovarian Cancer Research Fellow

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

The serum CA125 antigen is currently the 'gold standard' for ovarian cancer tumour markers. Unfortunately, the usefulness of CA125 as a marker for early detection is limited by the fact that less than 50% of cancers confined to the ovary are associated with detectable serum elevations of the protein; furthermore, CA125 is elevated in some benign diseases. A combination of serum CA125 and inhibin (with an enhanced detection of mucinous carcinomas and granulosa cell tumours) is an improvement on current procedures and useful for the initial diagnosis and monitoring recurrence of disease particularly in postmenopausal women

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RESEARCH REPORT - MAY 2003
By Dr Jane McNeilage,
National Australia Bank Ovarian Cancer Research Fellow

RESEARCH ACTIVITIES:

The overall aim of the research program is focused on the development of a sensitive and specific screening test for the detection of ovarian cancer. Work to this end continues in the laboratory in two different areas; improvement of the currently available tests for ovarian cancer and examination of the genes involved in the development of ovarian cancer.

Work by Associate Professor David Robertson continues on a more sensitive blood test for inhibin, which when combined with the existing blood test for Ca 125 (both are found in higher levels in the blood of individuals with ovarian cancer than in healthy women) increases detection of ovarian cancer. Whilst this is not the screening test for early disease that we are aiming for, it has increased the sensitivity and specificity of existing methods and so is a promising new development. Commercial development of this test is currently underway.

Our second approach to developing a screening test for ovarian cancer has involved examining the genes that produce the changes that occur in the ovary leading to the development of ovarian cancer. Our work has focused on identifying the genes that play a role in the process of cell death (apoptosis), some of which are unique to the different types of ovarian cancers and some of which are shared by all.

We have now extended this work to looking at much larger numbers of genes utilising the Microarray machine that has been purchased by the Foundation as a result of a very generous donation by the retailer, Witchery. This machine allows us to examine many thousands of genes in our ovarian cancer samples. Work to date has focused on refining the technique but our aim is to use this complex analysis of the expression of many thousands of different genes to identify genes that are unique to ovarian cancer.

CLINICAL RESEARCH ACTIVITIES:

The Gynaecological Oncology Unit at Monash Medical Centre continues to participate in two clinical trials at present. The aim of the first of these trials, the SMART Study is to evaluate a new treatment that may improve the long-term survival of women with ovarian cancer. The study involves the injection of an antibody, HMFG1 or Theragyn, into the abdomen of women with ovarian cancer who have been treated both surgically and with chemotherapy, and who have no visible signs of disease.

If suitable, the patient receives a single injection of the radioactive antibody into the abdomen. The theory is that the antibody will attach to any cancer cells that may still be present in the abdomen but not visible to the naked eye. The radioactive Yttrium, attached to the antibody, will destroy the remaining cancer cells. We have recruited 18 patients to the SMART study, a number that compares extremely favorably with other centres in Australia and overseas. Recruitment to this trial has now stopped.

The second trial, a direct extension of the first, the MIDAS Study, is designed to examine how the antibody is distributed after it is injected into the abdomen. The antibody in this study is labeled not with Yttrium but with a similar radioisotope, Indium. Again we are achieving a good level of recruitment to this study and continue to recruit suitable patients.

We are also founding members of the Australian and New Zealand Gynaecological Oncology Group and will be involved in clinical trials conducted by the American Gynaecologic Oncology Group. The first trial we will participate in will be the GOG 182 trial involving the comparison of five different chemotherapy treatment regimes for ovarian cancer.

The Annual Meeting of the Australian Society of Gynaecological Oncologists was held recently (April 24-27, 2003). Dr Jane McNeilage was the recipient of the Keith Free Memorial Prize for the best presentation by a Fellow.

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RESEARCH REPORT - DECEMBER 2002
By Dr Jane McNeilage,
National Australia Bank Ovarian Cancer Research Fellow

RESEARCH ACTIVITIES:

This year has seen some exciting new developments for the Foundation in the laboratory. Our overall aims are focused on the development of a sensitive and specific screening test for the detection of ovarian cancer in its early stages, and we have been attempting to tackle this from two different approaches.

Associate Professor David Robertson has developed a sensitive measurement for inhibin, which, when combined with the existing measurement for Ca 125 (both are markers found in higher levels in the blood of individuals with ovarian cancer than in healthy women) increases detection of ovarian cancer. Whilst this is not the screening test for early disease that we are aiming for, it has increased the sensitivity and specificity of existing methods and so is a promising new development.

The second approach to the development of a screening test for ovarian cancer has been to look at the genes that are involved in changes that occur to the ovary and lead to the development of ovarian cancer. Over the past 12 months we have continued to identify genes that play a role in the process of cell death (apoptosis), some of which are unique to the different types of ovarian cancers and some of which are shared by all.

Earlier in the year the Foundation was the recipient of a Microarray machine, as a result of a very generous donation by the retailer, Witchery. This machine allows us to examine many thousands of genes in our ovarian cancer samples. To date we have been refining the techniques required to perform such complex analyses, using two different ovarian cancer cell lines. The New Year will see us in a position to begin to look at a number of our ovarian cancer tissue samples that form part of the Monash Medical Centre and Prince Henry's Institute for Medical Research Gynaecological Tissue Bank. We will be looking for genes that are uniquely expressed in ovarian cancer, with the aim of identifying gene products that may serve as markers for early stage disease.

CLINICAL RESEARCH ACTIVITIES:

The Gynaecological Oncology Unit at Monash Medical Centre is involved in two clinical trials at present. The aim of the first of these trials, the SMART Study is to evaluate a new treatment that may improve the long-term survival of women with ovarian cancer. The study involves the injection of an antibody, HMFG1 or Theragyn, into the abdomen of women with ovarian cancer who have been treated both surgically and with chemotherapy, and who have no visible signs of disease.

If suitable, the patient receives a single injection of the radioactive antibody into the abdomen. The theory is that the antibody will attach to any cancer cells that may still be present in the abdomen but not visible to the naked eye. The radioactive Yttrium, attached to the antibody, will destroy the remaining cancer cells. We have recruited 18 patients to this study, a number that compares extremely favorably with other centres in Australia and overseas. Recruitment has now stopped and we are expecting a preliminary report of the early data very soon.

The second trial, the MIDAS Study, is a direct extension of the first. It is designed to examine the way in which the antibody mixes in the abdomen after it is injected, to see whether it is evenly distributed. The antibody in this study is labeled not with Yttrium, but with a similar radioisotope, Indium. Again we are achieving a good level of recruitment to this study.

Dr McNeilage recently attended a meeting of the international participants in this trial in London. Preliminary results have been collated and some of the technical problems seen in the first few patients on the trial were discussed and solutions proposed. We will continue to recruit to this study over the next 12 months.

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RESEARCH REPORT - JULY 2002

RESEARCH ACTIVITIES:MOLECULAR CHANGES IN OVARIAN CANCER

The progression of a normal cell to becoming a cancer cell requires the cell to develop a number of new characteristics. Six essential changes have been proposed (Hanahan and Weinberg, Cell 100: 58,2000):

The National Australia Bank Ovarian Cancer Research Foundation has chosen to focus on one of these aspects - evasion of cell death - in order to further understand the changes occurring in ovarian cancer.

All cells undergo a complex process of programmed cell death, called apoptosis, which involves a number of genes with actions that either support the process or prevent it. We are currently looking at the expression of a number of genes involved in apoptosis in human ovarian cancers and have identified several changes in gene expression between non-cancerous and cancerous ovarian tissue. These changes may play key roles in the generation of ovarian cancer.

MICROARRAY READER

The National Australia Bank Ovarian Cancer Research Foundation has recently acquired a Microarray Reader, purchased with funds donated by Witchery Australia. This will enable us to expand our current work, looking at the expression of genes in ovarian cancer, as it allows us to look at many thousands of genes.

CLINICAL RESEARCH ACTIVITIES

The Gynaecological Oncology Unit at Monash Medical Centre is continuing to recruit patients for the MIDAS study. This study is examining how a monoclonal antibody, HMFG1, (a molecule that recognizes and binds to ovarian cancer cells) spreads around the body after it has been injected into the abdomen of ovarian cancer patients who have completed chemotherapy and are disease-free. This study is a direct extension of the SMART study, which was designed to evaluate the efficacy of the HMFG1 antibody in the treatment of ovarian cancer.

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RESEARCH REPORT - DECEMBER 2001
by Dr Jane McNeilage,
National Australia Bank Ovarian Cancer Research Fellow

RESEARCH ACTIVITIES:

Previous studies performed at The Prince Henry’s Institute of Medical Research in collaboration with the Gynaecological Oncology Unit at Monash Medical Centre have demonstrated that the hormone inhibin is an excellent marker for women with two types of ovarian cancer, granulosa cell tumours and mucinous carcinomas. Over the past twelve months the group has developed a new, more sensitive test to detect inhibin levels in blood, allowing detection of 100% of granulosa cell tumours and helping to detect early recurrence of the disease. The test also detects 83% of mucinous ovarian tumours and 37% of serous papillary ovarian tumours.

Another marker found in the blood of patients with ovarian cancer is Ca-125, which is effective in detecting the majority of epithelial ovarian cancers. It has been found that by applying a combination of these two tests, it is possible to detect 95% of all ovarian cancers with a higher level of sensitivity than has been possible when used individually. Future work will focus on developing these tests as a potential method of screening for ovarian cancer. This will involve exploring in more detail the reasons why some subtypes of ovarian cancers do not show high inhibin levels, and why it can be raised in a small proportion of seemingly healthy women and women with benign (non-cancerous) ovarian growths.

The second aspect of the research efforts of the National Australia Bank Ovarian Research Foundation have involved examining the molecular changes that cause a normal ovarian cell to become cancerous. We are seeking to define and ultimately categorize ovarian tumours on the basis of the pattern of genes they express - their molecular fingerprint.

Earlier work from Prince Henry’s Institute examined several genes in ovarian cancers. This work has been continued to involve examination of the genes involved in a process known as ‘apoptosis’ or programmed cell death. This process plays a prominent role in the ovarian cycle and, as in other major organ systems, involves a number of genes and signaling pathways. We are particularly interested in any changes that may have occurred to the apoptosis pathways in granulosa cell tumours and in epithelial ovarian cancers as both the granulosa cells and the surface epithelium of the ovary are routinely involved in apoptosis.

Changes in the genes involved in apoptosis in granulosa cell tumours, serous papillary epithelial tumours and mucinous tumours have been analysed. To date, 205 genes, known to be part of the apoptotic pathways, have been probed and we have noted a number of differences between normal ovarian tissue and cancerous tissue. There are also differences between the types of ovarian cancers. Currently this work is being verified and will be further extended to a larger range of ovarian cancers.

CLINICAL RESEARCH ACTIVITIES:

The Gynacological Oncology Unit at Monash Medical Centre is involved in two clinical trials at present. The aim of the first, the SMART Study, is to evaluate a new treatment that may improve the long-term survival of women with ovarian cancer. The study involves injecting an antibody, HMFG1 or Theragyn, into the abdomen of women with ovarian cancer who have been treated both surgically and with chemotherapy, and have no visible signs of disease.

The theory is that the antibody will attach to any cancer cells that may still be present though not visible to the naked eye. The radioactive Yttrium, attached to the antibody, will then destroy remaining cancer cells. To date we have recruited 18 patients to the SMART study, a number that compares extremely favorably with other centers in Australia and overseas.

The second trial, the MIDAS Study, is a direct extension of the first, and recently commenced recruitment.

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RESEARCH REPORT - JULY 2001
by Dr Jane McNeilage,
National Australia Bank Ovarian Cancer Research Fellow

Current research involves looking at the normal process within the ovaries whereby certain cells undergo programmed cell death. This is of interest scientifically because it is believed that there may be a blockage in this natural process which may be associated with the development of ovarian cancer.

It is known from previous research on normal ovaries that a large number of genes are involved in this process, and it is through the use of technology used in the sequencing of the human genome that we are able to look at about 250 of them.

In programmed cell death some of the genes are involved in stopping the cell death from occurring. Our research is attempting to determine whether these particular genes are more active in ovarian cancers than in normal ovaries.

Research is conducted using human ovarian cancer tissue, removed during surgery for the disease. It is necessary to obtain a patient's consent before storing the tissue in a tissue bank, to be later made available research.

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