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Canary Scientific Programs Year-End Report 2008

The goal of the Canary Scientific Programs is to develop blood tests and imaging tests that together can be used to detect lethal cancers at a curable stage and thereby save more lives. Canary Foundation is focused on four cancer types, each of which presents a significant opportunity to save lives and prevent unnecessary treatments through early detection of otherwise lethal disease: cancer of the ovary, lung, prostate, and pancreas. Canary supports early detection research programs in each of these cancer areas as well as a Baseline Program and Collaborations Program. In 2008, Canary Foundation was delighted to announce a new addition to our Scientific Programs through the creation of the Canary Center at Stanford for Early Cancer Detection. This year's progress in all of the Canary Scientific Programs is described below.


CANARY OVARIAN CANCER PROGRAM

Canary envisions a world of simple tests that identify and isolate cancer at its earliest stages, when they are still curable. To accomplish this, we are developing a two-step screening process - a blood or other fluid test for cancer biomarkers followed by molecular imaging. The Ovarian Program has a complex and diversified research portfolio to achieve both steps of this screening process, and our team is beginning to translate that research into clinical application. Details of this research portfolio, including recent progress and future goals, are explained in the following six sections.

1) Biomarker Discovery

The goal of our biomarker discovery program is to identify and measure molecules in blood and other accessible body fluids that reliably distinguish women with ovarian cancer from healthy women. Through the end of 2008, Canary has focused on four classes of molecules to discovery biomarkers, with the following progress:

  • DNA methylation is a small chemical change made in many places in the genome without altering the DNA sequence and code. DNA methylation is often altered in cancer and can have deleterious effects, because it can affect how much of a gene is expressed (transcribed into RNA). Ovarian Team member Dr. Peter W. Laird measured methylation patterns at more than 1500 sites in the genome, using 15 ovarian cancer cell lines (human cancer cells able to grow in Petri dishes) and 27 primary tumors that include different subtypes of ovarian cancer. His team found that different ovarian cancer subtypes have different methylation patterns. Also, many sites in our genome are differentially methylated when comparing cancer and healthy tissue. The study, which also incorporated gene expression data, represents one of the largest methylation profilings of ovarian cancer to date and is being submitted for publication.

  • Gene expression is the process of transcribing messenger RNA from genes in our DNA. The messenger RNA molecules are then translated into proteins. To date, Canary investigator Dr. Patrick Brown has profiled gene expression in 15 ovarian cancer cell lines and 30 tumor tissues representing various cancer stages and subtypes. Work on ascites from cancer patients is still in progress. This study identified two cell lines out of the 15 that best resembled serous ovarian tumors (the most common ovarian cancer subtype). These two cell lines were subsequently chosen for further study, including mass spectrometry to identify proteins directly (see below). Our main goal for 2009 is to integrate the gene expression data with data generated from Canary's Baseline Project, particularly from healthy tissues near the ovaries, to predict protein biomarkers that have low background signal in healthy individuals. These candidate biomarkers will then be further developed for early detection.

  • DNA copy number variation, also measured in Dr. Brown's laboratory, refers to the fact that parts of the genome are amplified or deleted in tumors. If genes reside in these regions, this can alter their expression and therefore affect protein levels as well. Thus, measuring DNA copy number variation can be used to predict protein biomarkers. The experiments to obtain DNA copy number data from ovarian cancer cell lines and tumors have already been completed. For 2009, efforts will focus on computational analysis of the raw data and cross-comparison with other molecular data sets for biomarker identification.

  • Proteins are the result of gene expression in cells. Different tissues, including tumors, have unique gene expression profiles, and therefore differ in their protein constituency. Proteins that distinguish tumor tissue (and blood) from their healthy counterparts represent valuable biomarker candidates. Proteins can be detected using a technology called Mass Spectrometry (MS). Detecting and quantifying proteins is the focus of Ovarian Team members Drs. Sam Hanash and Martin McIntosh. A study led by Dr. Hanash (published in 2008) profiled cell surface and secreted proteins from three ovarian cancer cell lines and tumor cells from one patient. It observed high rates of shedding and secretion for some proteins, providing an encouraging basis for further biomarker exploration. In addition, Dr. McIntosh has profiled protein levels in blood and ascites fluid from ovarian cancer patients. The Ovarian Team is starting to make use of this data for biomarker prediction. Our major goal for 2009 is to integrate these data sets with results from our Baseline Program to identify and measure blood protein biomarkers in cancer that show low signal in healthy individuals. The most promising of these candidate biomarkers will then be added to our assay development pipeline.

    In addition to the just described biomarker discovery goals for 2009, Canary investigators will steer efforts toward discovering pathognomonic biomarkers, RNA or protein isoforms that are only found in cancer and never observed in healthy tissues. Such biomarkers would be ideal for detection, since they have virtually no background in healthy people. We expect to embark on a pilot project at the beginning of 2009, using high throughput sequencing methods to deeply examine ovarian cancer tissue samples for such cancer-specific biomarkers.

2) Biomarker Validation

Protein Biomarkers

Any biomarker that emerges from the above discovery projects must be thoroughly validated and pass clinical trials before it can be applied for early detection in clinical practice. Ovarian Team member Dr. Nicole Urban participated alongside four other research groups in a study managed by the NCI's Early Detection Research Network (EDRN) to validate the most promising ovarian protein biomarkers currently at hand. Earlier in 2008, Canary researchers published preliminary findings on a panel of 14 ovarian protein biomarkers, and several of these were included in the larger NCI-EDRN validation effort.

Canary investigators are now moving forward with a subset of the validated protein biomarkers that showed the greatest potential in these studies, to evaluate their detection performance in a prospective clinical trial. This too will be done in partnership with a larger government-funded ovarian cancer research effort. Canary Foundation will play a critical role in this clinical trial and provide resources and funding.

microRNA Biomarkers

Canary investigators are staying abreast of recent developments in basic research and technology that open avenues for additional types of biomarkers besides those mentioned in our Biomarker Discovery section. One promising kind are microRNAs, a class of small RNA molecules that are transcribed from DNA but do not get translated into proteins and instead help regulate gene expression. Beginning in 2009, Canary Foundation is proud to add a new member to its Ovarian Team. Dr. Muneesh Tewari, a young investigator at the Fred Hutchinson Cancer Research Center, is devoting his research career to uncovering the role of microRNAs in cancer and their potential for early detection and therapy. He has already identified several candidate microRNAs that are found at higher levels in ovarian cancer than in healthy tissues. His project as part of the Canary Ovarian Team will be to validate these microRNAs in blood samples of patients with ovarian cancer.

3) Biomarker Assay Development

DNA and RNA based biomarkers can be measured rather quickly using standard DNA and RNA amplification and sequencing methods. Measuring proteins requires significantly higher resource investment. For example, the commonly used ELISA assay requires two antibodies that must be specifically designed against an individual biomarker. One antibody captures the biomarker from a complex mixture; the other detects and quantifies it. Our assay development pipeline, headed by Dr. Brad Nelson at the BC Cancer Agency, has completed assays for six biomarkers. In 2009, we plan to add several new markers identified through gene expression and protein MS studies to this pipeline.

Because antibody generation is costly and time-consuming, biomarkers must be chosen with great care before they are added to the pipeline. In 2009, Canary plans to launch a pilot project that compares newly emergent technologies to measure biomarkers. While these new technologies may not take the place of common antibody-based detection in clinical applications any time soon, they require less resource investment and may improve and expedite the choice of biomarkers to focus our efforts to generate assays.

4) Molecular Imaging

Molecular imaging is the second stage of Canary's two-step early detection strategy. It relies on the identification of biomarkers that reside within the tumor tissue, and on the development of imaging probes specifically targeted to these biomarkers to make the cancer visible. For ovarian cancer detection, Canary is pursuing two types of molecular imaging strategies: PET imaging and targeted ultrasound. Canary investigators Drs. Sam Gambhir, Charles Drescher, and George Coukos have identified promising biomarkers and are in the process of developing imaging probes to target them. Imaging probe development represents a critical need for 2009. Thus, efforts toward that purpose will be stepped up in the coming year, using several technologies in parallel to ensure rapid progress.

Under Dr. Gambhir's leadership, targeted ultrasound experienced significant progress in 2008. This imaging method uses targeted microbubbles, small gas-filled spheres that are injected into the blood stream and designed to specifically adhere to biomarkers in tumor vasculature, increasing ultrasound signal at tumor sites. Two biomarkers found in tumor vasculature, VEGFR-2 and αVβ3 integrin, were successfully developed as microbubble imaging targets. Three publications (all published in 2008) from Dr. Gambhir's lab report that:

  • Targeted microbubbles produce specific and enhanced high-resolution ultrasound signal at tumor sites.
  • Microbubbles targeted against more than one biomarker produce increased signal compared to those targeted against a single biomarker.
  • Targeted microbubbles produce low background signal in most tissues and clear rapidly from the body.

These successful studies were performed in small animal models. In 2009, this work will continue, especially for newly identified microbubble targets. But our investigators are also actively working on pushing targeted ultrasound forward into human clinical trials to evaluate its safety and feasibility. Once the necessary government approvals have been obtained, Canary will take on a critical role in supporting this clinical trial.

5) Models of Ovarian Cancer

Models of ovarian cancer can help infer missing pieces, such as information that may be difficult to gain experimentally, and predict outcomes given a set of conditions. This is why cancer models are a valuable component of Canary's research portfolio. Through 2008, Canary supported two modeling efforts that attempt to answer different questions, both important for early detection.

The first, submitted for publication by Dr. Brown's laboratory, asks how small of a tumor we need to detect in order to make an impact on survival rates. It makes use of publically available data of occult serous ovarian cancers discovered in women who underwent prophylactic removal of the ovaries. It models cancer growth and progression during those stages when ovarian cancer usually has no symptoms but detection needs to occur. The model predicts that, in order to reduce ovarian cancer mortality by 50%, an annual screening test needs to detect a tumor of 5mm in diameter. Investigators on both the blood biomarker and imaging fronts now can establish more concrete goals for early detection.

The second model, published by Dr. Gambhir's laboratory, predicts the minimum tumor size detectable by a given blood-based biomarker. It takes into account many parameters that affect how much tumor-derived biomarker can be measured in blood, such as secretion rates from the tumor, half-life in blood, total blood volume, and others. The model was based on two established biomarkers, CA125 for ovarian cancer and PSA for prostate cancer. In the future, it will be applied to newly identified biomarkers and combinations of biomarkers.

For 2009, our investigators will begin integrating these and other modeling approaches to augment an existing cancer model by Dr. Urban geared at evaluating effectiveness and economics of various cancer screening strategies. Our goal is to apply these modeling strategies to our other cancer programs as well.

6) Collaborator Projects

Biomarker expression in subtypes of ovarian cancer

Based on tissue histology, different subtypes of ovarian cancer have long been recognized. Clinically however, ovarian cancer is still regarded and treated as a single disease. Our BC Cancer Agency collaborator Dr. David Huntsman assessed the levels of 21 candidate protein biomarkers in 500 ovarian tumors. The findings, published in 2008, clearly show that biomarker levels vary between subtypes, meaning they represent different diseases. The practical implication is that subtypes should be considered separately rather than combined as a single disease to achieve the most optimal detection and treatment results.

Proximate fluid collection

As an alternative to screening for biomarkers in blood, Canary is also pursuing local (or proximate) fluids such as uterine and vaginal lavage. Our collaborator Dr. Dianne Miller, also a member of the BC Cancer Agency, has been collecting these samples. Canary investigators have begun to profile their DNA methylation and gene expression. For 2009, we hope to continue collecting these valuable samples and to devise a validation strategy for candidate biomarkers in proximate fluids.

Mammographic breast density and ovarian cancer
Since ovarian hormones influence mammographic breast density, our BC Cancer Agency collaborators Drs. Richard Gallagher and Marilyn Borugian tested the hypothesis that mammograms could provide early warning signs of ovarian cancer. The study, which was completed in 2008, found no significant association between breast density and risk for ovarian cancer (measured over five years before diagnosis with ovarian cancer).

The Canary Ovarian Team enters 2009 with a strong and multi-faceted research program, and a new team member. Clinical trials are on the horizon for both blood biomarker screening and molecular imaging. Our continued biomarker discovery is benefiting from targeted sample collection, tactical application of new technologies, and data emerging from Canary's Baseline Project. We are gearing up to apply the knowledge we have gained from literature and experimentation to model the effectiveness and economics of different cancer screening strategies to further guide our search for the best working strategy. We are excited about all of these developments and look forward to reporting our progress.


CANARY LUNG CANCER PROGRAM

The objective of the Canary Lung Cancer Program is to develop tests to specifically detect early stages of lung cancers destined to be lethal. The year 2008 was both formative and productive for the Canary Lung Cancer Program. We assembled our team of expert scientists and clinicians, rapidly solidified the key program components, distributed our first research awards, and initiated projects aimed toward developing effective and cost-effective blood biomarker and molecular imaging tests. Later in 2008, we launched additional research projects focusing on lung cancer occurring in non-smokers and formed a major new partnership with the National Cancer Institute, putting us on track for another groundbreaking year in 2009.

The Canary Lung Cancer Program supports the development and maintenance of critical biospecimen resources, blood biomarker discovery and validation, molecular imaging projects for early detection of lethal lung cancer, and epidemiology and modeling for effective lung cancer screening. Here we document our recent progress in each of these areas.

1) Lung Cancer Biomarker Discovery

In order to discover and validate biomarkers for early detection of lung cancer, we will need access to carefully collected, well-annotated high quality specimens. Of particular interest are samples collected prior to disease diagnosis. Among the resources that Canary Foundation supports is the Carotene and Retinol Efficacy Trial (CARET) specimen collection. The CARET resource represents 18,000 individuals at high risk for lung cancer, including over 1400 individuals who developed lung cancer, with blood samples obtained prior to disease diagnosis. In addition to supporting maintenance of this resource, this year's Canary funding allowed the CARET repository to create an online database of specimens, available at www.compass.fhcrc.org/caretweb. The online version allows the research community to easily access all of the available information about samples of interest and facilitates searching by multiple different features to find the samples appropriate for their studies.

We have made great strides in our effort to combine the powerful genetics of mouse models of lung cancer with state-of-the-art proteomics techniques to discover blood biomarkers that detect potentially lethal lung cancer at an early stage. We have generated mouse models of human lung cancer in which lung cancer arises as a result of mutations in the EGFR and KRAS genes, which are commonly found in human lung cancers. Through genetic engineering, lung cancer in these mouse models can be induced to begin by a switch (administration of a drug called doxycycline), allowing serum samples to be drawn before tumor initiation and at both early and late stages of lung cancer development. This switch can be removed, allowing the lung tumors to regress, and blood can be drawn at various stages of tumor regression. In addition, mice with lung cancer can be treated with erlotinib, a drug that is currently used in the clinic to treat patients with lung cancer, to mimic treatment of tumors and find markers of response to therapy.

Our in-depth proteomic analyses on plasma from these mouse models have now identified several candidate proteins that were found in different levels in cancer versus control plasma. We have also identified proteins whose levels decrease in response to erlotinib treatment. We are in the process of prioritizing these candidates for further analysis and have begun to verify that some of these candidates are differentially expressed in tissue. Observations made with plasma from these mice have also been used to interrogate plasma samples from patients with lung cancer. Through comparison of our results from mouse and human samples, we are identifying the affected proteins that show the most promise as biomarkers for early detection of lung cancer or for monitoring tumor growth after surgery or during treatment.

The Canary Lung Cancer Program is focusing particular attention on early detection of lung cancer among non-smokers, a category representing people who have never smoked ("never smokers") or who have quit smoking ("former smokers"). We have approved plans for a new initiative aimed at discovering molecular differences in lung cancer among never smokers compared with lung cancer that develops among smokers or former smokers. The results of this research may provide a basis for biomarker tests for early detection of lung cancer applicable to all patients, regardless of smoking status. The Early Detection Research Network (EDRN) of the National Cancer Institute will be a cooperating partner on this large-scale project. The multi-institutional, multi-investigator lung cancer project will analyze carefully collected and annotated specimens-both tumor tissues and cell cultures-from patients who developed lung cancer as never smokers or as smokers. Platforms to be studied include serum proteomics, global gene expression, DNA mutations, and copy number analyses for a selected set of genes, genomic copy number alterations, and methylation analyses. All groups will do analyses on the same set of shared specimens, which is a powerful approach to biomarker discovery, followed up with massive integration of all datasets for identification of the most promising early detection biomarkers.

2) Biomarker Validation

We have found promising candidate genes that are modified by methylation in tumors of lung cancer patients but not in corresponding normal lung tissues. We have now further assessed the performance of these candidate biomarkers in additional tissue samples and are pleased to report that these candidates have withstood validation testing in the independent sample set. We are in the process of fine-tuning our technique for testing methylation of these genes in blood samples. If these candidates perform well in pilot analyses, we will test them in the high quality samples obtained from the Ontario Tumor Bank, which consist of paired tissue and blood samples obtained prior to surgery. Ultimately this DNA methylation panel may provide the basis for a blood-based early detection test for lung cancer.

Development of assays to measure protein biomarkers is a major bottleneck in accelerating biomarkers through the pipeline to a blood test. Canary has addressed this bottleneck by funding a dedicated team in Victoria, Canada to develop serum-based assays. Many of the candidates identified from our biomarker discovery studies do not have commercial assays available. The most promising candidates from our studies, as well as any promising candidates from the literature, are being thoughtfully prioritized for development of assays for validation, which will begin in early 2009.

3) Molecular Imaging of Lung Cancer

Molecular imaging allows us to "see" tumors based on specific, unique, molecular characteristics. We are pursuing a PET-CT imaging strategy for early detection of lung cancer. In order to detect cancer as early as possible, we will need the ability to visualize very small tumors (1-2mm in size), which is a challenge due to limited spatial resolution of even modern clinical scanners. We have recently developed a new image resolution recovery strategy for PET in order to visualize tracer uptake in small lung lesions, and testing of this new strategy is under way. We have also engineered a new tracer for lung cancer to target and attach strongly to a receptor molecule known as αVβ3 integrin on tumor cells and the blood vessels that feed tumors. We successfully completed the necessary steps to develop this specialized imaging probe and began testing in mouse models of lung cancer. Our initial studies with this probe have been very successful, with the tumors showing specific signal in mice by PET-CT. We are now in the process of confirming the findings on the PET-CT by testing the tumors removed from the mice for presence and quantity of the targeted probe. The amount of uptake of this new agent will help to answer if the lesion is likely malignant or benign with a high degree of accuracy. Through the use of novel PET imaging agent development and novel resolution recovery strategies, we hope to utilize PET-CT to detect much smaller lung cancer lesions and characterize their malignant potential. This work should help us to quickly move toward imaging studies in patients for the earlier detection of lung cancer.

4) Epidemiology and Modeling

We are now completing a mathematical model for predicting the size at which lung tumors make the transition that will lead them to spread and become lethal. In order to effectively screen for lung cancer to save lives, we must be able to catch tumors before this fatal transition. Incorporating data from the U.S. Surveillance Epidemiology and End Results (SEER) Program, our model predicts that for one of the common forms of lung cancer (adenocarcinoma), the probability of cure is high if the primary tumor is detected before it reaches even a relatively large size (0.8cm). The model was independently verified through data from the Mayo Lung Project. The findings from this model are encouraging because they suggest that otherwise lethal lung tumors can be found within feasible limits of detection, whether by imaging or by blood tests, to allow for cure. Manuscripts for publication of this work are in preparation.

The "natural history," or the course a cancer takes from its inception, is not well understood, despite its importance in predicting whether a cancer is likely to develop aggressive and potentially lethal characteristics. In order to better understand the natural history of lung cancer and its impact on screening practices, we have begun a comprehensive examination of cases from patients who received yearly CT screening for lung cancer. We have found that some patients died despite the yearly CT screening, suggesting that the window of opportunity may be smaller than that interval to catch aggressive tumors. Our preliminary findings have already resulted in a publication this year, and we are approximately halfway through the difficult process of accessing and reviewing the data from multiple centers so that we can confirm our results. These findings will inform future studies aimed at tests to find and isolate these aggressive lung cancers early, when there is the greatest opportunity to save lives.

We have also approved funding for a new epidemiological study that will address the need to identify risk factors among never smokers. The risk factors, and major causative and environmental factors, for lung cancer among people who have never smoked are largely unknown. For example, only a small percentage of cases is due to exposure to environmental tobacco smoke. The study will examine demographic, dietary, lifestyle, medical, family history, and reproductive risk factors in relation to risk of lung cancer and will comprehensively compare lung cancer risk factors among never, former, and current smokers. Knowing which people might be at high risk for lung cancer will aid in screening strategies for never smokers and may also lead to the identification of candidate lung cancer biomarkers for blood and imaging tests.


CANARY PROSTATE CANCER PROGRAM

The vision of the Canary Prostate Cancer Program is to accurately identify and isolate otherwise lethal prostate cancer at an early stage, when the likelihood of cure is greatest. During 2008, the Canary Prostate team has been largely focused on building the resources needed to enable discovery and validation of biomarkers of lethal prostate cancer, and has been extremely successful in that regard. The two resources that the team focused on are initiation of the Prostate Active Surveillance Study (PASS) clinical trial and creation of a tissue microarray resource. In addition, the team initiated the process of testing our first set of candidate blood biomarkers and began work on several strategies for molecular imaging of prostate cancer.

1) Prostate Active Surveillance Study (PASS) Clinical Trial

Discovery and validation of biomarkers is consistently limited by the availability of appropriate biospecimens. In the case of prostate cancer, we require specimens that are repeatedly and uniformly collected from men with early stage prostate cancer over an extended period of time. In order to address this critical need, the Canary Foundation is supporting a multi-institutional Prostate Active Surveillance Study (PASS). Men diagnosed with prostate cancer that opt to be closely monitored, with intervention if there are signs of progression, can enroll in this study. These men contribute biospecimens (blood, urine, biopsy tissue) at regular intervals for later analysis. Our goal is to enroll at least 400 men over 5 years and to follow them for 5 or more years. These specimens can then be used to both discover and validate biomarkers that predict lethal disease. More information about PASS is available at www.canaryfoundation.org/prostate-clinical-studies.cfm.

Coordination of such a multi-institutional effort is a major undertaking, but the Canary team has risen to the occasion. In 2008, we achieved several milestones:

  • Development and implementation of an active surveillance protocol: Six participating institutions (Stanford University, University of California San Francisco, University of Washington, Veterans Affairs Puget Sound Health Care System, University of Texas San Antonio, University of British Columbia) have developed the PASS protocol, as well as a Manual of Operations, to be used across all institutions. All sites filed and obtained the necessary human subjects permissions to allow implementation of the protocol.

  • Patient enrollment and follow-up: To date, five of the six participating institutions have begun patient enrollment. With interest high and enrollment strong since this fall when enrollment began, we are already two-thirds of the way to our accrual goals for the study's first year.

  • Establishment of partnership with Early Detection Research Network (EDRN) of the National Cancer Institute (NCI): We formalized a partnership with the EDRN (Early Detection Research Network) to leverage their experience in data management so that all PASS clinical sites will enter, store, and retrieve data using a common format. To expedite the storage and processing of patient information and biological specimens, the PASS study uses the EDRN's biospecimen management system and protocol oversight program. Canary Foundation and EDRN will jointly establish a Biomarker Evaluation Group with the goal of determining those biomarkers that are most promising for evaluation using the biologic materials collected in PASS.

  • Development of a clinical and biospecimen data repository: As part of the Canary-EDRN collaboration, the EDRN is providing critical support for the data management and specimen tracking required in PASS. Specimens and data are in a virtual repository as they are tracked in the secure Validation Studies Information Management Systems database managed by the Data Management Coordinating Center of the EDRN.

  • Establishment of a central specimen repository: The rich biospecimen resource created in PASS will be housed in a central specimen repository that has been established at the Fred Hutchinson Cancer Research Center. The central repository will provide a structure that will allow for ready access and distribution of specimens for approved research, and will endure over time even if the structure of the PASS study changes.

2) Canary Retrospective Tissue Microarray Study

Tissue microarrays (TMAs) are glass slides containing hundreds or thousands of tissue samples, and can be used to efficiently analyze tissue expression of candidate biomarkers. Construction of these arrays requires careful retrieval and processing of many banked radical prostatectomy tissue sections. The utility of a TMA is dependent on having thorough clinical data associated with each tissue so that tissue staining can be correlated with the outcome. We are supporting the construction and evaluation of uniformly well-annotated TMAs across all of our participating clinical sites. The primary goal of the Canary Retrospective TMA study is to evaluate tissue biomarkers for their ability to predict recurrent prostate cancer at the time of radical prostatectomy. TMAs resulting from this study will also serve as a resource for discovery of biomarkers that predict non-recurrent disease, as well as for assessing candidate biomarkers prior to selection for validation studies using the prospectively collected samples from PASS.

Thus far, we have successfully defined a set of common data elements for TMA annotation, identified a pathologist to lead construction at each site, completed an inventory of specimens available for TMA use, and developed a multi-institutional protocol for all sites. The EDRN has agreed to contribute its data management and coordinating center for this study, and we have established the means to allow data sharing online through the Stanford TMA database. Some sites have already begun to evaluate candidate markers using existing TMAs and in doing so have piloted our protocol for data analysis.

3) Blood Biomarker Assay Development and Validation

Many potentially useful candidate blood biomarkers of lethal prostate cancer remain unvalidated due to a lack of tools for testing. What is needed for validation studies is a test (or "assay") that specifically and sensitively measures the levels in the blood of a given candidate biomarker. Canary recognized this critical bottleneck early on and has addressed it by supporting an assay development team in Victoria, Canada. This team of full-time researchers has expertise in all portions of the biomarker development pipeline, and has begun to tackle an initial set of five candidate blood biomarkers of lethal prostate cancer. Even with a team of dedicated experts, the process is very time and labor intensive and thus we expect that it will be mid-2009 before these assays are completed and we can definitely assess their performance. In some cases, a commercial assay is readily available for a candidate biomarker and simply needs to be ordered and tested on an appropriately designed set of specimens. We have carefully designed our test sets and we have already initiated evaluation of a commercially available biomarker blood test. All commercial and custom assays will be tested on common serum sets so that performance of both individual biomarkers and biomarker combinations can be readily evaluated.

4) Molecular Imaging of Prostate Cancer

Molecular imaging allows us to "see" tumors based on specific, unique, molecular characteristics. We are pursuing several different molecular imaging approaches in our prostate cancer molecular imaging program, including PET-CT imaging of tumor cellular metabolism probes. Synthesis of probes, the first step to generate a molecular imaging agent, can be a difficult and time-consuming process, but our synthesis of the metabolic agent, FACBC, is now nearly complete. In previous studies, this agent has shown good tumor specificity and little background in the bladder (unlike traditional metabolic probes like FDG), so it is a promising molecular imaging agent for prostate cancer. Next, we will radiolabel the agent to produce 18F-FACBC and will proceed to pilot testing in mouse models of prostate cancer toward the eventual goal of obtaining RDRC/FDA approval to test in patients.


CANARY PANCREATIC CANCER PROGRAM

Canary's Pancreatic Cancer Program supports projects aimed at the goal of early detection of pancreatic cancer through a combination of blood-based biomarker tests and molecular imaging. Our program has gained great momentum in 2008 through focusing on our biomarker discovery and validation projects as well as building of critical biospecimen resources. In addition, we initiated a new project in molecular imaging and formed a partnership with The Lustgarten Foundation for Pancreatic Research to develop blood-based assays for candidate pancreatic cancer biomarkers. Our progress is detailed below.

1) Pancreatic cancer biomarker discovery

We have developed a protein extraction protocol that allows us to extract proteins from paraffin embedded, fixed tissues. The development of this technique not only allows us to assess a vast number of clinically valuable tissue samples, but also provides us a tool to perform proteomics experiments with better-defined samples for biomarker discovery.

Our ideal pancreatic cancer biomarker would be one that distinguishes patients with early stage pancreatic cancer from both healthy patients and those patients with chronic pancreatitis, a more common benign disease. In order to find such biomarkers, we have now completed large-scale quantitative global protein profiling experiments using different quantitative proteomics techniques. Through our proteomics experiments, we have compared pancreatic intraepithelial neoplasia (PanIN, which represents early-stage disease), pancreatic cancer, pancreatitis, and healthy control samples. We are still in the process of analyzing the large amount of quantitative proteomics data generated from our most recent profiling studies, but we have already assembled a list of candidate pancreatic cancer biomarkers that are being prioritized for verification and validation.

We are now enhancing our discovery process by selectively enriching for classes of proteins among which we are more likely to identify biomarkers that will work well in blood and imaging tests. Such classes include secreted and glycosylated proteins. We have developed a quantitative proteomics pipeline to achieve this enrichment and measure biomarker concentration within these protein classes. We are currently evaluating the robustness of the platform and have prepared all the needed samples for the study.

2) Biospecimen Resources

Assembly of appropriate serum sets is essential to our rigorous assessment of biomarker performance. The Canary pancreatic team joined forces with Dr. Hoda Anton-Culver, an expert epidemiologist and Director for the Center for Cancer Genetics Research and Prevention at the University of California at Irvine, to augment our specimen collections for biomarker testing. We have carefully assembled our validation sets from patients who are referred to the gastrointestinal (GI) clinic for evaluation of the pancreas, in which all patients and clinic controls underwent evaluation of the pancreas by endoscopic ultrasound. The population tested is derived from a setting that would be similar to the clinical setting in which a biomarker might be used.

Along with assembling samples from GI clinic patients, we also require large numbers of normal (disease-free) control samples, particularly for a disease like pancreatic cancer that has a low prevalence in the population. A biomarker for pancreatic cancer must have very high specificity (the ability to correctly identify people who do not have the disease), in order to avoid a high false positive rate when testing thousands of people. As such, all of our biomarker candidates must be rigorously tested against large numbers of age-and gender-matched controls to ensure that the specificity of the biomarker is high. Dr. Anton-Culver is currently working with the Canary team to collect the hundreds of required disease-free control samples.

3) Blood Biomarker Assay Development and Validation

Many biomarker candidates identified in our profiling studies are novel and have no assay available to specifically and sensitively measure the marker's levels in the blood. In order to address this bottleneck, we have turned to our full-time assay development group in Victoria, Canada, who are developing assays for our high priority candidates. Meanwhile, our common goal of early detection of pancreatic cancer has allowed Canary Foundation to form a partnership with The Lustgarten Foundation for Pancreatic Cancer Research. Canary and Lustgarten are now jointly supporting assay development for pancreatic cancer biomarker candidates -20 in total- at our Victoria facility. Because the process is very time and labor intensive, we expect that it will be the end of 2009 before these 20 assays are completed and we can definitely assess their performance.

We are also implementing alternative approaches to ELISA assay development in order to evaluate our candidate proteins in serum or plasma. We have established a mass spectrometry-based targeted protein quantification technology and selected four priority candidate proteins that have no ELISA available for analysis. We have synthesized a total of 14 stable isotopically-labeled reference peptides for the 4 biomarkers, which will serve as internal standards for the serum samples tested for those selected candidate proteins.

For some of our candidates, commercial assays, such as ELISA, are available for testing. We have tested 13 biomarker candidates using commercial ELISA assays. Initially, we conducted pilot studies using 10 serum samples of pancreatic cancer and 10 samples from normal controls. If the performance of the marker met or exceeded our threshold sensitivity and specificity values, then the marker was moved to expanded pilot studies. In these studies, the number of samples tested was enlarged and additional noncancer controls were included, such as samples from chronic pancreatitis. If the specificity and sensitivity continued to look promising in the expanded pilot results, then the biomarker passed into further validation studies with a new set of serum samples. To date, four candidate biomarkers have completed the validation set. We have now shown that proteomic analysis of tissue specimens is a valuable strategy for the identification of candidate biomarkers in serum, which may provide the basis for an early detection blood test based on a panel of biomarkers.

4) Molecular Imaging of Pancreatic Cancer

Molecular imaging allows us to "see" tumors based on specific, unique, molecular characteristics. We have initiated a molecular ultrasound approach for early detection of pancreatic cancer. We have engineered microbubbles to bind to specific biomarkers found in the blood vessels that feed tumors and are using ultrasound to image the labeled vessels. Ultrasound is a promising screening tool for early cancer detection because it is highly sensitive, non-invasive, does not use ionizing irradiation, and is routinely available in most imaging departments. We have begun pilot testing of these targeted microbubbles in mice bearing subcutaneous tumors derived from human pancreatic cancer. We are pleased to report promising preliminary results, whereby we can see early-stage (1-2mm) tumors with a very specific signal using a dedicated small animal high-resolution imaging system. We are still in the early stages of evaluating this technology, and our future studies will compare it with other molecular imaging technologies such as PET-CT. In addition, we plan to move into further preclinical testing with mouse models of pancreatic cancer. The long-term goal of this work is to translate molecular ultrasound into a highly sensitive clinical imaging modality for detection of early-stage pancreatic cancer using targeted microbubbles in patients.


CANARY BASELINE PROGRAM

Canary's Baseline Program was designed to study the molecular characteristics of the healthy human body. This knowledge is key to selecting biomarkers for early detection. A biomarker occurring at high levels in healthy individuals or varying greatly between them may not be very good at distinguishing cancer from healthy. On the other hand, a biomarker at low levels or varying little in healthy people will likely do a better job. Canary's Baseline Program has three major foci to help identify biomarkers with the desired low background noise: tissue sample collection, construction of a whole-body gene expression map, and quantification of protein concentration and variance in blood. All three systematically explore specimens from healthy individuals.

Blood sample collection

Systematic collection of healthy tissue specimens with comprehensive health annotation is a fundamental necessity for the Baseline Program. One such effort, carried out by our collaborators Drs. Richard Gallagher and Marilyn Borugian at the BC Cancer Agency, is the collection of serum, plasma, and DNA from healthy women who are enrolled in a mammography program. These samples are annotated with a wealth of socio-demographic, life-style, and other participant data. With 402 women recruited by the end of this pilot collection project in November of 2008, the initial goal of 100 women has been far surpassed. The resulting biobank represents a valuable resource for biomarker validation, as specimens with such comprehensive health annotations are hard to come by and in great demand. Through this pilot project, our collaborators were able to secure $6 million CAD, a 55-fold leveraging of Canary funds, to continue this cohort for at least five years as part of the Canadian Partnership for Tomorrow (CPFT). To read the announcement and learn more about CPFT, please visit www.canaryfoundation.org/bc-grant.cfm.

Gene Expression in the healthy human body

Gene expression studies have revolutionized the investigation of many diseases. Still, the coverage of the genome or the range of major tissues in the human body is far from complete, and often the technical quality of the currently available data sets is unsatisfactory. This represents a major gap in our knowledge about the healthy human body that the Baseline Program seeks to overcome.

Canary investigator Dr. Patrick Brown has taken on the task of constructing a detailed map of gene expression in the human body. More precisely, his goal is to profile four independent samples from each of at least 60 human tissue types. Furthermore, he plans on constructing tissue microarrays from fixed samples of these tissues to allow follow-up studies of candidate biomarkers. Samples are currently obtained from surgeries, but autopsy sources are also being considered, especially for difficult to obtain tissues. To date, Dr. Brown has obtained gene expression profiles from about 60 samples representing 25 different tissues. That is roughly one quarter of the anticipated number of samples and tissue types. Our goal for 2009 is to step up tissue collection for samples immediately relevant for the four Canary cancer programs, such as gynecological tissues. Furthermore, we will actively pursue autopsy sources for new materials, particularly for hard to obtain tissue types. We are currently in the process of establishing relationships with pathologists at different institutions who can give us access to these tissues. With these efforts underway, Canary's goal of establishing the human gene expression map moves closer within reach.

Profiling the healthy blood proteome

An understanding of the concentration and variation of proteins represented in healthy blood is particularly important for our efforts to identify blood-based biomarkers for cancer detection. Such information is currently unavailable, yet it is needed to define the background noise biomarkers need to exceed to reach clinical relevance.

Dr. Martin McIntosh is leading an effort to characterize the blood proteome (the set of proteins present in blood) with two important aims. Aim one is to determine the variation of proteins within and between women over time. Dr. McIntosh used blood from healthy women collected longitudinally, one year apart, to measure 75 proteins with available commercial ELISAs. The results, obtained earlier in 2008, indicate that biomarker levels tend to vary between individuals, but to remain constant within one person over time. This means that screening algorithms should rely on an individual's baseline levels of biomarkers rather than using a common biomarker threshold applied to the entire screening population.

The second aim is to quantify the range of concentrations of proteins in blood. Blood specimens from aim one will be used for IPAS (intact protein analysis system) interrogation to develop descriptive statistical models. These will allow for predicting how likely a protein will be detected or quantified, and for assigning concentration information to proteins identified in proteomics experiments. The work to achieve this aim will be performed in 2009.

Even though the Baseline Program was only recently begun and is not yet complete, it has already proven of great benefit for Canary's cancer detection programs. As soon as data become available from this program, they are made freely available to other Canary teams. The Ovarian Program, for example, is using the emerging gene expression and proteomics data to predict biomarkers that stand the greatest chance of succeeding. The usefulness the Baseline Program is providing to all Canary biomarker discovery teams is a greatly motivating experience. It is clear to us that this effort will be valuable for many other fields of research. Therefore, our goal is to make these data available to the rest of the world without restriction as soon as the data are published.


CANARY COLLABORATIONS PROGRAM

Canary Foundation believes that a collaborative, coordinated approach is necessary to achieve our goals. Our multi-disciplinary, multi-institutional Early Detection Program provides a great example of this philosophy in action. We created our Collaboration Program to foster this collaborative approach among the broader community of cancer researchers. We provide scientific program management to make sure that all funded research activities among the scientists and clinicians at the various partner institutions are on track and that any obstacles are rapidly addressed and overcome.

The specific collaboration programs that we support include: national and international workshops and symposiums, joint projects with government and foundations, a postdoctoral scientist training program, an online scientific journal, and a software standardization initiative.

Early Detection and Intervention (EDI) Symposium

2008 Highlights:

  • Keynote speaker Dr. John Niederhuber, Director of the National Cancer Institute
  • 192 scientists, doctors, administrators, industrialists, foundation representatives and philanthropists in attendance
  • Randy Scott, CEO of Genomic Health spoke on bringing diagnostic tests to market

The Fourth Annual Canary Foundation Early Detection Symposium was held from May 20-22 at the Arrillaga Alumni Center at Stanford University. We were pleased to welcome Dr. John Niederhuber, Director of the National Cancer Institute, as our keynote speaker. One hundred ninety-two scientists, doctors, administrators, industrialists, foundation representatives and philanthropists heard inspiring keynotes and cutting-edge scientific talks. The agenda focused on ovarian cancer as a model program for early detection research, covering biomarker discovery, validation, and translation. At the end of day one, Randy Scott, CEO of Genomic Health, shared the challenges and successes of driving diagnostic tests to market, providing a valuable outlook on Canary's mission to place effective cancer early detection tests into clinical practice. Symposium day two included overviews and progress updates on all of Canary's programs: ovarian, lung, prostate, pancreatic, and baseline. The 2008 symposium included a poster session for the second year. Scientists presented 50 posters featuring research from major Canary-supported labs, Canary-ACS fellowship recipients, and others.

Planning is already underway for the Fifth Annual Early Detection and Intervention Symposium to be held from May 4-6, 2009 at the Arrillaga Alumni Center at Stanford University.

International Cancer Biomarker Consortium (ICBC)

2008 Highlights:

  • International Cancer Biomarker Consortium (ICBC) supported by Canary Foundation
  • Meeting held February 20-22, 2008, Honolulu, Hawaii

The goal of the International Cancer Biomarker Consortium (ICBC) is to advance medical research and improve patient outcomes by discovering biomarkers for multiple types of cancer. The consortium is focusing on biomarkers for the assessment of disease risk, early detection of disease, therapeutic prognosis, and response to treatment, as well as disease recurrence. ICBC provides a structure for international teams to work together on global issues such as adoption of data standards and the sharing of data, as well as on scientific details such as the logistics of tissue sample sharing and investigation of mouse models of cancer.

Canary has supported the ICBC's international conference each year since its inception. The annual meeting provides a venue for scientists and technologists to get together to discuss their collaborations and provides technology-sharing opportunities.

Partnerships with Foundations and Government

2008 Highlights:

  • Canary Foundation and the Early Detection Research Network (EDRN) partnered on a study for the early detection of lung cancer among never smokers
  • Canary Foundation and the Early Detection Research Network (EDRN) of the National Cancer Institute partnered for statistical and data management support to the Canary Foundation Prostate Active Surveillance Trial
  • Canary Foundation and The Thomas G. Labrecque Foundation partnership launched the Lung Cancer Program
  • Canary Foundation and the Lustgarten Foundation for Pancreatic Cancer Research partnered to jointly support development of blood-based tests for the early detection of pancreatic cancer
  • Canary Foundation and the American Cancer Society (ACS) jointly awarded four postdoctoral fellowships in early detection research and renewed the postdoctoral fellowship program for 2009

The Early Detection Research Network (EDRN) is a collaborative research group funded by the National Cancer Institute. EDRN's goals align significantly with those of Canary Foundation. These goals include: 1) Development and testing of promising biomarkers or technologies for early detection of cancer, 2) Collaboration among academic and industrial leaders in molecular biology, molecular genetics, clinical oncology, computer science, public health and clinical application for early cancer detection. Canary Foundation and EDRN have come together to jointly fund a multi-investigator project for biomarker discovery in lung cancer among people who have never smoked. In addition, EDRN provides invaluable statistical and data management support to the Canary Foundation Prostate Active Surveillance Study of men with prostate cancer.

A shared recognition of the importance of early detection to save lives has brought Canary Foundation together with the Thomas G. Labrecque Foundation, which made the formation of the Lung Cancer Program possible, and the Lustgarten Foundation for Pancreatic Cancer Research, which allowed us to double our efforts to create blood tests for early detection of pancreatic cancer. These collaborations allow each organization to capitalize on their strengths and share valuable resources.

Canary Foundation has a long-standing relationship with the American Cancer Society (ACS) to provide funding for postdoctoral fellowships in early detection research. Nine postdoctoral fellowships have been funded over the past three years and in 2008 Canary and ACS awarded an additional four fellowships. Each recipient receives $138,000 in funding over three years.

David Joel Gorin, PhD - Harvard University - DNA-Encoded Libraries for Biomarker Identification

Christopher A. Maher, PhD - University of Michigan - The role of noncoding RNAs in early detection of prostate cancer

Joe Shuga, PhD - University of California Berkeley - High Throughput Detection of Mutations as Biomarkers of Hematologic Cancer

Robert William Sprung, PhD - Vanderbilt University - Medical Center Proteomics of Formalin-Fixed Paraffin-Embedded Colon Adenomas

The current group of Canary/ACS postdoctoral fellows published several scientific papers on their research (www.canaryfoundation.org/publications.cfm#acs-pr).

Canary Foundation and ACS also congratulated 2007 Canary/ACS fellow Jerel Garrison, PhD, who leveraged his fellowship to win a K99/R00 award from the National Cancer Institute (NCI). Such NCI awards support the transition of a mentored postdoctoral fellow to an independent faculty researcher. Dr. Garrison plans to pursue a faculty position in the development of cancer diagnostic and therapeutic agents.

Canary Foundation and ACS are committed to the postdoctoral program and expect to announce the 2009 fellows at the 2009 Canary Foundation Symposium.

Canary Journal

2008 Highlights:

  • Canary Foundation launched online journal to aggregate scientific papers and news articles about early detection of cancer

The Canary Journal is a web resource that serves as an aggregator of scientific papers and news articles about the early detection of cancer. Canary Foundation funding supports a part-time site editor who keeps the site current with the most up-to-date research papers. The journal format has the capability to record and display comments and recommendations so that viewers can read not only the papers but also responses from the scientific community. As the journal user community grows, we expect that input from the early detection community will add tremendous value to the papers and news items featured on the site. The journal can be accessed at: http://journal.canaryfoundation.org/.

Software & Standards: Computational Proteomics Analysis System Awards

In May 2007, the Canary Foundation awarded $225,000 to key labs around the world to customize and expand the Computational Proteomics Analysis System (CPAS), an open source proteomics data analysis and data management platform developed by LabKey Software and the Fred Hutchinson Cancer Research Institute. By encouraging labs to adopt the CPAS software, the Canary Foundation attempted to develop a standard bioinformatics platform for proteomics analysis. While the CPAS software was successfully deployed by awardees, adoption in the larger scientific community was not as rapid as the Canary Foundation had hoped. At this time, there are no plans for additional funding in this area.

Software & Standards: Genologics

2008 Highlights:

  • Canary Foundation partnered with Genologics to develop a Biomedical Informatics Suites for the Translational and Outcomes Research (TOR) laboratory at the Fred Hutchinson Cancer Research Center
  • Biomedical Informatics Suite may be exported to other Canary supported labs
  • Canary Foundation tested pilot Biomedical Information Suite for the Canary Center at Stanford

Canary Foundation has partnered with Genologics and the Translational and Outcomes Research (TOR) laboratory at the Fred Hutchinson Cancer Research Center. Genologics, a Canary supporter and partner for several years, is developing a Biomedical Informatics Suite to replace the TOR laboratory's current homegrown system. Canary is committed to accelerating and facilitating research through the use of common toolsets and has arranged for future Genologics credits for other Canary-supported labs, including the Canary Center at Stanford. Over the summer of 2008, we tested a pilot Biomedical Information Suite from Genologics for the Canary Center at Stanford. Initial installation at the Canary Center at Stanford is planned for 2009.


CANARY CENTER AT STANFORD FOR CANCER EARLY DETECTION

2008 Highlights:

  • Canary Foundation's partnerships with the William K. Bowes Jr. Foundation, Stanford University School of Medicine, the Stanford Department of Radiology and the Stanford Comprehensive Cancer Center launched the Canary Center at Stanford for Cancer Early Detection
  • Only facility in the world dedicated to the early detection of cancer
  • Dr. Sanjiv (Sam) Gambhir is first Scientific Director of the Canary Center at Stanford
  • Housed in a 60,000-square-foot facility which will contain four research cores: Chemistry, Proteomics, Bioinformatics and Clinical Trials
  • Operations commence in spring of 2009

On September 13, 2008, together with Dr. Bev Mitchell, Director of Stanford's Cancer Center, and Dr. Sanjiv (Sam) Gambhir, Director of Stanford's Molecular Imaging Programs (MIPS), Canary Foundation announced the creation of the Canary Center at Stanford. The Canary Center at Stanford will be the only facility in the world with dedicated full-time faculty focused on the early detection of cancer and will integrate both blood and imaging-based diagnostic research. This first class integrated facility will attract and develop the best minds in the world to the problem of cancer early detection. Operations will commence in the spring of 2009.

The Canary Center at Stanford has secured a major portion of a 60,000-square-foot facility sponsored by the School of Medicine and the Department of Radiology. The Center will house multiple faculty devoted to early detection of cancer and over 100 research affiliates. Dr. Sanjiv (Sam) Gambhir will lead the Center as Scientific Director. The Center will be supported by research "cores" that provide scientific capabilities to the research teams. The four initial cores will be Chemistry, Proteomics, Bioinformatics, and Clinical Trials.

The Canary Center at Stanford brings together multiple stakeholders, including the School of Medicine, the Radiology Department, the Cancer Center, and Canary Foundation. Canary Foundation has dedicated $25 million for start-up costs, including equipment, modest renovations to the existing research facility, staffing, and faculty recruitment. The Department of Radiology has committed to four new faculty members in molecular imaging, as well as $4 million to support faculty recruitment. The School of Medicine has also committed to four new faculty members for the Center and has committed to supporting facility renovations and lease costs.


CANARY FOUNDATION SUPPORTED PUBLICATIONS

To view publications supported by Canary Foundation, visit the Canary Foundation website at www.canaryfoundation.org/publications.cfm.