Brian Brummeler Haab - US grants

[unreadable] DESCRIPTION (provided by applicant): Serum markers hold great promise for improving the care and treatment of cancer patients. Although many proteins have serum levels associated with various cancers, each has limited clinical usefulness when measured individually at single time points. The lack of sensitivity and specificity of current serum markers stems from heterogeneity in the baseline levels of the marker proteins and heterogeneity in the tumors and patients. A biomarker discovery strategy that accounts for the heterogeneity in people and tumors is to use individualized thresholds, based on longitudinal measurements, to precisely define abnormal levels for each individual. Previous research has shown that biomarkers defined by longitudinal measurements could have greatly improved specificity and sensitivity over current markers. No systematic study of this topic has been performed, largely because of the lack of a convenient technology for that purpose. A well developed and validated antibody microarray technology in the laboratory of Dr. Haab now makes this exploration possible. The multiplex detection capability of the antibody microarray will allow us to test the performance improvement upon using longitudinal measurements for many different proteins and to establish the general principles that define the use of longitudinal markers. In addition, the multiplex detection capability ultimately will allow the use of combined longitudinal measurements for even further biomarker performance improvement. To test this strategy, we will evaluate the sensitivity, specificity and time of the detection of prostate cancer recurrence using both longitudinal and single-time-point measurements of many different prostate cancer-related proteins in serum. This new approach addresses fundamental issues in biomarker research and should result in valuable information for a wide variety of research areas. The successful demonstration of the approach to prostate cancer diagnostics will signal its potential usefulness for all types of biomarker studies. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The development of methods to accurately detect early pancreatic cancer and to better differentiate benign from malignant disease could greatly improve the outcomes for pancreatic cancer patients. It is known that malignant transformation of epithelial cells of the pancreas results in alterations in the carbohydrate chains of certain proteins secreted or released by these cells. Glycosylated proteins form the basis for current biomarkers for detecting pancreatic cancer and other adenocarcinomas, and refinement of these tests are predicted to enable detection of early pancreatic cancer. Our preliminary data has shown that a novel antibody-microarray technology allows the efficient detection of glycans on distinct proteins and the identification of specific glycan structures associated with pancreatic cancer. The method uses antibody microarrays to capture specific proteins from serum samples, followed by the incubation of a glycan-binding protein (such as a lectin) to quantify specific glycans on the captured proteins. Two classes of glycoproteins, mucins and carcinoembryonic-antigen-related proteins, are particularly associated with cancer, both in altered expression patterns and in altered glycan structures on the proteins. In the R21 phase, we will determine the levels of multiple specific glycans on members of those protein classes to test the hypothesis that the measurement of specific cancer-associated glycans on specific proteins, as opposed to measuring just protein or just glycan levels, will yield improved sensitivities and specificities for cancer detection. The R33 phase of the project will expand and thoroughly test the approach. The sensitivity and specificity of detecting pancreatic cancer using measurements of glycans on mucins, CEA proteins, and proteins identified in the R33 phase will be characterized in a large set of serum samples from subjects with pancreatic cancer, benign pancreatic disease, other cancers, and no disease. We expect to characterize the value of these measurements for disease diagnostics and to gain insights into the generality and frequency of specific glycan alterations on secreted proteins. Relevance to public health: The ability to more accurately diagnose cancers at earlier stages could lead to improved outcomes for many patients. This research could lead to significantly improved blood tests for the detection of cancer, as well as a powerful, generally- applicable platform for studying carbohydrate alterations on multiple proteins. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Glycan alterations are known to be critical for several processes that contribute to cancer progression, particularly in pancreatic cancer, where glycosylation alterations are highly prevalent. Little is known about the conditions that give rise to those alterations, or whether they distinctly occur in particular subpopulations of cancer cells. Preliminary data have shown that pro-inflammatory cytokine signaling induces glycan alterations in pancreatic cancer cells, suggesting the possibility that the inflammatory environment typically seen in pancreatic cancer induces cancer cells to modify their extracellular glycan structures. Furthermore, the preliminary data suggest that the induced glycan alterations are different between groups of cell lines, as defined by the presence or absence of cell-surface markers that are associated with tumorigenicity in pancreatic cancer. That finding suggests that induced glycan alterations are distinct between cell types, and that these glycan alterations contribute to the particular behavior of each cell type. In order to gain insight into the relevance of these behaviors to cancer biology, the next step is to investigate these hypotheses in primary tumor cells, rather than in the cell line models used for the preliminary studies. A good system for that work is mouse xenografts from human primary tumors, which have been established for pancreatic cancer and can provide enough material for experiments on sorted cell subpopulations. In the first aim, we will determine whether the glycosylation or expression status of several mucins is different between the tumorigenic and non-tumorigenic subpopulations. In the second aim, we will determine whether these subpopulations display different glycan alterations in response to cytokine signaling. A significant aspect of this project is the unique experimental approach of using antibody arrays with glycan detection to measure protein and glycan variation on several specific proteins. The micro-scale nature of that assay also is valuable for the study of small subpopulations of cells. This work is well suited to a pilot-project mechanism since it will give insights into a possibly transformative research direction. PUBLIC HEALTH RELEVANCE: Pancreatic cancer is a devastating disease with very low survival rates. It is known that alterations to the carbohydrates on the surfaces of cancer cells are important for disease progression, but the factors that give rise to those alterations, and the particular cancer cell types on which they appear, are incompletely understood. This proposal addresses that question, which has implications for treating or controlling pancreatic cancer.

DESCRIPTION (provided by applicant): The current best hope for the successful treatment of pancreatic cancer is the removal of pre-invasive lesions before they become malignant. The early detection of a portion of pancreatic cancer precursors- cystic neoplasms of the pancreas-is possible through high-resolution abdominal imaging. The increased use of abdominal imaging has led to a higher rate of identifying pancreatic cysts, but currently it is not possible to accurately determine which cysts have high malignant potential and should be removed. New biomarkers that could assist that determination would lead to more successful outcomes for patients with pancreatic cysts. Therefore, the goal of this research is to develop biomarkers for distinguishing pancreatic cysts with high malignant potential from those with low malignant potential. Our overall hypothesis is that the expression and glycosylation of specific proteins are significantly different between cysts with high malignant potential and cysts with low malignant potential, and that these molecules form accurate biomarkers for the diagnosis of pancreatic cysts. We have a comprehensive strategy for biomarker discovery, refinement, and validation. Multiple clinical sites will contribute the medical expertise and the high-quality sample sets, and experienced statistical expertise in biomarker research will guide the experimental design for biomarker discovery, pre-validation, and validation. Our technological strategy is built on the powerful combination of novel glycoproteomics biomarker discovery methods and complementary antibody array methods for the high-throughput and precise profiling of multiple protein and glycan candidates. Through the iterative characterization and testing of biomarker isoforms and glycoforms, the performance of the best candidate biomarkers will be refined and improved. Pivotal double-blind validation studies will provide accurate assessments of biomarker performance. The success of this project will result in biomarkers to be validated in clinical settings; high-quality sample sets to be used in ongoing EDRN-associated discovery and validation studies; and an improved understanding of the molecular alterations associated with pancreatic cystic neoplasms.

DESCRIPTION (provided by applicant): New biomarkers for pancreatic cancer are urgently needed on several fronts: screening among high-risk individuals, accurate diagnosis of suspected cancer, prognosis and treatment prediction, and monitoring the progress of tumors during the course of treatment. The CA 19-9 antigen is the best current marker for pancreatic cancer, yet its use is limited owing to its lack of expression in a significant fraction of patient. The goal of this research is to develop a panel of biomarkers for pancreatic cancer that specifically identifies patients that are either high or low in CA 19-9 and that would perform well enough to impact patient care. Research has shown that the lack of CA 19-9 elevation in certain patients is due to genetic or expression alterations in the glycosylation machinery not found in CA19-9-expressing patients. In addition, we have shown that certain patients who are low in CA 19-9 produce alternative glycans that can be used to specifically identify them. Our hypotheses are 1) the CA 19-9-low and CA 19-9-high tumors are distinct biological entities that produce divergent glycan structures; and 2) the detection of the glycans specific to CA 19-9-low tumors used in combination with the detection of CA 19-9 forms a highly accurate biomarker panel. We will use powerful glycomics tools guided by new biological/biochemical information to test these hypotheses. In Aim 1, we will use the development of new affinity reagents combined with Shotgun Glycomics to identify and characterize glycans that may specifically detect CA 19-9-low tumors. In Aim 2, we will derive biological information from gene expression analysis to further guide the testing of glycans for differential expression. In Aim 3, the identified affinity reagent will be used in the testing and development of biomarker panels. The completion of these aims will result in new biomarkers to improve the care of pancreatic cancer patients, the advancement of a new strategy for identifying and developing glycan-based biomarkers, and new resources for other glycobiology projects. PUBLIC HEALTH RELEVANCE: Pancreatic cancer patients typically have very short survival times after diagnosis. New diagnostic methods to better identify pancreatic cancer and guide treatment decisions could greatly benefit these patients. The goal of this research is to develop such biomarkers. The initial intended use of the biomarkers resulting from this project is to improve the accuracy of early-stage diagnosis among patients with suspected cancer. Success in that area would lead to the development of these or similar markers for other needs, such as screening among high-risk individuals or selecting the best therapy for patients with confirmed cancer.

? DESCRIPTION (provided by applicant): Pancreatic cancer is alone among the major cancers in its increasing prevalence, and it is predicted to be the second-leading cause of cancer death by 2030. A strategy to help reverse this trend is to identify and treat more pancreatic cancers at an early stage of the disease, when surgery and therapeutics are most effective. Currently no biomarker is good enough to be used for the detection of early-stage disease in people at elevated risk for pancreatic cancer. In our preliminary research, we have developed biomarkers a) that have better sensitivity and specificity for early-stage cancer than the current best biomarker for pancreatic cancer, CA 19-9, and b) that can detect cancers that are low in CA 19-9. The several individual biomarkers complement each other because each is elevated in distinct groups of patients. These encouraging results lead us to propose that a biomarker panel developed from these and related biomarkers will provide the necessary performance to enable clinical detection and diagnosis of early-stage pancreatic cancer. A second strategy for improving outcomes is to better identify those early-stage cancers that will rapidly progress. Some cancers that appear to be early-stage and resectable show progression within a short time after surgery. Biomarkers to identify such cancers would allow better treatment for those patients, as they could be spared the risk and recovery time of surgery and immediately begin the appropriate systemic treatment, likely leading to better results. Currently, we have no accurate indicators of rapid progression after surgery. One of the biomarkers discovered in our preliminary research is elevated in most patients who have rapid progression after surgery, suggesting the possibility of its use as a progression biomarker. Our goal is to develop biomarkers that will be effective for detecting early-stage pancreatic cancer (Aim 1) and for identifying the early-stage cancers that will rapidly progress (Aim 2). We will pursue this goal by optimizing the existing biomarkers and bolstering them with additional biomarkers, and by developing, testing, and validating panels of biomarkers that improve performance over the individual biomarkers. We hypothesize that the biomarker panels can achieve the performance required for further clinical validation. We are in a good position to achieve our goal, given the promising biomarkers already discovered and the convergence of all the other factors necessary for a successful project: we have assembled a team of top experts in the clinical, technological, and statistical fields, plus we have the sample resources, patient base, experimental methods, institutional environment and support, and passion as a team to deliver high-impact results.

PROJECT SUMMARY Recent research is uncovering unexpected ways that glycans contribute to biology, as well as new strategies for combatting diseases using approaches involving glycans. To make full use of the opportunities that are opening, we need methods that will enable biologists and clinicians who are not experts in glycobiology to conduct research in this field. The current methods require a high degree of expertise or specialized equipment. The methods also typically do not provide information relevant to disease-oriented research, such as the location, nature, and precise levels of specific glycans in clinical specimens. The goal of this project is to develop a method that will help meet this need. The method, which we call On-Chip Glycan Modification and Probing (On-Chip GMAP), will be accessible to non-specialists, usable with low sample volumes, and effective in providing precise measurements over many samples. We previously demonstrated micro-scale capture and affinity-based probing of glycans and glycoproteins from biological samples, as well as algorithms and software for interpreting the data. Here we will add enzymatic alterations of the captured glycans, followed by lectin/antibody probing of the altered glycans and automated interpretation of the data. The protocols are straightforward; reagents can be supplied in kit form; costs can be kept low because of the miniaturized scale; and the interpretation can be made readily understandable through the software. The information will be complementary to that derived from conventional glycan analyses using mass spectrometry and chromatography. Mass spectrometry and chromatography provide compositions and candidate structures for a limited number of samples, and the new method will provide motif information in a quantitative way over many samples. Therefore, On-Chip GMAP could be an important component of practical and comprehensive glycan analysis strategies, particularly for disease research. In addition, it could broaden the use of glycan analysis tools to researchers who are not experts in glycobiology, potentially facilitating new discoveries and applications.

PROJECT SUMMARY A difficulty in the development of effective treatments against pancreatic cancer is heterogeneity within and between tumors; a subset of cancer cells, rather than all cells in a tumor, is responsible for the behaviors of invasiveness and resistance to death. Drugs that act on the aggressive subpopulations promise to be more effective than drugs that act on the bulk of a tumor, but research to identify such drugs is hampered by the lack of biomarkers to detect, isolate, and study the aggressive subpopulations. The goal of this project is to identify biomarkers of aggressive subpopulations of pancreatic cancer cells. We hypothesize that distinct subtypes of pancreatic cancer cells exist, and that they differ in their invasiveness and resistance to death. We found support for this hypothesis in previous research through the identification of glycan biomarkers of subpopulations of cancer cells with differences in molecular characteristics and behaviors. We will build on the previous results to fully test the possibility that specific glycan markers can be used for detecting subtypes of cancer cells. We posit that aggressive subtypes will be associated with poor outcomes, and that they will display high invasiveness and resistance to death in model systems. To enable a complete evaluation of the candidate markers, we will employ valuable tissue resources such as primary tumors, patient-derived xenografts, and tumor organoids, and we will apply unique and powerful experimental systems?multimarker immunofluorescence and MALDI glycan imaging. Biomarkers that identify the aggressive subtypes of pancreatic cancer cells could pave the way for the development of truly effective therapies. They would provide a way to determine which model systems, or which cells within the models, are the important ones to target, and they would provide companion diagnostics to guide and monitor the use of the targeted therapies. Drugs arising from such research would, for the first time, strike at the critical point?the subset of cells giving rise to the lethal nature of the disease. We are in a good position to produce significant results given the leads from previous research, our technologies and resources, and the team of clinical, technological, and statistical experts.