Case Western Reserve University
Sanford D. Markowitz, M.D., Ph.D.
This Case GI SPORE proposal provides for a cutting edge Specialized Program of Research Excellence in gastrointestinal malignancies with emphasis on colorectal cancers and with additional attention to adenocarcinoma of the esophagus. This comprehensive program builds on the resources of the Case Comprehensive Cancer Center to propose 4 translational Research Projects to bring new molecular advances to patients with GI Cancers. A series of 4 core resources support these research projects and also establish a strong programmatic infrastructure for translational research in GI cancers. We further have developed a comprehensive infrastructure for identifying new Developmental Research Projects from basic science and clinical investigators from across the Case Cancer Center. Moreover, drawing on our strong track record of developing new faculty who emphasize translational research in GI cancers, we propose a targeted Career Development Program to further enhance the translational research cadre of SPORE faculty. The 4 SPORE translational research projects constitute novel and cutting edge approaches to GI cancers and include studies of: the predictive, prognostic, and therapeutic applications of the new 15-PGDH colon cancer suppressor pathway (Project 1, Drs. Markowitz and Willson); the noninvasive early detection of advanced colon adenomas through stool DNA testing for methylated DNA (Project 2, Drs. Cooper and Li); the comprehensive evaluation of colon cancer gene mutations as predictors of clinical outcome, via use of high throughput Next Generation genomic sequencing (Project 3, Drs. Willis and Wang); and the identification of esophageal adenocarcinoma susceptibility genes (Project 4, Drs. Chak and Elston). These projects are advantaged by special population and scientific resources developed by our SPORE faculty for: evaluation of colon cancer biomarkers, for high throughput screening for new drug discovery, for evaluation of colon cancer screening modalities, for Next Generation sequencing from archived formalin fixed tissue samples, and for study of familial Barrett's and adenocarcinomas of the esophagus. The strength of these investigators and the resources at their disposal will ensure this program leads to important advances.
PROJECT 1: TARGETING 15-PGDH IN COLON CANCER PROGNOSIS, PREDICTION, TREATMENT AND PREVENTION
Sanford D. Markowitz, M.D., Ph.D.
James K.V. Willson, M.D.
The Principal Investigators of this proposal are discovers of a novel and potent colon cancer suppressor pathway mediated by the colon cancer suppressor gene 15-Prostaglandin Dehydrogenase (15-PGDH). 15- PGDH controls the rate limiting step in the degradation of bioactive prostaglandins. As such, it is metabolically poised to antagonize the prostaglandin generating activity of the COX-2 oncogene, which is well characterized as being markedly up-regulated in most colon cancers and precancerous colon adenomas. The PIs have shown: i) that 15-PGDH is highly expressed by normal colonocytes, but that expression is dramatically lost in 90% of colon cancers; ii) that restoring 15-PGDH expression by gene transfection blocks colon cancer xenograft growth; iii) that gene knockout of murine 15-PGDH promotes development of murine colon tumors; iv) and that low levels of colonic 15-PGDH confers resistance to the colon tumor preventive effects of Celecoxib in murine models and in a pilot study of human subjects. Key translational research objectives of this project are now: i) to determine if levels of colonic 15-PGDH expression are a prognostic marker of individual's risk of developing colonic neoplasia; ii) to determine if 15- PGDH expression is a prognostic marker of colon cancer outcome; specifically, to determine whether the 10% of colon cancers that continue to express 15-PGDH represent either a less aggressive or a more aggressive form of the disease; iii) to determine if low levels of colonic 15-PGDH defines a cohort of individuals who are resistant to the chemopreventive effects of Celecoxib; specifically to extend a pilot analysis demonstrating this effect to a comprehensive study comparing colonic 15-PGDH levels versus outcome in over 450 individuals at high risk for colon neoplasia who were studied in the Adenoma Prevention with Celecoxib human trial; and iv) to identify and characterize small molecules that can re induce 15-PGDH in colon cancer cells and can increase 15-PGDH expression in normal colon; this to be done by performing a high throughput screen of a chemical compound library of over 200,000 small molecules in a sensitive cell based 15-PGDH reporter assay.
PROJECT 2: DETECTION OF ADVANCED ADENOMA VIA STOOL DNA (sDNA) METHYLATION TESTING
Gregory S. Cooper, M.D.
Li Li, M.D., Ph.D.
Colorectal carcinoma is largely preventable through the use of screening and removal of the precursor lesion, adenomas. Although colonoscopy is the most commonly used screening test, recent studies have cast serious doubt about its effectiveness in preventing right sided cancer. Furthermore, there is a significant proportion of eligible patients who decline colonoscopy or in whom colonoscopy is not readily available. Testing for aberrant molecular/genetic markers in stool DNA (sDNA) is emerging as a promising alternative to colonoscopy. The currently available sDNA test detects aberrant methylation of the vimentin gene and has reported an 80-85% sensitivity for the detection of colorectal carcinoma. A newly developed next-generation sDNA test with 3 additional methylated DNA markers that are complementary to vimentin promises to be more sensitive and specific. However, these newer markers have not been validated in a screening population and data on the efficacy of sDNA testing for advanced adenomas, hence prevention of colorectal cancer, are limited. Therefore, we propose a prospective screening study to systematically evaluate the accuracy and clinical relevance of this multi-marker panel sDNA testing for detecting advanced adenomas. Specifically, our study aims to: 1) evaluate the performance characteristics of sDNA testing and compare that to the fecal immunochemical test (FIT) for the detection of advanced adenomas in an asymptomatic average-risk screening population; 2) determine the concordance/discordance between tissue and stool gene aberrant methylation in patients with advanced adenomas; 3) investigate the persistence of positive sDNA testing after removal of advanced adenomas; and 4) assess the frequency of missed or occult colonic and upper gastrointestinal neoplasia in patients with a normal colonoscopy but persistently positive sDNA testing. These aims will be accomplished in a comparative study of 1,600 average risk patients undergoing screening colonoscopy. This project is highly translational and information gained from this study may have significant and immediate implication for the clinical practice of screening and primary prevention of colorectal cancer, and surveillance of patients with advanced adenomas.
PROJECT 3: IDENTIFYING MUTATIONAL DRIVERS OF LATE STAGE COLON CANCER
Joseph E. Willis, M.D.
Zhenghe (John) Wang, Ph.D.
This proposal seeks to identify cancer specific gene mutations that are linked with important clinical and epidemiological characteristics of colon cancer (CC). 'Driver' gene mutations are directly related to clinical behavior in virtually all cancers. However, mutations that drive CC progression, from the early curable disease to late stage incurable disease, are yet to be identified. Recent studies by our group and our collaborators have identified 140 candidate cancer genes (CAN genes) in CC. We also found that virtually all the CAN gene mutations found in metastatic CCs are also found in the antecedent primary cancer. This surprising result gives us an opportunity to explore CAN gene mutations in multiple clinical settings. To enable these studies, we have constructed a large biorepository containing CCs from patients treated at a large medical center over the last 20 years. The majority of these specimens are formalin-fixed paraffin embedded [FFPE]. We have developed sophisticated approaches to enhance the utility of DNA extracted from FFPE materials. These include reliable extraction of large DNA fragments. We also developed the technologies and processes to 'DNA capture' targeted exonal fragments and identify mutations from FFPE material using a 'next generation' sequencer. We have developed an infrastructure and knowledge base to support high throughput tissue processing and sequencing. We propose to use these methods to identify CAN gene mutations which appear only in late stage CCs. Also we will functionally dissect the role of these genes using cell line and xenograft mouse models. As CC is one of the cancers with significant disparities attributed to race and gender associated etiologic factors, we will compare CC CAN gene mutations between African American and white patients and between females and male patients, of similar cancer stage and outcome. The former studies will be facilitated by our use of an Ancestry Informative Marker- SNP classification of patients which will be incorporated directly into the DNA sequencing analyses. Our proposals have the potential to significantly advance the understanding of the biological basis for cancer metastasis and to gain insights into racial/gender based differences in etiology and outcomes among CC patients.
PROJECT 4: IDENTIFICATION OF ESOPHAGEAL ADENOCARCINOMA SUSCEPTIBILITY GENES
Amitabh Chak, M.D.
Robert Elston, Ph.D.
Barrett's esophagus is the precursor of most esophageal adenocarcinomas and a large proportion of esophagogastric junctional adenocarcinomas. These diseases are closely associated with gastroesophageal reflux. Screening endoscopy is therefore recommended for the evaluation of chronic gastroesophgeal reflux symptoms. Endoscopic surveillance, performed in those identified with Barrett's esophagus, detects cancer at an early stage. However, the large majority of these cancers occur in previously undiagnosed Barrett's esophagus and nearly half of these persons have no history of gastroesophageal reflux symptoms. Our research has characterized familial aggregation of Barrett's esophagus and its associated cancers, which we term Familial Barrett's Esophagus (FBE). We have developed a network of investigators who are actively identifying and accruing FBE families and biobanking lymphoblastoid cell lines in a centralized biorepository. Furthermore, our segregation analysis of pedigrees accrued to date indicates that FBE is the manifestation of autosomal dominant susceptibility alleles. Our central hypothesis: "FBE has a genetic basis" will now be tested in translational experiments designed to result in gene discovery. The specific aims of this proposal are: 1) to identify genomic loci linked to FBE trait in 46 multiplex multi-generational families already collected; 2) to narrow identified linkage regions in the larger cohort of all FBE families; and 3) to sequence genes of interest. Using the multidisciplinary approach of our collaborative team of investigators, these studies will result in novel information that explains the biological basis of FBE. Identification of genetic risk factors would lead to more effective screening programs and an improved understanding of the molecular pathogenesis of cancer.
CORE A: ADMINISTRATIVE CORE
Sanford D. Markowitz, M.D., Ph.D.
Nathan A Berger, M.D.
The Administrative Core will provide for overall direction, coordination, and administration of the Case GI SPORE. The Administrative Core will include: The SPORE Principal Investigator, the SPORE Co Principal Investigator, and the SPORE Administrative Coordinator. The Administrative Core will oversee the functioning of: the SPORE Executive Committee, the SPORE Internal Advisory Board, the SPORE External Advisory Board, and the SPORE Patient Advocates Advisory Board.
Responsibilities of the Administrative Core will include:
- Oversee conduct of all SPORE activities;
- Oversee administration and coordination of Core Facility utilization and accounting;
- Oversee quality control and quality improvement processes for core facilities;
- Oversee management and accounting of grant funds; assure monthly reconciliation of all accounts;
- Assure compliance and maintain documents for all institutional regulatory requirements for human experimentation, animal utilization, toxic material and safety training and utilization;
- Ensure compliance and coordination with all reporting regulations;
- Ensure compliance with all requirements for research progress reports;
- Manage all intellectual property affairs arising from SPORE-sponsored research;
- Coordinate SPORE seminars and retreats;
- Convene regular meetings of Steering Committee;
- Organize annual visits of External Advisory Committee;
- Coordinate solicitation and evaluation of pilot research studies;
- Coordinate solicitation and evaluation of career development awards;
- Represent the SPORE in interactions with other Centers, Departments, the School of
- Medicine, affiliated hospitals and community organizations;
- Coordinate and assure participation of the Case GI SPORE in National SPORE meetings.
CORE B: BIOSPECIMEN CORE
Joseph E. Willis, M.D.
John J. Pink, Ph.D.
The Biospecimen Core will work closely with SPORE investigators to enhance their projects by supplying quality controlled colon and esophagus tissues and expert consultation regarding multiple aspects of pathology. A longstanding collaboration between members of the Biospecimen Core and individual project investigators has resulted in the compilation of an extensive tissue archive of cancers from 2,443 patients with primary colon cancers of known pathological state and data based clinical follow-up. A substantial archive of paraffin blocks of normal, pre-cancer and cancer specimen, including metastases of regional lymph nodes, distant organs and subsequent recurrences is maintained. Frozen material with matched normal controls from 887 of these cases is banked. There are 444 esophageal cancers and Barrett's esophagus paraffin specimens and 202 frozen esophageal specimens. Historically, the Core has supplied GI SPORE investigators with microdissected esophageal and colon cancers suitable for analysis of the temporal sequence of gene mutations, colon cancer tissue arrays suitable for immunohistochemistry and in situ hybridization, frozen tissue suitable for RNA profiling, and blood specimens for genotyping in the discovery of novel inherited traits of colon cancer predisposition. The core also banks blood specimens for future biomarker development. Additionally the Core will: i) organize and distribute all prospective GI biospecimen procedures to SPORE investigators; ii) provide access to its existing tissue archive resource; iii) identify tissues of interest from archive and prospective accrual to investigators; iv) manage biospecimens obtained by individual projects for later targeted investigation; v) provide histopathological quality control for tissue sections with project specific morphology case reviews and oversee and provide longitudinal follow-up of clinical outcomes linked to these tissues; vi) provide tissue microarray sections for tissue targets of interest; and vii) provide expertise in immunohistochemistry assessment and samples appropriate for specific studies. The primary objective is to provide a resource that contributes significantly to individual project goals and cooperation between other GI SPOREs.
CORE C: BIOSTATISTICS CORE
Jill Barnholtz-Sloan, Ph.D.
Robert Elston, Ph.D.
Colorectal carcinoma is largely preventable through the use of screening and removal of precursor lesion, adenomas. Although colonoscopy is the most commonly used screening test, recent studies have cast serious doubt about its effectiveness in preventing right sided cancer. Furthermore, there is a significant proportion of eligible patients who decline colonoscopy or in whom colonoscopy is not readily available. Testing for aberrant molecular/genetic markers in stool DNA (sDNA) is emerging as a promising alternative to colonoscopy. The currently available sDNA test detects aberrant methylation of the vimentin gene and has reported an 80-85% sensitivity for the detection of colorectal carcinoma. A newly developed next-generation sDNA test with 3 additional methylated DNA markers that are complementary to vimentin promises to be more sensitive and specific. However, these newer markers have not been validated in a screening population and data on the efficacy of sDNA testing for advanced adenomas, hence prevention of colorectal cancer, are limited. Therefore, we propose a prospective screening study to systematically evaluate the accuracy and clinical relevance of this multi-marker panel sDNA testing for detecting advanced adenomas. Specifically, our study aims to: 1) evaluate the performance characteristics of sDNA testing and compare that to the fecal immunochemical test (FIT) for the detection of advanced adenomas in an asymptomatic average-risk screening population; 2) determine the concordance/discordance between tissue and stool gene aberrant methylation in patients with advanced adenomas; 3) investigate the persistence of positive sDNA testing after removal of advanced adenomas: and 4) assess the frequency of missed or occult colonic and upper gastrointestinal neoplasia in patients with a normal colonoscopy but persistently positive sDNA testing. These aims will be accomplished in a comparative study of 1,600 average risk patients undergoing screening colonoscopy. This project is highly translational and information gained from this study may have significant and immediate implication for the clinical practice of screening and primary prevention of colorectal cancer, and surveillance of patients with advanced adenomas.
CORE D: GENOMICS CORE
Mark D. Adams, Ph.D.
Martina L. Veigl, Ph.D.
The goal of the Genomics Core is to provide comprehensive support for genomic analyses carried out in each of the Case GI SPORE Projects. The Genomics Core will capitalize on the significant investment in common and shared infrastructure that has been created at CWRU, with support from the School of Medicine, the Case Comprehensive Cancer Center, and the Howard Hughes Medical Institute. CWRU and Cancer Center core resources that are part of this SPORE have been the subject of NIH support and are partially funded as such; thus, this SPORE will be leveraging the existing support to provide each project access to state-of-the-art genome-scale analysis expertise. The Genomics Core builds on existing core facilities that have a significant track record of performance and success, as well as the faculty interactions which have supported the initial work for components presented within each Project of this application. The Genomics Core will be a central resource for providing high-throughput genotyping and 'next-generation' DNA sequencing capability for two Projects. In addition, the Core will develop and implement additional methods as necessary to support all Projects, including Pilot Projects. The equipment and personnel available in the Genomics Core support research activities that require instrumentation and expertise that are not available or practical in the laboratories of the individual Project PIs, and for which centralized analyses are required in order to achieve process standardization and economies-of-scale. Specifically, the Genomics Core will perform region-specific DNA capture and high-throughput DNA sequencing and genotyping in support of the individual SPORE Projects. The Genomics Core will work closely with the Specimen and Tissue Core for access to quality assessment of materials for analysis and with the Biostatistics Core for data quality assessment and analysis.