Kenneth W. Kinzler - US grants

Colorectal tumors progress through a series of well defined clinical and histopathological stages. This progression is accompanied by a series of genetic changes which include inactivation of several tumor suppressor genes. Several studies have suggested that one or more tumor suppressor genes on chromosome 5q2l are important in the early stages of colorectal tumorigenesis. Recently, we have identified two candidate tumor suppressor genes (APC and MCC) from the chromosome 5q2l region. Both the APC and MCC genes were found to be somatically mutated in colorectal cancers. Furthermore, the APC gene was found to be mutated in the germline of several kindreds with either Familial Adenomatous Polyposis (FAP) or Gardner's Syndrome (GS), two inherited diseases which predispose to colon cancer. The studies outlined in this proposal are aimed at extending these results with APC and MCC in the following areas. First, the clinical significance of APC and MCC in colorectal cancer will be determined. This will include the determination of the prevalence of APC and MCC mutations in human colorectal tumors and in the germline of individuals with FAP and GS. Second, the biochemical properties of MCC and APC will be studied. This will include production of antibodies to APC and MCC, characterization of the APC and MCC protein products, and determination of APC and MCC expression in adult and developing tissues. Third, the functional properties of APC and MCC will be studied. This will include determining the effects of inducing expression of normal APC and MCC protein in colorectal tumor cell lines and determining the effects of reducing endogenous APC and MCC expression using anti-sense technology. The combination of the above studies should provide important insights into the role of APC and MCC in colorectal tumorigenesis.

DESCRIPTION (provided by applicant): Colorectal cancer represents a major public health issue but also provides exceptional opportunities for investigating human tumorigenesis. We have recently developed experimental approaches that allow us to analyze human cancers in unprecedented detail and address questions that were difficult or impossible to answer in the past. We intend to use these approaches to fill gaps in our understanding of the early events in colorectal tumor development. Aim #1 is directed at identifying the genetic basis of two polyposis syndromes, Serrated Polyposis Syndrome (SPS) and Atypical Adenomatous OligoPolyposis (AAOP). To determine the genetic basis for both these syndromes, we will perform whole genome sequence analysis of normal and tumor tissues from representative patients. Aim #2 is directed at identifying the clonal relationship and timing of genetic alteratios during colorectal tumorigenesis in the canonical Adenoma-Carcinoma and the Serrated Colorectal Cancer Pathways. We will perform genome-wide analyses of clonal somatic mutations in a unique collection of lesions including hyperplastic polyps, serrated adenomas, sessile serrated adenomas, small adenomatous polyps and, most importantly, invasive and adjacent non-invasive components (adenomatous and hyperplastic) from the same lesions. We will thereby be able to determine the clonal relationships among different components of the same tumor and evaluate the time periods required for tumor progression. Aim #3 is directed at characterizing non-clonal somatic mutations in normal and neoplastic colorectal tissues. The number and type of non-clonal somatic mutations can provide a window into past endogenous and exogenous mutagen exposures. We have recently developed a technique (SafeSeqS) that can reliably identify non-clonal somatic mutations in clinical samples. SafeSeqS will be used to assess such mutations in benign and malignant tumors as well as in normal colorectal tissues. Aim #4 is directed at identifying the full compendium of early events in colorectal tumorigenesis. Several lines of evidence support the idea that APC or CTNNB1 mutations are the initiating events in colorectal tumorigenesis. Is tumor development simply gated by inactivation of the APC pathway or are additional early changes required? Using technologies developed during the past funding period, we will pursue an integrated (i.e., genetic, epigenetic and transcriptomic) analysis of multiple synchronous adenomas removed from the same patients to look for recurring genetic and epigenetic changes that may act as early events in tumor development. Relevance: Our specific aims are directed at identification of the early events in colorectal tumorigenesis. It can be argued that understanding these early changes is essential for the development of more effective strategies for colorectal tumor prevention and treatment.

The two best described familial predispositions to colorectal cancer are familial adenomatous polyposis (FAP) and hereditary non-polyposis colon cancer (HNPCC). Recent advances make genetic diagnosis of these predispositions a reality. FAP is due to germline mutations in the APC tumor suppressor gene whereas HNPCC is caused by mutations in any one of at least four human DNA mismatch repair (MMR) genes. While these findings have paved the way for genetic diagnosis of FAP and HNPCC, many critical issues must be addressed before this work can be optimally translated to a clinical setting. The studies proposed in this application have three aims. The first is improving the sensitivity of testing for FAP and HNPCC. We have previously developed assays that can identify the genetic defect in about 80% of FAP and 50% of HNPCC patients. Recently, we have developed a sensitive and novel strategy for mutational testing (MAMA) which can detect mutations missed by conventional assays. In an effort to improve, screening sensitivity, we will use MAMA to test FAP and HNPCC patients who have tested negative with conventional analyses. Second, we will determine which patient populations warrant screening for APC and MMR gene alterations. Our previous studies have largely focused on patients who meet the classic criteria for FAP and HNPCC. However, we have identified APC and MMR mutations in several patients who do not meet these criteria. We will extend these studies to other similar patient populations who may have a predisposition to colorectal cancer. Third, we will attempt to improve the accuracy of testing by using functional tests to evaluate the disease- causing potential of selected mutations. A subset of the variants identified in the APC and MMR genes result in relatively subtle changes in the encoded protein. Determining whether these changes represent disease- causing mutations or harmless variations requires functional analysis. Together, these studies should provide information necessary for the translation of genetic testing for hereditary colorectal cancer syndromes to the clinic.

This project is a continuation of Project 1 A. Significant progress was made on each of the original aims of this project. Our goals related to improved presymptomatic diagnosis of individuals with inherited predispositions to cancer were essentially obtained and that aim has been retired. Our work related to early detection continued to show that somatic mutations can be sensitive and specific markers of neoplastic cells. Moreover, the development of a new technology called BEAMing, which allows hundred of thousands of individual PCRs to be performed in one tube, further improved our ability to detect somatic mutations. In total, our studies suggest that somatic mutations have the potential to significantly outperform conventional markers for early detection. Accordingly, our current proposal will focus on three aims related to the application of somatic mutations for the early detection of neoplasia. Aim #1 will focus on identification of somatic mutations in fecal DMAfor the early detection of colorectal cancers and adenomas. Specifically, we will develop and validate a Stool Mutation Test (SMT) capable of detecting clinically relevant adenomas and cancers with >70% and >90% sensitivity, respectively, and >99% specificity. Aim #2 will focus on identification of somatic mutations in plasma DMAfor the detection of colorectal cancers. The major goal of this aim is development and validation of a Plasma Mutation Test (PMT) capable of detecting early colorectal cancers (Dukes A and B) with >50% sensitivity and >99% specificity. Aim #3 will focus on identification of somatic mutations in plasma DMA for the early detection of pancreatic cancers. The goal of this aim is to develop and validate a PMT capable of detecting early pancreatic cancers (Stage I and II) with >50% sensitivity and >99% specificity. These studies will utilize samples from Cores 2 and 3 to translate discoveries made in former Projects 1A and 1B to patient care. We will provide tools for early screening of patients in Core 3 familial registries, help in the characterization of patients evaluated in Project 3A and aid development of new approaches by Projects 2B and 3B. The overall goal of the above studies is to provide clinically practical assays for early detection and management of colorectal and pancreatic cancers. From a public health prospective, such early detection strategies have the best chance of reducing the morbidity and mortality associated with colorectal and pancreatic cancers in the near term.

More than 2% of the adult population of the US harbors a pancreatic cyst and the increasing use of abdominal imaging is likely to increase the diagnosis of these lesions as incidental findings. Because these cysts can either be self-limiting lesions or represent true precursors to deadly invasive adenocarcinomas, they pose a difficult clinical management problem. There are three main forms of pancreatic cystic neoplasms, serous cystic adenomas (SCAs), intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs). SCAs are thought to be totally benign whereas IPMNs and MCNs are often associated with an invasive component. The ultimate goal of this proposal is to develop clinically useful biomarkers that can distinguish these cyst types and alter clinical decision-making. This goal is practical now due to previous successes of this SPORE in the areas of genomic analysis, biomarker development and pancreatic cyst analysis. To achieve this goal, we propose a detailed molecular-analysis of neoplastic cells from all three main types of pancreatic cyst neoplasms (Aim #1). These molecular findings will be used to develop cyst fluid biomarkers (Aim #2). Cyst fluid was chosen as the target clinical sample because of promising preliminary results and because cyst fluid is routinely available in the clinic at critical decision points. The best biomarkers developed in Aim #2 will be correlated with clinical findings and outcomes in both a retrospective (Aim #3) and prospective (Aim #4) manner. The above studies will identify molecular changes that underlie the pathogenesis of pancreatic cyst development and should allow the development of clinically useful cyst fluid biomarkers.

DESCRIPTION (provided by applicant): The focus of this proposal is to continue development of a unique panel of colon cancer associated nuclear matrix proteins (NMPs), discovered and developed during our previous EDRN-sponsored, Biomarker Development Laboratory funding. NMPs offer the potential serum-based biomarkers for the early detection of colorectal cancer. This work has progressed substantially from testing of un-blinded single institution convenience samples all the way to testing of blinded, EDRN sponsored multi-institution reference sets. Markers were tested in different laboratory settings, cancer subtypes, and patient population characteristics. These data provide compelling potential for application in clinical populations. Currently two markers, CCSA-2 and CCSA-4 are poised for Phase ll-lll validation studies. We propose, with the help of established experts in assay development, to further refine those assays to position them for definitive clinical testing. In addition, we will advance NMP markers related to liver metastasis. The two major objectives 1. To OPTIMIZE AND REFINE the CCSA-2 and CCSA-4 assays and test them in a series of small scale experiments to position them for definitive evaluation within the EDRN mechanism. In specific, we aim to (a) develop robust assays for the accurate and precise measurement of CCSA-2 and CCSA-4; (b) test the assays under a variety of clinical circumstances and conditions to understand, and anticipate sources of error and potential problems in implementation that might be encountered when testing the assays on a larger scale. The goal of this first objective is to deliver two assays to validation with ample preliminary testing to maximize the potential for success in the EDRN program. 2. To develop NMP markers for liver metastasis. We have identified proteins in liver metastasis tissue specimens that are neither present in adjacent uninvolved liver nor in liver from normal donors. These proteins are prime candidates for assay development for markers of advanced disease. We propose (a) to isolate, characterize and develop antibodies for serum detection of liver metastasis proteins, (b) to evaluate these antibodies in appropriate clinical specimens for eventual delivery for EDRN validation.

? DESCRIPTION (provided by applicant): A blood test for early detection of cancer would provide a valuable, additional means for screening the population at risk. Blood-based biomarkers for monitoring cancer could enhance care by earlier identification of subjects at risk of recurrence, better prognostic assessment, and potentially, improvements in survival or quality of life due to earlier implementation of alternative therapy. Thus, a liquid biopsy for early detection and for non-invasive assessment of tumor and tumor characteristics during treatment would represent a significant medical advance. Circulating tumor DNA (ctDNA) are small fragments of nucleic acid that originate from apoptotic or necrotic tumor cell turnover. Characteristic of the malignant process, ctDNA can be assessed in plasma, and offers the potential of a sensitive and specific biomarker for a host of applications including diagnosis or early detection of tumors, prognostic information on disease-free or overall survival, and predictive information on resistance and probability of lack of response to treatment. Previous ctDNA studies have been implemented by identifying characteristic mutations in the primary tumor and subsequently interrogating plasma DNA from the same patient for the presence of those mutations. The next step in evaluating ctDNA as a screening modality is to construct a panel of mutations amenable to detection in plasma (a PlasmaSeq panel) to assess the sensitivity and specificity of plasma ctDNA in identifying cancer without prior knowledge of which mutations are present in the tumor of the patient whose plasma is assayed. In Aim 1, we will prospectively recruit patients with colorectal cancer (CRC), disease-free controls, and subjects with adenomas prior to definitive surgical or endoscopic treatment and systematically evaluate a PlasmaSeq panel to explore the utility of ctDNA as a marker for early detection. Previous studies suggest that ctDNA can be used to monitor cancer in subjects under treatment or at risk for recurrent disease. In Aim 2, we will recruit newly diagnosed stage III CRC patients, determine their tumor mutational profile, and systematically collect high volume (>10 ml), serial plasma specimens every 3 months for up to 4 years for ctDNA and concurrent CEA measurement. Clinical outcome and survival will be tracked and parameters of ctDNA assessment, including absolute level, velocity of change, and degree of fluctuation will be evaluated in relation to clinical outcome, stratified by tumor stage classification (stage IIIA-III). The resulting data will permit assessment of ctDNA as a prognostic marker for disease-free and overall survival. This research will further advance study on the use of ctDNA in early detection and monitoring of CRC. ctDNA testing is also applicable to many other cancers. Thus, advancement in this technology is potentially of great impact to cancer care.