Stephen J. Meltzer - US grants

[unreadable] DESCRIPTION (provided by applicant): Chronic idiopathic inflammatory bowel disease (IBD) predisposes to the development of colorectal carcinoma. The molecular basis of this predisposition has been studied for many years, but much remains to be discovered. For example, we know that unique global gene expression patterns occur early in IBD-associated neoplasias (IBDNs), and that hypermethylation of certain promoter regions is a mechanism of gene inactivation in these lesions. But at which neoplastic stage do these alterations occur during IBD-associated carcinogenesis? Can individual genes be identified from global genomic screens of expression, methylation, or change in copy number? Which global patterns or individual gene alterations predict early neoplastic transformation or progression? The current proposal will answer these questions by developing the following unifying hypothesis: The hypothesis is that the study of IBDNs at all stages of evolution will benefit from global, comprehensive genomic approaches that will illuminate molecular genetic carcinogenetic pathways while simultaneously discovering clinically valuable neoplastic progression biomarkers. This hypothesis will be developed by pursuing the following Aims: 1. To perform a genome-wide characterization of the epigenetic signature of IBD-associated neoplasias (IBDNs), focusing on known as well as novel CpG islands in the promoter or upstream portions of genes. A. Known methylation targets will be analyzed, including E-cadherin (CDH 1), p 16, p 15, p 14-ARF, death-associated protein kinase (DAPK), O6-methylguanine DNA methyltransferase (MGMT), human mutL homolog 1 (hMLH 1), adenomatous polyposis coli (APC), RASSF1A, deleted in colon carcinoma (DCC), and 14-3-3-sigma. B. Searches for novel targets of methylation in IBDNs will be performed using CpG island microarrays. 2. To comprehensively scan the genome for alterations in gene copy number at each stage of IBD-neoplasia. A. To probe cDNA microarrays with genomic DNA in order to identify specific genes involved by DNA amplification and deletion in IBDNs. 3. To perform global gene expression studies of IBDNs using cDNA microarrays, a. To produce cDNA microarrays and probe them with RNAs from IBDNs at all stages of neoplasia, b. To use hierarchical clustering, significance analysis of microarrays (SAM) and artificial neural networks (ANNs) to identify global expression patterns and specific genes at each stage of IBD-associated neoplasia. 4. To perform clinical correlations with molecular data. A. Bioinformatics algorithms will be used to define gene expression patterns associated with neoplastic progression in IBDN. B. Clinical parameters will be correlated with gene expression, methylation and copy number data to delineate specific genes potentially relevant to neoplastic progression in IBD.

Gastric cancer has been epidemiologically linked to infection with the bacterium Helicobacter pylori (HP). Mechanisms underlying this possible connection are unknown. In HP infection, chronic gastritis occurs, characterized by the activation of mucosal macrophages and other immune cells. In this inflamed tissue, resident and recruited immune cells may be activated directly by bacterial products or indirectly by intracellular signaling. With stimulation, nitric oxide (NO) can be abundantly produced by the inducible form of the enzyme NO synthase (iNOS) in many cell types, especially immune cells. Our preliminary data indicate that HP dramatically increases iNOS mRNA and NO production in macrophages. NO has been shown to possess mutagenic activity. We therefore hypothesize that one mechanism by which HP contributes to the development of gastric adenocarcinoma is the chronic upregulation of NO production in gastric mucosa. Our proposal will have two main Specific Aims: I) To discover mechanisms of HP-induced NO production in macrophages, and II) To demonstrate that HP-induced NO production contributes to gastric carcinogenesis. In order to accomplish Aim I, we will: A) prove that functional iNOS is directly induced by HP in macrophages by measuring I) production of nitrite, a stable metabolite of NO, 2) iNOS enzyme activity, 3) iNOS mRNA expression by Northern blotting, and 4) iNOS protein expression by Western blotting; B) identify and purify HP-derived factors mediating iNOS induction in macrophages; C) determine whether luminal HP stimulates gastric epithelial-to-macrophage signaling using 1) a co- culture model with HP-treated epithelial cells grown on semipermeable membrane supports above macrophages, and 2) HP-free conditioned media from HP-stimulated and control epithelial cells; and D) study the mechanism of HP-induced activation of iNOS in macrophages, specifically mechanisms of transcriptional regulation of iNOS. For Aim II, we will: A) demonstrate mutagenic effects of NO on gastric epithelial cells, specifically microsatellite instability and mutations in the gastric cancer suppressor genes p53 and APC; B) correlate gastric mucosal iNOS gene expression with HP status in gastric biopsies of patients; and C) correlate in vivo HP status and iNOS expression with gastric carcinogenesis. To accomplish this last sub-Aim, we will: l) correlate HP serology with malignant tissue mutational events in gastric cancer patients from countries with relatively high (S. Korea) and low (U.S.A.) HP rates; 2) ascertain HP serology and tissue HP status, iNOS expression, and mutational events at discrete stages of gastric carcinogenesis, including active superficial gastritis, chronic atrophic gastritis, intestinal metaplasia of the stomach, gastric dysplasia, and frank adenocarcinoma. If iNOS induction participates in the initiation or progression of gastric cancer, an understanding of the factors mediating induction of iNOS by HP should provide insight into possible novel targets of pharmacologic intervention or prevention in gastric carcinogenesis as well as potential novel diagnostic markers of gastric neoplastic progression.

DESCRIPTION: (Applicant's Description) Carcinogenesis is a complex genome-wide process of mutation and altered genetic programing that is incompletely understood at the molecular level. Global molecular biologic approaches are needed if progress is to be made in our understanding of detection and treatment of this deadly group of diseases. Microsatellite instability (MI) occurs early and often in human carcinoma development, and it affects the entire genome, often known as the replication error-positive (RER+) phenotype. How can MI contribute to carcinogenesis? One prevailing notion is that by occurring within the coding regions of genes, MI causes gene innovation or dysfunction leading to cancer. The first of such targets to be identified was the transforming growth factor between beta 1 type II receptor gene (TGF-1RII), which mutates frequently at an intragenic microsatellicate in sporadic RER+ colorectal, gastric other neoplasms. We demonstrated that an identical mechanism exits in ulcerative colitis-associated premalignant lesions in that the insulin-like growth factor II receptor (IGFIIR), E2F-4, and phosphatase and tension homolog located on chromosome ten (PTEN 1) genes also mutate during RER+ human tumorigenesis. These data, along with evidence of MI within other cancer-related genes such as the anti-apoptotic gene BAX and some DNA mismatch repair genes themselves, appear to represent the "tip of the iceberg" for this category of molecular alteration. Using these data as a launching point, we propose to embark on the task of identifying all MI occurring within coding portions of genes in one group of MI-prone cancers, colorectal carcinomas. We call this global profile the colorectal tumor instabilitome. Our aim is to deepen our understanding of RER+ colorectal carcinogenesis by identifying all genes within which microsatellite instability occurs in these tumors. We will utilize powerful computer-based algorithms to discover microsatellites within open reading frames and will test these microsatellite sites for instability in RER+ colorectal tumor specimens. Multiplex calorimetric-labeled semi-automated PCR coupled with simultaneous electrophoresis will allow concurrent evaluation of 10 or more coding region microsate loci per single gel lane, greatly lowering the amount of genomic DNA and time need per locus assayed. The detailed objectives of this proposal are to 1) map the entire colorectal cancer coding region "instabilitome," i.e., to discover all the coding region targets of MI in colon cancers; 2) develop robust automatable methods of "instabilotyping" that will be generalizable to other tumor types, stages, and small tumor specimens; and 3) make available to the general scientific public a comprehensive database of coding region microsatellite loci and mutation for use in other laboratories.

DESCRIPTION: IGFIR and IGFII are antiapoptotic, growth-stimulatory receptor-ligand pair that play an important role in human tumorigenesis. The hypothesis of this proposal is that at least a subset of gastrointestinal tumorigenesis relies on IGFIR signal transduction to prevent apoptosis, and that inhibition of IGFIR signaling will cause cancer cells to undergo apoptosis. In addition, the P.I. hypothesizes that IGFII-catabolizing protein, the IGFII receptor (IGFIIR), is a growth-suppressive component of the IGFIR signaling pathway whose inactivation advances tumor growth. Finally the P.I. states that primary tumors will exhibit reciprocal alterations in p53 and IGFIR or its pathway components, and that these alterations have an effect on the biologic aggressiveness of gastrointestinal tumors. The specific aims of the proposal are: To test agents that effectively and specifically block signaling through the IGFIR with the final goal to augment apoptosis induced by chemotherapeutic regimens. To determine the involvement of IGFIIR in the IGFIR growth axis both in vitro and in primary gastrointestinal tumors in vivo To correlate IGFIR and IGFIIR expression and function with p53 status and clinical outcome in primary human gastrointestinal tumors in vivo.

Adenocarcinoma of the esophagus and gastroesophageal junction is rising faster than any other cancer in the United States. In order to arrest this trend, advances in early detection must be made. We hypothesize that unique molecular alterations occurring in premalignant and malignant esophageal and gastric epithelia will constitute biomarkers for earlier diagnosis and improved prognostication to direct screening, prevention, and treatment efforts. The studies proposed herein will identify molecular signatures of early and late esophagogastric adenocarcinogenesis. Aim number 1: To generate pure cellular populations from premalignant and frankly malignant primary human gastroesophageal lesions. Number 1a: Xenografts will be generated in nude mice from human esophageal and gastric adenocarcinomas. Number 1b: Pure populations of metaplastic and dysplastic Barrett s esophagus, intestinal metaplasia of the stomach, and primary gastric and esophageal adenocarcinomas will be produced using laser capture microdissection (LCM). Aim number 2: We will discover peptide biomarkers by employing a novel peptide sequencing methodology which utilizes tandem mass spectrometry. Peptide sequences found to be differentially expressed in tumors will be subsequently explored for known homology in protein databases. Antibodies to known and novel potentially secreted or cancer-related proteins will be obtained or generated and used for immunohistochemical analyses, Western blots, and to probe primary tissue microarrays containing all neoplastic stages. Aim number 3: We will identify genes whose expression levels are increased or decreased at each stage of esophageal or gastric carcinogenesis. Number 3a: We will utilize serial analysis of gene expression (SAGE) to generate and contrast global gene expression profiles. Number 3b: We will derive transcriptomes of each stage of esophageal or gastric neoplasia using large, comprehensive screening arrays created on nylon membranes in pilot studies, and then we will create multiple small signature arrays on glass slides, based on peptide studies, SAGE, and large nylon array data. These smaller signature arrays will be tested on our large bank of tissues and prospectively on a larger population of patients possessing all stages of esophagogastric neoplastic progression. Aim number 4: To identify signature allelic loss patterns (allelotypes) at each stage of esophagogastric neoplastic progression using novel automated technology. Aim number 5: To determine the complete sets of coding region microsatellite mutations (instabilotypes) of early and advanced esophageal and gastric neoplastic lesions. Aim number 6: To determine the clinical significance of biomarkers by correlating molecular findings with corresponding initial and followup clinicopathologic data, using appropriate statistical techniques.

[unreadable] DESCRIPTION (provided by applicant): Barrett's esophagus is a highly premalignant disease of unknown prevalence, but it predisposes to the development of esophageal adenocarcinoma, which is increasing at alarming rates in Western countries. The molecular genetics of esophageal adenocarcinoma and its precursor lesion, Barrett's esophagus, has been studied intensively in recent years. However, a better knowledge of the molecular alterations occurring in this setting will yield several benefits. Firstly, the discovery of novel molecular alterations will yield clues to biological pathways underlying Barrett's-associated neoplastic transformation, and these clues may lead to better in vitro and in vivo models of this disease. Secondly, molecular alterations themselves can be used as markers of early detection, disease progression, or ultimate prognosis in patients with Barrett's or cancer. Thirdly, these molecular alterations can be pursued as possible therapeutic targets for intervention, in both the prevention and treatment of this disease. The Aims of the current proposal will be to discover novel molecular alterations in Barrett's metaplasia and neoplasia, and to concentrate on the second of these benefits, i.e., to perform translational research to determine the potential value of these alterations as markers of disease progression. By using the same cDNA microarray platform to determine changes in DNA copy number, methylation status, and gene expression level, we will facilitate the translation of molecular genetic data from the genomic, to the epigenetic, to the transcriptomic, and finally to the protein (biomarker) level. This final level will employ tissue microarrays to test and validate specific candidate genes derived from the first three levels of study. These goals will be implemented by pursuing the following Aims: [unreadable] [unreadable] Aim 1. To perform global exploration for changes in DNA copy number in the Barrett's metaplasia-dysplasia-adenocarcinoma sequence (Barrett's neoplasia), using cDNA microarray-based comparative genomic hybridization (microarray-CGH). a) Global patterns of DNA amplification and deletion will be identified by microarray-CGH and characterized at each stage of Barrett's neoplasia, using hierarchical clustering, significance analysis of microarrays (SAM), and artificial neural networks, b) Specific cDNAs showing the most consistent and/or marked alterations in DNA copy number will be identified, characterized and validated using quantitative real-time PCR, for further study in Aim 4. [unreadable] [unreadable] Aim 2. To perform global epigenetic profiling at various stages in Barrett's neoplasia, using methylation-specific oligonucleotide microarrays. The genome will be screened for novel targets of DNA hypermethylation in various stages of Barrett's neoplasia, using methylation-specific oligonucleotide microarrays. [unreadable] [unreadable] Aim 3. To Using ANNs, to perform analyses of global gene expression data in Barrett's neoplasia. Results of genomic studies in Aims 1 and 2 will be correlated with global expression data in order to identify the genes most significantly different at both the genomic and transcriptomic levels at each stage of Barrett's neoplasia, for further study in Aim 4. [unreadable] [unreadable] Aim 4. Using tissue microarrays, to evaluate and validate potential biomarkers at the protein level in Barrett's neoplasia. Potential biomarkers identified in Aims 1-3 will be studied individually for expression at the protein level in all stages of Barrett's neoplasia, using tissue microarrays. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The molecular pathology of gastric carcinoma, the second most common cancer in the world, and of its precancerous precursor lesions has become much better elucidated in recent years, but much remains to be discovered. For example, tumor suppressor gene mutation, DNA hypermethylation, microsatellite instability (MSI), and altered gene expression have been shown to occur frequently during the origin and progression of this cancer. However, a greater understanding of these mechanisms and their timing in gastric carcinogenesis could lead to the ultimate development of early detection and staging biomarkers, as well as novel therapeutic approaches to this deadly disease. Therefore, in order to deepen our understanding of molecular mechanisms underlying gastric carcinogenesis, we will pursue the following Specific Aims: 1) To compare and contrast the global gene expression patterns of various subtypes and premalignant stages of gastric cancer, using cDNA microarrays and bioinformatics algorithms. 1a) cDNA microarrays will be produced and hybridized to amplified RNA from various histologic subtypes and premalignant stage precursor lesions of gastric cancer. 1b) The bioinformatics programs Cluster/Treeview, significance analysis of microarrays (SAM), artificial neural networks (ANNs), and support vector machines (SVMs) will be used to derive global gene expression patterns for each subtype of gastric cancer or precursor lesion. During this process, we will also identify genes that are significantly differentially expressed among the various subgroups. 2) To perform a limited methylotyping assay to ascertain epigenetic signatures of different types of gastric cancer and premalignant lesions, focusing on known as well as novel CpG islands in the promoter or upstream regions of genes. 2a) Known methylation targets will be analyzed, including E-cadherin (CDH 1), p 16, p 15, p 14-ARF, death-associated protein kinase (DAPK), O6-methylguanine DNA methyltransferase (MGMT), human mutL homolog 1 (hMLH1), adenomatous polyposis coli (APC), RASSFIA, deleted in colon carcinoma (DCC), and 14-3-3-sigma. 2b) Novel methylation target searches will be performed using CpG island microarrays, with alternative backup approaches to include methylation-sensitive genomic DNA digestion followed by arbitrarily primed PCR (AP-PCR), as well as single-gene MSP. 3) To correlate the results of, aims (1) and (2) with each other as well as with other clinical and biologic parameters of these gastric lesions.

DESCRIPTION (provided by applicant): Microsatellite instability (MSI), caused by defective DNA mismatch repair, occurs frequently in colorectal carcinomas (8, 9). MSI is categorized as high (MSI-H), low, (MSI-L), or negative (MSS, or microsatellite-stable), according to the frequency of microsatellite alterations at anonymous (non-coding) loci (10). There is strong evidence that tumors with high-frequency MSI (MSI-H tumors) have significant differences in their clinical behavior that distinguish them from MSS and MSI-L tumors (11, 12). In addition, evidence is accumulating that tumors with low-frequency MSI (MSI-L) tumors have unique features (13). Nevertheless, our understanding of both MSI-H and MSI-L tumors remains incomplete, and the existence of MSI-L tumors as a distinct subgroup has been questioned. Hypothesis: MSI-H, MSIL, and MSS gastrointestinal tumors are phenotypically unique. These distinct biologies can be better defined and understood through comprehensive genomic approaches, including instabilotyping and microarray-based bioinformatics. Moreover, valuable insights into molecular pathways underlying these entities can be gained by identifying and further studying candidate genes. This hypothesis will be explored with the following Specific Aims: 1) To broaden and extend unbiased instabilotyping of MSI-H colorectal cancers and cell lines, in order to identify additional genes targeted by frequent frameshift mutation; 2) To examine functional consequences of mutations in selected coding region targets of microsatellite instability; 2.a To demonstrate biallelic inactivation of genes showing frequent frameshift mutation by analyzing for loss of heterozygosity, point mutation, and altered expression; 2.b To determine functional differences between WT and mutant candidate proteins. To ascertain the effect(s) of mutant proteins on cell biology and behavior by transfecting WT candidate genes into biallelically mutated cells and ascertaining effect(s) on cell biology; 2.b.i To assess cell proliferation, anchorage-independent growth, invasion (Matrigel assay), mobility (wound assay), apoptosis (TUNEL, DAPI staining and caspase assays), and differentiation (morphology); 2.b.ii To evaluate the effects of WT transfected ACTR2 and other candidate genes on protein expression and signal transduction, including phosphorylated and total SMAD2, total SMAD4, caspase 1, and TTK; 2.b.ii) Using cDNA microarrays, to compare colon cancer cells before and after transfection with WT ACTR2, TTK, HDCMA18, CASP 1, and other as-yet unidentified genes containing frequently mutated microsatellites; 3 To increase our understanding of the biologies of MSI-H, MSI-L, and MSS colorectal carcinomas by comparing the transcriptomes of these cancers, using cDNA microarrays and bioinformatics strategies; 3.a To generate global gene expression data from MSI-H, MSI-L, and MSS status colorectal tumors; 3.a.i To produce and probe cDNA microarrays with RNAs extracted from MSI-H and MSS cells. 3.a.ii To hybridize microarrays to MSI-H, MSI-L, and MSS primary colorectal tumors; 3.b To determine whether MSI-L tumors comprise a biologically distinct subgroup; 3.b.i To apply bioinformatics strategies, including hierarchical clustering, significance analysis of microarrays (SAM) and principal components analysis (PCA) in order to confirm the existence and provide clues to the biology of a distinct MSI-L tumor subgroup; 3.c To identify genes defining molecular genetic pathways underlying MSI-H, MSS, and MSI-L tumors; and 3.c.i To utilize PCA components to find genes segregating with MSI status, and SAM to identify genes significantly differentially expressed among these three groups.

DESCRIPTION (provided by applicant): Esophageal and gastric carcinomas are among the top ten causes of cancer death worldwide. These diseases are usually detected at advanced stages, when available treatments are not very effective. Earlier detection of these cancers has been shown to make a significant impact on patient outcome. Our preliminary studies have discovered biomarkers that can diagnose synchronous cancer from normal or premalignant tissues. We will extend these studies to confirm that these same biomarkers can predict future (metachronous) esophagogastric cancer risk. This application will have as its ultimate goal the translation of these early detection biomarkers into clinical validation within 5 years. Aim 1. In pilot cohort Phase I discovery studies using cDNA microarrays, to develop biomarkers distinguishing between normal or metaplastic tissues of patients with vs. without cancer. Aim 1.a. In a cohort of 20 patients without Barrett's or cancer, 20 with Barrett's only, and 20 with Barrett's adenocarcinoma, using the shrunken nearest centroids predictor (SNCP) model, to identify gene expression panels and individual gene biomarkers to distinguish between patients with and without esophageal cancer. Aim 1.b. In 20 patients without and 20 with gastric cancer, using the SNCP model, to identify gene expression panels and individual gene biomarkers distinguishing between patients with and without gastric cancer. Aim 1.c. To determine the positive predictive value (PPV) of all potential biomarkers identified in Aims 1.a. and 1.b. Aim 2. In a pilot cohort Phase II validation study, to confirm expression panels and individual genes identified in Aim 1 with quantitative RT-PCR (Q-PCR) using a capillary microfluidics station-based method. Aim 2.a. To perform analytical validation of Q-PCR by checking its sensitivity in detecting a positive signal, its dynamic range, and its reproducibility in repeat analyses. Aim 2.b. To perform microfluidics measurement of gene expression levels from paraffin-derived esophageal tissues matched to the 60 frozen specimens studied in Aim 1.a. Aim 2.c. To perform microfluidics measurements of gene expression levels from paraffin-derived gastric tissues matched to the 60 frozen specimens studied in Aim 1.b. Aim 3. In larger cohorts, to perform Phase III cross-sectional retrospective longitudinal validation studies using the QPCR method analytically validated in Aim 2. Aim 3.a. To perform a Phase III study (n=400) of 200 esophageal cancer and 200 Barrett's patients, including 127 patients from whom tissues were obtained prior to a cancer diagnosis. Aim 3.b. To perform a Phase III study (n=200) of 100 gastric cancer and 100 noncancer patients. Aim 3.c. To perform statistical analyses of data from Aims 3.a. and 3.b. in order to determine their validity and significance.

DESCRIPTION (provided by applicant): R21Phase Our preliminary data indicate that freely circulating methylated DNA can be found in the plasma of patients with esophageal cancer. Specifically, these data show that methylated alleles of the APC, HPP1 and p16 genes can be detected in the bloodstream of esophageal cancer patients. Furthermore, our preliminary data show decreased survival in patients with high plasma levels of methylated APC DNA, which also parallel relapse of malignancy. Thus, these circulating methylated nucleic acids show promise as biomarkers for the prognostication and monitoring of esophageal cancer patients. The overall scope of this proposed project will be to move these circulating biomarkers from the laboratory into the clinic. In the initial R21 phase, precise assays of gene-specific methylated plasma DNA will be developed and refined using quantitative real-time methylation-specific PCR, with the established methylation targets p16 and APC serving as templates. In addition, during this exploratory R21 phase, additional novel methylation targets will be identified in neoplastic esophageal tissues and plasma. In the second, R33 phase, novel methylation targets identified in the R21 phase will be clinically validated on a larger, independent cohort by performing clinical correlations with tissue and plasma methylation levels. Aim 1. To develop and validate accurate, robust, standardizable, and scalable quantitative real-time plasma DNA methylation assays using the established tissue and plasma methylation targets p16 and APC. Assays will be analytically validated with known standards of plasma from normal subjects spiked with known levels of genomic DNA, as well as known but blinded positive and negative patient blood samples. Aim 2. To identify and measure 20 novel methylation events in 50 primary esophageal carcinomas and 50 normal esophageal tissues. A panel of candidate genes with known cancer and outcome relevance and reported frequent methylation in gastrointestinal and other human tumors will be evaluated using real-time quantitative methylation specific PCR (MSP). Genes methylated in at least 20% of tumors, but in less than 5% of normal specimens, will be further pursued in Aim 3. Aim 3. To study the same pilot group of 50 patients for plasma methylation of target genes identified in tissues during Aim 2. Genes methylated in the plasma of greater than 10% of these patients will constitute plasma targets for clinical validation during the R33 phase of the current proposal.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The goal of the UMB GI Research Training Program is to prepare pre- and post-doctoral fellows for academic careers in Gastroenterology by offering an integrated, interdisciplinary curriculum that emphasizes cancer genetics and genomics, mucosal biology and immunology, enteric pathogens and vaccine development, and epidemiology and preventive medicine research. Pre-doctoral trainees obtain Ph.D. or combined M.D./Ph.D. degrees in Molecular and Cell Biology, Pathology, or Microbiology/Immunology, whereas postdoctoral trainees earn a Master of Science degree in Epidemiology and Preventive Medicine, Clinical Research Track. The proposed training will consist of four programs: Cancer Genetics and Genomics, Mucosal Immunology and Microbiology, Epithelial Cell Biology, and Clinical Research Training in Epidemiology and Preventive Medicine. Each program is directed by a productive investigator and consists of expert, experienced mentors who interact both within and among these programs. Our training program also benefits from strong representation of underrepresented minority groups, particularly African-Americans, and by a high proportion of female trainees. Departments represented in this highly interdisciplinary program include Medicine, Pediatrics, Surgery, Pathology, Microbiology and Immunology, Biochemistry and Molecular Biology, Epidemiology and Preventive Medicine, the Center for Vaccine Development in the Medical School, and the Department of Oral Pathology in the Dental School. As evinced in the accompanying application, this program has been strengthened by the recruitment and retention of a number of talented, well-funded faculty members. Numerous mentees trained by our faculty have proceeded to obtain academic positions, earn peer-reviewed extramural funding, and publish important research papers. Although our faculty already have a strong track record in training GI researchers, there is a need at UMB for a formalized, integrated GI training structure such as that afforded by the NIDDK T32 granting mechanism. Organization of this interdisciplinary program under one umbrella will afford the advantages of a secure source of funding support, close monitoring of training progress, additional impetus for training in GI research, and the attraction of strong training candidates. We therefore submit this application to address these goals and to impact upon the shortage of physician-scientists and other researchers in the field of Gastroenterology [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Patients with ulcerative colitis (UC) are at increased risk of developing colorectal cancer. A more complete understanding of the molecular basis of UC-cancers and their precursor dysplastic lesions will result in several important benefits. Specifically, novel molecular alterations will provide clues to pathways underlying UC-associated neoplastic transformation, leading to better disease models. These events may evolve into therapeutic targets for both the prevention and treatment of this sequela. Recent technical and scientific advances, particularly explosive growth in the field of microRNAs (miRs), now enable us to delve more deeply and broadly than ever previously possible into the molecular underpinnings of UCN. By leveraging these advances, we can now evaluate the involvement of miRs in UC-associated inflamed, dysplastic, and cancerous lesions by discovering unique alterations in the expression of miRs, defining their functional impact both in vitro and in vivo, and defining pathways by which their dysregulation may be carcinogenic. Hypothesis: We hypothesize that miR-dysregulation is involved in UC-associated neoplastic progression. To prove this hypothesis, we will pursue the following Specific Aims: 1) To identify tumor-suppressive miRs (ts-miRs) and oncogenic miRs (oncomiRs) that are involved in UCN. 1a) To identify miRs that are dysregulated at each UC- neoplastic stage using miR microarray-based comparisons of non-neoplastic mucosae from non-UC controls vs. UC-associated non-neoplastic mucosa, dysplasia, and carcinoma. 1b) To confirm dysregulation and epithelial cell localization of prioritized significantly upregulated and downregulated miRs at each UC- neoplastic stage in Aim 1a, using qRT-PCR in a larger sample cohort and in situ hybridization assays. 2) To determine the biologic impacts of prioritized candidate ts-miRs and oncomiRs in UC-associated neoplastic progression in vitro and in vivo. 2a) To test the biologic impacts of prioritized dysregulated miRs in vitro by transfecting either miR-mimics (for ts-miRs) or antagomiRs (for oncomiRs) into UCN-derived cell lines, followed by growth, proliferation, cell cycle, and apoptosis assays. 2b) To test the biologic effects of in vitro effective miRs (Aim 2a) in vivo by transfecting miR-mimics or antagomiRs into UCN cells and implanting the cells in nude mice. 3) Using a two-pronged approach, to discover and investigate pathways involving UCN- miRs and their putative cognate UCN-gene transcripts. 3a) Starting from candidate miRs, to discover their target gene transcripts by performing mass spectrometric screening of iTRAQ-labeled proteins extracted from UCN cells that have been transfected with candidate miR-mimics or antagomiRs. 3b) Starting from previously established UCN-related gene transcripts, to document binding of their 3'-UTRs to putative cognate in silico- selected miRs that are also dysregulated in UCNs, using luciferase expression vectors and Western blotting. PUBLIC HEALTH RELEVANCE: The involvement of a unique set of microRNAs (miRs) in the development of ulcerative colitis-associated neoplastic lesions (UCNs) will be investigated. MiR microarray and quantitative reverse-transcriptase PCR (qRT-PCR) assays will establish miR dysregulation. In vitro and in vivo studies will be performed to determine the carcinogenic biologic effects of miRs dysregulated in UCNs, and in silico and in vitro methods will be used to show which messenger RNAs are targets of selected UCN-dysregulated miRs. Ultimately, the discovery and study of these carcinogenic mechanisms will establish a foundation for the future use of miR agonists and antagonists in the prevention and treatment of this disease.

DESCRIPTION (provided by applicant): Barrett's esophagus (BE), a sequela of chronic gastroesophageal reflux disease (GERD), is a premalignant condition that increases an individual's chance of developing esophageal adenocarcinoma (EAC) by 30-125- fold. The precise prevalence of BE among patients with GERD is unknown but has been estimated at 1-10% of the general population (2). EAC is one of the most rapidly increasing cancers in the United States. Therefore, subjects with BE are enrolled in surveillance programs in which they undergo endoscopy at regular intervals for the rest of their lives. Due to frequent endoscopic surveillance, BE has become, by default, a de facto human model of early human preneoplastic events. Unlike colorectal adenomas, the premalignant lesions at the other end of the GI tract, the at-risk organ is left in place for repeat serial observations, often for 30 or 40 years. This BE model lends itself quite readily to molecular genetic studies in which tissue is the issue. In human diseased tissue-based studies, there is no problem with clinical relevance, and one doesn't need to worry about being led down the (proverbial) garden path by the sometimes irrelevant findings (traps) that often crop up in nonhuman or in vitro models of human disease. Methylation has been reported in many human malignancies and premalignant syndromes, but was first reported in BE and EAC 11 years ago. Tumor suppressor genes affected by hypermethylation at various stages of BN include p16, p14, E-cadherin, APC, and others. However, these reports have all been candidate gene studies, based on the usual suspects, typically focusing on the tumor suppressor gene of the month. Our Preliminary Data suggest that an unbiased, epigenome-wide approach to this aspect of BN molecular genetics is likely to shift the paradigm in several ways: 1) the predominant epigenomic change in progression appears to be hypomethylation, rather than hypermethylation, implying the activation or unmasking of growth-stimulatory genes; 2) some genes change their methylation levels late during the run-up to progression, while others change earlier; this finding implies that by using arrays, we can a) for the future, find better early predictive biomarkers of progression; b) for the current project, dissect out the temporal epigenomic program of Barrett's neoplastic development. Hypothesis: The global methylation profile of Barrett's esophagus is in a constant state of flux and changes continuously as Barrett's evolves from early pre-progression, to later pre-progression, to LGD, to HGD, and finally to EAC. Changes that occur in this profile reflect changes in biology that cause or result from this process of preneoplastic and neoplastic evolution. By comprehensively harvesting genes that are epigenetically altered at different timepoints prior to and during progression, then feeding them into gene ontology programs and databases, we will gain novel insights into the cellular and biochemical pathways that become activated (or, in the case of hypermethylation, inactivated) as Barrett's pre-progression and its later neoplastic stages proceed.

DESCRIPTION (provided by applicant): Patients with Barrett's esophagus (BE) are at increased risk of developing esophageal adenocarcinoma (EAC), one of the most rapidly increasing cancers in developed nations. The molecular genetics underlying BE- associated neoplastic progression (BEAN) remain unclear, and a more thorough understanding of them would yield several benefits. These include: 1) clues to biological pathways underlying BEAN;2) useful biomarkers of early cancer detection, disease progression, or ultimate prognosis;and 3) therapeutic targets to intervene in the prevention treatment of this process. Small noncoding RNA species known as microRNAs (miRs) are involved in many human cancers, and miR-modulated translational regulation is an important gene-regulatory mechanism to consider along with transcriptional control of mRNA expression. Thus, miR expression analyses will provide biologic and clinical insights into BEAN. In addition, miRs themselves may eventually lead to targeted molecular therapies. We will evaluate the involvement of miRs in BE-associated metaplastic, dysplastic, and cancerous lesions by discovering unique alterations in the expression of miRs and by defining their biologic impact in vitro and in vivo. Hypothesis: We hypothesize that a unique set of miRs is involved in BEAN. To prove this hypothesis, we will compare miR expression levels at all stages of BE-associated metaplasia, dysplasia, and adenocarcinoma as well as in normal squamous esophagus. In addition, we will explore functional pathways by which these miRs are regulated and exert effects in BEAN. To achieve these broader goals, we will pursue the following Specific Aims: 1) To identify BEAN-specific tumor-suppressive miRs (ts-miRs) and oncogenic miRs (oncomiRs). 1a) To perform miR microarray-based comparisons of NE vs. BE vs. LGD vs. HGD vs. EAC to identify miRs that are differentially expressed at each preneoplastic transition. 1b) To confirm dysregulation of miRs identified by microarrays in Aim 1a, using miR RT-PCR. 1c) To evaluate potential mechanisms underlying dysregulation of miRs confirmed in Aim 1b, including DNA amplification and promoter methylation of miR mother genes. 2) To determine the biologic impact of key miRs on BE-associated neoplastic progression. 2a) To test the biologic effects of miRs -25, -93, -106b, - 100, -125b, and -205 in vitro by transfecting miR-mimics and antagomiRs into BEAN-derived cell lines, followed by proliferation, cell cycle, and apoptosis assays. 2b) To test the biologic effects of miRs -25, -93, - 106b, -100, -125b, and -205 in vivo by transfecting miR-mimics and antagomiRs into BEAN-derived cells and implanting the cells into nude mice. 3) Using complementary approaches, to explore interactions between key miRs and their target gene transcripts. 3a) To identify target gene transcripts of miRs -25, -93, -106b, - 100, -125b, and -205 by combining in-silico database searches, mRNA array data, and iTRAQ data. 3b) To study BEAN-miR target gene transcripts identified in Aim 3a, including p21 and Bim, using luciferase expression vectors containing the 3'-UTRs of these miR target mRNAs. PUBLIC HEALTH RELEVANCE: We will evaluate the involvement of miRs in BE-associated metaplastic, dysplastic, and cancerous adjacent transitions by discovering unique alterations in the expression of miRs and defining their functional impact in vitro and in vivo. In this fashion, we will gain comprehensive insights into the molecular basis of BEAN, while simultaneously establishing a foundation for future potential predictive and diagnostic assays and therapeutic intervention strategies.

DESCRIPTION (provided by applicant): The care of patients with inflammatory bowel disease (IBD) is complex. One crucial clinical dilemma is the management of the increased risk for colon cancer. Unfortunately, current knowledge does not allow for accurate determination of neoplastic risk and the best tailored management. This challenge is due, in part, to the lack of genetic information regarding somatic mutations occurring in colon cancer arising in IBD patients. Furthermore, the order of mutations and the significance of sequential mutations in regards to the course of neoplastic transformation are currently unexplored. We hypothesize that a comprehensive genetic survey of human IBD-associated neoplasia (IBDN), with detailed mathematical mapping and testing at discrete developmental/temporal stages, can be used to identify genetic alterations that drive neoplastic progression in IBD. We further hypothesize that by performing longitudinal analyses of genetic alterations in our IBDN porcine model, we will identify characteristic genetic oncogenic trajectories that drive cancer progression. Iterative biopsy specimens from neoplasia, as it develops and evolves within each animal, will be obtained via serial colonoscopic sampling, and genetic analyses will be performed. By integrating these human and animal datasets, we will learn which molecular events drive and/or predict the progression of early lesions to more advanced malignant disease in IBD. Throughout this grant, we will characterize small-scale exome alterations (point mutations, indels) and copy number alterations, using both whole-exome sequencing (WES) and SNP arrays. We will investigate these hypotheses by pursuing the following Specific Aims: Aim 1 - To identify and order genetic alterations in human IBD-associated neoplastic progression. A) Perform exome sequencing and SNP-array assays on a cross-sectional cohort of 100 IBD-Ca. B) Using a combination of algorithms, identify the most likely driver alterations and infer oncogenic trajectories. C) Test oncogenic trajectories by Sanger-sequencing and SNP-arraying 30 LGD, 30 HGD, & 30 IBD-Ca. Aim 2 - To characterize the temporal order and functional impact of genetic alterations in colon cancer arising in a porcine IBDN model. A) To determine the phenotypic impact of oncogenic trajectories determined in Aim 1. B) To identify temporal profiles of genetic alterations in longitudinal biopsies and compare to ordering of alterations in human IBDN. In toto, this novel integrated strategy is likely to provide insight into early and predictiv molecular events, since we will be able to temporally map these events in exquisite detail, as well as to catch them in the act as soon as they occur. Ultimately, the successful completion of this project will translate in better tailoring of curative and preventative treatments for IBD patients.

Esophageal squamous cell carcinoma (ESCC) ranks sixth among all cancers worldwide, with 450,000 new cases diagnosed per year and a very poor prognosis. Low-cost, minimally invasive point-of-care population screening for ESCC is badly needed, particularly in LMICs, where 5-year ESCC survival is less than 10%. Altered methylation occurs frequently in human malignancies, including EC, constituting an early event that can serve as an early cancer detection biomarker. However, for DNA methylation to be used in this manner, we need cost-effective, user-friendly and robust tests that permit clinical translation in LMICs. We propose an early ESCC diagnostic strategy comprising a single-use, swallowable sponge to collect esophageal specimens coupled with a smartphone-manipulated microfluidic chip for automated sample processing and methylation detection. This strategy does not require endoscopy (EGD), can be administered by healthcare workers without medical degrees, and uses an on-phone analytic app. The sponge is cheaper (approximately $30 each), less invasive, and easier to perform than EGD ($1500 total cost, including facility fees); there are no room charges, unlike EGD. The microchip integrates DNA extraction, bisulfite DNA conversion, and methylation analysis into a single device. In addition, the microchip interfaces with a cellphone, for both device control and methylation detection and analysis. The integrated device enables detection of DNA methylation from crude samples in a ?sample-to-answer? manner, without the need for sending data back to an analytic center off-site. Thus, the proposed platform promises a cost-effective and user-friendly POC strategy for early ESCC detection that is implementable in LMICs. We have also assembled a talented interdisciplinary, intercontinental team to execute this strategy. Our task will be achieved in 2 phases via the following Aims: UG3 PHASE: Aim 1: To assess a streamlined protocol for sample collection, processing and methylation detection. Aim 2: To implement DNA sample processing and methylation detection into a mobile phone- manipulated microfluidic chip system. Aim 3: To test a prototype ESCC diagnostic strategy integrating the DNA methylation detection system with the swallowable sponge for sample collection. UH3 PHASE: Aim 1: To improve the cost-effectiveness and robustness of the methylation diagnostic system for use in LMICs. Aim 2: To develop a method for ambient chip storage and perform on-chip QC tests to verify assay functionality. Aim 3: To conduct an ESCC diagnostic trial in Uganda using our point-of-care strategy.

Barrett?s esophagus (BE) is the dangerous obligate premalignant precursor lesion of esophageal adenocarcinoma (EAC), one of the most rapidly increasing and lethal malignancies in the United States and Europe(3). EAC is rarely detected before it becomes invasive and untreatable, and 95% of EACs develop in patients not previously diagnosed with BE(19)(20). Patients diagnosed with BE, in contrast, have an excellent prognosis, since neoplasia is detected very early by periodic endoscopic surveillance. There is, however, no currently available screening test for BE. Clinical translation of minimally invasive, low-cost biomarker-based approaches to BE diagnosis will improve early EAC detection and increase overall survival. By combining our swallowable, retrievable esophageal sample collection sponge (EsophaCapTM) manufactured by our industrial partner, Capnostics, with our patented BE DNA methylation markers and our enhanced processing technique, Methylation-On-Beads (MOB) to maximize extraction and bisulfite conversion of DNA; and by establishing this assay in the CLIA-compliant laboratory of our industrial partner, MyGenetx, we can now make this assay widely available. Preliminary data demonstrate high diagnostic accuracy of our sponge-based biomarker test for BE. We will apply this strategy by pursuing the following Specific Aims: Aim 1. To analytically validate our EsophaCapTM-based BE diagnostic assay. Aim 1a. First, using technical replicate EsophaCapTM specimens from 42 newly recruited BE patients and 42 non-BE controls, we will confirm the accuracy, robustness, and reliability of our BE diagnostic assay. Preliminary results in this context are also encouraging (see Table 2, Technical replicates). Aim 1b. Next, in these same 42 patients with known BE vs. 42 controls without BE, we will establish analytical concordance of EsophaCapTM-based data with matched tissue biopsy sample data. Aim 2. To conduct a pilot study to validate a multivariate model for EsophaCapTM-based diagnosis of BE. Based on our already-collected EsophaCapTM-derived specimen training dataset (see Fig. 5), we have constructed a 3-marker prediction model for BE (Fig. 6). We will apply this model and the chosen cut- off threshold to a newly collected set of 50 untested samples (independent of the Aim 1 samples). Aim 3. To prospectively test the combined sponge-methylation biomarker strategy in a test set cohort of BE patients vs. controls. Our assay will be performed in EsophaCapTM-derived samples from a prospectively- collected cohort of 80 BE patients and 240 controls. The multivariate model validated in Aim 2 will be applied to this test set of large patient population. Aim 4. To industrialize our EsophaCapTM assay protocol. In parallel and simultaneously with Aims 1-3, we will establish all steps in our MOB-based DNA extraction, bisulfite modification and quantitative methylation-specific PCR (qMSP) protocol in the CLIA-compliant laboratory at MyGenetx. Assays performed in Aim 3 will be repeated by obtaining repeat sponges from the same patients, this time in the CLIA lab, and checked for accuracy by comparison to Aim 3 results.