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R21 CA092739-01A1 2002 BAKER, LAURENCE HOWARD UNIVERSITY OF MICHIGAN AT ANN ARBOR Molecular Diagnosis of Sarcomas
We will elaborate serum quantitative-PCR (QPCR) and peripheral blood RT-QPCR tests to diagnose the presence of sarcomas. We aim to construct simple blood tests to screen people at increased risk for sarcoma, to evaluate the initial tumor, to assess therapy of patients with sarcoma and/or to follow people who have been treated for sarcoma. Sarcomas were chosen initially because we found appreciable amounts of lysed tumor cell DNA in the serum. This enabled us to propose a generic test based on quantitative differences of tumor suppressors and oncogenes in sarcoma. Subsequently, more specific tests have become available involving aberration of the c-kit gene in GI stromal tumors and specific translocations that characterize defined sarcoma types. We add these more specific tests to our previous application that was based on the generic tests only. 1. Test the primary sarcoma to assay the QPCR markers that show quantitative changes and the RT-QPCR markers that show specific translocations. 2. Monitor the serum simultaneously to determine the QPCR anomalies therein and/or the peripheral blood to screen for specific RT-QPCR markers of translocation. The usefulness of these determinations is supported by our preliminary results demonstrating the presence of QPCR serum anomalies in untreated sarcoma patients. 3. Monitor the serum and peripheral blood longitudinally to screen for these molecular anomalies and (if necessary) new anomalies to detect metastases and/or recurrence that might not be apparent clinically. If we detect useful serum QPCR anomalies and/or peripheral blood anomalies in particular situations in phase I, we will expand the studies in phase II to perform longitudinal QPCR assays useful in a given situation. We will use the test to perform diagnosis of at-risk and treated populations, to monitor cancer cell lysis before and during treatment, to monitor the efficacy of cancer treatments and to detect recurrences. This is proposed as an R21 (one year)/R33 (three year) grant to determine the feasibility of these studies for sarcomas in the R21 phase. If more studies are indicated, the R33 phase would support the longer-term longitudinal studies required to demonstrate how best to use this test to follow the course of sarcomas.
 
R21 CA086330-01A2 2002 BEITZ, ALVIN JAMES UNIVERSITY OF MINNESOTA TWIN CITIES In Vivo Analysis of Tumor Peptide Secretion
The interactions between a tumor and its local environment are critical to tumor development and progression, and ultimately to tumor metastasis. Most of our current knowledge of the signaling molecules secreted by tumor cells is based on in vitro studies that have largely ignored the dynamics of the in vivo tumor environment. The overall goal of this proposal is to study the dynamics of signaling peptide and protein secretion by several tumor types (fibrosarcoma, breast, prostate and myeloma) implanted in bone. In the R21 portion of the application we propose to combine a novel in vivo microperfusion procedure, which allows sampling of extracellular fluid from solid tumors over time, with new mass spectrometry methods allowing identification of proteins in mixtures. The R21 phase will be used to further develop MALDI-TOF, ESI-Q-TOF and ESI-MS/MS procedures and to optimize conditions for maximum detection and for identification of proteins and peptides in bone tumor microperfusates. The interactions of various tumor types with the unique micro-environment found in bone will be studied in the proposed R33 phase by analyzing the secretion of selected as well as unidentified proteins produced by breast, prostate and myeloma tumors over time. By permitting the in vivo identification of an array of peptides and proteins present in tumor extracellular fluid over time, this novel microperfusion approach will provide the opportunity to characterize the extracellular peptides and proteins secreted by different bone tumor types which are involved in the growth, progression, latency and metastasis of these tumors. The data obtained from these studies will facilitate cancer detection and diagnosis as well as define new targets or therapeutic and preventative agents.
 
R21 CA094392-01A1 2002 CHUONG, CHENG-MING UNIVERSITY OF SOUTHERN CALIFORNIA High Resolution Microarray Analysis of Cancer
The goal of cancer genomics is to determine the gene expression profile of normal, precancerous, and cancerous cells, with the hope of improving detection, diagnosis, and treatment for patients. The completion of the human genome project has accelerated this progression, and gene microarrays have set the stage for genomic pathology analysis. However, current technology to obtain RNA from specimens is inefficient and requires at least 2 million cells. This is especially problematic with tumors where little starting material is available, or where tumors are heterogeneous and require microdissection or similar methods that drastically limit the amount of starting material. To this end, invention of aRNA (amplified antisense) was a major step ahead but is not ideal for serial amplification or preparation for microarray analysis from a scant amount of sample. Based on the recently developed RNA-PCR Technology, we propose a format that can amplify mRNA from less than 100 cells. We postulate that further development and validation of this technology will lead to a routine procedure to obtain gene expression profiles (GEPs) from just a few or even single cells. In the R21 phase, we will use an NCI RNA standard and cultured sarcoma cell lines to compare amplified and non-amplified RNA with rigorous statistical analyses using Genetrix, a software tool developed here to analyze microarray data. Conditions will be optimized and worked out for pathological specimens. When the fidelity and efficacy of RNA-PCR amplification are validated, we will progress to the R33 phase to field test in cancer using childhood sarcoma as a model. We hope to achieve high resolution GEP using Laser Capture Micro-dissection microscopy to dissect histopathology sections. We will verify whether GEPs from a few cells are as predictive of biologic behavior as conventional GEPs, using the same clinical material used in Triche?s Director?s Challenge grant. We will also apply microarray analysis to skinny needle biopsies and cytological preparations, which were previously not possible. Bioinformatic analysis with Genetrix, which allows correlation of expression on a gene-by-gene basis with patients? clinical data (e.g., age, sex, pathological diagnosis, therapeutic protocol, etc.), may identify new markers of high predictive value that are obscured due to tissue heterogeneity, or unavailable due to limited amount of materials (e.g., those in tissue banks). This protocol is simple, fast, and reliable and uses minimal samples widely obtainable. The method can be applied to all cancers.
 
R21 CA097511-01 2002 LEVENSON, VICTOR V NORTHWESTERN UNIVERSITY AT CHICAGO DNA Methylation in Patients with Non-Hodgkin's Lymphoma
Technological advances of molecular biology are posed to bring new methods of diagnosis and treatment to clinical practice. Among other things these methods should improve on the sensitivity of existing techniques, be sufficiently robust to allow multi-parametric evaluation of clinical samples, and should not require invasive procedures for sample acquisition. In this proposal we will test a combination of existing modern techniques, which is designed to provide high-throughput, multi- parametric detection of diffuse large B lymphoma. In R21 phase of the project we will use cultured cell and paraffin-embedded tissues of patients to determine sensitivity, selectivity and variability of the assay. When the milestones of R21 phase are met we will move into clinical R33 phase of the project, testing the assay's ability to detect lymphoma- specific DNA in patient's plasma. We will test correlation of the assay's results with clinical diagnosis of diffuse large B lymphoma, and with clinical outcomes, including disease-free survival and drug resistence. When the assay is optimized for disease detection in patient's plasma we will also address the issue of residual disease detection and monitoring of treatment efficacy.
 
R21 CA094378-01 2002 MAYER, BRUCE J UNIVERSITY OF CONNECTICUT SCHOOL OF MEDICINE & DENTISTRY Profiling Tumor Cells by SH2 Domain Binding Pattern
This proposal describes a novel proteomic method to profile cells on the basis of their binding; sites for Src Homology 2 (SH2) domains. This molecular diagnostic approach takes advantage of a basic signal transduction mechanism whereby cells receive information from the extracellular environment. Many signals, including those from growth factors, cytokines, and the extracellular matrix, are transmitted by changes in the tyrosine phosphorylation of intracellular proteins. These phosphorylated proteins in turn serve as binding sites for proteins containing a small protein-binding module termed the SH2 domain. All SH2 domains recognize tyrosine-phosphorylated sites, but each SH2 domain also has a distinctive binding specificity based on the residues surrounding the phosphotyrosine. Thus to a large extent the cytosol of the cell interprets its environment by the presence or absence of binding sites for specific SH2 domains. Recent innovations that increase the specificity of SH2 binding in vitro and decrease nonspecific background now make it feasible to use the pattern of binding sites for a panel of SH2 domain probes to build a profile or "fingerprint" for any cell, including tumor cells. Preliminary work using hematopoietic cell lines demonstrates that this is a robust and sensitive method which provides a unique molecular insight into the signaling machinery of the cell. The SH2 binding profiles will provide molecular diagnostic data that can be used to sub-classify histologically similar tumors, in much the same way that cDNA profiling is beginning to be used to classify tumors on the basis of their transcriptional activity. Moreover the SH2 profiling method has the added advantage that the information it provides will in many cases be directly relevant to the signaling derangements that contributed to the outgrowth of the tumor. Therefore SH2 binding profiles may not only have prognostic value, but also provide novel insights that will rationally guide therapy. SH2 binding proteins that are highly correlated with disease outcome can be identified and may serve as novel targets for drug discovery. In this proposal the SH2 profiling method will be applied samples from patients with hematopoietic and other malignancies, and any correlation between disease course and SH2 profile will be determined. Variants of the existing profiling method will also be pursued, with the aim of developing a high-throughput method to quantitate binding sites for tens to hundreds of different SH2 domains in a single binding reaction.
 
R21 CA094341-01 2002 SMITH, LLOYD M UNIVERSITY OF WISCONSIN MADISON Single Droplet Electrospray Ionization
We propose to develop a new generation of mass spectrometric instruments with vastly increased sensitivity, approaching the single molecule level; greatly enhanced capability for the analysis of complex mixtures and multisubunit protein complexes. The research presented here focuses on development of a new time-of-flight mass spectrometer which utilizes single charged droplets as ion sources in combination with an aerodynamic lens to focus the ions onto the center axis of the time of flight region of the mass spectrometer. Single droplets will be generated by a piezoelectric dispenser and transmitted through an aerodynamic lens where they will be desolvated to produce analyte ions. The aerodynamic lens system will replace the standard nozzle-skimmer and collisional cooling systems commonly employed in ESI-TOF. The fact that the ions will travel along a single defined trajectory, rather than along a multitude of trajectories as in conventional electrospray, will permit the collection efficiency of the ions into the mass spectrometer to be greatly increased. Ions exiting the aerodynamic lens will be electrostatically accelerated and time-of-flight mass analyzed as in conventional matrix assisted laser desorption ionization (MALDI) mass spectrometry. Calculations of the increase in detection efficiency that in theory is attainable with this approach suggest that as much as a 10exp12 fold increase could be realized; although practical considerations may limit the achievable increase, it seams clear that a very substantial sensitivity gain is to be expected. Additionally, the proposed instrument greatly simplifies the spectrometer design which should make possible the construction of a simple, robust, and cost effective mass spectrometer.
 
R21 CA094408-01 2002 SOMMER, STEVE SEEV CITY OF HOPE/BECKMAN RESEARCH INSTITUTE Carbon Nanotubes: Artifical Gels for Mutations Detection
Our vision is to combine nanotechnology with proven genetic screening technology to provide non-invasive, low cost, rapid screening for specific molecular signatures of cancer. Highly sensitive and specific, DOVAM-S (Detection of Virtually All Mutations-SSCP) is capable of screening several human genes for mutations with virtually 100% accuracy. The key technology to be demonstrated is a nanofabricated, electrophoretic microdevice for detecting mutations and polymorphisms of virtually all types. With this technology, cost-effective screening for cancer predisposition could be implemented in the relatively near term. Based on the unique properties of carbon nanotubes, we envision an integrated nano-DOVAM-S system with significantly reduced reagent volumes, shortened amplification and separation times, and automated readout and interpretation of results. In this proposal, we present this vision, and the necessary steps toward a proof-of-concept demonstration: first proving that SSCP is possible with carbon nanotube arrays, followed by protocol development and comparison with standard DOVAM-S in clinical mutation screening experiments. Other efforts to develop on-chip DNA analysis technology lack the essential advantages that distinguish DOVAM-S from competing approaches to mutation screening. These are a virtually 100% detection rate and the ability to multiplex samples. In order to be effective, mutation screening by DOVAM-S requires electrophoretic separation of single- stranded DNA with and without mutations. Our approach is to use carbon nariotubes as a solid-state nanometer-scale sieve for electrophoretic separation of single-strand DNA, replacing the cross-linked polymers used in conventional gel electrophoresis. Competing efforts to provide on-chip DNA analysis retain the use of cross-linked polymers, and many are based on hybridization reactions, which cannot attain the 100% detection of mutations that has been demonstrated in the DOVAM-S approach advocated here. Nano-DOVAM- S will be validated using the human factor VIII and IX genes and the ATM gene, mutated in certain cancers.
 
R21/R33 CA094393-02 2002 CHODOSH, LEWIS A UNIVERSITY OF PENNSYLVANIA Temporal Analysis of Mammary Carcinogenesis
Breast cancer is the most common malignancy diagnosed among women in United States and is the second leading cause of cancer mortality. Despite intensive efforts aimed at improving the early detection and treatment of this disease, mortality due to breast cancer remains high. Consequently, strategies aimed at a more thorough understanding of the biology of this disease are needed. A cardinal feature of mammary tumorigenesis is that it occurs via a series of histologically recognizable intermediates that begin with normal tissue and progress through stages of hyperplasia, atypical hyperplasia, and carcinoma in situ, to invasive carcinoma and metastasis. Although this process has been intensively studies for decades, the molecular and cellular alterations that underlie this progression are poorly understood. As such, elucidating the molecular changes that are involved in tumor progression is a critical priority in breast cancer research. We have recently created a novel doxycycline-dependent bitransgenic mouse model system that permits oncogenes to be inducibly expressed in the mammary epithelium for a defined period of time, at a desired level, and during any desired developmental stage. Inducible expression of the c-MYC proto-oncogene using this system, for example, results in the formation of invasive mammary adenocarcinomas in a manner that is rapid, highly penetrant, mammary-specific, and absolutely dependent on transgene induction by doxycycline. The availability of rapid analytical tools that permit the parallel quantitative analysis of gene expression on a genome-wide scale, coupled with the ability to inducibly activate oncogenic pathways of known relevance to human breast cancer, makes it possible for the first time to provide a comprehensive description of the process by which mammary carcinogenesis occurs. A detailed analysis of the molecular changes that occur during successive stages of carcinogenesis will undoubtedly increase our understanding of the biological basis of breast cancer, lead to the ready identification of molecular differences between normal, precancerous, and cancerous cells, facilitate cancer detection and diagnosis, and ultimately identify potential new targets for therapeutic and preventative agents. This application is intended to demonstrate the suitability of oligonucleotide microarray technology for investigating mammary carcinogenesis by applying this technology to the analysis of a novel set of conditional bitransgenic model systems for breast cancer. The application consists of a first phase for technology evaluation, including proof -of -principle experiments demonstrating the accuracy, reproducibility and utility of the technology, and validating the transgenic animal models. In the second phase of this application, these novel animal models will be used in combination with high density oligonucleotide microarrays and novel analytical algorithms to define gene expression changes that occur during each of the stages of mammary carcinogenesis as induced by well- defined oncogenic stimuli relevant to human breast cancer.
 
R21/R33 CA091166-03A1 2002 CHU, WEI-SING AMERICAN REGISTRY OF PATHOLOGY, INC. Ultrasound-accelerated tissue formalin fixation and paraffin embedding
The first crucial step in cancer management is to assure timely and accurate pathological diagnoses. Formalin fixation and paraffin embedding (FFPE) has been a standard tissue preservation method employed in over 90% cases for clinical histology diagnosis. Though it provides superior morphology and easy long- term storage of clinical specimens, FFPE is time-consuming and does not fully support current molecular analyses. The long-term goal of our research is to apply modern techniques to medical practice and pathological diagnosis to effectively fight cancers and other diseases. Our proposed project is to further develop the ultrasound-accelerated tissue preservation (UTP) technology for multiple tissues and to study the mechanism of ultrasound (US)-facilitated formalin fixation. Our specific aims are: (1) to develop a multi- tissue preservation processor with an optional real-time digital system to monitor and standardize tissue fixation level; (2)to validate the UTP techniques by performing more statistical assessments on histopathology, macromolecule integrity, and their long-term stability; and (3) to conduct mechanism studies to elucidate the effects of formalin fixation with and without US on the formation of cross-linking, enzymatic activity, and protein conformational changes. We have demonstrated that in comparison to conventional FFPE, UTP provides similar preservation in tissue morphology with similar long-term storage stability, improved preservation of protein structure, antigen properties, and mRNA integrity. UTP allows easy general molecular profiling and analyses based on extracts from UTP-fixed tissues. UTP also provides a good opportunity to control and monitor fixation level by adjusting the time and strength of the ultrasound. We hypothesize that US-facilitated formalin fixation will greatly accelerate formaldehyde-induced macromolecule cross-linking in tissues and "freeze" macromolecules and their conformation due to accelerated fixation reactions. Since tissue preservation is still a standard and general requirement before histology diagnosis, the innovation should have great impact in economy and public health.
 
R21/R33 CA089841-02 2002 CLEVELAND, WILLIAM L ST. LUKE'S-ROOSEVELT INSTITUTE FOR HEALTH SCIENCES Robotic Preparation of cDNA Single Cells
The current state of the art for optical microscopy of living cells provides an array of techniques of extraordinary power. Living cells can now be studied in multiple dimensions (three spatial dimensions, time, multiple wavelengths, and multiple stage positions). A unique feature of microscopic approaches is the capability of observing transient, ephemeral structures and interactions on a cell-by basis. This includes the ability to monitor subcellular processes and to follow cell movements as well as cell interactions over time. We propose to develop an instrument that will couple the power of multidimensional microscopy with that of DNA array technology. Specifically, we envision an instrument in which individual cells selected on the basis of optically detectable features at critical time points in dynamic processes can be rapidly and robotically micromanipulated into reaction chambers to permit amplified cDNA synthesis and subsequent array analysis. In this way, "snapshots" of gene expression in single cells can be related to information obtained with multidimensional microscopy. The proposed instrument will incorporate an inverted research microscope capable of widefield deconvolution microscopy as well as a robotic system for manipulation of cells and reagents. An environmental chamber will provide conditions for optimal maintenance of cells. A laser ablation system will provide for automated cell lysis. Algorithms will be developed for automatic recognition and manipulation of cells, a requirement for high throughput. The planned system is expected to process 500-1000 cells per day. We believe that the proposed instrument will represent a genuine advance in technology that will be of great benefit to cell biology and the study of cancer cells. For example, the progression to malignancy involves the gradual accumulation of genetic changes in single cells, leading to heterogeneity among malignant cells. Studies of gene expression at the single level will permit an orderly dissection of this heterogeneity. In summary, we believe that an instrument which couples multidimensional microscopy with DNA array technology will be a spectacular tool that will be useful to many laboratories.
 
R21/R33 CA095941-02 2002 DYNAN, WILLIAM S GEORGIA HEALTH SCIENCES UNIVERSITY Laser Capture Microscopy and 2D-DIGE: Cancer Proteomics
Barriers to the analysis of proteins in tumor samples by traditional methods include the difficulty in accurate quantitation of the relative amounts of individual proteins in multiple samples and the inhomogeneity of most solid tumor specimens. This proposal overcomes these barriers by combining several new technologies in a cross-specialty collaborative effort. This strategy draws upon expertise from clinicians, pathologists, basic scientists, mass spectroscopists, and bioinformaticists. In order to obtain relatively homogeneous populations of tumors, cancer cells will be isolated by laser capture microscopy. While this technique permits isolation of specific subpopulations of cells within tumors, the total number of cells is often low. The proposal will therefore draw on a newly developed approach called 2-D DIGE (2-Dimensional Differential In-Gel Electrophoresis). In this method, protein extracts are labeled in vitro with reactive cyanine dyes that fluoresce at one of several chosen wavelengths. Up to three extracts labeled with different dyes are mixed and analyzed in the same large-format two-dimensional gel. The gel is imaged at multiple wavelengths and analyzed to determine the precise ratio of proteins migrating in various spots. The unique aspect of the technology is that it allows independent quantitation of proteins derived from two or three biological samples in the same gel, eliminating issues of gel-to-gel reproducibility and thus providing an exceptionally accurate proteome map. A new generation of dyes allows detection and quantitation of individual polypeptides present in picogram amounts. By combining this sensitive proteomic detection method with laser capture microdissection technology, it should be possible to derive a useful proteome map derived from cytologically homogeneous specimens containing as few as 1,000 to 10,000 cells. In this phase, we will focus on three goals for working with cancer samples obtained by laser capture microdissection (LCM): to directly compare the new generation of cysteine-reactive dyes to the lysine-reactive dyes for the labeling of LCM samples, to identify strategies that permit identification of protein species of interest by mass spectrometry, and to perform pilot experiments of LCM and 2D-DICE using the optimized techniques. In the next phase, broader clinical utility will be demonstrated using a larger sample set. Changes in the pattern of protein expression will be identified. These changes can serve as clinically useful markers of tumor progression.
 
R21/R33 CA091351-03 2002 FUTSCHER, BERNARD W UNIVERSITY OF ARIZONA Microarray Technology to Profile CpG Island Methylation
The long-term objective of this research project is to adapt microarray-based technology to measure CpG island methylation in human cancer cells. CpG islands are approximately 1kb stretches of DNA that have a high CG content, are enriched in the dinucleotide 5'-CG -3', are found at the 5' end of about 50% of all human genes, and participate in the transcriptional regulation of these genes. The cytosines in the CpG dinucleotides of CpG islands are unmethylated in normal tissue; however, CpG islands become aberrantly methylated during oncogenesis and has been linked to the transcriptional repression of the associated gene. In addition, from the limited number of CpG islands and tumors that have been analyzed to date, it appears that patterns of aberrant methylation occur in a tumor-specific and stage-specific fashion, suggesting that CpG island methylation profiles may be useful as a tumor- specific fingerprint to monitor disease activity and burden. Thus, a multiplexed assay where the cytosine methylation status of thousands of CpG islands can be determined simultaneously would be useful in the molecular profiling of human tumors, and will likely provide insights into the biology of cancer. To this end we have formed a multidisciplinary team to use human CpG island microarrays (CGI arrays) as a tool for determining CpG island methylation profiles in cancer, and from these profiles identify characteristic patterns of CpG island methylation that correlate with the tumor's clinical phenotype. The 4 integrated specific aims that follow are designed to reach our objective. 1) Construct CpG island microarrays for use in CpG island methylation analysis. 2) Optimize methylation analysis using CpG island microarrays 3) Determine CpG island methylation signatures in AML cell lines and in AML samples obtained from patients with known clinical outcome. 4) Develop and implement a public database for the dissemination and mining of the CGI array data
 
R21/R33 CA097502-02 2002 GARCIA-BLANCO, MARIANO A. DUKE UNIVERSITY Imaging Alternative Splicing During Tumor Progression
The broad long-term goal of the proposed work is to understand the molecular basis for tumor progression in prostate cancer. To that end we propose in the year of R21 funding to develop methods that can image gene expression patterns in living cells. We focus our study on the alternative splicing of fibroblast growth factor receptor type 2 (FGF-R2) transcripts. This alternative splicing results in the production of two FGF-R2 isoforms, FGF-R2(IIIb) and FGF-R2(lllc), which have very different ligand binding properties. The choice of FGF-R2(lllb) vs. FGF-R2(lllc) is highly regulated during development and is deregulated during prostate tumor progression. R21 Specific Aim No. 1: Construction and testing of real time reporters of alternative splicing of FGF-R2. We will design and construct reporter plasmids capable of imaging changes in gene expression due to alternative splicing. Alternative splicing regulation will be imaged by measuring fluorescence, luminescence and expression of MRI detectable proteins. Studies will be carried out in tissue culture to prove the utility of these plasmid reporters. In the three years of R33 support we propose to develop methods that can image gene expression patterns in tumors and in living animals. We focus our study on the alternative splicing of fibroblast growth factor receptor type 2 (FGF-R2) transcripts in prostate tumors in rats and in nude mice. We also propose to study the regulation of this alternative splicing in normal tissues in transgenic mice. In order to achieve these goals we propose to accomplish the following two specific aims: R33 Specific Aim No. 1: To evaluate the alternative splicing of FGF-R2 in prostate tumors in living animals. We propose to use the imaging reporters designed and built during the R21 year to study the transition from FGF-R2(lllb) to FGF-R2(lllc) that accompanies the progression of prostate tumors. We will evaluate two prostate tumor model systems, the Dunning rat prostate model in syngeneic immunocompetent rats and the LnCAP human xenograft in nu/nu mice. R33 Specific Aim No. 2: To evaluate the alternative splicing of FGF-R2 in normal tissues of transgenic mice. We will employ the aforementioned imaging reporters to study the regulation of alternative splicing in living animals. We hope to provide an anatomic map of gene expression based on alternative splicing regulation.
 
R21/R33 CA094356-03 2002 HEYDUK, TOMASZ SAINT LOUIS UNIVERSITY MOLECULAR BEACONS FOR PROTEIN DETECTION
The ability to rapidly detect and quantify specific proteins in a highly multiplexed manner is of great importance for basic science research, drug screening applications, and clinical diagnosis. In this application we propose the development of a novel methodology which will allow generation of reporter molecules capable at the same time to recognize and to signal the recognition of a specific protein molecule. The signal generated by these "molecular beacons" will be simple to measure fluorescence intensity, which will facilitate high-throughput use of this new methodology. The proposed method will allow design of truly homogenous assays. This methodology will be initially developed for proteins having natural sequence specific DNA binding activity. Subsequently, this methodology will be expanded to include the proteins which do not possess natural sequence specific nucleic acid binding activity. There will be two phases of this project. In the first phase we will use the model protein systems to provide "proof of principle" evidence that our approach can be developed for a DNA-binding protein and that it can be expanded to proteins lacking such activity. In the second phase of the project we will apply the concepts developed in the first phase of the project to generate "molecular beacons" which will recognize four cancer-related targets: p53 - a tumor suppressor whose inactivation is the most common defect in cancer cells, NF-kB - transcription factor involved in regulation of many genes and found to be constitutively active in many tumors, p16(INK4A) - a tumor suppressor involved in cell cycle regulation whose mutations have been found in greater than 70 different types of tumor cells, and p27(Kip1) - a cell-cycle inhibitor whose cellular levels were shown to be an important prognostic marker in breast cancer patients. We expect that the methodology we propose to develop will find broad applications in basic cancer research, molecular diagnosis of disease, identification of therapeutic markers and targets, and in characterization of response to pharmaceuticals.
 
R21/R33 CA094441-02 2002 HUANG, TIM H.-M. UNIVERSITY OF MISSOURI-COLUMBIA Novel Tool for Analysis of Promoter Hypermethylation
We propose to develop a novel DNA chip specifically for high-throughput analysis of promoter hypermethylation in primary tumors. It is now clear that abnormal DNA methylation frequently occurs in multiple promoter CpG islands in cancer cells. Increased density of CpG methylation is known to alter local chromatin structure within a promoter , resulting in transcriptional silencing of the corresponding gene. At present, bisulfite DNA sequencing is considered to be the" gold standard" for marring methylated CpG sites within a gene promoter. This method, as well as other related techniques, has provided important insights into the functional relationship of promoter methylation and transcriptional silencing on a "gene-by-gene" basis. Such approaches, however, have given limited pictures of complex epigenetic alterations in cancer and are restricted in throughput for routine clinical applications. In this proposal, we build on the approach of microarray techniques by developing a novel method, called Methylation-Specific Oligonucleotide (MSO) microarray, for high-throughput methylation analysis. Our goal aims at generating MSO chips in which thousands of short oligonucleotides are tethered to glass slide surfaces. These oligonucleotides specifically designed and tested are capable of differentiating methylated and unmethylated CpG sites at the specific locations of a promoter. Test (tumor) and reference samples are bisulfite-treated, PCR-modified products that may contain different pools of DNA fragments due to the hypermethylation status of the tumor genome. These DNA samples are co-hybridized to an MSO chip and quantitative differences in DNA methylation are determined by two-color fluorescence analysis. Distinct from the existing microarray technologies, the MSO approach allows simultaneous analysis of the anatomy of multiple promoter CpG islands in reference to the role of DNA methylation on gene silencing. In addition, this MSO chip can be directly applied to determine molecular signatures of different tumor subtypes. In the pilot R21 phase, we will apply a prototype MSO chip to analyze a small panel of tumor samples and determine its reliability in detecting DNA methylation. In the R33 phase, based on the experience we gain in the pilot study, we will design a full-fledged MSO chip for a comprehensive analysis of promoter methylation in multiple tumor types. An advanced computation system will be developed to handle a large set of methylation data and patients' clinicopathological information and to support heuristic queries. The MSO assay will furnish digital profiles of promoter hypermethylation for individual tumors and offers an alternative to cDNA microarray approaches for molecular classification of different cancer subtypes.
 
R21/R33 CA094365-02 2002 KENAN, DANIEL J. DUKE UNIVERSITY Endothelial cell molecular alterations in cancer
Despite the central role of endothelial cells (ECs) in cancer biology, relatively little is known about the molecular perturbations that occur in tumor endothelium and their relationship to disease. Tumor ECs are particularly difficult to study because their behavior is influenced by characteristics of the tumor microvascular environment that cannot be recapitulated ex vivo, including EC functional interactions with adjacent stromal and tumor cells and their dependence on blood flow and extracellular matrix for stable differentiation. This project seeks to develop a robust technology platform enabling analysis of tumor endothelium cell gene expression within native tissues. The technology is based on the ability to co-immunoprecipitate RNA binding proteins together with their associated mRNA species from cell and tissue extracts. The primary implementation of the technology will rely on engineering a transgenic mouse expressing an epitope-tagged poly(A) binding protein under control of an endothelium-specific promoter. Anti-epitope tag antibodies can then be used to recover endothelium mRNAs from both normal and tumor transgenic tissue extracts. Recovered mRNAs can be detected and quantitated in high throughput fashion using microarray technologies. In addition, the mRNAs will be used as input for construction of SAGE and EST libraries that will serve to validate the microarray data as well as to guide the construction of EC-targeted custom microarrays. Human homologues will be identified for any genes that are differentially expressed in murine tumor endothelium, and the homologues will be validated for specificity of expression in human tumor endothelium. As a secondary goal, a parallel effort will be conducted to identify naturally occurring human endothelium-specific ribonucleoprotein (RNP) epitope tags so that the technology can be practiced directly on human tissues. RNP-tag technologies will be used to study murine and human EC gene expression in a variety of tumors and normal tissues across a range of anatomic sites. Detected alterations in tumor EC gene expression that may affect disease progression or that can serve as diagnostic markers or therapeutic targets will be validated in ECs from different tumor and tissue types. This project will generate reagents, information, and technologies that are likely to lead to improved detection and treatment of cancer.
 
R21/R33 CA086285-02 2002 KNAPP, DANIEL R. MEDICAL UNIVERSITY OF SOUTH CAROLINA Microfluidic System for Cancer Proteome Analysis
This proposal is a combined R21/R33 application to develop an integrated multidimensional analysis system for characterization of complex mixtures of proteins, i.e. proteome analysis. The system will employ a microfluidic implementation of a 2D chromatography separation system directly interfaced via electrospray ionization to a mass spectrometer. The analytical approach is to digest the protein mixture prior to separation and then separate and analyze the resulting fragment peptides. Protein identification is done by computer matching of tandem mass spectra with predicted data from sequence databases. The separation system, which will employ strong cation exchange and reversed phase partition chromatography, is based upon a published capillary system which has been shown to be superior to conventional two dimensional gel electrophoresis methods for proteomic studies in yeast. The microchip implementation will enable improvements over the published approach to yield a more powerful, yet simpler to use and less expensive system. The ultimate implementation of the system will replace 2D gel separation with a relatively inexpensive, disposable injection molded plastic microfluidic device which interfaces directly to the mass spectrometer. The prototype devices will be produced using recently developed soft lithography methods which obviate the need for complex microfabrication facilities and yield designs directly transferable to large scale production by injection molding. This approach will facilitate the application of proteome analysis in cancer research by significantly reducing the labor and art required compared to 2D electrophoresis and eliminating the need for manual transfer and processing of gel for mass spectrometric analysis. The system will facilitate the molecular analysis of cancer by providing a readily accessible approach to determining the expression patterns of proteins in normal and neoplastic tissues as well as characterizing the protein content of clinical specimens. The fact that the system will be implemented as a microfluidic device makes it amenable to very small sample sizes and is potentially extendable to single cell analysis.
 
R21/R33 CA094354-02 2002 LARSON, DALE N HARVARD UNIVERSITY Protein Localization: Multi-aperture Near-Field Optics
The objective for this research is to develop a new form of microscopy, Surface Plasmon Enhanced Microscopy (SPEM). SPEM achieves resolution of 35 nm and is intended to be used for localization of multiple proteins in cells and determine molecular signatures of patients? tumors. The SPEM system generates a database of protein locations within a cell referenced to the position of cellular features (e.g., cell and nuclear membranes, organelles). SPEM can be used to identify molecular signatures of cancer based on protein expression levels and locations. Over time it is expected that a database of SPEM images will be assembled that will serve as a reference for the diagnosis of cancer at the molecular level. SPEM has been designed to seamlessly integrate into the clinical pathology laboratory. The steps required to implement a SPEM analysis are: (1) stain the slide (2) the pathologist reads the slide and marks cells for which a molecular analysis (using a digital coordinate system that is incorporated into the SPEM microscope) is desired (3) SPEM automatically generates the localization and expression level data for the marked cells, and (4) the pathologist analyzes the results in conjunction with the existing immunohistochemical and morphological data to determine the patients? prognoses and select the appropriate therapy. In the R21 research the optical performance of the system will be characterized and the applicability to paraffin-embedded tissue specimens will be demonstrated. In the R33 research the full microscope will be develop and used to generate protein localization and expression data for multiple proteins in a tissue sample.
 
R21/R33 CA094340-02 2002 MEHTA, ANAND S THOMAS JEFFERSON UNIVERSITY Identification of serum glycosylation markers of HCC
Infection with hepatitis B virus (HBV) (and/or hepatitis C virus) is the major etiology of hepatocellular cancer (HCC). In the United States, chronic hepatitis B and C account for about 30%- 40% of HCC and, worldwide, more than 350 million people are chronically infected with either HBV or HCV. Alteration in the oligosaccharides associated with glycoproteins and glycolipids is one of the many molecular changes that accompany malignant transformations. However, accurate and reliable detection and quantification of oligosaccharides has been technically very difficult or time consuming. It is well established that in HCC there is an elevation in the amount of fucosylated oligosaccharides associated with a-fetal protein (AFP). The increase of a l-6-fucosylated glycans is due, in part, to an abnormal secretion system and other proteins have been shown to be aberrantly glycosylated. However, no systematic analysis has been carried out to identify all the proteins that have an increase in fucosylation. Our hypothesis is that by combining our high throughput oligosaccharide sequencing methodology with our laboratory's proteome expertise, we will be able to use lectin extraction methods to purify and identify fucosylated glycoproteins from HCC sera. Hence, proof of principle experiments will be carried out in the R21 phase using cultured liver cells to demonstrate the utility of the technology; while in the R33 phase the technology will be evaluated in a pilot study using sera obtained from woodchuck, a well established animal model of HBV induced HCC. It is anticipated that this methodology will allow the identification of an array of molecules that show increased fucosylation in HCC. These proteins may be then further evaluated as members of a fucose index to act as a surrogate marker for HCC progression. Obviously the technology would be applicable to any cancer system where oligosaccharide changes are associated with development to neoplasia.
 
R21/R33 CA092731-03 2002 SESHI, BEERELLI LA BIOMEDICAL RESEARCH INSTITUTE/ HARBOR UCLA MEDICAL CENTER FUNCTIONAL PROTEOMICS OF LEUKEMIC MARROW STROMAL CELLS
Proteomics, which entails global gene expression analysis, is considered a nonbiased discovery-driven (as opposed to hypothesis-driven) approach to the analysis of protein expression. The long-term objective of the proposed research is to determine the role of human bone marrow (BM) stromal cells in normal and leukemic hematopoiesis. This proposal aims to: (1) Carry forward a critical previous discovery that shows that human BM stromal cells represent a unique pluridifferentiated mesenchymal progenitor cell type (MPC), coexpressing multiple mesenchymal lineage markers. (2) Apply a recently established method for purifying MPCs from leukemic BM stromal cell cultures, free of macrophages and hematopoietic cells. (3) Investigate MPC cell-specific protein expression profiles and how these profiles change in hematopoietic malignancies. (4) Establish the proof of principle and practice of high-resolution proteomics with relevance to normal and leukemic human BM stromal cells. Specific experimental design includes performance of the following. R21 Phase: Set up large-format 2-D gel electrophoretic system (2-D PAGE) for reproducible separation of MPC proteins. Prepare 2-D PAGE protein maps for normal BM-derived MPCs (untreated and treated with representative cytokines) and for MPCs derived from patients with representative leukemic conditions (AML, CML, MM). Using mass spectrometry (MALDI-MS and/or Nano ESI MS/MS), identify about 200 differentially-expressed MPC proteins (i.e., those that increased or decreased in intensity as compared to 2-D PAGE protein maps of normal, unstimulated MPCs). Construct a human BM MPC-2DPAGE database on WWW and publish it under WORLD-2DPAGE with links to the currently existing 2-D PAGE databases. In parallel studies, identify about 200 differentially expressed MPC proteins by an independent method, isotope-coded affinity tag (ICAT) labeling in conjunction with LC/MS/MS. Based on theoretical pI and MW, construct a "virtual" 2-D map of ICAT-identified MPC proteins for integration into 2-D PAGE database. R33 Phase: Add high-throughput robotics to the high-resolution 2-D PAGE proteomics established under R21 phase. Identify on a relatively large-scale (about 1,800) the MPC proteins that are differentially expressed following stimulation with different cytokines and in leukemias (AML, CML, MM). Facilitate understanding of the pathogenetic mechanisms by identifying the phosphoproteins potentially involved in cell signaling pathways. In parallel studies, identity about 1,800 MPC proteins by ICAT method. We plan to identify a total of about 2,000 functionally relevant BM stromal cell proteins by each method. Update the WWW database by including the proteins with pathologically altered expression. The database will be a valuable resource for researchers investigating the basic biology of hematopoiesis and leukemogenesis and for clinical hematologists/oncologists and pathologists alike.
 
R21/R33 CA097527-02 2002 SHIH, IE-MING JOHNS HOPKINS UNIVERSITY Development of Digital SNP Analysis in Cancer Detection
The objective of this project is to develop an innovative molecular genetic approach to analyze the allelic status in plasma for cancer detection. Allelic imbalance (AI), defined as losses or gains of particular chromosomal regions, is a unique genetic abnormality in almost all human cancers. Since tumors release substantial amounts of tumor DNA into the systemic circulation, AI in plasma DNA may represent a UNIVERSITYersal marker in cancer patients. Traditional methods to determine AI in plasma or serum using micro satellite markers are fraught with a variety of technical difficulties such as contamination of normal DNA, variable degradation of plasma DNA and difficulty for quantitation and interpretation. To overcome these obstacles, we propose to employ our recently developed PCR-based approach called Digital Single Nucleotide Polymorphism (SNP) analysis in which the paternal or maternal alleles within a plasma DNA sample are individually counted thus allowing a quantitative measure of such imbalance in the presence of normal DNA. With this technique, the alleles present in plasma can be directly counted to determine whether there is an imbalance. This R21/R33 combined proposal consists of two phases. The R2t phase is to develop Digital SNP analysis suitable for analysis using plasma DNA. The specific aims are: 1) select SNP markers for Digital SNP analysis in plasma; 2) optimize the conditions for Digital SNP analysis; and 3) improve the automation and throughput. The R33 phase is to test the feasibility of our innovated technology for practical application. The specific aims are: 1) demonstrate allelic imbalance in plasma DNA of cancer patients using Digital SNP analysis; and 2) assess the clinical potential of Digital SNP analysis for detection of asymptomatic cancer. In conclusion, the results generated from this proposal will provide a clear measurement of tumor-released DNA into circulation and whether tumor-released DNA can be detected by an efficient approach for a potential cancer screening. Thus, the encouraging results from this study will ensure the detection of AI in plasma a promising cancer research direction for further exploration.
 
R21/R33 CA094334-02 2002 SOMMER, STEVE SEEV CITY OF HOPE/BECKMAN RESEARCH INSTITUTE PAP: a technology platform
Pyrophosphorolysis activated polymerization (PAP) offers a novel approach for retrieving multiple types of information from nucleic acids. The exceptional specificity of PAP derives from an inactive dideoxy terminated oligonucleotide (P*). P* is activated by pyrophosphorolysis of the 3? terminal nucleotide, followed by extension of the activated oligonucleotide by DNA polymerization. The initial studies established proof of principle and recently the efficiency of PAP was improved greatly with genetically engineered polymerases that have a high affinity for dideoxy nucleotides. Herein, we propose to demonstrate that PAP is a platform technology. Three novel methods will be developed on the platform: i) allele-specific detection of a single nucleotide change in the presence of 10expl6-10exp9 normal alleles (PAP-A): ii) microarray-based resequencing (PAP-R) and iii) analysis of in vivo chromatin structure (LM-PAP). These methods have many applications including: i) ultra sensitive detection of minimal residual disease or measurement of mutation load (PAP-A); ii) molecular epidemiological analysis of sequence variants that predispose to complex disease or rapid molecular diagnosis (PAP-R); and iii) analysis of chromatin structure as a function of imprinting, X-inactivation and gene expression or quantitation of the level of methylation (LM-PAP).
 
R21/R33 CA097516-02 2002 VIDAL, MARC DANA-FARBER CANCER INSTITUTE A C. elegans localization-of-expression mapping project
The development of both cancer detection techniques and therapeutic strategies benefits tremendously from the identification of novel cancer genes. Since the discovery of oncogenes and tumor suppressor genes, about 300 human cancer genes have been identified. With two drafts of the human genome sequence in hand, it is now appropriate to aim to identify all cancer genes. Speculations on how many cancer genes remain to be found point to relatively high numbers. For example, epidemiological studies suggest there might be as many as 100 unknown genes involved in familial breast cancer. Once identified, novel cancer genes have to be organized into functional pathways and interaction networks. Hence, it is important to develop comprehensive research tools to identify and functionally annotate all cancer genes. It is well accepted that conventional genetics and biochemistry in model organisms such as yeast, nematodes, flies, amphibians, fish and mice can be highly advantageous in the discovery of orthologs of new cancer genes. One of the best illustrations of this statement is the discovery of cyclins and cyclin-dependent kinases in yeasts and sea urchins. Other examples of the role of C. elegans and Drosophila genetics include the elucidation of the Ras pathway and apoptosis. It has recently become clear that functional genomic and proteomic approaches in model organisms are also essential technological components for comprehensive searches for cancer genes. For example, recent work from a few labs including ours suggests that, in C. elegans, genome-wide expression profiling (transcriptome mapping), high-throughput gene knock-out analyses (phoneme mapping) and large-scale protein-protein interaction mapping (interactome mapping) can be used to identify orthologs of potential cancer genes. It has been suggested that such maps could provide even better functional information if they are integrated with each other and together with maps describing where [in what cell(s)] and when (at what stage(s) of development and in response to which external stimulus(i), worm proteins are expressed. In this context, the goals of this grant proposal are to develop innovative technologies for a C. elegans "Localization of Expression Mapping Project" (LEMP). Specifically, we will develop a novel high-throughput and 1 R21 CA97516-01 Marc Vidal, Ph.D. versatile cloning technology to clone worm promoters (R21) and use this technology to clone all worm promoters (R33). Using a complete set of worm promoters, we then propose to develop high-throughput methods to localize gene expression in vivo and initiate a protein-DNA interaction mapping project (R33).
 
R21/R33 CA091358-02 2002 WELSH, JOHN T SIDNEY KIMMEL CANCER CENTER Vertical Coverage Arrays in analyzing transcription
Microarray analysis provides a means by which the abundances of many thousands of transcripts can be followed simultaneously as genes respond to experimental or physiological cues. When experiments are more focused on a smaller number of genes that have been selected either by preliminary microarray analysis or by other means, standard microarrays become a cumbersome tool for further characterization. In the R21 phase, a novel microarray method is developed that would allow individual genes to be analyzed over many thousands of experimental conditions. The method is based on the development of low complexity representations of the mRNA of the cell. These low complexity representations are then spotted on a glass slide to produce a 'vertical' microarray, which can be probed with single fluorescently labeled probes. The low complexity representations are employed to increase signal. Multiple low complexity representations are used in order to include most of the complexity of the original mRNA population. Thousands of experimental variables can be explored on a single array, one gene at a time, or a few genes at a time using multi-color fluorescence. In the R33 phase, the vertical array method will be used to characterize the detailed responses of genes known to be controlled by a particular transcription factor to a collection of drugs and hormones. This data will be used to construct a class predictor that predicts whether or not a particular transcription factor is likely to be involved in the regulation of a gene.
 
R21/R33 CA095942-03 2002 YEUNG, EDWARD S. IOWA STATE UNIVERSITY Single-Molecule Immunoassay and DNA Screening
We will develop novel imaging technologies for real-time comprehensive analysis of molecular alterations in cells and tissues appropriate for automation and adaptation to high-throughput applications. With these techniques it should eventually be possible to perform simultaneous analysis of the entire contents of individual biological cells with sensitivity and selectivity sufficient to determine the presence or absence of a single copy of a targeted analyte (e.g. DNA region, RNA region, protein, small molecule), and to do so at relatively low cost. Since minimal manipulation is involved, it should be possible to screen large numbers of cells in a short-time to facilitate practical applications. The general scheme is based on novel concepts for single- cell and single-molecule detection and characterization recently demonstrated in our laboratory. Four distinct but interrelated goals are identified: (1) development of a single molecule detection system which permits rapid and highly confident detection of hybridization to a DNA probe of binding to an antibody in the presence of a large excess of unhybridized probe molecules of free antibodies, respectively. This will lead to single-event homogenous assays of molecular alterations of DNA, proteins, or small molecules in biological tissues; (2) development of high-speed high-throughput specialized data treatment software for rapid "on-line" analysis of the images with an emphasis on confidence in target recognition and reduction in post-imaging data work-up; (3) development of a microscale single manipulation and reaction system for the rapid detection and identification of specific target cells in the presence of a large excess of other cells. It may then be possible to detect directly targeted species in biological tissues without introducing additional probes; and (4) demonstration of actual analysis of human cells and tissue samples, with outside collaboration, to evaluate and to validate the technology developed, and to further optimize the performance regarding speed, ruggedness and accuracy. The first two goals will be the focus of the R21 Phase and the last two goals will be the focus of the R33 Phase of this application.
 
R33 CA094407-01 2002 ALBERTSON, DONNA G. UNIVERSITY OF CALIFORNIA SAN FRANCISCO Amplicon Profiling by Array CGH
Losses and gains of multiple portions of the genome occur in tumor cells, and in some cases amplifications, or high level copy number increases of portions of chromosomes are observed. Alterations in gene expression that result from these copy number aberrations provide a selective advantage to the tumor cells and specific copy number aberrations have been associated with inactivation of tumor suppressor genes or overexpression of oncogenes. The driver genes for many regions of recurrent amplification have not been identified and methods to facilitate their identification would be broadly applicable to increasing knowledge of the genetic events involved in cancer development. Recent implementation of a high resolution form of comparative genomic hybridization (CGH) using microarrays allows quantitative mapping of copy number across an amplicon such that the magnitude of the copy number increase and the extent of the amplicon can be mapped relative to the physical map of the human genome. The goal of this project is to further develop our understanding of the information that can be obtained from array CGH copy number profiles. In particular, we will determine if the copy number maxima as mapped by array CGH can be used to identify the driver gene(s) for an amplicon. This investigation was motivated by the observation that array CGH copy number profiles displayed peaks mapping to the locus of candidate oncogenes amplified at 20q13 in breast cancer, suggesting that these peaks in the copy number profile may result from selection for highest copy number of the genes conferring growth advantage. Therefore, quantitative mapping of amplicon structure by array CGH may provide a powerful new way to identify oncogenes in regions of recurrent amplification in tumors. In order to establish the generality of these observations, in this project, we will use array CGH to quantitatively map the amplicon structure of three well-established oncogenes, CMYC, ERBB2 and CCND1 in different tumor types. We will also map amplicons arising in cells in culture in response to selection for drug resistance. We will determine (a) the extent to which amplicon boundaries are clustered in the genome around these genes, (b) where amplification maxima occur in relation to the locus of the oncogene and (c) the similarities of differences in the amplicons as a function of tissue type. Characterization of the copy number profiles of amplicons containing these known oncogenes and drug resistance genes will provide the information required for interpretation of amplicon structure in other regions of the genome and facilitate identification of the critical genes they contain.
 
R33 CA095957-01 2002 BEACHY, PHILIP A JOHNS HOPKINS UNIVERSITY Genome-based targeting of the Hedgehog pathway in cancer
Advances in cancer therapeutics have occasioned a strategic shift away from generally cytotoxic drugs toward agents that specifically target tumor cells by modulating the pathways or processes that are inappropriately regulated in the tumor. Despite this shift in emphasis, the development of such mechanism-based therapies has been slow. We have recently developed cultured cell-based assays for signaling by the Hedgehog (Hh) family of secreted proteins that have the potential to significantly accelerate the discovery process. The Hh signaling pathway plays an important role in embryonic development, but when inappropriately activated after embryogenesis, is associated with human cancers such as basal cell carcinoma (BCC; 750,000 new cases per year in the U.S.), and medulloblastoma, the most common brain tumor in children. We propose to screen for cellular components with roles in the Hh pathway, using the complete Drosophila genome sequence as the basis for double-stranded RNA-mediated inactivation in Drosophila cells to systematically survey all genes. The availability of complete mouse and human genome sequences will make it possible to rapidly identify mammalian homologues that represent potential drug targets. Simultaneously, we will use our mammalian cell-based assays to screen chemical libraries for small molecules that modulate Sonic Hh (Shh) signaling. By mapping the action of these compounds and of mammalian components to points within the Hh pathway, we will assign potential drug leads to their cellular targets, thus facilitating further enhancement of drug potency and specificity. The value of such drug leads as potential chemotherapeutic agents is highlighted by our recent studies of cyclopamine, which demonstrate that this plant-derived compound is a specific inhibitor of Shh signaling, that adult mice tolerate prolonged treatment with this drug, and that this drug has anti-tumor activity in a mouse model of medulloblastoma. In addition to identification of new drug leads and cellular targets within the Hh pathway, our approach will serve as a general model for similar systematic approaches to anti-tumor therapies for tumors associated with the derangement of other cellular processes or signaling pathways.
 
R33 CA086243-01A2 2002 CAPRIOLI, RICHARD M VANDERBILT UNIVERSITY MEDICAL CENTER Molecular Analysis of Cancer--Imaging Mass Spectrometry
Imaging Mass Spectrometry, which takes advantage of the methodology and instrumentation of matrix-assisted laser desorption ionization (MALDI) mass spectrometry, is a relatively new technology. Preliminary work has shown that it can be used to locate specific molecules such as peptides and proteins up to about 80,000 Daltons, directly from fresh frozen tissue or blots of this tissue. The data from such an analysis is a pictograph in real x,y dimensions on the tissue sample of the location of a signal at any given molecular weight. It has been shown to be effective for the analysis of proteins and peptides in normal and tumor tissue in mouse models of cancer and some human tumor samples. We propose to further develop instrumentation, specifically targeted to imaging and profiling proteins in mouse models and appropriate human samples of prostate, colon and brain cancer. The goals of the technology development are increasing the speed of analysis and sample throughput, and developing advanced data analysis and interpretation methods. Application to cancer research will take place through close collaboration with three investigators who have routine access to tumor tissue. The general goals of these applications are; 1) comparison of molecular weight based protein patterns determined by mass spectrometry in tumors versus normal tissues; 2) identify and image potential tumor markers using mass spectrometry; and 3) compare protein patterns obtained by mass spectrometry and identify unique proteins in various stages of precursor lesions in these three cancer models.
 
R33 CA089837-01A1 2002 ISSA, JEAN-PIERRE J UNIVERSITY OF TEXAS MD ANDERSON CANCER CENTER Neoplasia and Methylated CpG Islands Amplification
CpG island methylation and associated gene silencing has been recognized as a very common event in human neoplasia, with potential to functionally contribute to tumor formation and progression. It has recently become apparent that CpG island methylation events are not random. Rather, there are substantial differences in methylation patterns between different tumors, as well as evidence of hypermethylator phenotypes in subsets of cases. These methylation differences are associated with distinct environmental exposures, distinct gene mutation patterns, and potentially important differences in progression and survival following standard therapies. Methylation profiling of human cancers may therefore prove useful both in etiologic studies of cancer, and in efforts to relate molecular changes to prognosis and treatment decisions. We have recently developed a new technique termed methylated CpG island amplification (MCA), which allows for the rapid methylation profiling of primary tumors. MCA is based on sequential restriction enzyme digestion with methylation-sensitive/insensitive isoschizomers, adaptor ligation and whole-methylated-genome PCR. In pilot studies, MCA appeared to be accurate, relatively high-throughput, and led to the identification of a new methylator phenotype in colorectal cancer. Here, we propose to continue developing MCA by simplifying the procedure to make it readily applicable to the analysis of primary tumors, and by developing tumor-specific CpG island arrays that can be used for profiling most of the major malignancies. Using MCA, the methylated CpG island pools of a given primary tumor can be labeled and hybridized to pre-made filters or DNA chips containing the CpG islands critical to the tumor type under study. This procedure could then reveal the methylation profiles of hundreds of CpG islands in a given tumor in less than 72 hours, without requiring sophisticated reagents or gel electrophoresis. None of the currently existing technologies for methylation analysis can achieve this rapidly or cost-effectively. Ultimately, methylation profiling may prove very valuable in identifying subsets of patients with distinct clinical courses and response to specific therapeutic interventions, including using methylation inhibitors.
 
R33 CA096309-01 2002 LI, HONGHUA RUTGERS ROBERT WOOD JOHNSON MEDICAL SCHOOL A Genetic Approach to Genome-scale Analysis of Cancers
Cancer development is caused by genetic alterations affecting the function of a number of genes. To understand the molecular mechanisms, it is necessary search for the affected genes exhaustively. In this phased innovation project, a highly efficient and cost effective genetic approach to genome scale analysis of cancers will be developed. In phase I, the capacity and sensitivity of a high-throughput multiplex genotyping system will be developed so that each assay may include 300 markers with a sensitivity of using 1200 cells from paraffin-archive tissue. In phase II, 12,000 genetic markers consisting of single nucleotide polymorphisms will be incorporated into this genotyping system so that as few as 40 assays will be sufficient for typing these markers. This system will be used for identifying chromosomal regions that may harbor tumor suppressor genes exhaustively and for revealing at least part of the regions that may contain protooncogenes in breast cancer. To obtain a high resolution, 1,000 carcinoma specimens and 200 metastatic specimens with paired specimens with paired specimens with paired specimens among the invasive specimens will be analyzed. The results will be used to select a small set of markers in the identified in the identified regions. The selected markers will be incorporated in to the high-throughput genotyping system so that one or very few assays will be needed for the future studies covering most, if not all, chromosomal regions that may harbor TSGs and some of the regions containing protooncogenes. Success of this project will make the high-throughput techniques available for genome-scale analysis of cancers. This will make many large-scale genetic studies that may need hundreds of years to complete if the single marker-base assays is used,, but only or few years with this high-throughput approach. The success of the project will also generate highly simplified and affordable experimental procedures for genome-scale analysis of breast cancer, which currently is technically challenging and not affordable for many of the laboratories.
 
R33 CA097528-01 2002 SCHNITZER, JAN EUGENIUSZ SIDNEY KIMMEL CANCER CENTER Technology to Unmask Accessible Tumor Vascular Targets
This grant application proposes to apply innovative, (either a well tested set or well-tested sets) yet reasonably well tested set of technologies to define the proteome of the vascular endothelial cell surface with emphasis on discovering protein differences between normal and tumor blood vessels. Much anticancer research has focused on the development of tumor cell-targeted drug therapies that are quite effective in destroying tumors cells grown in a culture dish but not when injected into the patient?s blood stream because various barriers prevent access to the cancer cells inside the tumor. An attractive alternative strategy for solid tumor therapy is to target the inherently accessible endothelial cells that line tumor blood vessels essential for tumor growth. This vascular targeting strategy is distinct from current anti-angiogenesis therapies because the goal is not preventing tumor blood vessel growth but rather tumor-specific destruction. A new addition to the vascular targeting strategy apparent from our recent work is the utility of a vesicular transport pathway (caveolae) discovered in endothelium for selectively overcoming this key cell barrier to permit delivery inside the tissue. Direct discovery of tumor vascular targets is now feasible because of novel technology that we have developed to facilitate the molecular mapping of the endothelial cell surface and its caveolae as they exist natively in tissue. Here, we will attempt the first in-depth antibody and proteomic analysis of the luminal endothelial cell surface and its caveolae in rat lung tumors using new tissue subfractionation techniques, comparative one- and two-dimensional gel electrophoresis, and mass spectrometric analysis. Data is provided that demonstrates the utility of this new approach in uncovering novel accessible tumor-induced targets expressed in multiple rat, mouse, rabbit, monkey and human tumors. Antibodies specific for such candidate targets will be generated to establish the degree of specificity for the tumor, endothelium, and caveolae as well as to test tumor immunotargeting in vivo. This work is likely to yield tumor-specific vascular targets that facilitate tumor-directed pharmaco delivery, overcome the endothelial cell barrier and, ultimately, provide improved localized tumor therapy with little damage to bystander organs. Discovery of tumor vascular targets may be useful in improving both the early detection and treatment of solid tumors in humans.
 
R33 CA097513-01 2002 SHAUGHNESSY, JOHN DAMIAN UNIVERSITY OF ARKANSAS MEDICAL SCIENCES, LITTLE ROCK Molecular Diagnosis and Prognosis of Multiple Myeloma
Multiple myeloma (MM) is a fatal, clonal B malignancy of terminally differentiated immunoglobulin-secreting plasma cells (PCs) that originate in germinal centers of lymph nodes and home to and expand in the bone marrow. Although other hematological malignancies have been molecularly classified by cytogenetic abnormalities, historically, molecular analysis of MM has been difficult. However, using high-density oligonucleotide DNA microarray (HDA) technology on RNA isolated from highly purified PCs, expression profiles of nearly 6800 genes in normal and malignant MM PCs, as well as monoclonal gammopathy of undetermined significance (MGUS), have been established. Building on preliminary data from more than 70 newly diagnosed MM patients and 7 MGUS cases, new molecular-based diagnostic and prognostic models of the plasma cell dyscrasia MGUS and MM will be established. This goal will be accomplished through 3 specific aims. 1. Define gene expression patterns that differentiate normal, pre-neoplastic, and neoplastic plasma cells. 2. Develop diagnostic and prognostic models for MGUS and MM by use of gene expression profiles. 3. Define the molecular signatures of disease evolution and resistance/relapse through longitudinal global gene expression studies. A new molecular classification of MM and related disorders, provided through the proposed work, will enable clinicians and bench scientists to better understand and combat plasma cell cancer on a genetic level. This knowledge will not only allow a better under- standing of the basic biological pathways that are altered in MM and MGUS but also will provide the framework for the creation new highly accurate molecular tests to diagnose and predict prognosis of these diseases as well aid in the design of novel therapeutics. Thus, the work proposed supports the goals of an R33 project funded through PAR-01-106 (Applications of Innovative Technologies for the Molecular Analysis of Cancer).
 
R33 CA094360-01A1 2002 SPEICHER, DAVID W. WISTAR INSTITUTE Development of Methods for Cancer Biomarker Detection
The overall goal of this three-year R33 proposal is to develop a novel proteomic strategy for quantitative comparisons of serum protein profiles capable of detecting proteins down to the low ng/ml level. This is about 100- to 1000-fold more sensitive than direct analysis of serum on either broad range or narrow range 2-D gels, and it represents the lower end of the concentration range for most known tumor serological markers. This high sensitivity proteome strategy will be developed and tested using chimeric mouse serum that contains human proteins secreted by tumors grown subcutaneously from injected human melanoma or pancreatic carcinoma cell lines. Systematic analyses of secreted human proteins using the new methods developed in this project should identify multiple new potential diagnostic targets for cancer screening. This proteome strategy will incorporate several novel supporting methods in addition to further optimization of a very promising proteome fractionation device and method recently invented in our laboratory. The fractionation method, "microscale solution isoelectrofocusing" (microsol-IEF) uses a variable number of tandem small volume chambers separated by semipermeable partitions containing immobilines at specific pH?s. Proof-of-principle experiments have been completed on several types of samples including mouse serum and demonstrate that this method can cleanly fractionate at least several mg of crude extracts into a small number of fractions. The resulting well-resolved fractions can be separated on a series of slightly overlapping narrow range 2-D gels using much higher protein loads than when unfractionated samples are used. Although the feasibility of this prefractionation method has been demonstrated, more reliable devices must be developed and separation parameters must be optimized to maximize resolution and reliability before this method can become a reliable robust proteomics method. Other novel features of the overall proteome analysis strategy include use of custom-made slightly overlapping narrow pH range gradients to ensure proteins are not lost at gel boundaries and use of dual sensitivity stains to increase dynamic range of detected spots. Proteins that appear to be specific to tumor-bearing mice will be identified and their species of origin (mouse = host or human = tumor) will be determined using LC-MS/MS methods. The Specific Aims are: 1) Develop an optimized microsol-IEF device and method, 2) Develop high sample capacity microsol-IEF devices, 3) Develop high sensitivity post microsol-IEF analysis methods, and 4) Systematically apply integrated proteome analysis strategy to serum samples containing human melanoma or pancreatic carcinoma tumors.
 
R33 CA097541-01 2002 WAGGONER, ALAN S CARNEGIE-MELLON UNIVERSITY Near IR fluorescent probes/molecular analysis of cancer
It is well known that fluorescence detection provides the basis of flow cytometry, fluorescence imaging, DNA sequencing, microarray detection, drug testing, diagnostics, and so on. It is our goal to extend the power of fluorescence detection in cancer research and diagnostics, by developing new fluorescent probes that absorb and fluoresce in the red and near infrared regions of the spectrum. These advances will: (1) increase the number of parameters that can be acquired in a single experiment (hence, more information becomes available through correlation of cell/tissue properties), (2) increase sensitivity (since cellular autofluorescence is much smaller in the red and near IR), and (3) allow imaging deeper into tissues (since absorbance and fluorescence will be away from hemoglobin absorption and in a range where light scattering and absorption is reduced). The new fluorescent reagents will be photostable, chemically stable, water soluble, brightly fluorescent and non-photo-toxic to cells and tissues. This will be accomplished by synthetic modifications in the molecular structure of cyanine (polymethine) dyes and by stabilizing dye structure through encapsulation methods including cyanine-cyclodextrin rotaxanes, nucleic acid encapsulation, sandwich complexation, and covalent self-encapsulation.