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R21 CA084739-01 2000 BENNETT, SCOTT W SRA INTERNATIONAL, INC. INNOVATIVE ALGORITHMS FOR MICROARRAY ANALYSIS
In order to better understand the vast quantity of microarray data currently being generated in a number of laboratories, new computational and analysis tools are needed. Pattern analysis of thousands of simultaneously expressed genes will provide insight into novel and progressive disease treatments. With the use of information technology tools, substantial opportunities exist for improving the ability to identify genetic anomalies. These tools will be critical in advancing the automation and interpretation of experimental results. We will develop an innovative computer-based analytical tool called MicroExplore that will bring together multiple methods for analysis of microarray data. Each method has unique properties that produce conceptually different results. In the first phase, we propose to compare conceptual clustering, hierarchical agglomerative and k-means algorithms. Evaluation of the quality of data will be assessed through a combination of factors that measure how well-known functional groupings are reflected in the output. Efficiency and scalability will be measured through timed runs of MicroExplore on a set of expression datasets of various sizes. Our study will focus on the complex dataset of lymphoid gene expression belonging to an intramural NCI laboratory. In the second phase, a novel hybrid approach will be designed to utilize the results of multiple clustering algorithms and configurations to achieve a ranked set of best overall clusters. Attempts to integrate external data sources will add new dimensions to the analysis tool and provide for powerful predictions. Improving the scientist's ability to accurately and efficiently identify which candidate genes would make good therapeutics will be a fundamental step for the advancement of cancer research and scientific discovery. MicroExplore will be a valuable resource for the continuance of gene discovery and characterization. This platform will have strong potential for the enhancement of clinical data analysis helping to characterize or profile the molecular changes found in normal, precancerous and malignant tissue samples.
 
R21 CA084708-01 2000 GAO, XIAOLIAN UNIVERSITY OF HOUSTON PARALLEL SYNTHESIS OF RNA OLIGONUCLEOTIDE MICROARRAYS
The objective of this proposal is to develop a novel technology for parallel synthesis of RNA microarray on solid surfaces (RNA-chips). Although DNA-chips have emerged as a high efficient and comprehensive tool for cancer gene analysis, drug discovery and a variety of bioassays, a parallel development of RNA-chips has not been possible, primarily because there is no practical method that can achieve efficient and economic parallel synthesis of RNA microarrays. In this proposal, a method of RNA-chip synthesis using novel photochemical reactions in combination with conventional chemistry of RNA synthesis will be developed. The method uses photogenerated reagents to allow optical control of a single reaction step, such as deprotection of blocking groups, in a sequence of reactions. Using a microarray reactor and a programmable digital optical projector, parallel synthesis of thousands of diverse RNA sequences can be achieved with high efficiency and low costs. Our Phase I goals are to obtain prototype RNA oligonucleotide arrays using the proposed method of light directed parallel synthesis and demonstrate hybridization patterns of the RNA-chips synthesized. A novel combinatory strategy using our chip technology will be applied to screen hundreds of photoreaction conditions. New applications of the light controlled RNA-chip synthesis will be explored. Our Phase II goals are to produce next generation of high quality RNA-chips containing oligonucleotides composed of 20-40 residues and establish the applications of RNA cancer gene-chips and RNA library-chips in identification of RNA targets by antisense oligonucleotides and potential antitumor agents. RNA-chips provide unambiguous sequence information and are indispensable and powerful tool for direct investigation of RNA interactions with a variety of ligand molecules in cancer analysis and drug development and in the study of many human diseases. It is expected that in keeping pace with rapidly mounting information in RNA genomics, there will be an escalated effort in identifying RNA sequences as specific diagnose and therapeutic targets. Our proposed research is to provide a highly efficient, flexible and easily accessible technology to accelerate these discovery processes.
 
R21 CA084712-01 2000 HADDAD, BASSEM R GEORGETOWN UNIVERSITY CYTOGENETIC SCREENING OF MAMMARY EPITHELIAL CELLS
Current methods for early diagnosis of breast cancer in premenopausal women are not optimal, for reasons related to breast density, tumor growth rates, and potential susceptibility to radiation-induced damage in women with germline predisposing mutations such as BRCA1/2. New methods for early detection in this population that can serve as an adjunct to mammography are urgently needed. We have developed a new approach that can be applied to early detection, based on cytogenetic analysis using comparative genomic hybridization (CGH) of epithelial cells derived from nipple aspirate fluid (NAF). This approach is based on the premise that women with early stages of neoplastic progression will have cytogenetic abnormalities (DNA gains or losses) that can be detected in the cells shed into NAF. In the R21 phase of the proposal we will validate this technique by (1) demonstrating that we can successfully expand NAF-derived cells in culture to produce sufficient DNA for CGH, (2) observe differences in cytogenetic profiles from these cells between women with normal breasts and those with stage I breast cancer (35 women in each group), and (3) that cytogenetic abnormalities detected by CGH in NAF-derived cells from stage I breast cancer patients are consistent with cytogenetic abnormalities detected in tumor tissue from the same patients. In the R33 phase of this research, we will evaluate the translational potential of this approach by further characterizing the pattens of cytogenetic abnormality across stages of neoplastic progression. We will compare CGH profiles in NAF-derived cells from the following four groups of 50 women each: (1) normal women, (2) women with atypical ductal hyperplasia, (3) women with ductal carcinoma in-situ, and (4) women with stage I breast cancer. This will help to determine the stage of neoplastic progression at which early detection based on this approach is feasible.
 
R21 CA086370-01 2000 OH, SEAJIN SRI INTERNATIONAL AFM BASED TECHNIQUE--ANALYZES EXPRESSION IN CANCER CELLS
We propose to develop and demonstrate an AFM-based technology involving a submicron scale probe array, and, by using the array, directly monitor protein interactions from cellular lysates. This technology will allow the analysis of the constituents of cells suspected or known to be involved in oncogenesis. In the technology, AFM tips pick up protein or nucleic acid probes from reservoirs, transfer them to a support, and deposit them at selected array positions. The process is repeated to complete a protein or nucleic acid array, which has submicron space between the array positions (e.g., a 100 x 100 array in about a 0.1 mm x 0.1 mm area). Target proteins are applied to the array, and the binding events of the arrayed molecules are monitored by AFM topography measurements. By carrying out these processes in a miniaturized format and on very small samples, the cost of the analysis will be minimized and the analysis rate will be maximized. Use of a multiple tip array further speeds the analysis rate. The proposed research consists of two phases. In the first phase (R21), we will demonstrate the manipulation of single molecules by the AFM tip and the detection of single molecular binding events by AFM topography measurement. A molecule involved in signal transduction will be selected and monitored by this technique. In the second phase (R33), we will extend the capability of the AFM technique by simultaneously monitoring different molecules associated with parallel signaling events. The immediate purpose of these studies is to determine the relative contributions of established parallel signaling pathways to the transmission of growth signals in cancer of transformed cells. Ultimately, this technique will be used to investigate and determine the assembly of multi-protein complexes isolated from cancer cells.
 
R21/R33 CA086305-02 2000 BROCK, GRAHAM J UNIVERSITY OF GLASGOW IDENTIFICATION OF M5CPG ALTERATIONS IN BREAST CARCINOMAS
The proposed research will identify modifications to specific CG- rich DNA sequences that occur at the earliest stages of development of human breast cancer. Identification of the scope and scale of these modifications will enable earlier diagnosis and thus increase the efficacy of subsequent treatment. Modifications (specifically changes in methylation) to CpG Islands (CGIs)occurs during the development of a significant subset of breast cancers. These changes affect the regulation of the gene associated with the CGI. Modifications to methylation patterns have also been reported in other CG-rich regions, for example in satellite or repetitive DNA. Disrupted methylation patterns in these sequences may be an early indicator of an aberration in normal cellular functions. Such alterations may either be a primary cause of malignant transformations or occur as a secondary effect. However once established these changes to the expression patterns are faithfully reproduced in progeny cells. A Methyl Binding Domain (MBD) column will isolate all the CG-rich methylated sequences from samples of normal tissue. Subsequent isolation of CG-rich methylated sequences will be from neoplastic samples at an early stage from the same tissue type. In an additional step a process of subtractive hybridisation will isolate those CG-rich sequences, which display divergent methylation patterns between the two types. The sequences will then be examined using a variety of molecular techniques to identify both the numbers of methylation changes during tumour progression and the earliest modification (s).
 
R21/R33 CA084713-03 2000 CHANCE, MARK R ALBERT EINSTEIN COLLEGE OF MEDICINE YESHIVA UNIVERSITY DYNAMICS OF MACROMOLECULAR INTERACTIONS
Deriving detailed structure and dynamics information for macromolecules and their complexes is a challenging and important step towards providing a molecular understanding of normal and malignant cells. New techniques of spectroscopy, microscopy, and crystallography have been developed over the last 10 years to address these questions. However, understanding the structure and dynamics of macromolecular assemblies is a problem of increasing importance. Although existing biophysical methods, such as fluorescence, single molecule techniques, crystallography and NMR have advanced our understanding greatly, it has been difficult to achieve high structural resolution, fast time-resolution, and the ability to monitor large assemblies while utilizing small amounts of precious samples. Time-resolved synchrotron x-ray footprinting is a relatively new technique developed to study the dynamics of nucleic acids. The method probes the solvent accessible surface of macromolecules and their complexes using hydroxyl radicals. The technique is coupled to stopped-flow initiation of reactions, and dynamics on timescales as fast as 5 milliseconds have been probed for nucleic acids. The technique has not yet been applied to analyzing the dynamics of proteins and their assemblies, or their complexes with nucleic acids. In the R21 phase of this proposal, we will develop a quantitative hydroxyl radical "footprinting" technique using synchrotron radiation to probe the structure of proteins. Successful examination of protein folding and protein-ligand complexes will provide milestones for the transition to the second phase of the grant. In the R33 phase, we will further develop the footprinting techniques to allow detailed examination of interactions of large macro molecular complexes. Specifically, the focus will be methodologies to examine the detailed pair-wise interactions of large binary complexes from the individual perspective of each member of the pair. The model systems used to develop the technology include: 1) examining the orientation and binding of cofilin and actin, 2) examining the time-resolved dynamics of actin filament disassembly catalyzed by gelsolin, and 3) probing the time-resolved dynamics of reverse transcriptase. These model systems will "drive" the technology to provide general methods relevant to studying a wide range of problems in cancer biology. Time-resolved protein footprinting, when perfected, will be applicable to studying macromolecular interactions critical to replication, transcription, signal transduction, translation, processing, and secretion.
 
R21/R33 CA088197-02 2000 CHEE, MARK S. ILLUMINA, INC. PROTEIN PROFILING ARRAYS
This proposal seeks to develop a new bead-based array technology for simultaneously measuring many proteins and their post-translational modifications in small volumes of cells or biological fluids. Technology of this type is needed to accelerate research in functional genomics by enabling molecular phenotyping of proteins, for many individuals in large populations. Methods will be developed that allow multiplexed immunoassays to be carried out on populations of beads, with each bead type in the population being specific to a particular immunoassay. Basic and clinical applications of this technology will enable careful examination of the molecular basis of cancer and an ability to identify individual characteristics that influence cancer development and progression.
 
R21/R33 CA088327-03 2000 CHURCHILL, GARY A JACKSON LABORATORY EXPERIMENTAL DESIGN & ANALYSIS FOR EXPRESSION MICROARRAY
This first phase of this project will develop statistical methods for the design and analysis of gene expression microarray experiments based on statistical principles. Our experimental designs will allow a scientist to carry out classical (ANOVA style) experiments using microarray technology. Designed experiments offer quality control, normalization and a method of analysis that takes multiple sources of variation into account. New analysis tools will be developed based on the experimental designs and on our experience with array data generated in this phase of the project. We will establish the basic infrastructure, including informatics tools, for microarray analysis at the Jackson Laboratory. We will develop a detailed strategy, that is both robust and efficient, for a large scale survey of gene expression in tissue samples obtained from mouse models of mammary and ovarian cancers. The second phase of our project will involve carrying out this survey with the goal of classifying and charactering tumor types based on expression data. The expression data will be analyzed together with available information on the pathology of the tumor and the genetic background in which the tumor type arises. The ultimate goal of this project is to provide a sound statistical basis for the routine practice of expression profiling of mouse tumors. It is our premise that experimental design and design-based analysis tools are essential to obtaining high quality expression information at a reasonable cost.
 
R21/R33 CA086248-02 2000 DABORA, SANDRA L BRIGHAM AND WOMEN'S HOSPITAL MUTANT ANALYSIS OF TSC1 & TSC2 IN TSC RELATED DISORDERS
The goals of this proposal are to develop robust, sensitive, and high throughput methods for mutation analysis for the tumor suppressor syndrome, tuberous sclerosis complex (TSC). These methods will be immediately useful for clinical purposes but will also be valuable research tools for analysing the molecular pathology in TSC and related disorders. The technology developed during this project will be directly applicable to the study of genetic variation in other disorders as well. TSC is a familial tumor syndrome characterized by the development of benign tumors (hamartomas) in multiple organs. Although it is inherited in an autosomal dominant pattern, the majority of new cases (about 65 percent) are sporadic without antecedent family history. Penetrance is high but expression is variable with both severely and mildly affected individuals. Organs most frequently involved are the brain, skin, kidneys and heart. Neurologic morbidity from seizures, mental retardation and behavior disorders is common. Two disease genes, TSC1 and TSC2 have been identified recently. Unfortunately the development of a genetic test has been hindered by the large size of the two genes and the diversity of the mutation spectrum in TSC. The R21 portion of this grant focuses on the development and optimization of a series of 3 assays which will allow for high throughput comprehensive genetic analysis for TSC. The R33 portion of this proposal focuses on using comprehensive mutation analysis for TSC to collect genotype and clinical data on a large cohort of TSC patients. Comprehensive mutation detection methods will also be used to study the role of TSC1 and TSC2 in related disorders as well as in TSC lesions and related non-TSC tumors. There is currently much demand from TSC patients and families for comprehensive mutation analysis for family planning and prenatal diagnosis, diagnosis confirmation, and prognostic information. The proposed plan of study will address this issue as well as help elucidate the role of TSC1 and TSC2 in the molecular pathology of TSC lesions and non-TSC tumors.
 
R21/R33 CA083232-03 2000 ELLINGTON, ANDREW D BRANDEIS UNIVERSITY APTAMER BASED DETECTOR TO QUANTIFY MACROMOLECULES
We propose to develop a technology to simultaneously detect and quantify large numbers individual proteins and post-translationally modified proteins. This technology will be based on a spatially-arrayed aptamer microchip and sensitive fluorescence-based optical detection techniques. The system will provide a powerful new tool for biomedical research and diagnosis of diseases and cancers. Currently, the levels of cellular proteins are inferred from cellular mRNA levels by using DNA oligonucleotide microchips. These microchips allow the detection of mRNA levels by base-pairing interactions to homologous DNA oligomers located at different sites in the microchip array. However, this indirect assay of protein levels suffers from two problems: First, mRNA levels may not accurately reflect the level of encoded protein because translation and degradation rates vary from protein to protein and are regulated in response to cell physiology. Second, post-translational modifications proteins often alter critical protein activities. This technology we propose to develop is comprised of three parts: 1) Aptamers with high-affinity and high-specifity for individual proteins. These aptamers will be designed to have ligand-dependent changes in fluorescence properties. 2) A microchip array with each array site occupied by aptamers against a different target molecule. 3) An evanescent field fluorescence detector which detects target molecules binding to aptamers at the array sites. Our proposal includes plans for: 1) selection of aptamers that can discriminate between different post-translational modifications of the same protein; 2) development of fluorescence-based techniques for detecting oligonucleotide:protein interactions; 3) design and construction of an optimized fluorescence detector; 4) scaling up methods to allow large numbers of independent aptamer selections against different proteins; 5) construction of a prototype protein chip.
 
R21/R33 CA084740-02 2000 ENGELWARD, BEVIN P. MASSACHUSETTS INSTITUTE OF TECHNOLOGY FLUORESCENCE DETECTION OF GENETIC INSTABILITY IN MICE
Recombinational repair protects against the cytotoxic effects of DNA damaging agents. However, a side effect of mitotic recombination is that it can also promote deletions and gene conversion events that contribute to tumorigenesis. Thus, it is critical to develop our understanding of the genetic and environmental factors that trigger mitotic recombination. Advanced tools are currently available for studying point mutations in mammals (e.g., the Big Blue mouse). However, an analogous high-throughput system for studying mitotic recombination in mammals is not yet available. Each of the few systems currently available for studying mitotic recombination in mice has a severe limitations, such as tissue type restrictions, requirement for high numbers of animals, and lack of sensitivity. Here, we propose to combine genetic engineering with mechanico- optical engineering to develop the technology to rapidly quantify homologous recombination events in multiple mammalian tissues types in situ. Mitotic recombination will be detected by reconstitution of expression green fluorescent protein in an engineered segment of DNA. Using high-throughput two-photon microscopy and state of the art computational analysis, it will be possible to quantify recombinant cells in diverse mouse tissues in situ in a matter of minutes per sample. This mouse system will provide an invaluable tool for identifying the genetic and environmental factors that modulate cancer-causing mitotic recombination events in mammals. Furthermore, the appearance of recombinant cells can be monitored over time in skin, thus revealing the differential susceptibility of stem cells and transition cells. Yet another important application will be in studying the effects of cancer chemotherapeutics on mitotic recombination and in determining how specific genetic traits effect cellular susceptibility chemotherapy-induced mitotic recombination. It is hoped that this line of research will ultimately aid in pharmacogenomics. The broad long term objective of this work is to develop technology that will improve our understanding of the genetic and environmental factors that cause cancer in people and improve our ability to develop better anti-cancer regimens.
 
R21/R33 CA083220-02 2000 FORTINA, PAOLO M CHILDRENS HOSPITAL OF PHILADELPHIA FOUR COLOR ARRAY BOUND SNP/MUTATION DETECTION IN CANCER
Development of cost-effective, high-throughput sampling coupled to rapid, parallel DNA profiling methods will facilitate molecular analysis of normal and diseased states. This R21/R33 initiative focuses on detection of single nucleotide polymorphisms (SNPs) and point mutations in cancer. Our goals are as follows: Milestones/Aims for the R21 phase include: 1) Development of a robust, 4-color rhodamine BigDye terminators-based single nucleotide extension (SNE) array assay which will allow DNA TaqFS-catalyzed, single nucleotide extension to detect a wide variety of SNPs/mutations associated with human cancer. We will increase signal intensity as well as eliminate the need for synthesis of costly spacers on array-bound probes by using dendrimer-coated surfaces; and, 2) Scale-up the assay to simultaneously monitor 30-50 SNPs/mutations on a single array. Multiplexing will be done both at PCR and SNE steps and use of total genomic DNA targets will be tested. Strategies will also be implemented to screen multiple individuals on a single chip for the same mutation. Aims for the R33 phase include: 1) Application of SNE assay to type SNPs within and flanking the minimal deleted region in lp, llq and 14q in neuroblastoma pediatric patients. The assay will facilitate identification of deletion location and extent and eventually will facilitate understanding the relationship between genotype and phenotype; and 2) Identification of single-base changes occurring within the mutation cluster region of adenomatous polyposis coli (APC) gene in patients with familial adenomatous polyposis (FAP) and in sporadic colorectal cancer. We will also evaluate genotype/phenotype relationships using information from SNE-based mutation analyses and results from array-based genome-wide mRNA expression profiles already funded by an NCI Contract. Array-based results generated from Aims 1 and 2 will be compared and contrasted to data available from previously typed individuals using more traditional DNA analysis approaches as well as to solution-phase results generated at PE-Biosystems. Development and validation of array-bound approaches for monitoring DNA changes in cancer should facilitate high throughput, parallel processing of samples for critical human cancer genes. In addition, it will help establish basic knowledge required to rapidly define the molecular basis of specific malignancies and eventually provide a foundation for rational molecular assessment of therapeutic.
 
R21/R33 CA088547-02 2000 FRESCO, JACQUES R PRINCETON UNIVERSITY FLUORESCENT TISH PROBES FOR CANCER REVELANT SEQUENCES
Fluorescent TISH Probes for Cancer-relevant Sequences Gene amplification, substitution and deletion mutations are associated with the molecular pathology of various malignancies, including breast, cervical and gastric cancers, as well as colon and lung cancers and tumors of the nervous system. Multidrug resistance of cancer cells is also associated with gene amplification. Amplification of erbB-2 (HER-2/neu) and N-myc genes is particularly correlated with poor prognosis in breast and cervical cancers and neuroblastoma, respectively. Methods that could simply and reliably detect such aberrations and quantitate them can therefore be of great value for diagnosis, for following the efficacy of treatment, and for reliable prognosis. Currently, detection of point-mutational events represents a major experimental effort. In the case of gene amplification as well, the methods are arduous, require relatively large samples, and they are of variable reliability. This project has a two-fold aim. One is to apply the methodology of TISH (third strand in situ hybridization of fluorescent probes via triplex formation, which avoids the need for DNA denaturation) for the cytogenetic quantitative analysis of these aberrations. TISH has three major advantages: greater sensitivity and quantitative reliability, and importantly, greater sequence specificity. The second parallel aim is to develop suitably intense and non-quenching fluorescent TISH probes. For this purpose, a major effort will be mounted to develop dendritic nuclei with fluors attached by rigid linkers that prohibit their interaction, therefore preventing the quenching of fluorescence. In this way, it is hoped to enhance the sensitivity of fluorescent probe detection by 1-2 orders of magnitude, thereby assuring that TISH probes can be reliably used for analyzing amplified genes and ultimately for detection of mutations in single-copy genes in situ.
 
R21/R33 CA088351-02 2000 FU, XIANG-DONG UNIVERSITY OF CALIFORNIA AT SAN DIEGO CANCER CLASSIFICATION BASED ON MRNA ISOFORMS
Messenger RNA precursors are often processed through alternative splicing into multiple mRNA isoforms, which in many cases encode proteins with distinct functions. It is estimated that as many as 30 percent of genes in humans exhibit alternative splicing and many alternative splicing events are known to be associated with human diseases. However, the current technology to analyze alternative splicing is slow and labor-intensive. Here we propose to develop a novel system to detect alternative splicing on a large scale and apply the technology to the molecular classification of cancer. Our experimental strategy is to synthesize DNA oligos that are selectively hybridized to spliced mRNAs through specific splice junction sequences. Each oligo will be linked to a unique 20 nt sequence to serve as an addressable "zip-code". Pooled oligos will be hybridized to total RNA from cells or tissues, and those annealed to mRNAs will be isolated by poly (A)+ selection followed by PCR-amplification. The products will then be hybridized to a UNIVERSITYersal zip-code array manufactured by Illumina based in San Diego. Individual splicing events will then be detected and quantified by imaging the array. This technology is different from any existing methodology in several important aspects: (1) Our experiments are designed to detect mRNA isoforms, rather than overall gene expression. This will allow us to specifically relate the post-transcriptional processing step to important biological processes such as cancer. (2) Our procedure, is highly scaleable, which will allow us to approach the alternative splicing problem at the genomic level. (3) Through a collaborative arrangement, we will have access to state-of-the-art array technology developed at Illumina, which is designed for large scale analyses in a "array of arrays" format. (4) Our strategy allows customization of the assay for specific applications and provides maximum flexibility in experimental design with minimal costs. We plan to approach the alternative splicing problem in a systematic way from database construction to assay development to application of the technology in cancer classification. For this purpose, we have assembled a team consisting of leading experts with highly complementary expertise. Because many alternative splicing events have been individually linked to specific cancer or cancer stages, it is almost certain that this combined approach will lead to efficient and accurate predictors for various diseases, which will be highly complementary to those based on monitoring gene expression. In fact, because alternative splicing is a general biological process, we believe that our technology will provide an urgently needed tool for both basic and clinical research, and therefore, will have a broad impact in many fields.
 
R21/R33 CA088323-02 2000 GALE, JAMES M UNIVERSITY OF NEW MEXICO MOLECULAR STAGING OF HUMAN SQUAMOUS CELL SKIN CANCER
Lifetime risk for developing skin cancer is currently l in 5 for the USA. While the non-melanoma skin cancers (basal and squamous cell) are generally not life threatening (2-5 percent of squamous cell carcinoma will become metastatic), they account for substantial morbidity and health-care expenditures. Our understanding of the molecular pathology and critical gene-networks involved with the development and progression of non-melanoma skin cancers is incomplete, resulting in poor markers for progression, prognosis and largely ineffective treatments for invasive stages. The broad objective of this proposal is to characterize the changes in gene expression in different stages of squamous cell carcinoma (SCC) to identify the different gene pathways involved. To characterize the most complete gene-expression profile currently possible, a set of reusable nylon microarrays containing 30,000 different human cDNA's (all of the approximately 7,000 "named" genes and EST's of unknown function) will be used. SCC is well suited for an initial human cancer study because (a) there is a better consensus about the clinical stages of the disease, (b) samples of SCC are relatively easy to obtain from the clinic, and (c) SCC is most associated with sun-exposure (most frequently found on sun-exposed skin and over 90 percent of SCC tumors have p53 mutations, mostly of the type caused by UV). Of the new technologies available, cDNA microarrays have ability to screen a huge portion of all human genes (30,000 of approximate 100,000 total human genes) with the expected potential to identify "all" the genes whose expression has been changed at different stages of cancer. The specific aim of the R33 component of this proposal is to profile the gene-expression levels in the three most distinct stages of the disease (actinic keratoses; a pre-malignant lesion, SCC and metastatic SCC) from 24 subjects (12 men and 12 women, 8 subjects in each lesion group). The necessary controls of normal-uninvolved skin will be obtained by punch biopsy from both the forearm (sun-exposed skin) and the buttocks (sun-protected skin). Having two normal skin controls will add power to the data analysis and help identify genes that are naturally responsive to sunlight (may be involved in early stages of skin cancer or show natural high variability in skin which would confuse the comparison with SCC lesions). Moreover, the potential power of reusable microarrays (multiple stripping and probing) is the ability to compare results between arrays. Once the variability-profile of comparing many microarrays is calculated (hybridization of 5 microarrays with the same probe, a milestone of this proposal), we will calculate the average and standard deviation for each of the 30,000 cDNA's for the sun-exposed skin controls and sun-protected skin controls form both the 12 men and 12 women in the study.
 
R21/R33 CA088364-02 2000 GASCOYNE, PETER RUSSELL CHARLES UNIVERSITY OF TEXAS MD ANDERSON CANCER CENTER INTEGRATED ISOLATION & MOLECULAR ANALYSIS OF TUMOR CELLS
Our goal for the combined R21 and R33 phases of this proposal is to make an integrated fluidic device that is capable of the automatic isolation of target cells from a cell mixture and their analysis for molecular markers for cancer detection and diagnosis. The final integrated device will be comprised of the following stages: (1) a cell separator able to process 2x106 cells/minute and to trap suspect tumor cells from them; (2) a cell fractionator that will separate suspect cells according to their dielectric and surface marker properties; (3) a microfluidic isolator stage that will collect and concentrate cell subpopulations emerging from the fractionator, combine them with a mixture of beads carrying multiple molecular probes, and burst them in the presence of the beads; and (4) an impedance sensor stage able to identify beads according to their dielectric fingerprints and thereby index them to the probes carried on their surfaces. Fluorescence of molecular probes on each bead will also be measured at this stage. By using a mixture of multiple bead types, several molecular assays will be run simultaneously. To achieve this goal we will develop and integrate fluidic and microfluidic technologies to automate the central problems of molecular diagnosis, namely the enrichment and isolation of a suspect cell subpopulation from a mixture of cells in a biopsy sample and the quantitative analysis of that subpopulation for molecular markers including proteins and mRNA's. The key technologies to be developed are: 1) A prefilter able to trap suspect tumor cells from large numbers of blood or lymph node cells; 2) A force balance method that exploits the dielectric and density properties, and, optionally, the immunomagnetic labeling properties, of target cells to enable the isolation; and 3) A dielectric indexing and manipulation method for carrier beads that, when combined with established molecular assays, will allow the simultaneous quantification of multiple molecular markers in parallel assays. The sample preparation aspects represent an extension of our successful dielectrophoretic trapping and dielectrophoretic field-flow-fractionation (DEP-FFF) methods that have been demonstrated for sorting and isolating different cell types from a cell mixture. The dielectric bead methods represent a novel approach to identifying individual molecular tests in a parallel molecular marker analysis scheme. Dielectric indexing of carrier beads will be used instead of the spatial indexing used on a gene chip. This will allow different subpopulations of beads, each carrying a different marker probe, to be identified and differentially manipulated by exploiting their dielectric signatures. The need to immobilize molecular probes in a tightly specified location on a fixed substrate, as demanded by spatial indexing is thereby eliminated and customized mixtures of probes, each carried on a separately-indexed bead type, may be identified as they emerge in a mixture from a reusable assay system. The study will be divided into a one year R21 phase and a three year R33 phase. During the R21 phase, proofs-of-concept will be developed for scaled-up dielectrophoretic trapping of suspect cells from large cell populations, for combined dielectrophoretic-magnetophoretic field flow fractionation, and for dielectric indexing of beads in mixed molecular assays. During the R33 phase, these technologies will be further developed and integrated in order to accomplish all steps of sample preparation and molecular analysis. The technologies will then be tested and refined using clinically relevant samples.
 
R21/R33 CA084641-03 2000 GIESE, ROGER WALLACE NORTHEASTERN UNIVERSITY CATION LABELING MASS SPECTROMETRY OF DNA ADDUCTS
We propose new methodology based on chemical labeling and mass spectrometry (MS) for the discovery and qualitative analysis of unknown DNA adducts in human samples. Novel, bulky, nonpolar cationic reagents will be synthesized and tested as labeling reagents for both nucleotide and nucleoside versions of DNA adducts. The primary purpose of the chemical labeling is to make all DNA adducts highly and equally (or near so) sensitive for analysis by MS. Especially laser desorption (including MALDI) but also electrospray forms of MS will be used. In the former case, large spot laser desorption on a Fourier transform mass spectrometer (FTMS) will be emphasized. The project is stimulated in part by our unpublished observation that bulky, nonpolar compounds that we have prepared are very sensitive by MALDI-MS. Potentially a DNA adduct labeled with a reagent of this type also can be detected with high sensitivity. If this can be demonstrated in an R21, then the ensuing R33 would involve further optimization of the technique, with eventual application in the project to DNA extracted from large, autopsy, human tissue samples to bring the overall methodology to full maturity.
 
R21/R33 CA086136-02 2000 GRIFFITH, JEFFREY K UNIVERSITY OF NEW MEXICO PROGNOSTIC VALUE OF TELOMERE DNA IN PROSTATE BIOPSY
Assessing prognosis of tumor growth potential in biopsy material is vital to choosing an appropriate therapy in prostate cancer. However, definitive molecular markers on which to base a prognosis are limited. Telomere DNA, the tandem six-base nucleotide repeats that cap and protect chromosome ends, are typically shorter in cancer cells. This suggests that telomere length may be an important determinant of tumor progression. A new slot blot assay which measures Telomere DNA Content (a proxy for telomere length) was developed by the Principal Investigator using tumor tissue. Results of this assay indicate that reduced telomere DNA content in breast cancer is correlated with aneuploidy (p less than 0.002) and metastasis (p less than 0.05) and in prostate cancer with reduced survival (p less than 0.004) and increased disease recurrence (p less than 0.0001). In Phase I, we will extend and refine the telomere DNA content assay to accommodate prostate needle-core biopsy specimens. In Phase II, we will utilize the assay to conduct a retrospective case-control study of archival prostate needle-core biopsies. The telomere DNA content data will be correlated to patient survival and the results evaluated to ascertain the predictive value of telomere DNA content in prostate biopsy samples.
 
R21/R33 CA088198-02 2000 HERLYN, DOROTHEE M WISTAR INSTITUTE MOLECULAR CLONING OF HLA CLASS II TUMOR ANTIGENS
Our major goal is to develop a novel technology to identify HLA class II tumor antigens using a unique cloning approach in filamentous phages which has not been applied to the cloning of class II antigens in the past. This approach has numerous potential advantage over existing approaches to class II antigen cloning or biochemical peptide isolation. The unique cloning approach proposed here will be applicable to a variety of tumor-associated HLA class II antigens derived from tumors of different histologic types, and also to class II antigens involved in infectious diseases. Active specific immunotherapy has great potential for the treatment of patients with melanoma or colorectal carcinoma (CRC). In light of the important role that CD4+, HLA class II-dependent helper T (TH) cells play in the control of tumor growth, approaches to active immunotherapy with TH cell-defined antigens need to be developed. Very few tumor- associated TH antigens or peptides with vaccine potential have been identified thus far. The long-term goal of the proposed studies is to identify TH antigens with vaccine potential for melanoma and CRC patients. The Th antigens are recognized by available Th lines and clones. A novel approach for the cloning of mammalian HLA class II-dependent Th antigens will be developed using filamentous phages. Tumor cells cDNA libraries will be expressed by the phages, followd by library phage presentation to TH cell by antigen- presenting cells and identification of the relevant Th antigen in cytokine release assay. To develop this approach, we have available a unique model system including Th cells against tetanus toxoid and a cDNA fragment encoding the tetanus toxoid-associated Th epitope. Specifically we will: 1) develop Th antigen cloning technology using filamentous phages and tetanus toxoid as a model system (R21 pilot study); and 2) optimize the filamentous phage HLA class II antigen cloning approach developed under the pilot study to clone melanoma and CRC-associated Th antigens; this will be followed by the identification and characrterization of these antigens (R33). The feasibility of the filamentous phage approach for the cloning of HLA class II antigens is emphasized by the demonstration of successful induction of antigen-specific, Th cell-dependent antibodies in mice by immunizing the mice with phages expressing various antigens. The proposed studies will develop a novel technology to identify HLA class II-dependent Th antigens using filamentous phages leading to novel approaches to active specific cancer immunotherapy using these antigens. The antigens also may be of diagnostic value.
 
R21/R33 CA086365-02 2000 HUNTER, IAN W MASSACHUSETTS INSTITUTE OF TECHNOLOGY ANDROGEN RECEPTOR BIOCHIPS--PROSTATE CANCER MANAGEMENT
Major recent advances in the clinical management of prostate cancer and in the molecular technologies available to dissect the action of the androgen receptor (AR) are re-shaping therapeutic goals, and new methods of monitoring patient hormonal status and response are needed. Rational design of new strategies for prostate cancer hormonal therapy and effective monitoring of patient response to hormonal manipulation is a priority area for clinical research, and one that is inadequately supported by current technologies. The major objective of the project described in this proposal is the design and fabrication of a battery of micro-scale yeast based bioassays or "biochips" that can be used to monitor AR ligands in sera and tissue extracts from patients with prostate cancer. Conceptually, this clinical application of yeast AR biochip technology is similar to the use of conventionally formatted yeast based assays in toxicological screens to detect environmental endocrine disrupters (or endocrine active compounds). However, the use of yeast based bioassays for steroid receptor ligands in a clinical environment is currently limited by the availability of human expertise and labor, the cost of reagents and the amount of biological sample. The MIT Bio-Instrumentation Lab (Professor Hunter's group) is currently actively engaged in the development of living chips, microdevices which use arrays of living cells for massively parallel detection of biochemical and biophysical events, including a two-dimensional microchannel array technology specifically for yeast-based assays used in drug discovery. In this grant we propose to adapt this technology for a particular clinical application: the automated screening of sera and tissue extracts for biologically active AR ligands. Specifically, we will use this new assay technology to measure changes in AR ligand profiles in prostate cancer patients as a function of clinical history, therapy, and changes in established indices of disease status. With the successful fabrication and validation of these yeast AR biochips, it will be possible to monitor one of the major biological determinants of prostate cancer progression - the bioavailable levels of androgen and other AR ligands - in a timely, cost effective manner.
 
R21/R33 CA086132-02 2000 MAJUMDAR, ARUNAVA UNIVERSITY OF CALIFORNIA BERKELEY OPTOMECHANICAL CHIPS FOR MOLECULAR PROFILING OF CANCER
The goal of this project is to develop the technology for biochemo-opto-mechanical (BioCOM) chips for high-throughput and highly sensitive proteomic and genomic analyses that are critical in early diagnosis, monitoring and prognostic evaluation of cancer. The chip will contain an array of pixels, with each pixel providing quantitative analysis for a certain analyte by producing a color-based optical signal. In contrast to existing microarray biochip technologies, the BioCOM chip would not require any external power, external or on-board electronics, or fluorescent dyes and associated optics for its operation, which will keep its costs low while keeping is simple to use in clinical research and laboratories. Each pixel of the BioCOM chip will contain an array of microcantilever springs, one surface of which will be derivatized with either an antibody coating for detecting cancer-associated antigens or a coating of single-stranded nucleic acid capture sequences complementary to cancer-associated mRNAs or mutant sequences. Recent experiments have confirmed that molecular binding on such derivatized cantilevers can generate sufficient chemomechanical force due to molecular conformational energy changes to produce an observable deflection of the cantilever springs. The underlying innovation in the BioCOM chip is to exploit this phenomenon using fabricated microstructures embedded in the springs that utilize interference and diffraction of background white light to produce iridescent colors in each pixel. Once optimized, the proposed technology will be able to screen literally hundreds of molecules, and these can be transcribed mRNAs or proteins. For protein expression, in particular, this technology would represent a new paradigm in the evaluation of multiple proteins from serum or from a single tissue, and could represent a cost-effective way to assess multiple cancer antigens in screening and monitoring programs. The approach is to first quantitatively demonstrate (R21 phase) the existence of chemomechanical signatures for two representative molecules - prostate specific antigen (PSA) and PSA coding DNA sequence. Subsequently, the R33 phase will involve the development of a comprehensive chemomechanical database for a large number of cancer-associated proteins and nucleic acid sequences. This, in combination with engineering mechanics of cantilever beams and wave optics of interference and diffraction, will be used to design the BioCOM chip. The chips will be microfabricated using processing techniques widely employed in microelectronics and microelectromechanical systems (MEMS). Optimization of the chips will occur through clinical tests by controlling the biochemistry of protein/DNA immobilization on cantilever surfaces and the mechanics and optics of cantilever beams.
 
R21/R33 CA083241-02 2000 MALKHOSYAN, SERGEI R SANFORD-BURNHAM MEDICAL RESEARCH INSTIT COMPARATIVE HYBRIDIZATION OF AP PCR ARRAYS
We applied an unbiased DNA fingerprinting technique, the Arbitrarily-Primed PCR (AP-PCR) to study tumor- specific genetic changes. AP-PCR is a PCR-based DNA fingerprinting method, which uses a single oligonucleotide primer of arbitrary sequence, and generates a profile of quantitative and qualitative differences between the fingerprints of tumor and matched-normal tissues. Our efforts to both automate the AP-PCR technique and make it more robust, gave us the idea of combining AP-PCR fingerprinting with DNA array hybridization technology. Developing this new technique, which we call Comparative Hybridization of AP- PCR Arrays (CHAPA), is the goal of this grant application. We propose to clone individual DNA fragments amplified by AP-PCR from human genomic DNA and array them on a solid base. The AP-PCR product can be viewed as a low- complexity representation of the genome. The array will be hybridized with AP-PCR products that are amplified from normal and tumor tissue DNAs, which have been labeled with green and red fluorescent dyes, respectively. The intensity ratio of the two colors at each hybridization spot will reflect tumor-specific losses, gains, or no change of corresponding genomic loci. For the Phase I application, we will develop a small array of 300 AP-PCR fragments and test the new technique to see if it is reproducible, sensitive, and reliable. Chromosomal and subchromosomal origins of the arrayed AP-PCR fragments will be determined by hybridizing the array with AP-PCR products from individual clones of radiation hybrid mapping panels. We will also test different arbitrary primers to obtain collections of AP-PCR products (representations) of the human genome with a degree of complexity that is optimal for a large scale array of AP-PCR fragments. We will also test AP- PCR's ability to produce quantitative fingerprints of genomic DNA isolated from minute amounts of fixed microdissected tissues. For the Phase II experiments, we propose to scale up the array to at least 5,000 nonredundant AP-PCR fragments. In its final form, the technique will allow the state of the tumor genome to be quickly and automatically analyzed with less than 1 Mbp of resolution. This high-density unbiased molecular karyotyping will facilitate the discovery of novel cancer genes and open new horizons for the diagnostic and prognostic analysis of cancer development.
 
R21/R33 CA084691-02 2000 MELDRUM, DEIRDRE R. UNIVERSITY OF WASHINGTON AUTOMATED MINIMAL RESIDUAL DISEASE QUANTIFICATION
Quantification of Minimal Residual Disease (MRD) is a general concern in oncology since this parameter is likely to give valuable information to clinicians to customize chemotherapeutic treatments and to anticipate possible relapses. An automated system will be developed for the quantification of MRD by using real-time PCR to quantify cancer cells in a background of non pathologic DNA. The system will be derived from an existing high-throughput sample handling system (developed by Meldrum and team) named Acapella. Acapella has a pipelined serial architecture which makes it possible to develop an adaptive PCR control algorithm ensuring a level of sensitivity and accuracy for the quantification results specified by the clinician. In the R21 phase of the project, a critical component of the system is the real-time thermocycler. It will provide DNA quantification results of greater precision than what is currently possible with commercial instruments such as the ABI PRISM 7700 Sequence Detector by taking advantage of a high performance custom fluorescence analyzer and sophisticated data analysis methods. The fluorescence analyzer will have a signal to noise ratio and a dynamic range of at least one order of magnitude larger than the optics used on commercial systems. This unique feature will permit precise measurements of the amplification kinetics during at least five cycles in the exponential phase of the reaction. The amplification yield will he derived from these data using statistical estimators customized to meet the requirements of real-time PCR data analysis. The sample DNA content will be derived from the amplification yield and the calibrated fluorescence measurements of the reaction kinetics. This new approach will make it possible to run series of real-time PCRs with more flexibility than would be possible if the assay was based on standard reactions required to have the same amplification yield as the clinical samples. PCR conditions will be adapted online without concerns about possible differences of amplification rate. The assay control algorithm will adapt the reaction DNA content, the primer selection, and the number of PCRs to meet the clinician requirements for particular patient DNA. During the R21 phase of the project a prototype of the real-time thermocycler will be developed to demonstrate the optic performance and the possibility to estimate the PCR amplification rates with 5% accuracy. A conceptual design of the fully integrated process from patient blood sample to MRD quantification data will be completed to allow assessment of expected performance, cost, and risks associated with the development of the fully engineered system. The development of the various hardware and software components along with their integration into the automated system will take place during the R33 phase of the project. Performance of the system will he evaluated on real biological samples provided by the UW Department of Laboratory Medicine. Results returned by the system will he compared with results of t(14; 18) PCR performed in this department with an ABI PRISM 7700 for the diagnosis of patients suffering from follicular lymphomas.
 
R21/R33 CA086135-02 2000 PALLAVICINI, MARIA G. UNIVERSITY OF CALIFORNIA SAN FRANCISCO PROTEIN SIGNATURES OF LEUKEMIC CELLS
The overall goal of this R21/R33 application is to develop and apply comparative "genome-wide" proteomic approaches to determine the extent to which protein/peptide signatures of malignant cells enhance information obtained from cytogenetic and histopathologic analyses. Specifically, we will evaluate the relationship between molecular cytogenetic aberrations and protein expression in myeloid leukemia and determine whether distinctive patterns of protein expression (e.g., protein/peptide signatures) predict treatment response. Two-dimensional (2-D) gel electrophoresis, time-of-flight mass spectrometry (MALDI-TOF-MS) and nanoliter/min flow rate electrospray mass spectromety (ESI-MS) will be combined in a novel approach to map low and high abundance proteins/peptides in cell and nuclear lysates from primary leukemic specimens. In the R21 application we will establish methodologies to optimize reproducible proteome sampling of clinical specimens. In the R33 application, we will pursue two parallel approaches, based on 2-D gel separation of proteins according to isoelectric points (pI) and mass, to investigate the proteome of clinical leukemic specimens. In one approach, we will quantify differentially expressed moderate-to high abundance proteins in cell and nuclei lysates from normal and leukemic specimens us sample processing and in gel protein detection optimized in the R21 application. In another approach we will create peptide maps of the entire 2-D gel of cell and nuclear lysates from clinical specimens to increase the dynamic range of protein measurements on 2-D gels by identifying low abundance proteins. Both of these approaches will be used to 1) establish the protein signature of primary t(15;17) APL specimens at diagnosis and relapse using low and high abundance protein/peptide maps and 2) determine the extent to which protein/peptide signatures of APL at diagnosis predict treatment response. Additionally, we will initiate studies to develop antibody-based reagents to test the clinical potential of the gel protein signature associated with poor prognoses APL. We anticipate that protein/peptide signatures will lend new insight into the physiologically active forms of proteins associated with molecular cytogenetic aberrations, increase understanding about regulators of tumor phenotype, and identify novel diagnostic and predictive tumor markers.
 
R21/R33 CA084697-03 2000 SHERRY, DEAN UNIVERSITY OF TEXAS DALLAS MRI AGENTS SENSITIVE TO BIOLOGICAL INDICATORS OF NEOPLAS
Rapidly dividing neoplastic cells characteristically have high metabolic rates, are O2 limited, and generate excess acid. Thus, physiological indices such as PO2, pH, and excess lactate are often targeted for detection and quantification as an index of tumor activity. Drugs that target cancer cells are often designed to take advantage of the unusually large pH gradient across the plasma membrane of neoplastic tissues, so knowing the extracellular pH status of a tumor prior to treatment would be extremely useful. Unfortunately, there is no simple, non-invasive method available to the clinician to monitor this biological indicator of cell activity. Numerous exogenous pH sensitive 'indicators' have been devised and demonstrated in animal models, yet none have so far have proven clinically useful in characterizing tumors in human studies. Our long-term objective is to develop pH and redox sensitive agents that report these biological indices by using the magnetic resonance (MR) signal of bulk water as an antenna. A new ligand was recently discovered that binds gadolinium in a macrocyclic cavity and protects a single metal ion-bound water molecule from exchanging rapidly with bulk water. This feature, historically considered detrimental to MR contrast agent design, appears to have beneficial characteristics for making biologically responsive or functional agents. The purpose of this grant is to explore the extent to which this property can be utilized in the design of agents that report pH, redox, and perhaps other biological indicators of cell activity directly in a MR image. Two classes of agents will be examined. In the first, proton exchange between a slowly exchanging gadolinium-bound water molecule and bulk water has been shown to be pH dependent in a model system. One goal here is to fine tune that system to generate the most efficient (high relaxivity), pH sensitive, contrast agent. A second goal is to use other lanthanide ions having smaller magnetic moments to prepare systems whereby magnetization is transferred via selective, single frequency, presaturation of the bound water resonance. This new type of MR contrast agent offers the promise of a direct quantitative measure of pH or cell redox without a separate measurement of agent concentration. If successful, both types of agents could be readily applied in human cancer detection and characterization using conventional, clinical MR imaging equipment.
 
R21/R33 CA086340-02 2000 SMITH, RICHARD D BATTELLE PACIFIC NORTHWEST LABORATORIES HIGH THROUGHPUT FTICR FOR MOLECULAR ANALYSIS OF CANCER
In this four year two phase (R21/R33) project we will integrate and apply new approaches for obtaining broad systems level views of protein expression in cancer research. The overall approach will advance the study of proteomes by more rapidly identifying proteins, precisely measuring the relative abundances for all detected proteins, and providing much greater sensitivity than existing methodologies. Our approach will utilize proteome-wide stable isotope and biotin labeling of cysteine-containing polypeptides combined with new approaches that use ultra-high sensitivity Fourier transform ion cyclotron resonance mass spectrometry. The approach will be at least 2 to 3 orders of magnitude more sensitive than existing 2-D PAGE methodologies and able to rapidly identify and measure relative expression levels for thousands of proteins in a single analysis. Phase 1 of this project will integrate and provide an initial demonstration of methods that include the sample processing for mouse B16 melanoma cells from culture, validate the use of new accurate mass tag and multiplexed-MS/MS methods for protein identification, and demonstrate the precise determination of relative protein abundances for all detected proteins from B16 and B16BL6 cell populations. Phase 2 will involve the pilot application of the technology to the study of proteome changes that occur as cells progress from low or nonmetastatic states, to a highly invasive and metastatic phenotype, using the B16 melanoma system as a model. The technology to be applied will enable ultra-sensitive (attomole level, and anticipated to be better) proteome-wide precise profiling of proteins from cells maintained in culture and from tissues (obtained by micro-dissection). The results will provide an abundance of new information on protein expression, and enable precise measurements of differences in relative protein expression levels as a function of cell type, developmental stage, metastasis, etc. A product of this research will be the first application of a high throughput technology for obtaining precise proteome displays that may be expected to illuminate the complex mechanisms and pathways relevant to cancer.
 
R21/R33 CA088335-03 2000 SUTHERLAND, JOHN CLARK BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB DNA DAMAGE QUANTITATION BY SINGLE MOLECULE LASER SIZING
Using single-molecule sizing by laser-induced fluorescence from DNA moving through a microfabricated channel, this project will develop novel technologies for quantifying DNA damage produced by ionizing radiation and other carcinogens. Damages include DNA strand breaks and oxidative damage to DNA bases, and clusters containing such lesions. This technology will reduce the cost and improve the sensitivity and throughput for quantifying DNA damage. It will quantify low levels of damage and can be applied to DNA damaged in situ, thus facilitating basic research on DNA damage and repair and the relationship of these processes to mutation induction and carcinogenesis. Potential clinical applications include determining the ability of normal and tumor cells to repair damage, thus permitting identification of individuals who may be at elevated risk or the optimum agents to use against a particular cancer. In the R21 phase, we shall assemble a laser system, optics and microfluidic DNA transport system and demonstrate its ability to count single DNA molecules and accurately determine their size. In addition, we shall demonstrate the ability of this system to determine the number average molecular lengths and frequencies of ionizing radiation induced strand breaks in populations of DNA molecules of known lengths. In the R33 phase, the emphasis will shift to focus on the human DNAs from cells and tissues found both in research and clinical settings. We will work with the much larger DNA molecules available from human cells, because the sensitivity of lesion detection increases with the size of the molecules that can be analyzed. We shall also extend the range of lesions that can be quantified, with particular emphasis on double-strand breaks and multiply damaged sites containing heterogeneous mixtures of strand breaks, oxidized bases and adducts, which are difficult for cells to repair accurately. We shall also work towards reducing the quantity of DNA required for a determination of lesion frequency to the level found in individual human cells. The basic technology for single-molecule sizing will be advanced in the R33 phase, first by improving the sensitivity of detection of fluorescence from single DNA molecules by photon counting and phase-sensitive detection. Improvements in fluorescence detection achievable with near-infrared fluorescence will be studied, and near-infrared fluorescent DNA labels developed, leading to a compact system optimized for high sensitivity and throughput and low cost.
 
R21/R33 CA088365-02 2000 TUNG, CHING-HSUAN MASSACHUSETTS GENERAL HOSPITAL IN VIVO IMAGING OF MATRIX METALLOPROTEASE
The ability to detect specific enzyme activities in vivo would have far reaching applications in diagnosing, characterizing and assessing novel treatments of cancer. We have recently developed and validated imaging probes for the in vivo sensing of specific proteases and have targeted enzymes that play key roles in different aspects of cancer growth, metastases formation and angiogenesis. The probes are based on 1) biocompatible long circulating graft copolymers which are efficiently internalized into tumor cells and 2) contain autoquenched near infrared fluorochromes (NIRF) positioned on cleavable peptide stalks attached to a delivery graft copolymer. When the peptide substrate is cleaved, released fluorochromes become highly fluorescent (up to 350-fold signal amplification documented so far). The overall goal of this proposal is to develop and test matrix metalloproteinase-2 (MMP-2) specific probes since the enzyme is prominently involved in angiogenesis and metastagenesis. During the R21 phase of the grant proposal (year 01), we will scale up the synthesis of a prototype MMP-2 probe, characterize it and perform key in vivo imaging experiments in a murine tumor model. In the subsequent R33 phase of the proposal (years 02-04 ) we will pursue the following aims: 1) optimize the substrate using combinatorial peptide libraries to further increase signal amplification; 2) test the in vivo imaging approach using different NIRF imaging systems; and 3) test the probes and imaging techniques during therapy in models of ovarian cancer disseminated to the peritoneal cavity. Regarding the latter we will ask the following clinically highly relevant questions: 1) can the methodology be applied to detect micrometastatic serosal tumor implants and 2) can the efficacy of MMP inhibitors be imaged at the molecular level before phenotypic changes become apparent? This proposal represents a multidisciplinary effort in developing a novel approach to detect minimal residual tumor and assess anti-MMP-2 treatment of cancer at the molecular level. The approach, if successful, is expected to have broad applications to a wide variety of biologic, immunologic, and molecular therapies designed to promote the control and eradication of cancer.
 
R21/R33 CA088343-03 2000 WANG, BINGHE NORTH CAROLINA STATE UNIVERSITY RALEIGH FLUORESCENT TAGS TARGETED ON CELL SURFACE CARBOHYDRATES
Malignant transformation is often associated with alteration of cell surface carbohydrates. The expression or over-expression of certain carbohydrates, such as sialyl Lewis X (sLex), sialyl Lewis a (sLea), Lewis X (Lex) and Lewis Y (Ley), has been correlated with the development of certain cancers. These cell surface carbohydrates can be used for cell-specific identification and targeting of carcinoma cells. The long-term goal of this project is the development of small molecule artificial receptors which can recognize target carbohydrate structures with high selectivity and affinity. Such receptors could be used for the development of fluorescent tags for cell- specific identification, tissue-specific imaging (such as MRI), and targeted delivery of therapeutic agents. In this study, we will use sLex as the model carbohydrate and use colon cancer as the model biological system because the expression of sLex is often associated with progression and metastasis of colon cancer. The short-term objective of this application is to develop tissue-specific fluorescent tags (sensors) which can recognize sLex with high affinity and selectivity. For the construction of such fluorescent sensors, we plan to use an integrated approach combining template-directed synthesis, combinatorial chemistry, and computer molecular modeling aided design. The sLex-specific artificial receptors have the potential to be used for cell identification, detection and tagging for the purpose of localization, staging, tissue biopsy, and fluorescence-directed surgical removal of colon cancer cells. Such tissue-specific compounds could also serve as vehicles for targeted delivery of cancer chemotherapeutic agents. These small molecule sensors may also have the following advantages over antibody-based detection/delivery systems: (1) greater stability during storage and in vivo; (2) increased permeability through biological membranes and, therefore, enhanced target accessibility; (3) intrinsic sensitivity to binding with significant fluorescence intensity increases, making detection and visualization easier and more suitable for high throughout screening, and (4) lower propensity to elicit undesirable immune responses. Similar methods, once developed, could also be used for the construction of fluorescent tags for other cell surface carbohydrates implicated in human malignancies.
 
R21/R33 CA088258-02 2000 WEIER, HEINZ-ULRICH G. UNIVERSITY OF CALIFORNIA-LAWRENCE BERKELEY LAB SPECTRAL IMAGING FOR PHENOTYPE ANALYSIS OF CANCER CELLS
This proposal seeks to develop a novel screening system to support cancer diagnosis, tumor staging, prognostication based on the measurement of gene expression at the RNA level. The underlying hypothesis hat tumors progress because of accumulation of genetic changes, and that the genetic changes manifest themselves in altered and measurable expression of one or several genes. Current techniques limit the measurement of RNA expression levels to a few genes per experiment. Sensitive tumor cell detection and prognostication will require methods that simultaneously profile the RNA expression levels of five or more suspected oncogenes/tumor suppressor genes. Furthermore, if an oncogene is overexpressed, then its gem dosage should be determined as well. The quantitative analysis of several marker genes is crucial for sensitive tumor cell detection and accurate cell classification as well as the development of novel anti-tumor strategies. This information should help discriminate benign vs. malignant neoplasms and define prognostic markers. We will develop multicolor fluorescence in situ hybridization (FISH) in conjunction with Spectral Ima (SIm) to perform these measurements. Existing SIm instrumentation can record fluorescence spectra from nm to 1100 nm with about 10 nm resolution. This allows the unique labeling and detection of eDNA probe with commercially available fluorochromes. SIm technology can be modified to investigate the correlation cancer gene expression with tumor progression. Spatial co-localization and spectral overlap will be add by software processing of digitally recorded images, termed "spectral un-mixing" (SUN). Using artificial mixtures of cells from existing thyroid tumor and breast cancer cell lines, we will develop the software modules needed to measure intracellular levels of multiple RNA species in the same cell and to determine the assay sensitivity, accuracy, and reproducibility. The work will be extended to thyroid and breast tumor tissues increasing the number of hybridization targets and correlating gene rearrangements/amplifications with gene expression. This technology, when applied biopsy tissue from fine needle aspirates, could help diagnose and predict the course of suspicious cells in a rapid, inexpensive, and minimally invasive manner.
 
R21/R33 CA088326-02 2000 WEISSMAN, SHERMAN MORTON YALE UNIVERSITY DISPLAY METHODS FOR SURVEYING MUTATIONS
We propose to develop a gel based approach that would display most of the single base substitution mutations present in any mRNA expressed in a cell type. The method would permit recovery of bands for sequencing and identification of the sites of mutation. We describe the application of this approach to the detection of somatic mutations in malignant cells. In Phase 2 we propose to apply the method to malignant melanomas, colon carcinoma, acute Myelocytic leukemia and myelodysplasia. In addition, we propose protocols for improving the sensitivity of the method, for extending it to small tissue samples, and for detecting mutations in a small fraction of cells in a sample.
 
R21/R33 CA081674-02 2000 WIGLER, MICHAEL H COLD SPRING HARBOR LABORATORY HIGH DENSITY GENE COPY NUMBER MICROARRAYS
We propose to expand a pilot project to develop a high density gene copy number micro-array based on low complexity genomic representations. Such a tool will lead to improved classification of cancers, which will likely impact all areas of cancer diagnosis and treatment, and be an enormous boon to the discovery of cancer causing genes. We are poised to scale up from arrays of a thousand probes to sets of probes in excess of 30,000 that can be rapidly mapped to very high resolution in array format. Our method will be able to resolve changes in the genome with a resolution of every 50 to 100 kilobases. Moreover, we believe that we can significantly enhance the closure of human genome sequencing project by providing independently derived BAC contigs and probes for gaps in the existing BAC maps.
 
R33 CA084698-01 2000 AEBERSOLD, RUEDI H. UNIVERSITY OF WASHINGTON ISOTOPE CODED AFFINITY TAGS FOR QUANTITATIVE PROTEOMICS
Global genome initiatives including the Human Genome Project have generated enormous amounts of information, spurned new technologies and catalyzed the emergence of a new type of biology which attempts to build biological knowledge from the global analysis of biological systems, pathways and cells. In this program we propose to develop a novel technology for the quantitative and global analysis of protein expression profiles in biological samples including biochemical and subcellular fractions, biological fluids, cells and tissues. Thus, this technology will extend the global approach to biological research to the analysis of proteins, the molecules that UNIVERSITYersally constitute the structure, function and control of biological systems. The basis of the technology is a new class of reagents we term isotope coded affinity tags. The tags which are introduced to specific functional groups in proteins post isolation serve as both, ligands for the isolation of tagged protein segments as well as a quantitative code which can be mass spectrometrically deconvoluted. The proposed program has three distinguishing features which satisfy the request for innovative technology development. First, the technology explores a new approach to quantitative protein analysis and has the potential to become the technical foundation of the emerging field of proteomics. Second, the research will be conducted by a multidisciplinary team constituted as an academic/industrial partnership. This partnership is expected to accelerate dissemination of the technology by developing, commercializing and supporting the required chemistries, instrumentation and software as mature, integrated products. Third, the research will be conducted in a unique research environment, the new Institute for Quantitative Systems Biology at the UNIVERSITY of Washington. The structure of this institute embodies the evolution of a technology from its inception to the integration into biological research programs and to the support of large-scale applications. The Institute also provides the interdisciplinary research environment and the facilities required for the academic/industrial partnership to flourish. Once developed, such a technology will be an essential tool for biologists' attempts to interpret the linear information of genomes in terms of function, control and mechanisms of biological systems. Applied to cancer research the method will permit the quantitative measurement and identification of the molecules that distinguish a particular cancer cell from a normal cell. The technology will thus significantly contribute to the molecular diagnosis, the assessment of the cancer risk and prognosis, and the understanding of the molecular basis of cancer.
 
R33 CA088303-01 2000 CONTAG, CHRISTOPHER H STANFORD UNIVERSITY VISIBLE ANIMAL MODELS OF NEOPLASTIC DISEASE
Imaging reporter gene expression in living animals provides critical spatiotemporal information about changes in cell growth, cell trafficking and gene expression during normal and disease processes. In addition to enabling non-invasive in vivo assays for cell migration and function, these bioluminescence-based methods permit real time analyses of gene expression patterns in neoplastic and normal cells. We have already shown that marking and transferring tumor cells with informative reporter gene constructs and creating transgenic mice expressing these constructs provides a clear, time-ordered view of in vivo tumor growth and of normal cell responses to stimuli. We propose here to add another dimension to this powerful in vivo assay technology by coupling it with equally powerful fluorescence methods for ex vivo identification of cells in suspension or tissue sections. Together, these technologies enable development of wholly new and vastly more effective methods for evaluating and improving cell-based and other anti-cancer therapies. In our first steps towards development of this dual methodology, we generated an unparalleled description of tumor-host immune interaction based on real time observations of trafficking and proliferation of immune and tumor cells in intact animals. Now, continuing along this path, we plan to capitalize on the ex vivo subset-discriminating capabilities of the multiparameter fluorescence-activated cell sorter (FACS) by developing multifunctional reporter genes (fusion proteins comprised of luciferases and fluorescent proteins) that are detectable in vivo by bioluminescence and ex vivo by fluorescence. By creating tumor cells and transgenic mice expressing these constructs, we will be able to monitor migration of cells into a tumor site (by imaging) and to characterize the cells at the site (by FACS). In addition, by creating appropriate constructs, we plan use this dual methodology in a novel "gene-trap" strategy that will enable isolation and identification of genes that are turned on or turned off in tumors and responding host cells. We will perfect and demonstrate these technologies here in three systems, one in which a tumor is killed by co-transferred natural killer cells, a second in which B cells naturally give rise to CLL-like tumors, and a third in which a radiation-sensitive B cell tumor is induced to give rise to radiation-resistant variants. The overall technology that we develop through these applications will enable high throughput analyses of immune cell and tumor interactions, rapid localization and genetic characterization of defined subsets of transferred immune cells active against tumors, and the facile identification of molecular determinants involved in potential tumor evasion from immune surveillance. 1 R33 CA88303-01 -3- Christopher Contag, Ph.D.
 
R33 CA084699-01 2000 GAASTERLAND, THERESA ROCKEFELLER UNIVERSITY TARGET SELECTION FOR THE STRUCTURAL GENOMICS OF CANCER
The broad aim of this proposal is to facilitate structural and functional genomics of cancer. The specific aims are to develop and apply computational tools for (i) identifying and annotating cancer-related protein sequences; (ii) prioritizing target proteins for the structural genomics of cancer; and (iii) maximizing structural information about cancer-related proteins. The first aim will be achieved by collecting cancer-related protein sequences from The Cancer Genome Anatomy Project at NCI and by identifying additional such sequences in the databases of metabolic and signaling pathways, and primary sequence databases. Proteins that occur in the same pathway or have similar regulatory patterns as cancer proteins, proteins that interact with cancer proteins, or proteins whose expression shares features with that of the cancer proteins will also be considered as cancer- related proteins. Queryable and up-to-date annotations of cancer-related proteins will be obtained by sensitive comparisons to all known protein sequences and structures. The annotations will include comparative protein structure models for all cancer-related proteins with assigned folds. The second aim is to identify and prioritize target protein domains for the AECOM/Brookhaven/Rockefeller Structural Genomics Research Consortium (SGRC) that will focus on developing high-throughput technology for structure determination of the cancer-related proteins by X-ray crystallography and NMR spectroscopy. The target domains will correspond primarily to the yeast homologs of the cancer-related proteins without known structure. The target list will be dynamically updated to maximize information from structure determinations. The third aim is to analyze and use the structures determined by SGRC for comparative structure modeling and comparative analysis of as many cancer-related proteins as possible. The annotation, modeling and analysis tools will build on the MAGPIE system for automated genome annotation, and on the MODELLER pipeline for large-scale comparative modeling. The annotations will be defined in the computer language Prolog through logical rules and relational facts, including rules to capture computed alignment data, domain definitions, and user preferences about properties of target domains. The ability to refer at the same time to the sequence, structure, and function of cancer-related proteins, organized in sequence and structure families, will allow cancer researchers to address questions that are currently not easily answered. This project will increase significantly the amount of protein structure information available to cancer biologists. The set of cancer- related proteins, their annotations, family membership, and structural models will be accessible efficiently over the web.
 
R33 CA084701-01 2000 HUANG, TIM H.-M. UNIVERSITY OF MISSOURI-COLUMBIA HIGH THROUGHPUT METHYLATION ANALYSIS IN CANCER
Aberrant DNA methylation frequently occurs in CpG islands, which are 0.2 to 2-kb GC-rich sequences located in the 5 ends of approximately 60 percent of all genes, in neoplasia. This type of epigenetic mutation is associated with the silencing of tumor suppressor genes, and plays an important role in promoting tumor development. Until recently, most methylation assays have been limited to analyzing a few CpG islands of known genes at a time, and suffer limited throughput for a genome-based study and for clinical applications. Therefore, the development of more efficient technology designed to detect CpG island hypermethylation has long been needed to dissect complex methylation changes during cancer development. With this in mind, in our exploratory phase (equivalent to R21) we have developed a novel DNA array-based technique, called differential methylation hybridization (DMH), that provides for the first time an opportunity to conduct a genome-based methylation analysis. The first part of this innovation is the generation of multiple CpG island tags as templates arrayed onto solid supports (e.g., nylon membranes). The second part involves preparation of amplicons, representing a pool of methylated DNA from the tumor and reference (control) genomes. Amplicons are used as probes in array-hybridization. Positive signals identified by the tumor amplicon, but not by the reference amplicon, indicate the presence of hypermethylated CpG island loci in cancer cells. We successfully applied DMH to identify multiple hypermethylated sequences in breast tumors. In this R33 application, we propose to upgrade DMH by reconfiguring it into a microarray-based assay and to develop an advanced information system in support of this high-throughput methylation analysis. The critical features of this full-scale development are 1) generation of a standard panel of approximately 15,000 CpG island tags broadly applicable in methylation analysis of solid tumors and leukemias, 2) improvement of amplicon generation from multiple clinical specimens, 3) implementation of sophisticated instruments (DNA arrayer and imaging device) for microarray assays, and 4) development of a robust data warehouse and computational tools for analyzing large-scale methylation data. We will catalog methylation changes of CpG islands in the clinical samples proposed for study, and the methylation data will be used to correlate with clinicopathological parameters of the patients. This type of analysis will provide a new tool for molecular staging of cancer. It is expected that high-throughput DMH will have a broad application in detecting DNA methylation changes in cancer. Moreover, the characterization of the corresponding biological relevant cDNAs will open unforeseen avenues for investigating the pathology of cancer and for better treatment of the disease.
 
R33 CA083219-01A1 2000 JARVIK, JONATHAN W CARNEGIE-MELLON UNIVERSITY TECHNOLOGIES FOR COMPREHENSIVE FUNCTIONAL PROTEOMICS
This project has two broad goals. The first is to develop CD-tagging technology into a robust tool for comprehensive analysis of the proteome of cultured cells, including primary tumor cells. The second is to develop discovery and analysis technologies that efficiently search CD-tagged cell libraries to identify and analyze genes and proteins that may have value in cancer diagnostics and therapeutics. Within this second goal we will focus on two areas: identifying cell surface receptors that trigger growth arrest or apoptosis, and identifying proteins whose levels rise in response to myc oncogene activation.
 
R33 CA086364-01 2000 LAM, KIT S UNIVERSITY OF CALIFORNIA DAVIS LIGANDS FOR ALTERED PHOSPHOPROTEINS IN CANCER
The long-term objective of my research is to apply combinatorial chemistry for basic research and drug development. In combinatorial chemistry, millions of compounds can be generated and screened for their ability to bind to a specific target macromolecule or to elicit a specific biological response. Over the same period, several molecular biology tools have emerged that rapidly analyze changes in gene expression. Differential display, serial analysis of gene expression or SAGE and more recently, cDNA microarrays are very powerful technologies enabling one to identify unique genes expressed in a disease state. Although 2- dimensional polyacrylamide gel electrophoresis was first described in 1975, it is only in the last few years that this technique resurfaced as a potential method for the identification of altered protein expressed in disease-tissues. Our laboratory has been awarded a two-year NIH R21 exploratory research grant (CA789909-01, 7/1/98-6/30/00) entitled "Peptide Ligands for Altered Protein in Disease-tissues." The project involves the application of the "one-bead one- compound combinatorial library method to differential genomic or protein display. Briefly, millions of peptide-beads are mixed with tagged extracts from both cancer and normal cells. We are developing methods to identify individual peptide-beads which interact with altered proteins of cancer cell. These peptide-beads can then be isolated to determine their chemical structures. In principle, this new approach will enable us to screen millions of ligands against thousands of proteins simultaneously (over a billion interactions) in "one-pot"; altered proteins that bind to specific ligands from the library are identified. This last year, we have reached two technical milestones essential for the development and application of this novel and potentially very powerful technology. The first technique enable us to effectively examine billion of phosphoprotein/ligand interactions simultaneously, and to identify the ligands that interact with unique phosphoproteins derived from cancer cells. The second technique involves the development of a chemical microarray system in which thousands of peptides or small molecules are chemically immobilized on a glass slide in a microarray format, and the use of these chemical microarrays to profile functional properties of whole cell extract of tumor tissue. Further development and application of this novel technology on human cancer cell lines and fresh biopsy specimens will be the subject of this grant proposal. Potential applications of this technology include (1) tumor profiling for unique phosphoproteins, (2) identification of cancer drug targets, (3) and identification of new leads for drug development.
 
R33 CA088347-01 2000 POOLA, INDIRA NONE HOWARD UNIVERSITY MOLECULAR ASSAY FOR ESTROGEN RECEPTORS IN BREAST TUMORS
Estrogen receptor (ER) is the most important prognostic marker recognized in breast cancer. The presence of ER indicates a good prognosis and the tumors likely to respond to anti-estrogen therapies. Recent studies have shown that some tumor cells express ERbeta and several spliced variants in addition to classical ERalpha and their presence correlate with prognosis and response/resistance to anti-estrogen therapies. In vitro studies have demonstrated that alpha, beta and the splice variants have distinct ligand binding and transcriptional properties. It strongly follows that the relative proportion of various ERs will result in different ligand- and DNA binding properties and the response to a particular anti-estrogen therapy depends on relative amounts of all Isoforms. Our long range goal is to improve patient survival by understanding how the expression profiles of various ERs in tumors can be applied for prognostic and therapeutic decisions. The objective of this application is develop a clinically applicable assay that can precisely quantify all ER isoforms from a small amount of tumor tissue and establish a correlation between ER profiles and disease outcomes. The rationale for the proposed research is that, once the composition(s) of ERs which correlates with disease outcomes are understood, they can be applied to select treatment options. Currently, the prognostic predictions and therapeutic decisions are made based on the presence of only ERalpha as detected by immunohistochemistry. The problem is that this method 1) cannot effectively distinguish all the known ER forms, 2) is not highly sensitive, and 3) requires a large amount of tumor tissue to quantify all ER forms. We propose to develop a Real-Time quantitative PCR assay that can accurately define the proportion of the total represented by each of the ER forms. To accomplish the objective of this application, we will pursue two specific alms: 1) Establish a correlation between the mRNA profiles of various ERs and ligand binding and response and 2) Identify the ER mRNA profiles by Real-time PCR that correlate with tumor stage, nodal status, histological type, prognosis and response/resistance to anti-estrogen therapies. We are poised to develop the above assay, because it capitalizes on the novel approaches for 1) quantifying various forms of ERs 2) specific methods to amplify spliced ERs which were developed by our group and 3) the availability of tumor tissues, in addition to successful completion of "Evaluation Phase of the assay development". It is our expectation that the resultant clinically applicable assay will permit us and others to generate data that allow definitive correlation between the status of various forms of ER with: 1) prognosis and 2) response to anti- estrogen as well as other therapies. Such outcomes will be significant for the breast cancer patients who are resistant or acquire resistance to anti-estrogen therapies.