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In a process called "Molecular Combing" DNA molecules attached at one end to a microscope slide are extended and aligned by a receding air-water interface and left to dry on the surface. The local action of the interface is the same on each of the molecules in solution: they are stretched in a reproducible manner to a constant value of 2kb/mum. Simple, controlled and reproducibleoptical mapping of total genomic DNA is made possible by applying Fluorescent Hybridization (FISH) or Immuno-Fluorescence (IF) to combed DNA. This technique using total human DNA easily prepared in agarose blocks allows combing of very high density of DNA molecules (103 genomes per slide) in a uniform and parallel fashion. These properties along with the scoring of single molecules allows for thorough staistical analysis of the hybridized clone sizes and distances yielding precise measurements. A physical map with a precision of a few kilobases (kb), can be obtained in this way, with no additional information from other techniques. An immediate application of screening of genomic DNA from patients for microdeliyions and translocation brekpoint at specific tumor susceptibility. The high level of resolution (2kb) within a range (5 - 300) not covered by the current methods (PCR, CGH) allows for more accurate diagnosis of specific loci. Molecular Combing is also more sensitive than CGH for the detection of low level amplifications (2-15) copies. Emphasis is now on automation in order to speed up data collection and analysis for high-throughput applications and dissemination of this of this novel technology. A second, more exploratory approach consists in scanning whole genomes of normal, precancerous cells for abnormal patterns of DNA replication units (replicons). Efficient DNA replication, dictated by regular replicon size, is necessary to maintain stable genomes. Many, if not most cancer cells show mutations in genes controlling the G1 phase of cell cycle, consequences of which might be a decrease of replication efficiency during the following S phase and increase in genomic instability. We will test this hypothesis by measuring replicon size using molecular combing in normal and transformed cells whose replication origins have been marked with bromo-deoxyuridine (BrdU). Deviation from standard values will be assessed as a general marker for genomic instability and malignancy.
Tumors are characterized by numerous genetic alterations ranging from point mutations in critical genes to a multitude of major alterations in the structure and number of chromosomes. Translocations, deletions, duplications and alterations in ploidy occur in almost all tumors and alter the expression of thousands of genes. The large number of alterations in the genome of a tumor cell can make it extremely hard to identify the specific genes whose change in expression is causal in the neoplastic process. Yet this knowledge is important since the identification of the causal genetic changes common to specific types of tumors will eventually lead to therapies based on a molecular profile. We propose to develop an automated method to generate a genome wide molecular profile of mouse and human tumors in a single experiment. This will enable the whole genome the whole genome to be analyzed in hundreds of tumors, which should facilitate the identification of common causal genetic changes. The technology to perform this analysis is based on comparative genomic hybridization (CGH) of fluorescently labeled DNA. We are applying CGH to high density microarrays of MAC clones of genomic DNA which have been covalently coupled on a glass microscope slide. To facilitate this we have developed a novel DNA-glass coupling chemistry which provides exquisite sensitivity. These arrays are co- hybridized with normal DNA and tumor DNA labeled with different colors and a laser scanner with photomultiplier detection is used to scan the array and convert the different colors and intensities of the fluorescence signals into an intensity ratio histogram. This provides a genome wide molecular profile of the tumor with respect to regions of the genome which are deleted or amplified compared with the normal genome at the resolution of a BAC (200kb). The "array analysis" of the genome of multiple tumor samples can be rapidly accomplished by this method. This technology will initially e developed for the mouse but will subsequently be developed for the human and applied to prostate cancer. Computational tools will be developed to analyze this data. This will enable the identification of alterations in key regions of the genome specific to certain tumor types. This can be correlated with knowledge of the response of the tumor to certain therapies to provide prognostic outcomes based on a molecular profile. This should also enable the specific genes which are causal in the neoplastic process to be identified.
We wish to create a technical infrastructure that would serve as a foundation for the study of pathophysiology of DNA CpG methylation in cancer. It has been shown that epigenetic modification of DNA in form of CpG methylation can result in mutations, cancer, and possibly aging. For example, a very clear mechanism has been established relating 5 MeC to an increase in mutational load through the transition of 5MeC to thymidine. In addition, changes in CpG methylation have been correlated with gene silencing, functional Loss Of Heterozygosity (LOH), and loss of epigenetic imprint. No current landscape allows for the study of changes in methylation at the single cell/single chromosome level. The central focus of this proposal is to establish a robust in situ technology for detection of sequence specific changes in CpG methylation on mammalian chromosomes. This technology would be sequence specific, capable of surveying multiple loci simultaneously, preserve the three dimensional organization of the metaphase chromosome, and be capable of ascertaining changes in cytosine methylation on metaphase chromosomes in single cells. To create this as a practical and transferable technology certain specific technical achievements can be defined. These incremental steps will be satisfied and the resulting technology evaluated in model systems to establish a base line of performance. This microscopic method will permit the investigator to distinguish the presence or absence of specific and/or global methylation in single cells at specific points in tumor development. This technology will also permit the investigator to develop an understanding of the normal development of the methylated genome in isolated single cells.
The immediate goal of this program is to develop a hyperspectral imaging spectrometer capable of measuring DNA microarrays which simultaneously utilize ten or more fluorophore-labeled nucleic acid probes, at a rate of 500,000 samples per hour. The device will greatly increase the speed and reduce the cost of gene expression profiling and mutation and sequence divergence detection. It is expected that the identification of genes that are specifically expressed in disease states will provides clues into the maintenance of these states and perhaps their etiology. Such information is needed for understanding and diagnosing different forms of cancer, and may ultimately lead to the design of therapeutic agents. It should also improve the analysis of the mechanisms of action and side effects of those drugs. Ultimately, though not part of this program, the long-term objective is to adapt this instrument to directly image multiple- fluorophore-tagged samples of tissues and cells. Such a step will dramatically enhance the ability to define the spatial distribution of many tens of molecules at or below the cellular level of resolution, simultaneously and in situ. This capability should increase the capacity to rapidly and reliably provide an assessment of the disease state from biopsies and other tissue samples, and should ultimately allow a more detailed analysis of the interactions between cancerous and pre-cancerous cells and their normal neighbors. The program is a collaboration between Raytheon Optical Systems and Yale UNIVERSITY. It is structured in two phases, R21 and R33. The first is a feasibility and concept definition effort. The specific aims are: to clarify the system requirements for a hyperspectral imager; to validate the design concept, and hence justify progressing to the R33 phase; to define an appropriate test and evaluation program in R33; and to define the scope of sample preparation work required for R33. A major objective will be to demonstrate that hyperspectral unmixingalgorithms developed for earth remote sensing and military reconnaissance are effective in discriminating multiple fluorophores. The second phase is primarily an electro-optical instrument design, manufacture, integration and test effort. However, it also involves the fabrication of DNA microarrays, labeled with suitable fluorophores. At completion, the project will result in two research/development instruments which will be in place at Yale and available to the cancer research community.
The ability to rapidly re-sequence a genomic region or to determine the differential expression profile of a large number of genes that are potentially implicated in cancer development will be extremely valuable for the cancer researcher. There is a tremendous amount of DNA sequence information being generated by the CGAP and human genome programs, more than can be effectively analyzed and then represented in contemporary 'chip' style re- sequencing or expression arrays. This severely limits the number of clinical samples that can be thoroughly inspected. We propose to construct dedicated arrays which have immediately reconfigurable gene sequences identified and designed by computer. This phased innovation award will answer the following questions: 1) Can the array design software, Digital Optical Chemistry (DOC) chip manufacturing device, the MAGNA/HIC readout device, and analysis/gene network software be constructed and ruggedized for routine analysis of cancer samples to differentiate cancer non-cancer cells and their progression by gene expression profiling and chip based resequencing? 2) Can the customizable arrays made possible by the DOC approach be continuously expanded and improved as new genomic data is amassed to generate chips dedicated to analysis of different cancer cell types? 3) Can candidate cDNA sequences (CGAP) and larger genomic regions identified by computer analysis such as Virtual Expression Array calculations be re-sequenced to identify informative gross variations down to SNPs in cancer patient populations to identify new oncogenes or tumor suppressor genes? The specific aims of this program are: 1) To develop bioinformatics tools for the identification and ellucidation of candidate cancer related genes including software for the design, readout and analysis of gene expression and re-sequencing arrays; 2) to develop and complete a Digital Optical Chemistry (DOC) array fabrication unit capable of synthesis of at least 100,000 custom oligonucleotide array members on a single chip with the capability of rapidly constructing chips with different arrays every 2 hours; 3) to develop and construct a custom DOC readout system by modifying/replicating an in-house developed hyperspectral imaging microscope; and 4) the integration of the entire system of software and hardware for the design, fabrication, and data analysis of DNA microarray chips and their subsequent testing on clinical samples relevant for cancer research. The testing of the integrated system for use in mutation detection, SNP discovery, allelotyping, and expression analysis will progress to use archival and prospectively collected cells from biopsies and fine needle aspirates that have been purified with the new laser capture microdissection system. This will integrate our system with current major technologies to produce a tool that should be widely available in translational and clinical trials cancer research.
Metastasis is one of the most devastating aspects of cancer. Thus, discovering therapies that inhibit metastasis is an important goal in cancer treatment. In order to do this, proteins involved in cancer cell invasiveness that could be targets for therapeutic drugs must be validated and this is often a rate-limiting step in drug discovery. The overall objective is to develop an enhanced high throughput screen (HTS) using chromophore assisted laser inactivation (CALI) for target validation of surface proteins that act in cancer cell invasiveness. CALI targets laser energy using a dye-labeled antibody to inactivate the function of the bound antigen. In the R21 phase, an HTS Transwell assay will be developed and used to show that CALI will increased markedly the number of phage display antibodies that disrupt invasiveness. Single chain Fv fusion phage libraries that bind specifically to surface proteins of HT-1080 human fibrosarcoma cells will be generated and used for a pilot screen (n=96 antibodies) using CALI. The achievement of these aims will establish the feasibility of an HTS using CALI for the validation of targets involved in cancer cell invasiveness. In the R33 phase, a full scale automated screen will be conducted (n= 10,000 antibodies) and protein targets validated by this screen will be identified using high resolution mass spectrometry. This proposed technology will provide a low cost and rapid means of target validation and will contribute a number of targets for anti-metastasis drug discovery. The methods developed are general; they will have an application to cell processes in cancer and other diseases. As such, they will be of great utility for pharmaceutical companies and academic labs.
Correlating cancer induction with specific mutations is critically dependent on the availability of technologies that can effectively screen for unknown mutations in several genes simultaneously. This proposal will optimize and streamline a newly developed technology (ALBUMS) for the sensitive and large scale screening of base substitutions, which are the mutations predominantly generated by a wide range of mutagens and are found in several human cancers. DNA from cancerous and normal cells are annealed and hybridized to generate mismatches at the positions of base substitutions. ALBUMS utilizes the covalent and specific binding of novel molecules at unique chemical groups (aldehydes) generated at the position of mismatches by highly specific mismatch - recognition enzymes, in order to isolate mutation - containing DNA. The microplate-based design of the assay allows to screen hundreds or thousands of diverse genes simultaneously, isolate those with mutations and apply them on existing large - scale hybridization DNA arrays for a single - step identification of the mutated genes. The R21 phase (feasibility) will (a) define the optimal operating conditions for detecting and isolating mutation-containing genes (genotypic selection). ALBUMS will be used to isolate cDNA from a single mutated gene (p53) in the presence of increasing amounts of normal alleles, and also to detect p53 mutants in cell lines known to contain p53 mutations. And (b) will establish feasibility for the single-step screening of several hundred or thousands of human genes by combining ALBUMS and commercial DNA hybridization arrays (chips). The second phase (R33) will develop technology to screen in a single step hundreds or thousands of genes in human tumor samples for mutations, on DNA chips. Sets of mutations will be mapped and verified by conventional sequencing in order to establish the utility of the new technology to define the molecular profile of cancer. In the last step, this high throughput technology will be streamlined to provide a procedure with easy access to researchers and clinicians for cost - effective, large - scale mutation screening of cancer samples. Further envisioned ALBUMS applications include genotyping and polymorphism studies and role of mutations in diseases other than cancer.
Cancer-relevant signal transduction involves a large number of signaling proteins with many parallel steps and interconnected feedback mechanisms. These properties of signal transduction processes cannot readily be measured by current techniques, which limits researchers ability to validate each of the more than 10,000 human signaling proteins as potential drug targets for cancer therapy. Over the last several years, my laboratory has made three separate developments that can now be integrated into a technology to systematically characterize complex signal transduction networks. In this approach, cell arrays will be made for simultaneously monitoring a large number of signaling processes. The proposed Evanescent Wave Cell Array Technology (ECAT) incorporates: 1) a microvolume electroporation method for RNA transfection, 2) a set of single cell GFP-tagged biosensors (such as Akt, PKC, Grb-2, GAP and Raf isoforms) that can monitor diverse signaling processes by their plasma membrane translocation or dissociation, and 3) an evanescent wave microscope setup to quantitatively monitor these plasma membrane translocation and dissociation events. In Phase I of the project, we will develop and test a 4 x 3 prototype cell array and, in Phase II, we will expand the cell array to 15 x 10. These two arrays will be used to simultaneously monitor different receptor or transformation induced signaling events in each of the 12, or 150, separate cell array segments on the same glass slide. While phase I includes a test of principle using existing biosensors and dominant negative and constitutively active interference proteins, Phase II will develop a library of such fluorescent translocation biosensors. Furthermore, we will develop two libraries of interference proteins by mutating a large number of serine/threonine kinases and small GTP-binding proteins into constitutively active and dominant negative constructs. As a test of the usefulness and the limitations of the ECAT method, we will determine the role of each member of these two interference libraries by testing them in the context of different receptor stimuli using the existing and newly developed fluorescent biosensors. Overall, this proposal will provide a new approach to signal transduction research and will provide a technological platform for the validation of cancer drug targets and the advancement of drug discovery.
This is a vertically integrated project that will advance the emerging technology of laser scanning cytometry (LSC) to the point that it will a) enable the performance of clinically relevant tissue profiling studies consisting of 50-100 fluorescent and immunofluorescent measurements per sample in human solid tumors, grouped in panels of 5-10 correlated measurements per cell on each of approximately 5,000 cells per panel, and, b) enable extensive analysis of the data, using hypothesis-testing and/or exploratory approaches. Starting with a commercially available instrument (CompuCyte Corp., Cambridge, MA) and multicolor protocols previously developed for lymphoid tissues, during the R-21 phase we will a) develop cell preparatory methods that are suitable for epithelial tumors, b) identify the best initial measurements for identifying and contouring individual cells for multiparameter analysis (light scatter vs cytokeratin, or tubulin), c) develop one or more dye combinations to serve as templates for subsequent development of additional multicolor panels, each encompassing 4-6 correlated measurements per cell, and d) determine the conditions under which individual cells can be revisited and restained with up to 5 additional fluorescent probes per cell. During the R-33 phase we will a) develop a core set of 5-10 multicolor immunofluorescent panels for tissue profiling, each consisting of 5-10 measurements per cell with restaining, using antibodies that have been optimized with respect to non-crossreactivity and binding affinity, b) develop software that will have the capability to capture, preprocess, display, analyze, store, and export mixed data sets consisting of mixtures of correlated, partially correlated, and uncorrelated data, c) extend the capabilities of the CompuCyte instrument by adding additional lasers, and doing custom dye development to increase the number of potential correlated measurements per cell, and d) expand interactions with clinical departments within our institution, in anticipation of devising specific translational clinical studies to explore tissue profiling for prognosis and treatment planning, and launching such studies by the time this grant period has been completed.
Over the last project period, we have achieved the goal of detecting the expression of many genes in a single cell using multiplexed probes to develop a barcode for eleven transcription sites for specific genes and their alleles. This single cell gene expression profiling method allows assessment of each cell's pattern of expression as well as allowing a population analysis of the regulation of these genes. The extensive data complexity that results from the gene-to-gene analyses indicates that this will be an informative approach to validate the gene expression patterns pertaining to cancer gene expression as well as to discover new correlations of genes with the cancer phenotype. The aim of this proposal is to apply this methodology we have developed to tissue samples in order to derive specific gene expression information, ultimately from patient specimens. The approach is to optimize this technology by developing reagents, protocols and imaging hardware and software to achieve a high signal of expressed genes and reduced background of tissue autofluorescence. Central to this development is the use of two new instruments, the Leica confocal microscope, and the VarispecTM liquid crystal tunable filter, which allow a complete spectral analysis of the hybridized tissue, and hence extraction of the principal dye components used for the probe. This provides a means by which we can multiplex the barcoding probes using more spectral bandwidth and obtain gene expression profiling of up to 247 genes in a single nucleus. The statistical analysis of the expression of these genes in tissue combined with a preserved tissue morphology will provide an eventual platform for interrogation of patient specimens where the outcomes are known. The expression patterns obtained from tumor cells in pathological samples will allow a correlation of specific genes with disease progression.
The capability for identifying proteins and measuring changes with good precision of proteomes (the complement of proteins produced by a given organism or cell) for model organisms would provide a powerful tool for understanding the interrelated roles of individual gene products underlying the molecular basis of cancer. In this four year two phase (R21/R33) project we will develop a new approach for obtaining such broad systems level views of differential protein expression. The approach would involve two-dimensional capillary electrophoretic separations for rapid proteome separations and advanced mass spectrometry for the rapid identification of proteins and their modifications. 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. A component of our approach involves on-line analysis using Fourier transform ion cyclotron resonance mass spectrometry for protein identification based upon very high mass measurement accuracy and multiplexed MS/MS measurements for polypeptides. We will also develop novel isotopic labeling (e.g., 13C, isotopic depletion or enrichment) methods allowing comparison of two proteomes in the same separation, effectively providing an internal standard, and enabling precise determination of expression levels. Phase 1 will involve initial development and validation of the instrumental approach. Phase 2 efforts will involve its extension to an automated format, further extension of its sensitivity, expansion of its applicability to more complex proteomes and the determination of modifications, and provide for computer-based "differential displays" of results. The eukaryotic yeast strain Saccharomyces cerevisiae will serve the model system for evaluation of the technology. The technology to be developed will enable rapid and sensitive differential proteome displays for the study complex mechanisms and pathways for research into the molecular basis of cancer.
An emerging theme in our current understanding of human oncogenesis is that cancers arise as mutations and epigenetic alterations accumulate in individual cells. The ultimate consequence of such changes is to alter the expression profile of the genome. Knowledge of the identity and function of genes whose expression is altered in particular types of cancer, would enable the correlation of specific genes and pathways to unique cancer induced phenotypes. This information would help our understanding of cancer biology and would provide new biomarkers for cancer detection as well as novel gene targets for therapeutic strategies. Thus, a high priority of current cancer research is to reveal the molecular variations that distinguish cancer cells from wild type and to determine the function of the affected genes. Of the possible genes whose altered expression may mark the molecular signature of cancer, primary candidates include those that encode proteins that function to regulate the expression of the genome and catalyze its replication and repair, collectively referred to as nucleic acid binding proteins (NBPs). Maintaining the integrity of the genome and regulating its appropriate expression are important first tier intracellular processes that must be controlled. Since genetic diseases such as cancer can result from alterations in the genome which ultimately change the expression profile of genes that function in nucleic acid house keeping and regulatory functions, our first goal, in the R21 phase of this proposal, is to examine the feasibility of developing a functional genomics technology aimed at identifying human genes selected by their ability to encode proteins that bind to DNA and/or RNA. Several additional components of the technology will be addressed in the R33 expanded development phase of the proposal. One encompasses a novel sorting phase that will rapidly distinguish between different classes of NBPs according to their (encoded) binding properties for particular nucleic acids types and conformations. We will also develop bioinformatic tools that can be used in both virtual and microarray based expression profiling to sample whether the expression of selected genes or gene clusters is altered in normal versus specific cancer cells. When developed, this new technology will enable the functional identification, categorization and expression profiling of genes that act to maintain and regulate the genome. It will directly improve the quality of data in the Cancer Genome Anatomy Project (CGAP) and it will likely identify new genes that confer a predisposition for cancer when their relative expression within a cell is altered. Such technology does not presently exist.
An important aspect of developing treatments against cancer consists in correcting the defects caused by the abnormal activity of oncogenes and tumor suppressor genes. This usually includes the assignment of cancer-related gene products to their respective biochemical pathways. It has been shown that the genetics available in model organisms such as yeast, nematodes, flies, and mice can be highly advantageous for these projects. Despite the power of model organisms, the number of genes with a function assigned is relatively small and thus the functional analysis of cancer-related orthologs is still relatively tedious. However, with the complete sequence of the genome of most model organisms anticipated to be available soon, several laboratories have initiated the development of genome-wide gene-function analysis projects. Such projects, collectively referred to as "functional genomics" include genome-wide expression analysis, gene knock-outs and protein-protein interaction mapping. They are expected to reveal gene functions at a drastically increased rate. In this context, the long-term goal of our laboratory is to generate a comprehensive protein-protein interaction map for the nematode C. elegans. This will be achieved in three steps: Step I: validation of our improved version of the yeast two-hybrid system to generate protein interaction maps (funded by an R01 grant from the NHRGI); Step II: development of new high-throughput technologies for protein interaction mapping (object of this grant); step III: production phase (planned for the long-term and not the object of this grant). In step II, we plan to automate most steps of the two-hybrid methodology using a new cloning method, referred to as Gateway cloning. Gateway allows the transfer of DNA inserts between donor plasmids (or PCR products) and recipient plasmids. It is based on an in vitro site- specific recombination event mediated by purified phage lambda proteins and thus eliminates the need for restriction enzymes and ligases. It is fast and reversible, and the whole procedure can take place in 96- well plates, which means that automation is possible. In summary, Gateway will allow us to go in and come out of the two-hybrid in a completely automated series of steps. We will first develop the Gateway technology in the context of the two- hybrid system (R21). Then we will develop high-throughput methods for protein interaction mapping based on the use of the Gateway/two-hybrid system (R33). We will also adapt the technology so that other worm functional projects can benefit from it.
As a step toward understanding the complex differences between normal and cancer cells, much research has been devoted to analyses of genes that are differentially expressed in particular cells. Though recent technological advances have made it possible to conduct serial and/or simultaneous analysis of the expression patterns of thousands of genes, no comprehensive study has been reported on how many genes are expressed differentially and whether most differences are cell line-specific. The long- term goal of this research is to develop intelligent data mapping and visual explanation technologies to improve information exploration and interpretation from high-throughput gene expression profiles for molecular analysis of cancer. Suggested by preliminary evidence from mRNA profiles of breast/prostate cancer cells that transcriptome patterns are rich in information about mechanisms that underlie cancer development, in the R21 research, multidisciplinary knowledge of molecular biology and computational intelligence are applied to (1) design cost effective molecular experiments to establish gene transcriptome distributions across cell lines, (2) pilot test the existence of transcriptome clusters in the molecular species space that correlate to cell phenotypes, and (3) identify key biomarkers that differentiate different cell lines with the highest prediction values. Since new knowledge can only be further acquired by exploring all of the interesting aspects of complex transcriptome data in high-dimensional space, in this R33 application a statistically principled hierarchical visual exploration technique is proposed to effectively reveal and interpret the intrinsic but hidden characteristics of transcriptome clusters that should better define the nature of cancer biology and therapeutic targets. A novel integration of information theory and computer graphics will permit (1) an automatic identification and modeling of biomarker clusters, (2) a probabilistic component analysis to form hierarchical visualization spaces allowing the complete data set to be analyzed at the top level with best separated sub-clusters analyzed at deeper levels, and (3) an interactive intelligent interface for task/hypothesis driven data mining and decision making. The innovative nature of the research relies on the concept of combining (1) a hybrid stepwise nonlinear discriminant analysis for biomarker identification and (2) a hierarchical visual exploration of multi-foci high-dimensional transcriptome distribution to interpret the complex relationships between molecular events and cell phenotypes.
Magnetic resonance microscopy (MRM), including microscopic imaging and spectroscopy are becoming increasingly important in cancer diagnosis and treatment. Both morphometric and metabolic changes in cells can be detected with MRM which may assist in early evaluation of therapeutic response. However, the results are often ambiguous due to heterogeneity of responses and the long measuring times circumvent the potential for following dynamic cellular processes. To improve this situation, we propose to develop and test a microscope in which proton MRM and optical microscopy (OM) can be performed simultaneously to study heterogeneous mammalian cell populations. With this technology, information from OM measurments will be used to guide MRM measurements, significantly improving the speed and accuracy by which MRM measurements can be obtained. High resolution OM (fluorescent) images will be used to select a subpopulation of cells undergoing a physiological response. These OM images will then be used to guide MRM experiments such that water images and metabolite spectra are obtained from only the cells of interest within the population. In the R21 phase of this proposal, an integrated OM/MR microscope in which 2 or more monolayers of cultured human cells growing within a perfusion system will be designed, constructed and tested. Each layer will have a field of view of 1 mm2. The probe will operate in a standard wide-bore (89 mm) vertical magnet with a field of 11.7 Tesla. Our goal is that this probe will be able to determine OM images with 1.5 muM spatial resolution in seconds, and measure proton MR metabolite spectra of 1,000-2,000 randomly distributed human cells within 15-60 minutes. In the R33 phase of this research, the utility and limitations of the integrated microscope will be evaluated by examining cells undergoing apoptosis, a process of critical importance to cancer therapy. Studies will be conducted to evaluate the ability of the instrumentation to identify subpopulations of cells at early and late phases of apoptosis induced by both chemicals and expression of specific genes. The ability of OM to guide MR measurements to specific cell populations or to neighboring populations will be determined. It is anticipated that the successful development of this instrumentation will, ultimately, greatly enhance the speed, specificity and utility of noninvasive MR methods in cancer research.
Certain tumors shed neoplastic cells into the blood before the primary tumor growth can be detected in the body. It has been shown recently that epithelial cells from the tumor can be detected in the blood at concentrations as low as 1 cell per ml of blood, and that the presence of rare cancer cells in blood has an important diagnostic value. The current screening methods are limited by relatively low speed of Fluorescent Activated Cell Scanning. (FACS),and complexity of preenrichment methods. The overall objective of this proposal is to develop a high-throughput, immunomagnetically based cell separating system to recover as many rare cancer cells in human blood as possible, for further molecular analysis (such as PCR, biological assays and other). The specific aims are as follows. First: the primary separation will be conduced using a novel continuos, flow-through immunomagnetic unit developed in our laboratories. Continuos units are intrinsically more efficient with respect to high throughput. We propose to develop experimental and theoretical basis for the next generation system which will sort cells at a rate of 10/7 cells/s, and allow a non- destructive screening of an entire volume of blood product used for cancer cell therapy ( typically 0.5 to 1.0 liters) in a short period of time (< 1hour). Second: the continuos cell separation process can be staged, unlike the currently used batch systems. We propose to develop experimental and theoretical basis for the continuos staged separation process which will further increase the purity and decrease the volume of the cell product, and thus increase the rate of success of analysis downstream of the cell separation step. Third: the current cell labeling procedure may require modifications and optimization for the best performance in targeting and isolating rare cancer cells. We propose to screen available monoclonal antibodies and colloidal magnetic labels for the highest sensitivity and specificity in targeting rare cancer cells using unique Cell Tracking Velocimetry instrument developed in our laboratories. In summary, this proposal focuses on the "front-end" of cancer screening namely a high-throughoutput device to rapidly isolate and concentrate rare cancer cells form large numbers of cells. This proposal is responsive to PAR-98-067, Innovative Technologies for the Molecular analysis of Cancer, and we believe it addresses the second objective of the PAR, namely "novel technologies that will allow high- throughput analysis of genetic alterations, expression of genome products, and monitoring of signal transduction pathways to cancer."
The word proteome was coined in 1995 to refer to the total protein complement of a genome. The human genome encodes roughly 100,000 genes, corresponding to a similar number of proteins. Not all genes are expressed in all tissues; roughly 10,000 proteins are found in any particular cell. The fraction of the proteome that is expressed by an organism varies between tissues and in response to the environment. Conventional proteome analysis is preformed by two-dimensional gel electrophoresis and requires the protein from roughly a million cells. We have improved the sensitivity of proteome analysis by six orders of magnitude. In preliminary work, we have demonstrated that we can perform a simple analysis of the proteome in a single human cancer cell. Our Preliminary work will be expanded in this R33 proposal. We will automate the manipulations of single cells, we will multiplex the instrument so that 96 cells can be analyzed simultaneously, we will expand our proteome analysis to two-dimensional electrophoresis, and we will evaluate the technology by monitoring the evolution of protein expression in mouse skin tumors.Single cell proteome analysis offers several important advantages. In particular, we can monitor the distribution in the expression of protein markers that are correlated with cancer stage. Like ploidy measurements, the distribution of protein expression may have valuable prognostic value. Sub-populations of metastatic or therapy-resistant cells may be identified at an early stage to guide treatment.
It is important to have the capability to perform precise measurements of gene expression levels in tumor cells. Available technology permits the assessment of levels of gene expression in samples containing a minimum of 10,000 cells. Thus, there is a need for methods that will permit such measurements in samples containing fewer tumor cells. A specific aim of this proposal is to complement a new approach that will yield quantitative data on the relative concentration of specific mRNA molecules in tissue samples containing less than 200 cells. Sequence detection is accomplished on oligonucleotide microarrays, using a target-directed DNA ligation step coupled to a Rolling Circle Amplification (RCA) unimolecular detection system. Each target detection event generates a primer that can be amplified by RCA. Each amplified DNA molecule generated by RCA remains localized on the array surface, and is imaged as a discrete fluorescent signal, indicative of a specific molecular ligation event. Expression profiles are generated as histograms of single molecule counts. Additionally, the DNA ligation step can e adapted to the detection of mRNAs containing point mutations. This capability will be developed for detection of one somatic mutant mRNA molecule in a background of 1000 wild type mRNA molecules, using K- ras mutations as an experimental model. Arrays for the analysis of 100 different gene products will include mRNAs known to be up- or down- regulated in cancer cells, wild type or mutant K-ra mRNAs, and housekeeping genes. Adequate controls will be incorporated in the system to insure its reliability. Another specific aim is to evaluate the coupling of this highly sensitive detection technology with laser- assisted tissue microdissection. We will combine these two technologies to demonstrate the capability for high resolution tissue analysis in: (a) normal and cancerous prostate, and prostate tissue with varying degrees of prostatic intraepithelial neoplasia; (b) normal colonic epithelium, as well as adenomatous epithelium with varying degrees of dysplasia, and colonic adenocarcinoma. The capability for measuring gene expression levels in samples containing as few as 10-20 cells, together with the capability for detection of somatic point mutations at several loci known to be altered with high frequency, will provide the infrastructure to address the question of possible microheterogeneity in gene expression profiles in small clusters of cells in dysplasia and cancer.
The reduced complexity and non-stoichiometric amplification intrinsic to RNA arbitrarily primed PCR (RAP-PCR) could be used to advantage to generate probes for differential screening of cDNA arrays. RAP-PCR fingerprints have a reduced complexity compared to the whole mRNA population because they sample only small parts of some of the mRNAs in a mixture. The reduced complexity should result in a lower background hybridization to non-homologous clones when compared to a full complexity probe. RAP-PCR fingerprints are non-stoichiometric because the probability of amplification of a particular cDNA PCR product is dependent on the match with the arbitrary primer. Thus, a rare mRNA may yield an abundant PCR product. This non-stoichiometry allows each different RAP-PCR fingerprint to amplify a different set of rate mRNAs that can then e detected easily on inexpensive arrays. We will experiment with various parameters to optimize the properties of the probe. We will (1) adjust the complexity of the non-stoichiometric probe, (2) bias sampling by the primers to increase the probability of sampling particular sequences of interest and, (3) explore stoichiometric methods to reduce probe complexity. These methods will be applied to simple cell culture systems and to a small set of samples from cancer patients. Our search for differentially expressed genes in these systems will help to compare the performance of the various strategies.
The objective of this project is to develop microfabricated devices for the comprehensive of cellular proteins to normal, precancerous and cancerous tissues. Specifically, we propose to develop lab-on-a-chip technologies as an alternative to the slow and labor-intensive two dimensional (2D) gel methods currently used for comprehensive protein analysis. The microchips will integrate on a single structure elements that enable multidimensional separations of protein mixtures, with either on-chip labeling for fluorescence detection or electrospray ionization (ESI) of the analytes for direct, on line interfacing with mass spectrometry (MS). Successful completion of the project will result in high throughput, automated devices for use in identifying disease markers and elucidating the molecular basis of cellular transformation. These devices will have research applications for understanding cellular transitions from normal to diseased states. In addition, these research tools will also find application in clinical diagnosis and therapy development.
We propose to develop an approach to allow rapid identification of proteins in complex mixtures suitable for protein expression mapping in total cell lysates. A robust approach to identify proteins as mixtures will advance cancer research in several areas. First, the approach has the potential to significantly improve the sensitivity of analysis of proteins. Benefits to cancer research will derive from the ability to identify proteins using smaller numbers of cells leading to direct examination of tumor biopsy samples. Rigorous molecular analysis at the protein level, encompassing separation, visualization, and identification, is currently difficult for small quantities of protein. The methodology proposed is general and therefore can be applied to the monitoring of gene produce expression, analysis and detection of cellular localization and post translational modification of proteins, and monitoring of major signal transduction networks involved in cancer. Second, the approach has great potential for automation allowing the technique to be transferred to laboratories involved in cancer research eliminating the need to collaborate with experts. Last, by developing the ability to quantitate and perform subtractive analyses changes in protein expression related to neoplastic transformation could be observed.
R41 CA081825 1999 Mach,Robert Anasazi Biomedical Research, Inc. Sigma 1 Receptor Probes For Imaging Tumor Proliferation
The goal of this STTR application is to develop 123I- and 99Tc-labeled labeled sigma-1 (sigma1) receptor imaging agents for Single Photon Emission Computed Tomography (SPECT) studies of solid tumors. This approach is based on the observation that sigma1 receptors are expressed in high density in a number of human tumors and are a suitable target for radiotracer development. Our preliminary data also indicate that SPECT- based sigma1 receptor imaging agents may also provide information regarding the proliferative status of solid tumors. An additional incentive for focusing on sigma1 receptors stems from our development of sigma1- selective compound, N-benzyl-4-yl phenylacetamide, that will serve as the lead compound for SPECT radiotracer development. The strategy for introducing 123I will be to incorporate an iodine atom into a region of the lead compound that does not result in a reduction in affinity for sigma1 receptors. The second year of this Phase I grant will address the feasibility of preparing a high affinity sigma1 receptors imaging agent containing a 99m/Tc-complex. Although the proposed radiopharmaceuticals will initially benefit the diagnosis and treatment of breast cancer, we believe the technology developed in this STTR application will translated to other human cancers that posses a high density of sigma1 receptors. PROPOSED COMMERCIAL APPLICATIONS: The radiopharmaceuticals developed from this STTR application have a high commercialization potential since they will use a nuclear medicine imaging procedure (SPECT IMAGING) having a widespread availability. The imaging strategy described in this application could provide a useful clinical tool for both the diagnosis, treatment strategy, and therapeutic monitoring of cancer patients.
R43 CA081860 1999 Chang,Hwai Stratagene Genetic Selection For Proteases And Their Substrates
The goal of the proposed research is to develop a genetic method, using the eukaryotic host Saccharomyces cerevisiae, to select for protease genes, identify their target recognition sequences, and select for tease inhibitors. This represents a novel approach in the functional cloning of these important cellular regulators and will enable rapid isolation of their genes, their targets, and their inhibitors.
R43 CA081870 1999 Miller,Jeffrey Ivs Technologies, Llc New Method For The Synthesis Of Cdna From Cancer Cells
A major obstacle to the identification and analysis of genes involved in cell transformation and tumor progression is the need for better methods for capture and stabilization of transcripts in tumor cells. Presently, cDNA populations synthesized from tumor cells are likely to incomplete, in terms of both the length of the product, and the representation of rare sequences. Therefore, cDNA probes and libraries, as well as the amplified products produced from biased and incomplete RNA populations, are less likely to accurately reflect the gene expression profile of the tumor. PROPOSED COMMERCIAL APPLICATION: When developed, in vivo cDNA synthesis is expected to be a broadly enabling technology, with applications in cDNA synthesis, cDNA cloning, sample preparation and stabilization, gene expression analysis and gene discovery, and molecular diagnostics.
R43 CA081890 1999 Neri,Bruce Third Wave Technologies, Inc. Specific And Sensitive Mutation And Snp Detection
The goal of this project, over Phases I and II, is to create a new, accurate and quantitative platform for massively parallel, ultra high throughput screening of SNPs, mutations, and gene expression on solid supports, e.g. chips. This platform, based on our novel, linear signal amplification technology, the Invader assay, will immediately impact the effort to associate SNPs and mutations with phenotypes and will be of increasing value in the molecular profiling of cancer. High throughput SNP analysis is an active area of technology development; nonetheless, most existing technologies are hampered by exclusive reliance on allele specific hybridization. The Invader assay is based on a highly specific enzyme-substrate reaction (i.e. cleavage of a precise structure formed by oligonucleotides hybridized to a target sequence). The unique accuracy of this method results from the combined specificity conferred by this sequence-specific probe hybridization and structure-specific enzymatic cleavage. This novel specificity enables direct analysis of genomic DNA or RNA, making possible simultaneous analysis of thousands of loci in a sample without intervening target amplification steps, thereby lowering costs and increasing throughput. In Phase I, we will establish the feasibility of adapting this technology to solid supports, including an initial demonstration of multiplexed analysis. PROPOSED COMMERCIAL APPLICATION: This project, over Phases I and II, could result in a low-cost, high-throughput, chip-based method for direct detection of mutations, Single Nucleotide Polymorphisms, and gene expression in a wide variety of genes. By obviating the need for costly and laborious target amplification, this simple, versatile technology the potential to replace many existing systems for routine repeat sequence-based analysis, such as array technologies and some PCR- based tests, and may contribute to the growth of the emerging molecular diagnostics market.
R43 CA081891 1999 Weaver,Daniel Genomica Corporation Analyzing Gene Expression For Cancer Gene Discovery
Gene expression analyses generate manipulation, and analysis. We propose to develop object-oriented, CORBA-compliant database software that is extensible and generally applicable to all array-based gene expression studies. We will design an object model to store and manage user-defined experimental variables and related data sets. We will incorporate CORBA-compliant client software to allow large, distributed research groups to distribute and share data. We will develop a query engine to filter quantitatively and qualitatively the stored expression data for those genes that display expression patterns of interest. Our software will help researchers to tailor these tools to the specific demands of their research interests and to automate the analysis of large expression projects. Finally, we will develop gene regulation modeling tools with which the researcher will develop hypotheses of gene regulation and will test those hypotheses against available expression data. Thus, we are proposing a flexible, integrated, and distributed software architecture for storing, manipulating, and analyzing array-based gene expression data using emerging industrial standards. PROPOSED COMMERCIAL APPLICATIONS: The software developed with this grant will be applied to manage and interpret data resulting from pharmaceutical, biomedical or basic research that employs array-based gene expression detection. It could also be used with expression-based disease diagnostic tests to compare test results to previously known diseased expression profiles.
R43 CA081903 1999 Ts'O,Paul Ccc Diagnostics, Llc Isolation And Profiling Of Circulating Pc Cells In Blood
The major long term goals of this project are 1) to standardize culture of cancer cells from individual prostate cancer patients, 2) to develop molecular profiling for cancer predictive characteristic such as chromosomal aneuploidy, circulating markers for cell proliferation and quiescence, androgen receptor gene copy. The molecular profiling is intended to provide a window into the metastatic potential of circulating cancer cell, as a prognostic tool. The patient-specific cultured cells, besides providing a wealth of research material for researchers studying prostate cancer, would serve the vast clinical market for (in-vitro out of body). Specific aims of Phase 1 are: 1. Seed cultured prostate cancer cells into white blood cell rich fraction (simulating leukapheresis-which concentrates circulating cancer cells) and enrich for live cancer cells. 2. Compare experimental approaches to further enrichment (to a 1:10 cancer cell to WBC ratio). 3. Culture the cancer cells. 4. Develop molecular profile assays for use on these samples. 5. Final goal is isolate, perform molecular characterization and culture cancer cells from 15 patients' leukaphereis samples. The technological innovation to the field of molecular analysis is a system to isolate, and characterize circulating cancer cells and provide circulating cancer cultures for further work. PROPOSED COMMERCIAL APPLICATION: Molecular profiling of cancer cells isolated directly from a patient's blood could have great commercial impact as a clinical test for prognosis and/or to directly monitor the effects of therapy on the circulating cancer cells. The second major commercial potential would be applications for cancer cell cultures isolated from individual patients.
R43 CA081949 1999 Oliner,Jonathan Affymetrix, Inc. Reverse-Engineering Signal Transduction Networks
Although numerous genetic alterations have been causally linked to cancer development, many of the affected genes appear to fall into common cellular pathways. With the advent of technologies capable of assessing the expression of thousands of genes simultaneously, the potential now exists to ""reverse- engineer"" cancer-related signaling pathways by computer modeling. Towards this end, the objective of this proposal is to develop experimental methods and analysis tools to elucidate the components of signaling networks functionally altered during tumorigenesis. To achieve this goal, prototype pathway modeling algorithms will be tested in Phase I for their ability to detect known relationships among genes manipulated in experimental systems. Specifically, recombinant wildtype versions of genes frequently mutated in cancer development will be inducibly expressed in cell lines, and the resulting expression changes in endogenous genes will be monitored by hybridization to oligonucleotide arrays. Analogous studies will also be performed using cells in which individual genes have been deleted. The protein products of genes that demonstrate altered expression in such studies serve as candidate functional mediators of the ectopically expressed genes. The commercial goal of these studies is to develop broadly applicable tools to facilitate the rational identification of novel diagnostic markers, prognostic markers, and therapeutic targets. PROPOSED COMMERCIAL APPLICATION: We will develop experimental and analytical tools to reverse-engineer signal transduction networks from gene expression profiles. These tools will facilitate the rational identification of novel diagnostic markers and therapeutic targets. As such, they will enhance the sale of high-throughput expression analysis systems and pathway analysis software.
R43 CA081951 1999 Xanthopoulos,Kleanthis Aurora Biosciences Corporation Id Of Novel Breast Cancer Agents By Functional Genomics
Our goals are to develop novel methods which rapidly identify genes and lead pharmaceutical compounds in the area of breast cancer. We have developed a gene-tagging method which uses highly sensitive B-lactamase (bla) reporter system to rapidly 1) screen for novel genes with expression profiles responsive to the presence or absence of estradiol, and 2) identify drug candidates with specificity/efficacy profiles different from existing drugs. We hypothesize that this gene tagging strategy can be used to create a set of stably transfected breast cell lines expressing inducible/repressible genes and to rapidly screen for novel anti- cancer agents which prevent modulation of those genes. PROPOSED COMMERCIAL APPLICATION: 1.4 million new cancer cases were estimated in 1997 in the U.S. with medical costs of $35 billion. Sales for breast cancer drugs tamoxifen and taxol are close to $500 million and $800 million respectively. However, standard endocrine drug therapy as well as chemotherapy and treatment with taxoids fail to treat recurring breast cancer. Hence, there is a need for more and improved agents for treatment of recurring and hormone independent breast cancer.
R43 CA081958 1999 Hagen,Frederick Icogenex Corporation Affinity And Transcriptase Technologies
One out of every three Americans will be afflicted with cancer in their life time. It is a devastating frightening disease which frequently ends in death after much suffering. Progress has been made in the treatment and cures of cancer but the progress has been incremental. Much time, effort, and expense has gone into research to understand and develop a cure for cancer but because of the complexity and resiliency of the disease, no ""magic bullet"" has been developed. The foregoing research has inspired the National Cancer Institute to develop the goal of obtaining a full set of complete cDNAs from normal, nant, and malignant cells to enable the molecular genetic events which predetermine, activate, and maintain malignancies to be determined. Once the differences in transcribed sequences are identified, the ucture, activity, and function of the encoded protein(s) will be of prime importance. To provide the greatest utility, the cDNA libraries to be studied should contain complete cDNA clones so that once differences are ascertained, the clones may be directly used for expression and determination of the function of the encoded protein. The present application describes the development of CAP affinity enrichment and enhanced reverse transcriptase technologies which will significantly contribute to the successful establishment of full sets of complete cDNAs of normal and cancerous tissues.
R43 CA081962 1999 Gerdes,John Xtrana, Inc. Quantitation And Archiving Of Gene Expression
Tumor malignant cell populations frequently occupy less than 5% of the tissue volume. Laser capture microdissection (LCM) provides a means of selecting malignant cells of similar morphology, but highly sensitive and accurate methods are needed for measuring DNA content, protein and mRNA expression levels ideally all within the same small number of microdissected cells. Molecular Innovations, incorporated (MII) will develop a precise method for simultaneous measurement of gene amplification and expression from LCM cells. Precise gene measurement is accomplished through the design of specific amplification primers and probes that provide a means of normalizing measurement fluctuations due to cell number, pseudogene sequences, specimen fixation, nucleic acid extraction, RNA degradation, and PCR amplification efficiency. Extracted Nucleic Acids are bound irreversibly to a solid phase material and can be repeatedly analyzed in series for multiple genes (archiving). The first gene targets include Estrogen and Progesterone Receptors (ER and PgR) and the c-erbB-2 oncogene. These genes are well accepted as important in the clinical management of breast cancer. Our assay will provide for more precise gene expression measurement localized in the specific tissue cells relevant to the breast tumor pathology. PROPOSED COMMERCIAL APPLICATIONS: Breast cancer is a leading cause of cancer deaths in women. An improved and more precise method of detecting gene expression levels that have been shown to be of prognostic and therapeutic importance has great commercialization potential.
R43 CA083382 1999 Monforte,Joseph Genetrace Systems, Inc. Expression Monitoring Of Multiplexed Cancer Gene Sets
There is a need for new methods in molecular genetics to provide insight into the many possible mechanisms of carcinogenesis and effects of therapeutic agents. This need extends to rapid screening methodologies in the field of pharmacology and oncology, where the evaluation and consequences of cancer progression and prevention in terms of gene expression and regulation is of major interest. We have developed a new methodology for quantitative, mulitplexed gene expression analysis based on novel labeling methods and mass spectrometry. This technology has much promise as an approach that will enable cancer researchers to acquire large volumes of information on the genetic effects of genetic mutations and environmental influences at a much lower cost than is currently possible. In Phase I, it is proposed to validate this technology for use in the study of gene expression of cancer-related genes. PROPOSED COMMERCIAL APPLICATIONS: High throughput gene expression monitoring has broad commercial application within pharmaceutical development. The methods being developed as part of this proposal may be used for primary and second chemical screens to identify new chemopreventive and chemotherapeutic agents. Further utility includes screens for chemical toxicity and the ability to decipher complex expression responses using multivariable factorial experiments.
R43 CA083384 1999 Richterich,Peter Codoncode Corporation Software For Sequencing Based Mutation Detection
CodonCode Corporation will develop a software program for mutation detection by fluorescence based sequencing. The program will combine accurate automatic detection of mutations with an intuitive graphical user interface that allows efficient interactive verification and annotation of mutations, as well as project management. Specifically, the software will be able to identify heterozygous nucleotide substitutions as well as short heterozygous insertions and deletions. The software will be written in Java to ensure system independence, and tested on both Windows98/NT and the Macintosh operating system. This will enable researchers to perform mutation analysis directly on the most commonly used desktop PCs, without the need to learn Unix.. Through fast and accurate detection of potential mutations and intuitive user interface, the software will support the rapid identification and analysis of DNA polymorphisms in clinical and basic research. CodonCode will build a product line of mutation detection software to support efficient sequence-based mutation detection in small as well as medium-size and large clinical settings. For improved support for large- scale projects, a back-end relational database and CORBA-based distributed processing will be added during Phase II of this SBIR grant. PROPOSED COMMERCIAL APPLICATIONS: The research and development will lead to a commercial software program for sequencing-based mutation detection. The software will be marketed directly CodonCode, as well as through collaborations with interested manufacturers of DNA sequencers, to researchers in clinical and basic biomedical research. The software will substantially increase the efficiency of sequencing-based mutation detection.
R43 CA083385 1999 Goldrick,Marianna Ambion, Inc. Detection And Quantitation Of Fusion Mrnas In Leukemia
The proposed work will result in diagnostic kits and assays for rapid high throughput detection and monitoring of abnormal mRNAs associated with leukemia. Hybridization-based molecular as says will be developed for rapid characterization of translocations involved in hematologic malignancies. The ability to more rapidly determine the particular translocation involved in a patient's leukemia will permit faster initiation of therapy at the time of initial diagnosis. Sensitive quantitative methods will also be developed for assessing amplified fusion mRNAs using a novel relative quantitative RT- PCR strategy. Improved assays able to provide quantitative values for fusion mRNAs will aid clinicians in determining the most effective treatment for leukemia patients at risk for relapse. The proposed assays will have a reduced chance of generating erroneous results due to sample cross-contamination, because they will provide the requisite sensitivity needed to detect minimal residual disease without the need to use sequential exponential amplification strategies, which are highly prone to contamination. The homogenous, non- gel-based as says proposed will have a reduced cost and faster turnaround time compared to current gel-based methods, which will result in cost-savings to the healthcare system as well as benefits to the patient. PROPOSED COMMERCIAL APPLICATIONS: Kits containing reagents and protocols to identify and monitor genetic abnormalities associated with leukemia and other cancers will be developed. The kits will be marketed by Ambion for use in both research and diagnostic settings.
R43 CA083388 1999 Cronin,Maureen Protogene Laboratories, Inc. Development Of Dna Microarray Fabrication Method
Our proposal is in response to NCI's solicitation PAR-98-066, Innovative technologies for the molecular analysis of cancer. We are developing a DNA microarray synthesizer capable of synthesizing DNA oligonucleotides in situ on two dimensional substrates. This technology will provide cost-effective, medium density, flexible design hybridization arrays not currently available in any other array format to support gene- expression monitoring, genotyping and sequence checking applications. Our system combines drop-on-demand inkjet reagent delivery with a chemically modified substrate. Surface tension constrains active chemistry to predefined locations. We plan to extend the method to include covalent immobilization of presynthesized nucleic acids. Preliminary results from experiments with a prototype synthesizer show successful in situ synthesis of 20mer homopolymers, 20mer mixed sequence oligonucleotides and a 40mer-oligonucleotide sequence. This was confirmed by capillary electrophoresis of synthesized products and by hybridizing with complementary oligonucleotides and PCR products. We have synthesized 400 and 1600 feature arrays on 16 cm2 surfaces. This DNA microarray technology is unique in its ability to support array design flexibility, manufacturing economy and incorporation of customized nucleotide chemistries. We propose to engineer the prototype synthesizer into a robust, automated manufacturing unit. We also plan to more fully characterize and optimize substrate surface-tension chemistry and oligonucleotide synthetic chemistry into robust manufacturing processes. PROPOSED COMMERCIAL APPLICATIONS: DNA microarrays have become the method of choice for performing highly parallel nucleic acid analysis whether it is for gene expression monitoring or genotyping applications. In addition, microarrays are proving to be useful adjuncts for rapid sequence checking and polymorphism discovery. Methods currently used to manufacture arrays are expensive, primarily due to set up costs for each array design. This may include collecting oligonucleotide or cDNA libraries or designing a set of photolithographic masks. The most cost-effective way to apply these methods is to make many copies of single array designs. The method we propose reduces array design and synthesis to a four-color printing process using inexpensive, commercially available reagents. The low expense and high flexibility should make arrays widely available to accelerate the pace of acquiring gene expression and genotype data. In addition, the array dimensions and feature sizes do not demand expensive, custom optical scanning instrumentation. These arrays are well suited to simple, generic data acquisition and analysis approaches that should enable wider accessibility and broader based adoption of hybridization array based genetic analysis.
R43 CA083398 1999 Chee,Mark Illumina, Inc. Parallel Array Processor
The major aims of this project are to develop and test a system for parallel processing of sensor arrays for analysis of biological samples. By processing multiple samples in parallel in a uniform manner, sources of signal.variation can be measured, characterized, and understood. This is particularly important for research and diagnostic analysis of human samples, because of the genetic and phenotypic diversity in human populations. An extreme example is the case of cancer cells, which have numerous mutations and are heterogeneous. Quantitative mRNA profiling and other array-based analyses of samples of this type can only be made fully effective by the analysis of many samples, in an experimentally well- controlled system. Unfortunately, commercially available array-based technologies have limitations that make it difficult to apply them to many samples in an automated way.This proposal aims to take advantage of conventional microtiter fluid handling and sample processing by marrying it to array-based experimentation. The proposal will allow 16 samples to be processed in parallel in this way, which is a capability that does not exist for any conventional array technology. PROPOSED COMMERCIAL APPLICATIONS: Parallel sample analysis can be applied directly to a number of important array-based analytical approaches. These include RNA and protein profiling and single nucleotide polymorphism genotyping. The technology has application in drug target discovery, toxicology, functional genomics research, cancer diagnostics and management of patient treatment. Potential customers include pharmaceutical companies, genomics companies, research laboratories, and providers of screening services.
R43 CA083402 1999 Beach,David Genetica, Inc. Technologies For Genetic Manipulation Of Tumor Cells
Genetica has developed a novel retroviral technology (MaRX technology) for the genetic manipulation of both normal and tumor cells. It is the objective of the present proposal to enhance the scope and applicability of the MaRX technology. Specifically, MaRX will be developed as a tool not only for gain-of-function genetic screens but also most importantly loss-of-function genetic screens. This will be achieved in the present proposal by developing a warhead technology that when coupled with an antisense DNA construct, will render it capable of catalytically degrading a specific mRNA species. In the phase I application, the feasibility of this approach will be assessed with regard to the tumor suppressor genes p53, p16 and p21 as molecular models. The long term objective is to develop a technology that provides deeper insight into potential drug targets for oncogenic disease in man. PROPOSED COMMERCIAL APPLICATIONS: The technology development program proposed herein has a wide range of commercial applications, most specifically, the deployment of the technology for the discovery of novel drug targets. In addition, the technology has value per se to both the pharmaceutical and biotechnology industries.
R43 CA083477 1999 Winzer-Serhan,Ursula Ambion, Inc. Development Of Rna Friendly, Histological Fixatives
There is a critical need for technology that will allow the isolation of high quality RNA from histologically preserved clinical samples. The most commonly used fixative, neural buffered formalin severely compromises subsequent isolation of intact nucleic acids. The need for improvement is most acute for techniques that require intact RNA for expression analysis. We proposed a three-track approach. First, we will develop and optimize procedures for the extraction and purification of high quality, reverse- transcription competent RNA from formalin-fixed samples. Second, we will develop new fixation technologies, fully compatible with nucleic acid isolation, by screening existing cross-linking reagents for suitability as fixatives. Finally, we propose synthesizing a novel reversible cross-linking, glutaraldehyde derivative. Ambion, a leading supplier of products for RNA quantitation and analysis, intends to market nucleic acid compatible tissue fixatives and RNA isolation products to the biomedical research community. We believe they will be widely utilized in both basic research and clinical laboratories. PROPOSED COMMERCIAL APPLICATIONS. Improvements to current technologies will allow high quality RNA to be isolated from histologically preserved samples. Ambion will develop and market this methodology in kit form.
R43/44 CA081835 1999 Mostert,Michael Echelon Biosciences, Inc. Phosphoinositide 3-Kinase Assays In Cancer Diagnosis
Phosphoinositide polyphosphates (PtdInsPns), biosynthesized by the interplay of kinases and phosphatases, are key signaling molecules in cellular communication. The lipid kinase, phosphatidylinositol 3-kinase (PI 3-K) has been identified in the control of diverse cellular processes such as proliferation and differentiation, tumorigenesis, and apoptosis. The use of PI 3-K and its lipid products, PtdIns(3,4,5)P2 and PtdIns(4,5)P2, as potential tumor markers in the early detection of cancer would be substantially enhanced with the development of rapid 96-well format assay kits for lipid kinase activity. Vast clinical isolets from epithelial cancer origin could be screened in such a high throughput assay format. Echelon Research Laboratories (ERL) has developed syntheses of commericalized PtdInsPns. In our second tier strategy, these PtdInsPns as well as licensed monoclonal antibodies against phosphoinositides will be employed to prepare assay plates containing a specific immobilized substrate using Flashplate(TM) and ELISA technologies, respectively. We will focus on the use of PtdIns(4,5)P2 as a substrate for PI 3-K to develop and optimize assay kits against benchmark techniques. Subsequent screening of various types of epithelial cancer isolets will be pursued to determine the significance of PI 3-K and its lipid products as a reliable diagnostic tool in cancer research. PROPOSED COMMERCIAL APPLICATIONS: The research would lead to production of 96-well assay kits for rapid testing of PI 3-K as a potential tumor marker in clinical isolets taken from various origins of epithelial cancer cells. Systematic screening of multiple cancerous cell extracts will allow rapid detection of tissue- or physiology- specific activities. Screening of libraries of compounds will allow detection of potential isozyme-specific inhibitors as therapeutic leads. Major pharmaceutical companies, numerous biotech start-ups, and hundreds of academic laboratories would employ such kits for basic and applied research.
R43/44 CA081952 1999 Chee,Mark Illumina, Inc. Gene Expression Analysis On Randomly Ordered Dna Arrays
A novel and highly innovative technology will be evaluated for high-throughput, low cost mRNA profiling. The technology has potential application in many areas of cancer research and drug discovery and development, as well as cancer diagnosis and prognosis and management of treatment. The proposal is based on the use of self-assembled arrays of beads on optical fibers. Self-assembly is rapid, which allows different arrays to be manufactured on demand. The arrays have 1-fold more elements per unit area than the most densely packed conventional arrays, and densities can be increased further. Small sample volumes are required by comparison to conventional arrays, allowing analysis of small amounts of material. The specific aims relate to hybridization to oligonucleotide probes on beads in fiberoptic arrays. The feasibility of detecting a specific mRNA species present in total cellular mRNA at a level of 1 part in 100,000 will be assessed, in a sample having a total target RNA concentration of not more than 50 ug/ml. The feasibility of detecting 2-fold differences in gene expression will be assessed, for a range of mRNA concentrations from 1:100,000 to 1:1,000 in a total sample of 50 ug/ml. PROPOSED COMMERCIAL APPLICATIONS: Cellular functional profiling for cancer has application in drug target discovery, toxicology, functional genomics research, cancer diagnostics and management of patient treatment. Potential customers include pharmaceutical companies, genomics companies, research analytical suppliers, research laboratories, providers of screening services (e.g. clinical diagnostic labs), and point- of-care patient monitoring services (e.g. hospitals).
R43/44 CA081965 1999 Bitter,Grant Bittech, Inc. Dna Mismatch Repair Functional Genetic Tests
Genomic instability has been well documented in both cancer cells and precancer cells. Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations in any one of four genes encoding proteins involved in DNA mismatch repair (DMR). Defects in DMR have also been demonstrated in several sporadic cancers as well as precancers, indicating that cellular defects in DMR may be a frequent early step in the evolution of a cancer cell. Genetic analyses of HNPCC kindreds reveal that approximately 25% of the observed alterations in DMR genes predict minor changes in the protein, such as amino acid replacements. With current genetic testing methods, it is not possible to unambiguously assign these sequence variations as either mutations or silent polymorphisms. This research grant application proposes development of functional genetic tests of DNA mismatch repair. This novel technology will have broad utility for basic, clinical and epidemiological cancer research. Defects in DMR predispose to cancer development, both when acquired in a precancer cell through somatic mutation or when inherited as a germline DMR mutation. The technology described in this research proposal will allow assessment of the in vivo function of DMR gene products, and will therefore allow definitive molecular characterization of genomic instability caused by mutations in specific DMR genes. PROPOSED COMMERCIAL APPLICATIONS: Genomic instability is a hallmark of both cancers, precancer cells and inherited predispositions to cancer. Defective DNA mismatch repair is a common source of genomic instability, but current genetic testing methods fail to adequately characterize approximately 25% of the gene variants observed. The technology to be developed in this research, in conjunction with other methods, will allow definitive characterization of cellular DNA mismatch repair competence.
R43/44 CA083390 1999 Wiktor,Peter Engineering Arts Piezo Electric Pipetting Technology For Dna Analysis
This grant brings together two innovative technologies: a novel fluid transfer device and a novel technique for detecting Single Nucleotide Polymorphisms (SNPs) on cDNA arrays. Integrated together, these two technologies will have an immediate and significant impact on the molecular analysis of cancer. Actuated by piezo-electric elements, the fluid transfer technology enables the pipetting of sub-nanoliter volumes of fluid. Additionally, novel sensing technology allows the operational state of the device to be continuously monitored. The sensing technology is indispensable for massively parallel DNA analysis applications requiring thousands of different samples to be reliably aspirated and dispensed. The ultimate goal is to integrate the fluid transfer and sensor technology into an automated pipetting instrument for cancer research and general laboratory applications. Initially the instrument will be applied to a novel technology making it possible for the first time to detect SNPs, deletions and insertions on readily available and relatively inexpensive, spotted cDNA arrays. An Invention Disclosure has been filed by the Collaborating Investigator with the Fred Hutchinson Cancer Research Center (F.H.C.R.C.), Seattle, WA for this exciting new technology. The detection of SNPs on DNA microarrays has heretofore only been possible using allele specific hybridization on oligonucleotide arrays (GeneChips) produced by Affymetrix. Limited access to these GeneChips and their prohibitive costs will make it difficult to apply this technology to large numbers of DNA samples. A major goal of the proposed study is to develop and demonstrate the feasibility of the new SNP detection methodology for high throughput detection of polymorphisms at multiple loci using the high-density cDNA arrays produced by the novel arrayer to be developed in this grant. These highly reproducible, high-density arrays are also expected to provide improved sensitivity and specificity in the analysis of mRNA expression. Experiments will be performed to demonstrate the advantages of the proposed technology over existing pin based mechanical arrayers for gene expression analysis. PROPOSED COMMERCIAL APPLICATIONS: Fueled by emerging technologies like bio-chips, micro-arrays and high throughput screening and the high costs of reagents in general, the bio- medical industry is going towards assays using smaller fluid sample volumes. The proposed research directly addresses this growing market by developing a general purpose automated laboratory pipetting instrument for these applications. By keeping development costs down, the new technology will make high quality, high density DNA arrays accessible to a much broader segment of the cancer research community.
R43/44 CA083396 1999 Gite,Sadanand Ambergen, Inc Novel Technology For The Analysis Of Gene Expression
The objective of this project is to develop new technology for the rapid screening of proteins that are expressed in vitro and derived from specific genes linked to cancer. Markers and photo cleavable linkers will be incorporated into proteins during cell-free synthesis with specially prepared misaminoacylated tRNAs. The incorporation of highly fluorescent amino acids into nascent proteins can significantly decrease the time needed to detect and characterize such proteins compared with conventional radioactive methods and is amenable to high throughput automation. The incorporation of proprietary photo cleavable linkers into nascent proteins facilitates their rapid affinity-based purification and downstream physical and functional analysis by a variety of techniques including gel-shift assays, mass spectrometry and capillary electrophoresis. This approach will be evaluated during Phase I by incorporating a variety of fluorescent markers and photo cleavable linkers into four model proteins, by analyzing the properties of these modified proteins and by testing various methods for the isolation and detection of these proteins. This technology will be evaluated at the clinical level for genetic diagnosis of chain terminating mutations in the APC gene during Phase I, and in Phase II will be extended to detect a variety of genetic mutations linked to other cancer syndromes. PROPOSED COMMERCIAL APPLICATIONS: The development of rapid methods for the detection and isolation of cell- free produced proteins will have commercial applications in many areas of cancer research and clinical diagnostics. Products include reagents, kits for incorporating fluorescent markers and PC-linkers into nascent proteins, and automated systems for the screening of genetic defects such as the protein truncation test (PTT).
R44 CA081954 1999 Lifshitz,Nadia Biophotonics Corporation High Throughput, High Resolution Dna Sequencing
Our goal is the development of a high-throughput automated sequencing instrument adapted for identification of rarely present mutations and polymorphism in mixed populations of DNA molecules and validation of the instrument using the p53 gene as a model system. Operation of the machine is based on a novel detection technique which offers the ultra-high resolution of mixed fluorescent markers. Main features of the machine: . analysis of mixed DNA samples with 1% resolution; . throughput of 6,000,000 bp/year; . low amount of labeled DNA material; . Easy-to-replace capillary cassette; . adapted to color sequencing with different dye sets; In Phase 1 we shall demonstrate a feasibility of 1% resolution of mixed DNA samples and test this detection system using clinical DNA material. In Phase II we shall develop a pilot prototype of the machine and validate the machines using the p53 gene as a model system. In Phase III we shall organize manufacturing of the 12-capillary cassettes and 12-lane automated sequence for diagnostics of mixed DNA samples. PROPOSED COMMERCIAL APPLICATION: The proposed automated sequencing machine will find applications in clinical diagnostics of genetic disease, primarily cancer. Its unique capability to precisely quantify contaminated DNA samples will ensure a dominant position in an important market segment.
R44 CA082038 1999 Anderson,N Large Scale Biology Corporation Proteomic Technologies For Cancer Research
Human serum and urine are each estimated to contain over 5,000 different proteins and peptides, many in trace concentrations. Disease-associated changes have been described for most of the less than 5% which have been quantitatively studied, but very few of these are useful cancer markers. Thus there is both an urgent need for new markers, and a forest of potential markers to be explored. We proposed concurrent development and testing of technology for systematically and sequentially fractionating proteins from serum and urine from normal individuals and cancer patients to discover useful tumor markers. The key analytical tools are quantitative high resolution two-dimensional electrophoresis under denaturing conditions, mass spectroscopy for protein identification, rapid recycling immunosubtraction, preparative and analytical gel filtration, high resolution chromatography based on ion exchange and hydrophobicity, and isoelectric focusing and conventional gel electrophoresis. In combination these can resolve thousands of proteins over a wide dynamic range. Phase I will concentrate on the demonstration that new proteins and a small set of candidate markers can indeed be found using these techniques in a manual format, while in Phase II each method will be mechanized or automated to allow higher throughput cancer marker discovery. PROPOSED COMMERCIAL APPLICATION: Identification of proteins or protein fragments unique to cancer would lead to the development of new clinical tests and to methods for imaging small tumor masses. If successful, this would revolutionize the diagnosis and treatment of cancer.