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R21 CA099089 2003 FERRARI, MAURO OHIO STATE UNIVERSITY Nanomechanical Method for Molecular Analysis of Cancer
This project aims at the development of a novel method for the quantitative, objective, and rapid analysis of the expression of the molecule Her-2/neu on breast tissue biopsies. Her-2/neu is a prognostic and predictive biomarker for breast cancer, and its level of expression is a basis for the determination of the optimal treatment modality in contemporary clinical practice. The technology platform proposed herein employs characterization-mode ultrasound, in conjunction with immunotargeted micro- and nanoparticles, for the amplification of the molecular signature. The centerpiece of the approach is a novel theoretical framework, which enables the identification of previously inaccessible information on the physical properties of the probed tissue, at different length scales. Preliminary studies have yielded encouraging results on both native and particle-modified tissue samples. The hypothesis underlying the proposal is that molecular information can be translated into mechanical properties at specified length scales by way of nanoparticle immunotargeting, and that information based on these properties can be detected and analyzed using the ultrasonic characterization system and software proposed herein. In the R21 phase, we propose to establish the feasibility of this approach, starting with the development and refinement of robust protocols for the derivatization of nanoparticles with antibody conjugates; the development of sample preparation methods that are most suited for quantitative interrogation by ultrasonic characterization; the completion of an ultrasonic characterization system for detecting the nanoparticle signals; and the finalization of the data analysis tool under the novel, multi-scale mechanical field theory. In keeping with the proof-of-principle nature of this phase, the milestones proposed for the transition into the R33 phase are the attainment of specificity and sensitivity figures that are no less than those that pertain to prevalent current pathological investigation techniques, i.e., in this context, IHC and FISH. Even at identical sensitivity and specificity, the proposed system is believed to be potentially advantageous over IHC and FISH in that it is very rapid, automated, and objective, has high potential for interobserver and interlaboratory agreement, and requires significantly reduced level of investment of a specialist's time. In the R33 phase, the objectives of the proposed program would be to optimize the system's performance and create an actual prototype for clinical testing in the context of breast malignancies.
 
R21 CA103071 2003 MAJUMDAR, ARUNAVA UNIVERSITY OF CALIFORNIA BERKELEY Nanocapillary Electrophoresis for Profiling Cancer
The goal of this project is to develop nanocapillary electrophoresis array technology (NEAT) as a label-free, quantitative, and high-throughput assay of proteins and nucleic acids that are critical in early detection, monitoring, diagnosis, and prognostic evaluation of cancer. The NEAT chips will contain an array of glass nanocapillaries whose inner surfaces are functionalized with receptors to capture specific ligands. We have developed a process for synthesizing glass nanocapillaries with lengths in the 1-10 micron range and diameters in the 5-20 nm range, which is on the order of biomolecular size. Hence, when analyte biomolecules are electrophoretically transported through functionalized nanocapillaries in response to an applied voltage (approx. 1 V), specific ligand-receptor binding will lead to reduction in the ionic current flow due to partial blockage of the nanocapillary. Recent experiments have shown that indeed when a transmembrane ion channel protein is functionalized with a single strand of DNA, binding of its complementary strand can be detected through modulation of the ionic current with single-molecule sensitivity, and specificity of single base pair mismatches. By using the same principles, the goal of the R21 phase is to demonstrate that ionic current modulation could be used as a label-free assay for PSA protein and p53 exonic sequence with specificity and sensitivity sufficient for cancer diagnostics and monitoring. The goal of the R33 phase is to build upon the R21 knowledge and develop the NEAT chip that will consist of an array of microfluidic cells, with each cell containing a single nanocapillary functionalized with a distinct probe/receptor. An electronic system will allow multiplexed addressing of individual nanocapillaries such that their respective ionic currents can be simultaneously measured. Such an integrated system will enable multiplexed assays of literally hundreds of molecules, such as transcribed mRNAs or proteins. For protein expression, in particular, this technology would represent a new paradigm in the evaluation of multiple proteins from a single tissue or from serum and could represent a cost-effective way to assess multiple cancer antigens in cancer screening and monitoring programs. Such routine screening is now only done for very few cancer antigens (primarily prostate specific antigen, PSA) due to the expense such large-scale screening would incur. This technology would directly address this important public health issue.
 
R21 CA102949 2003 MAKRIGIORGOS, G. MIKE DANA-FARBER CANCER INSTITUTE ERROR-FREE DNA AMPLIFICATION FOR MUTATION DETECTION
High selectivity mutation detection, which frequently relies on PCR, often falls short by 1-2 orders of magnitude of the selectivity required to identify cancer cells at an early stage, to investigate mechanisms of tumorigenesis, to detect mutations in single cells or to reliably detect minimal residual disease. A major cause of this deficiency is that PCR poses a selectivity limit, typically 1 mutant in 105- 106 wild type alleles, since all DNA polymerases invariably generate errors during DNA synthesis which can be misinterpreted as mutations (false positives). We have now developed hairpin-PCR, a novel method that allows elimination or major reduction (>100-fold) of PCR errors in a sequence of interest, and can supply existing mutation detection technologies with the necessary 'selectivity leap'. By converting a DNA sequence to a hairpin and performing PCR in a hairpin structure, true mutations can be separated from polymerase-generated misincorporations, thereby providing practically error-free DNA. This project will develop and optimize the technology to PCR amplify sequences from genomic DNA, eliminate error-containing sequences, and demonstrate its application for highly improved mutation detection using technologies previously limited by PCR errors. In the R21 phase we shall demonstrate amplification of hairpins up to 500 bp long directly from human genomic DNA, and we shall optimize isolation of hairpins containing genuine mutations (homoduplex hairpins) from hairpins containing polymerase errors (heteroduplex hairpins), using dHPLC separation. In the R33 phase the technology will be further developed to (a) reduce PCR errors by at least 1000 times, (b) demonstrate that hairpin PCR allows current mutation detection methods a major selectivity leap relative to their current limit, and (c) amplify large portions of the human genome in an error-free manner, for multi-gene mutation screening. By providing error-free amplified DNA for analysis, the present hairpin PCR will allow a major boost to almost every existing genotypic selection method and enable studies and diagnostic tests that were not possible with previous technology. The new technology will also improve the accuracy of microsatellite analysis and will have additional applications in molecular beacons and real time PCR, and in DNA cloning or protein functional analysis by in vitro translation.
 
R21/R33 CA101150 2003 ANGELETTI, RUTH HOGUE ALBERT EINSTEIN COLLEGE OF MEDICINE YESHIVA UNIVERSITY Proteomics of Hepatic Carcinogenesis
Hepatocellular carcinoma is the one of most frequent causes of death by cancer in the world. There is no reliable diagnosis prior to late stages of disease and no hope for cure except surgery. The overall goal of the proposal is to discover and identify distinctive alterations of protein expression in early precancerous lesions isolated from their physiological microenvironment. The data obtained will provide early molecular markers with diagnostic and therapeutic potential for early intervention in human liver cancer. We propose to apply innovative proteomics and laser capture microdissection microscopy (LCM) to study a well-characterized animal model of liver carcinogenesis (RH) that exhibits well-defined, synchronous stages of initiation and progression of liver cancer that are strikingly similar to those in liver cancer in humans. In the R21 phase, we will focus on demonstrating our ability to generate useful patterns/profiles with combined LCM and protein technologies using control tissue/serum and one preneoplastic stage of the biological model system. In the R33 phase, we will apply the pattern/profile generation technologies to the cells and sera of each of the early stages and control paradigms so that we can identify candidate markers and targets. We will use both global and targeted proteomics strategies to: 1) identify difference proteins in cells early in the development of liver cancer; 2) identify unique serum markers at early stages; 3) determine progressive changes in tubulin isotype composition, which is associated with development of drug resistance; and 4) identify changes in proteins associated with the cytoskeletal scaffold.
 
R21/R33 CA092752 2003 BARRON, ANNELISE EMILY NORTHWESTERN UNIVERSITY Fast Mutation Detection by Tandem SSCP/HA on Microchips
It is proposed to optimize, evaluate, and pilot rapid, scalable, and low-cost microchip electrophoresis technologies for sensitive and specific molecular detection of cancer by tandem single-strand conformational polymorphism (SSCP)/heteroduplex analysis (HA), using the p53 gene as a model system. We request a 1-year R21 phase and a 3-year R33 phase. The proposed project involves collaboration between members of Northwestern's Lurie Comprehensive Cancer Center, including researchers in Chemical Engineering, the Medical School, and Evanston Hospital. Microchannel "tandem" SSCP/HA is a novel mutation detection method recently developed in our laboratory, which involves the simultaneous generation and analysis of homo/heteroduplex DNA and SSCP conformers. Studies of a significant number of samples (32) indicate that tandem SSCP/HA allows for much higher-sensitivity mutation detection (100%) than SSCP alone (93%) or HA alone (75%), for p53 samples. We have developed and published optimized sample preparation protocols, gel formulations, and analysis conditions for capillary array electrophoresis (CAE). During the R21 phase, we will translate these methods to microfluidic electrophoresis chips, which offer a large increase in throughput and drop in cost of DNA analysis compared to CAE. The p53 gene, known to be mutated in >50% of human cancers, and whose mutation status can be predictive of patient response to chemotherapy, is the important model system chosen. However, microchip-based genetic analysis technologies to be developed should be easily applied to ANY cancer-related gene. In the R21 phase, we will analyze approximately 60 different DNA samples derived from tumor cell lines, representing a range of mutations in different p53 exons, to determine the impact of DNA sample characteristics and electrophoresis protocols on the sensitivity and specificity of the method, in a blinded study designed by collaborating biostatisticians. When optimized tandem SSCP/HA protocols have been developed for microchips, they will be piloted by the analysis of >200 selected samples amplified from frozen, solid tumors banked at Evanston Hospital. Via this blinded study, sensitivity and specificity (both expected to be at or near 100%) will be determined and reported for the first time using banked tumor tissue, providing necessary validation for clinical application of this technique, and making rapid, low-cost cancer genotyping technology widely available to physicians.
 
R21/R33 CA099139 2003 GOODLETT, DAVID ROBINSON INSTITUTE FOR SYSTEMS BIOLOGY Parallel Peptide Tandem Mass Spectrometry (MS)
It is the specific aim of the R21 phase of this proposal to develop the software tools necessary to interpret tandem mass spectra produced by collision induced dissociation (CID) of peptides in parallel rather than in series, as is commonly practiced in shotgun proteomics, and to prove its advantages over serial CID on samples of increasing complexity from peptide standards to proteins extracted from a medulloblastoma primary cell line lysate. Our proposed technology is referred to as shotgun CID and shotgun tandem mass spectrometry (MS/MS) to distinguish it from serial tandem MS and multiplex MS in a Fourier transform-ion cyclotron resonance-mass spectrometer (FT-ICRMS). It is the general aim of this combined R21-R33 proposal to provide a cost effective competitor to the advantages of multiplex MS in FT-ICR-MS. Our approach will use lower resolution and mass accuracy time-of-flight (TOF) mass analyzers and a continuously alternating data acquisition scheme of parent ion measurement followed fragment ion measurement throughout the chromatographic introduction of sample. Proteins will be identified by a combination of mass mapping of parent peptide ions across the entire chromatographic time, unique chromatographic constraints and their combined fragment ions. Primary brain tumors are the leading cause of cancer-related death in children. Medulloblastoma is a primitive neuroectodermal tumor that typically arises from the cerebellar vermis and shows variable degrees of arrested neural differentiation. The cerebellum requires endogenous retinoids for proper control of neuronal apoptosis and differentiation during development. In embryonal carcinoma and neuroblastoma cells, retinoids induce neural differentiation and cell-cycle arrest. Retinoids have recently been shown to induce extensive apoptosis and neuronal differentiation in medulloblastoma cell lines and freshly resected medulloblastoma cells. Together with Dr. Jim Olson of the Fred Hutchinson Cancer Research Institute, who is organizing a Phase III clinical trial on the effects of 13-cis retinoic acid in medulloblastoma therapy, we will develop shotgun CID within a model that seeks to: 1) facilitate identification of markers associated with retinoid-responsiveness in medulloblastoma cell lines and 2) define key components of the retinoic acid pathway modulated by treatment in retinoid-responsive medulloblastoma cells. This work will be done with medulloblastoma cell lines and primary cells lines derived from retinoid-sensitive and -resistant tumors.
 
R21/R33 CA103235 2003 KRON, STEPHEN J. UNIVERSITY OF CHICAGO BCR-ABL Kinase Assays for STI571 Sensitivity of Response
This phased innovation award proposal is focused on developing a robust approach to quantitative assay of specific protein tyrosine kinase activities from cancer cells. Our model is the oncogenic BCR-ABL fusion protein, the gene product of the t(9;22) Philadelphia chromosome (Ph1) translocation observed in the vast majority of Chronic Myelogenous Leukemia (CML) and in up to 30% of adults with Acute Lymphoblastic Leukemia and in other hematological neoplastic diseases. The activation of Abl kinase by fusion to BCR that is inferred to underly the malignant transformation of Phl positive CML is effectively opposed by the orally administered tyrosine kinase inhibitor (TKI) Imatinib Mesylate (IM, STI-571, Gleevec). The activity of IM as an Abl kinase inhibitor in vitro is thought to be the critical determinant of its efficacy in vivo. Nonetheless, a clinically useful assay for IM inhibition of BCR-ABL kinase activity in circulating CML leukemia cells is lacking. We propose to develop a protein/peptide chip-based assay for BCR-ABL that can detect the degree of inhibition by IM to evaluate dosing and drug resistance. Insofar as other activated tyrosine kinases may be critical mediators of malignancy in both leukemias and solid tumors, developing such an assay would be a powerful tool in evaluating other TKI drug candidates targeting these kinases for their efficacy in vivo. Thus, this project is directed at two major discovery objectives and three development objectives. First, in the initial project year, we intend to use our established methods for anti-phosphotyrosine antibody-based detection of purified Abl kinase activity on a peptide chip to 1) Recapitulate our Abl kinase assay with undiluted whole cell extracts from cell lines expressing BCR-ABL and 2) Use this assay to measure the inhibition of BCR-ABL by IM both in extracts and intact cells. During the development phase, we intend to use both BCR-ABL expressing cancer cell lines and circulating leukemic cells from treated patients as samples to 1) Optimize the BCR-ABL substrate and reaction conditions to enhance sensitivity and specificity of phosphorylation, 2) Examine alternative detection methods for BCR-ABL activity based on phosphospecific antibodies and thiophosphate targeted chemistry, and 3) Evaluate different chemistries for immobilizing BCR-ABL substrates on a surface and geometries for detection of phosphorylation. By these aims we intend to develop a highly versatile kinase assay system which can be applied to monitoring of patient response to IM and as a tool for discovery and testing of new TKI cancer drugs.
 
R21/R33 CA099191 2003 LABAER, JOSHUA HARVARD UNIVERSITY Functional Proteomics of Breast Cancer
In the past decade, biological research has witnessed a paradigm shift from focused reductionist approaches to a greater dependence on large "industrial-sized" projects. High-throughput (HT) biology began in earnest with the Human Genome Project, and increasingly these HT tools and approaches are being exploited for protein research. Given the importance of proteins in disease etiology and treatment, a major challenge facing biology is the elucidation of the physiological role of all proteins. In this light, the field of functional proteomics, a new approach to the HT study of proteins, will enable the expression and subsequent assay of proteins and their various properties such as subcellular location, interacting partners, biochemical activity or regulated modification at a scale of thousands at a time. A prerequisite for this approach is the need for large collections of cDNAs in a format conducive to HT protein expression. We and others (Walhout 2000, Brizuela 2001) have begun to create such collections of cDNAs using the novel technology of recombinational cloning that allows rapid transfer of DNA fragments from one vector to another, in frame and without mutation. However, high-quality collections of human clones are not yet available. Here we propose to build a collection of 1000 expression-ready human cDNA clones representing genes of significance to breast cancer (BC 1000). We are nearing completion of 100 such clones assembled in a pilot study and have already found them to be invaluable in the development of HT methods for both in vitro and in vivo studies. However, the relatively modest size of the collection prevents its application in any meaningful screening experiments. Thus it is important to expand this collection to a size that will enable more comprehensive screens and the development of experimental technologies that will truly exploit the HT setting. In order to exploit this resource, we have developed a novel method for creating protein microarrays that enables the HT functional study of proteins. This approach, called Nucleic Acid-Programmable Protein Array (NAPPA), replaces the complex process of spotting purified proteins with the simple process of spotting DNA. By exploiting the recombinational format of the BC1000, genes are then simultaneously transcribed/translated in a cell-free system and the resulting proteins are immobilized in situ, minimizing direct manipulation of the proteins and making this approach well suited to HT applications. Advantages of this approach include: the ability to express and interact proteins in a mammalian milieu, no requirement for HT expression-purification-storage of proteins, and real time collection of data, minimizing concerns about protein stability. We propose to adapt this method to a glass matrix and miniaturize it to allow for the screening of thousands of proteins simultaneously. We will demonstrate the effectiveness of this approach by executing a 1000 x 1000 interaction matrix with the BC1000.
 
R21/R33 CA099246 2003 SOPER, STEVEN ALLAN LOUISIANA STATE UNIVERSITY A&M COLLEGE BATON ROUGE Microsampling Unit for Capturing Low Abundant Cells
Our group will be developing modular microsystems that can be assembled in a variety of different configurations to carry out complex biomedical assays appropriate for monitoring both genetic and protein markers in clinical settings. Our focus in this R21/R33 application will be screening diagnostic markers associated with breast cancer. The focus of our project is to build upon our existing expertise in LIGA to fabricate BioMEMs devices possessing high-aspect ratio microstructures (HARMs) fabricated out of a variety of polymer materials. Specifically, we will be fabricating the following modular devices: (1) Microsampling unit with target preconcentration capabilities of rare cancer cells in circulating blood. Our microsampling unit will consist of capture beds prepared using surface imprinted polymers to semi-selectively capture pre-selected targets (cells expressing EpCAM). (2) Cellular lysis system. Captured cells will be electrodynamically lysed using microfabricated electrodes to induce cell rupture via electroporation. (3) Multiplexed hybridization association unit. To signal the presence of either a mutant gene or protein that is under- or over-expressed, we will build arrays of elements consisting of tethered nucleic acid probes or antibodies (Ab) to associate selectively with our targets. Since both genetic and protein markers can be effective in diagnosing a particular disease state, we will build hybrid systems (hybrid-biosensors) that will monitor simultaneously both genetic and protein markers to minimize false positives and negatives associated with the diagnosis. (4) Micro-optical bench with high sensitivity laser-induced fluorescence components. The transduction of the hybridization of target DNAs to our tethered probes or association with tethered Ab will be determined using near-IR fluorescence with excitation provided by a surface mounted vertical cavity surface emitting laser (VCSEL) and the sensing elements situated on a planar polymer waveguide. (5) Ultrasensitive probes for fluorescence transduction. Our fluorescence detectors will be configured to read luminescence generated in the near-IR by developing labeling probes that possess spectral properties in the near-IR (excitation/emission maxima > 700 nm).
 
R21/R33 CA099835 2003 WOODS, VIRGIL L UNIVERSITY OF CALIFORNIA AT SAN DIEGO Enhanced Crystallography of Cancer-Implicated Proteins
We aim to enhance the ability to design protein crystallographic constructs and, thereby, substantially speed protein structure determination with the use of information provided by innovative peptide amide hydrogen exchange techniques. In this resubmission, we present extensive preliminary studies, performed since review, that directly address all reservations regarding our prior submission. Determination of high-resolution structure at an increasingly high throughput (HT) pace is required for a fundamental understanding of how modifications of cancer- implicated proteins can promote oncogenesis and metastasis. Unfortunately, HT crystallographic efforts have a single, dominating roadblock: they produce suitable crystals for a small minority of target proteins. Floppy, unstructured regions of failed proteins play a major role in this problem. The exchange rates of the many peptide amide hydrogens within a protein are determined by the protein's stability at the individual amino acid scale. We have developed an enhanced form of amide hydrogen/deuterium exchange-mass spectrometry (DXMS) that can rapidly and precisely measure such rates. We propose that DXMS data can be used to identify and localize such unstructured regions within a protein and thereby guide the design of modified protein in which such regions are selectively removed. Furthermore, many proteins require tertiary-quaternary contacts, provided by binding partners, to induce structure in such regions. For these proteins, DXMS can be used to rapidly select binding partners that provide the needed stabilizing contacts, allowing focused protein-binding partner co-crystallization efforts. Importantly, repeat DXMS study of the modified protein(s) can rapidly determine how well they have retained the structured elements of the original protein. In our R21 year, we will demonstrate that DXMS can guide the re-design of protein constructs sufficiently to produce a 50% increase in overall crystallization success rates for target proteins, and do this at a high throughput pace. This result will establish the ability of DXMS to speed throughput of present HT crystallographic efforts, and likely similarly enhance construct definition for conventional, specific-protein focused crystallography, with obvious benefits for the structural study of cancer related proteins. In our R33 years we will establish a crystallography-dedicated DXMS facility and further refine our ability to guide construct design by analysis of the protein targets studied by our collaborators at the Joint Center for Structural Genomics, with an emphasis on those with cancer-relevance. This construct-refinement resource will then be broadly extended as a community service to NCI-funded investigators for application to both conventional and HT crystallographic efforts.
 
R33 CA103056 2003 ARAP, WADIH UNIVERSITY OF TEXAS MD ANDERSON CANCER CENTER STEM CELL-BRAIN TUMOR INTERPLAY & IN VIVO PHAGE DISPLAY
This proposal aims to develop innovative methods for brain cancer diagnosis and therapy that will combine the strengths of neural stem cell (NSC) biology and in vivo phage display technology. The proposal is based on our prior work that demonstrated a remarkable, apparently "magnetic" attraction of NSCs to glioblastoma brain tumor cells. When NSCs were injected into one cerebral hemisphere, and rat or human glioblastoma tumors into the other, the NSCs migrated across the midline and headed directly to the tumor masses. When the NSCs were injected intravenously, they entered the brain and selectively targeted on the tumor. NSCs attached even to single tumor cells which were in the process of invading normal brain tissue. When NSCs were engineered to deliver toxic molecules, tumor cells were killed. New experiments will build on these results. 1. Short- and long-term effects of NSCs will be analyzed on genetically-induced natural tumors, not only on grafted tumors. 2. Optimal cell numbers and optimal route of injection into mice will be explored with mouse and human NSCs, including determination of whether a carotid intra-arterial route might be more effective than intracerebral or intravenous routes. 3. As a step toward development of diagnostic procedures of higher sensitivity, for future use in humans, NSCs will be modified to carry molecules allowing radiological visualization, so that the NSCs will serve to delineate the positions, sizes, and number of tumor masses in the brain. 4. As model "proof-of principle" experiments, NSCs will be engineered genetically to synthesize and release agents that kill dividing cancer cells and/or other agents that may induce cancer cells to differentiate into stable, quiescent glial cells that no longer endanger life. 5. To uncover the basic molecular and cell biological mechanisms controlling the "cross-talk" between NSCs and tumor cells, the powerful phage display technology, which allows identification of ligands and their receptors without preexisting data about their natures, will be used in tissue culture and in intact mice to define host and tumor ligands that react with NSC receptors and attract NSCs to the tumor, as well as the reverse - - specific receptors on brain tumor cells and on their specialized blood vessels that bind peptide ligands released by NSCs.
 
R33 CA103068 2003 COLLINS, COLIN C UNIVERSITY OF CALIFORNIA SAN FRANCISCO Development of ESP: Structural & Functional Oncogenomics
The long-term objective of this proposal is to gain an enhanced understanding of the structural genomics of solid tumors through development of a novel, sequence-based method capable of identifying all types of structural rearrangements that occur in tumor genomes. Genome rearrangements can promote cancer development, progression and/or resistance to therapy by altering gene regulation and/or function, and the involved genes are potential therapeutic targets. This is well established in leukemia and lymphoma, but less so in solid tumors, in part because of the difficulty of identifying the genes involved in complex structural rearrangements. We describe here a powerful and high resolution, sequence-based analytical approach called End Sequence Profiling (ESP). ESP maps copy number aberrations and directly identifies and clones en masse genome breakpoints associated with genome rearrangements such as inversions, translocations, deletions and amplifications. ESP is accomplished by constructing a BAC library of a tumor genome, end sequencing a larger number of BAC clones, and mapping the BAC end sequences (BES) onto the normal genome sequence. Paired BES that map to different parts of the normal genome span structural rearrangements. Sequencing these clones will reveal exact breakpoints and involved genes. In Specific Aim 1 we will: Implement ESP as a cost effective sequence-based technology for determining the structural organization of tumor genomes and clone rearrangement breakpoints en masse. Determine the minimum sequencing depth needed to yield the maximum structural information. Determine if ESP can reproducibly identify recurrent rearrangements between tumors, and if so, whether specific sequence elements are associated with these rearrangements. In Specific Aim 2 we will: Develop robust computational methods for the analysis, visual representation, and integration of ESP data with the human reference sequence, making possible comparison of ESP data from independent tumors. Knowledge of how genome rearrangements such as inversions and translocations impact local gene expression is critical. Thus, we will integrate ESP-based structure data with expression microarray data and co-localize aberrantly expressed genes with genome rearrangement breakpoints. In Specific Aim 3: We will biologically and clinically validate key ESP findings. We believe ESP provides a rational framework for sequencing tumor genomes. In fact, ( 100 tumor genomes can be analyzed at ( 10 kb resolution for less than sequencing a single 3000 Mb genome yielding hundreds of novel biomarkers and therapeutic targets associated with translocations, inversions, and complex rearrangements. This is important because, just as a comprehensive systems-based knowledge of human biology is predicated on the structural organization and sequence of the human genome, a structure-based view of tumor genomes is essential for a comprehensive understanding of tumor biology.
 
R33 CA101136 2003 FURGE, KYLE A VAN ANDEL RESEARCH INSTITUTE Comparative Genomic Analysis of Microarray Data
Aneuploidy is a common feature of cancer and several lines of evidence suggest that cytogenetic aberrations can significantly influence cancer diagnosis, prognosis, and treatment. While molecular genetic based methods, such as comparative genomic hybridization (CGH), have traditionally been used to determine cell karyotypes, recent transcriptional profiling studies have suggested that it is possible to predict cytogenetic changes from microarray gene expression data. A technique we term comparative genomic microarray analysis (CGMA) is based on the observation that gene expression values show expression biases, either increased or decreased, in regions of chromosomal gain and loss, respectively. In tumor samples, CGMA predictions are made by mapping gene expression values to the public human genome assembly and scanning for genomic regions that contain a statistically significant upwards or downwards gene expression bias. While the first-generation of algorithms that identify gene expression biases produce reasonably good cytogenetic predictions, it is likely that more sophisticated algorithms could produce better results. The R21 phase of this proposal focuses on implementing and testing a set of refined CGMA algorithms to make more accurate and higher resolution cytogenetic predictions. Cytogenetic and transcriptional profiling data obtained from a small set of colon tumors will be used for algorithm testing. The R21 milestone is to establish a CGMA prediction method that matches CGH determinations with high accuracy. The R33 phase will focus on developing algorithms to make CGMA predictions across multiple samples and will test if frequently changed regions identified by CGMA match those regions previously identified by CGH. For the R33 phase, hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC) will serve as models because large sets of gene expression and cytogenetic profiling data are currently available. Historically, candidate genes have been identified by determining if a gene located within a region of frequent cytogenetic change is either mutated or misregulated. In this proposal, candidate genes will be identified from the HCC and RCC gene expression profiles by first using CGMA to locate frequently changed genomic regions and then by using traditional gene expression analysis to identify abnormally expressed genes located within these regions of frequent cytogenetic change.
 
R33 CA099136 2003 LAM, KIT S UNIVERSITY OF CALIFORNIA DAVIS Small molecule microarrays for intracellular proteins
This project involves the application of the "one-bead one-compound" encoded small molecule combinational library method and chemical microarray technique to study functional proteomics. Five enormous libraries of small molecule ligands (a total of over 1 million compounds) will be generated and screened against whole cell extracts derived from a B lymphoma cell line (Ramos). Billions of possible molecular interactions will be examined concurrently. Beads containing compounds that bind to cellular proteins or protein complexes will be isolated and the compound chemical structure determined by our novel decoding method. Selected small molecule ligands will be resynthesized on Sepharose beads and used as affinity matrix to capture the binding proteins or protein-complexes. The identity of the bound proteins will then be determined by protein separation and mass spectroscopy. Based on the chemical structure of these ligands, a small molecule microarray (approximately 1000 compounds) will be developed to probe the functional state of the whole cell extract. Our hypothesis is that with the above experimental scheme, we can systematically select a finite number of small molecule ligands and use them as capturing agents to probe the functional state of a B lymphoma cell. We further hypothesize that some of the ligands that bind to unique protein targets in lymphoma cell can be used as lead compounds for the development of anti-lymphoma agents. Once validated in a large number of lymphoid malignant cell lines, peripheral blood lymphocytes, and a limited number of primary malignant lymphoid tissues, this microarray technology can be applied to biopsy specimens obtained from a large number of patients with lymphoid malignancies. This technique, if successful, can readily be applied to other cancer types as well.
 
R33 CA099135 2003 LIZARDI, PAUL M. YALE UNIVERSITY Genetic Analysis of Amplified Genomic DNA Archives
Modern DNA analytical methods and microarray technologies are potentially enabling tools for the comprehensive genetic analysis of tumors as well as premalignant lesions in patients at risk for cancer. A significant obstacle to such analysis, however, is the need for relatively large DNA samples. A recently developed isothermal whole genome amplification method promises to eliminate these barriers to comprehensive genetic analysis. This research project aims to demonstrate the utility of simple and robust amplification procedures for generating archival copies of genomic DNA from small tissue samples. Head and neck cancer will be used as a model to optimize and validate procedures for DNA archiving of neoplastic and pre-neoplastic lesions. Samples will be collected prospectively, and cancer tissue, cancer tissue margins, as well as other lesions identified as candidates for pre-neoplasia will be collected. A subset of the samples will be isolated using laser capture microdissection. All collected tissue samples and microdissected samples will be amplified to generate a DNA archive of head and neck cancer and premalignant lesion specimens. A variety of methods, including microsatellite analysis, detection of human papillomavirus subtypes, and comparative genomic hybridization (CGH) on BAC microarrays, will be used to investigate and validate the utility of the archive of amplified DNA. CGH analysis on BAC microarrays enables the detection of genetic alterations at thousands of gene loci in the archived DNA samples, which are representative of different stages of cancer, preneoplasia, or benign dysplasia. Bioinformatics tools will be used to construct alternative classification schemes based on distance-based trees or clustering algorithms, utilizing the complete data set of microsatellite, array-CGH, and HPV infection status observations. This study is intended to serve as a demonstration and validation of the utility of DNA archives, generated by isothermal whole genome amplification, for comprehensive studies in cancer genetics. The novel capabilities established by this study should be extensible to any human cancer model and should greatly expand the utilization of genetic analysis at the level of the entire genome by eliminating many of the constraints related to limited availability of biological material.
 
R33 CA095996 2003 RONINSON, IGOR B ORDWAY RESEARCH INSTITUTE, INC. Function-based selection of target genes in tumor cells
This proposal uses a new methodology to identify human genes that are required for tumor cell growth. Such genes, which provide potential targets for cancer treatment, will be identified through expression selection of genetic suppressor elements (GSEs). GSEs are biologically active sense- or antisense-oriented cDNA fragments that inhibit the function of the gene from which they are derived. Genes that are essential for cell proliferation are expected to give rise to GSEs that inhibit cell growth. Such GSEs can be isolated by bromodeoxyuridine (BrdU) suicide selection from a normalized (reduced-redundance) library of human cDNA fragments in an inducible retroviral vector. In preliminary studies, selection for growth-inhibitory GSEs has been carried out in breast carcinoma cells, yielding growth-inhibitory GSEs from about 60 genes. Many of the genes identified by GSE selection are known oncogenes or positive regulators of cell growth, while other genes have no known function or had not been previously implicated in cell proliferation. This analysis will now be extended to several other types of tumor and normal cells. A normalized cDNA fragment library in an inducible retroviral vector will be generated from a mixture of RNA preparations from multiple human tumor cell lines. This library will be transduced into recipient cell lines derived from several major types of human cancer and into telomerase-immortalized lines of normal human cells. Prior to transduction, the recipient cell lines will be derivatized to provide for high efficiency of retroviral infection and for the ability to regulate gene expression from retroviral vectors. The transduced cells will be subjected to BrdU suicide selection for growth-inhibitory GSEs, and GSE-enriched population of cDNA fragments will be recovered from the selected cells. Genes enriched by GSE selection will be identified by sequencing, and representative GSEs from each gene will be tested by several functional assays. The role of GSE-cognate genes in cell proliferation will be confirmed via siRNA inhibition. Genes identified through GSE selection will be prioritized as potential targets by comparing the ability of their cognate GSEs to inhibit cell growth in different types of tumor and normal cells and by analyzing the ability of the GSEs to induce tumor cell death through mitotic catastrophe. This analysis will provide a database of potential new targets for the development of anticancer drugs.
 
R33 CA103455 2003 TAYLOR, CLIVE R UNIVERSITY OF SOUTHERN CALIFORNIA Retrieval of DNA, RNA, and Protein from Archival Tissue
The current phase I R21 study has accomplished three Specific Aims. The feasibility of retrieval/extraction of protein, RNA, and DNA from archival formalin-paraffin tissues by modified Antigen Retrieval (AR) methods was demonstrated. PCR based methods were adapted and applied successfully for the amplification and quantitation of RNA/DNA extracted by modified AR from formalin-paraffin tissues. Three model systems were developed to monitor the efficacy of the modified AR process, including novel simulated or 'faux tissues' and purified 'protein matrix pellets'. Continuation of these studies in this R33 proposal focuses upon the incorporation of these advances into an 'integrated systems approach' to the molecular analysis of cancer tissues that have been preserved as formalin-paraffin blocks. Currently, immunohistochemical (IHC), in situ hybridization (ISH) and PCR based assays are widely applied to formalin-paraffin cancer tissues in 'routine diagnosis' and in basic cancer research, but the reproducibility and the validity of the findings are open to serious question due to the lack of uniform methods, in particular the total lack of 'standard reference materials', which are considered essential to the use of analogous methods in the diagnostic clinical laboratory. This proposal addresses pre-analytical, analytical, and post-analytical aspects of the use of IHC, ISH and PCR based assays as applied to formalin-paraffin cancer tissues. Integral to this approach is the development of reference standards for key analytes that are, or may be, employed as cancer or prognostic markers, against which results obtained by the analysis of formalin-paraffin cancer tissues will be calibrated to yield strict quantitative findings for diagnostic and research purposes. To meet the goal of objective quantitation in tissue sections, spectral image analysis will be performed at the USC Image Core, for which we have shown the feasibility of quantitative analysis of up to four different analytes simultaneously in the same formalin-paraffin section. This proposal is multi-disciplinary, including collaborations with the USC Cellular Imaging Core at Childrens Hospital of Los Angeles, the Huntington Medical Research Institute in Pasadena, Roche Molecular Systems Inc., the National Institute of Standards and Technology (NIST), and the UNIVERSITY of California, San Francisco.
 
R41 CA103103 2003 Santangelo,Philip & Bao, Gang Vivonetics, Inc. In Vivo Gene Expression Imaging For Cancer Analysis
We propose to develop a novel dual FRET molecular beacons technology for the early detection of cancer in living cells with high specificity, sensitivity and efficiency. Molecular beacons (MBs) are single-stranded oligonucleotides with a stem-loop hairpin structure and dual-labeled with a fluorophore at one end and a quencher at the other. Delivering MBs into cells will result in a fluorescence signal if the MBs hybridize to target mRNAs. Thus, when the target mRNAs corresponding to the molecular markers of a cancer are detected in cells, cancer cells (bright) can be distinguished from normal cells (dark). However, the conventional design of MBs would suffer from false positives in cancer cell detection due to degradation by cytoplasmic nucleases and nonspecific interactions. To overcome this difficulty, we have created the dual FRET MBs concept (i.e., to hybridize a pair of donor and acceptor MBs on the same target and detect the resulting FRET), and demonstrated its potential to significantly reduce or even eliminate the false positives. We have studied the energy transfer between MBs with different dye molecule pairs, developed lanthanide dyes and performed time-resolved FRET to further reduce the background noise. To guide the design of molecular beacons, we have synthesized MBs with various molecular structures and performed in-solution thermodynamic and kinetic studies of MB-target binding. We have also studied the feasibility of detecting K-ras codon 12 mutant mRNA and survivin mRNA in pancreatic cell lines. To further develop the new dual FRET molecular beacons technology for clinical applications, in this Phase I STTR project, we will enhance the intracellular stability of molecular beacons by modifying the backbone with 2'-O-methyl and phosphorothioate (PS) chemistry. We will synthesize 5 types of molecular beacons with a random sequence and compare their stability in both cell lysates and living cells. We will also study the effect of such modifications on hybridization kinetics and thermodynamics. We will determine the signal-to-noise ratio and specificity of dual FRET molecular beacons by detecting mRNA expression in both cell lysates and living cells. We will synthesize dual FRET MBs targeting K-ras codon 12 mutations and survivin, deliver the MBs into pancreatic cancer cell lines and HDF cells and establish the detection specificity and signal-to-noise ratio using fluorescence imaging and spectroscopy. We will determine the detection sensitivity by systematically varying the relative ratios of normal and cancerous cells in a mixture in vitro and seek out the cancer cells based on MB-induced fluorescence using a confocal microscope and a FACS cell sorter. The goals are to develop the dual FRET molecular beacons technology for early cancer detection and diagnosis, and to commercialize this technology for a wide range of biomedical applications including medical research, cancer analysis, drug discovery, and in vivo detection of gene expression.
 
R41 CA103120 2003 Guthold,Martin Nanomedica, Inc. Novel, Single-Molecule Aptamer Selection Method
Molecular recognition is the underlying principle in many biomedical applications, such as labeling and imaging specific proteins, organelles, cells, tissues and organs, detecting and quantifying clinical analytes, and discovering target-specific drug candidates. Currently, the primary technology for identifying research and diagnostic ligands is monoclonal antibody selection by hybridoma screening. Aptamers - an emerging class of antibody mimetics - have been used to a much lesser extent but are gaining attention in academic circles. Aptamers are short oligonucleotides that fold into target-specific 3D structures. Although aptamers have many potential advantages over antibodies, their adoption has been slow, because 1) many researchers presume that SELEX is the only way to select aptamers, 2) SELEX is regarded by many as a cumbersome, labor-intensive and time-consuming method that requires specialized expertise, and 3) SELEX is heavily protected by patents that are aggressively enforced. The long-term goal of this proposal is to develop a convenient and fully automated instrument-based system for rapid selection of aptamers useful in the molecular analysis of cancer. NanoMedica plans to commercialize an affordable, modular system that will empower bench scientists to select and characterize high-affinity aptamers for studying cancer, signal transduction, and protein-protein interactions. If successful, this system will be fast, efficient and much more amenable to broad dissemination than SELEX. At the core of this technique is unique instrumentation that combines a nanoManipulator-Atomic Force Microscope (nM-AFM) with an inverted optical microscope to achieve fast imaging, high resolution and single-molecule detection and manipulation capabilities. The instrument will be able to detect single aptamers specifically bound to immobilized target molecules by fluorescence microscopy. The fluorescence signal will be used to guide the AFM tip towards the bound aptamer to enable single-molecule detection and aptamer extraction. Before extraction, the aptamer's affinity for its target will be measured in situ by dynamic force spectroscopy. After extraction, the aptamer molecule will be amplified by PCR and further characterized by traditional biochemical methods.
 
R41 CA103467 2003 Chen,Wen-Tien Vitatex, Inc. Gene Expression Of Viable Ovarian Cancer Cells
Molecular profiling of gene expression is a powerful tool that can be used to stage human tumors and categorize them with respect to projected prognosis and therapeutic responsiveness. This technology has been applied most effectively to leukemias and lymphomas for which it is relatively easy to obtain highly enriched pools of tumor cells. Similar approaches to study solid tumors have been less successful because solid tumor tissue samples contain many non-tumorigenic types of cells, because many of the tumor cells found in them are dead or dying at any given moment, and because of the low abundance emigrating metastatic cells. Current technology to enrich for metastatic ovarian cancer cells from ascites at the time of surgery involves using a combination of centrifugation and positive- and negative-selecting immuno-affinity steps. This cumbersome protocol yields pools of metastatic cells less than 50% pure and viable that are contaminated by non-tumor leukocyte and epithelial cells and dead and dying tumor cells. In this STTR Phase I application, we propose to optimize and validate a novel and rapid cell separation technology for enrichment of viable metastatic tumor cells from the ascites of patients with ovarian cancer. The approach is anticipated to yield a collection of viable tumor cells greater than 99% pure. In parallel, we will also purify tumor-associated leukocytes to enable expression profiling comparisons. This will provide a measure of the efficiency of the enrichment procedure and a proof-of-principle demonstration of the proposed profiling methods. In Phase II, the integrated technologies optimized in this first phase will be translated into broad gene expression studies on ovarian and uterine cancer: We will perform microarray analysis to identify genes up-regulated in metastatic ovarian and uterine tumor cells from blood and ascites in comparison to non-tumorigenic ovarian and uterine epithelial cells and primary (in situ) ovarian and uterine tumor cells. Ultimately, the combination of specific metastatic tumor cell enrichment and correctly targeted molecular profiling will create a technology capable of staging and predicting prognosis and therapeutic responsiveness for multiple types of epithelial tumors.
 
R42 CA099123 2003 Ching,Jesus Cepheid Automated Rna Isolation And Rt-Pcr For Cancer Detection
Cepheid develops integrated sample preparation and detection systems for rapid molecular diagnostic applications at a point of use environment. Currently, Cepheid has the GeneXpert system, which is a cartridge and instrument platform for integrated sample preparation and real-time PCR detection of target nucleic acids in biological samples. The current configuration of the GeneXpert system filters bacteria from samples and then lyses the bacteria for PCR detection of endogenous DNA. The next level in the evolution of the GeneXpert platform is to incorporate a nucleic acid purification function to address biological samples such as frozen tissue sections, where the presence of inhibitors may stop PCR. We believe that this automated system with nucleic acid purification will be invaluable in clinical diagnostic laboratories and will find many uses in molecular oncology applications. Specifically, our hypothesis is that a disposable fluidic cartridge, with integrated functions of RNA isolation and real-time QRT-PCR, can accomplish sample preparation and analysis of tissue cross-sections for detection of cancer marker expression in 25 minutes or less.
 
R43 CA099053 2003 Dunker,Alan Molecular Kinetics, Inc. Computational And Experimental Tool For Cancer Protein
In the predominant sequence-to-function paradigm, 3-D structure is an obligatory prerequisite for protein function. Even though over 100 counterexamples can be found in literature, generalization of the functions associated with nonfolded (disordered) protein has been mostly ignored. Application of our proprietary bioinformatics software, PONDR, to a comprehensive set of oncogenes revealed that these proteins are likely to contain significantly more disorder than other protein types. This result, combined with indications that disorder is crucial to many protein functions, demand that disorder be considered explicitly in the study of human disease. Thus, cancer-specific data mining will produce ""Cancer DisProt,"" containing correlations of disorder/order with protein function, is proposed. Disorder/order predictions and existing structural knowledge will be correlated with functions of cancer-associated proteins and augmented with local interaction networks to provide an interactive resource tool for cancer-related research. A companion methods manual will facilitate the integration of order/disorder knowledge into novel experiments. Cancer DisProt will be a useful bioinformatics tool for functional annotation of entire genomes. When augmented with methods for studying counter-example proteins, it will provide the basis for design of novel approaches to development of cancer treatments or drug discovery.
 
R43 CA099103 2003 Agnew,Brian Molecular Probes, Inc. Proteomic Analyses Of Breast Cancer Refractoriness
Molecular Probes, Inc., has developed a fluorescence-based, Multiplexed Proteomics (MP) platform that allows for the simultaneous identification and characterization of glycosylation, phosphorylation and total protein in a single 2-D gel. With subpicomolar sensitivity, the system provides for the rapid quantification of differentially regulated and/or post-translationally modified proteins, with subsequent mass spectrometry based identification. The central objective of this proposal is to utilize this technology to discover the important protein targets which confer pregnancy-induced protection against mammary carcinogenesis. Full-term pregnancy early in life is the most effective natural protection against breast cancer in women, and in experimental models, rats are similarly protected. It is proposed that these pregnancy-specific changes in susceptibility to mammary tumorigenesis are ultimately due to changes in the mammary epithelial proteome and include altered post-translational regulation and expression of key signaling proteins involved in maintaining cellular homeostasis and protection from cellular transformation. There have been no published comprehensive proteomic studies aimed at determining the molecular origin of pregnancy-induced mammary carcinogenesis protection. Our MP platform is uniquely capable of rapid detection and quantitation of changes in the posttranslational modification state of proteins, an event which we believe is crucially linked to the understanding of this effect. Phase I studies will characterize protein-based changes that take place in the virgin rat mammary epithelial cell proteome in response to full-term pregnancy or treatment with pregnancy levels of ovarian hormones. A fully integrated approach to sample preparation, 2-D gel/mass spectrometry techniques, and data analyses is demonstrated. A minimum of three protein targets linked to pregnancy-induced protection will be identified. Using a combination of laser-tissue microdissection and antibody-based blotting techniques, these initial protein targets will be independently validated. Building upon this validated proteomics-based discovery platform, Phase II of this program will expand its efforts to characterize and classify the important signaling cascades and protein targets linked to the pregnancy-induced cancer protection phenotype. These protein targets will be ideal candidates for the rational design of diagnostic indicators and/or therapeutic intervention.
 
R43 CA099111 2003 Shen,Li Superarray Bioscience Corporation Id-Tag Protein Profiling Array For The Molecular Charact
The long-term objective of this proposal is to develop a simple, reliable, sensitive, and cost-effective technological platform that will allow the simultaneous and quantitative analysis of multiple antigens in human tumor samples derived from archived tissue specimens. Such a platform is urgently needed in the clinical laboratory setting to thoroughly characterize cancer. Multiplex analysis of molecular markers can augment and improve conventional methods for determining the primary anatomical site of tumor origin, predicting tumor behavior, and formulating effective therapy. SuperArray Inc.'s proprietary ID-Tag Protein Profiling technology is an antibody-based multiplex detection. Multiple ID tags are used to track multiple protein targets simultaneously. ID-tag signal can be further amplified to increase assay detection limit. Therefore, it is very promise to develop a simple clinical diagnostic tool that will be supersensitive and allow the simultaneous analysis of multiple antigens from archival material such as paraffin-embedded sections and alcohol-fixed cytology specimens. In this phase I proposal, SuperArray, Inc., will collaborate with Dr. Jian Yu Rao, Department of Pathology, University of California at Los Angeles, to evaluate the feasibility and application of the ID-Tag Protein Profiling Array technology for the molecular characterization of human cancers.
 
R43 CA099113 2003 Zauderer,Maurice Vaccinex, Inc. A Method To Identify Upstream Regulators Of Oncogenes
A comprehensive understanding of all transduction partners, transcription factors, and their targets responsible for each type of primary tumor is lacking, despite intense effort. Furthermore, only a limited number of known ""points of regulatory convergence,"" covering multiple cancer types, exists. Vaccinex has developed a highly efficient, proprietary technology for the identification of genes based on functional selection within a mammalian background. This method utilizes cDNA libraries inserted into the vaccinia virus (vv) genome and has been successfully applied to the isolation of novel tumor antigens. The ability to make libraries in vaccinia virus represents a major advance, since vaccinia virus is the only mammalian expression vector in which it is possible to construct a diverse and representative cDNA library whose elements can be efficiently recovered from the cytoplasm of infected cells. We propose to apply a variant of this functional gene selection technology to use known oncogenes and tumor cell markers as 'bait' to identify proteins that modulate their activity. This approach represents a refreshing, functional alternative to global transcript monitoring experiments performed with cDNA and oligonucleotide microarrays. Specifically, where microarray experiments monitor global transcriptional events related to cellular environment, our technique chooses a well-defined end point of transformation, either in the form of an up-regulated transcript or an over-abundant protein and proposes to uncover proteins that regulate these targets. Vaccinex technology, therefore, has the unique capability to functionally scan upstream in a transduction pathway. Moreover, the technique is designed to identify common points of regulation from overlapping pathways. Defining these regulatory mechanisms directly in the context of cancer is a critical step for generating more effective therapeutics.
 
R43 CA101121 2003 Ferre,Francois Althea Technologies, Inc. Development Of A Multiplexed Gene Expression Screen
The pace at which the genetics of cancer is being deciphered has been accelerating. Researchers have begun to characterize in detail multiple genetic mechanisms that give rise to cancer, as well as numerous functional pathways associated with cancer, such as damage response, cell cycle, cell proliferation, and cell death. This exponential growth in our knowledge base of cancer genetics has led to the identification of a large array of genes, proteins and pathways that potentially play a central role in carcinogenesis and/or may be potential targets for therapeutic intervention. The challenge now is to experimentally delve deeper, both into how these genes function and interrelate in vivo and in vitro, and also into how different compounds and compound classes influence these genes. Althea Technologies proposes the development of a new, production-oriented, high-throughput, rtPCR-based approach to gene expression analysis. The proposed methods involve performing multiplexed rtPCR using a universal primer strategy, and further integrating the rtPCR step with the high-density capabilities of the microarray-format readout for analysis. The potential advantages of the method include (a) high throughput sample processing, (b) high detection sensitivity, opening the door to multiplexed gene expression analysis of very small amounts of tissue, and (c) a targeted cost per data point of 1 or a few pennies per gene. The Specific Aims for Phase I are focused on (1) demonstrating the proof of principle of the approach and (2) providing the roadmap for automating much of the process, a process that manages samples from cells and/or cell culture through to data acquisition and analysis.
 
R43 CA101271 2003 Diatchenko,Luda Attagene, Inc. Profiling Of Signal Transduction Pathways In Cancer
A hallmark of cancer is deregulated activation of the signal transduction pathways controlling proliferation, apoptosis, and differentiation. The signal transduction pathways culminate in activation of transcription factors (TFs), proteins that bind promoter regions of genes, thereby controlling transcription initiation. To understand the complex regulation of the signal transduction network, it is necessary to put in place technologies for assessment of numerous TFs. It is currently possible to monitor the specific changes occurring in cancer cells in the expression and function of genes and gene products at the DNA, RNA, or protein level. However, very few tools are available for examining the molecular changes at the TF level. ATTAGENE has developed a proprietary technology enabling the profiling of the transcriptional activities of multiple TFs in vivo. Based on this technology, we will develop an assay suitable for the profiling of several TFs that are critically involved in carcinogenesis, including c-Myc, E2F, p53, AP-1, NF-kB, CREB, and beta-catenin/TCF. The utility of this assay will be tested in several human cancer cell lines. Our technology allows for a comprehensive analysis of alterations in the signal transduction pathways occurring in cancer and for an identification of TFs that play a key role in a particular type or stage of cancer. The availability of this tool will advance understanding of cancer cell anatomy.
 
R43 CA102986 2003 Krizman,David Expression Pathology, Inc Protein Arrays For Molecular Analysis Of Cancer Tissue
The objective of this Phase I proposal is to demonstrate feasibility of the innovative laser induced forward transfer (LIFT) technology called Matrix Assisted Pulsed Laser Evaporation-Direct Write (MDW) for high throughput manufacturing of protein chips to achieve the long term objective of developing an innovative, fully integrated protein array system for molecular profiling of cancer progression directly from EPI's extensive archive of formalin fixed, paraffin embedded (FFPE) human tumor tissues. The Specific Aims of the Phase I proposal are: 1. Deposit, via MDW technology, sub-microgram amounts of a known protein in a microarray format and detect that protein to determine level of sensitivity 2. Demonstrate capability to assay individual proteins within MDW-deposited total cellular protein lysates Once these clear and appropriate experimental milestones are achieved, EPI intends to transition this technology to the much broader systems approach as described in the Phase II proposal. The long term goal of this grant application is to establish MDW as the pivotal technology in an innovative protein array system designed for high throughput molecular profiling of the cancer process directly from fixed archived human tumor tissues blocks. Development of this integrated system will enable rapid and cost effective determination of changing patterns of protein expression across hundreds to thousands of tissue samples simultaneously, resulting in direct and definitive correlations of protein expression profiles with progression of specific cancers. This will lead to commercial advances in the discovery of new drug and diagnostic targets directly from human tumor tissue, as well as commercial application within the pharmaceutical industry, to improve the validation and prioritization process of novel and existing drug and diagnostic targets.
 
R43 CA103085 2003 Schneider,Luke Target Discovery Idbest Differential Display On Protein Chips
Differential display analysis on DNA and protein chips has proven useful in the identification of biomarkers for cancer. Isotope tagging methods, such as ICAT, have the potential to significantly improve the precision of these methods, but cannot currently be applied to protein chips where the tagged peptides cannot be effectively separated from untagged peptides. However, by incorporating one of the elements with atomic numbers between 35 (Br) and 63 (Eu) into isotopic tags, the resulting tagged peptides are shifted in the mass spectrum by 0.1 amu. As our preliminary work shows, these isotope differentiated binding energy shift tags (IDBEST) allow direct detection of the tagged peptides from untagged species in the mass spectrum without the need for affinity or liquid chromatography clean up of the sample. The goal of this project is to develop isotope differentiated binding energy shift tags (IDBEST) and associated mass spectral filtering algorithms that allow high precision (< 10% standard deviation) differential display to be conducted directly on affinity protein chips.
 
R43 CA103086 2003 Balgley,Brian Calibrant Biosystems, Inc. Itp-Based Selective Enrichment Of Low Abundance Proteins
Probably the greatest challenge presently facing comprehensive proteome analysis is related to the large variation of protein relative abundances (>6 orders of magnitude), having potential biological significance in mammalian systems. Additionally, limited sample amounts ranging from 1,000-100,000 cells are available in mammalian proteomics, corresponding to a total protein content of 0.1-10 micrograms. For example, the use of laser capture microdissection techniques yields tissue volumes in cubic micrometers and sample sizes in the sub-microgram range. Furthermore, the heterogeneous nature of cells and tissues also contributes to the requirement for analyzing limited subpopulations. The R43 Phase of this project aims to develop and demonstrate a transient capillary isotachophoresis/zone electrophoresis (CITP/CZE)-based multidimensional separation platform, capable of providing significant analyte concentration through electrokinetic stacking (400-5000 fold) and extremely high resolving power toward peptide and protein mixtures. Most importantly, the final concentration of the analyte achieved in CITP is largely proportional to the molarity of the leading buffer. Thus, the result of the CITP process is that major components may be diluted, but trace compounds are concentrated. Such selective enhancement toward low abundance proteins can drastically reduce the range of relative protein abundances in complex proteomes, and greatly improve proteome coverage using the proposed CITP/CZE-based multidimensional separation technology. In comparison with the displacement chromatography, the only reported approach in the literature to this point for the selective enrichment of low abundance peptides, transient CITP/CZE offers the benefits of speed, straightforward manipulation/switching between the stacking (CITP) and separation (CZE) modes, and no need for column regeneration, including the removal of bound displacers. Research efforts in the R43 Phase will address early implementation, optimization, and demonstration of the proposed CITP/CZE-based multidimensional separation platform for proteomic analysis of yeast tryptic peptides. The results of R43 Phase studies will allow validation of technology, and provide information necessary to guide the improvement, design, and completion of an automated, high throughput, robust, sensitive, and ultrahigh resolution proteome instrument in the R44 Phase studies.
 
R43 CA103151 2003 Bazar,Leonard Trevigen, Inc. Lateral Flow Assay For Oncogenic Strains Of Hpv
Trevigen/TreviMed has developed the Mismatch Identification DNA Analysis System (MIDAS) that relies on SNIPase (genetically-optimized DNA mismatch repair enzymes) for the detection of pathogen DNA without the need for PCR amplification. The purpose of the research outlined in this Phase I proposal is to adapt MIDAS to a lateral-flow dipstick platform. To this end, we propose the following specific aims: (1) to design and test immobilized MIDAS probes to high-risk oncogenic strains of human papilloma virus (HPV strains 16 and 18) that incorporate biotin and various haptens such as dinitrophenyl (DNP), digoxigenin (DIG), fluorescein isothiocyanate (FITC), or bromodeoxyuridine (BrdU). In the presence of HPV target DNA and SNIPase, cleavage of the probe occurs, leading to the formation of a colored band on a lateral-flow dipstick. The MIDAS probes will be optimized initially with single-stranded oligonucleotides as targets, corresponding to HPV-16 and HPV-18 DNA sequences; (2) to optimize Dipstick-MIDAS for detecting the presence of cloned HPV-16 and HPV-18 DNA, and genomic DNA derived from HPV-16 and HPV-18-positive cell lines. These efforts will culminate in a rapid, sensitive, multiplexed, cost-effective, and accurate assay for oncogenic strains of HPV that cause cervical cancer.
 
R43/44 CA101106 2003 Rush,John Cell Signaling Technology, Inc. Immunoaffinity Isolation Of Phosphopeptides
Among post-translational modifications, protein phosphorylation is particularly relevant to cancer biology and therapy. However, despite advances in proteomics, it is still difficult to pinpoint phosphorylation sites in proteins. The long-term goal of this project is to develop and commercialize a multiplexed method for isolating and identifying phosphorylation sites based on phosphorylation-specific antibodies. This method would contribute to the development of a new generation of drugs tailored to inhibit specific protein kinases with roles in cancer by identifying new phosphorylation sites that could become targets for cancer diagnosis and treatment. During Phase I we will develop an immunoaffinity method for multiplexed phosphopeptide sorting. We will address key issues qualitatively to establish the method and its tools. We will show that the method can isolate phosphopeptides from simple model systems and complex biological samples, evaluating results with MALDI-TOF mass spectrometry. We will show that isolated peptides can be further analyzed to identify sequences and phosphorylation sites using capillary LC-MS/MS, MS3, chemical modification, and enzymatic treatment as needed to generate informative spectra. Novel, ambiguous, or especially important phosphorylation sites will be confirmed by comparing the spectra of peptides synthesized to have the identified sequences and phosphorylation sites to the spectra of the isolated, natural phosphopeptides.
 
R44 CA099117 2003 Nedelkov,Dobrin Intrinsic Bioprobes, Inc. Affinity-Based Mass Spectrometry Protein Assays
The objective of this Phase I - Phase II Fast Track Research Proposal is to advance the development of a combined Mass Spectrometric Immunoassay (MSIA) - Bioreactive Mass Spectrometer Probes (BRP) approach to a robust multiplexed technology that will ultimately be used in population screening applications and in studies relevant to cancer research. Individually, the MSIA and BRP technologies had been under development at Intrinsic Bioprobes Inc. for the last several years. Each technology brings a unique perspective to protein analysis. The MSIA approach utilizes affinity captures to selectively retrieve proteins from biological samples for subsequent MALDI-TOF MS analysis. When internal standards are incorporated in the analysis, absolute and/or relative quantitation of the assayed proteins can be achieved. On the other hand, the Bioreactive Probes aid in the thorough structural protein characterization via utilization of MALDI target surface-immobilized enzymes capable of digesting minute amounts of proteins. When combined together, the two technologies can yield a complexed approach that enables sensitive detection, quantitation and structural analysis of specific proteins from complex biological samples. In the Phase I Research, we propose to evaluate and demonstrate the reproducibility and sensitivity of the MSIA technology via execution of well-designed experiments using several model proteins and MSIA assays. In Phase II, we will demonstrate the robustness and the high-throughput capability of the combined MSIA-BRP approach. We will also proceed with the development of additional MSIA-BRP assays for proteins endogenously present in plasma and urine, some of which have been indicated as potential cancer biomarkers. A number of these proteins and assays will be utilized for the development and evaluation of a novel, relative quantitation MSIA approach. And finally, the assays and protocols designed in the Phase I and Phase II will be tested in a large-scale setting (200-300 samples, both urine and plasma) to validate the potential of the MSIA-BRP technology for population screening experiments. The ultimate product resulting from this Phase I-Phase II Research will be a combined MSIA-BRP platform and assays capable of assessing both qualitative and quantitative modulations in proteins analyzed directly from plasma, urine, or other biological fluid or tissue.
 
R44 CA099126 2003 Zebala,John Syntrix Biosystems, Inc. Artificial Antibody Mimetics
Among the many mechanisms by which cancer arises, it is now appreciated that inappropriate phosphorylation is a key pathway. Detection agents capable of binding discrete phosphoproteins with high specificity will be important tools for research, diagnostics, and drug discovery. Currently available detection agents are most commonly antibodies or antibody mimetics made using biologic or enzymatic systems. Detection agents that rely on biology or enzymes suffer from several inherent shortcomings that include a restricted biomolecule repertoire that is time-consuming and costly to manufacture. Syntrix proposes to develop a MetaMorph technology that completely bypasses biologic and enzymatic sources with novel synthetic mimetics comprising PNA-cyclopeptide heterotetramers. MetaMorphs will be identified using a novel and purely chemical discovery paradigm termed PNA-display. We hypothesize that PNA-display will permit us to screen very large heterotetramer populations and identify at least moderate- to high-affinity mimetics in a facile system. High-affinity MetaMorphs will provide a phosphoprotein detection capability equivalent to that of antibodies and other biologically derived mimetics, but without the disadvantages. The SBIR Phase I proposal is designed to prove the feasibility of using PNA-display to identify at least moderate affinity MetaMorphs. Demonstrating feasibility will set the stage to move into an aggressive Phase II program to develop high-affinity MetaMorphs capable of detecting phosphotyrosine embedded within peptides having numerous different primary sequences. Such a MetaMorph collection will be capable of detecting hundreds of known and unknown phosphoproteins and will be of significant utility in basic science, diagnostics, and efforts to develop drugs that modulate protein kinases and phosphatases.
 
R44 CA099333 2003 Zebala,John Syntrix Biosystems, Inc. Instrumentation For High-Resolution Analysis Of Cancer
The analysis of cancer within complex heterogeneous tissues represents special challenges posed by the presence of infiltrating non-neoplastic cells. We have developed biolithography, a novel and proprietary method (U.S. Pat. No. 6,159,681 to Syntrix, Inc.) for high-resolution tissue microdissection that employs lithography directly on biologic samples. Biolithography offers several advantages over laser-based methods that include improvements in spatial resolution, reliability, and contamination risk. Importantly, there is the potential for simplified and less costly instrumentation because the method requires only low-energy incoherent light. In this Phase I/II FAST-TRACK proposal, we propose to develop an automated biolithography system that will integrate the advantages of biolithography within an automated system that offers both attractive performance and cost characteristics. The automated biolithography system will consist of a novel PhotoBlaster instrument that projects user-defined microscopic light patterns during biolithography using low-energy incoherent light and a silicon chip-based microdisplay device. Consumables will consist of adhesive polypropylene reaction wells and validated reagents and photofilms suitable for isolating DNA, RNA and protein from microscopic regions of interest. Phase I is designed to prove the feasibility of the proposed PhotoBlaster design to generate photopatterns of sufficient intensity and minimum feature size. The Phase II program will yield a final system prototype. The proposed automated system of biolithography offers the potential to provide a facile and cost-effective method to address whole organism analysis for a wide audience of researchers and clinicians engaged in cancer analysis. Syntrix will commercialize the PhotoBlaster system that results from this research either through direct sales or through third-parties.