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R21 CA140143-01 2009 BURBULIS, IAN E VTT/MSI MOLECULAR SCIENCES INSTITUTE PCR for carbohydrate cancer biomarkers
The identity and abundance of glycans attached to specific circulating blood proteins change during cancer but the value of using these changes for diagnosis is largely unavailable because state-of-the-art methods to decipher these residues requires expensive equipment and technically challenging protocols. The long-term objective of this proposed work is to create an analytical tool for quantifying these glycosylation changes so that informative glycan biomarkers may be discovered and used to aid in cancer diagnosis. Here, we propose to label unique glycans with sequence-specific oligonucleotides and count each adduct by real-time polymerase chain reaction (RT- PCR); in effect, converting different carbohydrates on a specific glycoprotein into identifying DNA sequences that enable ultra-sensitive detection and precise glycan profile measurement. This measurement constitutes a unique and informative biomarker signature. The specific aims of this R21 are to, (i) develop affinity probes for labeling glycan residues on four different human serum glycoproteins known to change in glycosylation pattern during cancer, (ii) measure the numbers of different glycans attached to each of the glycoproteins to derive a ""glycan profile"", and (iii) evaluate whether this technique is effective at deciphering the glycan profiles of these same glycoprotein found in human clinical samples. If successful, this technique will enable the discovery of carbohydrate biomarkers that comprise clinically relevant signatures and enable capturing the diagnostic value inherent to changes in protein glycosylation for cancer diagnosis. The goal of this R21 is to develop general-purpose methods to detect and quantify unique carbohydrates attached to individual glycoproteins. Specific glycan residues are converted into identifying DNA oligonucleotides that constitute a unique signature read by real-time polymerase chain reaction (RT-PCR). If successful, this work will enable the discovery of carbohydrate biomarkers that comprise clinically relevant signatures and enable capturing the diagnostic value inherent to changes in protein glycosylation for cancer diagnosis.
 
R21 CA140096-01 2009 CHAREST, JOSEPH LEO CHARLES STARK DRAPER LABORATORY Microfluidic 3D Scaffold Assay for Cancer Cell Migration and Intravasation
Migration through extraular matrix (ECM) and intravasation across a cellular barrier comprise the initial, rate-limiting steps of cancer metastasis. Physiologically relevant and well-controlled models that mimic the in vivo tumor microenvironment will enable better understanding of the initial steps of metastasis and evaluation of potential therapy efficacy. In vivo models have physiological relevancy, yet inherently lack a high level of control. In vitro cancer migration models have high levels of control, yet lack critical components of the tumor microenvironment. We propose a new technology, a microfluidic migration and intravasation assay (?MIA). The ?MIA replicates essential components of the in vivo tumor microenvironment, including a 3D ECM and a vasculature, while providing tight control of biochemical and biophysical parameters. To further establish the ?MIA, we propose to use it to investigate a specific biophysical factor - interstitial flow - which has not previously been studied in the context of metastatic disease. The objective of the proposed work is to evaluate the metastatic potential of cancerous cells by developing the ?MIA and identifying novel extent of invasion metrics (Specific Aim 1), and applying them to study the influence of interstitial flow on cancer cell metastasis (Specific Aim 2). The ?MIA will have an input channel for the cancer cells, a 3D collagen gel to simulate native ECM, and an endothelial cell (EC) layer adherent to the gel in a second channel. The configuration will permit migration of cancer cells either from the input channel or within the gel towards the second channel. Optimized gel parameters will present appropriate chemotactic gradients and physical parameters simulating a tumor microenvironment and inducing cancer cell migration. The EC layer will mimic the in vivo vascular barrier allowing observation of cancer cell intravasation. Optical access from two vantage points will permit real time observation of cancer cell migration and intravasation. The optical access combined with image processing techniques will quantify cancer cell morphological and migratory parameters, leading to identification of novel extent of invasion metrics that will quantify the metastatic potential of cancer cells. Finally, we will leverage the microfluidic capability of the ?MIA to induce interstitial flow across the gel, and quantify the effects of this biophysical parameter on cancer cell invasion. Taken together, the two aims establish the ?MIA as an excellent platform for quantitative research of molecular mechanisms governing cancer cell invasion. For example, therapies capitalizing on altered vascular morphology near tumors would clearly benefit from using the ?MIA as a development platform, as the system provides a characterized EC layer in conjunction with a well-controlled system. Future development will enable the ?MIA to serve as a cancer cell diagnostic device and a high throughput drug development tool. Cancer spreads and invades through a process called metastasis, often resulting in patient death. The metastasis process is not well understood, since there is a shortage of well-controlled models that realistically represent the tumor microenvironment and its blood supply. This application seeks to develop a well-controlled and realistic tumor environment model to aid cancer metastasis research and eventually provide a platform to more efficiently develop and evaluate cancer therapies.
 
R21 CA134368-01A1 2009 DORSEY, KATHLEEN CONWAY ; THOMAS, NANCY E (contact) UNIVERSITY OF NORTH CAROLINA at CHAPEL HILL DNA-Methylation Profiling from Fixed Melanocytic Tissues
Melanoma, which is increasing in incidence, has the capacity to metastasize early and its course is rarely impacted by medical intervention. Because of the pronounced difference in survival between localized and metastatic disease, it is imperative to diagnose melanoma in its earliest form; however, early diagnosis is confounded by the overlap of the clinical and histopathological appearances of melanomas with highly prevalent benign nevi (moles). Molecular pathology has proven useful as an adjunct to diagnosis rendered by histopathologists for enhancing early cancer detection. Tumor DNA-methylation holds promise as a tool for molecular pathology because aberrant promoter methylation, which often results in the abnormal silencing of tumor suppressor genes, has been shown to occur widely in human melanomas. High-throughput methylation arrays, a new technology which can simultaneously evaluate promoter methylation in many cancer-related genes, has potential for discovery of candidate DNA-methylation sites useful for melanoma diagnosis. However, these arrays have been developed for use on unfixed tissues, and their validity and reproducibility has not been determined on formalin-fixed paraffin-embedded (FFPE) tissue, which is typically the only diagnostic tissue available for primary melanomas and nevi. The central hypothesis of our proposal is that DNA methylation patterns exist that can discriminate melanomas from benign moles with high sensitivity, specificity, and reproducibility; and high-throughput DNA-methylation assays are a feasible method for discovery of these patterns in diagnostic FFPE tissues. A goal for this R21 is to assess whether formalin-fixed tissues are a suitable source of DNA for high-throughput methylation array profiling by assessing the reproducibility of results between matched formalin-fixed and frozen melanoma specimens and between formalin-fixed duplicates. Importantly, as a second aim, a 'dose response curve' will be determined to assess the proportion of melanocytic tumor to surrounding non-melanocytic tissue necessary for tumor DNAmethylation detection using high-throughput arrays, and these results will establish the proportion tumor below which selective procurement using laser capture microdissection will be done prospectively. Furthermore, this application proposes to identify a 'proof-of-principle' methylation-signature algorithm which will differentiate melanomas from benign moles. This study will be a first step toward the development of diagnostic methylation assays that could be used to standardize melanoma diagnosis, thereby decreasing under- and over-treatment of melanocytic lesions.
 
R21 CA137704-01 2009 DRAKE, RICHARD R EASTERN VIRGINIA MEDICAL SCHOOL Proximal Prostate Fluids for Protein and miRNA Biomarkers
Detection of prostate specific antigen (PSA) levels in serum as a biomarker of prostate cancer continues to evolve to reflect new knowledge about disease-specific isoforms and free versus bound complexes with serum proteins. While there are many known advantages to PSA testing, the well documented problems with PSA being an excellent organ-specific marker, but not a cancer-specific marker, will continue to be compounded with the aging population of the U.S. Proximal fluids are found adjacent to a given tissue or organ and represent a repertoire of secreted proteins and shed cells reflective of the physiological state of that tissue. Hence, proximal fluids are rapidly emerging as a potential source of protein and genetic biomarkers for cancers. Seminal plasma and expressed-prostatic secretion (EPS) fluids are proximal fluids of the prostate. In this proposal, we describe the clinical collection and characterization of prostate proximal fluids to meet the increasing demand for improved prostate cancer biomarkers. EPS represents the fluid being secreted by the prostate following a digital rectal prostate massage, which in turn can be collected in voided urine post-exam. This collection is not disruptive to a standard urological exam, nor does it add excessive time to the visit. Currently, a commercial genetic assay for prostate cancer detection, based on the presence of a non-coding RNA, PCA3, uses EPS urines as a source of shed epithelial cells collected as a sediment after low speed centrifugation. Identification of prostate-associated microRNA (miRNA) species from the epithelial cell pellet has not been reported. The remaining urine, which contains many prostate-derived secreted proteins, has not been well characterized. Over the past year, clinicians and researchers at the Virginia Prostate Center have been collecting and expanding an existing biorepository of EPS urine samples, as well as more pure prostatic fluids obtained prior to prostatectomy. Over 250 EPS urines from healthy clinic controls, benign prostatic disease, and risk stratified prostate cancers (low, intermediate, high and metastatic) are available for this study. A pooled sample of EPS urines from each condition was used for comprehensive proteomic analysis, leading to identification of over 600 prostate enriched constituent proteins. We are also using these fluids to establish a comprehensive database of glycans from secreted PSA and other prostatic proteins, and the sedimented pellet material is being evaluated for prostate disease specific miRNA signatures. We hypothesize that optimization and standardization of EPS collection consistent with current urological exam practices, and definition of the prostate specific components of this fluid, will lead to the discovery of new biomarkers and application of new diagnostic assays for prostate cancer. This will be accomplished in the following Specific Aims: Aim 1. Establish the prostate specificity of potential protein and miRNA biomarkers from expressed prostatic secretions. Aim 2. Determine standards for what constitutes an acceptable and unacceptable sample for secreted protein content and miRNA Aim 3. Establish a standard operating procedure for collection of post-DRE and pre-prostatectomy expressed prostatic secretions. Our goal is to optimize the clinical information obtained from EPS urines, using both the cellular sediment for miRNA and the prostatic protein fluid phase for glycoproteomic characterizations. Both miRNA and glycoproteins are inherently stable molecules. The EPS urines can be collected across the entire clinical spectrum of prostatic cancers and benign disease. Collection of EPS urine at the time of DRE during office examinations does not add significant time or delays to these visits, either for the physician or patient. Defining the constituent proteins and cellular miRNAs, standardization of collection procedures and establishing stability and storage parameters will facilitate incorporation of EPS urine based assays into widespread clinical use. PUBLIC HEALTH RELEVANCE: Testing of prostate specific antigen levels in the blood of men over 50 has improved prostate cancer detection and treatment rates. Unfortunately, there are an increasing number of problems with PSA being an excellent organ specific marker, but not a specific cancer marker, a problem that will continue to worsen with the aging male population. We propose to characterize a different type of fluid termed expressed prostatic secretions for improving current PSA testing. Defining the constituent proteins and cellular miRNAs, standardization of collection procedures and establishing stability and storage parameters will facilitate incorporation of EPS urine based assays into mainstream clinical use.
 
R21 CA134359-01A1 2009 FOWLER, CAROL B AMERICAN REGISTRY OF PATHOLOGY, INC. Recovery of Proteins from Formalin-fixed Tissues under High Hydrostatic Pressure
Cancer is a leading cause of morbidity and premature mortality in the United States; it led to over 550,000 deaths in 2005. High-throughput genomic and proteomic methods hold great promise for developing knowledge of the molecular characteristics of cancer, which can be translated into practical interventions for the diagnosis, treatment, and prevention of this disease. Unfortunately, proteomic studies using fresh or frozen tissues cannot be related directly to the clinical course of disease in a timely way. Tissue repositories world-wide contain millions of formalin-fixed, paraffin-embedded (FFPE) specimens that could overcome this limitation by providing large numbers of tissues for which the clinical course of disease and treatment have been established. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hampered by the adverse effects of formalin fixation. We recently demonstrated nearly quantitative reversal of formaldehyde-induced protein adducts and cross-links when lysozyme tissue surrogates, a model for FFPE tissues, were processed under high hydrostatic pressure (45,000 psi) at 65-80 oC in Tris buffer, pH 4, containing 2% SDS. We have also identified by MS all of the proteins in a 5-protein FFPE tissue surrogate extracted under high pressure with excellent sequence coverage, comparable to an identical native protein mixture. These results indicate that elevated hydrostatic pressure is a promising approach for recovering proteins from FFPE tissues in a form suitable for proteomic analysis, and thus is worthy of further study. The objective of this proposal is to use elevated hydrostatic pressure at moderate temperatures (=65 oC) to develop a significantly improved method for recovering proteins from FFPE tissues for proteomic analysis. In Aim 1, we will use elevated pressure to optimize a method for the quantitative extraction of proteins from complex tissue surrogates and the reversal of their formaldehyde-induced modifications. In Aim 2 we will use methods developed in Aim 1 to conduct a series of proteomic analyses comparing proteins identified in fresh cell lysates with those from the same cells processed as FFPE agarose plugs. In Aim 3, we will confirm that the optimized pressure extraction methods can be used to perform proteomic analyses with real FFPE tissue. The use of high hydrostatic pressure supplemented with moderate heating (=65 oC)is an innovative and highly effective approach for the extraction of whole proteins from FFPE tissues and the reversal of their formaldehyde-induced modifications. The rationale for these studies is that their successful completion will ultimately lead to improved practical interventions for the diagnosis, treatment, and prevention of cancer and facilitate the development of therapeutic agents.
 
R21 CA138310-01 2009 GODLEY, LUCY ANN UNIVERSITY OF CHICAGO A chemical crosslinking strategy to determine DNA methylating protein complexes
Epigenetic changes alter chromatin structure, thereby regulating gene transcription. In normal cells, repetitive DNA is hypermethylated and transcriptionally silent, whereas transcribed gene promoters are undermethylated and associated with open chromatin. Cancer cells are characterized by abnormal DNA methylation: repetitive DNA sequences and some gene promoters are hypomethylated and transcriptionally active, whereas many tumor suppressor gene promoters are hypermethylated and transcriptionally inactive. Although many studies have focused on categorizing which genes have altered DNA methylation patterns in cancer cells, the precise components of the DNA methylation machinery mediating those changes have not been established. We propose to develop a unique method of chemical cross-linking and protein complex identification to identify the factors involved in promoter hypermethylation in breast cancer. Our new strategy is based on the development of novel chemical compounds synthesized within the He Laboratory and has significant advantages over other approaches in that it assembles a protein complex directly on a specific biologically relevant DNA. We envision applying this strategy to determine a quantitative molecular signature for DNA methylating complexes in cancer cells. To develop our new technology, we propose focusing on determining the protein complexes that mediate the hypermethylation of the promoters of BRCA1, a classic tumor suppressor gene, and CDH1, which encodes a protein important for cell adhesion, using two Specific Aims: (1) To incorporate novel chemical cross-linking compounds into oligonucleotides corresponding to the BRCA1 and CDH1 promoters, perform cross-linking to protein extracts from breast cancer cells, and identify the cross-linked proteins by mass spectroscopy; and (2) To compare the DNA methylating complexes quantitatively in human breast tumors versus normal tissue using our technique. In the future, a detailed understanding of the DNMT/other protein contacts at particular gene promoters could lead to the development of hypomethylating agents targeted to these promoters in a sequence-specific manner, thereby avoiding the consequences of genome-wide hypomethylation. In addition, the new chemistry allows formation of DNMT-DNA complexes at high efficiency, which could facilitate the structural characterization of human DNMT-DNA complexes.
 
R21 CA137707-01 2009 GRZYBOWSKI, BARTOSZ ANDRZEJ NORTHWESTERN UNIVERSITY Microassay Systems for Diagnosis of Cancer Cell Motility and Metastasis
Motility of cancer cells is a hallmark of metastasis - that is, a complex, multistage process, in which these cells acquire a tissue-invasive, directional motility phenotype, detach from primary tumors, migrate to distant sites, and colonize them to start new loci of disease. Although metastasis is the major cause of mortality in cancer patients, there are currently no approved drugs available that target motility of invasive cells, and only a handful of lead compounds are being tested in Phase I or II clinical trials. In the absence of effective drugs, early detection/diagnosis of metastasis becomes a critical element in the ongoing battle against cancer. This application aims to develop a conceptually novel technological platform with which to correlate motility and/or cytoskeletal dynamics of cancer cells to their metastatic potentials. Specifically, a combination of micro/nanotechnology, surface chemistry and cell and cancer biology will be used to prepare materials and systems with which to (i) create populations of micropatterned, ""designer"" cells of desired shapes and (ii) elicit specific cell phenotypes. The micropatterns will be used to probe and distinguish metastatic from non-metastatic cancer cells based on their differential abilities to polarize and/or to rearrange cytoskeletal organization/dynamics in response to the applied geometries. Statistics over populations of designer cells will then provide analytical measures of the cells' invasiveness. The assays thus developed will be tested with cells from cell lines as well as those from cancer patients. Multiplexing the assays with microfluidics will allow for screening many samples/substances on one micropatterned ""chip"". It is expected that the proposed systems will be more robust and accurate than the existing measures of invasiveness, such as counts of circulating cells or mRNA expression microarrays. The long term goal of this work is to develop the technology from the proof-of-concept experiments to clinical applications, where it would help save lives of cancer patients.
 
R21 CA138308-01 2009 HELD, JASON M. BUCK INSTITUTE FOR RESEARCH ON AGING OxMRM: A Technique to Quantify Oxidation of Endogenous Redox-Sensitive Cysteines
Thiol signaling by reactive nitrogen and oxygen species (RN/OS) regulates aspects of tumor growth, migration, invasion, survival, angiogenesis, and metastasis; however the key modifications and mechanisms of thiol signaling in cancer are relatively obscure. Although chronic exposure to RNOS has traditionally been thought to be deleterious, thiol signaling is essential for normal cellular function, suggesting that RNOS play a complex role in cancer biology. Insights into cysteine oxidation signaling in cancer will further our understanding of cancer progression, as well as aid development of new anti-cancer therapeutics. A variety of RNOS molecules oxidize thiols leading to a diverse array of modifications. The biological consequences of these modifications, however, are not well understood, since the chemistry that governs their formation is often transient and complex. The difficulty of cysteine oxidation analysis has led to a technological void in which the function of many oncogenes such as p53, NF?B, and HIF1a are known to be regulated by key thiols, though little is known about their oxidation status. In addition, the mechanism by which oxidation is regulated, and how it changes during cancer initiation and progression are not well understood. Using p53 as a model redox sensitive protein, this study will address this challenge by proposing a technology, (OxMRM), capable of sensitively quantifying cysteine oxidation status of potentially any protein by integrating differential thiol alkylation, protein purification, and analysis by multiple reaction monitoring (MRM). The advantage of MRM is that it is the most sensitive and quantitative mass spectrometry (MS) technique available, and will allow thiol oxidation analysis of even low level endogenous proteins such as p53 from both cellular and in vivo sources. OxMRM can address the interface between essential thiol oxidation signaling and the chronic effects of increased RNOS production by examining the reversible redox status of proteins as well as potentially irreversible oxidation. This study aims to uncover insights into the functional consequences of oxidation and whether, in the case of p53, disruption leads to increased susceptibility to DNA mutations.
 
R21 CA137708-01 2009 KANDEL, EUGENE S ROSWELL PARK CANCER INSTITUTE CORP Array-Assisted Insertional Mutagenesis for Molecular Analysis of Cancer
We propose to develop and validate a technology that will enable unequivocal genome-wide functional identification of cancer-related genes using either a positive or negative selection in conditions of either low or high stringency. This will extend the reach of forward genetics to a principally new set of phenomena. Identification of the genes and their products, which form the molecular basis of cellular phenotypes, is the fundamental problem in molecular analysis of cancer. The cellular factors that control proliferation, death, response to therapy, motility, interaction with other cells and the environment have emerged as the diagnostic and prognostic markers, as well as the targets of therapeutic intervention. Forward genetics is a popular methodology that uses genetic tools to uncover the modulators of various biological processes. Insertional mutagenesis based on random insertion of a strong and regulated promoter is a versatile, cost-efficient, unbiased and comprehensive approach to forward genetics in somatic cells. However, the current implementations of this approach apply exclusively to the conditions of stringent positive selection, and are inapplicable to a wider range of clinically-significant phenomena. Moreover, large-scale mapping and validation of multiple relevant insertional targets remains the technical bottleneck of the whole approach. To overcome these limitations, we will develop a new technology, which combines the elements of insertional mutagenesis with those of microarray analysis. The microarray component will allow high-throughput mapping of the inserts in a highly complex pool of cells. Positive or negative changes in the representation of individual inserts could be used to identify the loci, which affect the cell behavior under various selective conditions. The dependence of the changes on the function of the inserted promoter could be used for large-scale validation of the findings. We will construct a series of appropriate lentiviral vectors and will confirm their activity as insertional mutagens. We will optimize the procedure to monitor the frequency of individual mutant alleles in a high-throughput format. Finally, we will apply the newly developed tools and procedures to the discovery of genes whose products determine the response of prostate carcinoma to Taxol, the only chemotherapeutic compound known to extend the life of patients with androgen-independent prostate cancer. We will identify the events that either sensitize or protect cells from the drug. The clinical significance of these findings will be pursued in future studies, while our data, tools and procedures will be shared with the research community.
 
R21 CA138365-01 2009 KRON, STEPHEN J. UNIVERSITY OF CHICAGO Imaging DNA damage dynamics for diagnostics, screening and target discovery
Radiation remains an important treatment modality in the therapy of breast cancer. Ionizing radiation induces DNA double strand breaks, which initiates a characteristic response. ATM and related protein kinases recognize each break and phosphorylate adjacent histone H2AX, promoting assembly of ionizing radiation induced foci (IRIF), multiprotein signaling and repair complexes spread over megabases of the surrounding chromatin. The resulting amplification mediates its effects by activating downstream kinases to induce cell cycle arrest, DNA repair, and apoptotic responses. This project is directed toward developing a novel reporter of DNA damage response that will allow tracking of the formation and resolution of DNA damage foci in normal and breast cancer cells. Toward these ends, we will develop fluorescent protein fusions based on 53BP1 that accumulate at double strand breaks, and use these fusion proteins as probes to identify proteins that constitute IRIF. This new technology can be used to determine the radiation responses of tumors and normal tissues and to discover new biological targets to enhance radiation effects.
 
R21 CA131884-01A2 2009 LARSON, DALE N CHARLES STARK DRAPER LABORATORY The Development of a Chip-Scale Nano-Calorimeter
The study of binding interactions is a central aspect of basic biology research and pharmaceutical R&D and there are numerous analytical methods available to study various aspects of these interactions. Each has its own strengths and weaknesses. Calorimetry is currently used, not as a screening tool, but as a tool to understand a specific reaction and is very important in the study of binding interactions. A calorimeter measures the energy released or absorbed by a reaction over a range of reactant concentrations to determine the relative contributions of enthalpically driven processes (related to the number and types of bonds) and entropically driven processes (related to the shapes of the binding site and the ligand). Unfortunately, the need for a large amount of protein (0.5 to 5mg) limits its usage. Additionally, there are some reactions where the amount of heat is too small for the current generation of calorimeters to measure. We are developing a chip scale calorimeter based on extraordinary optical transmission (EOT) through an array of nanometric apertures. Stark et al and Brolo et al have shown that these nanohole array devices can be used as affinity sensors where one of the binding partners is immobilized on the surface of the nanohole array device. With these nanohole array sensors the signal is temperature dependent due to the dielectric function of the buffer changing the plasmon excitation conditions. Holding the concentration constant in an approximately 100nm thick layer of dielectric directly above the nanohole array surface enables the use of EOT as a fast and sensitive temperature sensor to measure the heat of reaction (enthalpy, ¨H) from binding events. The inherent ability to multiplex many nanohole array sensor devices on a single chip enables the simultaneous measurement of controls to characterize confounding effects (e.g. buffer dilution, mixing, presence of DMSO in the buffer) and deconvolution of these effects to determine the true heat of reaction. This multiplexing also indicates the possibility of using this for high throughput screening as well as expanding on the current role of calorimetry. Early results indicate that a nanohole array calorimetry system has the potential to reduce the amount of protein required by 1000-fold and increase sensitivity by 100-fold. This will expand the use of calorimetry in pharmaceutical R&D. Our research plan consists of three specific aims to demonstrate proof-of-principle for this technology. Aims 1 and 2 explore the fundamental design options and tradeoffs involved in nanohole array device design and sample delivery. Aim 3 integrates these results into a calorimetry system and assesses the resulting measurement performance against quantitative milestones. In this application we propose to develop a new chip-scale nanocalorimeter that addresses the key limitations (compound usage, sensitivity, and analysis time) of current calorimetry technologies. The two primary performance goals for this project are to decrease compound usage by at least 1000-fold and to increase sensitivity by at least 100-fold while ensuring compatibility with existing liquid handling equipment.
 
R21 CA138295-01 2009 LESSNICK, STEPHEN L. UNIVERSITY OF UTAH Antibody-based detection of translocations in pediatric cancer
Many cancers are associated with recurrent abnormalities of chromosome structure. One abnormality, a chromosomal translocation, occurs when portions of two normal chromosomes are exchanged. In the cancer setting, translocations may change the expression of genes near the chromosomal breakpoint, or may cause an abnormal ""fusion oncogene"" to be formed when portions of two genes at the breakpoint become linked together in an abnormal way. The products of these fusion oncogenes induce cancer development in the cells where they occur. In many instances, the presence of these fusion oncogenes may provide diagnostic, prognostic, or therapeutic information about the tumor. A highly sensitive and specific approach for detecting these chromosomal translocations would allow new information to be developed about cancers that contain them, and would also be of great use to the clinical care of these patients with these tumors. Unfortunately, although current approaches have been useful in detecting translocations and their products, they have a number of shortcomings that limit their use. We now propose to develop a new approach for translocation detection that avoids many of the difficulties associated with current methods: antibody detection of translocations (ADOT). We plan to develop two versions of this technology, one that can analyze thousands of potential translocations at a time, but is somewhat costly, and one that can analyze a limited number of translocations at once, but is relatively cheap. We will determine the functional characteristics of these approaches, and compare them to current methodologies. At the end of these studies, we will have developed and characterized a technique for detecting these important chromosomal abnormalities that may be ready for clinical and research applications.
 
R21 CA137706-01 2009 LIOTTA, LANCE ALLEN GEORGE MASON UNIVERSITY Nanotechnology for One Step Concentration and Preservation of Labile Biomarkers i
Cancer-associated blood biomarkers exist in exceedingly low concentrations within complex mixtures of high-abundance proteins such as albumin and immunoglobulins. Moreover, biomarkers in the blood may be subjected to degradation during transportation and storage. Such degradation is a significant source of bias for cancer biomarker measurement and discovery. We have created N-isopropylacrylamide porous sieving core shell ""smart"" nanoparticles containing an internal affinity bait to perform three independent functions within minutes, in one step, in solution (serum or plasma): a) molecular size sieving with complete separation from high abundance residence proteins such as albumin and immunoglobulin, b) affinity capture of all solution phase target molecules, and c) complete protection of harvested proteins from enzymatic degradation. The captured analytes can be readily electroeluted for analysis. In our preliminary studies we have manufactured large quantities of uniform porous nanoparticles containing specific bait chemistries that bind highly labile low abundance target cancer biomarker proteins (e.g., platelet derived growth factor). We have demonstrated that the particles can capture and concentrate target proteins from serum such that the target molecule is completely removed from the bulk solution within 5 minutes, with completed exclusion of albumin and immunoglobulins. The captured proteins, even if denatured, are protected from degradation by high concentrations of proteases (trypsin or chymotrypsin) even at 37 oC overnight. The deliverable goal of this proposed feasibility R21 study is to combine this ""smart"" nanoparticle technology with standard blood collection tubes, with or without additives/anticoagulants, to rapidly encapsulate cancer biomarkers known to be very rare and labile. The envisioned technology is a panel of dry lyophilized sub-micron sized harvesting particles that carry specific bait for known biomarkers. Following introduction of the blood or body fluid, the respective particle populations will remove all of their target molecules, in one step, in solution, from the entire volume of the sample and concentrate the sequestered analytes inside the particles. Analytes will then be eluted from the particles to yield a much higher concentration and purification compared to the starting sample. Depending on the starting volume of the blood, this technology can concentrate a biomarker many hundred fold, and prevent degradation, within minutes. PUBLIC HEALTH RELEVANCE: Biomarkers for early stage cancer are urgently needed so that treatment can be initiated prior to metastasis. Unfortunately, cancer biomarkers exist in very low concentrations and are highly labile. This project will deliver a novel nanotechnology that can rapidly harvest, concentrate, and protect from degradation, labile biomarkers in whole blood or serum, in one step, in the blood collection tube.
 
R21 CA138359-01 2009 LIU, QIANG ; YEN, YUN (contact) CITY OF HOPE/BECKMAN RESEARCH INSTITUTE Ultrahigh sensitive, personalized monitoring of lymphoma relapse using blood spec
Diffuse large B lymphoma (DLCL) is a common lymphoid malignancy in adults, accounting for 30,000 new cases each year and nearly 40% of all non-Hodgkin lymphomas. Although modern treatment can lead to complete remission in a considerable proportion of DLCL patients, many ultimately relapse, probably due to the presence of minimal residual DLCL cells. In this project, we propose to identify relapse of lymphoma at a much earlier time point through detection of personalized lymphoma-specific markers in blood. Specifically, we will use Pyrophosphorolysis Activated Polymerization (PAP), an ultrahigh sensitive nucleic acid amplification technology, to detect as few as one copy of lymphoma-specific somatic mutations in 5 ml of plasma. This assay will be 1000-fold more sensitive than current PCR-based methods. Besides its ultrahigh sensitivity, the testing is non-invasive. The success of this proposal will open new opportunities for personalized monitoring other cancers using similar strategies.
 
R21 CA138370-01 2009 LIU, YANG UNIVERSITY OF PITTSBURGH AT PITTSBURGH Partial-wave spectroscopic microscopy for the diagnosis of cholangiocarcinoma
The major objective of this R21 application is to demonstrate the feasibility of using an emerging biophotonics technology - partial-wave spectroscopic microscopy (PWS) for the diagnosis and surveillance of cholangiocarcinoma (CCA). CCA is a primary malignancy in bile duct epithelium. Although it is a rare cancer, the global incidence of this cancer is on the rise. It is a deadly disease with a median survival of months. However, significant improvements in survival rates are possible when the cancer is diagnosed at an early localized stage. Current imaging techniques suffer from either low sensitivity and sometimes lead to significant complications such as pancreatitis and cholangitis. The histopathological confirmation is notoriously difficult, with a suboptimal sensitivity of 9% up to 67% even when the advanced molecular techniques (e.g., fluorescence in situ hybridization (FISH)) were applied. These low rates are in large part due to the challenges of identifying malignant cells with conventional microscopic techniques in the setting of inflammation, and highlight a potential role for a novel biophotonic approach to diagnose malignancy by detecting subtle changes within a cell. PWS represents a novel molecular analysis of cell nano-architectural refractive index fluctuation arising from the changes in the concentration of intracellular solids (DNA, RNA and proteins, etc.) at a single cell level beyond what conventional microscopy reveals. We have completed successful pilot human studies to demonstrate the feasibility of PWS to improve the diagnostic accuracy and stratify risk for pancreatic cancer. This study will be guided by three specific aims: Aim 1 will assess the ability of PWS to improve the diagnostic accuracy of cytology by identifying CCA in patients with suspected biliary strictures in a retrospective study using archival cytologic specimens. Aim 2 will validate the optical signatures from PWS identified in Aim 1 in a prospective human study in an independent set of human subjects. Aim 3 will demonstrate the potential of PWS to diagnose as well as stratify risk for CCA through the identification of the ""field-effect"" of CCA, a concept of detecting a neoplastic alteration far away from the lesion, through the assessment of the duodenal epithelial cells in the vicinity of the bile duct. We believe that reasonable outcomes of the proposed project are to demonstrate the possibility of our optical technique to identify the presence of CCA in the duodenum and to improve the cytologic diagnosis of CCA. The long term goal is to develop a minimally invasive surveillance strategy to identify high risk individuals in whom intensive examination using more invasive procedures is warranted to detect a CCA at a stage when curable treatment is available, without the need for direct interrogation of the bile duct.
 
R21 CA138280-01 2009 MAKRIGIORGOS, G. MIKE DANA-FARBER CANCER INSTITUTE Technology for sensitive and reliable mutational profiling in pancreatic cancer
Mutational profiling of pancreatic cancer holds major promise for early detection, prognosis and therapeutic management of this disease. However, as with many other cancers, while reliable screening methods for germline or prevalent somatic mutations already exist, detection of low-prevalence somatic mutations in heterogeneous, multifocal pancreatic cancers with stromal contamination, or in bodily fluids remains problematic. Thus, for a substantial fraction of clinical pancreatic cancer samples, the new powerful mutation detection technologies ""lose steam"" and their advantages cannot be exploited. We developed co-amplification at lower denaturation temperature polymerase chain reaction (COLD-PCR), a new form of PCR that amplifies preferentially the ""minority alleles"" from mixtures of wild-type and mutation-containing sequences, irrespective of where the mutation lies, providing a 10-100-fold enrichment of the mutated sequences during PCR. Because PCR comprises the ubiquitous first step in genetic analysis, COLD-PCR provides a general platform to improve sensitivity for essentially all diagnostic assays. In this application we propose to develop further, optimize and adapt COLD-PCR for increasing the sensitivity of two established mutation detection methods, such that they can be applied for reliable identification of clinically-relevant, somatic mutations in heterogeneous, multifocal pancreatic cancers: matrix assisted laser desorption ionization-time of flight (MALDI-TOF) for known mutations, and single molecule sequencing for high-throughput sequencing of somatic mutations. The combination of COLD-PCR with these two technologies, each tackling a different aspect of mutation detection, will boost the sensitivity of patient- specific mutational profiling, and is suited for application to pancreatic cancer. A comprehensive list of genes mutated in pancreatic cancers will be compiled and COLD-PCR will be adapted for parallel screening of somatic mutations in pancreatic surgical specimens and plasma samples using the selected technologies. In the forthcoming era of molecular medicine, clinical decisions will increasingly rely on molecular tumor profiling, and the reliability of identifying somatic mutations in diverse clinical specimens must be high. This application tackles the problem of molecular analysis in heterogeneous cancers. We focus the new technology on pancreatic cancer, a heterogeneous cancer that currently has very low cure rates and for which molecular biomarkers can make a difference.
 
R21 CA137695-01 2009 MALLICK, PARAG KUMAR; MANALIS, SCOTT R (contact) MASSACHUSETTS INSTITUTE OF TECHNOLOGY Developing a single cell growth monitor for classifying therapeutic response
Clinical oncology and cancer biology are challenged by the lack of assay platforms for measuring changes in cancers' growth kinetics in response to therapeutic intervention. Cell growth kinetics can be measured in a number of ways, such as by DNA copy number, volume, mass, density, shape, or by expression of particular proteins. Here we define growth kinetics as changes in a cell's mass and density over time. We propose to develop an instrument for concurrently monitoring single cell growth kinetics and cell surface protein expression. We hypothesize that changes in growth kinetics and in cell surface protein expression can be used as a surrogate for response to pathway-directed therapeutic agents. As a validation of the instrument, we will monitor mass, density and cell-surface protein expression (determined by fluorescence) in single A431 cells in response to intervention with the pathway targeted therapy gefitinib, a small molecule inhibitor of the epidermal growth factor receptor (EGFR), and appropriate controls. Single cell mass and density will be measured by a previously validated device known as the suspended microchannel resonator (SMR). The SMR can measure the mass of a mammalian cell with a resolution near 0.01% (1 Hz bandwidth). In order to achieve a continuous measurement of mass and density, silicon posts will be used for capturing a single cell within the sensitive region of the microchannel resonator. To enable simultaneous detection of protein expression, by fluorescence of coupled antibodies, the SMR will be modified to have optical transparency within the region where the cell is captured. The proposed instrument will leverage our prior proteomics work with the Center for Cancer Nanotechnology Excellence focused on Therapeutic Response (CCNE-TR) where we used proteomic methods to discover cell-surface protein biomarkers indicative of therapeutic response. Single measurement of the abundance of these proteins, concurrently with cell mass and density kinetics will provide a new approach for characterizing and monitoring cell response to therapy on a physiological and molecular level.
 
R21 CA138373-01 2009 MARTIN, JENNIFER M; YIN, HANG HUBERT (contact) UNIVERSITY OF COLORADO AT BOULDER Probing EBV-LMP-1s Transmembrane Activation Domain with Synthetic Peptide Antago
Although many therapeutic strategies exist for molecular targets accessible from the outside of the cell (e.g. therapeutic antibodies) or within the cytoplasm (e.g. small molecule inhibitors), they are not applicable to molecular targets that lie within the membrane bilayer. The hydrophobic phospholipid bilayer imposes an impenetrable barrier to water-soluble polar therapeutic agents. The Yin lab recently developed a computational method, Computed Helical Anti-Membrane Protein (CHAMP), to rationally design peptide probes that recognize protein transmembrane domains with high affinity and selectivity. This study utilizes this cutting edge technology to study the activation mechanism of oncogenic Latent Membrane Protein 1 (LMP-1) of Epstein-Barr virus (EBV). EBV is a human tumor virus associated with a number of malignancies and lymphoproliferative syndromes. EBV's ability to infect and immortalize B lymphocytes underlies its contribution to human disease. EBV's transforming activity depends on the expression and activity of LMP-1, the viral oncoprotein expressed in many EBV-dependent lymphomas and lymphoproliferative syndromes. LMP-1 functions as a constitutively active Tumor Necrosis Factor Receptor (TNFR) whose activity requires the function of its hydrophobic transmembrane domain. LMP-1 most resembles the TNFR CD40 in its signaling. Unlike CD40, whose activity requires activation by ligand, LMP-1's activity is constitutive and ligand-independent. Constitutive homo-oligomerization and lipid raft association, activities of LMP-1's transmembrane domain, play a key role in activation of downstream signaling. This proposal focuses on LMP-1 as a model membrane protein target for the design of peptide inhibitors because of LMP-1's essential role in EBV-dependent B cell transformation, LMP-1's contribution to EBV-dependent lymphoma and lymphoproliferative syndromes, and EBV's dependence on LMP-1's hydrophobic transmembrane domain for activity. This study aims to develop an innovative approach to target LMP-1's transmembrane domain, using CHAMP-designed anti-peptide antagonists as probes to study the contribution of oligomerization and raft association to LMP-1 activation, with the goal of inhibiting downstream signaling. Results of this research will provide insight into the molecular interactions within the membrane environment and the mechanisms underlying constitutive/oncogenic receptor signal transduction across membranes, will reveal the mechanism of LMP-1's constitutive activation of signaling, and will be applicable to the future development of novel therapeutics targeting diseases dependent on critical transmembrane proteins. Specifically, this proposal addresses the following Aims: 1) Can anti-TMD-1 peptides probes be developed that have high affinity and specificity for LMP-1's TMD-1? 2) Do identified peptides bind specifically and with affinity to LMP-1's TMD-1 in vitro? and 3) Can peptides that target TMD-1 (identified in Aims 1 and 2) interfere with LMP-1 homo-oligomerization, raft association, and constitutive signaling in intact cells?
 
R21 CA134419-01A1 2009 MC GOWN, LINDA B. RENSSELAER POLYTECHNIC INSTITUTE Hybridization of Promoter DNA Targets in Chromatin to Discover Regulatory Protein
Chromatin immunoprecipitation (ChIP) is a powerful tool for investigating the mechanisms through which nuclear proteins influence gene regulation. ChIP can be used to determine whether or not a particular protein is present in the in the chromatin of a living cell, to localize the region of interaction of a protein within the genomic DNA, and to isolate chromatin fragments that contain a particular target. However, since the basis of ChIP is the recognition of particular proteins or functional groups, it is limited in its ability to interrogate the full complement of proteins in a specific genomic region. In order to overcome this limitation, we propose to develop new approaches to analysis of chromatin that uses hybridization probes to target specific DNA sequences rather than antibody-based recognition. Our particular interest is in genomic DNA that may form alternate structures during nuclear processes, specifically in this application in gene promoter regions that contain G-rich sequences known to form G-quadruplex structures in vitro. These regions are the focus of much speculation and growing investigation in the cancer research community. The specific aims for this project period are to (1) demonstrate feasibility and selectivity and optimize conditions for in vivo capture of target chromatin DNA and associated proteins using chromatin antisense rehybridization (CAR) and chromatin sense rehybridization (CSR) with the model system of the insulin- linked polymorphic region (ILPR), and (2) evaluate the effectiveness of CAR and CSR to probe the proteins associated with the human c-myc oncogene promoter region in both cancer and normal cells. The proposed CAR/CSR techniques should be adaptable to isolation of target DNA and associated proteins in any genomic region of interest, and will be an important addition to the chromatin analysis toolbox. They will complement ChIP techniques by targeting specific DNA sequences rather than specific antibody targets such as proteins, thereby enabling discovery of new protein interactions that participate in gene regulation. There is exciting potential for discovery in epigenetic regulation as well. We propose to develop new approaches to analysis of chromatin, the genetic material in the nucleus of the living cell that uses hybridization probes to target specific DNA sequences rather than antibody-based recognition of immunochemical targets. Identification of the full complement of proteins associated with a particular DNA target of interest such as a gene promoter region in chromatin will lead to a better understanding of gene regulation and to the discovery of new biomarkers and drug targets.
 
R21 CA128718-01A2 2009 MESSMER, DAVORKA UNIVERSITY OF CALIFORNIA AT SAN DIEGO Real time quantitative multiplex analysis of cell interactions
Though it has been known for long that tumor cells can be self-sufficient for growth and survival, recent studies highlight the importance of interactions between the tumor and cells of the microenvironment, which can play a supportive role for tumor development and sustained growth. Thus a key to understanding cancer development and develop more effective therapeutics is to gain a complete picture of tumor cell interactions with cells of its environment. Cell interactions are a complex process that can involve several surface molecules as well as molecules that are released from cells. To develop an understanding of cancer microenvironment biology, a new technique must be developed for the simultaneous analysis of multiple secretants and surface molecules from different cells that are interacting. Our long-tern goal is to develop a generic device for cell interactions that can be custom tailored by the end user for analysis of a high number of secretants and cell surface molecules of choice. The objective of this application is the development of a new technology for the analysis of cell communication, which will allow us to measure several secretants and surface molecules on single cells that are interacting with one another in real time. We will use this novel technology to investigate interactions between cells from chronic lymphocytic leukemia (CLL) patients and cells of their microenvironment. We chose to use CLL as model system for this application, because it is an easily accessible liquid tumor and our team member Dr. Kipps has more than 600 characterized samples in his tissue core that are available to us. Furthermore, we have data showing that CLL cells can release secretants that cause cells of their environment to acquire changes which in turn are of benefit for leukemia cell survival. Understanding what secretants are released by CLL cells will allow us to design novel therapeutic approaches. The specific aims of this application are: Construct and validate a novel tool that can make quantitative and qualitative measurements of many molecules at the same time on two different cell types that are interacting. Use the new device to investigate the communication process between CLL cells and cells of their environment. Research Design and Methods: Chemical and bioengineering methods will be used to build a collection of tiny chambers, that each holds one cell. These chambers will be connected with small tunnels that will allow molecules to pass through and thus enable the cells that are next to each other to communicate. Small detectors will be build into the chambers that will report what factors are released from the different cells. Cells will be distributed into the wells and secretants and surface molecules will be measured using a microscope. Recent evidence demonstrates that tumors can manipulate surrounding ""non-tumor"" cells which in turn play a critical role in tumor development and sustained growth. We will develop a new technology that will allow us monitor the complex communication process between cancer cells and cells of their environment in real time, for which there is currently no tool available. Understanding these interactions will reveal possible new approaches for therapeutic intervention.
 
R21 CA140068-01 2009 SHAPIRO, BENJAMIN (contact); SMELA, ELISABETH UNIVERSITY OF MARYLAND COLLEGE PARK CAMPUS Spatial, High-Accuracy, Multiplexed Mapping of Genes in Human Tissue Sections
The aim of the proposed research is to demonstrate a technology to visualize the spatial distribution of genes and gene methylation in tissue sections. Our novel approach combines the key advantages of existing techniques: the amplification of in-situ PCR, the per-fews resolution of laser capture microdissection, and the multiplexing of FISH. We have already demonstrated that our technology can extract, amplify, and detect DNA in a spatially resolved manner using a substrate with mini-vials (so to a resolution of 1.6 millimeter per spot). Our aims now are to Aim 1 (achieve DNA methylation): Map GSTP1 promoter methylation over whole tissue sections from prostates with cancer. Aim 2 (achieve high spatial resolution): Miniaturize the technology to achieve a resolution of 100 ¨m. Aim 3 (multiplexing): Map multiple genetic alterations at once. We will demonstrate 3 but hundreds at once are possible in principle. It is also possible to spatially map gene expression (mRNA) by reverse-transcriptase PCR, and to quantify the amount of mRNA (by quantitative real-time PCR). Though we have initial results for both these directions and the path to them is open, we must meet aims 1 to 3 before we can move on to these more challenging areas. Consequently, multiplexed mRNA mapping and quantification will be done in future work. We will demonstrate a technology to spatially map genetic changes in tissue sections. Our method will allow the mapping of gene deletions, mutations, insertions, and methylations (silencing) in tumors, neighboring abnormal cells, and surrounding healthy tissue. The technology will be validated on frozen and paraffin fixed tissues supplied to us by Dr. Emmert- Buck at NCI and Dr. Olga Ioffe at UMB Medical School.
 
R21 CA137689-01 2009 SHEN, LANLAN UNIVERSITY OF TEXAS MD ANDERSON CAN CENTER Massively Parallel Sequencing Technology for Cancer Epigenome.
Methylated CpG island amplification (MCA) is a DNA library construction technique that permits amplification of 146,148 methylated CpG rich regions throughout the whole genome, covering 76% of all genes and 70% of all bona fide CpG islands. When coupled with microarrays, this technique has proven to be highly specific and very useful for the high-throughput analysis of genome-wide methylation in normal and cancer cells. Problems such as non-uniform probe performance, cross-reactivity, difficulties of normalization, and issues of relevant controls, however, prevent the full quantitative potential of MCA from being realized. We propose to develop and validate a high-resolution tool for DNA methylation profiling by coupling MCA with 'next generation' Solexa 1G sequencing technology (MCA-Seq). During the R21 phase of the project, we will apply various strategies to optimize MCA-Seq to improve coverage and minimize the quantity of initial DNA required. Additionally, we will build quality controls for MCA-Seq and develop optimized algorithms for data analysis. We will then validate our optimized MCA-Seq protocols, and evaluate the sensitivity, specificity, and quantitative accuracy using an innovative approach that simulates biological variation in methylation. Our proposed research will result in the development of a simple, robust, and reliable genome-wide assay for DNA methylation which will have broad utility in cancer research. Our long term goal is to utilize this technology to test biological hypotheses. Therefore, in an R33 phase of this project, we plan to fully implement this emerging technology by applying MCA-Seq in a set of well characterized tumor cell lines and primary cancer patient samples. These future studies will further validate the ability of MCA-Seq to accurately profile highly variable and aberrant cancer epigenomes, and generate preliminary biological data. The development and validation of MCA-Seq will enable major advances in our understanding of the basic biological mechanisms that contribute to cancer, and likely contribute to the design of future cancer therapies.
 
R21 CA137721-01 2009 SIMBERG, DIMITRI UNIVERSITY OF CALIFORNIA AT SAN DIEGO Isolation of rare circulating tumor cells from blood using microbubbles
It is well established that metastatic tumor cells are present in blood and bone marrow throughout the course of cancer disease (1-4). These circulating tumor cells (CTCs) are thought to seed the disease and distant metastases in the body (5,6). Isolating and characterizing the CTCs could provide invaluable information for diagnosis, treatment and prognosis of different stages of cancer (2,3,7). The concentration of CTCs in blood is typically low and can be as low as 1 cell/ml of blood (8). An ideal isolation technique has to be able to specifically capture and isolate single CTCs from billions of blood cells with close to 100% efficiency, without significant contamination by non-specific cells. We propose to use targeted microbubbles (hereafter ¨bubbles) to develop a method for isolation and analysis of rare tumor cells. ¨Bubbles could offer potential advantages over magnetic beads as a cell isolation technique. We show in our preliminary data that targeted ¨bubbles efficiently attach to their target cells in blood, so that the cells become buoyant and easily separate from all other cells using gravity/centrifugation. The goal of this proposal is to test whether the ¨bubble separation can achieve the sensitivity of magnetic bead separation, but with higher specificity, speed and productivity (ability to process >10 ml blood). These goals are achievable within the time frame of this award to allow us to assess whether further development is warranted. PUBLIC HEALTH RELEVANCE: To develop and to test tumor cell isolation technique for diagnosis of cancer. The technique will be based on the use of gas microbubbles that will attach to metastatic tumor cells in blood sample and will separate by buoyancy.
 
R21 CA140030-01 2009 SODERLING, SCOTT H. DUKE UNIVERSITY Mapping the Architecture of Cancer Signaling Pathways
To appreciate how cells migrate, establish polarity, and adopt cell shape is fundamental to understanding cancer biology. Almost one percent of the human proteome is dedicated to Rho GTPase signaling, which regulates key aspects of each of these processes. It is well known that Rho GTPases are ""signaling switches"" that are turned ""on"" or ""off"" by GEF (Guanine nucleotide Exchange Factors) and GAP (GTPase Activating Proteins) proteins. What is not known are the molecular mechanisms that determine Rho GEF and GAP specificity. Because GEFs and GAPs regulate Rho-GTPases in specific cellular contexts, they are promising candidates for future therapeutic interventions to modulate GTPase activity in different types of cancers. This proposal develops an innovative and integrated approach to map the architecture of Rho GTPase signaling in vivo. Multiple independent methods will be used, including novel proteomic screens using cellular proteins with unnatural amino acids as well as high throughput binding assays and informatics- based experiments. We have recently established this approach in our laboratory and our preliminary data validates our rationale that this approach will uncover novel links between Rho GTPase signaling and cancer biology. At the conclusion of this pilot project we expect to demonstrate that this approach can work on a larger scale to map interaction networks for regulators of this pathway. These results will fundamentally advance our understanding of how Rho GTPases are modulated to regulate processes such as migration, polarity, and cell morphology. It can also be expected to provide the basic information necessary for the future development of therapeutic strategies to alter cancer outcomes.
 
R21 CA132723-01A1 2009 TAINSKY, MICHAEL A WAYNE STATE UNIVERSITY Serum Biomarkers for Colorectal Cancer Detection
When cancer is identified at the earliest stages, cancer survival rates dramatically increase and therefore diagnostic screening tests that can detect early stage cancer are crucial. The overall goal of our research is to develop such an early detection screening test. We intend to prospectively accrue a large well-defined independent cohort of colorectal cancer (CRC) cases where we collect extensive data on this cohort at the time of diagnosis. We will generate a comprehensive database that includes data on demographics, personal and family history, tumor characteristics, and comorbidities. Our preliminary research efforts have been to develop a detection assay utilizing the sera of our discovery cohort of CRC patients and healthy controls. We have developed a high throughput method to isolated cDNA clones of antigens which can be used to identify cancer cases by detecting the presence of auto-antibodies to tumor proteins in the serum of the test subject. Our first aim is to identify the minimal number of antigen clones that are critical in distinguishing sera from patients with colorectal cancer from healthy controls utilizing our initial discovery cohort. We will eliminate antigen clones from our discovery set of 3800 clones that react with sera from patients with other cancers, benign gastrointestinal conditions, duplicates or do not react with any CRC sera. Our second aim is to determine the test characteristics (sensitivity, specificity, accuracy) on these newly selected antigen markers for distinguishing colorectal cancer cases using newly acquired sera samples not previously used in the development of the marker set. Lastly, we intend to determine the test characteristics of these antigen markers on a large independent well-defined cohort of colorectal cancer patients and healthy controls. In addition, due to the size, racial/ethnic makeup of the study population, and captured patient data, we will be able to evaluate the expression of these markers in relationship to important subgroups.
 
R21 CA137694-01 2009 TANNER, SCOTT DAVID UNIVERSITY OF TORONTO Development of (Prototype) Bead Array Flow Cytometer with Mass Spectrometer Detec
The unambiguous identification of a rare, diseased cell for the purpose of early diagnosis requires the quantitative determination of many biomarkers simultaneously in individual cells at high throughput. A similar requirement for multi-parameter assay is shared by genomic and proteomic researchers, who need to determine simultaneously the presence and quantity of the many genes, proteins and small molecules involved in the translation of gene to cell activity. We propose a novel solution for a broad spectrum of bio-analytical challenges. The new technology takes advantage of the high resolution and sensitivity of inductively coupled plasma mass spectrometry (ICP-MS), combined with the many available stable isotopes of elements of the periodic table, to simultaneously determine many proteins and gene transcripts in samples through the quantification of stable isotope tags bound to a wide variety of bioaffinity molecules. Beads are an attractive option for supporting surface chemistries for immunoassays and oligonucleotide hybridization assays. One of the advantages of beads is the ability to increase the reaction surface area per volume of the reaction mixture, which provides a reliable means of increasing the capacity and dynamic range potential of an assay, as well as miniaturizing the reaction. We expect that our technology will be able to recognize many thousands of distinguishable analytical beads created by the incorporation of various concentrations and ratios of metal ions. We hope that this integrated massively multiplexed technology based on our prototype flow cytometer-mass spectrometer (FC-MS) and reagent support/encoding system will simplify and enhance the diagnostic, prognostic and therapeutic efficacy available to physicians and their patients, increasing the effectiveness of healthcare while substantially reducing the human and financial costs of treatment. The objective of this application is the development of analytical instrumentation, an advanced element (metal) encoding system and methodology for rapid recognition of functionalized encoded beads - the bead array mass spectrometer (BA-MS). The combination with an elemental detector will provide researchers and clinicians with massively multiplexed analytical capabilities. The focus of this application is on the development of an individual (cytometric) bead analysis at high throughput (employing the FC-MS invented, developed and built in our lab). FC-MS continues to undergo extensive development in our lab and is not yet available elsewhere. Our Specific Aims include: 1) development of these technologies to the engineering beta-level, suitable for placement in laboratories for routine use and evaluation. These beta instruments will be made available within 12 months of the completion of the proposed effort. 2) development of a statistically significant unambiguous encoding system with massive variability (>105 individual beads) and unsupervised algorithms to recognize encoding in a rapid and dependable manner. 3) development of demonstrative bio-analytical methods using the technology in a multiplexed format and validation against existing fluorochrome bead-based technologies. Our team has long experience in research and pre-commercial analytical instrument development, is completely committed to the goals and is poised to advance rapidly.
 
R21 CA125461-01A2 2009 TYCKO, BENJAMIN COLUMBIA UNIVERSITY HEALTH SCIENCES Optimizing MSNP for profiling DNA methylation in cancers and precursor lesions
Alterations in DNA methylation are hallmarks of cancer cells, and epigenetic markers are increasingly viewed as having great potential for diagnosing, classifying and prognosticating cancers and cancer precursor lesions. Thus, a technical challenge is to develop and apply efficient and high-coverage methods to profile DNA methylation genome-wide in human cancers and in the normal precursor tissues of these cancers. We have developed such a method, called MSNP, to characterize DNA methylation genome-wide using Affymetrix single nucleotide polymorphism DNA microarrays. In addition to profiling gains and losses of net DNA methylation (GOM, LOM), a particular strength of MSNP is that it also queries allele-specific DNA methylation (ASM). Here we hypothesize that MSNP can be further developed and optimized as a high resolution method to reveal differences in methylation patterns not only between cancer and normal tissues, but also between normal tissues and the early atypical or dysplastic precursor tissues which eventually give rise to cancers. In this collaborative R21 proposal, with an experienced team of investigators from the Institute for Cancer Genetics and the Departments of Pathology and Biostatistics, we will advance the methodology and applications of MSNP in several ways. Aim 1 is to optimize MSNP for very high density Affymetrix 1.8M (6.0 array) SNP chips, vetting this method by profiling net and allele-specific DNA methylation in human breast cancers and normal breast epithelium. The results of will be verified by independent assays, including high throughput bisulfite sequencing. In this Aim we will develop bioinformatics approaches for tumor class prediction from MSNP data, and develop formats for data annotation and data sharing. Aim 2 is to miniaturize the MSNP method so that high quality genetic and epigenetic data can be obtained from the small amounts of genomic DNA available from laser-capture microdissection (LCM) or manual microdissection (MM). We will establish conditions allowing ASM and net DNA methylation to be determined using genomic DNA obtained by LCM or MM from normal and cancerous breast epithelium. In this aim we will particularly evaluate breast cancer precursor lesions, namely atypical duct epithelial hyperplasias (ADH), which are associated with a high risk for subsequent breast cancer development. Aim 3 is to correlate MSNP data with expression profiling data, to determine whether MSNP can produce a list of candidate DNA sequences, both promoter-associated and non- promoter-associated, in the human genome that may act as novel methylation-sensitive regulatory elements controlling gene expression in normal and cancer tissues.
 
R21 CA133275-01A2 2009 WATERMAN, MARIAN L UNIVERSITY OF CALIFORNIA IRVINE LEF-1 translation in chronic myelegenous leukemia
A two year plan is proposed to apply a new tagging system to the analysis of specific RNAs in cancer. This new applied technology will enable rapid isolation and quantitative mass spectrometry analysis of RNA/protein complexes. The development of this tagging system will be performed using Chronic Myelogenous Leukemia, a cancerous hematopoietic stem cell disorder driven by the oncogene BcrAbl. The specific focus is on Lymphoid Enhancer Factor-1 mRNA, a transcription factor that mediates Wnt signaling and is an essential factor in CML. We have recently linked BcrAbl action to LEF-1 translation in CML cells, a novel observation since LEF-1 protein is produced by the actions of two internal ribosome entry sites that direct cap-independent initiation of translation. The goal is to analyze LEF-1 mRNA/protein complexes in CML and identify proteins that are sensitive to BcrAbl action. Since a key activity of BcrAbl oncogenic action is misregulated translation, the expectation is that new BcrAbl target proteins will be revealed. The first aim of this proposal is to create a series of LEF-1 mRNAs tagged by stem loops for strong and specific association to epitope-specific matrices and resins. Expression systems in CML cells will be devised such that the tagged RNA can be rapidly isolated either under physiological or denaturing conditions. Negative controls and proof-of-principle experiments will be carried out to make sure that the LEF1 mRNA complexes are authentic. The second aim will refine the expression system for the use of isotope-labeling media (12C6-lysine/12C6-arginine vs. 13C6-lysine/13C6-arginine), such that SILAC-based analyses can be used for quantitative mass spectrometry. Beyond the specific focus of LEF-1 translation in this application, the RNA-tagging method will be developed so that it can be applied to the analysis of different RNP complexes in many contexts: normal cells, cancer, and other aberrant, diseased cell states. The goal is to develop the method to make it reproducible, reliable and able to be established in cell lines, primary cells and whole organisms. Vectors for tagging RNA and proteins will be developed for general use.
 
R21 CA137686-01 2009 WIRTZ, DENIS JOHNS HOPKINS UNIVERSITY High-throughput intracellular microrheology: a new tool for cancer research
Cancer mortality and morbidity are critically related to tumor invasion and metastasis in which the molecular mechanisms are poorly understood. Until their etiology is better revealed, attempts to develop new cancer therapeutics would remain empirical. Cell motility, which drives cancer metastasis, involves dynamic and regulated re-arrangements of the cytoskeleton. Our work and that of several other groups have shown that cytoskeleton phenotypes are typically accompanied by drastic changes in the viscoelastic properties of the cytoskeleton, which in turn modulate the ability of the cytoskeleton to generate net pushing forces at the leading edge and allow the cell to change its shape. Changes in cell mechanical properties have long been predicted to correlate with metastatic potential. However, current cell-mechanics approaches suffer from serious drawbacks - including time of measurement, lack of multiplexing, ambiguity of measurements - which prevent a direct test of this important hypothesis. The objective of this study is to: develop a highly-optimized high-throughput ballistic injection nanorheology (htBIN) technological platform to measure the micromechanical properties in cancer cells rapidly (< 30 seconds per cell) and reliably, and to assess these biophysical properties as a function of cell migration and invasion by comparing ovarian cancer cells of low and high invasive nature to normal cells, all obtained from patients at the Johns Hopkins Hospital. The proposed instrument, which is based on multiple-particle microrheology, presents key advantages over current approaches to cell mechanics. Our device will serve as a new tool for cancer research to study cell mechanics in the context of cancer cell migration and adhesion, and may ultimately serve as a diagnostic tool for patients who are at high risk for ovarian cancer, complementing more conventional biomolecular markers of cancer in a clinical setting While our proposed approach to cell mechanics is a priori applicable to detect intracellular mechanical differences in any type of cancer cells, a primary focus of this project is ovarian cancer. Ovarian cancer was selected as the disease model in this study because it represents one of the most aggressive cancers in women.
 
R21 CA137651-01A1 2009 ZENG, GANG UNIVERSITY OF CALIFORNIA LOS ANGELES Auto-antibody plus PSA assay for patients with prostate cancer
The lack of sufficient sensitivity and specificity of PSA as a screening modality for the diagnosis of prostate cancer underscores the importance of improving its operating characteristics to considerably improve the quality of predicting individual prostate cancer risk. Recently, serum-derived autologous antibodies (Ab) to individual prostate cancer-associated antigens (PCAA) received renewed attention as potential biomarkers for prostate cancer. The overall hypothesis of the proposal is that a multiplex approach that measures circulating autoantibodies against individual PCAA complement serum PSA tests to improve the prediction value of individual prostate cancer risk. Featuring an open-architecture design, the flow-cytometry based Luminex xMAP technology can be configured to detect autologous Ab to as many as 100 independent PCAA. However, to individually purify recombinant PCAA proteins and then conjugate with xMAP microspheres is difficult in practical terms. My lab has been focusing on identification of peptide epitopes from clinically relevant PCAA for the detection of autologous Ab present in serum samples. The simplicity of manipulating peptides over full-length proteins provides a unique opportunity to conjugate with microspheres applicable for the Luminex xMAP technology. To establish the Luminex-based multiplex assay for prostate cancer, we propose the following specific aims: 1) To configurate xMAP microsphere sets conjugated with B cell epitopes from a panel of clinically relevant PCAA previously defined in my lab; 2) To assess the sensitivity and specificity for retrospectively differentiating prostate cancer as well as to prospectively monitor tumor progression by the multiplex assay. Serum sample from 75 subjects in each cohort of healthy donors, BPH patients, patients with prostatitis as well as prostate cancer patients will be available for the proposed study. This study will provide a novel multiplex assay to serve as a potential biomarker for prostate cancer in areas such as monitoring disease recurrence.
 
R21 CA134391-01A1 2009 ZHONG, JOHN UNIVERSITY OF SOUTHERN CALIFORNIA Microfluidic Devices for Molecular Characterization of Cancer Cells at the Single
The goal of this proposal is to develop microfluidic devices for effective single analysis in biomedical laboratories without the requirement of specialized expertise. The lack of ability to perform biochemical and molecular characterization at the single level is a critical obstacle in cancer research and diagnosis. We have developed microfluidic devices for efficient large-scale single analysis to overcome this obstacle. The technology has broad applications in cancer research and diagnosis for identification, enumeration and characterization of the rare cells including 1) circulating tumor cells (CTC) in occult metastases; 2)T-regulatory cells, 3) tumor-specific T-cytotoxic cells; 4) cancer stem cells; 5) malignant cells captured by laser capture microdissection (LCM); and 6) suspicious malignant cells in needle biopsy. Isolation methods have been developed to capture rare cancer cells from blood and other tissues for cancer diagnosis. However, there is still a lack of robust methods to perform biochemical and molecular characterization on these captured cells at the single levels. Here, we propose to develop an inexpensive and integrated microfluidic device that can simultaneously profile 100 singles within 3 hours. The proposed device can be fabricated and used in general laboratories without the need of specialized expertise, and has a 5 fold higher mRNA-to-cDNA efficiency than current bulk assays. With our microfluidic devices, multiple biochemical analyses can now be performed at the single levels with a limited number of target cells such as the rare cancer cells listed above. We propose to optimize our devices for single biochemical characterization of CTC captured by our membrane filters. Characterization of these captured CTC is likely to lead to the identification of molecular therapeutic targets. After developing and optimizing the device with this R21 proposal, a R33 proposal will be submitted to apply this device for molecular characterization of CTC captured from bloods of cancer patients at the single level.
 
R21/R33 CA132075-03 2009 HALLEN, HANS ; RIEHN, ROBERT (contact) NORTH CAROLINA STATE UNIVERSITY RALEIGH Epigenetic profiling by near-field UV-Raman scattering in nanochannels
Epigenetic programming has emerged as a central theme in cancer development. Here we propose a new technique for monitoring the methylation state, chromatin status, and histone modification of genomic sized molecules, ideally whole chromosomes. The proposed method combines stretching of genetic material in nanofluidic channels, and subsequent optical readout by UV-resonant Raman scattering under near-field enhancement. We expect a resolution of about 1 kbp or better. The technique will provide a tool for single-molecule studies probing the epigenetic diversity of cell populations, and should be able to yield information about the importance and evolution of progenitor cell within tumors. The proposed research is exploratory in nature, but will provide significant improvements over the current art because of its capability to handle long molecules, high resolution, and label-free detection. Our strategy toward our goal is to establish a number of milestones along the way, which by themselves are significant achievements. In the first stage, we will nanochannel stretching of DNA that is labeled using methylation-specific fluorescent markers. We also aim at demonstrating chromatin stretching, and visualization of histones using specific antibodies. In the next stage, we will demonstrate that near-field enhancement of fluorescence by metal structures that are integrated with the nanofluidic device. The resolution should improve by a factor of 10. We will then investigate the near-field UV-resonant Raman signatures of specific DNA and chromatin constructs on test structures on wafers. Finally, we will integrate our findings to demonstrate near-field UV-resonant Raman of DNA scanned through nanochannels. Further, we will use the best technology proven in the course of the project, Raman or fluorescence, chromatin or bare DNA, to construct an epigenetic map of genomic model DNA or reconstituted chromatin from yeasts. We propose a new technique for monitoring the epigenetic programming state of genomic sized molecules, ideally whole chromosomes. The proposed method combines stretching of genetic material in nanofluidic channels, and subsequent optical readout by UV-resonant Raman scattering under near-field enhancement. We expect the method to become a valuable tool in cancer research.
 
R33 CA137673-01 2009 BEEBE, DAVID J UNIVERSITY OF WISCONSIN MADISON Microchannel cell-based assays to enable cancer research
It is becoming increasingly clear that cancer initiation and progression is actively regulated by signaling between neoplastic cells and their non-neoplastic neighbors. Yet current tools are not well suited to dissecting these multicell type conversations. While microfluidic-based tools have shown promise, typical embodiments present barriers to wide spread use. Here we propose to apply a microfluidic culture platform that utilizes surface tension effects to manipulate fluids allowing seamless integration with ubiquitous pipetting methods (manual and automated) eliminating the need for new infrastructure/equipment. Using this approach, we have developed a number of devices/ assays to probe cell communication via soluble factors. Here we propose to rigorously validate the platform biologically and apply the technology to two important areas of inquiry in cancer biology - hormonal regulation and stromal-epithelial interactions. The long term goal of the hormonal regulation studies is to improve prediction of responsiveness to hormone therapies. Towards this goal we will first validate the microchannel assay using a panel of stress assays. This will be followed by determining the sensitivity of the assay, the use of a one way signaling system to explore ER? reciprocal signaling and finally a study of the ability of the assay to measure hormone resistance. Regarding stromal-epithelial interactions, altered and activated cancer-associated fibroblasts (CAF) promote breast cancer growth and progression, whereas normal fibroblasts (NF) may keep cancer growth in check. Currently, our understanding of the molecular pathways involved in heterotypic stromal-epithelial signaling is limited. The systematic examination of these signaling pathways is hindered by the lack of a suitable in vitro assay platform. We propose to apply a microchannel three dimensional co-culture assay to examine the heterotypic interactions between human mammary fibroblasts and breast carcinoma cells. Broadly, we will apply the technology to monolayer (2D) culture, culture in matrices (3D) and primary cell culture. By evaluating the technology across both experimental and biological models we aim to enhance researchers' ability to identify and characterize molecular factors that influence cancer risk and progression.
 
R33 CA140039-01 2009 ELLEDGE, STEPHEN J BRIGHAM AND WOMEN'S HOSPITAL A multiplex genome-wide shRNA screening platform for cancer-lethal gene discovery
A major bottleneck in devising effective targeted therapies for cancer treatment lies at the identification of relevant drug targets. Genetic screens that measure the outcome of inhibiting individual genes in cancer cells and in normal cells on a genome-scale are powerful experiments that could identify such drug targets. Until recently these screens could not be performed in human cells. To perform such screens, my laboratory has developed new methods, based on the principle of RNA interference (RNAi), to individually inactivate every gene in the human genome, approximately 32,000 genes, one at a time. Furthermore, we have developed technologies to carry out these screens in high throughput fashion. Through this funding opportunity, I aim to further develop our RNAi technology platform to enhance its throughput and fidelity. I will use lung cancer cell lines as a discovery paradigm for this technology development effort. The ultimate goal is to enable the genome-wide interrogation of large numbers of cancer cell lines to comprehensively identify the dependencies and vulnerabilities specific to cancer cells but not normal cells. This approach should uncover many previously unrecognized targets for drug discovery and has the potential to provide additional, more effective treatment options for cancer patients.
 
R33 CA134304-01A1 2009 SHI, HUIDONG GEORGIA HEALTH SCIENCES UNIVERSITY Application of 454 Sequencing to Cancer Epigenomics
Although epigenetic components play a major role in driving tumor progression in many human cancers, the methylation landscape in cancer epigenomes is still largely unexplored. Systematic sequence-based methylation analyses are notably absent and as a result, the potential clinical value of specific methylation differences and their biological impacts in cancers remain largely untapped. By identifying the aberrant methylation ""hot spots"" in the cancer epigenome, we can target these genes for therapeutic intervention and develop them into DNA methylation biomarkers for early detection, diagnosis, prognosis, and monitoring the response to therapy. However, to fully understand the interactions between methylation and clinical behaviors, new methods are needed to determine single-base-level specific methylation patterns across the genome. As an important clinical model for our work, we will examine subsets of chronic lymphocytic leukemia (CLL) to discover DNA methylation alterations that distinguish the sub-types of CLL and suggest underlying mechanisms for differential clinical behaviors and tumor progression. Successful completion of this study will substantially influence the clinical management of CLL patients and allow ""up-front"" administration of epigenetic therapies. To accomplish this, we will develop a high-throughput, large-scale, sequencing-based approach to provide efficient methods for deeply exploring the CLL methylome. In our preliminary study, we demonstrated that bisulfite sequencing can be carried out using an innovative massively parallel sequencing system (454-sequencing) that is capable of analyzing millions of DNA bases in a single run. This new generation of bisulfite sequencing will provide highly quantitative single methyl-cytosine resolution for specific methylation mapping in multiple CpG islands (CGIs). In this R33 application, we propose to optimize and develop a prototype high-throughput bisulfite sequencing method for ultra-deep analyses of DNA methylation patterns in primary CLL samples from CD38+ and CD38- CLL Bs and test the hypothesis that the clinical behavior of subclasses of CLL can be defined in part by their distinct DNA methylation profiles that in turn affect multiple genes and signaling pathways. Specifically, we will: (1) develop a multiplexed amplicon preparation method for high-throughput, ultra-deep bisulfite sequencing; (2) develop a genome-scale approach for bisulfite sequencing of methylation-enriched genomic DNA libraries; (3) apply the innovative high-throughput bisulfite sequencing method to investigation of the CLL methylome. We believe that the technology developed will revolutionize the current analytical methods of DNA methylation, provide digital profiles of aberrant DNA methylation for individual human diseases and offer a deep-sequencing, robust method for epigenetic classification of disease subtypes.