2nd Principal Investigators Meeting Abstracts

Donna Albertson1,^{2, 3}, Antoine Snijders1, ³, Richard Segraves³, Stephanie Blackwood³, Nils Brown³, Joe Gray^2,3, Anna Katherine Hindle³, Greg Hamilton³, Bing Huey³, Sindy Law³, Ken Myambo³, Bauke Ylstra1, ³, Jingzhu Yue³, and Daniel Pinkel^2,3

¹Cancer Research Institute, ²Department of Laboratory Medicine, and ³UCSF Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA

We have assembled arrays of genomic clones for analysis of DNA copy number alterations by comparative genomic hybridization (CGH). Relative copy number is measured at these specific loci by hybridization of fluorescently labeled test and reference DNAs as in conventional CGH. In array CGH, the resolution is determined by the genomic spacing of the target clones. Currently, we are printing arrays of > 2500 BAC clones for the human genome and 1000 clones for the mouse. Each BAC contains an STS and has been verified to be single copy by FISH mapping. Copy number abnormalities can be readily linked to the physical map and the genome sequence by the sequence tags. The use of BACs provides sufficiently bright signals so ratio variation among the clones in a comparison 10‰. We have validated the capability of?of two normal genomic DNA samples is these arrays to quantitatively distinguish between heterozygous and homozygous deletions and trisomies by analysis of cell lines and patient samples containing known gains or losses involving one or more chromosomes. High level amplifications can also be quantitatively measured on the same arrays. This high level of measurement precision and sensitivity has been achieved both through the development of procedures for preparation of BAC DNA for spotting and the development of high density printing technology. The arrays are currently being used for mapping copy number alterations occurring in cancers, developmental disorders and for cross species comparisons.

Bryan Alexander, Greg Tafoya and James Gale (P.I.)

University of New Mexico

The lifetime risk for developing skin cancer is currently 1 in 5 for the USA. While only 2-5% of non-melanoma skin cancers (basal and squamous cell) become metastatic and potentially life threatening, they account for substantial morbidity and health-care expenditures. Our understanding of the molecular pathology and critical gene-networks involved in the development and progression of non-melanoma skin cancers is incomplete, resulting in poor markers for progression, prognosis and largely ineffective treatment for invasive stages. The broad objective of this project is to characterize the changes in gene expression in different stages of squamous cell carcinoma (SCC) and to identify different gene pathways involved. The first phase of this project will characterize UV-induced gene expression in cultured, primary human keratinocytes using cDNA microarray containing 4000 named genes. The second phase of the project will profile the gene-expression levels in the three most distinctive phases of the disease (actinic keratoses; SCC; and metastatic SCC). To more completely characterize these expression profiles, a microarray containing ~30,000 human cDNAs will be used (containing all ~7000 named genes and ~23,000 ESTs of unknown function). For control purposes, we will additionally collect biopsies from normal, uninvolved skin from both the arm (sun-exposed) and buttocks (sun-protected).Having two normal skin controls will add another dimension of analysis by allowing for the identification of genes that are responsive to sunlight and that may be involved in early stages of induction of SCC.

During this initial part of the project, our emphasis has been protocol optimization and control milestone experiments. Initially, our efforts were concentrated on optimization of protocols for RNA isolation and array hybridization/wash conditions. Within the array protocol, we experimented with many factors in order to maximally reduce background without losing signal intensities. Additionally, we tested a system developed by Ambion that was aimed at prolonging the life of a filter array (ie. increase the number of times a filter may be re-used without substantial lose of signal. By optimizing each step in the process (RNA isolation, RNA labeling, array hybridization/washing, array imaging and array stripping), we have robust system that allows for maximal analysis of our experiments. Having an optimized working protocol, we then focused on control experiments designed to measure the reproducibility, noise, and limitations of the Research Genetics' filter arrays. In brief, these control experiments include the following: 1. Probing a set of filters with the same probe to determine the reliability of intra-filer comparisons 2. Determining the lowest amount of RNA (in nanograms) that can be labeled and still produce adequate signal intensity 3. Stripping and reprobing a single filter repeatedly in order to see how many times it may be reliably re-used. In addition to finalizing the control experiments, we are in the process of generating expression profiles of keratinocytes and melanocytes at various time points following exposure to solar simulating light (SSL). Additionally, we intend to repeat the same time course on cells that have had repeated doses of SSL prior to the time course in order to see if the cells have an adapted response.

Nancy L. Allbritton¹, Christopher E. Sims¹, Eric. J. Stanbridge²,and David L. Van Vranken³

¹Dept. of Physiology and Biophysics, ²Dept. of Microbiology and Genetics, and ³Dept. of Chemistry, University of California, Irvine, CA

The molecular analysis of the signal transduction pathways driving uncontrolled growth in tumor cells will have a dramatic impact upon cancer biology and patient care. New technologies such as those to identify the genome and proteome of cells hold great promise. However these methods do not provide direct measurements of the activity of molecules involved in signal transduction. Ultimately it is the activation state of molecules such as enzymes that control cell behavior, for example, fueling the growth of tumor cells. A new technology and biochemical assay, the Laser Micropipet System (LMS), has the potential to perform simultaneous biochemical analysis of the activation state of multiple signal transducing enzymes within a single cell. Such data will enable misregulated signaling of tumor cells to be assessed in both linear signaling pathways and in interconnected networks of signaling proteins. The goal of this research is to apply the LMS to the Ras signaling cascades which are of immense importance in both the basic and clinical investigation of cancer. The research will draw on methods from analytical chemistry to analyze, separate, and detect kinase substrates from single cells. The strengths of combinatorial chemistry and synthetic organic chemistry will be brought to bear on the development of new kinase substrates to be used as specific reporters of Ras-regulated kinase activation. Molecularly engineered tumor cell lines in which individual proteins have been selectively mutated will be used to demonstrate the capabilities of the LMS in measuring the activation of kinases in the Ras-regulated signaling cascades. The successful completion of this work will provide a new and powerful tool for basic research, drug discovery and screening, cancer classification, and potentially clinical decision making.

Minerva Biotechnologies Corporation

With SBIR Phase I funding, Minerva Biotechnologies Corp. developed novel nano technologies that enable detailed study of cell surface receptor-ligand interactions that were heretofore not possible.

The technology is based on proprietary nano particles, that we call nano-probes. These are 10 nm gold colloids that we coat with modular self-assembled monolayers (SAMs). The SAM-coated nano-probes present moieties that bind to commonly used affinity tags, to facilitate the attachment of virtually any biological probe. We chose to modify gold nano particles because they have the intrinsic optical property that when dispersed in a homogeneous solution, the solution appears pink, but if the particles are drawn into close proximity, ie, via interaction of two biological species immobilized on different particles, the solution turns blue. Sensitivity can be increased dramatically by incorporating auxiliary signaling entities (optical or electronic) into the SAM-coating on the nano particles.

The technology is fast and sensitive. The assays are simple, 1-step, no-wash assays, which allow the study of weak interactions. The pre-formed particles are ready to use and customizable. To immobilize a probe peptide, protein, antibody or nucleic acid, one merely adds an aliquot of the probe to an aliquot of the pre-formed particles. Using our electronic detection assays, we were able to study ligand-receptor interactions on the surfaces of live, intact cells. It is critical to study cell surface receptors in their native state. The interactions and behaviors of receptors, which have been taken out of the context of the whole cell, may be irrelevant.

We used our new technologies to attack a very interesting an elusive problem: how does the MUC1 receptor function and how is it linked to tumorigenesis?

Peter E. Barker, DeLoise Gambrel-Hocker, Donald H. Atha and Catherine D. O’Connell

Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD

Within the NIST DNA Technologies Group, a new program for analytical validation and technology development has been initiated as a component of the NCI’s Early Detection Research Network (EDRN). Validation studies focus on nucleic acid analytes in EDRN-supported biomarker assay development for early detection or risk assessment, especially those that parallel ongoing NIST research and technical expertise in DNA diagnostics. Collaborative validation studies in progress include a FISH assay of lung cancer risk that measures molecularly defined, clastogen-induced cytogenetic changes. Prevalidation technology development collaborations between NIST staff and EDRN researchers focus on high throughput screening for serum telomerase activity and high throughput sequence analysis for homoplasmic mutations in mtDNAs for early detection of solid tumors. (Supported by NIST-NCI interagency agreement #Y1-CN-0103-01.)

Robert Basedow¹, Douglas Kankel², Michael Snyder², Peter Miller¹, and John Russo¹

¹BFGoodrich Corp (formerly Raytheon), ²Molecular, Cellular and Developmental Biology Department, Yale

BFGoodrich, in collaboration with Yale University, is developing an imaging spectrometer which will measure, in just a few seconds, the relative quantities of a mixture of up to 10 different fluorophore-tagged molecular species in each site of an array containing approximately 104 to 105 sites. It is primarily intended for use in fields of cancer research and genomics, but will also be capable of being used in a variety of applications, such as the evaluation of assays in high throughput drug discovery experiments. It will be affordable to the average researcher, and versatile enough to measure all microarray formats now available or anticipated. The effort is primarily one of technology development and technology transfer. An imaging spectrometer design, of the type built by BFGoodrich (then Raytheon) for airborne surveillance and reconnaissance applications will be adapted to the laboratory environment. Data analysis techniques, which have been developed for unmixing the spectra of a complex earth scene, will be applied to resolving the signatures in a mixture of fluorophores.

A feasibility and conceptual design study has demonstrated the soundness of the basic approach. A suitable subset of probes has been identified, and key representative characteristics of these have been measured. A radiometric model, tailored to measured probe behavior, has been developed. It has been iterated with the initial instrument concept design to produce an end-to-end model, including spectral unmixing, and an improved hardware design. In the design process, cost has been included as a major constraint. Thanks to recent commercialization of aerospace technologies, almost all components and subassemblies can now be of the commercial-off-the-shelf variety. Hardware trades and selections have been made, and manufacturers published data have been included in the model. The probes themselves can also be made using established techniques, but protocols have been developed to optimize the specific activity of each. Preliminary syntheses of amino-allyl-dUTP labeled DNA has also been carried out, with the objective of increasing the pool of flours suitable for a multi-fluor capability.

Uncertainties of the modeling approach largely involving lack of knowledge of the basic properties of the probes have been estimated. Variability of the kind observed in sets of repeated laboratory measurements on sample probes has also been included, using Monte Carlo methods. The model has yielded false alarm rates as a function of relative concentrations in a 7-fluor mixture, quantification accuracy as a function of one fluor’s concentration varying in a mixture of 7 fluors, and system dynamic range (of fluor concentration) as a function of threshold accuracy of quantification. The model has also been used to identify the system’s limiting parameters (e.g. detector saturation, signal-to-noise) and refine system design parameters (e.g. laser power, integration time).

Design, fabrication, and in-house testing of the multi-probe instrument are proceeding. The spectral unmixing algorithms which have formed part of the model are being refined and incorporated into the overall data analysis system. The resulting prototype instrument will be field-evaluated by Yale, and any critical product improvements will be implemented as part of the overall project.

Samuel W. Beenken, Neal Poulin, Ph.D, James Crowell, Ph.D, Andra Frost, M.D, William Grizzle, M.., Ph.D, Kirby I Bland, M.D.

University of Alabama

The specific aims of a recent study of 40 archival specimens of ductal carcinoma in situ of the breast (DCIS) and/or microinvasive ductal carcinoma included the development of morphometric and architectural indices of cancer and DCIS progression. The analytic strategy for data analysis assumed a continuous model of progression between normal breast tissue, different grades of DCIS and invasive cancer.

5-micron tissue sections were stained using a Feulgen-Thionin stain. Tumor grade was measured according to the Scharf-Bloom-Richardson scale and DCIS was classified according to low, intermediate, and high nuclear grades. Using a high-resolution image cytometer, a digital image of selected nuclei was captured, which contained between 500 and 2,000 individual pixels depending on nuclear size. From the distribution of the individual pixel measurements, over 100 nuclear features were calculated for each nucleus, including DNA content, size, shape, roughness of the nuclear boundary, etc. The nuclei of neoplastic cells as well as the nuclei of surrounding normal-appearing cells were analyzed.

Quality control studies were carried out in order to assess the relative magnitude of errors from various sources, including temperature control of the staining reaction, reproducibility of the staining reaction, effect of section thickness on digital capture, cytometer calibration and stability, and reproducibility. These studies will be presented. Following the quality control studies, discriminant function analysis was used to identify differences in the grade of nuclei from pooled cell measurements. Linear discriminant functions with stepwise variable selection were developed to classify high grade vs. intermediate vs. low-grade tumor nuclei and the discriminant functions so derived were evaluated. These results will also be presented.

Aaron Bensimon¹, John Herrick¹, Ekaterina Svetlova¹, Chiara Conti¹

Department of Biotechnology, the Biophysics laboratory, Pasteur Institut, Paris, France

The intellectual stance of these studies is the goal of providing a quantitative analysis of events at the level of the single DNA molecule or even the single cell.

The research being pursued concerns the mechanisms underlying the control of DNA replication and genome stability with particular emphasis on the mechanisms and consequences of oncogene amplification. We have developed a technological platform for the genomic study of DNA replication and genetic alterations. This technology involves a method called molecular combing, which is used to straighten and align molecules of genomic DNA on a solid surface.

Molecular combing relies on the action of a receding aie/water interface, or meniscus, to uniformly straighten and align DNA molecules on a solid surface. The ability to comb large molecules at a high density makes long range genomic studies feasible (over 700 kb). The advantages of this approach reside in the reproducibility of the results, their precision (1-4 kb resolution) and the relative ease of analysis afforded by the ability to directly visualize the molecules. Beyond its applications to genomic studies and genetic diseases, it creates new experimental possibilities for research into cancer. We have demonstrated that this is an attractive approach to the study of those phenomena underlying the mechanisms of carciogenesis including microdeletions, inversions and amplifications of specific genetic loci. In a recent study we examined the genomic organization of the proto-oncogene met in renal cell carcinoma. We intend to develop a more general procedure which may contribute for an understanding of the genetic reasons for the tumor development and permit following its evolution in time. Since the density of stretched fibers is high, hundreds of signals are rapidly collected and precisely measured, yielding a statistically reliable analysis. Consequently, the complex organization of a genomic region can be visualized and micro-dissected allowing a molecular genomic classification of a tumor and its evolution in time.

Indeed, as a tool, molecular combing is a versatile approach to a wide range of subjects and question of fundamental interest. This is especially true for the multifaceted domain of DNA replication in higher eucaryotes. Molecular combing, in combination with other higer resolution techniques, permits the identification and mapping of origins of replication on a genome wide basis. Consequently the replication programs of higher eucaryotes can be reliably elucidated by determining the distribution and activities of replication origins over wide regions of the genome. To that effect, we have recently undertaken the mapping and analysis of all origins of replication in the MHC region to verify the validity of this approach for its eventual application in cancer genomic. The longer term goal of our research is to investigate those factors that are implicated in the aberrant replication of the genome in transformed cells. The objective is to elucidate the mechanisms underlying the replication programs in both normal and cancerous cells. Indeed the replication program differs from one tissue type to another depending on the respective cell?s transcription profile. Hence, our ultimate interest is to establish integrated replication/transcription maps of different tumor type.

The molecular combing method, its biophysical characteristics, and its biological applications to cancer genomic studies will be discussed.

Grant A. Bitter, Aaron R. Ellison and Joan Lofing

BitTech, Inc.

Genomic instability is associated with cancer progression, and defects in the process of DNA mismatch repair (MMR) is one pathway leading to genomic instability. Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant inherited disease caused by defects in MMR, and mutations in the hMLH1 or hMSH2 genes are responsible for the majority of HNPCC. Defects in MMR have also been observed in a variety of sporadic cancers. In addition to clear loss-of-function mutations conferred by nonsense or frameshift alterations in the coding sequence or by splice variants, genetic screening has revealed a large number of missense codons (25% of all alterations observed) with less obvious functional consequences. The ability to discriminate between a loss-of-function mutation and a silent polymorphism is important for genetic testing for inherited diseases like HNPCC where there exists opportunity for early diagnosis and preventive intervention. In this project, quantitative in vivo DNA mismatch repair (MMR) assays in the yeast Saccharomyces cerevisiae are utilized to determine the functional significance of amino acid replacements observed in the human population.

Missense codons previously observed in human genes were introduced at the homologous residue in the yeast MLH1 or MSH2 genes. This study also demonstrated feasibility of constructing genes that encode functional hybrid human-yeast MLH1 proteins in which regions of the yeast protein were replaced with the homologous region from the human protein. The genes encoding functional hybrid MMR proteins allow determination of the in vivo effects of codon changes at residues that are not conserved in the yeast gene. Three classes of missense codons were found: 1) complete loss-of-function, i.e. mutations; 2) variants indistinguishable from wild-type protein, i.e. silent polymorphisms; and 3) functional variants which support MMR at reduced efficiency i.e. efficiency polymorphisms. There was a good correlation between the functional results in yeast and available human clinical data regarding penetrance of the missense codon in HNPCC. The results of this study raise the intriguing possibility that differences in the efficiency of DNA mismatch repair exist between individuals in the human population due to common polymorphisms. Future work will investigate this possibility by further mutation analysis. If individual differences in the efficiency of MMR exist due to common polymorphisms, it would predict differential sensitivities to cancer development. Elucidation of such relationships will facilitate implementation of cancer prevention strategies.

Samuel W. Beenken, M.D., Neal Poulin, Ph.D., James Crowell, Ph.D., Andra Frost, M.D., William Grizzle, M.D., Ph.D., Kirby I. Bland, M.D.

University of Alabama

The specific aims of a recent study of 40 archival specimens of ductal carcinoma in situ of the breast (DCIS) and/or microinvasive ductal carcinoma included the development of morphometric and architectural indices of cancer and DCIS progression. The analytic strategy for data analysis assumed a continuous model of progression between normal breast tissue, different grades of DCIS and invasive cancer.

5-micron tissue sections were stained using a Feulgen-Thionin stain. Tumor grade was measured according to the Scharf-Bloom-Richardson scale and DCIS was classified according to low, intermediate, and high nuclear grades. Using a high-resolution image cytometer, a digital image of selected nuclei was captured, which contained between 500 and 2,000 individual pixels depending on nuclear size. From the distribution of the individual pixel measurements, over 100 nuclear features were calculated for each nucleus, including DNA content, size, shape, roughness of the nuclear boundary, etc. The nuclei of neoplastic cells as well as the nuclei of surrounding normal-appearing cells were analyzed.

Quality control studies were carried out in order to assess the relative magnitude of errors from various sources, including temperature control of the staining reaction, reproducibility of the staining reaction, effect of section thickness on digital capture, cytometer calibration and stability, and reproducibility. These studies will be presented. Following the quality control studies, discriminant function analysis was used to identify differences in the grade of nuclei from pooled cell measurements. Linear discriminant functions with stepwise variable selection were developed to classify high grade vs. intermediate vs. low-grade tumor nuclei and the discriminant functions so derived were evaluated. These results will also be presented.

Steven A. Bogen and Greg Testa

CytoLogix Corporation, Cambridge, MA

Modern methods for the genetic analysis of cancer include hybridization assays to tissue sections, cells, and arrays. Each of these techniques has in common the fact that they are performed on an optically clear flat surface, such as a microscope slide. These assays require that a small amount of reagent is spread over a planar surface and heated while preventing evaporation. Traditionally, this has often accomplished by sealing a coverslip over the sample with rubber cement or nail polish. However, that process is not readily amenable to automation. Although the problem seems relatively straightforward, it has been particularly difficult to solve because the assay procedure entails a high surface to volume ratio. Both friction and evaporation occur at liquid-surface interfaces, making it difficult to obtain even reagent spreading with low reagent volumes, without evaporation in a fashion that can be performed robotically. Under Phase I SBIR funding, CytoLogix demonstrated feasibility of a microfluidics technology that addresses these technical constraints. This microfluidics technology involves a means for fluid exchange in a capillary-thick chamber. The main technical barrier has been filling the microchamber with reagent without entrapping air bubbles over the sample. CytoLogix plans to further develop this technology into a walk-away, completely automated instrument.

Gerry Boss¹, Anna Dreilinger², David Gough³, James Harrell¹, Stephen Jones⁴, Stephen Qualman⁵, Anne Wallace⁶, and Linda Wasserman²

¹Departments of Medicine, ²Pathology, ³Bioengineering, and ⁶Surgery, and ⁴Electronics Shop, University of California, San Diego, La Jolla, CA, and ⁵Department of Pathology, Ohio State University, Columbus, OH

Ras and Rho transmit pro-proliferative and cellular transforming signals when appropriate ligands bind to growth factor receptors on the plasma membrane; both proteins cycle between an active GTP-bound state and an inactive GDP-bound state. We devised a method to measure Ras activation (the ratio of Ras-bound GTP over Ras-bound GTP plus GDP) in human tumors and found that Ras is highly activated in a significant number of neuronal tumors and in breast, lung, and ovarian cancers, even in the absence of a genetic mutation in the ras gene. Assessing Ras activation provides information not only about the basic biology of a tumor, but it may also have therapeutic importance because a large number of Ras inhibitors are under development or are already in clinical trials; in cell culture and animal models, these drugs are most effective when Ras is in an activated state. Some of the agents designed to disrupt Ras function have been found to exert their inhibitory effects on cell growth by inhibiting Rho and Rho may be involved in the development of metastases. It is likely, therefore, to be of clinical value to assess the activation states of Ras and Rho as part of developing specific chemotherapeutic regimens based on the molecular alterations found in a tumor.

Graham J.R.

Brock Institute of Biomedical and Life Sciences; Dept. Molecular Genetics, University of Glasgow, UK

Objectives
A: To fractionate DNA fragments retaining those with the large numbers and densities of m5CpGs (~5% of the genome). B: To establish the efficiency of a subtractive hybridization procedure. C: To isolate the densely methylated fraction from matched pairs of normal and tumor tissue then use the subtractive hybridization procedure to isolate those sequences with altered methylation patterns.

During tumourigenesis, normal genomic methylation patterns are altered by the addition of a methyl group to the 5 cytosine of CpG dinucleotides. Well-characterized examples include the hypermethylation of the ordinarily unmethylated GC-rich regions known as CpG islands (CGIs). Whether methylation changes occur as a cause or effect is currently unclear since the earliest changes have yet to be defined. However, once established, aberrant methylation changes are faithfully reproduced in all progeny cells, making such alterations potential biomarkers for tumour identification and classification.

To examine those GC-rich regions, altered during tumourigenesis, a novel method was developed which utilized a methyl CpG-binding domain (MBD) column. The MBD column has a strong affinity for densely methylated sequences, allowing these to be purified from bulk genomic DNA. However, in addition to methylated CGIs large numbers of repetitive sequences (e.g. LINEs and SINES) are both GC-rich and methylated and therefore bind to the MBD column. To remove both high and low copy number repeats a process of subtractive hybridization followed by linker attachment and amplification was used. Following optimisation of the method through recovery of a plasmid spike, a library of sequences (whose methylation status is altered in DNA extracted from a poorly differentiated tumour and its adjacent normal tissue counterpart) was then generated. With the DNA derived from the tumor being used as tester and DNA derived from normal tissue used as driver. Of 61 clones fully sequenced to date, 31 have the GC content and CpGobs/exp of a CGI. Database searches with these sequences indicate no homology to known CGIs, however, analysis of regions from which the clones are derived (using the GRAIL/cpg program) demonstrates that many of these sequences are fragments of predicted CGI’s. Analysis of the methylation status of these regions in matched pairs of tumour and adjacent normal samples is currently underway.

These results and future potential uses of libraries derived from matched pairs of normal and tumour DNA will be discussed. The libraries will be used to generate genomic arrays of sequence whose methylation status is altered during tumourigenesis. These arrays will then be utilized in the genome wide analysis of alterations in methylation patterns in different grades of tumours and to identify specific patterns of methylation and any association with clinically defined tumour stage and grade. Finally the method will be employed to investigate loss of methylation by using the DNA derived from a tumor as the driver at the subtractive hybridisation stage.

R. Hugh Daniels, Marcel Bruchez, Xingyong Wu, Kari Haley, Christopher Ng, Yanzheng Xu, and Huayong Yong

Quantum Dot Corporation

Semiconductor quantum dots are a new class of fluorescent probe that can be used for simple multiplexing with a single excitation source. These materials have been developed for detection of progesterone and estrogen receptors, P53, cytokeratin 8/18 and Her-2 in sub-nanogram quantities, and have been used to specifically stain these markers in both human breast cancer cell lines and tissue sections. These probes have been used in model assays to demonstrate the ability and simplicity of multiplexing, and multiplexed analysis of breast cancer tissues are currently underway. The probes developed show substantially improved photostability in the cellular milleu, substantially higher sensitivity and are significantly more stable to archiving than similarly stained materials using fluorescent dyes. These quantum dot probes will be developed into a more complete platform for cancer sub-classification and diagnosis.

Stephen Weeks, Hiep L. Tran, and Tauseef R. Butt

Research and Development, LifeSensors Inc, Malvern, PA

Application of human nuclear receptor as sensors in molecular profiling individuals with high risk for progression of specific type of cancer is very promising. In addition, quantifying response to therapy with Biosensors also holds a great promise. Ligand dependent function of many human nuclear receptors has been established in yeast. Many of the receptors and their ligands are important therapeutic and diagnostic markers. The distinguishing feature of our technology is that panel of yeast cells engineered with several human nuclear receptors (Biosensors) are ultra-sensitive to ligands and hormones. Receptor regulating activities from human sera of control and cancer patients can be. We have? monitored using the panel of functional microarrays called LifeSensors used human estrogen receptor as a model system to develop an ultra-sensitive estradiol and phytoestrogens?estrogen sensor. It has been shown that 17 transactivation rank order of potency was remarkably similar in yeast and human HepG2 cells. Yeast cells demonstrated an order of a magnitude higher efficacy for variety of compounds as compared to human cells. Application of this technology to monitor pre-disposition to disease and therametrics will be discussed.

Ali Al-Timimi, Eric Cahoon, Susan Castillo, Venkat Chalasani, and Scott Bennett

SRA International, Fairfax, VA

The amount of data generated by microarray gene expression experiments can be enormous. To better understand this data, computationally intensive analysis tools are needed. MultiCluster brings together multiple methods for analysis of microarray data.

As it becomes more and more imperative for researchers to share these data cooperatively, the need for a standard method for data exchange becomes paramount. Recently, a number of XML standards for the exchange of gene expression data along with the associated gene and experiment annotation have been proposed. Among these standards is MAML (Microarray Markup Language) (http://www.mged.org).

We have added to MultiCluster a Java transformation tool that maps MultiCluster data sources to the MAML DTD and subsequently converts the data to XML. With this tool, MultiCluster users can rapidly create MAML XML documents through a user-friendly interface.

Jeffrey J. Chalmers¹, Masa Nakamura¹, Keith Decker¹, Julia Chosy¹, Kristie Melnik¹, Kara McCloskey¹, Lee Moore², Maciej Zborowski²

¹Department of Chemical Engineering, Ohio State University, ²Department of Biomedical Engineering, Cleveland Clinic Foundation

Magnetically based cell separation technologies have become commonly used techniques to enrich and/or separate cells of interest from a heterogeneous cell population. Recently, significant interest has been generated in the potential to separate rare cancer cells from blood. The analysis of this separation approach can be broken down into three aspects: 1) the specificity and selectivity of the immunomagnetic labels for the target cell of interest, 2) the ability of the paramagnetic label to impart on the target cell a magnetophoretic mobility sufficient to allow an effective separation, and 3) the effectiveness of the actual separation system to remove the immunomagnetically labeled target cell from a heterogeneous mixture of cells.

This presentation will attempt to summarize the current state of knowledge in each of these three aspects. With respect to the first aspect, we have conducted studies which characterizes the degree to which several human cancer cell lines, and tissue from a primary tumor, can be labeled with both fluorescently labeled, and magnetically labeled antibodies. We used both FACS and a new type of instrument that we have developed, Cell Tracking Velocimetry, CTV, to conduct these studies.

With respect to the second and third aspect, we have developed two flow-through magnetic cell separators. One separator, the Quadrupole Magnetic cell separator, QMS, can separate immunomagnetically labeled cells at a feed rate of 107 cells/s. The second separator, the Dipole Magnetic Flow Sorter can "Fractionate" cells in to different sub-populations based on the cells magnetophoretic mobility (degree of immunomagnetic labeling). Various cell mixtures are being separated with these two systems as well as commercial, batch systems. Measures of the performance of these systems are being developed using a variety of analytical methods including FACS and CTV.

Wing C. (John) Chan

University of Nebraska Medical Center

Tumors derived from the same cell type and having similar morphology may nevertheless have a distinctly different clinical behavior and response to therapy. Differences in the genetic lesions in these tumors, as reflected by their gene expression profiles, will provide insight into the mechanisms underlying the divergent clinical spectrum that is observed. Comparative genomic hybridization (CGH) and spectral karyotyping (SKY) and highly complementary novel techniques that examine the entire genome for genetic abnormalities and can supplement and extent conventional cytogenetic studies. In addition, the recently developed high density cDNA microarray technology is a very promising method for displaying the pattern of gene expression in tumor tissues. These powerful technologies with their associated informatic systems are now available for translational research. In order to evaluate the information generated by these technologies, an adequate number of well-characterized tumors with detailed clinical data must be available. We propose a multi-institutional, comprehensive molecular analysis of a large series of B-cell non-Hodgkin’s lymphoma (NHL).

Britton Chance

University of Pennsylvania

The remarkable scope of the IMAT meeting, together with the NCI commitment to the program, the perspectives of Dr. Klausner, together with the teachings on RNA, protein expression, protein structure and function make this a must for anyone attempting to do today's biochemistry of cancer.

The fact that molecular beacons are now planned to recognize specific genetic expressions of cancer makes this program of special interest to those working on the UIP. It is hoped that not only the PI, but also those designing contrast agents would be able to attend this fascinating session.

Michael Brenowitz and Steve Almo

Center for Synchrotron Biosciences, An NIH Resource Center and Albert Einstein College of Medicine, Bronx NY 10461

Radiolysis of water by synchrotron X-rays generates oxygen-containing radicals that undergo reactions with solvent accessible sites of macromolecules inducing stable covalent modifications or cleavage on millisecond timescales. The extent and site of these reactions are determined by gel electrophoresis for nucleic acids and mass spectrometry analysis in the case of proteins. The Albert Einstein Center for Synchrotron Biosciences (www.aecom.yu.edu/home/csb/) has been developing synchrotron footprinting technology for the last several years with the aim of determining detailed structure and dynamics information in solution for large macromolecules and their complexes (Sclavi et al., Science, 279: 1940-1943 1998, Maleknia, et al. Anal. Biochem. 289: 103-115, 2001).

The footprinting data is used to construct a quantitative map of solvent accessibility at individual reactive sites. The experiments can be performed in both in an equilibrium configuration with a matrix of proteins to examine protein-protein interactions in a high-throughput fashion and in a time-resolved manner to provide kinetic rate constants for formation of protections for individual sites within a macromolecule. The application of this synchrotron footprinting technique to the study of DNA-protein complexes and interactions of actin binding proteins will be presented. These model systems will drive the technology to provide general methods relevant to studying protein-protein interactions in cancer biology. Specifically, we will examine a library of C. elegans proteins implicated in DNA damage and cell-cycle checkpoint that have been selected by high-throughput screening and expressed and purified by high-throughput proteomics methods.

Zunxue Chang¹, Patricia Flatt², William H. Gerwick², and David Sherman¹

¹Department of Microbiology, University of Minnesota, Minneapolis, MN, ²College of Pharmacy, Oregon State University, Corvallis, Oregon

Curacin A was isolated as a major lipid component of a marine cyanobacterium strain Lyngbya majuscula for its high brine shrimp toxic and anti-proliferative activities. Its ability to inhibit cell growth and mitosis and to bind rapidly and irreversibly to the colchicine site of tubulin make it a potential anticancer drug lead. A chlorinated lipopeptide, barbamide, was also extracted from the same strain for its high ichthyotoxicity. Screening of the genomic library of L. majuscula constructed in cosmid vector pOJ446 using the PCR-amplified polyketide synthase (PKS) fragments as a probe identified sixty positive clones. Two groups (A and B) were assigned to be responsible for the biosynthesis of curacin A and barbamide, respectively by three rounds of Southern hybridization using the PCR-amplified PKS and non-ribosomal peptide synthetase (NRPS) fragments as probes, and their DNA sequence was determined. Analysis of the 80-kb assembled DNA sequence of three cosmids (group A) revealed nine large ORFs responsible for ten modules of PKS and NRPS, and three small ORFs for HMG-CoA formation, which is presumed to be an intermediate in curacin biosynthesis. Domain organization of the PKS and NRPS modules is consistent with the chemical structure of curacin A. Analysis of the DNA sequence of another cosmid (group B) revealed three modules of NRPS and one module of PKS. Combined with the domain organization, this gene cluster is believed to be the barbamide gene cluster. The several small ORFs upstream of the first NRPS module is presumed to specify chlorination and modification of leucine, the initiation unit of barbamide biosynthesis. The heterologous expression of curacin A, the adenylation domains and the whole gene cluster of barbamide in E. coli and Streptomyces venezuelae are in progress.

Mark Chee, Tim McDaniel, Shawn Baker, Semyon Kruglyak, Francisco Garcia, Kenneth Kuhn, Csilla Fenczik, Kevin Gunderson, and Jian-Bing Fan

Illumina, Inc., San Diego, CA

Self-assembled arrays of bead-based sensors have been developed. Each bead contains oligonucleotide probes that can hybridize with high specificity to complementary sequences in a complex nucleic acid mixture. The identity of each bead in the random array is determined by a hybridization-based decoding procedure. By formatting the miniaturized arrays into a matrix that matches a 96-well microtiter plate, many samples can be processed efficiently in parallel. We are using this platform to develop assays in the areas of SNP genotyping, gene expression profiling, and protein analysis. Progress in the area of gene expression profiling will be discussed.

Gary A. Churchil

The Jackson Laboratory

Gene expression microarrays are an innovative technology with enormous promise to reveal the function of genes in normal and diseased tissues. Although the potential of this technology has been clearly demonstrated, many important and interesting statistical questions persist.

The problem of making relative comparisons among large numbers of samples using heterogeneous experimental conditions is not new. Indeed there is a history of 100 years of research in agricultural experimentation which is relevant and can be applied to the problem of designing microarray experiments.

We advocate greater attention to experimental design issues and a more prominent role for the ideas of statistical inference in microarray studies.

It is our premise that good bioinformatics begins before one goes to the bench. Experiments that are well designed can be used to answer not only the questions which they were specifically designed to address they can also be "mined" for useful information which was not anticipated by in the original experiment. Sound statistical design is crucial to ensure that these goals can be achieved. We will illustrate these points by contrasting the analysis of microarray data obtained on the same set of samples using both standard and novel experimental designs.

J. Condeelis

Aecom

We have developed metastasis models in rats and mice that permit real time multiphoton-based imaging of the behavior and interactions of metastatic tumor cells in the primary tumor in vivo. Certain mouse models are clinically relevant in that they resemble human breast tumors in etiology and histology. Intravital imaging results indicate that tumor cell chemotaxis leads to the accumulation of tumor cells around, and their polarization toward, blood vessels in primary breast tumors and is correlated with efficient intravasation (Cancer Research 60:2504-2511, 2000). Viable tumor cells can be collected from the blood of rats with metastatic tumors with 90-fold greater frequency than from rats with non-metastatic tumors. Intravital imaging of GFP labeled tumor cells was used to identify blood burden in the vessels at the edge of the tumor. Six-fold more tumor cells are observed in vessels per minute per 250 um square field of metastatic tumor compared to matched preparations of non-metastatic tumors. Since tumor cell polarization toward blood vessels and chemotaxis are not observed in matched non-metastatic tumors, polarization and chemotaxis of tumor cells toward blood vessels are proposed to be important in invasion and metastasis. To extend these observations to a mechanistic level, we have used chemotaxis of tumor cells in vivo to advantage to collect subpopulations of motile and chemotactic tumor cells from primary breast tumors. Needles containing chemoattractants and matrigel are used to collect motile tumor cells in vivo as a pure population suitable for further analysis.

Christopher H. Contag¹, Leonard A. Herzenberg², Robert S. Negrin³, Leonore A. Herzenberg²

¹Department of Pediatrics and Microbiology and Immunology, ²Department of Genetics, and ³Department of Medicine, Stanford University, Stanford CA

Imaging reporter gene expression in living animals provides critical spatiotemporal information about changes in cell growth, cell trafficking and gene expression during normal and disease processes. We have added another dimension to this powerful in vivo assay by coupling it with equally powerful fluorescence methods for ex vivo identification of cells in suspension or tissue sections, through the use of multifunctional reporter genes. Together, these technologies enable effective methods for evaluating immune surveillance of neoplastic disease, and improving cell-based and other anti-cancer therapies. We have described in vivo tumor-host immune interactions based on real time observations of trafficking and proliferation of immune and tumor cells in intact animals. We have designed a build a series of multifunctional reporter genes that can be used for in vivo trafficking studies and in ex vivo assays (fusion proteins comprised of luciferases and fluorescent proteins) that are detectable in vivo by bioluminescence and ex vivo by fluorescence.

Daniela Ladner¹, David Ward², Paul Lizardi1, and Jose Costa¹

¹Department of Pathology, Yale University School of Medicine, ²Department of Genetics, Yale University School of Medicine, New Haven, Connecticut

There is an urgent need for biomarkers capable of identifying patients at risk during early phases neoplasia in pancreatic, breast, and colon tumors. Having access to surrogate samples for the analysis of these tissues provides an opportunity for the development of noel biomarkers whose status can be assessed through non-invasive to minimally invasive procedures, using currently available technologies. We propose that quantitating the proposition of mutated cancer alleles (cells) in a population of somatic cells, and measuring (with sufficient statistical power) the degree of diversity at specific gene loci, will accurately reflect the risk of cancer and is likely to emerge as a biomarker that can be validated prospectively and applied widely. We refer to this analysis as Mutational Load Distribution Analysis (MLDA). Surrogate tissue samples containing a sufficiently small number of cells, will enable us to perform MLDA analysis during the preneoplastic stages of tumor development. We will use technologies that lend themselves well to quantitative analysis.

Sandra L. Dabora¹, Sergiusz Jozwiak², David Neal Franz³, Penelope S. Roberts¹, Andres Nieto¹, Joon Chung¹), Yew-Sing Choy1,⁴, Mary Pat Reeve¹, Elizabeth Thiele⁴, John C. Egelhoff³, Jolanta Kasprzyk-Obara^x2, Dorota Domanska-Pakiela², and David J. Kwiatkowski¹

¹Brigham and Women’s Hospital, Boston, MA, ²Children's Memorial Hospital, Warsaw, Poland, ³Children's Hospital Medical Center, Cincinnati, OH, and ⁴Children's Hospital, Boston, MA

Tuberous sclerosis (TSC) is a multisystem hamartoma syndrome, caused by mutations in either of two tumor suppressor genes, TSC1 and TSC2. We have performed comprehensive mutation analysis in 224 index TSC patients and correlated mutation findings with clinical features. DHPLC, long-range PCR and quantitative PCR were used for mutation detection. Mutations were identified in 186/224 (83%) of cases, comprising 138 small TSC2 mutations, 20 large TSC2 mutations, and 28 small TSC1 mutations. A standardized clinical assessment instrument covering 16 TSC manifestations was used. Sporadic patients with TSC1 mutations had on average milder disease compared with patients with TSC2 mutations despite being of similar age. They had a lower frequency of seizures and moderate-severe mental retardation, fewer subependymal nodules and cortical tubers, less severe kidney involvement, no retinal hamartomas, and less severe facial angiofibroma. Although there was overlap in the spectrum of many clinical features of patients with TSC1 versus TSC2 mutations, some features (grade 2-4 kidney cysts or angiomyolipomas, forhead plaques, retinal hamartomas, and liver angiomyolipomas) were very rare or not seen at all in TSC1 patients. Thus both germline and somatic mutations appear less common in TSC1 than in TSC2.

Norman J. Dovichi, Shen Hu, Amy Dambrowitz, Le Zhang, David Michels

Department of Chemistry, University of Washington, Seattle, WA

The objectives of this proposal are to generate two-dimensional protein map from single cancer cell, to correlate the map with the phase of the cell in the cell cycle, and to multiplex the instrument to analyze several cells in parallel.

Our methods focus on the development of capillary electrophoresis instrumentation with ultrasensitive laser-induced fluorescence detection.

We have achieved several results over the past two years. We have generated the first protein map of a single cell. This map was based on 1-dimensional free solution electrophoresis of an HT29 adenocarcinoma cell. Since that work, we have also generated single cell protein maps based on 1-dimensional free solution electrophoresis of a single-cell C. elegans embryo and single E. coli cells. In addition to the free solution electrophoresis experiments, we have generated single cell proteome maps based on 1-dimensional capillary gel electrophoresis of HT29 cells. We have modified our instrument to determine the phase of the cell in the cell cycle before analysis, and we have correlated cell cycle with capillary gel electrophoresis protein maps. Last, we have developed a prototype 2-dimensional capillary electrophoresis instrument for fully automated protein analysis.

Our plans for the last year of this grant are to incorporate our two-dimensional electrophoresis technology into the single cell analytical instrument. We will also develop a multiple capillary system to analyze several cells simultaneously.

Daniel L. Farkas ^1,2, Elliot Wachman ², and Stanley Shackney³

¹Univ. of Pittsburgh, ²Carnegie Mellon University , and ³Allegheny General Hospital, Pittsburgh PA Department of Biotechnology, the Biophysics laboratory, and Pasteur Institut, Paris, France

Objective: Develop the capability to study the accumulation of multiple abnormalities per cell at the molecular level, for improved cancer diagnosis and individualized treatment.

Methods: By focusing our proprietary technologies towards the program’s goals we will (a) build the next-generation imaging cytometry instrument, able to bypass previous limitations; (b) concentrate on the imaging of 5-10 molecular species simultaneously within the same cancer cell, with a new multispectral imaging instrument incorporating our acousto-optic tunable filters (AOTFs) and their unique simultaneous spatio-temporal and spectral capabilities. Geared towards the imaging of human solid tumor-derived cells, with sub-cellular resolution, this will be a highly flexible, computer-controlled instrument combining the best features of a research microscope, a spectrometer and an imaging cytometer. The innovation consists in bringing versatile tunability to multi-parameter imaging cytometry, to increase the number of entities resolved per cell and their quantitation far beyond what is possible today, for breast and lung cancer research. The research being pursued concerns the mechanisms underlying the control of DNA replication and genome stability with particular emphasis on the mechanisms and consequences of oncogene amplification. We have developed a technological platform for the genomic study of DNA replication and genetic alterations. This technology involves a method called molecular combing, which is used to straighten and align molecules of genomic DNA on a solid surface.

Results: We concentrate on the improvement of digitally controlled spectral devices using AOTF technology, and integrate them on both the excitation and emission sides of a research microscope. The emphasis is on fluorescence, due to its very high specificity for labeling intracellular features, and we aim to reproducibly resolve probes with highly similar but non-identical spectral signatures. Moreover, background subtraction will be based on spectral features, yielding a more elegant way of disposing of unwanted contributions arising from autofluorescence or the presence of unconjugated dye. The system compares very well in performance with all alternative technologies, and the difference between multi-wavelength and spectral imaging goes beyond that pertaining to the number of image acquisition wavelengths utilized. Spectral imaging is differentiating between various contributions by their global spectral signature, thus being a more quantitative, and sophisticated method. Multispectral imaging is, in our definition, spectral imaging with added versatility: both excitation and emission wavelengths could be spectrally selected, scanning could be done with either or both, including a tandem scanning, where excitation and emission are varied simultaneously, with a fixed, but selectable wavelength gap between them. We will demonstrate the unique discrimination thus achieved by imaging four very closely spaced dyes under the same filter set. Additionally, in multispectral imaging one can vary critical experimental parameters such as exposure times at various wavelengths, very helpful when trying to equalize signal-to-noise in datasets by longer exposure at less sensitive wavelengths.

Plans: We aim to (1) build a prototype instrument with the desired new functionality; (2) implement a second, more user-friendly workstation in our cancer research laboratories; (3) test the latter instrument in experiments focusing on molecular-level prognostic factors for tumor progression within single cells of individuals’ tumors; (4) elucidate critical sequences of genetic evolutionary changes in solid tumors that are responsible for increasing cancer aggressiveness; (5) identify the steps in the sequence that are most closely associated with cellular acquisition of the capacity to metastasize; (6) develop a productized, customizable version of the instrument embodying the new technology, multispectral imaging, ready for use on an array of problems by other researchers; and (7) develop a practical overall approach for the timely performance of relevant measurements on individual tumors, analysis of the data, and characterization of each tumor with respect to the degree of its advancement along its particular genetic evolutionary pathway, so that this information can be used for prognosis and adjuvant treatment planning. The ultimate clinical challenge is the elimination of false negatives and false positives in diagnostics, leading to individually optimized treatment and very significant savings. Overall, our focus, innovation and expected competitive advantage will consist in bringing together the specificity of fluorescence with the relevance and diagnostic importance of measuring multiple labels in the same cell, and with the ability to do so for 5-10 fluorophores, using the new technology of AOTF multispectral imaging.

David A. Fishman

Northwestern University

Epithelial ovarian cancer (EOC) is the fourth leading cause of death in American women due to our present ability to detect only late (III/IV) stage disease. Currently 75% of all EOC patients are diagnosed with late stage disease with only a 12-15% 5-year survival despite aggressive cytoreductive surgery and chemotherapy. However, 90% of women diagnosed with early disease (Stage I) are alive 5 years after less morbid surgery and chemotherapy. The National Ovarian Cancer Early Detection Program was specifically established to clinically apply our understanding of the biochemical, genetic and molecular basis of ovarian carcinogenesis, invasion and metastasis to address the problem of early detection of epithelial ovarian cancer. This research consortium includes over 100 clinicians and scientists worldwide. The enhanced understanding of ovarian cancer biology has led to the identification and detection of specific genetic, molecular and serum biomarkers in women with ovarian cancer that may have clinical utility in the evaluation of women deemed at increased risk for the development of this disease. Increased risk is assigned to those women with either a personal history of breast cancer (4X increase), a family history of affected first-degree relatives (2-7x increase), membership within a recognized inherited malignancy syndrome (40-60% increase), or the presence of an inherited BRCA mutation (16-100%). The newly developed Ovarian Pap test provides cytological samples for pathological examination as well as molecular, genetic, and biochemical analysis.

The biochemical and molecular analysis of tissue and tumor samples for changes in patterns of gene expression of a select subgroup of growth regulatory molecules that already have been implicated in EOC growth and tumor progression is under investigation. One experimental approach used is the high throughput molecular genetic analysis of tissue and tumor samples using state-of-the-art methodologies. As our studies progress, we intend to correlate the results of these high throughput studies with our expression and functional analyses, thereby maximizing the clinical application of this new information. The metastatic process of cellular adhesion, migration, extracellular matrix degradation, invasion into host parenchyma, proliferation, and neovascularization are influenced by numerous regulatory molecules, such as epidermal growth factor (EGF) and receptors (EGF-R/ErbB isoforms such as p110), urinary-type plasminogen activator (uPA) and receptor (uPAR), matrix metalloproteinases (MMP), and lysophospholipids (such as LPA, LPC). Levels of lysophosphatidic acid (LPA) are elevated in the plasma of patients with ovarian carcinoma including 90% of patients with stage I disease, suggesting that LPA may promote early events in ovarian carcinoma dissemination. Activation and cell surface expression (yet not gene, RNA, or protein expression) of MMPs are also upregulated in malignant ovarian epithelium, and we have reported a direct role of MMPs in intraperitoneal invasion and metastasis. Using three-dimensional type I collagen cultures or immobilized a1 integrin subunit-specific antibodies, we previously demonstrated that a1 integrin clustering promotes activation of proMMP-2 and processing of membrane type 1 (MT1-)-MMP in ovarian cancer cells. LPA increased cellular membrane fluidity and adhesion to type I collagen and a1 integrin expression with significant upregulation of MMP-dependent proMMP-2 activation, leading to enhanced pericellular MMP activity.

As a result of increased MMP activity, haptotactic and chemotactic motility, in vitro wound closure, and invasion of a synthetic basement membrane were enhanced. These data indicate that LPA contributes to metastatic dissemination of ovarian cancer cells via upregulation of MMP activity and subsequent downstream changes in MMP-dependent migratory and invasive behavior. It is our hope that the ability to simultaneously evaluate a single patient for multiple biomarkers, all of which have been demonstrated to be significant in ovarian carcinogenesis and metastases, should translate into a means for identifying early stage disease.

Paolo Fortina¹, Taku Sakazume¹, David Graves², Marty Johnson³, Penny Dong³, Kathleen Delgrosso⁴, and Saul Surrey⁴

¹ Department of Pediatrics, The Children's Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, PA, ² Department of Chemical Engineering, University of Pennsylvania School of Engineering and Applied Science, Philadelphia, PA, ³Applied Biosystems, Foster City, CA, and ⁴ Department of Medicine, Cardeza Foundation for Hematological Research, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA

Development of cost-effective, high-throughput genotyping methods will facilitate molecular analysis of normal and diseased states. This initiative focuses on detection of SNPs and point mutations in cancer. Milestones for phase 1 are as follows: 1) Development of cost effective 4-color array bound single nucleotide primer extension (SNE) with signal enhancement, including algorithms for deconvolution of spectral overlap; 2) Scale-up for simultaneous detection of 30-50 interrogated sites and technology validation compared to other methods; and, 3) Use of dendritic surfaces or other methods to reduce costs.

We recently reported a 2-color array-based SNE assay for mutation detection (Eur. J. Hum Genet. 8: 884-894, 2000), and are working on a 4-color assay which will require spectral deconvolution due to dye emission overlaps. A deconvolution algorithm was developed by first ascertaining whether dyes in solution spectrally behaved like array-bound oligo-extended dyes after SNE. Spectral properties were compared after excitation at 488 nm for each of the 4 separate Big Dyes (ABI/PE) in solution, as well as following single color array-based SNE. We found similar normalized spectral emissions comparing the 2 different approaches, but only when the array was scanned in a hydrated state. We next investigated whether there was any quenching when the 6 possible 2-color dye combinations were mixed. We tested this by doing all 6 possible 2-color array-bound extensions so that both dyes were incorporated at the same register. Our results showed minimal quenching; and, therefore deconvolution software was developed, and is being used for assessing spectral overlap for multicolor SNE on arrays.

We next investigated use of polyamidoamine starburst dendrimers as spacer molecules that can be attached to glass, to increase surface density of oligonucleotides on arrays and to enhance SNE signals. Surface coverage was varied using different generations of dendrimers whose size and number of reactive groups were directly related. Adsorption of PCR products indicated optimal dendrimer concentration versus generation utilizing this surface chemistry. We are now applying use of these surfaces in array-bound multicolor SNE reactions, and are testing whether spacers and amino-modification can be eliminated when using various slide surfaces in an effort to reduce assay cost.

We are also testing a new ASO DNA chip which allows melting temperatures at each array register to be independently controlled. Preliminary results with a prototype thermal gradient chip indicate optimum hybridization conditions can be defined for each probe/target reaction on the array.

Aims for phase 2 include: 1) Application of SNE compared to other genotyping strategies to type SNPs within and flanking the minimal deleted region in neuroblastoma patients; and, 2) Identification of DNA changes in the mutation cluster region of the APC gene in patients with colorectal cancer.

Development and validation of array-bound approaches for monitoring DNA changes in cancer should facilitate high throughput, parallel processing of samples for critical human cancer genes. In addition, it will help to rapidly define the molecular basis of specific malignancies and eventually provide a foundation for rational molecular assessment of therapeutic approaches for treating these diseases.

Jacques R. Fresco¹, Nina Dolinnaya¹, Olga Amosova¹, Alex Weis², and Tamas Bakos²

¹Department of Molecular Biology, Princeton University, Princeton, N.J., ²Lipitek International, San Antonio, TX

Gene amplification, substitution and deletion mutations are associated with the molecular pathology of various malignancies, including breast, cervical and gastric cancers, as well as colon and lung cancers and tumors of the nervous systems. Multidrug resistance of cancer cells is also associated with gene amplification. Amplification of erbB-2 (HER-2/neu) and N-myc genes is particularly correlated with poor prognosis in breast and cervical cancers and neuroblastoma, respectively. Methods that could simply and reliably detect such aberrations and quantitate them can therefore be of great value for diagnosis, for following the efficacy of treatment, and for reliable prognosis. Currently, detection of point-mutational events represents a major experimental effort. In the case of gene amplification as well, the methods are arduous, require relatively large samples, and they are of variable reliability. This project has a two-fold aim. One is to apply the methodology of TISH (third strand in situ hybridization of fluorescent probes via triplex formation, which avoids the need for DNA denaturation) for the cytogenetic quantitative analysis of these aberrations. TISH has three major advantages: greater sensitivity and quantitative reliability, and importantly, greater sequence specificity. The second parallel aim is to develop suitably intense and non-quenching fluorescent TISH probes. For this purpose, a major effort will be mounted to develop dendritic nuclei with fluors attached by rigid linkers that prohibit their interaction, therefore preventing the quenching of fluorescence. In this way, it is hoped to enhance the sensitivity of fluorescent probe detection by 1-2 orders of magnitude, thereby assuring that TISH probes can be reliably used for analyzing amplified genes and ultimately for detection of mutations in single-copy genes in situ .

In the first year of this investigation, one aim is to develop probes for the HER-2/neu gene. Nine different deoxynucleotide probes have been designed for the homopyrimidine targets present in the gene. Three of these have?9 homopurine already been synthesized and tested.

Simultaneously, an amplified fluor with three fluorescent moieties linked to a rigid adamantane core has been prepared, and efforts are underway to synthesize a more amplified adamantane dendrite with 9 fluoresceins. Moreover, alternative means of linking the fluors to the oligonucleotide probe are being explored. One involves use of probes with a biotin terminus and fluor-labeled avidin. The other makes use of a probe with a reactive terminus to which we plan to link an activated fluor directly once the probe strand is already bound to the target.

Once these goals are met, work will proceed to cytogenetic comparison of the degree of amplification of the Her2/neu gene in normal and malignant tissue culture cell lines and then in fixed pathological tissues.

Xiang-Dong Fu¹, Joanne M. Yeakley¹, Jian-Bing Fan², Mark Chee², David Tarin³, Michael Gribskov⁴, and Michael Zhang⁵

¹Department of Cellular and Molecular Medicine, University of California, San Diego, ²Illumina, Inc., San Diego,³UCSD Cancer Center, ⁴San Diego Supercomputer Center, and ⁵Cold Spring Harbor Laboratory

Recent completion of the human genome project has set the stage for understanding network regulation of gene expression in biology. One of the major surprises is the large number of mRNA isoforms generated by alternative splicing (60% of human genes express more than one transcript and this percentage is likely underestimated), which undoubtedly contributes to molecular diversity in complex biological pathways. In our battle against cancer, variations in mRNA isoforms may underlie many previously unrecognized mechanisms for cellular transformation, and therefore may prove valuable for cancer diagnosis and characterization of potential therapeutic targets.

Our project aims at developing a genomic approach to alternative splicing because current techniques for assaying alternative splicing rely on low throughput methods that are not suitable for genome-wide analysis. The technology utilizes the addressable zip-code strategy on fiber optic-based microarrays developed at Illumina. Briefly, DNA oligos are used to cover specific splice junction sequences and each isoform-sepcific oligo is linked to a unique zip-code sequence. The oligos are first hybridized to total RNA and those detecting specific alternative splicing events are selectively amplified by PCR. Individual splicing events are then sorted and quantified on a universal Zipcode array. Using this novel microarray technology, we can detect specific alternative splicing events with unprecedented specificity and sensitivity, reaching to the single cell level in certain cases. Furthermore, semi-quantitative data are obtained from a panel of experimental cell lines and 90% of data points match the results obtained by conventional RT-PCR. Although we have been focusing on technology development in the initial R21 phase, our research has already led to the discovery of a number of striking cell-specific alternative splicing events in our model systems.

In the future, we plan to refine the assay to determine optimal experimental conditions for large scale application of the technology. We are interested in coupling our technology with sample preparation methods, such as immunofractionation of cells and laser capture microscopy. This will allow us to analyze alternative splicing in cell populations associated with distinct cancer stages. In particular, we plan to examine several hundred prostate cancer samples available at the UCSD Cancer Center to explore the utility of variations in alternative splicing for cancer classification. Parallel to our experimental efforts, our collaborators on the team will develop computational tools to aid database construction and analysis.

Suzanne A. W. Fuqua

Baylor College of Medicine

The Baylor Breast SPORE Program has permitted the rapid translation and development of new basic research findings into clinical practice. There are five major projects in the SPORE program. These include: Project 1 (Mechanisms of Tamoxifen Resistance), Project 2 (Heat Shock Proteins and the Pathogenesis of Breast Cancer), Project 3 (Identification of Surrogate Markers in a Tamoxifen-Prevention Trial), Project 4 (Molecular Genetics of Pre-Malignant Breast Disease) and Project 5 (Liposomal Gene Therapy). The SPORE also has two core resources (National Tissue Resource, and the Family Registry Cores). The National Tissue Resource has grown to more than 100,000 tumors. Last year, more than 2,000 specimens were distributed to thirteen investigators at various institutions. The Familial Breast Cancer Registry is an important resource for studies of new breast cancer genes and for identifying patients for prevention trials.

The five research projects have made excellent progress toward our translational objectives. In Project 1 we identified several new estrogen receptor interacting proteins via the yeast two-hybrid screen, and we are characterizing these proteins to see if they can explain tamoxifen-stimulated growth. We also found FKHR as a receptor-interacting protein that has bi-functional activity, inhibiting steroid receptors but stimulating thyroid and retinoic acid receptors. Most interesting, FKHR is found on a locus on chromosome 13 that displays a high frequency of LOH, suggesting the possibility that it could function as a tumor susceptibility gene, with loss of its function resulting in over-activity of the estrogen receptor. In Project 2 we have determined that overexpression of hsp27 confers resistance to doxorubicin and that the key underlying mechanism for resistance is hsp27’s ability to inhibit drug-induced apoptosis, and thus to increase the survival of breast cancer cells. Utilizing microarray technologies, we have identified several novel targets of drug resistance, and we hypothesize that hsp27 is a key signaling intermediate.

In Project 3 we have collected tissue samples from a tamoxifen prevention study and they are a unique resource to study the biological mechanisms of how tamoxifen reduces breast cancer in women with premalignant lesions. We are assessing by IHC and microarray for changes in biomarkers between first and second biopsies and between controls (placebo) and tamoxifen-treated patients. In Project 4 we have shown that in hyperplasia from non-cancerous breasts, Loss Of Heterozygosity (LOH) at any given locus is rare. Hyperplastic lesions however showed at least one LOH, suggesting a complex interaction of tumor suppressor genes and environment in the early development of these lesions. In cancerous breasts, many of the precancerous lesions shared LOH with the synchronous cancers, supporting the idea that these premalignant lesions are in fact precursors of invasive breast cancer. RNA based microarray experiments are underway, as well as DNA array studies in collaboration with other SPORE groups to examine the molecular profiles of these early lesions. In Project 5, we are developing adenoviral vectors for liposome mediated gene transfer. In summary there are a number of projects utilizing powerful molecular analysis technologies applied to translational research objectives.

Xiaolian Gao¹, Erdogan Gulari², and Xiaochuan Zhou³

¹Department of Chemistry, University of Houston, Houston, TX, ²Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, MI, 3Xeotron Cooperation, Houston, TX

Oligonucleotide microarrays are effective decoding and analytical tools for genomic sequences and are useful for an enormously broad range of applications. Therefore, it is highly desirable to have synthesis methods of DNA/RNA chips that are highly flexible in sequence design and also provide high quality and general adoptability. We report herein DNA microarray synthesis based on a flexible biochip technology. Our method simply uses photogenerated acid (PGA) in solution to trigger deprotection of the 5'-OH group in conventional nucleotide phosphoramidite monomers (i.e. PGA gated deprotection), with the rest of the reactions in a synthesis cycle the same as those used for routine synthesis of oligonucleotides. The complete DNA chip synthesis process is accomplished on a regular DNA synthesizer that is coupled with a UV-VIS projection display unit for performing digital photolithography. Using this method, DNA chips containing probes of newly discovered genes can be quickly and easily synthesized at high yields in a conventional laboratorial setting. The PGA gated chemistry is applicable to microarray syntheses of a variety of combinatorial molecules, such as peptides and organic molecules. Our latest progress on RNA chip synthesis will be reported.

R. Balog, G. DeMasellis, K. Luebke, D. Mittelman, E. Ponce De Souza, J. Minna, and H.R. Garner

Center for Biomedical Inventions, University of Texas Southwestern Medical Center, Dallas, TX 75390-8573, and McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, TX 75390-8591

A rapid method for creating custom DNA microarrays is described. Oligonucleotide microarrays are routinely used for resequencing and gene expression studies. In resequencing studies, custom oligonucleotide arrays can be used to discover new single nucleotide polymorphisms, genotype different cancer lines, and aid in determining inherited alleles responsible for familial predisposition toward cancer. Light directed fabrication of oligonucleotide arrays allows for highly parallel synthesis, yielding high feature-density arrays. Ultraviolet light is spatially directed to promote chemical reactions on a solid glass surface. In our approach, a Texas Instruments Digital Light Processor (DLP) is used to direct light, as opposed to traditional photolithographic methods. The DLP consists of ~800,000 digitally controlled microscopic mirrors, which are used to instantaneously create digital masks rather than machining physical photolithographic masks. This technique allows the researcher to quickly customize arrays for their uses and to quickly modify array designs.

John Gerdes, Jeffrey Marmaro, Shannon Beard, and Craig Sampson

Xtrana, Incorporated, and Denver CO Xtrana, Incorporated, Denver CO

Breast carcinomas are heterogeneous in their biological and clinical behavior. Cell populations where molecular markers will be of most relevance frequently occupy less than 5% of the tissue volume of a tumor or biopsy. Laser Capture Microdissection (LCM) is a method for procuring pure cell populations from specific microscopic regions of tissue sections. Although LCM provides a means of selecting malignant cells of similar morphology, it also presents challenges to accurate analysis of cancer markers due to the relatively small numbers of cells. Sensitive methodologies are needed for measuring low copy mRNA expression levels ideally for several different genes all within the same small number of microdissected cells.

Xtrana is developing a sensitive and precise method for the relative measurement of gene expression following solid phase capture of nucleic acid onto our proprietary Xtra BindTM solid phase matrix. Capture occurs in an Xtra Bind coated PCR tube (Xtra Amp) that is used directly for RT PCR. DNase treatment prior to binding insures that only RNA is captured. We have observed that solid phase RT works best using the AMV RT. Commercially available kits containing protocols using AMV including the Roche Titan 1-step and Promega Access RT PCR kits also provide robust results. Initial investigations have utilized the breast cancer T47D cell line. The optimal cell lysis / Xtra Bind binding buffer is LiCl based.

Solid phase capture offers the advantage of rendering the mRNA stable. Bound mRNA has been stored at room temperature and analyzed 8 weeks later. When interfaced with RT PCR, robust amplification is observed by gel electrophoresis, TaqMan, or other standard detection methods. Extraction, amplification and detection can be performed in the same tube from as few as 10 to as many as 104 cells. The RNA is bound irreversibly and will not elute even following rigorous aqueous washes or following multiple PCR cycles. This property can be exploited for repeated or extended RT PCR reactions from bound RNA from the same specimen.

The interface of Xtra Amp captured mRNA with LCM cell nucleic acid extraction and quantitative fluorescence measurement of gene expression should enable extensive analysis of multiple genes from the same few cells. Briefly, we envision capture of LCM cells onto a cap that fits a standard PCR tube. This tube contains cell lysis/Dnase/nucleic acid binding buffer and a nucleic acid binding matrix coating the inside of the bottom of the tube (Xtrana’s Xtra Amp tubes). By simply mixing, the mRNA is bound inside the tube. The solid phase bound nucleic acid is amplified directly in this tube by RT PCR with real time TaqMan detection.

Roger W. Giese, Jianxin Gao, Guodong Li, Gang Shao, Chi-Yu Kao, Olga Shimelis, Changming Yang, Aijian Liu, and Poguang Wang

Bouve College and Barnett Institute,Northeastern University, Boston, MA

Nearly all of the substances that are classified as human carcinogens cause direct or indirect damage to DNA (DNA adducts). Improved measurements of DNA adducts in human samples may contribute to advances in cancer prevention. Our work is intended to help in filling the following gaps in what current assays provide. (1) Screening and identification of unknown DNA adducts in human samples. For trace adduct screening and identification, we are studying cation labeling mass spectrometry. The purpose of the cation labeling is to establish uniform, high sensitivity detection across a diversity of adduct structures. With current mass spectrometry methodology, sensitivity can depend on the adduct. Towards this goal, we have set up a large laser spot, laser desorption Fourier transform mass spectrometer, and have begun to optimize the detection of cationic tags. Reproducible detection of a model cation tag deposited as a 2 mm diameter spot on the probe target has been achieved at the 0.5 fmol level, with signal persistence for a few shots. This result, especially the high reproducibility, advances the performance of laser desorption mass spectrometry for detecting a favorable small molecule by about 100-fold.

Work on attaching this and other cation tags to DNA adducts is in progress. (2) Comprehensive detection of DNA adducts. For comprehensive adduct detection we are studying chemical labeling with a fluorescent dye, based on the use of a dye-imidazole/carbodiimide reaction that specifically labels a phosphomonoester such as the phosphate group of a deoxynucleotide (Lan, Wang, Giese, Rapid Commun. Mass Spectrom. 13 [1999] 1454). A BODIPY-IMI dye was employed in our initial work. Currently, we are studying xanthamide dyes (prepared recently in our laboratory) because of their enhanced chemical and photostability, and cyanine dyes, because their absorption/emission in the near-infrared region escapes background signals. Recently we have detected a model near-infrared dye in a practical way by injecting 0.5 uL containing 0.5 amol of dye into a capillary electrophoresis laser-induced fluorescence (CE-LIF) instrument.

(3) High sensitivity detection of oxidative sugar DNA adducts. The initial analyte of interest in our sugar oxidation work is phosphoglycolate, which can be released from DNA by a repair enzyme. We have set up a method involving use of the electrophore-tag reagent, AMACE1 (Lu and Giese, Anal. Chem., 72 [2000] 1798), which detects one picogram (13 fmol) of glycolate in a procedure that terminates with gas chromatography electron capture mass spectrometry (GC-EC-MS). Current work involves the extension of this method to real samples, and broadening to related analytes based on the ability of AMACE1 to label four functional groups (carboxyl, lactone, aldehyde, ketone) in a single procedure. (4) Simultaneous detection of multiple small DNA adducts such as alkyl and hydroxyalkyl. For the detection of small DNA adducts as a class, we are relying on electrophore labeling GC-EC-MS, and studying N7-hydroxyethylguanine as a representative analyte. Our current methodology detects 10 picograms of this compound, and our next analytical goal is to get closer to the detection limit of 10 femtograms for a diluted standard of the final derivatization product. Detecting a diversity of adducts of this type in a single procedure is our long-term goal. Thus, these emerging CE-LIF and MS techniques potentially will advance the measurement of DNA adducts in human samples.

V. Golovlev¹, C.H. Chen², S. Dai², and K. Matteson³

¹Sci-tec, Inc., Knoxville, TN, ²Oak Ridge National Laboratory, Oak Ridge, TN, and ³University of Tennessee Medical Center, Knoxville, TN

Fast growing applications of microarray in biomedical fields has created strong demand of affordable and reliable methods for preparing and reading DNA micro-arrays. To address this demand, Sci-Tec, Inc. is currently working on development of ultra-sensitive and cost-efficient micro-array system for bio-medical applications. The core of the approach is to detect metal clusters attached by a DNA molecule to the surface of magnetic media (magnetic disk). The spatial location of hybridized probe-target complexes can be determined by monitoring magnetic properties of the surface. The information about location of the bounded DNA then can be used to identify the presence of target DNAs with specific sequence. An important feature of this approach is that the DNA detection is based on the attachment of relatively large tagging particles. Since the detection signal is proportional to the amount of tagging material, the sensitivity of DNA detection can be improved by controlling the size of the particles.

With our approach, magnetic detection can be employed using either magnetic or non-magnetic particles for DNA labeling. To detect the change of magnetic field triggered by the binding reaction on the surface we have developed a reader system based on a floppy drive of a personal computer (PC). An electronic circuitry was modified to acquire high-resolution analog signal from the magnetic head of the drive. The head, controlled by a computer, scans the surface of the disk and produces 2D magnetic map of the array surface.

Resolution, i.e., system’s ability to distinguish large number of closely located spots, is very important issue for high-density micro-arrays. In order to assure our reader can be used with high-density micro-arrays we have tested system’s performance by preparing and reading array with the size of spots from 1500 to 30 um. Reliable detection was achieved down to the size of probe spots of 60 um and separation between two neighboring spots of 100 um. The experimental results show the system has the potential to detect up to 45,000 DNA probes on a single 3.5 diskette.

Currently the work is in progress for optimizing and using the disk array system for gene mutation analysis. Hybridization experiments are pursued using a set of synthetic probe-target oligos designed from the locus HSU62962 of the IL6: Interleukin 6 (7p21). The set of probes will be expanded in the future to focus on the diagnostics of mutations relevant to different cancers.

Jeffrey Griffith and Colleen Fordyce

Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, NM

Telomeres are protein-DNA complexes located at the ends of linear chromosomes that protect them from degradation, recombination and end-to-end fusions. Telomeres are progressively shortened every time a cell divides--ultimately leading to genomic instability and phenotypic variability. Our previously published studies have demonstrated that reduced telomere DNA content in invasive human breast carcinomas is associated with aneuploidy and metastasis (p‹0.002, p‹0.05 respectively) and in prostate adenocarcinoma with death and disease recurrence (p ‹0.0001, p‹0.0001, respectively). However, our initial assay for telomere DNA content lacked the sensitivity needed to analyze biopsy material, required 32P-labelled probes, and took 7-10 days, making it poorly suited to large-scale investigations and clinical settings. The goal of the initial phase of this project was to develop an assay for telomere DNA content that had the sensitivity needed for use with biopsy materials and was suited for large-scale investigations and clinical settings. DNA was purified from archival samples or obtained commercially and quantitated using a commercially available fluorescent reagent, PicoGreen. DNA was denatured, fixed to a positively charged membrane and hybridized with a telomere specific oligonucleotide (5TTAGGG3)4 labeled with fluorescein.

Michael Gruid

H. Lee Moffitt Cancer Center and Research Institute at USF

This group of ten investigators focuses on developing assays to detect preclinical lung cancer proteins and altered DNA in body fluids. They have already identified one potential biomarker, expression of hnRNP A2/B1 in exfoliated airway epithelial cells, which is currently in clinical trials. New markers from collaborating laboratories at Moffitt are being developed, refined and compared on common paired tumor/normal specimens from the Moffitt core tissue bank. These biomarkers include: a difucosylated ceramide, lacto-N-fucopentose III; markers of the TGF-beta signaling pathway, TGF-beta receptor Type II, SMAD 2, SMAD 4, and SMAD 7; and markers of tumor suppressor genes silenced by promoter methylation and by allelic loss. Technical approaches include Enzyme-linked immunosorbent assays, Western blot analysis, methylation specific PCR, immunostaining, thin Layer chromatography, automated DNA sequencing and laser-scanning immunofluorescence. These panels of assays are being developed as complementary technologies to helical CT detection of pre-clinical lung cancer. Promising biomarker assays from this project will be applied to archived sputum specimens collected during the ongoing Moffitt helical CT lung cancer screening trial "Markers of Transformation in airways Epithelial Cells from a Cohort of Obstructed smokers and Former Smokers". This archive will provide preclinical material with subsequent known cancer outcome for final biomarker case-control assay. The final assay will be conducted on a high-throughput screening platform currently under development by a Collaborative Research and Development Agreement industrial partner. Comparisons of additional biomarkers on these specimens will be facilitated through interactions with the Lung Cancer SPORE programs at Johns Hopkins, University of Colorado and MD Anderson/Texas SW.

Baochuan Guo and Xiyan Sun

Department of Chemistry, Cleveland State University Cleveland, OH 44115

Point mutations are one of the most common genetic changes leading to cancer and therefore the detection of point mutations is essential to cancer research and diagnosis. Solid tumor specimens often contain a significant number of normal cells and the mutant cells present in specimens may be less than 1%. The specimens from other sources may contain an even smaller percentage of mutant alleles. Thus, assays for tumor detection need to be highly sensitive and specific, such that mutated alleles can be readily detected in a large background of wild-type alleles. Moreover, cancers arise from mutations in multiple genes and therefore, a good assay should allow the simultaneous identification of multiple mutated genes.

This primary focus of our proposed research is the development of mass spectrometric based technologies for automated, multiplexed, high-throughput, sensitive, and specific detection of a small population of point mutation tumor cells in a large background of wild-type cells. The technology developed in this work consists of three major steps. First, the clinical DNA samples are amplified using the peptide nucleic analogues (PNA) directed PCR clamping reactions in which mutant DNA are preferentially amplified; second, the PCR amplified DNA fragments are extended through mini-sequencing to generate diagnostic products; and third, diagnostic products are identified using matrix-assisted-laser-desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry and therefore the presence and nature of mutations are determined. Our preliminary results have demonstrated that this approach could identify mutant alleles in the presence of over 1000-fold excess of normal alleles. This preliminary work was performed using both tumor and bronchoalveolar lavage fluid (BAL) specimens from lung cancer patients. Thus, the next logical step is to develop this method and to explore its potential in cancer research and detection. Two experimental goals will be achieved in this project. First, we will prove the feasibility of this new technology to identify various cancer-causing point mutations using both single and multiplexed assays. Second, we will develop the proven assays for the detection of the hotspot point mutations in both k-ras and p53, two of the most important genes related to cancers.

Bassem R. Haddad, Marie Pennanen, Janice D. Rone, Luciane R. Cavalli, Bruce Trock, and Robert B. Dickson

Lombardi Cancer Center, Georgetown University Medical Center

Objectives: Our project involves the development of a new, non-invasive approach for early detection of breast cancer, based on chromosomal analysis of mammary epithelial cells shed into the nipple aspirate fluid (NAF). Methods: The proposed research involves the use of a state-of-the-art molecular cytogenetic technique, comparative genomic hybridization (CGH), to detect chromosomal aberrations in NAF-derived epithelial cells that have been expanded in short term cell culture. This approach circumvents the two major limitations to conducting cytogenetic analysis in NAF-derived cells: (1) the inability to obtain good quality metaphase preparations from NAF cells, and (2) the low cellularity of NAF samples. The DNA isolated from the short term culture with expanded cell number (250-500 cells) is amplified using a universal DNA amplification protocol, degenerate oligonucleotide primed PCR (DOP-PCR), prior to CGH evaluation.

Methods: The proposed research involves the use of a state-of-the-art molecular cytogenetic technique, comparative genomic hybridization (CGH), to detect chromosomal aberrations in NAF-derived epithelial cells that have been expanded in short term cell culture. This approach circumvents the two major limitations to conducting cytogenetic analysis in NAF-derived cells: (1) the inability to obtain good quality metaphase preparations from NAF cells, and (2) the low cellularity of NAF samples. The DNA isolated from the short term culture with expanded cell number (250-500 cells) is amplified using a universal DNA amplification protocol, degenerate oligonucleotide primed PCR (DOP-PCR), prior to CGH evaluation.

Results to date: Here we report on our preliminary findings with the analysis of 50 NAF samples. We will discuss the advantages of this approach and highlight the major hurdles encountered. Our preliminary results clearly support the feasibility of this approach. However, the results also indicate that future development of this method heavily rely on the ability to improve the number of mammary cells available for testing. Efforts towards that end are underway in our laboratory.

Plans for the Future: One very promising, new approach to overcoming low NAF cellularity has recently been designed and tested: Breast Ductal Lavage. It permits the collection of a large number of mammary cells (in the thousands) by introducing a very small catheter to the breast duct and performing a "ductal lavage" with saline. This procedure is now available at our institution. At the present time, all collected fluid is sent for cytologic evaluation. Once the procedure is validated at our institution, we plan to use an aliquot of the fluid to perform our genetic evaluation (CGH). Breast ductal lavage promises to provide a much higher success rate than nipple aspiration.

Dorothee Herlyn, Rajasekharan Somasundaram, and Kapaettu Satyamoorthy

The Wistar Institute, Philadelphia, PA

Our major goal is to develop a novel technology to identify HLA class II tumor antigens as potential vaccines for cancer patients. These vaccines may induce a T helper (Th) response important for the activation of cytolytic T lymphocytes and other effector cells of the innate immune system in patients. To clone HLA class II-dependent Th tumor antigens, tumor cell cDNA libraries are expressed by the phages, followed by library phage presentation to Th cells by antigen-presenting cells and identification of the relevant Th antigen in cytokine release assay. This approach has numerous potential advantages over existing approaches to class II antigen cloning or biochemical peptide isolation. To develop this approach, we have available a unique model system including Th cells against tetanus toxoid (TT) and a cDNA fragment encoding the TT-associated Th epitope. Using this model system during the R21 phase of this study, we have: i. developed phage vector with TT insert; ii. expressed TT in phages; iii. demonstrated that control phages do not act as superantigens in Th cell stimulation assays; iv. shown that anti TT Th cells specifically proliferate after stimulation with TT-phages presented to the Th cells by autologous Epstein Barr virus-transformed B cells; and v. determined that the sensitivity of Th cell stimulation by TT-phages is one TT-phage in fifty irrelevant phages. In future studies (R33) the described technology will be used to clone melanoma and colorectal cancer-associated Th antigens using available Th lines and clones.