Abstracts

American Registry of Pathology, Inc.

2 R33 CA091166-03A1

The first crucial step in cancer management is to assure timely and accurate pathological diagnoses. Formalin fixation and paraffin embedding (FFPE) has been a standard tissue preservation method employed in over 90% cases for clinical histology diagnosis. Though it provides superior morphology and easy long- term storage of clinical specimens, FFPE is time-consuming and does not fully support current molecular analyses. The long-term goal of our research is to apply modern techniques to medical practice and pathological diagnosis to effectively fight cancers and other diseases. Our proposed project is to further develop the ultrasound-accelerated tissue preservation (UTP) technology for multiple tissues and to study the mechanism of ultrasound (US)-facilitated formalin fixation. Our specific aims are: (1) to develop a multi- tissue preservation processor with an optional real-time digital system to monitor and standardize tissue fixation level; (2) to validate the UTP techniques by performing more statistical assessments on histopathology, macromolecule integrity, and their long-term stability; and (3) to conduct mechanism studies to elucidate the effects of formalin fixation with and without US on the formation of cross-linking, enzymatic activity, and protein conformational changes. We have demonstrated that in comparison to conventional FFPE, UTP provides similar preservation in tissue morphology with similar long-term storage stability, improved preservation of protein structure, antigen properties, and mRNA integrity. UTP allows easy general molecular profiling and analyses based on extracts from UTP-fixed tissues. UTP also provides a good opportunity to control and monitor fixation level by adjusting the time and strength of the ultrasound. We hypothesize that US-facilitated formalin fixation will greatly accelerate formaldehyde-induced macromolecule cross-linking in tissues and “freeze” macromolecules and their conformation due to accelerated fixation reactions. Since tissue preservation is still a standard and general requirement before histology diagnosis, the innovation should have great impact in economy and public health.

Pathology and Urology Department, Keck School of Medicine, University of Southern California

1 R21 CA123027-01

Metastasis is probably the most important event for determining outcome in cancer patients. The detection of occult metastases in the bone marrow, while known to be clinically important, has not become routine clinical practice. This is due to the technical difficulties and costs involved in the current methods for their collection and detection. Detection of circulating tumor cells (CTC) in the blood is less sensitive than in bone marrow and suffers from the same technical barriers as the detection of tumor cells in the bone marrow, but offers the distinct advantage of being less invasive and better for patient compliance. Therefore, sensitive detection of earliest metastatic spread of tumor in a minimally invasive and user-friendly manner will have a great impact on the clinical management of cancer patients. The currently available methodologies for CTC capture and identification face significant barriers including multiple procedural steps, substantial human intervention, extremely high cost, and importantly, lack of reliability and standardization for the detection methods. We have demonstrated the potential for sized-based tumor cell capture using a parylene-based micropore membrane. We propose to develop this into a microchip device for processing blood, and eventually bone marrow and other fluids like pleural effusions or ascites. This microdevice, coupled with microfluidics, has the potential to revolutionize the approach to tumor cell capture and identification. Further, we propose to develop methods for on-chip characterization of the captured cells. First, in R21 Phase, we will develop and optimize the capture device using a model system to isolate and molecularly characterize cultured cancer cells admixed in blood, followed by a pilot study to examine blood from 45 actual cancer patients with metastatic disease for breast, prostate or bladder cancer. In R33 Phase, we will extend the application of microdevice to assess about 310 patient samples from the same three malignancies, and we will also assess the molecular characteristics of the CTC using the Quantum Dots to understand the biological features of these otherwise rare cells (such as existence of putative stem cell sub-population which may be more malignant). At completion, studies in this project will develop a cost effective on-chip system for capture, identification, and characterization of CTC, easily usable in the clinical setting.

Hematology Department of Clinical Pathology, Cleveland Clinic Foundation

1 R21 CA123006-01

Protein phosphorylation is an important mechanism of regulating protein function and activity that depends on a competing system of kinases and phosphatases. It is a dynamic processes that is altered in many disease states. For example, activated tyrosine kinases are central to the pathogenesis of chronic myelogenous leukemia (BCR-ABL1) and gastrointestinal stromal tumors(KIT). Detection of phosphoproteins (PPs) in fixed tissues by in situ immunohistologic methods may have diagnostic, prognostic, and therapeutic implications for cancer patients. Initial studies have shown that PPs are quite labile. Little is known regarding methods to preserve phosphorylation status in tissues. The purpose of this application is to develop optimal tissue handling methods that will be suitable for detection of PPs in fixed tissues, keeping in mid practical limitations in the clinical setting. To this end we intend to (1) develop a quantitative immunofluorescence (IF) method using quantum dots to quantitate PP status in fixed cell blocks; (2) characterize optimal fixation conditions (time, fixative, requirement of phosphatase inhibitors) in murine xenografts of human cell lines as a controlled model of available control material that is assayed both by quantitative Western blot and IF; and (3) show proof of principle of in a murine model of BCR-ABL1 containing cell line xenograft treated with imatinib mesylate (IM) and bone marrow biopsies from patients suspected of chronic myeloproliferative disorder harboring the JAK2 V617F mutation. Phospho-STATS is known to be increased in both these systems. Decreased expression by phospho-STAT5 immunostaining in IM-treated xenografts and increased expression in JAC2 V617F+ bone marrow megakaryoctyes is expected in optimally handled tissues. This application has relevance in the diagnosis, prognosis, and therapy of malignancies and other diseases that have altered PP levels as part of their pathogenic pathways. It will define tissue handling conditions that adequately preserve in vivo PP status for subsequent diagnostic and prognostic testing. Furthermore, control material with defined relative expression levels of many PPs will result from this application and allow laboratories to assess performance of their individual assays.

Cancer Genomics Laboratory, Mitchell Cancer Institute, University of South Alabama

1 R21 CA114043-01

Transcriptional regulation has been the main focus for gene regulation in the past. However, a tremendous amount of evidence from recent studies also indicates that translational regulation plays a key role during development, cell cycle control, and mechanisms related to acute drug resistance. Gene expression analysis on actively translated mRNA transcripts provides a unique approach to study post transcriptional regulation. Previous studies have relied on a traditional sucrose gradient ultracentrifugation procedure to isolate polysome complexes and requires a large amount of cells (up to 500 million cells). As a result, this still remains a major bottleneck for the investigation of post transcriptional regulation with limited quantities of clinical samples. Therefore, there is an urgent need to develop a novel approach to isolate actively translated polysomes from a small number of cells (10 to 500 cells). The new approach will allow us to systematically study potential translational regulation with limited clinical samples. It has been shown that actively translated mRNAs are associated with multiple units of ribosomes and the newly synthesized polypeptides are closely associated with molecular chaperones such as hsp73. These molecular chaperones assist in the proper folding of nascent polypeptides into higher ordered structures. These chaperones will provide the anchor to separate actively translated mRNAs associated with polysomes from free mRNAs. Affinity antibody capture beads will be developed to capture hsp73 chaperones associated with the polysome complexes so that all polysomes can be separated from monosomes and free mRNAs. The isolated actively translated mRNAs will be used for high throughput gene expression analysis. The specific aims of the proposed project are: (1) Develop antibody conjugated affinity capture magnetic beads and conditions to capture actively translated mRNAs associated with the polysome complex from a small number of cells. (2) Validate the antibody affinity capture approach for polysome isolation by comparing with traditional polysome isolation protocols via quantitative RT-PCR gene expression analysis. (3) Identify potential translationally regulated genes that are responsible for determining chemosensitivity during 5- fluorouracil (5-FU) treatment from human colon cancer samples.

Department of Chemistry, University of Virginia

1 R33 CA116115-01A1

With an ever-increasing interest in the molecular typing of cells from histologic tissue sections, the ability rapidly and efficiently extract DNA from the selected cells will be of paramount importance. The overall goal of this project is to develop a microchip-based sample preparation method for high efficiency, low-cost extraction of DNA from tissue samples - this microdevice will easily accommodate blood or other cells sources. The microdevices will be created using state-of-the-art microfabrication techniques coupled with fluidically controlled on-chip cell lysis and solid phase extraction chemistries. This project couples the industrial capabilities of HT Micro for facile fabrication of complex, high surface area microstructures, with surface modification chemistries developed at the University of Virginia that enable efficient and high capacity DNA extraction. The microdevices will be tested using a variety of sample varying in type and quality, and extraction efficiency will be determined using real-time PCR. As a demonstration of integration with current laser-capture microdissection instruments, the microdevice will be fabricated to directly accept the cap from the Arcturus Pixcell IIe which will contain the selected cells bound to an ethylene vinyl acetate polymer membrane on its bottom surface. The tissue samples will be collected and laser microdissected by our surgical pathology collaborator here at UVa - samples of normal and malignant cells will be analyzed. The final device will offer the rapid analysis, high extraction efficiency, and high-throughput advantages of microdevices and, in addition, is expected to offer higher capacity, and lower cost-per-device than current conventional or microchip techniques.

American Registry of Pathology, Inc.

1 R21 CA118477-01

High-throughput molecular biologic and proteomic methods provide several promising approaches for relating genetic changes, such as mutation or altered gene expression, to metastasis, to treatment outcomes, and to survival. In cancers where the interval between initial diagnosis and treatment and the appearance of metastases is long, clinical correlations would be more readily obtained if formalin-fixed paraffin-embedded (FFPE) tissues could be used instead of fresh or frozen specimens. Large scale multiplex techniques, such as serial analysis of gene expression (SAGE), and gene chip methods yield experimental results that are somewhat different for FFPE tissue and unfixed tissue. The long term goal of our research program is to use high-throughput molecular biologic screening methods to identify the molecular and genetic basis of cancer origins and behavior. The objective of this application is to identify the formaldehyde-induced chemical modifications that occur to nucleic acids during histologic tissue processing and to develop methods to reverse these modifications. Our central hypothesis is that formaldehyde adducts and cross-links formed during tissue processing can be sequentially reversed by a series of heating and dialysis steps, carried out under appropriate salvation conditions. We formulated this hypothesis on the basis of preliminary data which show that the reversal of formaldehyde-induced chemical changes in proteins and nucleic acids is relatively facile in aqueous solutions, but less so following dehydration in the presence of organic solvents. The rationale for these studies is that their successful completion will provide a foundation for applying high-throughput screening methods to FFPE tissues. This will lead to improved practical interventions for the diagnosis, evaluation, treatment, and prevention of cancer and facilitate the development of therapeutic agents. Our studies are innovative in that we have pioneered a novel model system (tissue surrogates) ideally suited to identify the formaldehyde-induced modifications to proteins and nucleic acids that occur during tissue processing. At the completion of this project it is our expectation to have established a comprehensive understanding of the formaldehyde-induced chemical modifications to mRNA that occur during tissue histology, and methods for optimally reversing these modifications. This knowledge should result in an ability to carry out genomic analysis on FFPE tissue, significantly expanding our capability to conduct genomic research and opening important new areas to practical investigation.

Biochemistry Department, Vanderbilt University

1 R33 CA116123-01

Direct tissue profiling and imaging mass spectrometry (MS) provides a molecular assessment of numerous expressed proteins within a tissue sample. MALDI MS (matrix-assisted laser desorption ionization) analysis of thin tissue sections results in the visualization of 500- 1000 individual protein signals in the molecular weight range from 2000 to over 200,000. These signals directly correlate with protein distribution within a specific region of the tissue sample. The systematic investigation of the section allows the construction of ion density maps, or specific molecular images, for virtually every signal detected in the analysis. Ultimately, hundreds of images, each at a specific molecular weight, may be obtained. To date, profiling and imaging MS has been applied to multiple diseased tissues, including human non-small cell lung tumors, gliomas, and breast tumors. Interrogation of the resulting complex MS data sets using modern biocomputational tools has resulted in identification of both disease-state and patient-prognosis specific protein patterns. These studies suggest that such proteomic information will become more and more important in assessing disease progression, prognosis and drug efficacy. Molecular histology has been known for some time and its value clear in the field of pathology. Imaging MS brings a new dimension of molecular information that specifically focuses on the disease phenotype. One important aspect of the MALDI MS imaging technology is sample preparation and processing. We propose here to further optimize the existing methodologies to maximize the information recovered from the MS analysis of fresh frozen sections, and develop and validate new approaches to investigate solvent fixed biopsies. Next, we propose to further develop and optimize methodologies to measure pharmaceutical compounds by MS in tissue sections. We also propose to further develop and validate protocols for the molecular analysis by MS of cancer cells in fine needle aspirates. Finally, we propose to automate some key aspects of these methodologies.

Division of Urology, Department of Surgery, Beth Israel Deaconess Medical Center

1 R21 CA112220-01

Molecular profiling has emerged as an important strategy for identifying marker “signatures” associated with the biological changes that characterize specific cancers. To realize the full potential of the wealth of new biomarker information, it is essential to develop strategies for profiling human tissue and tumor specimens that are workable in a clinical setting. Clinical specimens are heterogeneous, and tissue heterogeneity is one of the major sources of complexity that must be addressed in the application of molecular profiling to the analysis of human cancers. We have developed a set of novel “tissue print” techniques that allow us to profile the molecular markers over extended areas of human tissue and tumor samples without damaging the specimen. We first applied our new tissue print techniques in the profiling of protein markers associated with capsular invasion in radical prostatectomy specimens. More recently, we have discovered that, during tissue print collection, we can peel a layer of cells off of the specimen and that this process does not cause detectable tissue damage (as determined by surgical pathologists), and thus does not interfere with routine clinical surgical pathology. We have also shown that the cells collected in the tissue print “micropeel” are adequate for PCR and quantitative rt-PCR analysis, allowing us to score multiple molecular markers and assemble the results in “tiling patterns” corresponding to the specimen surface. In the project outlined in this proposal, we will work closely with the research and development team at Qiagen Inc. to optimize the yield of mRNA and DNA from our tissue print micropeels collected from human prostate and breast tissue/tumors specimens. We will then develop a proof-of-principle pilot application of the tissue-print micropeel sampling technique for prostate needle biopsies, one of the classes of specimens that must be conserved intact for clinical diagnosis. Our long term goal is to utilize tissue print techniques in the clinical setting to simplify the process of obtaining an adequate representation of human cancers in biopsies and surgical specimens, and to develop protocols for this tissue sampling platform to support both proteomic analysis and PCR-based DNA and mRNA profiling techniques. In addition to facilitating basic and translational research, the tissue-printing platform can also be utilized as a tool for dedicated clinical applications, to provide “molecular sections” of extended areas of the specimen when the marker profile is itself of potential diagnostic importance.

Department of Biological Chemistry and Molecular Pharmacology, Harvard University Medical School

1 R21 CA114167-01

Biorepositories are a valuable resource in translational research for cancer and there are many such repositories in both academic and industrial settings. While these repositories are valuable, they are not immune from cost constraints and the approach to storing biological specimens (e.g., serum and plasma) involves a fundamental cost tradeoff between storing the samples in a larger number of vials each with volumes (100 fl to 400 fl) suitable for assaying or storing the samples in larger volumes (2 ml or 4 ml) to save freezer space. The first approach avoids downstream aliquotting and has only one freeze/thaw cycle but requires labor to aliquot the fresh sample and is volumetrically inefficient. The second approach requires aliquotting when the samples are requested and as a result the sample experiences a freeze/thaw cycle when it is processed for a study but is volumetrically efficient. A hybrid approach is also pursued where fresh samples are initially stored in a volumetrically efficient format until they are requested for a study and then returned to storage in the volumetrically inefficient format to avoid subsequent freeze/thaw cycles. This project will develop an automated instrument that will offer a new approach, combining the volumetrically efficient storage with the single freeze/thaw cycle. This instrument will extract aliquots from frozen samples without thawing the samples. Not only will this approach reduce the cost of operating these repositories while eliminating the second freeze/thaw cycle associated with the hybrid approach, but because it is automated it will increase the throughput in processing samples, reducing the 6 1/2 weeks to one week for a 1000 sample study, which is typical for the Nurses’ Health Study. This team has demonstrated the ability to: (1) maintain the sample at -70 °C during processing; (2) extract aliquots from normal saline frozen at -80 oC, and (3) manage frost buildup during the procedure. The R21 project and the milestones have been developed to address the key technical risks associated with this instrument and the R33 project will deliver a prototype instrument that is suitable for use by the Nurses’ Health Study to process samples for their collaborators. The design at this point will be documented well enough to enable its production in our lab in small quantities for use in other biorepositories.

Radiation Oncology Department, Dana-Farber Cancer Institute

1 R21 CA111994-01A1

Genomic, epigenetic and gene expression analysis from archived formalin-fixed paraffin embedded (FFPE) tissue samples with known clinical outcomes provides a unique opportunity for extraction of genetic information leading to improved cancer diagnosis, prognosis and therapy. However, extensive genotyping or microarray profiling on homogeneous cell populations within these samples often requires whole genome/mRNA amplification prior to screening. Major hurdles to this process are the introduction of amplification bias and the inhibitory effects of formalin fixation on DNA/RNA amplification. We have developed (RCA-RCA), a novel method based on isothermal rolling-circle amplification, that overcomes the limitations and promises to provide the needed link between obtaining a minute biopsy from partially degraded, FFPE samples and genotyping or micro-array screening. RCA-RCA enables whole genome/mRNA amplification that can be adjusted to the degree of FFPE sample degradation, as this is assessed via real time PCR. Thereby RCA-RCA enables retrieval of the maximum possible amount of information from the degraded sample. In the revised application, apart from adopting the Study Section’s recommendations, a further enhancement of RCA-RCA is included, mRCA-RCA. mRCA-RCA amplifies DNA while retaining epigenetic modifications on a genome-wide basis (‘whole methylome amplification’), thereby allowing highly expanded detection of methylation in fresh or FFPE samples. The R21 phase will examine the maximum capabilities of the technology and establish criteria for adjusting RCA-RCA to conform to the condition of the specific FFPE sample. The R33 phase will develop the technology for obtaining minute cancer biopsies from FFPE samples, assessing sample quality, and amplifying the whole genome/methylome (DNA) or transcriptome (RNA) without introducing amplification bias. Subsequently it will establish criteria and will validate the utility of the amplified material as input for the most frequently used molecular assays (mutation/SNP detection, microsatellite instability/LOH, array-CGH, expression profiling and methylation detection). By removing problems associated with sample degradation and biases associated with amplification this project will enable application of the newest technologies to the analysis of minute biopsies from archived tissue with known outcomes, thereby accelerating the process of candidate gene discovery.

Department of Electrical Engineering, University of Washington

1 R21 CA112149-01

3D microscopy represents a powerful new cell analysis tool for early detection and diagnosis of cancer, but its future use may be limited because methods for preparation of samples are cumbersome, inefficient, labor intensive and generally imprecise. Current methods for cytological sample collection are manual and distributed in nature through various physicians’ office laboratories and local hospitals, with the actual analysis being centralized at regional clinical laboratories. Among the cytological specimens are sputum, gynecological and colorectal scrapes, fine needle aspirates, urinary tract, and gastrointestinal samples. We propose the development of a new automated system that will transform these difficult and messy clinical specimens into an optimal format for 3D microscopy morphological and molecular analysis. The model and method we propose is comprised of three sequential steps. First, at the distributed site, an automated sample processor dissociates and fixes cells and debris for shipment in an automation compatible canister. Second, after shipment to a centralized clinical laboratory, the specimen canisters are loaded into an automatic processor that performs cleanup (debris removal), specimen/assay specific staining (and counterstaining), and finally embedding of cells of interest in glass microcapillary tubes (about 50 mu m ID), with cells being spaced at regular intervals (about 200 mu m) within a tube. This preparation format is uniquely suited for integration with multiple 3D imaging platforms for true 3D volumetric assessment of cell morphology and molecular probe and/or stain density distribution. The proposed system also enables use of cytometric flow sorting for enrichment of cells of interest at an intermediate stage of the sample preparation process. The potential impact to improved human health through rapid diagnostic screening will be illustrated using a high impact emerging technology, optical tomography. In summary, the aim of the proposed project is to develop, design, and build a complete sample processing system that automates the process of sample cleanup, assay specific staining, and mounting of cells into glass microcapillary tubes, and a tube positioning and rotation scanner mechanism for 3D microscopy analysis of cell morphology for the early detection of cancer.

Department of Electrical Engineering, University of Washington

1 R21 CA112149-01

3D microscopy represents a powerful new cell analysis tool for early detection and diagnosis of cancer, but its future use may be limited because methods for preparation of samples are cumbersome, inefficient, labor intensive and generally imprecise. Current methods for cytological sample collection are manual and distributed in nature through various physicians’ office laboratories and local hospitals, with the actual analysis being centralized at regional clinical laboratories. Among the cytological specimens are sputum, gynecological and colorectal scrapes, fine needle aspirates, urinary tract, and gastrointestinal samples. We propose the development of a new automated system that will transform these difficult and messy clinical specimens into an optimal format for 3D microscopy morphological and molecular analysis. The model and method we propose is comprised of three sequential steps. First, at the distributed site, an automated sample processor dissociates and fixes cells and debris for shipment in an automation compatible canister. Second, after shipment to a centralized clinical laboratory, the specimen canisters are loaded into an automatic processor that performs cleanup (debris removal), specimen/assay specific staining (and counterstaining), and finally embedding of cells of interest in glass microcapillary tubes (about 50 mu m ID), with cells being spaced at regular intervals (about 200 mu m) within a tube. This preparation format is uniquely suited for integration with multiple 3D imaging platforms for true 3D volumetric assessment of cell morphology and molecular probe and/or stain density distribution. The proposed system also enables use of cytometric flow sorting for enrichment of cells of interest at an intermediate stage of the sample preparation process. The potential impact to improved human health through rapid diagnostic screening will be illustrated using a high impact emerging technology, optical tomography. In summary, the aim of the proposed project is to develop, design, and build a complete sample processing system that automates the process of sample cleanup, assay specific staining, and mounting of cells into glass microcapillary tubes, and a tube positioning and rotation scanner mechanism for 3D microscopy analysis of cell morphology for the early detection of cancer.