In this project research is conducted to characterize and develop new animal models of human disease and to develop the means to better characterize a model's relevance for a human condition. The project aims include the evaluation of research technologies and biomarkers. Progress was made in developing cancer diagnostics and in research resources useful in developing and characterizing new models of human cancer. Methods and reagents were developed and technologies were applied for use in validating animal models of human cancer. This research project included developing capabilities in molecular diagnostics for cancer models, developing methods for automated morphmetric image analysis of cancer specimens for quantitative pathology, investigating the role of S100 in ovarian cancer and cell cycle regulation in nondividing cells undergoing Ras oncogene activation. Research advances were made in the area of image analysis which were applied to develop quality assurance methods applicable to tissue biobanking. Biorepository supported translational research depends upon high-quality, well-annotated specimens. Histopathology assessment contributes insight into how representative lesions are for research objectives. Feasibility of documenting histological proportions of tumor and stroma was studied in an effort to enhance information regarding biorepository tissue heterogeneity. Using commercially available software, unique spatial-spectral algorithms were developed for applying automated pattern recognition morphometric image analysis to quantify histologic tumor and non-tumor tissue areas in biospecimen tissue sections. Measurements were acquired successfully for 75/75 (100 percent) lymphomas, 76/77 (98.7 percent) osteosarcomas and 60/70 (85.7 percent) melanomas. The percentage of tissue area occupied by tumor varied among patients and tumor types, and was distributed around medians of 94 percent (interquartile range (IQR) = 14 percent) for lymphomas, 84 percent for melanomas (IQR = 24 percent), and 39 percent for osteosarcomas (IQR = 44 percent). Within-patient comparisons from a subset including multiple individual patient specimens revealed less than or equal to 12 percent median coefficient of variation for lymphomas and melanomas. Phenotypic heterogeneity of osteosarcomas resulted in 33 percent median coefficient of variation. Uniformly applied, tumor-specific pattern recognition software permits automated tissue feature quantification. Furthermore, dispersion analyses of area measurements across collections, as well as of multiple specimens from individual patients, support using limited tissue slices to gauge features for some tumor types. Quantitative image analysis automation is anticipated to minimize variability associated with routine biorepository pathologic evaluations and enhance biomarker discovery by helping to guide the selection of study-appropriate specimens. Determination of disease relevant proteomic profiles from limited tissue specimens, such as pathological biopsies or tissues from small model organisms, remains an analytical challenge. A transgenic mouse model of inducible cardiac-specific H-Ras-G12V hypertrophic cardiomyopathy provided a system to explore the potential of using mass spectrometry (MS)-based proteomics to obtain disease-relevant molecular profiles from limited specimens, routinely used in pathological diagnosis. A method that relies on two-stage methanol-assisted solubilization was developed to digest lysates prepared from 8 micrometer-thick fresh-frozen histological tissue sections of cardiomyopathic and normal hearts. A nano-flow reverse-phase liquid chromatography (nfRPLC), coupled to a hybrid LIT- FTICR, led to identification of a total of 704 and 752 proteins in hypertrophic and wild-type (control) myocardium, respectively. Protein network analyses on a total of 106 differentially expressed proteins in diseased hearts revealed an association with cardiovascular diseases. Biologically relevant expression of H-Ras and vimentin were identified by MS and cross-validated in diseased myocardium by immunohistochemistry and Western blot. More importantly, the MS identification and further validation of Wnt-3a and beta-catenin, in conjunction with IHC labeling of phosphorylated GSK-3beta accompanied by nuclear localization of beta-catenin, provided evidence of canonical Wnt/beta-catenin pathway activation during pathogenic myocardial hypertrophy, secondary to Ras activation. Our results indicate that the proteomic approach described permits molecular discovery and assessment of differentially expressed proteins implicated in pathological processes from histopathological tissue sections;the findings can be further validated with immunohistochemical techniques in serial tissue sections from the specimens analyzed by MS. The significant materials, equipment or methods in this project include use of recombinant DNA technology, in vitro cell culture, DNA sequence analysis, immunodiagnostics, molecular imaging, morphometrics, computer assisted image analysis, optical imaging, mass spectrometry, molecular pathology, and veterinary medical diagnosis.
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