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Syftet med studien var att undersöka det histopatologiska utseendet hos humana periimplantit-lesioner i relation till lesionens kliniska utseende. Periimplantit-vävnad lokaliserad runt 15 implantat avlägsnades kirurgiskt från 13 individer. De 15 kirurgiskt avlägsnade vävnadsproverna skiljde sig betydligt i utseende kliniskt och delades in i två grupper beroende på om de var inkapslade och välavgränsade mot omgivande vävnad eller mer diffust avgränsade. Vävnaderna preparerades histokemiskt (MAYERS HTX) och immunohistokemiskt (MACH 4 Universal HRP-Polymer Detection System). De celler som undersöktes med immunohistokemiska metoder var T-celler (CD3+), B-celler (CD20+), plasmaceller (CD138+), M1-makrofager (CD68+) och M2-makrofager (CD163+). Förekomsten av neutrofila granulocyter identifierades morfologiskt med rutininfärgning. Därefter scannades och analyserades proverna kvalitativt och kvantitativt. Ingen statistiskt signifikant skillnad kunde påvisas mellan tätheten av infiltrerade lymfocyter (P=0,613) och neutrofila granulocyter (P=0,336) samt arean av inflammationsinfiltratet (P=0,613) mellan de två studerade grupperna. Vidare kunde inget samband observeras mellan den histologiska sammansättningen av de studerade cellerna och det kliniska utseendet vilket dock kan förklaras av otydliga kliniska kriterier. Däremot kan det konstateras att en skillnad föreligger mellan proverna utifrån den histologiska bilden. Däribland varierar antalet plasmaceller avsevärt mellan proverna samt förekomst av fibrös bindvävszon mot benet. Ytterligare studier behövs för att förklara skillnaden i det kliniska och histologiska utseendet. / The purpose of this study was to examine the histopathological composition of human peri-implantitis lesions in relation to the clinical appearance of the lesion. Peri-implantitis tissue located around implants was surgically removed from 13 individuals. The surgically removed tissue samples differed significantly in appearance clinically and were divided into two groups, depending on whether they were encapsulated and well-bound to surrounding tissue or more diffusely delimited. The tissues were histochemically prepared (MAYERS HTX) and immunohistochemical (MACH 4 Universal HRP-Polymer Detection System). The cells examined by immunohistochemical methods were T cells (CD3 +), B cells (CD20 +), plasma cells (CD138 +), M1 macrophages (CD68 +) and M2 macrophages (CD163 +). The presence of neutrophilic granulocytes was examined morphologically by routine staining. The samples were then scanned and analyzed qualitatively and quantitatively. No statistically significant difference could be detected between the density of infiltrated lymphocytes (P = 0.613) and neutrophil granulocytes (P = 0.336) and the area of ​​the inflammatory infiltrate (P = 0.613) between the two studied groups. Furthermore, no relationship could be observed between the histological composition of the cells studied and the clinical appearance, which however, can be explained by unclear clinical criteria. The results indicated that there was a difference between the samples based on the histological composition, including the number of plasma cells which varied considerably between the samples and the presence of fibrous connective tissue zone close to the bone. Further studies are required to explain the difference in the clinical and histological appearance.

Corneal transplantation carries a high rate of success in the domestic cat and is an indicated treatment for specific corneal diseases in this species. The potential for iatrogenic transmission of viral diseases is a well-recognized problem in human corneal transplantation programs and screening donors for certain diseases is routine. Feline leukemia virus (FeLV) is a common agent of disease in domestic cats and available blood tests are highly effective in identification of infected individuals. This study investigates the presence of FeLV within corneal tissues of FeLV infected cats. Seventeen cats were identified to be positive for serum p27 antigen by enzyme-linked immunosorbent assay (ELISA). Twelve of these individuals were found to be positive on peripheral blood by immunofluorescent antibody (IFA) testing. Seventeen ELISA negative cats were identified to serve as negative controls. Full thickness corneal specimens were collected from all subjects and analyzed for the presence of FeLV proviral DNA and gp70 antigen by polymerase chain reaction (PCR) and immunohistochemical (IHC) testing, respectively. Eleven (64.7%) positive corneal PCR results were obtained from 17 ELISA positive cats. Of 12 cats which were both ELISA and IFA positive on peripheral blood, 10 (83.3%) had positive corneal PCR results. All corneal tissues from ELISA negative subjects were PCR negative. IHC staining of corneal sections revealed the presence of FeLV gp70 in corneal tissues of nine (52.9%) ELISA positive cats. Of the 12 cats which were both ELISA and IFA positive on peripheral blood, 8 (66.7%) had positive corneal IHC results. Positive IHC staining was localized to the corneal epithelium. Corneal tissues of all ELISA negative cats and all IFA negative cats were negative on IHC testing. This study reveals FeLV to be present within the corneal epithelium of some FeLV infected cats. Screening potential corneal donors for this virus is warranted. This work was funded by grants from the American College of Veterinary Ophthalmologists, the Virginia Veterinary Medical Association Pet Memorial Fund, and the DSACS Quick Response Fund. / Master of Science

Translational biomedical informatics is the application of computational methods to facilitate the translation of basic biomedical science to clinical relevance. An example of this is the multi-step process in which large-scale microarray-based discovery experiments are refined into reliable clinical tests. Unfortunately, the quality of microarray data is a major issue that must be addressed before microarrays can reach their full potential as a clinical molecular profiling tool for personalized and predictive medicine. A new methodology, titled caCORRECT, has been developed to replace or augment existing microarray processing technologies, in order to improve the translation of microarray data to clinical relevance. Results of validation studies show that caCORRECT is able to improve the mean accuracy of microarray gene expression by as much as 60%, depending on the magnitude and size of artifacts on the array surface. As part of a case study to demonstrate the widespread usefulness of caCORRECT, the entire pipeline of biomarker discovery has been executed for the clinical problem of classifying Renal Cell Carcinoma (RCC) specimens into appropriate subtypes. As a result, we have discovered and validated a novel two-gene RT-PCR assay, which has the ability to diagnose between the Clear Cell and Oncocytoma RCC subtypes with near perfect accuracy. As an extension to this work, progress has been made towards a quantitative quantum dot immunohistochemical assay, which is expected to be more clinically viable than a PCR-based test.

HER2 breast cancer is an aggressive disease that occurs in 20 - 30% of the breast cancer population. Treatment for HER2 breast cancer includes use of an anti-HER2 monoclonal antibody, trastuzumab. Testing for HER2 is of critical importance due to the adverse side effects and substantial costs associated with this anti-HER2 treatment. Currently, two kinds of tests, Fluorescence In Situ Hybridization (FISH) and Immunohistochemistry (IHC), are FDA approved for determination of HER2 status in breast cancers. Clinical and non clinical factors that affect the choice HER2 test and the use of anti-HER2 therapy in breast cancer were analyzed using a data set containing information from six outpatient oncology clinics in the United States. The analysis showed that geographic location, cancer stage, and diagnosis date (pre- or post-publication of testing guidelines) have significant effects on choice of test. With regard to trastuzumab prescription, geographic location and HER2 status have significant effects on the prescription of trastuzumab. In addition, there was a non-significant trend for certain Medicare patients not to receive trastuzumab therapy. These findings indicate that disparities are present in breast cancer care based on geography and cancer stage, and highlight the importance of testing guidelines. The cost effectiveness of FISH vs. IHC was determined, by considering the financial and health-related costs associated with testing and subsequent treatment as well as the accuracy of each test. The results show that FISH is the optimal choice for HER2 testing and is more cost-effective than IHC.

Automated tissue image analysis aims to develop algorithms for a variety of histological applications. This has important implications in the diagnostic grading of cancer such as in breast and prostate tissue, as well as in the quantification of prognostic and predictive biomarkers that may help assess the risk of recurrence and the responsiveness of tumors to endocrine therapy. In this thesis, we use pattern recognition and image analysis techniques to solve several problems relating to histopathology and immunohistochemistry applications. In particular, we present a new method for the detection and localization of tissue microarray cores in an automated manner and compare it against conventional approaches. We also present an unsupervised method for color decomposition based on modeling the image formation process while taking into account acquisition noise. The method is unsupervised and is able to overcome the limitation of specifying absorption spectra for the stains that require separation. This is done by estimating reference colors through fitting a Gaussian mixture model trained using expectation-maximization. Another important factor in histopathology is the choice of stain, though it often goes unnoticed. Stain color combinations determine the extent of overlap between chromaticity clusters in color space, and this intrinsic overlap sets a main limitation on the performance of classification methods, regardless of their nature or complexity. In this thesis, we present a framework for optimizing the selection of histological stains in a manner that is aligned with the final objective of automation, rather than visual analysis. Immunohistochemistry can facilitate the quantification of biomarkers such as estrogen, progesterone, and the human epidermal growth factor 2 receptors, in addition to Ki-67 proteins that are associated with cell growth and proliferation. As an application, we propose a method for the identification of paired antibodies based on correlating probability maps of immunostaining patterns across adjacent tissue sections. Finally, we present a new feature descriptor for characterizing glandular structure and tissue architecture, which form an important component of Gleason and tubule-based Elston grading. The method is based on defining shape-preserving, neighborhood annuli around lumen regions and gathering quantitative and spatial data concerning the various tissue-types.

Bladder cancer is a potentially curable malignancy; however in regards to the state of current therapy regimens, a plateau has been reached in both the non-muscle and muscle invasive types. To obtain effective treatment, and consequently a decreased mortality, it has become imperative to test and understand aspects affecting therapy response. The aim of this thesis is to illustrate a better understanding of clinical factors affecting therapy response using new drug combinations and new tumor markers alongside established risk criteria. In Paper I we reported the 5 year follow up from a multicenter, prospectively randomized study and we evaluated the 5-year outcomes of BCG alone compared to a combination of epirubicin and interferon-a2b in the treatment of patients with T1 bladder cancer. Treatment, tumor size and tumor status at second resection were independent variables associated with recurrence. Concomitant Cis was not predictive of failure of BCG therapy. Independent factor for treatment failure was remaining T1 stage at second resection. In Paper II &III we investigated the validity of emmprin, survivin and CCTα proteins as biomarkers for response and survival before neoadjuvant cisplatin chemotherapy. Bladder tumor specimens were obtained before therapy from a total of 250 patients with T1-T4 bladder cancer enrolled in 2 randomized trials comparing neoadjuvant chemotherapy before cystectomy with a surgery only arm. Protein expression was determined by immunohistochemistry (IHC). Patients in the chemotherapy cohort with negative emmprin and CCTα expression had significantly better overall survival (OS) than those with positive expression. In Paper IV primary end point was examining STMN1 as prognostic factor in bladder cancer.  Analysis was performed on three bladder cancer patient cohorts using IHC, western blot and a bladder cancer cell line. High levels of STMN1, expression correlated to shorter disease-specific survival and the growth and migration of the cells were significantly reduced when transfecting the cells with STMN1 siRNA. Conclusion Risk assessment and predictors of outcomes could help in individualized treatment and follow up.  Biomarkers will become more important for treatment choices in bladder cancer management.

Despite recent advances, prognosis in metastatic prostate cancer remains poor. As with other cancers, tumor heterogeneity is an increasingly evident contributor in prostate tumorigenesis and developed resistance. Using in vitro and in vivo model systems, we examined novel diagnostic and therapeutic strategies in prostate cancer. In these studies, combination treatment with amuvatinib, a receptor tyrosine kinase inhibitor, and erlotinib, an epidermal growth factor inhibitor, was assessed for its ability to differentially modulate growth signaling in pathway diverse LNCaP (PTEN⁻) and DU-145 (PTEN⁺) human prostate cancer cell and mouse xenograft models. Our results suggest both individual mechanistic signaling activities, as well as benefits of the combination therapy though modulations of MAPK (pERK) and 4EBP1/cyclin D1 in growth signaling divergent PTEN+ and PTEN- prostate cancer cells. Additionally, despite the importance preanalytical tissue preservation on downstream diagnostic assays, exact protocols are not well defined and highly variable clinically and, as such, critical diagnostic information is lost. We show that a novel 2+2 fixation method induces target- and cell-specific alterations in immunostain intensity and efficacy. Importantly, cyclin D1 is increasingly utilized for as a clinical prognostic/diagnostic marker and demonstrated improved immunohistochemical staining efficacy with 2+2 fixation compared with treatment-matched xenograft protein alterations as assessed by western analysis. Finally, pentoxifylline (PTX) is a clinically utilized and well tolerated PDE inhibitor that has shown promise as a radio-/chemo-sensitization and anti-cancer agent against a variety of cancers. In these studies, we demonstrate that PTX induces cell and tumor growth inhibition in LNCaP prostate cancer cells. Mechanistically, PTX induces transient cellular signaling modulations of both the AMPK metabolic and AKT/mTOR growth pathways, while inducing autophagy. Also, PTX sensitizes LNCaP prostate cancer to cytotoxicity induced by first line chemotherapy docetaxel, inducing significant cellular apoptosis and reducing effective docetaxel concentrations by >10 fold for equivalent toxicity in viability assays. These findings nominate PTX as an adjunct therapy for the treatment of prostate cancer. In summary, these studies characterize the targeted signaling modulation by combination erlotinib and amuvatinib therapy, as well as pentoxifylline, for their use as therapies for prostate cancer. A novel fixation protocol was also assessed for improved diagnostic tissue preservation of critical signaling proteins. Further understanding in these areas will aid and expand the development of effective diagnostics, as well as emphasize the benefits of these and similar therapeutics for the treatment of prostate cancer.