T-Cell Immunogenicity and Dysfunction in Cancer and Viral Diseases

abstract: CD8+ T-lymphocytes (CTLs) are central to the immunologic control of infections and are currently at the forefront of strategies that enhance immune based treatment of a variety of tumors. Effective T-cell based vaccines and immunotherapies fundamentally rely on the interaction of CTLs with peptide-human leukocyte antigen class I (HLA-I) complexes on the infected/malignant cell surface. However, how CTLs are able to respond to antigenic peptides with high specificity is largely unknown. Also unknown, are the different mechanisms underlying tumor immune evasion from CTL-mediated cytotoxicity. In this dissertation, I investigate the immunogenicity and dysfunction of CTLs for the development of novel T-cell therapies. Project 1 explores the biochemical hallmarks associated with HLA-I binding peptides that result in a CTL-immune response. The results reveal amino acid hydrophobicity of T-cell receptor (TCR) contact residues within immunogenic CTL-epitopes as a critical parameter for CTL-self/nonself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy.Dissertation/ThesisDoctoral Dissertation Biological Design 201

Einfluss von Chemotherapie auf das Tumorwachstum und die metastatische Progression im Computermodell

Diese Arbeit konzentriert sich auf die Rolle der Blutgefäßgeometrie bei der Behandlung von Tumorerkrankungen. Zur Untersuchung wurde eine Kombination von mathematischen Modellen, Computersimulationen und experimentellen Daten verwendet. Die Anwendung einer Cisplatin-Chemotherapie reduziert signifikant die Blutgefäßdichte und beeinflusst somit das Ausbreitungsverhalten von Metastasen. Der Zeitpunkt einer Chemotherapie ist ein kritischer Faktor für den Behandlungserfolg, da sich der behandelbare Anteil aufgrund der begrenzten Verteilung von Chemotherapeutika mit der Zeit rapide verringert

First-order statistical speckle models improve robustness and reproducibility of contrast-enhanced ultrasound perfusion estimates

Contrast-enhanced ultrasound (CEUS) permits the quantification and monitoring of adaptive tumor responses in the face of anti-angiogenic treatment, with the goal of informing targeted therapy. However, conventional CEUS image analysis relies on mean signal intensity as an estimate of tracer concentration in indicator-dilution modeling. This discounts additional information that may be available from the first-order speckle statistics in a CEUS image. Heterogeneous vascular networks, typical of tumor-induced angiogenesis, lead to heterogeneous contrast enhancement of the imaged tumor cross-section. To address this, a linear (B-mode) processing approach was developed to quantify the change in the first-order speckle statistics of B-mode cine loops due to the incursion of microbubbles. The technique, named the EDoF (effective degrees of freedom) method, was developed on tumor bearing mice (MDA-MB-231LN mammary fat pad inoculation) and evaluated using nonlinear (two-pulse amplitude modulated) contrast microbubble-specific images. To improve the potential clinical applicability of the technique, a second-generation compound probability density function for the statistics of two-pulse amplitude modulated contrast-enhanced ultrasound images was developed. The compound technique was tested in an antiangiogenic drug trial (bevacizumab) on tumor bearing mice (MDA-MB-231LN), and evaluated with gold-standard histology and contrast-enhanced X-ray computed tomography. The compound statistical model could more accurately discriminate anti-VEGF treated tumors from untreated tumors than conventional CEUS image. The technique was then applied to a rapid patient-derived xenograft (PDX) model of renal cell carcinoma (RCC) in the chorioallantoic membrane (CAM) of chicken embryos. The ultimate goal of the PDX model is to screen RCC patients for de novo sunitinib resistance. The analysis of the first-order speckle statistics of contrast-enhanced ultrasound cine loops provides more robust and reproducible estimates of tumor blood perfusion than conventional image analysis. Theoretically this form of analysis could quantify perfusion heterogeneity and provide estimates of vascular fractal dimension, but further work is required to determine what physiological features influence these measures. Treatment sensitivity matrices, which combine vascular measures from CEUS and power Doppler, may be suitable for screening of de novo sunitinib resistance in patients diagnosed with renal cell carcinoma. Further studies are required to assess whether this protocol can be predictive of patient outcome

Predicting local recurrence following breast conserving therapy for early stage breast cancer : the significance of a narrow (less than or equal to 2mm) surgical resection margin

Introduction Controversy continues over the extent of surgical resection margin required to minimize the risk of local recurrence (LR) in breast conserving therapy (BCT) for stage I and II breast cancer. This thesis explores whether or not a narrow (less than or equal to 2 mm) but negative resection margin in BCT for stage I and II breast cancer affects LR. Methodology To address the question, all patients registered at the Saskatoon Cancer Center between January 1, 1991 and December 31, 2000 with a diagnosis of stage I or II invasive duct carcinoma of the breast treated with BCT were examined. All charts and pathology reports were reviewed with a review of the pathology for all cases where the resection margin was unclear in the original report. Other factors know or thought to effect LR (age, radiation boost, grade, extensive duct carcinoma in situ, ER/PR receptor status, tumor size, and systemic adjuvant therapy) were considered in the statistical analysis. Results Amongst the 200 narrow margin cases 19 LR were detected (19/200=9.5%) while 52 LR were detected in the 491 wide margin cases (52/491=10.6%). This difference was not statistically significant. Conclusions A narrow (less than or equal to 2 mm) surgical resection margin does not result in an increase in local recurrence compared to a surgical resection margin greater than 2 mm in breast conserving therapy for early stage duct carcinoma and does not warrant re-excision

Cell-Free Nucleic Acids

The deficits of mammography and the potential of noninvasive diagnostic testing using circulating miRNA profiles are presented in our first review article. Exosomes are important in the transfer of genetic information. The current knowledge on exosome-associated DNAs and on vesicle-associated DNAs and their role in pregnancy-related complications is presented in the next article. The major obstacle is the lack of a standardized technique for the isolation and measurement of exosomes. One review has summarized the latest results on cell-free nucleic acids in inflammatory bowel disease (IBD). Despite the extensive research, the etiology and exact pathogenesis are still unclear, although similarity to the cell-free ribonucleic acids (cfRNAs) observed in other autoimmune diseases seems to be relevant in IBD. Liquid biopsy is a useful tool for the differentiation of leiomyomas and sarcomas in the corpus uteri. One manuscript has collected the most important knowledge of mesenchymal uterine tumors and shows the benefits of noninvasive sampling. Microchimerism has also recently become a hot topic. It is discussed in the context of various forms of transplantation and transplantation-related advanced therapies, the available cell-free nucleic acid (cfNA) markers, and the detection platforms that have been introduced. Ovarian cancer is one of the leading serious malignancies among women, with a high incidence of mortality; the introduction of new noninvasive diagnostic markers could help in its early detection and treatment monitoring. Epigenetic regulation is very important during the development of diseases and drug resistance. Methylation changes are important signs during ovarian cancer development, and it seems that the CDH1 gene is a potential candidate for being a noninvasive biomarker in the diagnosis of ovarian cancer. Preeclampsia is a mysterious disease—despite intensive research, the exact details of its development are unknown. It seems that cell-free nucleic acids could serve as biomarkers for the early detection of this disease. Three research papers deal with the prenatal application of cfDNA. Copy number variants (CNVs) are important subjects for the study of human genome variations, as CNVs can contribute to population diversity and human genetic diseases. These are useful in NIPT as a source of population specific data. The reliability of NIPT depends on the accurate estimation of fetal fraction. Improvement in the success rate of in vitro fertilization (IVF) and embryo transfer (ET) is an important goal. The measurement of embryo-specific small noncoding RNAs in culture media could improve the efficiency of ET

Doctor of Philosophy

dissertationComplex cancer phenotypes are defined by their aggressive nature and lack of known or accessible therapeutic targets. My dissertation focuses on the use of a personalized medicine approach for the identification of novel therapies against two complex cancer phenotypes: Basal-like/Claudin-low breast cancer and RAS-active nonsmall cell lung cancer. RAS-active cancer is characterized by the activation of the complex signaling network of RAS, which lacks effective therapeutics capable of inhibiting the RAS protein itself or the overall pathway. Further complicating treatment is the ability of the RAS pathway to be activated independent of the presence of an activating mutation in the RAS protein. To broadly characterize pathway activation independent of RAS protein mutation, I used a gene-expression-based biomarker for RAS network activity in nonsmall cell lung cancer (NSCLC) cell lines, and identified RAS activation in both RAS-mutant and wild-type lines. I then screened for drugs whose efficacy significantly correlated to RAS network activity and showed that EGFR and MEK co-inhibition is an effective treatment personalized against RAS-active NSCLC. Finally, I demonstrated that EGFR and MEK co-inhibition induced apoptosis and blocked both EGFR-RAS-RAF-MEK-ERK and EGFR-PI3K-AKT-RPS6 nodes simultaneously in RAS-active, but not RAS-inactive NSCLC. Secondly, I identified a novel compound effective against Basal-like and Claudin-low breast cancer (BL-CL). BL-CL is a molecular subtype of breast cancer characterized by an aggressive, recurrent and nonluminal nature, epitomized by the lack of known therapeutic targets and poor patient prognosis. Using high- iv throughput screening of a marine invertebrate compound library and sequential purification of crude fractions, I identified a previously uncharacterized sulfated sterol, Topsentinol L Trisulfate (TLT), purified from a marine sponge, and showed that it inhibits AMPK and CHK1 but activates p38. Furthermore, I indentified the potential use of known AMPK and CHK1 inhibitors, alone or in combination, as an effective therapy against BL-CL. Lastly, sensitivity to TLT was projected against various human tumors by generating a gene-expression signature that predicted breast and bladder cancer as the cancer types most receptive to TLT therapy. This work describes the identification of novel treatments personalized against BL-CL and RAS-active NSCLC, providing a framework for future pre-clinical studies