A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Recent advances in cancer immunology, highlighted by immune checkpoint inhibitors, have demonstrated that immunotherapy is a viable option in the oncologist’s armamentarium. Despite these advances, many patients are nonresponders. Preliminary studies have suggested that non-responders lack a de-novo anti-tumor antigen immune response that can be unmasked by checkpoint blockade; thus, strategies to induce anti-tumor immune responses are needed. We hypothesized that many tumor associated antigens (Ag) are readily susceptible to immune attack, but only in the context of identifying the tumor antigen epitopes that can reliably initiate an immune response, regardless of individual patient human leukocyte antigen (HLA) haplotype restrictions. We further hypothesized that epitope prediction strategies which seek to identify pan- or highly promiscuous-HLA binding epitopes would reduce the number of potential candidates and be more likely to accurately identify high-priority tumor Ag epitopes. Utilizing known HLA-serotype frequencies and setting a threshold of ninety percent of population coverage, regardless of race or ethnicity, twenty-nine different HLA-DRB1 haplotypes were chosen for antigen prediction utilizing the open source epitope prediction algorithm netMHCIIpan. Predictions were also performed for HLA-A serotypes utilizing the open source algorithm netMHCpan. Predicted epitopes were synthesized in the form of synthetic long peptides and tested in immune system sensitization assays involving unfractionated peripheral blood mononuclear cells (PBMC). Briefly, PBMC were subjected to a two-step culture, first synchronizing their exposure to the long peptides with aggressive surrogate activation of innate immunity, followed by IL-7-modulated T-cell hyperexpansion. Predictions resulted in identification of highly promiscuous-HLA binding epitopes. Unexpectedly, these epitopes clustered together forming high priority regions: unique “hot spots” with high densities of promiscuous HLA-binding epitopes from the widely expressed oncoproteins MUC1, HER2/neu and CMV-pp65 (p<0.0001, for predicted HLA-DRB1 binding affinities, compared to non-hot spot regions). Added synthetic long peptides (>20aa) derived from “hot spot” regions of MUC1, HER2/neu, and CMVpp65 reliably produced selective and sustained expansion of both CD4+ and CD8+ peptide-specific, interferon-γ (IFNγ)-producing Tcells when synchronized with step 2 exposure to exogenous IL-7 (p<0.0001 and p=0.0048, for CD4+ and CD8+ Ag-specific T-cells, respectively, compared to T-cells directed against peptides from non-hot spot regions). “Hot spot” peptide Ag-specific T-cells preferentially recognized endogenous tumor derived MUC1, either in MUC1 expressing tumor cell killing assays (p=0.038, compared to non-peptide Ag-specific T-cells) or as MUC1 tumor lysate when pulsed onto restimulatory PBMC (p=0.022 and 0.025, for CD4+ and CD8+ T-cells, respectively, compared to T-cells directed against peptides from non-hot spot regions). This mechanistically rational antigen selection sequence, effective even for unvaccinated donors, regardless of HLA-haplotype, enables rapid identification of tumor protein regions relevant for cancer immunology, including adoptive immunotherapy, vaccines, and even identification of tumor neo-antigens unique to each patient.

Conjugated linoleic acid (CLA) exhibits multiple biological and molecular activities that have made it the subject of considerable nutrition-related research. Numerous studies support broad acting anti-tumor effects including anti-inflammatory, anti-proliferation, and pro-apoptosis in a variety of model systems. CLA’s ability to influence multiple tumor promoting pathways, without toxicity, may prove valuable in the chemoprevention of breast cancer. The overall objective of this dissertation research was to investigate the potential of CLA in the chemoprevention of breast cancer in a subgroup of women at risk of developing estrogen receptor (ER) negative disease. Overexpression of either the ERBB2 oncogene or the epidermal growth factor receptor (EGFR) is a common event in ER negative breast cancer. To respond to this association, the stated research objective was pursued in relevant model systems. The primary hypothesis was that CLA would downregulate the ERBB2 receptor in vitro and inhibit mammary tumorigenesis in vivo. The t10c12 CLA isomer significantly reduced ERBB2 protein expression in the ERBB2 overexpressing cell line SKBr3. This was accompanied by a decrease in NFκB nuclear localization, cyclooxygenase-2 (COX2)-derived prostaglandin (PG) E2 production, increased apoptosis, and inhibition of proliferation. In contrast to the in vitro data, however 1% dietary CLA had pro-tumor effects in the PyV-mT transgenic mouse model, Mammary gland whole mounts indicated a significant loss of adipose in the CLA-treated group compared to controls that was confirmed by the downregulation of adipocyte-specific genes including PPARγ and adiponectin. CLA’s effect on the adipose was supported by decreases in fatty acid synthase at the protein and mRNA level. cDNA microarray revealed significant downregulation of cytoskeletal and adhesion-related genes in the CLA-treated group. These data suggest CLA’s combined effect on the adipose and epithelial architecture may have promoted tumor growth in this model While the large body of evidence supporting an anti-tumor effect of CLA can not be discounted, the studies herein demonstrate the complexity of its action that may not be captured in simple model systems. Reports of adverse effects of mixed isomers or the t10c12 purified isomer lend caution to supplementation that is supported by our in vivo data.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature cells at various stages of differentiation. These cells are broadly characterized by the simultaneous expression of the surface markers CD11b and Gr1 and have been found to accumulate in large numbers in response to many different tumors in both mice and humans, including HER2/neu+ breast cancers. The adoptive immunotherapy of cancers has been a promising field, yet the clinical efficacy of adoptive immunotherapies targeted against human breast cancers and many other cancers has been extremely limited. Given the influx of MDSC in tumor-bearing individuals, we hypothesized that these cells were the reason for the failure of adoptively transferred T cells to effectively reject primary tumors. Using either monoclonal antibodies or the chemotherapeutic drug, gemcitabine, we aimed to eliminate MDSC cells in vivo to determine if adoptively transferred T cells would be more effective in the absence of these cells. We further aimed to characterize the mechanism of T cell suppression by MDSC and the tumor-derived soluble factor(s) responsible for their accumulation. We have found that the elimination of MDSC in vivo does result in significant tumor inhibition when adoptively transferred T cells are administered. Furthermore, the use of gemcitabine in conjunction with adoptively transferred T cells resulted in complete tumor rejection in 100% of mice and was accompanied by large antibody titers against HER2/neu as well as strong recall responses characterized by IFN-g release and subsequent rejection of further tumor challenges. We report herein that suppression by MDSC is contact dependent and affects the proliferation of both CD4+ and CD8+ T cells. The accumulation of MDSC in tumor-bearing mice can be entirely attributed to tumor-derived soluble factors, with GM-CSF specifically causing the generation and maintenance of these cells. Our findings suggest that the adoptive immunotherapy of breast carcinomas in a clinical setting should be combined with the use of gemcitabine, and that the use of GM-CSF as an adjuvant in cancer vaccines should be carefully re-evaluated as this cytokine may result in increased MDSC accumulation in vivo.

HER2/neu is an oncogenic tumor-associated antigen over-expressed in several human tumors including breast and ovarian cancer. The selective expression of HER2/neu and its role in epithelial carcinogenesis makes HER2/neu an ideal target for immunotherapy. Tumor-derived chaperone-rich cell lysate (CRCL), containing numerous heat shock proteins, has successfully been used to generate tumor-specific immunity against a wide range of murine tumors and is a great candidate for an effective vaccine against HER2/neu positive tumors. In the first part of this study, the potency of human ovarian cancer-derived CRCL to activate dendritic cells (DCs) and to generate tumor-specific T cells in vitro has been investigated. Chaperone-rich cell lysate was generated from primary ovarian cancer tissues and SKOV3-A2, a HER2/neu, Wilm's tumor gene 1 (WT1) and HLA-A2 positive human ovarian tumor cell line. T cells from healthy donors and from ovarian cancer patients secreted higher amounts of interferon-&#947; following in vitro re-stimulation with ovarian cancer-derived CRCL compared to HER2/neu or WT1 peptide-pulsed DCs. We were also able to generate cytotoxic T lymphocyte activity against cancer-specific antigens such as HER2/neu and WT1 from all healthy donors, but from only one of the four ovarian cancer patients with bulky disease. In the second part of the study, the potency of tumor-derived CRCL to elicit the humoral immune response against a murine HER2/neu positive tumor (TUBO) has been examined. Vaccination of mice bearing a palpable tumor efficiently delayed the development of the tumor. In the vaccinated mice, CRCL vaccination induced significant anti-HER2/neu antibodies. Using B cell deficient mice and antibody transfer experiments, we have shown that the induction of anti-HER2/neu antibodies is both necessary and sufficient for the anti-tumor effect. Further, we have demonstrated that serum from TUB0 CRCL-vaccinated mice stimulated the internalization of the HER2/neu molecules, resulting in the down-regulation of their surface expression. Moreover, antibody-dependent cellular cytotoxicity has been observed against TUBO cells when presented with sera from vaccinated mice. These results indicate that CRCL may be a potent adjuvant for women suffering from HER2/neu positive ovarian or breast cancer and that this personalized vaccine may be a promising approach for active immunotherapy.

Synthetic xenoestrogens have differential estrogenic properties. Research has shown that exposures to xenoestrogens could promote breast cancer by disrupting normal function of the human epidermal growth factor receptor 2 (Her2) gene. Although animal models demonstrated a connection between xenoestrogen exposure and Her2 activity, no study using human cells has systematically examined their carcinogenic potential influencing the Her2 gene expression. Furthermore, breast cancer cells are phenotypically disparate (ER+, Her2+), with some phenotypes (Her2+), leading to more aggressive disease. This study aimed to dosimetrically assess the carcinogenic potential of commonly used xenoestrogens influencing Her2 gene expression, and delineate cellular phenotypes at greater risk of more aggressive disease. The study assessed whether the composition, concentrations, and exposure duration of BPA, EE, NPH, and DDT significantly altered Her2 copy numbers in estrogen and Her2 receptor positive or negative breast cancer lines. Each line was randomly assigned to cases (exposed) and control (unexposed) groups using a randomized block design. Fluorescent in-situ hybridization measured Her2 gene copies. Mann Whitney, Kruskal Wallis, and Incidence Rate Ratios revealed Her2 copy gains in all 4 xenoestrogens and receptor types with persistent exposures. A 44% increase in Her2 was observed in the normal ER and Her2 line, marking a shift in its Her2 status, and a 30-times greater risk was noted in the Her2+ lines. These findings promote positive social change by revealing all 4 xenoestrogens as risk factors for breast cancer. This information can be used by breast cancer advocacy groups, health educators, and steering committees to educate women and formulating policies.

No description available.

Thesis (M.Med. (Radiation Oncology)) -- University of Limpopo, 2022 / Background Breast cancer is known to be a heterogeneous disease that demands patient centered care. Establishing the clinicopathological characteristics of breast cancer patients is a vital step in an effort to individualize their treatment. Aim The aim is to evaluate the clinicopathologic features of the different subtypes of breast cancer when classified according to immunohistochemistry markers in women attending Pietersburg hospital. Methods A retrospective review of medical records of women treated at Pietersburg hospital between 2010 and 2011 was done. Data collection was extracted on a customized data collection sheet. Chi square was used to determine association between clinicopathologic features and molecular subtypes. Analysis of variants was used to assess association between molecular types and age. Results The mean age of the population was 55.3 years (+/-14 standard deviation). The majority of patients were in stage III (46.9%) and IV (33.5%). The ER, PR, HER2/neu positive rate was 50.6%, 30% and 14,3 % respectively with a negative rate of 13,4%, 19,5% and 23,4% respectively. ER, PR and HER2/neu was unknown in 18%, 19, 5% and 23,4% respectively. The most common molecular subtype was luminal A (53,6%) followed by triple negative (27.2%), HER2/neu (11, 4%) and luminal B (7. 9%).There was no association between the subtypes and tumour stage (p=0.578).The rate of distant metastasis was similar across the subtypes being 37,9%,35%, 32,4% and 31,9% in HER2/neu, luminal B ,luminal A and TNBC, respectively. All four molecular subtypes had high rate of axillary lymph node involvement (p=0.886) Luminal A had the least percentage of high grade tumours with TNBC having the highest. Five-year overall survival for the cohort was 25, 6% with luminal A and B having a better 5 year overall survival of 27,2% and 25% respectively, whereas HER2/neu and TNBC had lower 5 year OS of 24% and 23,3%. Conclusion The findings of this study suggest that luminal A subtype is the most predominant and the majority might benefit from hormonal therapy. However, some patients could not be classified due to missing IHC marker test results. The outcome across all four subtypes is poor and more effort should be put towards improving the diagnosis and treatment individualization and follow-up in these patients.

No description available.

In the era of personalized cancer treatment, adoptive T cell therapy (ACT) shows promise for the treatment of solid cancers. However, partial or mixed responses remain common clinical outcomes due to the heterogeneity of tumours. Indeed, in many patients it is typical to see a response to ACT in one tumour nodule, while others show little or no response. Thus, defining the tumour features that distinguish those that respond to ACT from those that do not would be a significant advance, allowing clinicians to identify patients that might benefit from this treatment approach. The first chapter of this thesis provides the necessary background to understand the principals behind and components of ACT. This chapter also offers selected historical advances contributing to the current state of the field. The second chapter introduces a novel murine model of breast cancer developed to investigate the tumour-specific mechanisms associated with immune evasion in an ACT setting. The third chapter describes the in vivo characterization of mammary tumour cell lines derived from our mouse model that reliably showed complete, partial or no response to ACT. Using these cell lines, we were able to characterize in vivo tumour-specific differences in cytotoxic T cell trafficking, infiltration, activation, and proliferation associated with response to ACT. In the fourth chapter, we used bioinformatics approaches to develop a preliminary predictive gene signature associated with response to ACT in our mammary tumour model. We used this signature to predict outcome and then test a number of murine mammary tumours in vivo, with promising results, wherein 50% of tumours responded to ACT as predicted based upon gene expression. Thus, using an innovative model for breast cancer, these results suggest that there are tumour-specific features that can be used a priori to predict how a tumour will respond to adoptive T cell therapy. Importantly, these findings might facilitate the design of immunotherapy trials for human breast cancer. / Graduate

More than half of all cases of breast cancer occur among women without any known risk factors. More research is needed on suspected risk factors in order to refine current breast cancer screening tools. The objectives of this thesis were: to determine the breast cancer risk associated with known risk factors (ethnicity, family history, breast biopsy, age at menarche, age at first birth, alcohol, HRT, and BMI), suspected risk factors (smoking, second-hand smoke exposure, smoked/grilled foods, and NSAID use), and to examine the above associations according to tumour receptor status, histologic grade, and menopausal status, with potential confounders also considered. This thesis project was conducted within the framework of the Molecular Epidemiology of Breast Cancer study, a case-control study of women in Vancouver, BC and Kingston, ON, with 1140 cases and 1169 controls recruited from 2005-2010. Information was collected from a detailed questionnaire. Cases and controls were similar in terms of age at menarche, age at first birth, smoking history, second-hand smoke exposure, lifetime smoked/grilled food consumption, HRT, and BMI. Among cases, there were significantly less Europeans and more Chinese, Japanese, and Filipino subjects compared to controls. Cases were more likely to have a first degree relative with breast cancer, as well as a previous benign breast biopsy. Alcohol consumption and past NSAID usage was higher among controls. The level of education completed was higher among controls. Cases were also more likely to be postmenopausal. Family history was associated with breast cancer risk (OR=1.59, CI=1.30-1.94), as was BMI (OR=1.28, CI=1.05-1.58 for overweight and OR=2.28, CI=1.35-3.86 for obese class II). Second-hand smoke was also found to be associated with breast cancer risk (OR=1.42, CI=1.02-1.97 for individuals with a less than 10 pack-year smoking history). Due to reduced sample size with stratification and marginally significant results, it is not possible to draw definitive conclusions regarding pathology sub-types. In summary, these results provide support for the association between several risk factors and breast cancer risk. More research is needed to ascertain how receptor status, histologic grade, and menopausal status affect these associations. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2011-08-15 19:43:06.689