Dissertations / Theses on the topic 'Breast cytology'

The HER family of receptors plays a major role in a variety of cancers including breast cancer. Several researchers have shown that HER family overexpression in breast cancer is a significant prognostic factor, especially for survival and relapse. Therefore, many therapeutics are being developed to test the impact of HER family blockade in breast cancer. Although numerous therapies have been developed, many have not been very successful in the clinic. This is often a consequence of cancer cells developing new mechanisms to activate HER family signalling indirectly through cross talk with compensatory pathways. Thus, it is vital to consider the biology of the HER signalling network to a greater extent, which includes RAS/MAPK, PI3K/AKT, mTOR, JAK/STAT, ER and AhR pathways and, also identify breast cancer patient populations that will benefit from specific targeted therapies that target these pathways. In the current study, 6 breast cancer cell lines (MCF7, T47D and ZR-75-1, SKBR3, MDA-MB 468 and MDA-MB 231) representing distinct molecular subtypes of breast cancer have been used to investigate anti-cancer effects of a variety of agents. These agents include clinical as well as currently experimental and entirely novel pharmacological agents alone or in combination. Among the clinical agents studied, it was found that EGF and Gefitinib were significantly potent against the HER2 overexpressing SKBR3 cell line, out of the panel of cell lines studied. EGF and Gefitinib showed a slightly different spectrum of activity from each other against the SKBR3 cell line. However, more research is needed to determine whether EGF could be used as a therapy for HER2 overexpressing breast cancer. Even though Gefitinib is currently used as a treatment in the clinic, the therapeutic window of this agent is drastically narrowed by its poor bioavailability, acquired resistance and systemic toxicity. Thus, in the current study, encapsulation of Gefitinib within the cavity of human heavy chain (H) apoferritin (AFt), provided a route for sustained release of Gefitinib from the H-AFt cavity, which demonstrated enhanced anti-tumour activity, at a longer duration against the SKBR3 cell line compared to Gefitinib alone. Overexpression of HER2 is considered to confer a more aggressive phenotype in breast cancer. Many patients have shown resistance to existing clinical agents such as Trastuzumab, demonstrating the need for novel therapies. Hence, 2 novel HER2 targeting human H and light chain (L)-AFt-fusion proteins were tested, and it was found that the nanoagent - H-AFt-fusion protein was very potent against the SKBR3 cell line compared to the L-AFt-fusion protein. This novel H-AFt-fusion protein abolished SKBR3 colony formation completely, caused a G1 arrest and a reduction in the orchestration of S and G2/M cell cycle events and also induced a large SKBR3 apoptotic population demonstrating its potent cytotoxic effects. Furthermore, this agent down-regulated the HER2 protein remarkably which resulted in significant down-regulation of the RAS/MAPK, PI3K/AKT and JAK/STAT signal transduction pathways in SKBR3 cells. Previous research has shown that a combination of pharmacological agents are more effective against cancer than individual agents due to up-regulation of compensatory signalling pathways which cancer cells use to thrive and acquire resistance to agents. Thus, several agents were tested in combination. Out of the agents tested it was found that 3 dual PI3K/mTOR inhibitors were potent against the triple negative breast cancer cell line - MDA-MB 468 and the HER2 overexpressing SKBR3 cell line. Further, Gefitinib in combination with an experimental AhR ligand - 5F 203, showed synergistic growth inhibition against the SKBR3 cell line by inducing CYP1A1, thereby resulting in a large apoptotic population. It was observed that the effect of Gefitinib was mainly potentiated by the effect of 5F 203 within the agent combination. There is a momentous unmet medical need for the development of effective therapies that can stabilise or slow the progression of breast cancer, therefore, these results may contribute to existing knowledge or enhance further understanding of the HER signalling network and therapies targeting this network. It may also guide potential treatment options which might lead to significant improvements in breast cancer therapy in the clinic thereby personalising therapy for patients with breast cancer.

The induced pluripotent stem cells (iPSCs) technology has revolutionized disease modelling by enabling the generation of patient-specific pluripotent stem cells for the study of complex disorders such as cancer. Somatic cell reprogramming through iPSCs induces global epigenetic reconfiguration of the chromatin which converts cancer cells to an embryonic stem cell-like state with potential reversion of tumorigenicity. Therefore, reprogramming can be used to answer the question as to whether epigenetic alterations alone can be sufficient to induce carcinogenesis, independent of genetic defects. In addition, it can used to dissect the relative contribution of genetics and epigenetics and epigenetics to tumorigenicity. In this study, the triple negative breast cancer (TNBC) cell line BT-549 and oestrogen receptor positive (ER+) cell line MCF7 were successfully reprogrammed by using the non-integrative episomal vectors expressing OCT4, SOX2, L-MYC, KLF4, LIN28, EBNA1, shRNA against TP53, and microRNA-302/367 cluster together with treatment of sodium butyrate. Pluripotency of cancer-derived iPSCs was confirmed by RT-PCR, RT-qPCR and immunofluorescence staining for expression of pluripotency markers. Differentiation potential of iPSCs was also assessed by using in vitro differentiation either spontaneous or directed to the mammary lineage. Functional assays indicated potential loss of tumorigenicity in re-differentiated cells derived from cancer iPSCs. The same approach was applied to study an immortalised, non-malignant mammary epithelial cell line MCF10A and two of its derived isogenic lines harbouring the two most frequent mutations in breast cancer, PIK3CAH1047R (+/-) and TP53(-/-), created by using CRISPR-Cas9 gene editing. Reprogramming induced a tumorigenic phenotype in iPSCs (PIK3CAH1047R (+/-) isogenic line only) and re-differentiated progenies (in both wild type MCF10A and PIK3CAH1047R (+/-) cell lines), suggesting the contribution of PIK3CA mutation in enhancing malignant transformation. Results in this study suggested that epigenetics alone and/or its interaction with genetic defects (e.g. PIK3CA mutation) has significant impact on breast cancer carcinogenesis. The dissection of the molecular mechanisms underlying the loss and gain of tumorigenicity using the iPSC models generated in this study could provide general understandings on breast carcinogenesis, which in turn could have important clinical implications.

ABSTRACT INVESTIGATON OF CHEMOPREVENTIVE PROPERTIES OF URTICA DIOICA L., IN MCF-7 AND MDA231 BREAST CANCER CELL LINES. G&uuml<br>ler, Elif Ph.D., Biological Sciences Department Supervisor : Prof. Dr. Mesude

In this study, meta-analysis of diagnostic tests, Summary Receiver Operating Characteristic (SROC) curve, bivariate random effects and Hierarchical Summary Receiver Operating Characteristic (HSROC) curve theories have been discussed and accuracy in literature of Fine Needle Aspiration (FNA) biopsy that is used in the diagnosis of masses in breast cancer (malignant or benign) has been analyzed. FNA Cytological (FNAC) examination in breast tumor is, easy, effective, effortless, and does not require special training for clinicians. Because of the uncertainty related to FNAC&lsquo<br>s accurate usage in publications, 25 FNAC studies have been gathered in the meta-analysis. In the plotting of the summary ROC curve, the logit difference and sums of the true positive rates and the false positive rates included in the meta-analysis&lsquo<br>s codes have been generated by SAS. The formula of the bivariate random effects model and hierarchical summary ROC curve is presented in context with the literature. Then bivariate random effects implementation with the new SAS PROC GLIMMIX is generated. Moreover, HSROC implementation is generated by SAS PROC HSROC NLMIXED. Curves are plotted with RevMan Version 5 (2008). It has been stated that the meta-analytic results of bivariate random effects are nearly identical to the results from the HSROC approach. The results achieved through both random effects meta-analytic methods prove that FNA Cytology is a diagnostic test with a high level of distinguish over breast tumor.

<strong>Background:</strong> Our laboratory identified ELTD1, an orphan GPCR belonging to the adhesion GPCR family (aGPCR), as a novel regulator of angiogenesis and a potential anti-cancer therapeutic target. ELTD1 is normally expressed in both endothelial cells and vascular smooth muscle cells and expression is significantly increased in the tumour vasculature. The aim of this project was to analyse ELTD1's function in endothelial cells and its role in breast cancer. <strong>Method:</strong> 62 sequenced vertebrate genomes were interrogated for ELTD1 conservation and domain alterations. A phylogenetic timetree was assembled to establish time estimates for ELTD1's evolution. After ELTD1 silencing, mRNA array profiling was performed on primary human umbilical vein endothelial cells (HUVECs) and validated with qPCR and confocal microscopy. ELTD1's signalling was investigated by applying the aGPCR ‘Stinger/tethered-agonist Hypothesis'. For this, truncated forms of ELTD1 and peptides analogous to the proposed tethered agonist region were designed. FRET-based 2^nd messenger (Cisbio IP-1;cAMP) and luciferase-reporter assays (NFAT; NFÎoB; SRE; SRF-RE; CREB) were performed to establish canonical GPCR activation. To further investigate ELTD1's role in endothelial cells, ELTD1 was stably overexpressed in HUVECS. Functional angiogenesis assays and mRNA array profiling were then performed. To investigate ELTD1 in breast cancer, a panel of cell lines representative of all molecular subtypes were screened using qPCR. Furthermore, an exploratory pilot study was performed on matched primary and regional nodal secondary breast cancers (n=43) which were stained for ELTD1 expression. Staining intensity was then scored and compared with relapse free survival and overall survival. <strong>Results:</strong> ELTD1 arose 435 million years ago (mya) in bony fish and is present in all subsequent vertebrates. ELTD1 has 3 evolutionary variants of which 2 are most common: one variant with 3 EGFs and a variant with 2 EGFs. Additionally, ELTD1 may be ancestral to members of aGPCR family 2. HUVEC mRNA expression profiling after ELTD1 silencing showed upregulation of the mitochondrial citrate transporter SLC25A1, and ACLY which converts cytoplasmic citrate to Acetyl CoA, feeding fatty acid and cholesterol synthesis, and acetylation. A review of lipid droplet (fatty acid and cholesterol) accumulation by confocal microscopy and flow cytometry (FACS) revealed no changes with ELTD1 silencing. Silencing was also shown to affect the Notch pathway (downregulating the Notch ligand JAG1 and target gene HES2; upregulating the Notch ligand DLL4) and inducing KIT, a mediator of haematopoietic (HSC) and endothelial stem cell (ESC) maintenance. Signalling experiments revealed that unlike other aGPCRs, ELTD1 does not couple to any canonical GPCR pathways (Gαi, Gαs, Gαq, Gα12/13). ELTD1 overexpression in HUVECS revealed that ELTD1 induces an endothelial tip cell phenotype by promoting sprouting and capillary formation, inhibiting lumen anastomoses in mature vessels and lowering proliferation rate. There was no effect on wound healing or adhesion to angiogenesis associated matrix components. Gene expression changes following ELTD1 overexpression included upregulation of angiogenesis associated ANTRX1 as well as JAG1 and downregulation of migration associated CCL15 as well as KIT and DLL4. In breast cancer, none of the representative breast cancer cell lines screened expressed ELTD1. ELTD1 breast cancer immunohistochemistry revealed higher levels of vascular ELTD1 staining intensity within the tumour stroma contrasted to normal stroma and expression within tumour epithelial cells. Additionally, ELTD1 expression in tumour vessels was differentially expressed between the primary breast cancer microenvironment and that of the matched regional node. Due to the small size of the pilot study population, survival comparisons between the various subgroups did not yield significant results. <strong>Conclusion:</strong> ELTD1 is a novel regulator of endothelial metabolism through its suppression of ACLY and the related citrate transporter SLC25A1. ELTD1 also represses KIT, which is known to mediate haematopoietic and endothelial progenitors stem cell maintenance, a possible mechanism through which endothelial cells maintain terminal endothelial differentiation. ELTD1 does not signal like other adhesion GPCRS with CTF and FL forms of ELTD1 not signalling canonically. Additionally, ELTD1 regulates various functions of endothelial cell behaviour and function, inducing an endothelial tip cell phenotype and is highly evolutionarily conserved. Lastly, ELTD1 is differentially expressed in tumour vessels between primary breast cancer and regional nodal metastases and is also expressed in a small subset of breast cancer cells in vivo despite no cancer cell lines expressing ELTD1. The pilot study investigating ELTD1 in the primary breast cancer and regional involved nodes will be followed up with a larger study including the investigation of ELTD1 in distant metastases.

Mucin deficiency is a possible cause of tear film instability. The important sources of mucin are the conjunctival goblet cells and the non-goblet conjunctiva! epithelial cells where the mucin-like glycoproteins are stored in the mucous secretory vesicles. This thesis addresses the relationship between the quantity of the mucin sources and tear film physiology in a group of normal to marginal dry eye Chinese subjects. Chinese subjects were used in this study because they were found to have a lower tear break-up time than Caucasians (Cho and Brown, 1993). Impression cytology techniques were used to obtain conjunctiva! cells and goblet cells from the bulbar conjunctiva! of 61 subjects in order to study the relationship of the goblet cell density and tear film stability. The tear function tests included subjective dry eye symptoms, non-invasive tear break-up time, phenol red thread test and tear break-up time. A second source of mucin supply and tear film stability was also studied. This mucin supply comes from the mucous secretory vesicles of the conjunctival epithelial cells. To study the anatomical structure, the ultrastructure of the conjunctiva! epithelial cells was investigated under the transmission electron microscope. The findings revealed that goblet cell density did not show any correlation with severity of dry eye symptoms, non-invasive tear break-up time, phenol red thread test or tear break-up time test. There are three possibilities for this finding: Deficiency in goblet cell density is not associated with mucin deficiency but mucin de2. Mucin deficiency is not associated with low tear stability but deficiency in goblet cell density remains associated with mucin deficiency. 3. Deficiency in goblet cell density is not associated with mucin deficiency and mucin deficiency is not associated with low tear stability.ficiency remains associated with low tear stability. The negative finding could also be due to the relatively normal and healthy subjects used in the present study. They did not show a great reduction in goblet cell density that could cause an impact on the result. In the counting of goblet cell density using the impression cytology technique, the imprints of the goblet cells might be obtained instead of the whole goblet cells. There was also no significant correlation between dry eye symptoms and tear function tests such as the non-invasive tear break-up time, phenol red thread test and tear break-up time test. This study also found that the gender of the age group between 18 to 28, and contact lens wear of up to three years, would not affect goblet cell density, dry eye symptoms, non-invasive tear break-up time, phenol red thread test and tear break-up time. There was no significant difference in the number of mucous secretory vesicles, microvilli and cell invaginations in the conjunctiva} epithelial cells of subjects with high and low tear stabilities. Therefore, the tear film function may not be associated with the presence of the number of mucous vesicles, microvilli and cells invaginations. On the other hand, the finding could also be due to a small subject sample and the inter-group difference in tear stability might be too small to reflect a difference in the assessment of the ultrastructures. However, a new technique of cell processing for transmission electron microscopy work was also developed for specimens obtained using the impression cytology technique.

Research Doctorate - Doctor of Philosophy (PhD)<br>Breast cancer is the most prevalent cancer in women in the western world including Australia. Early detection and accurate diagnosis of new breast lesions is essential for appropriate medical management. Fine needle aspiration (FNA) is a cytological investigative tool commonly used to provide the initial pathological diagnosis of breast lesions. An atypical cytology report (C3) is an ambiguous or equivocal result. This uncertainty creates a dilemma and a more invasive investigative procedure such as core biopsy or incisional biopsy may be required, which comes at greater cost and anxiety to the patient. The aims of this project were to understand the true nature of C3, to determine the underlying causes of C3 and to devise a strategic approach to minimise its use without compromising the other cytological categories. The practical aims were to create a greater understanding of the issue and to produce a collective uniform approach to reporting atypical breast cytology cases thereby refining its use. The results of a blind rescreen of 256 consecutive C3 cases were statistically analysed. From these results, a cytomorphological approach to assess the risk of malignancy was developed and tested against a validation set of 230 subsequent C3 cases. Various strategies have been developed to reduce the incidence of the C3 category. Extrinsic factors can be easily reduced by greater involvement by cytology staff in the FNA procedure. Intrinsic factors can be understood and considered when allocating cases into C3. Specific diagnoses, such as papillary neoplasm can direct more appropriate definitive management. The cytologists in our institution have gained greater awareness of the atypical category by actively participating in the project and by having access to teaching resources and examples. The benefits manifest as financial, medical and social enhancements. It is hoped that some of these approaches will be taken up by other institutions in Australia and internationally.

碩士<br>國立中山大學<br>醫務管理研究所<br>96<br>Objective: The incidence rate and mortality of breast cancer are increasing in Taiwan during recent years. The incidence rate of breast cancer is ranked number one among top ten female cancers, and the mortality of breast cancer is ranked fourth among cause of death for female cancer sufferers. The most common age group for breast cancer is between 40 and 50 years old. Breast cancer causes huge disease burdens for individual, family and society. The breast sonography and fine needle aspiration cytology (FNAC) are common screening methods for breast cancer diagnosis. Nevertheless, little study has focused on the benefits of combing these two methods in clinical application. This study aims to fill such research gap. Method: This study conducted medical chart reviews and collected 2,776 observations that were under breast sonography and FNAC examination from a regional hospital locates in southern Taiwan. The diagnosis categories for sonography include: malignant, benign, and probably benign tumor. The diagnosis categories for FNAC include: malignant, benign, and suspicious for malignant. Results: Among 2,776 observations, there were 555 observations (20%) had operation in the studied hospital. The operation results indicated that 205 (36.9%) observations were with malignant status, and 350 (63.1%) observations were with benign status. The diagnosis categories of both sonography and FNAC were significantly associated with the operation results (p<0.001). The FNAC had specificity in 100%, false positive ratio in zero, and positive predictive value in 100%. The Odds ratios for sonography diagnosis categories, age groups, and tumor sizes were OR=4.132 (95%CI: 1.5–11.6), OR=31.957 (95% CI: 3.7–272.4), OR=0.457 (95% CI: 0.1–1.5), respectively. When combining sonography and FNAC in parallel tests, the diagnosis accuracy was 89.2%, sensitivity was 90.2%, specificity was 88.6%, positive predictive value was 82.2%, and negative predictive value was 93.9%. When combining sonography and FNAC in serial testing, the diagnosis accuracy was 88.1%, sensitivity was 67.8%, specificity was 100%, positive predictive value was 100%, and negative predictive value was 84.1%. Conclusion: Combining sonography and FNAC in breast cancer diagnosis can increase the accuracy, decrease false positive ratio and false negative ratio. These two methods can be conducted during outpatient visit and are fast, accurate and cost-effective tools for breast cancer diagnosis. These two methods particularly appropriate for younger female patients for early screening, early intervention, and may increase the survival rates.

碩士<br>國立嘉義大學<br>資訊工程學系研究所<br>95<br>According to the statistic data from the Department of Health, Executive Yuan, R.O.C, breast cancer is the fourth leading cancer for Taiwan women. It causes great threat to women’s life in Taiwan. There how to identify the breast cancer as early as possible and remedy efficient is very important to women. The most important clinical feature of breast cancer is the touchable knot in women’s breast. When a woman finds the knot of breast by herself, she should go to accept examination as soon as possible, and if the doctor discovers possible symptoms of breast cancer, a further inspection should be arranged. The doctor could use fine needle aspiration or core needle aspiration to obtain specimens of breast tissue, identify the disease based on cytology and histology and arrange treatment as early as possible. The main goal of our study is to analyze the cell images of breast cancer by using cytology and image processing techniques. First, nuclei were isolated from the sampled image. Features related to the diagnosis of breast carcinoma were extracted to identify the sampled image whether contains normal or abnormal cells. Therefore, it may assist physicians evaluate the breast carcinoma as early as possible. A sequences of cell images were acquired from the fine needle smears or core needle biopsy on microscope. We utilize many image processing techniques such as color space transformation, thresholding, contrast enhancement, mathematical morphology, multiple active contour model in order to detect the nucleus. Then we separate the overlapped nuclei, measure some nucleus features such as area, curvature, compactness and texture respectively, and determine the thresholds for normal or abnormal cell, in terms of these features according to pathologist’s discussions. Then we compare the identification results with the macrography result evaluated by pathologist, as the accuracy, sensitivity and specificity in order to evaluate the identification performance of the proposed system. Finally we selected all of the feature parameters as training data and test data, and feed these datas into support vector machine (SVM) to evaluate the performance of the proposed system. This system provides diagnostic and screening information to the pathologist to decrease the load of the pathologists. The experimental results also reveal that the proposed system can obtain good accuracy.

Inflammatory Breast Cancer (IBC) is a rare, lethal, and understudied form of breast cancer. Although affecting 1-2% of the population, the remission rate is half that of the spectrum of other breast cancers, and most cases present in the advanced stages due to rapid undetectable development. Of the diagnosed cases, systemic chemotherapeutics are relatively ineffective in comparison to non-IBC breast cancer cases, indicating other unique mechanisms driving IBC progression. Historically, the specific sensitivities of a particular tumor type or subtype have been linked to genetic alterations that represent addiction hubs, such as hyperactivation of oncogenes due to mutation. Although some efforts have been made to characterize the molecular fingerprint of inflammatory breast cancers (IBCs), unfortunately, no clinical application has emerged from these studies. Thus, we decided to utilize a different strategy to identify the Achilles' heel of IBC cells. Using shRNA libraries, we performed an unbiased genome-wide loss-of-function screen comparing the gene functions required for survival of IBC and non-IBC cells. Histone deacetylase 6 (HDAC6) emerged as one of the top genes required for IBC cell survival and was further validated. HDAC6 is vital in the cell's unfolded protein response (UPR) to clear misfolded or toxic protein, and IBC cells proved to be preferentially sensitive to this aspect of HDAC6 inhibition, displaying increased protein accumulation, ER stress indicators, and subsequent apoptosis upon failure to clear or refold accumulated proteins. These data indicate HDAC6 is a crucial gene required for IBC cell line survival, in part due to its function in IBC cell UPR. Furthermore, emerging orally bioavailable agents for HDAC6 inhibition make it a promising candidate towards tailored therapeutic implementation in IBC patient trials.

The aim of this thesis is to establish a simple physical framework that captures and predicts key aspects of the spatial organization of cells in models of breast cancer, while also probing the downstream consequences of particular tumor composition and cell organization within tumors on disease progression. An in vitro model of tumor heterogeneity and a complementary minimal computational model of cell sorting were used to accomplish these goals. We evaluated the tendencies of cells to sort and segregate, the factors driving the sorting process, and the mechanisms of invasion that cells exhibited as a result of different composition and cellular organization. Chapter 1 presents background information on breast cancer progression, the origins and consequences of heterogeneity in tumors and their local microenvironment, existing theoretical and computational approaches to explain cell segregation in tissues, and commonly employed experimental models of cancer invasion. Chapter 2 explores cell sorting of healthy and cancerous breast cells in an in vitro tumor model. This work was motivated by previous observations that mixing genetically distinct breast cancer cells results in cell sorting and the formation of sharp boundaries between cell types, analogous to the segregation of cells during embryonic development. We examined cell segregation among six different breast cell lines and found that more invasive breast cancer cells tended to sort to the outside of mixed cell-type aggregates, such that more aggressive cells were poised to invade the surrounding extracellular matrix. The particular sorting among all binary sets of breast cells studied was found to follow predictions of the differential adhesion hypothesis, which predicts cell sorting to be dependent on a combination of available adhesion proteins and actomyosin contractility. Differential adhesion was found to be a useful lens for not only rationalizing cell sorting tendencies but also directing the assembly of cells. In fact, we showed that through use of a simple contractility inhibiting agent, invasive cell types could be made to sort inside mixed-type aggregates, reducing subsequent invasion. In Chapter 3, we further probed the applicability of differential adhesion frameworks for explaining cell segregation in cancer by employing a Cellular Potts model. Experimentally observed sorting patterns were replicated using a minimal model and varying only two parameters across simulated cell types – one that governs cell morphology and one that governs cell adhesion, thus validating the differential adhesion hypothesis as a useful minimal model to rationalize sorting in this system. Less invasive cell types were found to have more fluid-like character that drives the sorting process and leads to their positioning in the interior of simulated aggregates, surrounded by more invasive cells. We observed evidence of non-equilibrium behavior in certain less adhesive cell types, as well as the capacity for more adhesive cell types to enhance motility and fluidize those that otherwise demonstrate non-equilibrium, slow dynamics. Chapter 4 summarizes these findings and suggests future studies. Throughout this work, we show the value of applying existing views of cell sorting for rationalizing cell segregation and tumor organization in breast cancer. We find that cells sort in a predictable manner that relates to aspects of their adhesive character as captured by differential adhesion, which is shown experimentally to depend on co-regulation of adhesion protein function and actomyosin contractility. These same properties also dictate cell invasive strategy and efficiency, making this a critical area of study to enhance understanding of cancer invasion and metastasis. The drivers of spatial organization of cells in tumors and the consequences of particular organization remain an underexplored topic in breast cancer research. We argue that continued study in this area can yield improved understanding of the impacts of tumor heterogeneity on cancer progression.

Introduction: Despite intense research efforts and improvements to mortality rates, breast cancer remains the leading cause of premature cancer death for women. Identifying women at highest risk is vitally important for screening decisions that may have a critical impact on diagnosis and prognosis. A family history of breast cancer is a well-established risk factor, but it can be unreliable. An easily measured and accurate biomarker of breast cancer risk would be a significant advancement to public health by allowing targeted screening of women who would benefit most. It may also reveal underlying molecular mechanism that could illuminate pathways to prevention. Incidence rates of breast cancer have remained unmoved owing to the lack of known modifiable risk factors. This may be due in part because most breast cancer research has focused on behaviors and exposures in, or recalled from, adulthood. The studies detailed in this dissertation seek to investigate the associations between oxidative stress and breast cancer risk in both adult women and adolescent girls with a family history of breast cancer. Methods: To determine the relationship between oxidative stress and breast cancer risk in adult women with a family history of breast cancer we measured and compared urinary levels of 8-OxodG and F2-Isoprostane in a prospective matched case control study nested within the New York Breast Cancer Family Registry. Cases (N=73) were individually matched with 2 controls on age, year of urine donation, menopausal status, and race. Conditional logistic regression methods were used to determine the odds of breast cancer from oxidative stress controlling for other risk factors for breast cancer and potential confounders. To better understand how oxidative stress levels change during puberty in girls and if such change is modified by a family history of breast cancer, we measured and compared levels of those same urinary biomarkers of oxidative stress in adolescent girls with and without a family history of breast cancer from the New York site of the Lessons in Epidemiology and Genetics of Adult Cancer from Youth cohort (LEGACY). Oxidative stress levels were measured both cross-sectionally at baseline and longitudinally every 6-months for up to 18-months. Linear regression was used for the cross-sectional analysis and repeated measures analysis using mixed models was employed for the longitudinal analysis. In both studies, biomarker levels were measured using well-established ELISA methods and adjusted for hydration status using specific gravity. Results: In the case control study of adult women we found that both 8-OxodG and F2-Isoprostane levels were significantly associated with a reduced risk of breast cancer after adjusting for BRCA1/2 mutation status, time between menarche and parity or menopause, and BMI (8-OxodG: β10-unit= -0.14, OR=0.87, p=0.03; F2-Isoprostane: β10-unit = -0.53, OR=0.59 , p=0.03). This inverse association was strongest among women under 50 and in women with a BMI below 25 for both biomarkers, and among women who reached menarche before age 14 for F2-Isoprostane. Overall, women in the highest tertile of either oxidative stress biomarker had approximately 50% reduced odds of breast cancer diagnosis. In our cross-sectional study of adolescent girls, we found that there was no significant difference in either oxidative stress biomarker in girls based on their family history of breast cancer. F2-Isoprostane levels were significantly associated with breast development measured by Tanner stage even after adjusting for age, age-specific BMI category and race (β=0.28, p=0.01). 8-OxodG levels were not significantly associated with age, BMI, race or Tanner stage at baseline but they were significantly associated with overweight/obese BMI but only among girls with a breast cancer family history (β=0.47, p=0.01). Change in 8-OxodG levels was significantly higher over the follow-up period in girls with a family history of breast cancer. This result remained significant after categorical measures of age, BMI, Tanner breast stage and race were added to the longitudinal model. F2-Isoprostane levels significantly increased in all girls over follow-up but this increase did not differ by family history of breast cancer, and the change was no longer significant our multivariate longitudinal analysis. Discussion: In both adult women and adolescent girls we found significant associations between oxidative stress and breast cancer risk. In adult women, low levels of urinary biomarkers of oxidative stress may promote cancer progression. During adolescence, girls with a family history of breast cancer may be exposed to higher rates of DNA oxidation that could result in genetic mutations. The relationships between oxidative stress, breast development, family history, and BMI should be the focus of future investigations.