Tumor initiation and progression is dependent on the acquisition and accumulation of multiple driver mutations that acti­vate and fuel oncogenic pathways and deactivate tumor suppressor networks. This complex continuum of non-stochastic genetic changes in accompaniment with error-prone mitoses largely explains why tumors are a mosaic of different cells. Contrary to the long-held notion that tumors are dominated by genetically-identical cells, tumors often contain many different subsets of cells that are remarkably diverse and distinct. The extent of this intratumor heterogeneity has bewildered cancer biologists’ and clinicians alike, as this partly illuminates why most cancer treatments fail. Unsurprisingly, there is no “wonder” drug yet available which can target all the different sub-populations including rare clones, and conquer the war on cancer. Breast tumors harbor ginormous extent of intratumoral heterogeneity, both within primary and metastatic lesions. This revelation essentially calls into question mega clinical endeavors such as the Human Genome Project that have sequenced a single biopsy from a large tumor mass thus precluding realization of the fact that a single tumor mass comprises of cells that present a variety of flavors in genotypic compositions. It is also becoming recognized that intratumor clonal heterogeneity underlies therapeutic resistance. Thus to comprehend the clinical behavior and therapeutic management of tumors, it is imperative to recognize and understand how intratumor heterogeneity arises. To this end, my research proposes to study two main features/cellular traits of tumors that can be quantitatively evaluated as “surrogates” to represent tumor heterogeneity at various stages of the disease: (a) centrosome amplification and clustering, and (b) mitotic frequency. This study aims at interrogating how a collaborative interplay of these “vehicles” support the tumor’s evolutionary agenda, and how we can glean prognostic and predictive information from an accurate determination of these cellular traits.

Like with many cancers, a single ovarian tumor can display remarkable diversity in genetics, epigenetics, expression profiles, microenvironment and cell differentiation and plasticity. This so-called intratumoral heterogeneity (ITH) is thought to greatly increase mortality by enabling tumors to adapt quickly to therapy, metastasize, and recur, thus the study of ITH holds great clinical significance. Clonal evolution and cancer stem cell (CSC) theory are two models for the initiation and propagation of a tumor, which offer differing views on the way that ITH is developed and maintained. In the clonal evolution model, cancer arises from a single cell and, through genetic instability, proliferates into a diverse population of daughter cells, which develop additional mutations and undergo Darwinian selection under the influence of the tumor microenvironment. Each cell of the clonal evolution model may be capable of initiating a tumor independently. In CSC theory, cancer arises from the transformation of a stem cell that has the capacity to self-renew and differentiate into a diverse population of daughter cells. Each cell is NOT capable of tumorigenesis as most are terminally differentiated and do not harbor self-renewing capabilities. According to CSC theory, small, rare subpopulations of CSCs persist throughout chemotherapy and are responsible for repopulating the heterogeneous tumor post-treatment. The hypothesis that CSCs may play a role in ovarian cancer progression is the subject of this thesis. Many studies have detected the presence of stem cell markers and dysregulated stem cell signaling pathways in ovarian cancer, but doubts remain as to the existence of ovarian CSCs; critics have pointed out inherent flaws in experimental designs meant to identify and characterize CSCs. For example, the presence of cancer cells which express the stem cell marker CD133 has been correlated to both positive and negative impacts on prognosis. Further challenging the study of ovarian CSCs is the lack of consensus on the true cell of origin for ovarian cancer - whether it be from the fallopian tube epithelium or ovarian surface epithelium, or elsewhere in the peritoneal cavity - this will have important implications for the identification and characterization of tumorigenic ovarian CSCs. Advocates of clonal evolution theory have put forth incredible effort to reveal the extent of inter and intra-tumoral heterogeneity in ovarian cancer, and from these data there has arisen a general consensus that cancer cell populations do evolve in a step-wise fashion, accumulating additional mutations over time. The involvement of cancer stem cells in this progression and how exactly they fit in (as a cell of origin or arising from genetic mutations), as well as their significance for different cancer types, is a question worth answering. Despite the challenges facing the study of ovarian CSCs, the clinical impact of cells with stem-like properties has been repeatedly demonstrated, especially with regard to metastatic processes and chemoresistance. Moreover, new drugs which target stem cell pathways have proven effective in the treatment of ovarian cancer. The existence of a rare subset of cells that have enhanced tumor-initiating properties is apparent in ovarian cancer, and more work is needed to characterize the unique identifiers and behavior of these cells in vivo. Future experiments involving lineage tracing promise to deepen our understanding of the nature of ovarian CSCs and address whether normal stem cells might serve as the cell of origin.

BACKGROUND: Breast cancer is the most commonly diagnosed cancer and second leading cancer death cause among females in the U.S.A. About 1 in 8 women in U.S will develop invasive breast cancer over the course of her lifetime. In 2013, 234,580 new invasive breast cancer cases are expected to occur in women within the US and approximately 64,640 non-invasive carcinomas in situ were diagnosed in 2013, most of which were ductal carcinoma in situ (DCIS). Along with technological advances, a wide variety of candidate biomarkers have been proposed for cancer diagnosis and prognosis, including DNA content and non-coding RNA. Current techniques for detecting DNA content abnormalities in formalin-fixed, paraffin-embedded (FFPE) tissue samples by flow cytometric analysis have used cells recovered from ≥50µm whole tissue sections. Here, in our first study, a novel core punch sampling method was investigated for assessing DNA content abnormalities and intratumoral heterogeneity in FFPE specimens. Secondly, long non-coding RNAs (lncRNAs) has been examined. LncRNA participates in a broad spectrum of biological activities by diverse mechanisms and its dysregulation is associated with tumorgenesis. Some lncRNAs may function as oncogenes (O) and others as tumor suppressor genes (TSG). To date, lncRNA has been investigated primarily by qRT-PCR and RNA sequencing. This study has examined the relationship of lncRNA expression patterns to breast tumor pathology by chromogenic in situ hybridization (CISH). METHODS: Firstly, FFPE breast carcinoma specimens were selectively targeted using 1.0 mm diameter punch needles. Extracted cores were assayed by flow cytometry using a modified-Headley method. Secondly, the lncRNA expression levels of 6 lncRNAs: HOTAIR, H19, KCNQ1OT1, MEG3, MALAT11 and Zfas1, was examined by RNAscope® CISH using FFPE breast tissue microarrays (TMAs) comprising normal adjacent epithelia (NA), DCIS, and invasive carcinoma (IC) from 46 patients. LncRNA associate polycomb complex protein EZH2 was evaluated by immunohistochemistry (IHC). LncRNA data was also compared to standard breast tumor data including ER, PR, Her2 and Ki67 IHC. SYSTAT version 11 statistical package was used to perform for all the tests. RESULTS: Following optimization experiments of the core punch flow cytometric approach, DNA index and percent S-phase fraction intratumoral heterogeneities were detected in 10/23 (44%) and 11/23 (47%) specimens respectively. The lncRNA CISH study utilized a TMA that contained 36 spots of NA breast tissues, 34 DCIS spots and 43 IC spots. HOTAIR CISH staining was significantly stronger in IC than DCIS (p CONCLUSION: Core-punching is an effective alternative to whole specimen sectioning and shows that macro-level genomic heterogeneity is common even within a single FFPE block. The interrelationship of DNA content heterogeneity to other forms of heterogeneity requires further study. RNAscope CISH supports bright-field microscopy investigations of lncRNA expression in FFPE tissue specimens. HOTAIR, H19 and KCNQ1OT1 may be potential breast cancer biomarkers, both HOTAIR and H19 may be a marker for DCIS at increased risk of progression to invasive cancer. HOTAIR, in particular, may be a predictor for invasive cancer grade.

Résumé : Nous avons d’abord souhaité, dans ce travail de thèse, préciser les mécanismes moléculaires conduisant à l'expression ectopique du récepteur du GIP (glucose-dependent insulinotropic polypeptide receptor, GIPR) dans des adénomes somatotropes provenant de patients présentant une acromégalie avec une réponse paradoxale (stimulation) de l’hormone de croissance au glucose par voie orale. Nous avons montré que l’expression ectopique de GIPR se produit par une activation transcriptionnelle hypomorphe du gène GIPR associée à des anomalies de méthylation dans le corps du gène. L’activation de la voie AMP cyclique par le GIP postprandial dans les adénomes exprimant le GIPR peut représenter un mécanisme alternatif de la tumorigenèse somatotrope en l’absence de mutations de l’oncogène GNAS.Nous rapportons d’autre part une analyse cytogénétique approfondie des adénomes somatotropes, qui nous a permis de définir deux groupes d'adénomes, un groupe à faible altération du nombre de copies et un groupe à forte altération du nombre de copies. Deux tumeurs présentaient des réarrangements chromosomiques complexes avec une signature typique de chromothripsis, et une architecture sous-clonale incluant jusqu’à six populations cellulaires différentes, témoignant d’une hétérogénéité intratumorale importante.Dans une collection d'adénomes hypophysaires invasifs comportant la portion intrasellaire et la portion envahissante le sinus caverneux, nous avons montré par RNA-seq des profils d'expression génique divergents, apportant des arguments supplémentaires en faveur de l'hétérogénéité intratumorale dans ces tumeurs bénignes. Les échantillons tumoraux provenant de portions invasives ont montré une surexpression de la voie de transition épithélio-mésenchymateuse et des marqueurs de cellules souches cancéreuses soulignant leur rôle potentiel dans l’acquisition du phénotype invasif des cellules adénomateuses hypophysaires. / AbstractIn this work, we explored the molecular mechanisms of ectopic glucose-dependent insulinotropic polypeptide receptor (GIPR) expression in somatotroph adenomas from patients with acromegaly displaying a paradoxical GH increase to oral glucose. We showed that ectopic GIPR expression occurs through hypomorphic transcriptional activation of GIPR gene likely driven by DNA methylation changes. Activation of the cAMP pathway by postprandial GIP may represent an alternative tumorigenic mechanism in GIPR expressing somatotroph adenomas without driver mutations in GNAS oncogene. Cytogenetic profiling defined two groups of adenomas, a low-copy-number alteration (CNA) group and a high-CNA group.Two tumor samples displayed complex chromosomal rearrangements compatible with chromothripsis and showed subclonal architecture with up to six distinct cell population in each tumor, demonstrating an important intratumor heterogeneity.In a collection of invasive pituitary adenomas including the non-invasive intrasellar portions and the portions invading the cavernous sinuses, we showed by RNA-seq different gene expression profiles, providing supplemental evidence for the intratumoral heterogeneity in these benign tumors. Tumor samples from invasive portions showed up-regulation of the epithelial-mesenchymal transition pathway and increased expression of cancer stem-cell markers highlighting their potential role in pituitary tumor cell invasive behavior.