Prostate Tumor Volume Measurement on Digital Histopathology and Magnetic Resonance Imaging

An accurate assessment of prostate tumour burden supports appropriate treatment selection, ranging from active surveillance through focal therapy, to radical whole-prostate therapies. For selected patients, knowledge of the three-dimensional locations and sizes of prostate tumours on pre-procedural imaging supports planning of effective focal therapies that preferentially target tumours, while sparing surrounding healthy tissue. In the post-prostatectomy context, pathologic measurement of tumour burden in the surgical specimen may be an independent prognostic factor determining the need for potentially life-saving adjuvant therapy. An accurate and repeatable method for tumour volume assessment based on histology sections taken from the surgical specimen would be supportive both to the clinical workflow in the post-prostatectomy setting and to imaging validation studies correlating tumour burden measurements on pre-prostatectomy imaging with reference standard histologic tumour volume measurements. Digital histopathology imaging is enabling a transition to a more objective quantification of some surgical pathology assessments, such as tumour volume, that are currently visually estimated by pathologists and subject to inter-observer variability. Histologic tumour volume measurement is challenged by the traditional 3–5 mm sparse spacing of images acquired from sections of radical prostatectomy specimens. Tumour volume estimates may benefit from a well-motivated approach to inter-slide tumour boundary interpolation that crosses these large gaps in a smooth fashion. This thesis describes a new level set-based shape interpolation method that reconstructs smooth 3D shapes based on arbitrary 2D tumour contours on digital histology slides. We measured the accuracy of this approach and used it as a reference standard against which to compare previous approaches in the literature that are simpler to implement in a clinical workflow, with the aim of determining a method for histologic tumour volume estimation that is both accurate and amenable to widespread implementation. We also measured the effect of decreasing inter-slide spacing on the repeatability of histologic tumour volume estimation. Furthermore, we used this histologic reference standard for tumour volume to measure the accuracy, inter-observer variability, and inter-sequence variability of prostate tumour volume estimation based on radiologists’ contouring of multi-parametric magnetic resonance imaging (MPMRI). Our key findings were that (1) simple approaches to histologic tumour volume estimation that are based on 2- or 3-dimensional linear tumour measurements are more accurate than those based on 1-dimensional measurements; (2) although tumour shapes produced by smooth through-slide interpolation are qualitatively substantially different from those obtained from a planimetric approach normally used as a reference standard for histologic tumour volume, the volumes obtained were similar; (3) decreasing inter-slide spacing increases repeatability of histologic tumour volume estimates, and this repeatability decreases rapidly for inter-slide spacing values greater than 5 mm; (4) on MPMRI, observers consistently overestimated tumour volume as compared to the histologic reference standard; and (5) inter-sequence variability in MPMRI-based tumour volume estimation exceeded inter-observer variability

Wnt-Targeted Therapy to Treat Myeloma-Induced Bone Loss and Prevent Disease Relapse

Multiple myeloma (MM) is characterised by an expansion of malignant plasma cells in the bone marrow, systemic bone loss and destructive osteolytic bone lesions. These are mediated by an imbalance in bone remodelling, in which bone resorption is exacerbated and bone formation is suppressed. More than 90% of MM patients present with osteolytic lesions that can lead to pain and increased risk of fracture, significantly impacting quality of life. Bone-targeted treatments currently used in the clinic can suppress lesion progression and reduce fracture risk, however these agents cannot replace lost bone and patients continue to fracture. The canonical Wnt/beta-catenin signaling pathway is a potent regulator of bone formation. We sought to examine whether Wnt-targeted agents could be of therapeutic benefit to rebuild lost bone and improve bone strength in MM patients. Utilising the 5TGM1eGFP murine model of myeloma, this thesis first demonstrated that a novel anti-LRP6 antibody, which potentiates LRP6-mediated Wnt1 class signaling, prevented myeloma-induced bone disease primarily through reduced bone resorption. The greatest protection against myeloma-induced bone loss however was shown when anti-LRP6 was used in combination with the bone anabolic agent, anti-DKK1, in the 5TGM1eGFP murine model of myeloma; compared to anti-LRP6 single treatment approaches. Importantly, stimulation of the Wnt/beta-catenin signaling pathway through these therapeutic strategies does not exacerbate tumour growth, which was a concern raised previously in the literature. Moreover, this thesis established in vivo murine models of bone repair following myeloma-induced bone destruction utilising the 5T2MM and Vk*Myc cell lines. Improving upon bone repair methods published recently, this thesis provides the first evidence that anti-SOST antibody treatment could repair osteolytic lesions within the 5T2MM model of myeloma. Importantly, this thesis is the first to characterise the skeletal destruction and osteolytic lesions associated with the Vk*Myc murine model of myeloma. Results arising from this thesis will define novel therapeutic strategies that could be beneficial to address the skeletal complications associated with MM. Murine models of bone repair established in this investigation will provide opportunities for the field to explore the potential for these Wnt-targeted agents to repair bone following cancer-induced bone destruction

Comparative Analysis of Tissue Reconstruction Algorithms for 3D Histology

Motivation: Digital pathology enables new approaches that expand beyond storage, visualization or analysis of histological samples in digital format. One novel opportunity is 3D histology, where a three-dimensional reconstruction of the sample is formed computationally based on serial tissue sections. This allows examining tissue architecture in 3D, for example, for diagnostic purposes. Importantly, 3D histology enables joint mapping of cellular morphology with spatially resolved omics data in the true 3D context of the tissue at microscopic resolution. Several algorithms have been proposed for the reconstruction task, but a quantitative comparison of their accuracy is lacking. Results: We developed a benchmarking framework to evaluate the accuracy of several free and commercial 3D reconstruction methods using two whole slide image datasets. The results provide a solid basis for further development and application of 3D histology algorithms and indicate that methods capable of compensating for local tissue deformation are superior to simpler approaches.publishedVersionPeer reviewe

Registration of histology and magnetic resonance imaging of the brain

Combining histology and non-invasive imaging has been attracting the attention of the medical imaging community for a long time, due to its potential to correlate macroscopic information with the underlying microscopic properties of tissues. Histology is an invasive procedure that disrupts the spatial arrangement of the tissue components but enables visualisation and characterisation at a cellular level. In contrast, macroscopic imaging allows non-invasive acquisition of volumetric information but does not provide any microscopic details. Through the establishment of spatial correspondences obtained via image registration, it is possible to compare micro- and macroscopic information and to recover the original histological arrangement in three dimensions. In this thesis, I present: (i) a survey of the literature relative to methods for histology reconstruction with and without the help of 3D medical imaging; (ii) a graph-theoretic method for histology volume reconstruction from sets of 2D sections, without external information; (iii) a method for multimodal 2D linear registration between histology and MRI based on partial matching of shape-informative boundaries

Tumour heterogeneity during the progression of metastatic breast cancer and anti-tumour effects of the novel FAK inhibitor BI 853520 in breast cancer

Over the past decades, reasonable progress has been made in the understanding of breast cancer biology and the treatment of the primary tumour. However, the molecular contribution of multiple cancer cell clones on the various steps of tumour progression is still poorly understood. Aside from that, standard of care treatments, like the chemotherapeutic reagents cyclophosphamide and docetaxel, are rarely able to cure breast cancer patients and the overall survival rates for metastatic disease remain poor. Only in 2018, more than 620,000 women lost their live to breast cancer, mostly due to the presence of tumour heterogeneity, an emerging drug resistance and the formation of secondary lesions. This exemplifies the unmet medical need to an in-depth understanding of tumour heterogeneity during the progression of metastatic breast cancer to finally develop new targeted therapies for this presently incurable disease. The first project has aimed to assess clonal heterogeneity during tumour progression using the MMTV-PyMT mouse model of metastatic breast cancer expressing the Confetti lineage reporter. For this purpose, mammary epithelial cells have been induced to express one of the four Confetti reporter fluorescent proteins. The outgrowth of clonal cell populations has been analysed when the maximum tumour volume comprising all stages (normal, hyperplasia, adenoma, carcinoma, pulmonary metastases) had been reached. The Confetti lineage tracing system initially visualized the emergence of clonal heterogeneity, which culminated in clonal restriction during carcinogenesis and pointed towards a polychromatic metastatic spread. Laser capture microdissection, RNA sequencing and comparative gene expression analysis of various clonal lesions indicated a substantial level of heterogeneity across and also within the various stages of tumour progression. This intra-stage tumour heterogeneity manifested by differences in proliferation, oxidative phosphorylation and cell death and could also be observed in human breast cancer biopsies. This novel understanding of clonal variation and intra-stage heterogeneity needs to be implemented in diagnosis and therapeutic options. In the past years, rising efforts have been made to develop agents targeting molecules and signalling pathways that are specifically present in breast cancer cells. Previous studies have linked an overexpression of focal adhesion kinase (FAK) – a cytoplasmic tyrosine kinase – with the initiation and progression of a wide variety of malignancies, including breast cancer. This correlation of FAK and cancer, together with its role in cell migration, invasion, and proliferation, propose FAK as an attractive target for cancer therapy. In collaboration with a pharmaceutical company, we have assessed and characterized the therapeutic potential and the biological effects of BI 853520, a novel, potent and selective small chemical inhibitor of FAK, in vitro and in several preclinical mouse models of breast cancer. We observed a significant reduction in primary tumour growth driven by an anti-proliferative effect of BI 853520. In contrast, dissecting its influence on metastasis revealed heterogenous effects at different levels of the metastatic cascade. Hence, manipulation of FAK activity with the novel FAK-inhibitor BI 853520 offers a promising anti-tumour approach for breast cancer therapy. In summary, my Ph.D. work delivered new insights into: - The existence of an intra-stage tumour heterogeneity, which is conferred by clonal variations in proliferation, oxidative phosphorylation and cell death. This novel understanding of an intra-stage heterogeneity could have a significant impact on a patient’s diagnosis and therapeutic response and should be implemented in clinical decision-making. - The therapeutic potential and biological effects of the novel FAK-inhibitor BI 853520 in vitro and in preclinical mouse models of breast cancer. This highlighted BI 853520 as a promising anti-proliferative approach for cancer therapy

Head and Neck Cancer Invasion: Contributions of Actin Regulatory Proteins and the Microenvironment

Metastasis of primary tumor lesions is the leading cause of cancer-related death. In head and neck cancer, a local-regional disease, metastasis is achieved mainly through invasion into surrounding tissue and spreads to cervical lymph nodes. Movement from the initial tumor site requires dynamic reorganization of the actin cytoskeleton, which utilizes the coordinated action of many actin regulatory proteins. However, there is increasing evidence that the tumor microenvironment is also a driver of invasion. This work aims to determine the contributions of proteins which regulate the actin cytoskeleton during head and neck cancer invasion both in vitro and in vivo, and provide details on how the HNSCC tumor microenvironment influences progression. This was accomplished, by the following Studies. In Study one, the actin binding protein coronin 1B is found to be amplified and overexpressed in invasive HNSCC patient samples, and a novel function in the regulation of protrusive membrane structures called invadopodia is described. Study two defines an in vivo role for the actin regulatory protein cortactin, which has been previously associated with more aggressive cancers in vitro and in patients. This work finds that cortactin expression is dispensable for tongue tumor invasion in a transgenic model of oral cancer, implicating the tumor microenvironment as being the major contributor to driving oral cancer invasion. Study three describes a technique for monitoring and biopsying cervical lymph nodes of mice using high frequency ultrasound. By using this technique, alterations in cervical lymph node size and blood flow were discovered in mice given the carcinogen 4-NQO to induce oral carcinogenesis. Collectively, these studies shed light on the importance of choosing comprehensive model systems for studying roles of actin binding proteins in cancer invasion

The role of endoglin in angiogenesis and its potential as an anti-angiogenic therapeutic target

Tumour growth and metastasis depend on the vascularization of tumours by angiogenesis. This is regulated by the combined action of several growth factors (e.g. vascular endothelial growth factor, VEGF) that are secreted by the growing tumour, and activate VEGF receptors (VEGFR) expressed on the surface of endothelial cells to stimulate new blood vessel formation. Therapies that target VEGF/VEGFR signalling have indicated that anti-angiogenic therapy may be a useful supplementary anti-cancer treatment in the clinic. In addition to VEGF, malignant cells secrete transforming growth factor (TGF)-β, which is thought to stimulate new blood vessel formation by interacting with endoglin, an endothelial co-receptor for TGF-β that regulates angiogenesis. However, it is not yet clear whether this property could also be utilised to inhibit angiogenesis and metastasis, consistent with endoglin acting as a therapeutic target in a clinical setting. Therefore, the aim of my project was to investigate the role of endoglin in tumour angiogenesis and metastasis and its potential as an anti-angiogenic therapeutic target. I used a conditional endoglin knockout mouse model, that was generated by combining a floxed endoglin allele with a tamoxifen inducible vascular specific Cre (Cdh5(PAC)Cre-ERT2). Angiogenesis was tested using the matrigel subdermal plug assay and was significantly less in endoglin-deficient adult mice compared with tamoxifen treated control mice. Subsequently, angiogenesis and metastasis were investigated using a subdermal lewis lung carcinoma (LLC) model. The growth of the primary tumours was initially reduced, suggesting that targeting endoglin may delay tumour progression at an early stage. However, there was no significant effect of endoglin loss on primary tumour growth at later stages of tumour progression. Furthermore, loss of endoglin was associated with a significant increase in metastases, in a similar way to recent findings for other anti-angiogenesis treatments. The reasons for this are not yet clear. iii In terms of animal health, endothelial specific loss of endoglin alone did not appear to cause any major adverse effects. Endoglin inducible knockout (Eng-iKOe) mice did not lose weight and appeared healthy (over two months). However, Eng-iKOe mice did exhibit abnormal venous enlargement close to matrigel plugs supplemented with angiogenic growth factors compared to control mice. There was no evidence for a similar response in the peritumoral vasculature. In parallel to the in vivo studies, I took advantage of combining the conditional Eng- iKO line and the „immortomouse‟ line to create conditionally immortalised Eng-iKO mouse lung endothelial cell lines (MLECs) to investigate the role of endoglin in regulating endothelial cell viability, proliferation and migration. In standard media, MLECs showed normal cell viability, proliferation and migration in the absence of endoglin. However, titration of the growth factor supplements did result in significant reduction in viability in the absence of endoglin, suggesting endoglin is important for maintaining endothelial cell viability. Although the exact mechanisms regulating the role of endoglin in angiogenesis are still unclear, this study has increased our understanding of the endothelial cell phenotype in pathophysiological conditions in the absence of endoglin. In particular, the finding that endoglin depletion delays tumour progression in the early stage but is associated with increased metastatic risk is important when considering appropriate utilisation of anti-endoglin therapy, which is already being given to cancer patients in phase I/II clinical trials.EThOS - Electronic Theses Online ServiceCancer Research UK (CRUK)GBUnited Kingdo

Analysis of tumour angio-architecture and blood flow using microcomputed tomography and lattice Boltzmann simulations.

The precise architecture of the vascular system is critical to its many specialised functions. In sharp contrast tumour vascular architecture is highly disorganised and dysfunctional. The reason for this is the grossly abnormal angiogenic signalling prevalent in the tumour microenvironment. Aberrant tumour vasculature is a key determinant of spatial and temporal heterogeneities of blood flows. Additionally, irregularities in the tumour vascular wall, a lack of functional lymphatics and a severely retarded trans-mural hydrostatic pressure gradient also diminish convective transport out of the vessels. Diffusion therefore remains the dominant transport mode in tumours and presents a considerable barrier to macromolecular therapy (e.g. Antibody-directed enzyme prodrug therapy (ADEPT)). A number of recent studies of vascular morphology in both clinical and xenograft tumours have demonstrated the existence of type-specific architectures. Precisely how these type-specific architectures translate to blood flow through the vascular system had not been determined. To address this we have developed a method for studying the 3D architecture of the tumour and simulating flows through it. This technique uses corrosion casts to capture the 3D tumour vascular system. 3D morphometry was determined by stereoimaging and X-ray micro-computed tomography. A computational fluid dynamics model was then used to study the hydrodynamics of the vascular networks. My results show that vessel structure and architecture varies in clinical colon cancers, but these differences were substantially smaller than those of two human colorectal xenografts (LS147T and SW1222) commonly used in pre-clinical studies. The results also provide evidence that LS147T is, in general, a closer model to most clinical colorectal tumours than SW1222. To our knowledge this is the first attempt to utilise X-ray micro-computed tomography to study vascular corrosion casts of tumours, and using this data, produce 3D flow profiles