Colorectal cancer (CRC) is common in Ontario. This study described the processes of care following CRC resection, and identified CRC relapse from administrative data. Methods: CRC patients aged 18-80 from 1996-2001 with a colorectal resection were identified from the Ontario Cancer Registry. Linked discharge abstracts and physician billings were examined for physician visits, body imaging and endoscopy over the 5 year follow-up period. Administrative codes suggesting disease relapse were compared with patient charts. Results: Overall, 12,804 patients were identified and 8,804 had no evidence of relapse. Most (96.2%) patients had general practitioner follow-up, while 49.3% had medical oncology and 80.4% had general surgery follow-up. Greater than 90% of patients received endoscopy, while only 68.7% of patients received body imaging. Detecting disease relapse was 87.5% sensitive and 93.0% specific. Conclusions: There is potential for improving post-resectional follow-up in CRC patients. It is possible to detect relapse through administrative databases.

The Proximity Ligation Assay (PLA) is a relatively new method which utilizes the strength of both immunoassays and DNA detection. PLA has the capacity of high multiplexing due to the high specificity achieved with both dual protein-binding and dual primer binding during detection with Real-Time PCR. We developed a multiplexed PLA protocol that can measure 28 biomarkers in human EDTA plasma. The method was tested on 46 individuals diagnosed with colorectal cancer and 48 age matched healthy controls. The results are very promising as we re-discover the most well-known biomarkers for colorectal cancer and also find some potential new markers (significance tested with students T-test with p<0.05). Further improvements of the protocol are needed to decrease the variation.

Le cancer colorectal est issu des cellules de l’épithélium et se définit comme une pathologie induite par une accumulation d’altérations génétiques. Des mutations de type gain-de-fonction dans les gènes KRAS, NRAS et BRAF sont retrouvées dans plus de 60% des cancers colorectaux entraînant potentiellement l’activation constitutive de la signalisation en aval, notamment le sentier MEK/ERK. Dans cette thèse, nous avons évalué les mécanismes moléculaires par lesquels l'activation du sentier MEK/ERK régularise la différenciation épithéliale et la tumorigénèse intestinale. Dans les cellules épithéliales intestinales (CEIs), nos résultats montrent que les kinases ERK1/2 doivent être inactivées pour permettre l’induction du processus de différenciation entérocytaire. En effet, l'expression d’une forme constitutive active de MEK1 (caMEK1) est suffisante pour bloquer la différenciation tant morphologique que fonctionnelle. Une augmentation de la phosphorylation du facteur de transcription Cdx2 sur la sérine 60, qui diminue son activité transcriptionnelle, constituerait un des mécanismes impliqués. Dans des cellules cryptales intestinales indifférenciées, l’expression du caMEK1 induit une transition épithélium-mésenchyme (EMT), un processus associé au cancer colorectal. Cette EMT confère aux CEIs des capacités invasives et métastatiques in vivo associées à la sécrétion de protéases extracellulaires (MMP2/9). Nous avons identifié plusieurs autres cibles moléculaires de l’activité MEK/ERK impliquées dans l’EMT et l’invasion tumorale. Nous avons démontré l’implication des cascades Fra-1/Snail2 et EGR-1/Snail1 dans la répression transcriptionnelle de la E-cadhérine, une étape clé de l’EMT. Ce mécanisme de répression est également présent dans les lignées cancéreuses colorectales humaines possédant la mutation oncogénique de KRAS. L’analyse comparative par micropuce d'ADN Affymetrix dans les CEIs transformées par caMEK1 a permis d’identifier le gène encodant pour la serpineE2, un inhibiteur de protéases, comme le gène le plus fortement induit. Nous avons démontré que la serpineE2 est une cible moléculaire directe de l'activité oncogénique de la voie KRAS/BRAF/MEK/ERK et démontré son importance dans la migration et l’invasion tumorale par l'utilisation d'ARN interférents. On observe d’ailleurs une très forte expression de la serpineE2 dans des tumeurs colorectales humaines en comparaison à la marge saine adjacente. Nous avons aussi démontré que la transformation des CEIs induite par l’expression des formes actives de KRAS ou de MEK1 stimule la voie signalisation Wnt/β-caténine, dont la fréquente dérégulation constitue un évènement majeur menant au développement du cancer du côlon. La phosphorylation MEK-dépendante de LRP6 (S1490/T1572) semble être responsable de l’augmentation de l’activité transcriptionnelle de la β-caténine, associée à une augmentation de sa présence au noyau. Cette interconnexion entre les voies KRAS/MAP kinase et Wnt/β-caténine a également été observée dans des cellules cancéreuses colorectales humaines mutées pour KRAS ou BRAF. Finalement, une augmentation du niveau de phosphorylation de LRP6 a été observée dans les adénomes et tumeurs colorectales humaines, supportant l’idée que la phosphorylation de LRP6 puisse être impliquée dans la progression tumorale.

Background and ObjectiveColorectal cancer (CRC) remains the second deadliest cancer in the United States. Several screening methods exist; however, detection of small polyps remains a challenge. Optical coherence tomography (OCT) has been demonstrated to be capable of detecting lesions as small as 1mm in the mouse colon, but detection is based on measuring a doubling of the mucosa thickness. The colon microvasculature may be an attractive biomarker of early tumor development because tumor vessels are characterized by irregular structure and dysfunction. Our goal was to develop an endoscopic method of detecting and segmenting colon vessels using Doppler OCT to enable future studies for improving early detection and development of novel chemopreventive agents. MethodWe conducted in vivo colon imaging in an azoxymethane (AOM)-treated mouse model of colorectal cancer using a miniature endoscope and a swept-source OCT system at 1,040nm with a 16kHz sweep rate. We applied the Kasai autocorrelation algorithm to laterally oversampled OCT B-scans to resolve vascular flow in the mucosa and submucosa. Vessels were segmented by applying a series of image processing steps: (i) intensity thresholding; (ii) two-dimensional matched filtering; and (iii) histogram segmentation. ResultsWe observed differences in the vessels sizes and spatial distribution in a mature adenoma compared to surrounding undiseased tissue and compared the results with histology. We also imaged flow in four young mice (two AOM-treated and two control) showing no significant differences, which is expected so early after carcinogen exposure. We also present flow images of adenoma in a living mouse and a euthanized mouse to demonstrate that no flow is detected after euthanasia. ConclusionWe present, to the best of our knowledge, the first Doppler OCT images of in vivo mouse colon collected with a fiber-based endoscope. We also describe a fast and robust image processing method for segmenting vessels in the colon. These results suggest that Doppler OCT is a promising imaging modality for vascular imaging in the colon that requires no exogenous contrast agents.

El diagnóstico histológico de los pólipos colorrectales determina la conducta que tomará el médico especialista con el paciente. Con la aparición de nuevos pólipos en los últimos años, la clasificación histológica se ha tornado más compleja y amplia. Nuestro objetivo es actualizar los conceptos en el diagnóstico histológico de pólipos de colon de una manera clara y de fácil comprensión, especialmente para gastroenterólogos y patólogos.

Introduction The adjuvant treatment of colorectal cancer (CRC) has seen little improvement in terms of mortality of the disease in the last 40 years. There has been a resurgence in research into the use of monoclonal antibodies in the treatment of CRC. Carcinoembryonic antigen (CEA) is a useful target in cancer immunotherapy. The distribution of CEA in CRC differs from that in normal colorectal tissue. In normal colorectal tissue CEA is found only on the luminal surface of the cell which is inaccessible to intravenous antibody, whereas in CRC, CEA is found on all borders of the cell membrane and so becomes accessible to intravenous antibody. However, anti-CEA antibodies are prone to sequestration by circulating CEA. The anti-CEA antibody, PR1A3, binds only membrane-bound CEA and thus is able to overcome this problem. The aim of my research was to assess whether PR1A3 is suitable to be considered as a therapeutic agent in the treatment of CRC and what its mechanism of action might be. Methods The level of expression of CEA on a panel of cell lines was determined under different conditions using a solid-phase ELISA and FACS analysis. Humanized PR1A3 (hPR1A3) was assessed in a variety of in vitro cytotoxicity assays with colorectal cell lines expressing varying levels of CEA, using peripheral blood mononuclear cells and purified natural killer cells as sources of effector cells. The mechanism of action of PR1A3 was investigated by modifying the Fc fragment of the antibody and using antibodies to block the FcIIIA receptor on the effector cells. PR1A3 was also investigated in combination with a humanised A33 antibody. Results A panel of colorectal cell lines was found to have a range of CEA expression which could be upregulated in certain cell lines by growing the cell line beyond confluence and by treatment with the chemotherapeutic agent, 5-fluorouracil. The in vitro assays demonstrated hPR1A3 antibody-dependent and CEA-specific killing of tumour cell lines by human PBMC. The effect increased with increasing concentration of antibody and was lost by using the parent murine IgG1 PR1A3. Using 50&mu;g/ml hPR1A3, tumour cell lysis was increased by more than 3-fold above spontaneous killing (p < 0.001) in a high CEA-expressing cell line. Both antibody-dependent and antibody-independent (spontaneous) killing was blocked by using whole antibody to the Fc-&gamma;IIIA receptor, although the spontaneous killing was restored when a F(ab')2 was used instead of whole antibody. hPR1A3 and the A33 antibody showed potential synergy when used in combination against a high-CEA and a moderate-A33 expressing cell line. Conclusion The monoclonal antibody hPR1A3 causes CEA-specific lysis of human colorectal cancer-derived cell lines in the presence of human PBMCs. This lysis is dependent on the dose of the antibody, requires a compatible Fc-receptor and is inhibited by blockade of the FcγIIIA receptor. These findings show that hPR1A3 can kill tumour cells by antibody-mediated cellular cytotoxicity (ADCC) and implicate NK cells as a major contributor to this effect. The results support the development of hPR1A3 for therapy of colorectal cancer.

BACKGROUND: Radiation exposure to the abdominal region causes intestinal toxicity and is also capable of inducing colorectal cancers (CRC). Genotype-phenotype studies provide some evidence explaining the variation in familial adenomatous polyposis (FAP) patients caused by modifiers of adenomatous polyposis coli (APC). This study aims to extend our understanding of irradiation-induced modifiers of ApcMin/+ mice and CRC. METHODS: By using a pre-existing backcross between recombinant inbred line of ApcMin/+ mice to the irradiation sensitive inbred BALB/c mouse, we obtained panels of 2Gy-irradiated and sham-irradiated N2 ApcMin/+ mice for genotyping with a genome-wide panel of microsatellites markers. Using the number of adenomas in different intestinal segments to represent polyp multiplicity, we carried out a genome wide quantitative trait loci (QTL) scan followed by statistical epistasis modelling and bioinformatics analysis. RESULTS: We identified five significant QTLs responsible for radiation induced tumour multiplicity in the upper small intestine defined as Mom (Modifier of Min) radiation-induced polyposis (Mrip1-5) on chromosome 2 (LOD 2.8, p = 0.0003), two regions within chromosome 5 (LOD 5.2, p=0.00001, 6.2, p=0.00001) and two regions within chromosome 16 (LOD 4.1, p=4x10-5 and 4.8, p=0.00001). Suggestive QTLs were found for sham-irradiated mice on chromosomes 3, 6 and 13 (LOD 1.7, 1.5 and 2.0 respectively; p,0.005). Two significant QTLs were detected in the 2large intestine on chromosome 2 and 7 (LOD 2.7, p=1.2x10-3 and 2.2, p=1.2x10-3, 12 respectively). Using statistical epistasis modelling and logical selection of target genes though in silico sequence based on BALB/c specific non-synonymous polymorphisms which are predicted deleterious we selected target genes and further eliminated genes by sequencing and mRNA expression. CONCLUSIONS: Our study locates the QTL regions responsible for increased radiation-induced intestinal tumorigenesis in ApcMin/+ mice and identifies candidate genes with predicted functional polymorphisms that are involved in spindle checkpoint and chromosomal stability (Bub1b, Bub1r, and Casc5), Wnt pathway (Tiam1, Rac1), DNA repair (Recc1 and Prkdc) and inflammation (Duox2, Itgb2l and Cxcl5).

Background: Colorectal cancer is the result of dysregulation within classic regulatory pathways in epithelial stem-cell(s): the precursors giving rise to all other intestinal lineages. The resulting cancer stem-cell (CSC) generates tumours utilising its innate properties, e.g. self-renewal and lineage plasticity. CSCs appear to persist within a tumour as a distinct subtype responsible for local recurrence/metastasis. Therefore, therapies targeting colorectal CSCs may lead to improved cancer-specific outcome measures. The PROM-1/CD/AC133 cell surface marker has been associated with colorectal CSCs and its expression is reported as an independent negative prognostic marker. Therefore, this thesis sought to investigate the role of PROM-1/CD/AC133 in colorectal cancer. Methods: Tissue-culture, RT-qPCR, IHC, Western blotting, siRNA, PCR-array and FACS analyses were used to quantify and profile mRNA/protein expression patterns. Results: PROM-1/CD/AC133 was widely expressed in patient-matched colorectal tumour, adjacent normal epithelium, vascular invasion, lymph node metastases with significantly decreased expression in liver metastases. Furthermore, PROM- 1/CD/AC133 expression was not found to enrich colorectal cancer cell line populations for additional stem cell phenotypes (expression of ABCB1/ABCG2/BMIJ Murphy 1/CD44/LGR5/MSI-1). The data confirm the presence of alternative splice variants of PROM-1, and show that transcripts specifying PDZ binding predominate in colorectal cancer cell lines. Concomitantly, siPROM-1 was shown to modulate the expression of several key transcripts in colorectal tumourigenesis as well as regulate signal transduction pathways including the central cancer, colorectal cancer, NF-kB and p53 signalling cascades. Conclusions: PROM-1/CD/AC133 does not identify rare colorectal cancer cells responsible for tumourigenesis. However, it is associated with the regulation of signalling networks associated with cell growth, differentiation and apoptosis suggesting a potential role for this marker in colorectal tumourigenesis. The identification of specific target genes and signalling pathways in this thesis provides a springboard for further investigations into the functional role of this marker in colorectal cancer, with the potential for better treatments for this disease.

This research presents the design, fabrication and controller development of a robotic retractor which driven by a robotic manipulator for laparoscopic colorectal surgery. The system consists of a dual-head fan retractor and a manipulator. The dual-head fan retractor comprises two fan devices, retractor wrist, tubular element and handle. The fan device is facilitated with a fan end-effector, an expansion mechanism and a clutchspring mechanism. Two fan devices have been used in the system to provide an anthropoid hand-holding shape which is specifically advanced for surgical purpose because intestine tends to slip when subject to disturbance and the anthropoid handholding shape can effectively halt that. One of the two fan devices is rotatable which makes the anthropoid hand-holding shape achievable. The retractor wrist possesses a triggering device, based on clutch-spring mechanism, for rotating the rotatable fan device. The clutch-spring mechanism has an impact on rotating the palms of the fan devices. In front of the handle, it is the so called front body which includes two fan devices, retractor wrist and tubular element. The front body can be controlled and is motorised using two motors fixed to the tubular element. The dual-head fan retractor is modelled in SolidWorks, and stress analysis of the retractor has been carried out by SolidWorks Simulation. Then, the mathematical model of the fan blades is developed. A 3-joint manipulator is modelled and controlled by a computed torque PD control approach as part of an investigative study to fit such a system to the retractor for robotic manipulation. Based on this investigation, the retractor is attached to a 2-joint robotic manipulator which has one rotational joint and a prismatic joint. This manipulator is mathematically modelled, and the dynamic equations are obtained. Control methods from Azenha and Khatib are simulated and compared. Azenha & Machado's method has fewer input parameters and less oscillation when utilising the same control gains. Timeoptimal control is then successfully developed for the above 2-joint manipulator. This study clearly indicates that a retractor to be used for laparoscopic surgery can be effectively controlled using a multi-joints and multi degrees of freedom robotic manipulator.

Introduction: Van Gogh-Like 2 (VANGL2) is a scaffolding planar cell polarity protein involved in non-canonical Wnt signalling. It has been shown to have crucial roles in regulating epithelial development and homeostasis. Moreover, VANGL2 has been implicated in human cancers, with increased expression and copy number amplification seen in several cancer contexts. Many related components within this pathway have also been linked to cancer development, with VANGL2 expression known to regulate factors involved in cell migration and extracellular matrix (ECM) remodelling in cell lines. These cellular processes tend to be erroneously activated in cancer. VANGL2 is known to inhibit the classical driver pathway of colorectal cancer (CRC), canonical, or β- catenin dependant, Wnt signalling, in CRC cell lines. The aim of this thesis is to determine the expression of VANGL2 in CRC, and to investigate how VANGL2 may act to regulate intestinal homeostasis and disease. Methods: Transcriptional verification of VANGL2 expression in the mouse intestine was carried out by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and transcripts localised within the murine colon using RNA-In Situ Hybridisation (RNAISH). Expression and localisation of the VANGL2 protein and related non-canonical Wnt signalling components was confirmed using immuno-histochemistry (IHC). Furthermore, using a combination of human Tissue Micro-Array (TMA), transcriptional data and genomic data, we determined an association between VANGL2 on tumour grade and disease-free survival. To functionally validate the effects of VANGL2 on colorectal biology, we used a model in which VANGL2 is selectively deleted from the colonic epithelium using Villin-CreERT Vangl2flox mouse lines. Using a combination of molecular biology methods, we identified the ECM as differentially regulated following VANGL2 modulation. To test the role of VANGL2 in colorectal cancer, we used a murine colorectal cancer model in which adenomatous polyposis coli (APC) is deleted from colonic epithelium resulting in the formation of cancer concurrently with deletion of Vangl2. We evaluated survival of these mice as well as tumour number and size. Tumour tissue was analysed using IHC, qRT-PCR and 3-Dimensional organoid culture. Results: Within this thesis I have illustrated that the murine colonic epithelium expresses Vangl2, and other components known to interact with VANGL2 including Vangl1, Wnt5A, and Protein Tyrosine Kinase 7 (Ptk7). I have also shown that VANGL2 is expressed within the human colonic epithelium. I go on to show that 9.2% of human CRC possesses VANGL2 transcriptional alterations which correlates with a worsened disease-free survival (DFS) rate among patients. Using IHC, I also show that higher grade CRC is associated with increased VANGL2 expression. In our murine cancer model, mice with single or dual-copy loss of VANGL2 were found to have a reduced number of colonic tumours, while maintaining similar tumour size. Investigations to identify how VANGL2 may have control of tumour initiation were carried out focussing on the ECM. I found that, contrary to what I have discovered in the healthy murine colon, tumours from VANGL2-deficient mice had increased transcription of the ECM markers Secreted protein acidic and rich in cysteine (Sparc) and Decorin (Dcn), as well as increased expression of the ECM regulators Matrix Metallopeptidase 9 (Mmp9) and Tissue Inhibitor of Metalloproteinases 1 (Timp1). Changes in the ECM was also seen at the protein level, with increases in staining for the ECM components Col1 (Collagen, type I), and Laminin in VANGL2-deficient tissue. The ECM modulator Connective Tissue Growth Factor (Ctgf), is implicated in multiple cancers including CRC and is increased within VANGL2-deficient tumours at both the transcript and protein level, implicating Ctgf in increasing the ECM of these tumours.