Dissertations / Theses on the topic 'Early colorectal Cancer'

Colorectal cancer (CRC) is the third most common cancer worldwide, with about 1.2 million new cases diagnosed each year. CRC derived from the gradual accumulation of both genetic and epigenetic changes that transform the normal intestinal glandular epithelium into invasive cancer. While the genetic alterations are already used as prognostic and predictive markers, epigenetic alterations are currently the subject of intense research in the biomedical field because are considered as common and early molecular events in carcinogenesis that potentially could be used as molecular markers. The aims of this study were: to identify the alterations that characterize the CRC methylome; verify that these changes represent early events in the development of CRCs; explore the use of ultra-sensitive molecular techniques to track these alterations in biological matrices suitable for a non-invasive assessment (blood and stool); correlate the methylation alterations with the associated genes expression. The methylome analysis, conducted by Infinium HumanMethylation450 BeadChip on CRC and adenoma samples, has allowed us to delineate both the CRC methylation profile and that associated with precancerous stages. The gene-set/pathway enrichment analysis conducted by Toppgene and based on case/control differential methylation analysis results of CRCs and adenomas, allowed the identification of pathways involved in CRC carcinogenesis. The contribution of these pathways had never been widely emphasized and discussed in the literature. A very important result, emerged from the comparison of the genes belonging to the most altered significant pathways both in CRCs and adenomas, has been the identification of methylation alterations of regions, known as CpG islands, since the earliest stages of precancerous lesion suggesting that the alteration of specific pathways can lead the tumorigenic process. The selection of these regions has allowed us to identify a panel of biomarkers that can discriminate, with high specificity and sensitivity, CRCs and adenomas from peritumoral / normal counterpart. This panel has been extensively validated in silico in over 600 samples. We also evaluated the gene expression associated with these regions; more than 70% of hypermethylated CpG islands correlated with a downregulation in tumor tissue. To evaluate the usefulness of these biomarkers as a potential tool for non-invasive early diagnosis of CRC in clinical practice, we tried to trace through the use of ultra-sensitive techniques (methyl_BEAMING), the hypermethylation of three selected biomarkers in DNA extract from blood and stool. The hypermethylation of these regions, due to the presence of tumoral DNA, has been traced with great sensitivity and specificity in both matrices confirming the usefulness of these regions as possible biomarkers for the early diagnosis and traceability of residual disease of CRC.

Colorectal cancer (CRC) is one of the deadliest cancers in the world. The early stage of the disease is usually asymptomatic and therefore screening methods for colorectal cancer need to improve. There is a need for early detection of CRC as treatment is less effective in the advanced stage of the disease.  The current standard screening methods are endoscopy and fecal immunochemical blood tests. Endoscopy is a commonly used method to diagnose the patient, but it is costly, time consuming, and rather unpopular for the patients. An alternative could be to develop targeted molecular imaging probes that specifically deliver agents for example magnetic resonance imaging to colon adenomas and adenocarcinomas. This alternative would be non-invasive and able to detect the disease before morphological changes become evident. Biomarkers are used as an objective indicator of an altered biological process. Here, a literature study was conducted to identify protein biomarkers that are overexpressed in early stages of CRC. This study has focused on biomarkers that could be used to target imaging agents to cancerous lesions. Thus, the biomarkers need to be membrane-bound and expressed on the luminal side of the gastrointestinal tract. This will help future research to develop orally administered targeted imaging probes. Furthermore, a smaller literature search was conducted to identify cell and mouse models representing early stages of CRC. This was done to facilitate translational research going from in vitro to in vivo. Ideally the same protein is available in cell lines, mouse models and humans to enable translational research. This work has resulted in the selection of 7 different proteins that are upregulated during early stages of CRC. These proteins are potentially apically located and therefore possible targets for monoclonal antibodies. These findings might lead to a novel way for preventive patient screening and hopefully reduce the mortality for colorectal cancer.

Three cross-sectional surveys of acceptance of faecal occult blood testing for colorectal cancer screening, one survey of non-acceptors and one randomised controlled trial of an information leaflet were conducted. These were workplace based schemes in the private and public sectors and opportunistic screening using blood donors as a model. Simple educational leaflets explaining the high frequency of colorectal cancer and screening which addressed reasons for non-compliance were investigated. Subjects completed faecal occult blood tests at home and those with positive results underwent colonoscopy. Completion of tests in general practice in those aged 51 to 70 years was 33% (665/2029) in men and 42% (900/2147) in women. In private industry in subjects aged 41 to 65 years, compliance in men was 25% (425/1703) and in women 32% (40/125). In public industry in subjects aged 41 to 65 years compliance was 32% (53/165) in men and 46% (376/820) in women. With opportunistic screening at the blood donor centre compliance in those offered screening aged 51 to 65 years was 66% (75/114) in men and in women 59% (41/70). The health educational leaflets increased awareness of cancer and screening and raised intention to participate in a 100 subjects accompanying patients to hospital clinics. Reasons for non-compliance addressed in the leaflet, were identified from an interview survey of 81 non-compliers in Market Harborough. Common reasons were the unpleasantness of stool collection, lack of appreciation that healthy subjects should participate, fear of further tests and surgery and intercurrent illness. After piloting the leaflet it was tested in a randomised community controlled trial in general practice in subjects aged 61 to 70 years. The leaflet increased compliance in men from 25% (91/360) to 38% (143/381) (X2=12.9, p 0.001), but was ineffective in women (33%, 134/405, vs 34%, 145/425, X2=0.1,ns). Organisers of screening should consider opportunistic approaches and health education leaflets to increase participation. As compliance in this study was lower than in some other programmes, more work is needed to identify other reasons for non-compliance.

Colorectal cancer (CRC) has a relatively poor prognosis when detected at a later stage, therefore understanding its development to allow prevention or early detection is key to improving outcomes. Bowel cancer screening allows for the detection of tumours and precursor lesions. Even earlier changes could potentially be detected; genetic aberrations within the normal bowel before phenotypic changes occur. Early lesions may develop independently throughout the bowel or through a cancer field effect. FAP adenomas are a useful model due to adenomas developing within the same environment and all incurring the same first “hit” within APC. Using next generation sequencing the genetic profiles of FAP adenomas can be compared to better understand the development of these lesions. Firstly the sensitivity of pyrosequencing and NGS was determined as was the value of PCR-based mutant enrichment techniques. Samples of carcinoma, adenoma and their associated normals alongside normal mucosa from patients with normal colonoscopies were tested for mutations in commonly mutated genes in CRC using NGS. Multiple FAP adenomas from four patients were also tested with this mutation panel. Alongside this, copy number analysis was performed. The mutational and copy number data was used to ascertain the pattern of adenoma development throughout the bowel. Mutations were detected in carcinoma associated normal in APC, KRAS, CTNNB1 and SMAD4. The KRAS mutations in carcinoma associated normal differed to the KRAS mutation in the matched tumour. No mutations were detected in oncogenes in adenoma associated normal or normal mucosa from patients with normal colonoscopies. Studying mutations and copy number aberrations in FAP adenomas revealed that some adenomas shared specific lesions, indicating that they were clonally related. These results have confirmed previously findings of KRAS mutations in carcinoma associated normal mucosa as well as describing mutations in APC, CTNNB1 and SMAD4. This combined with the large amount of similarity in terms of mutations and copy number seen in adenomas from the same patient provides evidence for the cancer field theory.

Colorectal cancer is second only to lung cancer as a leading cause of internal cancer death. Individuals over 65 years of age are most at risk yet least likely to engage in screening for colorectal cancer. The purpose of this descriptive-correlational study using a modified Pender Health Promotion Model was to identify motivations of elderly individuals to engage in health protective behavior. A convenience sample of 90 subjects answered a four-part motivations questionnaire in which three subscales--early detection, powerful others, and chance--met reliability standards (alpha >.70). Chance was significantly related to compliance (r = -.28; p =.003); Hemoccult compliers believed less in chance and powerful others than did non-compliers (p =.005;.002). The 88 percent who performed a Hemoccult stool test as a screening method for early detection of colorectal cancer demonstrated that these elders willingly engaged in health protective behavior and supported the nurses' role in promoting primary prevention in elderly clients.

Colorectal cancer (CRC) is the third most common cancer in the UK and Adenomatous Polyposis Coli (APC) mutations are the most common genetic abnormality encountered in the sporadic form of this disease. In this study, we have used an Apc fl/fl/ mouse model which has a conditionally regulated Apc gene in the intestinal epithelium. Our hypothesis was that analysis of the changes in protein expression which occur in the intestinal epithelial cells of adult mice following Apc deletion would provide insights into signalling pathways which are also involved in early human colorectal carcinogenesis. The aims of this study were to use proteomic analysis to identify potential biomarkers for the early stages of CRC and to identify key proteins or processes involved in early colorectal carcinogenesis. iTRAQ-QSTAR proteomic analysis of intestinal epithelial extracts from Apc+/+ and Apc f//f/ mice identified 125 proteins which were differentially expressed, 50 being upregulated and 75 downregulated. We focused our efforts on the upregulated proteins as the detection of a positive signal is better for a biomarker. Ingenuity Pathway Analysis identified 19 proteins that could be detected in serum/blood, of which 13 were selected for further validation based on review of the literature. Immunohistochemistry, Western blotting and qRT-PCR identified 7 of these proteins as potential serum biomarkers of colorectal carcinogenesis. Integration of our iTRAQ data with a previous cDNA microarray performed using this mouse model identified c-Myc-dependent proteins. These included the 7 proteins identified in earlier studies and 4 additional proteins that might also play roles in colon carcinogenesis. The resulting 11 potential biomarkers were HMGB1, NCL, KRT18, RPL6, DDX5, PHB, SFRS2, FABP6, NAP1 L 1, NPM-1 and CBX3. These were further validated using another transgenic mouse model with the conditional regulation of both Apc and c-Myc genes (ApcfllfiMycfllfi), for which another iTRAQ (8- plex) analysis was performed. Confirmation studies were then carried out using the ApcMin/+ mouse model which shows more resemblance to human CRC. qRT-PCR studies comparing colonic polyp tissue samples from 6 month old ApcMin/+ mice and colonic tissue samples from their Apc+/+ wild-type counterparts showed increased expression of our candidate biomarkers in polyp tissue. For one of our candidates, HMGB1, a commercial ELlSA kit was then used to assess its serum concentration in various mouse models. This showed a statistically significant increase in serum HMGB1 concentration in Apc fl/fl mice compared to Apc+/+ mice. At 6 months of age, serum HMGB1 concentration was shown to be 1.69 fold higher in ApcMin/+ than in Apc+/+ mice. Our candidate biomarkers have also subsequently been validated using human serum and colonic tissue samples. The proteomic analysis of samples from mouse models with aberrant Apc expression has therefore generated a series of candidate biomarkers which have been demonstrated to be transferable to human CRC, thus validating our overall approach.

The current issue of Visceral Medicine focuses on colorectal cancer. Despite intense screening efforts, colorectal cancer still belongs to the most frequent cancers in both males and females in the EU. Particularly in rectal cancer there have been major developments in the surgical techniques over the last years that have led to a substantial reduction in local disease recurrence.

Colorectal cancer (CRC) is one of the most common causes of death from malignant disease. Nevertheless, no ideal screening method exists and there is a lack of prognostic and predictive factors to support clinical decisions and to aid the development of a more individualized treatment for patients with CRC. The aim of this thesis was to investigate early detection, prognostic and predictive factors of CRC. In the first paper, a novel method to collect cells for DNA quantification from the rectal mucosa was investigated. The sensitivity and specificity of this test to detect CRC or any pathology in colon and rectum were ultimately too low to be acceptable. In the second paper, the prognostic value of growth differentiation factor 15 (GDF 15) was evaluated in patients curatively operated for colorectal cancer. GDF 15 expression was demonstrated to be associated with a negative prognosis in patients with stages I-III and III disease. In the third paper, the prognostic value of BRAF, PIK3CA KRAS and MSI was evaluated in a cohort of patients with CRC stratified by disease and recurrence. The results indicated that patients with CRC stage III without recurrence have a higher frequency of BRAF mutation compared to stage III patients with recurrence. In the fourth paper, histopathological predictors of pathologic complete response (pCR) as well as the association between pre-treatment carcinoembryonic antigen (CEA) levels and pCR in non-smoking and smoking patients receiving preoperative chemo-radiotherapy for rectal cancer were evaluated. Only in non-smokers was a low CEA level significantly associated with pCR, suggesting that the predictive value of CEA for pCR in rectal cancer in smokers can be limited. In sum, this research has investigated a new method for CRC detection and further evaluated the clinical use of prognostic and predictive markers in CRC.

Le processus de dissémination du cancer colorectal (CCR) est un processus extrêmement peu efficace mais qui de par sa fréquence, parvient à initier des métastases. Elle débute par le départ de quelques cellules depuis la tumeur primaire selon différents modes de migration et des stratégies individuelles ou collectives. Parvenues à entrer dans un vaisseau sanguin, les cellules cancéreuses deviennent des cellules tumorales circulantes (CTCs). Ces CTCs profitent de la circulation sanguine pour se propager dans l’organisme. Cependant chacune de ces étapes les confronte à un micro-environnement qui peut être un avantage ou un désavantage. Certaines cellules tumorales disséminées (CTDs) parviennent cependant à éduquer le micro-environnement en un micro-environnement pro-tumoral. Si ce n’est pas le cas, elles peuvent entrer en dormance et attendre que ce dernier change et devienne pro-tumoral. Chacune des étapes de ce processus peuvent faire l’objet d’une stratégie thérapeutique visant à empêcher la dissémination colorectale. Il est donc nécessaire de mieux comprendre l’hétérogénéité tumorale et les interactions quelles ont avec le micro-environnement. De plus, le processus de dissémination est un phénomène dynamique et qui doit être pris en compte dorénavant très tôt afin d’intégrer la notion de dissémination précoce du cancer colorectal
The colorectal cancer (CRC) dissemination is an extremely inefficient multistep, but despite this, metastasis can be successfully formed. Dissemination starts by few cells that leave the primary tumor and use different type of migration individually or collectively. Once they enter into blood vessels, cancer cells become circulating tumors cells (CTCs). CTCs use the blood stream circulation to disseminate to distant organs. However, in each of the dissemination step, CTCs can face a new microenvironment that can give them advantages or disadvantages. Some of these disseminated tumor cells (DTCs) transform the microenvironment to a pro-tumoral microenvironment. If not, they can enter in dormancy and wait until that the microenvironment changes and became permissive. Each of these steps could be used to develop a targeted therapeutic strategy in order to impede the colorectal cancer dissemination. We face thus an urgent need to better understand the tumoral heterogeneity and their microenvironmental interactions. Furthermore, the dissemination process is a dynamic phenomenon and it must be considered earlier in order to integrate the early dissemination of the colorectal cancer

The present PhD research project starts from the need of deeply investigating some methodological aspects related to the identification, validation and application, in a routine clinical setting, of new non-invasive biomarkers for the (early) detection of cancer. Specifically, I investigated the statistical-methodological issues related to the identification and validation of molecular-based signatures detected with qPCR-based platforms by using the colorectal cancer as disease model (CRC-INT study). Colorectal cancer (CRC) is one of the major causes of cancer death in western countries1,2. More than 90% of CRC cases occur after the age of 50 years2 and, on the basis of the natural history of CRC progression and of the long time-interval of progression from normal mucosa to invasive cancer many efforts have been focused on the implementation of screening programs for CRC prevention and detection in its early stage, especially in this cohort of subjects. Currently adopted screening programs are based on colonoscopy (invasive, but still considered as the gold standard for both detection and removal of lesions) or on tests that search the human haemoglobin in stool (i.e. faecal occult blood test, FOBT/FIT). The latter are less invasive and easier to carry out, but showed low sensitivity values for polyps identification. In Italy, screening programs are based on FOBT/FIT - offered every 2 years to residents of 50-69/74 years-old - or on flexible sigmoidoscopy (FS) offered to a single age cohort, generally at 58-60 years-old. As concerns FIT programmes, quantitative haemoglobin analysis is performed in a centralized reference laboratory using the threshold of 100ng/ml of faecal haemoglobin as cut-off value to determine the positivity to the test. People with a negative test are invited to repeat the test after 2 years. Subjects with a positive test (FIT+) are contacted in order to perform a total colonoscopy (TC) in referral centres during dedicated sessions. According to 2011-2012 data, colonoscopy is performed by the 81% of FIT+ subjects and a diagnosis of carcinoma is formulated in 5% of FIT+ subjects and that of advanced adenoma in a further 25%. Subjects with non cancerous lesions are enrolled in follow-up programs according to the colonoscopy’s output, whereas subjects with a screen-detected CRC undergo surgery(www.giscor.it). MicroRNAs have been studied intensively in the field of oncological research and several studies have highlighted their easy detectability in plasma and serum3, suggesting their possible role as non-invasive biomarkers for the diagnosis and monitoring of human cancers. However, their detection and quantification could be influenced by haemolysis, i.e. pink discoloration of serum or plasma due to the release of the red blood cells into the fluids4,5,6,7. In addition, several Authors are now highlighting the need of shared workflows for the entire process of miRNA identification (pre-analytical and analytical) as well as for the statistical analysis. Accordingly, we recently proposed a workflow that tries to schematize all the key phases involved in biomarkers studies, from biomarker discovery to their analytical and clinical validation, including the issues related to the development of operative procedures for their analysis8. The process usually begins with a discovery phase, followed by a validation one and ultimately by the clinical application of the identified biomarker signature. Two additional assay-oriented steps (assay optimization and assay development) could be introduced in the workflow, before and/or after the validation phase. From a statistical-methodological point of view, the main issues involved in this workflow are those related to (i) data normalization of high-throughput qPCR data and the (ii) building and validation of miRNA-based signatures. Data normalization represents a crucial pre-processing step aimed both at removing experimentally-induced variation and at differentiating true biological changes. Inappropriate normalization strategies can affect the results of the subsequent analysis and, as a consequence, the conclusion drawn from the results. In the miRNA context, there are no yet verified and shared reference RNA in serum and plasma that can be used for data normalization. Several methods have been proposed to solve the problem of reference RNA selection. Currently, the most accepted and widely used method for data normalization of circulating miRNAs is that proposed by Mestdagh9, based on the computation of the global mean of the expressed miRNAs. This method is valid if a large number of miRNAs are profiled, but it is almost never applicable in validation studies focused on a limited number of miRNAs. To overcome this issue, the same Authors proposed to search the set of reference miRNAs that resembles the mean expression value of all the miRNAs, and use that set for data normalization. Starting from this, we developed a comprehensive data-driven normalization method for high-throughput qPCR data that identifies a small set of miRNAs to be used as reference for data normalization in view of the subsequent validation studies10,11, using results obtained with the global mean method as reference method. This algorithm was also implemented in a R-function (NqA algorithm). As concerns the biomarker studies based on the use of high-throughput assays, it should be considered that in such setting a wide range of weak biomarkers are constantly identified. In such setting, a single molecular biomarker alone may not achieve satisfactory performance for patients classification, so that the linear combination of these biomarkers in a more powerful composite score could represent a suitable approach to achieve higher diagnostic performances. As reported by Yan12, several methods are available, such as those based on the searching of the alpha value that maximizes the AUC value of the linear combination of p-biomarkers (grid-search methods), or the conventional logistic regression model when the purpose is to guide healthcare professionals in their decision-making. Prediction model studies are usually organized in a model development and in a model validation phase, or in a combination of both. In the first phase the aim is to derive a multivariate prediction model by selecting the most relevant predictors and combing them into a multivariate model, whereas the model validation consists in the evaluation of the performance of the model on other data, not used for model development. As far as miRNAs are concerned, the process starts with the identification of the candidate miRNAs that should be included in the initial multivariate model, as reported in (8). Once identified these candidates, the model development phase could start with the fitting of the initial multivariate model, by opportunely taking into consideration the number of event-per-variable (EPV). Penalized regression models (PMLE) 13, in which the beta value are obtained by maximising the penalized log-likelihood, can be used to prevent overfitting when a large number of covariates are present in a model with respect to the number of outcome events. According to the penalty term (i.e. the functional form of the constraints) and the tuning parameter (i.e. the amount of shrinkage applied to the coefficients), different penalized regression models can be fitted13 Once defined the initial model and fitted it with the proper procedures, another important theme is the definition of the final model, which could correspond to the full initial model or to a reduced one. For standard regression models, backward elimination or forward selection can be used for this purpose, even if the latter does not provide a simultaneous assessment of the effects of all the candidates in the model14, whereas for PMLE, a reduced model can be obtained using the R-square method15. An alternative approach to the standard stepwise/backward methods is the all subsets regression, which can discover combinations of variables that explain more variation in patients outcome than those obtained by using the standard stepwise/backward algorithms8. This approach has several potential advantages, but also some drawbacks, including the possibility of selecting models which omit important predictors (i.e. pre-existing evidences). The performance of a developed model should be then assessed by evaluating discrimination and calibration. Discrimination refers to the ability of the model to distinguish individuals with the disease from those without the disease (measured with the c-index or the equivalent area under the ROC curve), whereas calibration refers to the agreement between the probability of developing/having the outcome of interest as estimated by the model, and the observed outcome. In addition, it is important to consider that the performance of the developed model could be too optimistic because the same data are used for developing and testing the model14,17. In fact, when applied to new subjects, the performance of the model is generally lower than that observed in the development phase. Therefore, it is necessary to evaluate the performance of a developed model in new individuals before its implementation and application in clinical practice. Two different types of validation can be adopted, according to the design of the study and the data available: internal and external validation. Internal validation can be adopted when only one sample is available: approaches vary from the single splitting (one time) of the study data in a training and a testing set to the repeated splitting of the data a large number of times (i.e leave-one-out, k-fold and repeated random split cross-validation). Alternatively, the bootstrapping can be adopted when the development sample is relatively small and/or a large number of candidate predictors is under investigation14. The bootstrapping procedure allows the use of all the data for model development, and it also provides information about the level of model overfitting and optimism as well as about what can be expected when the model is applied to new individuals from the same theoretical source population. Even if these internal validation methods can correctly control overfitting and optimisms, they cannot substitute external validation, which consists in testing the model on new subjects17. The objective is to apply the original model to new data and to quantify the model’s predictive performance, without any re-estimation of the parameters included in the model. The new set of individuals may come from the same institution but be recruited in a different period (i.e. temporal validation), or they may come from other institutions/contexts (i.e. geographical validation)17. When a poorer performance of a prediction model is obtained on new individuals, instead of developing a new model (sometimes by repeating the entire selection of predictors), a valid alternative is to update the existing model by adjusting (or recalibrating) the model by the local circumstance or setting of the validation sample at hand17,18. In this way, the updated model combines the information captured in the original model on the development dataset with information from new individuals, theoretically improving the transportability to other individuals. Moreover, to improve the performance of a clinical prediction model, new marker(s) can be incorporated in the existing model19. The above-described methodology was applied to the CRC-INT study, aimed at identifying plasma circulating miRNAs to be used as biomarkers for the early detection of CRC lesions, using the FIT positive individuals as target population. The study included three cohorts of FIT+ subjects: discovery (DC), internal validation (IVC) and external validation (EVC). Blood was collected before colonoscopy and circulating miRNAs extracted from plasma were analyzed by using PCR assays. The principal aim of the discovery phase was to investigate the suitability of searching miRNAs in plasma from FIT+ individuals as well as identify a set of reference and candidate miRNAs to be deeply investigated in the subsequent phases focused on prospectively enrolled subjects. During the discovery phase, the expression levels of human miRNAs on a cohort of already available plasma samples from FIT+ individuals who have performed a screening colonoscopy at INT was used. As output, a subset of reference miRNAs was identified using the NqA R-function and a total of candidate miRNAs were identified as showing significantly different expressions in subjects with proliferative lesions vs subjects without lesions, or in subjects with a specific proliferative lesions. Based on these results, a custom micro fluidic card including the candidate and reference miRNAs was designed to be used in the following internal validation phase. Before moving to the custom-made assay, we performed a technical validation phase (on the same DC samples) aimed at evaluating the level of reproducibility between the involved assays (i.e. the high-throughput assay used in the discovery phase and the custom-made one to be used in the IVC/EVC20). In addition, an ad-hoc in-vitro controlled haemolysis experiment was implemented by artificially introducing different percentages of red blood cells (RBCs) in a haemolysis-free plasma sample21. Results evidenced that miRNAs known as haemolysis-related in literature were confirmed also in our experiment as influenced by haemolysis, whereas all our reference and 70% of candidate miRNAs were not influenced by haemolysis. Candidate miRNAs, showing relevant changes with respect to the haemolysis-free plasma sample were not considered in the subsequent statistical analysis. In addition, by taking advantage of the availability, for each contaminated tube, of haemolysis indexes (spectrophotometrically measured) and known RBC concentration, a calibration curve was generated with the aim to estimate the unknown percentage of RBCs in new plasma samples. For the analysis of the IVC data, we adopted an approach based on the all-subset analysis and the PMLE method to estimate the miRNA-based signatures, in order to take into consideration the peculiarity of the scenario under investigation, such as many weak biomarkers measured in plasma using platforms developed for research purposes only. We then performed a signature selection (i.e EPV > 3, significant AUC and finite shrinkage value) in order to select on the IVC only few signatures to be tested on the EVC. The latter includes FIT+ subjects enrolled at our Institute and also in other Hospitals joining the CRC-screening program of the Local Health Authority of Milan. Statistical analysis of the EVC cohort is ongoing: preliminary results confirmed the predictive capability of some of the identified signatures, even if lower performance with respect to that obtained on the IVC. Further analysis will be performed to evaluate a possible effect of the variable “Hospital” as well as to apply different model validation and model updating approaches. The evaluation of the added value of the developed miRNA-based signatures to pre-existing prediction models and the gain brought in by the introduction of the miR-test in the existing CRC screening diagnostic workflow will be eventually evaluated. References 1 Jemal, A., Bray, F., Center, M. M., Ferlay, J., Ward, E., & Forman, D. (2011). Global cancer statistics. CA: A Cancer Journal for Clinicians, 61(2), 69-90. 2 Mazeh, H., Mizrahi, I., Ilyayev, N., Halle, D., Brucher, B., Bilchik, A., et al. (2013). The diagnostic and prognostic role of microRNA in colorectal cancer - a comprehensive review. Journal of Cancer, 4(3), 281-295. 3 Chen, X., Ba, Y., Ma, L., Cai, X., Yin, Y., Wang, K., et al. (2008). Characterization of microRNAs in serum: A novel class of biomarkers for diagnosis of cancer and other diseases. 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(2016). Developing miRNA signatures: A multivariate prospective. British Journal of Cancer, 115(1), 1-4. 9 Mestdagh, P., Van Vlierberghe, P., De Weer, A., Muth, D., Westermann, F., Speleman, F., et al. (2009). A novel and universal method for microRNA RT-qPCR data normalization. Genome Biology, 10(6), R64-2009-10-6-r64 10 Pizzamiglio, S., Bottelli, S., Ciniselli, C. M., Zanutto, S., Bertan, C., Gariboldi, M., et al. (2014). A normalization strategy for the analysis of plasma microRNA qPCR data in colorectal cancer. International Journal of Cancer, 134(8), 2016-2018. 11 Verderio, P., Bottelli, S., Ciniselli, C. M., Pierotti, M. A., Gariboldi, M., & Pizzamiglio, S. (2014). NqA: An R-based algorithm for the normalization and analysis of microRNA quantitative real-time polymerase chain reaction data. Analytical Biochemistry, 461, 7-9. 12 Yan, L., Tian, L., & Liu, S. (2015). Combining large number of weak biomarkers based on AUC. 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E., Royston, P., Vergouwe, Y., Altman, D. G., et al. (2012). Risk prediction models: II. external validation, model updating, and impact assessment. Heart (British Cardiac Society), 98(9), 691-698. 18 Vergouwe, Y., Nieboer, D., Oostenbrink, R., Debray, T. P., Murray, G. D., Kattan, M. W., et al. (2016). A closed testing procedure to select an appropriate method for updating prediction models. Statistics in Medicine, 19 Nieboer, D., Vergouwe, Y., Ankerst, D. P., Roobol, M. J., & Steyerberg, E. W. (2016). Improving prediction models with new markers: A comparison of updating strategies. BMC Medical Research Methodology, 16(1), 128. 20 Verderio, P., Bottelli, S., Ciniselli, C. M., Pierotti, M. A., Zanutto, S., Gariboldi, M., et al. (2015). Moving from discovery to validation in circulating microRNA research. The International Journal of Biological Markers, 30(2), e258-61. 21 Pizzamiglio, S., Zanutto, S., Ciniselli, C. M., Belfiore, A., Bottelli, S., Gariboldi, M., et al. (2017). A methodological procedure for evaluating the impact of hemolysis on circulating microRNAs. Oncology Letters, 13(1), 315-320.

Cyclin O, a novel identified Cdk1 and Cdk2-binding cyclin, has been demonstrated to be required for apoptosis induced by intrinsic apoptotic stimuli. Because proteins implicated in apoptosis are often deregulated in cancer, we aimed to study the expression and role of Cyclin O in Colorectal carcinomas. We demonstrate that Cyclin O is overexpressed in CRC already during early stages of tumour development. Cyclin O expression correlated with a better prognosis of the CRC patients. Furthermore, we could show that loss of one Cyclin O allele leads to a higher adenoma count in a mouse model of CRC, the APCmin/+ mouse, suggesting that Cyclin O has tumour suppressor functions. Addtionally, we observed Cyclin O expression in the histologically normal APCmin/+ mouse- and in human peritumoral- intestinal epithelium. Accordingly, we hypothesise that Cyclin O could act as an early indicator of epithelial cell transformation to CRC.
La Ciclina O, una ciclina que ha estat identificada recentment i que s'uneix amb Cdk1 i Cdk2, s'ha demostrat ser necessària per a l'apoptosis induïda per estímuls intrinsecs. Donat que les proteïnes implicades en apoptosis solen estar desregulades en el càncer, nosaltres hem volgut estudiar l'expressió i la implicació de la Ciclina O en carcinomes colorectals (CRC). Hem demostrat que la Ciclina O es sobreexpressa en estadis prematurs de CRC. L'expressió de la Ciclina O correlaciona amb una millor pronòstic en pacients amb CRC. A més, hem demostrat que la pèrdua d'un al·lel de la Ciclina O comporta l'aparició d'un major nombre d'adenomes en un model de ratolí de CRC, els ratolin s APCmin/+. Això ens indica que la Ciclina O funciona com un gen supressor de tumors. També hem observat expressió de la Ciclina O en teixits no tumorals de l'epitel·li intestinal dels ratolins APCmin/+ i en teixit peritumorals de pacients amb CRC. Amb aquestes observacions hem arribat a formular la hipòtesi de que la Ciclina O podria ser útil com a indicador precoç de la transformació de cèl·lula epitel·lial a tumoral en los pacientes de CRC.

Colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer related deaths in the UK. Research has shown that the five year survival rate for patients if diagnosed at an early stage is 83% however, only 11% of cases are diagnosed at this stage. The aim of this study was to use proteomic approaches to investigate secreted proteins from colorectal cancer cell lines to identify candidate biomarkers for early stage diagnosis. Microvesicles (MVs) are a mixed population of vesicles that are released by a wide range of cells and are thought to play a role in tumour development and progression. Stable Isotope Labeling of Amino Acids in Culture (SILAC) was used to investigate the relative abundance of proteins secreted in MVs released by two cell lines that are used as a model of early tumour progression. This study identified 86 potential candidates that demonstrated increased release and six of these proteins (AGR2, OLFM4, SBP1, HSP90α, HSP90β and CEACAM5) were selected for further investigation by Western blot analysis. These proteins show potential as markers of early stage CRC and would be suitable for further validation in patient serum samples.

Liver resection, combined with modern chemotherapy, is considered the standard treatment for patients with resectable CRLM. However, the recurrence of hepatic metastasis after liver resection remains a concern worldwide. About twenty years ago, important studies overwhelmed the historical concept that 1.0-cm margin was not an absolute requirement for a curative approach in the treatment of patients with colorectal cancer liver metastases. This is a prospective observational study, performed at the Oncological Surgery, Hospital Policlinic San Martino, Genoa, Italy from 1st April 2014 to the 1st June 2019. Patients undergoing primary hepatic resection for colorectal liver metastasis with curative intent and having a minimum follow-up period of 6 months were included. Several clinical, pathological, and surgical factors have been tested for correlation with early recurrence and disease-free survival (DFS) in univariate analyses with a specific focus on the impact of resection margin depth. Microscopically and in line with the histological reports, the widths were stratified as coincidental margins if the tumor was in contact with the surgical margin (0 mm); widths of less than, or equal to, 1 mm or greater than 1 mm. During the follow up period, recurrence after liver resection was documented in 24 patients (48%). Early recurrence (within 6 months after liver resection) occurred in 11 patients (22% of the sample and 46% of the total recurrences), including 4 patients (36%) with liver-only recurrence and 7 patients (63%) with systemic recurrence (with or without liver recurrence). One-year and two-year mortality were 12% and 22%, respectively. According to univariate analysis, no significant differences were found in early recurrence and DFS between gender, location of the primary tumor, number and size of resected liver metastases, growth pattern and KRAS wild type. Time of diagnosis of liver metastases was the only significant prognostic factor for both DFS and for early recurrence. Moreover, histological grade of primary tumor (G2:33% vs. G3:86% vs. G4:100%; p<0.040) and synchronous presentation of liver metastases (80% vs. 20%; p<0.037) were associated with shorter DFS. No significant differences were found in the early recurrence rates and DFS in R1 versus R0 patients and even between the stratification of surgical margin size. Indeed, patients with wider-margin groups showed similar trend of recurrence in comparison with the narrow-margin group. Additionally, there was a slightly significant association between the severity of postoperative complication and the occurrence of a recurrence disease (p<0.08). In conclusion, in the present study, the lack of association between R1 status and DFS or early recurrence disease suggested that R1 margin status may be a surrogate indicator of advanced and/or more extensive disease. Even exploratory in nature, the present study suggests that the tumor biology (in term of grading and synchronous metastasis) rather than R1 resection was associated recurrence disease. So, up to date, the preferred surgical technique should be a parenchymal-sparing non-anatomic resection using modern surgical devices to keep as much liver parenchyma as possible. Furthermore, the risk of an R1 resection should not be considered a contraindication to surgery with curative intent, as neoadjuvant chemotherapy may destroy peripheral micrometastases before liver resection, minimizing consequently the residual micro-metastatic disease.

Colorectal cancer (CRC) is one of the most frequent malignancies worldwide and it is considered an important healthcare problem. Moreover, colorectal carcinogenesis is a long stepwise process through a known precursor, the adenoma, providing a perfect scenario for prevention and the identification of biomarkers. In this sense, my thesis dissertation aimed at giving a better understanding of CRC progression by evaluating both early precursor lesions and molecular changes leading to malignant transformation. Aberrant crypt foci (ACF), the earliest visible lesions in the colorectum, and LINE-1 methylation levels have been proposed as predictive biomarkers during CRC development. Nevertheless, their role in the colorectal carcinogenesis is still under debate. A wide cohort of ACF samples and paired normal mucosa extracted from patients at different risk of CRC was used to evaluate the mutational status of CRC key genes, microsatellite instability, and aberrant DNA methylation patterns. As a result, none of the potential biomarkers were found to be suitable to predict CRC development. Understanding the adenoma to adenocarcinoma transition is a decisive step when investigating CRC. In this sense, adenocarcinoma in adenoma samples were used to perform single-cell multi-color FISH in order to assess chromosomal heterogeneity and identify drivers of transition and progression. Interestingly, the gain of chromosomes 7p, 13q and 20q, as well as whole genome duplication, demonstrated to lead CRC evolution. Finally, the effects of whole genome duplication on cellular behavior were evaluated for two CRC cell lines, DLD-1 and RKO. Transcriptomic profiling revealed the dysregulation of genes related with replication in tetraploid cancer cells. Moreover, these cells showed replication stress, which triggered genomic instability and chromosome aberrations. Furthermore, polyploidization was found to be associated with an increased capacity for mobility, which explained the presence of tetraploid cells in the invasive fronts of colon adenocarcinomas.
El cáncer colorrectal (CCR) representa un destacado problema para la salud, ya que es uno de los tumores más frecuentes a nivel mundial. La carcinogénesis colorrectal se desarrolla de forma progresiva a través de una lesión precursora conocida, el adenoma, lo que proporciona el escenario ideal para su prevención y la identificación de biomarcadores. En este sentido, el objetivo de esta tesis doctoral fue profundizar el conocimiento sobre la progresión de CCR a través de la evaluación de tanto lesiones precursoras como cambios moleculares que promueven la transformación maligna. Los focos de criptas aberrantes (FCA) son las primeras lesiones visibles del colón y recto, y los niveles de metilación de los elementos móviles LINE-1 han sido propuestos como posibles biomarcadores predictivos de CCR. Sin embargo, el papel de ambos en la carcinogénesis colorrectal aún guarda varias incógnitas. Por lo tanto, se utilizó una amplia cohorte de muestras FCA procedentes de pacientes con diferente riesgo de CCR (control, adenoma y CCR) para evaluar el estado mutacional de los principales genes asociados con CCR, la inestabilidad de microsatélites, y patrones de metilación aberrantes. Como resultado, no se observó ninguna asociación entre las alteraciones evaluadas en FCA y el riesgo de desarrollar CCR, indicando que estas lesiones no son buenos biomarcadores predictivos para esta enfermedad. Comprender la transición de adenoma a adenocarcinoma es un paso decisivo cuando se investiga la naturaleza del CCR. Así, lesiones de adenoma con adenocarcinoma in situ fueron utilizadas para realizar FISH multi-color a nivel de célula única y poder evaluar la heterogeneidad cromosómica y identificar drivers de transición y progresión. Curiosamente, las ganancias de los cromosomas 7p, 13q y 20q, así como la duplicación del genoma completo, demostraron que conducen a la evolución del CCR. Finalmente, se estudiaron los efectos de la duplicación del genoma completo sobre el comportamiento de las células de cáncer en dos líneas celulares de CCR, DLD1 y RKO. El perfil transcriptómico reveló la desregulación de los genes relacionados con la replicación en células de cáncer tetraploides, es decir, aquellas con el genoma duplicado. Además, estas células mostraron estrés replicativo, lo que desencadenaba en inestabilidad genómica y aberraciones cromosómicas. La duplicación del genoma también se asoció con una mayor capacidad de movilidad, de hecho, se demostró la presencia de células tetraploides en los frentes invasivos de los adenocarcinomas de colon.

Background: Circulating cell-free DNA (cfDNA) in plasma is under investigation as a “liquid biopsy” for monitoring cancers as a surrogate for the primary tumour. The aim of the study was to evaluate the potential of cfDNA for early detection and follow up of patients with colorectal cancers (CRC). Methods: 55 colonic and 29 rectal operable carcinomas were investigated comparing matched tumour tissue and cfDNA. A 69bp ALU repeat qPCR assay was used to quantify fragmented DNA. Allele specific locked nucleic acid/peptide nucleic acid (AS-LNA/PNA qPCR assays were used to detect hot spot mutations in KRAS, BRAF and PIK3CA, optimised using cell lines with known mutation status. Results for cfDNA were compared with clinicopathological features of CRC and overall survival. Results: Total cfDNA levels correlated with disease stage (p=0.003). Cell lines studies demonstrated a limit of mutant DNA detection of 1 genome equivalent (6pg total DNA). Using this threshold, at least one mutation was detected in cfDNA of 40% of cases of CRC. Mutations in cfDNA were more common in colonic cases than rectal cases (p=0.01) and with significantly higher levels of mutant cfDNA (p=0.02).). In colonic tumours, cfDNA levels correlated with tumour stage (p=0.003), size (p=0.04) and LN status (p < 0.005). However, only 6 (86%) with BRAF, 6 (45%) with KRAS and 3 (20%) with PIK3CA matched to the mutations found in the tumour tissue suggesting clonal evolution. PIK3CA mutations were detected for the first time in cfDNA in localised primary tumours including early stage of CRC. Potential use of cfDNA total levels to screen for early lymph nodes involvement demonstrated a ROC of 0.76, with a 75% sensitivity and 86% specificity, suggesting early detection of cancer prior to metastasis. No significant survival differences were observed for cohort cancers for mutant cfDNA. However, mutation in cfDNA of colonic cancers was associated with poor overall survival (p < 0.05). Conclusion: This study has shown that cfDNA has a potential for use as a surrogate for the primary tumour in CRC, and for early detection of colonic cancer prior to metastasis. However, further study in a larger cohort is required to validate these findings.

I have investigated the pattern of β-catenin staining in a spectrum of apparently normal epithelium from animals bearing mutations in Apc (the Min mouse), p53 and Msh2 and compared this with patterns of Apc and E-cadherin protein immunohistochemistry staining. In general, β-catenin and E-cadherin expression was found at the lateral intercellular membrane in all types of epithelium, whereas Apc staining was found in the cytoplasm of epithelial cells in each organ but also showed strong positivity at the apical surface in intestinal epithelium. In animals bearing heterozygous mutation of Apc, enhanced β-catenin expression was identified in intestinal and pancreatic lesions in Apc^Min +/- and (Apc +/-, p53 -/-) mice and in a subset of intestinal lesions in Msh2-/- mice. This thesis characterised in detail the pattern of β-catenin expression in intestinal and pancreatic lesions arising in mice singly or multiple singly or multiply mutant for Apc, p53 and Msh2. These lesions were first graded on the basis of morphology. Overexpression of β-catenin was found in the majority of early intestinal lesions arising on an Apc^Min +/- background. In all categories of lesion studied mosaic patterns of β-catenin expression were observed with the proportion of cells showing a decrease in staining intensity associated with increasing lesion size. p53 status did not have any effects on these patterns. Mice mutant for the mismatch repair gene, Msh2, also showed increased expression of β-catenin in intestinal adenomas and microadenomas. However, in contrast with Apc^Min +/- mice, a subset of these lesions retained apparently normal expression. β-catenin upregulation was also found in the pancreas in foci of histologically abnormal cells in Apc^Min +/- and (Apc +/-, p53 -/-) mice. PCR analysis of microdissected materials confirmed that β-catenin overexpression was consistently associated with loss of the remaining wild type Apc allele. Subsequently analysis and adenocarcinomas of the pancreas arose and were characterised by an increased staining intensity of β-catenin. Taken together, these findings demonstrated that increased expression of β-catenin is an efficient marker of early neoplasic change in both murine intestine and pancreas in Apc mutant mice.

Introduction: Early growth response 1 (EGR1) is a zinc-finger transcription factor involved in the regulation of cell growth. It can act as either a tumour suppressor or a tumour promoter with a role in the induction of apoptosis in cancer cells by various pathways and is likely to play a role in colorectal cancer (CRC). EGR1 also appears to play a significant role in inflammatory pathways, therefore a possible role in Inflammatory Bowel Disease (IBD) is hypothesised. Patients with IBD have a greater risk of developing CRC, which is increased with duration of symptoms and severity of inflammation and dysplasia. The aim of this study is to determine whether EGR1 is differentially expressed in diseased colon tissue and to investigate novel EGR1-protein interactions in CRC cell lines. Methods: The relative EGR1 expression in CRC cell lines and in normal mucosa and tumours of colorectal cancer patients was determined by qRT-PCR. IBD patient samples were also examined for differential EGR1 expression levels by qRT-PCR, before and after stimulation with inflammatory mediators. Statistical analysis of the data was performed using ‘R’ statistical package, with the mixed-model ANOVA. Statistical significance was set at < 0.05. The genotype of three EGR1 variants was determined in the samples using PCR and sequencing, and the methylation status of regions of the EGR1 promoter was determined using bisulfite sequencing. A yeasttwo hybrid screen was conducted with EGR1 as bait, and screened against a SW480 CRC cell line library. Interesting novel interactions were investigated using immunocytochemistry and immunoprecipitation, as was the novel interaction between EGR1 and NOD2 and between EGR1 and components of the cytoskeleton. Results: Investigation into the relative EGR1 mRNA expression in CRC has shown that there is differential expression of EGR1 between matched normal mucosa and tumour. EGR1 expression is decreased in IBD patients compared with healthy controls. Induction of EGR1 by inflammatory stimuli also appears to be aberrant in these patients. The differential expression of EGR1 was not associated with aberrant methylation of a large region of the EGR1 promoter in either the CRC or IBD patients or with the genotype of EGR1 variants. EGR1 localises to both the cytoplasm and the nucleus in CRC cell lines and this study demonstrate interactions with the IBD susceptibility protein NOD2 and with components of the cyotskeleton. A yeast-two hybrid screen conducted with EGR1 as bait using a CRC cell line library has identified several other novel protein interactions of EGR1 in CRC cell lines. Conclusion: EGR1 is differentially expressed in both CRC and IBD, and in the case of IBD shows aberrant activity, suggesting that EGR1 may play a role in both colorectal diseases. EGR1 interacts with the IBD protein NOD2, and components of the cytoskeleton in CRC cells. Several novel protein interactions with EGR1 have been identified and warrant further study.

This thesis aims to develop a new non-invasive tool based on faecal bacterial markers for the early detection of colorectal cancer, which is capable of detecting precancerous lesions before the onset of clinical signs. In this work three tools have been developed: (1) RAID-CRC, based on the combination of four bacterial species with FIT, capable of reducing FIT false positive results in a 50% in a population with symptoms compatible with CRC; (2) RAID-CRC Screen, based on the combination of six bacterial species, capable of reducing I a 20% FIT false positive results in an asymptomatic population aged between 50 and 69 (current CRC screening population); and finally, (3) RAID-LS, based on the combination of three bacterial species, capable of detecting colorectal neoplastic lesions absence in Lynch syndrome carriers (subjects with CRC genetic predisposition) with a 100% sensitivity.
Aquesta tesi té l’objectiu principal de desenvolupar una nova eina no invasiva basada en marcadors bacterians fecals per a la detecció precoç del càncer colorectal, que sigui capaç també de detectar lesions precanceroses abans que apareguin signes clínics. En aquest treball s’ha aconseguit desenvolupar tres eines: (1) RAID-CRC, basada en la combinació de quatre espècies bacterianes amb el TSOF, capaç de reduir el nombre de falsos positius del TSOF en un 50% en una població amb símptomes compatibles amb el CCR; (2) RAID-CRC Screen, basada en la combinació de sis espècies bacterianes, capaç de reduir en un 20% els falsos positius del TSOF en una població sense símptomes d’entre 50 i 69 anys d’edat (població actual de cribratge de CCR); i per últim, (3) RAID-LS, basada en la combinació de tres espècies bacterianes, capaç de detectar l’absència de lesions neoplàsiques colorectals en portadors de la síndrome de Lynch (persones amb predisposició genètica a patir CCR) amb una sensibilitat del 100%.

Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths in the UK, and patient mortality is strongly correlated to the cancer stage at diagnosis. Circulating cell-free DNA (cfDNA) are short DNA fragments released into the blood from dying cells, and the use of cfDNA as a predictive and diagnostic biomarker for CRC patients has gained traction recently. The work undertaken in the thesis aimed to assess whether cfDNA could be a suitable biomarker for detection and monitoring of early colorectal lesions in preclinical and clinical settings. Custom qPCR and ddPCR assays were designed and implemented for quantitative cfDNA analysis and for detection of tumour-derived mutations in plasma samples. Preclinical studies investigating the cfDNA dynamics during early tumourigenesis were conducted with the Lgr5-EGFP-IRES-creERT2+/0;Apcfl/fl mouse model of CRC. In parallel, plasma cfDNA was also assessed in an adenoma patient cohort (n=76) against a ‘polyp-free’ control group (n=37). Analysis of mice failed to show a correlation between total cfDNA levels and the adenoma development in three separate preclinical studies, whereas detection of a surrogate tumour-derived mutation in animal plasma samples showed sensitivities between 16 and 25%. Similarly, total plasma cfDNA concentrations between the adenoma patient and control group showed no significant difference (p=0.1012). Targeted detection of BRAF and KRAS mutations in the plasma was achievable, but hampered by low sensitivities (0-25%). Multi-regional targeted next-generation sequencing (NGS) was performed on selected patient cases. The data unmasked extensively and previously unappreciated intra-tumour heterogeneity in colorectal adenomas. Nonetheless, identification and plasma targeting of tumour truncal mutations did not improve the detection rate. In conclusion, these results suggested that further methodological optimisation is necessary to achieve improved diagnostic sensitivity with plasma cfDNA liquid biopsy for the early detection of colorectal lesions.

Colorectal cancer (CRC) is the third most common cancer amongst South Koreans. Indirect evidence suggested CRC can be prevented, if not cured through the early detection and the subsequent removal of the precursor of CRC using colonoscopy (COL), the colorectal adenoma (polypectomy). The main aim of this thesis is to identify cost-effective strategies in the follow-up of people with confirmed colorectal adenomas (COL surveillance) for the prevention and early detection of colorectal cancer in the colorectal cancer screening (CRCS), National Health Insurance in Korea. To fulfil the main aim of this study, the following specific objectives were carried out: Estimation of adenoma recurrence post-polypectomy, identification of resources used in the CRCS and CRC treatment and the mapping of common pathways in the CRCS – this was achieved through a collaboration with a researcher in Korea by constructing a CRC cohort utilising the NHI data (2009-2012); Examination of the relevant cost-effectiveness evidence of COL surveillance in individuals with adenomas – this was achieved by conducting a review of the cost-effectiveness evidence in the prevention and early detection of CRC; A literature review of the Health State Utility Values (HSUVs) was conducted to identify methodologically robust HSUVs with health states of interest, this information was used for economic evaluation of COL surveillance; Identification of cost-effective strategies for COL surveillance utilising the findings from previous objectives. Results from a de novo cost-utility analysis indicated that a 0LR3HR (a COL 3 years post- polypectomy for high-risk) strategy is expected to be the most cost-effective in the follow-up of people with confirmed adenomas in the CRCS, NHI. The findings of this study will inform the COL surveillance policy in the CRCS, NHI. Approaches taken in this study and the findings can provide a foundation for further comparative policy analyses in other Asian countries where similar rates of CRC are observed.

Colorectal cancer is a potentially curable disease if diagnosed at its earliest stages. However, as the current tools for early diagnosis of colorectal cancer are suboptimal, this condition is still a major health issue in the UK and the whole world. Each year nearly 40,000 new cases are diagnosed in the UK. Approximately half of these patients have advanced disease at the time of diagnosis and this is associated with a less favourable prognosis. Therefore, there is still an urgent need for better colorectal cancer screening tools. Moreover, it has long been suggested that APC deletion is an early and major event in the initiation of more than 80% of all colorectal cancers. However, the molecular events that occur following APC deletion are yet to be fully understood. Working on an acute Apc deletion animal model, our group has identified several candidate biomarker proteins. It is postulated that studying these proteins will reveal new aspects about early colorectal tumourigenesis. We hypothesised that these proteins are upregulated very early following Apc deletion and are involved in various aspects of colorectal cancer development, therefore they are potential biomarkers and/or therapeutic targets for this disease. Most of the project’s aims were addressed using immunohistochemical assessment of the expression of several candidate biomarkers in a novel in vivo model of acute Apc deletion (AhCre+Apcfl/fl mouse), a model of established early intestinal neoplasia (ApcMin/+ mouse) and a model of invasive disease (AhCreERT+Apcfl/+Ptenfl/fl mouse) as well as clinical samples from patients with early and advanced colorectal cancer. Mechanistic studies were carried out using the human colonic adenocarcinoma cell lines, HCT116 and HT29. In the animal models, deletion of Apc resulted in an early activation of the Wnt signalling pathway as indicated by nuclear localisation of Beta catenin. Six (NAP1L1, RPL6, SFRS2, PHB, FABP6 and NCL) out of the nine candidate biomarker proteins tested and two proposed partner molecules (Cyclin E and CDC5L), demonstrated obvious differential patterns of expression in areas where Apc loss had induced Wnt pathway activation. Specific knockdown studies of selected members of this protein list in human colon adenocarcinoma cell lines using siRNA identified important effects of these proteins on critical cellular functions such as proliferation and apoptosis. NAP1L1 and RPL6 knockdown had an inhibitory effect on cell proliferation and survival and caused a simultaneous increase in apoptosis. SFRS2 knockdown caused increased abundance of nuclear CDC5L and vice versa. SFRS2 down regulation had marginal effects on cell proliferation and cell survival and resulted in a small reduction in the amount of apoptosis. In contrast, CDC5L knockdown caused a dramatic inhibition of cellular proliferation, loss of the G2 cell cycle peak and a significant increase in the amount of apoptosis and caspase 8 activity. HCT116 cells with a p53 deletion were more sensitive to toxicity of experimental (transfection) reagents and SFRS2 knockdown in these cells caused obvious inhibition of cell proliferation with increased apoptosis. Immunohistochemical staining of the candidate biomarker proteins in human samples of colorectal cancer generally supported the results shown in animal studies for early stages of the disease. Moreover, it demonstrated reduced nuclear expression of Beta catenin, nuclear relocalisation of CDC5L and cytoplasmic displacement of SFRS2 in the more advanced stages of colorectal cancer. PHB showed increased cytoplasmic staining in Dukes’ stage A and B cancers. These results were backed up by appropriate scoring systems. Our results concerning these proteins in colorectal cancer are novel and agree with several studies by other research groups describing their roles in other cancers or cancer models. Due to the relatively late detection of lesions in humans, the early changes which were observed in animals might have been missed in humans. NAP1L1, RPL6, SFRS2 and NCL showed early overexpression during colorectal tumourigenesis. These proteins therefore have the potential as screening biomarkers. Due to their role in regulating cellular proliferation, NAP1L1, RPL6 and CDC5L are potential therapeutic targets for colorectal cancer. PHB is also a potential marker for Dukes’ stage A and B cancers. Mechanistically, the interplay between SFRS2 and CDC5L during colorectal tumourigenesis is a promising case to follow. Based on this study and other studies conducted in our group, the above proteins are promising screening, predictive or prognostic markers as well as potential therapeutic targets for colorectal cancer.

Colorectal cancer remains one of the most common cancers in the United Kingdom with around 40,000 new cases being diagnosed each year. Around 25% of patients will have liver metastases at the time of presentation, with up to 50% developing metastases at some point in their life. Advances in surgical technique and developments in chemotherapy have increased the number of patients with advanced disease for whom potentially curative treatment is possible. The use of chemotherapy in a neo-adjuvant setting has improved the outcome for patients with liver metastases who have initially irresectable or borderline disease. Chemotherapy-induced hepatotoxicity affects up to 78% of patients receiving standard chemotherapy for colorectal cancer and can lead to increased morbidity and mortality. Current gold standard serum-based biomarkers of drug-induced hepatotoxicity have their limitations and there remains a need for more sensitive and specific novel biomarkers to detect early hepatotoxicity. The use of serum-based microRNAs, in particular microRNA-122 (miR-122), a hepatocyte-specific molecule has been proposed as a possible biomarker for chemotherapy-2induced hepatotoxicity. The work described in this thesis assessed the characteristics of serum miR-122 in a healthy human population. It also assessed serum levels of miR-122 in different diseases including primary liver cancer. In order to assess the role of serum miR-122 in chemotherapy-induced hepatotoxicity, a pilot study was carried out in patients receiving chemotherapy for advanced colorectal cancer. In a healthy human population (n=129) serum levels of miR-122 were measured to investigate the degree of variation and define a normal reference range that could be used to assess changes found in patients with hepatic injury and disease. In addition to miR-122, two endogenous (U6snRNA/let27d) and one exogenous controls (c.lin24) were measured. Inter-individual variation was low for miR-122 (CV% 5.21) and also for all three controls (CV% <4%). There was no circadian variation in serum miR-122 (ANOVA p=0.1254). Analysis of intra-patient variation over three consecutive days was similarly low (p=0.66). In a human population with underlying chronic liver disease (n=90) and primary liver cancer (n=104), serum miR-122 was significantly raised in the both cohorts (p=<0.001) but no difference was seen between the chronic disease and cancer cohorts (p=0.338). Patients with an underlying inflammatory condition had significantly raised serum miR-122 (p=<0.001) compared to those with underlying cirrhotic or fibrotic change (p=0.372). ROC analysis supported this finding (AUC 0.79 vs 0.54). In a pilot study of serum miR-122 during neo-adjuvant chemotherapy for colorectal cancer liver metastases, 11 patients were recruited. Serial blood sampling during chemotherapy treatment revealed a non-significant rise in miR-122 (p=0.14). Clinically insignificant levels of liver toxicity were seen in the ten patients who completed the treatment and had surgery. In those with histology changes known to be associated with chemotherapy, there was a significant rise in serum ALT (p=0.0082 and 0.0085) while the miR-122 did not rise significantly (p=0.053). This work confirmed the low variation in serum miR-122 that is a requirement for a novel biomarker. Furthermore, it confirmed the detectable increase of serum miR-122 in patients with liver disease, particularly those with an inflammatory pathophysiology. Finally, in a human model of chemotherapy-related hepatotoxicity, the role of miR-122 remains unclear at present, but the non-significant changes in level related to clinically insignificant liver perturbation compared to the significant changes in ALT suggest that, although more work is required, it may be a valuable biomarker with potential in this field.

Background: Very recently a gene marker panel that allows the mutational analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientific literature. Methods: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243–1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. Results: It could be shown that about 80% of early stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No significant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. Conclusions: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80–90% of human sporadic CRC samples analyzed.

Background The increased detection of pT1 colorectal cancers (CRC) in the National Health Service Bowel Cancer Screening Programme (NHSBCSP) raises new concerns for clinicians. The aim of this study is to investigate the phenotypic features and biology of screened and symptomatic pT1 CRC and to assess current and new high risk features associated with lymph node metastasis (LNM). The second aim of this study is to investigate the inter-observer variation of reporting screened pT1 CRC between pathologists. Methods Symptomatic and screened pT1 CRC were identified from two databases (Northern and Yorkshire Cancer Registry and Information Services [NYCRIS] and NHSBCSP database). Phenotypic features of the pT1 CRC were evaluated and compared from both cohorts. The second part of the study investigated the inter-observer variability in the qualitative and quantitative assessments of screened pT1 CRC. Participating pathologists were asked to perform quantitative and qualitative assessments on 41 screened pT1 CRC. The level of agreement was determined using Fleiss Kappa statistics and intraclass correlation coefficient testing. Results Symptomatic CRC with LNM had a significantly wider area of invasion (p=0.001), a greater area of submucosal invasion (p < 0.001) and a higher proportion of tumour stroma (p = 0.005) compared to CRC without LNM. Symptomatic pT1 CRC were also significantly bigger in size than screened pT1 CRC. The inter-observer variation study showed that quantitative factors had better levels of agreements than qualitative factors. Conclusion This study has shown that screened pT1 CRC are quantitatively smaller to their symptomatic counterparts suggesting that the NHS BCSP detects earlier pT1 CRC. This study also showed that novel quantitative factors such as width of invasion, area of submucosal of invasion and PoTS could be used as valid parameters in determining the rate of LNM. Finally, this study highlights the need for better guidelines/definitions in the evaluation of screened pT1 CRC.

The increasing occurrence of CRC developing in young patients with no identified genetic predisposition, defined as sporadic early onset colorectal cancers (EOCRCs), demands maintaining a high index of suspicion when people below 50 years of age present with symptoms. To define the clinicopathological features and the stage at presentation of EOCRCs, as well as to understand whether the histological, immunohistochemical and molecular analyses were associated with particular clinicopathological parameters and oncologic outcome, a total of 94 cases of EOCRCs diagnoses between 2006 and 2014 at the Sant’Orsola University Hospital were studied. Indeed, EOCRCs appear frequently as an aggressive disease located in the sigmoid colon and rectum, and most patients are symptomatic at the time of presentation, mainly presenting with rectal bleeding, haematochezia or abdominal pain. The genetic basis in the majority of early onset colorectal carcinomas remains unknown, however, most EOCRCs, not related hereditary syndromes, appear to arise through the same pathways as sporadic CRCs, such as the classical adenoma-carcinoma sequence, but with only rare involvement of the methylator pathway. Taken together, the analyses described in this study suggest that, in the absence of screening programs for patients under 50 years of age, the risk factor of a family history and the presence of symptoms may be considered as an indication for prompt endoscopic investigation in these patients, since this may reduce the stage of disease at diagnosis and likely have an impact on improving survival.

The increasing occurrence of CRC developing in young patients with no identified genetic predisposition, defined as sporadic early onset colorectal cancers (EOCRCs), demands maintaining a high index of suspicion when people below 50 years of age present with symptoms. To define the clinicopathological features and the stage at presentation of EOCRCs, as well as to understand whether the histological, immunohistochemical and molecular analyses were associated with particular clinicopathological parameters and oncologic outcome, a total of 94 cases of EOCRCs diagnoses between 2006 and 2014 at the Sant’Orsola University Hospital were studied. Indeed, EOCRCs appear frequently as an aggressive disease located in the sigmoid colon and rectum, and most patients are symptomatic at the time of presentation, mainly presenting with rectal bleeding, haematochezia or abdominal pain. The genetic basis in the majority of early onset colorectal carcinomas remains unknown, however, most EOCRCs, not related hereditary syndromes, appear to arise through the same pathways as sporadic CRCs, such as the classical adenoma-carcinoma sequence, but with only rare involvement of the methylator pathway. Taken together, the analyses described in this study suggest that, in the absence of screening programs for patients under 50 years of age, the risk factor of a family history and the presence of symptoms may be considered as an indication for prompt endoscopic investigation in these patients, since this may reduce the stage of disease at diagnosis and likely have an impact on improving survival.

Les tumeurs sont composées de cellules malignes et d'une grande variété de cellules non-tumorales, en particulier des cellules immunitaires qui forment le micro-environnement tumoral (MET). Il a été démontré que la composition du MET était associée au devenir clinique des patients, en termes de survie et de réponses thérapeutiques. Avec le développement récent des immunothérapies qui ciblent des éléments spécifiques du MET, l'immunité anti-tumorale a soulevé un intérêt majeur. Plusieurs méthodologies ont été mises au point afin d'étudier la composition du MET, avec une précision toujours plus grande. En particulier, des méthodes comme MCP-counter permettent d'exploiter les données transcriptomiques de la tumeur entière afin de quantifier les différentes populations qui composent le MET. Le volet méthodologique de ce travail de thèse a ainsi consisté à proposer une amélioration de MCP-counter, en particulier pour l'analyse de données RNA-Seq. Une adaptation de la méthode pour des données issues de modèles murins (mMCP-counter) est également proposée. MCP-counter permet d'analyser rapidement le MET de larges séries de tumeurs. Un second volet de cette thèse consiste en l'application de cette méthode pour établir une classification immunitaire des sarcomes des tissus mous, un type de cancer rare, hétérogène et agressif. Cette classification immunitaire a permis de mettre en évidence des groupes de tumeurs faiblement ou fortement infiltrés, ainsi qu'un groupe marqué par une forte vascularisation. De manière intéressante, la classification immunitaire permet de prédire la réponse des patients aux immunothérapies. Ce travail a aussi démontré un rôle important des structures lymphoïdes tertiaires (SLT). Les SLT sont des structures de type noeud lymphatique composées de lymphocytes B et T qui se forment dans la tumeur ou à proximité de celle-ci. Au sein des SLT, une réponse immunitaire anti-tumorale peut se former et maturer. L'intérêt porté aux SLT est de plus en plus important pour de nombreux types de cancers. Dans la plupart des types de cancer, une forte infiltration de la tumeur par des lymphocytes T, en particulier CD8+, est associée à une meilleure survie des patients. Cependant, le carcinome rénal à cellules claires et le cancer de la prostate sont des exceptions à cette règle. En effet, dans ces deux cancers urologiques, la présence dans la tumeur de lymphocytes T est associée à une survie plus courte des patients, ainsi qu'à une rechute et une progression plus précoce. Ces exceptions sont détaillées dans une troisième partie de cette thèse, par une description minutieuse du MET, ainsi que par l'analyse de l'implication du système du complément. Dans leur ensemble, les résultats présentés dans cette thèse démontrent qu'en combinant différentes méthodes d'analyse, in silico, in situ et in vivo, il est possible d'obtenir une vision extrêmement complète du MET. La connaissance des types cellulaires présents dans la tumeur ainsi que leur orientation fonctionnelle permet de guider le soin apporté aux patients et d'améliorer leur devenir clinique. La description complète du MET ouvre la voie à une médecine personnalisée pour les patients atteints de cancer
Tumors are composed not only of malignant cells but also contain a vast variety of non-malignant cells, notably immune cells forming the tumor microenvironment (TME). The composition of the TME was shown to be associated with clinical outcome for cancer patients, in terms of survival and therapeutic responses. With the relatively recent development of immunotherapies targeting specific elements of the TME, tumor immunology has risen a strong interest and holds a strong therapeutic potential. Several methodologies have been developed to study the composition of the TME with an increased precision. Notably, some methods such as MCP-counter enable the use of the tumor bulk transcriptome to quantify cell populations composing the TME. The methodological aspect of this PhD project consisted in setting up an enhanced version of MCP-counter that can be readily applied to RNA-Seq data, as well as propose an adaptation of the method for mouse models. Using MCP-counter, the TME of large series of tumors can be easily analyzed. The application part of this PhD work consisted of applying MCP-counter to establish an immune-based classification of soft-tissue sarcoma, a rare, aggressive and heterogeneous cancer type. The immune classification notably allowed to identify immune low and high groups, and a group characterized by a strong vasculature. Interestingly, the classification was notably found to be predictive of the patients' response to immunotherapies. It also highlighted an important role of tertiary lymphoid structures (TLS). TLS are lymph-node-like structures composed of T and B cells that form within the tumor or in close proximity. They are a site of formation and maturation of antitumoral immune responses. TLS are raising a growing interest in many malignancies. In most cancer types, a strong infiltration by T cells, in particular CD8+ T cells, is associated with a favorable clinical outcome. However, clear-cell renal cell carcinoma and prostate cancer are exceptions to this general rule. Indeed, in these urological cancers, an increased infiltration by T cells is associated with a decreased patient survival and with earlier relapse and disease progression. In a third part of this thesis, these exceptions are investigated with more details by scrutinizing the TME, and questioning the implication of the complement system. Overall, this thesis presents how the combination of several analysis methods, in silico, in situ and in vivo, can help achieve an extremely precise description of the TME. Knowing accurately what cell populations and what their functional orientation can help guide patients care and improve clinical outcome. Complete description of the TME opens the way towards personalized medicine for cancer patients

碩士
國立臺灣大學
護理學研究所
103
The patients with colorectal cancer usually suffer from multiple symptoms after surgery. Pain, nausea, vomit, abdominal fullness, low appetite, anxiety, depression and various postoperative complications are experienced by the patients . The symptoms not only cause the discomfort, prolong the postoperative recovery and hospitalization period, but also worsen the psychological condition. Early postoperative ambulation is thought to be a useful treatment to improve the postoperative recovery. But how it really works and how to apply on clinical condition remain unclear. The study aims to understand the correlation between postoperative ambulation and the recovery of patients with colorectal cancer. We conducted a longitudinal correlation study with a set of structured questionnaire survey with consecutive sampling in a medical center in Northern Taiwan. The population was postoperative patients with colorectal cancer. The questionnaire included: (1) Preoperative part: demographic profiles, Taiwan International Physical Activity Questionnaire (IPAQ), Hospital Anxiety & Depression Rating Scale (HADS); (2) Postoperative part: walking diary, physical symptoms scale and the heart-rate sensor ring were applied every day. HADS was recorded on the day before discharge. Data were analyzed by descriptive statistics and generalized estimating equation (GEE). Finally, 150 patients were recruited in 13 months. The results indicated that: (1) the higher the frequency of postoperative ambulation is, the time of flatus and tolerating to solid food is earlier, and the degree of physical symptoms and anxiety are lower; (2) the longer the distance of postoperative ambulation is, the time of tolerating to solid food is earlier, the hospitalization period is shorter, the degree of physical symptoms and anxiety are lower. In conclusion, the increase of frequency and distance of postoperative ambulation certainly lead to a better postoperative recovery. Further studies on the exact amount of ambulation are recommended for developing the clinical guidelines for this population.

Early diagnosis represents an effective way to improve patient prognosis in cancer. New opportunities for cancer diagnosis and screening may arise from identification of cancer-specific epigenetic alterations in the cell-free circulating DNA (cirDNA). This study investigated biomarkers at the level of DNA methylation in the plasma cirDNA of individuals affected with prostate cancer or colorectal cancer. A methylation-sensitive restriction enzyme-based method was used to enrich methylated DNA fractions, which were interrogated on CpG island and human genome tiling microarrays. A number of genes and non-coding loci exhibited differential methylation between prostate cancer patients and controls. The candidate loci identified from these microarray experiments underwent verification by bisulfite modification coupled with pyrosequencing. Our results suggest that microarray-based studies of DNA methylation in the cirDNA can be a promising avenue for the identification of epigenetic biomarkers in cancer.

Research Doctorate - Doctor of Philosophy (PhD)
The contents of this thesis by publication include an introduction, a critical review, five data-based manuscripts and a general discussion providing implications and conclusions. The papers examined the early detection and prevention of colorectal cancer (CRC) in the community-based setting and among first-degree relatives of CRC patients. At timing of thesis submission, three papers (two data-based and one review paper) had been accepted for publication. The remaining three are under editorial review. The burden of disease, early detection and prevention of colorectal cancer (CRC) is presented in the Introduction. It provides a general overview of CRC-related global burden of disease, its aetiology and the efficacy of screening in reducing incidence and mortality. It also examines current levels of CRC screening uptake and the populations currently experiencing inequality in CRC screening. This chapter also provides an overview of the current state of medical consultation and delay in seeking medical advice for primary symptoms of CRC (i.e. rectal bleeding and change in bowel habit). Paper One provides a critical review of methodically sound community-based approaches to increasing CRC screening levels. Paper Two is a cross-sectional study which identified the current levels of CRC screening uptake and screening in accordance with National Health and Medical Research Council (NHMRC) screening guidelines among an at-risk community cohort of persons aged 56-88 years. Paper Three is a cross-sectional cohort study using the aforementioned sampled population which assessed the socio-demographic and provider-level factors associated with ever receiving CRC testing and CRC screening in accordance with guideline recommendations. A secondary analysis was conducted to examine National Bowel Cancer Screening Program eligibility on each aforementioned outcome. Paper Four is a population-based study among first-degree relatives (FDRs) of CRC patients examining across varying levels of risk, the proportion of FDRs (i) ever receiving any CRC testing in their lifetime and (ii) screened in accordance with NHMRC screening guidelines. Socio-demographic and provider-level predictors of (i) and (ii) were also evaluated. Paper Five reports on a cross-sectional study examining, for two primary symptoms of colorectal cancer (i.e. rectal bleeding and change in bowel habit), rates of (i) non-consultation and (ii) delay in seeking medical advice for both symptoms. Additionally, the reasons for non-consultation and delay in seeking medical advice for each symptom as well as the triggers for consulting a doctor following symptom episode were investigated. Paper Six: The purpose of this study using the aforementioned Hunter Community Study cohort was to identify the socio-demographic and provider-related characteristics associated with (i) ever seeking medical advice for primary symptoms of CRC and (ii) early medical-advice-seeking behaviour for primary symptoms of CRC. Discussion and implications for future research and practice: In conclusion, this dissertation has provided insight into the current levels of CRC screening in compliance with NHMRC screening guideline recommendations at a community level and among an increased-risk population (i.e. first-degree relatives of CRC patients). Previously, little was known about community CRC screening levels across varying levels of risk. The low rates of screening in accordance with guidelines and the identified screening inequalities across individual and socio-demographic characteristics highlights the need for systematic population-based approaches to increase the rate of risk-appropriate CRC screening. This thesis also highlighted the poor receptivity of community members to prompt medical advice seeking for potential symptoms of CRC. Both high rates of delay and non-consultation for primary symptoms were evident, with little appreciable improvements indicated through a direct comparison with an earlier at-risk community data set. The current work highlights the need for systematic population-based approaches that are tested in methodically rigorous interventions, if improvements in the earlier presentation of primary symptoms and the level of risk-appropriate CRC screening are to occur. The direction of future research stemming from this dissertation and the possible pathways for future research initiatives are discussed.

碩士
國立臺北護理健康大學
護理研究所
107
Colorectal cancer is the most prevalent cancer and the third leading cause of cancer-related death in Taiwan. Surgical resection is the most common treatment for colorectal cancer. Postoperative early enteral nutrition can promote recovery, reduce the risk of complications and ease the medical burden. However, there remains a substantial reluctance to utilize early enteral feedings due to the fear of the increased risk of anastomotic dehiscence and complications. The purpose of this study is to evaluate the safety and tolerability of early enteral feeding after colorectal anastomosis surgeries and postoperative outcomes. This is a retrospective, propensity score-matched case-control study. We retrospectively reviewed the medical records of 708 adult patients undergoing colorectal surgery with GI anastomosis from January 2013 to June 2018 in a medical center of North Taiwan. Among them, 249 cases received early enteral feeding. Another propensity score 1:1 matched 227 patients who did not receive early enteral feeding were also include. Data were collected from patients’ medical records, including gastrointestinal symptoms, the timing of first flatus, timing of passaging the first face, feeding interruption, anastomotic leakages, infection rate, reoperation rate, calorie intake, length of hospital stay and unplanned seven-day readmissions. Chi-square and T-tests were used to compare between-group differences in outcome variables. Results of this study show that early enteral feeding can shorten the length of postoperative hospital stay for 1.53 days (10.94 ± 5.27 vs 12.48 ± 6.75, p < .01) and shorten the time to achieve energy goals for 1.26 days (7.90 ± 2,98 vs. 9.16 ± 3.63, p < .001), compared with the non-early enteral feeding group. There were no significant difference in post-operative complications, including most concerned issue: anastomotic leakage. It is recommended that colorectal surgeons can start enteral nutrition as soon as possible within two days after regular surgery without special concerns. This will not only bring better medical quality but also reduce hospital cost. Because this is a case-control study, it is recommended that randomized controlled trials can be conducted in the future. Take Enhanced recovery after surgery (ERAS) guidelines for colorectal cancer operation as a reference, including perioperative nutrition supplement, management of postoperative ileus and the use of mechanical bowel preparation, it is helpful for postoperative recovery and treatment.

碩士
國立中山大學
企業管理學系醫務管理碩士班
107
Objectives: Helicobacter pylori (H.p) infection is common in Taiwan, with approximately 50% of Taiwanese nationals infected. H.p causes gastric ulcer, gastritis, duodenal ulcer, gastric cancer, and gastric lymphoma. The main objective of this study was to address the research gap in Taiwan on medical resource utilization for early and late antibacterial treatments. In addition, cancer has consistently been the leading cause of death in Taiwan for numerous years. Therefore, this study also aimed to determine the correlation between the early antibacterial treatment for H.p and the risk of developing colorectal cancer based on the relationship between this bacterium and cancer. Methodology: This study employed a retrospective cohort design and secondary data analysis of outpatient and admission records from the 2000 Longitudinal Health Insurance Database obtained from the National Health Insurance (NHI) Research Database of the National Health Research Institutes, Taiwan. Patients that were newly diagnosed with gastric ulcer between 2000 and 2005 and were receiving treatment for H.p were selected. Patients receiving treatments within a year of diagnosis was defined as receiving early antibacterial treatment whereas those receiving treatment later than 1 year after diagnosis were considered as receiving late antibacterial treatment. An analysis was conducted on the mean number of clinical visits and medical expenses of patients in short and long term (1, 2, and 4 years) periods. A 10-year cumulative risk chart of both early and late antibacterial treatment groups was generated using a Kaplan–Meier survival curve, and then the variables of age, sex, comorbidity, and severity of illness were controlled for by applying Cox proportional hazard regression to determine the differences in risk between early and late antibacterial treatment on developing colorectal cancer. In terms of medical resource utilization and the number of return visits, multiple linear regression was applied to control for comorbidities (other confounders such as age, sex, and comorbidities) in order to determine if early and late antibacterial treatments caused statistically significant differences. Results: For patients that received early antibacterial treatment for H.p, the number of return visits within the first and second year was significantly lower than for those who received late treatment. In terms of mean medical expenses, the patients that received early antibacterial treatment spent considerably less on return visits within the first and second year than those who received late antibacterial treatment. Regarding the mean admission expenses, significant differences were observed within the period of 2–4 years. No significant differences were noted regarding the mean number of return visits within 4 years and the mean outpatient and admission expenses within 4 years and 1 year, respectively. Moreover, early or late antibacterial treatment for H.p was not significantly correlated with a risk of developing colorectal cancer. Conclusion: This study revealed that early antibacterial treatment for H.p resulted in fewer return visits compared with late antibacterial treatment, which proved to be effective in lowering medical resource utilization, thus decreasing registration fees and copayments for the NHI. Additionally, in contrast to other studies, the results of this study did not indicate any significant correlation between early antibacterial treatment for H.p and the development of colorectal cancer. This inconsistency in results might have been caused by the use of different diagnostic tools for H.p, research design differences, research bias, and different confounders. According to this study, on the basis of applying preventive health care to reduce NHI expenses and ensuring its sustainability, encouraging patients to receive early antibacterial treatment for H.p is recommended. This measure can lower medical resource utilization and reduce NHI expenses, which will bring substantial health benefits to the Taiwanese population. Key Word: Helicobacter pylori、Colorectal Cancer、Helicobacter pylori eradication、Resources utilization

碩士
國防醫學院
公共衛生學研究所
101
Background: Colorectal cancer is the most increased frequency of new cases among all cancer patients in Taiwan and ranks second incidence rate of all cancer. Recently, due to well prognosis in early stage but no high compliance and accurate colorectal cancer screening tool, the concept through use of epigenetic inheritance of DNA promoter methylation status in feces to predict the progress of colorectal cancer is necessary. However, existing data is not sufficient to assess temporal association between the progression of CRC and aberrant DNA methylation. Therefore, it is necessary to develop new genes as biomarkers. Methods: 20 male SD rats enrolled in our animal studied which collected the stools and given saline injections of either DMH in a dosage of 20 mg/kg body weight (n = 10), and the same volume of saline (n = 10) every week. Collecting feces and isolating the DNA, we use DNA microarray to choose target genes, and the DNA methylation status of colon tissue (tumor/normal) within the target genes was analyzed using methylation-specific PCR. Result: Nr5a2, Pomt1, Hes1 have been choose, but only Nr5a2 has well predictable power. The average sensitivity of the methylated Nr5a2 gene detected in stools in ACF phase, adenoma phase and tumor phase are 5%, 29%, 93%, respectively. Conclusion: Our result shows that Nr5a2 has well prediction on progress of adenoma phase and tumor phase for colorectal cancer, and also confirm that DAN microarray can be used as a more consummate colorectal cancer screening tool.

碩士
國立臺灣大學
流行病學與預防醫學研究所
103
Background As the incidence rate of colorectal cancer (CRC) has been increasing in Taiwan, early detection of CRC through fecal immunochemical test (FIT) screening first and then colonoscopy examination and hospitalization of CRCs cannot be overemphasized. However, the arrival rate of screenees, the non-compliers of undergoing colonoscopy, the waiting time (WT) for undergoing colonoscopy, and the length of stay (LOS) for CRCs has rendered the conventional queue model infeasible. Aims The objective was to integrate the queue process, hurdle model, and Coxian phase-type model into a unifying framework that was applied to two empirical datasets, one relating to the WT of undergoing colonoscopy from Taiwanese nationwide screening program, and the other pertaining to the LOS on hospitalized CRCs enrolled from one medical centre. Methods The hurdle model was developed in combination with a mixture of the logistic regression model that dealt with the non-compliance part and the truncated Poisson regression model pertaining to the WT distribution. The Coxian phase-type was further developed to identify the optimal hidden phase of WT. To further consider the arrival rate of screenees, we developed the queue hurdle Coxian phase-type model which is the combination of the Poisson process, hurdle model and Coxian phase-type model. Data on the LOS of 178 CRCs were modelled by the Coxian phase-type model to identify the optimal number of hidden phases. Results Part I : From 2004 to 2009, the results of the hurdle model indicate the factors associated with non-compliance for colonoscopy included female, older age group, eastern Taiwan or offshore islands area, rural area, hospital screening unit and prevalent screening rounds, and the factors associated with shorter WT for colonoscopy included middle Taiwan area, main urban area, public health centers screening unit and subsequent screening rounds. Part II : The queue hurdle 2-phase Coxian phase-type model was classified as short- and long waiting phase. The arrival rate was 0.00021 per person-days and the probability of non-compliance with colonoscopy was 0.26. Annually, around 15% subjects were so hesitant to be referred to undergo colonoscopy that they were trapped in long waiting phase. The mean WT of short waiting phase and long waiting phase were 32 days and 169 days, respectively. Further to consider the effect of risk score on the model, the queue hurdle 2-phase Coxian phase-type model indicates the mean WT in short waiting phase were 36 days and 30 days for the low score group and the high score group, separately and 167 days in longer waiting phase among these two groups. Part III : For hospitalization, the LOS with 178 CRCs was modelled by the 3-phase Coxian phase-type model classified as short-stay, medium-stay and longer-stay phase. In the short-stay phase, the expected LOS was 10 days whereas both the medium- and longer-stay phases were 49 days. When gender was taken into account, the LOS was modelled as a 2-phase Coxian phase-type model, short- and long-stay care. It shows that male would discharge or die earlier than female. Regarding age, it shows the elderly would discharge or die earlier than the young. Conclusions A new queue hurdle Coxian phase-type model was developed to solve the queue process, the hurdle issue in relation to the problem of non-compliance with the referral of positive results of screenees to have confirmatory diagnosis, and to identify hidden phases during the WT for undergoing colonoscopy among the referrals and LOS in hospitalization for the treated CRCs.