In most randomised controlled trials (RCTs), a large number of patients need to be followed over many years, for the clinical benefit of the drug to be accurately quantified (1). Using an early proxy, or a surrogate endpoint, in place of the direct endpoint of overall survival (OS) could theoretically shorten the duration of RCTs and minimise the exposure of patients to ineffective or toxic treatments (2, 3). This thesis examined the relationship between surrogate endpoints and OS in metastatic colorectal cancer (CRC), advanced non-small cell lung cancer (NSCLC) and metastatic breast cancer (MBC). A review of the literature identified 144 RCTs in metastatic CRC, 189 in advanced NSCLC and 133 in MBC. The publications were generally of poor quality with incomplete reporting on many key variables, making comparisons between studies difficult. The introduction of the CONSORT statement was associated with improvements in the quality of reporting. For CRC (337 arms), NSCLC (429 arms) and MBC (290 arms) there were strong relationships between OS and progression free survival (PFS), time to progression (TTP), disease control rate (DCR), response rate (RR) and partial response (PR). Correlation was also demonstrated between OS and complete response (CR) in CRC and duration of response (DOR) in MBC. However, while strong relationships were found, the proportion of variance explained by the models was small. Prediction bands constructed to determine the surrogate threshold effect size indicated that large improvements in the surrogate endpoints were needed to predict overall survival gains. PFS and TTP showed the most promise as surrogates. The gain in PFS and TTP required to predict a significant gain in overall survival was between 1.2 and 7.0 months and 1.8 and 7.7 months respectively, depending on trial size and tumour type. DCR was a better potential predictor of OS than RR. The results of this study could be used to design future clinical trials with particular reference to the selection of surrogate endpoint and trial size.

Background: Laparoscopic surgery for colorectal cancer is a novel healthcare technology, for which much research evidence has been published. The objectives of this work were to compare the oncologic outcomes of this technology across different study types, and to define patterns of adoption on the basis of the literature. Methods: A comprehensive systematic review of the literature was conducted using 1) existing systematic reviews, 2) randomized controlled trials (RCTs), and 3) observational studies. Outcomes of interest were overall survival, and total lymph node harvest. Outcomes were compared for congruence. Adoption was evaluated by means of summary expert opinions in the literature. Results: 1) Existing systematic reviews were of low to moderate quality and displayed evidence of overlap and duplication. 2) Laparoscopy was not inferior to open surgery in terms of oncologic outcomes in any study type. 3) Oncologic outcomes from RCTs and observational studies were congruent. 4) Expert opinion in the literature has been supportive of this technology, paralleling the publication of large RCTs. Conclusions: The evaluation of laparoscopic surgery for colorectal cancer in RCTs and observational studies suggests that it is not inferior to open surgery. Adoption of this technology has paralleled RCT evidence.

Germline mutations in mismatch repair genes predispose individuals to Lynch Syndrome, the most common colorectal cancer predisposition syndrome. MLH1 is a key mismatch repair gene that is mutated in Lynch syndrome and about a third of the genetic alterations identified in MLH1 are missense variants of unclassified clinical significance. We hypothesize that missense alterations in distinct domains of MLH1 likely affect its expression and function(s) to varying degrees. To address this we utilized several approaches to investigate the molecular basis of the pathogenicity of a panel of unclassified variants. Our results demonstrate that the MLH1 variants p.R265C and p.K618A significantly decrease the stability of the MLH1 protein, while the variant p.L749Q compromises heterodimerization of the MLH1-PMS2 complex. Given the limitations and complexity of in vitro assessment strategies, we conducted a proof-of-principle study to investigate whether missense variants in MLH1 lead to allelic imbalances at the transcriptional level. Our analysis using the PeakPicker software indicated that the missense variants c.350C>T, c.793C>T, c.1852_1853AA>GC, as well as the truncating variant c.1528C>T were associated with significantly unbalanced allelic expression. This illustrates a novel method of investigating the pathogenicity of unclassified genetic variants, which has the potential to be applied in the diagnostic setting. Previous genetic epidemiology studies from our laboratory have demonstrated that the MLH1 c.-93G>A promoter variant is strongly associated with the microsatellite instability phenotype in colorectal tumours. Additionally, this promoter variant was associated with an elevated risk of endometrial cancer in case-control studies. Results from our functional studies indicate that the c.-93G>A variant significantly alters the promoter activity of MLH1. The MLH1 promoter is bi-directional with the EPM2AIP1 gene located on the antisense strand. Interestingly, we observed that this variant significantly affected EPM2AIP1 transcription as well. Furthermore, our experiments suggest that c.-93G>A variant affects transcription by altering the affinity of nuclear factors that bind this region. Combined, these findings shed light on the possible mechanisms by which missense variants affect MLH1 expression and function, which in conjunction with results from other functional assays will help increase the accuracy and efficiency of genetic testing of inherited cancers.

Germline mutations in mismatch repair genes predispose individuals to Lynch Syndrome, the most common colorectal cancer predisposition syndrome. MLH1 is a key mismatch repair gene that is mutated in Lynch syndrome and about a third of the genetic alterations identified in MLH1 are missense variants of unclassified clinical significance. We hypothesize that missense alterations in distinct domains of MLH1 likely affect its expression and function(s) to varying degrees. To address this we utilized several approaches to investigate the molecular basis of the pathogenicity of a panel of unclassified variants. Our results demonstrate that the MLH1 variants p.R265C and p.K618A significantly decrease the stability of the MLH1 protein, while the variant p.L749Q compromises heterodimerization of the MLH1-PMS2 complex. Given the limitations and complexity of in vitro assessment strategies, we conducted a proof-of-principle study to investigate whether missense variants in MLH1 lead to allelic imbalances at the transcriptional level. Our analysis using the PeakPicker software indicated that the missense variants c.350C>T, c.793C>T, c.1852_1853AA>GC, as well as the truncating variant c.1528C>T were associated with significantly unbalanced allelic expression. This illustrates a novel method of investigating the pathogenicity of unclassified genetic variants, which has the potential to be applied in the diagnostic setting. Previous genetic epidemiology studies from our laboratory have demonstrated that the MLH1 c.-93G>A promoter variant is strongly associated with the microsatellite instability phenotype in colorectal tumours. Additionally, this promoter variant was associated with an elevated risk of endometrial cancer in case-control studies. Results from our functional studies indicate that the c.-93G>A variant significantly alters the promoter activity of MLH1. The MLH1 promoter is bi-directional with the EPM2AIP1 gene located on the antisense strand. Interestingly, we observed that this variant significantly affected EPM2AIP1 transcription as well. Furthermore, our experiments suggest that c.-93G>A variant affects transcription by altering the affinity of nuclear factors that bind this region. Combined, these findings shed light on the possible mechanisms by which missense variants affect MLH1 expression and function, which in conjunction with results from other functional assays will help increase the accuracy and efficiency of genetic testing of inherited cancers.

Background: Laparoscopic surgery for colorectal cancer is a novel healthcare technology, for which much research evidence has been published. The objectives of this work were to compare the oncologic outcomes of this technology across different study types, and to define patterns of adoption on the basis of the literature. Methods: A comprehensive systematic review of the literature was conducted using 1) existing systematic reviews, 2) randomized controlled trials (RCTs), and 3) observational studies. Outcomes of interest were overall survival, and total lymph node harvest. Outcomes were compared for congruence. Adoption was evaluated by means of summary expert opinions in the literature. Results: 1) Existing systematic reviews were of low to moderate quality and displayed evidence of overlap and duplication. 2) Laparoscopy was not inferior to open surgery in terms of oncologic outcomes in any study type. 3) Oncologic outcomes from RCTs and observational studies were congruent. 4) Expert opinion in the literature has been supportive of this technology, paralleling the publication of large RCTs. Conclusions: The evaluation of laparoscopic surgery for colorectal cancer in RCTs and observational studies suggests that it is not inferior to open surgery. Adoption of this technology has paralleled RCT evidence.

The use of colonoscopy has demonstrated to be the most effective method to reduce colorectal cancer(CCR) mortality . A wide variety of screening modalities have been developed to date and the majority demonstrated to be effective to reduce CCR cancer incidence and mortality ratesColonoscopy is presently the gold standard examination method of the colon. This unique feature explains why its practice is done in most hospitals of the present healthcare systems. Quality measurements of any process require firstly, the definition of valid and reliable indicators which enables its assessment . In the recent years, many attempts have been tried in order to define useful criteria for this purpose. There is strong evidence which asserts colonoscopy performance varies among different centers and between endoscopists. We present the process of application and adaption of several parameters to a specific context in order to evaluate the quality of a CCR screening program. / La práctica de la colonoscopia ha demostrado ser el método más eficaz en la reducción de mortalidad por cáncer colorectal(CCR). Se han diseñado un número importante de estrategias de cribado en los últimos años, todas ellas son eficaces en la reducción de la incidencia y mortalidad por este tipo de cáncerLa colonoscopia es el método de exploración de referencia del colon. Esta singularidad explica el hecho de que su práctica se lleva a cabo en prácticamente todos los niveles de asistencia hospitalarios. Existen varias líneas de evidencia que sugieren que la calidad de la colonoscopia en la práctica clínica habitual varía de manera considerable, el problema planteado la inexistencia de herramientas capaces de medir estos niveles.En el presente estudio se plantea el proceso de definición de una serie de indicadores que posteriormente serán aplicados en el análisis del nivel de calidad de un programa de cribado de CCR.

Vascular endothelial growth factor (VEGF-A) was first described in 1989 for its angiogenic and mitogenic properties. Early studies indicated that VEGF-A acts primarily in a paracrine pathway which is limited to vascular endothelium. Further investigation showed that VEGF-A and VEGF receptor-2 (VEGFR-2) are expressed by many solid tumors and improve cell growth and survival. Therefore, VEGF-A may act via an autocrine pathway that effects tumor cellular proliferation by binding VEGFR-2 at the cell surface. This study utilizes small interfering RNA (siRNA) technology to investigate the presence of an autocrine loop in human RKO colorectal cancer cells. RT-PCR demonstrated the expression of VEGF-A, VEGF-B, VEGF-D, placental growth factor (PlGF), VEGFR-2, neuropilin-1 (NP-1) and neuropilin-2 (NP-2) in vitro by RKO cells. Transfection with siRNA against VEGF-A resulted in a 94% knockdown of VEGF-A expression by ELISA. Northern blot, quantitative real time PCR and semiquantitative RT-PCR confirmed the knockdown data. In addition, transfected RKO cells showed a 67% decrease in cellular proliferation by WST-1 assay. This data correlated to the ELISA results. In summary, the presence of VEGF-A and VEGFR-2 argues in favor of an autocrine loop in human colorectal cancer cells. siRNA targeting of VEGF-A remains a promising anti-tumor therapeutic strategy.

Carcinogenesis is a progressive process involving multiple genetic alterations in tumor cells and complex interactions in the tumor-host microenvironment. To better understand the contribution of molecular alterations in tumor cells and stromal variables to the development of colorectal cancer (CRC) and identify prognostic factors, in this study we examined the clinicopathological and biological significance of stromal variables, including particularly interesting new cysteine-histidine rich protein (PINCH), inflammatory infiltration, angiogenesis and lymphangiogenesis, as well as hRAD50/hMRE11/hNBS1 proteins and hRAD50 mutation in tumor cell in CRC. PINCH protein expression in the stroma was increased from normal mucosa to primary tumors and further to lymph node metastases. In particular, PINCH expression was most intense at the tumor invasive margin, which was related to low inflammatory infiltration and independently related to an unfavorable prognosis. Low inflammatory infiltration at the tumor invasive margin was related to advanced tumor stage, worse differentiation and microsatellite instability (MSI). Further, it was independently related to an unfavorable prognosis. Increased blood and lymphatic vessel density was observed in the primary tumors compared with the corresponding normal mucosa. However, neither angiogenesis nor lymphangiogenesis was associated with tumor stage and patients’ survival. Moreover, PINCH was present in a proportion of endothelial cells of the tumor vasculature, and PINCH expression in tumor-associated stroma was positively related to blood vessel density. In primary tumor cells of CRC, strong expression of hRAD50, hMRE11 or hNBS1 was related to microsatellite stability (MSS). A high percentage of hMRE11 expression was associated with less local recurrence and high apoptotic activity. Further, we observed that the expression of hRAD50, hMRE11 or hNBS1 among normal mucosa, primary tumors and metastases in MSS CRC differed from that in MSI CRC. In MSS CRC, the expression intensity of hRAD50, hMRE11 and hNBS1 was consistently increased with respect to normal mucosa, but there was no difference between the primary tumors and metastases. In the primary MSS tumors, the expression of individual or combination of hRAD50/hMRE11/hNBS1 was associated with a favorable prognosis in the same series of the CRCs. Moreover, strong/high hRAD50 in MSS primary tumors was related to earlier tumor stage, better differentiation and high inflammatory infiltration, whereas strong hNBS1 expression tended to be independently related to a favorable prognosis in MSS CRC with earlier tumor stage. However, in MSI CRC, there were neither differences in the expression of hRAD50/hMRE11/hNBS1 among normal mucosa, primary tumors and metastases, nor any association of the protein expressions with clinicopathological variables. On the other hand, frameshift mutations of (A)9 at coding region of hRAD50 were only found in MSI CRC. Our study indicates that 1) PINCH is likely a regulator of angiogenesis, and PINCH expression at the tumor invasive margin is an independent prognostic indicator in CRC. 2) Inflammatory infiltration at the tumor invasive margin is also an independent prognostic indicator in CRC. The lack of association between high inflammatory infiltration and MSI may help to explain the non-association of MSI with survival in CRC patients. 3) Angiogenesis and lymphangiogenesis occur in the early stage of CRC development, but do not associate with CRC progression and patients’ prognosis. 4) hRAD50/hMRE11/hNBS1 may act dependently and independently, playing different roles in MSS and MSI CRC development. In MSS CRC, the strong expression of the three proteins, associated with a favorable prognosis, may present the cellular response against tumor progression. Expression of hNBS1 may be a prognostic indicator for MSS CRC patients in the earlier tumor stage. In MSI CRC, the frameshift mutations at the coding region of hRAD50 may contribute to tumor development.

Background: Genetic and environmental factors have been associated with colorectal cancer (CRC) risk. However, their association with prognosis has been less studied. Methods: Path analysis was employed to examine causal pathways from education to environmental (diet, alcohol, smoking, physical activity) and personal factors (screening), and then to obesity and ultimately to tumour characteristics (stage, grade, microsatellite instability (MSI), and site) that are associated with CRC prognosis. Data came from the Ontario Familial Colon Cancer Registry. Pathways were evaluated for effect modification by sex and two indicators of CRC genetic susceptibility (Bethesda criteria and newly identified familial cancer clusters). Results: Four food patterns (healthy foods, high-fat foods, sweet and processed foods, and oriental foods) and four nutrient patterns (total macronutrient, fat vs. carbohydrate, and micronutrients from supplements and from foods) were identified. Education was associated positively with healthy lifestyle factors (e.g. healthy foods factor) and negatively with unhealthy factors (e.g. smoking). As expected, high body mass index (BMI) was associated with lower physical activity and higher fat vs. carbohydrate factor. Unexpectedly, BMI was positively associated with the healthy foods factor among Bethesda positive patients and men. An association between education and BMI was mediated by the healthy foods factor and by physical activity. Important poor prognostic factors, higher grade and stage, were associated with smoking and not being screened. However, unexpected associations included a positive association of physical activity with tumour grade among Bethesda positive patients and a positive association of healthy foods with stage among Bethesda negative patients. Patients with right-sided tumours were more likely to receive micronutrients from supplements, and screening and less likely to smoke, and for men, to have a high BMI, high fat diet and healthy food diet. Conclusion: Some unhealthy lifestyle factors, such as smoking and a high fat food dietary pattern, are associated with adverse CRC tumour characteristics and so may affect the prognosis. Family history may modify some associations though the findings require independent confirmation.

Background: Genetic and environmental factors have been associated with colorectal cancer (CRC) risk. However, their association with prognosis has been less studied. Methods: Path analysis was employed to examine causal pathways from education to environmental (diet, alcohol, smoking, physical activity) and personal factors (screening), and then to obesity and ultimately to tumour characteristics (stage, grade, microsatellite instability (MSI), and site) that are associated with CRC prognosis. Data came from the Ontario Familial Colon Cancer Registry. Pathways were evaluated for effect modification by sex and two indicators of CRC genetic susceptibility (Bethesda criteria and newly identified familial cancer clusters). Results: Four food patterns (healthy foods, high-fat foods, sweet and processed foods, and oriental foods) and four nutrient patterns (total macronutrient, fat vs. carbohydrate, and micronutrients from supplements and from foods) were identified. Education was associated positively with healthy lifestyle factors (e.g. healthy foods factor) and negatively with unhealthy factors (e.g. smoking). As expected, high body mass index (BMI) was associated with lower physical activity and higher fat vs. carbohydrate factor. Unexpectedly, BMI was positively associated with the healthy foods factor among Bethesda positive patients and men. An association between education and BMI was mediated by the healthy foods factor and by physical activity. Important poor prognostic factors, higher grade and stage, were associated with smoking and not being screened. However, unexpected associations included a positive association of physical activity with tumour grade among Bethesda positive patients and a positive association of healthy foods with stage among Bethesda negative patients. Patients with right-sided tumours were more likely to receive micronutrients from supplements, and screening and less likely to smoke, and for men, to have a high BMI, high fat diet and healthy food diet. Conclusion: Some unhealthy lifestyle factors, such as smoking and a high fat food dietary pattern, are associated with adverse CRC tumour characteristics and so may affect the prognosis. Family history may modify some associations though the findings require independent confirmation.