Rozprawy doktorskie na temat „Colorectal cancer”

Colorectal cancer (CRC) is the third most common cancer in the world, with about 5000 new cases in Sweden every year. CRC is caused by mutation (inherited or acquired) in genes, by gene variants and changed expression of proteins. The primary way to achieve a curative result for CRC is to remove the tumor by surgery. To reduce risk of recurrence chemo- or radiotherapy are given as a complement to surgery. p73 is a structural and functional homologue of tumor suppressor p53. However, p73 is rarely mutated in tumors, but rather overexpressed as compared to normal tissue. There are two main isoforms of p73, the transactivation capable TAp73 and the truncated ΔNp73, which are involved in an autoregulatory loop with TAp73 and p53. The aim of this study was to investigate the role of p73 and related proteins in the development and treatment of CRC. A G4C14-to-A4T14 polymorphism of p73 was studied in CRC patients and healthy controls (Paper I), and rectal cancer patients who were randomized to treatment with either surgery alone or preoperative radiotherapy and surgery (Paper II). The AT/AT genotype of the p73 polymorphism may increase risk of CRC development and CRC patients with the AT allele had a better prognosis. When dividing the cases into colon and rectal cancer it was seen that in colon cancer the AT allele tended to be more favorable for overall survival, while in rectal cancer the GC allele seemed to be more favorable. Rectal cancer patients, with a combination of GC/GC genotype, wild type p53 and weak survivin expression survived longer after preoperative radiotherapy. This was not observed in the patients only receiving surgery. The protein expression of p73 was further studied in the rectal cancer patients randomized to treatment with either surgery alone or preoperative radiotherapy and surgery (Paper III). p73 was expressed higher in tumor tissue than in normal mucosa. Patients with p73 negative tumors had a lower risk of local recurrence after radiotherapy, as opposed to patients that had p73 positive tumors or patients with p73 negative tumors that did not receive radiotherapy. Effects of γ-radiation was further studied in colon cancer cell lines KM12C, KM12SM and KM12L4a regarding cell cycle, survival fraction (clonogenicity), apoptosis and protein expression patterns of mutated p53, TAp73, ΔNp73, survivin and PRL-3 (Paper IV). KM12C displayed low survival fraction, low apoptosis, no cell cycle arrest and an upregulation of the antiapoptotic ΔNp73 after irradiation. KM12L4a showed a high survival fraction, but high apoptosis, arresting of the cell cycle and upregulation of the radio-resistance factor survivin. The effects of overexpression and knockdown of survivin on TAp73, ΔNp73 and p53 expression in colon cancer cell lines HCT-116p53+/+ and HCT-116p53-/- with and without γ-radiation were studied (Paper V). Overexpression of survivin decreased wild type p53, whilst downregulation of survivin lead to a simultaneous downregulation of TAp73 and ΔNp73, mRNA and protein, both with and without γ- radiation. Knockdown of survivin also demonstrated an increase in apoptosis. In conclusion, we showed that the G4C14-to-A4T14 polymorphism of p73 and p73 protein expression may be involved in CRC development, radiotherapy response and survival. We further showed that TAp73, ΔNp73 and p53 were regulated by survivin in colon cancer cells.
Cancer i tjocktarmen (kolon) och ändtarmen (rectum) är den tredje vanligaste cancerformen i världen och i Sverige drabbas varje år ca 5000 personer av tjockoch ändtarmscancer. Antalet drabbade individer är högre i de industrialiserade länderna än övriga världen, vilket tyder på att vissa omgivnings- och livsstilsfaktorer påverkar risken för tjock- och ändtarmscancer. Även vissa genetiska faktorer påverkar risken för insjuknande i dessa cancerformer. En gen är mallen för ett eller flera proteiner och en förändring (mutation) i en gen kan innebära att motsvarande protein förändrar eller förlorar sin normala funktion. En del gener har också nedärvda naturliga variationer, så kallade polymorfier, vilka i sin tur kan leda till variation i funktionen hos motsvarande protein. Proteiner som förändrat eller förlorat sin normala funktion bidrar till de speciella egenskaper som finns hos cancerceller. Cancerceller till skillnad från normala celler delar sig okontrollerat, är motståndskraftiga mot den programmerade celldöd (apoptos) som normalt förstör skadade celler och kan sprida sig via blodbanan till andra organ (metastasera). Tjock- och ändtarmscancer behandlas främst med kirurgi, cellgifter och strålning och tack vare förbättrade behandlingar har dödligheten minskat de senaste årtiondena. Genom att studera olika genetiska varianter, proteiner och förhållandet mellan proteiner i cancerceller får vi större insikt i vilka faktorer som ökar risken för tjock- och ändtarmscancer, men också vilka faktorer som påverkar hur effektiv en behandling är för varje individuell patient. Målet med denna avhandling var att studera proteinet p73 och hur den påverkar risken för tjock- och ändtarmscancer och behandlingseffekten av sjukdomen. p73 tillhör samma proteinfamilj som “genomets väktare” p53 och båda har förmågan att skydda cellen genom att stoppa celldelning och inducera apoptos i skadade celler. Dock är p53 muterat i ungefär 50 % av alla tjock- och ändtarmscancrar. Genom att studera en polymorfi i p73 hos tjock- och ändtarmscancerpatienter och friska blodgivare såg vi att de individer som har dubbel uppsättning av variantgenen har ökad risk att utveckla tjock- och ändtarmscancer och att de patienter som är bärare av variantgenen hade en längre överlevnad (Paper I). Ändtarmscancerpatienter som fått strålbehandling överlevde längre om de hade dubbel uppsättning av den ursprungliga p73-genen, icke-muterat p53 och lågt uttryck (mängd) av survivin. Survivin är ett protein som normalt hindrar cancerceller från att dö (Paper II). De ändtarmscancerpatienter som behandlats med strålning och som hade p73 negativa tumörer fick mycket färre återfall än de patienter som hade p73 negativa tumörer men inte fått strålbehandling, eller som hade p73 positiva tumörer (Paper III). För att närmare studera tjocktarmscancercellers motståndsmekanismer mot strålbehandling mättes bland annat olika typer av celldöd och uttrycket av två olika varianter av p73, kallade TAp73 och ΔNp73, survivin och PRL-3. PRL-3 är ett protein som uttrycks starkast i metastatiska tumörer och leder till ökat motstånd mot strålbehandling. Den cellinje som inte dog genom apoptos hade högt uttryck av ΔNp73, vilket är en variant av p73 som hindrar celler från att dö, medan den som trots strålning i hög grad fortsatte dela sig hade ett högt uttryck av survivin (Paper IV). Efter indikationer om att uttrycket av p73 proteinerna TAp73 och ΔNp73 är kopplade till survivin undersöktes ett eventuellt förhållande mellan dessa proteiner i tjocktarmscancerceller. I de fall där uttrycket av survivin minskades i cellerna minskade också uttrycket av TAp73 och ΔNp73. Detta fenomen sågs även i celler som behandlats med strålning. En minskning av survivin i cellerna ledde även till att fler celler dog genom apoptos (Paper V). Sammanfattningsvis pekar våra resultat på att p73 spelar en viss roll i utvecklingen av tjock- och ändtarmscancer, men också avseende effekterna på strålbehandling av ändtarmscancer. Vi har också visat att TAp73, ΔNp73 och p53 regleras av survivin.

Le champ de la pharmacogénétique est d'une importance cruciale en oncologie pour optimiser la sélection du traitement à utiliser en fonction du profil génomique du patient et de la tumeur. En effet, au-delà des caractéristiques spécifiques de la tumeur, le génome de l'individu explique une grande partie de la variation de la réponse du observée à des agents chimiothérapeutiques à la fois en terme d'efficacité et de toxicité. Les patients atteints de cancer colorectal (CCR) sont susceptibles de recevoir une ou plusieurs lignes de chimiothérapie avec une efficacité variable et de faire l'expérience des effets secondaires connexes. Il est donc essentiel d'optimiser l’arsenal thérapeutique pour améliorer l’efficacité des traitements en évitant au maximum les effets indésirables. Le but des études de pharmacogénétique est d'étudier spécifiquement pour chaque médicament les voies métaboliques impliquées et leurs variations interindividuelles potentielles secondaires à des mutations génomiques ou somatiques. Cette recherche vise ainsi à identifier les biomarqueurs pronostiques et prédictifs qui aideront à une meilleure sélection de traitement. Pour les patients atteints de CRC, le bénéfice de survie de l'administration de la chimiothérapie adjuvante chez les patients de stade II et III reste à évaluer. En effet, l'avantage de survie donné par la perfusion de 5-fluorouracile en adjuvant (avec ou sans oxaliplatine) a été montré pour les patients de stade III CRC mais est encore indéterminé pour les patients de stade II CRC. Par définition, les mutations somatiques sont détectées dans le génome des cellules tumorales et ont été associées à la réponse aux agents chimiothérapeutiques. En outre, plusieurs rapports ont suggéré l'importance du rôle des variations héréditaires (génome constitutionnel) pour la réponse aux médicaments et à la prévision des effets secondaires. Dans ce travail, je me suis concentrée sur la relation entre les polymorphismes et la réponse aux chimiothérapies chez les patients de stade II - III CRC. J'ai observé que la cycline D1 (CCND1), les canaux sodique voltage-dépendants (SCN1A), les régulateurs de la voie WNT / β - caténine, KSR et les gènes de cellules souches cancéreuses pouvaient prédire la réponse et la survie de patients traités pour CCR en situation adjuvante
The pharmacogenetics field is of crucial importance in oncology to optimize the selection of which chemotherapy regimen to use according to the patient’s and tumor’s genomic profile. Indeed, beyond the specific tumor characteristics, the individual’s inherited genome accounts for a large proportion of the variation in patient’s response to chemotherapeutic agents both in term of efficiency and toxicity. Patients with colorectal cancer are likely to receive one or several lines of chemotherapy with variable efficacy and to experience some related side effects. It is therefore critical to tailor the best therapeutic arsenal to improve treatments efficacy meanwhile avoiding adverse reactions susceptible to lead to treatment disruption as much as possible. The purpose of pharmacogenetics studies is to specifically investigate for each drug the implicated metabolic pathways and their potential individual variations related to genomic or somatic mutations. This research aims at identifying both prognostic and predictive biomarkers that will help for the best treatment selection. In CRC, one important issue remains to evaluate the survival benefit of adjuvant chemotherapy administration in patients with stage II and III CRC. In this setting, the survival advantage given by adjuvant 5-fluoruracil-infusion (with or without oxaliplatin) has been shown for patients with stage III CRC but is still undetermined for patients with stage II CRC. By definition somatic mutations can be found in tumor cells genome and have been related with response to chemotherapeutic agents. In addition, several reports suggested the important role of inherited variations (constitutional or germ line) for drug response and side effects prediction

Colorectal cancer (CRC) is one of the commonest malignancies in the United Kingdom and tumour cell invasion and metastasis is the main cause of death. The transcriptional repressor ZEB1 has been shown to be expressed in several epithelial malignancies and embryonic epithelial-mesenchymal transitions (EMT). The understanding of cellular signalling cascades should allow the discovery of novel targets for potential future therapeutic manipulation in the treatment of colorectal cancer and other malignancies. Here, in order to further investigate the role of ZEB1 in CRC, in vitro investigations and immunohistochemical analysis of 101 colorectal cancers and their matched lymph node and liver metastases are performed. The Wnt-Inducible Signalling Proteins and Plakophilin-3 are also investigated for their relationship with ZEB1 signalling. ZEB1 is expressed in tumour cell lines with mesenchymal characteristics and over-expression in an epithelial-phenotype cancer cell line causes down-regulation of epithelial markers such as E-cadherin and Plakophilin-3. Knockdown of ZEB1 in mesenchymal cell lines caused up-regulation of PKP-3 expression. In vitro attempts at manipulation of WISP expression were unsuccessful. In CRC tumours ZEB1 was noted to be up-regulated at the tumour invasive front, with concomitant loss of Plakophilin-3 expression. However expression of these markers did not correlate with disease stage or survival on multivariable analysis. Increased WISP-1 nuclear and cytoplasmic expression were noted at the tumour invasive front and correlated with disease stage and several pathological factors on univariable analysis. Increased nuclear WISP-1 expression at the invasive front correlated with survival on uni- and multivariable analysis. This study demonstrates that ZEB1, Plakophilin-3 and WISP-1 are expressed in CRC and may be involved in tumour cell invasion and dissemination at the invasive front. Expression of nuclear WISP-1 is an independent predictor of poor survival and is worthy of further investigation as a target of future therapeutic intervention.

Thrombosis and colorectal cancer have a bi-directional relationship. The presence of a colorectal malignancy results in an increased risk of developing a thrombosis and the presence of a thrombosis results in a worse cancer prognosis. The physiology causing this is at present unclear but it is proposed that proteins from the tissue factor (TF) pathway may be the instigator of this bi-directional relationship. The in-vitro studies have shown that in colorectal cancer TF impairs that action of colorectal cancer stem cells as demonstrated by reduced cancer sphere formation and also lower expression of the stem cell marker ALDH. The ability for a colorectal cell to avoid anoikis is impaired by a reduced TF level. Proliferation is affected by the level of expression of TF with a significant increase in proliferation with additional expression of TF. The increase in proliferation is further increased by the presence of TF’s ligand factor VIIa. Paradoxically reduced expression of TF also increases colorectal cancer expression. The ERK1/2 pathway offers a possible method by which TF and factor VIIa may exert their proliferative effects. In the prospective clinical cohort study (CHAMPion) abnormal expression of TF pathway proteins (TF, PAR1, PAR2 and thrombin) by both malignant epithelial and cancer associated stromal cells has been demonstrated. The stromal expression was independent of the epithelial expression and was only in stroma in close contact (0.1mm) with epithelial cells suggesting that the TF pathway proteins may have a role in stromal/epithelial communication. There was no link between the expression of TF pathway proteins and clinicopathological markers of a poor prognosis. The plasma expression of markers of TF pathway activation did not demonstrate any role as a biomarker for colorectal cancer or prognosis. The CHAMPion study has demonstrated that 7% of patients undergoing surgery for colorectal cancer have asymptomatic pre-operative DVTs present. A further 6% who were DVT free pre-operatively developed a DVT in the peri-operative period despite receiving venous thromboprophylaxis in line with current national guidelines. Pre-operative d-dimer may have the potential to identify those patients at risk of a post-operative VTE.This thesis establishes the role that TF has in promoting proliferation and anoikis resistance. It also confirms the abnormal expression of TF pathway proteins by colorectal cancer epithelial cells and for the first time demonstrates abnormal expression by the cancer associated stroma. The interaction between the stroma and epithelial cells, combined with the cellular effects of TF suggests that targeting this interaction may have a therapeutic role. The incidence of DVTs pre-operatively suggests that screening patients for the asymptomatic presence of a DVT may have an impact on their clinical outcome. The development of DVTs despite prophylaxis suggests that the level of anticoagulation is insufficient and current guidelines need to be revisited.

Colorectal cancer (CRC) remains a significant burden in contemporary society due to an aging population, unhealthy dietary choices and an increasingly sedentary lifestyle. While the underlying defects for many hereditary forms of CRC have been determined, many genetic and epigenetic changes promoting common sporadic CRCs have yet to be identified. The Mutated in Colorectal Cancer (MCC) gene, identified in 1991, was initially thought to be responsible for the hereditary form of CRC, familial adenomatous polyposis, before the discovery of the susceptibility gene Adenomatous Polyposis Coli (APC), which then became the focus of intense research. Recent data, however, suggests that MCC may also be important in the development of CRC. I have investigated the mechanism of MCC gene silencing, the putative structure, and multiple functions of MCC. MCC was frequently silenced by promoter hypermethylation in CRC cell lines and primary tumours. MCC methylation showed strong molecular and clinicopathological associations with hallmarks of the serrated neoplasia pathway. Furthermore, MCC methylation was more frequent in serrated precursor lesions compared with adenomas, thus occurring early during carcinogenesis. MCC is highly conserved in complex multicellular organisms. Re-introduction of MCC in CRC cell lines resulted in partial G1 to S phase, and G2/M phase cell cycle blocks, potentially by upregulating cell cycle inhibitor gene transcription and interfering with the process of mitotic checkpoints and division, respectively. Changes in MCC levels also modulated NF?B pathway signalling, the pathway required for maintaining cell viability and proliferation in colonic epithelial cells. In particular, MCC overexpression suppressed both TNF? and LPS-induced NF?B activation, decreasing both the magnitude and rate of cellular responses. Overexpression also resulted in downregulation of proteins involved in canonical NF?B pathway signalling, while increasing the transcription of non-canonical NF?B genes. Therefore, MCC may direct activation of this pathway to a specific subset of NF?B-regulated genes. These data provide a molecular basis for the role of MCC as a tumour suppressor gene in CRC. MCC may have multiple functions, regulating cell cycle progression and modulating NF?B pathway signalling, either through direct involvement in pathway signalling cascades, or by providing a scaffold on which signalling events can occur.

Anthocyanins, polyphenolic phytochemicals which render fruit and vegetables bright red or blue, possess anticarcinogenic properties in preclinical models of carcinogenesis. The aim of this study was to elucidate whether consumption of mirtocyan, a standardised anthocyanin extract, would cause pharmacodynamic changes consistent with chemoprevention and generate measurable levels of anthocyanins in blood, urine and target tissue. Twenty-five patients with either primary colorectal cancer or colorectal liver metastases received 1.4, 2.8 or 5.6 g of mirtocyan (containing 0.5-2.0 g anthocyanins) daily for 7 days prior to colon/liver resection. Anthocyanin levels were measured by high performance liquid chromatography. Proliferation (Ki-67), apoptosis (caspase-3) and inflammation (COX-2) were measured in colorectal tumour tissue. Effects on the insulin-like growth factor (IGF) axis were evaluated in plasma and markers of oxidative DNA damage were assessed in blood and urine. Consumption of up to 5.6 g of mirtocyan daily was well tolerated. Analysis of colorectal tumour tissue revealed that consumption of mirtocyan was associated with a 7% decrease in proliferation (p=0.003) and a 1.7% increase in apoptosis (0.044). A trend towards a reduction in circulating IGF-1 levels was observed (p=0.168). Mirtocyan anthocyanins and methyl and glucuronide metabolites were identified in plasma, urine and colorectal tissue, but not in liver. Anthocyanin concentrations in biomatrices were approximately dose-dependent. Following consumption of 5.6 g of mirtocyan daily anthocyanin levels in plasma, urine and colorectal tumour tissue were 117 ng/ml, 3 μg/ml and 179 ng/g, respectively. Mirtocyan did not affect levels of COX-2 or markers of oxidative DNA damage. Administration of mirtocyan furnished levels of anthocyanins in colorectal tumour tissue comparable to those capable of mediating chemopreventive effects in vivo. Consumption of only 1.4 g of mirtocyan daily may exert pharmacodynamic effects commensurate with colorectal cancer chemoprevention. These data support further clinical development of anthocyanins as potential colorectal cancer chemopreventive agents.

Mitjançant tècniques d’anàlisi de tot el genoma hem sigut capaços d'identificar regions diferencialment metilades entre teixit de colon normal i tumoral provinents del mateix pacient. Un dels gens més importants és AKR1B1, l'illa CpG associada al promotor d’aquest gen esdevé hipermetilada en un 90% dels càncers colorectals estudiats (més de 200), aquesta troballa ha estat confirmada en dos sets de mostres independents. Sorprenentment, la hipermetilació no està acompanyada per una clara baixada de l’expressió d’AKR1B1, de fet vam observar que aquesta hipermetilació estava associada amb el silenciament dels gens AKR1B10 i AKR1B15 (tots ells membres de la mateixa família d’aldo-keto reductases), localitzats a 60 Kbs de l'illa CpG. Utilitzant tècniques per esbrinar l'estructura de la cromatina dins del nucli vam observar que l'illa CpG es troba en contacte amb el promotor de AKR1B10, el que indica una possible funció d’enhancer per a l'illa CpG. Aquesta funció enhancer va ser corroborada mitjançant assajos de luciferasa i demostrada in situ mitjançant l’alteració de la regió enhancer, utilitzant la tècnica d’edició genòmica CRISPR. A més a més, l’acció enhancer va ser recuperada en models cel·lulars gràcies a l’acció de diverses drogues epigenètiques, que produïen l’activació de l’enhancer (observable per la reaparició de la marca H3K27ac) i la reactivació de l’expressió d’AKR1B10 i AKR1B15. En aquest treball també s’avalua l’impacte de la hipermetilació en la via de l’àcid retinoic, a la qual pertanyen els enzims de la família AKR1B. Observem que la via està fortament hipermetilada i, en conseqüència, silenciada. El fet més remarcable a nivell clínic és que aquestes alteracions poden servir com a marcador de pronòstic de la malaltia i com a marcador de diagnòstic no invasiu.
By applying a genome-wide approach to detect differential methylation between colorectal tumors and their paired normal tissue we identified a number of genes that suffer hypermethylation in colorectal cancer. One of the most interesting genes was AKR1B1, which promoter CpG island became hypermethylated in about 90% of all the colorectal cancers samples analyzed. Unexpectedly, this hypermethylation was not accompanied by a clear downregulation of the AKR1B1 transcript. After extending the analysis to the neighboring genes, we realized that this hypermethylation was actually associated with silencing of AKR1B10 and AKR1B15 genes (all of them members of the same aldo-keto reductase gene family), located 60 Kb upstream of the methylated CpG island. Using techniques to elucidate the chromatin structure within the nucleus, we realized that the CpG island was in close contact with the AKR1B10 promoter, what indicates a putative enhancer role for the CpG island. This enhancer function was checked using luciferase assays and was demonstrated in situ through the alteration of the enhancer region, using the genome editing technique CRISPR. Moreover, the enhancer activity was recovered using cellular models through the treatments with different epigenetic drugs, which produced the activation of the enhancer (with the characteristic H3K27ac mark) and the reexpression of AKR1B10 and AKR1B15 genes. In this work we also evaluated the impact of the hypermethylation in the retinoic acid pathway, where AKR1B enzymes belong to. We observed a strong hypermethylation of the retinoic acid pathway and, as a consequence, silencing. The most remarkable finding is that those alterations can be used to predict the outcome of the disease and also as a non-invasive diagnostic marker.

The Wnt signalling pathway is involved in stem cell maintenance, differentiation and tissue development, and in so doing plays a key role in controlling the homeostasis of colorectal crypts. In response to an external Wnt stimulus, the intracellular levels of the protein beta-catenin are regulated by the proteins which make up the Wnt signalling pathway. Abnormalities in the Wnt signalling pathway have been implicated in the initiation of colorectal and other cancers. In this thesis we analyse and simplify existing models of the Wnt signalling pathway, formulate models for Wnt's control of the cell cycle in a single cell, and incorporate these into a multiscale model to describe how Wnt may control the patterns of proliferation in a colorectal crypt. A systematic asymptotic analysis of an existing ODE-based model of the Wnt signalling pathway is undertaken, highlighting the operation of different pathway components over three different timescales. Guided by this analysis we derive a simplified model which is shown to retain the essential behaviour of the Wnt pathway, recreating the accumulation and degradation of beta-catenin. We utilise our simple model by coupling it to a model of the cell cycle. Our findings agree well with the observed patterns of proliferation in healthy colon crypts. Furthermore, the model clarifies a mechanism by which common colorectal cancer mutations may cause elevated beta-catenin and Cyclin~D levels, leading to uncontrolled cell proliferation and thereby initiating colorectal cancer. A second model for the influence of the Wnt pathway on the cell cycle is constructed to incorporate the results of a recent set of knockout experiments. This model reproduces the healthy proliferation observed in crypts and additionally recreates the results of knockout experiments by additionally including the influence of Myc and CDK4 on the cell cycle. Analysis of this model leads us to suggest novel drug targets that may reverse the effects of an early mutation in the Wnt pathway. We have helped to build a flexible software environment for cell-based simulations of healthy and cancerous tissues. We discuss the software engineering approach that we have used to develop this environment, and its suitability for scientific computing. We then use this software to perform multiscale simulations with subcellular Wnt signalling models inside individual cells, the cells forming an epithelial crypt tissue. We have used the multiscale model to compare the effect of different subcellular models on crypt dynamics and predicting the distribution of beta-catenin throughout the crypt. We assess the extent to which a common experiment reveals the actual dynamics of a crypt and finally explain some recent mitochondrial-DNA experiments in terms of cell dynamics.

Colorectal cancer is common with a high mortality rate despite significant advances in its management. While outcomes for rectal cancer have improved over recent years, outcomes for low rectal cancer. and colon cancer have not improved to the same degree. Studies were designed to investigate the optimal operations for both low rectal cancer and colon cancer using tissue morphometry and assessment of the surgical planes. Additionally, the utility of tumour cell density as a prognostic marker and a method of assessing the response to pre-operative treatment was investigated. Extralevator 'abdominoperineal excision for low rectal cancer was shown to be associated with better pathological outcomes and planes of surgery when compared to standard abdominoperineal excision. Careful mesocolic plane surgery for colon cancer was associated with better survival when compared to disrupted specimens. Complete mesocolic excision with central vascular ligation and Japanese D3 resection resulted in an oncologically superior specimen when compared to standard surgery. They could both be performed laparoscopically and were easily adopted by a group of highly motivated surgeons. Tumours with a low tumour cell density appeared to have an independently worse prognosis, and the same technique generated a useful measure of response to pre-operative therapy in both colon and rectal cancer. Outcomes for low rectal cancer and colon cancer could be improved by surgical education programmes backed up by pathological audit. Tumour cell density could be introduced as a new prognostic marker and could be used to define the degree of response to pre-operative treatment in colorectal cancer.

A genomic microarray with a genome wide resolution of ~1Mb was constructed, consisting of approximately 3500 large insert clones selected from the “golden path” sequencing clone set. Also included on the array were clones chosen to represent 142 cancer-associated genes and a human chromosome specific subtelomeric clone set. The 1Mb array was validated and used to investigate genomic copy number changes in 48 colorectal cancer cell lines and 37 primary colorectal cancers. The samples were divided for analysis according to type of genomic instability; microsatellite (MSI) or chromosomal (CIN). Consistent copy number changes were identified, including gain of chromosomes 20, 13 and 8q and smaller regions of amplification such as chromosome 17q11.2-q12. Loss of chromosome 18q was a recurrent finding, along with deletion of discrete regions such as chromosomes 20p12.1-p11.23 and 4q34-q35. A greater number of aberrations were detected in CIN+ than MSI+ samples, as well as differences in the particular changes reported. Expression analysis using Affymetrix whole genome oligonucleotide arrays was carried out on ten of the colorectal cancer lines and changes in expression of genes located in the recurrent regions of gain and loss were identified. The deletion of chromosome 20p12.1-p11.23 was analysed at higher resolution using a specifically designed array with tiling path coverage of the region; a recurrent homozygous deletion was identified and the minimum region of loss refined to ~200Kb. Two non-coding RNA genes which map to this region, bA318C17.1 and dJ974N19.1, were investigated using mutation analysis and quantitative PCR. Sequence changes in bA318C17.1 and reduced expression of both genes were detected in colorectal cancer cell lines. A number of genes of interest have been identified, displaying altered expression and located within the common regions of copy number change, which are likely to be of importance in the development of colorectal cancer.

Colorectal cancer is the third most common cancer in the United Kingdom after breast and lung cancer. In Britain, approximately 20,000 patients die from colorectal cancer each year [Reeves et aI., 1999]. The lifetime risk of developing a colorectal cancer is about 6-7% and the probability of dying from it is about 3 % [Marshall, 1996]. It is uncommon before the age of 40 years but the risk increases at that point, rising sharply from 50 years, approximately doubling with each successive decade of life. The peak incidence occurs in the seventh decade. The incidence of left sided tumours in females is less than in males, a possible protective effect of female hormonal factors in the development of colorectal carcinoma is suggested by [dos, I and Swerdlow, 1996]. The 5-year survival rate in these cancers is approximately 43 % [Gatta et aI., 1998]. While progress has been made in understanding cancer development and spread and improving treatment, the mortality from colorectal cancer has remained almost unchanged in the last two decades. In order to reduce the mortality in patients with this cancer, an effective screening test to identify cancers at an earlier stage is necessary. In spite of being one of the commonest cancers there is no established screening programme available to the general population.

Colorectal cancer (CRC) is the second largest cause of cancer deaths in the UK, with approximately 16,000 per year. Over 41,000 people are diagnosed annually, and 43% of those will die within ten years of diagnosis. The treatment of CRC patients relies on pathological examination of the disease to identify visual features that predict growth and spread, and response to chemoradiotherapy. These prognostic features are identified manually, and are subject to inter and intra-scorer variability. This variability stems from the subjectivity in interpreting large images which can have very varied appearances, as well as the time consuming and laborious methodology of visually inspecting cancer cells. The work in this thesis presents a systematic approach to developing a solution to address this problem for one such prognostic indicator, the Tumour:Stroma Ratio (TSR). The steps taken are presented sequentially through the chapters, in order of the work carried out. These specifically involve the acquisition and assessment of a dataset of 2.4 million expert-classified images of CRC, and multiple iterations of algorithm development, to automate the process of generating TSRs for patient cases. The algorithm improvements are made using conclusions from observer studies, conducted on a psychophysics experiment platform developed as part of this work, and further work is undertaken to identify issues of image quality that affect automated solutions. The developed algorithm is then applied to a clinical trial dataset with survival data, meaning that the algorithm is validated against two separate pathologist-scored, clinical trial datasets, as well as being able to test its suitability for generating independent prognostic markers.

Colorectal cancer (CRC) is the second most prevalent cancer. Although not typically considered a hormonally responsive malignancy, oestrogens are linked to disease risk and survival. Here, it is hypothesised that oestrogen synthesis, via steroid sulphatase (STS) and 17β-hydroxysteroid dehydrogenases (HSD17β), is promoted in CRC leading to oestrogen-driven tumour proliferation. To identify potential novel treatment targets oestrogen metabolism was characterised in CRC. Oestrogen metabolism was characterised in CRC cell lines and human colon tissue using Western blotting, qRT-PCR, STS activity assay and proliferation assays. A mass spectrometry method to quantify oestrogen metabolites was developed and validated for cell culture medium. CRC exhibited increased STS activity indicating increased intratumoural oestrogen availability. In CRC a drive towards potent oestradiol production through decreased HSD17β2 expression (oxidative enzyme) and increased HSD17β7 and 12 expression (reductive enzymes) was seen. STS over-expression increased proliferation in CRC cell line HCT116, which was at least in part mediated through G protein-coupled oestrogen receptor action. Additionally, STS activity was regulated in vitro and in vivo by the inflammatory modulator TNFα. This thesis demonstrates that increased STS activity and oestrogen synthesis in CRC increases tumour proliferation via GPER. Thus, multiple novel targets have been identified to impede CRC proliferation.

Colorectal cancer (CRC) continues to cause significant global health burden, despite advances in our understanding of tumour biology, the development of screening programs and increasing public awareness about the disease. Previous studies investigating CRC pathogenesis have been criticised for focussing on the tumour tissue itself. Investigators have proposed that if early biomarkers of disease are to be identified, efforts need to be undertaken in examining pre-neoplastic tissue prior to malignant transformation. Based on the field cancerisation concept, the research hypothesis was that the macroscopically normal mucosa (MNM) around colorectal cancer and polyps is biologically altered. The aims of the study were to determine if the presence of colorectal adenomas at time of cancer diagnosis was predictive of future risk of colonic neoplasia and to characterise the global gene expression profile of MNM adjacent to CRC and adenomas. A retrospective cohort study of CRC patients demonstrated that synchronous adenomas were associated with a higher risk of future adenomas at short term follow up but were not predictive of local recurrence. Thus, other more reliable biological markers of field effect need to be identified. Global gene expression profiles of MNM around cancer, polyps and in control subjects were significantly different when evaluated with micro-array. The differentially expressed genes were involved in immunity, metabolism, epithelial-mesenchymal transition and RNA transcription. CXCL2 and FGF7 were identified as being upregulated in MNM adjacent to CRC suggesting that they could be utilised as markers of field cancerisation in the colon. Further investigation demonstrated that the FGF7-FGFR2 axis was disrupted only at the tumour site with downregulation of some of its downstream targets emphasising the potential role of this signalling axis in CRC formation. Collectively, these findings support the field cancerisation concept in CRC and highlight the importance of signals released by stromal cells in facilitating epithelial growth. These genes may be utilised to develop early biomarkers of disease or could be targeted with pharmacotherapy to modulate future CRC risk.

Introduction: Colorectal cancer is the second most common UK cancer. Biomarkers which predict survival may be valuable for targeting adjuvant therapy and can provide insights into tumour biology. Small and early cancers are being diagnosed more commonly in the UK population due to the introduction of population-based colorectal cancer screening in 2005. Analysis of resected small (≤20mm across) tumours in Liverpool has established that flat and depressed morphology can predict advanced stage at presentation. Proteomic analysis of small cancers was conducted with the aim of generating biomarkers which correspond to morphology, stage and patient survival. Patients and Methods: Laser capture microdissection was used to procure enriched matched benign and malignant colorectal epithelial cell populations. Laser captured proteins were extracted into lysis buffer, normalised against a reference standard, separated using 2D SDS-PAGE and visualized with silver staining. Comparison was made between the tumour gels, (n=10) and matched normal colonic gels, (n=9) by two different observers and gel analysis software, Progenesis SameSpots. Differentially expressed proteins were identified using tandem mass spectrometry and included redox proteins peroxiredoxin 2, peroxiredoxin 6 and SH3 binding glutamic acid-rich protein-like 3; and cytoskeletal protein cofilin1. Also identified were the anti-apoptotic protein heat shock protein 27 and inflammatory protein S100A8, which had been previously identified in 2D gel analysis of undissected colorectal cancer in our Institution (n=12 gels) and previously validated in a small cohort of paraffin-embedded colorectal cancers (n=98). In this study, HSP27 was further evaluated in a large cohort of paraffin-embedded colorectal cancer tissue (n=404). S100A8 and related proteins S100A9 and Smad4 were similarly evaluated in a large cohort (n=313). Results: High HSP27 levels were strongly associated with poor cancer-specific survival in rectal cancer (n=205, P=0.0063) but not colon cancer; (n=199, P=0.7385). Multivariate Cox regression confirmed nodal metastases (P=0.0001) and HSP27 expression (P=0.0233) as independent markers of survival in rectal cancer. HSP27 levels remained unchanged in the majority of cases 65/80 (81%) between diagnostic biopsies and matched surgical samples, regardless of whether patients had undergone preoperative radiotherapy. S100A8 expression co-localised with a subset of S100A9-positive monocytes. S100A9 was co-expressed with CD14 in tumour-associated monocytes, but not with CD68 in tissue macrophages. Smad4 was expressed in the tumour cytoplasm of 262/304 (14%) tumours. Loss of Smad4 expression correlated with a reduction in the stromal S100A8-positive, but not S100A9-positive cell count, (P=0.034, Mann-Whitney U test) and was associated with a poorer overall survival in patients with stage I-II disease, but not stage III disease. Antibodies to cofilin1 and cofilin-phospho(ser3) were assessed in colorectal cancer cell and tissue lysate and found to be specific on 1D and 2D western blot. Conclusion: Elevated HSP27 is an independent marker of poor prognosis in rectal cancer whose expression is not altered by neo-adjuvant radiotherapy. Smad4-negative tumours are associated with fewer infiltrating S100A8 positive stromal monocytes. In node-negative tumours, loss of Smad4 expression in associated with a poorer prognosis. These findings provide a sound platform for further investigation of both S100A8 and HSP27 proteins in colorectal cancer.

Colorectal cancer (CRC) remains a major public health problem with advanced and recurrent disease being a management challenge due to the lack of efficacy of currently available monitoring tools. Carcinoembryonic antigen is used as a marker for recurrent disease but has limited sensitivity. Early sensitive markers of disease severity and recurrence are required. The aim of this thesis was to identify potential metabolic biomarkers of CRC patients using ‘Metabonomics’ based on proton nuclear magnetic resonance (1H NMR) spectroscopy. The breadth of 1H NMR metabonomics exploring different aspects of CRC pathogenesis was investigated. A global approach profiling a large number of metabolites was undertaken in cell line and chemoprevention studies with subsequent targeted profiling experiments using human polypoid tissue samples. The studies primarily exhibited changes in choline and lipids to be associated with CRC. Although these metabolites are well reported, this thesis presents contemporary metabolic profiling using systems not previously reported. Tumour extracts of CRC xenografts showed higher levels in the ratio of phosphocholine and glycerophosphocholine between poorly and well-differentiated cell lines. Human tissue and biofluids were used to define metabolic phenotypes. Normal and polypoid colorectal tissues were also metabolically characterised by changes in choline using magic angle spinning (MAS) 1 H NMR. Investigating chemoprevention strategies with bile acids in adenoma patients indicated alterations in short chain fatty acids, related to gut microflora metabolism. In clinical studies, higher levels of lactate and glycoproteins were identified in rectal cancer patients, as potential metabolic predictors of response to chemoradiotherapy. A metabolically translational feature of the thesis in a heterogeneous population confers a significant increase in the risk of CRC incidence with metabolic syndrome (MetS). Thus, inferring identification of MetS components as part of screening measures. In vivo studies provide a functional way of networking metabolic pathways to pathological states, thereby unifying perspectives post hoc.

Microsatellite instability (MSI) has been a promising molecular marker in colorectal cancer over the past twenty years. However, within the literature, there has been a degree of uncertainty in the effect of MSI status on prognosis, recurrence and treatment decision-making. The thesis consists of seven peer-reviewed publications on different aspects of MSI in clinical management including: 1. A nationwide survey of medical oncologists and colorectal surgeons showing that MSI status is considered useful in selecting patients for adjuvant therapy as well as prognostication of colon cancer. 2. Meta-analysis evaluating the prognostic significance of MSI in CRC demonstrating that MSI is associated with an overall survival benefit with lower risk of dissemination, with survival benefit most evident in stage II and stage III colorectal cancer. 3. Cohort study evaluating the prognostic significance of sporadic MSI-H CRC. After adjustment for death from other causes as a competing risk, no statistically significant difference in cancer specific survival nor overall survival was demonstrated. 4. Lab-based study evaluating the tumour microenvironment of CRC by MSI status, showing increased tumour infiltrating lymphocytes (TILs), Programmed Death-1 (PD-1) expression and the presence of T resident memory (TRM) cells. 5. Lab-based study examining circulating tumour cell (CTC) release based on MSI status. Prior to this study, it was not known if the immunogenicity of MSI-H CRC decreased the risk of dissemination by reducing the release of CTCs. This study reports a trend towards increased CTC (rather than decreased CTC) release. 6. Systematic review on the role of MSI status in identifying CRC patients suitable for immunotherapy, demonstrating that immunotherapy has the potential to benefit immunogenic MSI-H CRCs. 7. Lab-based study developing a cost-effective and reliable method of determining MSI status with an MSI scoring system based on automatic computational algorithms.

Los receptores EPH (receptores de efrina) constituyen la subfamilia más grande de los receptores tirosina quinasas en mamífero. Se unen a las efrinas, ancladas a las membranas celulares mediante un enlace glicosilfosfatidilinositol (GPI) (efrinas de tipo A) o mediante una dominio transmembrana seguido por un dominio citoplasmático (efrinas de tipo B). El complejo receptor-ligando activa una cascada de señalización tanto en las células que expresan los receptores EPH como en las células adyacentes, que expresan las efrinas. Este mecanismo se denomina “forward” y “reverse signalling”, respectivamente. La interacción entre receptor y su ligando puede desencadenar una amplia gama de respuestas celulares incluyendo la adhesión, repulsión y la segregación/posicionamiento celular durante la separación de los tejidos en las primeras fases de desarrollo embrionario. Los mecanismos que regulan estas distintas respuestas dependen en gran medida del tipo celular y del tejido. Aunque, los receptores EPH han sido ampliamente caracterizados para sus funciones durante el desarrollo embrionario, se encuentran re-expresados en una variedad de tumores malignos humanos. No obstante, dependiendo del tipo de tumor, de su estadio y de la actividad quinasa, los receptores EPH pueden actuar induciendo o inhibiendo la progresión del cáncer. Los receptores EPH de tipo B están involucrados en la homeostasis del epitelio intestinal y la pérdida de función de estos receptores es un evento clave en la progresión del cáncer de colon, sobretodo de adenoma a carcinoma. El receptor EPHA3, identificado y aislado por primera vez ellas membranas de células pre-B de leucemia linfoblástica aguda, se encuentra ampliamente expresado durante el desarrollo embrionario, particularmente en el tejido nervioso y corazón. Tal como se espera de su primera identificación, se ha demostrado que también está involucrado en muchos tipos de cáncer. Los efectos inactivantes de distintas mutaciones somáticas encontradas en varios tipos de cáncer, sugieren fuertemente que la proteína EPHA3 suprime las propiedades de las células cancerígenas a través un mecanismo que dependería de su unión con la efrina y de su actividad quinasa. Dos estudios independientes de secuenciación de ADN empleando técnicas de nueva generación, descubrieron una alta frecuencia de mutaciones en EPHA3 en una amplia cohorte de tumores de colon humano y, más importante, la mayoría de estas mutaciones se descubrieron afectar la actividad quinasa o el dominio de unión con el ligando. Sin embargo, la función de EPHA3 en la tumorigenesis intestinal no ha sido investigada a fondo. Por lo tanto, en este estudio se utilizaron dos líneas celulares de cáncer de colon humano, LS174 y DLD1, para sobre-expresar de forma inducible por doxiciclina el receptor EPHA3. Hemos observado que la reintroducción de EPHA3 en líneas de cáncer de colon no afecta el crecimiento de las células cultivadas en sustratos sólidos, en medio semi-solido (soft-agar) o como xenotrasplantes en ratones inmunodeprimidos. Además, se utilizó un modelo transgénico de ratón en el que el gen EphA3 ha sido suprimido y descubrimos que la inactivación específica de uno o los dos alelos de este gen no inicia el proceso tumorígenico en el intestino de los ratones. Tampoco observamos cambios en el tamaño o en el número de tumores intestinales en ratones donde la tumorigenesis intestinal se inició genéticamente (modelo de ratón Apcmin/+) o farmacológicamente (tratamiento con azoximetano). También hemos observado que la sobreexpresión de EPHA3 en células de cáncer de colon no afecta su capacidad de migración o de invasión en ensayos in vitro. Para profundizar el papel de EPHA3 en el proceso metastático, se usó un modelo murino de metástasis de pulmón observando que la reintroducción de EPHA3 en células de cáncer de colon inyectadas en las colas de ratones inmunodeprimidos no altera su capacidad migratoria y de generar metástasis en otros órganos in vivo. Estos resultados confirman los datos de inmunhistoquimica donde observamos ausencia de diferencias en los niveles de tinción de EPHA3 en los nódulos linfáticos respeto a los tumores primarios de pacientes con cáncer colorrectal localmente avanzado (estadio Dukes C). Por último, el análisis inmuhistoquimico de los niveles de tinción de EPHA3 en pacientes con cáncer colorrectal (estadio Dukes C) no reveló ninguna correlación con la supervivencia y otras características clinicopatologicas de estos pacientes. En conclusión, en este estudio, reportamos que el mecanismo de señalización quinasa dependiente del receptor EPHA3, aunque está frecuentemente inactivado en cáncer colorrectal, no juega un papel como supresor de la tumorigenesis intestinal durante ningún estadio de la progresión del tumor, tanto in vitro como in vivo. Estos resultados, esclarecen el papel de EPHA3 en cáncer colorrectal y evidencian la importancia de estudios funcionales para confirmar el papel de los genes identificados como posibles candidatos de la progresión tumoral empleando técnicas avanzadas de secuenciación genómica de tumores.
EPHs, which make up the largest family of mammalian receptor tyrosine kinases, bind cell surface–associated Ephrins that have either a glycosylphosphatidylinositol (GPI) membrane anchor (A-type Ephrins) or a transmembrane region followed by a conserved cytoplasmatic tail (B-type Ephrins). Clustering of EPH receptors and their ligand at the surface of adjacent cells activates downstream signaling in both the EPH- and Ephrin-expressing cells, termed forward and reverse signaling, respectively. Eph–ephrin interactions can trigger a wide array of cellular responses including cell adhesion, repulsion and cell sorting/positioning during tissue boundary and the mechanisms leading to these different responses are highly dependent on the cell type and tissue context. Although most extensively characterized for their roles in development, Eph receptors are re-expressed in a variety of human malignancies. However, depending on the tumor type, disease stage and kinase function, EPH overexpression can promote or inhibit tumor progression. The role of EPHB receptors was discovered to be involved in the homeostasis of the normal intestinal epithelium and loss of function of B-type EPHs is a key event of colorectal cancer progression, mainly from adenoma to carcinoma. The EPHA3 receptor, originally recognized and isolated from membranes of pre-B acute lymphoblastic leukemia cells, is widely expressed during embryonic development, with the highest levels occurring in the nervous system and heart. As expected from its first identification, it was then implicated as having a key role in many cancers. The inactivating effects of many somatic mutations found in different cancers, strongly suggest that wild-type EPHA3 receptor suppresses the malignant properties of cancer cells in an ephrin-and kinase-dependent manner. Two independent studies of next-generation DNA sequencing using large cohorts of human colorectal cancer samples showed unexpected high mutation frequency in EPHA3 and, importantly many of these mutations were found to impair the kinase activity or ligand binding domain. However, the function of EPHA3 in intestinal tumorigenesis has not been thoroughly investigated. Thus, in this study we generated a doxycycline-dependent EPHA3 overexpression system in two different human colon cancer cell lines, LS174 and DLD1. Importantly, we found that the reintroduction of EPHA3 in colon cancer cells did not affect their growth on a solid substrate, soft agar or as xenografts in immunodeficient mice. Moreover, using a full knockout mouse model we show that targeted inactivation of one or both alleles of EphA3 did not initiate the tumorigenic process in the murine intestine, and had no effects on tumor size/multiplicity after tumor initiation either genetically or pharmacologically. Restoring EPHA3 levels in colon cancer cells did not modulate their motility/invasion in vitro. In addition, to more closely investigate the role of EPHA3 in the metastatic process, we used a mouse model of lung metastasis and found that the ectopic overexpression of EPHA3 in human colon cancer cells, injected in to the tail vein of immunodeficient mice, does not affect their capability in vivo to migrate and colonize distant organs. These results are consistent with our immunohistochemistry data showing no EPHA3 expression differences in primary Dukes C tumors and matched lymph node metastases. Finally, immunohistochemical analysis of EPHA3 tumor levels did not reveal associations with survival or clinicopathological features of Duke C colorectal cancer patients. In conclusion, here we report that the kinase dependent signaling of EPHA3, despite frequently inactivated in colorectal cancer, does not play a tumor suppressive function in intestinal tumorigenesis and, importantly, in any stage of the cancer progression using either in vivo or in vivo experimental models. These results significantly contribute to our understanding of the role of EPH signaling during colorectal carcinogenesis, and highlighting the need for detailed functional studies to confirm the relevance of putative cancer driver genes identified in sequencing efforts of the cancer genome.

Abstract Colorectal cancer is the second most common malignancy in the Western World. The overall 5-year survival is still only 50–60%. Thus, better prognostic markers are needed to improve survival of the disease. Most colorectal cancers develop from pre-existing adenomas including conventional, flat and serrated adenomas. The most important prognostic factors include tumour stage, histologic subtype and poor differentiation. The prognosis of colorectal cancer depends mainly on tumour stage. The growth of colorectal cancer is determined by cell proliferation, differentation and apoptosis. The progression of colorectal cancer is associated with the growth pattern of colorectal cancer and its invasive margin. Cancer cell budding means the presence of cells scattered in the stroma at the invasive margin, and is associated with β-catenin, an adhesion protein involved in the nuclear Wnt/β-catenin pathway. Hormones may be directly involved in the growth of a cancer, for example sex hormones play an important role in the development of most gynaecological cancers. The knowledge about the dependency of cancers on other hormones, such as thyroid hormones, is limited. This thesis focuses on factors affecting growth and prognosis in colorectal cancer. Antibodies for Ki-67, caspase cleavage site for keratin 18, β-catenin and TRβ1 were used to determine their possible associations with colorectal cancer growth patterns and the characteristics of the invasive margin. Apoptosis and proliferation were decreased at the invasive margin, particularly in serrated adenocarcinomas. The invasive margin showed a presence of budding cell clusters in 24.0% of the cases and this predicted a very poor 5-year-survival (15.4%, P < 0.00001), but nuclear β-catenin accumulation did not predict budding. Thyroid hormone receptor TRβ1 was associated with polypoid growth, presence of KRAS mutations and also with a higher WHO histological grade and advanced Dukes' stage, and in in vitro analysis, thyroid hormone T3 had a modulatory effect on colorectal cancer cell protein synthesis and apoptosis. In conclusion, the growth type of colorectal cancer, i.e. conventional polypoid, flat or serrated, has an association with the characteristics of the invasive margin. Budding margin is associated with poor prognosis in colorectal cancer, and could be utilised in diagnostic pathology. Association of TRβ1 expression with polypoid growth pattern and the presence of KRAS mutations suggest that abnormalities in thyroid hormone signalling involving TRβ1 play a role in the development of some types of colorectal adenocarcinomas.

Introduction: Advanced colorectal cancer (CRC) has a poor prognosis with a 5-year survival of only 5% despite treatment with chemotherapeutic agents. The ATP- Tumour Chemosensitivity Assay (ATP-TCA) has been used to demonstrate heterogeneity of chemosensitivity between tumours of the same tissue type, but this has been difficult to establish in colorectal cancer due to infection of cells in culture. Methods: The ex vivo ATP-TCA was modified with antibiotics for use in CRC, and with immunohistochemistry and quantitative RT-PCR, has been used to assess the chemosensitivity and resistance of CRC tumour-derived cells. Results: (a) The addition of 2.5 mug/ml amphotericin B and 1 mug/ml metronidazole to culture media did not effect the cytotoxicity of all drugs tested on SK-MEL-28 melanoma cell lines. (b) The metabolite of irinotecan, SN38, was found to be inactive in the ATP-TCA (c) The ATP-TCA was performed on 71 CRC samples, 58 of which were evaluable (82%). There was considerable heterogeneity for individual samples and drugs tested. (d) Mitomycin C + gemcitabine was the most effective combination in 78% of specimens, with all but one sample showing sensitivity. The synergistic effect between these two drugs was not found to be schedule-specific. (e) Molecular studies determined the expression of a number of molecular targets which were correlated with the ATP-TCA results. The only correlation found was between positive staining for topoisomerase I and sensitivity to irinotecan. (f) Using qRT-PCR it was found that cyclo-oxgenase2 is up-regulated by short-term exposure to 5-fluorouracil (3-fold), but down-regulated by irinotecan (2.5-fold). Conclusion: The results show that it is possible to perform the ATP-TCA on CRC tumour-derived cells with a high evaluability rate. The changes in gene expression after short-term drug exposure have important implications for the use of sequential therapy in the treatment of colorectal and cancers.

We aimed to identify genetic factors that alter colorectal cancer (CRC) risk, patient survival and response to treatment. Using a cohort of 2,186 advanced CRC (aCRC) cases and 2,176 healthy controls, we validated a role for loci at 18q21, 8q23, 15q13 and 8q24 in CRC susceptibility. We also identified a variant in OGG1 that may act as a novel low penetrance risk allele. We identified four germline SNPs from 14 genome wide associated CRC risk loci that independently altered patient survival (rs16892766 at 8q23, HR 1.28, 95% CI 1.13-1.45, P<0.001 rs9929218 at 16q22, HR 1.46, 95% CI 1.23-1.74, P=0.002 rs10411210 at 19q13, HR 1.23, 95% CI 1.08-1.39, P=0.001 and, rs10795668 at 10p14, HR 0.71, 95% CI 0.59-0.86, P=0.001). We found that somatic mutations of the KRAS (HR 1.34, 95% CI 1.18-1.52 P<0.001) and BRAF (HR 2.00, 95% CI 1.61-2.50, P<0.001) oncogenes altered patient survival independent of treatment. Through the COIN trial, we found no evidence for improved response to cetuximab in patients' wild-type for KRAS (OS HR 1.04, 95% CI 0.87-1.23, P=0.67) or all wild-type for KRAS, BRAF and NRAS (OS HR 1.02, 95% CI 0.83-1.24, P=0.86). Similarly, PIK3CA mutation status did not correlate with response (OS 1.03, 95% CI 0.86-1.24, P=0.89). However, we identified a 'most responsive' cohort (patients wild-type for KRAS, BRAF and NRAS, with £1 metastatic sites and that received OxFU) that showed improved response (PFS HR 0.55, 95% CI 0.35-0.87, P=0.01). Analysis of individual somatic variants revealed no significant associations with response. We found that rs9929218 altered response to (OR 0.48, 95% CI 0.31-0.75, P=0.001) and side effects from (OR 4.68, 95% CI 1.84- 11.9, P=0.001) standard chemotherapy and response to cetuximab in a KRAS, BRAF and NRAS mutant dependent manner (OR 1.69, 95% CI 0.61-4.74, P for interaction 0.004). We elucidated the underlying mechanisms at the 8q23 and 16q22 loci. These data may help to tailor future therapies for patients with aCRC.