Interrogating the genetic profile of sporadic colorectal cancer

Background and aims: Colorectal cancer (CRC) is one of the most common types of cancers. Prognosis for CRC is dependent on the stage of the disease at diagnosis and a variety of molecular features including expression of biomarkers and gene mutation. This study sought to (i) develop molecular tests for use in molecular profiling of formalin-fixed paraffin embedded tissue (FFPE) and (ii) interrogate the molecular profile and biomarker expression in multiple cohorts of sporadic CRC. Materials and methods: Firstly, we developed the Quick Multiplexed Consensus (QMC) PCR protocol – a method used in conjunction with High Resolution Melting (HRM) analysis for screening of multiple mutational hotspots (10 hotspots in this study were screened) in the CRC candidate genes using DNA from FFPE tissue. The performance of the assay for the detection of single nucleotide variants (SNV) and small insertion-deletions (indels) was compared with Sanger sequencing, pyrosequencing and real-time PCR. In addition, a PCR/HRM assay was developed for testing microsatellite instability (MSI) using a panel of six quasi-monomorphic mononucleotide markers (five of which have been previously described: BAT25, BAT26, NR21, NR22, and NR24; the sixth marker, B-CAT25, is a novel in-house developed marker) and this assay was validated against the Bethesda commercial MSI PCR kit and against immunohistochemistry (IHC). Once QMC-PCR was optimised, we sought to ascertain whether diagnostic biopsies yield sufficient information for clinical decision making. The mutation profiles of CRC candidate genes (KRAS, BRAF, PIK3CA and TP53) and MSI status were evaluated in the diagnostic biopsy specimen of 30 cases and compared with the profile in matched blocks from the resection specimen. The classification of CRC into tumours with MSI and chromosomal instability (CIN) is well described. However tumours which show neither MSI nor CIN have been described and are known as microsatellite and chromosomal stable CRCs (MACS). We sought to evaluate the frequency of CIN and MACS in a series of 89 sporadic microsatellite-stable CRCs. PCR/HRM was used to exclude MSI tumous, while CIN tumours was discriminated from MACS tumours using flow cytometry. These tumours were also compared for mutations in KRAS/BRAF/TP53/PIK3CA by QMC-PCR. Some rectal tumours may receive neoadjuvant chemoradiotherapy (CRT) and we tested whether the CIN or MACS ploidy status could predict response in a group of 62 rectal cancers treated with neoadjuvant CRT. We next evaluated the clinical utility of deficient Mismatch Repair (dMMR) protein and aberrant P53 protein expression, both individually and in combination, as prognostic and predictive of response to therapy biomarkers in CRC. A total of 884 tumours from the VICTOR trial (a large phase III trial of rofecoxib in stage II and III CRC) were tested for expression of four mismatch repair (MMR) proteins (MLH1, PMS2, MSH2, MSH6) and p53 by IHC. The expression was correlated with outcome. Results: QMC-PCR worked on DNA from FFPE tissue. Spiking experiments showed the protocol could detect a minimum of 2.5% of mutant alleles compared to 20% detectable for Sanger sequencing. Precision tests showed that there was little intra-assay and inter-assay variation. Forty-three FFPE colorectal tumours were initially sequenced for hotspots in KRAS and PIK3CA and then screened by QMC-PCR. In these tests, QMC-PCR showed a sensitivity of 100%, specificity of 71%, PPV of 76% and NPV of 100%. All 43 samples were then screened for mutations in all 10 hotspots. Of 430 tests, 43 (10%) showed aberrant melting and 36 were confirmed mutant (PPV 84%). Since our technique is more sensitive than direct sequencing, the remaining 7 tests are probably sequencing false negatives. HRM analysis of KRAS (codon12/13) and BRAF (V600E) showed that 3% and 1.5% mutant alleles respectively could be reliably detected whilst pyrosequencing reliably detected 6% mutant alleles in each case. Of 110 tests performed on 22 DNA samples, in 109 cases HRM and pyrosequencing gave identical results. The results of KRAS (codon12/13) screening from the comparative study of 468 cases with a real-time PCR test (DxS), showed 89.9% concordance with the DxS test. In comparison with real-time PCR, the analysis by HRM showed 87.2% sensitivity, 91.6% specificity, 86.7% positive predictive value, 91.9% negative predictive value. After refinement of the protocol, re-testing of 100 randomly selected cases showed 99% concordance between QMC-PCR/HRM and the real-time PCR test. The performance of the HRM based MSI assay showed 100% accuracy on both cell lines and FFPE tissue in comparison to PCR and IHC. Pertaining to the results from the paired biopsy and resection specimens work, a total of 570 paired PCR tests were performed and identical results were obtained in both biopsy and resection specimens in 569 tests (>99% concordance). Four cases (13%) showed microsatellite instability, and, in all four cases, instability was seen at identical mononucleotide markers in both biopsy and matched resection specimens. Regarding the immunostaining of MMR proteins, the staining was more intense and easier to interpret in biopsies and that it faithfully replicates the diagnosis in the resection specimen. The results from the DNA ploidy study showed, in addition to the two classical forms of genetic instability (MSI, CIN), a significant third group of colorectal cancers (CRC) without either MSI or CIN i.e. Microsatellite and Chromosome Stable (MACS) CRCs. Fifty-one of 89 CRCs (57%) were aneuploid and 38 (43%) were diploid. There was no significant association seen between mutations in TP53/KRAS/BRAF/PIK3CA with ploidy. Testing of association between mutations revealed only mutual exclusivity of KRAS/BRAF mutation (p<0.001). Of the 62 rectal cancers treated with neoadjuvant chemoradiotherapy, 22 had responded (Mandard Tumour Regression Grade 1/2) and 40 failed to respond (Grade 3-5). Twenty-five of 62 (40%) tumours were diploid but there was no association between ploidy and response to the neoadjuvant CRT therapy. Evaluation of TP53 and MMR protein expression in the VICTOR trial, showed that there was dMMR in 12% (87/735) of patients. It was associated with female gender (p=0.001), proximal location of tumour (p<0.001), poor differentiation (p<0.001) and stage II disease (p<0.001). dMMR was not associated with either disease free survival (DFS) or overall survival (OS) in the group overall or when stratified by stage. However, unexpectedly, proficient MMR (pMMR) was associated with significantly improved OS (hazard ratio 0.28 (0.11, 0.68), p=0.005) and DFS (hazard ratio 0.47 (0.22, 0.99), p=0.047) in patients who did not receive chemotherapy. Aberrant p53 expression was found in 65% (482/740) of patients. It was associated with distal location (p<0.001) and stage III disease (p<0.001) and it was negatively associated with dMMR (p<0.001). No effect was observed on DFS or OS and there was no interaction with chemotherapy or radiotherapy. When assessed in combination, dMMR and aberrant p53 expression had no effect on DFS, OS or responsiveness to adjuvant therapy. Conclusions: The following conclusions can be drawn from this research work: 1) QMC-PCR with HRM is a simple, robust and inexpensive technique which had greater sensitivity than Sanger sequencing. It allows multiple mutation hotspots to be rapidly screened and is thus highly suited to mutation detection in DNA derived from FFPE tissues. Both HRM and pyrosequencing can detect small numbers of mutant alleles although HRM has a lower limit of detection. Both are suitable for use in mutation detection and are both more sensitive than Sanger sequencing. QMC-PCR with HRM performs very well against real-time PCR based tests for detection of KRAS mutation. Although the real-time PCR has a lower limit of detection, HRM has greater flexibility overall. Both tests are suitable for KRAS codon 12/13 mutation detection in patients being considered for anti-epidermal growth factor receptor (EGFR) biological therapy. 2) PCR-HRM/MSI assay is simple, straightforward and highly sensitive technique for MSI testing which can be easily incorporated in a single closed-tube panel whether to screen for Lynch syndrome, or to test sporadic CRC for tumour MSI status in order to inform the decision maker to administer 5-fluorouracil-based chemotherapy to the patient or not. 2) This was the first study to show that diagnostic biopsy specimens, even though they are a tiny sample of the tumour, are sufficiently representative for use in predictive testing for early driver mutations in colorectal cancer, especially important when neoadjuvant therapy is being considered. 3) MACS-CRCs form a significant proportion of microsatellite-stable CRCs with a mutation profile overlapping that of CRCs with CIN. A diploid genotype does not however predict the responsiveness to radiotherapy in rectal cancers. This finding leads us to conclude that there are other factors in addition to ploidy that define response to radiation therapy. 4) Analysis of MMR status and p53 expression in the patients recruited to the VICTOR trial confirmed that dMMR and p53 expression are associated with site and stage of CRC. Neither biomarker has prognostic or predictive utility in this cohort of patients overall although in sub-group analysis dMMR was indicative of poor outcome in chemo-naive patients

DNA content analysis of colorectal cancer defines a distinct ‘microsatellite and chromosome stable’ group but does not predict response to radiotherapy

Colorectal cancers (CRC) are thought to have genetic instability in the form of either microsatellite instability (MSI) or chromosomal instability (CIN). Recently, tumours have been described without either MSI or CIN, that is, microsatellite and chromosome stable (MACS) CRCs. We investigated the (i) frequency of the MACS-CRCs and (ii) whether this genotype predicted responsiveness to neoadjuvant chemoradiotherapy. To examine the frequency of MACS-CRCs, DNA content (ploidy) was examined in 89 sporadic microsatellite-stable CRCs using flow cytometry. The tumours were also screened for mutations in KRAS/BRAF/TP53/PIK3CA by QMC-PCR. To examine the value of tumour ploidy in predicting response to chemoradiotherapy, DNA content was tested in a separate group of 62 rectal cancers treated with neoadjuvant chemoradiotherapy. Fifty-one of 89 CRCs (57%) were aneuploid and 38 (43%) were diploid. There was no significant association between mutations in TP53/KRAS/BRAF/PIK3CA and ploidy. Testing of association between mutations revealed only mutual exclusivity of KRAS/BRAF mutation (P < 0.001). Of the 62 rectal cancers treated with neoadjuvant chemoradiotherapy, 22 had responded (Mandard tumour regression grade 1/2) and 40 failed to respond (Grade 3–5). Twenty-five of 62 (40%) tumours were diploid, but there was no association between ploidy and response to therapy. We conclude that MACS-CRCs form a significant proportion of microsatellite-stable CRCs with a mutation profile overlapping that of CRCs with CIN. A diploid genotype does not, however, predict the responsiveness to radiotherapy

Advantages of automated immunostain analyses for complex membranous immunostains: An exemplar investigating loss of E-cadherin expression in colorectal cancer

As pathology moves towards digitisation, biomarker profiling through automated image analysis provides potentially objective and time-efficient means of assessment. This study set out to determine how a complex membranous immunostain, E-cadherin, assessed using an automated digital platform fares in comparison to manual evaluation in terms of clinical correlations and prognostication.Tissue microarrays containing 1000 colorectal cancer samples, stained with clinical E-cadherin antibodies were assessed through both manual scoring and automated image analysis. Both manual and automated scores were correlated to clinicopathological and survival data.E-cadherin data generated through digital image analysis was superior to manual evaluation when investigating for clinicopathological correlations in colorectal cancer. Loss of membranous E-cadherin, assessed on automated platforms, correlated with: right sided tumours (p = <0.001), higher T-stage (p = <0.001), higher grade (p = <0.001), N2 nodal stage (p = <0.001), intramural lymphovascular invasion (p = 0.006), perineural invasion (p = 0.028), infiltrative tumour edge (p = 0.001) high tumour budding score (p = 0.038), distant metastasis (p = 0.035), and poorer 5-year (p= 0.042) survival status. Manual assessment was only correlated with higher grade tumours, though other correlations become apparent only when assessed for morphological expression pattern (circumferential, basolateral, parallel) irrespective of intensity. Digital assessment of E-cadherin is effective for prognostication of colorectal cancer and may potentially offer benefits of improved objectivity, accuracy, and economy of time. Incorporating tools to assess patterns of staining may further improve such digital assessment in the future

Immuno-Interface Score to Predict Outcome in Colorectal Cancer Independent of Microsatellite Instability Status

Tumor-associated immune cells have been shown to predict patient outcome in colorectal (CRC) and other cancers. Spatial digital image analysis-based cell quantification increases the informative power delivered by tumor microenvironment features and leads to new prognostic scoring systems. In this study we evaluated the intratumoral density of immunohistochemically stained CD8, CD20 and CD68 cells in 87 cases of CRC (48 were microsatellite stable, MSS, and 39 had microsatellite instability, MSI) in both the intratumoral tumor tissue and within the tumor-stroma interface zone (IZ) which was extracted by a previously developed unbiased hexagonal grid analytics method. Indicators of immune-cell gradients across the extracted IZ were computed and explored along with absolute cell densities, clinicopathological and molecular data, including gene mutation (BRAF, KRAS, PIK3CA) and MSI status. Multiple regression modeling identified (p < 0.0001) three independent prognostic factors: CD8+ and CD20+ Immunogradient indicators, that reflect cell migration towards the tumor, were associated with improved patient survival, while the infiltrative tumor growth pattern was linked to worse patient outcome. These features were combined into CD8-CD20 Immunogradient and immuno-interface scores which outperformed both tumor-node-metastasis (TNM) staging and molecular characteristics, and importantly, revealed high prognostic value both in MSS and MSI CRCs

CD10 inhibits cell motility but expression is associated with advanced stage disease in colorectal cancer

Introduction CD10 is a cell membrane-bound endopeptidase which is expressed in normal small bowel but not in normal colon. It is aberrantly expressed in a small proportion of colorectal cancers (CRC) and this has been associated with liver metastasis and poor prognosis. We sought to investigate the mechanism of CD10 activity and its association with clinicopathological features. Material and methods CD10 was stably knocked down by lentiviral shRNA transduction in the CRC cell lines SW480 and SW620 which are derived from a primary tumour and its corresponding metastasis respectively. Expression of epithelial – mesenchymal transition (EMT) markers was tested as well as the effect of knockdown on cell viability, migration and invasion assays. In addition, immunohistochemical expression of CD10 in primary colorectal tumours (N = 84) in a tissue microarray was digitally quantified and analysed for associations with clinicopathological variables. Results Knockdown of CD10 did not alter cell viability in SW480, but migration and invasion levels increased (P < 0.001 for each) and this was associated with a cadherin switch. In SW620, CD10 knockdown caused a reduction in cell viability after 72 h (P = 0.0018) but it had no effect on cell migration and invasion. Expression of epithelial CD10 in primary tumours was associated with presence of lymph node invasion (P = 0.001) and advanced Duke's stage (P = 0.001). Conclusions Our results suggest that the function of CD10 may change during tumour evolution. It may inhibit cell motility in early-stage disease whilst promoting cell viability in late-stage disease. It has a complex role and further studies are needed to elucidate the suitability of CD10 as a prognostic marker or therapeutic target

Ran GTPase is an independent prognostic marker in malignant melanoma which promotes tumour cell migration and invasion

Aims Ran GTPase is involved in nucleocytoplasmic shuttling of proteins and is overexpressed in several cancers. The expression of Ran in malignant melanoma (MM) and its functional activity have not been described and were investigated in this study.Methods The prognostic value of Ran expression was tested in a series of 185 primary cutaneous MM cases using immunohistochemistry. The functional activity of Ran was investigated in the two melanoma cell lines. Ran expression was knocked down using two siRNAs and the effect on the expression of the c-Met oncogene, a potential downstream target of Ran, was tested. Functional effects of Ran knockdown on cell motility and cell proliferation were also assessed.Results Positive Ran expression was seen in 12.4% of MM and was associated with advanced clinical stage and greater Breslow thickness. Positive expression was an independent marker of shorter overall survival (p=0.023). Knockdown of Ran results in decreased expression of c-Met and the downstream c-met signalling targets ERK1/2. There was a significant reduction in cell migration (p [less than] 0.001) and cell invasion (p [less than] 0.001). c-Met knockdown decreased the expression of Ran through MAPK and PI3K-AKT in A375 cell line, inhibited the cell viability and migration of both A375 and G361 melanoma cell lines while invasion was enhanced.Conclusion Ran is a poor prognostic marker in cutaneous MM. It upregulates expression of the oncogene c-Met and, possibly through this, it promotes cell motility which may in turn promote metastasis

A novel Nav1.5-dependent feedback mechanism driving glycolytic acidification in breast cancer metastasis

The authors wish to acknowledge the roles of the Breast Cancer Now Tissue Bank in collecting and making available the samples and data, and the patients who have generously donated their tissues and shared their data to be used in the generation of this publication. The authors also thank Prof. Miles Whittington (Hull-York Medical School, UK), Dr. John Davey and Dr. Katherine Newling (Technology Facility, University of York, UK), and Prof. Lýdia Vargová (Charles University, Czechia) for providing invaluable advice. For the purpose of open access, a Creative Commons Attribution (CC BY) licence is applied to any author accepted manuscript version arising from this submission.Peer reviewe

Activated tissue resident memory T-cells (CD8+CD103+CD39+) uniquely predict survival in left sided “immune-hot” colorectal cancers

Introduction: Characterization of the tumour immune infiltrate (notably CD8+ T-cells) has strong predictive survival value for cancer patients. Quantification of CD8 T-cells alone cannot determine antigenic experience, as not all infiltrating T-cells recognize tumour antigens. Activated tumour-specific tissue resident memory CD8 T-cells (TRM) can be defined by the co-express of CD103, CD39 and CD8. We investigated the hypothesis that the abundance and localization of TRM provides a higher-resolution route to patient stratification.Methods: A comprehensive series of 1000 colorectal cancer (CRC) were arrayed on a tissue microarray, with representative cores from three tumour locations and the adjacent normal mucosa. Using multiplex immunohistochemistry we quantified and determined the localization of TRM.Results: Across all patients, activated TRM were an independent predictor of survival, and superior to CD8 alone. Patients with the best survival had immune-hot tumours heavily infiltrated throughout with activated TRM. Interestingly, differences between right- and left-sided tumours were apparent. In left-sided CRC, only the presence of activated TRM (and not CD8 alone) was prognostically significant. Patients with low numbers of activated TRM cells had a poor prognosis even with high CD8 T-cell infiltration. In contrast, in right-sided CRC, high CD8 T-cell infiltration with low numbers of activated TRM was a good prognosis.Conclusion: The presence of high intra-tumoural CD8 T-cells alone is not a predictor of survival in left-sided CRC and potentially risks under treatment of patients. Measuring both high tumour-associated TRM and total CD8 T-cells in left-sided disease has the potential to minimize current under-treatment of patients. The challenge will be to design immunotherapies, for left-sided CRC patients with high CD8 T-cells and low activate TRM,that result in effective immune responses and thereby improve patient survival

N_LyST: a simple and rapid screening test for Lynch Syndrome

Aims: We sought to use PCR followed by high-resolution melting (HRM) analysis to develop a single closed-tube screening panel to screen for Lynch Syndrome. This comprises tests for microsatellite instability (MSI), MLH1 methylation promoter and BRAF mutation.Methods:For MSI-testing, 5 mononucleotide markers (BAT25, BAT26, BCAT25, MYB, EWSR1) were developed. In addition, primers were designed to interrogate Region C of the MLH1 promoter for methylation (using bisulphite-modified DNA) and to test for mutations in codon 600 of BRAF. Two separate cohorts from Nottingham (n = 99, 46 with MSI, 53 being microsatellite stable (MSS)) and Edinburgh (n=88, 45 MSI, 43 MSS). Results:All the cases (n=187) were blind tested for MSI and all were correctly characterised by our panel. The MLH1 promoter and BRAF were tested only in the Nottingham cohort. Successful blinded analysis was performed on the MLH1 promoter in 97 cases. All MSS cases showed a pattern of non-methylation whilst 41/44 cases with MSI showed full methylation. The three cases with MSI and a non-methylated pattern had aberrations in MSH2 and MSH6 expression. BRAF mutation was detected in 61% of MSI cases and 11% of MSS cases. Finally, 12 cases were blind screened by using the whole panel as a single test. Of these, 5 were identified as MSS, 4 as MSI/non-LS and 3 as MSI/possible LS. These results were concordant with the previous data.Conclusion: We describe the Nottingham Lynch Syndrome Test (N_LyST). This is a quick simple cheap method for screening for Lynch Syndrome

Interrogating the genetic profile of sporadic colorectal cancer

Background and aims: Colorectal cancer (CRC) is one of the most common types of cancers. Prognosis for CRC is dependent on the stage of the disease at diagnosis and a variety of molecular features including expression of biomarkers and gene mutation. This study sought to (i) develop molecular tests for use in molecular profiling of formalin-fixed paraffin embedded tissue (FFPE) and (ii) interrogate the molecular profile and biomarker expression in multiple cohorts of sporadic CRC. Materials and methods: Firstly, we developed the Quick Multiplexed Consensus (QMC) PCR protocol – a method used in conjunction with High Resolution Melting (HRM) analysis for screening of multiple mutational hotspots (10 hotspots in this study were screened) in the CRC candidate genes using DNA from FFPE tissue. The performance of the assay for the detection of single nucleotide variants (SNV) and small insertion-deletions (indels) was compared with Sanger sequencing, pyrosequencing and real-time PCR. In addition, a PCR/HRM assay was developed for testing microsatellite instability (MSI) using a panel of six quasi-monomorphic mononucleotide markers (five of which have been previously described: BAT25, BAT26, NR21, NR22, and NR24; the sixth marker, B-CAT25, is a novel in-house developed marker) and this assay was validated against the Bethesda commercial MSI PCR kit and against immunohistochemistry (IHC). Once QMC-PCR was optimised, we sought to ascertain whether diagnostic biopsies yield sufficient information for clinical decision making. The mutation profiles of CRC candidate genes (KRAS, BRAF, PIK3CA and TP53) and MSI status were evaluated in the diagnostic biopsy specimen of 30 cases and compared with the profile in matched blocks from the resection specimen. The classification of CRC into tumours with MSI and chromosomal instability (CIN) is well described. However tumours which show neither MSI nor CIN have been described and are known as microsatellite and chromosomal stable CRCs (MACS). We sought to evaluate the frequency of CIN and MACS in a series of 89 sporadic microsatellite-stable CRCs. PCR/HRM was used to exclude MSI tumous, while CIN tumours was discriminated from MACS tumours using flow cytometry. These tumours were also compared for mutations in KRAS/BRAF/TP53/PIK3CA by QMC-PCR. Some rectal tumours may receive neoadjuvant chemoradiotherapy (CRT) and we tested whether the CIN or MACS ploidy status could predict response in a group of 62 rectal cancers treated with neoadjuvant CRT. We next evaluated the clinical utility of deficient Mismatch Repair (dMMR) protein and aberrant P53 protein expression, both individually and in combination, as prognostic and predictive of response to therapy biomarkers in CRC. A total of 884 tumours from the VICTOR trial (a large phase III trial of rofecoxib in stage II and III CRC) were tested for expression of four mismatch repair (MMR) proteins (MLH1, PMS2, MSH2, MSH6) and p53 by IHC. The expression was correlated with outcome. Results: QMC-PCR worked on DNA from FFPE tissue. Spiking experiments showed the protocol could detect a minimum of 2.5% of mutant alleles compared to 20% detectable for Sanger sequencing. Precision tests showed that there was little intra-assay and inter-assay variation. Forty-three FFPE colorectal tumours were initially sequenced for hotspots in KRAS and PIK3CA and then screened by QMC-PCR. In these tests, QMC-PCR showed a sensitivity of 100%, specificity of 71%, PPV of 76% and NPV of 100%. All 43 samples were then screened for mutations in all 10 hotspots. Of 430 tests, 43 (10%) showed aberrant melting and 36 were confirmed mutant (PPV 84%). Since our technique is more sensitive than direct sequencing, the remaining 7 tests are probably sequencing false negatives. HRM analysis of KRAS (codon12/13) and BRAF (V600E) showed that 3% and 1.5% mutant alleles respectively could be reliably detected whilst pyrosequencing reliably detected 6% mutant alleles in each case. Of 110 tests performed on 22 DNA samples, in 109 cases HRM and pyrosequencing gave identical results. The results of KRAS (codon12/13) screening from the comparative study of 468 cases with a real-time PCR test (DxS), showed 89.9% concordance with the DxS test. In comparison with real-time PCR, the analysis by HRM showed 87.2% sensitivity, 91.6% specificity, 86.7% positive predictive value, 91.9% negative predictive value. After refinement of the protocol, re-testing of 100 randomly selected cases showed 99% concordance between QMC-PCR/HRM and the real-time PCR test. The performance of the HRM based MSI assay showed 100% accuracy on both cell lines and FFPE tissue in comparison to PCR and IHC. Pertaining to the results from the paired biopsy and resection specimens work, a total of 570 paired PCR tests were performed and identical results were obtained in both biopsy and resection specimens in 569 tests (>99% concordance). Four cases (13%) showed microsatellite instability, and, in all four cases, instability was seen at identical mononucleotide markers in both biopsy and matched resection specimens. Regarding the immunostaining of MMR proteins, the staining was more intense and easier to interpret in biopsies and that it faithfully replicates the diagnosis in the resection specimen. The results from the DNA ploidy study showed, in addition to the two classical forms of genetic instability (MSI, CIN), a significant third group of colorectal cancers (CRC) without either MSI or CIN i.e. Microsatellite and Chromosome Stable (MACS) CRCs. Fifty-one of 89 CRCs (57%) were aneuploid and 38 (43%) were diploid. There was no significant association seen between mutations in TP53/KRAS/BRAF/PIK3CA with ploidy. Testing of association between mutations revealed only mutual exclusivity of KRAS/BRAF mutation (p<0.001). Of the 62 rectal cancers treated with neoadjuvant chemoradiotherapy, 22 had responded (Mandard Tumour Regression Grade 1/2) and 40 failed to respond (Grade 3-5). Twenty-five of 62 (40%) tumours were diploid but there was no association between ploidy and response to the neoadjuvant CRT therapy. Evaluation of TP53 and MMR protein expression in the VICTOR trial, showed that there was dMMR in 12% (87/735) of patients. It was associated with female gender (p=0.001), proximal location of tumour (p<0.001), poor differentiation (p<0.001) and stage II disease (p<0.001). dMMR was not associated with either disease free survival (DFS) or overall survival (OS) in the group overall or when stratified by stage. However, unexpectedly, proficient MMR (pMMR) was associated with significantly improved OS (hazard ratio 0.28 (0.11, 0.68), p=0.005) and DFS (hazard ratio 0.47 (0.22, 0.99), p=0.047) in patients who did not receive chemotherapy. Aberrant p53 expression was found in 65% (482/740) of patients. It was associated with distal location (p<0.001) and stage III disease (p<0.001) and it was negatively associated with dMMR (p<0.001). No effect was observed on DFS or OS and there was no interaction with chemotherapy or radiotherapy. When assessed in combination, dMMR and aberrant p53 expression had no effect on DFS, OS or responsiveness to adjuvant therapy. Conclusions: The following conclusions can be drawn from this research work: 1) QMC-PCR with HRM is a simple, robust and inexpensive technique which had greater sensitivity than Sanger sequencing. It allows multiple mutation hotspots to be rapidly screened and is thus highly suited to mutation detection in DNA derived from FFPE tissues. Both HRM and pyrosequencing can detect small numbers of mutant alleles although HRM has a lower limit of detection. Both are suitable for use in mutation detection and are both more sensitive than Sanger sequencing. QMC-PCR with HRM performs very well against real-time PCR based tests for detection of KRAS mutation. Although the real-time PCR has a lower limit of detection, HRM has greater flexibility overall. Both tests are suitable for KRAS codon 12/13 mutation detection in patients being considered for anti-epidermal growth factor receptor (EGFR) biological therapy. 2) PCR-HRM/MSI assay is simple, straightforward and highly sensitive technique for MSI testing which can be easily incorporated in a single closed-tube panel whether to screen for Lynch syndrome, or to test sporadic CRC for tumour MSI status in order to inform the decision maker to administer 5-fluorouracil-based chemotherapy to the patient or not. 2) This was the first study to show that diagnostic biopsy specimens, even though they are a tiny sample of the tumour, are sufficiently representative for use in predictive testing for early driver mutations in colorectal cancer, especially important when neoadjuvant therapy is being considered. 3) MACS-CRCs form a significant proportion of microsatellite-stable CRCs with a mutation profile overlapping that of CRCs with CIN. A diploid genotype does not however predict the responsiveness to radiotherapy in rectal cancers. This finding leads us to conclude that there are other factors in addition to ploidy that define response to radiation therapy. 4) Analysis of MMR status and p53 expression in the patients recruited to the VICTOR trial confirmed that dMMR and p53 expression are associated with site and stage of CRC. Neither biomarker has prognostic or predictive utility in this cohort of patients overall although in sub-group analysis dMMR was indicative of poor outcome in chemo-naive patients