Advances in molecular analysis have led to the development of patient-tailored cancer treatments. In the majority of cancer patients, successful treatment is achieved using conventional methods such as surgery, radiotherapy and/or chemotherapy. However, for those patients who do not respond to conventional treatments, the development of tailored treatments that target particular molecular pathways involved in cancer development and progression is increasingly emerging [1] . The promise of targeted therapy has been to be more specific, with improved global cancer control in the individual, thus improving survival, whilst having less impact in terms of toxicities and quality of life. Interestingly, oncologists have been learning to cope with a range of new toxicities which have not been relevant with more traditional chemotherapy agents, with less risk of immunosuppression but higher chances of skin, eye or cardiac effects. To date, the results of these target-specific treatments have been mixed. As single agents, results in some tumors have been disappointing, suggesting that too little is known about the agents and the molecular pathways they are deemed to impact. A notable example is the targeting of the BRAF axis, in various tumor types. Tumors that harbor a BRAF mutation in colorectal cancer commonly have a worse prognosis in the metastatic setting. Agents such as Encorafenib (a BRAF inhibitor), targeted at the altered BRAF protein arising from the mutated gene, appear to have limited single agent effect in colorectal cancer, whilst single agent BRAF inhibition in BRAF mutant metastatic melanoma often has a dramatic effect. When Encorafenib is combined with Cetuximab (an EGFR inhibitor), it on its own is deemed to be ineffective in BRAF mutant colorectal cancer, then we see a positive effect from the dual blockade such that the combination of Cetuximab and Encorafenib is now licensed and found in guidelines as a standard of care second-line therapy treatment for patients with BRAF mutant metastatic colorectal cancer.

In recent years, advances in molecular screening technologies have provided evidence of the genomic alterations that can occur in cancers. These acquired genetic mutations can result in changes in protein expression of the mutated gene leading to abnormal expression levels that can influence the response of the tumor to various treatments available [1] . Understanding these changes in the genome and proteome has resulted in the development of tailored treatments for multiple cancers including colorectal, lung and breast cancer [2] [3] [4] .

Globally, CRC is the third most common cancer in males and the second in females, with an estimated 1.4 million cases and 693,900 deaths occurring in 2012 [5] . Within Wales >2300 patients are diagnosed per year and it is the second biggest cancer killer. Approximately, 90% of CRC cases are sporadic without a family history or genetic predisposition [6] . Extensive studies analyzing the genetics of CRC have identified mutations in the DNA sequence in both oncogenes and tumor-suppressor genes, predominantly the KRAS and p53 genes [7] .

One of the key pathways that have been identified in the progression of CRC is the Mitogen Activated Phospho Kinase (MAPK) pathway [8] . The MAPK signaling pathway controls cell proliferation, differentiation and apoptosis [8] . In normal tissue, the activation of the MAPK pathway is controlled through the interaction of an external growth factor such as epidermal growth factor (EGF), with its receptor (EGFR). Mutations that occur within genes that regulate the interaction can result in the inability of cells to switch this mechanism off resulting in continual activation of the pathway.

In advanced colorectal cancer (CRC), one of the main treatments for patients is anti-EGFR therapy (Cetuximab [9] or Panitumumab [2] ). Anti-EGFR drugs bind to the EGFR that is present on tumor cells and limits the growth of these cells by inhibiting the RAS signaling pathway. The identification of mutations in the RAS (KRAS or NRAS) gene has determined that patients harboring a mutated RAS will unlikely benefit from anti-EGFR therapy [10] and as a result all patients with advanced CRC are now routinely screened for RAS mutations. Also, present in the MAPK pathway is the BRAF protein, a single point mutation in the gene accounts for most of the cancer associated aberrations in this gene resulting in a valine to glutamine change at residue 600 (V600E). As a consequence of this mutation, the BRAF protein is constitutively activated and in advanced disease has a significant detrimental impact on survival. Whilst specific BRAF inhibitors have been developed which are effective in other cancers, in metastatic CRC the use of BRAF inhibitors has been ineffective with a response rate of approximately 5% confirming that there is still plenty to learn about BRAF mutation in mCRC [11] [12] [13] .

Over the last 12 years, the Wales Cancer Biobank (WCB) has consented more than 2000 patients with colorectal cancer [14] . From these patients, clinical data has been collected and follow-up has been regularly performed during this period. We have analyzed the data from these patients to identify any correlation between disease grade and outcome. In a cohort of patients, WCB has performed genetic analysis on patient primary tumor samples. Our aim at the WCB is to identify the proportions of colorectal cancers that are currently banked within the WCB that, contain the common mutations that have been identified as potential drivers in CRC and are the targets for developing anti-cancer drugs. We are also able to link the presence of these mutations with clinical parameters and outcomes, identifying any variance in the Welsh cohort in comparison to internationally published data.

Between February 2005 and November 2016, 2217 patients with colorectal cancer were recruited to the WCB (~10% of the overall CRC population over 11 years). Of these, 97 patients (4%) were diagnosed post-procedure with a benign tumor and 20 patients with a neuroendocrine tumor. One hundred and thirty-nine (6%) patients had less than 12 months follow-up and were listed as alive (Figure 1). These patients were excluded from the analysis (Figure 1). In addition, ten patients (<1%) were also excluded as they had been lost to follow-up (Figure 1). The resulting 2005 patients with colorectal cancer that met all the eligibility criteria were included in the final analysis (Figure 1). Median follow-up was 36 months, ranging from 12 - 225 months. As of 31^st December 2016, 1385 (69%) patients were listed as alive. These patients had a median follow-up time of 41 months (range 12 - 225).

The WCB colorectal cancer patient cohort is representative of the Welsh population, accruing ~10% of patients diagnosed over an 11 years. This powerful dataset including demographic, treatment and outcome data represents a unique resource linked to tissue samples, to support translational research in a disease that has seen little progress in therapies over the last 15 years. Molecular analysis has

*Mutations listed were found in more than 2 patients within the WCB cohort.

further indicated that on a national level mutation profiling is similar to data provided from clinical trials datasets internationally. At the Wales Cancer Biobank (WCB), the recruitment of patients with colorectal cancer has been underway for more than 13 years. To date, tissue samples from 2217 colorectal patients have been collected along with clinical and follow-up data. Out of these, samples from 407 patients that had a minimum of 12 months follow-up data were characterized for mutations in the key genes that are known to play a role in cancer development. The analysis of these samples provides insight into the biology of CRC at a national level.

As a cost recovery, not for profit organization, the Wales Cancer Biobank along with other biobanks offers an unrivalled resource to further our understanding of cancer biology, its impact on patient outcomes and our ability to identify targets for future therapeutic intervention. Since the inception of WCB, 19 research groups have applied to WCB for colorectal samples ranging from fresh tumor tissue for the creation of 3D modeling systems for drug discovery, whole blood samples for analyzing the immune cell signatures and plasma and tumor DNA for analyzing temporal changes in circulating biomarkers during treatment.

Statistical analysis of the patient demographics of the WCB colorectal cohort determined that CRC is more prevalent in males within the Welsh population (60% vs 40%) as confirmed using data derived from the Welsh Cancer Intelligence and Surveillance Unit (WCISU) [16] . Compared to other UK countries, the WCB cohort is slightly higher towards men, where the UK average is 55% of newly diagnosed CRC occurring in men [17] [18] . This is also observed when compared to other countries such as the US [19] . Interestingly, within the WCB cohort, only 8% were diagnosed with metastatic colorectal cancer at presentation. This is considerably lower when compared to the UK average, where 23-26% of patients have metastases at diagnosis [20] . This almost certainly relates to the impact of non-removal of the primary tumor in patients who have synchronous metastatic disease, in the WCB cohort of patients, as we have avoided analysis of biopsy only material. As expected, the higher the stage of disease the worse the prognosis. The UK average for net survival for bowel cancer at two- and five-years are 67.9% and 58.7%, respectively [20] . This compares to the disease specific survival rates at two- and five-years for the WCB cohort, of 87.4% and 75.3% respectively. The Wales Cancer Registry WCISU indicates a five-year survival rate of 58.2% [16] . Evidently, factors such as patient consenting are impacted by emergency presentation and synchronous metastatic disease in which no surgical removal of the tumor is planned and this inherently impacts upon prognosis. WCB consented patients’ data is heavily reliant on the data recorded in the Cancer Network Information System Cymru (CaNISC) and the Office of National Statistics (ONS). If data was missing or not recorded, patients were excluded from the analysis.

During the lifetime of this research project and over the period these patients have experienced their disease, there have been some significant adaptations in the use of molecular evaluation of tumors to inform clinical practice. Notably, in the adjuvant setting, it has become routine to perform an assessment of microsatellite instability (MSI) in patients with stage II disease. MSI-High tumors are predominantly right sided and often poorly differentiated yet are of better prognosis and thus may not gain a significant advantage from adjuvant chemotherapy. Nationally MSI testing for all colorectal cancers commenced in June 2019. In the metastatic setting it has become a standard of care to evaluate RAS and BRAF mutation status and to consider the use of cetuximab or panitumumab in combination with chemotherapy in the first-line setting in those patients with wild type tumors. Further advances have seen the introduction of administering the immunotherapies, Nivolumab plus ipilimumab to patients with MSI-high metastatic CRC. Studies have demonstrated high response rates with increased OS in these patients [21] . The continued screening for genetic aberrations and advances in immunotherapy, especially in metastatic patients, has demonstrated a benefit to patients that in previous years may have had limited treatment options available.

With regard to the mutational analysis, the WCB cohort was significant by its similarities to published data. However, we must accept the limitations of the molecular analysis performed. Analysis of the genes, KRAS and NRAS were sufficient for the detection of known mutations, but analysis of BRAF and PIK3CA were limited due to the region covered by the pyrosequencing. Analysis of TP53 was also limited by the Limit of Detection (LoD) of Sanger sequencing. At the time of analysis, molecular diagnostics was rapidly evolving, although compared to current methods of molecular analysis the techniques utilized was limited, the results are still comparable.

In the WCB cohort, there was a significant decrease in the disease specific survival in patients that harbored a BRAF mutation than patients with the wild-type gene. Previous studies have identified BRAF mutations in approximately 8% - 15% of all colorectal cancers [22] and have been associated with poor prognosis [12] , indicating the importance of BRAF mutations in the development and prognosis of CRC. The prominence of a BRAF mutation in CRC has previously been shown to affect the response of patients with metastatic disease to targeted therapies such as the anti-EGFR treatments cetuximab or panitumumab [2] [23] . Patients with a wild type KRAS gene but harboring a BRAF mutation did not respond to the EGFR inhibitors confirming that the BRAF mutation plays a critical role in the signaling pathway for EGF [13] [24] . The clinical implications of these findings suggest the need for BRAF screening in wild type KRAS CRC patients before EGFR inhibitors are administered. In addition, these results demonstrate the further need for research into the pathways involved with the targeted drugs in order to improve the efficacy of these therapies [25] .

Mutations in RAS family members are frequently found in human cancers including non-small cell lung cancer [26] , pancreatic cancer [27] and colorectal cancer [25] . Three RAS genes have been identified and although they are functionally distinct they are highly homologous within their genetic sequence [28] . The function of these proteins has a critical role in cell proliferation, survival, and differentiation [28] . The majority of the mutations for the RAS genes occur within codons 12, 13 or 61 and the activating mutations result in constitutive activation leading to a sustained proliferation signal within the cell [28] [29] .

Of the three RAS family members, studies have shown that mutations within the KRAS gene are more frequent in solid tumors, mainly adenocarcinomas, whilst NRAS mutations are more prevalent in leukemia [29] [30] . Studies have shown that KRAS mutations occur within 40% of CRC whilst NRAS mutations occur within 1% - 6% of CRC [31] [32] . Of the 407 CRC cases sent for mutational analysis, 99% of patients had been diagnosed with adenocarcinoma and 41.2% of patients that were tested for KRAS, harbored a mutation. In comparison, mutations in NRAS were only detected in 4.7% of patients confirming that mutations within the Welsh population are comparable to other populations. Although NRAS mutations are rare in CRC, within the WCB CRC cohort the presence of codon 13 mutations was found only in Dukes C1 and C2 but due to low numbers further investigation to determine any correlation will be required. Furthermore, little is known about NRAS mutations and their relationship to clinical, pathologic, and molecular features remains uncertain.

As previously mentioned, KRAS mutations are more prevalent in codons 12, 13 and 61. In the WCB CRC cohort, the majority of mutations occurred within these regions, however, 4% of the mutations were located with codon 146. In these patients, a single nucleotide change from cytosine to thymine at cDNA position 437 resulted in a protein change from Alanine to Valine. The impact of this mutation is currently unknown and to date, few reports have investigated codon 146 mutations and the clinical relevance of this mutation [32] [33] . Interestingly, when disease specific survival was compared against mutations based on codon location, results suggested that both patients with codon 12 and 13 mutations had a worse prognosis when compared to wt KRAS, codon 61 and codon 146 mutations. When disease specific survival was compared for wt KRAS and mutated KRAS, patients harboring a mutation had an inferior survival but this was not significant. Similar findings have been published by others and therefore the effect of KRAS mutations appears to have a greater impact on responses to EGFR treatments than prognosis [25] [34] [35] .

Mutations harbored in the PIK3CA gene have been implicated in the pathogenesis of multiple cancers including CRC [36] . Somatic mutations in the phosphatidyl 3-kinases (PI3K) family member, PIK3CA, result in over activation of the gene which has a role in various cellular processes that can regulate cell proliferation and survival [36] . In CRC, PIK3CA mutations are thought to occur within 10% - 30% of cancers with the mutations occurring usually within exon 9 and exon 20 of the gene [37] . In the WCB CRC cohort tested for PIK3CA mutation, 13.2% of patients harbored a mutation within exon 9 and/or exon 20. Although patients with a mutated form of PIK3CA had an unfavorable prognosis for disease specific survival, this was not significantly different from patients with wtPIK3CA. These findings are consistent with other studies that have analyzed PIK3CA mutations [38] . A number of studies have reported on the different functions of exon 9 and exon 20 of the PIK3CA gene and the effects of these in cancer [39] [40] [41] . Analysis of the disease specific survival for PIK3CA comparing wtPIK3CA with exon 9 and exon 20 mutations separately, suggested that exon 20 mutations resulted in a worse prognosis. This has also been observed in other studies for CRC and other cancers [39] [40] however, due to the low frequency of these mutations the prognostic role of PIK3CA mutations, both exon 9 and exon 20, in CRC remains uncertain.

One of the most commonly mutated genes in human cancers is the p53 gene [42] . It has been reported that more than 50% of CRC will harbor a mutation within the p53 gene [43] . These mutations are known to drive oncogenic events in CRC, however the mechanisms that p53 mutations use to exert these events are still unclear. In recent years the development of p53 targeting agents has been explored but due to the complexity of the oncogenic and biological effects that may occur, few have been translated out of the laboratories [44] . Within the WCB cohort, 62% of patients harbored a p53 mutation, however most of the mutations detected were present in only one patient confirming the complexity of p53 mutations. Of the mutations identified, seven mutations were found to be present in more than 3 patients. These mutations are distributed within exon 4-9 which encode the DNA-binding domain [45] . Within this domain, there are six common mutational hotspots, residues R175, G245, R248, R249, R273 and R282 [46] . The most common mutation within the WCB cohort tested, residue R175 (c.524G > A p.(Arg175His)), was identified in 15 patients. Although little is known about the effect this mutation has in CRC, it has been implicated in the activation of c-Met receptor tyrosine kinase in Esophageal squamous cell carcinoma (ESCC) mediating tumor cell invasion [47] . It has also been implicated in endometrial cancers, increasing the invasive phenotypes through activation of the EGFR/PI3K/AKT pathway [48] . All of the mutations observed within the WCB cohort have been identified by various other cancer studies identifying mutations of the p53 gene. Interestingly p53 mutations within the common hotspots were identified in patients with more advanced disease (Dukes A 8.8% vs Dukes C2 54.5%) suggesting that p53 aberrations occur late in tumorigenesis. Studies comparing p53 mutations in colorectal healthy tissue, adenomas and carcinomas suggest that p53 mutations develop at late stage adenoma and increase with frequency as carcinomas progress [46] . This has also been documented in other cancers where p53 mutations have been studied [49] [50] . Studies such as these suggest that p53 aberrations may be used to determine prognosis, however the clinical significance of p53 aberrations has long been debated and remains one of the most controversial areas of p53 research. A review performed in 2010 looking at p53 mutation and prognosis in multiple cancers suggested that p53 aberrations within breast, head and neck, liver and haemopoietic cancers were associated with a worse prognosis. This correlation was not conclusive for bladder, brain, lung or ovarian cancer [51] .

In addition to the p53 mutational hotspots, studies have also focused on the haplotype of p53 searching for links with cancer prognosis however the results are still inconclusive although most have described a weak association between the SNP c.215C > G p.(Pro72Arg) and an increased risk of CRC [43] . In our cohort, 85.3% of the patients tested had the SNP c.215C > G. When disease-free survival was compared between patients with and without the SNP, we found no significant differences that would suggest that the SNP indicated a worse prognosis.

In the light of precision medicine and improved genetic testing, screening of cancers for gene aberrations has revolutionized the monitoring and treatment of the disease. Our increased knowledge of the mechanisms that tumors use to ensure their progression has enhanced drug development. Genetic alterations in CRC have been studied extensively and with increased sensitivity in screening and detection methods, it continues to advance. Further research on this ever-expanding repertoire of mutations will ensure that future drug development can be tailored to the specifics of the disease rather than a one drug fits all approach.

The sample collection by the Wales Cancer Biobank was funded by Welsh Government through Health and Care Research Wales and the research was financed by Cancer Research Wales, CRW Program Grant 2011 (DOI:10.5334/ojb.46). WCB would like to acknowledge all the patients that have donated their samples to the biobank over the years. WCB would also like to acknowledge the Wales NHS, their staff and the continued support that they provide.

The sample collection by the Wales Cancer Biobank was funded by Welsh Government through Health and Care Research Wales. All research performed was financed by Cancer Research Wales, CRW Program Grant 2011.

The authors declare no conflicts of interest regarding the publication of this paper.

Spary, L.K., DeLoyde, K., Roberts, H., Martin, F., Lee, C.P., Butler, R., Mason, M.D., Thomas, G.A., Parry-Jones, A. and Adams, R.A. (2023) Clinical Analysis of the Colorectal Cohort within the Wales Cancer Biobank: A Study of Outcomes and Genetic Screening. Journal of Cancer Therapy, 14, 317-344. https://doi.org/10.4236/jct.2023.147027