A Systematic Review and Meta-analysis on the Occurrence of Biomarker Mutation in Colorectal Cancer among the Asian Population

Globally, colorectal carcinoma (CRC) is the third most common cancer and the third major cause of cancer-related death in both sexes. KRAS and BRAF mutations are almost mutually exclusively involved in the pathogenesis of CRC. Both are major culprits in treatment failure and poor prognosis for CRC. Method. A systematic review and meta-analysis of various research was done following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. This trial is registered with PROSPERO CRD42021256452. The initial search included 646 articles; after the removal of noneligible studies, a total of 88 studies was finally selected. Data analysis was carried out using OpenMeta Analyst and Comprehensive Meta-Analysis 3.0 (CMA 3.0) software to investigate the prevalence of KRAS and BRAF mutations among patients with CRC in Asia. Results. The meta-analysis comprises of 25,525 sample sizes from Asia with most being male 15,743/25525 (61.7%). Overall prevalence of KRAS mutations was (59/88) 36.3% (95% CI: 34.5-38.2) with I2 = 85.54% (P value < 0.001). In 43/59 studies, frequency of KRAS mutations was majorly in codon 12 (76.6% (95% CI: 74.2–78.0)) and less in codon 13 (21.0% (95% CI: 19.1-23.0)). Overall prevalence of BRAF mutations was 5.6% (95% CI: 3.9-8.0) with I2 = 94.00% (P value < 0.001). When stratified according to location, a higher prevalence was observed in Indonesia (71.8%) while Pakistan has the lowest (13.5%). Conclusion. Total prevalence of KRAS and BRAF mutations in CRC was 36.6% and 5.6%, respectively, and the results conformed with several published studies on KRAS and BRAF mutations.


Introduction
Globally, cancer is a serious medical burden, and it is one of the main causes of death and morbidity throughout the world [1,2]. With more than 1.8 million new CRC diagnoses and 0.86 million deaths globally in 2018 [3], colorectal carcinoma (CRC) is the third most common cancer and the third major cause of cancer-related death in both sexes [4]. The occurrence of CRC differs globally; the overall highest incidence rates of CRC may be seen in the United States, Canada, Europe, and Australia, whereas the lowest rates can be found in South-Central Asia and Africa [2]. However, the prevalence is rising exponentially in Asia [5], especially as the number of new cases of CRC is rapidly growing in Asia-Pacific thus, accounting for more than half of all new cases diagnosed globally [5,6]. CRC prevalence rates vary due to several factors such as ethnicity, genetics, regions, and lifestyles. It is reported to be 38 percent among Caucasians, 40 percent among Asians, and just 21 percent among Africans [6]. Pathogenesis of CRC involves gene mutation, mostly involving the MAPK-ERK cascade, for which the KRAS and BRAF are exclusively involved.
Nation-wise, the prevalence of KRAS and BRAF mutation varies regionally, and this is majorly due to genetic changes from heterogeneously related races [6]. From the World Health Organization (WHO) regional grouping, the prevalence of KRAS mutation among constituting nations with CRC is 30.23%, 35.12%, 31.83%, 33.17%, and 32.64% for the EMRO, EURO, PAHO, SEARO, and WAPRO, respectively, [5]. Colorectal carcinogenesis is a multisignalling process involving four major pathways: the Wnt-β catenin pathway, MAPK/ERK pathway, PI3K/Akt pathway, and p53 pathway. Each pathway involves several sequential genetic modifications, such as chromosomal anomaly, gene mutations, and/ or epigenetic changes, that turn normal colonic epithelium into colorectal cancer [7] [8]. Like the KRAS gene, BRAF is also part of the Ras family that targets the RAS/RAF/MEK/ ERK pathway; together, they both account for 7-25% and 5-20% of all cancers as well as 30-45% and 8-10% of CRC for both KRAS and BRAF, respectively [8,9]. Mutations in KRAS and BRAF are almost mutually exclusive. The detection or testing for KRAS and BRAF gene mutation presents a blueprint and change to standard diagnostic guidelines for inpatient care and creates a major development in early decisionmaking in personalizing cancer care. The identification of this mutation would be crucial for the prognosis of CRC patients. Early diagnosis and treatment will improve patients' standard of health, increase their chances of survival, and lower morbidity and mortality. The poor prognosis of metastatic CRC has fuelled continued efforts to identify therapeutic options that will improve patient outcomes via detailed gene profiling such as in KRAS and BRAF mutations.
Ras proteins are tiny guanosine triphosphatases (GTPases) to which the KRAS and BRAF genes belong. Through the GTPase cascade, they govern a variety of intracellular activities such as proliferation, differentiation, immune response, and survival rate [10]. The understanding of genetic alterations (such as in KRAS and BRAF mutations) in metastatic CRC (mCRC) via the use of gene profiling can be a catch point in explaining the gene's resistance to antiepidermal growth factor receptor (EGFR) antibody management [11] and as a prognostic predictor in arresting the progress of CRC [12] [8,13]. Both KRAS and BRAF are downstream EGFR oncogenes, for which their mutations can activate EGF receptor signalling in cancer cells and are linked with poor prognosis in the CRC. Hence, certain aberrations or mutations that have a well-established prognostic and predictive blueprint in CRC are now regularly examined as a component of clinical therapy [14]. Through this study, the authors intend to determine the prevalence of KRAS and BRAF gene mutations in CRC and whether the prevalence of mutated KRAS and mutated BRAF genes in colorectal carcinoma differs among patients in Asia via literature review and meta-analysis to provide a very accurate KRAS and BRAF mutation estimates.

Materials and Methods
The present study is a systematic review and meta-analysis of various researched and published papers that were carried out following the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) [15]. The study protocol was registered with PROSPERO (registration number: CRD42021256452).
2.1. Literature Search and Selection Criteria. In the study, articles were retrieved from three electronic databases (PubMed, Scopus, and ScienceDirect); the eligible studies were searched and assessed using a combination of relevant keywords: ("colorectal cancer" OR "colon cancer" OR "metastatic colon cancer" OR "metastatic colorectal cancer" OR "CRC" OR "Rectum") and ("BRAF" OR "BRAF" OR "c-BRAF" OR "KRAS" OR "K-RAS" OR "c-KRAS") and ("Asia"). The full details of the search strategies for this study are in the supplementary search strategic file. A comprehensive search for the most relevant studies was carried out by combing through titles, keywords, and abstracts of various papers. The initial search included 646 articles ( Figure 1) which were performed on 10 April 2021 via the EndNote X9 software; references of all assessed studies were exported to the software after which, duplications were then removed. The inclusion criteria for the studies selected for this meta-analysis include cross-sectional studies, cohort studies, or case series carried out to investigate the frequency of KRAS or BRAF gene mutations in colorectal cancer patients in Asia. Also included are studies on KRAS and BRAF gene mutations from fresh frozen, formalin-fixed paraffin-embedded (FFPE), or biopsied colorectal carcinoma specimens. Also, KRAS and BRAF studies involving more than one sample size and all related papers published at valid international summits were included. No limit is set on methods for determining gene mutations. The exclusion criteria include (1) studies not associated with frequency of KRAS and BRAF gene mutations, (2) studies that investigated just one of either codon 12 or codon 13 of KRAS gene mutation, (3) reviews and case reports, (4) KRAS and BRAF gene mutations that are related to cell lines and animal studies, and (5) studies that investigated BRAF gene mutation through KRAS-positive patients [16]. All authors were involved in the study screening, selection, and assessment criteria. Two authors (A.H.A. and A.A.I.) independently screened the articles based on title and abstract. This was proceeded by the assessment of the full texts. Any discords during the screening process were resolved by discussion with other authors in the study.

Data
Extraction and Quality Assessment. The data extraction was carried out by using an Excel spreadsheet. Two reviewers (H.A.A. and A.A.I.) independently vetted the titles and abstracts and extracted crucial information required; study I.D, publication year, period and design, gender, data on the mutation of KRAS, and BRAF prevalence among patients diagnosed with colorectal cancer in Asia were diligently extracted. Any inconsistencies were handled through conversation with a third reviewer (S.M.S) to prevent any sort of prejudice, and any discrepancies were sorted out via dialogue involving other reviewers to avoid any kind of bias. The quality of the methodological approach for the studies included was appraised independently by two authors (A.H.A. and Y.W.) via the Joanna Briggs Institute (JBI) critical appraisal checklist for prevalence data [17] (Supplementary JBI file). A score of 1 for "yes" and 0 for other parameters was allotted to obtain a sum quality score that ranges between 0 and 9. Studies with a final score of 7-9 were chosen to be of desirable quality. The studies within the latter acceptable score range were included in the data extraction phase of the meta-analysis.

Data Synthesis and Analysis.
Data analysis was carried out via the use of OpenMeta Analyst and Comprehensive Meta-Analysis 3.0 (CMA 3.0) software [18]. The prevalence of KRAS and BRAF gene mutations among patients with colorectal carcinoma in Asia was calculated, and subgroup analysis was also carried out on location, tumour stage, tumour grade, and period of study. A random-effect model through the DerSimonian-Laird method of the metaanalysis was employed to obtain the pooled estimates of the reported KRAS and BRAF gene mutation cases. Further, to ascertain the study quality, possible publication bias was scrutinized by creating a funnel plot. The asymmetry of the plot was further investigated via Egger's regression test [19]. The heterogeneities of study-level estimates were determined by Cochran's Q test and quantified using I 2 statistics. I 2 values of 25%, 50%, and 75% were considered low, moderate, and high heterogeneities, respectively [20]. For all tests, a P value < 0.001 was labelled statistically significant.

Result
This section is divided into subheadings to provide a concise and precise description of the experimental results and their interpretation, as well as the experimental conclusions that can be drawn from the outcomes.

Search Results and Study Selection.
A total of 646 records were obtained by searching three electronic databases. After eliminating duplications and studies that do not favour the inclusion criteria, the remaining 498 were screened via titles and abstracts and by further excluding 261 records that satisfied the exclusion criteria and another 115 records that were done outside Asia; then, 122 records were left. Upon further scrutiny, another 34 records that did not merit the inclusion criteria as depicted in Figure 1 above were  Figure 1: Summary of article identification and selection process.
3 BioMed Research International excluded. Finally, a total of 88 unique records were confirmed eligible to be included in the meta-analysis. Among these later 88 eligible studies, 59 reported on KRAS gene mutation, and 29 reported on BRAF gene mutation. Thus, a total of 88 studies were selected for this meta-analysis.
3.2. Characteristics of the Eligible Studies. The characteristics of studies on KRAS and BRAF mutations are illustrated in Tables 1 and 2, respectively. The meta-analysis study comprises of 25,525 sample sizes; all studies were from the Asian region with the male patient being most of the total participants, 15,743 out of 25525 (61.7%). The comprehensive characteristics of the included studies are illustrated in Table 1.

Prevalence of KRAS Gene Mutation in Colorectal Cancer
Stratified by Study Location and Period of Study. To determine the prevalence of KRAS mutation in CRC patients from various regions in Asia, a subgroup meta-analysis was undertaken. Data were available for nineteen locations from the listed studies, with the largest number of studies coming from Iran (n: 12) ( Table 3; Supplementary Figure SF3).

Subgroup Analysis of the Prevalence of BRAF Gene Mutation in Patients with Colorectal Cancer Stratified by Study Location and Period of Study Conduct.
To determine the prevalence of BRAF in colorectal cancer CRC patients from various regions in Asia, a subgroup meta-analysis was undertaken. Data were available for fourteen locations from the listed studies, with the largest number of studies coming from China (n: 7) ( Table 4; Supplementary Figure SF12).

Analyses of Sensitivity and Publication Bias.
A funnel plot of random effects was generated to observe evidence of publication bias among the studies reporting KRAS gene mutation ( Figure 4) and BRAF gene mutation ( Figure 5) among Asian CRC patients. There was no clear evidence of publication bias in both KRAS and BRAF mutation studies.  BioMed Research International

Discussion
Several research today showed that mutations in the RAS family of genes especially the KRAS are linked to around a third of all malignancies; however, the incidence of the gene mutations varies greatly depending on the kind of cancer: often seen to be 40% in colorectal cancer, 15-20% in nonsmall-cell lung cancer, and 95% in pancreas carcinoma [44]. Only a few individuals diagnosed with colorectal cancer would be opportune to receive curative surgery if detected early because, at the time of consult with the surgeon, it is already in the late stage wherein the prognosis is poor. More so, the illness involves no specific early presenting features, and the long-term disease period is usually associated with probable organ metastases [86,88]. Also, because colorectal cancer is thought to grow progressively over time due to the buildup of genetic abnormalities, the threat of reoccurrence and mortality from colorectal cancer is significantly linked to the stage of the disease at diagnosis [86]. Although there is a tremendous advance in the CRC treatment via the use  11 BioMed Research International of cytotoxic agents, i.e., monoclonal antibodies to targeted therapy such as on EGF receptor [78], CRC still poses a significant threat to life as KRAS gene mutation is reported as a major cause of treatment failure in cancer therapy [89].
Colorectal cancer (CRC) is the third most frequent cancer in the world, with 2.0 million new cases in 2020, accounting for 11% of all new cancer cases [90]. It was estimated as 1.9 million of all new cases and 880,000 deaths in 2018 [91]. The incidence and mortality rates of colorectal cancer (CRC) differ significantly around the globe, i.e., differs in various regions. From a total of 646 eligible papers that were filtered in this study, 88 studies were finally selected to investigate the prevalence of KRAS and BRAF gene mutations in this analysis. During this analysis, approx-imately 115 articles reporting KRAS and BRAF gene mutations in CRC outside Asia were identified, but they were, however, excluded because they did not fulfill the study's inclusion criteria. This plethora of articles discovered spanned almost every corner of the world. Balschun et al. [92] documented the prevalence case of KRAS and BRAF in German patients in Europe. Di Fiore et al. [93] reported the first instance of KRAS and BRAF mutations in CRC in the United Kingdom. Raskin et al. [94] and Osasan [95] studies were done in Africa. Altogether, these illustrated the different prevalence of KRAS mutation existence in CRC around the globe.
The dynamic of gene expression patterns on gender and age was investigated by some studies as a possible risk for developing CRC [120,121]. In this study, the age of the participants was also taken into consideration, the bulk of the recruited participants were adults, with most of them being over 50 years old, implying that KRAS gene mutation predominates in adult CRC. It was indeed as anticipated, given that older age has hitherto been identified as a health risk for CRC in numerous investigations [122]. Although data on gender were not reported for some studies in the included studies for this analysis, CRC was found to be more common in male patients (60.7%) than female patients (39.3%). This information points to the importance of gender predilection in the occurrence of CRC which is consistent with findings from other studies around the world [123,124]. On the location of the tumour, the cancer was mostly found in the colon (82.1%) which is a similar finding in several studies [39], probably because the patient would present at the latter stage of cancer [125].
Although human scientific knowledge has greatly advanced compared to decades ago, however, our study found that the prevalence of KRAS gene mutation was higher among patients screened "after 2010," 36.7% (95% CI: 34.6-38.8), than when compared to those screened "before 2010," 32.3% (95% CI: 28.8-36.0), probably due to medical advances and more medical screening [126]. During the shedding of tumour cells or apoptosis, small DNA fragments flow into the blood system, leading to the detection of this ctDNA mutation in almost all cancer types and in the late stages of the tumour or the malignancy, hence, more frequency of the DNA mutation detection on screening.
Another reason could range from lifestyle evolution to dietary choice, synergically working together to modify our body biocomposition and genetic make-up [127]. The latestage (stages 3 and 4) recorded more KRAS gene mutation (68%) than the early stage (30%) but this could be associated with discrepancies in the time of consultation and stages of  On the location of KRAS and BRAF gene mutations, the colon (61% and 68%), respectively, was the most recorded mutation site which on the contrary is the rectum [128]; however, this is as expected as the main physiological function of the intestinal lumen of the colon includes water absorption and stool storage. Therefore, the contents contained inside the colon are relatively desiccated which is the tumour-conducive condition for gene mutation detection [129,130]. In this present research, the majority of KRAS mutations occurred in codon 12, 76.6% (95% CI: 74.2-78.8), than in codon 13, 21.0% (95% CI: 19.1-23.0). These findings are comparable to those of previous research [105,116,117]. For example, in a Belgian research, 36.3 percent of people had KRAS mutations, with 91 percent of mutations in codon 12 [131]. Another study published in Dobre et al. [132] found that KRAS mutations in codons 12 and 13 were found in 79.3 percent and 19.7 percent of people, respectively. A similar study in Brazil reported that 87% of KRAS mutations were in codon 12 and 13% in codon 13. However, research in the Greek population found that KRAS mutations at codon 12 are uncommon (29.3%) [133]. Only 3 studies of the colorectal cancer patients in our analysis had a KRAS codon 61 mutations [26,67,68] which is not surprising given that the majority of KRAS mutations reported in human tumours are in codon 12, with mutations in codons 13 and 61 accounting for only 1.7-9 percent [27].
BRAF is also a member of the RAF gene subfamily that, like KRAS, performs its function in the EGFR downstream cascade, but their mutations are less frequent than the KRAS gene mutations. Among the BRAF gene, BRAFV600E mutation is the most prevailing [134], and in this present study, BRAFV600E mutation is used to examine the prevalence of BRAF gene mutation in CRC. The frequency of BRAF mutation varies globally, approximately 1.1-25% [16,49,[135][136][137][138][139]. The prevalence of BRAF mutation obtained in this study was 5.6% (95% CI: 3.9-8.0), and this is conforming 14 BioMed Research International with the several existing findings, i.e., 1.1 to 5.8% in Asian studies and 5-21% in western studies [112,131,134,[140][141][142]. Another reason for these prevalence similarities could be associated with genetic homogeneity as the studies involve certain regions, and their lifestyles and diet are almost similar [143]. BRAF-activating mutations are frequently exclusive with KRAS mutations, accounting for 5-15% of mCRC cases, and are linked to a poor prognosis in stages II, III, and IV [144]. This mutation causes a constant stimulation of the mitogen-activating protein kinase MAPK pathway, which controls the transcriptase activity of regula-tory genes in the cell cycle by modulating cell growth stimuli, a nonfunctioning condition that predisposes to cancerous growth [145]. This study possesses several merits and strengths. First, to the best of the author's knowledge, it has been the first systematic review and meta-analysis carried out on the prevalence of KRAS and BRAF mutations among Asians with CRC. Also, a well-detailed and comprehensive search strategy ensures that elaborate all-inclusive papers are included, thus leading to a very large population size of 25,525. This also ensures high confidence in the outcomes obtained since  the included studies were of high methodology quality. However, this analysis was not without some limitations, with many linked to the data from the literature of the included studies such as small sample size, incomplete reports on sex, mean age, period of study conduction, differentiation, and location of the tumour, and lastly, mutation screening was done just for the BRAFV600E. All these parameters/characteristics that would be crucial in upholding the study appraisal were not reported in some of the studies analysed in this meta-analysis, thus accounting to some of the heterogeneity seen in the studies.

Conclusions
This systematic review and meta-analysis study, which to the best of our knowledge, is the first to report on the prevalence of KRAS (36.6%) and BRAF (5.6%) mutations in CRC patients in Asia. The result showed that the rate of KRAS and BRAF gene mutations in CRC among Asians is rising. The adult age was more associated with CRC prevalence, and the males have increase fold and poorer outcome than their female counterparts. Despite some limitations, the meta-analysis yielded impressive results. The total prevalence of KRAS and BRAF mutations, 36.6% and 5.6%, respectively, differs in various countries in Asia according to this meta-analysis. Furthermore, when the findings of this study were compared to those of other studies, it was discovered that the prevalence of these mutations obtained in our analysis conformed with them.

Data Availability
All data accessed and analysed in this study are available in the article and its Supplementary Materials.

Disclosure
The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Conflicts of Interest
The authors declare no conflict of interest.