==== Front Ann Saudi MedAnn Saudi MedAnnals of Saudi Medicine0256-49470975-4466King Faisal Specialist Hospital and Research Centre 3007802310.5144/0256-4947.2018.251asm-4-251Original ArticleMolecular characteristics of colorectal cancer in a Middle Eastern population in a single institution Ibrahim Tony aeSaer-Ghorra Claude bTrak-Smayra Vivane bNadiri Samah bYazbeck Charbel cBaz Maria dKattan Joseph G. e a Department of Medical Oncology, Gustave Roussy, Villejulf, France b Department of Pathology, Faculte de Medecine, Universite Saint-Joseph, Beirut, Lebanon c Department of Gastroenterology, Faculte de Medecine et des Sciences Medicales, Universite Saint-Esprit de Kaslik, Jounieh, Lebanon d Department of Genetics, Faculte de Medecine Site Cochin, Universite Paris Descarte, Paris, France e Department of Hematology-Oncology, Faculte de Medecine, Universite Saint-Joseph, Beirut, LebanonCorrespondence: Tony Ibrahim,, Deparment of Medical Oncology, Gustave Roussy, 114 Rue Edouard Valliant, Villejulf 94800, France, T: 075 504 4905, tony.ibrahim@gustaveroussy.fr, ORCID: http://orcid.org/0000-0001-9728-8554Jul-Aug 2018 02 8 2018 38 4 251 259 25 3 2018 15 7 2018 Copyright © 2018, Annals of Saudi Medicine2018This is an open access article under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND). The details of which can be accessed at http://creativecommons.org/licenses/by-nc-nd/4.0/BACKGROUND The few studies of the molecular biology of colorectal cancer (CRC) in Middle Eastern populations have included only small samples of patients. OBJECTIVE Evaluate the frequency and prognostic effect of RAS, BRAF, PIK3CA, PTEN, and EGFR somatic mutations as well as mismatch repair (MMR) deficiency in Lebanese Middle Eastern patients. DESIGN Retrospective single-center descriptive study. SETTING Lebanese Middle Eastern patients in a tertiary medical center. METHODS We included all patients diagnosed with CRC between January 2010 and December 2015, in whom RAS mutational status and the expression of MLH1 and MSH2 proteins were available. MAIN OUTCOME MEASURES Genetic mutations detected by direct sequencing while MMR protein expression was evaluated by immunohistochemistry. SAMPLE SIZE 645 patients. RESULTS RAS, BRAF, EGFR, PI3KCA, and PTEN mutation rates were 38.5%,12.9%, 0%, 11.1% and 0% respectively. The MMR deficiency rate was 20.6%. No factor was associated with RAS mutation whereas MMR-deficient tumors were less likely to be metastatic at diagnosis. Among patients with wild-type RAS females fared better than males (median overall survival [OS]=1734 vs 1079 days respectively, P=.015) even after adjustment for confounding factors by Cox regression analysis. This finding was not reproduced in the RAS-mutated group. The median OS of patients with MMR-deficient tumors was not reached, while the median OS was 2475 days in patients who had maintained expression of both MLH1 and MSH2. CONCLUSION The RAS mutation rate was similar to Western and East Asian countries, but not for the BRAF mutation and MMR deficiency. We also found a prognostic effect for sex in the RAS wild-type group, a finding worthy of further exploration. LIMITATIONS Retrospective, single center and small sample size. Expression of MSH6 and PMS2 not analyzed. ==== Body Colorectal cancer (CRC) is a global burden; its incidence has increased significantly over the last decade.1 Progress in the molecular biology of cancer has led to better understanding of the mutational landscape of CRC and the possibility of developing targeted therapies.2,3 Adding anti-epidermal growth factor receptor (EGFR) antibodies to chemotherapy has shown a clear benefit in metastatic CRC.4–7 However, genetic mutations conferring resistance to these drugs have been identified; the mutations mainly affect RAS (KRAS and NRAS) and BRAF genes.5,6 Thus, genetic analysis of RAS and BRAF have become part of standard of care in metastatic CRC.7 Microsatellite instability (MSI) is a hypermutability status resulting from a deficient DNA mismatch repair (MMR).8 MSI is mainly due to somatic hypermethylation of the promoter of the MLH1 gene, but can arise through inherited mutations in one of the four genes implicated in DNA repair (MLH1, MSH2, PMS2, and MSH6). It has become crucial to define the MSI status of a tumor, on the one hand to detect patients with Lynch syndrome and on the other hand to adapt the treatment.6,9 Immunohistochemistry (IHC) is a valid method to detect MMR protein deficiency and serves as a surrogate for MSI with a high sensitivity and specificity.6 The frequency of RAS and BRAF mutations as well as MMR deficiency have been reported in large epidemiological studies in western and Asian countries.10–12 However, the few studies that have been reported in Middle Eastern populations included only small samples of patients.13–20 With the movement toward personalized cancer management, there has been significant interest in additional biomarkers such as PIK3CA, PTEN, and EGFR mutations. Some authors have suggested that these mutations could be associated with a lack of response to anti-EGFR antibodies.21 These molecular alterations are potential targets for new drugs in the near future.6 The aim of this descriptive study was to retrospectively report the frequency and the prognostic effect somatic mutations of RAS (KRAS, NRAS), BRAF, PIK3CA, PTEN and EGFR genes, as well as the frequency of MMR deficiency in Lebanese Middle Eastern patients in a real-life setting. PATIENTS AND METHOD This study retrospectively included all patients diagnosed with metastatic CRC in a single tertiary care Lebanese medical center, between January 2010 and December 2015, in whom RAS mutational status and the expression of MLH1 and MSH2 proteins were available. RAS mutations were investigated either at the time of diagnosis or during the course of the disease for the purpose of adjusting the treatment. The expression of MLH1 and MSH2 was analysed in patients who were suspected to have Lynch Syndrome, or in order to adapt the treatment. Finally, mutations of the BRAF, PIK3CA, EGFR, and PTEN genes were analysed in patients who had refractory metastatic CRC to look for a potentially targetable mutation. Ethical committee approval was unnecessary since there was no direct contact with patients. Gene mutation analysis was performed on paraffin-embedded tissue. DNA was extracted from selected tumor tissue area (containing at least 50% neoplastic cells) after hematoxylin-eosin (H&E) staining. Deparaffinization was performed using xylene baths followed by 100% ethanol solution then by proteinase K digestion. DNA was isolated using the QIAamp DNA Extraction kit (Qiagen, Crawley, UK) according to the manufacturer’s instructions, followed by PCR using specific primers for KRAS and NRAS codons 12, 13, 59, 61, 117 and 146, BRAF exon 15, PIK3CA exon 9 and 20, EGFR exons 19 and 20, and PTEN exon 5 to 9.22 Mutations were detected by standard Sanger sequencing using BigDye Terminator v3.1 (Applied Biosystems, Foster City, CA, USA) and interpreted via the ABI 3130 Genetic Analyser. Each detected mutation was verified by two independent cycle sequencing PCR reactions and bidirectional DNA analysis. The expression of MLH1 and MSH2 was evaluated by IHC on formalin-fixed paraffin-embedded tumor tissue and adjacent normal mucosa after deparaffinization of 5-μm thick sections. Mouse monoclonal antibodies against the MLH1 protein (clone G168–728; PharMingen, San Diego, CA) and MSH2 protein (clone FE11; Oncogene Research Products, Cambridge, MA) were used according to manufacturer’s instructions, both diluted 1:100. Detection was made possible by the UltraVision streptavidin-biotin peroxidase detection kit (TP-060-HL; Lab Vision Corporation, Fremont, CA). The diaminobenzidine tetrachloride was used as chromogen during the peroxidase reaction. Slides were finally counterstained lightly with Mayer hematoxylin. Nuclear immunostaining of normal epithelial cells and lymphocytes served as internal positive controls. Samples were evaluated simultaneously by two pathologists according to international recommendations (diffuse 1+, focal or diffuse 2+, and 3+ defining positive staining).23 All information was collected from medical records including age at diagnosis, sex, primary tumor site (right ascending, transverse, left descending colon, sigmoid/high rectal area and middle/low rectum), macroscopic tumor size (defined as maximal diameter), grade, TNM stage according the AJCC staging system seventh edition,24 number of positive lymph nodes, and number of resected lymph nodes. Statistical analysis was performed using the IBM SPSS software version 22 (IBM, Armonk, NY). Continuous variables are represented by their means (or medians) and standard deviations. Discrete variables are represented by proportions. RESULTS The study included 644 patients with a male: female ratio of 1.35:1 and a mean (SD) age at diagnosis of 62 (14) years. All but 14 patients had surgical resection of the primary tumor. The sigmoid colon/high rectum and the right ascending colon were the most frequent primary tumor sites [n=192 (29.7%) and n=163 (25.3%) respectively], followed by the middle/lower part of the rectum [n=155 (24%)], the left descending colon [n=106 (16.4%)], and the transverse colon [n=30 (4.6%)]. At diagnosis, 58 (9%), 161 (25%), 206 (32%) and 220 (34%) patients had stage I, II, III, and IV cancers. Table 1 shows the distribution of patients by biomarkers and Table 2 shows the molecular biology of the tumor cells. The RAS mutation rate was equal to 38.5%, and the frequency of loss of expression of MSH2 or MLH1 proteins was equal to 20.6%. No factor was associated with RAS mutation (Table 3). MMR-deficient tumors (loss of expression of MLH1 or MSH2) were less likely to be metastatic at diagnosis and occurred more frequently in the right ascending colon (Table 4). We could not analyse the association between RAS mutations and MMR deficiency since the expression of MLH1 and MSH2 was maintained in all patients in whom RAS mutations and MMR status were analysed simultaneously. Due to the small number of patients harboring BRAF, PIK3CA, PTEN or EGFR mutations, we could not analyze the association between these biomarkers and tumor stage at diagnosis. Survival After a median follow up of 1770 days, median overall survival (OS) of the whole sample was 1556 days, 883 days in the RAS-mutated group versus 1149 days in the wild-type group (log Rank P=.90). Among the wild-type RAS group, females fared better than males (median OS: 1734 days vs 1079 days, respectively, log rank P=.015) even after adjustment for confounding factors (Table 5). This finding was not reproduced in the RAS-mutated group (median OS: 874 days vs 883 days in females and males respectively, log rank P=.42) (Figure 1). The median OS of patients with MMR-deficient tumors was not reached, while the median OS was 2475 days in patients who had maintained expression of both MLH1 and MSH2. The median OS of the 4 patients harboring a BRAF mutation was equal to 1100 vs 900 days in the wild type group (log rank P=.33). The median OS of the 3 patients harboring the PIK3CA mutation was equal to 800 vs 890 days in the wild type group (log rank P=.64). Since no patient had a PTEN or EGFR mutation, we could not compare the median OS between a wild-type and a mutated group for these two genes. DISCUSSION In this study, the RAS mutation rate was equal to 38.5%, which is in the range reported by large studies conducted in the USA,10 Europe11,25,26 and East Asia,27,28 as well as in international surveys.29 Besides, the frequency of the BRAF mutation and loss of MLH1 or MSH2 protein expression were also in the range reported in Western populations.6,8,18,19 Conversely, BRAF mutations and MMR deficiency seem to be much less frequent in East Asia.30–32 The fact that the frequency of mutated-RAS was similar between different populations whereas the rates of mutated-BRAF and MMR deficiency differed is intriguing. Colorectal carcinogenesis is influenced by both genetic and environmental factors.25,33,34 It is induced by multiple pathways, among others the serrated and the epigenetic pathways. The former is the MAPK-ERK pathway in which RAS mutations are acquired during early steps, promoting tumor invasion and metastasis.35–37 On the other hand, tumors harboring BRAF mutation or MMR deficiency are triggered by epigenetic alterations mainly CpG islands hypermethylation. This phenomenon could explain why MMR-deficient tumors are less likely to harbor RAS mutations.38–40 Furthermore, Shen et al identified three distinct CRC subgroups based on genetic and epigenetic profiling, which were designated as CpG island methylator phenotype (CIMP) 1, CIMP2, and CIMP negative.40 CIMP1 is characterized by a high frequency of MMR deficiency and BRAF mutation, but low KRAS and P53 mutation rates. CIMP2 frequently harbors RAS mutations, but rarely BRAF, p53 mutations and MMR deficiency.40 Therefore, CIMP2 might occur at the same rate between different populations whereas CIMP1 is mostly affected by genetic and environmental variations. This is only a hypothesis since epidemiological studies relating geographical variants, ethnicity and lifestyle to RAS, BRAF mutations and MMR deficiency reported inconsistent results.25,41,42 Besides, large studies in the Western world have suggested a slightly higher prevalence of RAS mutation in females as well as in patients with right-side CRC,10,25,29 which is not the case in our study probably because of sample size. Concerning the MMR-deficient CRC, we confirmed what have been published in the literature concerning its low aggressiveness and the right-side predominance.32 Some reports associated female gender with MMR deficiency,43 which is not the case in our study. The prognostic value of RAS mutations in non-metastatic CRC is not well established. This study included patients with localized and metastatic CRC at diagnosis and showed a non-statistically significant trend toward poorer survival among patients with mutated RAS. In a study by Phipps et al patients with invasive CRC harboring KRAS mutations had poorer disease-specific survival.44 Yet, a systematic review and meta-analysis that included stage II and III tumors did not show a significant difference in survival.45 One potential explanation could be derived from the expanded analysis of the RASCAL collaborative study, which reported that G12V was the only codon 12 mutation associated with worse prognosis in patients with node-positive disease.46 The most interesting finding in our study was the higher survival rate of females compared to males in the RAS wild-type group, even after adjustment for age, stage and primary tumor site at diagnosis. To our knowledge, no study had focused on the prognostic role of gender in patients with wild-type RAS. Controversial results relating gender to CRC survival have been reported.47 In a German population-based study which included 164 966 cases, young females with localized CRC survived better than males from the same age group.48 In another study, females aged less than 50 years fared better than males from the same age group, whereas the opposite was true in older patients.49 Finally, Kooh et al reported a higher rate of sporadic right side MSI-H cancer among women in a systematic review, but there was no difference in post-adjuvant chemotherapy survival or postradiotherapy.43 We found no study reporting a potential difference in survival between women and men who were treated with anti-EGFR therapies for mCRC.4 Hormonal status might affect tumor growth and responsiveness to chemotherapy and should be further elucidated. Thus, the finding that females fared better than males in the RAS wild-type group deserves to be investigated even though results should be interpreted with caution. Expression of MSH6 and PMS2 were not analysed in this study, which could be considered a limitation. However, the contribution of MSH6 and PMS2 to MSI is much less important than MLH1 and MSH2. In addition, loss of expression MSH6 and PMS2 are related to loss of MSH2 and MLH1 respectively, and would not affect the rate of MMR deficiency.50 The sample size is another limitation of this study compared to the large studies carried out in developed countries. However, taking into account the size of the Lebanese population and the incidence of CRC of nearly 400 patients per year,51 the size of the study sample is relatively satisfactory. Furthermore, this study is among the largest ones that have been performed in the Middle East and Arab world.13–16,18–20 Finally, this study is mostly limited by being retrospective. The results should be interpreted with caution especially since the biomarkers were not analysed completely in all the patients, which introduced the possibility of selection bias. However, the rationale behind this study was the lack of data in the literature in Middle Eastern populations, especially in Lebanon. Therefore, its goal was to describe the frequency of molecular alterations in Lebanese CRC patients who have benefited from an analysis of these biomarkers in a real-life setting. To our knowledge, this is the first report describing the frequency and the prognostic effect of RAS and MMR deficiency in a Lebanese Middle Eastern CRC population. In conclusion, the present study evaluated the incidence of RAS and BRAF mutations as well as MMR deficiency by IHC and found similar rates compared to Western countries. No factor was associated with mutated-RAS status, whereas MMR deficient tumors were mainly localized in the right ascending colon and were less aggressive. We also found an independent relationship between sex and survival in the RAS wild-type group. Finally, the prognostic effect of BRAF, PI3K, PTEN and EGFR mutations could not be elucidated in this study and requires a larger sample. Funding: None. CONFLICT OF INTEREST: None. Figure 1 Survival curves from diagnosis until death comparing female with males in RAS wild (Panel A) and mutated groups (Panel B). Table 1 Number of patients by biomarkers (n=644). Patients by biomarkers N (%) RAS mutation analysis 222 (34.4) RAS mutation analysis and MSH2 and MLH1 expression analysis 52 (8.1) MSH2 and MLH1 expression analysis 370 (57.4) Table 2 Molecular biology of tumor cells. Gene Type Number of patients Percentage KRAS Wild 172 62.8 Mutated 102 37.2a NRASb Wild 70 94.6 Mutated 4 5.4 BRAF Wild 27 87.1 Mutated 4 12.9 PIK3CA Wild 24 88.9 Mutated 3 11.1 EGFR Wild 22 100 Mutated 0 0 PTEN Wild 22 100 Mutated 0 0 MLH1 Conserved 348 82.5 Lost 74 17.5 MSH2 Conserved 409 96.9 Lost 13 3.1 a Exon 2 codon 12 mutation in 70 cases (68.6%), exon 2 codon 13 mutation in 25 cases (24.5%), exon 4 codon 146 mutation in 4 cases (3.9%) and exon 3 codon 61 mutation in 3 cases (2.9%). b In patients with KRAS wild type mutation. Table 3 Association between RAS status (KRAS and NRAS) and patient tumor characteristics. RAS status P Wild, n (%) Mutated, n (%) Male sex 98 (58.3) 64 (61.0) .67a Age at diagnosis: mean (standard deviation) 59 (17) 62 (13) .08b Size of the tumor at diagnosis (in cm) 4.5 (2.4) 5 (1.9) .23b Tumour localization Right ascending colon 39 (24.4) 29 (28.2) .67a Transverse colon 6 (3.8) 4 (3.9) Left descending colon 29 (18.1) 13 (12.6) Sigmoid colon/high rectum 59 (36.9) 35 (34) Middle/lower rectum 27 (16.9) 22 (21.4) Tumor stage at diagnosis T2 7 (6.2) 2 (3.1) .61a T3 55 (48.7) 30 (46.9) T4 51 (45.1) 32 (50) Node stage at diagnosis N0 25 (21.9) 16 (27.1) .56a N1 39 (34.2) 22 (37.3) N2 50 (43.9) 21 (35.6) Number of positive lymph nodes 5 (7) 4 (5) .28b Lymph node ratio 0.3 (0.3) 0.2 (0.2) .09b Stage IV at diagnosis No 82 (50) 42 (40.8) .14a Yes 82 (50) 61 (59.2) a Chi-square test of independence. b Independent samples t test. Table 4 Association between loss of MLH1 or MSH2 status and patient tumor characteristics. MLH1 and MSH2 conserved, n (%) Loss of expression of MLH1 or MSH2, n (%) P value Male sex 193 (57.4) 46 (52.9) .44b Age at diagnosis: mean (standard deviation) 63 (14) 60 (15) .07a Size of the tumor at diagnosis (in cm) 4.4 (1.9) 4.8 (1.9) .09a Tumor localization Right ascending colon 77 (23.3) 29 (34.1) .04b Transverse colon 14 (4.2) 6 (6.9) Left descending colon 50 (15.2) 16 (18.4) Sigmoid colon/high rectum 95 (28.8) 13 (14.2) Middle/lower rectum 94 (28.5) 23 (26.4) Tumor stage at diagnosis T1 20 (6.7) 3 (3.6) .32b T2 43 (14.3) 12 (14.3) T3 161 (53.7) 40 (47.6) T4 76 (25.3) 29 (34.5) Node stage at diagnosis N0 171 (55.9) 41 (48.2) .37b N1 84 (27.5) 25 (29.4) N2 51 (16.7) 19 (22.4) Number of positive lymph nodes 2 (4) 3 (5) .08b Lymph node ratio 0.2 (0.3) Not available Not applicable Stage IV at diagnosis No 232 (78.6) 74 (90.2) .02b Yes 63 (21.4) 8 (9.8) a Independent samples t test; b Chi-square test. Table 5 Cox regression model for overall survival among the wild-type RAS group. B P Exp (B) 95.0% CI for Exp (B) Lower Upper Age at diagnosis 0.01 .60 1.01 0.98 1.03 Sex 0.75 .04 2.12 1.03 4.36 AJCC staging at diagnosis T 0.15 .65 1.17 0.60 2.28 N 0.42 .11 1.52 0.91 2.53 M 0.68 .09 1.96 0.90 4.29 Primary tumor localization (right versus left) −0.37 .37 0.69 0.31 1.55 ==== Refs REFERENCES 1 Arnold M Sierra MS Laversanne M Soerjomataram I Jemal A Bray F Global patterns and trends in colorectal cancer incidence and mortality Gut 2017 4 66 4 683 91 26818619 2 Ibrahim T Yazbeck C Maalouly G Baz M Haddad F Sabbagh C TGFBR1*6A polymorphism in sporadic and familial colorectal Carcinoma: a case-control study and systematic literature review J Gastrointest Cancer 2014 12 45 4 441 7 24880985 3 Formica V Roselli M Targeted therapy in first line treatment of RAS wild type colorectal cancer World J Gastroenterol 2015 3 14 21 10 2871 4 25780283 4 Lucas AFH Molecular genetics of colorectal cancer UpToDate [Internet] UpToDate Waltham, MA 2018 Available from: https://www.uptodate.com/contents/molecular-genetics-of-colorectal-cancer?source=search_result&search=molecular/biology/colorectal/cancer/selectedTitle=1~150 5 Allegra CJ Rumble RB Hamilton SR Mangu PB Roach N Hantel A Extended RAS Gene Mutation Testing in Metastatic Colorectal Carcinoma to Predict Response to Anti-Epidermal Growth Factor Receptor Monoclonal Antibody Therapy: American Society of Clinical Oncology Provisional Clinical Opinion Update 2015 J Clin Oncol 2016 1 10 34 2 179 85 26438111 6 Furtado LV Samowitz WS Colorectal cancer molecular profiling: from IHC to NGS in search of optimal algorithm Virchows Arch 2017 8 471 2 235 42 28551725 7 Benson AB Venook AP Cederquist L Chan E Chen Y-J Cooper HS Colon Cancer, Version 1.2017, NCCN Clinical Practice Guidelines in Oncology J Natl Compr Cancer Netw 2017 15 3 370 98 8 Roseweir AK McMillan DC Horgan PG Edwards J Colorectal cancer subtypes: Translation to routine clinical pathology Cancer Treat Rev 2017 6 57 1 7 28505475 9 Le DT Uram JN Wang H Bartlett BR Kemberling H Eyring AD PD-1 Blockade in tumors with Mismatch-Repair Deficiency N Engl J Med 2015 6 25 372 26 2509 20 26028255 10 Basu GD Xiu J Arguello D Feldman RA Millis SZ Bender RP Prevalence of KRAS, BRAF, NRAS, PIK3CA, and PTEN alterations in colorectal cancer: Analysis of a large international cohort of 5,900 patients J Clin Oncol 2014 1 20 32 3_suppl 399 399 11 Piton N Lonchamp E Nowak F Sabourin J-C KRAS group Real-Life Distribution of KRAS and NRAS Mutations in Metastatic Colorectal Carcinoma from French Routine Genotyping Cancer Epidemiol Biomark Prev 2015 9 24 9 1416 8 12 Ye J-X Liu Y Qin Y Zhong H-H Yi W-N Shi X-Y KRAS and BRAF gene mutations and DNA mismatch repair status in Chinese colorectal carcinoma patients World J Gastroenterol 2015 2 7 21 5 1595 605 25663779 13 Mehdi I Abdulmonem E Al Bahrani BJ KRAS mutations: Does ethnicity play a role? J Clin Oncol 2014 5 20 32 15_suppl e14628 e14628 14 Zahrani A Kandil M Badar T Abdelsalam M Al-Faiar A Ismail A Clinico-pathological study of K-ras mutations in colorectal tumors in Saudi Arabia tumori 2014 2 100 1 75 9 24675495 15 Al-Allawi NA Ismaeel AT Ahmed NY Merza NS The frequency and spectrum of K-ras mutations among Iraqi patients with sporadic colorectal carcinoma Indian J Cancer 2012 3 49 1 163 8 22842183 16 Alqahtani M Grieu F Carrello A Amanuel B Mashour M Alattas R Screening for Lynch Syndrome in Young Colorectal Cancer Patients from Saudi Arabia Using Microsatellite Instability as the Initial Test Asian Pac J Cancer Prev APJCP 2016 17 4 1917 23 27221876 17 Siraj AK Bu R Prabhakaran S Bavi P Beg S Al Hazmi M A very low incidence of BRAF mutations in Middle Eastern colorectal carcinoma Mol Cancer 2014 7 8 13 168 25005754 18 Ashktorab H Brim H Al-Riyami M Date A Al-Mawaly K Kashoub M Sporadic colon cancer: mismatch repair immunohistochemistry and microsatellite instability in Omani subjects Dig Dis Sci 2008 10 53 10 2723 31 18299982 19 Siraj AK Prabhakaran S Bavi P Bu R Beg S Hazmi MA Prevalence of Lynch syndrome in a Middle Eastern population with colorectal cancer Cancer 2015 6 1 121 11 1762 71 25712738 20 Elbjeirami WM Sughayer MA KRAS mutations and subtyping in colorectal cancer in Jordanian patients Oncol Lett 2012 10 4 4 705 10 23205087 21 De Roock W De Vriendt V Normanno N Ciardiello F Tejpar S KRAS, BRAF, PIK3CA, and PTEN mutations: implications for targeted therapies in metastatic colorectal cancer Lancet Oncol 2011 6 12 6 594 603 21163703 22 Perrone F Lampis A Orsenigo M Di Bartolomeo M Gevorgyan A Losa M PI3KCA/PTEN deregulation contributes to impaired responses to cetuximab in metastatic colorectal cancer patients Ann Oncol 2009 1 20 1 84 90 18669866 23 Shia J Immunohistochemistry versus Microsatellite Instability Testing For Screening Colorectal Cancer Patients at Risk For Hereditary Nonpolyposis Colorectal Cancer Syndrome: Part I. The Utility of Immunohistochemistry J Mol Diagn 2008 7 1 10 4 293 300 18556767 24 Hari DM Leung AM Lee J-H Sim M-S Vuong B Chiu CG AJCC Cancer Staging Manual 7th edition criteria for colon cancer: do the complex modifications improve prognostic assessment? J Am Coll Surg 2013 8 217 2 181 90 23768788 25 Peeters M Kafatos G Taylor A Gastanaga VM Oliner KS Hechmati G Prevalence of RAS mutations and individual variation patterns among patients with metastatic colorectal cancer: A pooled analysis of randomised controlled trials Eur J Cancer 2015 9 51 13 1704 13 26049686 26 Neumann J Zeindl-Eberhart E Kirchner T Jung A Frequency and type of KRAS mutations in routine diagnostic analysis of metastatic colorectal cancer Pathol Res Pract 2009 205 12 858 62 19679400 27 Zhang J Zheng J Yang Y Lu J Gao J Lu T Molecular spectrum of KRAS, NRAS, BRAF and PIK3CA mutations in Chinese colorectal cancer patients: analysis of 1,110 cases Sci Rep 2015 12 22 5 18678 26691448 28 Kawazoe A Shitara K Fukuoka S Kuboki Y Bando H Okamoto W A retrospective observational study of clinicopathological features of KRAS, NRAS, BRAF and PIK3CA mutations in Japanese patients with metastatic colorectal cancer BMC Cancer 2015 4 11 15 258 25886136 29 Basu GD Gatalica Z Millis SZ Braiteh FS Molecular abnormalities of 17 types of gastrointestinal cancer in an international cohort of 11,324 patients J Clin Oncol 2014 5 20 32 15_suppl 11053 11053 30 Jones JC Renfro LA Al-Shamsi HO Schrock AB Rankin A Zhang BY Non-V600 BRAF Mutations Define a Clinically Distinct Molecular Subtype of Metastatic Colorectal Cancer J Clin Oncol 2017 8 10 35 23 2624 30 28486044 31 Zhang R Qin W Xu G-L Zeng F-F Li C-X A meta-analysis of the prevalence of somatic mutations in the hMLH1 and hMSH2 genes in colorectal cancer Colorectal Dis 2012 3 14 3 e80 89 21988782 32 Kim JH Kang GH Molecular and prognostic heterogeneity of microsatellite-unstable colorectal cancer World J Gastroenterol 2014 4 21 20 15 4230 43 24764661 33 Slattery ML Curtin K Anderson K Ma KN Edwards S Leppert M Associations between dietary intake and Ki-ras mutations in colon tumors: a population-based study Cancer Res 2000 12 15 60 24 6935 41 11156393 34 La Vecchia C Mediterranean diet and cancer Public Health Nutr 2004 10 7 7 965 8 15482626 35 Giehl K Oncogenic Ras in tumor progression and metastasis Biol Chem 2005 3 386 3 193 205 15843165 36 Miranda E Destro A Malesci A Balladore E Bianchi P Baryshnikova E Genetic and epigenetic changes in primary metastatic and nonmetastatic colorectal cancer Br J Cancer 2006 10 23 95 8 1101 7 16969349 37 Moon B-S Jeong W-J Park J Kim TI Min DS Choi KY Role of oncogenic K-Ras in cancer stem cell activation by aberrant Wnt/β-catenin signaling J Natl Cancer Inst 2014 2 106 2 djt373 24491301 38 Chan AO-O Broaddus RR Houlihan PS Issa J-PJ Hamilton SR Rashid A CpG island methylation in aberrant crypt foci of the colorectum Am J Pathol 2002 5 160 5 1823 30 12000733 39 Weisenberger DJ Siegmund KD Campan M Young J Long TI Faasse MA CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer Nat Genet 2006 7 38 7 787 93 16804544 40 Shen L Toyota M Kondo Y Lin E Zhang L Guo Y Integrated genetic and epigenetic analysis identifies three different subclasses of colon cancer Proc Natl Acad Sci U S A 2007 11 20 104 47 18654 9 18003927 41 Gay LJ Arends MJ Mitrou PN Bowman R Ibrahim AE Happerfield L MLH1 promoter methylation, diet, and lifestyle factors in mismatch repair deficient colorectal cancer patients from EPIC-Norfolk Nutr Cancer 2011 63 7 1000 10 21875327 42 Razzak AA Oxentenko AS Vierkant RA Tillmans LS Wang AH Weisenberger DJ Associations between intake of folate and related micronutrients with molecularly defined colorectal cancer risks in the Iowa Women’s Health Study Nutr Cancer 2012 64 7 899 910 23061900 43 Koo JH Leong RWL Sex differences in epidemiological, clinical and pathological characteristics of colorectal cancer J Gastroenterol Hepatol 2010 1 25 1 33 42 19874446 44 Phipps AI Buchanan DD Makar KW Win AK Baron JA Lindor NM KRAS-mutation status in relation to colorectal cancer survival: the joint impact of correlated tumor markers Br J Cancer 2013 4 30 108 8 1757 64 23511557 45 Rui Y-Y Zhang D Zhou Z-G Wang C Yang L Yu Y-Y Can K-ras gene mutation be utilized as prognostic biomarker for colorectal cancer patients receiving chemotherapy? A meta-analysis and systematic review PloS One 2013 8 10 e77901 24205021 46 Russo A Bazan V Agnese V Rodolico V Gebbia N Prognostic and predictive factors in colorectal cancer: Kirsten Ras in CRC (RASCAL) and TP53CRC collaborative studies Ann Oncol 2005 5 16 Suppl 4 iv44 49 15923428 47 Grundmann RT Meyer F Gender-specific influences on incidence, screening, treatment, and outcome of colorectal cancer Zentralbl Chir 2013 8 138 4 434 41 23950079 48 Majek O Gondos A Jansen L Emrich K Holleczek B Katalinic A Sex differences in colorectal cancer survival: population-based analysis of 164,996 colorectal cancer patients in Germany PloS One 2013 8 7 e68077 23861851 49 Koo JH Jalaludin B Wong SKC Kneebone A Connor SJ Leong RWL Improved survival in young women with colorectal cancer Am J Gastroenterol 2008 6 103 6 1488 95 18510616 50 Young J Simms LA Biden KG Wynter C Whitehall V Karamatic R Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis Am J Pathol 2001 12 159 6 2107 16 11733361 51 Adib SM Tabbal N Hamadeh R Ammar W Geographic epidemiology in a small area: cancer incidence in Baakline, Lebanon, 2000–2008 East Mediterr Health J Rev Sante Mediterr Orient Al-Majallah Al-Sihhiyah Li-Sharq Al-Mutawassit 2013 4 19 4 320 6