158 J Contemp Med Sci | Vol. 9, No. 3, May-June 2023: 158–162 Original Molecular Detection of Mononucleotide Biomarkers of Microsatellite Instability in Sporadic Colorectal Carcinoma Patients with Clinicopathological Correlation Wed Thamer Salman Al-Jumaili1*, Bassam Musa Sadik Al-Musawi2 1Al-Amal National Hospital for Cancer Management, Baghdad, Iraq. 2Department of Pathology and Forensic Medicine, College of Medicine, University of Baghdad, Baghdad, Iraq. *Correspondence to: Wed Thamer Salman Al-Jumaili *(E-mail: widthamir1984@gmail.com) (Submitted: 10 April 2023 – Revised version received: 09 May 2023 – Accepted: 11 June 2023 – Published online: 26 June 2023) Abstract Objectives: To identify the frequency and types of microsatellite instability among a group of sporadic CRC patients and to correlate the findings with clinicopathological characteristics. Methods: During an 8-month period, all patients with sporadic CRC who attended to two teaching hospitals in Baghdad, Iraq were recruited to this cross-sectional study regardless of age, sex, ethnicity, or tumor characteristics. Demographic, clinical, and histopathological features were recorded. DNA was extracted from FFPE-blocks of the resected tumors and normal tissues. PCR amplification of five microsatellite mononucleotide repeat loci (BAT25, BAT26, NR-21, NR-24, and MONO-27) and 2 pentanucleotide repeat control markers (Penta C and Penta D) was performed to determine the MSI status. Capillary electrophoresis and Genetic Analyzer 3500 (Applied Biosystem, Japan) were used to separate and examine the products. Data were analyzed by Genescan software (Promega, USA). Instability of two or more loci is considered MSI-H. Result: In this study, ages of the 45 recruited patients ranged between 20–80 years, with a mean ± SD of 55 ± 12.3 years; of them, 31(68.9%) were ≥50 years; 25 (55.6%) were males. Rectal bleeding was the most frequent presenting feature [22 (48.9%)] patients; 23 (51.1%) of CRCs were located at recto-sigmoid region, 29 (64.4%) were T3 tumors, 34(75.5%) were non-mucinous adenocarcinoma, 39(86.7%) were moderately differentiated, 17 (37.8%) patients had stage III tumors; and 25 (55.5%) had lymphovascular invasion. MSI-H was seen in 5/45 (11.1%) patients; 3(60%) of them were ≥50 years, 4(80%) were males, 3(60%) were smokers, 2 (40%) presented with intestinal obstruction and altered bowel habits each; 4(80%) had T3 tumors, 3(60%) had mucinous adenocarcinomas [P = 0.004], 2(40%) had stage II tumor and stage III each. Conclusion: The frequency of MSI-H among the recruited patients with CRC was 5/45 (11.1%) and it was significantly associated with mucinous adenocarcinoma subtype. NR-24 and NR-21 were the most prevalent instable markers. Keywords: Colorectal neoplasms, adenocarcinoma, microsatellite instability, polymerase chain reaction, iraq ISSN 2413-0516 Introduction Colorectal cancer (CRC) is the second most prevalent cancer in women following breast cancer, and the third most frequent cancer in men following lung and prostate cancers.1–3 Around the world, both incidence and mortality rates of CRC had sig- nificantly risen.4 Rectal and colon cancer deaths are predicted by 2035 to reach 60% and 71.5%, respectively.5 CRC is a heter- ogeneous disease caused by the interaction of environmental and genetic factors that turn healthy colonic and rectal cells into invasive cancer.6 Several factors may increase the risk of developing CRC including age, environmental factors such as unhealthy diet, alcohol, obesity, smoking, digestive disorders (Crohn’s disease and ulcerative colitis), and genetic factors such as familiar adenomatous polyposis (FAP), and hereditary non-polyposis colorectal cancer (HNPCC).7–9 Chromosome instability, microsatellite instability (MSI), and CpG island methylator phenotype (CIMP) are the three main types of genomic instability seen in CRC.10 MSI, seen in 15% of CRC cases, can develop due to genetic or epigenetic changes. Genetic modification includes germline mutations in the MMR genes (MLH1, MSH 2, MSH6, and PMS2), which typi- cally manifests in inherited CRCs e.g. HNPCC – Lynch syn- drome. The second process involves an epigenetic alteration that results in hypermethylation of the MLH1 gene’s promoter region, which causes an accumulation of DNA mutations and the production of the mutant MMR protein. Three primary forms of colorectal cancer can be distinguished by MSI testing: MSI-high (MSI-H), MSI-low (SI-L) and MSI-stable (MSS).11 MSI-H has particular clinical and prognostic significance for CRC.12 Compared to other types of CRCs, MSI-H tumors exhibit less metastasis and respond better to immunotherapy than chemotherapy, particularly 5FU.13 The aims of this study were to identify the frequency and types and of MSI among a group of sporadic CRC patients and to correlate MSI status with demographic and clinicopathological characteristics. Methods This is a cross-sectional study that recruited 45 patients with sporadic colorectal adenocarcinoma from two major teaching hospitals in Baghdad, Iraq, namely: Baghdad Teaching Hos- pital and Gastroenterology & Hepatology Teaching Hospital, Medical City between September 2021 and April 2022. All patients, regardless of age, sex, race, residence, tumor charac- teristics, who underwent surgical resection of their tumors, were enrolled to this study. The diagnosis and histopathologic findings were reviewed and confirmed by a second histopa- thologist. For each patient, the basic demographic, clinical and histopathologic findings were recorded from hospital records. Two slices were cut from the formalin-fixed, paraffin-embedded (FFPE) tissue blocks: one from the tumor with the highest concentration of tumor cells and the other from nearby 159J Contemp Med Sci | Vol. 9, No. 3, May-June 2023: 158–162 W. T. S. Al-Jumaili et al. Original Detection of MSI Biomarkers in Sporadic Colorectal Cancer non-tumorous tissue (safe margin). DNA was extracted using the QIAamp DNA FFPE Tissue Kit® (50) by (QIAGEN / German), according to manufacturer’s instructions. Five single nucleotide repeat loci (BAT25, BAT26, NR-21, NR-24, and MONO-27) and two pentanucleotide repeat control markers (Penta C and Penta D) were tested. The mononucleo- tide markers were used for MSI determination while the pent- anucleotide markers were used for specimen identity i.e. the normal and the tumor specimens originate from the same individual. The MSI Analysis System, Version 1.2 kit (Promega, USA), which is a fluorescent PCR-based assay, was used for detection of microsatellite instability. PCR amplification was carried out in the following steps: initial denaturation at 95°C for 11 min; 10 cycles at 94°C for 30 sec., 58°C for 30 sec., and 70°C for one min., followed by 20 cycles at 90°C for 30 sec., 58 °C for 30 sec., and 70°C for one min., and finally, an exten- sion step at 60°C for 30 minutes. The PCR products were sep- arated by capillary electrophoresis (CE) using Genetic Analyzer 3500 (Applied Biosystem, Japan). The data were exported and analyzed with Genescan software (Promega, USA) to determine MSI status. Operational Definitions MSI-High (MSI-H) is used when >2 of the 5 markers exhibit instability, MSI-Low (MSI-L) is used when only one of the five markers exhibits instability, and MS-Stable (MSS) is used when none of the markers exhibit instability.11 For the purpose of this study, both MSI-L and MSS were considered in one category, referred to as MSS, in accordance to ESMO guidelines as no clinical differences were observed between MSS and MSI-L tumors.14 Statistical Analysis The computer software IBM-SPSS ver. 26 was used for statis- tical analysis. Qualitative data were described by frequency and percentage. Fisher Exact Test was used to determine if there are non-random associations between two categorical variables of small numbers. A P-value <0.05 was considered statistically significant. Results Patients’ Characteristics Ages of the 45 recruited patients ranged between 20–80 years, with a mean ± SD of 55 ± 12.3 years. Patients were divided into two age groups: <50 years [14 (31.1%)], and ≥50 years old [31 (68.9%)]. CRC was more frequently observed in males 25 (55.6%) than in females 20 (44.4%), with an M:F ratio of 1.25:1. The most frequent presenting feature was rectal bleeding as seen in 22 (48.9%) patients. Twenty (44.4%) patients were smokers, while only 2 (4.4%) drink alcohol. Twenty-three (51.1%) of CRCs were located at recto-sigmoid region, 29 (64.4%) were T3 tumors, 34 (75.5%) were non- mucinous adenocarcinoma, 39 (86.7%) were moderately dif- ferentiated, 17 (37.8%) patients had stage III while 14 (31.1%) had stage IV tumors; and 25 (55.5%) had lymphovascular invasion; Table 1. Table 1. Clinicopathological characteristics and MSI status of the 45 recruited CRC patients Parameter To ta l N o. M SS No . ( % ) M SI -H No . ( % ) P- v al ue Age <50 14 12(30 %) 2(40.0%) 0.639 > = 50 31 28(70 %) 3(60%) Sex Male 25 21(52.5%) 4(80%) 0.362 Female 20 19(47.5%) 1(20%) Smoking Non-smoker 25 23(57.5%) 2(40%) 0.642 Smoker 20 17(42.5%) 3(60%) Alcohol consumption No 43 38(95%) 5(100%) 1.0 Yes 2 2(5%) 0(0%) Presentation Rectal bleeding 22 22(55%) 0(0%) 0.15 intestinal obstruction 8 6(15%) 2(40%) Abdominal pain 6 6(15%) 0(0%) Altered bow- el habits 5 3(7.5%) 2(40%) Anemia 2 2(5%) 0(0%) Constipation 2 1(2.5%) 1(20%) Tumor location Right-sided 11 9(22.5%) 2(40%) 0.255 Lt-sided colon 11 9(22.5%) 2(40%) Recto-sig- moid 23 22(55%) 1(20%) Histological subtype of adenocarci- noma Non-muci- nous 34 33(82.5%) 1(20%) 0.004* Mucinous 10 7(17.5%) 3(60%) Signet ring 1 0(0%) 1(20%) L.V. invasion Present 25 23(57.5%) 2(40%) 0.642 Absent 20 17(42.5%) 3(60%) Tumor grade Well 2 1(2.5%) 1(20%) 0.125Moderately 39 36(90%) 3(60%) Poorly 4 3(7.5%) 1(20%) TNM-T stage T1 1 1(2.5%) 0(0%) 0.664 T2 6 5(12.5%) 1(20%) T3 29 25(62.5%) 4(80%) T4 9 9(22.5%) 0(0%) TNM-N stage N0 15 13(32.5%) 2(40%) 0.751 N1 16 15(37.5%) 1(20%) N2 13 11(27.5%) 2(40%) N3 1 1(2.5%) 0(0%) TNM-M stage M0 30 26(65%) 4(80%) 0.651 M1 15 14(35%) 1(20%) L.V. = Lymphovascular; TNM=Tumor, Node, Metastasis Staging System * = Statistically significant (Fischer exact test) 160 J Contemp Med Sci | Vol. 9, No. 3, May-June 2023: 158–162 Detection of MSI Biomarkers in Sporadic Colorectal Cancer Original W. T. S. Al-Jumaili et al. MSI Status Results In this cross-sectional study, 5/45 (11.1%) patients had MSI-H. Three (60%) of them were ≥50 years, 4(80%) were males, 3(60%) were smokers, 2 (40%) presented with intestinal obstruction and 2(40%) with altered bowel habits. Regarding the tumor characteristics, 4/5 (80%) had T3 tumors, 3(60%) had mucinous adenocarcinomas [P = 0.004], and 3(60%) with moderately-differentiated adenocarcinoma. Two (40%) had stage II tumor and 2(40%) with stage III, 2(40%) with lympho- vascular invasion; Table 2. Regarding the gene frequency of MSI panel, NR-24 showed the highest percentage as detected in 4 (80%) patients, followed by NR-21 that was detected in 3/5 (60%)], while all 3 remaining markers were detected in 2 (40%) patients each. Two (40%) patients showed the combination of BAT26, NR-21, and NR-24 as the instable markers; Table 3. Discussion Various chromosomal and epigenetic alterations are seen in colorectal cancers. Repeated sequences known as microsatel- lites are highly susceptible to misalignment during replication, which raises their mutation rate to 1.2 × 10-3 15 and causes MSI. Recent studies indicate that individuals with CRC who display MSI have a favourable prognosis, but is usually absent in 80–85% of CRCs.16 MSI-H CRC is associated with immune checkpoint upregulation such as programmed cell death receptor (PD-1) and programmed death ligand (PDL-1), the expression of which is usually used as an indication to use immunotherapeutic PD-1 blocking agents e.g. pembroli- zumab (Keytruda) and dostarlimab (Jemperli), the former is the first-line therapeutic agent in advanced metastatic or inop- erable CRCs.17 MSI testing has remained an excellent therapeutic and prognostic tool for CRCs.18 Three methods can be used to detect the deficient mismatch repair (dMMR) or MSI: immu- nohistochemistry (IHC), polymerase chain reaction (PCR) followed by capillary electrophoresis (CE), or next generation sequencing (NGS). There is a sufficiently compelling evidence to warrant the use of PCR as the most accurate and efficient method of MSI detection and is considered the gold standard test.19 MSI testing is performed by comparing the length of the amplified product of the target microsatellite in cancer tissues with those of matched normal tissues via capillary electrophoresis.19 Only a few regional studies have reported MSI status among CRC patients, like UAE,20 Egypt,21 North part of Iran,22 Turkey,23 and India.24 Assessment of MSI in different solid tumors, including CRCs, is still under research and remains out of the daily routine practice in many countries. In this study, the mean age of the 45 CRC patients was 55 ± 12.3 years. These findings were similar to many locore- gional studies from Baghdad-Iraq,4 Turkey.25 and Saudi Arabia.26 The study also showed male predominance [25 (55.6%)]. These results are comparable to results of other studies from Al-Najaf city in Iraq27 and Saudi Arabia.28 The majority of CRCs [23 (51.1%)] were located in the rectosig- moid region, which is similar to a regional study from Saudi Arabia.26 On histological examination, 34 (75.5%) CRCs were non-mucinous adenocarcinoma, and 39 (86.7%) were Table 2. Clinicopathological features of the five MSI-H CRC patients Parameter No. of MSI-H (%) (n = 5) Age <50 2(40.0%) > = 50 3(60%) Sex Male 4(80%) Female 1(20%) Smoking Non-smoker 2(40%) Smoker 3(60%) Alcohol consumption No 5(100%) Yes 0(0%) Presentation intestinal obstruction 2(40%) Altered bowel habits 2(40%) Constipation 1(20%) Tumor location Right-sided 2(40%) Lt-sided colon 2(40%) Recto-sigmoid 1(20%) Histological subtype of adenocarcinoma Non-mucinous 1(20%) Mucinous 3(60%) Signet ring 1(20%) L.V. invasion Present 2(40%) Absent 3(60%) Tumor grade Well 1(20%) Moderately 3(60%) Poorly 1(20%) TNM-T stage T1 0(0%) T2 1(20%) T3 4(80%) TNM-N stage N0 2(40%) N1 1(20%) N2 2(40%) TNM-M stage M0 4(80%) M1 1(20%) Gene frequency of MSI NR-24 4(80%) NR-21 3(60%) BAT-26 2(40%) BAT-25 2(40%) MONO-27 2(40%) L.V. =Lymphovascular; TNM=Tumor, Node, Metastasis Staging System moderately differentiated; this is similar to another regional study from Northern Saudi Arabia29 and Jordan.30 In this study, 5/45 (11%) sporadic CRCs showed MSI-H, which is less than the percentage reported in the northern parts of Iran (22.9%)22 and Egypt (57.9%)21 but comparable to results from different countries e.g. North America, Europe, and East Asia.31 For the 5 MSI-H patients, age ≥50 years was more fre- quently seen than <50 years, similar to another study.32 There is a male predominance in both MSI-H and MSS groups, 161J Contemp Med Sci | Vol. 9, No. 3, May-June 2023: 158–162 W. T. S. Al-Jumaili et al. Original Detection of MSI Biomarkers in Sporadic Colorectal Cancer contrary to what was reported in another study,31 but is com- parable to findings from Iran and India and other parts of the world.22, 24 They found a higher incidence of CRC in males compared to females and stated that young women (18–44 years) with CRC have a better survival outcome compared to men of the same age or compared to older women.33 In addi- tion, MSI behaves in a sex-dependent manner, where the mutation rate is five times higher in males than in females.34 Regarding smoking, 60% of MSI patients and only 42.5% of MSS patients smoke, with no statistically significant differ- ence. This is consistent with another study conducted from university of Utah, USA.35 Statistical analysis showed no significant difference between MSI-H and MSS groups of CRC patients in terms of presenting features, tumor location, tumor grade, tumor stage, and lymphovascular invasion. Tumor type, however, showed a statistical significance in favour of mucinous adenocarci- nomas in MSI-H; this latter finding is consistent with other studies.36–37 The small sample size of MSI-H in this study averts making comparisons and conclusions to these findings, awaiting larger-scale future studies on this topic. Generally, these findings are similar to other studies;38–39 the advanced presentation is common in our population due to lack of a national screening programs. NR-24 marker was the most frequently instable marker among the MSI-H patients, being reported in 4(80%) patients, followed by NR-21. A previous Iraqi study using a different panel of MSI genes reported that the most frequent instable marker among the MSI-H patients was BAT-25 (48.93%) fol- lowed by BAT-26 (44.68%).37 The type of tested MSI genes and the method used has changed with time, starting from dinucleotide markers that showed inconsistencies in results, to mononucleotide markers requiring comparison to normal tissues, to the more recent use of MSI kits e.g. Idylla MSI kit that is quick, fully automated, does not require normal tissues, and is as sensitive and specific as the currently used kits e.g. Promega MSI Analysis System version 1.2.40 A novel panel of seven monomorphic biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, and SULF2) is currently recommended to be used in a diagnostic test to identify the MSI status in CRC.40 The recent inclusion of MSI assessment in the investiga- tion of newly diagnosed CRC patients in the specialized centers, would allow a clearer vision to the true incidence of MSI-H and characteristics among different population groups in Iraq in the near future. Conclusion The clinical benefits of detecting MSI status accurately and affordably are clear. The frequency of MSI in sporadic CRC patients in this study was 5/45 (11.1%) and was significantly associated with mucinous type. NR-24 and NR-21 were the most prevalent instable markers. These findings help physi- cians and oncologists decide whether to give immunotherapy or chemotherapy, and to predict prognosis based on result of MSI testing. Declarations Ethics Approval and Consent to Participate • This study was approved by the ethics research committee at the Dept. of Pathology and Forensic Medicine, College of Medicine, University of Baghdad (Issue No. 200 at November, 10th, 2021). Availability of Data and Material • The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing Interests • The authors declare that they have no competing interests. Funding • This study was not funded by any organization, institute, or body what so ever. Acknowledgements The authors would like to acknowledge the kind assistance of Dr. Sazan Abdulwahab Mirza Al-Atrooshi in sample collec- tion, and Dr. Mohammed Ghanim M. Al-Hilal for assistance in molecular analysis in this study.  Table 3. Types, frequencies, and patterns of the detected mononucleotide markers among 5 MSI-H CRC patients Mononucleotide marker P1 P2 P3 P4 P5 Gene frequency BAT25 + + 2/5(40%) BAT26 + + 2/5(40%) NR-21 + + + 3/5(60%) NR-24 + + + + 4/5(80%) MONO-27 + + 2/5(40%) Total 3(60%) 3(60%) 3(60%) 2(40%) 2(40%) 162 J Contemp Med Sci | Vol. 9, No. 3, May-June 2023: 158–162 Detection of MSI Biomarkers in Sporadic Colorectal Cancer Original W. T. S. Al-Jumaili et al. This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i3.1348 References 1. Arnold, M., et al., Global patterns and trends in colorectal cancer incidence and mortality. Gut, 2017. 66(4): p. 683–691. 2. Baraaj, A. and H. Mahmood, Role Some Risk Factors : Age ,Sex And Lipid Profile In Colo-rectal Cancer In Iraqi Patient. Systematic Reviews in Pharmacy, 2021. 12: p. 1–5. 3. Mattiuzzi, C., F. Sanchis-Gomar, and G. Lippi, Concise update on colorectal cancer epidemiology. Ann Transl Med, 2019. 7(21): p. 609. 4. Al Dahhan, S.A. and F.H. Al Lami, Epidemiology of Colorectal Cancer in Iraq, 2002-2014. Gulf J Oncolog, 2018. 1(26): p. 23–26. 5. Douaiher, J., et al., Colorectal cancer-global burden, trends, and geographical variations. J Surg Oncol, 2017. 115(5): p. 619–630. 6. Binefa, G., et al., Colorectal cancer: from prevention to personalized medicine. World J Gastroenterol, 2014. 20(22): p. 6786–808. 7. Xi, Y. and P. Xu, Global colorectal cancer burden in 2020 and projections to 2040. Transl Oncol, 2021. 14(10): p. 101174. 8. Alrubaie, A., N. Alkhalidi, and S. Abd-Alhusain, A clinical study of newly- diagnosed colorectal cancer over 2 years in a gastroenterology center in Iraq. Journal of Coloproctology, 2019. 39(3): p. 217–222. 9. De Rosa, M., et al., Genetics, diagnosis and management of colorectal cancer (Review). Oncol Rep, 2015. 34(3): p. 1087–96. 10. Grady, W.M. and S.D. Markowitz, The molecular pathogenesis of colorectal cancer and its potential application to colorectal cancer screening. Dig Dis Sci, 2015. 60(3): p. 762–72. 11. Zeinalian, M., et al., Clinical Aspects of Microsatellite Instability Testing in Colorectal Cancer. Adv Biomed Res, 2018. 7: p. 28. 12. Nguyen, L.H., A. Goel, and D.C. Chung, Pathways of Colorectal Carcinogenesis. Gastroenterology, 2020. 158(2): p. 291–302. 13. Webber, E.M., et al., Systematic review of the predictive effect of MSI status in colorectal cancer patients undergoing 5FU-based chemotherapy. BMC Cancer, 2015. 15: p. 156. 14. Luchini, C., et al., ESMO recommendations on microsatellite instability testing for immunotherapy in cancer, and its relationship with PD-1/PD-L1 expression and tumour mutational burden: a systematic review-based approach. Ann Oncol, 2019. 30(8): p. 1232–1243. 15. Söreide, K., et al., Microsatellite instability in colorectal cancer. British Journal of Surgery, 2006. 93(4): p. 395–406. 16. Loukola, A., et al., Microsatellite Marker Analysis in Screening for Hereditary Nonpolyposis Colorectal Cancer (HNPCC)1. Cancer Research, 2001. 61(11): p. 4545–4549. 17. Ali, E., et al., Jemperli (Dostarlimab-gxly): An unprecedented cancer trial. Ann Med Surg (Lond), 2022. 79: p. 104047. 18. Chung, C., Predictive and prognostic biomarkers with therapeutic targets in colorectal cancer: A 2021 update on current development, evidence, and recommendation. 2022. 28(4): p. 850–869. 19. 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. 10(4): p. 293–300. 20. Kamat, N., et al., Microsatellite instability and loss of heterozygosity detected in middle-aged patients with sporadic colon cancer: A retrospective study. Oncology letters, 2013. 6(5): p. 1413–1420. 21. Kassem, N.M., et al., Clinicopathological features of Egyptian colorectal cancer patients regarding somatic genetic mutations especially in KRAS gene and microsatellite instability status: a pilot study. Egyptian Journal of Medical Human Genetics, 2019. 20(1): p. 20. 22. Faghani, M., et al., The Correlation between Microsatellite Instability and the Features of Sporadic Colorectal Cancer in the North Part of Iran. Gastroenterol Res Pract, 2012. 2012: p. 756263. 23. Deligonul, A., et al., Prognostic Significance of Microsatellite Instability in Turkish Patients with Stage II and III Colorectal Cancer. 2021. 5(1). 24. Rai, P.R., et al., A study on the frequency and clinicopathological correlates of mismatch repair-deficient colorectal cancer. J Cancer Res Ther, 2020. 16(Supplement): p. S183–s188. 25. Aykan, N.F., et al., Epidemiology of colorectal cancer in Turkey: A cross-sectional disease registry study (A Turkish Oncology Group trial). Turk J Gastroenterol, 2015. 26(2): p. 145–53. 26. Ayyub, M.I., et al., Clinicopathological trends in colorectal cancer in a tertiary care hospital. Saudi Med J, 2002. 23(2): p. 160–3. 27. H.;, H.M., et al., Age and Gender in Relation to Colorectal Cancer in Najef Province: A Histopathological Study. Journal of Clinical and Laboratory Research, 2021. 2 (1): p. 10. 28. Alsanea, N., et al., Colorectal cancer in Saudi Arabia: incidence, survival, demographics and implications for national policies. Ann Saudi Med, 2015. 35(3): p. 196–202. 29. Alharbi, S.H., et al., Patterns and grades of presentation of colon cancer in Northern Saudi Arabia. Prz Gastroenterol, 2021. 16(3): p. 235–239. 30. Sharkas, G.F., et al., Colorectal Cancer in Jordan: Survival Rate and Its Related Factors. J Oncol, 2017. 2017: p. 3180762. 31. Kim, J.H. and G.H. Kang, Molecular and prognostic heterogeneity of microsatellite-unstable colorectal cancer. World J Gastroenterol, 2014. 20(15): p. 4230–43. 32. Goksu, S.Y., et al., Clinicopathologic variables and outcomes in elderly colorectal cancer patients with microsatellite instability and multiple primary malignancies. 2021. 39(15_suppl): p. e15516-e15516. 33. Abancens, M., et al., Sexual Dimorphism in Colon Cancer. 2020. 10. 34. Vargas Jentzsch, I., et al., Evolution of microsatellite DNA. 2013. 35. Slattery, M.L., et al., Associations between cigarette smoking, lifestyle factors, and microsatellite instability in colon tumors. J Natl Cancer Inst, 2000. 92(22): p. 1831–6. 36. Fleming, M., et al., Colorectal carcinoma: Pathologic aspects. J Gastrointest Oncol, 2012. 3(3): p. 153–73. 37. Mohymen, N., B. Hanon, and A.S. Mahmood, The Correlation between Microsatellite Instability and the Features of Sporadic Colorectal Cancer in Sample of Iraqi Patients. JOURNAL OF ADVANCES IN BIOTECHNOLOGY, 2014. 4: p. 301–312. 38. Popat, S., R. Hubner, and R.S. Houlston, Systematic review of microsatellite instability and colorectal cancer prognosis. J Clin Oncol, 2005. 23(3): p. 609–18. 39. Pritchard, C.C. and W.M. Grady, Colorectal cancer molecular biology moves into clinical practice. Gut, 2011. 60(1): p. 116–29. 40. Zwaenepoel, K., et al., Clinical Performance of the Idylla MSI Test for a Rapid Assessment of the DNA Microsatellite Status in Human Colorectal Cancer. The Journal of Molecular Diagnostics, 2020. 22(3): p. 386–395.