Donia OUNISSI, Marwa WESLATI, Rahma BOUGHRIBA, Meriam HAZGUI, Saadia BOURAOUI

Clinicopathological characteristics and mutational profile of KRAS and NRAS in Tunisian patients with sporadic colorectal cancer

Background/aim: Colorectal cancer (CRC) is a major public health problem worldwide and in Tunisia due to its increasing rate of incidence. KRAS and NRAS mutations have become a pivotal part of CRC diagnosis, given their association to treatment resistance with antiepidermal growth factor receptor (EGFR) monoclonal antibodies. In this study, we aimed to screen for mutations in KRAS and NRAS genes in Tunisian patients with CRC and explore their correlations with clinicopathological features. Materials and methods: AmoyDx KRAS and NRAS mutation real-time PCR kits were used to screen for mutations in KRAS (exon 2) and NRAS (exons 2, 3, and 4) in 96 CRC tumors. Results: KRAS exon 2 mutations were found in 41.7% (40/96) of the patients. Codon 12’s most abundant mutations were G12D and G12V, followed by G12A, while G13D is the predominant mutation in codon 13. KRAS exon 2 mutations were associated with older patients (P = 0.029), left-sided tumors (P = 0.037), and greater differentiation (P = 0.044). The prevalence rate of NRAS mutations was 7.3%, mostly in exon 2. These mutations were associated with early stages of the disease (P = 0.039) and the absence of lymph node metastasis (P = 0.045). Conclusion: It can be inferred from this study that Tunisian CRC patients have a similar frequency of KRAS and NRAS mutations compared to those observed in other populations. Consequently, screening for KRAS and NRAS mutations is crucial for the orientation of therapies and the selection of appropriate candidates, while also helping to avoid unnecessary toxicity and increased costs for patients.

PDF

___

1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. A Cancer Journal for Clinicians 2018; 68 (6): 394- 424. doi: 10.3322/caac.21492
2. Sayagués JM, Del Carmen S, Del Mar Abad M, Corchete LA, Bengoechea O et al. Combined assessment of the TNM stage and BRAF mutational status at diagnosis in sporadic colorectal cancer patients. Oncotarget 2018; 9 (35): 24081-24096. doi: 10.18632/oncotarget.25300
3. Khiari H, Ayoub HWB, Khadhra HB, Hsairi M. Colorectal cancer incidence trend and projections in Tunisia (1994 - 2024). Asian Pacific Journal of Cancer Prevention 2017; 18 (10): 2733-2740. doi: 10.22034/APJCP.2017.18.10.2733
4. Missaoui N, Jaidaine L, Abdelkader AB, Trabelsi A, Mokni M et al. Colorectal cancer in central Tunisia: increasing incidence trends over a 15-year period. Asian Pacific Journal of Cancer Prevention 2011; 12 (4): 1073-1076.
5. Kassab A, Landolsi S, Miled A, Ben Ahmed S, Olfa G. Nutrition and colorectal cancer relationship in Tunisian population; beginning an answer. Immuno-analyse & Biologie Spécialisée 2013; 28 (5-6): 327-334. (in French with an abstract in English). doi: 10.1016/j.immbio.2013.05.003
6. Othman RB, Ksira I, Smida A, Berriche O, Mahjoub F et al. Malnutrition and risk factors in tunisian patients with colorectal cancer. Ibnosina Journal of Medicine and Biomedical Sciences 2018; 10 (3): 88. doi: 10.4103/ijmbs.ijmbs_79_17
7. Fearon ER, Vogelstein B, A genetic model for colorectal tumorigenesis. Cell 1990; 61 (5): 759-767.
8. Nguyen HT, Duong H-Q, The molecular characteristics of colorectal cancer: implications for diagnosis and therapy (Review). Oncology Letters 2018; 16 (1): 9-18. doi: 10.3892/ ol.2018.8679
9. Margetis N, Kouloukoussa M, Pavlou K, Vrakas S, MariolisSapsakos T. K-ras mutations as the earliest driving rorce in a subset of colorectal carcinomas. In Vivo 2017; 31 (4): 527-542. doi: 10.21873/invivo.11091
10. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC et al. Genetic alterations during colorectal-tumor development. The New England Journal of Medicine 1988; 319 (9): 525-532. doi: 10.1056/NEJM198809013190901
11. Xie M, Li J, Cai Z, Li K, Hu B. Impact of primary colorectal cancer location on the KRAS status and its prognostic value. BioMed Central Gastroenterology 2019; 19 (1): 46. doi: 10.1186/s12876-019-0965-5
12. Chang Y-Y, Lin P-C, Lin H-H, Lin J-K, Chen W-S et al. Mutation spectra of RAS gene family in colorectal cancer. American Journal of Surgery 2016; 212 (3): 537-544. doi: 10.1016/j.amjsurg.2016.02.013
13. Hong SN, Genetic and epigenetic alterations of colorectal cancer. Intestinal Research 2018; 16 (3): 327-337. doi: 10.5217/ ir.2018.16.3.327
14. Bai B, Shan L, Xie B, Huang X, Mao W et al. Mutations in KRAS codon 12 predict poor survival in Chinese patients with metastatic colorectal cancer. Oncology Letters 2018; 15(3): 3161-3166. doi: 10.3892/ol.2017.7709
15. Sinha R, Hussain S, Mehrotra R, Kumar RS, Kumar K et al. Kras gene mutation and RASSF1A, FHIT and MGMT gene promoter hypermethylation: indicators of tumor staging and metastasis in adenocarcinomatous sporadic colorectal cancer in Indian population. Public Library of Science ONE 2013; 8 (4): e60142. doi: 10.1371/journal.pone.0060142
16. Sirisena ND, Deen K, Mandawala DEN, Herath P, Dissanayake VHW. The pattern of KRAS mutations in metastatic colorectal cancer: a retrospective audit from Sri Lanka. BioMed Central Research Notes 2017; 10 (1): 392. doi: 10.1186/s13104-017- 2731-5
17. Karapetis CS, Khambata-Ford S, Jonker DJ, O’Callaghan CJ, Tu D et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. The New England Journal of Medicine 2008; 359 (17): 1757-1765. doi: 10.1056/ NEJMoa0804385
18. Allegra CJ, Jessup JM, Somerfield MR, Hamilton SR, Hammond EH et al. American Society Of Clinical Oncology provisional clinical opinion: testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology 2009; 27 (12): 2091- 2096. doi: 10.1200/JCO.2009.21.9170
19. Engstrom PF, Arnoletti JP, Benson AB, Chen Y-J, Choti MA et al. NCCN Clinical practice guidelines in oncology: colon cancer. Journal of the National Comprehensive Cancer Network 2009; 7: 778-831. doi: 10.6004/jnccn.2009.0056
20. Zhang H, Song J, Ren H, Xu Z, Wang X et al. Detection of low-abundance KRAS mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions. Public Library of Science One 2013; 8 (1): e54510. doi: 10.1371/journal.pone.0054510
21. Li W, Li H, Liu R, Yang X, Gao Y et al. Comprehensive analysis of the relationship between RAS and RAF mutations and MSI status of colorectal cancer in northeastern China. Cellular Physiology and Biochemistry: International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology 2018; 50 (4): 1496-1509. doi: 10.1159/000494649
22. Negru S, Papadopoulou E, Apessos A, Stanculeanu DL, Ciuleanu E et al. KRAS, NRAS and BRAF mutations in Greek and Romanian patients with colorectal cancer: a cohort study. British Medical Journal Open 2014; 4 (5): e004652. doi: 10.1136/bmjopen-2013-004652
23. Kodaz H, Kostek O, Hacioglu M, Erdogan B, Elpen Kodaz C et al. Frequency of RAS mutations (KRAS, NRAS, HRAS) in human solid cancer. Eurasian Journal of Medicine and Oncology 2017; 1 (1): 1-7. doi: 10.14744/ejmo.2017.22931
24. De Roock W, Claes B, Bernasconi D, De Schutter J, Biesmans B et al. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. The Lancet Oncology 2010; 11 (8): 753-762. doi: 10.1016/S1470-2045(10)70130-3
25. Douillard JY, Oliner KS, Siena S, Tabernero J, Burkes R et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. The New England Journal of Medicine 2013; 369 (11): 1023-1034. doi: 10.1056/NEJMoa1305275
26. Allegra CJ, Rumble RB, Schilsky RL. Extended RAS gene mutation resting in metastatic colorectal carcinoma to predict response to anti-epidermal growth gactor receptor monoclonal antibody therapy: American Society of Clinical Oncology Provisional Clinical Opinion Update 2015 Summary. Journal of Oncology Practice 2016; 12 (2): 180-181. doi: 10.1200/ JOP.2015.007898
27. Bosman FT, Carneiro F, Hruban RH, Theise ND. WHO Classification of Tumours of the Digestive System. 4th ed. Lyon, France: International Agency for Research on Cancer Press; 2010.
28. Amin MB, Edge S, Greene F, Byrd DR, Brookland RK et al. (editors). AJCC Cancer Staging Manual. 8th ed. Berlin: Springer International Publishing: American Joint Commission on Cancer; 2017.
29. Rimbert J, Tachon G, Junca A, Villalva C, Karayan-Tapon L et al. Association between clinicopathological characteristics and RAS mutation in colorectal cancer. Modern Pathology: An Official Journal of the United States and Canadian Academy of Pathology 2018; 31 (3): 517-526. doi: 10.1038/ modpathol.2017.119
30. Abdelmaksoud-Dammak R, Saadallah-Kallel A, MiladiAbdennadher I, Khabir A, Sallemi-Boudawara T et al. Mutations du gène KRAS chez les patients du sud tunisien atteint de cancer colorectal: signification clinique. Journal de l’Information Médicale de Sfax 2015; 21: 39-44 (in French).
31. Aissi S, Buisine M-P, Zerimech F, Kourda N, Moussa A et al. KRAS mutations in colorectal cancer from Tunisia: relationships with clinicopathologic variables and data on TP53 mutations and microsatellite instability. Molecular Biology Reports 2013; 40 (11): 6107-6112. doi: 10.1007/s11033-013- 2722-0
32. Karim B, Florence C, Kamel R, Nadia K, Ines O et al. KRAS mutation detection in Tunisian sporadic coloractal cancer patients with direct sequencing, high resolution melting and denaturating high performance liquid chromatography. Cancer Biomarkers 2011; 8 (6), 331-340. doi: 10.3233/CBM2011-0222
33. Ouerhani S, Bougatef K, Soltani I, Elgaaied ABA, Abbes S et al. The prevalence and prognostic significance of KRAS mutation in bladder cancer, chronic myeloid leukemia and colorectal cancer. Molecular Biology Reports 2013; 40 (6): 4109-4114. doi: 10.1007/s11033-013-2512-8
34. Neumann J, Zeindl-Eberhart E, Kirchner T, Jung A. Frequency and type of KRAS mutations in routine diagnostic analysis of metastatic colorectal cancer. Pathology, Research and Practice 2009; 205 (12): 858-862. doi: 10.1016/j.prp.2009.07.010
35. Rako I, Jakic-Razumovic J, Katalinic D, Sertic J, Plestina S. Mutation pattern of KRAS and BRAF oncogenes in colorectal cancer patients. Neoplasma 2012; 59 (4): 376-383. doi: 10.4149/ neo_2012_049
36. Zhang J, Zheng J, Yang Y, Lu J, Gao J et al. Molecular spectrum of KRAS, NRAS, BRAF and PIK3CA mutations in Chinese colorectal cancer patients: analysis of 1,110 cases. Scientific Reports 2015; 5 (1). doi: 10.1038/srep18678
37. Jouini R, Ferchichi M, BenBrahim E, Ayari I, Khanchel F et al. KRAS and NRAS pyrosequencing screening in Tunisian colorectal cancer patients in 2015. Heliyon 2019; 5 (3): e01330. doi: 10.1016/j.heliyon.2019.e01330
38. Cushman-Vokoun AM, Stover DG, Zhao Z, Koehler EA, Berlin JD et al. Clinical utility of KRAS and BRAF mutations in a cohort of patients with colorectal neoplasms submitted for microsatellite instability testing. Clinical Colorectal Cancer 2013; 12 (3): 168-178. doi: 10.1016/j.clcc.2013.04.005
39. Shen Y, Wang J, Han X, Yang H, Wang S et al. Effectors of epidermal growth gactor receptor pathway: the genetic profiling of KRAS, BRAF, PIK3CA, NRAS mutations in colorectal cancer characteristics and personalized medicine. Public Library of Science One 2013; 8 (12): e81628. doi: 10.1371/journal.pone.0081628
40. Tong JH, Lung RW, Sin FM, Law PP, Kang W et al. Characterization of rare transforming KRAS mutations in sporadic colorectal cancer. Cancer Biology & Therapy 2014; 15 (6): 768-776. doi: 10.4161/cbt.28550
41. Wang J, Yang H, Shen Y, Wang S, Lin D et al. Direct sequencing is a reliable assay with good clinical applicability for KRAS mutation testing in colorectal cancer. Cancer Biomarkers 2013; 13 (2): 89-97. doi: 10.3233/CBM-130334
42. Guo F, Gong H, Zhao H, Chen J, Zhang Y et al. Mutation status and prognostic values of KRAS, NRAS, BRAF and PIK3CA in 353 Chinese colorectal cancer patients. Scientific Reports 2018; 8 (1). doi: 10.1038/s41598-018-24306-1
43. Ye J-X, Liu Y, Qin Y, Zhong H-H, Yi W-N et al. KRAS and BRAF gene mutations and DNA mismatch repair status in Chinese colorectal carcinoma patients. World Journal of Gastroenterology 2015; 21 (5): 1595-1605. doi: 10.3748/wjg. v21.i5.1595
44. Imamura Y, Lochhead P, Yamauchi M, Kuchiba A, Qian Z et al. Analyses of clinicopathological, molecular, and prognostic associations of KRAS codon 61 and codon 146 mutations in colorectal cancer: cohort study and literature review. Molecular Cancer 2014; 13 (1): 135. doi: 10.1186/1476-4598-13-135
45. Baran B, Mert Ozupek N, Yerli Tetik N, Acar E, Bekcioglu O et al. Difference between left-sided and right-sided colorectal cancer: a focused review of literature. Gastroenterology Research 2018; 11 (4): 264-273. doi: 10.14740/gr1062w
46. Estrada P, Rojas-Atencio A, Zabala W, Borjas L, Soca L et al. Frequency and clinicopathological associations of K-ras mutations in Venezuelan patients with colo-rectal cancer. Investigacion Clinica 2009; 50 (1): 55-63 (in Spanish with an abstract in English).
47. Poehlmann A, Kuester D, Meyer F, Lippert H, Roessner A et al. K-ras mutation detection in colorectal cancer using the pyrosequencing technique. Pathology - Research and Practice 2007; 203 (7): 489-497. doi: 10.1016/j.prp.2007.06.001
48. Veldore VH, Rao MR, Prabhudesai SA, Tejaswi R, Kakara S et al. Prevalence of KRAS mutations in metastatic colorectal cancer: a retrospective observational study from India. Indian Journal of Cancer 2014; 51 (4): 531-537. doi: 10.4103/0019- 509X.175371
49. Zhang X, Ran W, Wu J, Li H, Liu H et al. Deficient mismatch repair and RAS mutation in colorectal carcinoma patients: a retrospective study in Eastern China. Peer The Journal of Life and Environmental Sciences 2018; 6: e4341. doi: 10.7717/ peerj.4341
50. Song G, Deng G, Bell I, Kakar S, Sleisenger M et al. Mucinous carcinomas of the colorectum have distinct molecular genetic characteristics. International Journal of Oncology 2005; 26 (3): 745-750. doi: 10.3892/ijo.26.3.745
51. Tanaka H, Deng G, Matsuzaki K, Kakar S, Kim GE et al. BRAF mutation, CpG island methylator phenotype and microsatellite instability occur more frequently and concordantly in mucinous than non-mucinous colorectal cancer. International Journal of Cancer 2006; 118 (11): 2765-2771. doi: 10.1002/ijc.21701
52. Schirripa M, Cremolini C, Loupakis F, Morvillo M, Bergamo F et al. Role of NRAS mutations as prognostic and predictive markers in metastatic colorectal cancer. International Journal of Cancer 2015; 136 (1): 83-90. doi: 10.1002/ijc.28955
53. Jauhri M, Bhatnagar A, Gupta S, BP M, Minhas S et al. Prevalence and coexistence of KRAS, BRAF, PIK3CA, NRAS, TP53, and APC mutations in Indian colorectal cancer patients: next-generation sequencing–based cohort study. Tumor Biology 2017; 39 (2). doi: 10.1177/1010428317692265
54. Irahara N, Baba Y, Nosho K, Shima K, Yan L et al. NRAS mutations are rare in colorectal cancer. Diagnostic Molecular Pathology: The American Journal of Surgical Pathology 2010; 19 (3): 157-163. doi: 10.1097/PDM.0b013e3181c93fd1
55. Russo AL, Borger DR, Szymonifka J, Ryan DP, Wo JY et al. Mutational analysis and clinical correlation of metastatic colorectal cancer. Cancer 2014; 120 (10): 1482-1490. doi: 10.1002/cncr.28599
56. Wong SQ, Scott R, Fox SB. KRAS mutation testing in colorectal cancer: the model for molecular pathology testing in the future. Colorectal Cancer 2016; 5 (2): 73-80. doi: 10.2217/crc-2015- 0009
57. Matsunaga M, Kaneta T, Miwa K, Ichikawa W, Fujita K-I et al. A comparison of four methods for detecting KRAS mutations in formalin-fixed specimens from metastatic colorectal cancer patients. Oncology Letters 2016; 12 (1): 150-156. doi: 10.3892/ ol.2016.4576
58. Zhang H, Zheng X, Ji T, Fu L, Bai D et al. Comparative screening of K-ras mutations in colorectal cancer and lung cancer patients ssing a novel real-time PCR with ADx-K-ras kit and sanger DNA sequencing. Cell Biochemistry and Biophysics 2012; 62 (3): 415-420. doi: 10.1007/s12013-011-9318-x
59. Chat-Uthai N, Vejvisithsakul P, Udommethaporn S, Meesiri P, Danthanawanit C et al. Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues. Public Library of Science ONE 2018; 13 (6): e0198795. doi: 10.1371/journal.pone.0198795