Studying the Impact of Tp53 Mutations on Colorectal Cancer Pathogenesis and Therapeutic Response in a Tunisian Population

Studying the Impact of Tp53 Mutations on Colorectal Cancer Pathogenesis and Therapeutic Response in a Tunisian Population | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Studying the Impact of Tp53 Mutations on Colorectal Cancer Pathogenesis and Therapeutic Response in a Tunisian Population Asma Bouafif This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9381448/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: Colorectal cancer has been ranked as the second most deadly cancer and the third most diagnosed cancer for the year 2020.TP53 a pivotal tumor suppressor gene, is frequently mutated in colorectal cancer (CRC), influencing tumorigenesis and therapy response. This study investigates the mutational spectrum of TP53 in CRC, elucidates its effects on tumor progression, and examines its correlation with clinical outcomes and chemotherapy efficacy. The primary objective was to study the correlation between p53 expression by immunohistochemistry and TP53 mutational status in Tunisian colorectal cancer patients. The secondary objective was to characterize TP53 mutations in CRC and assess their impact on tumor development, clinical parameters, and response to 5- fluorouracil (5-FU)-based chemotherapy. Methods: A cohort of 59 CRC samples obtained from formalin-fixed paraffin-embedded (FFPE) tissue blocks were analyzed. DNA was extracted and polymerase chain reaction (PCR) targeting TP53 exons 5-8 followed by Sanger sequencing was performed to identify mutations. Immunohistochemistry was used to assess p53 protein expression and its association with mutation status. In silico tools, including SIFT, PolyPhen-2, Mutation Taster, PROVEAN, FATHMM, RNAfold, and Project Hope, were used to predict the functional consequences of identified mutations. The association between TP53 mutations and clinicopathological features such as tumor stage, grade, lymphovascular and perineural invasion, RAS mutation status, overall survival (OS), and progression-free survival (PFS) were analyzed. Results: TP53 mutations were identified in 42.37% of the analyzed CRC samples, encompassing 14 different mutation types. The mutations consisted of 10 missense mutations (71.4%) and 4 synonymous mutations (28.5%). A novel pathogenic variant (L252R, c.755T>G) was also discovered. Initially, a general analysis of TP53 mutations revealed a significant association with increased p53 protein expression (P=0.04); however, no direct correlation was found with clinicopathological parameters such as tumor stage or metastasis at this stage of analysis. In a more focused investigation, pathogenic TP53 mutations were specifically analyzed. This analysis demonstrated that pathogenic mutations were strongly associated with more aggressive clinical features, including advanced tumor stage (P=0.008), lymph node metastasis (P=0.015), poorer overall survival (OS) (P=0.047) and progression-free survival (PFS) (p=0.04) in patients treated with 5-FU-based chemotherapy. In silico predictions further confirmed that these pathogenic mutations significantly disrupted the stability and function of the p53 protein, highlighting their potential role in driving tumor aggressiveness and resistance to standard therapies. Conclusions: TP53 mutations, especially pathogenic variants, play a crucial role in the pathogenesis of colon cancer as they influence tumor aggressiveness and resistance to therapy. The identification of specific mutations and their functional consequences has improved our understanding of CRC biology and highlighted the potential of TP53 as a biomarker for personalized treatment strategies. Incorporating TP53 mutation analysis into clinical practice could significantly improve patient stratification and treatment outcomes. Cancer Biology Molecular Genetics Molecular Biology Oncology Colorectal cancer TP53 mutation profile Sanger sequencing immunohistochemistry Full Text Additional Declarations The authors declare no competing interests. Ethics approval:This retrospective study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Institut Pasteur de Tunis, Tunisia. The study was carried out at the Department of Human and Experimental Pathology in collaboration with the Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis.Participant consent:Due to the retrospective nature of the study and the use of anonymized data, the requirement for informed consent was waived by the Ethics Committee. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9381448","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":621096549,"identity":"823bcd48-4ba9-48da-ab58-9e91354c9c34","order_by":0,"name":"Asma Bouafif","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYBACAxBRYSAHohgffACSbOzEaDljYAyimA1ngLQwE6WFAayFTZoHrJOAFnP2HsMPBwoM5OWnHX4mbfNrmzwfMwPjh485uLVY9pwxljhgYGC44XaasXVu323DNmYGZsmZ2/A47EaOgfQHgz+MG6QTDG/n9txmBGphY+bFr8X4B9AW+/mz0z9IW/bctidGixnIYYkNt3OMpBl+3E4kqMWy51iZBVBL8obbOcWGvQ23k9uYGZvx+sWcvXnzjQN/DGyBDtv44Mef27bz25sPfviIRwsqYGwDkw3EqgeBP6QoHgWjYBSMgpECAJDiUwampKWxAAAAAElFTkSuQmCC","orcid":"","institution":"Institut Pasteur de Tunis","correspondingAuthor":true,"prefix":"","firstName":"Asma","middleName":"","lastName":"Bouafif","suffix":""}],"badges":[],"createdAt":"2026-04-10 15:58:32","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9381448/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9381448/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107705092,"identity":"10349662-9698-4084-8f4e-77f280bf2145","added_by":"auto","created_at":"2026-04-24 09:07:59","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":476753,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptTP53CRC.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9381448/v1_covered_735b64a0-9bb0-4c53-9fdb-95acb3eed8c1.pdf"}],"financialInterests":"\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eEthics approval:This retrospective study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Institut Pasteur de Tunis, Tunisia. The study was carried out at the Department of Human and Experimental Pathology in collaboration with the Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis.Participant consent:Due to the retrospective nature of the study and the use of anonymized data, the requirement for informed consent was waived by the Ethics Committee.\u003c/p\u003e","formattedTitle":"\u003cp\u003eStudying the Impact of Tp53 Mutations on Colorectal Cancer Pathogenesis and Therapeutic Response in a Tunisian Population\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Institut Pasteur de Tunis","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Colorectal cancer, TP53, mutation profile, Sanger sequencing, immunohistochemistry","lastPublishedDoi":"10.21203/rs.3.rs-9381448/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9381448/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction:\u003c/strong\u003e Colorectal cancer has been ranked as the second most deadly cancer and the third most diagnosed cancer for the year 2020.TP53 a pivotal tumor suppressor gene, is frequently mutated in colorectal cancer (CRC), influencing tumorigenesis and therapy response. This study investigates the mutational spectrum of TP53 in CRC, elucidates its effects on tumor progression, and examines its correlation with clinical outcomes and chemotherapy efficacy. The primary objective was to study the correlation between p53 expression by immunohistochemistry and TP53 mutational status in Tunisian colorectal cancer patients. The secondary objective was to characterize TP53 mutations in CRC and assess their impact on tumor development, clinical parameters, and response to 5- fluorouracil (5-FU)-based chemotherapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e A cohort of 59 CRC samples obtained from formalin-fixed paraffin-embedded (FFPE) tissue blocks were analyzed. DNA was extracted and polymerase chain reaction (PCR) targeting TP53 exons 5-8 followed by Sanger sequencing was performed to identify mutations. Immunohistochemistry was used to assess p53 protein expression and its association with mutation status. In silico tools, including SIFT, PolyPhen-2, Mutation Taster, PROVEAN, FATHMM, RNAfold, and Project Hope, were used to predict the functional consequences of identified mutations. The association between TP53 mutations and clinicopathological features such as tumor stage, grade, lymphovascular and perineural invasion, RAS mutation status, overall survival (OS), and progression-free survival (PFS) were analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e TP53 mutations were identified in 42.37% of the analyzed CRC samples, encompassing 14 different mutation types. The mutations consisted of 10 missense mutations (71.4%) and 4 synonymous mutations (28.5%). A novel pathogenic variant (L252R, c.755T\u0026gt;G) was also discovered. Initially, a general analysis of TP53 mutations revealed a significant association with increased p53 protein expression (P=0.04); however, no direct correlation was found with clinicopathological parameters such as tumor stage or metastasis at this stage of analysis. In a more focused investigation, pathogenic TP53 mutations were specifically analyzed. This analysis demonstrated that pathogenic mutations were strongly associated with more aggressive clinical features, including advanced tumor stage (P=0.008), lymph node metastasis (P=0.015), poorer overall survival (OS) (P=0.047) and progression-free survival (PFS) (p=0.04) in patients treated with 5-FU-based chemotherapy. In silico predictions further confirmed that these pathogenic mutations significantly disrupted the stability and function of the p53 protein, highlighting their potential role in driving tumor aggressiveness and resistance to standard therapies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e TP53 mutations, especially pathogenic variants, play a crucial role in the pathogenesis of colon cancer as they influence tumor aggressiveness and resistance to therapy. The identification of specific mutations and their functional consequences has improved our understanding of CRC biology and highlighted the potential of TP53 as a biomarker for personalized treatment strategies. Incorporating TP53 mutation analysis into clinical practice could significantly improve patient stratification and treatment outcomes.\u003c/p\u003e","manuscriptTitle":"Studying the Impact of Tp53 Mutations on Colorectal Cancer Pathogenesis and Therapeutic Response in a Tunisian Population","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-15 19:27:16","doi":"10.21203/rs.3.rs-9381448/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"659b6bfa-59e6-446a-9f89-be867cbc8801","owner":[],"postedDate":"April 15th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":66395976,"name":"Cancer Biology"},{"id":66395977,"name":"Molecular Genetics"},{"id":66395978,"name":"Molecular Biology"},{"id":66395979,"name":"Oncology"}],"tags":[],"updatedAt":"2026-04-25T09:36:40+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-15 19:27:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9381448","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9381448","identity":"rs-9381448","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}