Analiza p53 gena i HPV infekcija u karcinomima grlića materice i jajnika

Abstract

Uvod: Ginekološki maligniteti predstavljaju veoma raznoliku grupu kancera, među kojima su najučestaliji karcinomi grlića materice a najsmrtonosniji karcinomi jajnika. Podaci o povezanost p53 mutacija (TP53 po HUGO nomenklaturi), polimorfizma kodona 72 i infekcije humanim papiloma virusima (HPV) sa kliničko-histopatološkim karakteristikama, nastankom ovih maligniteta, kao i odgovorom na antikancersku terapiju su kontradiktorni. Malo se zna o međusobnoj povezanosti TP53 mutacija, polimorfizma kodona 72 i HPV infekcije. Zato postoji potreba za ispitivanjem pomenutih potencijalnih biomarkera ovih maligniteta. Cilj: Ispitivanje povezanosti TP53 mutacija, polimorfnih varijanti kodona 72 i HPV infekcije sa demografskim karakteristikama, kliničko-histopatološkim karakteristikama karcinoma grlića materice i karcinoma jajnika i karakteristikama bolesnica sa ovim karcinomima, kao i ispitivanje međusobne povezanosti pomenutih potencijalnih biomarkera i njihovog uticaja na antikancersku terapiju. Materijal i metode: U radu su analizirana 53 uzorka karcinoma grlića materice i 54 uzorka karcinoma jajnika. Kontrolnu grupu činilo je 95 uzoraka briseva grlića materice žena sa urednim ginekološkim i normalnim Papa nalazom, kao i odsustvom prethodne istorije postojanja prekancerskih i kancerskih lezija ginekološke regije. DNK je izolovana metodom isoljavanja. Egzoni 4-8 TP53 gena su amplifikovani lančanom reakcijom polimeraze (PCR). Preliminarni skrining mutacija vršen je metodom konformacionog polimorfizma jednolančane DNK (SSCP), a automatskim sekvenciranjem DNK je potvrđivano prisustvo i utvrđivan tip mutacija. Polimorfizam kodona 72 TP53 gena je ispitivan analizom polimorfizma dužine restrikcionih fragmenata (RFLP). Prisustvo HPV infekcije je detektovano putem amplifikacije dela L1 virusnog gena. HPV16 i HPV18 tipovi u karcinomima grlića materice su detektovani amplifikacijom dela E7, odnosno E1 virusnih gena, dok je genotipizacija HPV u karcinomima jajnika vršena sekvenciranjem DNK. Za statističku obradu podataka korišćeni su Fišerov egzaktni, χ2 , odds ratio i Log-Rank test...

Introduction: Gynecological malignancies present a various group of cancers; among them the most frequent are carcinomas of cervix and the most aggressive are ovarian carcinomas. Conflicting data about correlation of TP53 mutations, codon 72 polymorphism and HPV infection with clinicopathological characteristics, the origin of these malignancies and with the response to anti-cancer therapy, as well as correlation between mentioned biomarkers, are pointing out the necessity of analysis of these biomarkers. Goal: Examination of correlation of TP53 mutations, codon 72 polymorphic variants and HPV infection with demographic features, clinicopathological characteristics of ovarian and cervical carcinomas, patient’s characteristics, as well as examination of interconnection of these biomarkers and their possible predictive values to anti-cancer therapy. Material and methods: 53 samples of cervical carcinomas and 54 samples of ovarian carcinomas were analyzed. Control group was consisted of 95 cervical smears of gynecological healthy women with normal Papa test results and without previous history of pre- and cancer lesion of gynecological region. DNA was extracted by salting-out procedure. Exons 4-8 of TP53 gene were amplified by polymerase chain reaction (PCR). Preliminary screening of mutations was done by single strand conformation polymorphism (SSCP) and automatic DNA sequencing was employed to confirm the presence and establish the type of mutation. Codon 72 polymorphism was assessed by restriction fragment-length polymorphism (RFLP). Presence of HPV infection was detected through amplification of one part of L1 viral gene. Types HPV16 and HPV18 in cervical carcinomas were detected by amplification of one part of E7 or E1 viral gene, while HPV genotyping in ovarian carcinomas were performed by DNA sequencing. Fisher exact, χ2, odds ratio and Log-Rank tests were employed for statistical analysis...