Detekcija mutacija u p53 genu kod bolesnica sa primarnim operabilnim karcinomom dojke

Abstract

Uvod: Zbog svoje uloge u očuvanju genomskog integriteta, produkt normalnog p53 gena se često naziva i "čuvar genoma". Pokazano je da u ćeljama gde je p53 izmenjen dolazi do aberantne replikacije DNK, stoje praćeno genomskom nestabilnošću. Gubitak funkcije p53 povezan je ne samo sa nemogućnošću popravke DNK, već i sa apoptozom. Podaci ukazuju da p53 ima kritičnu ulogu u aktiviranju apotoze pri odgovoru na različite vidove hemioterapije u karcinomu dojke. Cilj ovog rada bio je da se utvrdi da li p53 mutacije utiču na odgovor na hemioterapiju i da li p53 mutacije mogu dati informaciju o prognozi bolesnica sa karcinomom dojke. Bolesnice i metode. U rad je bilo uključeno 100 bolesnica, od toga 70 sa metastazama u regionalnim limfnim čvorovima i 30 bolesnica bez metastaza u regionalnim limfnim čvorovima. Sve osim jedne bolesnice sa metastazama u limfnim čvorovima su primile adjuvantnu hemioterapiju. 65/70 bolesnica je primilo CMF (ciklofosfamid, metotreksat, 5-fluorouracil) po modifikovanom intravenoznom protokolu, i 5/70 je primilo FAC (5-fluorouracil, adriablastin, ciklofosfamid). Bolesnice su praćene u intervalu 6-127 meseci, medijana 48 meseci. DNK je izolovana fenolskom ekstrakcijom iz uzoraka rutinski prikupljanih za odredjivanje receptora za steroidne hormone. Specifični DNK regioni p53 gena koji odgovaraju eksonima 5-8, umnoženi su lančanom reakcijom polimeraze (PCR). Detekcija mutacija izvršena je SSCP (konformacioni polimorfizam jednolančane DNK) elektroforezom. Gelovi su bojeni srebrom. Amplifikacija c-myc gena odredjena je diferenijalnim PCR-om. Receptori za steroidne hormone odredjivani su biohemijskom saturacionom metodom sa aktivnim ugljem. Hi-kvadrat i Fisher-ov egzaktni test, kao i Kaplan-Meir-ov i Log-Rank test su korišćeni za statističku obradu podataka. Rezultati: p53 mutacije detektovane su kod 21/100 bolesnica. Ukupno je pronadjeno 25 mutacija (po dve mutacije detektovane su kod 4 bolesnice), i njihova raspodela po ispitivanim eksonima nije bila jednaka: 10 u eksonu 5, 6 u eksonu 6, 8 u eksonu 7 i samo jedna mutacija u eksonu 8. Učestalost p53 mutacija bila je znatno viša kod bolesnica sa metastazama u limfnim čvorovima u odnosu na bolesnice bez metastaza u limfnim čvorovima. p53 mutacije bile su predominantno locirane kod bolesnica sa invazivnim duktalnim tipom karcinoma dojke. S obzirom da je kod bolesnica bez metastaza u regionalnim limfnim čvorovima pronadjena samo jedna mutacija, korelacija izmedju detektovanih mutacija i klničkog toka bolesti analizirana je samo kod bolesnica sa metastazama u limfnim čvorovima. Od ovih bolesnica, 32/70 je imalo ponovno javljanje bolesti (relaps). U prvih 24 meseci praćenja, bolesnice sa p53 mutacijama su imale značajno kraći period do ponovnog javljanja bolesti (DFI), u odnosu na bolesnice bez p53 mutacija. Takodje, u grupi bolesnica sa relapsom pokazan je značajno kraći period do progresije bolesti (PFI) kod bolesnica sa p53 mutacijama u odnosu na bolesnice bez mutacija. U odnosu na ukupno preživljavanje (OS), značajno kraće preživljavanje zabeleženo je kod bolesnica sa p53 i/ili c-myc alteracijama u odnosu na bolesnice bez detektovanih genskih alteracija. Zaključak: Pojava ranog relapsa kod bolesnica sa p53 mutacijama koje su primile hemioterapiju, ukazuje na prediktivni značaj p53. Rezultati dobijeni za OS, ističu značaj istovremenog odredjivanja alteracija više gena u nastojanju da se definiše molekularni profil svakog tumora, što za krajnji cilj ima individualni terapijski pristup.

Background: the main function of normal p53 gene product is to preserve genome integrity by acting as the "guardian of the genome". It has been shown that in p5 3-defective cells DNA aberrant replication is followed by genome instability. Loss of p53 function seems to be connected not only with abrogation of DNA reapair, but also with abrogation of apoptosis. The evidence indicates that p53 play a critical role in implementing apoptosis in response to treatment with different chemotherapeutics in breast cancer. The aim of this study was to evaluate whether tumor's p53 mutations affect response to chemotherapy, and whether p53 mutations provide prognostic information for breast cancer patients. Patients and methods: 100 breast cancer patients, among 70 lymph node-positive and 30 lymph node-negative were included. All except one node-positive patients underwent chemotherapy as adjuvant treatment. 65/70 patients received CMF (cyclophosphamide, methotrexate, 5-fluorouracil) modified intravenous protocol, and 5/70 FAC (5-flurouracil, adriablastine, cyclophosphamide) protocol. The patients were followed-up from 6 to 127, median 48 months. DNA was isolated by phenol extraction from samples routinely collected for steroid receptor determinations. Specific DNA regions corresponding to exons 5-8 were amplified by polimerase chain reaction (PCR). Detection of mutations was done by single stranded conformation polymorphism - SSCP - electrophoresis. Gels were silver stained. C-myc amplification was detected by differential PCR. Steroid receptors were measured by five- point dextrane coated charcoal (DCC) assay. Chi-square and Fisher exact test, as well as Kaplan-Meir and Log-Rank tests were used for statistical evaluation. Results: p53 mutations were detected in 21/100 patients. Totally 25 mutations were found (in 4 patients double mutations were detected). They were not equally spread within examined exons - 10 in exon 5, 6 in exon 6, 8 in exon 7 and only one mutation in exon 8. The incidence of p53 mutations was significantly higher in lymph node-positive than in lyph node-negative patients. Further, p53 mutations were predominantly located in invasive ductal histological type. Since the node-negative patients only one mutation was found, the correlation between detected mutations and the course of disease was examined in lymph node-positive group of patients. Among them, 32/70 patients developed relapse of disease. In the first 24 months of follow-up, patients with p53 mutations had significantly shorter disease free interval (DFI), than the patients without p53 mutations. Also, significantly shorter progression free interval (PFI) in relapsed patients with p53 mutations than in relapsed patients without p53 mutation was found. Concerning overall survival (OS), significantly shorter OS was found in the patients with p53 and/or c-myc gene alterations than in the patients without gene alterations. Conclusion: In conclusion, occurrence of early relapse in chemotherapy treated patients with p53 mutations, supports data about p53 predictive value. The results obtained for OS indicate importance of simultaneous screening for multiple gene alterations in attempt to define specific tumor molecular profile, finally resulting with individual therapeutic approach.