HER2 and c-MYC mutational status and INK4a/ARF methylation status in tumors, tumor margins and unaffected oral mucosa of patients with oral squamous cell carcinoma

Abstract

The development of malignant diseases is due to the accumulation of genetic and epigenetic changes. Oral squamous cell carcinoma (OSCC) is an aggressive and very common malignancy of the oral cavity. Patho-histological methods lack sensitivity in terms of the evaluation of the risk of OSCC recurrence and metastases. This issue can potentially be overcome by assessing molecular changes in OSCC and its margins. Aims: (a) to determine the presence of oncogene amplification (c-MYC and HER2) and tumor suppressor gene methylation (P14 and P16) in tumor, tumor margin and healthy oral mucosa of patients with OSCC; (b) establish a potential association between molecular and clinical parameters. Material and methods: DNA was isolated from tumor, margin and oral mucosa tissue of 40 patients with OSCC, operated at the Clinic for Maxillofacial Surgery, School of Dental Medicine. The presence of C-MYC and HER2 gene amplification was determined by real-time PCR, and P14 and P16 methylation by methyl-specific PCR. Statistical analysis with SPSS was applied to estimate the association between molecular and clinical findings. Results: Tumor tissues showed the highest prevalence of alterations and oral mucosa the lowest. Multiple alterations were significantly more frequent in tumors and tumor margins compared to unaffected oral mucosa (P<0.001 and P=0.027, respectively). HER2 amplification in margin tissue (P < 0.001) and swabs (P = 0.013), as well as the existence of three co-alterations in margins and unaffected oral mucosa were correlated with shorter survival (P = 0.035 and P=0.027, respectively). Conclusion: HER2 amplification, as well as the presence of three co-alterations in margins and unaffected oral mucosa proved to be markers of poor outcome in OSCC.

Razviće malignih oboljenja uslovljeno je akumulacijom genetičkih i epigenetičkih promena. Oralni skvamocelularni karcinom (OSCK) je najčešći malignitet usne duplje. Patohistološkim metodama evaluacije rizika od pojave recidiva i metastaza nedostaje senzitivnost, a taj problem može potencijalno da bude prevaziđen analizom molekularnih promena u OSCK-u i njegovim marginama. Ciljevi: (a) utvrditi prisustvo amplifikacije onkogena (c-MYC i HER2) i metilacije tumor supresorskih gena (P14 i P16) u tumoru, margini i zdravoj oralnoj sluzokoži pacijenata sa OSCK; (b) ustanoviti postojanje asocijacije između molekularnih i kliničkih parametara. Materijal i metode. DNK je izolovana iz tkiva tumora, margina i oralne sluzokože 40 pacijenata sa OSCK-om, operisanih na Klinici za maksilofacijalnu hirurgiju Stomatološkog fakulteta. Prisustvo amplifikacije C-MYC i HER2 gena određeno je metodom PCR u realnom vremenu, a P14 i P16 metilacije metodom metil-specifičnog PCR-a. Statistička analiza SPSS paketom je primenjena za procenu asocijacije između molekularnih i kliničkih nalaza. Rezultati: Najveća učestalost promena pokazana je u tumorskom tkivu, a najmanja u zdravoj oralnoj sluzokoži. Višestruke promene (ko-alteracije) su bile znatno češće u tumorima i tumorskim marginama nego u sluzokoži (P <0.001 odnosno P = 0.027). Amplifikacija HER2 gena u tkivu margina (P <0.001) i sluzokoži (P = 0.013), kao i postojanje tri ko-alteracije u marginama i neizmenjenoj oralnoj sluzokoži korelisane su sa kraćim preživljavanjem (P = 0.035 odnsono P = 0.027). Zaključak: HER2 amplifikacija, kao i prisustvo tri ko-alteracije u marginama i zdravoj oralnoj sluzokoži pokazale su se kao prediktori lošeg ishoda u OSCK-u.