Okužba s človeškimi papilomavirusi pri bolnikih z invertiranimi papilomi nosu in obnosnih votlin ter s pridruženim ploščatoceličnim karcinomom

Jenko, Klemen (avtor), Žargi, Miha (mentor)

Invertirani papilomi (IP) nosu in obnosnih votlin so redki benigni epitelni lokalno agresivni tumorji. Za IP so značilne nagnjenost k ponovitvam, povezava s prehodom v ploščatocelični karcinom (PCK) in zmožnost destrukcije kostnine. Etiologija IP še ni pojasnjena. Podatki številnih raziskav o povezavi okužbe s človeškimi papilomavirusi (HPV) in nastankom IP so nasprotujoči si in še vedno nezadostni, da bi potrdili povezavo. Opazna odsotnost klasičnih genskih sprememb v IP je nakazala možnost, da maligno alteracijo in nastanek PCK v IP lahko povzročajo visokorizični genotipi HPV. Do danes vloga HPV v PCK v IP še ni pojasnjena. Skupino genotipov virusov iz družine Papillomaviridae, ki so pomembni v humani medicini, imenujemo človeški papilomavirusi. Izmed petih rodov, v katere uvrščamo genotipe HPV, je najpomembnejši rod alfa. Glede na onkogeni potencial jih delimo na nizko- in visokorizične genotipe, ki izražajo tropizem za sluznico ali kožo. Povzročajo lahko navadne ali ploščate, kožne ali anogenitalne bradavice, papilome v ustni votlini ali grlu in rak materničnega vratu, zadnjika, penisa, vulve, nožnice, ustnega žrela in nosne votline. Namen naše raziskave je bil ugotoviti prisotnost HPV DNA pri bolnikih z IP, PCK v IP in pri kontrolni skupini. Samo dokaz HPV DNA v IP in PCK v IP ne zadošča za opredelitev HPV kot enega od etioloških dejavnikov v nastanku bolezni. HPV je lahko namreč le naključno prisoten (by stander). Zato smo želeli ugotoviti tudi morebitno integracijo virusne DNA v človeški genom in s tem povezano transkripcijsko aktivnost HPV v IP in PCK v IP. Dodatno smo pri naših bolnikih pridobili podatke o lokalizaciji IP in vplivu vrste kirurškega zdravljenja na ponovitev bolezni. V raziskavo smo vključili biopsijske vzorce 83 bolnikov z IP, 5 bolnikov s PCK v IP in 61 bolnikov kontrolne skupine, pri katerih smo ob operaciji nosnega pretina odstranili tudi del kompenzatorno zadebeljene sluznice v spodnji nosni školjki. Z dvema občutljivima različicama PCR (GP5+/GP6+ in CPI/CPIIg) na 218 tkivnih vzorcih 146 bolnikov z IP, PCK v IP ali kontrolne skupine nam je HPV DNA uspelo dokazati v tkivnih vzorcih 22 bolnikov z IP, 3 bolnikov s PCK v IP in 7 bolnikov iz kontrolne skupine. Ugotovili smo, da je okužba s HPV pri bolnikih z IP ali PCK v IP v nosu in obnosnih votlinah statistično pomembno pogostejša kot v kontrolni skupini. Z metodo ISH mRNA E6/E7 HPV smo transkripcijsko aktivnost dokazali pri dveh bolnikih z IP. HPV je bil pri naših bolnikih z IP redko prisoten v integrirani transkripcijsko aktivni obliki in najverjetneje ni eden od pomembnih dejavnikov v etiologiji IP. Prisotnost HPV DNA pri bolnikih z IP, pri katerih je prišlo do ponovitve IP po kirurškem zdravljenju, ni bila pogostejša kot pri bolnikih, pri katerih ni prišlo do ponovitve bolezni, tako da ni napovedni dejavnik za ponovitev. V naši raziskavi nismo ugotovili statistično pomembnih razlik med pogostostjo HPV DNA med bolniki s PCK v IP in bolniki z IP. Z metodo ISH mRNA E6/E7 HPV smo transkripcijsko aktivnost dokazali pri enem bolniku s PCK v IP. Glede na rezultate ISH mRNA E6/E7 HPV menimo, da HPV najverjetneje ni eden od glavnih etioloških dejavnikov v nastanku PCK v IP, da pa pri PCK v IP kljub temu obstajajo primeri, v katerih je integriran in transkripcijsko aktiven HPV lahko vpleten v etiopatogenezo PCK. S kombinacijo imunohistokemičnega izražanja beljakovine p16 in odsotnosti izražanja beljakovine pRb smo v naši raziskavi potrdili z metodo ISH za E6/E7 mRNA dokazano integrirano in transkripcijsko aktivno okužbo s HPV pri bolniku s PCK v IP. Imunohistokemično izražanje beljakovine p16 je bilo prisotno pri dveh naših bolnikih s PCK v IP. Samo pri enem je bila z metodo ISH za E6/E7 mRNA dokazana integracija in transkripcijska aktivnost. Pri naših bolnikih s PCK v IP glede na samostojen izvid p16 torej ne moremo sklepati o morebitni integraciji in transkripcijski aktivnosti HPV. Metoda izražanja p16 je lahko samo komplementarna metodi ISH za E6/E7 mRNA. Pri naših bolnikih z IP so bile ponovitve po kirurškem zdravljenju z zunanjim pristopom pogostejše kot pri bolnikih, zdravljenih endoskopsko. Odstotek ponovitev je približno enak odstotku ponovitev v objavljenih raziskavah, zato kirurško zdravljenje naših bolnikov z IP lahko ocenimo kot uspešno.

• Jezik: Slovenski jezik
• Ključne besede: invertirani papilomi, človeški papilomavirusi, nos, obnosne votline, ploščatocelični karcinom
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: MF - Medicinska fakulteta
• Leto izida: 2018
• PID: 20.500.12556/RUL-101466
• COBISS.SI-ID: 3911700
• Datum objave v RUL: 07.06.2018

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Human papillomavirus infection in patients with inverted papilloma’s of nose and paranasal sinuses and with associated squamous cell carcinoma

Izvleček

Inverted papilloma (IP) is a rare benign epithelial sinonasal lesion with a propensity for locally invading the surrounding tissues. They have strong tendency to recur and malignant transformation is reported. Pressure erosion of bone may also be apparent. Several studies have proposed human papillomaviruses (HPV) infection to be involved in the etiology of IPs, but the reports are contradictory and still insufficient to prove the connection. The notable absence of conventional genetic alterations in IPs has suggested the possibility of high-risk human papillomavirus (HPV) as an alternative driver of malignant transformation. To date, however, the role of HPV in carcinomatous transformation of IP is unresolved. The group of viruses from Papillomaviridae family which are important in human medicine are named human papillomaviruses. Out of five viral genera in which this family is further divided, the alpha genus is the most important. They are further divided in low-risk and high-risk genotypes which can show tissue tropism or predilection for infecting mucous membrane or skin. They can cause common or flat warts, skin or anogenital warts, mouth papilloma, laryngeal papillomatosis and cancer of the cervix, anus, penis, vulva, vagina, oropharynx and sinonasal tract. The aim of our study was to find out the presence of HPV DNA in IPs, IPs associated with squamous cell carcinoma (IPsSCC) and in control group. However, simply finding HPV DNA cannot distinguish HPV infections that are truly causative from those that are clinically insignificant (ʺpassengerʺ HPV). That is why we wanted to identify integration of viral DNA and HPV transcriptional activity in IPs and in IPsSCC. In our study we have additionally collected the data on location of IPs and the influence of type of the treatment on recurrence after surgical treatment. Our study included tissue samples from 83 patients with IP, 5 patients with IPsSCC and 61 patients from control group in whom we removed a part of inferior turbinate during the surgery for deviated nasal septum. Using two primer sets (GP5+/GP6+ in CPI/CPIIg), PCR amplification was performed on 218 tissue samples from 146 patients with IPs, IPsPCC or control group and HPV DNA was detected in tissue samples from 22 patients with IP, 3 patients with IPsCC and 7 patients from control group. The presence of HPV DNA was statistically higher in patients with IPs and patients with IPsSCC in comparison with the control group. Using the in situ hybridization (ISH) for detecting HPV E6/E7 mRNA transcripts we found transcriptional activity in 2 patients with IP. In our patients with IPs HPV was rarely present in integrated and transcriptionally active form and most probably isn’t one of the important etiological factors of IPs. The presence of HPV DNA in patients with recurrence of IP after surgical treatment was not statistically higher in comparison to patients with no recurrence and is not a risk factor of the recurrence. In our study we did not find statistically significant differences in the presence of HPV DNA between patients with IPs and patients with IPsSCC. Using detection of the HPV E6/E7 mRNA transcripts we have demonstrated integration and transcriptional activity in one patient with IPsSCC. According to the results of ISH mRNA E6/E7 HPV we believe that HPV is not one of the most important etiological factors in development of IPsSCC but furthermore we do believe that there are the cases in which integrated and transcriptionally active HPV can be involved in the etiopathogenesis of IPsSCC. Protein expression was analysed by immunohistochemistry. In our study the combination of high expression of p16 and low expression of pRb additionally confirmed integrated and transcriptionally active HPV infection in patient with IPsSCC. Expression of p16 was immunohistochemically present in 2 patients with IPsSCC. Only one had with ISH mRNA E6/E7 HPV proven integration and transcriptional activity. In our patients with IPsSCC immunostaining for p16 cannot substitute ISH mRNA E6/E7 HPV for HPV testing. Immunostaining for p16 can only be used as a complimentary procedure for HPV testing in IPsSCC. In our patients with IP the recurrences after surgery were more common after external approach than after endoscopic treatment. The percentage of recurrences and treatment success is comparable to published studies and we consider the treatment of our patients as successful.

• Ključne besede: inverted papilloma, human papillomavirus, paranasal sinuses, squamous cell carcinoma