Učestalost visokorizičnih tipova humanoga papiloma virusa u karcinomima usne šupljine u bolesnika bez rizičnih čimbenika

Dediol, Emil (2013) Učestalost visokorizičnih tipova humanoga papiloma virusa u karcinomima usne šupljine u bolesnika bez rizičnih čimbenika. PhD thesis, Sveučilište u Zagrebu.

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Abstract

Oral cavity cancer is usually the result of heavy smoking and longstanding alcohol consumption. Only around 10% of oral cancer patients neither smoke nor drink. In western countries, there is a rising trend in oral cancer in those who neither smoke nor drink alcohol. Human papilloma virus (HPV) has already been linked to oropharyngeal cancer, especially in those who neither drink nor smoke. This relationship between HPV and oral cancer has not yet been established, with previous results being controversial. We have hypothesised that the frequency of high risk HPV types, especially types 16 and 18, is increased in non smokers non drinkers (NSND) who develop oral cancer. This would therefore link HPV involvement in oral carcinogenesis in this population. Only patients with oral squamous cell carcinoma treated primarily with surgery in the Department of Maxillofacial Surgery, University Hospital Dubrava, Zagreb, from 1995 to 2010 were included in the study. Patients were divided into two groups. The first group consisted of smokers of more than 10 pack/year who were also daily drinkers. The second group consisted of NSND and included those of less than 10 pack/year active smokers. HPV status, age, gender, anatomic locations of the tumour, stage, second primary tumours, recurrence and survival were compared between the two groups. HPV was detected directly with PCR method and indirectly with p16INK4a immunohistochemistry. There were 77 non smokers non drinkers and 77 controls (smokers and drinkers, SD). Women were more frequent among NSND group (75%) versus more males among SD (90%), p<0,001. The age range was greater among NSND, with the youngest patient 24 years old and the oldest one 90 years old. More than half of the patients were younger than 45 or older than 70 years. The SD group were middle aged (45-69 years) in 79%. The tongue was the most frequent tumour site in oral cavity among NSND (42%) followed by the sublingual region (17%), upper gum (13%) and lower gum (12%). In the control group (smokers and drinkers), sublingual cancer was the most frequent site (39%), followed by tongue cancer (34%) and then retromolar cancer (17%). The NSND group had smaller cancers (T1 and T2) without regional metastasis (N0), although there was no statistical significance. No difference in recurrence or second primary tumour (SPT) was observed between the two patient groups. HPV detected by PCR was less frequent among the NSND group. Only 22% of NSND were positive for HPV, compared with 31% of SD. HPV was positive in 27% of all oral cancer patients which is similar to previous published results. Men were more frequently HPV positive (68%) than women. HPV positive patients tended to be younger than HPV negative patients (56 years vs 60 years) but without statistical significance. Regional metastasis (N) correlated significantly more with HPV positivity (39% vs 23%) although there was no difference in primary tumour (T) or stage. Similar rate of SPT was observed between the two groups. HPV positive patients had a greater incidence of recurrences and worse survival, but this was not statistically significant. If p16INK4a expression is considered as surrogate marker of HPV infection, we have confirmed the hypothesis of the study that high risk HPV groups are more frequently seen in NSND oral cancer patients than in SD patients with oral cancer. p16INK4a was positive in 27% of NSND and only 10% of SD (p=0,0064). However, PCR results did not support the above hypothesis and the overall correlation between p16INK4a and HPV was weak. All p16INK4a positive patients should be HPV positive if it is sensitive as a surrogate marker, but this was the case in only 7 out of 29 patients. 22 out of 29 patients were p16INK4a positive and HPV negative. Based on our findings and those of other studies, we cannot support the use of p16INK4a immunostaining as the only test for HPV in oral cancer. No statistically significant difference was observed between p16INK4a positive and negative patients according to age, sex, anatomic tumour locations, stage, SPT or HPV status. A higher rate of recurrences was associated with stronger p16INK4a expression, and this trend was also observed in survival curves. Stronger p16INK4a expression had worse survival outcomes, particularly among tongue cancers. Both HPV PCR and p16INK4a may be extrapolated for survival and prognostic purposes. When both are positive, survival outcomes are worse while best survival is associated with negative HPV PCR and p16INK4a. NSND that is HPV positive has 2.8 hazard ratio and strong p16INK4a expression carries 2.9 hazard ratio for the same population. According to this study, correlation between HPV and p16INK4a is very weak. In clinical practice, patients should be routinely tested for HPV using PCR and p16INK4a immunohistochemistry in order to give us valuable information regarding their prognosis.

Abstract in Croatian

Karcinom u usnoj šupljini obično nastaje kao posljedica dugogodišnjeg konzumiranja alkohola i pušenja cigareta. Samo u oko 10% slučajeva nalazimo karcinom usne šupljine kod bolesnika koji ne konzumiraju alkohol i ne puše cigarete. Unatoč smanjenom trendu pušenja i konzumiranja alkohola primjećen je porast karcinoma usne šupljine. Humani papiloma virus (HPV) je već povezan s orofaringealnim karcinomom, pogotovo kod nepušača i nealkoholičara. Takva povezanost još uvijek nije sa sigurnošću utvrđena za karcinom usne šupljine kod nerizične populacije. Hipoteza istraživanja je da je učestalost visokorizičnih tipova HPV-a, poglavito tipova 16 i 18, veća kod nerizične populacije s karcinomom usne šupljine nego kod bolesnika s tipičnim rizičnim čimbenicima, što bi ukazalo na povezanost HPV-a i karcinoma usne šupljine kod nerizične populacije. Svi uključeni bolesnici su primarno kirurški liječeni na Klinici za kirurgiju lica, čeljusti i usta, KB Dubrava, Zagreb od 1995. g. do 2010. g. i imali su histološki dokazan planocelularni karcinom u usnoj šupljini. Podijeljeni su u dvije grupe: oni koji svakodnevno konzumiraju alkohol i/ili puše više od 10 kutija na godinu (kontrole) i oni koji nemaju te rizične navike u anamnezi (ispitanici). Obje skupine bolesnika su uspoređene s obzirom na HPV status, dob i spol, preživljenje, pojavu drugih primarnih tumora, recidiva te lokalizacije u usnoj šupljini gdje se karcinom pojavljuje. U dokazivanju prisutnosti HPV-a koristili smo se indirektnim dokazivanjem imunohistokemijskom metodom usmjereno na protein p16INK4a te direktnim dokazivanjem PCR genotipizacijom virusa metodom amplifikacije i analize DNA. Bilo je 77 ispitanika i 77 randomiziranih kontrola. Među ispitanicima su prevladavale žene (75%), dok su među kontrolama prevladavali muškarci (90%) te je razlika bila statistički značajna (p<0,001). Raspon dobi među ispitanicima je bio veći. Najmlađi bolesnik je imao 24 a najstariji 90 godina te su, gledano po dobnim skupinama, nešto više od polovice činili mlađi od 45 i stariji od 70 godina. Među kontrolama su pak prevladavali bolesnici srednjih godina (79%). Ispitanici su imali i nešto drugačiju raspodjelu karcinoma u usnoj šupljini. Najčešći je bio jezik (42%), zatim sublingvalno (17%) te gingiva maksile (13%) i mandibule (12%) dok su kontrole imale najčešće sublingvalni karcinom (39%), karcinom jezika (34%), retromolarni karcinom (17%). Ispitanici su se nešto češće javljali s manjim tumorima (T1 i T2) bez regionalnih metastaza (N0). Nije bilo razlike u stadiju, broju recidiva ili drugih primarnih tumora. Suprotno hipotezi nije bilo statistički značajne razlike u HPV PCR statusu. Čak su kontrole bile pozitivnije na HPV od ispitanika (31% prema 22%). Ukupno je 27% bolesnika s karcinomom usne šupljine bilo pozitivno na HPV. Muškarci su češće bili HPV pozitivni (68%). HPV pozitivni su bili nešto mlađi od negativnih, ali ne statistički značajno (56 prema 60 godina). HPV pozitivni bolesnici su češće imali regionalne metastaze (39% prema 23%). U veličini primarnog tumora, ukupnom stadiju i drugim primarnim tumorima nije bilo značajne razlike. Iako su u pojavi recidiva i preživljenju HPV pozitivni bolesnici imali nešto lošiji ishod, nije bilo statistički značajne razlike. Ako shvatimo ekspresiju p16INK4a kao indirektni pokazatelj HPV infekcije onda se hipoteza istraživanja potvrdila, odnosno p16INK4a je bio pozitivan kod 27% ispitanika te 10% kontrola (p=0,0064), tj. HPV je u karcinomu usne šupljine češći kod nepušača i onih koji ne konzumiraju alkohol. Međutim rezultati PCR analize nisu ovo potvrdili niti u velikoj mjeri korelirali s rezultatima imunohistokemijskog ispitivanja. Svi koji su p16INK4a pozitivni bi trebali biti i HPV pozitivni PCR-om. To je bio slučaj kod samo sedam bolesnika dok ih je 22 bilo p16INK4a pozitivno, a HPV negativno. Može se dakle zaključiti da za karcinom usne šupljine p16INK4a imunohistokemijsko dokazivanje nije pouzdan indirektni test za HPV. Nije bilo statistički značajnih razlika između p16INK4a pozitivnih i negativnih u spolu, dobi, dobnim skupinama, anatomskim lokalizacijama tumora, TNM stadiju, broju drugih primarnih tumora ili HPV statusu. Značajno su više recidiva imali oni koju pokazivali jaču ekspresiju p16INK4a i bili su povezani sa značajno lošijim preživljenjem, a pogotovo karcinomi jezika. I HPV i p16INK4a imaju određenu prediktivnu ulogu u preživljenju. Kada su pozitivna oba, preživljenje je najlošije za razliku od slučaja kada su oba negativna. Osobito negativni prognostički predznak nose za bolesnike bez rizičnih čimbenika. Rizik od umiranja je čak 2,8 puta veći ako je HPV pozitivan, a 2,9 puta veći ako tumor pokazuje izrazitu p16INK4a eskpresiju. S obzirom da prema ovoj studiji ne koreliraju, trebalo bi u kliničkoj praksi određivati i HPV direktnom metodom (PCR ili ISH) i p16INK4a imunohistokemijski jer time možemo dobiti vrijedne podatke o prognozi bolesnika.

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