Dejavniki, ki vplivajo na pojav in klinični potek papilomatoze grla

Gluvajić, Daša (avtor), Hočevar Boltežar, Irena (mentor), Poljak, Mario (komentor)

Laringealna papilomatoza (LP) je zelo redka bolezen, pri kateri se pojavljajo papilomi v grlu in je etiološko povezana z okužbo s človeškimi papilomavirusi (angl. human papillomavirus, HPV), saj jo v več kot 90% primerov povzročata HPV-6 in -11. Ker način aktivacije virusa še ni bil opredeljen, še ni povsem jasno, zakaj po okužbi s HPV pride do pojava LP in kateri dejavniki vplivajo na klinični potek bolezni. Ta je nepredvidljiv in pri nekaterih bolnikih lahko poteka agresivno s pogostimi ponovitvami in s širjenjem papilomov v distalne dele dihal. Pri manjšem deležu bolnikov lahko v papilomih nastane visoko-rizična intraepitelijska sprememba (VR-IPS), redko pa lahko pride tudi do nastanka ploščatoceličnega karcinoma v papilomih grla (PCC ex-LP). Namen doktorske naloge je bil opredeliti dejavnike, ki so povezani z agresivnim potekom LP, oceniti incidenco VR-IPS v tkivu papilomov in PCC ex-LP ter opredeliti dejavnike tveganja, ki značilno vplivajo na pojav teh epitelijskih sprememb. V prvem delu longitudinalne retrospektivne raziskave smo na podlagi vzorcev 59 bolnikov z LP s časom sledenja več kot 10 let ocenili možne dejavnike tveganja za agresivni potek bolezni, pri čemer smo se osredotočili na klinične dejavnike, histopatološko stopnjo intraepitelijskih sprememb (IPS), imunohistokemično stopnjo izražanja receptorja za epidermalni rastni faktor (angl. epidermal growth factor receptor, EGFR) in število Langerhansovih celic (LC) v tkivu LP, prejeto adjuvantno zdravljenje in cepljenje proti HPV ter pod(tip) in podtipske različice HPV-6 in -11, ki smo jih opredelili z molekularnimi metodami. Tako smo pri 59 bolnikih z LP, z mediano časa sledenja 18 let (IQR: 14–27) in v povprečju 10,4 (standardni odklon (SD) 9,4, razpon: 2–49) kirurškimi posegi, agresivni potek LP opredelili pri 26/59 (44,1%) bolnikov. Adjuvantno zdravljenje je prejelo 26/59 (44,1%) bolnikov, najpogosteje sistemsko protivirusno zdravljenje z aciklovirjem ali valaciklovirjem (16/26, 61,5%), medtem ko je cepljenje proti HPV prejelo 12/59 (20,3%) bolnikov. Izražanje EGFR smo dokazali v vseh tkivnih vzorcih LP, odvzetih pri prvem kirurškem posegu (58/58, 100,0%), najpogosteje v spodnjih 2/3 epitelija (40/58, 67,8%). Stopnja izražanja EGFR v tkivu LP se ni razlikovala glede na kajenje (p=0,453) ali stopnjo IPS v tkivu (p=0,071), prav tako pa se pri posameznem bolniku med prvim in zadnjim posegom ni spremenila glede na sledeče klinične dejavnike: juvenilna ali odrasla oblika LP (p=0,945), agresivni potek bolezni (p=0,143), prejemanje adjuvantnega zdravljenja (p=0,111) in cepljenje proti HPV (p=0,620). Ugotovili smo tudi, da se stopnja izražanja EGFR v LP ni statistično značilno spremenila v tkivu, ki je bilo odvzeto s kirurškim posegom, ki je sledil adjuvantnemu zdravljenju (sistemsko protivirusno zdravljenje (p=0,257), indol-3-carbinol (I-3-C) (p=1,000), cidofovir (p=1,000)), cepljenju proti HPV (p=0,157) ali v tkivu, odvzetem pri posegu, kateremu je nato sledilo klinično poslabšanje zaradi hitrejše rasti papilomov (p=0,317). Mediana števila LC/mm2 v tkivu LP iz prvega kirurškega posega je bila 139,2 (interkvartilni razmik (IQR): 96–176) in se ni statistično značilno razlikovala glede na kajenje (p=0,951) ali stopnjo IPS (p=0,103). Po prejetem adjuvantnem zdravljenju (sistemsko protivirusno zdravljenje (p=0,173), cidofovir (p=0,327), I-3-C (p=0,225)), cepljenju proti HPV (p=0,142) in poslabšanju poteka s pogostimi ponovitvami LP (p=1,000) se pri posameznemu bolniku število LC/mm2 ni statistično značilno spremenilo. Smo pa v tkivu LP iz zadnjega posega ugotovili statistično značilno višje število LC/mm2 v primerjavi z vzorci, odvzetimi pri prvem posegu: mediana 180,8 (IQR: 123,2–260,8) proti 142,8 (IQR: 96–187,2) (p=0,019). HPV DNA smo v tkivu LP dokazali pri 58/59 (98,3%) bolnikov, najpogosteje genotip HPV-6 (40/58, 68,9%) in (pod)tip HPV-6a (27/40, 67,5%). V dveh ločenih tkivnih vzorcih istega preiskovanca smo pri 96,5% (56/58) bolnikov dokazali enak (pod)tip HPV-6 in -11 in dodatno pri izbranih 15/15 (100,0%) bolnikov enako podtipsko različico HPV v tkivu iz prvega in zadnjega kirurškega posega (mediana 17 let, IQR: 12–24). Pri HPV-6 smo sicer najpogosteje opredelili podtipsko različico linije A (6/12, 50,0%), pri HPV-11 pa podtipsko različico podlinije A2 (7/8, 87,5%). Med naslednjimi preiskovanimi dejavniki: (pod)tip (p=0,544) in podtipska različica HPV (p=0,217), moški spol (p=0,127), kajenje (p=0,356), VR-IPS (p=0,405), višja stopnja izražanja EGFR (p=0,060) in višje število nezrelih LC (p=0,205) v tkivu LP pri prvem kirurškem posegu smo kot statistično značilni dejavnik tveganja za agresivni potek LP opredelili le juvenilno obliko LP, pri kateri so bili relativni obeti za agresivni potek bolezni dvanajstkrat višji v primerjavi z odraslo obliko LP (p<0,001). V drugem delu naloge smo v skupini 163 bolnikov z LP s časom sledenja več kot eno leto opredelili dejavnike tveganja za nastanek VR-IPS v LP in PCC ex-LP, pri čemer smo zraven kliničnih dejavnikov pri vseh bolnikih v vzorcu LP in PCC ex-LP z molekularnimi metodami preverili še prisotnost HPV ter v HPV-pozitivnih PCC ex-LP z in situ hibridizacijo opredelili še integracijo in transkripcijsko aktivnost HPV. Dodatno smo pri izbrani skupini bolnikov opredelili še prisotnost mutacij v gostiteljevih genih (npr. TP53), ki so pogosto prisotne pri malignih tumorjih glave in vratu. Tako smo pri 163 bolnikih z LP s povprečnim časom sledenja 11,4 let (SD 11,3, razpon: 1–62) VR-IPS v LP dokazali pri 35/163 (21,5%) bolnikov, PCC ex-LP pa pri 7/163 (4,3%) bolnikov. VR-IPS se je statistično značilno pogosteje pojavljala v LP pred nastankom PCC ex-LP (p<0,001) in v primerjavi z NR-IPS, predstavlja dvaindvajsetkrat višje relativno tveganje za PCC ex-LP. Odsotnost HPV DNA v LP pri 9/36 (25,0%) bolnikov se je izkazal kot statistično značilen dejavnik tveganja za VR-IPS in/ali PCC ex-LP (p=0,049), HPV-negativna biopsija LP pa, v primerjavi s HPV-pozitivno biopsijo, predstavlja skoraj dvakrat višje relativno tveganje za nastanek VR-IPS in petnajstkrat višje relativno tveganje za PCC ex-LP. Medtem ko je višja starost ob diagnozi LP bila statistično značilen dejavnik tveganja za nastanek VR-IPS (p=0,013) in PCC ex-LP (p=0,017), ostali analizirani potencialni dejavniki tveganja ((pod)tip nizko-rizičnega HPV (NR-HPV) (p>0,005), visoko-rizični HPV (VR-HPV) (p=0,123), odrasla oblika LP (p=0,684), moški spol (p=0,403), agresivni klinični potek LP (p=0,738), višje število posegov (p=0,754), subglotisna rast papilomov (p=0.066), traheotomija (p=0,499), kajenje (p=0,698) in adjuvantno zdravljenje s cidofovirjem (p=0,100)) niso imeli statistično značilnega vpliva na stopnjo epitelijske spremembe v LP. Pri obeh (2/7) bolnikih s HPV-pozitivnim PCC ex-LP smo z in situ hibridizacijo dokazali integracijo HPV v genom gostiteljeve celice, dodatno pa smo pri bolniku z integrirano okužbo HPV-6b v PCC ex-LP dokazali tudi patogeno različico gena TP53. Na podlagi naših rezultatov smo zaključili, da najpomembnejši dejavnik tveganja za agresivni potek LP predstavlja juvenilna oblika LP. Kljub temu, da smo ugotovili, da se EGFR izraža v vseh tkivih LP in da so v papilomih pogoste LC, nismo uspeli opredeliti, kateri dejavniki vplivajo na stopnjo izražanja EGFR in na spremembo števila LC v tkivu LP. Dokazali smo, da pri posameznem bolniku v dveh časovno ločenih tkivnih vzorcih LP vztraja enak genotip HPV in dodatno da pri vseh bolnikih vztraja enaka podtipska različica HPV-6 in -11, ne glede na prejeto adjuvantno zdravljenje, klinični potek bolezni in več kot 10-letni čas sledenja. Nadalje smo ugotovili, da sta najpomembnejša dejavnika tveganja za nastanek VR-IPS v LP in PCC ex-LP višja starost ob diagnozi LP in odsotnost dokazljive HPV DNA v tkivu ter da prisotnost VR-IPS v LP poveča tveganje za nastanek PCC ex-LP. Nenazadnje smo zaključili, da je integracija HPV v gostiteljev genom možna tako pri VR-HPV kot tudi pri NR-HPV, vendar pri slednji na rakavo preobrazbo celic najverjetneje vplivajo še dodatni dejavniki tveganja.

• Jezik: Slovenski jezik
• Ključne besede: laringealna papilomatoza, agresivni potek, juvenilna papilomatoza, ploščatocelični karcinom
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: MF - Medicinska fakulteta
• Leto izida: 2022
• PID: 20.500.12556/RUL-137495
• COBISS.SI-ID: 132298499
• Datum objave v RUL: 19.06.2022

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Factors influencing the occurence and clinical course of laryngeal papillomatosis

Izvleček

Laryngeal papillomatosis (LP) is a very rare disease of the larynx caused by human papillomaviruses (HPV), mainly HPV-6 and 11 in more than 90% of cases. It is not entirely clear what activates the virus to cause papillomas and which are the factors that influence the clinical course of the disease, that is typically unpredictable and can take an aggressive course with numerous recurrences and distal spread in the respiratory system. High-grade squamous intraepithelial lesion (HG-SIL) is found upon histopathological examination of LP in a minority of patients, while squamous cell carcinoma in LP (PCC ex-LP) occurs rarely. Therefore, the goal of this study was to identify the factors that influence the aggressive clinical course of LP, to define the incidence of HG-SIL in LP and PCC ex-LP, and to determine the risk factors for dysplastic and malignant epithelial changes of the mentioned disease. In the first part of the longitudinal retrospective study, risk factors for the aggressive clinical course of LP were analyzed in a cohort of 59 patients with LP and more than a 10-year-long follow-up. An aggressive clinical course was identified in 26/59 (44.2%) patients with an average of 10.4 (standard deviation (SD) 9.4, range: 2–49) surgical procedures for LP and a median follow-up time of 18 years (interquartile range (IQR): 14–27). The investigated factors were clinical data, grade of dysplasia in LP, grade of epidermal growth factor receptor (EGFR) expression and the number of Langerhans cells (LC) in LP, evaluated by immunohistochemistry staining, previously prescribed adjuvant therapy, vaccination against HPV and the (sub)type and genomic variants of HPV-6 and -11, identified using molecular methods. Adjuvant therapy was prescribed to 26/59 (44.1%) patients, most commonly antiviral systemic therapy with acyclovir or valacyclovir (16/26, 61.5%), while 12/59 (20.3%) patients were vaccinated against HPV. EGFR expression was confirmed in all tested LP tissue samples obtained during the first surgical procedure (58/58, 100%), in the majority of LP samples (40/58, 67.8%) in the lower 2/3 of the epithelium. Based on the smoking status of the patients (p=0.453) and grade of SIL in LP (p=0.071), statistically significant difference of EGFR expression in LP was not identified. Compared to the first surgical procedure, no statistically significant change in EGFR expression was identified in the last surgical procedure, considering the following clinical factors: juvenile or adult LP (p=0.945), aggressive clinical course (p=0.143), adjuvant therapy (p=0.111) and vaccination against HPV (p=0.620). Furthermore, EGFR expression did not change in the LP tissue samples surgically obtained after receiving adjuvant therapy (systemic antiviral therapy (p=0.257), indol-3-carbinol (I-3-C) (p=1.000), cidofovir (p=1.000)), vaccination against HPV (p=0.157) or at the beginning of a period with more recurrences of LP (p=0.317). A median of 139.2 (IQR: 96–176) LC/mm2 were detected in the LP tissue samples from the first surgical procedure and no statistically significant difference in LC number was identified based on smoking status (p=0.951) or grade of dysplasia in LP (p=0.103). Similarly, no statistically significant change in the number of LC/mm2 was identified after adjuvant therapy (systemic antiviral therapy (p=0.173), cidofovir (p=0.327), I-3-C (p=0.225)), vaccination against HPV (p=0.142) or at the beginning of a period with more recurrences of LP (p=1.000). Nonetheless, a statistically significant higher number of LC/mm2 was detected in the LP tissue sample from the last surgical procedure compared with the first one (median 180.8 (IQR: 123.2–260.8) versus 142.8 (IQR: 96–187.2) (p=0.019)). HPV DNA was isolated in LP tissue samples of 58/59 (98.3%) patients, most commonly HPV-6 (40/58, 68.9%) and subtype HPV-6a (27/40, 67.5%). In 96.5% (56/58) of patients the same HPV genotype was identified in two different LP tissue samples and in all 15/15 (100%) patients the same genomic variant of HPV-6 or -11 was identified in the tissue samples from the first and last surgical procedure (median 17 years, IQR: 12–24). HPV-6 lineage A (6/12, 50%) and HPV-11 sublineage A2 (7/8, 87.5%) were the most commonly identified genomic variants in LP. The analysis of risk factors for the aggressive clinical course of LP included: HPV (sub)type (p=0.544), genomic variants of HPV-6 and -11 (p=0.217), male gender (p=0.127), smoking status (p=0.356), HR-SIL (p=0.405), higher EGFR expression (p=0.060) and a higher number of LC (p=0.205) in LP tissue samples from the first surgical procedure. However, the only statistically significant risk factor identified was juvenile LP, in which relative odds for the aggressive clinical course were twelve times higher in comparison to the adult LP (p<0.001). In the second part of the study, the incidence and risk factors for HG-SIL and PCC ex-LP in 163 LP patients with a follow-up longer than one year were studied. HPV genotype, identified with molecular methods in LP and PCC ex-LP tissue samples, and clinical factors were analyzed in all LP patients. In the HPV-positive PCC ex-LP tissue samples in situ hybridization (ISH) was performed to identify a transcriptionally active HPV infection. Furthermore, genotyping of the host genes commonly mutated in head and neck tumors (for example TP53) was performed in PCC ex-LP patients. In the group of 163 patients with a mean follow-up time of 11.4 years (SD 11.3, range: 1–62), HG-SIL was identified in 35/163 (21.5%) patients and PCC ex-LP in 7/163 (4.3%) patients. Compared to low-grade-SIL (LG-SIL), HG-SIL was more common in LP before malignant transformation (p<0.001) and represented twenty two fold increase in relative risk for PCC ex-LP. HPV negativity in LP in 9/36 (25.0%) samples was a statistically significant risk factor for HG-SIL and/or PCC ex-LP (p=0.049) and represented, compared to an HPV-positive LP, an almost two-fold increase in relative risk for HG-SIL in LP and a fifteen-fold increase in presenting PCC ex-LP. Older age at diagnosis of LP was a statistically significant risk factor for HG-SIL (p=0.013) and PCC ex-LP (p=0.017), while the other analyzed factors ((sub)type of low-risk HPV (LR-HPV) (p>0.005), high-risk HPV (HR-HPV) (p=0.123), adult LP (p=0.684), male gender (p=0.403), aggressive clinical course of LP (p=0.738), higher number of surgical procedures (p=0.754), subglottic spread of LP (p=0.066), tracheotomy (p=0.499), smoking status (p=0.698) and therapy with cidofovir (p=0.100)), did not have a statistically significant influence on the occurrence of epithelial changes in LP. Using ISH, a transcriptionally active HPV infection was identified in both (2/7) patients with HPV-positive PCC ex-LP and additionally genotyping has identified a mutation of the TP53 gene in the patient with a transcriptionally active HPV-6b infection in PCC ex-LP. Based on the results of our research, we can conclude that juvenile LP is the most important risk factor for the aggressive clinical course of LP. We have confirmed that in LP tissue the EGFR is expressed and LC are common, but we have failed to identify the factors that influence the grade of EGFR expression and the change in the number of LC. In the majority of patients, we have found that the same HPV genotype persists in two different LP tissue samples of the same patient. Furthermore, in a selected group of patients, the same HPV-6 and -11 genomic variants in the LP tissue samples from the first and last surgical procedure were confirmed. The identified genomic variants did not change based on clinical course, adjuvant therapy and long-term follow-up of more than 10 years. Our study has identified older age at LP diagnosis and HPV negativity in LP as the most important risk factors for HG-SIL in LP and PCC ex-LP. Moreover, HG-SIL in LP is an important risk factor for malignant transformation in LP. We have identified integration in the host genome of both HR- and LR-HPV DNA in PCC-ex LP, but most probably in cases of LR-HPV infection, malignant transformation is dependent on additional carcinogenic factors.

• Ključne besede: laryngeal papillomatosis, aggressive course, juvenile papillomatosis, squamous carcinoma