| Abstract | Cilj istraživanja: Istražiti međusobnu povezanost i prognostički značaj ekspresije dvojnog biljega p16/Ki-67, HPV genotipa i citomorfologije kod pacijentica s citološkim nalazom abnormalnih stanica pločastog epitela vrata maternice graničnog, blagog i umjerenog stupnja.
Materijal i metode: U studiju su uključene pacijentice s inicijalnim citološkim nalazom vrata maternice atipičnih skvamoznih stanica neodređenog značenja (ASCUS, N=160), skvamozne intraepitelne lezije niskog stupnja (LSIL, N=155) i nalazom skvamozne intraepitelne lezije visokog stupnja u smislu umjerene displazije (HSIL-M, N=129). Analizirane su citomorfološke osobine koilocitoze, parakeratoze, makrocitoze, nezrele metaplazije i atipične nezrele metaplazije te morfološki tipovi: zreli, metaplastični, nezreli i mješoviti. Detekcija biljega p16/Ki-67 pomoću CINTec PLUS imunocitokemijskog kita (Roche, Švicarska) izvršena je na reprocesiranim citološkim uzorcima. Sve pacijentice testirane su pomoću grupnog visokorizičnog HPV DNA testa (hybrid capture 2, QIAGEN, Njemačka), a za pozitivne nalaze određen je genotip HPV-a pomoću molekularne metode INNO-LiPA HPV Genotyping v2 (Innogenetics, Belgija). Pacijentice su praćene prosječno 26 mjeseci pomoću citologije, kolposkopije i patohistologije te je zabilježena regresija, stagnacija i progresija bolesti.
Rezultati: Najzastupljeniji HPV genotip je HPV 16 (24,32%), a njegova učestalost značajno je veća kod žena mlađih od 30 godina (p<0,001). p16/Ki-67 dvojno bojenje bilo je pozitivno u 46,62% slučajeva, češće u HR HPV pozitivnim u odnosu na HR HPV negativne grupe (p<0,05). Pojedinačni biljezi p16 i Ki-67 u odnosu na HR HPV nalaz značajno su češće izraženi samo u grupi HR HPV pozitivnog ASCUS-a. Slučajevi s HPV 16 genotipom imali su 72,22%, a HPV ne-16 genotipom 52,23% pozitivan nalaz p16/Ki-67, a nađena razlika statistički je značajna samo za grupu LSIL (p=0,015). Makrocitoza i nezrela metaplazija češće se nalaze kod HR HPV negativnog ASCUS-a i LSIL-a (p<0,05). Koilocitoza i parakeratoza nisu pokazale značajne razlike u odnosu na HR HPV status. Odsutnost koilocitoze znatno je češća kod HPV 16 genotipa u odnosu na HPV ne-16 genotip, (p=0,001), a taj nalaz najizraženiji je kod LSIL-a. Parakeratoza se češće nađe u HPV ne-16 grupi, ali razlika je statistički značajna samo kod LSIL-a (p=0,038). Ostale morfološke osobine nisu značajno povezane s prisustvom HPV 16 genotipa. U zrelom morfološkom tipu prevladava nalaz HPV ne-16 genotipa, a p16/Ki-67 dvojno bojenje češće je negativno. Kod HPV 16 genotipa prevladava metaplastični morfološki tip (p<0,001), a pozitivan nalaz p16/Ki-67 dvojnog bojenja češće se nađe u metaplastičnom, nezrelom i mješovitom morfološkom tipu (p=0,001). Nepovoljan ishod bolesti zabilježen je u 25.32 % slučajeva ASCUS-a, 13,79 % LSIL-a i 57,6 % HSIL-M. Progresija bolesti statistički je značajno povezana uz HR HPV pozitivitet (p<0,001), prisustvo HPV 16 genotipa (p<0,001) i p16/Ki-67 pozitivitet (p<0,001). U grupi HSIL-M ishod bolesti nije povezan uz HPV genotip. Prisustvo koilocitoze, parakeratoze, makrocitoze i nezrele metaplazije upućuju na vjerojatan regresivni ishod, ali statistička značajnost u većini grupa nije dostignuta, dok porastom intenziteta nalaza atipične nezrele metaplazije raste i stopa progresije bolesti (p<0,001). Metaplastični, nezreli i mješoviti morfološki tip značajno češće završavaju progresijom kao ishodom bolesti u odnosu na zreli morfološki tip citoloških promjena (p<0,001). Usporedbom svih ispitivanih varijabli nađeno je da je najsnažniji prediktor progresivnog ishoda bolesti p16/Ki-67 dvojno bojenje čiji pozitivitet znači 15 puta veću vjerojatnost za progresiju bolesti u grupi ASCUS, osam puta u grupi LSIL i šest puta u grupi HSIL-M. Pozitivan rezultat HR HPV testa i nalaz HPV 16 genotipa tu vjerojatnost povećavaju tri puta. Povišenje stupnja citološke dijagnoze, nalaz atipične nezrele metaplazije i nezreliji morfološki tipovi od 1,5 do gotovo dva puta povećavaju izglede za progresijom. Nasuprot tome, nalazi nezrele metaplazije i makrocitoze gotovo dva puta smanjuju izglede za progresivnim ishodom bolesti.
Zaključak: Dvojno bojenje p16/Ki-67 pokazalo se kao najjači prediktor progresije bolesti u svim citološkim grupama. HPV 16 genotip povezan je s višim rizikom od progresije u grupama ASCUS i LSIL, ali ne i u grupi HSIL-M. Ispitivane citomorfološke osobine pokazale su najslabiju povezanost s kliničkim ishodom bolesti. |
| Abstract (english) | Objective: To analyse characteristics and prognostic value of p16/Ki-67 dual stain expression, HPV genotype and cytomorphology in the patients with cervical cytology finding of borderline, mild and moderate abnormalities of squamous epithelium.
Material and methods: Patients with initial cytology finding of atypical squamous cells of undetermined significance (ASCUS, N=160), low-grade squamous intraepithelial lesion (LSIL, N=155) and high-grade squamous intraepithelial lesion favour moderate dysplasia (HSIL-M, N=129) were included. Cytomorphological characteristics of koilocytosis, parakeratosis, macrocytosis, immature metaplasia and atypical immature metaplasia and four morphological types were analysed. P16/Ki-67 detection was made using CINTec PLUS immunocytochemical assay (Roche, Switzerland) on reprocessed slides. In all patients HPV DNA test was performed using high risk probe of hybrid capture 2 test (Qiagen, Germany). In positive samples additional HPV genotyping was done using INNO-LiPA HPV Genotyping v2 assay (Innogenetics, Belgium). Patients were followed by cytology, colposcopy and histology for average of 26 months. Three possible clinical outcomes: regression, stagnation or progression was recorded.
Results: The most frequent HPV genotype was HPV 16 (24,32%), and it was found more frequently in women under 30 years of age (p<0,001). P16/Ki-67 dual stain was positive in 46,62% samples, more frequently in HR HPV positive vs. negative cases (p<0,05). Separate positivity of p16 and Ki-67 were found to be more frequent only in ASCUS depending of HR HPV status. Cases with HPV 16 genotype were p16/Ki-67 positive in 72,22% of the cases and cases with HPV non-16 genotype in 52,23%, significantly different only in LSIL (p=0,015). Separate expression of p16 and Ki-67 showed no correlation with HPV genotype. Macrocitosis and immature metaplasia are more frequent findings in HR HPV negative cases in ASCUS and LSIL (p<0,05). Koilocytosis and parakeratosis in LSIL and HSIL-M didn't differ according to HR HPV status. When analysed according to HPV genotype, koilocytosis was more frequent finding in HPV non-16 genotype compared to HPV 16 genotype (p=0.001), and that difference is the most visible in LSIL. Parakeratosis is more frequently found in HPV non-16 genotype cases, but statistically significant difference is found only in LSIL (0.038). Other analysed cytomorphological features didn't show correlation with HPV genotype. p16/Ki-67 expression didn't show association with koilocytosis and parakeratosis. Macrocytosis was more frequently found in the cases with negative p16/Ki-67 dual stain (p<0,001), and immature and atypical immature metaplasia in the cases of positive p16/Ki-67 dual stain (p<0,001). Mature morphologycal type was associated mostly with HPV non-16 genotype and negative p16/Ki-67 dual stain, and metaplastic and immature type with HPV 16 and positive p16/Ki-67.
Unfavourable disease outcome was recorded in 25.32 % of ASCUS, 13.79% LSIL and 57.6% HSIL-M cases. Disease progression was associated with HR HPV positivity (p<0,001), HPV 16 genotype (p<0,001) and p16/Ki-67 positive dual stain (p<0,001). Only in the HSIL-M group HPV genotype didn't showed association with the disease outcome. Separate p16 staining and separate Ki-67 staining showed increased progression rate only in the group of HR HPV positive ASCUS (p=0.046 and p<0.013, respectively). Presence of koilocytosis, parakeratosis, macrocytosis and immature metaplasia are probably more associated with the regression but without statistical significance. But, atypical immature metaplasia was connected with disease progression (p<0,001). So, metaplastic and immature morphological types more frequently progress compared to mature type with regression as the most frequent clinical outcome (p<0,001). Using the logistic regression model the best predictor of the clinical outcome was found to be p16/Ki-67 dual stain. Positivity of p16/Ki-67 increased risk of progression for 15 times in ASCUS, eight times in LSIL and six times in HSIL-M. HR HPV positivity and HPV 16 genotype increased probability for progression for three times, and cytological diagnosis, finding of atypical immature metaplasia and metaplastic and immature morphological types for 1.5 to two times. In contrary, immature metaplasia and macrocytosis increase the probability of negative outcome.
Conclusion: p16/Ki-67 was the best and strongest predictor of the progressive clinical outcome for cytological categories of ASCUS, LSIL and HSIL-M. HPV 16 genotype was associated with the disease progression in ASCUS and LSIL group, but not in HSIL-M group. Cytomorphology features showed the weakest association with the disease outcome. Immature metaplasia and macrocytosis were connected with favourable and atypical immature metaplasia with unfavourable disease outcome. Koilocytosis and parakeratosis was associated mostly with HPV non-16 genotypes, but not with disease outcome. |
