Liquid-based cytology - new possibilities in the diagnosis of cervical lesions [Tekuća citologija - nove mogućnosti u dijagnostici lezija vrata maternice]

Jurič, Danijela and Mahovlić, Vesna and Rajhvajn, Sanda and Ovanin-Rakić, Ana and Škopljanac-Mačina, Lada and Barišić, Ana and Šamija Projić, Ivana and Babić, Damir and Suša, Martina and Ćorušić, Ante and Orešković, Slavko (2010) Liquid-based cytology - new possibilities in the diagnosis of cervical lesions [Tekuća citologija - nove mogućnosti u dijagnostici lezija vrata maternice]. Collegium Antropologicum, 34 (1). pp. 19-24. ISSN 0350-6134

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Abstract

Liquid-based cytology (LBC) enables the use of supplementary methods in the diagnosis and prognosis of cervical lesions. The aim of this study was to analyze the correlation between p16INK4a immunoexpression in ThinPrep cervical cytologic samples and human papillomavirus (HPV) detection by polymerase chain reaction (PCR) from the same sample. LBC-ThinPrep (Cytyc, USA) cervical cytology samples, prepared and stained by Papanicolaou method, were analyzed using modified Bethesda cytologic classification named »Zagreb 2002«. A second ThinPrep slide, prepared from the same sample, was immunostained for p16INK4a using CINtec p16INK4a Cytology Kit (DakoCytomation, Denmark). Increased expression of the high-risk (HR) HPV E6 and E7 oncogenes results in a highly specific increase in p16 protein expression and overexpression of p16INK4a acts as a potential biomarker for cervical cancer progression from premalignant lesions. Brown nuclear and/or cytoplasmic staining of abnormal cells was considered a positive result. Residual material was used for 13 HR HPV-DNA detection by the PCR based AMPLICOR HPV test (Roche Molecular Systems). A total of 120 ThinPrep Pap tests with the following cytologic diagnoses: 17 within normal limits, 17 atypical squamous cell (ASC) (7 ASC of undetermined significance /ASCUS/ and 10 ASC of high-grade squamous intraepithelial lesions cannot be excluded /ASC-H/), 26 low-grade squamous intraepithelial lesions (LSIL) corresponding cervical intraepithelial neoplasia (CIN) I, 57 high-grade SIL (HSIL) i.e. 24 CIN II and 33 CIN III and 3 squamous cell carcinoma (SCC) were included in the study. All CIN III (n=33) and SCC (n=3) specimens expressed p16INK4a immunoreactivity, whereas the HR HPV test was positive in 97% (32/33) of CIN III and 100% (3/3) of SCC specimens. The p16INK4a biomarker was positive in 87.5% (21/24) of CIN II and 69% (18/26) of CIN I, while the HR HPV was positive in 75% (18/24) of CIN II and 50% (13/26) of CIN I. In ASCUS cytology, p16INK4a and HR HPV showed the same rate of positivity (28.5%; 2/7). Expression of p16INK4a was detected in all cytologic (10/10) ASC-H lesions, in contrast to HR HPV detected in only 20% (2/10) of ASC-H cases. These data suggest the p16INK4a evaluation in ThinPrep cervical samples to be significantly associated with HR HPV testing by PCR in the same sample for the diagnosis of HSIL lesions and cervical carcinomas. A prospective study with longer follow up may clarify the predictive values in the management of LSIL and ASC diagnosis.

Abstract in Croatian

Metoda tekuće citologije (engl. »liquid-based cytology«, LBC) omogućuje primjenu dopunskih metoda u dijagnostici i prognozi lezija vrata maternice. Cilj rada bio je usporedba imunocitokemijske ekspresije biomarkera p16INK4a u LBC-ThinPrep cervikalnim uzorcima u odnosu na HPV tipizaciju metodom polimeraze lančane reakcije (engl. »polymerase chain reaction«, PCR). Ukupno 120 ThinPrep (Cytyc, USA) cervikalnih uzoraka obojenih metodom po Papanicolaouu primarno su se analizirali primjenom citološke klasifikacije »Zagreb 2002«, a na drugom ThinPrep preparatu istoga uzorka napravljena je procjena ekspresije biomarkera p16INK4a pomoću CINtec p16INK4a Cytology Kit (DakoCytomation, Denmark). Povećana ekspresija onkogena E6 i E7 humanog papilomavirusa visokog rizika (engl. »high-risk human papillomavirus«, HR HPV) rezultira porastom ekspresije proteina p16INK4a, čiji se pozitivitet očitava pojavom smeđih granula u jezgri i/ili citoplazmi abnormalnih stanica. Preostali LBC uzorak rabio se za HR HPV-DNA tipizaciju metodom PCR (AMPLICOR, Roche). Citološki je klasificirano: 17 negativnih nalaza, 17 atipičnih pločastih stanica (ASC) (7 ASC -neodređenog značenja /ASCUS/ i 10 ASC- ne može se isključiti skvamozna intraepitelna lezija visokog stupnja /ASC-H/), 26 skvamoznih intraepitelnih lezija niskog stupnja (LSIL) tj. cervikalnih intraepitelnih neoplazija (CIN) I, 57 SIL visokog stupnja (HSIL) koji uključuju 24 CIN II i 33 CIN III, te 3 karcinoma pločastih stanica. Imunocitokemijska reakcija na p16INK4a bila je pozitivna u svim CIN III lezijama (11/11) i karcinomima (3/3), dok je HR HPV tipiziran u 97% (32/33) CIN III i 100% (3/3) karcinoma. Citološki je identificiran p16INK4a u 87,5% (21/24) CIN II, te 69% (18/26) CIN I, dok je HR HPV bio pozitivan u 75% (18/24) CIN II i 50% (13/26) CIN I. ASCUS promjene imale su istu stopu pozitiviteta na p16INK4a i HR HPV (28,5%; 2/7), dok je u ASC-H promjenama p16INK4a bio pozitivan u svim slučajevima (100%, 10/10), a HR HPV je tipiziran u svega 20% (2/10) lezija. Na temelju naših prvih rezultata može se zaključiti da imunocitokemijska ekspresija biomarkera p16INK4a u ThinPrep cervikalnim uzorcima usko korelira s HR HPV tipiziranim metodom PCR iz istoga uzorka u otkrivanju HSIL lezija i karcinoma vrata maternice, dok su u procjeni LSIL i ASC lezija potrebna daljnja morfološko-molekularna praćenja.

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