LAUSR

Dear Editor, Although human papillomavirus (HPV) testing has been implemented for primary cervical cancer screening in many countries, primary cytology screening with Pap test still remains the most widespread despite its lower sensitivity. HPV testing has much higher sensitivity but is not very helpful in young women due to the high prevalence of infections, the majority of them being transient. Therefore, an additional test that would identify young women with high risk for high-grade cervical intraepithelial lesion (HSIL) would be very helpful. In the recent years, the number of studies reporting on the performance of p16/Ki-67 dual immunostaining in the detection of cervical intraepithelial neoplasia (CIN) 2+ lesions in women with low-grade cytologic changes has been increasing. The first studies indicate that the 5-year risk of cervical precancer in women with negative p16/Ki-67 reaction is low. According to the American Society for Colposcopy and Cervical Pathology guidelines, women with ASC-US cytology should have HPV testing and if this test is positive, and they should be referred to colposcopy. In women between 21 and 24 years of age, a repeat cytology in 12 months is preferred. Women with low-grade cervical intraepithelial lesion (LSIL) cytology and negative HPV testing should have repeat cytology in 12 months. If HPV test is positive or not available, they should be referred to colposcopy. Women with ASC-H cytology should be referred to colposcopy regardless of HPV status. Young women with repeated low-grade Pap test results represent a sensitive population group, and possible overtreatment can be harmful for the forthcoming pregnancies. Preterm birth, premature rupture of the membranes, low birth weight, and caesarean sections have been reported after cervical excisional treatment. On the other hand, the regression rate of CIN 2 is high in this age group. Also, younger women with biopsy-proven HSIL are more likely to have CIN 1 or no dysplasia after excisional biopsy. With this study, we aimed to evaluate the performance of dual p16/Ki-67 immunostaining in women under 30 years with repetitive low-grade ASC cytology. A retrospective analysis on prospectively collected samples was performed. All patients under 30 years of age who attended the national cervical cancer screening program and had their cervical smears examined in General Hospital Celje in a 1-year period (January 1, 2017-December 31, 2017) with repeated LSIL/ASC cytological results were included in the study. The Bethesda system for reporting cervical cytology was used for the interpretation of Pap smears. The list of patients was obtained from our computerized database together with subsequent histology results. The Pap smears were destained and p16/Ki-67 dual immunostaining was performed using CINtec PLUS (CINtec PLUS, Cytology CE; Ventana Medical Systems, Inc 2015, Tucson, Arizona) according to the manufacturer's instructions. A positive reaction was defined as p16 signal (brown) and Ki-67 signal (red) present in the same cell with red-stained nucleus and brown-stained cytoplasm (Figure 1 red arrow). The blue arrow in Figure 1 shows a negative reaction. One dual-stained cell was an indicator of positive result and the result of immunostaining was independent of morphological criteria. The slides were assessed by a cytopathologist and a cytotechnologist who were blinded for the final pathology results. Statistical analysis was performed using the SPSS software version 22.0 (IBM, Armonk, New York). A total of 53 patients met the inclusion criteria. Of which, 4 patients (7.5%) had their Pap smear interpreted as atypical squamous cells of undetermined significance (ASC-US), 16 patients (30.2%) as atypical squamous cells—cannot exclude HSIL (ASC-H), and 33 patients (62.3%) had LSIL. Then, 19 patients (35.8%) had negative p16/Ki-67 staining and 34 patients (64.2%) had positive p16/Ki-67. Positive immunostaining was present in 75% of patients with ASC-US, 75% of patients with ASC-H, and 58% of patients with LSIL (Table 1). In total, 79.2% of patients with histologically confirmed CIN2+ had positive p16/Ki-67 immunostaining. Of which, 51.7% of patients with subsequent CIN1 or no dysplasia had positive p16/Ki-67 staining (χ 4.300, P .038), and 24 patients had histologically proven CIN2+ on end histology. Among the patients with the final diagnosis of CIN2+, 12.5% had a previous cytologic diagnosis of ASC-US, 41.7% ASC-H, and 45.8% LSIL (Table 2). CIN2+ was the final histological diagnosis in 75% of patients with ASC-US, 62.5% of patients with ASC-H, and 33.3% of patients with LSIL (Table 2). The sensitivity and specificity of p16/Ki-67 immunostaining for the detection of CIN2+ were 79.2% and 48.3%, respectively. The positive and negative predictive values were 55.9% and 73.7%, respectively. These results indicate the rather high rate of false-positive p16/Ki-67 immunostaining in women under 30 years. On the other hand, given the relatively high sensitivity, it could be argued that young women with repetitive LSIL/ASC cytology and negative p16/Ki-67 could be followed without immediate referral for more Received: 3 August 2019 Revised: 2 October 2019 Accepted: 23 October 2019