Abstract: Background and purpose: At present, there is a lack of effective shunt measures for the patients with negative cervical cytology and positive high-risk human papilloma virus (HR-HPV). At the same time, it is not clear whether the patients with low-grade squamous intraepithelial lesion (LSIL) will progress to high-grade cervical lesions. The purpose of this study was to explore the role of immunocytochemical p16/Ki-67 double staining in the shunt of cervical cytology negative and HR-HPV positive patients and the predictive value of LSIL outcome. Methods: From Apr. 2017 to Jul. 2018, 708 married women over the age of 30 were selected from Shantou Central Hospital and Sun Yat-sen University Cancer Center. Cervical cancer screening was negative, and HR-HPV was positive in those women. All patients underwent primary colposcopy and p16/Ki-67 double staining. The relationship between the results of p16/Ki-67 double staining and the histological results of primary colposcopy was observed. The efficacy of p16/Ki-67 double staining in the diagnosis of cervical intraepithelial neoplasia grade 2/3 (CIN2/CIN3) was discussed. The difference in the positive rate of p16/Ki-67 double staining between different HPV subtype infection groups was compared. Two hundred and twenty-three patients with LSIL were followed up for one year. The predictive value of p16/Ki-67 double staining test results for the outcome of LSIL was analyzed. Results: Under biomicroscopy, the results of double staining of p16/Ki-67 in cervical cells were divided into two categories: negative and positive. The incidence of CIN2+ was 33.33% (55/165) in the patients with positive double staining of p16/Ki-67 and 1.10% (6/543) in the patients with negative double staining of p16/Ki-67 (χ ² =166.94，P<0.001). The incidence of CIN3+ was 26.06% (43/165) in patients with positive double staining of p16/Ki-67 and 0.37% (2/543) in patients with negative double staining of p16/Ki-67 (χ ² =140.35，P<0.001). χ ² trend test showed the positive rate of p16/Ki-67 in the other 12 subtypes of HPV, 16/18 subtypes of HPV and the simultaneous positive group was increased (χ ² =29.119, P<0.001). The positive rates of p16/Ki-67 double staining were significantly different among the three groups (P<0.01). Among 8 cases of LSIL with progressive disease, 6 cases were positive for p16/Ki-67, and 2 cases were negative for p16/Ki-67. The positive rate of p16/Ki-67 double staining was 3 times higher than the negative rate of p16/Ki-67 double staining. Among 22 cases of LSIL with persistent disease, 12 cases were positive for p16/Ki-67, and 10 cases were negative for p16/Ki-67. The persistence rate of p16/Ki-67 double staining positive diagnosis of LSIL was 1.2 times that of p16/Ki-67 double staining negative diagnosis. Among 29 patients with regression of LSIL, 3 were positive for p16/Ki-67, and 26 were negative for 16/Ki-67. The regression rate of p16/Ki-67 double staining positive diagnosis of LSIL was 0.12 times that of p16/Ki-67 double staining negative diagnosis. Conclusion: p16/Ki-67 double staining test has high efficiency in the diagnosis of CIN2/CIN3. The positive rate of p16/Ki-67 double staining test is related to high-risk HPV infection subtypes. p16/Ki-67 double staining test is an effective method for cases with negative cervical cytology and positive HR-HPV. It has high predictive value for the prognosis of tissue LSIL and potential for clinical application.

Key words: Immunocytochemistry, p16/Ki-67, Double staining, Cervical cell, High-risk human papilloma virus, Tissue, Cervical intraepithelial neoplasia