ASCUS is a term that abnormal characteristics of cells are significantly more than inflammatory cell changes, but the number and quality were not enough to diagnose CIN. Frequency of ASCUS lesions is between 1.6 and 9.0% [
24]. Our previous research found that Pathology of 160 ASCUS included inflammation, CIN lesions at all levels and cervical cancer [
25]. Marwa Fakhreldin et al. [
26] retrospectively analyzed 297 cases of ASCUS and found that pathology of ASCUS contained normal cervical tissue, CIN1–3 and cervical cancer. In view of this, if women with ASCUS can’t be treated properly, high grade cervical intraepithelial neoplasia lesions or cervical cancer lesions will be missed. However, as found in this study, CIN2+ just only accounts for 18.0% and CIN3 + for 8.3% and most of ASCUS are CIN1 or cervical inflammation, ASCUS will be overtreated if all women with ASCUS undergo colposcopy. For different tests in our study, the positive rates were statistically different in different cervical lesions, which can be confirmed by many studies [
27,
28]. The positivity of HPV DNA test is 85.3%, HPV E6/E7 mRNA testing is 62.7% and p16/Ki67 immunocytochemistry is 32.0%, thus if we use HPV E6/E7 mRNA testing or p16/Ki67 immunocytochemistry instead of HPV DNA assay, the rate of colposcopy referral will be reduced. A low positivity rate can be translated into a low referral rate for colposcopy which is very appealing in a triage setting.
The present study also evaluates the performance of HPV E6/E7 mRNA testing, p16/Ki67 immunocytochemistry and HPV DNA assay in triaging women with ASCUS in a cross-sectional clinical setting. HPV E6/E7 mRNA testing and p16/Ki67 immunocytochemistry were identified in recent studies to increase specificity for the detection of high grade cervical disease compared to HPV DNA detection [
29]. In women with HR-HPV-positive ASCUS and LSIL, sensitivity and specificity of p16/Ki67 immunocytochemistry for detection of CIN3 were 90.6 and 48.6%, respectively [
22]. Five eligible studies were identified in a meta-analysis, which found that in the ASCUS subgroup, taking CIN2+ as the endpoint, sensitivity ranged from 0.64 to 0.92 (p16/Ki67 test) versus 0.91 to 0.97 (HPV DNA test); specificity ranged from 0.53 to 0.81 versus 0.26 to 0.44, respectively [
30]. Nicolas Wentzensen et al. [
22] suggested that the sensitivity and specificity of p16/Ki67 immunocytochemistry for detection of CIN2+ were 85.5 and 59.4% respectively and had a sensitivity close to HR-HPV DNA testing
(P = 0.32), but a higher specificity(
P = 0.0001). Increasing the p16/Ki67 threshold to two or more dual stain-positive cells led to a substantial reduction in test positivity compared with cytology and the dual stain assay at the usual cutoff. At a cutoff of five or more dual stain-positive cells, the sensitivity for detection of CIN3+ was statistically significantly lower compared with both cytology and the dual stain assay at the usual cutoff, while the specificity and the PPV were statistically significantly increased [
31]. In our study, the specificity of p16/Ki67 immunocytochemistry for detection of CIN2+ was higher than HPV DNA test (82.5% vs. 17.5%, χ
2 = 208.13,
P < 0.001). These finding support that p16/Ki67 can be a viable option for ASCUS triage. In a review that compared HPV E6/E7 assay versus HPV DNA test (Hybrid Capture 2 method) in triage of women with ASCUS or LSIL cervical cytology, the pooled sensitivity and specificity of HPV E6/E7 assay to triage ASCUS to detect underlying CIN3 or worse was 96.2% (95% CI: 91.7–98.3%) and 54.9% (95% CI: 43.5–65.9%), respectively. HPV E6/E7 assay and HPV DNA test showed similar pooled sensitivity; however, the specificity of the former was significantly higher (ratio: 1.19; 95% CI: 1.08–1.31 for CIN2+) [
32]. In our study similarly found that HPV E6/E7 mRNA testing have greater specificity than HPV DNA test (42.7% vs. 17.48%, χ
2 = 37.147,
P < 0.001), while the sensitivity is similar (98.2% vs. 87.0%). HPV assays for detecting the mRNA of 5 hrHPV types may reduce the over-diagnosis of women who have minor cytologic abnormalities. However, given the lower sensitivity, women with negative mRNA test results cannot be considered free of CIN2+ and require further surveillance [
33]. It is important to consider the trade-off between sensitivity and specificity of the diagnostic test when designing screening algorithms [
34]. The data presented in this study confirm these previous observations reported by our previous research that HPV E6/E7 mRNA testing have higher specificity than HPV DNA test (40.3, 95%CI: 32.4–48.8 vs. 15.1, 95%CI: 10.3–22.7) [
25]. These results showed greater specificity without loss in sensitivity for HPV E6/E7 mRNA testing in triage of ASCUS compared to HPV DNA assay.
From our study, we can see that both p16/Ki67 immunocytochemistry and HPV E6/E7 mRNA testing have higher specificities than HPV DNA test, when we group analysis by 30 years old, the accuracy of each test is higher in the group over 30 years old. However, we didn’t find a study that solve such a problem: which is better in triaging ASCUS for p16/Ki67 immunocytochemistry and HPV E6/E7 mRNA testing. Our present data suggested that p16/Ki67 immunocytochemistry has a higher sensitivity than HPV E6/E7 mRNA testing (82.5, 95%CI: 77.3–86.8 vs. 42.7, 95%CI: 36.7–48.9). ROC curve was used to further demonstrate the diagnostic performance of p16/Ki67 immunocytochemistry, HPV E6/E7 mRNA testing and HPV DNA test for detecting CIN2+, we can see that the AUC of p16/Ki67 immunocytochemistry is the largest (AUC = 0.903, 95%CI: 0.866–0.940). The figure found that when the optimal cut-off value of HPV E6/E7 mRNA testing is selected at 882.53 copies/ml, the specificity of HPV E6/E7 mRNA testing is improved. Tong-Yu Liu’s and Ye-li Yao’s finding also supported this result [
23,
35]. However, the diagnostic performance of HPV E6/E7 mRNA testing was still lower than p16/Ki67 immunocytochemistry. Miriam Reuschenbach et al. [
28] conducted a clinical experiment which compare the accuracy of CINtec p16INK4a-cytology, HPV E6/E7 mRNA and HPV DNA, they found that the specificity to detect high grade dysplasia was highest for CINtec p16INK4a-cytology (60.6% (52.7–68.0) in CIN3+ and 74.8% (65.5–82.3) in CIN2+), followed by HPV E6/E7 mRNA (56.4% (48.4–64.0) in CIN3+ and 71.2% (61.7–79.2) in CIN2+) and HPV DNA (49.1% (41.3–56.9) in CIN3+ and 63.4% (53.7–72.1) in CIN2+). As we all know, some normal cells may express p16, observation of p16-positive cells in cytology preparations requires additional morphologic evaluation to achieve adequate specificity. An assay of p16/Ki67 has been developed that combines staining for p16 with staining for the proliferation marker Ki67 on cytological slides. Theoretically, co-expression of p16 and Ki67 in the same cell should indicate HPV-related transformation obviating the need for morphological interpretation and the diagnostic accuracy of p16/Ki67 is higher than p16 alone. In summary, we can give the answer that p16/Ki67 immunocytochemistry may be better in triaging ASCUS than HPV E6/E7 mRNA testing.