1273 patients with complete results for HPV genotyping and liquid-based cytology tests met the initial inclusion criteria. Of these 1273 patients, 79 were excluded for no available cervical biopsy results. Of the remaining 1194 patients, 106 underwent endocervical curettage, and 52 consisted of vaginal HPV and cytology tests followed by vaginal biopsy. 1194 cases were available for evaluation, including 534 diagnosed with no intraepithelial lesion or malignant lesion (NILM), 289 diagnosed with CIN1, 173 diagnosed with CIN2, 160 diagnosed with CIN3 and 38 diagnosed with cancer. Patient ages ranged from 30 to 60 years old, and the median age was 40 years old.
The 1194 cases comprising the HPV16/18+ (n = 606), other HPV + and ASCUS (n = 339), and LSIL (n = 381, including 105 with HPV16/18 + and 249 with other HPV+) were analyzed. The associations between HPV genotype, cytology and corresponding histology are listed in Table 1. For HPV16/18+, CIN2 + was confirmed in 32.2% (195/606) of cases. For other HPV + and ASCUS, CIN2 + was confirmed in 20.4% (69/339) of specimens. For LSIL, CIN2 + was confirmed in 24.6% (87/354) of cases. Of the 1194 cases, 76 cases showed discrepant results among different IHC markers and were adjudicated by a third pathologist. Finally, 516 (43.2%), 336 (28.1%), 348 (29.1%), 369 (30.9%), and 255 (21.4%) samples were positive for p16, Ki-67, RELA, YAP1 and SMAD3, respectively. The positive rates of p16, Ki-67, RELA, YAP1 and SMAD3 were also significant different in biopsy < CIN2 and CIN2 + cases (p < 0.05, Fig. 2). The positive rate of each combination of p16, Ki-67, RELA, YAP1 and SMAD3 staining was also determined. There were 285 (23.9%), 255 (21.4%), 243 (20.4%), 162 (13.6%), 312 (26.1%) and 345 (28.9%) cases that were positive for p16 combined with Ki-67, p16 combined with RELA, p16 combined with YAP1, p16 combined with SMAD3, p16 combined with YAP1 or RELA, and p16 combined with YAP1 or SMAD3 or RELA, respectively (Table 2). The sensitivity and specificity of each marker or combination of potential markers for predicting biopsy CIN2 + are also shown in Table 2. The sensitivity, specificity and Youden’s Index of p16 combined with Ki-67 for predicting CIN2 + were 62.1%, 89.5% and 51.6%, respectively. However, the addition of SMAD3, YAP1 and RELA to p16 as a concurrent stain provided a better sensitivity (70.9%, vs. p16 + combined with Ki-67+, p = 0.184), considerable specificity (89.5%, vs. p16 + combined with Ki-67+, p = 1) and better Youden’s Index (60.4%, vs. p16 + combined with Ki-67+) for p16 + combined with YAP1 + and/or RELA+ (p16+, YAP1+/RELA+), better sensitivity (72.8%, vs. p16 + combined with Ki-67+, p = 0.102), considerable specificity (86.4%, vs. p16 + combined with Ki-67+, p = 0.255) and better Youden’s Index (59.2%, vs. p16 + combined with Ki-67+) for p16 + combined with YAP1 + and/or SMAD3 + and/or RELA+ (p16+, YAP1+/SMAD3+/RELA+) in the colposcopy referral population (except for HSIL).
Table 1
Association between HPV genotyping, cytology, and histology in the enrolled patients
| Histology | | |
| NILM | CIN 1 | CIN 2 | CIN 3 | Ca | Total | Rate of CIN2+ (%) |
HPV16/18+ | 318 | 93 | 78 | 87 | 30 | 606 | 32.2 |
HPV16/18+, NILM | 153 | 18 | 9 | 16 | 18 | 222 | 23.0 |
HPV16/18+, ASCUS | 129 | 48 | 39 | 54 | 9 | 276 | 35.9 |
HPV16/18+, LSIL | 36 | 27 | 30 | 12 | 0 | 105 | 40.0 |
other HPV+, ASCUS | 204 | 66 | 39 | 30 | 0 | 339 | 20.4 |
other HPV+, LSIL | 117 | 87 | 21 | 24 | 0 | 249 | 18.1 |
LSIL | 153 | 114 | 51 | 36 | 0 | 354 | 24.6 |
Total | 639 | 246 | 138 | 141 | 30 | 1194 | 25.9 |
Table 2
The positive rate of p16, Ki-67, SMAD3, YAP1 and RELA expression and association with biopsy CIN2 + in the enrolled patients.
| Positive rate n (%) | Sensitivity (%) | Specificity (%) | Youden’s Index (%) |
p16+ | 516(43.2) | 82.5 | 70.5 | 53.0 |
Ki-67+ | 336(28.1) | 62.1 | 83.7 | 45.8 |
RELA+ | 348(29.1) | 70.9 | 85.4 | 54.6 |
YAP1+ | 369(30.9) | 68.9 | 82.4 | 51.3 |
SMAD3+ | 255(21.4) | 41.7 | 85.8 | 27.5 |
p16+, Ki-67+ | 285(23.9) | 62.1 | 89.5 | 51.6 |
p16+, RELA+ | 255(21.4) | 63.1 | 93.2 | 56.3 |
p16+, YAP1+ | 243(20.4) | 61.2 | 93.9 | 55.1 |
p16+, SMAD3+ | 162(13.6) | 35.0 | 93.8 | 28.8 |
p16+, YAP1+/RELA+ | 312(26.1) | 70.9 | 89.5 | 60.4 |
p16+, YAP1+/SMAD3+/RELA+ | 345(28.9) | 72.8 | 86.4 | 59.2 |
Moreover, the performances of the five markers for predicting CIN2 + in different subgroups are shown in Table 3. Of the 606 cases with HPV16/18 + in an HPV genotyping test, the sensitivity and specificity of p16 + combined with Ki-67 + for predicting CIN2 + were 67.7% and 87.6%, respectively, whereas those of RELA + were 76.9% (p = 0.240) and 90.5% (p = 0.439), respectively, and p16 + combined with YAP1 + and/or RELA+ (p16+, YAP1+/RELA+) were 69.2% (p = 0.850) and 91.2% (p = 0.326), respectively. Of the 339 cases that were diagnosed with other HPV + combined with ASCUS, the combination of p16+, YAP1+/RELA + produced a better performance for predicting CIN2+, which had better sensitivity (86.7% vs. 52.2%, p = 0.028) and considerable specificity (86.7% vs. 90.0%, p = 0.486) than were achieved by p16 + combined with Ki-67+. In addition, in the LSIL group, RELA + also produced better sensitivity (82.8% vs. 58.6%, p = 0.043) and considerable specificity (92.1% vs. 88.8%, p = 0.444) than were achieved by p16 + combined with Ki-67+. Moreover, the RELA + provided the Max AUC area (0.902, 0.837 and 0.875) in the colposcopy referral population (except for HSIL) (Fig. 4A, Table S1), HPV16/18 + group (Fig. 4B, Table S2) and other HPV + combined with ASCUS group (Fig. 4C, Table S3), respectively. The (p16+, YAP1+/RELA+) provided the Max AUC area (0.847) in the LSIL group (Fig. 4D, Table S4).
Table 3
The association between p16, Ki-67, SMAD3, YAP1 and RELA expression and biopsy CIN2 + in each subgroup.
| HPV16/18+ | | | other HPV+, ASCUS | | | LSIL | |
| Sensitivity (%) | Specificity (%) | Youden’s Index (%) | | Sensitivity (%) | Specificity (%) | Youden’s Index (%) | | Sensitivity (%) | Specificity (%) | Youden’s Index (%) |
p16+ | 81.5 | 65.7 | 47.2 | | 82.6 | 75.6 | 58.2 | | 89.7 | 69.7 | 59.4 |
Ki-67+ | 73.8 | 75.2 | 49 | | 60.9 | 80 | 40.9 | | 62.1 | 82.0 | 44.1 |
RELA+ | 76.9 | 90.5 | 67.4 | | 52.2 | 76.7 | 28.9 | | 82.8 | 92.1 | 74.9 |
YAP1+ | 64.6 | 89.1 | 53.7 | | 69.6 | 82.2 | 51.8 | | 75.9 | 74.2 | 50.1 |
SMAD3+ | 40.0 | 89.8 | 29.8 | | 34.8 | 84.4 | 19.2 | | 51.7 | 82.0 | 33.7 |
p16+, Ki-67+ | 67.7 | 87.6 | 55.3 | | 52.2 | 90.0 | 42.2 | | 58.6 | 88.8 | 47.4 |
p16+, RELA+ | 64.6 | 95.6 | 60.2 | | 52.2 | 88.9 | 41.1 | | 72.4 | 97.8 | 70.2 |
p16+, YAP1+ | 55.4 | 95.6 | 51 | | 65.2 | 94.4 | 59.6 | | 69.0 | 89.9 | 58.9 |
p16+, SMAD3+ | 33.8 | 94.9 | 28.7 | | 26.1 | 95.6 | 21.7 | | 48.3 | 91.0 | 39.3 |
p16+, YAP1+/RELA+ | 69.2 | 91.2 | 60.4 | | 82.6 | 86.7 | 69.3 | | 72.4 | 87.6 | 60 |
p16+, YAP1+/SMAD3+/RELA+ | 72.3 | 86.9 | 59.2 | | 82.6 | 80.0 | 62.6 | | 72.4 | 84.3 | 56.7 |
The follow-up interval for 711/885 cases which were diagnosis of NILM and CIN1 ranged from 36 to 60 months, contained 93 cases of (p16+, YAP1+/RELA) and 618 cases of others except for (p16+, YAP1+/RELA+). During the follow-up period, 45 cases were developed to CIN2+ (Fig. 3). 38, 21, 34,18, 17, 15, 32, 14, 15, 37 and 37 of the 45 cases were positive for p16+, Ki-67+, RELA+, YAP1+, SMAD3+, (p16+, Ki-67+), (p16+, RELA+), (p16+, YAP1+), (p16+, SMAD3+), (p16+, YAP1+/RELA+) and (p16+, YAP1+/SMAD3+/RELA+) at the initial IHC staining. The (p16+, YAP1+/RELA+) provided the Max AUC area (0.878) for CIN2 + predicting of NILM and CIN1 group in the colposcopy referral population (except for HSIL) (Fig. 4E, Table S5).