For many decades, cervical cancer prevention has been based on screening with cervical cytology.1 This method has two major drawbacks: high variability in interpretation among cytopathologists and relatively low sensitivity, which requires shorter screening intervals.2 The interpretation of cervical cytology requires experience and long-term training.3
Inevitable factor in development of cervical cancer is infection with high-risk human papillomavirus (HPV)4, but it is not sufficient. However, other cofactors, such as smoking, have been identified to increase the risk of cervical cancer in HPV positive women as well.5, 6 Some European countries have already implemented primary HPV screening in women aged 30–35 years and older due to the higher sensitivity of validated HPV tests compared to cytology, taking into account the lower specificity of HPV tests due to high HPV prevalence in younger women.1, 7
Due to the challenges of cytology and HPV cervical screening, novel biomarkers have been studied. Dual p16/Ki-67 immunostaining (p16/Ki-67 DS) has shown promising sensitivity and specificity for the detection of high-grade cervical intraepithelial neoplasia (CIN2+).8, 9, 10, 11, 12 Tjama
The presence of 1 or more cervical epithelial cell(s) showing p16/Ki-67 double immunoreactivity is defined as a positive test result for p16/Ki-67 DS cytology, independent from morphology interpretation.10 This study has been designed to evaluate the diagnostic accuracy of p16/Ki-67 DS for detection of high-grade cervical intraepithelial neoplasia (CIN2+) and the possible diagnostic role of the number of p16/Ki-67 positive cells. The goal was to determine whether taking a different number of positive cells as the cut-off in the p16/Ki-67 DS test has a statistically significantly different result in detection of CIN2+.
We performed the analysis on a subset cohort of women enrolled within a large-scale HPV self-sampling project within the organised, population-based Cervical Cancer Screening Programme ZORA in Slovenia that was conducted in 2013–2016 in two Slovenian regions.21 The project was approved by the National ethics committee (Approval Nos. 154/03/13, 136/04/14 and 102/11/15). All enrolled women with permanent residence in the Celje region, who had p16/Ki-67 DS of the cervical smear and colposcopy in the Celje General Hospital region were included in the analysis.
Women were invited to colposcopy to Celje General Hospital either due to high-grade cytology or HPV-positive triage test after low-grade cytology or during follow-up after treatment of CIN2+ according to national cervical cancer screening guidelines or due to a positive HPV-self sampling result from an open label, multi-arm trial with a randomised design. A cervical smear was taken prior to the colposcopy. Conventional cytology with split sample technique was used. The first smear was stained with the standard Papanicolaou method and assessed according to national guidelines (Bethesda classification). The second smear was stained with p16/Ki-67 DS (CINtec PLUS, Cytology CE; Ventana Medical Systems, Inc 2015, Tucson, Arizona USA) according to the manufacturer’s instructions.22, 23 All women underwent colposcopy. In the case of an abnormal colposcopy result, a biopsy was taken, and the result was included in the analysis. If the patient had a negative colposcopy, no biopsy was taken, and she was regarded as negative for CIN2+. All patients were managed according to the national guidelines.24 p16/Ki-67 DS was performed in the cytopathology laboratory of the Institute of Oncology Ljubljana and sent to the cytopathology laboratory of Celje General Hospital for assessment. All slides were blinded at the Institute of Oncology Ljubljana and independently assessed by a cytotechnologist and cytopathologist in Celje General Hospital.
The cytopathologist’s result was included in the analysis. A positive reaction was defined as a p16 signal (brown) and a Ki-67 signal (red) present in the same cell with red stained nucleus and brown stained cytoplasm (Figure 1). One dual-stained cell was an indicator of a positive result.10 All evaluators recorded the number of positive or suspicious cells (one to five). A suspicious category was introduced to identify cases that were difficult to interpret. For the purpose of these analyses, suspicious DS results were considered positive, and inadequate as negative.22, 23
Number of p16/Ki-67 DS positive cells and CIN2+ according to Pap test results were calculated. The diagnostic accuracy of p16/Ki-67 DS for the detection of CIN2+ was assessed with sensitivity (true positive rate), specificity (true negative rate), positive predictive value (PPV) and negative predictive value (NPV). The association between the number of p16/Ki-67 positive cells and the detection of CIN2+ was evaluated with Mann–Whitney U test. Statistical analysis was performed with R version 4.0.5. A p value of less than 0.05 was considered statistically significant.
Of 212 enrolled women from the Celje region, 38 were excluded due to the lack of p16/Ki-67 DS, leaving 174 women who had both p16/Ki-67 DS and colposcopy performed to be included in the analysis. The average age of women was 45.1 years. 73 women (42.0%) had a pathologic smear, and 101 women (58.0%) had a normal smear. The types of pathologic smears were high-grade intraepithelial lesion (HSIL) in 29 women (16.7%), ASC-US in 24 women (13.8%), LSIL in 14 women (8.0%), atypical squamous cells-cannot exclude high-grade squamous intraepithelial lesion (ASC-H) in 4 women (2.3%), invasive squamous cell carcinoma in 1 woman (0.6%) and atypical glandular cells, not otherwise specified (AGC-N) in 1 woman (0.6%).
The smear was interpreted as p16/Ki-67 DS positive in 83 women (11 of which were originally evaluated as suspicious) and negative in 91 (1 of which was initially inadequate). The analysis of p16/Ki-67 DS positivity among different smear results is presented in Table 1.
p16/Ki-67 dual immunostaining (p16/Ki-67 DS) positivity and number of positive cells among different smear results
Cervical cytology | Number of p16/Ki-67 positive cells (n, [%]) |
Total | ||||||
---|---|---|---|---|---|---|---|---|
0 (Negative) | 1 | 2 | 3 | 4 | ≥ 5 | 1+ (Total Positive) | ||
Normal | 13 (12.9) | 9 (8.9) | 1 (1.0) | 1 (1.0) | 7 (6.9) | 101 | ||
ASC-US | 0 (0.0) | 1 (4.2) | 2 (8.3) | 1 (4.2) | 6 (25.0) | 24 | ||
LSIL | 3 (21.4) | 0 (0.0) | 0 (0.0) | 1 (7.1) | 3 (21.4) | 14 | ||
AGC-N | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 1 (100.0) | 1 | ||
HSIL | 1 (3.4) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 28 (96.6) | 29 | ||
ASC-H | 0 (0.0) | 0 (0.0) | 1(25.0) | 0 (0.0) | 3 (75.0) | 4 | ||
Inv. cancer | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 1(100.0) | 1 | ||
Total | 17 (9.8) | 10 (5.7) | 4 (2.3) | 3 (1.7) | 49 (28.2) | 174 |
ASC-H = high-grade squamous intraepithelial lesion; AGC-N = atypical glandular cells, not otherwise specified; ASC-US = atypical squamous cells of undetermined significance; HSIL = high-grade intraepithelial lesion; LSIL = low-grade intraepithelial lesion
Among the 83 women with a positive p16/Ki-67 DS result, 17 women (20.5%) had one positive cell, 10 women (12.0%) had two positive cells, 4 women (4.8%) had three positive cells, 3 women (3.6%) had four positive cells, and 49 women (59.0%) had at least five positive cells (Table 1).
Among analysed women, 42/174 (24.1%) had histologically confirmed CIN2+, 92 women (52.9%) had CIN1 or normal histology and 40 (23.0%) women had only colposcopy performed. Among the CIN2+ women, 37 (88.1%) had a p16/Ki-67 DS positive smear, and among the women without CIN2+, 46 (34.8%) had a p16/Ki-67 DS positive smear.
The analysis of the number of p16/Ki-67 DS positive cells according to CIN2+ outcome is presented in Table 2 and Figure 2. Among the 91 women with negative p16/Ki-67 DS, 5 women (5.5%) had CIN2+. Among p16/Ki-67 DS positive women, the risk for CIN2+ was higher in those with more positive cells (p < 0.001: one cell: 2/17 [11.8%], two cells: 0/10 [0.0%]; three cells: 1/4 [25.0%]; four cells: 1/3 [33.3%], five or more cells: 33/49 [67.3%]).
Cervical intraepithelial neoplasia (CIN2+) according to the number of p16/Ki-67 dual immunostaining (p16/Ki-67 DS) positive cells
p16/Ki-67 | Histology | ||
---|---|---|---|
Positive cells | n | < CIN2 n (%) | CIN2+ n (%) |
0 | 91 | 86 (94.5) | 5 (5.5) |
1 | 17 | 15 (88.2) | 2 (11.8) |
2 | 10 | 10 (100.0) | 0 (0.0) |
3 | 4 | 3 (75.0) | 1 (25.0) |
4 | 3 | 2 (66.7) | 1 (33.3) |
≥ 5 | 49 | 16 (32.7) | 33 (67.3) |
Total | 174 | 132 (75.9) | 42 (24.1) |
The diagnostic accuracy of p16/Ki-67 DS for the detection of CIN2+ is presented in Table 3. For the total population, sensitivity was 88.1% (50% for women with ASC-US or LSIL), specificity was 65.2% (61.1% for women with ASC-US and 50% for LSIL), PPV was 44.6% and NPV was 94.5%.
Diagnostic performance of p16/Ki-67 dual immunostaining (p16/Ki-67 DS) according to cytology results and according to different cut-offs (number of positive cells) in detecting cervical intraepithelial neoplasia (CIN2+)
CIN2+(n) | p16/Ki-67 |
||||
---|---|---|---|---|---|
Sensitivity (%, 95% CI) | Specificity (%, 95% CI) | PPV† (%, 95% CI) | NPV‡ (%, 95% CI) | ||
Negative | 66.7 | 70.4 | 6.5 | 98.6 | |
(n = 101) | 3 | (9.4–99.2) | (60.3–79.2) | (0.8–21.4) | (92.3–100.0) |
ASC-US | 6 | 50.0 | 61.1 | 30.0 | 78.6 |
(n = 24) | (11.8–88.2) | (35.7–82.7) | (6.7–65.2) | (49.2–95.3) | |
LSIL | 2 | 50.0 | 50.0 | 14.3 | 85.7 |
(n = 14) | (1.3–98.7) | (21.1–78.9) | (0.4–57.9) | (42.1–99.6) | |
HSIL | 100.0 | 0.0 | 89.7 | ||
(n = 29) | 26 | (86.8–100.0) | (0.0–70.8) | (72.6–97.8) | / |
1+ | 88.1 | 65.2 | 44.6 | 94.5 | |
(n = 174) | 42 | (74.4–96.0) | (56.4–73.2) | (33.7–55.9) | (87.6–98.2) |
2+ | 42 | 83.3 | 76.5 | 53.0 | 93.5 |
(n = 174) | (68.6–93.0) | (68.4–83.5) | (40.3–65.4) | (87.1–97.4) | |
3+ | 42 | 83.3 | 84.1 | 62.5 | 94.1 |
(n = 174) | (68.6–93.0) | (76.7–89.9) | (48.5–75.1) | (88.2–97.6) | |
4+ | 42 | 81.0 | 86.4 | 65.4 | 93.4 |
(n = 174) | (65.9–91.4) | (79.3–91.7) | (50.9–78.0) | (87.5–97.1) | |
5+ | 78.6 | 87.9 | 67.3 | 92.8 | |
(n = 174) | 42 | (63.2–89.7) | (81.1–92.9) | (52.5–80.1) | (86.8–96.7) |
†positive predictive value; ‡negative predictive value; ASC-US = atypical squamous cells of undetermined significance; HSIL = high-grade intraepithelial lesion; LSIL = low-grade intraepithelial lesion; NPV = negative predictive value; PPV = positive predictive value
We evaluated the diagnostic accuracy of p16/Ki-67 DS to detect CIN2+ at different cut-offs defined by the number of positive cells.
Our analysis showed 88.1% sensitivity of p16/ Ki-67 DS for the detection of CIN2+, which is comparable to several other studies that reported sensitivity between 86.4 and 98.2% 3,10,12,25, 26, 27, 28 and somewhat higher than some other reported data, including previous data from our group where were analysed postmenopausal women with low-grade cytology.9, 29, 30 Our group also reported that additional training contributes to higher sensitivity of p16/Ki-67 DS for detecting CIN 2+ without a decrease in specificity.22, 23 Additional analyses showed only 50% sensitivity in women with LSIL, which might reflect the low number of enrolled patients (95% CI: 1.3–98.7%). Other authors reported p16/Ki-67 DS as an effective triage of patients with LSIL.2, 10, 26 Peeters
In our study, the specificity of p16/Ki-67 DS was 65.2%, while the specificity in ASC-US was 61.1% and the specificity in LSIL was 50.0%. Triage studies reported similar results.2, 25, 26, 33, 35, 36 Schmidt
PPV and NPV for the detection of CIN2+ in our study were 44.6% and 94.5%, respectively. Killeen
The major limitation of our study is the small number of participants.
Only one positive cell is required for a positive result of the p16/Ki-67 DS.10 Ziemke reported in his study that using a score of 10 p16/Ki-76 DS positive cells as a positive result instead of one led to significantly higher specificity (89.0
We have observed a statistically significant increase in p16/Ki-67 DS specificity at the cut-off for p16/Ki-67 DS positivity at 3 cells compared to 1 cell, with statistically insignificant decrease in sensitivity (Figure 3). This finding opens a new research question, whether changing the cut-off in p16/Ki-67 DS test could improve performance of p16/Ki-67 DS triage in terms of a further increase in specificity, which would lower the colposcopy referrals even further without a significant loss in longitudinal sensitivity and NPV, which would still enable a safe prolongation of follow-up intervals.