The Value of P16 Protein Detection in the Screening for High‐Grade Squamous Intraepithelial and Higher Lesions of the Cervix by the Combined Detection of HR‐HPV and TCT

ABSTRACT

Liquid‐based thin layer cytology (TCT) and HR‐HPV detection are the most important screening methods for cervical cancer. These two methods have limited sensitivity and specificity, so some cervical lesions are still missed or misdiagnosed. This paper mainly discusses the value of P16 protein detection in cervical cancer screening. In particular, it is effective and practical in high‐grade squamous epithelial and above cervical lesions (CINII+). In this retrospective study, the diagnostic specificity and positive predictive value (PPV) of P16 protein detection for cervical CINII+ lesions were significantly higher than that of TCT and HR‐HPV detection, and the accuracy was the highest. P16 protein detection can also reduce the rate of missed diagnoses in HR‐HPV‐negative patients and reduce unnecessary colposcopic biopsies. Our data highlight the feasibility and significance of P16 protein detection in cervical disease screening.

Abbreviations

ASCCP

American Society for Colposcopy and Cervical Pathology

ASC‐H+

high‐grade squamous intraepithelial lesions and cervical squamous cell carcinoma

AUC

area under the curve

HR‐HPV

high‐risk human papillomavirus

HSIL

high‐grade squamous intraepithelial lesions

LSIL

lower‐grade squamous intraepithelial lesion

NILM

negative for intraepithelial lesions or malignancy

NPV

negative predictive value

PPV

positive predictive value

SCC

squamous cell carcinoma

TCT

thinprep cytologic test

1. Introduction

Cervical cancer is a gynecological malignancy that poses a serious threat to women's health. It has a high incidence and mortality rate, with a trend toward younger incidence. As it is a global health problem, the World Health Organization currently proposes an elimination threshold of 4 cases per 100,000 women by 2030 through vaccination, screening, and treatment of pre‐invasive and invasive cervical cancer [1]. Cervical cancer is the only malignant tumor with a clear etiology, high‐risk human papillomavirus (HR‐HPV). Persistent infection with HR‐HPV is an important factor in the occurrence and development of cervical cancer, but precancerous lesions will exist for a long time after infection with HR‐HPV, and early screening and effective treatment can reverse precancerous lesions and save patients' lives [2, 3]. Therefore, early screening for cervical cancer is of great significance in reducing the incidence and mortality rate of cervical cancer. How to improve the sensitivity and specificity of screening, increase the detection rate and accuracy of precancerous lesions, and reduce misdiagnosis and underdiagnosis is the key to the prevention and treatment of cervical cancer. According to the 2019 American Society for Colposcopy and Cervical Pathology (ASCCP) risk‐based management consensus guidelines for abnormal cervical cancer screening tests and cancer precursors, for non‐pregnant patients aged 25 years and older, when the immediate risk of CIN3+ is ≥ 60% (such as cytology HSIL, HPV16+), rapid treatment is recommended as a priority; treatment is performed directly without colposcopic biopsy [4]. The proposal of this management program has undoubtedly put more diagnostic pressure on cytology diagnosticians, especially for the direct diagnosis of cytology HSIL+. Currently, HR‐HPV combined with thinprep cytologic test (TCT) is the main screening method for cervical cancer [5]. However, clinical studies [6, 7] have shown that HR‐HPV testing cannot differentiate between transient and persistent infection, which can be potentially harmful and lead to unnecessary colposcopies and biopsies, especially in young women, where HPV infection usually resolves spontaneously [8]. TCT detection is limited by the level of the reader, and there are many small cells with high nuclear‐to‐plasma ratios in cytology, making it impossible for the pathologist to accurately interpret high‐grade lesions, resulting in an overdiagnosis of atypical squamous cells of undetermined significance (ASC‐US), which have unclear clinical significance, and the reproducibility is also low. The excessive colposcopic referral rate during combined HR‐HPV and TCT screening creates a large psychological and economic burden for patients. Unnecessary invasive tests and overtreatment of some reversible lesions may adversely affect disease progression and reproductive outcomes. It is evident that an adjunct with high specificity is needed to compensate for the shortcomings of combined TCT and HR‐HPV testing and to more accurately identify patients at high risk or potential for cervical cancer and precancerous lesions. P16 (p16INK4A), encoded by CDKN2A, is an important regulator of the cell cycle. As a tumor suppressor protein, down‐regulation of P16 expression is usually associated with an increased risk of cancer [9]. Relevant studies [10] have shown that the overexpression of P16 protein is closely associated with HR‐HPV integration status, viral oncogene activation expression, and virus‐induced cell cycle dysregulation, thus indicating an increased risk of cervical cancer. Therefore, P16 protein detection is an alternative method for cervical cancer screening [11, 12, 13, 14, 15, 16]. To verify the efficacy and utility of P16 protein in the detection of high‐grade squamous epithelial lesions and above lesions of the cervix(CINII+), in this paper, we retrospectively analyzed the data of TCT‐positive (ASC‐US and above defined as cytologically positive, ASC‐US+) and/or high‐risk HPV‐positive cases, all of which had the results of P16 protein detection, TCT, and high‐risk HPV test. We compared the efficacy of these methods in screening for the detection of CINII+ with histopathological diagnosis as the gold standard and analyzed the value of P16 protein detection in cervical pre‐cancer lesions and cervical cancer screening, with the aim of providing a more convenient and efficient screening method.

2. Materials and Methods

2.1. Participants

TCT‐positive and/or high‐risk HPV detection‐positive patients of women at Hunan Maternal and Child Health Hospital from January 2024 to December 2024 who followed the following inclusion and exclusion criteria, with P16 protein detection results and cervical histopathological diagnostic data within 6 months, were selected for the study. A total of 300 cases that met the above criteria were included in the study.

Inclusion criteria: (1) age ≥ 20 years; (2) history of sexual intercourse; (3) non‐menstrual, non‐lactation, non‐pregnancy; (4) no history of definite cervical disease, surgery, and pelvic radiotherapy; (5) no coitus or vaginal medication 2 days before the examination.

Exclusion criteria: (1) those who have been diagnosed with cervical cancer, precancerous lesions, or postoperative review of cervical cancer; (2) those who have combined acute inflammation of the reproductive system; (3) those who have combined other malignant tumors; (4) those who have combined autoimmune diseases or cognitive disorders; (5) those who have been in pregnancy or postpartum for < 8 weeks.

The age of the included subjects ranged from 20 to 67 years old, with a mean age of (44.22 ± 12.66); 275 were married and 25 were unmarried. The study was approved by the Medical Ethics Committee of Hunan Maternal and Child Health Hospital, and all subjects signed an informed consent form.

2.2. Specimen Collection

There was no sexual intercourse, vaginal examination, or vaginal medication for 2 days before specimen collection. After emptying the bladder, the patient should take a conventional position, and the cervical discharge was swabbed with a sterile cotton swab without applying acetic acid or iodine solution.

Liquid‐based cytology (TCT) sampling: Gently insert the brush in the sampler into the cervical orifice, tighten the outer edge of the cervical orifice, and rotate it in a clockwise direction for 5–10 turns to collect the exocervical orifice and exocervical cells in the cervical canal, place the collected exocervical specimen into a special preservation bottle for preservation, and screw the cap on tightly; the specimen can be used for both TCT and P16 protein detection.

HPV sampling: A special HPV high‐risk sampling swab was used, which was left in the cervical orifice for 1 min, and then the sampling swab was quickly put into a special EP tube for storage.

2.3. Liquid‐Based Cytology (TCT) Preparation and Diagnosis

Two thin smears were prepared using the liquid‐based preparation method, one for Pap cytology staining and the other for P16 protein detection. According to the third edition of the Bethesda system (TBS) [9], squamous epithelial lesions were classified into: Negative intraepithelial lesion or malignancy (NILM), Atypical squamous epithelial cell (atypical epithelial cell), ASC‐US, low‐grade squamous intraepithelial lesion (LSIL), Atypical squamous cell cannot rule out high‐grade lesion (ASC‐H), High‐grade squamous intraepithelial lesion (HSIL), and squamous cell carcinoma (SCC). In this study, the diagnosis was defined as negative when the result was NILM, and the rest of the results were judged as positive, and ASC‐H and above lesions were defined as high‐grade squamous intraepithelial lesion (ASC‐H+). Due to the relative rarity of cervical adenoepithelial lesions, only squamous epithelial lesions were selected for this study.

2.4. HR‐HPV Testing

Thirteen high‐risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) were detected using the second‐generation hybridization capture test. The second‐generation hybridization capture kit (Digene, USA) was applied in this study. Diagnostic criteria: HPV DNA relative light unit (RLU)/RLu of standard positive control in the samples ≥ 1.0, which indicated that the detected HPV load was ≥ 1.0 pg/mL, which was considered HR‐HPV positive.

2.5. P16 Protein Detection and Interpretation

Automatic immunohistochemical staining system (SY7300, etc.) (Shenzhen Senying Biotechnology Co., LTD.) was used for P16 staining of cytological specimens of patients, and specific operations were carried out according to the instructions of the P16 antibody staining kit. After preparing the slides, primary antibody, secondary antibody, and 3,3‐diaminobenzidine(DAB) color developing solution were added successively, followed by dehydration, transparency, and finally sealing the slides. Microscopic examination was performed. All specimens were individually reviewed by 2 pathologists with more than 5 years of experience. Test results: Brown‐yellow cytoplasm or nucleus of one or more epithelial cells is positive, and colorless cytoplasm and nucleus of epithelial cells are negative.

2.6. Colposcopic Cervical Biopsy for Histopathological Examination

It was performed 3–4 days after menstrual cleansing. Using a photoelectric integrated electronic colposcope for examination, first observe the appearance of the cervix, morphology, color, and the presence of bleeding, etc.; if there is no suspected lesion, then perform routine cervical sampling biopsy at points 3, 6, 9, and 12, and perform multi‐area and multi‐point sampling if there is any abnormality. All specimens were fixed in 3.7% neutral formaldehyde, routinely dehydrated, embedded in paraffin, sectioned, and stained with HE. Pathological sections were read and diagnosed by two senior pathologists, and the histological morphology was observed by light microscopy, and the results were referenced to pathological diagnostic criteria [11], which classified cervical squamous epithelial tissues into normal, CIN I, CIN II, CIN III, and squamous carcinoma according to the degree of lesions. In this stud, CIN IIincluding normal, inflammation, and CIN I was judged as negative (CIN II‐); ≥ CIN II including CIN II, CIN III, and squamous carcinoma was judged as positive (CIN II+). The diagnosis of TCT and histopathology was made by senior pathologists respectively.

2.6.1. Statistical Processing

SPSS 24.0 statistical software was applied to analyze the data. Counts and percentages were used for general statistical description, and mean ± standard deviation (x ± s) were taken as measurement data. The chi‐square test was used to compare whether the difference between P16, TCT, and HR‐HPV tests in identifying CIN II+ was statistically significant. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of P16, TCT, and HR‐HPV tests were evaluated using histopathologic test results as the gold standard. The receiver operating characteristic (ROC) curves were plotted, the area under the curve (AUC) was calculated, and the comparisons were made with the Z‐test. p < 0.05 was regarded as the statistically significant difference.

3. Results

3.1. Analysis of P16, TCT, and HR‐HPV Results and Pathological Findings

Of the 476 patients, 338 were positive for any of the P16, TCT, and HR‐HPV results, and a total of 300 colposcopies were recalled in 6 months. 36 cases were positive for P16, with a positivity rate of 12.0%, and the coincidence rate with the biopsy results was 91.3% (274/300). 205 cases were positive for TCT, with a positivity rate of 68.3%, and a coincidence rate with biopsy results was 39.0% (117/300). 220 cases were positive for HR‐HPV, with a positivity rate of 73.3%, and the coincidence rate with the biopsy results was 35.7% (107/300). Among the three tests, P16 had the lowest positivity rate but the highest compliance rate with biopsy results, and the difference was statistically significant (p < 0.05, Table 1).

TABLE 1.

Comparison of three methods in the detection of cervical lesions.

Methods	Groups	Histopathologic biopsy results [n (%)]			χ 2	p
		NILM	LSIL	ASC‐H+
P16	Negative	164 (62.1)	90 (34.1)	10 (3.8)	96.8	< 0.001
	Positive	6 (16.6)	10 (27.8)	20 (55.6)
TCT	Negative	75 (78.9)	16 (16.8)	4 (4.2)	28.2	< 0.001
	Positive	95 (46.3)	84 (41.0)	26 (12.7)
HR‐HPV	Negative	60 (75.0)	18 (22.5)	2 (2.5)	16.4	< 0.001
	Positive	110 (50.0)	82 (37.3)	28 (12.7)

Abbreviations: ASC‐H+, high‐grade squamous intraepithelial lesions and cervical squamous cell carcinoma; HR‐HPV, high‐risk human papillomavirus; LSIL, lower‐grade squamous intraepithelial lesion; NILM, negative for intraepithelial lesions or malignancy; TCT, thinprep cytologic test.

3.2. Analysis of the Positive Rate of P16 Protein Detection in Different TCT Results and Histopathological Results

According to the TCT results, 300 patients were divided into the NILM group (95 cases), ASC‐US group (107 cases), LSIL group (48 cases), and ASC‐H+ group (including ASC‐H, HSIL, and SCC) (50 cases). The positive rates of P16 in each group were 0.00%, 1.87%, 12.50%, and 56.00%, respectively. The positive rate of P16 increased with the severity of cytological lesions (P trend < 0.001, Table 2). According to the histopathological results, 300 patients were divided into the CINII‐ group (including inflammation, normal, and CINI) with 270 cases and the CINII+ group (including CINII, CINIII, and SCC) with 30 cases. The positive rates of P16 in the two groups were 5.93% and 66.67%, respectively. The positive rate of P16 protein detection in CINII+ was significantly higher than that in CINII‐, and the difference was statistically significant (p < 0.001, Table 3).

TABLE 2.

Positive rate of P16 protein detection in different TCT results.

TCT	n	P16+ n (%)
NILM	95	0 (0.0)
ASC‐US	107	2 (1.9)
LSIL ASC‐H+	48 50	6 (12.5) 28 (56.0)

Abbreviations: ASC‐H+, high‐grade squamous intraepithelial lesions and cervical squamous cell carcinoma; ASC‐US, atypical squamous cells ofundetermined significance; NILM, negative for intraepithelial lesions or malignancy; TCT, thinprep cytologic test.

TABLE 3.

Positive rate of P16 protein detection in histopathological results.

Histology	n	P16+ n (%)
< CINII (Negative)	270	16 (5.9)
≥ CINII (Positive)	30	20 (66.67)

Abbreviations: < CINII, negative for intraepithelial lesions or malignancy and low‐grade squamous intraepithelial lesions; ≥ CINII, high‐grade squamous intraepithelial lesions and cervical squamous cell carcinoma.

3.3. Analysis of the Efficiency of P16, TCT, and HR‐HPV in Screening CIN II+

We calculated the sensitivity, specificity, PPV, NPV and accuracy of P16, TCT, and HR‐HPV screening for CIN II+. The sensitivities of P16, TCT, and HR‐HPV screening for CIN II+ were 66.7%, 86.7%, and 93.3%, respectively, of which HR‐HPV had the highest, but the differences among the three were not statistically significant. The specificity and PPV of P16 were the highest, higher than those of TCT (33.7% and 12.7%) and HR‐HPV (28.9% and 12.7%), respectively, and the difference was statistically significant (p < 0.001). The accuracy of P16 screening for CIN II+ was 91.3%, higher than that of TCT (39.0%) and HR‐HPV (35.3%), and the difference was statistically significant (p < 0.001, Tables 4 and 5).

TABLE 4.

Three methods of screening results statistics.

Methods	Groups	Histopathologic biopsy results [n (%)]		Total (n)
		Negative (n = 270)	Positive (n = 30)
P16	Negative	254 (96.2)	10 (3.8)	264
	Positive	16 (44.4)	20 (55.6)	36
TCT	Negative	91 (95.8)	4 (4.2)	95
	Positive	179 (87.3)	26 (12.7)	205
HR‐HPV	Negative	78 (97.5)	2 (2.5)	80
	Positive	192 (87.3)	28 (12.7)	219

Abbreviations: HR‐HPV, high‐risk human papillomavirus; TCT, thinprep cytologic test.

TABLE 5.

Three methods of screening effectiveness analysis.

Methods	Sensitivity/%	Specificity/%	PPV/%	NPV/%	Accuracy/%
P16	66.7	94.1	55.6	96.2	91.3
TCT	86.7	33.7	12.7	95.8	39.0
HR‐HPV	93.3	28.9	12.7	97.5	35.3

Abbreviations: HR‐HPV, high‐risk human papillomavirus; NPV, negative predictive value; PPV, positive predictive value; TCT, thinprep cytologic test.

3.4. ROC Curve Analysis of P16, TCT, and HR‐HPV Methods

Using the results of cervical biopsy with CINII+ as the dependent variable (0 = no; 1 = yes), ROC curve plotting and Z‐test analysis were performed based on the results of TCT, HR‐HPV, and P16, with sensitivity as the vertical axis and the false‐positive rate (1‐specificity) as the horizontal axis. The AUCs of the P16, TCT, and HR‐HPV assays were 0.804, 0.602, and 0.611, respectively; among them, P16 had the highest area and Youden's index, indicating that it had the highest diagnostic efficacy (Table 6, Figure 1).

TABLE 6.

ROC curve analysis of three methods.

Methods	AUC	SE	95% CI		Youden's index
			Lower limit	Upper limit
P16	0.804	0.053	0.700	0.907	0.608
TCT	0.602	0.049	0.506	0.699	0.205
HR‐HPV	0.611	0.047	0.518	0.704	0.222

Abbreviations: 95% CI, 95% confidence interval; AUC, area under the curve; HR‐HPV, high‐risk human papillomavirus; SE, standard error; TCT, thinprep cytologic test.

FIGURE 1.

ROC curves of three methods. [Color figure can be viewed at wileyonlinelibrary.com]

3.5. P16 Detection Helps to Reduce the Underdiagnosis of CINII+ in HPV‐Negative Patients

There were 76 HR‐HPV‐negative patients among 300 patients, including 72 cases of CIN II‐ and 4 cases of CIN II+. The positive rates of P16 of HR‐HPV‐negative CIN II‐ patients were 2.8%, while the positive rates of P16 of HR‐HPV‐negative CIN II+ patients were 50.00%, with a statistically significant difference (p < 0.001, Table 7).

TABLE 7.

Association of P16 with HR‐HPV infection in histology category.

Histology	HR‐HPV	P16 [n (%)]
		Positive	Negative
< CINII (n = 270)	Negative (n = 72)	2 (2.8)	70 (97.2)
	Positive (n = 198)	14 (7.1)	184 (92.9)
≥ CINII (n = 30)	Negative (n = 4)	2 (50.0)	2 (50.0)
	Positive (n = 26)	18 (69.2)	8 (30.8)

Abbreviations: < CINII, negative for intraepithelial lesions or malignancy and low‐grade squamous intraepithelial lesions; ≥ CINII, high‐grade squamous intraepithelial lesions and cervical squamous cell carcinoma; HR‐HPV, high‐risk human papillomavirus.

4. Discussion

Currently, in domestic clinical practice, cervical lesion screening is primarily performed by TCT and/or HR‐HPV testing. In economically better areas, it is usually implemented to conduct combined screening with both methods. Both screening methods have their limitations. TCT reports show an excessive number of ASC‐US and have poor reproducibility. The high sensitivity of HR‐HPV leads to excessive colposcopic referral rates. Among HR‐HPV‐positive women, nearly 50% of patients have negative colposcopic biopsy results [17]. High‐grade and higher lesions of the cervix (CIN II+) are a high‐risk group for cervical cancer, and targeted interventions should be provided in clinical practice. In cervical cancer screening, timely and effective identification of the CIN II+ population is a prerequisite for reducing the incidence and mortality of cervical cancer [18]. With the application of artificial intelligence, the sensitivity of TCT testing is increasing, and how to more accurately identify and interpret the CIN II+ population has become the key to screening. Some studies have shown that cervical cytology immunocytochemistry has great prospects in the early screening for cervical cancer, especially in the triage of cervical cancer screening.

Our results showed that the positive rate of p16 protein detection increased with the severity of cytological and histological lesions, suggesting a positive correlation between P16 and the degree of cervical lesions, which is in agreement with the report of wentzensen et al. [19]. In this study, we found that the specificity of TCT was lower and similar to the HR‐HPV test, which may be related to the excessive number of ASC‐US reports. For detecting CIN II+ cervical lesions, the specificity and PPV of P16 protein detection were significantly higher than those of TCT and HR‐HPV detection, and the accuracy was the highest, indicating that P16 protein detection is the most reliable for screening out CIN II+ lesions and can be used as an effective method for the initial screening of cervical cancer. This is consistent with the findings of Cohen et al. [20].

Our results found that the positive rate of P16 protein detection in HR‐HPV‐negative CIN II+ cases was as high as 50%. This experiment has some limitations due to the small amount of specimens, but it also indicates to a certain extent that, regardless of the results of the HR‐HPV detection the P16 protein positivity reflects the severity of the cervical lesions very well and helps to reduce the number of HR‐HPV‐negative patients with CIN II+ lesions, highlighting the importance of P16 protein detection in screening for CIN II+ lesions.

Reviewing the history of the 300 patients in this study, we found three patients with positive HR‐HPV and P16 protein detection and a cytologic diagnosis of ASC‐H. However, colposcopic biopsies lacked features of high‐grade lesions. After clinical repeat biopsies or conization, the pathologic findings were CIN II. This suggests that when P16, TCT, and HR‐HPV are positive, even if the colposcopic biopsy shows no pathological lesions, further treatment or strict follow‐up should be performed. This is because irregular colposcopic sampling or negligence of pathologic embedded sections may lead to the absence of pathologic tissue. In addition, two patients with a positive cytologic diagnosis of HSIL and P16 protein but negative HR‐HPV were diagnosed with CIN II after 12 months of follow‐up.

Early screening for cervical cancer aims to identify patients with HSIL and those who are likely to progress to HSIL [21]. Studies [22] have confirmed that in LSIL, those who are positive for P16 protein have a higher risk of progression than those who are negative for P16 protein. Different interpretations of TCT detection have a significant impact on clinical management. However, small cells with atrophied, repaired, or high nucleoplasmic ratios are sometimes seen in cytologic sections with altered chromatin patterns. It is still difficult for experienced cytologists to differentiate between reactive changes, small cells with endothelial detachment, histiocytes, or lesions above ASC‐H, which may easily lead to overreporting of ASC‐US, high referral rates for colposcopy, or missed diagnosis. Therefore, we suggest that for this group of patients, P16 protein detection can be used as an effective objective indicator to assist in differential diagnosis, reduce the rate of ASC‐US reports, improve the accuracy of the diagnosis of CIN II+ lesions, reduce underdiagnosis, and avoid overtreatment. Compared to triple detection for all patients, it can also save patients' costs to a certain extent.

Of course, our research has some limitations. Diagnosis error cannot be excluded entirely because of inconsistencies in the interpretation of P16 by cytopathologists. Due to the majority diagnosis from two expert gynecologic cytopathologists and our study samples being sufficient, we considered this diagnosis bias only to influence results to a minor degree. The study of LF Duan et al. [23] has shown that there was little difference in the subjective interpretation of P16 by cytopathologists, and the interpretation of positive and negative P16 is highly reproducible.

With the research and clinical application of P16/ki‐67 double staining, it shows high specificity and sensitivity to CINII+, and reduces the false‐positive rate of P16 protein detection to a certain extent [24, 25, 26]. However, this study has proven that P16 protein detection has high accuracy and specificity in screening for CINII+ lesions. It can still be used as an effective triage tool in resource‐limited settings.

Author Contributions

W.C., C.L., J.Z., and M.T. contributed to the study conception and design. Material preparation and data collection and analysis were performed by Wei Chen. The first draft of the manuscript was written by Jiayu Zhou. All authors read and approved the final manuscript.

Ethics Statement

The study protocol was approved by the Ethics Committee of the Hunan Provincial Maternal and Child Health Care Hospital, Changsha, China. The need for written informed consent was waived by the ethics committee of the Hunan Provincial Maternal and Child Health Care Hospital because of the retrospective nature of the study. All experiments were performed in accordance with the Helsinki Declaration.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.