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Relationship between p16/ki67 immunoscores and PAX1/ZNF582 methylation status in precancerous and cancerous cervical lesions in high-risk HPV-positive women

BMC Cancer volume 24, Article number: 1171 (2024) Cite this article

Abstract

Background

The risk of cervical cancer progression in high-risk human papillomavirus (HR-HPV)-positive women is associated with cervical lesion severity and molecular heterogeneity. Classification systems based on p16 and Ki67 expression cumulative scores (0–3 each)—p16/Ki67 collectively known as an immunoscore [IS]—are an accurate and reproducible method for grading cervical intraepithelial neoplasia (CIN) lesions. Meanwhile, DNA methylation is an early event in the development of cervical cancer. Hence, this study evaluated the relationship among CIN, p16/Ki-67 IS, and PAX1/ZNF582 methylation.

Methods

In this study, 414 HPV-positive paraffin-embedded specimens were collected, and PAX1/ZNF582 methylation and the p16/ki67 IS were determined. A total of 43 invalid samples were excluded and 371 were included in the statistical analyses. There were 103 cervicitis, 95 CIN1, 71 CIN2, 89 CIN3, and 13 squamous cell carcinoma (SCC) cases. The association between PAX1/ZNF582 methylation and p16/Ki6 immunohistochemical staining scores was analyzed.

Results

The ΔCp of PAX1m (PAX1 methylation) and ZNF582m (ZNF582 methylation) decreased with cervical lesion severity (Cuzick trend test, all P < 0.001). The severity of the cervical lesions and p16, Ki67, and p16/Ki67 IS showed an increasing trend (Multinomial Cochran-Armitage trend test, all P < 0.001). The prevalence of PAX1m/ZNF582m increased with an increase in the IS of p16, Ki67, and p16/Ki67 (Cochran-Armitage trend test, all P < 0.001). In cervical SCC, the IS was 5–6, and the PAX1m/ZNF582m was positive. Meanwhile, heterogeneity was observed in CIN lesions: 10 cases had an IS of 3–4 and were PAX1m/ZNF582m-positive in ≤ CIN1; 1 case had an IS of 0–2 and was PAX1m/ZNF582m-positive in CIN2/3.

Conclusions

Significant heterogeneity was observed in CIN lesions for p16 and Ki67 immunohistochemical staining scores and PAX1/ZNF582 methylation. This may help clinicians personalize the management of CIN based on the predicted short-term risk of cancer progression, minimizing the rate of missed CIN1 diagnoses and incorrect treatment of CIN2/3.

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Background

Persistent infection with high-risk human papillomavirus (HR-HPV) is the main cause of cervical cancer. Most patients with grade 3 cervical intraepithelial neoplasia (CIN) develop cervical cancer [1]. CIN lesions can be classified into grades 1, 2, and 3 based on the area in which the normal epithelium has been replaced by atypical epithelium, determined by histological analysis, and the area in which abnormal cells are atypical. However, classical histological grading of CIN remains subjective, with considerable inter- and intra-observer variation and moderate reproducibility [2]. The pathological results of colposcopy biopsies are also inconsistent with those after coning, with ∼ 10% of CIN3 coning pathological results diagnosed as cervical cancer [3].

The accurate evaluation of cervical lesions is critical to selecting clinical treatments. The current standard practice is to treat CIN3 and most CIN2 lesions with macrocyclic or conical resection of the cervical transformation zone to prevent cervical cancer development. Spontaneous regression of CIN2 (40–50%) and CIN3 (∼ 30%) lesions frequently occurs [4, 5]. Therefore, excision of all high-grade cervical lesions (CIN2/3) could lead to overtreatment. Clinically, cervicitis or CIN1, as determined through colposcopy biopsy, can progress to cervical cancer within a short period without developing CIN2 and CIN3 [6, 7]. In these patients, cervical cancer may develop during normal follow-up.

CIN is a heterogeneous lesion categorized by the production of new viral particles (pCIN) or transforming CIN lesions (tCIN) that integrate HPV DNA into host cells. Biomarkers can distinguish between pCIN and tCIN, with the latter considered to have a higher risk of rapid progression to cervical cancer [8]. p16 is an important biomarker for detecting transforming HPV and is used as a surrogate indicator of HPV E7 transforming activity [8]. Currently, p16 immunohistochemical staining is used to morphologically identify suspected cases of low- and high-grade CIN [2]. Additionally, Ki67 is a marker of cell cycle activity and CIN classification [9, 10]. The co-expression of p16 and Ki67 is associated with cellular dysfunction and occurs in dysplastic cervical cells. Hence, their expression is considered a useful tool for detecting progressing cervical disease [11, 12]. In fact, classification systems based on p16 (0–3) and Ki67 (0–3) expression cumulative scores (i.e., the immunoscore) have proven more accurate and reproducible for grading CIN than hematoxylin–eosin (H&E) staining alone. The cumulative score also reduces the diagnosis rate of grade 2 CIN with inconsistent clinical management [13].

DNA methylation in the promoter regions of tumor suppressor genes precedes cervical cancer development [8, 14, 15]. Several studies have shown that methylation markers can be used to identify transforming CIN lesions and predict CIN lesion degeneration or progression [16,17,18,19,20,21,22,23]. Additionally, HR-HPV infections that persist for at least 5 years are associated with CIN2/3 disease with cancer-like methylation patterns, suggesting a higher risk of short-term cervical cancer progression [8, 24]. Thus, combining DNA methylation analysis with p16 and Ki67 immunohistochemical staining may provide a biomarker phenotype to improve clinical guidance for women with CIN and prevent overtreatment of reversible lesions and potential obstetric complications.

This study evaluates the relationship among CIN, p16/Ki67 immunoscores, and PAX1/ZNF582 methylation. To assess CIN heterogeneity, all cases were stratified according to the p16/Ki-67 immunoscores and PAX1/ZNF582 methylation status of patients. The primary objective of this study was to provide insights to further the development of individual treatment regimens for CIN based on the predicted cancer progression risk.

Methods

Study population and sample collection

In this retrospective study, HR-HPV-positive patients with cervicitis, CIN1, CIN2, CIN3, or squamous cell carcinoma (SCC) who were diagnosed via histopathological analysis (colposcopy biopsy) at Changsha Central Hospital from October 2022 to July 2023 were included in the study population. The inclusion criteria included (1) HR-HPV-positive and (2) agreeing to participate and signing informed consent. The exclusion criteria included (1) women with a previous cancer history, (2) previous treatment of CIN or cancer, (3) immunodeficiency, and (4) HPV vaccination history.

Paraffin-embedded biopsies were cut into five slices, two of which were used to evaluate p16 and Ki67 expression. The remaining three sections were tested for PAX1 and ZNF582 methylation. This study was approved by the Medical Ethics Committee of the Affiliated Changsha Central Hospital, Hengyang Medical School, University of South China (approval number: 2022-S0165), and informed consent was obtained from all patients.

HR-HPV testing

HR-HPV was detected using the HPV gene array test kit (HybriBio Ltd., Guangzhou, China), including 14 h-HPV genotypes (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and genotyped according to the manufacturer’s instructions.

Scoring of p16/Ki67

Two pathologists, blinded to patient clinical information and DNA methylation results, performed p16 and Ki67 immunohistochemical staining of the paraffin sections. If the scores of the two pathologists differed, a third expert was asked to score the results, and the preference of the final third expert prevailed.

For p16, a score of 0 was defined as positive cells or small cell clusters without p16 positive reaction or local dispersion; 1 represented a diffusively positive response limited to the lower third of the epithelium, 2 represented positive responses only in the lower half of the epithelium, and 3 represented responses throughout the epithelial thickness. Immunohistochemical staining for nuclear Ki67 was performed in keratinocytes and as scored as follows: a score of 0 indicated a normal expression pattern and scattered staining of nuclei in the basal layers; scores of 1, 2, and 3 were defined as having expression distributed in the lower third, lower half, and more than two-thirds of the epithelium, respectively. The cumulative immunohistochemical staining scores for p16/Ki67 (ranging from 0 to 6) were designated the “immune score” (IS), which could be categorized into three groups: an IS of 5–6 was associated with CIN3 + lesions, 3–4 was associated with CIN2 lesions, and 0–2 was associated with CIN1 lesions.

Measurement of PAX1/ZNF582 methylation

The methylation status of PAX1 and ZNF582 was evaluated using a cervical cancer gene methylation test kit (Hoomya, Changsha, China) on the Cobas 480 II PCR platform. The COL2A1 gene was used as an internal reference gene; a Cp value > 35 indicated an insufficient sample size and an invalid methylation test. The methylation value was obtained by subtracting the Cp value of COL2A1 from that of the target gene to calculate the difference (ΔCp). The lower the ΔCp value of PAX1m and ZNF582m, the higher the methylation level.

Statistical analysis

Data were analyzed using R V 4.3.2. The CIN grades were original local diagnoses used in the hospital. Continuous variables were recorded as medians [IQR], and categorical variables were recorded as counts (%). The distribution of methylation (ΔCp), determined by pathology and IS, was mapped using the ggpubr package. The ggplot2 package was used to create the stacking plot of the IS and pathology results. The pROC package was used to map the receiver operating characteristics (ROC) curve for the diagnostic performance of DNA methylation for CIN3+ (Supplementary Fig. 1). Following the Youden index of the ROC maximization principle, the cut-off value for PAX1m was 11.9; hence, the text was deemed positive when ΔCp ≤ 11.9 and negative when ΔCp > 11.9. The cut-off value for ZNF582m was 10.8. Cuzick’s test was performed to identify trends between continuous and ordered categorical variables using the PMCMRplus package. Trends between two ordered categories were analyzed using the DescTools and multiCA packages. A mosaic plot was constructed using the VCD package. Two-sided P values < 0.05 were considered statistically significant.

Results

Case series

In this study, 414 paraffin specimens from HR-HPV-positive patients were collected from the pathology department, of which 33 were invalid for the PAX1m assessment, and 43 were invalid for the ZNF582m assessment. Thus, 43 cases were excluded, and 371 patients were included in the statistical analysis, with a median age of 42 years (IQR: 32–54). Supplementary Table 1 shows the cytology results of p16, Ki67, PAX1m, and ZNF582m. There were 103 cases of cervicitis (27.8%), 95 cases of CIN1 (25.6%), 71 cases of CIN2 (19.1%), 89 cases of CIN3 (24.0%), and 13 of cervical SCC (3.5%).

DNA methylation increased with increased cervical lesion severity

The ΔCp values for PAX1m and ZNF582m decreased with increasing lesion degree (Cuzick trend test, all P < 0.001, Table 1). The distribution of PAX1m and ZNF582m in cervical lesions significantly differed between CIN2 and CIN3 (all P < 0.05; Fig. 1A and B). The severity of cervical lesions (cervicitis, CIN1, CIN2, CIN3, and SCC) and PAX1m, ZNF582m, and PAX1m/ZNF582m were positively correlated (Cochran–Armitage trend test, all P < 0.001; Table 1). The positivity rates of PAX1m and ZNF582m in all patients were 23.7% and 28.3%, respectively, and their positivity rates in cervical cancer were 100%. The positivity rates for PAX1m/ZNF582m in cervicitis, CIN1, CIN2, CIN3, and SCC were 17.5%, 18.9%, 35.2%, 65.2%, and 100.0%, respectively.

Table 1 Relationship among cervical lesions and PAX1m, ZNF582m, p16, and Ki67 expression
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Fig. 1
figure 1

Violin plot of PAX1/ZNF582 methylation distribution in cervical lesions and p16/Ki67 immune scores. A, Distribution of PAX1m (ΔCp)  grouped according to pathology results. B,  Distribution of ZNF582m (ΔCp)  grouped according to pathology results.C, Distribution of PAX1m (ΔCp)  grouped according to IS results. D, Distribution of ZNF582m (ΔCp)  grouped according to IS results. NS., no significance; * P < 0.05; ** P < 0.01; *** P < 0.001

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Relationship among p16/Ki67 immunostaining scores, cervical lesions, and PAX1/ZNF582 methylation

The severity of cervical lesions (cervicitis, CIN1, CIN2, CIN3, and SCC) exhibited a clear linear correlation with the immunostaining scores of p16 and Ki67 and the IS (multinomial Cochran–Armitage trend test, all P < 0.001; Table 1). The immunostaining scores for p16 and Ki67 were 3, and the IS was 6 in all cervical SCC cases (Fig. 2). In cervicitis, the IS was < 5 in all cases. The ΔCp of PAX1m and ZNF582m was statistically significantly different between cases with an IS of 3–4 and 5–6, respectively (all P < 0.0001, Fig. 1C and D). The positive rates of PAX1m, ZNF582m, and PAX1m/ZNF582m increased with an increase in p16, Ki67, and IS (Cochran–Armitage trend test, all P < 0.001). The positivity rates of PAX1m/ZNF582m in cases with p16 and Ki67 immunostaining scores of 3 were 59.9% and 72.0%, respectively. The positivity rate for PAX1m/ZNF582m was 78.5% in patients with an IS of 6. The positivity rates of PAX1m/ZNF582m in patients with an IS of 0–2, 3–4, and 5–6 were 17.3%, 26.8%, and 62.4, respectively (Table 2).

Fig. 2
figure 2

Stack plot of p16/Ki67 immune scores in cervical lesions. A, p16. B, Ki67. C, IS. D, IS group.

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Table 2 Relationship between p16/Ki67 immunostaining scores and PAX1/ZNF582 methylation
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Subsets of PAX1 m /ZNF582 m and IS in different cervical lesions

Heterogeneity was detected in PAX1m/ZNF582m and IS among different cervical lesion clusters. Cervical lesions (cervicitis, CIN1, CIN2, CIN3, and SCC) were stratified according to IS and PAX1m/ZNF582m status (Table 3). There were 36 cases of CIN2 with an IS of 5–6, of which 14 were PAX1m/ZNF582m-positive and 22 were PAX1m/ZNF582m-negative. There were 81 cases of CIN3 with an IS of 5–6, among which 56 were PAX1m/ZNF582m-positive and 25 were PAX1m/ZNF582m-negative. An additional 52 cases of CIN1 had an IS of 0–2, of which 9 were PAX1m/ZNF582m-positive and 43 were PAX1m/ZNF582m-negative. Supplementary Fig. 2 shows the distribution ratios of IS and PAX1m/ZNF582m in different cervical lesions.

Table 3 PAX1/ZNF582 methylation and p16/Ki67 immune score heterogeneity in cervical lesions
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Discussion

In this study, we evaluated the relationship between cervical lesions with various histopathological characteristics and p16/ki67 immunohistochemical staining and PAX1/ZNF582 methylation in HR-HPV-positive women. The abnormal expression of P16 and Ki-67—two commonly used cyclin markers—is associated with CIN and cervical cancer [25]. In this study, we evaluated the relationship between cervical lesions with various histopathological characteristics and p16/ki67 immunohistochemical staining and PAX1/ZNF582 methylation in high-risk HPV-positive women. P16 and Ki67 are two commonly used cyclin markers and their abnormal expression is associated with CIN and cervical cancer [26,27,28,29,30]. Overall, p16 and Ki67 single and double (IS) molecular immunohistochemical staining scores increased with increasing cervical lesion severity. However, the p16 and Ki67 immune scores were 0 and 1 for CIN2 and CIN3, respectively. DNA methylation is an important biomarker that reflects molecular events in the pathophysiology of HPV infection to distinguish cervical lesions that may be at high risk of progression from those that are potentially degenerative [8, 17]. Indeed, many studies have confirmed the clinical performance of DNA methylation for triaging HR-HPV-positive women [23, 31,32,33,34,35,36]. PAX1 and ZNF582 are the most important candidate genes. PAX1 suppresses the malignant phenotype under oncogenic stress, such as persistent infection by HR-HPV virus. PAX1 activated a group of phosphatases, including DUSP1, 5, and 6, and inhibited EGF/MAPK signaling to suppress cancer development [37]. Many methylated differential genes were identified in cervical cancer and normal cervix using MeDIP-on-chip technology. One of them, ZNF582, located at chromosome 19q13.43, encodes the zinc finger protein 582, which contains one KRAB-A-B domain and nine zinc-finger motifs that repress transcription of the gene. May be involved in a variety of biological processes related to DNA damage response, proliferation, cell cycle control and tumor transformation. Methylation specific PCR (MSP) methods confirmed that ZNF582 is frequently methylated in CIN3 and more severe lesions [38]. In this study, we found that the ΔCp values of PAX1 and ZNF582 methylation decreased with increasing cervical lesion severity (Fig. 1A and B). Furthermore, PAX1m and ZNF582m were observed in all cervical cancer cases, whereas approximately 10% were positive for PAX1m and ZNF582m in pathological cervicitis (Table 1).

Our study found that the positivity rate for PAX1m/ZNF582m increased with increasing p16 and Ki67 immunohistochemical scores and IS (Table 2). A similar relationship between p16/Ki67 immunohistochemical staining and FAM19A4/miR124-2 methylation has been reported in CIN2/3 [19]. High-risk HPV infects the cervical cells of the host and achieves viral multiplication, resulting in CIN (pCIN), which is easily cleared by the immune system. However, HPV DNA integration into host cell DNA is called transforming CIN (tCIN); abnormal expression of E6 and E7 viral proto-oncogenes leads to high-grade lesions and cancer [8, 14]. Additionally, p16/Ki67 co-expression reflects cell dysfunction and is a marker of CIN transformation [8, 17]. Under the action of E6/E7 oncoproteins, the key tumor suppressor proteins p53 and pRb are degraded by proteasomes and p53, as well as the Rb-related transcription factors Sp1 and E2F, bind DNMT1 promoters to induce its overexpression, and numerous tumor suppressor genes are methylated [39].

Two hypotheses have been proposed regarding the mechanism of cervical precancerous lesion formation. The “progressive progression” model centers on the gradual sequential progression from normal tissue to CIN1, CIN2, and CIN3 [40]. Meanwhile, the “molecular switch” model suggests that DNA methylation serves as a “molecular switch,” allowing CIN3 to evolve directly from HPV-infected normal epithelial cells without progressing through CIN1 and CIN2 [6, 7, 41]. In the current study, nine CIN1 cases had immune scores of 3–4 and were PAX1m/ZNF582m-positive, while three cases had immune scores of 5–6. The presence of CIN1 molecular heterogeneity in these 12 cases suggests the need for closer follow-up every 6 months to avoid short-term progression to CIN3 + and underdiagnosis.

In previous studies, methylation at the CpG sites of PAX1 progressed to CIN2 + with an HR of 12.62 (95%CI = 1.35, 117.88) over 5 years [18]. Additionally, negative methylation of FAM19A4/miR124-2 is associated with clinical regression of CIN2/3 (odds ratio: 2.72, 95% CI = 1.41–5.26) [19]. Meanwhile, in the current study, among the CIN2/3 cases, four had an IS of 0–2 and were PAX1m/ZNF582m-negative, corresponding to a low-grade risk. Thus, for these patients, treatment should be postponed to protect the fertility of women of childbearing age; however, they should be followed up every 12 months. As for the 26 cases with an IS of 3–4 who were PAX1m/ZNF582m-negative, follow-up every 6 months was recommended.

The relationship among the risk of CIN1 progression, CIN2/3 lesion regression, and biomarker lineage expression requires further verification in prospective studies. Accurate prognostic biomarkers can identify women at high risk for CIN, which could improve cervical cancer screening programs. This allows for more precise interventions for lesions with real progression potential, reduces repeat testing, and eliminates the need for treatment in patients with subsided CIN.

The primary strength of this study was the inclusion of different grades of cervical lesions, i.e., cervicitis, CIN1, CIN2, CIN3, and cervical SCC, enabling a comprehensive analysis of the relationship among PAX1/ZNF582 methylation, p16/Ki67 immunohistochemical scores, and cervical lesions. One limitation of the study was the use of paraffin specimens; the processes of DNA extraction and deparaffinization tend to lead to DNA fragmentation, which may affect DNA methylation results. The paraffin specimens were divided into two groups: one for p16/Ki67 immune score analysis and the other for the DNA methylation analysis. Intra-sample differences may have led to a bias in the results. Another limitation is the small sample size and lack of long-term follow-up to assess the risk of progression in low-grade lesions that were PAX1m/ZNF582m- and p16/Ki67-positive and the likelihood of regression in high-grade lesions that were both PAX1m/ZNF582m- and p16/Ki67-negative. However, in this study, immunohistochemistry was performed by a senior technician. The histopathology results were interpreted by two chief physicians, and in the event of inconsistencies, a third pathologist interpreted the results to ensure the accuracy of the results. Overall, we found heterogeneous molecular marker expression in different CIN lesions, providing a new direction for subsequent research on preventing cervical cancer and preventing the overtreatment of cervical lesions.

Conclusion

A correlation among PAX1/ZNF582 methylation, p16/Ki67 immunohistochemical staining scores, and cervical lesions, with considerable heterogeneity, was observed across CIN cases. This heterogeneity may help clinicians improve personalized management of CIN based on the predicted short-term risk of cancer progression, avoiding missed CIN1 diagnoses and CIN2/3 overtreatment.

Data availability

The datasets used during the current study are available from the corresponding author on reasonable request.

Abbreviations

CIN:

Cervical intraepithelial neoplasia

HPV:

Human papillomavirus

H&E:

Hematoxylin/eosin staining

IS:

Immune score

PAX1 m :

PAX1 methylation

ZNF582 m :

ZNF582 methylation

ROC:

Receiver operating characteristics curve

References

  1. Balasubramaniam SD, Balakrishnan V, Oon CE, Kaur G. Key molecular events in cervical cancer development. Med (Kaunas). 2019;55:384.

    Google Scholar 

  2. Reuschenbach M, Wentzensen N, Dijkstra MG, von Knebel Doeberitz M, Arbyn M. p16INK4a immunohistochemistry in cervical biopsy specimens: a systematic review and meta-analysis of the interobserver agreement. Am J Clin Pathol. 2014;142:767–72.

    Article  PubMed  Google Scholar 

  3. Fu K, Lei M, Wu LS, Shi JC, Yang SY, Yang WQ, et al. Triage by PAX1 and ZNF582 methylation in women with cervical intraepithelial neoplasia grade 3: a multicenter case-control study. Open Forum Infect Dis. 2022;9:ofac013.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Tainio K, Athanasiou A, Tikkinen KAO, Aaltonen R, Cardenas J, Hernandes, et al. Clinical course of untreated cervical intraepithelial neoplasia grade 2 under active surveillance: systematic review and meta-analysis. BMJ. 2018;360:k499.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Loopik DL, Bentley HA, Eijgenraam MN, IntHout J, Bekkers RLM, Bentley JR. The natural history of cervical intraepithelial neoplasia grades 1, 2, and 3: a systematic review and meta-analysis. J Low Genit Tract Dis. 2021;25:221–31.

    Article  PubMed  Google Scholar 

  6. Larson AA, Liao SY, Stanbridge EJ, Cavenee WK, Hampton GM. Genetic alterations accumulate during cervical tumorigenesis and indicate a common origin for multifocal lesions. Cancer Res. 1997;57:4171–6.

    CAS  PubMed  Google Scholar 

  7. Castle PE, Gage JC, Wheeler CM, Schiffman M. The clinical meaning of a cervical intraepithelial neoplasia grade 1 biopsy. Obstet Gynecol. 2011;118:1222–9.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Steenbergen RD, Snijders PJ, Heideman DA, Meijer CJ. Clinical implications of (epi)genetic changes in HPV-induced cervical precancerous lesions. Nat Rev Cancer. 2014;14:395–405.

    Article  CAS  PubMed  Google Scholar 

  9. Sun X, Kaufman PD. Ki-67: more than a proliferation marker. Chromosoma. 2018;127:175–86.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wentzensen N, von Knebel Doeberitz M. Biomarkers in cervical cancer screening. Dis Markers. 2007;23:315–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Silva DC, Gonçalves AK, Cobucci RN, Mendonça RC, Lima PH, Cavalcanti GJ. Immunohistochemical expression of p16, Ki-67 and p53 in cervical lesions - a systematic review. Pathol Res Pract. 2017;213:723–9.

    Article  CAS  PubMed  Google Scholar 

  12. Shiraz A, Crawford R, Egawa N, Griffin H, Doorbar J. The early detection of cervical cancer. The current and changing landscape of cervical disease detection. Cytopathology. 2020;31:258–70.

    Article  PubMed  Google Scholar 

  13. van Zummeren M, Leeman A, Kremer WW, Bleeker MCG, Jenkins D, van de Sandt M, et al. Three-tiered score for Ki-67 and p16(ink4a) improves accuracy and reproducibility of grading CIN lesions. J Clin Pathol. 2018;71:981–88.

    Article  PubMed  Google Scholar 

  14. Wilting SM, Steenbergen RDM. Molecular events leading to HPV-induced high grade neoplasia. Papillomavirus Res. 2016;2:85–8.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Lorincz AT. Virtues and weaknesses of DNA methylation as a test for cervical cancer prevention. Acta Cytol. 2016;60:501–12.

    Article  CAS  PubMed  Google Scholar 

  16. Lindroth Y, Pedersen L, Alssamaray J, Berglund T, Sundqvist A, Borgfeldt C, et al. Cervix cytology samples revealed increased methylation of the human markers FAM19A4/miR124-2 up to 8 years before adenocarcinoma. Acta Obstet Gynecol Scand. 2024;103:378–86.

    Article  CAS  PubMed  Google Scholar 

  17. Burdier FR, Waheed DE, Nedjai B, Steenbergen RDM, Poljak M, Baay M, et al. DNA methylation as a triage tool for cervical cancer screening - A meeting report. Prev Med Rep. 2024;41:102678.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Bukowski A, Hoyo C, Vielot NA, Graff M, Kosorok MR, Brewster WR, et al. Epigenome-wide methylation and progression to high-grade cervical intraepithelial neoplasia (CIN2+): a prospective cohort study in the United States. BMC Cancer. 2023;23:1072.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kremer WW, Dick S, Heideman DAM, Steenbergen RDM, Bleeker MCG, Verhoeve HR, et al. Clinical regression of high-grade cervical intraepithelial neoplasia is associated with absence of FAM19A4/miR124-2 DNA methylation (CONCERVE study). J Clin Oncol. 2022;40:3037–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Vink FJ, Lissenberg-Witte BI, Meijer C, Berkhof J, van Kemenade FJ, Siebers AG, et al. FAM19A4/miR124-2 methylation analysis as a triage test for HPV-positive women: cross-sectional and longitudinal data from a Dutch screening cohort. Clin Microbiol Infect. 2021;27:125.

    Article  Google Scholar 

  21. De Strooper LMA, Berkhof J, Steenbergen RDM, Lissenberg-Witte BI, Snijders PJF, Meijer C, et al. Cervical cancer risk in HPV-positive women after a negative FAM19A4/mir124-2 methylation test: a post hoc analysis in the POBASCAM trial with 14 year follow-up. Int J Cancer. 2018;143:1541–8.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Dick S, Kremer WW, De Strooper LMA, Lissenberg-Witte BI, Steenbergen RDM, Meijer C, et al. Long-term CIN3 + risk of HPV positive women after triage with FAM19A4/miR124-2 methylation analysis. Gynecol Oncol. 2019;154:368–73.

    Article  CAS  PubMed  Google Scholar 

  23. Zhang L, Zhao X, Hu S, Chen S, Zhao S, Dong L, et al. Triage performance and predictive value of the human gene methylation panel among women positive on self-collected HPV test: results from a prospective cohort study. Int J Cancer. 2022;151:878–87.

    Article  CAS  PubMed  Google Scholar 

  24. Kremer WW, Steenbergen R, Heideman D, Kenter GG, Meijer C. The use of host cell DNA methylation analysis in the detection and management of women with advanced cervical intraepithelial neoplasia: a review. BJOG. 2021;128:504–14.

    Article  CAS  PubMed  Google Scholar 

  25. Voidăzan ST, Dianzani C, Husariu MA, Geréd B, Turdean SG, Uzun CC, et al. The role of p16/Ki-67 immunostaining, hTERC amplification and fibronectin in predicting cervical cancer progression: a systematic review. Biology (Basel). 2022;11:956.

    PubMed  Google Scholar 

  26. Castle PE, Adcock R, Cuzick J, Wentzensen N, Torrez-Martinez NE, Torres SM, et al. Relationships of p16 immunohistochemistry and other biomarkers with diagnoses of cervical abnormalities: implications for LAST terminology. Arch Pathol Lab Med. 2020;144:725–34.

    Article  CAS  PubMed  Google Scholar 

  27. Li Y, Liu J, Gong L, Sun X, Long W. Combining HPV DNA load with p16/Ki-67 staining to detect cervical precancerous lesions and predict the progression of CIN1-2 lesions. Virol J. 2019;16:117.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Diouf D, Diop G, Fall C, Sarr S, Diarra CAT, Ngom AI, et al. The association of molecular biomarkers in the diagnosis of cervical pre-cancer and cancer and risk factors in Senegalese. Asian Pac J Cancer Prev. 2020;21:3221–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. El-Zein M, Gotlieb W, Gilbert L, Hemmings R, Behr MA, Franco EL. Dual staining for p16/Ki-67 to detect high-grade cervical lesions: results from the screening triage ascertaining intraepithelial neoplasia by immunostain testing study. Int J Cancer. 2021;148:492–501.

    Article  CAS  PubMed  Google Scholar 

  30. Zhang SK, Jia MM, Zhao DM, Wu ZN, Guo Z, Liu YL, et al. Evaluation of p16/Ki-67 dual staining in the detection of cervical precancer and cancer in China. Cancer Epidemiol. 2019;59:123–8.

    Article  PubMed  Google Scholar 

  31. Yang L, Tao H, Lin B, He X, Chen Y, Fan X. Utilization of PAX1 methylation test for cervical cancer screening of non-HPV16/18 high-risk HPV infection in women. Future Oncol. 2023;19:1917–27.

    Article  CAS  PubMed  Google Scholar 

  32. Wang Y, Chuan J, Zhu B, Zu Y, Zhang R, Tang L, et al. Evaluation of PAX1/ST6GALNAC5 methylation as a triage test for cervical intraepithelial neoplasia and cervical cancer. Epigenomics. 2023;15:131–45.

    Article  CAS  PubMed  Google Scholar 

  33. Salta S, Lobo J, Magalhães B, Henrique R, Jerónimo C. DNA methylation as a triage marker for colposcopy referral in HPV-based cervical cancer screening: a systematic review and meta-analysis. Clin Epigenetics. 2023;15:125.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Verhoef L, Bleeker MCG, Polman N, Steenbergen RDM, Meijer C, Melchers WJG, et al. Performance of DNA methylation analysis of ASCL1, LHX8, ST6GALNAC5, GHSR, ZIC1 and SST for the triage of HPV-positive women: results from a Dutch primary HPV-based screening cohort. Int J Cancer. 2022;150:440–9.

    Article  CAS  PubMed  Google Scholar 

  35. Su Y, Tang Y, Zhang T, Xu Y, Zhang Y, Shen Y, et al. Methylated ZNF582 as a triage marker for occult cervical cancer and advanced cervical intraepithelial neoplasia. Future Oncol. 2022;18:2583–92.

    Article  CAS  PubMed  Google Scholar 

  36. Liang H, Li GL, Liu J, Fu M, Huang H, Zhao K, et al. The application value of PAX1 and ZNF582 gene methylation in high grade intraepithelial lesion and cervical cancer. Clin Transl Oncol. 2021;23:283–8.

    Article  CAS  PubMed  Google Scholar 

  37. Su PH, Lai HC, Huang RL, Chen LY, Wang YC, et al. Paired box-1 (PAX1) activates multiple phosphatases and inhibits kinase cascades in cervical cancer. Sci Rep. 2019;9(1):9195.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Huang RL, Chang CC, Su PH, Chen YC, Liao YP, et al. Methylomic analysis identifies frequent DNA methylation of zinc finger protein 582 (ZNF582) in cervical neoplasms. PLoS ONE. 2012;7(7):e41060.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Sen P, Ganguly P, Ganguly N. Modulation of DNA methylation by human papillomavirus E6 and E7 oncoproteins in cervical cancer. Oncol Lett. 2018;15:11–22.

    PubMed  Google Scholar 

  40. Snijders PJ, Steenbergen RD, Heideman DA, Meijer CJ. HPV-mediated cervical carcinogenesis: concepts and clinical implications. J Pathol. 2006;208:152–64.

    Article  CAS  PubMed  Google Scholar 

  41. Nedjai B, Reuter C, Ahmad A, Banwait R, Warman R, Carton J, et al. Molecular progression to cervical precancer, epigenetic switch or sequential model? Int J Cancer. 2018;143:1720–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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Funding

This work was supported by the Changsha Municipal Natural Science Foundation [kq2208440].

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Author notes

  1. Haijun Luo and Yixiang Lian contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology, The Affiliated Changsha Central Hospital, Hengyang Medical School, University of South China, Changsha, 410004, China

    Haijun Luo, Yixiang Lian, Yan Zhao, Zhigan Wang, Jing Zhou, Zirong Zhang & Shali Jiang

  2. Department of Medical Statistics, Hunan Hoomya Gene Technology Co., Ltd., Changsha, 410205, China

    Hong Tao

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  1. Haijun Luo
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Contributions

S Jiang, H Luo designed conception, S Jiang, Y Lian, H Tao developed methodology , S Jiang, H Luo, Y Lian, Y Zhao, Z Wang, J Zhou, Z Zhang collected data, S Jiang, H Luo, Lian, S Jiang wrote, reviewed, and/or revised the manuscript, S Jiang supervised study.

Corresponding author

Correspondence to Shali Jiang.

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Ethics approval and consent to participate

This research project was approved by the Medical Ethics Committee of the Affiliated Changsha Central Hospital, Hengyang Medical School, University of South China (approval number: 2022-S0165). Informed consent was obtained from all patients.

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Luo, H., Lian, Y., Tao, H. et al. Relationship between p16/ki67 immunoscores and PAX1/ZNF582 methylation status in precancerous and cancerous cervical lesions in high-risk HPV-positive women. BMC Cancer 24, 1171 (2024). https://doi.org/10.1186/s12885-024-12920-4

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  • DOI: https://doi.org/10.1186/s12885-024-12920-4

Keywords

BMC Cancer

ISSN: 1471-2407

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