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HPV integration status conversion and CIN2 + cancer risk stratification based on HPV integration levels among HPV integration-positive women: a 1-year follow-up study

BMC Cancer volume 25, Article number: 885 (2025) Cite this article

Abstract

Background

HPV integration is a crucial genetic step in cervical carcinogenesis and the level of HPV integration increases with the grade of precancerous lesion. This study aimed to conduct risk stratification based on HPV integration levels and HPV integration status conversion among HPV integration-positive women after 1 year of follow-up.

Methods

This prospective cohort study was conducted in Tongji Hospital between June 2020 and August 2022 and included 1297 consecutive HPV-positive women. The level of integration reads was stratified for risk assessment.

Results

In total, 194 women were HPV integration-positive and followed for at least 1 year. The immediate risk of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) increased from 36.2% (25/69) among women with 6–20 integration reads to 93.8% (30/32) among women with more than 1000 integration reads (Ptrend < 0.001). The 1-year cumulative risk of CIN2 + increased from 39.1% (27/69) among women with 6–20 integration reads to 96.9% (31/32) among women with more than 1000 integration reads (Ptrend < 0.001). The 1-year cumulative risk of CIN2 + with HPV integration reads more than 40 was 93.8% (90/96), which was significantly higher than that of HPV integration reads less than 40 (38/85, P < 0.001). Among women with HPV integration reads more than 40, 99.0% (95/96) of women progressed with positive outcomes after one year of follow-up (persistent integration at the same site, immediate CIN2+, and 1-year CIN2+). The progression rate of women with persistent integration at the same site was 41.6% (5/12), which was significantly higher than those of HPV integration-negative conversion (0/41, 0%, P < 0.001).

Conclusion

The number of HPV integration reads may help CIN2 + risk stratification and facilitate the clinical management of high-risk patients.

Peer Review reports

Background

Cervical cancer is the fourth most common malignancy worldwide, with a morbidity and mortality rate of 9.89 and 3.05 per 10,000, respectively, in China [1, 2]. Several studies have shown that human papillomavirus (HPV) infection is highly associated with the development of cervical cancer, with 95% of patients with cervical cancer being positive for HPV infection [3,4,5]. After infection with HPV, the virus can be integrated into the human genome under certain circumstances. For instance, a consistent HPV infection imposes a high viral load [6]. HPV integration can lead to various genetic alterations, including oncogene activation, tumor suppressor gene inactivation, inter-chromosomal or intra-chromosomal rearrangements, and genetic instability [7, 8]. This host genetic transformation can change the expression levels of proteins, promoting cancer cell viability while providing a selective growth advantage [9]. Therefore, HPV integration may be an important event in oncogenic progression.

The integration of HPV into the human genome was first detected in 1987 using a technique based on restriction digestion/Southern blot hybridization [10]. Many techniques have emerged in recent decades [11,12,13]. The advent of next-generation sequencing (NGS) enabled the detection of HPV-human reads through whole-genome sequencing (WGS) [14,15,16]. Using these techniques, many studies have found a significant difference in the number of HPV integration events between normal, precancerous lesions, and invasive cervical cancer, suggesting that HPV integration may be a potential marker for precancerous progression [17,18,19]. Furthermore, it has been reported that HPV virus integration levels (number of integrations per genome or relative number of the supporting reads) are positively correlated with the severity of cervical lesions [20, 21]. Therefore, HPV integration levels may contribute to risk stratification for cervical intraepithelial neoplasia (CIN).

HPV integration events can be detected in all stages of the natural progression of cervical cancer, even among HPV-positive women with normal lesions [22, 23]. Previous studies have shown that the HPV integration rate increases with the grade of CIN lesions [17,18,19]. Among patients with HPV-positive cervical cancer, the prevalence of HPV integration is 84.3–97.8% [21, 24]. However, whether the HPV integration status persists among HPV integration-positive women over time has not been reported.

Here, we conducted a prospective cohort study to investigate risk stratification based on HPV integration levels and assess the HPV integration status conversion of HPV integration-positive women. To our knowledge, this is the first study to propose a critical threshold for HPV integration level and risk stratification.

Methods

Study design

This was a prospective cohort study conducted at Tongji Hospital, Tongji Medical College, and Huazhong University of Science and Technology in Wuhan, China. Between June 2020 and August 2022, we enrolled 1297 HPV-positive women aged from 21 to 75 in our outpatient department, all of whom underwent HPV integration testing. Among them, 194 women were HPV integration-positive and were invited to participate in this study. The exclusion criteria were as follows: (1) CIN1 with surgical treatment; (2) a history of cervical cancer; and (3) no colposcopy was conducted at the baseline. At baseline, women all underwent colposcopy and biopsy within 1 month after HPV integration testing. Women with histological results ≤ CIN1 at baseline were recommended to repeat cervical cancer screening and another HPV integration test at a one-year follow-up. We conducted a repeat HPV integration test on the discarded Thinprep cytology specimen at a one-year follow-up. The flow chart is shown in Fig. 1. Ethics Ethical approval was obtained from the Ethics Committee of Tongji Hospital of Huazhong University of Science and Technology, and patients’ consent was waived (TJ-IRB20211110).

Fig. 1
figure 1

The flow chart of study enrollment. Abbreviations: CIN2, cervical intraepithelial neoplasia grade 2; CIN3+, cervical intraepithelial neoplasia grade 3 or worse; HPV, Human papillomavirus

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HPV Test

The HPV DNA test was conducted using the Cobas 4800 system (Roche Molecular Diagnostics), which detects HPV16, HPV18, and 12 other high-risk HPV types (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68).

HPV integration test

The sampling method for HPV integration test was the same as that of the HPV test, which used cervical exfoliated cells. After DNA extraction, 500 ng of genomic DNA was used from each patient for DNA library preparation. Based on our previous study, the HPV integration test was conducted using high-throughput viral integration detection (HIVID) [19]. This technique detected 18 HPV integration types, including HPV16, 18, and 12 other high-risk HPV types. Low-quality bases of each read were trimmed using an average Q value < 20. Window size = 4 bases from left and right ends, and reads with length > 100 bp were retained for downstream analysis. These clean reads were filtered using BWA and paired-end reads (partial read sequence aligned to human genome and partial read sequence aligned to HPV genome) were reserved [25]. All integrated sites were verified using PCR and Sanger sequencing. Sequencing reads containing both HPV and human genome sequences (both not shorter than 20 bp in length) was labeled as 1 integrated read. Samples with more than 5 reads were considered HPV integration positive. Samples with discrepant HPV infection results between the HPV test and HPV integration test were excluded.

Study endpoint

All subjects underwent at least one colposcopy examination. Histologic diagnosis was considered the gold standard, and the lesions were categorized based on the CIN grade: no indication for biopsy, normal, CIN grade 1 (CIN1), CIN grade 2 (CIN 2), CIN grade 3 (CIN3), adenocarcinoma in situ (AIS), and cervical cancer. Two pathologists at our hospital reviewed all histological results. The clinical endpoint included CIN2+ (including CIN2, CIN3, AIS and cervical cancer) and CIN3+ (including CIN3, AIS and cervical cancer).

In this study, we divided patients’ outcomes into positive outcomes and negative outcomes. The positive outcomes included: (1) immediate CIN2+: women with CIN2 + at the baseline; (2) 1-year CIN2+: women with normal or CIN1 at baseline and histologically diagnosed with CIN2 + at 1-year follow-up; (3) persistent integration at the same site: women with the same breakpoint in two consecutive HPV integration tests at different time points (at a minimum of 3 months). The negative outcomes included negative conversion and HPV integration at a different site. Women with positive HPV integration results at baseline and negative results at 1-year follow-up were defined as a negative conversion. Women with a different breakpoint in two consecutive HPV integration detection sessions (at a minimum interval of 3 months) were regarded as having HPV integration at a different site.

Statistical analysis

All analyses were conducted using SPSS (version 26). All P values are from a two-sided test, and the results were deemed statistically significant in the presence of P < 0.05. In the present study, integration reads were defined as the number of supporting reads of the most frequent breakpoint per 5,000,000 sequencing reads. Contingency tables and χ [2] trend tests were used to determine the percentage of positive outcomes and 1-year cumulative CIN2 + rate among HPV integration reads (6–20, 21–40, 41–200, 201–1000, and > 1000) strata. The CIN2 + progression rates of different HPV integration statuses (negative conversion, HPV integration at a different site, and persistent integration at the same site) were also measured. Categorical variables were analyzed using Pearson’s chi-square test or Fisher’s exact test. The odds ratio (OR) with its 95% confidence interval (95% CI) was used to express the statistical correlation between the number of HPV integration reads and 1-year cumulative risk of CIN2+.

Results

Study population

We included 194 HPV integration-positive women in this study. In this cohort, we excluded 7 women with CIN1 who received surgical treatment, 3 women with a history of cervical cancer, and 3 women without colposcopy at the baseline. Finally, 181 women were enrolled in this study (Fig. 1). Baseline characteristics of participants are shown in Table S1. The median age of participants was 42 years (interquartile range, 35–52 years). The median follow-up duration was 10 months (interquartile range, 5.5–13 months). Among patients, 28 had AIS, 79 had CIN3, 16 had CIN2, 13 had CIN1, and 45 had benign lesions at baseline. The most common integrated HPV types were HPV16 (99/181, 54.7%), HPV18 (18/181, 9.9%), and HPV52 (18/181, 9.9%) (Table S1).

Risk stratification for CIN2 + and CIN3 + based on HPV integration reads

The immediate risk of CIN2 + increased as the number of HPV integration reads increased. The immediate risk of CIN2 + was 36.2% (25/69) among women with 6–20 integration reads, 62.5% (10/16) among women with 21–40 integration reads, 85.7% (30/35) among women with 41–200 integration reads, 96.6% (28/29) among women with 201–1000 integration reads, and 93.8% (30/32) among women with more than 1000 integration reads (Ptrend < 0.001, Fig. 2; Table 1). The same patterns were found for the immediate risk of CIN3+, which was 30.4% (21/69) among women with 6–20 integration reads, 50.0% (8/16) among women with 21–40 integration reads, 74.3% (26/35) among women with 41–200 integration reads, 82.8% (24/29) among women with 201–1000 integration reads, and 87.5% (28/32) among women with more than 1000 integration reads (Ptrend < 0.001).

Fig. 2
figure 2

The immediate risk of CIN2 + and CIN3 + and the 1-year cumulative risk of CIN2 + and CIN3 + at different number of HPV integration reads. Abbreviations: CIN2+, cervical intraepithelial neoplasia grade 2 or worse; CIN3+, cervical intraepithelial neoplasia grade 3 or worse; HPV, Human papillomavirus. The dashed line corresponds to the treatment referral threshold

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Table 1 The number of immediate risk of CIN2+, 1-year cumulative risk of CIN2+, immediate risk of CIN3+, 1-year cumulative risk of CIN3 + of different HPV integration reads strata
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The 1-year cumulative risk of CIN2 + and CIN3 + also increased with the number of HPV integration reads. The 1-year cumulative risk of CIN2 + was 39.1% (27/69) among women with 6–20 integration reads, 68.8% (11/16) among women with 21–40 integration reads, 88.6% (31/35) among women with 41–200 integration reads, 96.6% (28/29) among women with 201–1000 integration reads, and 96.9% (31/32) among those with more than 1000 reads (Ptrend < 0.001, Fig. 2; Table 1). The 1-year cumulative risk of CIN3 + was 33.3% (23/69) among women with 6–20 integration reads, 56.3% (9/16) among women with 21–40 integration reads, 77.1% (27/35) among women with 41–200 integration reads, 86.2% (25/29) among women with 201–1000 integration reads, and 90.6% (29/32) among women with more than 1000 integration reads (Ptrend < 0.001).

After 1 year of follow-up, the 1-year cumulative CIN2 + risk of women with integration reads more than 40 was 93.8% (90/96), which was much higher than that of women with integration reads less than 40 (38/85, 44.7%, OR = 18.6, 95%CI, 7.3–47.0, P < 0.001).

Effect of integrated HPV type on the immediate/1-year cumulative CIN2 + risk of women stratified based on HPV integration reads

Among women with immediate CIN2 + risk, we observed no significant differences between HPV16/18 integration women and non-HPV16/18 integration women with 6–20 integration reads (P = 0.206), 21–40 integration reads (P > 0.99), 41–200 integration reads (P = 0.139), 201–1000 integration reads (P > 0.99), and more than 1000 integration reads (P > 0.99, Table 2). The same patterns were observed for 1-year cumulative risk of CIN2 + between HPV16/18 integration women and non-HPV16/18 integration women with 6–20 integration reads (P = 0.204), 21–40 integration reads (P > 0.99), 41–200 integration reads (P = 0.477), 201–1000 integration reads (P > 0.99), and more than 1000 integration reads (P > 0.99).

Table 2 The relationship between HPV integration type and CIN2 + risk in each HPV integration reads strata
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HPV integration status conversion of HPV integration-positive women after 1 year of follow-up

After 1 year of follow-up, 71.4% (5/7) of women with persistent integration at the same site had more than 40 integration reads. In addition, 97.6% (40/41) of women with negative conversion and 100% (5/5) of women with HPV integration at a different site had less than 40 integration reads (Table 3).

Table 3 The relationship between participants’ positive outcomes and HPV integration reads
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In this study, persistent integration at the same site, immediate CIN2 + and 1-year CIN2 + were considered positive outcomes, and HPV integration at a different site and negative conversion were considered negative outcomes. Of 181 participants, after 1 year of follow-up, the proportion of positive outcomes was 42.0% (29/69) among women with 6–20 integration reads, 68.8% (11/16) among women with 21–40 integration reads, 100% (35/35) among women with 41–200 integration reads, 96.6% (28/29) among women with 201–1000 integration reads, and 100% (32/32) among women with more than 1000 integration reads (Ptrend < 0.001, Table 3). We observed that the proportion of negative outcomes decreased as the integration reads increased. In contrast, the percentage of positive outcomes increased with the increase in HPV integration reads (Fig. 3). After 1 year of follow-up, 99.0% (95/96) of women had positive outcomes among women with more than 40 integration reads (P < 0.001). Among women with negative outcomes, 87.0% (40/46) of women had integration reads less than 20.

Fig. 3
figure 3

The 1-year outcomes of HPV integration-positive women at different integration reads Abbreviations: CIN2+, cervical intraepithelial neoplasia grade 2 or worse; HPV, Human papillomavirus

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The association between persistent HPV integration at the same site and the development of precancerous lesions

Among 181 participants, 58 women had < CIN2 histologic results at baseline. After 1 year of follow-up, the CIN2 + progression rate of women with negative conversion and the CIN2 + progression rate of women with HPV integration at a different site were 0%. The CIN2 + progression rate of women with negative conversion was significantly lower than that of women with persistent integration at the same site (5/12, 41.6%, P < 0.001, Table S2). However, there were no significant differences in CIN2 + progression rates between women with persistent integration at the same site and women with HPV integration at a different site.

Discussion

Previous studies have suggested that HPV infections can be classified into productive infections that clear spontaneously and transforming infections that lead to cervical precursor lesions [26]. HPV integration is a crucial event in HPV-mediated malignant transformation and is associated with transforming infections [27, 28]. Previous studies have demonstrated that HPV integration can lead to genome instability, structural rearrangement, and copy number variation [29,30,31]. The level of HPV integration increases with the grade of CIN and has been suggested as a biomarker for cancer progression [32,33,34]. Moreover, recent studies have found that the number of HPV integration reads increases with the severity of CIN [21, 35]. Therefore, HPV integration reads may be a potential marker for risk stratification among HPV-positive women.

HPV integrated testing has the advantages of easy sampling, simple operation, and minimal trauma, as it uses cervical exfoliated cells similarly to standard HPV testing. Therefore, it can be effectively used as a cervical cancer screening method in clinical practice. In this study, we investigated the prognostic role of HPV integration reads in cervical precancer risk prediction. As the number of HPV integration reads increased, the immediate risk of CIN2 + and CIN3 + and the 1-year cumulative risk of CIN2 + and CIN3 + increased. In our study, compared to non-HPV16/18 infection, HPV16/18 type is more easily integrated into the human genome, which is consistent with what has been reported before [36, 37]. However, in each integration read strata, there were no significant differences in CIN2 + risk between women with HPV16/18 integration and non-HPV16/18 integration. This finding raises the question of whether the oncogenicity of different HPV types is solely associated with their ability to integrate, rather than the number of integration reads. This question warrants further studies. Furthermore, we found that the 1-year cumulative CIN2 + risk with HPV integration reads more than 40 was 93.8%, which was significantly higher than the 1-year cumulative CIN2 + risk with HPV integration reads less than 40. Therefore, our findings suggested that HPV integration reads more than 40 can indicate an extremely high risk for CIN2+. These women should be referred to expedited treatment based on the guideline [38].

We explored the integration status conversion among HPV integration-positive women during a 1-year follow-up period. The results showed that women with less than 40 integration reads were more likely to have negative conversion or HPV integration at a different site. On the contrary, women with integration reads more than 40 were more likely to maintain persistent HPV integration at the same site. For women with persistent integration at the same site, the number of integration reads increased during the follow-up. On this basis, we investigated whether this persistent integration status was associated with cervical cancer progression. The results demonstrated that women with persistent integration at the same site had a significantly higher progression rate than those with negative conversion. Thus, persistent integration at the same site may be a potential biomarker for CIN2 + progression. Previous studies have shown that HPV integration occurs in two forms: productive integration and silent integration [39]. The former is closely associated with the development of cervical precancerous lesions, while the latter rarely progresses to cervical cancer. Therefore, we hypothesized that HPV integration at different sites may be the result of natural clearance of a silent and non-oncogenic integration site. However, in our study, the difference in progression rates between women with persistent integration at the same site and women with HPV integration at a different site was not statistically significant, probably due to the small sample size. After one year of follow-up, 99.0% of women with more than 40 integration reads had persistent integration at the same site, immediate CIN2 + or 1-year CIN2+, suggesting that women with integration reads more than 40 might have a high risk of CIN2+. Therefore, close attention should be paid to women with more than 40 integration reads or persistent integration at the same site in clinical settings.

HPV integration test may help with risk management after colposcopy. In our study, we discovered two women with negative cytology results and normal histology, one with HPV35 infection and the other with HPV16 infection. Based on the guidelines, they were referred to 1-year follow-up. However, both patients had more than 40 integration reads and progressed to AIS within 1 year. Several researchers have found that AIS has rapid progression, is difficult to identify, and can be easily misdiagnosed [40,41,42]. The immediate CIN3 + risk for these two women was more than 70% based on our risk stratification results, while the strategy recommended by the American Society for Colposcopy and Cervical Pathology (ASCCP) suggested that women with an immediate CIN3 + risk of 60% were referred to treatment instead of colposcopy [38]. Therefore, based on our results, women with more than 40 HPV integration reads should be referred to cervical conization, and women with integration reads between 20 and 40 should be referred to diagnostic conization if follow-up is inconvenient. Li et al. reported that the HPV integration test can prevent the misdiagnosis of cervical cancer [43]. Besides, some rare combinations, such as high-grade squamous intraepithelial lesion (HSIL) or atypical squamous cells, cannot rule out HSIL (ASC-H) cytologic results with histological results ≤ CIN1, the risk assessment according to the guideline can be less reliable, because of the occult diseases after any high-grade cytology [44]. For these rare combinations, HPV integration reads can help colposcopists assess the risk of lesions, and together with biopsy, can prevent missed diagnoses.

To our knowledge, this is the first study to evaluate the CIN2 + risk and HPV integration status conversion among HPV integration-positive women. The strengths of this study were as follows. Previous techniques for identifying integration events based on the E2/E6 ratio may be inaccurate, as the E2 gene may not be affected in some events, while HIVID could overcome this problem in this study [45, 46]. Compared to WGS, identifying HPV integration events using HIVID can detect more HPV integration sites in cervical exfoliated cells [19]. Therefore, our approach provided accurate integration data for CIN2 + risk assessment and HPV integration status, suggesting that HPV integration can precisely predict the risk of developing cervical precancerous lesions. Technically, the number of integration supporting reads correlates with the number of cells in which HPV integration occurs, while cervical squamous epithelial cells were usually exfoliated once every 4–5 days [47]. Thus, we hypothesized that a higher number of integrated cells more robustly supports the abnormal proliferation of the same clone. In summary, HIVID is promising in the clinical stratification and clinical management of high-risk CIN lesions and is affordable for the majority of patients in our country.

There were also some limitations to this study. First of all, the follow-up period was short. Considering only 5 women progressed into CIN2 + and 12 women had persistent integration, it was hard to calculate the statistical power. Only a small proportion of HPV integration was productive integration, increasing the risk of transformation from cervical precancerous lesions to cancer. In addition, HPV can be integrated into different human chromosomes and different gene loci (Table S3). The integration reads and the persistence of HPV integration are the results of the natural selection of different integration sites. Many studies have shown that the frequency of distinct integration sites is different and the ability to promote cancer development may also depend on the integration site [48]. These findings should be verified in a bigger population. Finally, the sample size was relatively small and a larger sample size is needed to investigate whether HPV integration test can safely stratify follow-up intervals and prevent excessive treatment. For example, women with HPV integration reads more than 40 integration reads need shorter follow-up intervals if diagnostic cervical conization is not conducted. A multicenter cohort study based on a large population with longitudinal follow-up led by our hospital is being conducted.

Conclusions

This study was the first to explore risk stratification based on HPV integration levels and HPV integration status conversion among HPV integration-positive women. The CIN2 + risk increased with the number of HPV integration reads. After 1 year of follow-up, the majority of women with more than 40 integration reads experienced persistent integration at the same breakpoint. Women with more than 40 integration reads or persistent integration at the same site need to be vigilantly monitored in clinical practice. In conclusion, the number of HPV integration reads may be a potential biomarker for early warning and precise identification of high-risk CIN lesions.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

95% CI:

95% confidence interval

AIS:

Adenocarcinoma in situ

CIN:

Cervical intraepithelial neoplasia

CIN1:

Cervical intraepithelial neoplasia grade 1

CIN2:

Cervical intraepithelial neoplasia grade 2

CIN2 +:

Cervical intraepithelial neoplasia grade 2 or worse

CIN3:

Cervical intraepithelial neoplasia grade 3

HIVID:

High-throughput viral integration detection

HPV:

Human papillomavirus

NGS:

Next-generation sequencing

OR:

Odds ratio

WGS:

Whole genome sequencing

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Acknowledgements

The authors would like to express their gratitude to EditSprings (https://www.editsprings.cn) for the expert linguistic services provided.

Funding

This work received funding from the National Key R&D Program of China (2021YFC2701201).

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Author notes

  1. Xiaoyuan Huang and Ting Hu are co-corresponding authors and contributed equally to this work.

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, China

    Kexin Li, Fanwei Huang, Tao Zhang, Ting Hu & Xiaoyuan Huang

  2. New Technology Platform, Wuhan KDWS Biological Technology Co., Ltd., Wuhan, Hubei, 430074, China

    Fan Yang, Weitao Duan & Shimin Chen

  3. Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Anv, Wuhan, 430030, China

    Ting Hu & Xiaoyuan Huang

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  1. Kexin Li
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  3. Tao Zhang
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Contributions

Kexin Li: Conceptualization, Formal analysis, Validation, Writing—original draft and Writing—review and editing.Ting Hu: Conceptualization, Formal analysis, Validation, Supervision, Writing—original draft and Writing—review and editing. Fanwei Huang: Formal analysis, Validation and Writing—review and editing. Tao Zhang: Validation and Writing—review and editing. Fan Yang: Data curation and Validation. Weitao Duan: Data curation and Validation. Shimin Chen: Data curation and Validation. Xiaoyuan Huang: Supervision, Validation and Writing—review and editing.

Corresponding authors

Correspondence to Ting Hu or Xiaoyuan Huang.

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Li, K., Huang, F., Zhang, T. et al. HPV integration status conversion and CIN2 + cancer risk stratification based on HPV integration levels among HPV integration-positive women: a 1-year follow-up study. BMC Cancer 25, 885 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12885-025-14138-4

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BMC Cancer

ISSN: 1471-2407

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