Testing for viral DNA integration among HPV‐positive women to detect cervical precancer: An observational cohort study

Human papillomavirus (HPV) integration is a crucial genetic step in cervical carcinogenesis. This study aimed to evaluate the performance of an HPV integration test for the triage of HPV‐positive women.

4][5][6] The American Society for Colposcopy and Cervical Pathology (ASCCP) recommends HPV testing or co-cytology testing as the primary approach for cervical cancer screening. 7][6]8,9 In addition, HPV testing detects significantly more CIN1 and CIN2 lesions, resulting in a 20% increase in loop electrosurgical excision procedures (LEEP). 10][13][14] Next-generation sequencing (NGS) technology coupled with advanced computational approaches have identified promising biomarkers for clinical use. 15In 2015, our team first conducted whole-genome sequencing and high-throughput viral integration detection (HIVID) to investigate the genome-wide profiling of HPV integration in cervical cancer. 168][19] A recent clinical case report indicated that an NGS-based HPV integration test can help avoid misdiagnosis in cervical cancer patients. 20Furthermore, evidence suggests that HPV integration is related to CIN levels in the cervix and may serve as a potential biomarker for assessing cervical cancer risk in HPVpositive women. 16,18,21Notably, the frequency of HPV integration increases with cervical disease progression. 22e report the results of a large observational cohort study that we conducted to evaluate the clinical performance of the HPV integration test by hybrid capture-based NGS among HP-positive women.

| Study population
Between July 2020 and April 2021, a total of 2093 consecutive HPV-positive women were enrolled in the present study.All were tested for HPV integration and cytology within 3 months after the positive HPV test.The inclusion criteria were as follows: women aged 25-65 years, a positive HPV screening test and HPV integration test, and no prior history of CIN2 or more severe cervical dysplasia.Women with any one of the following were excluded: pregnancy; without colposcopy when indicated; undetermined pathology; discrepant HPV infection results between screening and HPV integration test; and loss to follow-up.Patients who met the criteria underwent a 1-year prospective follow-up.
Clinical management in our study was based on the 2019 ASCCP guidelines.Women were immediately referred to colposcopy and biopsy (within 3 months) if they had abnormal cytologic results or were positive for HPV16 or 18. Women with negative cytologic results and high risk HPV strains other than 16 or 18 were tested after 1 year and were referred to colposcopy if either HPV or cytologic test results were positive.This cohort study was approved by the Ethics Committee of Tongji Hospital of Huazhong University of Science and Technology (TJ-IRB20211110) and a waiver of informed consent was granted.Clinical information and laboratory data were collected from electronic medical records without personal identifying information.

| Clinical routine cytologic test
Pap samples were collected using a cytology brush and stored in tubes filled with ThinPrep® PreservCyt Solution (Hologic).Slides were prepared, stained and processed on Thinprep 5000 Processor (Hologic).All the slides were reviewed by experienced pathologists who had access to patients' clinical history through computer-aided reading (Hologic).According to the Bethesda system, the cytologic results were broadly divided into the following categories: no intraepithelial lesion/malignant lesion cell (NILM), atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), atypical squamous cells -cannot rule out HSIL (ASC-H) and high-grade squamous intraepithelial lesion (HSIL).

| HPV integration test
A human papillomavirus integration test was performed using HIVID as described in our previous study. 16In this report, samples with more than five sequencing reads supporting HPV integration were considered HPV integration-positive.The integrated region was verified by PCR and Sanger sequencing.This HPV integration test can also detect HPV infection types; patients with discrepant HPV infection results between the HPV integration test and HPV genotyping were excluded.

| Clinical endpoints
The histologic findings of the biopsy were the gold standard for the final diagnosis, and the classification was based on the CIN scale: no indication for biopsy, normal, CIN grade 1 (CIN1), CIN grade 2 (CIN2), CIN grade 3 (CIN3) and adenocarcinoma in situ (AIS) or cancer.All the histological results were reviewed by two pathologists at our hospital.Women without colposcopy or biopsy indications were considered free of disease in the present study.
The primary endpoint was CIN3 or more severe dysplasia (CIN3+), including CIN3, AIS and cervical cancer.The second endpoint was CIN2 or more severe dysplasia (CIN2+), which included CIN2, CIN3, AIS and cervical cancer.

| Statistics
Statistical analyses were performed using SPSS Statistics, version 24.0 (IBM Corp.) and R (version 3.3.1).All P-values were two-sided and a P-value <0.05 was considered statistically significant.The chi-square trend test was used to evaluate HPV integration positivity in cytology (NILM, ASCUS/ LSIL, ASC-H and HSIL) and histology (benign/no biopsy, CIN1, CIN2, CIN3/AIS, cancer) strata.Categorical variables were analysed by Pearson's chi-square test or Fisher's exact test.We calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the detection of CIN2+ and CIN3+ by various triage strategies.Differences in positivity, sensitivity and specificity were evaluated using an exact McNemar's Chi-square, and differences in PPV and NPV were evaluated using the method developed by Leisenring and Pepe, with the R package 'DTComPair'. 23e calculated the risk of CIN3+ for different screening strategies and compared these with the internal risk benchmarks in our study population using the method reported by a previous study. 24The risk of CIN3+ in HPV-positive women with ASCUS (6.1% [14/230]) was the benchmark for immediate risk of colposcopy referrals, and the risk of CIN3+ in HPV-positive women with NILM (3.3% [28/856]) was the internal benchmark for a 1-year return.These risks differ.

| Baseline characteristics of study population
A total of 1393 women with positive HPV results were enrolled in the study (Figure 1).The median age of participants was 42 years (interquartile range 34-51; see Figure S1 for distribution).Of these women, nine had cervical cancer, 86 had CIN3 or adenocarcinoma in situ, 24 had CIN2, 140 had CIN1, and 1134 had benign lesions or did not require colposcopy (Table S1).The most common HPV infection types were HPV16, 52, 58 and 18.There were 352 HPV16positive and 109 HPV18-positive women (with negative HPV16 results), and 932 women with non-HPV16/18 positive results (Table S2).

| Risk stratification of CIN3+ for different screening strategies
Risk stratification allows more low-risk populations to be identified and thus reduces referral rates for colposcopy.Figure 2 shows the risk stratification of CIN3+ for the combination of HPV16/18 genotyping and cytologic triage, HPV16/18 genotyping and HPV integration triage, HPV16 genotyping and HPV integration triage, and the HPV integration triage alone.Compared with the lowest risk population in the ASCCP screening strategy who were NILM and HPV16/18-negative (10/564, 1.8%), women with HPV16-negative and HPV integrationnegative results had a similar low risk of CIN3+ (16/965, 1.7%) and a larger population size (69.3%versus 40.5%) (Table S4, Figure 2).Women with HPV16-positive and HPV integration-positive results had the highest risk of CIN3+ (38/62, 61.3%) among all combinations, more than twice as high as those with ASCUS+ and HPV16/18positive results (42/169, 24.9%, Figure 2).When stratified by HPV integration, more women showed a low risk of CIN3+ below the threshold for colposcopy referral compared with the combination of HPV16/18 genotyping with cytology (1255 [90.1%] versus 564 [40.5%], Figure 2).
We also investigated the performance of HPV integration in the triage of specific populations (HPV16/18 − and ASCUS/ LSIL, NILM, and HPV16/18 + ).Results showed that the risk of CIN3+ in HPV integration-negative women in all populations was below the 1-year return threshold (Figure S2, Table S5).

| One-year follow-up
After 1-year follow-up, eight of 69 women (12.0%) with HPV integration and benign/CIN1 at baseline developed highgrade cervical lesions, including two with CIN2 and six with CIN3.In addition, 25 of 1205 women (2.1%) without HPV integration developed high-grade lesions after 1 year -15 with CIN2 and 10 with CIN3 (Figure 1).The progression rate of HPV integration-positive women was significantly higher than that of HPV integration-negative women (OR 5.6, 95% CI 2.6-11.9,P < 0.001).
Two women with CIN2 with positive HPV integration results and 12 of 22 (54.5%) with negative HPV integration results were treated with local excision of the cervix.In 10 conservatively managed integration-negative CIN2 patients, all showed spontaneous regression and seven (70%) showed HPV clearance after 1 year (Table S6).

| Main findings
In this study, we demonstrated that the HPV integration test had lower positivity and better accuracy than cytology in  HPV-positive women including in with HPV16 or 18. Notably, compared with the lowest risk population in the ASCCP screening strategy who were NILM and HPV16/18negative, women with HPV16-negative and HPV integrationnegative results had a similarly low risk of CIN3+, and included a large portion of all HPV-positive patients.These results indicated that HPV integration-based screening strategy could safely reduce unnecessary colposcopy referrals.Furthermore, our results demonstrated that the CIN2 lesions of women with negative HPV integration results spontaneously regressed after 1 year of follow-up.These findings suggest that an HPV integration-based screening strategy could safely triage a larger proportion of HPV positive women to repeat testing in 1 year, and thereby reduce excessive colopscopic referrals.

| Interpretation
Although early human papillomavirus (HPV) vaccination may substantially reduce cervical cancer incidence and mortality, to date the coverage rate is relatively low. 25Thus, early screening remains the most important intervention in the prevention of cervical cancer in China.There are approximately 245 million women aged 35-64 years in China requiring cervical cancer screening, 26 leading to a heavy burden of colposcopic referral on the healthcare system.Meanwhile, in clinical settings with primary HPV-based cervical screening, the positivity of colposcopy examination is relatively low. 27A more precise triage method is urgently needed to avoid unnecessary colposcopy referrals in China and worldwide.HPV integration is a crucial genetic step in cervical carcinogenesis.Our study suggests that HPV integration testing is a robust stratification method for HPVpositive women, which is also a prognostic tool to predict progression to high-grade dysplasia.
Research suggests that HPV infections can be classified into productive infections that clear spontaneously, and transforming infections that lead to precursor lesions or larger lesions. 28HPV integration is associated with transforming HPV infection and may lead to instability and The risk of CIN3+ for different screening strategies.The risks of CIN3+ for combinations of cytologic tests with HPV16/18 genotyping, HPV integration with HPV16/18 genotyping, HPV integration with HPV16 genotyping, and HPV integration alone are plotted on the y-axis, with the number and percentage of women indicated.The dotted line corresponds to the 1-year return threshold (negative for intraepithelial lesions or malignant neoplasm [NILM] in HPV-positive women, 3.3%) and the dashed line corresponds to the colposcopy referral threshold (atypical squamous cells of undetermined significance [ASCUS] in HPV-positive women, 6.1%).
aberrant expression host genome, so it is a crucial genetic step in the occurrence and development of cervical cancer. 29,30Consistent with previous studies, our study confirmed that the frequency of HPV integration is positively correlated with the severity of cervical precancerous lesions. 22,31,32e observed that 100% of CIN2 diseases with negative HPV integration results spontaneously regressed within 2 years in our study, which was higher than the 50% regression rate reported by a systematic review and meta-analysis of studies from 1973 to 2016. 33The overtreatment of CIN2 may lead to adverse outcomes such as miscarriage and premature birth, which will decrease the newborn birth rate, an issue that is especially salient in China.Our results indicated that CIN3+ may be more suitable as the main endpoint instead of CIN2+ in cervical cancer screening protocols, as recommended by ASCCP. 7Furthermore, for the detection of CIN3+, our study demonstrated that HPV integration was a more robust stratification tool in HPV-positive women compared with cytology, especially in HPV16/18-positive women.
In the 2019 ASCCP recommendations for cervical cancer screening, all women with HPV16 or 18 are recommended to receive colposcopies immediately.However, over 90% of HPV16 and 18 infections will clear spontaneously after 2 years. 34,357][38] Our study observed a high sensitivity (80%) and a high specificity (90.8%) of the HPV integration test for detection of CIN3+ in HPV16/18-positive women, and the immediate CIN3+ risk of HPV16/18-positive women with negative HPV integration results was lower than the internal benchmark for immediate risk of colposcopy referrals.Considering these results, HPV integration might play an important role in the future, including the triage or guiding the management of HPV16/18-positive women in conjunction with patient screening history and other risk factors.
It is worth noting that a prospective cohort study (EXPL-HPV-002) of 688 women in the Czech Republic has been conducted to evaluate the diagnostic and prognostic value of HPV integration by molecular combing method since 2016. 22However, the threshold and accuracy for triage and the results of the longitudinal phase have not been reported.

| Strengths and limitations
To the best of our knowledge, this is the first study to evaluate the performance of HPV integration by an NGS-based method for the triage of HPV-positive women.Our study was based on a large population with well-organized screening and management and limited loss to follow-up, thus providing precise estimates of precancerous lesion risk.In addition, all procedures were carried out in parallel with routine clinical cervical precancerous lesion screening protocols (ASCCP-recommended strategies), thus allowing the direct comparison of our novel HPV integration test with established management strategies.
Several study limitations should be noted.First, not all women underwent colposcopy or biopsy at the baseline stage, and the CIN3+ risk of women without colposcopy or biopsy indications may be underestimated in our study.Secondly, the follow-up period of this study was short and the HPV integration test was not performed during the 1year follow-up phase.A multi-centre prospective cohort study initiated by our centre is ongoing to investigate a long-term screening strategy incorportating HPV integration.Thirdly, the vaccination coverage was relatively low in our population, and the performance of the HPV integration test cannot be evaluated among women who have been vaccinated against human papillomavirus.Fourthly, because there was a short period of time (up to 3 months) between the Cobas test and the HPV integration test, 4.9% of the HPV-positive patients had discrepant HPV infection results and were excluded, which might have had some effect on the results.

| CONCLUSIONS
Our findings suggested that HPV integration testing, alone or combined with HPV genotyping, could play an important role in risk stratification in HPV-based cervical cancer screening programmes.Compared with cytology, HPV integration testing may greatly reduce colposcopy referrals and could reduce overtreatment of patients with CIN2.Furthermore, among HPV-positive patients without highgrade dysplasia at baseline, HPV integration predicts progression to high-grade dysplasia.

AU T HOR C ON T R I BU T ION S
PW, XH and DM conceived, designed and directed the study.HW, PW and DM acquired funding.TH, LH and FH acquired and analysed the data.TH and KL wrote the draft of the article.FY and SC provided technical support.PW, XH, HW and DM reviewed and revised the article.All authors read and approved the final article.The order of the co-first authors was assigned based on the relative contributions of the individuals.

AC K NO W L E D GE M E N T S
We are grateful to the patients who participated in the study.

DATA AVA I L A BI L I T Y S TAT E M E N T
The corresponding authors had full access to all of the data in the study and had the final responsibility to submit the article for publication.Research data in this study are not shared.

E T H IC S A PPROVA L
This cohort study was approved by the Ethics Committee of Tongji Hospital of Huazhong University of Science and Technology (TJ-IRB20211110).

T A B L E 1
Performance of cytology and human papillomavirus (HPV) integration among HPV-positive and HPV16/18-positive women for detection of CIN2+ and CIN3+.

F
U N DI NG I N FOR M AT IONHW was supported by the National Key R&D Program of China (grant 2021YFC2701204).PW was supported by the National Natural Science Foundation of China (82072895) and the National Key R&D Program of China (grant 2021YFC2701201).DM was supported by the National Natural Science Foundation of China (grant 82141106).FY was supported by the Wuhan Scientific and Technical Achievements Project (grant 2020030603012339).The funding grant for this study did not include external peer review for scientific quality and priority assessment.

C
ON F L OF I N T E R E S T S TAT E M E N TDM is an inventor of a patent for the HPV integration test.DM and XH have stock ownership in Wuhan KDWS Biological Technology Ltd.All other authors declare no competing interests.
Performance of different combined screening strategies among 1393 women for detection of CIN2+ and CIN3+.
Note: Thresholds: Cytology ≥ ASCUS; HPV integration >5 reads.Abbreviations: ASCUS+, Atypical Squamous Cells of Undetermined Significance or more severe cytologic diagnosis; CIN2+, cervical intraepithelial neoplasia grade 2 or worse; CIN3+, cervical intraepithelial neoplasia grade 3 or worse; HPV, human papillomavirus; NPV, negative predictive value; PPV, positive predictive value.aComparison between HPV integration and cytology was calculated using McNemar's Chi-square test for positivity, sensitivity and specificity, and the method developed by Leisenring and Pepe for predictive values.All statistical tests were two-sided.TA L E 2