Preliminary study of HPV integration status on the occurrence and development of Vaginal intraepithelial neoplasia

doi:10.21203/rs.3.rs-3142728/v1

Download PDF

Research Article

Preliminary study of HPV integration status on the occurrence and development of Vaginal intraepithelial neoplasia

https://doi.org/10.21203/rs.3.rs-3142728/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 10 Dec, 2023

Read the published version in Journal of Obstetrics and Gynaecology Research →

Version 1

posted

You are reading this latest preprint version

Vaginal intraepithelial neoplasm (VaIN), a precancerous lesion of vaginal cancer, increased distinctly in the past decade, while its occurrence after performing hysterectomy came to attention nowadays. As hr-HPV DNA integrates into host chromosome, transient HPV infection could transform into persistent infection to trigger high-grade intraepithelial neoplasm or HPV-related cancer. High-throughput Viral Integration Detection (HIVID) is a novel genetic sequencing method detecting HPV integration breakpoints for more precise sequence. There is no systematic study on the mechanism and risk assessment prediction system of VaIN in the vaginal stump after total hysterectomy for CIN. In this study, 24 cases suffered from VaIN were involved. HPV integration was detected in 11 cases, furthermore, we discovered HPV 16 and 73, chromosome 1 and 2 possessed most HPV integration sites while EMBP1, CLO5A1, EHF, ELF5 as dominate hot spots. Taken clinical outcome into account, we found a significant difference between HPV integration occurrence and VaIN(p = 0.011). HPV integration could serve as a predictive marker for monitoring and management of VaIN in a way.

Vaginal intraepithelial neoplasm

High-throughput Viral Integration Detection

HPV integration

integration sites

clinical outcome

Vaginal intraepithelial neoplasm(VaIN), a condition that is considered to be the precancerous lesions of vaginal cancer. Sui L et al[1] indicates that the morbidity of VaIN in lower genital tract intraepithelial neoplasia is 13.08%༈834/6374)during 2015, increased distinctly compared with that of 2013. Previous studies show that the incidence of VaIN in USA is 0.2–200/100000[2], while its occurrence after performing hysterectomy is 0.9%~6.8%. Due to the specific anatomical characteristics of the vagina and the low incidence of VaIN, there is a lack of awareness and serious underestimation of VaIN nowadays, leading to insufficient attention to the diagnosis, treatment and follow-up. Some clinical risk factors that we know for the occurrence of VaIN are age, persistent hr-HPV infection, history of cervical cancer and cervical intraepithelial neoplasia, history of radiation therapy, immune deficiency, etc[3, 4].

Groves et al[5] found in up to 85% of cervical squamous cell carcinomas that hr-HPV DNA could integrate into host chromosome, turning "transient infection" into “persistent infection”. Hr-HPV DNA integration is an important event to initiate cervical carcinogenesis. When HPV is integrated, it is not easily detected by the host immune system, thus clearance became difficult, which is more common in high-grade intraepithelial neoplasia and cervical cancer[6–8]. As integrated hr-HPV increases and episomal HPV decreases, the lesion severity increased. However, the relationship between integration status and the development of VaIN are still unclear.

High-throughput Viral Integration Detection (HIVID), as a genetic sequencing method for HPV integration breakpoints, is available for detection of more precise sequence[9]. It can measure the expression level of oncogene in addition to detect the integration site and HPV status at the same time, confirmed as a cost-effective methodology with high specificity and sensitivity to identify viral integration in genome[10].

There is no systematic study on the mechanism and risk assessment prediction system of VaIN in the vaginal stump after total hysterectomy for CIN. Herein, the lesion of vaginal stump after hysterectomy due to cervical lesion or single VaIN is a hotspot and urgent clinical problem nowadays. The current study mainly focuses on the impact of HPV integration in cervical intraepithelial lesion, the relationship of HPV integration and VaIN is not enough[7]. This study aimed to explore the association of integration breakpoints and HPV integration in VaIN.

1. Sample collection

24 frozen vaginal tissues were collected at the First Affiliated Hospital of Nanjing Medical University, between October 2019 and June 2020. Medical history of every included patient were recorded, the following demographic data were documented including age, gravidity, parity, history of hysterectomy and cervical dysplasia. The hr-HPV and Pap tests, colposcopic findings, histologic diagnoses were also noted in our study. Pathological diagnosis was deemed as golden standard in our study. This study was approved by the ethics committee of First Affiliated Hospital, Nanjing Medical University (approval No.2021-SR-423).

2.HPV library sequencing bioinformatic analysis process

Prepare HPV DNA whole-genomic library

In order to establish the whole genomic library for HPV, based on Sure Select XT Target Enrichment System, we prepared a library of DNA small fragments, including the following steps:

(1) Using Covaris Ultrasonic Crusher on genomic DNA (qualified in quality inspection) for fragmentation process, sizes of 150-200bp DNA fragments were obtained. (2) Perform DNA fragment end repair and joint connection method (adapter) to construct the library. (3) We applied ligation-mediated PCR(LM-PC) to conduct pre-amplification and quality control, the eligible libraries were captured for exon hybridization and chip hybridization. The captured samples were re-amplified by LM-PCR at the end.

3. Bioinformatics analysis method

The raw image data obtained by running the double-end sequencing program (PE150) on the Illumina HiSeq sequencing platform is converted into raw sequence data (raw reads) by Illumina Base Calling software, and then stored in the form of FASTQ file, named after raw data. Information on the human genome and HPV sequences can be assessed through informatics analysis of high-throughput sequencing offline files. The bioinformatics analysis comprises quality control and preliminary processing of the offline data, calculating the sequencing data output, and then matching the data to the human and HPV reference genomes using software such as BWA[11], searching for integration sites using the HIVID method[10], and annotating the data using ANNOVAR software. The analysis of HPV sequencing data is mainly as follows: (1) basic quality control of offline data, including de-junctioning and removal of low-quality reads, etc.; (2) alignment of quality-controlled data to the reference genome by BWA software, GRCh37/hg19 and HPV sequence databases; (3) filter out reads that are perfectly aligned to human and HPV genomes, and keep the reads that match to both human and HPV; (4) assemble the retained paired reads with FLASH software to obtain PE-assembled reads; (5) align PE-assembled reads to the reference genome by BWA software, GRCh37/hg19 and HPV sequence databases; (6) Keep reads that match both human and HPV genomes, detect integration sites and integrate sites within 20bp and standardize them; (7) Apply the HIVID method to filter the integration sites and obtain high-confidence integration sites (8) Annotate the integration sites using ANNOVAR software.

4. PCR amplification and Sanger sequencing

Based on the Whole-Exome Sequencing (WES) results, 25 HPV integration sites were screened. We downloaded the gene sequences of integration sites exploiting the information of genes provided by GRCH37 database, and primers were designed for PCR using the software Primer4.0 (v.0.4.0). All primers were synthesized by Shanghai Biotech Bioengineering Co. PCR products were soaked in 5 µl 1% agarose gel electrophoresis. Electrophoresis parameters were set as 150 V, 100 mA, 10 ~ 20 min (see electrophoresis figure). The PCR products were recovered according to the size of the primer PCR products and conducted Sanger sequencing for validation.

5. Statistical analysis

Continuous data are described using the mean standard deviation and compared between groups using the two-sample t test. Categorical variables are presented as counts with percentages and compared between groups using Fisher exact test. All statistical analyses were conducted using SPSS 25.0. Two-tailed p < 0.05 was considered as statistically significant.

1. Study design and Patient identification

1.1 Baseline and clinicopathologic characteristics

Among 24 patients (age: 53.0\(\pm\)11.4 years), 11 vaginal tissues were collected while performing hysterectomy for CIN before the diagnosis of VaIN. In six-month follow-up, 3 of them developed into VaIN and others remained normal, named after newly discovered VaIN and normal group. Other cases defined as original VaIN group were diagnosed at inclusion. The distribution of histologic grade at inclusion was as follows: 4 cases were diagnosed as VaIN 1, 3 cases as VaIN 2 and 6 cases as VaIN 3. During the follow-up, 6 of them regressed to normal, 6 showed persistent lesion, while the other one progressed to VaIN 3 (Table 1).

Table 1

Baseline and demographic characteristics (n = 24)
Pathology	hysterectomy		history of CIN		HPV types
Pathology	yes	no	yes	no	HPV16/18	OT12	HPV16/18 + OT12
normal(n = 11)	11	0	11	0	3	3	5
VaIN(n = 13)	7	6	11	2	5	3	5
OT12: 12 other hr-HPV genotypes including HPV 31,33,35,39,45,51,52,56,58,59,66, 68

1.2 Pap and HPV results

Cytology reports of VaIN included as follows: NILM (negative diagnoses for intraepithelial lesion or malignancy): 5 cases, ASC-US (atypical squamous cells of undetermined significance):6 cases, LSIL 4 cases, ASC-H (atypical squamous cells that cannot exclude HSIL):3 cases, and HSIL:6 cases. As to HPV genotype detected by cobas 4800, HPV16/18 is the most prominent types in these samples, affecting 16 patients. Followed by other 12 genotypes, which account for 18 cases in total. Overall, there were no statistically significant differences in patient characteristics among the different grades of VaIN.

2. Detection of HPV integration breakpoints

We applied HIVID method on these tissues to obtain HPV integration breakpoints. Among the 24 patients enrolled, HPV integration was detected in 11 cases. No HPV integration was found in newly discovered VaIN group, 37.5% cases detected integration in normal group (3/8), and 92.3% original VaIN group possessed integration (12/13). Based on this, we divided them into two subgroups as integrated group and non-integrated group. 92 gene integration events were detected in 24 cases for 20 HPV gene subtypes, which involved 109 distinct genes.

2.1 distribution of HPV integration sites in host chromosome

HPV integration breakpoints were distributed among all different 24 human chromosomes in Fig. 1. Chromosomes 1 and 2 were inspected the most HPV integration sites in our study, accounting for 11.9%. Followed by chromosome X, making up 11% separately. We detected chromosome 13 comprises only 0.9% integrated events, ranking the last one in all chromosomes. 7 HPV integration sites were detected in normal group, which scattered among chromosome 9,11, and 17. On the other hand, chromosomes 1 and 2 took the lead in original VaIN group, which accounted for 12.7% and 12.7%, respectively.

2.2 distribution of HPV integration sites in host genes

In accordance with previous studies, what we observed is that integration sites were randomly scattered in the host genome. Among these 103 HPV breakpoints, EMBP1(5.5%) was the most frequent integration hot spots in our analysis, CLO5A1(1.83%), EHF (1.83%), ELF5(1.83%), LOC101927406(1.83%), LOC441666(2.75%) were other leading hot spots in these 24 cases. COL5A1, ELF5, EHF and CUEDC1 were HPV breakpoints regarding to normal group. In original VaIN group, however, EMBP1(5.9%) and LOC441666(2.9%) were the dominant host gene. Other detailed hot integration gene sites were expressed in Fig. 2.

2.3 distribution of integration sites in HPV types

Regarding the integrated HPV type in these 103 genes, HPV 16 occupied the most integration sites unexpectedly, for HPV 16 and HPV 18 are two main subtypes during carcinogenesis. HPV73 was the second integrated genotype in this study, which accounted for 14.5%. Our analysis indicated that apart from HPV16, HPV 52 and HPV 58 are found integrated, taking up 12% integration sites (Fig. 3).

2.4Association of HPV integration and clinical outcome

By conducting Fisher exact test based on whether neoplasia happened in vaginal tissues, we found a significant difference between HPV integration occurrence and VaIN(p = 0.011). Based on the histopathologic findings of six-month follow-up, the results showed 14 patients regressed or remained free from VaIN, 6 patients remained persistent while the other four were progressed or newly discovered.

Regarding cases that remained disease-free or regressed, HPV integration events were found in five tissues, corresponding to different HPV integration types, with high-frequency types such as HPV 16, 32, 52, 35, 56, 58, 62, and 84, respectively, and showed a diversity in integration sites.

The four cases with progressive or newly discovered lesions were then further analyzed, all of which were vaginal lesions found at follow-up after hysterectomy. 11 HPV integration events were found in two of them, and the follow-up pathology was VaIN 3 in both cases, while the other non-integrated cases were VaIN 1. The corresponding HPV integration types were HPV16 and HPV73, integrated on chromosomes 2, 4, and 8. The four cases had no significant specificity of integration loci, including the previously detected genes like LOC441666, LOC101927406, ELF5, EHF. Among them, the relationship of integration loci for vaginal pathogenesis progression revealed the presence of HPV16 and HPV33 infections in all this specimen, and the integration loci were present in HPV73.

VaIN, as the inevitable stage of vaginal cancer, long-term surveillance is still in need considering the persistence and progression rate of VaIN. Careful observation of the vagina is often neglected by colposcopists, resulting in some patients being miss diagnosed or delayed in treatment[12, 13]. Studies have shown that nearly 96% of VaIN 2/3 are associated with HPV infection. The reason for this regional effect is that the upper 1/3 of the vagina has the same embryological origin as the cervix[14], and tissues of common embryonic origin are susceptible to tumor or precancerous lesions due to similar oncogenic stimuli. Given the fact that hysterectomy for CIN is a common procedure worldwide, co-test of cytology and HPV in vaginal stump ought to be performed routinely to monitor occurrence of vaginal lesion. According to 2019 ASCCP[15], 3 consecutive annual HPV-based tests combined with cytology tests before entering long-term surveillance is recommended. Nevertheless, our study also put a novel insight on the relationship between HPV integration and clinical outcome in VaIN.

Zhang et al[16] found that the most common HPV genotype among VaIN in China were HPV 16,33,81,53,18,58, and 66. Due to the limits of HPV cobas 4800 method, we were unable to identify other HPV genotypes besides HPV 16 and 18, which requires a better differentiation method to further investigate epidemiology of VaIN. It’s common in clinical work to detect co-infection of HPV 16/18 with other genotypes, such as HPV33 and 58 in vaginal lesions[17]. However, it’s common knowledge that HPV16 has the most tumorigenicity. One possible explanation is that HPV test swab foliating ample cells for analyzing, instead of obtaining accurate precancerous tissue by biopsy. This reminds us when comes to integration, HPV genotypes also take part in predicting clinical outcome.

Huang’s[18] research showed that chromosome 2 had the most integration events, our study also supported this theory. The possible explanation for this could be length of chromosome 2 is in the lead of all. The results of our HPV integration breakpoints showed diversity from other studies, which supports previous conclusion that HPV is randomly integrated into the host genome at the beginning. However, the recurrent loci of hot genes in every individual provided a growth advantage for carcinogenesis. Previous studies majored in HBV and HPV demonstrated that Common Fragile Sites (CFS) were widely located among human chromosomes, which provided a weakness in human genome that enables carcinogenic virus to integrate. Integration of virus creates a chromosomal instability via chromosomal translocation[19], leading oncogenesis in hosts. Previous sequencing methods have disadvantages against HIVID such as longer sequencing time, more samples required, low sensitivity, time-consuming, poor results in amplifying HPV, and higher probes cost. With the development of sequencing method, we may have the ability to detect novel hot genes that promote carcinogenesis or facilitate the development of vaginal precancerous lesion.

Here we reviewed previous literature on hot genes ETS homologous factor (EHF), E74-like factor 5 (ELF5) and lncRNA embigin pseudogene 1 (EMBP1). EHF, ELF5 and EMBP1 were all found integrated in original VaIN group. As a gene expressed in differentiated epithelial tissues, EHF located on chromosome 11p13 close to ELF5, can be a transcriptional activator or repressor, studied in lung and breast cancer[20, 21]. We found ELF5 had most missense mutations, which was predicted to regulate EHF expression[22]. Regarding EMBP1, it had significant higher methylation levels in HPV-positive OPSCC samples[23]. To our knowledge, EHF and ELF5 were never discussed in cervical nor vaginal precancerous lesion.

Previous studies showed HPV integration may occur only at the beginning of precancerous lesions (e.g., CIN 1, VaIN 1), most of which regress in the natural course of disease thereafter. It has been well illustrated in cervical cancer that HPV integration can function as predictive marker in disease free survival and clinical outcome[24, 25]. For low-grade cytology results, besides HPV genotyping and pathologic results, HPV integration acts as an innovate tool in predicting the progression of neoplastic lesions. Although follow-up suggested that most patients with low-grade VaIN had remission of clinical outcomes during the 6-month follow-up period, HPV integration acts as hierarchical system in management of VaIN to avoid high-grade precancerous lesion. Further analysis of progressive cases revealed that specimens with a follow-up result of VaIN 3 were all detected with HPV integration, which is consistent with the findings in previous studies. It’s obvious that HPV integration could serve as a predictive marker in the future, however, due to the limited sample size in our study, we found no significant difference between HPV integration and clinical outcome of VaIN to screen out patients who need more frequent monitoring.

Contribution

YX, CQ and SNY contributed to conception and design of the study. HLZ organized the database. SNY performed the statistical analysis. SNY wrote the first draft of the manuscript. CQ and SNY wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

Acknowledgments

This study was supported by Jiangsu Commission of Health (General Program) (No H2019009).

Competing interests

The authors report there are no competing interests to declare.

Ethics approval

This study was approved by the ethics committee of First Affiliated Hospital, Nanjing Medical University (approval No.2021-SR-423).

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent for publication

Patients signed informed consent regarding publishing their data and photographs.

Availability of data and material

The data generated or analyzed during this study are available from the corresponding author on reasonable request.

Code availability

Not applicable.

Cong Q, Wang Q, Gao SJ, et al. [Detection trend of vaginal intraepithelial neoplasia diagnosed by colposcopy guided biopsy from 2013 to 2015]. Zhonghua Fu Chan Ke Za Zhi. 2017;52(4):239-43.
Murta EF, Neves Junior MA, Sempionato LR, et al. Vaginal intraepithelial neoplasia: clinical-therapeutic analysis of 33 cases. Arch Gynecol Obstet. 2005;272(4):261-4.
Lamos C, Mihaljevic C, Aulmann S, et al. Detection of Human Papillomavirus Infection in Patients with Vaginal Intraepithelial Neoplasia. PLoS One. 2016;11(12):e0167386.
Li H, Guo YL, Zhang JX, et al. Risk factors for the development of vaginal intraepithelial neoplasia. Chin Med J (Engl). 2012;125(7):1219-23.
Groves IJ, Coleman N. Human papillomavirus genome integration in squamous carcinogenesis: what have next-generation sequencing studies taught us? J Pathol. 2018;245(1):9-18.
Guo M, Sneige N, Silva EG, et al. Distribution and viral load of eight oncogenic types of human papillomavirus (HPV) and HPV 16 integration status in cervical intraepithelial neoplasia and carcinoma. Mod Pathol. 2007;20(2):256-66.
Hu Z, Zhu D, Wang W, et al. Genome-wide profiling of HPV integration in cervical cancer identifies clustered genomic hot spots and a potential microhomology-mediated integration mechanism. Nat Genet. 2015;47(2):158-63.
Oyervides-Munoz MA, Perez-Maya AA, Rodriguez-Gutierrez HF, et al. Understanding the HPV integration and its progression to cervical cancer. Infect Genet Evol. 2018;61:134-44.
Chen X, Kost J, Sulovari A, et al. A virome-wide clonal integration analysis platform for discovering cancer viral etiology. Genome Res. 2019;29(5):819-30.
Li W, Zeng X, Lee NP, et al. HIVID: an efficient method to detect HBV integration using low coverage sequencing. Genomics. 2013;102(4):338-44.
Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25(14):1754-60.
Aho M, Vesterinen E, Meyer B, et al. Natural history of vaginal intraepithelial neoplasia. Cancer. 1991;68(1):195-7.
Gunderson CC, Nugent EK, Elfrink SH, et al. A contemporary analysis of epidemiology and management of vaginal intraepithelial neoplasia. Am J Obstet Gynecol. 2013;208(5):410 e1-6.
Alemany L, Saunier M, Tinoco L, et al. Large contribution of human papillomavirus in vaginal neoplastic lesions: a worldwide study in 597 samples. Eur J Cancer. 2014;50(16):2846-54.
Perkins RB, Guido RS, Castle PE, et al. 2019 ASCCP Risk-Based Management Consensus Guidelines for Abnormal Cervical Cancer Screening Tests and Cancer Precursors. J Low Genit Tract Dis. 2020;24(2):102-31.
Long W, Yang Z, Li X, et al. HPV-16, HPV-58, and HPV-33 are the most carcinogenic HPV genotypes in Southwestern China and their viral loads are associated with severity of premalignant lesions in the cervix. Virol J. 2018;15(1):94.
Akagi K, Li J, Broutian TR, et al. Genome-wide analysis of HPV integration in human cancers reveals recurrent, focal genomic instability. Genome Res. 2014;24(2):185-99.
Yang-Chun F, Sen-Yu W, Yuan Z, et al. Genome-Wide Profiling of Human Papillomavirus DNA Integration into Human Genome and Its Influence on PD-L1 Expression in Chinese Uygur Cervical Cancer Women. J Immunol Res. 2020;2020:6284960.
Li W, Qi Y, Cui X, et al. Characteristic of HPV Integration in the Genome and Transcriptome of Cervical Cancer Tissues. Biomed Res Int. 2018;2018:6242173.
Albino D, Longoni N, Curti L, et al. ESE3/EHF controls epithelial cell differentiation and its loss leads to prostate tumors with mesenchymal and stem-like features. Cancer Res. 2012;72(11):2889-900.
Fossum SL, Mutolo MJ, Tugores A, et al. Ets homologous factor (EHF) has critical roles in epithelial dysfunction in airway disease. J Biol Chem. 2017;292(26):10938-49.
Luk IY, Reehorst CM, Mariadason JM. ELF3, ELF5, EHF and SPDEF Transcription Factors in Tissue Homeostasis and Cancer. Molecules. 2018;23(9).
Ren S, Gaykalova D, Wang J, et al. Discovery and development of differentially methylated regions in human papillomavirus-related oropharyngeal squamous cell carcinoma. Int J Cancer. 2018;143(10):2425-36.
Das P, Thomas A, Mahantshetty U, et al. HPV genotyping and site of viral integration in cervical cancers in Indian women. PLoS One. 2012;7(7):e41012.
Shin HJ, Joo J, Yoon JH, et al. Physical status of human papillomavirus integration in cervical cancer is associated with treatment outcome of the patients treated with radiotherapy. PLoS One. 2014;9(1):e78995.

Download PDF

Journal Publication

published 10 Dec, 2023

Read the published version in Journal of Obstetrics and Gynaecology Research →

Version 1

posted

You are reading this latest preprint version

Preliminary study of HPV integration status on the occurrence and development of Vaginal intraepithelial neoplasia

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Material and methods

Results

1. Study design and Patient identification

2. Detection of HPV integration breakpoints

2.1 distribution of HPV integration sites in host chromosome

2.2 distribution of HPV integration sites in host genes

2.3 distribution of integration sites in HPV types

2.4Association of HPV integration and clinical outcome

Discussion

Declarations

References

Status:

Journal Publication

Version 1