# The significance of human papillomavirus integration in carcinogenesis and the development of specific diagnostics and countermeasures

Jiaxu Ying, Gary Wong, Nicolas Berthet

► To cite this version:

Jiaxu Ying, Gary Wong, Nicolas Berthet. The significance of human papillomavirus integration in carcinogenesis and the development of specific diagnostics and countermeasures. Global Translational Medicine, 2023, 2 (4), pp.2034. 10.36922/gtm.2034. pasteur-05065247

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REVIEW ARTICLE

# The significance of human papillomavirus integration in carcinogenesis and the development of specific diagnostics and countermeasures

Jiaxu Ying<fnref n="1" />, Gary Wong<fnref n="1" />*, and Nicolas Berthet<fnref n="2" />,<fnref n="3" />*

## Abstract

Human papillomavirus (HPV) infection is associated with various tumors, notably in the cervix, oropharyngeal region, and anus. As the disease progresses, integration of the viral genome into the host genome is often observed, yet the means of integration and its intrinsic relationship to carcinogenesis remain unclear. To address this gap, novel sequencing technologies have been developed to enhance the accuracy, intuitiveness, and cost-effectiveness of identifying integration breakpoints. HPV genome integration is thought to induce imbalances or dysfunctions in gene expression, epigenetic changes, chromosomal translocation, and genetic instability. The precise mechanisms underlying the changes in gene expression caused by genome integration offer avenues for tumor risk prediction and prognosis assessment. Personalized precision medicine, grounded in the integration patterns unique to each patient, holds promise for cancer treatment. This review summarizes and discusses the current state of knowledge regarding the mechanisms, carcinogenesis, detection and analysis, and clinical significance of HPV integration. It synthesizes existing information to offer a comprehensive overview, potentially enhancing our understanding of HPV-related tumorigenesis and aiding in the development of more effective diagnostic and therapeutic strategies.

**Keywords:** Human papillomavirus; Integration mechanism; Carcinogenesis; Integration sites identification; Clinical significance

## 1. Introduction

Globally, cervical cancer ranks as the fourth most common cancer among women, with an estimated 604,000 new cases reported in 2020. Out of the approximately 342,000 cervical cancer-related deaths in the same year, roughly 90% were concentrated in low- and middle-income countries[1]. In Africa and Southeast Asia, human papillomavirus (HPV)

*Corresponding authors:

Gary Wong
(garyckwong@ips.ac.cn)
Nicolas Berthet
(nicolas.berthet@pasteur.fr)

**Citation:** Ying J, Wong G, Berthet N, 2023, The significance of human papillomavirus integration in carcinogenesis and the development of specific diagnostics and countermeasures. *Global Transl Med*, 2(4): 2034.
https://doi.org/10.36922/gtm.2034

**Received:** October 13, 2023

**Accepted:** November 30, 2023

**Published Online:** December 18, 2023

**Copyright:** © 2023 Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution License, permitting distribution, and reproduction in any medium, provided the original work is properly cited.

**Publisher's Note:** AccScience Publishing remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<footnote n="1">¹Viral Hemorrhagic Fevers Research Unit, Chinese Academy of Sciences Key Laboratory of Molecular Virology and Immunology, Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences, Shanghai, China</footnote>

<footnote n="2">²Environment and Infectious Risks Research and Expertise Unit, Institut Pasteur, Université Paris-Cite, Paris, France</footnote>

<footnote n="3">³Epidemiology and Physiopathology of Oncogenic Viruses Unit, Institut Pasteur, Université Paris-Cite, Paris, France</footnote>
stands out as the main infectious oncogenic pathogen, accounting for over 50% of infection-related cancers[2]. The identification of over 200 distinct HPV types has been a significant advancement in understanding this complex viral landscape. At present, 231 HPV reference types have been cataloged and submitted to the International HPV Reference Center (https://www.hpvcenter.se/human_reference_clones/). Five genera of HPV have been defined: *Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Mupapillomavirus,* and *Nupapillomavirus*. The alpha genus is particularly well-characterized, as a subset of these mucosal viruses has been classified as “high-risk” or carcinogenic. The World Health Organization (WHO) identifies at least 12 genotypes of HPV (genotype 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) as high-risk, with HPV16 and 18 being the most prevalent high-risk HPVs (HR-HPVs) worldwide[3]. Among the numerous HPV types, 20 display a notably higher prevalence in cases of cervical cancer when compared to women with normal cervical cytology. All HPVs infect epithelial cells, usually through the mucosal or cutaneous route. HR-HPVs are associated with various human mucosal surface cancers, including penile, vulvar, vaginal, anal, oropharyngeal, and cervical cancers (CC)[4]. Notably, it has been suggested that at least 90% of CC and over 70% of oropharyngeal cancers are associated with HR-HPV infection[5,6].

HPVs are non-enveloped, double-stranded circular DNA viruses characterized by a 7-8 kb genome comprising three distinct regions: The upstream regulatory region (URR), the early coding region (E), and the late coding region (L). Within the beta genus, HPVs lack the E5 open reading frame (ORF), and the size or position of ORFs can vary. However, HPVs generally contain highly conserved regions for replication (E1 and E2) and packaging (L1 and L2), with other genes displaying greater sequence diversity between genotypes[5]. E4 is thought to influence tropism and may affect virus release or transmission[7]. In the alpha genus, the E5, E6, and E7 proteins serve as accessory proteins supporting the HPV life cycle. They manipulate the balance of cell proliferation and differentiation while antagonizing innate immune pathways[8]. The E6 and E7 proteins of HR-HPVs interact with numerous proteins involved in cell cycle regulation, including p53 and pRB.

Persistent HPV infection is associated with cancer development, yet the precise mechanisms through which HPV induces carcinogenesis remain unclear. A critical oncogenic step involves the escape of HPV from immune system clearance and detection. Different proportions of T-helper (Th) cells, including Th17 cells, have been identified in different cervical lesion grades, exerting

regulatory control over the immune microenvironment[9,10]. In the context of cervical carcinogenesis, the integration of the extrachromosomal viral genome into the host chromosome emerges as a common occurrence. Notably, in cervical cancer, HPV integration events can be detected in premalignant lesions, with the percentage of cells containing integrated HPV genome escalating with disease progression[11]. The integration event is, therefore, thought to transpire at a later stage in the progression of high-grade cervical dysplasia[12]. In cervical cancer cases, approximately 70% of HPV16-positive cases and almost all HPV18-associated cases exhibit detectable viral integration[5,13]. Similarly, about 75% of HPV16-associated oropharyngeal squamous cell carcinomas (OPSCC) and uterine cervical squamous cell carcinoma (UCSCC) display viral genome integration[14,15]. The significance of HPV integration is underscored by its potential utility as a biomarker for diagnosis, prognostic monitoring, survival prediction, and even in HPV-related cancer screening. Consequently, the integration status of HR-HPV has become a focal point in the realm of early diagnosis and precision treatment of cervical lesions.

It has been suggested that integration may confer a selective growth advantage to the virus and trigger host cell transformation through dysregulation of *E6/E7* viral oncogenes, resulting in an imbalance between cell proliferation and apoptosis and an increase in genetic instability[11,12]. However, it is crucial to recognize that *E6/E7* overexpression is not the sole factor underlying carcinogenesis. A series of structural and epigenetic changes in the host genome, resulting from viral genome integration, may also play an important role. Much remains to be elucidated about the mechanisms of integration and the ensuing patterns and alterations to the human genome. In this review, we will briefly describe the current understanding of the mechanism of HPV integration, the role of HPV in the neoplastic transformation and carcinogenesis process, recent advances in the detection and analysis of HPV integration, and the clinical significance of HPV integration in cancer prediction, prognostic evaluation, and the treatment for HPV-associated disease.

## 2. Mechanism of HPV integration

Little is currently understood about the mechanism through which the HPV genome integrates into the host genome. However, it is evident that this integration is an active process within the virus life cycle rather than an incidental or arbitrary occurrence. HPV integration exhibits a propensity to take place at random points throughout the human genome, with a notable preference for regions proximate to CpG sites, transcriptionally active regions, and common fragile sites[16]. It has been
suggested that HPV initially integrates into the host genome at random sites based on genome accessibility. However, over the course of extended carcinogenesis, there appears to be a propensity for integration to concentrate at specific loci, at which recurrent observations of integration have been made. This phenomenon may confer selective advantages to host cells, resulting in the formation of integration hotspots in different samples[17]. Several possible mechanisms have been proposed to elucidate HPV integration, including a DNA damage response, a looping model, and microhomology-mediated integration. Importantly, multiple integration mechanisms may concurrently operate. The following sections briefly introduce the principal mechanisms of integration that have been proposed.

## 2.1. DNA damage response

The frequency of integration is thought to be related to the degree of DNA damage, as the integration process necessitates the creation of double-strand breaks in both the viral and host DNA[18]. Notably, several proteins pivotal in DNA double-strand break repair pathways exhibit significant upregulation in various tumors. The integration into related genes further facilitates the insertion of the viral genome[19,20]. HPV infection and genome replication within cells induce the recruitment of excessive DNA damage factors, creating favorable conditions for the accidental integration of viral DNA[21]. Chromatin enriched in E2 and Brd4 is frequently situated proximal to common fragile sites and nucleic acid replication foci. This observation supports the theory that HPV exploits the host DNA repair mechanism, thus promoting its own replication[22]. Beyond its role in viral replication, E1 has been found to suppress the growth of host cells through a mechanism associated with the activation of DNA damage responses[23]. Consequently, E1 expression is hypothesized to promote HPV integration, with its interruption during integration resulting in the loss of the growth-suppressing properties associated with the E1 protein. Moreover, HPV16 E6*, the spliced isoform of E6, has been shown to elevate levels of reactive oxygen species, inducing chronic oxidative stress and cellular DNA damage. This process, in turn, increases the frequency of HPV integration, a relationship confirmed in both oral and cervical keratinocytes[24].

## 2.2. Looping model

In this model, HPV integration is mediated through DNA replication and recombination, providing insight into the structural aspects of HPV insertions flanked by host copy-number variations. Akagi et al.[25] observed a recurrent pattern of focal amplification and rearrangement adjacent to HPV integration sites in HPV-positive cancer

cell lines. Their hypothesis posits that HPV integrants bridge discontinuous host sequences by connecting nicked sites. This connection leads to a focused amplification of breakpoints, including viral and flanking host sequences. Such amplification occurs through the replication of transient circular structures and circularized genomic segments. The subsequent recombination of distal free ends with noncontiguous sequences, coupled with repair processes, further explains the tandem insertion of HPV fragments. This tandem insertion exhibits one identical breakpoint shared across multiple fragments, while unique breakpoints manifest on the opposite side.

## 2.3. Microhomology-mediated integration

Zones of microhomology (MH) with a maximum length of 4 bp between the viral genome and the host genome can be detected in proximity to the integration sites. Initially, it was suggested that homologous stretches of DNA facilitate the annealing of partially dissociated strands, thereby facilitating recombination. Importantly, sequence homology at the integration site was posited as not strictly necessary[26]. Two probable mechanisms, FoSTes[27] (fork stalling and template switching) and MMBIR (microhomology-mediated break-induced replication), have been identified in this context. Both mechanisms are triggered by local genomic instability and integration sites have been significantly enriched in several genomic instability-related elements, such as satellite and short interspersed nuclear element (SINE)-Alu repeats[17]. These findings suggest that HPV might leverage the zones of MH flanking the breakpoint to hijack the DNA repair pathway as a means of inserting the viral genome into the damaged host genome.

## 3. The role of HPV in cellular transformation and carcinogenesis

Integration alone proves insufficient to affect terminal cell differentiation or induce malignant transformation; additional alterations to host-cell genes and accompanying epigenetic modifications are required for tumorigenesis[28]. In the context of HPV-positive primary tumors, numerous structural variants and expression patterns, along with differential DNA methylation, have been associated with viral integration. Notably, many of these changes are shared between head-and-neck squamous cell carcinomas (HNSCC) and CC[13,19,29]. Earlier studies characterized viral integration patterns as Type I (a single integrated copy) or Type II (multiple tandem head-to-tail repeats of the viral genome). Recent studies, however, have confirmed that host-cell flanking sequences are often coamplified and rearranged. They have introduced a third integration type (Type III), where tandem copies of the HPV
genome are interspersed with cellular DNA, resembling
a Brd4-dependent super-enhancer-like element[30]. The
role of HPV integration in cell transformation and
carcinogenesis is described below (Figure 1), considering
both the direct and indirect effects of E6/E7 dysregulation.
This exploration encompasses the influence of imbalanced
cancer gene expression, epigenetic changes such as
abnormal methylation, and changes in chromosomal
structure.

**3.1. E6/E7 oncoprotein overexpression**

The abolition of E2 protein-mediated transcriptional
repression of the E6 and E7 promoters leads to the
overproduction of the E6 and E7 proteins, culminating in
the inactivation of important cell cycle checkpoints and
propelling cancer development[21]. Notably, screening tests
for cervical cancer based on HPV E6/E7 mRNA detection
are commercially available[31]. The affinity of the E2 protein
for various E2 binding sites (E2BSs) is contingent on E2
concentration. At low concentrations, E2 binds to the
high-affinity E2BS1 (promoter-distal) site, activating the
expression of E6/E7. In contrast, binding to E2BS3 or
E2BS4 (promoter-proximal) results in the transcriptional

repression of *E6/E7*<sup>[32,33]</sup>. During integration, the early
3' region of the virus is always deleted completely or
truncated, resulting in the deletion or disruption of *E2* and
the loss of the polyadenylation site. Conversely, the *E6* and
*E7* genes remain intact<sup>[34]</sup>. Transcription in cells harboring
integrated HPV DNA generates more stable virus-host
fusion transcripts, conferring growth advantages on these
cells compared to those harboring only extrachromosomal
HPV<sup>[34-36]</sup>. Breakpoints also frequently occur in *E1*,
influencing the downstream function of *E2*<sup>[12]</sup>.

In CC samples in which the HPV-coiled coil domain-
containing 106 (CCDC106) sequence integrates into the
host genome, alternative splicing of *E6* occurs, leading
to the generation of *E6*I. In this context, CCDC106
is strongly expressed, while there are low levels of p53
expression[37]. While E6 is generally implicated in p53
degradation in CC, the overproduction of *E6*I inhibits
this degradation, and the overexpression of CCDC106
has an opposing effect. Consequently, the p53 oncogenic
pathway is not only regulated by HPV oncogenes but is also
influenced by alterations in host gene expression resulting
from HPV integration. A more detailed understanding of
the relationship between the splicing of *E6/E7* transcripts

<figure><img src="image_2.png" /><figcaption>**Figure 1.** The role of HPV integration in the process of cell transformation and carcinogenesis.
Abbreviation: HPV: Human papillomavirus.</figcaption></figure>
and HPV integration necessitates further investigation. In addition, as detailed below, other influences may affect the expression of these two proteins[38]. However, it is essential to note that breakpoints can occur in any region of the viral genome. Several studies have reported that differences in *E6/E7* transcript levels between samples of different histopathological grades or between different physical states of the viral genome are not statistically significant. These observations suggest that high levels of E6/E7 may not be imperative for tumorigenesis. Conversely, secondary genetic events, such as copy number variation, might be required to induce cancer[12,39].

## 3.2. Imbalance in the expression of tumor-related genes and the affected key cellular pathways

As mentioned above, the majority of the integration sites cluster in regions of open chromatin and common fragile sites. Identified as “integration hotspots,” locations such as 3q28, 4q13.3, 8q24.21, 13q22.1, and 17q21 have been documented[4,26,40,41]. HPV integration disrupts host genes through various mechanisms, including genome rearrangements (amplifications, deletions, inversions, translocations, and others), intragenic insertions, and the introduction of promoters, splice acceptors, splice donors, and transcription termination elements[15]. The disruption of host genes by HPV integration can result in gene dysfunction through a complete loss of expression of the affected genes at the RNA or protein level[42]. Integration within 50 kb of known oncogenes or tumor repressor genes can occur, often resulting in the downregulation of tumor suppressor genes and the upregulation of proto-oncogenes[41]. DNA from HPV16 or 18 has been detected downstream, upstream, and within the *MYC* gene (8q24.21 integration hotspot) at distances ranging from 0 to 513 kb from this gene. This results in *MYC* overexpression at both the mRNA and protein levels, suggesting a potential contribution of the *MYC* activation triggered by the insertion of HPV viral DNA to cervical carcinogenesis[28,43]. In HeLa cells, the integrated HPV fragment is 3300 kb away from the 8q24.22 region in the linear distance, but the integrated HPV DNA can be brought into the close vicinity of the 8q24.22 region by chromatin folding, which is required for colocalization of the *MYC* locus and 8q24.22 region[44]. *MYC* and *PDL1* are also frequently integrated and overexpressed in HNSCC[15,45].

The proteins belonging to the apolipoprotein B mRNA-editing catalytic (APOBEC) polypeptide family increase the likelihood of DNA breaks. Levels of the APOBEC3B polypeptides are significantly higher in HPV-positive HNSCCs than in HPV-negative HNSCCs[46], and *APOBEC3A* is overexpressed in biopsy samples from oropharyngeal cancer displaying HPV16 integration[20].

A study of 25 HNSCC cases exhibiting HPV integration identified integration sites within the gene encoding the DNA repair protein RAD51 homolog 2 (RAD51B), resulting in an amplified chimeric segment encoding a non-functional RAD51B protein[19]. Li et al.[47] found that the genes repeatedly targeted by HPV integration were enriched not only in genes related to DNA repair pathways but also in genes associated with the pathways of xenobiotics metabolism by cytochrome P450, chemical carcinogenesis, and steroid hormone biosynthesis. Integration ratios increased with the progression of CC. The *ERBB2* gene (which enhances the MAPK and PI3CA pathways) is another reported site of HPV integration[13]. It has been suggested that even though integration can occur at multiple separate loci in tumor genomes, there is usually only one transcriptionally active locus[48]. MicroRNAs (miRNAs) close to integration sites may also be influenced. One study identified 47 integration sites and showed that 32 of these sites were within 3 Mb of a miRNA locus, and 19 of the 75 affected miRNAs were associated with tumors, influencing cell proliferation, differentiation, and apoptosis[26].

## 3.3. Epigenetic changes

Epigenetic changes may occur in both the HPV and host genomes, providing integrated viruses with a selective advantage. Therefore, it is necessary to consider both integration and epigenetic changes[4]. Most studies on epigenetic changes to date have focused on methylation. Epigenetic and genetic alterations to *E2BSs* in the HPV16 URR (with *E2BS3/4* becoming more methylated than *E2BS1* in CC) may also block the suppressive function of *E2*. A remarkably high degree of heterogeneity has been observed for URR methylation levels in squamous cell carcinoma samples displaying HPV genome integration[32,49]. The internal copies of the viral genome are always epigenetically silenced, and Type 2 integration events are associated with greater hypermethylation of the long control region[50]. The integration of HPV16 viral DNA into the host genome is associated with hypermethylation in the *L1/L2* and *E2* regions in vulval intraepithelial neoplasia[51]. In one study, the methylation status of the integrated HPV DNA was found to be correlated with the methylation status of the host genome close to the integration site in three HNSCC cell lines[52]. Brd4-enriched transcriptional regulatory hubs have been identified in cells displaying HPV16 genome integration. These hubs act as a super-enhancer-like element, promoting the transcription of *E6* and *E7*[30,53]. The expression of the *E7* gene in HR-HPVs leads to the extensive epigenetic reprogramming of cells. In HPV16-E7 expressing cells and HPV16-positive cervical intraepithelial neoplasia (CIN) specimens, levels
of H3K27me3, an important class of transcriptional repressive post-translational modifications, may decrease, while levels of the H3K27-specific demethylases (KDM6A and KDM6B) increase[54]. The induction of transcription in the genes encoding these enzymes is reminiscent of what occurs in the wound-healing model, suggesting that HPV tends to maintain a wound-healing state favorable for the viral life cycle. In HNSCC samples, HPV integration sites also colocalize with H3K27ac (active histone marks) enhancers upstream from several genes related to HNSCC tumorigenesis, such as *TP63*, *FOXE1*, *NOTCH1*, and *EGFL7*[55].

### 3.4. DNA instability and changes in chromatin structure

Integration of the HPV genome results in structural changes in the host genome at the insertion sites. HPV integration is frequently accompanied by somatic copy number variations, including focal amplifications, deletions, and intra and interchromosomal rearrangements[12]. For example, interchromosomal translocation between chromosomes 3 and 13, resulting from HPV insertion in HNSCCs, leads to elevated levels of RNA for *KLF5*, *TP63*, and *TPRG1*[19]. In cell lines displaying HPV integration, chromatin interactions between the integrated viral genome and host chromosomes occur both 500 kb away from integration sites, potentially causing dysregulation of host genes[56]. In a prospective cohort of 272 patients with annotated CC, the *MACROD2* gene emerged as a hotspot for viral genome integration. The intronic integration site in *MACROD2* may underlie genomic instability, given its association with a common fragile site[57].

## 4. Methods for detecting, identifying, and analyzing HPV integration sites

Technological advancements have introduced more efficient and accurate methods for the identification of viral integration sites. Traditional methods for the identification of integration sites relied on fluorescent in situ hybridization (FISH) and immunohistochemistry. Morphological approaches remain the preferred choice for the visually localizing integration sites in cells, enabling the observation of the state and location of viral DNA and its interaction with chromatin[58]. However, the genes affected by integration can only be identified by molecular methods. The following sections provide a brief description of the molecular methods available for detecting, identifying, and analyzing integration sites.

### 4.1. Polymerase chain reaction-based techniques

Polymerase chain reaction (PCR)-based techniques can be classified into two groups depending on whether the

amplification product is derived from the transcriptome or the nucleic acid of HPV itself: those that are RNA-based and those that are DNA-based. One such RNA-based technique is an amplification of papillomavirus oncogene transcripts (APOT), designed to detect HPV-derived fusion transcripts. These transcripts undergo reverse transcription, and the assay relies on a 3'-rapid amplification of cDNA ends in a nested PCR format. Utilizing HPV E7 primers as forward primers, an adapter primer complementary to the linker sequence serves as the first reverse primer, followed by a shifted primer as the second nested primer[59]. The assay is universally adopted for detecting viral-cellular fusion transcripts and analyzing integration sites carrying transcriptionally active viral genes[40].

Unlike RNA-based methods, which exclusively detect transcriptionally active integrants, DNA-based methods detect all integrated virus genomes, regardless of their transcriptional activity. Integrated papillomavirus sequences have been detected through ligation-mediated PCR (DIPS-PCR) to pinpoint the locus of HPV genome integration within the DNA[60]. This method involves the ligation of endonuclease-specific double-stranded adapters, followed by linear amplification with specific primers, resulting in the production of DNA fragments containing HPV. Finally, double-stranded PCR products are generated through PCR amplification with HPV-specific and adapter-specific primers.

### 4.2. Next-generation sequencing (NGS)-based techniques

At present, NGS stands as the principal method for the accurate identification of insertion sites. The NGS approaches include unbiased, untargeted techniques, such as whole-genome sequencing (WGS) and RNA sequencing (RNA-seq), as well as the utilization of specific probes and primers for targeted enrichment. Different probes can be designed to capture different HPV genomes. Subsequently, NGS is performed, and information about integration is derived from HPV-enriched DNA libraries. This method is considered unbiased and applicable to any sequence containing the HPV genome, be it episomal or integrated into a chromosome[61]. The integration analysis can be further enriched by combining DNA and RNA sequencing, offering comprehensive information about HPV integration at both genomic and transcriptomic levels[19,62]. A previous review discussed the various applications of NGS in HPV integration studies[63].

The high-throughput viral integration detection method, initially developed for detecting HBV integration[64], utilizes viral probes to capture inserted viral genome fragments in the host genome. Employing an effective bioinformatic
pipeline with a pair-assembling strategy, integration sites are identified through data analysis. This approach has proven successful in several studies for pinpointing meaningful integration sites[65]. Another strategy, tagging, enrichment, and NGS of HPV16 (TEN16), relies on targeted next-generation DNA sequencing[66]. The process involves Nextera *in vitro* transposition for DNA fragmentation and adapter-tagging, followed by multiplex PCR with HPV16-specific primers for HPV16 DNA enrichment. This approach successfully identified 75 HPV16 integration sites in a mixed tumor sample pool. RNA-sequencing stands out as another method of choice for detecting viral-host fusion transcripts. Koneva et al.[67] successfully identified 320 integration breakpoints distributed across the human and HPV genomes using RNA-seq data from 84 HNSCC tumors. In addition, whole-exome sequencing and high-throughput chromosome conformation capture sequencing have been adapted for the identification of HPV integration sites[56,65,68].

Parsing the NGS data is crucial for the identification of integration sites, and various novel bioinformatic pipelines have been developed to process the data to obtain the necessary information. ViFi, a computational approach in 2018, utilizes phylogenetic methods combined with reference-based read mapping for calling integration sites[69]. ViFi achieves not only high precision and sensitivity in identifying fusion reads but also detects circular extrachromosomal DNA (ecDNA) human-viral fusion structures. SearcHPV is another novel pipeline based on targeted capture technology, specifically designed for the analysis of targeted capture DNA sequencing data. Notably, SearcHPV demonstrates higher sensitivity and specificity for detecting HPV-host integration sites compared to VirusFinder and VirusSeq, two integration callers developed earlier. It is important to acknowledge that a significant limitation of SearcHPV is its validation on only three samples[70]. In addition to SearcHPV, deep-learning models such as DeepHPV have been designed for predicting HPV integration sites[71]. A comprehensive database is essential for systematic searches, comparisons, and analyses of viral integration sites. The Viral Integration Site Database was developed as a user-friendly web tool for browsing, searching, analyzing, visualizing, and downloading data[72]. The utilization of well-organized, comprehensive, and continuously updated databases, along with the availability of various evolving intelligent analysis pipelines, has significantly alleviated the burden of analyzing large amounts of sequencing data in advanced biomedical research.

### 4.3. Third-generation sequencing-based techniques

Short reads are suitable for investigating specific structural variations at insertion sites, such as deletions or chromosomal translocations. However, they lack the

capacity to reveal more extensive changes, such as changes in gene copy number resulting from the integration of the HPV genome into the host genome. The development of long-read single-molecule sequencing technologies by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) has introduced new possibilities for the application of direct nucleic acid enrichment tools. Long-read sequencing enables the elucidation of complex gene rearrangements, and Nanopore analysis requires smaller amounts of sequencing data compared to Illumina methods while maintaining similar accuracy[73,74].

One study successfully identified four new candidate target genes for CC by integrating multi-omics data from The Cancer Genome Atlas with patient-matched long-read sequencing of HPV integration sites. The identified genes are *BNC1*, *RSBN1*, *USP36*, and *TAOK3*. The study employed the HPV-targeted PacBio long-read sequencing method and validated the potential involvement of these genes in tumorigenesis by assessing their expression levels. It is essential to note that the study had a limited sample size, including samples from only eight women, and as such, its findings may not be broadly representative[75].

Pooled CRISPR inverse PCR sequencing (PCIP-seq) is a method employing a pool of CRISPR guide RNAs for selective cleavage of circular DNA fragments carrying viral DNA. Subsequently, reverse long-fragment PCR and multiplex sequencing are performed using the MinION platform. The DNA is then sheared into approximately 8 kb fragments, followed by intramolecular ligation and removal of remaining linear DNA through digestion. To selectively linearize circular DNA containing viral sequences, specific regions of the HPV18 genome are targeted for selective CRISPR-mediated cleavage, and PCR amplification is performed before sequencing. The identified insertion sites support the hypothesis that rearrangements occur through a “looping” mechanism, as the two sets of breaks share a common breakpoint[76].

Xdrop technology is a novel microfluidics system designed for targeted enrichment in low-input and long-fragment DNA. The technology relies on the isolation of long DNA fragments within millions of droplets, wherein the droplets containing a fluorescently labeled target sequence of interest are sorted using fluorescence-activated cell sorting. The sorted DNA fragments undergo amplification through multiple displacement amplification (MDA) and are subsequently subjected to sequencing. This approach successfully resolved three HPV18-chr8 integration sites at base-pair resolution[77]. Table 1 summarizes the studies conducted on the identification of HPV integration sites for various types of
<table><caption>Table 1. Methods used to identify HPV integration sites across various cancers in studies published from 2020 onwards</caption><thead><tr><th>Cancer type</th><th>Technology</th><th>Method (s) used</th><th>Number of samples</th><th>Infected HPV types</th><th>Number of integration sites identified</th><th>Genes into which HPV integrates at high frequency</th><th>References</th></tr></thead><tbody><tr><td rowspan="10">Cervical cancer</td><td>FISH</td><td>Dual-color FISH</td><td>96</td><td>16, 18</td><td>N/A<br>(62 cases of cervical cancer displayed HPV integration)</td><td>FHIT, MYC</td><td>[58]</td></tr><tr><td rowspan="3">2<sup>nd</sup>-generation sequencing</td><td>HPV double capture and Illumina sequencing</td><td>272</td><td>16, 18, 31, 33, 39, 42, 45, 52, 56, 58, 59, 68, 70, 73, 82</td><td>308</td><td></td><td>MACROD2, MIPOLA/TTC6, TP63</td><td>[57,94]</td></tr><tr><td rowspan="2">Virus capture sequencing</td><td>1,466</td><td>6, 11, 16, 18, 31, 33, 34, 35, 39, 45, 52, 56, 58, 59, 66, 68, 69, 82, etc</td><td>1,002 in 24.8% of non-cancer samples, 588 in 38.0% of precancer samples, and 1597 in 69.0% of cancer samples</td><td></td><td>LINC00392, LRP1B, DIAPH 2, PROS1, LSP1P5, ANKRD26P1, FHIT, MACROD2, ERBB2, RREB1 and more (24 hotspots identified)</td><td>[95]</td></tr><tr><td></td><td></td><td></td><td>1</td><td>N/A</td><td></td><td>[96]</td></tr><tr><td rowspan="4">3<sup>rd</sup>-generation sequencing</td><td>Tagmentation-assisted multiplex PCR enrichment sequencing (TaME-seq2)</td><td>36</td><td>51, 52, 59</td><td></td><td></td><td></td><td>[96]</td></tr><tr><td>HPV DNA hybridization capture plus Illumina sequencing and nanopore sequencing, FISH used to visualize the integration site</td><td>1</td><td>70</td><td>1</td><td>BCL11B</td><td></td><td>[97]</td></tr><tr><td>Nanopore sequencing and Illumina sequencing</td><td>1</td><td>16</td><td>83</td><td>Intergenic region of CHMP48 and RALY-AS1</td><td></td><td>[73]</td></tr><tr><td>HPV-enriched DNA for PacBio sequencing with matched multi-omics data on TCGA</td><td>8</td><td>16</td><td>267</td><td>BNC1, RSBN1, USP36, and TAOK3</td><td></td><td>[75]</td></tr><tr><td rowspan="2">3<sup>rd</sup>-generation sequencing combined CRISPR</td><td>Nanopore sequencing</td><td>16</td><td>16, 58</td><td>63</td><td>LINC00290, LINC02500, and LENG9</td><td></td><td>[86]</td></tr><tr><td>Nanopore sequencing</td><td>1</td><td>16, 35</td><td>448 in CaSki and 60 in clinical samples</td><td>N/A</td><td></td><td>[98]</td></tr><tr><td>3<sup>rd</sup>-generation sequencing combined CRISPR</td><td>Pooled CRISPR inverse PCR sequencing (PCIP-seq), nanopore sequencing</td><td>2</td><td>18</td><td>74</td><td>NISCH, PBRM1</td><td></td><td>[76]</td></tr><tr><td>3<sup>rd</sup>-generation sequencing combined with microfluidics</td><td>DNA enriched through sequencing, PacBio sequencing, nanopore sequencing and Illumina sequencing</td><td>0 clinical sample, HeLa cell DNA used</td><td>18</td><td>4</td><td>N/A</td><td></td><td>[77]</td></tr></tbody></table>

(Cont'd...)
<table><caption>Table 1. (Continued)</caption><thead><tr><th>Cancer type</th><th>Technology</th><th>Method (s) used</th><th>Number of samples</th><th>Infected HPV types</th><th>Number of integration sites identified</th><th>Genes into which HPV integrates at high frequency</th><th>References</th></tr></thead><tbody><tr><td rowspan="10">Small cell cervical carcinoma</td><td rowspan="10">Multi-omics</td><td>Whole-genome, RNA, chromatin immunoprecipitation and high-throughput chromosome conformation capture (Hi-C) sequencing, HiVID</td><td>61</td><td>16, 18, 52, 53, 56, 58</td><td>109</td><td>CCDC106</td><td>[37,65]</td></tr><tr><td>HPV-capture sequencing, RNA sequencing, and whole-genome bisulfite sequencing</td><td>50</td><td>16, 18, 31, 33, 45, 56, 58, 59, 73, 82</td><td>1,470</td><td>LINC00486, LINC02425, LLPH, PROS1, KLF5, LINC00392, MIR205HG, NRG1</td><td>[99]</td></tr><tr><td>High-throughput HPV capture sequencing, whole-exome sequencing, transcriptome sequencing, proteomics, phosphoproteomics, and single-cell RNA sequencing</td><td>106</td><td>6, 16, 18, 31, 33, 34, 45, 52, 58, 66</td><td>762</td><td>FHIT, TP63, KLF5, RASSF6, LRBA, TCERG1L, and more (21 recurrent integration regions identified)</td><td>[100]</td></tr><tr><td>Hi-C sequencing, whole-genome sequencing, RNA sequencing, ChIP-seq and CUT&Tag, virus capture second-generation sequencing, nanopore whole-genome and ecDNA sequencing</td><td>0 clinical sample, 9 cell lines</td><td>16,18</td><td>135</td><td>N/A</td><td>[101]</td></tr><tr><td>High-throughput HPV capture sequencing, whole-genome sequencing, whole-transcriptome sequencing, and OncoScan microarrays</td><td>214</td><td>16, 18, 31</td><td>381</td><td>MYC, SOX, NR4A, ANKRD, CEA family genes</td><td>[102]</td></tr><tr><td>Whole-genome sequencing and whole-exome sequencing</td><td>20</td><td>16, 18, 21, 27, 40, 51, 52, 56, 57, 59, 61, 62, 81, 123, 127, etc.</td><td>N/A (1,036 genes with somatic mutations in samples presenting HPV integration)</td><td>N/A (PIK3CA, KRAS, TRAPPC12, NDN, GOLGA6L4, and BAAP3 predicted as driver genes)</td><td>[103]</td></tr><tr><td>HPV16 capture-based targeted DNA sequencing and RNA sequencing</td><td>48</td><td>16</td><td>22</td><td>N/A (13 host genes displaying HPV integration)</td><td>[104]</td></tr><tr><td>Double capture-HPV method followed by Illumina sequencing</td><td>80</td><td>16, 26, 33, 35, 56</td><td>267</td><td>PDL1/PDL2/PLGRKT, MYC/ PVT1, MACROD2, KLF5/KLF12</td><td>[45]</td></tr><tr><td>Endocervical adenocarcinomas</td><td>Second-generation sequencing</td><td>Whole-genome sequencing and whole-exome sequencing</td><td>20</td><td>16, 18, 21, 27, 40, 51, 52, 56, 57, 59, 61, 62, 81, 123, 127, etc.</td><td>N/A (1,036 genes with somatic mutations in samples presenting HPV integration)</td><td>N/A (PIK3CA, KRAS, TRAPPC12, NDN, GOLGA6L4, and BAAP3 predicted as driver genes)</td><td>[103]</td></tr><tr><td>Mucoepidermoid carcinomas</td><td>Second-generation sequencing</td><td>HPV16 capture-based targeted DNA sequencing and RNA sequencing</td><td>48</td><td>16, 26, 33, 35, 56</td><td>22</td><td>N/A (13 host genes displaying HPV integration)</td><td>[104]</td></tr><tr><td>Head and neck squamous cell carcinoma</td><td>Second-generation sequencing</td><td>Double capture-HPV method followed by Illumina sequencing</td><td>80</td><td>16, 26, 33, 35, 56</td><td>267</td><td>PDL1/PDL2/PLGRKT, MYC/ PVT1, MACROD2, KLF5/KLF12</td><td>[45]</td></tr></tbody></table>

(Cont'd...)
<table><caption>Table 1. (Continued)</caption><thead><tr><th>Cancer type</th><th>Technology</th><th>Method (s) used</th><th>Number of samples</th><th>Infected HPV types</th><th>Number of integration sites identified</th><th>Genes into which HPV integrates at high frequency</th><th>References</th></tr></thead><tbody><tr><td>Oropharyngeal cancer and gastric cancer</td><td>Sanger sequencing</td><td>3' rapid amplification of cDNA ends for the detection of E6/E7 fusion RNA</td><td>2 oropharyngeal cancer tissues and 1 gastric cancer tissue</td><td>16</td><td>1 (in oropharyngeal cancer)</td><td>KIAA0825</td><td>[105]</td></tr><tr><td rowspan="2">Oropharyngeal cancer</td><td>Second-generation sequencing</td><td rowspan="2">Whole-genome sequencing</td><td rowspan="2">105</td><td>16, 18, 33, 35, 59, 69</td><td rowspan="2">874</td><td rowspan="2">SOX2, TP63, FGFR, MYC, CD274</td><td rowspan="2">[15]</td></tr><tr><td>Third-generation sequencing</td><td>16, 18</td></tr><tr><td>Penile squamous cell carcinoma</td><td>Second-generation sequencing</td><td>Long-read sequencing (PacBio HiFi and nanopore sequencing) and whole-genome sequencing</td><td>4-cell lines and 105 clinical samples</td><td>16, 18</td><td>N/A (focus on resolving the structures of genomic rearrangements flanking integration sites)</td><td>MYC</td><td>[106]</td></tr><tr><td>Anal squamous cell carcinoma</td><td>Second-generation sequencing</td><td>Whole-exome sequencing</td><td>72</td><td>6, 16, 18</td><td>2,252</td><td>N/A (associated with CNVs)</td><td>[108]</td></tr></tbody></table>

Abbreviations: ChIP-seq: Chromatin immunoprecipitation sequencing; ecDNA: Circular extrachromosomal DNA; FISH: Fluorescence in situ hybridization; HIVID: High-throughput viral integration detection; HPV: Human papillomavirus; PCR: Polymerase chain reaction.

tumors since 2020, with a predominant focus on cervical cancer in most publications. Emerging studies employing multi-omics techniques (such as genomics, proteomics, transcriptomics, and epigenomics) hold promise for offering multidimensional, integrative approaches to unveil new associations between biological entities and relevant biomarkers.

## 5. Clinical significance

In the latest WHO guidelines for the screening and treatment of cervical precancer lesions aimed at preventing CC, HPV DNA testing is recommended as the principal screening approach for diagnosis and treatment[78]. However, genotyping alone proves effective solely in CC prevention. A more clinically relevant strategy involves a combination of genotyping, evaluation of integration status and sites, analyses of epigenetic changes, and examination of tumor-related immune molecules (such as HLA-G)[79]. It is important to note that nonvalent HPV vaccines, such as Gardasil 9, covering HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, hold the potential to prevent up to 90% of all cases of cervical cancer. While preventive vaccination against HPV, especially HPV16 and HPV18, proves effective in halting the spread of HPV-related cancers, there is a pressing need to expand vaccination efforts in developing countries. Moreover, the development of new treatments, such as therapeutic vaccines, remains imperative for addressing the unvaccinated aging population suspected of HPV infection. Analyzing the landscape of HPV integration patterns has unveiled clinical and molecular associations, along with altered signaling pathways that could serve as predictive, prognostic, or therapeutic biomarkers for HPV-associated mucosal neoplasia[80].

### 5.1. Cancer risk assessment and predictive markers

Many studies have consistently demonstrated that HPV integration is a prevalent event in the initial stages of cervical carcinogenesis (such as CIN1). As the disease progresses from CIN to cancer, both the rate and number of integration events increase. This underscores the potential value of these markers as predictors of cancer progression[17]. The molecular profile of entire genomes displaying HPV integration can serve as an indicator of the duration of CIN. Notably, utilizing a methylation marker panel has proven to be a highly sensitive method for screening CIN undergoing transformation. In a study conducted by Liu *et al.*[81], it was observed that methylated CpG sites frequently co-occurred with integration sites in the HPV16 genome. Clinical samples validated that a panel of CpG sites (nt5606, nt5609, nt5615, and nt5378)
could potentially serve as biomarkers for the diagnosis of
CC. Assessing the methylation status of *MAL* and *miR-124-2* has demonstrated similar efficacy to cytological
tests in detecting CIN2+ in a study involving over 500
HR-HPV-positive women[82]. Another study revealed
that the hypermethylation of four genes, including
*hsa-miR-124*, was meaningful for predicting precursor
lesions in CC[83]. As *E2* is systematically disrupted during
integration, the *E2/E6* mRNA ratio can provide valuable
information about the integration status. An *E2/E6* ratio
below 1 is indicative of integrated viral DNA. In a study
of 248 patients diagnosed with only HPV16 infection,
it was found that an HPV16-*E2/E6* ≤0.6471 or relative
levels of *POU5F1B* mRNA ≥1.0310 in cervical exfoliated
cells could reliably predict CIN2+ tumors[84]. *POU5F1B*
is located on chromosome 8q24, a frequent site of HPV
integration. However, it is important to note that hybrid
virus-human episomes may also occur, and in such
cases, the *E2/E6* ratio is around 0.5, even in the absence
of integration. Thus, the *E2/E6* ratio may be irrelevant if
*E2* is not the principal gene displaying integration and
disruption[85]. If effective biomarkers can be identified
and utilized in clinical practice, it may be possible to
avoid invasive colposcopy in cases where the risk of CC
is sufficiently low, indicated by low levels of molecular
aberrations and negative results for methylation
markers[38].

## 5.2. Prognostic markers

Determining the physical status of HPV and identifying
integration sites and affected genes could facilitate
individualized patient follow-up, evaluation of disease-
free survival, and early clinical diagnosis of recurrent
disease. HPV-positive HNSCC generally has a more
favorable prognosis than HPV-negative HNSCC, and
patients with integration-negative tumors exhibit better
survival than those with integration-positive tumors,
especially coupled with a strong HPV-specific immune
response[6,67]. Multiple integration events are significantly
associated with poor prognosis[86]. Individualized
tests targeting the junction between viral and cellular
genomes, when combined with cytology, exhibit a
lower false positive rate for predicting recurrence
compared to standard HPV DNA/cytology cotesting
(18.2% and 12.1%, respectively). However, this strategy
lacks sensitivity, detecting integration in only 10.8% of
patients[87]. Shin *et al*. [88] observed lower disease-free
survival in CC patients with completely integrated HPV
genomes compared to those with both integrated and
episomal HPV, supporting the use of integration patterns

as prognostic indicators. Combining HPV integration
with proteomic data identified RAB2A as a potential
predictive biomarker for longitudinal surveillance and
prognosis evaluation in HPV-positive patients. RAB2A
levels are significantly elevated in primary cancers
compared to adjacent normal tissues, and high RAB2A
levels are associated with poor survival[89].

## 5.3. Therapeutic options

In addition to traditional treatments such as surgery,
radiotherapy, and chemotherapy, an increasing number
of clinical trials now concentrate on addressing the
carcinogenic factors associated with HPV infection
(Table 2). Potential therapeutic methods related to
virus-host interactions include knocking out integrated
HR-HPV DNA, inhibiting early virus gene transcription,
utilizing epigenetic methods (demethylation agents), and
employing specific drugs targeting identified genomic
alterations.

Decreasing or eliminating viral transcripts through
vaccination is a potential approach that could complement
traditional chemoradiotherapy methods for HPV-
associated tumors. Therapeutic vaccines targeting HPV
E6/E7 have already been developed, demonstrating
the ability to activate strong cytotoxic T-lymphocyte
responses[90]. For instance, Advaxis (ADXS11-001, Advaxis
Inc., New Jersey, USA), expressing the HPV16-E7 antigen,
exhibits promising therapeutic efficacy[91]. In addition,
RNA-based therapies aimed at silencing *E6/E7*, including
short-interfering RNA, CRISPR gene editing, miRNAs,
long non-coding RNAs, and mRNA-based HPV vaccines,
have also been developed.

The feasibility of translating these technologies
into clinically relevant modes of treatment for HPV-
derived cancers and potentially other virus-driven
human cancers has been clinically or experimentally
validated[92]. In W12 20861 cells (characterized by
tandemly repeated HPV16 integration), the use of
bromodomain inhibitors to displace Brd4, disrupting
super-enhancer function, has demonstrated a decrease
in the transcription of HPV16 *E6/E7* and the induction
of cellular senescence. This suggests that cancer cells
harboring integrated HPV could be a potential treatment
target[53]. Precision medicine is also applicable to certain
gene products already shown to be altered in cancer. For
instance, PI3K inhibitors are currently under clinical
trials for treating HNSCC[12]. In CC, *ERBB2* may be
activated by HPV integration in some patients, making
them potential candidates for inclusion in clinical trials
of *ERBB2* inhibitors[93].
<table><caption>Table 2. Clinical trials with novel therapies for HPV-associated cancers</caption><thead><tr><th>Clinical trial No.</th><th>Cancer type</th><th>Name</th><th>Principle</th><th>Treatment effect</th><th>Status</th><th>References</th></tr></thead><tbody><tr><td>NCT05686226</td><td>Metastatic or refractory/ recurrent HPV16+cancer</td><td>E7 TCR-engineered T cells</td><td>Infused T cells express the HPV16 E7 TCR and bind the E7<sub>11-19</sub>-HLA-A*02:01</td><td>Robust tumor regression was observed with objective clinical responses in 6 of 12 patients, including four of eight patients with anti-PD-1 refractory disease (in phase I clinical trials)</td><td>Phase II recruiting</td><td>[109]</td></tr><tr><td>NCT02280811</td><td>Metastatic or refractory/ recurrent HPV16+cancer</td><td>E6 TCR-engineered T cells</td><td>Infused T cells express the HPV16 E6 TCR and bind the E6<sub>29-38</sub>-HLA-A*02:01</td><td>Two of the 12 patients achieved objective tumor responses. A patient with three lung metastases experienced complete regression of one tumor and partial regression of the other two</td><td>Phase I/II completed</td><td>[110]</td></tr><tr><td>NCT02379520</td><td>High-risk HPV-infected cancer</td><td>HPV16/18 E6/E7-specific T lymphocytes</td><td>HPV16/18 E6/E7-specific T cells</td><td>Unknown</td><td>Phase I</td><td>[111]</td></tr><tr><td>NCT04713046</td><td>Metastatic or refractory/ recurrent HPV16+cancer</td><td>HPV-specific T cells</td><td>CD8 depleted peripheral blood cells taken from related donors vaccinated against HPV16</td><td>Unknown</td><td>Phase I/II</td><td>https://clinicaltrials.gov/study/ NCT04713046</td></tr><tr><td>NCT03912831</td><td>Refractory/recurrent HPV16+cancer</td><td>KITE-439</td><td>HPV16 E7 T-cell receptor-engineered T-cells</td><td>Unknown</td><td>Phase I</td><td>https://clinicaltrials.gov/study/ NCT03912831</td></tr><tr><td>NCT04672980</td><td>Metastatic or refractory/ recurrent HPV16+cancer</td><td>RTX-321</td><td>RTX<sup>TM</sup>-321 is an engineered antigen-presenting red blood cell that expresses human leukocyte antigen (HLA)-A*02:01 with human papillomavirus (HPV) 16 E7 peptide<sub>11-19</sub> (HLA-A2-HPV), 4-1BBL, and IL-12</td><td>RTX-321 activates HPV-specific T cells and promotes effector function in vitro</td><td>Phase I</td><td>[112]</td></tr><tr><td>NCT02853604</td><td>Advanced cervical cancer</td><td>ADXS11-001</td><td>Immunotherapy based on <i>Listeria monocytogenes</i> bioengineered to secrete a tLLO-HPV-16E7 fusion protein</td><td>Better overall survival was observed in ADXS11-001 treatment groups in a Phase 2 trial</td><td>Phase III</td><td>[113]</td></tr><tr><td>NCT03946358</td><td>HPV-positive anal cancer, head-and-neck carcinoma, or cervical and vulvar carcinoma</td><td>UCPVax and atezolizumab</td><td>UCPVax is a CD4 Th1-inducing anti-cancer vaccine composed of two separate peptides, UCP2 and UCP4, derived from telomerase. Atezolizumab is a PD-L1 inhibitor</td><td>The UCPVax vaccine is currently under evaluation in a multicenter phase I/II study in non-small cell lung cancer (NSCLC) (NCT2818426). It appears to be safe and immunogenic</td><td>Phase II</td><td>[114,115]</td></tr></tbody></table>

(Cont'd...)
<table><caption>Table 2. (Continued)</caption><thead><tr><th>Clinical trial No.</th><th>Cancer type</th><th>Name</th><th>Principle</th><th>Treatment effect</th><th>Status</th><th>References</th></tr></thead><tbody><tr><td>NCT03618953</td><td>Recurrent or metastatic HPV-associated tumor</td><td>MG1-E6E7 and Ad-E6E7</td><td>Oncolytic Maraba virus with Adenovirus vaccine both expressing mutant HPV16/18 E6 and E7</td><td>Induction of multifunctional, specific antitumor CD8<sup>+</sup>T cells capable of eradicating advanced tumors in a durable manner in mice</td><td>Phase I</td><td>[116,117]</td></tr><tr><td>NCT05108870</td><td>HPV 16<sup>+</sup> head and neck squamous cell carcinoma of the oropharynx</td><td>HB-201 and HB-202</td><td>Engineered replication-competent arenavirus vectors expressing HPV16 E7 and E6 genes</td><td>A single injection of HB-201 or HB-202 monotherapy is highly immunogenic in several patients, as demonstrated by an increase in inflammatory cytokines/chemokines and antigen-specific CD8<sup>+</sup>T cell responses</td><td>Phase I/II recruiting</td><td>[118]</td></tr><tr><td>NCT03978689</td><td>HPV16 Recurrent/ metastatic head-and-neck squamous cell carcinoma</td><td>CUE-101</td><td>CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced-affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain</td><td>CUE-101 displays selective binding, activation, and expansion of HPV16 E7<sup>+</sup>-specific CD8<sup>+</sup>T cells from PBMCs relative to nontarget cells. Anticancer efficacy and immunologic memory were demonstrated in TC-1 tumor-bearing mice treated with a murine surrogate (mCUE-101)</td><td>Phase I recruiting</td><td>[119]</td></tr><tr><td>NCT02866006</td><td>Metastatic or recurrent HPV16 or 18-positive cervical cancer</td><td>BVAC-C</td><td>BVAC-C is an immunotherapeutic vaccine based on B cells and monocytes transfected with recombinant human papillomavirus (HPV) 16/18 E6/E7 gene and loaded with alpha-galactosyl ceramide</td><td>The ORR was 11%, and disease stabilization was observed in five patients. The median progression-free survival (PFS) in the total population of patients was 6.8 months, and the PFS rate at 6 months was 56%</td><td>Phase I completed</td><td>[120]</td></tr><tr><td>NCT02865135</td><td>HPV 16<sup>+</sup> oropharyngeal, cervical and anal cancer</td><td>DPX-E7</td><td>A DepoVax<sup>TM</sup> (DPX)-based vaccine containing fusion peptide and adjuvant. The HPV16 E7 (H-2Db) peptide RAHYNIVTF49-57 (R9F) containing the CTL epitope was fused to a PADRE-containing CD4 T-helper epitope to generate the fusion peptide</td><td>The DPX-E7-vaccinated group contained the highest proportion of tumor-free C57BL/6 mice (>80%). Larger numbers of CD8<sup>+</sup>-T cells were found to infiltrate tumors from DPX-E7-immunized mice than tumors from PBS control mice.</td><td>Phase I/II</td><td>[121]</td></tr></tbody></table>

(Cont'd...)
<table><caption>Table 2. (Continued)</caption><thead><tr><th>Clinical trial No.</th><th>Cancer type</th><th>Name</th><th>Principle</th><th>Treatment effect</th><th>Status</th><th>References</th></tr></thead><tbody><tr><td>NCT00257738</td><td>MAGE-A3 positive tumor and/or HPV 16 positive head and neck squamous cell carcinoma</td><td>MAGE-A3/HPV 16 Trojan peptides 0001 and 0002</td><td>Two novel Trojan peptide complexes, composed of MAGE-A3 and HPV 16 epitopes</td><td>None of the 5 patients immunized displayed a complete or partial response</td><td>Phase I completed</td><td>[122]</td></tr><tr><td>NCT04892043</td><td>HPV16+metastatic solid tumors</td><td>SQZ-AAC-HPV</td><td>SQZ® Activating Antigen Carriers (AACs), derived from engineered red blood cells, are designed to transport tumor-specific antigens and adjuvants to a patient's own professional antigen-presenting cells. SQZ-AAC-HPV targets E6 and E7 oncoproteins</td><td>In preclinical studies, SQZ® AACs have been shown to induce robust immune responses to CD8 T-cell infiltration and were correlated with tumor reduction</td><td>Phase I recruiting</td><td>https://sqzbiotech.com/ patients-2/</td></tr><tr><td>NCT05357898</td><td>HPV16+metastatic solid tumors</td><td>SQZ-eAPC-HPV</td><td>Delivering five different mRNAs (targeting E6 and E7) to the patient's monocytes</td><td>In preclinical studies, SQZ® eAPCs have been shown to generate robust CD8-T cell responses to multiple antigens through simultaneous expression of the antigens CD86, membrane-bound IL-2 and membrane-bound IL-12</td><td>Phase I/II recruiting</td><td>https://sqzbiotech.com/ patients-2/</td></tr><tr><td>NCT04084951</td><td>HPV16+metastatic solid tumors</td><td>SQZ-PBMC-HPV</td><td>SQZ-PBMC-HPV is a novel cancer vaccine generated from peripheral blood mononuclear cells (PBMC) squeezed with HPV16 E6 and E7 antigens, resulting in delivery into the cytosol</td><td>Three patients (of 17 patients evaluated) displayed an increase in CD8+ tumor-infiltrating lymphocytes (TILs) and FoxP3+ cell density</td><td>Phase I completed</td><td>[123,124]</td></tr><tr><td>NCT03260023</td><td>Metastatic or refractory/ recurrent HPV16+cancer: cervical, vulvar, vaginal, penile and anal</td><td>TG4001 and avelumab</td><td>TG4001 is a suspension of MVATG8042 vector particles containing modified E6 and E7 genes from HPV16; avelumab is a fully human IgG1 anti-PD-L1 mAb</td><td>48% of patients were clinical responders 6 months after TG4001 treatment. Avelumab had already been approved by the FDA following clinical trials</td><td>Phase I/II recruiting</td><td>[125,126]</td></tr><tr><td>NCT05232851</td><td>High-risk human papillomavirus-associated oropharynx cancer</td><td>PDS0101 and pembrolizumab</td><td>PDS0101 is a lipid-based vaccine containing six HLA-unrestricted epitopes against HPV16 E6 and E7; pembrolizumab is a mAb with a high affinity for PD-1 for inhibition of PD-L1 binding to tumorigenic cells</td><td>PDS0101 has been shown to be safe in phase I clinical trials (NCT02065973) and has met the secondary endpoints for the regression of cervical dysplasia</td><td>Phase II recruiting</td><td>[127,128]</td></tr><tr><td>NCT04260126</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

(Contd..)
<table><caption>Table 2. (Continued)</caption><thead><tr><th>Clinical trial No.</th><th>Cancer type</th><th>Name</th><th>Principle</th><th>Treatment effect</th><th>Status</th><th>References</th></tr></thead><tbody><tr><td>NCT02426892</td><td>HPV16+solid tumors</td><td>ISA101 and nivolumab</td><td>ISA101 is a synthetic long-peptide anti-HPV16 vaccine covering the complete sequence of E6 and E7; nivolumab is used for PD-1 immune checkpoint blockade</td><td>This treatment gave a promising response rate of 33% and remains promising in long-term follow-up</td><td>Phase II completed</td><td>[129,130]</td></tr><tr><td>NCT04646005</td><td>HPV16+cervical cancer</td><td>ISA101 and cemiplimab</td><td>Cemiplimab is an anti-PD-1 monoclonal antibody</td><td>N/A</td><td>Phase II</td><td>https://clinicaltrials.gov/study/NCT04646005</td></tr><tr><td>NCT04432597</td><td>HPV positive cancers</td><td>PRGN-2009 alone or in combination with M7824</td><td>PRGN-2009 is a therapeutic gorilla adenovirus vaccine containing multiple cytotoxic T cell epitopes from the viral oncoproteins HPV16/18 E6 and E7; M7842 is an anti-PD-L1/ TGF-β trap fusion protein</td><td>PRGN-2009 treatment reduced tumor volume and increased CD8+ and CD4+ T cell levels in the tumor microenvironment of humanized mice bearing the human cervical tumor</td><td>Phase I/II</td><td>[131]</td></tr><tr><td>NCT03162224</td><td>HPV-associated recurrent/ metastatic head-and-neck cancer</td><td>MEDI0457 plus durvalumab</td><td>MEDI0457 is a DNA vaccine targeting HPV16/18 E6/E7 with IL12 adjuvant</td><td>The objective response rate was 27.6%. However, 28 (80.0%) patients had treatment-related adverse events</td><td>Phase I/II completed</td><td>[132]</td></tr><tr><td>NCT0344376</td><td>HPV16- or HPV18- positive cervical cancer</td><td>GX-188E and pembrolizumab</td><td>GX-188E (tirvalimogene teraplasmid) is a therapeutic HPV DNA vaccine that encodes HPV-16 and HPV-18 E6 and E7</td><td>At 24 weeks, 11 (42%) of 26 patients achieved an overall response; four (15%) had a complete response and seven (27%) had a partial response</td><td>Phase II ongoing</td><td>[133]</td></tr><tr><td>NCT05286060</td><td>HPV16- and/or HPV18- positive head-and-neck cancer</td><td>GX-188E, GX-I7 and pembrolizumab</td><td>GX-I7 (hyleukin-7) is a long-acting form of recombinant human IL-7 fused to a hybrid Fc</td><td>GX-I7 promotes the proliferation, persistence and cytotoxicity of human CAR T cells in xenogeneic mouse models</td><td>Phase II recruiting</td><td>[134]</td></tr><tr><td>NCT04405349</td><td>HPV16- positive cervical cancer</td><td>VB10.16 and atezolizumab</td><td>VB10.16 targets the cancer-specific full-length HPV16 E7 and E6 antigens</td><td>A phase I/IIa trial of the use of VB10.16 for high-grade cervical intraepithelial neoplasia has been completed. In this phase II trial, VB10.16 plus atezolizumab gave an ORR of 19% with a median response duration of 17.1 months and a disease control rate of 60%</td><td>Phase II ongoing</td><td>[135]</td></tr><tr><td>NCT05799144</td><td>PD-L1- and HPV- positive oropharyngeal cancer</td><td>pBI-11 and TA-HPV</td><td>pBI-11 is a circular DNA vaccine that expresses HPV16 E6/E7 and HPV18 E6/E7. TA-HPV is a vaccinia virus vaccine that expresses HPV16 and HPV18 E6-E7 fusion proteins</td><td>Priming with pBI-11 DNA followed by boosting with TA-HPV in conjunction with anti-PD-1 can generate significant antitumor effects</td><td>Phase II recruiting</td><td>[136]</td></tr></tbody></table>

Abbreviation: ORR; Overall response rate
# 6. Conclusion

Due to the low vaccination rates in developing countries and the considerable number of individuals still susceptible to HR-HPV infections, there is a pressing need for studies investigating the pathogenic mechanisms of HPV infection. Exploring clinical treatments targeting the oncogenic mechanisms of HPV should thus be a high priority in research. The integration of the HPV genome into the host DNA is a key step in the induction of carcinogenesis by HPV. In this review, we briefly explore the potential mechanisms of HPV integration and its effects on the host genome. The emergence of novel sequencing methods has provided robust technical support for identifying integration sites. With the rapid development of third-generation sequencing, the complex effects of HPV integration on host genes and chromosomes can increasingly be elucidated in greater detail. However, further studies involving larger cohorts are required to validate the accuracy of these novel methods. Drugs targeting the genes affected by integration could be employed in the treatment of HPV-related tumors within the framework of personalized medicine. Identifying new tumorigenic genes through integration analyses, along with assessing the levels of the molecules they encode, holds promise as effective biomarkers for diagnostic purposes, tumor risk assessment, and prognostic surveillance.

# Acknowledgments

None.

# Funding

The study was performed with the support of a grant from the Science and Technology Commission of Shanghai Municipality (Grant numbers: 2019SHZDZX02).

# Conflict of interest

The authors declare no conflicts of interest.

# Author contributions

Conceptualization: Jiaxu Ying, Nicolas Berthet

Writing – original draft: Jiaxu Ying

Writing – review and editing: Jiaxu Ying, Gary Wong, Nicolas Berthet

All authors have read and approved the manuscript.

# Ethics approval and consent to participate

Not applicable.

# Consent for publication

Not applicable.

# Availability of data

Not applicable.

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