TRMOME 2039 No.

of Pages 13

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Review

HPV integration and cervical cancer: a failed

evolutionary viral trait
Mariano A. Molina 1,2,*, Renske D.M. Steenbergen 1,2, Anna Pumpe 3, Angelique N. Kenyon 3, and
Willem J.G. Melchers 3

Countless efforts have been made to eradicate cervical cancer worldwide, in- Highlights
cluding improving disease screening and human papillomavirus (HPV) vaccina- Human papillomavirus (HPV) infections
tion programs. Nevertheless, cervical cancer still claims the lives of more than are common among reproductive-age
women. The use of primary HPV testing
300 000 women every year. Persistent infections with high-risk HPV genotypes
has proven more effective in identifying
16 and 18 are the main cause of cancer and may result in HPV integration into high-grade precancerous lesions, lead-
the host genome. The central dogma is that HPV integration is an important ing to better clinical outcomes in women.
step in oncogenesis, but in fact, it impedes the virus from replicating and spread-
HPV integration into the host genome is
ing. HPV causing cervical cancer can therefore be perceived as a failed evolu-
a multifactorial process. Various host,
tionary viral trait. Here we outline the occurrence and mechanisms of HPV viral, and environmental cofactors, such
integration and how this process results in oncogenic transformation. as smoking, cervicovaginal microbiota,
estrogen levels, and coinfection with
HIV, influence this process.

HPV-based screening and infection biology The disruption of the E2 gene is identi-
In middle-aged women, cervical cancer (see Glossary) has the fourth largest mortality rate fied as a hallmark of HPV integration
and cancer development, leading to
followed by breast, colorectal, and lung cancers. Cervical cancer has a higher prevalence in
the overexpression of oncogenic
women living in low- and middle-income countries (LMICs) due to poor accessibility to vaccina- proteins (E6/E7) and the formation of
tion, screening, and healthcare [1,2]. Higher-income countries, however, have observed a decline superenhancers, thereby contributing
in the number of women affected annually by cervical cancer. The use of a Papanicolaou (Pap) to cellular transformation and cervical
neoplasia.
test has particularly led to earlier detection of the disease, resulting in better treatment strategies,
and a decrease in the overall mortality rate [3]. In a Pap test, cytological abnormalities are de- HPV integration results in host
tected in a cervical smear sample, thereby determining the risk for cervical cancer development genomic instability, chromosomal
(Box 1). Regular screening is important for women because it can most likely detect the disease rearrangements, loss of function in
tumor suppressor genes, and epi-
before onset of symptoms. Although Pap testing has been successful in reducing the cervical
genome dysregulation.
cancer death toll, the test has a suboptimal sensitivity [3,4]. Thus, screening for human pap-
illomavirus (HPV) (Box 2), the causative agent of most cervical cancer cases, started replac-
ing Pap testing and became the primary screening tool for cervical cancer in many countries
due to its superior performance over cytology in detecting high-grade precancerous lesions
[3,5,6].
1
Department of Pathology, Amsterdam
UMC, Location Vrije Universiteit
HPV infections are common for reproductive-age women. In reproductive-age women, the
Amsterdam, Amsterdam, The
chances of being infected within the first year of sexual intercourse is 20% and an overall 80% Netherlands
2
throughout their lifetime. The prevalence of HPV infections ranges between 5 and 60% depend- Cancer Centre Amsterdam, Imaging
and Biomarkers, Amsterdam, The
ing on, among others, the geographical region [7–10]. In most cases, the host immune system
Netherlands
can clear the infection without giving any sign that the infection was ever present (asymptomatic 3
Department of Medical Microbiology,
infection) [11]. Nonetheless, HPV can persist in many women and around 10% of all HPV-infected Radboud University Medical Center,
6500, HB, Nijmegen, The Netherlands
women exhibit signs of oncogenic transformation in the cervix. During the HPV infection life
cycle, the virus reaches the basal membrane of the epithelium via microwounds and infects kera-
tinocytes. HPV can be found in its episomal form in these basal epithelial cells, but it only repli-
*Correspondence:
cates in the upper differentiated epithelial layers [12,13]. HPV mainly resides in the stratified [email protected]
epithelia, where the virus exploits the mechanism of tissue renewal to complete its life cycle. (M.A. Molina).

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© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Box 1. Precancerous cervical lesions

Glossary
Precancerous lesions or CIN lesions are graded 1–3 and represent an estimation of dysplastic cells in the cervix. CIN1 is
Alternative oncogenic routes: non-
low-grade cervical neoplasia characterized by less than one third of the epithelium being dysplastic. CIN1 lesions show
HPV DNA integration pathways leading
normal maturation with only a few nuclear deformities. Both CIN2 and 3 are high-grade cervical neoplasia, which are char-
to cervical cancer, such as the episomal
acterized by greater than two thirds and up to the full epithelium being dysplastic, respectively. While CIN2 shows dysplas-
E2, E4, and E5 routes.
tic cellular changes and many more nuclear deformities than CIN1, CIN3 often lacks complete differentiation of cervical
Cervical cancer: malignant growth of
cells and has more nuclear deformities [94]. HPV infections contribute to all CIN grades, however, when a CIN3 has been
cells in the cervix, often linked to
established there is a higher risk of progression to cervical cancer.
persistent HPV infection.
DNA damage repair (DDR): cellular
mechanisms repairing damaged DNA,
implicated in HPV integration.
When HPV has replicated and expressed its oncoproteins, it can persist and integrate into the E2: viral gene involved in HPV replication
host genome (Box 2). and regulation of viral oncogenes.
Episomal form: circular viral DNA
existing independently in the host cell,
However, HPV DNA integration is not a standard course of its life cycle, as once integration has not integrated into the genome.
taken place, the virus loses its ability to fully replicate and spread to another person (Box 3) Genomic instability: increased
[14,15]. Therefore, it remains essential to investigate the biology of HPV integration and under- tendency for errors in the genome, often
observed after HPV integration.
stand the mechanisms underlying this process to better comprehend, diagnose, and treat pre- Human papillomavirus (HPV): DNA
cancerous cervical lesions and cancer in women (Box 1). Throughout this review we describe virus causing common infections, with
the current landscape of HPV integration in cervical cells and identify the interplay between high-risk types associated with cervical
virus and host in oncogenic transformation. We delve into the multifaceted process of HPV inte- cancer.
HPV DNA integration: insertion of
gration, elucidating viral and host (co)factors that shape cervical cancer development. We high- HPV DNA into the host genome,
light the complexities underlying HPV integration by describing diverse molecular mechanisms, influencing cellular functions and
such as DNA damage repair (DDR) pathways and the role of the viral proteins E1 and E2 potentially leading to cancer.
Long-read sequencing: sequencing
(Figure 1). Furthermore, we explore the outcomes of HPV integration, including effects on host
method that provides longer DNA reads
genomic stability, oncogenic transformation, and resistance to radiotherapy, as well as alter- than conventional sequencing methods,
native oncogenic pathways. With advancements in sequencing technologies and deeper insights offering insights into complex genomic
into HPV integration mechanisms, there is promise for improved screening and targeted thera- events like HPV integration.
Microhomologies (MHs): short
pies in combating cervical cancer.
nucleotide sequences shared between
viral and host DNA, implicated in HPV
The course towards HPV integration into the host genome integration.
HPV DNA integration is a multifactorial process that depends on viral factors and cofactors within Oncogenic transformation: process
by which normal cells acquire
the host and cervicovaginal microenvironment. It is thought that one of the major cofactors of
characteristics of cancer cells.
HPV integration is smoking [16]. Smoking causes genotoxic effects in the host DNA [17], which Papanicolau (Pap) test: cervical
together with cellular oxidative and nitrosative stresses lead to DNA damage. In women, smoking cancer screening method involving the
can also cause a stimulation and accumulation of mutagens in the cervicovaginal mucus, examination of cervical smear samples
for cytological abnormalities.
compromising the integrity of the cervical epithelium and local immunity [18,19]. Additional Resistance to radiotherapy: reduced
responsiveness of cervical cells to
radiation treatment, observed in cases
Box 2. Molecular biology of HPV with disrupted E2 after HPV integration.
HPV infection is considered the main driver of oncogenesis in the cervix. HPV is a non-enveloped DNA virus with a double- Single-cell RNA sequencing:
stranded genome containing ~8000 base pairs (Figure I). More than 400 HPV genotypes in the Papillomaviridae family technique analyzing gene expression at
have been identified to date, which have been categorized into the five genera Alpha, Beta, Gamma, Mu, and Nu the individual cell level, enhancing
[15,95]. Of particular interest are the Alpha HPVs because of their significant contribution to oncogenesis, in which the on- precision in HPV research.
cogenicity varies per genotype. The HPV genome consists of three major regions. The LCR or upstream regulatory region Superenhancers: clusters of
is noncoding, and plays an important role in the regulation of viral replication and transcription of viral DNA. The other two enhancers driving gene expression,
regions, the early and late, encode eight open-reading frames (ORFs) for various proteins, including six early ORFs and two associated with HPV integration
late ORFs. The early HPV genes are E1, E2, E1^E4, E5, E6, and E7. E1 and E2 are replication factors, while E1^E4, E5, E6, outcomes.
and E7 are accessory proteins. The late ORFs are L1 and L2, both of which encode viral capsid proteins [96]. HPV geno-
types have also been categorized into high-risk (hrHPV) or low-risk (lrHPV) groups. This classification is based on their on-
cogenic potential or disease association. hrHPVs are associated with cancer, while lrHPVs cause benign lesions. The
hrHPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 are defined by the World Health Organization
(WHO) as cancer-causing types [97]. From these hrHPV genotypes, types 16 and 18 are responsible for more than 70% of
global cervical cancer cases. In general, an HPV infection is acquired during sexual intercourse, but even skin-to-skin con-
tact may be sufficient to become infected [98].

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PE PL
E7
LCR Ori E6

8000/0

L1 E1

HPV
6000 2000
genome

4000 E4 E2

L2
E5

Oncoproteins Capsid proteins LCR: Long control region

Trends in Molecular Medicine

Figure I. The human papillomavirus (HPV) genome. HPV is a double-stranded DNA virus of 8000 base pairs with
three regions: long control region (LCR); early region encoding viral E1–E7; and late region encoding viral L1 and L2.
The early region includes those genes essential for viral replication and oncogenesis and its expression is driven by the
viral early promoter PE, whereas L1 and L2 are capsid proteins needed to form new viral particles and their expression
is driven by the viral late promoter PL. This figure was made using BioRender (www.biorender.com). Abbreviation: ori,
origin of DNA replication.

cofactors of HPV integration might include the composition of the cervicovaginal microbiota, par-
ticularly highly diverse microbiomes with abundant pathogenic anaerobic species [20,21], estro-
gen levels [22,23], mental stress [24], and coinfection with HIV [25]. These cofactors also increase
cellular stress in the microenvironment that promotes DDRs that eventually facilitate HPV integra-
tion. Furthermore, the host genome exhibits site-specific susceptibility for HPV integration. By
evaluating DNA breakpoints, it was observed that even though HPV integration occurred at ran-
dom in the host, there were specific host genes that acted as hotspots and were more prone to
integration [26]. In particular, the genes FHIT, KLF5, and LINC00392 were suggested as hotspot

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Box 3. HPV DNA integration: a double-edged sword for the virus

HPV DNA integration into the host genome is a complex process with advantages and drawbacks for the virus. DNA in-
tegration allows HPV to establish a persistent infection within host cells by ensuring the maintenance and replication of viral
DNA along with host cellular DNA. Integration can also lead to increased expression of E6 and E7, which promotes viral
replication and propagation within the stratified cervical epithelium. Likewise, integration may facilitate immune evasion
mechanisms by downregulating viral antigen expression or interfering with host immune responses, thereby allowing
HPV-infected and HPV-transformed cells to evade detection and clearance by the host immune system [46]. Alternatively,
loss of E2-mediated control over viral replication and gene expression can lead to dysregulated viral replication, genomic
instability, and increased risk of host cell transformation. Integration therefore decreases the infectivity of HPV, as inte-
grated viral DNA is not efficiently packaged into virions and transmitted to new host cells, limiting the spread of the virus
within the host population. Moreover, HPV-induced cancer represents a dead-end for the virus, as it may ultimately lead
to the death of the host and the cessation of viral transmission.

genes based on an accumulation of integrated HPV [26]. Similarly, another study assessed
possible hotspots with evidence of HPV integration and found the gene MACROD2 as a
main integration hotspot for HPV [27]. Interestingly, MACROD2 can lead to chromosomal in-
stability and dysregulate PARP1, an enzyme involved in cellular differentiation, proliferation,
and tumor transformation [27].

In terms of viral factors, HPV18 has been defined as more commonly integrated into the host ge-
nome compared with HPV16 [28,29]. Moreover, the early HPV genes E1 and E2, involved in the
replication of the viral genome, play a significant role in the integration process (Figure 2, Key figure).
E1 is a DNA helicase, which by means of ATP hydrolysis requires energy [30]. As it is ATP-
dependent, it also has the capability to take part in initiation and catalysis of DNA synthesis, partic-
ularly viral DNA. E1 binds to the origin of DNA replication (ori), initiating the cascade. Once the
cascade has started, E1 regulates replication of the viral episome located within the nucleus
of basal epithelial cells [31]. Consequently, there is an increase in viral DNA copy number that
remains constant while cells differentiate [32]. E1 is also known to promote the replication of
both viral and host genomes simultaneously as well as DDR pathways, which play a crucial
role in HPV integration [33].

The replication factor E2 interacts with E1, as without E2, E1 is unable to bind to the ori to initiate
replication (Figure 1) [34]. Essentially, E2 recruits E1 [35] and is one of the main regulators of the
HPV replication cycle. E2 also has additional roles, such as the transcriptional activation and re-
pression of HPV early promoters and early polyadenylation signal (PAS). HPV16 early promoter
p97 contains four E2 binding sites (E2BSs) upstream of the p97 transcription start site in the

Figure 1. E1 and E2 are essential

E1 N-terminal OBD Helicase/ATPase
for human papillomavirus (HPV)
replication. E1 consists of an
origin-binding domain (OBD) and
functions as an ATP-dependent
E2 TAD H DBD helicase. E1 is involved in replication
of the viral genome but can only
bind the ori with the help of E2,
which consists of a transactivating
TAD domain (TAD) connected to a DNA-
ATPase E1
E2 binding domain (DBD) via a hinge
OBD region (H). Therefore, E2 can bind
DBD the viral DNA and recruit E1 to the
ori, initiating replication. This figure
HPV genome
was made using BioRender (www.
Trends in Molecular Medicine biorender.com).

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Key figure
Human papillomavirus (HPV) integration disrupts the function of E2

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Figure 2. During the infection life cycle, the HPV genome is initially in its episomal form (A). The expression of E1/E2 (red arrow)
drives viral amplification of the viral episome, and particularly, the expression of E2 results in suppression of the viral early
promoter (red line, B). In cervical cancer cases or derived cell lines, the viral genome linearized through E1 or E2, and HPV
integration disrupts E1 or E2 expression (C). Since E2 acts as a suppressor for the transcription of the viral early promoter for
expression of E6 and E7 (B), disruption of the E2 open reading frame (ORF) for viral DNA integration leads to a loss E2
control of E6 and E7 expression (C), thereby contributing to oncogenic transformation. This figure was made using
BioRender (www.biorender.com). Abbreviations: LCR, long control region; ori, origin of DNA replication.

long control region (LCR). E2 binding to the proximal E2-binding site negatively regulates the
p97 promoter activities, thereby suppressing the viral oncoproteins E6 and E7 [36,37]. HPV
integration by disruption of the E2 open-reading frame (ORF) results in loss of E2 regulation
of the viral early promoter, loss of the viral PAS, and increases E6 and E7 expression, thereby
resulting in oncogenesis and cervical cancer development (Figure 2) [38]. Importantly, to be
expressed from integrated HPV DNA, E6/E7 need to have a host PAS downstream of the in-
tegrated viral DNA. Thus, the path towards HPV integration elucidates a complex interplay of
viral, host, and environmental factors, underscoring the multifaceted nature of this process in
cervical cancer development. Understanding the dynamics of this event sets the stage for
assessing the molecular biology of HPV integration.

Mechanistic biology and models of HPV integration

The disruption of E2 during the viral life cycle is considered a hallmark of HPV integration and
cancer and many studies have performed in-depth analyses into the biology of this event
(Box 3). Characteristically, E2 is disrupted in the HPV integration process, but for integration
to occur, there must be breakpoints in both viral and host genomes. One model that explains
this event involves the E1-induced DDR pathways [33]. Cellular replication is generally con-
trolled by the DDR pathways following DNA damage. HPV integration can promote genome

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damage via partial replication of the integrated genomes via E1 and E2. Uncontrolled nuclear
expression of E1 can trigger DNA damage and the activation of DNA damaging signaling
kinases, such as ATR and ATM [39,40]. This creates an ideal environment to promote
double-stranded DNA breaks (DSBs) in the replicating HPV genome, thereby facilitating
HPV integration. E2 can also target nucleases to its binding sites in the LCR, which can gen-
erate DBSs at the E2BSs in the HPV genome. This may explain the disruption of E2, the
DSBs in the viral genome, and activity of the nucleases may further explain the truncated
HPV genomes found in cancer cases [41]. The model offers a plausible explanation for how
the manipulation of the cellular DDR machinery by HPV could lead to integration. However,
it may oversimplify the complexity of the integration process and the contribution of other fac-
tors. Another hypothesis involves the hyperactivity of the apolipoprotein B mRNA-editing en-
zyme catalytic polypeptides (APOBEC, A3), which can lead to hypermutation, disruption, and
reduced expression of E2, thereby promoting integration of HPV DNA into the host genome
[28,42]. Although the hypermutation induced by APOBEC has been implicated in various can-
cers, the model may not fully account for the mechanisms underlying HPV integration, partic-
ularly the specific interactions between viral and host factors. Further studies have also shown
that E1 promotes focal genomic instability by inducing DNA overamplification while E2 in-
hibits cellular growth [43] and regulates E6-mediated apoptosis [44], suggesting a mecha-
nism in which HPV (in)directly targets E1/E2 to prevent cellular death and promote cellular
growth and differentiation to complete its life cycle, thereby causing DNA breakpoints in the
viral genome and facilitating integration.

The conventional classification of HPV integration included two main types [45]. In this classification,
type 1 HPV integration occurs when only a single complete viral genome (containing E6/E7) is incor-
porated into the host genome, while type 2 HPV integration entails multiple tandem head-to-tail
repeats (concatemers) [45]. Nevertheless, advances in sequencing technologies have shown that
more types of HPV integration do exist, and the existence of non-truncated viral genomes is incon-
sistent with the HPV DNA integration mechanisms previously described. By employing long-read
sequencing, four types of HPV integration were described in cervical cancer cases, and their clas-
sification clarified the discovery of truncated and complete HPV DNA integrated genomes [41]. Like-
wise, a recent study described the occurrence of silent and productive HPV integration [46].
Productive HPV integration is specifically associated with higher E6/E7 proteins and enhanced
tumor aggressiveness and immunoevasion, which is in line with a type 1 HPV integration with intact
copies of the viral oncogenes. Alternatively, a silent HPV integration failed to produce sufficient
E6/E7 oncoproteins to drive clonal expansion [46]. These classifications can be unified into
three main types of HPV integration that are consistent with the biology of HPV integration. In
this unified HPV integration classification, type 1 HPV integration occurs when only a single
truncated viral genome has been incorporated within the host genome. This genome is incom-
plete at the 5′ and 3′ ends and may possess intact copies of viral (onco)proteins (Figure 3A).
Alternatively, type 2 HPV integration involves multiple truncated repeats and type 3 a combina-
tion of both HPV integration types 1 and 2 (Figure 3B,C). This classification is also consistent
with the occurrence of productive and silent HPV integration. Productive HPV integration can
result from any type of integration with intact viral oncogenes and typically involves a single
active integration event, whereas silent integration may occur when the oncogenes are missing
or they have been silenced due to DNA methylation or other epigenetic modifications [46]. High
viral load and formation of viral–host fusion transcripts may lead to an overexpression of E6/E7
in tumors with episomal or integrated HPV, respectively, which should be considered when
categorizing productive or silent integration events [45,46]. This classification therefore reflects
the diversity of HPV integration patterns observed in cervical cancer cases and provides a more
nuanced understanding compared with the traditional classification.

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(A) (D)
HPV integration type 1: a single-truncated copy HPV genome
Host genome E5 L2 L1 LCR E1 Host genome

Host genome Breakpoint

E2 E6 E7 E2
PRODUCTIVE

Host genome E5 L2 L1 LCR Host genome

E2 HPV
SILENT
integration Intermediate
(B) HPV integration type 2: multiple complete viral copies circular
DNA
Host genome Host genome
LCR E1 E4 E5 L2
E6 E7 E2 L1
n
PRODUCTIVE
SILENT
(C) Looped DNA
HPV integration type 3: combination of types 1 and 2
Host genome Host genome
E5 L2 LCR E1 E4 E5 L2 LCR E1

E2 L1 E6 E7 E2 L1 E6 E7 E2
n

(E) HPV genome Concatemers

Host genome MHs Breakpoint

FoSTeS, MMBIR, MMEJ

Host genome HPV integration

Linearization and incorporation into the host genome

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Figure 3. Human papillomavirus (HPV) integration types and models within the host genome. HPV integration
can be observed as three different types, either as (A) a single truncated genome; (B) as multiple tandem head-to-tail
repeats; or (C) as a combination of both. Broken lines indicate truncation. (D) The looping-based model of HPV integration
proposes that HPV integrates into the host genome at sites of genomic instability. This process involves the integration of
viral DNA at breakpoints, forming a circular structure that replicates, generating concatemers. The rearrangement of the
host genome disrupts genes and can cause translocations between chromosomes. The repair of transient loops then
incorporates linearized concatemers into the host genome. The mechanism is mediated by rolling circle replication.
(E) Microhomology (MH)-mediated HPV integration. The HPV genome exhibits MHs with the host genome and functions
as the new template for replication when a breakpoint occurs in both DNAs. The host DNA damage repair pathways fork
stalling and template switching (FoSTeS), MH-mediated break-induced replication (MMBIR), or MH-mediated end joining
(MMEJ) are triggered, thereby facilitating HPV DNA integration into the host genome. This figure was made using
BioRender (www.biorender.com). Abbreviation: LCR, long control region.

Additional mechanisms have been suggested to be involved in the replication of viral DNA that
have an influence on E2 disruption, and therefore, HPV integration. Besides the classical semi-
conservative pathway, replication may occur as another repair mechanism for DSBs, named
break-induced replication (BIR), which is independent of starting sites like the ori, E1, and E2
[47]. Moreover, genome-wide analysis of HPV integration has resulted in the proposal of a looping
model mechanism for integration (Figure 3D) [48]. Although not yet experimentally confirmed, the
looping model explains why HPV integrates at regions of host genomic instability, such as focal
amplifications, rearrangements, deletions, and translocations. This mechanism is also consistent
with the HPV integration types 2 and 3 (Figure 3). The model assumes that HPV integration is me-
diated by replication and recombination, specifically by rolling circle replication [49]. In summary,
when a breakpoint in the host genome occurs, the viral DNA is integrated at that position and cre-
ates an intermediate circular structure (Figure 3D). Subsequently, replication of this DNA leads to
the generation of viral–host and host–host concatemers because the looped structure is repli-
cated several times. The rearrangement of the host genome then results in the disruption of
host genes. This process can take place at several chromosomes at once, which can cause
translocations between them. These looped structures and associated rearrangements are
thought to be byproducts of aberrant replication of integrated HPV DNA induced via E1/E2. Ulti-
mately, the repair of these transient loops incorporates the concatemers in its linearized form into

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the host genome (Figure 3D) [48]. This type of integration has been associated with a structural
variation termed heterocateny, which is characterized by diverse, interrelated, and repetitive pat-
terns of concatemerized virus and host DNA segments within a tumor. These variations are driven
by the dynamic, aberrant replication and recombination of HPV, thereby promoting intratumoral
heterogeneity and clonal evolution [50]. This model therefore offers insights into how HPV may ex-
ploit host DDR pathways. However, experimental validation is needed to confirm their relevance
in vivo and it remains essential to determine the clinical relevance of looped patterns in cervical
cancer development.

Another model suggests that HPV integration is mediated by microhomologies (MHs) between
the viral and host genomes (Figure 3E) [51]. MHs are short nucleotide sequences with a length of
1–10 bp that are enriched from the viral–host junction in both directions [51]. Overall, two different
types of integration mediated by MHs have been observed known as fork stalling and template
switching (FoSTeS) and MH-mediated BIR [52]. Both pathways are involved in DNA repair and
are triggered by genomic instability, which may explain the genomic events occurring around
the HPV integration sites. The viral genome exhibits MHs with the host DNA and functions as
the new template for replication, thereby facilitating HPV DNA integration into the host genome
[51]. This model may explain how HPV initially integrates in the looped model (Figure 3D) and is
further supported by another DDR pathway known as MH-mediated end joining (MMEJ)
(Figure 3E) [53]. MMEJ also occurs when there are MHs between the host and viral DNAs.
After a DSB occurs, one strand is excised in both directions from the breakpoint until the MHs re-
gions are reached. Consequently, the HPV genome anneals to the host genome and the remain-
ing nucleotides stick out of the double stranded (ds)DNA as flaps [53]. Finally, the flaps are cut off
by nucleases leading to deletion of this genomic region, which may also explain the truncated in-
tegrated HPV genomes (Figure 3) [41]. Other integrating viruses, such as adeno-associated virus
and Merkel cell polyomavirus, can also induce the MMEJ pathway [54]. This model provides a
plausible mechanism for how HPV DNA integrates into the host genome. The enrichment of
MHs in the viral–host junctions suggests a functional role in mediating the integration process.
Nevertheless, further experimental evidence is required to validate its significance in HPV-
positive cancers [14].

Overall, functional, in vivo, and bioinformatic studies agree on the involvement of the host DDR and
recombination pathways, induced by HPV, in viral integration and genomic instability [54,55]. Al-
though there have been many advances in the investigation of HPV integration in the past few
years, more studies are required, and the complete mechanisms still need to be elucidated.

HPV integration outcomes

HPV integration can be found in all grades of cervical intraepithelial neoplasia (CIN), but the per-
centage of integration differs by the different stages towards cervical cancer development,
which indicates that in earlier stages of transformation, the HPV genome is mainly present in its
episomal form [27,56,57]. Various studies assessed the integration of mainly HPV16/18 and
found consistent results showing that the percentage of HPV integration increases as the lesion
progresses. However, the range of percentage varied greatly among different studies. Whereas
some studies did not find any evidence for HPV integration in patients with CIN1, others found
greater than 50% integration [51,58,59]. A similar range have been observed for CIN2 (5–71%)
and CIN3 (16–82%) lesions, while the integration rates are more similar in cervical cancer (66–
100%) [59,60]. These differences in the frequency of HPV integration can be explained by the ge-
netic backgrounds on the study populations, the methodologies used and their sensitivities, and
the type of assessment of cervical samples (cytology and histology) [51,58,59,61]. In the past,
methods like detection of integrated papillomavirus sequences (DIPS) by ligation-mediated

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PCR [62,63] and amplification of HPV oncogene transcript (APOT) assays [64,65] were used to Clinician’s corner
measure integration, while long-read sequencing and single-cell RNA sequencing ap- With advancements in the assessment
proaches have been used more recently [14,41,46,66]. These sequencing methods can read of HPV integration, clinicians are en-
couraged to stay updated on the latest
longer genome lengths and analyze cell–cell variations, respectively, thereby allowing a compre-
technologies, such as sequencing, for
hensive characterization of large-range virus–human integration events in cell lines and cervical more precise identification of inte-
tumors. Thus, methods with a higher sensitivity are needed to fully clarify the association of grated HPV DNA.
HPV DNA integration with cervical disease progression.
The emergence of personalized
medicine requires therapeutic
HPV integration leads to several effects in the host that are mainly caused by the disruption of E2, approaches aimed at HPV integration
the use of a host PAS downstream of the integrated DNA, and the subsequent overexpression of events and E6/E7 overexpression,
E6 and E7. HPV integration can form Brd4-dependent and extrachromosomal DNA (ecDNA) such as small RNA molecules
targeting viral-host transcripts and
[50,67] superenhancers, which eventually overexpress E6 and E7. The expression of these CRISPR/Cas-based editing.
oncoproteins further promote HPV integration by stimulating DDR pathways, cellular proliferation,
dysregulation of cell cycle control, growth of transformed cells, and progressive genetic instability, As research progresses on the role of
all of which are hallmarks of cancer development (Figure 2) [67,68]. Moreover, productive HPV in- host genetic factors in HPV integra-
tion, clinicians should anticipate the
tegrations may allow tumor cells to evade immune surveillance by inducing the PVR–TIGIT path- development of personalized triage
way, which skews the immune response toward an immunosuppressed phenotype and strategies based on individual sus-
abrogates T cell cytotoxicity, and by regulating the extracellular matrix (ECM) and multiple immune ceptibility, enabling targeted preven-
tive measures.
checkpoint ligands and HLAs [46]. Additionally, HPV integration was found to cause a decrease in
the overall survival of cervical cancer patients [69]. Disruption of E2 has been associated with re- Recognizing the impact of cofactors like
sistance to radiotherapy. Cervical cells with disrupted E2 are harder to treat with irradiation be- smoking and microbiota composition
cause the disruption leads to increased radioresistance [38]. Irradiation generally generates on HPV integration, clinicians are
urged to integrate lifestyle interventions
more lethal DNA-damage, which the cell is unable to repair leading to cell death. It has been hy- into patient care plans, aiming to reduce
pothesized that cells with disrupted E2 gene have a higher capability of repairing sublethal DNA- cervical cancer risk.
damage, which can be explained by the stimulation of DDR pathways by E6 and E7 following
Understanding the mechanisms of
HPV DNA integration [38,70,71].
resistance to radiotherapy in cases
with disrupted E2 highlights the need
HPV integration results in host genomic instability in the chromosomes and gene alterations, par- for further research into tailored
ticularly loss of function of tumor suppressor genes, chromatin reorganization, chromosomal re- therapeutic approaches. Clinicians
should explore innovative treatment
arrangements, and epigenome dysregulation [66,72–74]. Genomic instability contributes to the
strategies to improve outcomes in
progression of precancerous lesions to invasive carcinoma and facilitates the acquisition of addi- such cases.
tional genetic alterations necessary for malignant transformation [75,76]. Disruption of tumor sup-
pressor genes or activation of oncogenes through proximity to integration events can drive Acknowledging alternative routes for
oncogenic transformation, clinicians
tumorigenesis and influence the clinical behavior of cervical cancer [74,77]. In addition, clonal
should be vigilant in cases where
evolution driven by HPV integration may result in the emergence of subclones with distinct phe- HPV-integrated DNA is absent. Explor-
notypic characteristics, including differences in aggressiveness, metastatic potential, and re- ing alternative oncogenic pathways
sponse to therapy [14,46,50,78]. These overall findings imply a timeline in which HPV may enhance diagnostic precision
and guide therapeutic decisions.
integration initiates a cascade of events that eventually triggers oncogenesis. Once the oncogenic
pathways are triggered, cervical cancer develops.

Nonetheless, there are known alternative non-HPV DNA integration routes for oncogenic trans-
formation. These alternative routes are less common, but they can also induce cervical cancer.
These routes include the episomal E2, E4, and E5 alternative oncogenic pathway [79], the
AP1/Wnt/β-catenin pathway [46,80], and the PI3K/AKT/mTOR signaling pathway [81,82].
These alternative oncogenic routes may explain why some cervical cancer cases exhibit an
intact E2 gene and lack HPV-integrated DNA. This outcome has been particularly associated
with an increased methylation of the E2BSs 3 and 4, which impedes E2 binding to LCR, thereby
abrogating the E2-mediated p97 promoter inhibition of E6 and E7 expression. Consequently,
methylation of the E2BSs within the viral LCR results in only a loss of E2 function and E6 and
E7 overexpression [83]. This event is supported by the fact that HPV integration and aberrant

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methylation of the E2BSs are negatively correlated in most HPV-induced cancers [84]. Thus, it is Outstanding questions
important to understand and consider these alternative routes when investigating viral integration What are the specific host,
and developing screening and therapeutical strategies against cervical cancer. environmental, and viral factors that
contribute to the persistence of HPV
infections and to viral integration,
HPV integration as a therapeutic approach and how can this knowledge inform
One therapeutic strategy involves targeting the expression or function of viral oncogenes, partic- interventions to prevent oncogenic
ularly E6/E7, which are overexpressed following HPV integration. Small molecules, peptides, or transformation?

gene editing-based approaches (e.g., CRISPR/Cas systems) could be developed to disrupt What are the basic mechanisms
E6/E7 interactions with cellular targets such as p53 and pRb, restoring their tumor-suppressive underlying HPV integration, consider-
functions [85,86]. Therapeutics could also target molecular interactions between integrated ing the diverse types and models pro-
posed in this review, and how can
HPV DNA and host cellular factors involved in viral replication, transcription, or integration
this knowledge be translated into
(e.g., viral–host fusion transcripts) [87]. more effective diagnostic and thera-
peutic strategies?
Inhibition of the DDR pathways represents another therapeutic avenue. Inhibitors targeting DDR ki-
What are the roles of E1 and E2 in the
nases, DNA repair enzymes, or checkpoint proteins could sensitize HPV-positive cancer cells to
molecular biology and models of HPV
DNA damage-inducing therapies, such as radiation or chemotherapy [88–90]. Immunotherapeutic integration?
approaches aimed at enhancing the host immune response against HPV-positive cancer cells
could exploit the immunogenicity of viral oncoproteins expressed following integration [46,91]. Can the association between
disrupted E2 and resistance to
Therapies targeting immune checkpoint molecules, therapeutic vaccines, or oncolytic viruses radiotherapy be further elucidated,
could stimulate antitumor immunity and eradicate cancer cells with HPV integrated DNA [92]. and are there potential therapeutic
Last, stratifying patients based on HPV integration status, viral oncogene expression levels, and in- targets to overcome this resistance in
tegration patterns could also facilitate personalized therapeutic interventions [80]. Biomarker-driven cervical cancer treatment?

clinical trials could identify patients most likely to benefit from specific targeted therapies or combi- How do alternative non-HPV DNA inte-
nation treatment regimens tailored to their individual tumor and HPV integration biology. gration routes contribute to cervical
cancer, and what implications do
these routes have for screening and
Concluding remarks
therapeutic strategies?
HPV DNA integration is a crucial step towards oncogenic transformation. Nevertheless, the pro-
cess is a dead end for the virus because it impedes viral replication, and therefore, HPV causing Are there specific genomic events or
cancer can be perceived as a failed evolutionary viral trait. The analysis into HPV integration in cer- signatures associated with HPV inte-
gration sites that could serve as bio-
vical cancer has revealed a complex interplay between the virus and the host, unraveling intricate markers for disease progression and
essential mechanisms in oncogenic transformation. This multifaceted process, influenced by var- treatment response?
ious cofactors, such as cellular stress in the microenvironment and the viral proteins E1 and E2,
How do next-generation sequencing
unfolds a course marked by diverse integration types and models, from DNA damage repair path-
technologies impact the identification
ways to looping mechanisms and MH-mediated pathways. The outcomes of HPV integration, and analysis of integrated HPV DNA,
disrupting E2, hijacking a host PAS, and promoting superenhancers formation, fuel uncontrolled and how can these technologies be fur-
oncoprotein expression, contributing to cellular transformation and cervical neoplasia. Novel ther optimized for clinical applications?
techniques like single-cell RNA sequencing [14,54] and long-read sequencing [41,66] as well
as databases for HPV mutations and integration sites [93] show promise for more precise iden-
tification and analyses of integrated HPV DNA (see Clinician’s corner).

Future research should prioritize elucidating the specific factors influencing HPV integration and
cervical cancer development, particularly the relationships between host genetics, microenviron-
ment, and viral dynamics that contribute to HPV persistent infections and integration (see
Outstanding questions). A deeper understanding of the mechanisms underlying the disruption
of E2 and its association with resistance to radiotherapy might be crucial for developing person-
alized therapeutic strategies. Exploring alternative non-HPV DNA integration routes and identify-
ing genomic events associated with HPV integration sites as potential (pre)cancer biomarkers are
areas that warrant in-depth investigation. Further development of sequencing techniques re-
mains essential for assessment of their sensitivity, feasibility of implementation, and cost-
effectiveness in cervical cancer research and prevention programs. Notably, HPV vaccination is

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Trends in Molecular Medicine
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associated with a substantially reduced risk of invasive cervical cancer and therefore is consid-
ered the best available preventive measure against HPV-induced carcinogenesis. Overall, this
deepens our understanding of the dynamics between HPV and the host, offering in-depth in-
sights that could pave the way for improved screening methods and targeted therapeutics in
the fight against cervical cancer.

Acknowledgments
We thank Zhi-Ming Zheng, National Cancer Institute/National Institutes of Health, for his critical review of the manuscript.

Declaration of interests
The authors declare that they have no competing interests.

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