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Global Translational Medicine The research advances in HPV integration

of H3K27me3, an important class of transcriptional amplification product is derived from the transcriptome or
repressive post-translational modifications, may decrease, the nucleic acid of HPV itself: those that are RNA-based
while levels of the H3K27-specific demethylases (KDM6A and those that are DNA-based. One such RNA-based
and KDM6B) increase . The induction of transcription technique is an amplification of papillomavirus oncogene
[54]
in the genes encoding these enzymes is reminiscent of transcripts (APOT), designed to detect HPV-derived fusion
what occurs in the wound-healing model, suggesting that transcripts. These transcripts undergo reverse transcription,
HPV tends to maintain a wound-healing state favorable for and the assay relies on a 3’-rapid amplification of cDNA
the viral life cycle. In HNSCC samples, HPV integration ends in a nested PCR format. Utilizing HPV E7 primers as
sites also colocalize with H3K27ac (active histone marks) forward primers, an adapter primer complementary to the
enhancers upstream from several genes related to HNSCC linker sequence serves as the first reverse primer, followed
tumorigenesis, such as TP63, FOXE1, NOTCH1, and by a shifted primer as the second nested primer . The
[59]
[55]
EGFL7 . assay is universally adopted for detecting viral-cellular
fusion transcripts and analyzing integration sites carrying
3.4. DNA instability and changes in chromatin transcriptionally active viral genes .
[40]
structure
Unlike RNA-based methods, which exclusively
Integration of the HPV genome results in structural changes detect transcriptionally active integrants, DNA-based
in the host genome at the insertion sites. HPV integration methods detect all integrated virus genomes, regardless
is frequently accompanied by somatic copy number
variations, including focal amplifications, deletions, of their transcriptional activity. Integrated papillomavirus
and intra and interchromosomal rearrangements . sequences have been detected through ligation-mediated
[12]
For example, interchromosomal translocation between PCR (DIPS-PCR) to pinpoint the locus of HPV genome
[60]
chromosomes 3 and 13, resulting from HPV insertion in integration within the DNA . This method involves
HNSCCs, leads to elevated levels of RNA for KLF5, TP63, the ligation of endonuclease-specific double-stranded
and TPRG1 . In cell lines displaying HPV integration, adapters, followed by linear amplification with specific
[19]
chromatin interactions between the integrated viral primers, resulting in the production of DNA fragments
genome and host chromosomes occur both 500 kb away containing HPV. Finally, double-stranded PCR products
from integration sites, potentially causing dysregulation of are generated through PCR amplification with HPV-
host genes . In a prospective cohort of 272 patients with specific and adapter-specific primers.
[56]
annotated CC, the MACROD2 gene emerged as a hotspot 4.2. Next-generation sequencing (NGS)-based
for viral genome integration. The intronic integration site techniques
in MACROD2 may underlie genomic instability, given its
association with a common fragile site . At present, NGS stands as the principal method for
[57]
the accurate identification of insertion sites. The NGS
4. Methods for detecting, identifying, and approaches include unbiased, untargeted techniques, such
analyzing HPV integration sites as whole-genome sequencing (WGS) and RNA sequencing
(RNA-seq), as well as the utilization of specific probes and
Technological advancements have introduced more primers for targeted enrichment. Different probes can be
efficient and accurate methods for the identification designed to capture different HPV genomes. Subsequently,
of viral integration sites. Traditional methods for the NGS is performed, and information about integration is
identification of integration sites relied on fluorescent in derived from HPV-enriched DNA libraries. This method
situ hybridization (FISH) and immunohistochemistry. is considered unbiased and applicable to any sequence
Morphological approaches remain the preferred choice for
the visually localizing integration sites in cells, enabling the containing the HPV genome, be it episomal or integrated
[61]
observation of the state and location of viral DNA and its into a chromosome . The integration analysis can be
interaction with chromatin . However, the genes affected further enriched by combining DNA and RNA sequencing,
[58]
by integration can only be identified by molecular methods. offering comprehensive information about HPV
[19,62]
The following sections provide a brief description of the integration at both genomic and transcriptomic levels .
molecular methods available for detecting, identifying, A previous review discussed the various applications of
[63]
and analyzing integration sites. NGS in HPV integration studies .
The high-throughput viral integration detection method,
4.1. Polymerase chain reaction-based techniques initially developed for detecting HBV integration , utilizes
[64]
Polymerase chain reaction (PCR)-based techniques can viral probes to capture inserted viral genome fragments in
be classified into two groups depending on whether the the host genome. Employing an effective bioinformatic

Volume 2 Issue 4 (2023) 6 https://doi.org/10.36922/gtm.2034