Fonc 12 864820

REVIEW
published: 27 April 2022

doi: 10.3389/fonc.2022.864820
Molecular Detection Methods in

HPV-Related Cancers
Jordana Williams , Morris Kostiuk and Vincent L. Biron *
Division of Otolaryngology-Head and Neck Surgery Research Laboratory of Alberta, Department of Surgery, University of
Alberta, Edmonton, AB, Canada
Human papillomavirus (HPV) is responsible for most cervical cancers and some head and
neck cancers, including oropharyngeal squamous cell carcinoma and sinonasal
carcinoma. Cervical cancer is commonly diagnosed by liquid-based cytology, followed
by HPV testing using commercially available DNA polymerase chain reaction (PCR), p16
immunohistochemistry (IHC), or DNA/RNA in situ hybridization. HPV in head and neck
cancers is commonly diagnosed by p16 IHC or by RT-qPCR of HPV-16 E6 and E7
oncoproteins. Droplet digital PCR has been reported as an ultrasensitive and highly
precise method of nucleic acid quantification for biomarker analysis and has been used to
detect oncogenic HPV in oropharyngeal and cervical cancers.
Edited by:
Nerina Denaro, Keywords: human papillomavirus, diagnostic tools, p16, droplet digital polymerase chain reaction, polymerase
Azienda Sanitaria Ospedaliera S.Croce chain reaction, immunohistochemistry, in situ hybridization
e Carle Cuneo, Italy
Reviewed by:
Mirella Fortunato,
Azienda Sanitaria Ospedaliera S.Croce INTRODUCTION
e Carle Cuneo, Italy
Paolo Boscolo-Rizzo, Human papillomavirus (HPV) is the most common sexually transmitted infection (STI) in the
University of Trieste, Italy world (1). and classified as a carcinogenic infectious agent by the International Agency for Research
*Correspondence: on Cancer (2). Both sexually active men and women will be infected at least once without
Vincent L. Biron developing any symptoms or cancerous diseases in their lifetime (1). However, only some HPV
vbiron@ualberta.ca strains are oncogenic. These have been shown to cause most cervical cancers and some head and
neck cancers, particularly in the oropharynx (3, 4) and, to a lesser extent, in the sinonasal region (5).
Specialty section: HPV testing is important clinically for the accuracy of diagnosis, patient-centered treatment, and
This article was submitted to prognostication (3, 6–11).
Head and Neck Cancer, Cervical cancer screening and diagnosis is minimally invasive. It combines liquid-based cytology
a section of the journal
stained Papanicolaou stain (Pap smear) and HPV testing using DNA/RNA PCR-based methods (12,
Frontiers in Oncology
13). The association between cervical squamous cell carcinoma (CSCC) and HPV is well established,
Received: 28 January 2022 as HPV is known to cause most cervical cancers (1–3, 12, 13). In developed countries, cervical
Accepted: 15 March 2022
cancer has been effectively controlled by cytological screening, which involves physician-
Published: 27 April 2022
administered cervical samples and directed cervical exams which are interpreted by a trained
Citation:
cytopathologist. However, in low- and middle-income countries where the burden of cervical cancer
Williams J, Kostiuk M and Biron VL
(2022) Molecular Detection Methods in
is the highest (1, 2), such established screening programs are not available nor feasible. Some of the
HPV-Related Cancers. barriers that affect the success of the screening programs include the availability of physicians,
Front. Oncol. 12:864820. trained personnel that can interpret the sample results, access to equipment and technology, and
doi: 10.3389/fonc.2022.864820 social and cultural issues (14). To overcome these drawbacks, recent studies have investigated the
Frontiers in Oncology | www.frontiersin.org 1 April 2022 | Volume 12 | Article 864820

Williams et al. Molecular Detection Methods in HPV-Related Cancers
use of self-sampling swabs for HPV detection to replace Pap EPIDEMIOLOGY

smears and cervical exams as first-line screening. Their results
showed that self-sampling has greater sensitivity compared to HPV infection is recognized as one of the major causes of viral-
traditional cytology and similar sensitivity to clinician-collected related cancers in both men and women. It is classified into two
specimens (14–16). The studies suggested that self-sampled HPV categories: low-risk HPVs (LR-HPVs), which are responsible for
testing can be cost-effective and can be used as a primary skin warts on the hands, feet, and around the genitals and the
screening strategy or in addition to existing screening anus, and high-risk HPVs (HR-HPVs) associated with
programs. By self-sampling, the cost of testing can be lowered anogenital (cervical, anal, vulvar, vaginal, and penile) and head
and the level of screening attendance will be increased, and it can and neck cancers (mainly oropharyngeal and sinonasal) (1).
attract long-term under-screened women or never-screened There are more than 200 genotypes of HPV, but only a few are
women to participate (17). However, the HPV assays that have considered carcinogenic. There are as many as 15 HR-HPV types
been developed have limited sensitivity, specificity, and (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and
replicability in resource-limited settings (12, 13, 18). 82), and globally, HPV 16 is the most frequent oncogenic type
For head and neck cancers, p16INK4a (p16) (1–4). It is estimated that 4.5% of all cancers worldwide (630,000
immunohistochemistry (IHC) is a widely used surrogate new cancer cases per year) are attributable to HPV infection:
marker for oncogenic HPV (19, 20). Since HPV-related SCC in 8.6% in women and 0.8% in men. Presented in Table 1 is a
the head and neck region is predominantly seen in the summary of the epidemiology of HPV-associated Cervical
oropharyngeal zone, p16 IHC testing is considered an cancer, OPSCC and Sinonasal carcinoma.
acceptable clinical standard for the diagnosis of oropharyngeal Cervical cancer (CC) which includes the two major histology
SCC. Although sinonasal SCC is thought to be associated with types squamous cellcarcinoma (SCC) and adenocarcinoma (AC),
HPV in many cases, p16 or direct HPV testing is not routinely is the fourth most common cancer among women worldwide (3,
done for these cancers (21, 22). Most methods of HPV detection 15, 28, 29), affecting women under 50 years of age (4) and with
in head and neck SCC, including p16 IHC, require a fine needle approximately 570,000 new cases in 2018 (13.1/100,000 women)
aspirate (FNA) or tissue biopsy (19, 20). This can often be (1, 27). Almost all cervical SCCs (CSCC) and some cervical ACs
limiting because special equipment is needed to acquire FNA (CAC) are HPV-related and AC is rare compared with SCC
samples and tissue biopsies are often invasive and resource- (29, 30). Globally, HPV 16 and 18 together account for 71% of
intensive, because special equipment is needed to acquire cervical cancer, and this percentage rises to 90% for HPV 6/11/
FNAsamples and they are obtained under general anesthesia. 16/18/31/33/45/52/58 (4). HPV 16 is the more dominant type in
Droplet digital polymerase chain reaction (ddPCR) is a CSCC while HPV18 is more prevalent in CAC (29). In 2018, CC
promising technology for the minimally invasive detection of was responsible for 3.3% of deaths due to cancers by causing
oncogenic HPV. It allows for the quantification of the absolute more than 300,000 deaths, with more than 85% of the deaths
amount of target nucleic acid present with high precision and occurring in low- to middle-income countries (1). About 98% of
reproducibility (23). ddPCR involves partitioning a single nucleic CC deaths are attributed to HR-HPVs (1). It is estimated that the
acid sample into up to 20,000 uniform, nanoliter-sized water-in- highest CC attributable to HR-HPV is in Africa (31.5/100,000
oil droplets, amplifying them by PCR, analyzing each droplet women/year), specifically in sub-Saharan Africa (75.3/100,000
individually, and reporting the results digitally (23, 24). This women/year), and lowest in Asia (10.2/100,000 women/year) (1).
method quantifies the absolute amount of target nucleic acid HR-HPVs are more prevalent in developing countries, mostly
present with high accuracy and reproducibility that is several due to shortage and/or lack of healthcare access, higher
orders of magnitude higher than traditional PCR (23, 24). prevalence of immunocompromised patients, a paucity of
ddPCR is a highly sensitive method for the identification of screening programs, and low vaccination rates (1).
oncogenic HPV as it is able to quantify gene expression with Head and neck squamous cell carcinoma (HNSCC) is the
extremely low copy numbers (25–27). This method can be sixth most common malignancy worldwide (7, 31, 32) with
applied in the early detection of oncogenic HPV in swabs from 710,000 cases per year (7). HNSCC represents a large and
the oropharynx, sinonasal, and cervix. diverse group of malignancies, which have been historically
TABLE 1 | Epidemiology summary of HPV-associated cervical cancer, OPSCC and sinonasal carcinoma.
Cervical cancer OPSCC Sinonasal carcinoma
Incidence Decreasing Increasing Decreasing

Prevalence Higher in developing Higher in developed countries Higher in developed countries
countries
Sex 100% female >70% male Male and female about similar rates
Age Under 50 Under 60 50s
Etiology Almost all are caused Tobacco and alcohol remain important causes, along Environmental toxins such as tobacco and wood dust, etc., along
by HPV with HPV with HPV
HPV 50% HPV16, 20% HPV18 >90% HPV16, HPV18 82% HPV 16, 12% HPV 31/33, 6% HPV 18
genotype

attributed to tobacco and alcohol consumption (3, 4). Although The HPV oncoproteins E5, E6, and E7 play a role in
the incidence of HNSCC is declining in some parts of the world, infiltrating many signaling pathways to create favorable
largely due to a decrease in tobacco use, developed countries (e.g., conditions for cellular transformation. The E5 protein has been
United States, Canada, Australia, Sweden) have experienced an demonstrated to play an important role during the productive
increase in the incidence of oropharyngeal cancer over the past viral life cycle of HPV (43). The role of E6 and E7 in the initiation
two decades due to HPV infection, especially in men under 60 and progression of HPV-related cancers has been extensively
years of age (7). HNSCC accounts for about 6% of HPV- demonstrated, and together they have been shown to be
attributable cancer (38,000 cases globally), most of which are necessary but not sufficient for HPV-driven cellular
located in Northern America and Europe (3, 4). HPV 16 and 18 transformation (44). E6 targets p53 by forming a complex with
are responsible for 85% of HPV-related cancers of the head and the E3 ubiquitin-protein ligase E6-associated protein (E6AP) for
neck (4, 7). Most HPV-related HNSCC arise in the oropharynx proteasomal degradation and can also bind p53 and block
(>90%) but has also been detected in other sites, including the transcription of tumor-suppressive genes (39, 41, 45). The
oral cavity, larynx, nasopharynx, and sinuses (3, 4). degradation of p53 aids in productive viral replication and
Although sinonasal malignancies are rare, accounting for allows for the accumulation of genetic mutations which can
approximately 0.2% of all cancers and 3 to 5% of head and lead to transformation, dysplasia, and cancer (45). Both LR and
neck cancers (5, 33), the sinonasal tract is the second anatomic HR E6 oncoproteins are able to bind to p53, but LR E6 cannot
subsite of the head andneck for HPV-related carcinomas induce degradation (40, 45). HR HPV E7 binds a cell cycle
(34, 35). The mean age of patients with sinonasal malignancies regulator, retinoblastoma protein (Rb), and other retinoblastoma
is about 62 years, and it is more prevalent in Caucasian men (5). pocket proteins—p105, p107, and p130—for degradation, which
The overall incidence is estimated to be 5 to 9 per million for results in the release and activation of transcription factor E2F
males and 2 to 5 per million for females based on WHO statistics (45). This promotes the expression of S-phase genes, inducing
taken from the GLOBOCAN dataset for 9 countries. cell proliferation and increased viral gene transcription (45). E7
Environmental toxins, such as tobacco, and industrial agents, further induces cell proliferation by promoting the G1–S phase
such as wood dust, thorium dioxide, formaldehyde, isopropyl entry of the cell cycle through the inhibition of cyclin-dependent
oils, lacquer paints, solder, and welding materials, are risk factors kinase (CDK) inhibitors p21 and p27, leading to the increased
for developing sinonasal malignancies (5, 33). The incidence activity of CDK2 (41, 45). The degradation of Rb and the
of sinonasal cancer has been declining in most countries due increased E2F activity result in a feedback loop, causing an
to decreasing tobacco use and efforts to reduce occupational increased expression of the biomarker p16INK4a (p16) which
exposures (5, 33). However, there is increasing acknowledgment controls the crucial G1–S phase transition (46). LR HPV E7
that a subset of malignancies isHPV-related but how the virus is proteins are still able to target Rb, but with a lower affinity
transmitted remains unclear (35). HPV type 16 (82%) is themost compared to HR HPV E7 proteins, possibly contributing to their
prevalent, followed by type 31/33 (12%) and type 18 (6%) (34). difference in progression to cancer (45).
The most commonsinonasal histologic type is SCC (SNSCC)
which accounts for about 60-75% and it is estimatedthat 20% to
62% of SNSCC is HPV positive (36).
HPV ATTRIBUTES, SCREENING,
DIAGNOSIS, TREATMENT, AND
HPV CARCINOGENESIS PREVENTION
An understanding of transformation processes initiated by HPV Almost all cervical cancers are caused by persistent infections
infection has relied on the study of premalignant uterine cervical with oncogenic strains of HPV, leading to the development of
cells and has led to a recognized model of HPV carcinogenesis. premalignant lesions and, eventually, invasive cancer (40). Since
The model parallels the normal HPV life cycle with initial HR-HPV is well established as the main cause of almost all
infection, establishment, and maintenance, but with persistent cervical cancers, it has been effectively controlled by screening
infection of basal or stem cells, carcinogenesis can be initiated and diagnosis. Primary screening involves Pap smears that detect
(37). Persistent infection with HPV, causing genomic instability, morphologic changes in the cervical epithelium (such as
is considered a necessary but not sufficient event for the abnormal cells and precancerous and cancerous lesions) caused
development of cancer (38). There are a variety of molecular by early HPV infections (30). It is followed by HPV DNA testing
mechanisms involved in HPV-associated carcinogenesis that if the Pap smear results showed malignancy or co-screening
include the overexpression of HPV oncoproteins E6 and E7 together with HPV DNA testing on the same cytology sample,
altering multiple signaling pathways and inducing genomic which gives greater sensitivity and specificity (30). HPV-related
instability. Cancer-associated phenotypes are caused by HPV cervical cancer histology includes cervical squamous cell (70%),
DNA integration in the host genome, immune evasion, changes cervical adenocarcinoma (25%), or mixed-histology tumors (30).
in global DNA methylation (39–41), and the buildup of genetic Non-HPV-related cervical cancer is rare, representing <1% of
and epigenetic modifications or mutations in genes whose newly diagnosed cases, with histologies including cervical
encoded proteins act in diverse signaling pathways (42). neuroendocrine, small cell, and large cell carcinomas (30). In

comparison of the two major histologies, SCCdevelops from the radiotherapy (RT) (30). Patients with more advanced disease or
ectocervix's squamous epithelia and AC develops from the presence of extensive nodal metastases are generally treated
the endocervix'sglandular epithelia (29) Studies suggest that the with combined modalities, including surgery, radiation, and/
incidence of AC appears to be increasing in some countries while or chemoradiation.
SCC incidence is decreasing (29, 47). The rise is seen among While the incidence of sinonasal carcinoma is low, their
young women, partly due to cohort effect and partly due to histology is among the most diverse ofall head and neck sites
cytology screening, which is less effective for detection of AC with several uncommon and distinct subtypes, several SCC
compared to SCC (29). Although there is growing evidence that variants,interesting etiologic lesions and HPV- related tumors
ACs have different epidemiology, prognostic variables, patterns (107). HPV-related sinonasal carcinomahistologic types are SCC
of dissemination, and treatment failure following therapy and variants (non- or partially-keratinizing, papillary,
compared to SCCs, both are staged and treated similarly (47). adenosquamousand basaloid), small cell carcinoma,
Silvaclassification, which stratifies invasion in three patterns, is undifferentiated and carcinoma with adenoid cystic-like features
used to determine HPV-related CAC (47, 48). Even though p16 (34), which is now known as HPV-related multiphenotypic
expression is considered to be a surrogate marker for sinonasal carcinoma (HMSC) (53–56). HMSC is rare and
HPVassociation, p16 IHC testing is not absolutely necessary histologically characterized by multiple patterns of
for the classification, and HPV analysisis not necessary for the differentiation, including squamoid, ductal, and myoepithelial,
diagnosis (48). HPV-related CSCC causes pre-cancerous lesions similar to adenoid cystic carcinoma (10, 51). There is increasing
but there is no known precancerous lesion in the very rare non- histologic and epidemiologic evidence suggesting that a subset of
HPV-related CSCC (48). Accordingto WHO guidelines, HPV SNSCC may be caused by HPV and detection may be a biomarker
DNA testing is used to detect HPV-related CSCC but p16 IHC is for improved survival similar to HPV positive OPSCC but
also recommended since morphology alone cannot distinguish definitive conclusions are hampered by small sample sizes and
the two types (48). Cervical cancer is a continuous single disease inconsistent HPV detection methods (57). The available literature
process advancing gradually from mild cervical intraepithelial has shown conflicting results with some studies showing that
neoplasia (CIN1) to more severe degrees of neoplasia and micro- HPV-related SNSCC is associated with better outcomes,while
invasive lesions (CIN2 or CIN3) and finally to invasive disease others have reported that HPV status is not a significant
(30). The primary treatment for early-stage cervical cancer is prognostic factor (36). However, HPV testing in these cancers
surgery and for later-stage type are chemotherapy and/or is not widely performed by pathologists. The primary treatment
radiation (37). modality is surgery with or without adjuvant RT, with some
HPV-related OPSCC is clinically distinct, affecting younger evidence suggesting that adjuvant RT may prolong the disease-
patients with fewer comorbidities, responding favorably to free interval among patients who develop local recurrence (53,
treatment, and portending survival outcomes compared to 58). Table 2 shows the summary of a few attributes of HPV-
HPV-negative OPSCC, affecting older patients with a related Cervical cancer, OPSCC and Sinonasal Carcinoma.
significant history of tobacco use and alcohol consumption (49, Because almost all cervical cancers and rising proportions of
50). HPV 16 induces over 90% of HPV-related OPSCC, followed OPSCCs are attributable to HPV infections, universal access to
by HPV 18 and 45 which presented at less than 2% each (44). vaccination against HPV could effectively reduce the incidence of
Most HPV-related OPSCC present with small primary tumors these and other HPV-associated cancers (49). By reducing the
but often cystic, multilevel nodal disease. The histology is incidence and transmission of anogenital HPV, the vaccine
predominantly non-keratinizing SCC with basaloid should also indirectly reduce the incidence and sexual
morphology (9, 51). OPSCC is usually tested for HR-HPV by transmission of oral HPV and thereby decrease the incidence
surrogate marker p16 IHC, and discretionally, additional of HPV-positive OPSCC (30). Universal HPV vaccination has
molecular HPV-DNA testing may also be performed (9, 22). been introduced into national immunization programs in most
For early-stage OPSCC with minimal or no nodal disease, the developed countries. In Canada, HPV2, HPV4, and HPV9 are
treatment is generally either primary surgery and/or definitive available for both sexes from the age of 9 or Grade 6 and are
TABLE 2 | Summary of attributes of HPV-related Cervical cancer, OPSCC and Sinonasal carcinoma.
Cervical cancer OPSCC Sinonasal carcinoma
Histopathology Keratinizing SCC, AC, large cell Non-keratinizing SCC Squamoid, ductal myoepithelial non- or partially-keratinizing, papillary,
nonkeratinizing, with adenosquamous,basaloid, small cell
small cell nonkeratinizing basaloid morphology
neuroendocrine
Molecular diagnosis HPV-DNA testing p16 p16 Not recommended
immunohistochemistry immunohistochemistry
(and HPV-DNA)
Early-stage primary treatment Surgery Surgery and/or RT Surgery and/or RT
Treatment sensitivity to Moderate High High
chemotherapy and radiation

administered as a two-dose series as part of the national squamous intraepithelial lesion (HSIL), and squamous cell
immunization program (59). Overall, HPV vaccination has carcinoma (SCC) (12, 13, 63, 64).
been effective in the prevention of persistent HPV16 and Cytology screening is one of the most successful public health
HPV18 infections (39). However, immunization programs are prevention activities worldwide. It has led to significant
not established in developing countries, and the uptake of the reductions in cervical cancer incidence and mortality, but it
HPV vaccine is low; hence HPV-related diseases continue to rise. has significant limitations, such as low sensitivity and poor
HPV vaccination has the potential to prevent almost 90% of reproducibility (60). HPV testing was more advantageous than
cervical and other HPV-related cancers worldwide (30) and will cytology largely due to its ability to direct early detection further
provide the ultimate prevention against HPV-associated diseases upstream in cervical carcinogenesis (60). Some of the benefits
among young adults. However, screening and HPV testing will include the following: (1) higher sensitivity and reproducibility
continue to play a key role, as prophylactic vaccines are most but somewhat lower specificity, (2) ability to be automated,
effective prior to HPV exposure, and the eradication of HPV centralized, and be quality-checked for large specimen
through vaccinations is still decades away (30, 60). throughput, (3) more cost-effective than cytology, if deployed
for high volume testing, and (4) the ability to use self-sampling,
which has the potential to increase screening in remote areas or
to women who are not directly reached by primary healthcare in
CERVICAL CANCER SCREENING urban areas (60, 65). In 2008, the 3-year prospective study
AND DIAGNOSTIC TOOLS FOR ATHENA (Addressing the Need for Advanced HPV
HPV DETECTION Diagnostics) was initiated in the US, and it is the first and
largest screening study to evaluate the performance of HPV
Cervical cancer screening and diagnosis is combined liquid- primary screening (66). The results indicated that co-testing,
based cytology stained Papanicolaou stain (also known as Pap cytology, and HPV provided minimal increased protection
smear) and HPV testing using DNA/RNA PCR-based methods against the development of CIN2 or worse compared to HPV
(12, 13, 61). Papanicolaou carried out the first prospective studies primary screening. This led the FDA to approve, in 2014, HPV
of the vaginal cycle by working with guinea pigs, and in 1943, primary screening tests for women ages 25–65. Women tested
jointly with Traut, he outlined detailed studies of cycle- for HR-HPV 16 and/or 18 are referred for colposcopy, and those
dependent epithelial changes in the vaginal epithelium of the positive with the other HR-HPVs should be triaged with
human female (62). Epithelial cells are collected from the cytology; if the latter is positive (ASC-US or worse),
external surface of the cervix and lower part of the cervical colposcopy is recommended. The important development was
canal using a cervical sampling brush or spatula, processed into a that the majority of women who tested HPV-negative are to be
thin layer on a glass microscope slide, stained with Papanicolaou screened no sooner than 3 years later (60, 61, 66, 67). Table 5
stain, and evaluated by a cytopathologist using a microscope shows the cervical cancer screening recommendations from the
(62). The cytopathologist evaluates the sample by comparing the American College of Obstetricians and Gynecologists (ACOG),
histologic structure to the normal squamous epithelium from American Society for Colposcopy and Cervical Pathology
the vagina and ectocervix (62). Höffken et al. (62) summarized (ASCCP), and US Preventive Services Task Force (USPSTF).
the histology and cytology of a normal squamous epithelium Primary HPV testing followed by cytology was accepted in
from the vagina and ectocervix as shown in Table 3. Canada and Europe because of its safety relative to co-testing and
The current reporting system for Pap smears is the Bethesda reduction of required tests nearly in half, with a consequent
System, which was introduced in 1988 and amended in 1991 to reduction in the cost for screening programs (60). Combining
replace the cervical intraepithelial neoplasia (CIN) system. Burd primary HPV screening with cytology triage provides greater
et al. (13) summarized the cytology and histology terminology reassurance of the absence of cervical lesions and supports
for HPV-associated squamous lesions of the cervix, as shown in increased intervals between screening rounds for up to almost
Table 4. The histologic diagnoses are reported as normal, atypia, double the maximum duration allowed by conventional cytology
low-grade squamous intraepithelial lesion (LSIL), high-grade (60, 66).
TABLE 3 | Histology and cytology of normal squamous epithelium from the vagina and the ectocervix.
Histology Cytology Cytometry C = cell diameter Proliferation

N = nuclear diameter grade
Basal cell layer (stratum basale) Basal cells, basophilic with dense cytoplasm, nucleus round or oval C: 12–20 mm N: 8–10 mm Not seen in
normal smears
Parabasal cell layer (stratum Parabasal cells, basophilic with dense cytoplasm, nucleus round or oval C: 15–25 mm N: 8–10 mm 1
spinosum profundum)
Intermediate cell layer (stratum Small intermediate cells, polygonal, basophilic, pale-staining cytoplasm, C: 20–40 mm N: 7–9 mm 2
spinosum superficiale) nucleus vesicular, with fine granules
Superficial cell layer (stratum a) Large intermediate cells, polygonal, basophilic, eosinophilic, nucleus still C: 40–60 mmN: 6–8 mm 3
superficiale) vesicular
b) Surface cells, polygonal, eosinophilic, basophilic, nucleus pyknotic C: 40–60 mmN: 6 mm 4

TABLE 4 | Cytology and histology for HPV-associated squamous lesions of the cervix.
Bethesda system CIN system Interpretation
No epithelial abnormalities or benign cellular changes Normal Normal

Atypical squamous cells (ASC): ASC-US (undetermined Atypia, squamous cells with abnormalities greater than those
significance), ASC-H (cannot exclude HSIL) attributed to reactive changes but not meeting the criteria for a
squamous intraepithelial lesion
Low-grade squamous intraepithelial lesion (LSIL) CIN 1 Koilocytosis, mild dysplasia, and mild abnormalities caused by
HPV infection
High-grade squamous intraepithelial lesion (HSIL). (perform p16 CIN 2-3 Moderate dysplasia, severe dysplasia, carcinoma in situ, suspicious;
IHC to upgrade or downgrade; if negative, classify as LSIL and more severe abnormalities that have a higher likelihood of
if positive, classify as HSIL) progressing to cancer if left untreated
Squamous cell carcinoma Invasive squamous cell Invasive squamous cell carcinoma (cervical cancer) Atypia, glandular
carcinoma, invasive glandular epithelial cells
cell (adeno) carcinoma
Immunohistochemistry (IHC) for p16INK4a (p16) is In situ hybridization (ISH) is a method used to detect
commonly used as a surrogate marker for the presence of HR- nucleotide sequences based on the complementary binding of a
HPV E7 in tumor tissues and has become the clinical standard nucleotide probe (cDNA, cRNA, or synthetic oligonucleotide) to
for HPV testing (9, 22, 68). Most routine laboratories testing a specific target sequence of RNA or DNA in cells, tissue sections,
surgical pathologies usually have accessible IHC with or an entire tissue (73). The hybrids that form between the
pathologists that can easily perform the methods and labeled probe and the specific target sequences can be visualized
adequately interpret the staining reactions (69). The IHC assay and detected by various methods (73). Tissue samples are
is widely used in the diagnosis of abnormal cells to identify its prepared by the treatment with proteases to facilitate access of
origin based on the binding of antibodies (Ab) to specific the target nucleic acid to increase hybridization efficiency and
antigens (Ag) in tissue sections. It is visualized by a reduce nonspecific background staining (73). The probes used
histochemical chromogen reaction or by fluorochromes visible have radioisotope labels or non-isotope labels (biotin,
by using conventional microscopy or fluorescence microscopy fluorescein, digoxigenin, alkaline phosphatase, or
(70). IHC is generally performed on 4–6-mm-thick formalin- bromodeoxyuridine are used) (73). Radioisotope labeling is
fixed, paraffin-embedded (FFPE) tissue slices or on frozen fresh considered as the most sensitive, but others believe that
tissue with thickness of 8–90 mm (70). IHC assays detect distinct nonisotopic methods are just as sensitive (73). The
tissue components by capturing target antigens, with specific radioisotope labeling hybridization sites are observed by
antibodies tagged with proper labels binding to the tissues, and autoradiography with an X-ray film or liquid emulsion, and
the reaction is visualized using fluorochrome (a substance that the nonisotopic labeling hybridization sites are observed by
absorbs or emits light) or chromogens (substances that produce histochemistry or immunohistochemistry (73). The HPV
distinct colors) (70). While most pathologists use strong nuclear detection procedure in ISH occurs within the nuclei of infected
and cytoplasmic expression for a positive result, a few interpret cells, which makes it the only molecular method that reliably
only cytoplasmic staining as positive (68). The College of detects and identifies the location of specific nucleic acid
American Pathologists (CAP) and the 8th edition of the sequences in tissues, which is evaluated microscopically (74).
American Joint Commission on Cancer (AJCC8) recommend The presence of HPV in tissue samples being tested is indicated
that, for a result to be considered positive, a threshold of at least by the development of appropriate precipitate within the nuclei
70–75% of tumor cells must show moderate to strong nuclear of the epithelial cells, and the condition of the virus can be
and cytoplasmic staining of the neoplastic cells. The threshold of classified as integrated or episomal by the presence of
at least 70% of positive tumor cells might be too high because it punctuating signals and diffuse signals, respectively (74). ISH is
was found that there is a presence of nuclear and cytoplasmic highly specific (100%) but not sensitive (83%) for HPV infection
staining in 50–70% of tumor cells associated with HR-HPV in a compared with p16 immunohistochemical staining (73, 74).
subset of patients (71). IHC for the detection of p16 expression is Polymerase chain reaction (PCR) is a widely used technique
a highly sensitive surrogate marker for transcriptionally active that allows a specific stretch of DNA to be copied exponentially
HR HPV infection in CSCC (in the triage of women with positive in a short amount of time (75–77). There are five primary
screening results and to identify pre-cancer biopsies) (72). components of PCR, and it is summarized in Table 6. They
TABLE 5 | Cervical cancer screening recommendations from ACOG, ASCCP, and USPSTF.
Testing ACOG ASCCP USPSTF
Pap only Every 3 years Every 3 years Every 3 years

Pap–HPV co-test Every 5 years, age 30–65 Every 5 years, age 30–65 Every 5 years, age 30–65
High-risk HPV only Every 3 years, age >25 Every 3 years, age >25 Every 5 years, age 30–65

TABLE 6 | Summary of PCR components and description.
Component Description
Template DNA Double-stranded DNA segment to be copied

dNTPs The building blocks of DNA. The 4 nucleotides are ATP, TTP, GTP, and CTP
Polymerase enzyme Taq DNA polymerase enzyme which joins the nucleotides together, creating a mirror image of the template
Oligonucleotide primers DNA sequence complementary to the target DNA where DNA polymerase binds and initiates DNA synthesis
Buffer solution A solution to contain the DNA sample of favorable ionic strength and pH
are as follows: (1) template DNA, the double-stranded target the L1 gene or the E6 and E7 oncogenes (78). PCR primers
DNA segment to be copied; (2) deoxynucleotide triphosphates directed at the E6 or E7 regions have been described as preferable
(dNTPs), the building blocks of DNA [adenine triphosphate because the L1/E1 regions are often lost during the integration of
(ATP), thymine triphosphate (TTP), guanine triphosphate viral DNA into host genomic DNA, and targeting the L1 or E1
(GTP), and cytosine triphosphate (CTP)]; (3) polymerase region may miss advanced disease (77).
enzyme, Taq DNA polymerase joins the nucleotides together; The most current HPV detection methods that are
(4) oligonucleotide primers, DNA sequence complementary to the commercially available are type-specific target amplification
target DNA; and (5) buffer solution of favorable ionic strength and DNA PCR and signal amplification DNA ISH, which are
pH (75). approved for cervical samples (77, 78). HPV DNA PCR is a
PCR uses Taq DNA polymerase derived from the target amplification technique that effectively amplifies small
thermophilic bacterium Thermus aquaticus for its heat amounts of DNA sequences in a biological specimen containing
stability, as it allows the enzyme to withstand the heating diverse cell types, using primers that can be specific for a single
needed to denature DNA and maintain activity at relatively HPV type or target sequence shared by multiple types (78). HPV
high temperatures which improve primer specificity (75). DNA PCR can also be used as a non-quantitative technique, but
There are three core steps involved in PCR, as summarized in information about the abundance of a particular DNA species is
Table 7—step 1: denaturation is heating the PCR tube not provided (78). DNA ISH is a signal amplification technique
components at high temperatures (94–96°C), which weakens that utilizes labeled DNA probes (that can be type specific to one
the DNA and breaks the two complementary strands apart; step HPV type or multiple HPV types or mixed in a single reaction to
2: annealing is cooling the PCR tube components (55°C), which cover a range of HPV types) that bind to a specific target
allows the DNA primers to bind themselves to the sequence of DNA-forming hybrids visualized using microscopy
complementary sites on the template strands; and step 3: (73, 78). The performance of DNA PCR and DNA ISH is
extension is heating the PCR tube components (72°C), which comparable, but a direct comparison suggests that DNA ISH
permits the DNA polymerase to copy the template strands by may be more practical as a diagnostic tool due to its ability to
adding nucleotides onto the ends of the primers and producing reliably differentiate relevant HPV infection from passenger virus
two molecules of double-stranded DNA (75). The process is or contaminant (78). Furthermore, DNA ISH adaptation to
normally repeated through a number of cycles, thereby FFPE tissues makes it compatible with standard tissue
increasing the amount of the target DNA exponentially (75). processing procedures, using nonfluorescent chromogens that
PCR is an integral component of many protocols and is allow hybridized DNA to be visualized using conventional light
perhaps the key technique of molecular biology (75). PCR has microscopy and the introduction of various signal amplification
broad applications, including medical diagnostics, and as such, it steps that has increased sensitivity (78).
is used to detect HPV. PCR is a selective technique capable to Hybrid Capture 2 (HC2) HPV DNA test was developed by
reproduce and increase the amount of target HPV sequences Digene Corporation (Gaithersburg, MD) and is now marketed
present in biological specimens exponentially, following repeated by Qiagen (Germantown, MD) and approved by the FDA in
cycles of amplification (77). PCR-based assays have wide-ranging 1999, and it replaced the original Hybrid Capture (HC1) tube-
specificity and sensitivity determined by a few factors such as the format assay, which was approved in 1995. It was the only test
size of the PCR product, the spectrum of HPV DNA amplified available until 2009. The HC2 is a microtiter-format nucleic acid
and ability to detect multiple types, the primer sets chosen, the hybridization assay with signal amplification for cervical
reaction conditions, and the performance of the polymerase specimens collected using the HC2 DNA collection device or
enzymes in the reaction (77). Most primer sets are designed to HC cervical sampler (cervical broom) (13). The specimen release
TABLE 7 | Summary of the steps and events in PCR.
Steps Event
Denaturation A very small PCR tube is heated to 94–96°C, which denatures the DNA and splits the two complementary strands apart
Annealing The tube is cooled, which allows the DNA primers to bind themselves to the complementary sites on the template strands
Extension The DNA polymerase copies the template strands by adding nucleotides onto the ends of the primers and producing two molecules of
double-stranded DNA

and denature target DNA after treatment, and a mixture of moves to the side of the tube by the utilization of magnetic
multigene RNA probes specific for 13 high-risk HPV—16, 18, 31, fields, and the supernatant is aspirated and then washed (79, 81).
33, 35, 39, 45, 51, 52, 56, 58, 59, and 68—is added (13). If HR The captured HR HPV mRNA is amplified by transcription-
HPV is present, it combines with the probes, and the resultant mediated amplification, detected by hybridization protection
DNA–RNA hybrids are captured onto the wells of a microtiter assay using chemiluminescent labels (13, 79). A luminometer is
plate that are coated with monoclonal antibodies to DNA–RNA used to measure the resultant signal in RLUs, and the results are
hybrids (13). The addition of a second monoclonal antibody interpreted based on the analyte signal-to-cutoff (S/CO) value
conjugated to the alkaline phosphatase binds to the captured (79). Internal control (IC) is added to each reaction, and the
hybrids in multiples, resulting in dephosphorylation of a signal in each reaction is distinguished from the HPV signal by
chemiluminescent substrate which produces light (13). The the differential kinetics of light emission from probes with
alkaline phosphatase acts on many copies of the substrate, different labels (79). Target RNA amplification is detected
creating an amplified target/signal level, and the emitted light using probes with a slow emission of light (glowers), and IC
is measured in relative light units (RLU) on a luminometer (13). amplification is detected using probes with a rapid emission of
HR probe may cross-react with LR HPV that is not in the probe light (flashers) (79). The dual kinetic assay is a method used to
mixture, which will adversely affect the sensitivity (77, 79). The differentiate between the signals from the flasher and glower
HC2 test has a cutoff of 1 RLU, and an RLU greater than or equal labels (79). The analyte S/CO is calculated from the analyte RLU
to 1 indicates the presence of HR HPV DNA, while an RLU less of the test sample and the analyte cutoff for the run (79). If the S/
than 1 indicates either the absence of HR HPV DNA or HR HPV CO ratio is <0.50, a negative result is generated, and if the S/CO
DNA levels below the limit of detection of the test (13). The test ratio is ≥0.50, a positive result is generated (79). The system is
has a sensitivity of 0.2 to 1 pg/ml, which is equivalent to 1,000 to automated with high output, and the full process from sample
5,000 genome copies of HPV, but does not distinguish the preparation to result detection can be automated on the TIGRIS
specific HPV genotype present (13, 79). It is not possible to system (Hologic) (13, 79).
determine the quality of the specimen or the presence of The Cobas 4800 HPV test (Roche, Pleasanton, CA, USA) was
potentially interfering substances because HC2 test does not approved by the FDA in 2011 but has been available in the
contain an internal control (13, 79). European market since 2009. It is a target amplification assay that
The Cervista HR HPV test (Third Wave Technologies, detects the same 14 HR HPV types as the Cervista and APTIMA
Madison, WI, USA; now Hologic/Gen-Probe, San Diego, CA) tests but also distinguishes HR-HPV types 16 and 18 (13, 79, 80).
was approved by the FDA in 2009. It utilizes proprietary Invader It simultaneously detects the L1 gene of HPV16 and HPV18 as
Chemistry to generate signal amplification of a fluorescent probe individual reactions and the other 12 HR-HPV as a pooled result
to detect HPV DNAs from 14 HR types, including the same 13 by using multiplex real-time PCR and nucleic acid hybridization
types detected by the HC2 test plus HPV66 (13, 79). The analytical with four different fluorescent reporter probes (13, 79). There are
sensitivity of the Cervista HPV HR test varies depending on HPV four fluorescent-labeled cleavage primer probes used for detection
type, with limits of detection of 1,250 to 2,500 copies per reaction of amplification of the HPV DNA that target the L1 region: one
for HPV16, 18, 31, 45, 52, and 56, 2,500 to 5,000 copies per specific for HPV 16, one specific for HPV 18, one for non-16/18
reaction for HPV33, 39, 51, 58, 59, 66, and 68, and 5,000 to 7,500 genotypes, and one for b-globin (79). The test is automated, and
copies per reaction for HPV35 (13, 79). Similar to HC2, it does not the system consists of two separate instruments: the Cobas z 480
identify the individual HPV type (13, 79). Cervista uses a lower instruments for automated nucleic acid extraction and the Cobas x
sample requirement of 2 ml (vs. 4 ml) and has lower cross- 480 analyzers for PCR amplification and detection reactions in a
reactivity with some LR HPV types compared to HC2 (13, 79). Its single tube (13). The system is designed to process up to 280
analytical sensitivity is comparable to HC2, but it uses the human cervical specimens collected in PreservCyt solution in 1 day (13).
histone 2 gene as an internal control to ensure the efficacy of the False negatives can occur though since the L1 gene is lost upon
specimen and eliminate false-negative results (80). integration into the human genome in a considerable proportion
The APTIMA HPV assay (Hologic Gen-Probe Inc., San of cancers (13, 79). The overall intra-laboratory agreement is
Diego, CA, USA) was approved by the FDA in late 2012. The 98.3%, and genotyping agreement is 98.2%. Inter-laboratory
assay qualitatively detects E6/E7 mRNA transcripts of 14 high- reproducibility studies showed 94.6% overall agreement and
risk HPV types and uses a noninfectious RNA transcript as 93.7% genotyping agreement (79).
extrinsic process control (13). The assay performs pooled HR The OncoE6™ Cervical Test (Arbor Vita Corporation,
HPV detection that does not distinguish between the 14 targeted Fremont, CA) is a qualitative lateral flow assay (strip test) that
HR types like HC2 and Cervista HR HPV assays. The 3 main detects the elevated level of E6 oncoprotein expressed from HPV
steps in the assay, which occur in the same tube, involve target infected cells associated with the most common oncogenic HPV
capture, target amplification using transcription-mediated types 16 and 18 (82, 83). The presence of elevated E6 oncoprotein
amplification, and detection (79, 81). The assay uses 1 ml of levels suggests that there is an existing malignant cell or an
liquid-based cytology, and a lesser amount is inadequate for increased risk of future malignancy (82, 83). The assay uses cell
testing (79, 81). The cells are lysed so that mRNA can be released lysates samples from cervical swab specimens or from specimens
and allowed to hybridize to capture oligonucleotides attached to collected in PreservCyt® solution (82). The lysate is incubated with
magnetic microparticles. (79, 81) The bound target mRNA highly specific mouse monoclonal antibodies (mAbs) to E6

oncoprotein from HR-HPV types 16 and 18 bound with alkaline specificity compared to the other assays. They all have similar
phosphatase (AP) (82). The test strip made out of nitrocellulose sensitivity for the detection of cervical dysplasia (79).
with two capture lines consisting of the immobilized mAbs to E6
16/18 is placed in the lysate/mAb-AP mix (82). By capillary action,
the lysate/mAb-AP mix pass through the test strip, and a complex
(capture mAb-E6-detector mAb) may form if E6 16 and/or 18 is OPSCC SCREENING AND DIAGNOSTIC
present and becomes visible as a purple line at the respective TOOLS FOR HPV DETECTION
locations (either 16 or 18) when the enzyme substrate is added
(82). If the test is valid and a purple test line at any intensity is seen, The current recommendation for HPV testing for OPSCC from
the result is positive and no line indicates a negative result (82). the College of American Pathologists (CAP) and American Society
The assay was validated in several clinical studies. Valdez et al. (84) of Clinical Oncology Guidelines (ASCO) is p16 IHC, and
conducted cervical cancer screening study in rural China and their additional molecular HPV-DNA testing may also be
results showed that OncoE6TM Test had a 70.3% sensitivity and performed at the physician’s discretion. However, HPV testing
98.9% specificity for CIN3 detection compared to HPV DNA is not recommended for other HNSCC (9, 21, 22). There is
testing (careHPV) and visual inspection with acetic acid (VIA). evidence that p16 IHC shows strong diffuse cytoplasmic and
Torres et al. (85) performed a cervical cancer screening in remote nuclear staining in >70% of the tumor cells in SNSCC, though a
areas in Brazil and their results showed that OncoE6™ has overall lower rate than that for OPSCC (86) can be used as a surrogate
50% sensitivity and 99% specificity for CIN3+ and specificity is a marker (21, 34, 86). Since SNSCC is not studied as much as
high priority in remote geographic settings due to the difficulties of OPSCC due to its rarity, the favorable effect of HPV diagnosis is
follow up. Krings et al. (83) demonstrated that OncoE6™ has a inconclusive and therefore p16 IHC testing is not a routine
high sensitivity in the detection of HPV 16or 18 in 3 different types practice (52, 87). Future research studies are essential to better
of self-sampled specimens and their results showed 90% sensitivity understand the role of HR HPVs in sinonasal carcinoma. p16
with the Delphi Screener lavage and the cytobrush sample in IHC is currently used as a highly sensitive surrogate marker for
PreservCyt media and 95% sensitivity for the swab sample. They detecting transcriptionally active HPV in OPSCC (both primary
suggested that using OncoE6 ™ testing and self-sampled and metastatic sites) (21). Other HPV testing methods are also
specimens will allow highly effective cervical cancer screening in utilized, such as viral DNA detection by PCR or ISH; the
remote areas, thereby increasing the effectiveness of preventive combined detection of p16INK4a IHC and HPV DNA-PCR is
strategies (83). The comparison of the diagnostic tools of CSCC is frequently applied as well (68). The E6 oncoprotein testing has
summarized in Table 8. In testing women with abnormal cytology, also been used to detect HPV in HNSCC. Menegaldo et al. (88)
HPV testing is more sensitive (97.4 vs. 56.4%) and more detected HPV16/18 E6 oncoproteins in 34 OPSCC and (cancer
reproducible (Cohen’s kappa coefficient k = 0.60–0.93 vs. k = of unknown Primary) CUP usingOncoE6TM and their results
0.46) but less specific (94.3 and 97.3%) compared to cytology for showed 94% and 88% sensitivity when applied to the primary
the detection of cervical pathology (13, 79). For the detection of tumorand neck nodes respectively and 100% specificity in both
CIN2 + in women with abnormal cytology, p16 IHC sensitivity primary and neck lesions. Cherneskyet al. (89) evaluated HPV E6
compared to cytology is 85.7 vs. 54.7% and for specificity 88 and oncoproteins and nucleic acids in FNA and oral samples
61%, respectively (79). All of the FDA-approved assays for HPV frompatients with OPSCC using commercial assays. Their
detection use either target or signal amplification techniques and results showed that for FNA samples, theoverall agreements of
are approved for use with liquid-based cytology. For the sensitivity p16 antigen staining of tumor were 81.4% (k 0.53) for OncoE6™,
comparison of HC2, APTIMA, and Cobas 4800 (96.3, 95.3, and 94.9%(k 0.83) for Aptima HPV E6/E7 mRNA and 91.1% (k 0.73)
95.2%, respectively), HC2 is most sensitive, and for specificity for cobas HPV DNA (89). Therewere lower agreements with
(19.5, 28.8, and 24.0%), APTIMA is more specific (79). The HC2, tumor markers for saliva and oral swab samples; 23.7–24.0%
Cervista, and Cobas 4800 tests target HPV DNA, while the (k0.02) for OncoE6™, 55.9–68.0% (k 0.24–0.37) ) for Aptima
APTIMA tests target E6/E7 mRNA and have improved HPV E6/E7 mRNA and 78.9–86.9% (k 0.49–0.58) for cobas HPV
TABLE 8 | Summary of performances of the tests for CSCC.
Test Sensitivity, % Specificity, % Reproducibility
Cytology 53.3 92
p16 IHC 85.7 (88a) 54.7 (61a)
OncoE6™ 50-70 99
Cytology 53.3 92 k = 0.46
HPV testing 73.0 56.9 k = 0.60–0.93
HPV testing methods
HC2 96.3 19.5
APTIMA 95.3 28.8
Cobas 4800 95.2 24.0
OncoE6TM 50-70 99
a
If performing p16 on HPV-positive women only.

DNA (89). The E6 oncoprotein testing has also been used to New HPV biomarkers have been studied in the management
detect HPV in HNSCC. Menegaldo et al. (88) detected HPV16/ of HPV-related OPSCC. Antibodies against E6 protein have been
18 E6 oncoproteins in 34 OPSCC and (cancer of unknown associated with a 132-fold increased risk in developing OPSCC
Primary) CUP using OncoE6TM and their results showed 94% and develop more than 10 years before HPV-related OPSCC
and 88% sensitivity when applied to the primary tumor and neck diagnosis (71). Research showed that these E6 antibodies are
nodes respectively and 100% specificity in both primary and neck detectable in less than 1% of healthy individuals, but other
lesions. Chernesky et al. (89) evaluated HPV E6 oncoproteins studies have shown that most HPV-positive OPSCC patients
and nucleic acids in FNA and oral samples from patients with (>90%) present an HPV16 E6 antibody response in blood at the
OPSCC using commercial assays. Their results showed that for time of their HPV16-OPSCC diagnosis (71). Some researchers
FNA samples, the overall agreements of p16 antigen staining of suggest that E6 serology could be considered for HPV OPSCC
tumor were 81.4% (k 0.53) for OncoE6™, 94.9% (k 0.83) for monitoring, especially in tracking a residual disease or
Aptima HPV E6/E7 mRNA and 91.1% (k 0.73) for cobas HPV recurrence, but more validation and research is needed before
DNA (89). There were lower agreements with tumor markers for consideration for clinical routine application (71).
saliva and oral swab samples; 23.7–24.0% (k 0.02) for OncoE6™, The detection of HPV circulating tumoral DNA (ctDNA)
55.9–68.0% (k 0.24–0.37) ) for Aptima HPV E6/E7 mRNA and from plasma by using ultra-sensitive droplet digital PCR
78.9–86.9% (k 0.49–0.58) for cobas HPV DNA (89). Agustin (ddPCR) has garnered a growing clinical interest in HNSCC
et al. (71) summarized the benefits and drawbacks of HPV and CSCC. HPV ctDNA detection in the plasma of HPV-related
detection techniques for OPSCC, as shown in Table 9 with the OPSCC patients using ddPCR is highly sensitive and specific in
addition of OncoE6™ testing. p16 IHC sensitivity in OPSCC is identifying HPV16 and HPV33 subtypes in a similar distribution
around 80–90%, and specificity varies from 80 to 90% (71). p16 as reported in major genomic profiling studies (90). Their results
IHC is a cost-effective method, and its diagnostic performance is suggested that HPV16 and HPV33 ctDNA ddPCR could be used
considered high enough to diagnose HR HPV infection in in early detection screening trials and in disease response
OPSCC (71). DNA PCR techniques are known to be stable monitoring. The HPV ctDNA in CSCC detection using ddPCR
and reproducible and have a sensitivity of 98% and specificity of may predict relapse, and their results suggest that monitoring
84% (68, 71). RT PCR detection of HPV mRNA E6/E7 has a HPV ctDNA could help evaluate treatment options for patients
sensitivity of 97% and specificity of 100% and is considered by with residual HPV ctDNA after treatment (91). ddPCR and RT-
some authors to be the gold standard to diagnose HPV-related PCR performances were compared in the detection of HPV
OPSCC, but it requires fresh/frozen specimens and is technically ctDNA in cervical neoplasia at different stages of the disease, and
demanding and therefore not useful for routine screening (68, ddPCR offers sensitive detection and absolute quantification of
71). HPV DNA ISH allows for direct visualization of the virus low target DNA compared to RT-PCR (92).
within the tumor cells and minimizes the risk for a false-positive The quantitative method of ddPCR is characterized by its
test result that may derive from tissue contamination with viral high sensitivity, its accuracy, and its inter-laboratory and intra-
DNA. HPV DNA ISH has a sensitivity of 85% and specificity of laboratory reproducibility (31, 71). The ultrasensitive ddPCR can
88% (68). be operated at a very low cost compared to other innovative
TABLE 9 | Summary of HPV detection techniques used in OPSCC.
Detection method Advantages Disadvantages Sensitivity, Specificity,

% %
p16 IHC High sensitivity Inexpensive FFPE tissues Moderate specificity 80–90 80–90
manageable
DNA PCR HPV genotype information High sensitivity FFPE No information about viral transcription High risk of contamination 98 84
tissues manageable Easy and inexpensive (intrinsic and extrinsic)
E6/E7 mRNA High sensitivity and specificity Detects active Time-consuming Non-FFPE tissues manageable (fresh or frozen 97 100
RT-PCR viral infection Gold standard for research tissue only) RNA fragility RNA degradation over time, expensive
E6/E7 mRNA ISH High specificity and sensitivity In situ detection RNA degradation over time Expensive 87–100 88–100
of a transcriptionally active HPV infection FFPE
tissues manageable
HPV DNA ISH In situ detection of HPV DNA High specificity Low sensitivity 85 88
FFPE tissues manageable
OncoE6™ High specificity, easy to use Low sensitivity, only for HPV 16 and 18, needs to be validated 88-94 100
with a larger cohort
Serology for Present in more than 90% of patients with Lack of clinical data and retrospective
antibodies against E6 OPSCC related to HPV16 Easy to set up
protein
HPV circulating Early detection of recurrences in post treatment Needs to be validated
tumoral DNA by monitoring High sensitivity and specificity,
ddPCR low cost

technologies (71). These properties of ddPCR can be applied to is a useful tool in preventive strategies as well as a biomarker for
detect samples in swabs with very low amounts of DNA. monitoring treatment response and prognosis estimation in HPV-
related diseases (96, 97). ddPCR was used to detect HPV in CSCC
by using FFPE tissues, cervical liquid cytology samples, and cell
lines. Malin et al. (96) detected HPV VL in FFPE tissues and
DROPLET DIGITAL POLYMERASE CHAIN cervical liquid cytology, and their results showed that ddPCR was
REACTION FOR HPV DETECTION highly sensitive in detecting HPV and VL at the lowest dilution
level, there was no difference in VL between tumors with multiple
ddPCR quantifies the absolute amount of target nucleic acid and single HPV infections and women’s age, and HPV genotype
molecules encapsulated in discrete, volumetrically defined water- and genera were associated with VL (96). Larsson et al. (97)
in-oil droplet partitions (23, 93). It was first commercially available compared ddPCR with qRT-PCR in quantifying HPV VL in FFPE
in 2011 (94), but the concept of ddPCR was first raised by Sykes in tissues and liquid-based cytology (LBC). Their results showed that
1992, in which DNA molecules are quantified using Poisson DNAs extracted from FFPE tissue samples yielded lower
distribution and diluting templates to single-molecule level (95). amplification signals compared to LBC samples, and ddPCR was
The samples are prepared in a similar manner as the PCR reactions found to quantify copy numbers that are 1 to 31 times higher than
that use TaqMan hydrolysis probes or DNA binding dyes (Eva qRT-PCR numbers (97). Rotondo et al. (27) used ddPCR to
Green®) but in smaller volume-precise reactions or partitions which quantify HPV DNA in CIN specimens and human cell lines,
are then run individually. Positive reactions are checked and and their results showed the reliability of ddPCR in the
calculated among each partition using Poisson distribution (95, simultaneous detection and quantification of different HPV
96). The system involves 3 main parts as follows (also summarized types in one experimental run and low-template-copy-number
in Table 10): (1) droplet generation, in which the samples are placed conditions (27). ddPCR exhibited high sensitivity, accuracy, and
in a droplet generator to partition each sample into 20,000 uniform, specificity in quantifying HPV DNA sequences, and the method
nanoliter-sized droplets, enabling precise target amplification; (2) was repeatable and reproducible (27).
amplification, in which samples are placed in a thermal cycler to HPV detection using ddPCR has been demonstrated in
amplify each droplet, following the PCR principle involving OPSCC FFPE tissues, tissue biopsy, fine needle aspiration (FNA)
denaturation, annealing, and extension; and (3) droplet reading, biopsy, and swabs. Schiavetto et al. (98) detected HPV DNA in
in which the droplet reader reads spaced-out individual droplet OPSCC FFPE tissues and showed comparable results to the
fluorescence in two channels (93). clinical standard technique p16 IHC (98). Antonsson et al. (99)
ddPCR has a broad range of applications, as summarized in detected HPV 16 VL in OPSCC FFPE tissues and showed large
Table 11, in both research and clinical diagnostic applications, variations among HPV 16-positive OPSCC ranging from 1 copy
such as (1) absolute quantification for target DNA measurements, per cell to over 900 per cell compared to CSCC where high VL is
viral load analysis, and microbial quantification, (2) genomic associated with an increased risk of CIN progression (99). Biron
alterations such as gene copy number variations (CNV), (3) et al. (26) detected HPV 16 in OPSCC tissues, FNA, and swabs,
detection of rare sequences, (4) gene expression and microRNA and they showed that adequate amounts of RNA were extracted
analysis, (5) next-generation sequencing, (6) single-cell analysis, using commercially available kits, and the sensitivity and
and (7) genome edit detection (93). specificity of HPV E6 and E7 ddPCR for the detection of p16
positivity was 91.3 and 100%, respectively, compared against p16
ddPCR HPV Detection in CSCC and IHC (26). Isaac et al. (25) detected HPV 16 in OPSCC swabs
OPSCC showing 92% sensitivity and 98% specificity against fresh tissue
The high sensitivity, specificity, and absolute quantification for p16 IHC, which is the clinical reference standard (25). The
target DNA measurement by ddPCR are particularly of interest for excellent sensitivity and specificity of HPV detection using
HPV detection. Several studies have used ddPCR to detect HPV ddPCR in swabs without the need for invasive tissue biopsy
DNA and viral load (VL) in CSCC. HPV VL is an important have several potential applications for both diagnosis and
determinant of virus persistence, and therefore VL quantification disease surveillance. Furthermore, the ddPCR method is
TABLE 10 | Summary of the steps and events in ddPCR.
Steps Events
Droplet The samples are placed in a droplet generator using specially developed reagents and microfluidics to partition each sample into 20,000 uniform,
generation nanoliter-sized droplets, enabling precise target quantification. The target and background DNA are distributed randomly into the droplets. Figure 1
shows the partitioning of discrete droplets and the distribution of target and background DNA (93)
Droplet The droplets are transferred in a thermal cycler to amplify each droplet. The amplification of target molecules follows a similar principle of RT-PCR which
amplification involves denaturation, annealing, and extension (93)
Droplet The droplets are streamed in a single file in the reader which calculates the target DNA concentration by counting the fluorescent positive and negative
reading droplets in two channels. The positive droplets containing at least one copy of the target DNA molecule demonstrate increased fluorescence compared to
negative droplets. Figure 2 shows the separation of individual droplets and readings measured in two channels (93)

TABLE 11 | Summary of the applications and capabilities of ddPCR.
Applications ddPCR capabilities
Absolute quantification ddPCR’s immense droplet partitioning provides quantification of DNA copies without standard curves, giving more precise and
reproducible data and making it ideal for target DNA measurements, viral load analysis, and microbial quantification (93)
Genomic alterations such as gene CNVs are deletions and amplifications of genome segments involved in phenotypic variability, complex behavioral traits, and disease.
copy number variation (CNV) ddPCR’s droplet partitioning provides a large number of replicates that precisely measure copy numbers (23, 93)
Detection of rare sequences ddPCR increases sensitivity by partitioning the target mutant DNA away from highly homologous wild-type DNA (93)
Gene expression and ddPCR provides stand-alone absolute quantification withsensitivity and precision of expression levels, especially low-abundance
microRNA analysis microRNAs (93)
Next-generation Absolute quantification and accuracy of NGS sample preparations and validated sequencing results or CNVs (93)
sequencing (NGS)
Single-cell analysis ddPCR enables the quantification of low copy number (93)
Genome edit detection dPCR provides fast, accurate, and cost-effective evaluation of homology-directed repair and non-homologous end joining generated
by CRISPR-Cas9 or other genome editing tools (93)
reported to be accurate, repeatable, reproducible (27, 94, 100), and smear cytology. Irregular or absenteeism to cervical screening is a
cost-effective (23, 25, 26, 90). major barrier to eliminating cervical cancer, and there are many
reasons for low participation, such as cultural reluctance (14, 16,
ddPCR for Cervical HPV Self-Sampling 104), limited access to healthcare or geographical isolation (105),
Several studies have demonstrated the effectiveness of self-sampling lack of health insurance, low health literacy, language barriers, and
vaginal swabs as a screening tool for CSCC in the minorities and lack of awareness (16). HPV self-sampling is a great tool to increase
lower socioeconomic groups, remote or hard-to-reach areas, and cervical screening, and several studies reported high uptake in
low-resource settings. The HPV self-sampling was effective in participation (14, 16, 101, 105–108). Moses et al. (107) reported
detecting HPV and as sensitive as clinician cytology samples to that there was a high uptake of self-sampling HR-HPV testing, and
detect CIN2 or higher (15, 16, 101–103). The study of Wright et al. it was highly acceptable in the community for cervical cancer
(18) found that HPV testing of the self-sampled vaginal swab is less screening which exceeded 99%, whereas the standard of care, visual
specific but as sensitive as cytology for detecting high-grade cervical inspection with acetic acid, reached only 48.4% in a low-resource
disease in women age 35 years and older, while the study of setting. Women have positive experiences and a highly accepted
Sancho-Garnier et al. (103) found that the sensitivity and specificity HPV self-sampling screening strategy (14, 15, 106). Furthermore,
of HR-HPV testing using self-sampled vaginal swabs is very similar in a randomized trial performed by Haguenoer et al. (108), they
to that of clinician-collected cervical specimens. Gustavsson et al. showed that HPV self-sampling is a cost-effective method to
(104) showed that self-sampling and repeated HPV tests detected increase participation in a cervical cancer screening program.
more than twice as many women with CIN2+ compared to Pap With the substantial amount of studies performed on HPV
FIGURE 1 | The ddPCR sample is partitioned into 20,000 uniform, nanoliter-sized droplets, and the target and background DNA are distributed randomly
into the droplets. (93).

FIGURE 2 | The droplet reader reads spaced-out individual droplet fluorescence in two channels in positive droplets with at least one copy of the target DNA
molecule demonstrating increased fluorescence compared to negative droplets. (93).
testing of self-sampled specimens with positive outcomes, it has tract is the second anatomic subsite of the head andneck for
been proposed to be considered as a screening tool (14, 15, 101, HPV-related carcinomas and favorable HPV prognosis is
105, 107, 108). Self-sampling at home followed by HR-HPV testing unresolved, therefore moreresearch studies is essential to better
has been proposed to increase screening recruitment among understand the role of HR HPVs in sinonasal carcinoma.
underserved groups for convenience and to avoid the need for a Self-sampling HPV testing could be used in the future to
gynecological clinical exam in women with negative tests (103). replace Pap smears and cervical exams as first-line screening for
Most of the HPV self-sampling was tested using commercially cervical cancer. However, to ensure similar or better accuracy
available HC2 (18, 103), Cobas (15), and other PCR-based compared to clinician-collected samples, a test with high
methods, particularly RT-PCR (101, 102, 104, 105, 107, 108), and analytical sensitivity and specificity is required. For HPV-
PCR-based testing is preferred to HC2 as it is more sensitive (108). related HNSCC, swabs will be sufficient for diagnosis, without
Because the viral load in the vagina is lower than the cervix, a test the need for highly invasive tissue biopsy. p16 IHC is the most
with high analytic sensitivity appears to be required for self- widely used method due to its availability in laboratories, but the
sampling to ensure equivalent accuracy between clinician and results can be highly variable, as the criteria for interpretation are
self-sampled specimens (108). It has been demonstrated that not standardized. The commercially available HPV testing
ddPCR exhibits high sensitivity, accuracy, specificity, methods approved for cervical samples, including HC2,
repeatability, and reproducibility compared to RT-PCR in Cervista, Aptima, and Cobas 4800, all have comparable
quantifying HPV DNA (31, 71, 92), and therefore it can be used sensitivity and specificity. In comparison to cytology and p16
to test the self-sampled swabs. Since ddPCR method is reported to IHC, they have higher sensitivity but lower specificity.
be accurate, repeatable, reproducible (27, 94, 100), and cost- The new generation of HPV assay, such as ddPCR, is highly
effective (23, 25, 26, 90), it is an ideal method for routine sensitive and can be performed on non-invasive samples, such as
diagnostic testing. those obtained using swabs. ddPCR has the potential clinical
applicability in early HPV detection for screening, diagnosis, and
disease surveillance. It has the ability to amplify a target sequence
from minimal RNA samples and provides significantly higher
CONCLUSION precision and sensitivity for specific DNA/RNA compared to
The routine practice for cervical cancer diagnosis is minimally traditional PCR.
invasive and utilizes liquid-based cytology, followed by HPV
testing using commercially available p16 IHC, DNA/RNA ISH, AUTHOR CONTRIBUTIONS
or DNA/RNA PCR. For OPSCC, the main HPV detection
method available is for fresh, frozen, or FFPE tissues using p16 JW designed the manuscript and wrote the first draft. MK and VB
IHC and/or DNA/RNA PCR. For other HPV-related HNSCC, edited the first draft. JW and VB edited and revised the manuscript. All
however, HPV testing is not a standard procedure. The sinonasal authors contributed to the article and approved the submitted version.
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published: 27 April 2022

Molecular Detection Methods in

Frontiers in Oncology | www.frontiersin.org 1 April 2022 | Volume 12 | Article 864820

use of self-sampling swabs for HPV detection to replace Pap EPIDEMIOLOGY

Cervical cancer OPSCC Sinonasal carcinoma

Incidence Decreasing Increasing Decreasing

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Cervical cancer OPSCC Sinonasal carcinoma

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Histology Cytology Cytometry C = cell diameter Proliferation

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Bethesda system CIN system Interpretation

No epithelial abnormalities or benign cellular changes Normal Normal

Testing ACOG ASCCP USPSTF

Pap only Every 3 years Every 3 years Every 3 years

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TABLE 6 | Summary of PCR components and description.

Template DNA Double-stranded DNA segment to be copied

TABLE 7 | Summary of the steps and events in PCR.

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TABLE 8 | Summary of performances of the tests for CSCC.

Test Sensitivity, % Speciﬁcity, % Reproducibility

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TABLE 9 | Summary of HPV detection techniques used in OPSCC.

Detection method Advantages Disadvantages Sensitivity, Speciﬁcity,

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TABLE 10 | Summary of the steps and events in ddPCR.

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TABLE 11 | Summary of the applications and capabilities of ddPCR.

Applications ddPCR capabilities

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REFERENCES 2. Plummer M, de Martel C, Vignat J, Ferlay J, Bray F, Franceschi S. Global

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