JCM 01403-22

MINIREVIEW
Testing for Human Papillomaviruses in Urine, Blood, and Oral

Specimens: an Update for the Laboratory
Mario Poljak,a Kate Cuschieri,b Laia Alemany,c,d Alex Vorsterse
a Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
b Scottish HPV Reference Laboratory, Royal Infirmary of Edinburgh, Edinburgh, Scotland, United Kingdom
c Cancer Epidemiology Research Program, Catalan Institute of Oncology, L’Hospitalet de Llobregat, Barcelona, Spain
d Centro de Investigación Biomédica en Red: Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III, Madrid, Spain
e Centre for Evaluation of Vaccination, Vaccine & Infectious Disease Institute, University of Antwerp, Antwerp, Belgium
ABSTRACT Twelve high-risk alpha human papillomavirus (HPV) genotypes cause

approximately 690,000 cancer cases annually, with cervical and oropharyngeal cancer
being the two most prominent types. HPV testing is performed in laboratory settings
for various applications of a clinical, epidemiological, and research nature using a
range of clinical specimens collected by clinicians or by individuals (self-collected
specimens). Here, we reflect on the importance and justification of using the right
test for the right application and provide practical updates for laboratories either Editor Romney M. Humphries, Vanderbilt
participating in or anticipating involvement in HPV testing in three specimen types, University Medical Center
namely, urine, blood, and oral specimens, which are considered “alternative” speci- Copyright © 2023 Poljak et al. This is an open-
access article distributed under the terms of
mens by many. In addition to clinician-collected cervical samples and self-collected
the Creative Commons Attribution 4.0
cervicovaginal samples, first-void urine is emerging as a credible specimen for HPV- International license.
based cervical cancer screening, triage of HPV screen-positive women, monitoring Address correspondence to Mario Poljak,
HPV vaccine impact, and HPV testing in groups for which a less invasive sample is [email protected].
preferred. Detection of cell-free DNA (including HPV DNA) in blood has great prom- The authors declare a conflict of interest. M.P.
institution received research funding, free-of-
ise for the early detection of HPV-attributable oropharyngeal cancer (HPV-AOC) charge reagents, and consumables to support
and potentially other HPV-driven cancers and as an adjunct prognostic marker in research in the last 3 years from Qiagen,
long-term tumor surveillance, including treatment response. The moderate sensitiv- Seegene, Abbott, and Roche, all paid to his
employer. K.C. institution received research
ity of HPV testing in oral rinses or swabs at HPV-AOC diagnosis prevents its use funding, reagents, and consumables to
in HPV-AOC secondary prevention but represents a promising prognostic tool in support research in the last 3 years from
Cepheid, Euroimmun, GeneFirst, Self-screen,
HPV-AOC tertiary prevention, where the HPV persistence in oral rinses throughout
Hiantis, Seegene, Roche, Abbott, Hologic, and
treatment may predict early HPV-AOC recurrences and/or the development of Vaccitech, all paid to her employer. K.C. also
secondary HPV-AOC. The increasing sophistication of specific collection devices attended an advisory board meeting of
Hologic where UK-based travel expenses were
designed for alternative samples and the enhanced precision of novel molecular
supported and is currently on the advisory
technologies are likely to support the evolution of this field and catalyze potential board of Vaccitech (with any associated
translation into routine practice. reimbursement paid to employer). L.A.
institution received funding to support
research in the last 3 years from Merck Sharp &
KEYWORDS HPV, urine, blood, oral specimens, cervical cancer, oropharyngeal cancer
Dohme, Roche, GSK, Vitro, Hologic, and
Seegene, all paid to her employer. A.V.
H uman papillomaviruses (HPVs) have a remarkably stable DNA genome and are classi- institution received funding to support
research and setting up professional meetings
fied by the homology of their genome into five genera (Alpha-, Beta-, Gamma-, Mu-, in the last 3 years from Merck, Roche, GSK,
and Nupapillomavirus), several species, and numerous genotypes (1, 2). Genotypes are Hologic, Abbott, Novosanis and Becton,
numbered chronologically in order of characterization, and 223 distinct HPV genotypes Dickinson and Company, all paid to his
employer. A.V. is the co-founder of Novosanis,
were officially recognized as of the end of June 2023 (https://www.hpvcenter.se/human_ a spin-off company of the University of
reference_clones/) (3). The 12 HPV genotypes from the Alphapapillomavirus genus with Antwerp, Belgium, and a subsidiary of OraSure
Technologies, Inc. since 2019, and he was a
highest oncogenic potential are often referred to as high-risk HPV genotypes (hrHPVs) and
board member and minority shareholder until
are as follows: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, and -59 (3, 4). January 2019.
Most hrHPV infections follow a subclinical course, but some persistent infections are Published 13 July 2023
etiologically linked with benign and malignant lesions of the epithelia (4, 5). Human
August 2023 Volume 61 Issue 8 10.1128/jcm.01403-22 1

Minireview Journal of Clinical Microbiology
cancers attributable to hrHPVs represent 4.5% of all cancers worldwide (8.8% and 0.9%
of all cancers in women and men, respectively), with approximately 690,000 new cancer
cases identified each year (6). hrHPVs cause virtually all cervical and anal cancers, a sub-
stantial proportion of vaginal cancers, and a component of oropharyngeal, penile, and
vulvar cancer (5, 6). Consequently, laboratory detection of HPV to support screening for
(or management of) associated disease has been the subject of research and develop-
ment since the 1980s, of which some has translated into large-scale screening practice.
Furthermore, general improvements in the precision of molecular testing and in our
understanding of the natural history of HPV infection have ensured that the laboratory
detection of HPV is still a highly dynamic field.
In this review, we reflect on the importance of “tailoring” the HPV test to the appli-
cation and discuss the following three key and expanding areas in the laboratory
detection of HPV: testing of urine, testing of blood, and testing of oral samples.
Implications of this expansion for the detection and management of the two most
common hrHPV driven cancers, namely, cervical and oropharyngeal, are our focus.
THE BURDEN OF CERVICAL AND OROPHARYNGEAL CANCER AND THE IMPORTANCE

OF EVIDENCE-BASED INTERVENTIONS
Cervical cancer, with 570,000 new annual cases worldwide, is the hrHPV-driven can-
cer associated with the greatest morbidity and mortality (6). Because hrHPVs are found
in nearly all cases of cervical cancer, the etiological association between persistent
hrHPV infection and this cancer is very strong, consistent, specific, and universal, mak-
ing cervical cancer a highly credible target for public health preventive interventions,
including through vaccination and screening. Although primary prevention via prophy-
lactic HPV vaccination is a key tool for the elimination of cervical cancer (and other
HPV-related cancers), the secondary prevention of cervical cancer via sensitive HPV-
based screening using molecular tests is crucial and will be necessary for at least the
next 50 years and will significantly accelerate the pace of cancer reduction (7–9).
Increased access to HPV-based screening in “hard to reach” populations, including
those in low- and middle-income countries will be essential if the goal of cervical can-
cer elimination is to be reached (7). The use of self-taken samples, including urine sam-
ples, is a clear way to support equitable access as will be discussed.
Oropharyngeal cancer is the second most common hrHPV-related human cancer, which
is traditionally associated with chronic exposure to tobacco, heavy alcohol consumption,
and deprivation (10). However, in the last 2 to 3 decades, hrHPV has emerged as an etiologi-
cal agent for a subset of oropharyngeal cancer (6). Although the attributable fraction of
HPV-associated oropharyngeal cancer varies according to geography, in many settings, it
has increased markedly (11, 12). HPV-attributable oropharyngeal cancers (HPV-AOCs) show
specific epidemiological, clinical, and molecular features that clearly differentiate them from
those linked to tobacco and alcohol, including significantly improved prognosis (13). This dif-
ference is recognized in the recently updated tumor, node, metastasis (TNM) staging rules,
which acknowledge the HPV-associated and HPV-independent pathway to oropharyngeal
cancer (14). In 2018, a total of 140,000 new cases of oropharyngeal cancers were recorded
worldwide (6). While the natural history of cervical cancer is relatively well understood and
usually involves a preinvasive phase, which is detectable on screening, this is not the case
for HPV-AOC. Thus, screening for HPV-AOC does not exist routinely, and patients generally
present with clinical symptoms, frequently with advanced disease. Given the morbidity and
increased incidence of this disease research to support improved prevention, screening and
management strategies are areas of intense activity; laboratory-based biomarkers and tools
to support the diagnosis and risk stratification are a cornerstone of this activity (15, 16).
PRINCIPLES OF HPV TESTING—CHOOSING THE RIGHT TEST FOR THE RIGHT

APPLICATION
While HPV testing is relevant for population-based screening of the healthy popula-
tion, increasing evidence suggests it may be relevant for individual prognostication in

the patient population (15). To serve both healthy and patient populations, a range of
biological specimens (biospecimens) and HPV tests with different performance charac-
teristics are required (17, 18). The characteristics of HPV tests used and the required
specimen types differ considerably depending on whether the application for use
serves a screening, diagnostic, epidemiological, or research objective (17, 18).
In routine clinical laboratory settings, testing for hrHPV is used most frequently as a
screening tool in HPV-based primary cervical cancer screening programs (18). For this
purpose, HPV testing must be performed using validated molecular tests with clinical
sensitivity and specificity calibrated to the detection of high-grade cervical intraepithe-
lial lesions rather than minute quantities of virus (17, 18). Until recently, this testing
was performed almost exclusively using cervical samples collected by a trained health
care worker (19, 20).
Compared with HPV screening tests, HPV assays required for epidemiology, includ-
ing vaccine impact monitoring, have different characteristics with respect to analytical
performance, HPV genotype range, and the biospecimen applied (5, 17, 18). For epide-
miological purposes, archived specimens with partially degraded nucleic acids are of-
ten required, e.g., formalin-fixed, paraffin-embedded tissues. Tests suitable for archived
material require higher analytical sensitivity, absolute analytical specificity, precise
type-specific resolution of several HPV genotypes without cross-reactivity (leading to
false-positive calls), and frequently an expanded or alternative composition of targeted
HPV genotypes (17). A composition that includes additional HPV genotypes is clearly
important for determining vaccine impact on low-risk genotypes (included in the HPV
vaccine) and for investigating potential genotype replacement.
Similarly, HPV assays used for individual prognostication at the time of diagnosis
and for monitoring treatment success require high analytical sensitivity and specificity,
including in the context of head and neck disease (15). As will be discussed, tumor tis-
sue is the specimen type used most frequently for determining the HPV status of oro-
pharyngeal cancer, whether through PCR-based detection of HPV DNA or HPV mRNA
in situ hybridization for viral sequences or (more commonly in routine settings) immu-
nohistochemistry for an HPV-associated biomarker (p16INK4a) (21). While in situ hybrid-
ization and immunohistochemistry allow for the detection and identification of HPVs
in topographical relation to their pathological lesions, its sensitivity can be lower than
that of PCR (21, 22). Note, that when testing tissue specimens, strict anticontamination
procedures are required at all levels—including meticulous histologic sectioning to
prevent false-positives, a “sandwich” sectioning method for lesion verification, and
optimized nucleic acid extraction protocols for HPV (21–23).
In line with this information, when it comes to HPV testing in blood, where virus
quantities may be low and labile, highly sensitive PCR-based and alternative amplifica-
tion approaches confer dividends on performance (24, 25). Digital droplet PCR (ddPCR)
or partitioning PCR uses microfluidics to divide template DNA into many individual
reactions (or droplets), which are then amplified separately. The fraction of PCR-posi-
tive droplets/reactions (out of the total number) is used to generate an absolute quan-
tity. Whereas quantitative PCR (qPCR) generally offers one data point per reaction,
ddPCR can offer several thousand and provides a sensitivity and precision that can be
suitable for low-copy targets, particularly for early-stage disease (24). Its increased
application is evident in the substantial number of publications in the last 3 years.
Next-generation sequencing (NGS) has also been used for the detection of both HPV
and cellular biomarkers in tissue and blood, and this technology allows precision and
sensitivity for target detection and also offers possibilities for biomarker discovery
depending on how it is applied (26).
Oral rinse and gargles using sterile saline or alcohol-based mouthwash are used
most frequently for sampling the oral cavity or oropharynx for epidemiological and
research purposes (27). Again, HPV tests required for these applications require high
sensitivity, and the general acceleration of molecular diagnostics with improved sensi-
tivity (including ddPCR and NGS) is likely to enhance this field (21).

As mentioned above, this review summarizes recent evidence and provides updates
for the laboratories already participating or anticipating involvement in testing for HPV
using the following three specimen types: urine, blood, and oral specimens. The three
specimen types targeted by this review are considered “nonstandardized,” “alterna-
tive,” or “extragenital” by many; this language is not accepted uniformly, and the main
dispute is whether urine is an “extragenital” or “genital” specimen type.
HPV TESTING IN URINE

HPV self-sampling. To improve equitable access to screening, women in many cer-
vical cancer screening programs or projects are now given the option of providing a
self-collected sample (28–30). Self-taken samples for HPV testing have a similar accu-
racy to that of clinician-collected samples for the detection of high-grade cervical intra-
epithelial lesions when a validated PCR-based assay is used (20). In most self-sampling
scenarios, women collect the self-sample by inserting a swab, brush, or more compli-
cated collection device into the vagina to collect cervicovaginal secretions (containing
exfoliated epithelial cells and debris of disintegrated cells) by turning or rubbing or
through washing or lavage (21). However, urine as the least invasive self-collected sam-
ple has strong potential for use in self-sampling exercises for screening and epidemio-
logical applications.
Background. Our understanding regarding the use of urine as a valid specimen for
testing for HPV has evolved substantially in the last decade (Table 1). In the early days
of investigation, major technical and interpretational challenges were identified and
suboptimal analytical and clinical sensitivity were observed particularly when urine
was compared with clinician-collected cervical samples (31). The mechanism through
which HPV DNA “contaminated” urine was poorly understood and was a major limita-
tion for advancing the field. An initial review on the performance of urine as a biospeci-
men for HPV testing demonstrated that the conflicting results obtained in early studies
were largely a consequence of a lack of standardization and an imprecise broad definition
of the term urine (31). In certain pioneering papers, investigators did not differentiate
between the different “types” of urine, for example, initial-stream (first-void), midstream,
total-void, any-void, first urine of the day, or urine collected later in the day. In addition,
many investigators did not realize the considerable difference between female and male
urine specimens and (wrongly) expected equivalent performance in both sexes.
Rationale for the preferable use of first-void urine for HPV testing in women.
When different types of urine specimens were assessed from a cohort of HPV-positive
women, substantial differences were observed in the reported copy number of HPV
and human DNA when comparing first-void (or initial-stream) urine versus the subse-
quent fractions (32). It is now generally agreed that first-void urine contains consider-
ably more epithelial cells and consequently higher concentrations of both HPV and
human DNA than subsequent void fractions. This observation is in fact consistent with
an opinion forged in the early 1940s, where Traut and Papanicolaou (33) suggested
that for the detection of cancer of the uterus a self-collected vaginal smear would be
appropriate and could be used for routine diagnostic purposes. Their basic rationale
for this suggestion was that all epithelial tissues have superficial cell layers that are
subject to continuous exfoliation. This debris of epithelial tissues and exfoliated cells
subsequently mix with the secretions of the uterus and cervix and descend into the va-
gina, explaining why they can be detected in a vaginal sample. Subsequent to this in-
formation came the realization that female genital secretions, including the debris of
superficial cell layers, further descend and exit the vagina, where they are accumulated
between the labia minora and around the urethra opening (Fig. 1). This natural biologi-
cal cleaning process of the female genital tract is completed when a woman urinates
and flushes away (mostly with first-void urine) said accumulated secretions. Thus, in
the context of HPV testing, we should not categorize first-void urine as a urine speci-
men but rather consider it a mixture of genital tract secretions collected through urina-
tion. In Fig. 1, this important rationale is shown graphically, and four selected examples

TABLE 1 What is known, knowledge gaps, and how far are we away from implementation of first-void urine, blood and oral specimens as routine samples for HPV testinga
How far away are we from implementation as a
Minireview
Sample type What is known Knowledge gaps routine specimen for HPV testing
Urine First-void urine contains considerably more epithelial cells Widespread clinical implementation of urine-based HPV tests has HPV assay(s) approved for cervical cancer
and their debris and consequently higher concentrations been hindered by the lack of commercial standardized HPV screening by stringent regulatory authority
of both HPV and human DNA than those of subsequent tests. As for self-collected cervicovaginal samples in general, using self-collected cervicovaginal samples and
void fractions. also for first-void urine, no HPV assay approved by FDA, EMA, first-void urine are expected on the market in
When using first-void urine, it is important to consistently or other stringent regulatory authority is available on the the next 3 yrs.
use sample collection/transport medium containing market. However, several countries have already implemented HPV testing of first-void urine samples using fully
August 2023 Volume 61 Issue 8

urine-conservation preservative to stabilize HPV DNA to HPV-based cervical cancer screening using home collected integrated, automated, sample-to-result
prevent its degradation by nucleases. cervicovaginal samples as an off-label screening strategy. molecular analyzers that allow continuous
If HPV testing is performed using clinically validated PCR- Urine samples have been used for a number of in certain loading of samples and high-throughput testing
based HPV DNA assays and on appropriately collected regions in France as an off-label screening strategy. are expected in the next 3 yrs.
and preserved first-void urine, similar clinical accuracy The whole workflow from urine sample collection to result Some countries in Europe and selected low- and
(CIN21, CIN31) as for/on self-collected cervicovaginal or interpretation needs further optimization and standardization, middle-income countries are expected to adopt
clinician-collected cervical samples can be obtained. including the determination of optimal sample volume and first-void urine and other self-collected samples
First-void urine sampling, using a validated collection cut-off value for the interpretation of results. Including an as a valid alternative to clinician-collected
device, is well accepted by women, is convenient, is internal control to monitor the whole workflow may be cervical samples in their population-based
noninvasive, and allows home collection of a sample for beneficial. organized screening programs in the next 5 yrs.
cervical cancer screening. HPV testing of urine samples has still not been incorporated into Some countries in Europe and some low- to
First-void urine is a valuable source of other female fully integrated, automated, sample-to-result molecular middle-income countries are expected to adopt
reproductive and genital tract biomarkers, such as analyzers that allow continuous loading of samples and high- first-void urine as the valuable source of female
methylation markers, providing opportunities for triage of throughput testing. reproductive and genital tract biomarkers, such
HPV-screen positives using (the same) self-collected Only one first-void urine collection device is currently as methylation markers, for triage of HPV-screen
sample. commercialized. Since this device is more expensive than positives in the next 3 yrs.
First-void urine has great potential to provide information devices for self-collected cervicovaginal samples, more devices Novel anti-HPV assay(s) with enhanced analytical
on the HPV vaccination status of the women. for first-void urine collection are needed on the market for sensitivity will enable the collection of valid
First-void urine is emerging as a credible specimen for HPV- further cost optimization. information regarding natural infection-induced
based cervical cancer screening, triage of HPV screen- Preliminary evidence regarding the acceptance and cost- anti-HPV antibodies in/from urine samples in
positive women, and monitoring of HPV vaccine impact effectiveness of cervical cancer screening using urine vs the next 3 yrs.
and for HPV testing in groups where a less invasive cervicovaginal self-collected samples in population-based
sample is preferable (including transgender individuals). organized screening settings have been collected in the last
years. Pilot randomized clinical trials on the topic are ongoing
in France (CapU4 study) and Belgium (ScreenUrSelf study).
Blood Tumors can shed nucleic acid fragments into the circulating Due to a poor positive predictive value as a result of the low It is likely that the first routine application of liquid
blood of patients; detection of these fragments is burden of HPV-AOC in the general population, the lack of an biopsies in the HPV field will be for the
technically possible, particularly when using assays with accepted algorithm for follow-up of positive serology cases, monitoring of treatment response in patients
high precision and sensitivity. and the need for further optimization of the assay(s), the with HPV-AOC. Private entities offer liquid
Detectable nucleic acid fragments include ctDNA, miRNA potential of anti-HPV-16 E6 antibody testing for screening and biopsy testing (for HPV in blood) for the
and/or circulating viral DNA. These fragments are early detection of HPV-AOC is unclear at present. management of HPV-AOC, although it is not
sometimes referred to collectively as “cell-free DNA” or Anti-HPV-16 E6 antibody presence in blood may be useful for considered standard of care.
“liquid biopsies.” annotation of the HPV status of oropharyngeal cancer in Reports on the use of liquid biopsies to measure
Although HPV-AOC is rising, no validated screening strategy selected clinical scenarios and may serve as a potential treatment response in longitudinal clinical trials
is currently available. To improve the screening and prognostic marker of HPV-AOC, but greater research in these compared with other more standard clinical
management of HPV-AOC, the following two approaches areas is warranted. modalities will be essential. These will help
have been explored, of which both utilize blood as a Studies with larger sample sizes, optimized clinical protocols determine how the technology can be applied
biospecimen: (i) detection of antibodies against HPV-16 with clear longitudinal outcomes, and refined, consistent optimally to deintensify follow-up and are likely
E6 protein and (ii) liquid biopsies or the detection of cell- technical methodology are needed to better establish the to report in the next 3–5 yrs.
free DNA (including HPV DNA). clinical utility of liquid biopsies or the detection of cell-free Significant improvements in molecular detection
Antibodies against HPV-16 E6 protein are detectable 10 yrs DNA (including HPV DNA) in blood samples. technology (and growing academic and
prior to the diagnosis of HPV-AOC. Those individuals who A specific tumor signature for the component of HPV-unrelated commercial interest in the area) are likely to lead
10.1128/jcm.01403-22
are antibody positive are at significantly greater risk of oropharyngeal cancers (that can be detected consistently in to standardized HPV assays for liquid biopsies in
HPV-AOC. blood) has so far proven elusive, which is likely due to the 1–3 yrs. Assays applicable to liquid biopsies
5
Journal of Clinical Microbiology
(Continued on next page)

TABLE 1 (Continued)
How far away are we from implementation as a
Sample type What is known Knowledge gaps routine specimen for HPV testing
Minireview
Detection of HPV in liquid biopsies is indicative of disease heterogeneity of tumors with a nonviral etiology. already exist in the commercial sector for non-
recurrence posttreatment in HPV-AOC. There is evidence to suggest that liquid biopsies may be suitable HPV targets and is likely to facilitate
Nucleic acid fragments in liquid biopsies have a short half- for the management of other HPV-associated anogenital development.
life; swift processing is thus key to prevent degradation. cancers, including cervical and anal cancer, particularly in
Improved (blood) capture and processing protocols in high-risk populations; however, larger follow up studies are
addition to a greater application of highly sensitive needed to confirm a definitive clinical use case. Very little is
detection techniques, such as digital droplet PCR (ddPCR) known about the performance of liquid biopsy for the
August 2023 Volume 61 Issue 8

and next-generation sequencing (NGS), are likely to detection and prognosis of vaginal, vulvar, and penile disease.
enhance the performance of the technology overall. Greater research in this area is warranted.
Oral samples HPV-AOC is a distinct disease, markedly different from The key knowledge gaps in the natural history of HPV-AOC that Standardized, clinically validated oral rinse and
oropharyngeal cancer etiologically associated with hinder the implementation of secondary prevention lie gargle collection protocols and standardized
tobacco and alcohol at the following three levels: around the existence (or not) of HPV-related preneoplastic collection devices allowing reliable HPV testing
epidemiological, clinical, and molecular. Compared with lesions and, if such lesions exist, whether they can be are expected in the next 3 yrs.
cancers etiologically associated with tobacco and alcohol, identified early enough to be treated in a safe, effective, and Commercial HPV assay(s) approved by stringent
HPV-AOC has a substantially better prognosis and acceptable way to prevent development of HPV-AOC, as in the regulatory authority using oral samples are
superior response to treatment. case of cervical cancer. expected on the market in the next 5 yrs.
The incidence of HPV-AOC is rising in many countries even No validated screening strategy for the secondary prevention of
surpassing the incidence of cervical cancer in some HPV-AOC aiming for early disease detection in the healthy
regions. Overall, 30% of oropharyngeal cancers are population is available at present.
currently attributable to HPV, with substantial Nontissue oral sample alternatives for differentiation of HPV-AOC
geographical variability in HPV-attributable fractions from HPV-non-attributable oropharyngeal cancers, including
being as high as 80% in North America and northern (i) oral rinse and gargle using sterile saline or alcohol-based
Europe. mouthwash and (ii) swabbing or brushing of surface of visible
Differentiation of HPV-AOC from HPV-non-attributable lesions, have been studied intensively in the past decade, but
oropharyngeal cancers is currently a major clinical the moderate sensitivity limits their use as a valid diagnostic
application of HPV testing. Annotation of the HPV status tool currently.
of oropharyngeal cancer is best ascertained by testing The whole workflow from oral sample collection to result
tumor tissue for the overexpression of p16INK4a using interpretation needs further optimization and standardization,
immunohistochemistry ideally in combination with the including the determination of optimal sample volume and
presence of HPV DNA or HPV mRNA discerned either by cut-off value for an interpretation of the results.
PCR-based methods or in situ hybridization.
Detection of HPV persistence in oral rinses and gargles using
sterile saline or alcohol-based mouthwash (and/or by
detecting cell-free DNA in blood) throughout treatment
and posttreatment represent encouraging tools for
monitoring response and for predicting disease
recurrences after therapy.
The use of preservative added immediately to the collection
tube after oral sample collection improves the stability of
the oral sample. Oral samples should be processed as
close to collection as possible or alternatively immediately
frozen at 220°C.
Because HPV viral load in oral (and also blood) samples is
significantly lower than that in genital sites, testing oral
specimens requires the use of HPV tests with substantially
higher analytical sensitivity.
aAbbreviations:
HPV, human papillomavirus; CIN21, cervical intraepithelial lesion grade 2 or higher; CIN31, cervical intraepithelial lesion grade 3 or higher; FDA, United States Food and Drug Administration; EMA, European
Medicines Agency; ctDNA, circulating tumor DNA; miRNA, microRNA; ddPCR, digital droplet PCR; NGS, next-generation sequencing; HPV-AOC, HPV-attributable oropharyngeal cancer.
10.1128/jcm.01403-22
6
Journal of Clinical Microbiology
FIG 1 Rationale for the use of first-void urine as source of female reproductive and genital tract biomarkers and HPV-related biomarkers
detectable in first-void urine. (A) Presents the anatomy of the female reproductive and genital tract and bladder, with the black arrow
depicting the physiological movement of secretions of the uterus, cervix, and vagina subsequently mixed with debris of superficial cell
layers of the epithelium covering the entire surface of the genital tract, exfoliated cells, and transudated or exudated immunoglobulins
(mainly IgG). All secretions further drift and exit the vagina, where they are accumulated between the labia minora and around the urethra
opening and flushed away by the urine originating from the bladder (yellow arrow). (B) Shows a first-void urine collection device (Colli-Pee;
Novosanis, Wijnegem, Belgium) that can be prefilled with a urine preservative and is able to capture a prespecified volume of first-void or
initial-stream urine without interrupting the urine void. Once the collection tube is filled, an outlet ensures that subsequent urine volume
exits the device into the toilet. (C) Shows several relevant biomarkers detectable in first-void urine, as follows: (i) cell-free and cell-associated
HPV DNA; (ii) transudated or excudated immunoglobulins, including HPV genotype-specific antibodies; (iii) methylated viral and human
DNA; and (iv) HPV virions in the case of a productive HPV infection. (D) Shows selected examples of urine specimens collected by four out
of eight women that captured their initial or first-void urine (FVU) and the subsequent void (often referred to as midstream urine [MSU])
directly into urine collection containers. In six of the eight women, macroscopically visible differences between FVU versus MSU samples
due to substantially more debris derived from superficial layers of the epithelium, exfoliated cells, and genital tract secretions present in
FVU were noticed. In the samples from women 3 and 4, who were menstruating at the time of sample collection, the FVU samples also
contained menstrual blood visible with the naked eye compared with an almost transparent MSU sample(s).
of macroscopically visible differences between first-void urine versus subsequent urine

fraction(s) are presented.
The type of urine specimen or the choice of void fraction used for HPV testing does
have a profound impact on the amount of HPV DNA present in the sample and, in
turn, the probability of accurate HPV detection. The superiority of first-void urine for
HPV testing was confirmed in the first meta-analysis on the subject, published in 2014,
but without understanding the underlying reasons and mechanism (34). Surprisingly,
although the fraction of the void exerted an impact on testing accuracy, as well as use
of a tailored device to capture the initial void, no difference between (first-void) urine
collected during first morning urination versus later in the day was observed (35, 36).
Consequently, in applied projects where women are asked to collect urine for HPV

DNA testing, they are asked not to wash their genitals thoroughly in advance of the
sample and to wait at least 1 h after the previous urination before providing a first-
void urine specimen for HPV testing (34).
Other technical considerations. Another important observation described in pilot
experiments is the relative instability of HPV DNA in certain urine samples and the
requirement for a preservative in the sample collection/transport media to enhance
stability and minimize degradation. Even with well-established DNA extraction proto-
cols, which have been found to garner a high yield and quality of HPV DNA in more
cellular samples, significant reductions of HPV DNA in urine specimens have been
observed in the absence of a preservative applied immediately after collection of first-
void urine (32). This finding is consistent with the fact that, due to nucleases present in
unpreserved urine, HPV DNA may become undetectable (32). The instability of HPV
DNA in urine is its main drawback compared with cervical samples, where HPV DNA
remains stable at room temperature for months. Therefore, it is important when using
first-void urine to incorporate a sample collection system that includes urine-conserva-
tion preservative to stabilize HPV DNA. In addition, human DNA present in urine may
not be optimal as an internal control for sample validity in urine because it can remain
positive even if HPV DNA is degraded substantially, running the risk of false-negative
results (32). Instead, the use of an external DNA control to monitor sample validity is
recommended for urine (37).
Use of urine in primary HPV-based cervical cancer screening. By applying the
“lessons learned” for the optimal capture and preservation described above, good ana-
lytic concordance between such first-void urine versus clinician-collected cervical sam-
ples has been demonstrated in pilot studies, with respect not only to the spectrum of
HPV genotypes detected but also to genotype-specific viral load (38). Such studies
clearly demonstrated the significant potential of first-void urine as a valid alternative to
clinician-collected cervical and self-collected cervicovaginal specimens for HPV-based
cervical cancer screening programs (Table 1). Subsequent to these pilot studies, clinical
validation trials have been performed, showing positive results. The international vali-
dation of human papillomavirus assays and collection devices for HPV testing on self-
samples (VALHUDES) diagnostic test accuracy study follows a design where the clinical
performance of validated PCR-based HPV DNA assays on optimally collected and pre-
served first-void urine samples is compared with clinician-collected cervical samples,
with the latter considered standard of care (39). Existing results are very promising,
and a number of commercially available HPV DNA assays, including the US FDA-
approved Onclarity HPV assay (BD Diagnostics, Sparks, MD) and Cobas 6800 HPV assay
(Roche Molecular Systems, Branchburg, NJ), as well as the WHO-prequalified RealTime
high-risk HPV test (Abbott Molecular, Des Plaines, IL), showed comparable clinical sen-
sitivity and specificity for cervical intraepithelial neoplasia grade 2 or higher (CIN21)
using at-home self-collected first-void urine samples versus clinician-collected cervical
samples (40–44). Compared with clinician-collected cervical samples, first-void urine is
relatively easy to self-collect, is noninvasive, and allows home collection of a sample
for cervical cancer screening. In addition, qualitative research comparing urine-based
self-sampling to cervicovaginal self-sampling has shown that some women prefer urine
sampling, and in a study by De Pauw et al. (42), substantially more women were confi-
dent they had performed the sampling correctly when providing a urine sample.
However, the preference and affordability of a particular sampling method are impor-
tant aspects that are clearly context specific.
One potential operational consideration for “scaling-up” urine-based screening is
that, compared with cervical samples, HPV testing of urine samples has still not yet
been fully integrated into automated, sample-to-result molecular analyzers that allow
the continuous loading of samples and high-throughput testing.
Use of urine for triage of HPV screen-positive women. A further promising appli-
cation when using self-collected samples, including first-void urine, is the potential to
use the same sample to screen for hrHPV infection and to detect biomarkers that triage
the risk of that infection to cause significant disease, such as DNA methylation markers

(Fig. 1). Multiple studies have shown the high accuracy of DNA methylation assays as a
triage tool in hrHPV-positive vaginal swabs for detecting high-grade lesions (45). DNA
methylation assays have shown equivalent performance as cytology for detecting
underlying CIN21 lesions and a greater longitudinal negative predictive value for the
absence of disease at follow-up than cytology-negative women (46). Recently, the clini-
cal value of DNA methylation markers to discern clinically relevant disease was demon-
strated in first-void urine samples (47, 48). This knowledge further supports self-samples,
including first-void urine, as credible specimens for cervical cancer screening programs
with the potential to support underserved women and avoid overreferral through mo-
lecular triage.
Monitoring the impact of HPV vaccination using urine. In addition to screening
applications, first-void urine samples are a highly practical means to sample young
individuals to ascertain the population-level impact of HPV vaccination. Indeed, in
cohorts of young women, who may or may not yet be sexually active, urine sampling
is generally well accepted. The large HPV vaccination monitoring impact trials per-
formed in Rwanda and Bhutan targeting young women between the ages 17 and 22
showed that 94% of the 4,463 participants returned their (at-home) self-collected urine
samples for HPV testing (49). In these trials, urine samples were self-collected by partic-
ipants using a specific device (Colli-Pee; Novosanis, Wijnegem, Belgium) designed to
collect the first 14 mL of first-void urine immediately into 7 mL of a urine-conservation
medium while the subsequent urine volume exits into the lavatory (Fig. 1).
Detection of anti-HPV antibody in urine. As mentioned, a first-void urine specimen
contains female genital secretions, including those harboring locally produced immuno-
globulins or immunoglobulins transferred from blood by transudation or exudation (50).
Perhaps unsurprisingly, it was thus confirmed that first-void urine also contains func-
tional neutralizing anti-HPV antibodies, and a statistically significant correlation between
the concentration of genotype-specific anti-HPV antibody in serum samples and in first-
void urine samples was demonstrated (51). This information provides an opportunity to
use a first-void urine sample to detect active HPV infection as well as immune status after
HPV vaccination or after natural HPV infection (by testing for anti-HPV antibodies).
During a productive HPV infection, new infectious viral particles are released from the
outer layers of the epithelium and mix with the genital secretions. In HPV-vaccinated
women, these genital secretions also contain vaccine-induced neutralizing anti-HPV anti-
bodies that may bind to the infectious HPV viral particles. This process may substantially
impact infectivity, help prevent transmission to a sexual partner, and reduce the chance
of autoinoculation, or self-infection of other parts of the body (52). Because a mixture of
the genital secretions can be collected easily using first-void urine, this specimen has sig-
nificant potential for further research on the impact of HPV vaccination and may assist in
defining a much-needed correlate of protection (the minimum concentration of anti-
HPV antibody required to prevent transmission) especially in view of the recent imple-
mentation of one-dose HPV vaccination schedules.
Use of urine for HPV testing in males. In contrast to women, for whom a first-void
urine specimen is a credible alternative to both clinician-collected and self-collected
specimens, urine specimens collected from males (including first-void urine) are less
appropriate and accurate for the detection of anogenital HPV infections because the
anogential secretions “collected” by urination differ substantially due to anatomical dif-
ferences (53). Consequently, a significantly lower HPV positivity rate and also smaller
amounts of human DNA are reported in male first-void urine specimens than those col-
lected from women.
Use of urine for HPV testing in transgender individuals. There are limited data
on HPV prevalence, the natural history of HPV-related tumors, and screening options in
transgender individuals due to the low acceptance rate of traditional sample collection
approaches. A recent pilot study on 200 transgender individuals showed a high accep-
tance rate (98.5%) of first-void urine self-collected using the Colli-Pee FV-5000 collec-
tion device for HPV testing, providing a significant opportunity for natural history, epi-
demiological, and screening studies in this hard-to-reach population (54).

TESTING FOR HPV IN BLOOD

Background. Blood as a specimen has attracted significant recent attention for the
detection and management of HPV-associated disease because it can contain detecta-
ble biofragments that may be indicative of underlying or recurrent neoplastic disease
(25, 55). Such fragments include microRNA (miRNA), circulating tumor DNA (ctDNA),
and/or circulating HPV DNA (cHPV DNA), sometimes referred to collectively as “liquid
biopsies” or “cell-free DNA.” In addition, the detection of antibodies against the HPV-16
E6 protein in blood has been explored extensively in the last decade as a potential
screening, diagnostic, and prognostic tool for the early detection and monitoring of
HPV-AOC (15, 56).
The use of liquid biopsy for detecting HPV-AOC. To date, the bulk of studies that
have explored the utility of liquid biopsies for detecting HPV-associated disease have
focused on oropharyngeal cancer, particularly using cHPV DNA as a target. As we will
go onto describe, the mutational landscape of oropharyngeal cancer is complex, and
so the determination of a core tumor target set to cover HPV-independent tumors
generically has thus far proved elusive. In a study by Warlow et al. (57), the detection
of HPV DNA in pretreatment plasma using ddPCR showed high (.90%) agreement
with p16INK4a and the HPV PCR status of the pretreatment (solid) biopsy samples in oro-
pharyngeal cancer patients. The detection of cHPV DNA has also been shown to be in-
dicative of disease recurrence posttreatment. In a study of over 100 HPV-AOC patients
treated with curative intent, of the 87 patients that had undetectable cHPV DNA, none
had recurrent disease during follow-up (median 23 months), whereas the sequential
detection of cHPV in two posttreatment samples exhibited a positive predictive value
of 94% (58). A recent meta-analysis on the performance of cHPV DNA in monitoring
treatment response in 457 HPV-AOC patients showed a pooled diagnostic sensitivity
and specificity of 65% (95% confidence interval [CI], 40 to 84) and 99% (95% CI, 96 to
99), respectively (59). This range reflects the heterogeneity of inclusion criteria, proto-
cols, and detection technology summarized by the authors, who stated that “Larger
sample sizes and the homologation of study protocols and methodology are needed
to better establish its utility in the clinical practice” (59).
The use of liquid biopsy for detecting cervical cancer. After oropharyngeal can-
cer, liquid biopsies have been researched most extensively for cervical cancer studies.
The targets evaluated have included cHPV DNA, the more common cancer mutations
(e.g., PIK3CA), and host and viral methylation targets, including CADM1 and EPB41L3
as recently reviewed by Herbst et al. (60). Most studies in the cervical context have
focused on the detection of invasive disease rather than preinvasive disease. Given
that the extent of vascularization is likely to influence the secretion of relevant biomo-
lecules into the blood, it is logical that the sensitivity of liquid biopsy for detecting
CIN2 and CIN3 may be limited, although proof-of-concept studies exist to show it is
possible, including for methylated targets (61). The 2020 meta-analysis assessed the
diagnostic performance of cHPV DNA for detecting invasive cervical cancer across 10
different studies conducted between 2001 and 2017, and it reported a pooled sensitiv-
ity of 27% (95% CI, 24 to 30) (62). This modest sensitivity reflected a range of diagnostic
approaches and techniques used with respect to chemistry, the biospecimen (serum
versus plasma) target molecule, and HPV genotype coverage. Because cervical cancer
is etiologically associated with a range of hrHPV genotypes (as described earlier), geno-
type coverage is not trivial. In common with the meta-analysis on oropharyngeal can-
cer (59), the authors called on the scientific community to optimize and standardize
the technical approach. This endeavor seems worthwhile; although investment in bio-
markers for cervical disease in the healthy screening population is of course essential,
there is a comparative lack of research in the posttreatment population, which remains
at a higher risk of recurrent disease up to 20 years after initial treatment (63, 64).
Applications of liquid biopsy for other HPV-related cancers. There are a smaller
but growing number of studies on the use of liquid biopsy for detecting anal cancer; in
a study of 57 patients with advanced anal cancer, HPV ctDNA was detected in 91.1%
when an HPV-16 genotype-specific HPV assay was employed (65). In a more recent

FIG 2 Process and rationale for the detection of cell-free DNA (including HPV DNA). Abnormal lesions and cancers with a vascular
component contain nuclear material that can be shed into the blood through various pathways, including necrosis, apoptosis, and secretion.
This material, referred to as cell-free DNA (cfDNA), can be double or single stranded, and the concentration and stability depend on a
number of factors, including lesion size, extent of proliferation, and extent of vascularization. cfDNA can be host or viral in origin, and it is
feasible for a lesion to exude both. After a blood draw and centrifugation, nucleic acid that contains cfDNA can be extracted from the
plasma component. Given the short half-life of some cfDNA targets, efficient sample transfer to the laboratory for processing and extraction
is important. cfDNA can be used to detect viral and/or host sequences, specific mutations, integration hallmarks, methylation targets, and
microsatellite alterations. Although a range of amplification technologies, such as quantitative PCR (qPCR), have been used for detecting
cfDNA, those methods that can offer high analytical sensitivity are valuable, such as next-generation sequencing (NGS) and droplet digital
PCR (ddPCR). The translation of cfDNA into routine diagnostic laboratory practice is at a relatively early stage for HPV-associated disease, but
it holds promise for various applications, including diagnosis/prognostication and monitoring treatment response. In addition, cfDNA
determination is valuable in natural history studies and epidemiological surveys.
study in which the authors employed NGS technology for eight hrHPV genotypes, the
sensitivity increased to 100% in 20 patients with histologically confirmed disease (26).
Although the data on the use of liquid biopsies for managing anal disease are relatively
scant, they are encouraging—particularly because this disease can be challenging to
treat, and screening protocols are generally not well developed or established, even in
high-risk populations. In addition, there are very few data on the feasibility and appli-
cation of liquid biopsy for diagnosing and monitoring vulvar, penile, and vaginal can-
cer and its precursors; given the morbidity of these diseases and their potential for re-
currence, this area would benefit from more attention.
Technical challenges of liquid biopsy and opportunities for improvement.
Circulating DNA can be single or double stranded and is generally secreted into the
blood at an early stage of cancer development through mechanisms that include apo-
ptosis or tumor necrosis (60) (Fig. 2). The target may have a short half-life of between
20 min and a few hours, which means that the capture of the sample and processing
of the blood specimen should be well controlled and validated. Aspects to consider
are the blood capture volume and collection tube type, transport conditions, maxi-
mum time from blood capture to separation of cell-free component (usually plasma),
method for capture of plasma (including centrifugation conditions), volume-input for
nucleic acid extraction, and method of nucleic acid extraction. It is notable that,
although these conditions are important, particularly if liquid biopsy testing is to be
integrated into routine service, this level of technical detail is frequently missing from
published articles and may be part of the reason why there is variation in diagnostic
performance between different laboratories. A similar lack of important technical detail

in the published literature was also described in a recent systematic review of the
detection of various urine biomarkers, including HPV (66).
After nucleic acid extraction from plasma, the extract may be frozen at 220°C prior
to testing, but again, the impact of any longer-term storage on target deterioration
should be quantified. The technology is likely to be supported by the increase in the
availability of commercial kits, which have been optimized to extract circulating nucleic
acid of high quality.
The evolution of technology for liquid biopsy detection. Diagnostic targets in liq-
uid biopsies vary and reflect the virus, host, or both; with regard to the virus, the focus has of-
ten been only on HPV-16 and/or HPV-18, although there are examples of broader HPV panels
(26). Methylated targets have also been investigated in liquid biopsies of patients suffering
from HPV-associated cancers (59). The systematic reviews of oropharyngeal and cervical dis-
ease incorporate a range of technologies and reflect the molecular evolution of diagnostics
in general (59, 62). Some of the earliest techniques applied were standard PCR or nested
PCR followed by Southern blotting or agarose electrophoresis. These methods preceded the
application of quantitative PCR (qPCR) and, more recently, NGS and ddPCR.
NGS can detect insertions, deletions, somatic single nucleotide polymorphisms, and
gene copy number variations, and it has been applied in an exploratory manner to
define biomarkers of interest in cancer patients and, in turn, to create cancer-target
“panels,” including for cervical cancer patients (67). NGS also offers the opportunity for
the design and application of a test that is specifically tailored to the mutation profile
within an individual patient. This prospect is an exciting one, although bioinformatic
and technical infrastructure clearly need to be in place; furthermore, NGS can be
deployed in a simpler manner to detect viral sequences in blood (26).
Molecular developments in areas such as NGS and ddPCR will clearly help refine the
technology and will support quality and consistency. The update of digital minimum in-
formation for publication of quantitative digital PCR experiments (dMIQE) is helpful in
this regard (24). Although translation and assay commercialization of the end-to-end process
of liquid biopsy is more developed for other cancers—for example, epidermal growth factor
receptor (EGFR) mutation testing for lung cancer patients—the experience from such appli-
cations is also likely to enhance the HPV field (Table 1).
Anti-HPV-16 E6 antibody as a screening, diagnostic, and prognostic marker of
HPV-AOC. In the last decade, several investigators have shown that anti-HPV-16 E6
antibody presence in blood may serve as a potential screening, diagnostic, and prog-
nostic marker of HPV-AOC (68). A pioneering study showed that anti-HPV-16 E6 sero-
positivity was present up to 10 years before the diagnosis of HPV-AOC (69). In a recent
systematic literature review and meta-analysis, the sensitivity of HPV-16 E6 seropositiv-
ity for underlying HPV-16-driven HPV-AOC was 83.1% (95% CI, 72.5 to 90.2) with a
specificity of 94.6% (95% CI, 89.0 to 97.4) (68). However, due to a poor positive predic-
tive value due to the low burden of HPV-AOC in the general population, the lack of an
accepted algorithm for the follow-up of positive serology cases, and the need for fur-
ther optimization of the assay(s), the potential of anti-HPV-16 E6 antibody testing for
screening/early detection of HPV-AOC is unclear at present. The anti-HPV-16 E6 anti-
body has also been piloted for diagnostic purposes, e.g., annotation of the HPV status
of oropharyngeal cancer in selected clinical scenarios, but greater research in this area
is warranted (13, 15). A few studies have also explored the use of anti-HPV-16 E6 anti-
body titer dynamics as a prognostic marker of tumor recurrence in the tertiary preven-
tion of HPV-AOC but with mixed and inconclusive results (15).
TESTING FOR HPV IN ORAL SPECIMENS

Rationale for detecting HPV in oral specimens. As discussed, strong evidence
exists to show that HPV-AOC is a distinct disease and is markedly different from oro-
pharyngeal cancer etiologically associated with tobacco and alcohol at three levels, as
follows: epidemiological, clinical, and molecular (13).
The incidence of HPV-AOC is rising in many countries and is even surpassing the
incidence of cervical cancer in some regions (11, 12). Overall, 30% of oropharyngeal cancers

are currently attributable to HPV, although with substantial geographical variability in HPV-
attributable fractions (as high as 80% in North America and northern Europe) (70). Globally,
more than 80% of HPV-AOC cases are caused by HPV-16 and approximately 3% each by
HPV-18, HPV-33, and HPV-35 (70). Clinically, compared with cancers etiologically associated
with tobacco and alcohol, HPV-AOC has a substantially better prognosis and superior
response to treatment (13). Regarding differences at the molecular level, the carcinogenesis
of HPV-AOC is driven mainly by HPV overexpressed proteins interacting with host tumor-
suppressor genes. The HPV oncoprotein E6 inhibits the host tumor-suppressor gene p53,
and the HPV oncoprotein E7 binds to the host tumor-suppressor gene pRb, promoting its
degradation (13). Overexpression of p16INK4a, a tumor suppressor which is upregulated by
high-level expression of E7, is critical for cell survival in all HPV-related tumors, whereas it is fre-
quently inactivated in HPV-independent tumors. Therefore, p16INK4a overexpression is a surro-
gate marker for transcriptionally active HPV infection. In addition to the presence of HPV
oncoviral proteins, the most common genetic changes in HPV-AOC are in the phosphoino-
sitide-3-kinase (PI3K) pathway, particularly involving activating mutations and amplifica-
tions of the PIK3CA oncogene (71). The host genes TP53 and cyclin-dependent kinase
inhibitor 2A (CDKN2A)—most frequently affected in HPV-independent head and neck can-
cers, including oropharyngeal cancer—are largely unaffected in HPV-AOC (71).
The differences described between HPV-AOC and its counterpart associated with
tobacco and alcohol have recently led to a new classification of p16INK4a-positive HPV-
AOC for the eighth edition of the American Joint Committee on Cancer Staging Manual
(14), a substantial revision of diagnostic approaches for oropharyngeal cancer, and the
launch of several clinical trials of deintensified treatment. A recent large international
study on 7,654 patients with oropharyngeal cancer showed that detection of p16INK4a over-
expression should be complemented with testing for HPV DNA or HPV mRNA to reduce the
risk of misclassification of HPV-non-attributable tumors (11). Specifically, sometimes
tumors can be p16INK4a positive in the absence of HPV infection and behave clinically as
p16INK4a/HPV-double-negative tumors in terms of poorer survival, particularly compared
with p16INK4a/HPV-double-positive cases (11). Because oropharyngeal cancers are often
detected at an advanced stage with low tumor size but high nodal dissemination (spread
of cancer into lymph nodes), timely and accurate differentiation of HPV-AOC from HPV-non-
attributable oropharyngeal cancers by HPV testing of tumor tissue using reliable HPV tests is
of utmost importance (Table 1).
Natural history of HPV-AOC. There are some similarities but also crucial differen-
ces in the natural history of HPV-AOC and HPV-related neoplasms in the anogenital
context with important consequences for primary, secondary, and tertiary prevention
approaches (Fig. 3). Similar to HPV-related anogenital disease, the first necessary step
in the natural history of HPV-AOC is undoubtedly persistent HPV oral infection. In the general
population, the prevalence of oral HPV infection with any HPV has been estimated to be
around 5% and that for HPV-16 is around 1% (72). Regarding determinants of oral HPV infec-
tion, males have a higher prevalence than females, whereas the influence of age is not entirely
clear (some studies show a bimodal distribution and others a slight increase of prevalence by
age). Additionally, a history of smoking and sexual behavior has also been associated with oral
HPV infection (73, 74). However, there remain knowledge gaps, and further research is needed
to establish the incidence, clearance, and persistence rates of oral HPV infection, as well as its
determinants, more precisely.
A small number of studies have explored the intraperson HPV genotype concordance
(the same HPV genotype[s] present at different body sites) between anogenital and oral
sites with conflicting results but generally showing low concordance. Also, intraperson
hrHPV genotype concordance in males has been reported to be low, although it is higher
in males who have sex with males than that in males who have sex with females (only)
(75). Similar results have also been obtained in females, with hrHPV genotype concord-
ance in anogenital and oral sites at around 5% only (76, 77). In practical terms, this infor-
mation means that HPV status established in one body site is not a reliable indicator of
the genotype(s) at other body sites. Site-specific HPV testing is therefore warranted.

FIG 3 Primary, secondary, and tertiary prevention of HPV-attributable oropharyngeal cancer (HPV-AOC). Primary prevention of HPV-AOC by
HPV vaccination might work for HPV-AOC, pending the results of a postapproval confirmatory study, which is still ongoing. No validated
screening strategy for the secondary prevention of HPV-AOC aiming for early disease detection in the healthy population is available at
present, but the following two approaches have been extensively explored in the last decade: (i) the detection of antibodies against HPV-16
E6 protein in blood and (ii) “liquid biopsies,” or the detection of cell-free DNA (including HPV DNA) in blood samples. Differentiation of HPV-
AOC from HPV-non-attributable oropharyngeal cancers is currently a major clinical application of HPV testing in the field. Annotation of the
HPV status of oropharyngeal cancer is best ascertained by testing tumor tissue (formalin-fixed, paraffin-embedded or fresh-frozen tissues) for
overexpression of p16INK4a using immunohistochemistry and the presence of HPV DNA or HPV mRNA by either in situ hybridization or PCR-
based methods. Oral sample nontissue HPV-annotation alternatives, including (i) oral rinse and gargle using sterile saline or alcohol-based
mouthwash and (ii) swabbing or brushing of surface of visible lesions, have been evaluated, and yet, the moderate sensitivity of these
alternatives currently limits their use as diagnostics. In addition, nonoral alternatives, e.g., detection of cell-free DNA and antibodies against
HPV-16 E6 protein in blood, have been also piloted for HPV-annotation purposes in selected clinical scenarios but with mixed and
inconclusive results. The detection of HPV persistence in oral rinses and/or by detecting cell-free DNA in blood throughout treatment and
posttreatment represent valid treatment response monitoring tools and promising predictive tools in the tertiary prevention of HPV-AOC
(prevention of disease recurrences after definite therapy and/or prevention of the development of secondary HPV-AOC).
The key knowledge gaps in the natural history of HPV-AOC that hinder the imple-
mentation of preventive measures similar to those used for decades for cervical cancer
lie around the existence (or not) of HPV-related preneoplastic lesions that precede
HPV-AOC and, if such lesions exist, whether they can be identified early enough to be
treated in a safe, effective, and acceptable way to prevent the development of HPV-
AOC, as in the case of cervical cancer (Fig. 3).
Sampling oral cavity/oropharynx and testing for HPV. Oral rinse and gargle
using sterile saline or alcohol-based mouthwash are used most frequently for sampling
the oral cavity or oropharynx for epidemiological, research, and clinical purposes (15, 78). The
method used most frequently relies on 15- to 30-s rinsing followed by 15- to 30-s gargling
(16, 79), although there is some heterogeneity in the literature. As is the case for first-void
urine, evidence suggests that the use of preservative added immediately after sample cap-
ture can improve the stability of the oral sample for downstream molecular work (79). Oral
samples should be processed promptly after the collection (especially in the absence of
preservative) or frozen immediately at 220°C to minimize degradation (16). However, the

impact of storage time (including at frozen temperatures) on sample quality/target deteri-

oration remains unknown.
As an alternative to the rinse/gargle biospecimen, if oral lesions are visible, the mu-
cosal surface has been sampled using a swab or brush. Saliva is not considered a ro-
bust specimen for HPV testing nor are specimens obtained by swabbing or brushing
tonsils or the oropharynx without visible lesions (15, 78).
Relatively few studies have directly compared different sampling approaches in the
oral cavity. One study compared oral gargles versus tonsil brushings in a sizeable age-
stratified sample of oral/oropharyngeal cancer-free individuals and found that HPV
detection was rare when using tonsil brushings from both children and adults (80). In
contrast, HPV infection was detected more frequently in gargle specimens, particularly
those of adults, but with poor agreement with the paired tonsillar sample (80).
In addition to obtaining a robust oral specimen, the choice of the appropriate test
for detecting and genotyping HPV in oral samples is clearly important. Because HPV vi-
ral load in oral samples is significantly lower than that in genital sites, testing oral
specimens requires the use of HPV tests with substantially higher analytical sensitivity
(23). In the largest study to date to compare the performance of different HPV tests in
oral specimens, 1,455 oral gargle samples from the HPV Infection in Men (HIM) study were
tested in parallel using the Linear Array HPV genotyping test (Roche Molecular Systems,
Alameda, CA) versus the SPF10 PCR-DEIA-LiPA25 system (DDL Diagnostic Laboratory, Rijswijk,
Netherlands) with an overall HPV positivity of 1.9% versus 8.6%, respectively (81). The observed
(significant) difference in HPV positivity is most likely a consequence of the substantially
shorter region of HPV DNA targeted by SPF10 PCR-DEIA-LiPA25 (65 bp) than that of the
Linear Array HPV genotyping test (approximately 450 bp). This difference in analytical per-
formance becomes important in samples with very low viral load (such as gargle oral sam-
ples) or in samples with partially degraded DNA.
Primary, secondary, and tertiary prevention of HPV-AOC. Primary prevention by
HPV vaccination might work equally well in the case of HPV-AOC as for anogenital HPV-
related carcinomas but with a potential caveat. Namely, although the US FDA recently also
conditionally approved an expanded indication of HPV vaccine for primary prevention of
oropharyngeal and other head and neck cancers caused by targeted HPV genotypes, the
final vaccine approval for this specific indication is dependent on the results of an ongoing
postapproval confirmatory study (16).
As described above, no validated screening strategy for the secondary prevention
of HPV-AOC to support early disease detection in the healthy population is routinely
available at present (27). In contrast, promising results in the tertiary prevention of
HPV-AOC were obtained recently (15). The tertiary prevention of HPV-AOC aims to pre-
vent disease recurrence after definite therapy and/or to prevent the development of
secondary HPV-AOC either by detecting cfDNA in blood (as presented above) or by
detecting HPV in posttreatment oral rinses (as presented below).
HPV testing of oral specimens in clinical practice. Differentiation of HPV-AOC from
HPV-non-attributable oropharyngeal cancers is currently a major clinical application of
HPV testing in the field (27, 78). Annotation of the HPV status of oropharyngeal cancer is
best ascertained by testing tumor tissue (fresh-frozen or formalin-fixed, paraffin-embedded
tissue) for overexpression of p16INK4a using immunohistochemistry and the presence of HPV
DNA or HPV mRNA by either in situ hybridization or PCR-based methods (Fig. 3).
The use of nontissue alternatives for the annotation of the HPV status of oropharyn-
geal cancer has been studied intensively in the past decade. The accuracy of HPV testing
in oral rinses or swabs for diagnosis of underlying HPV-AOC was assessed in a recent system-
atic review (82). Eight studies met the eligibility criteria, with five restricted to patients with
oropharyngeal cancers. Five studies tested oral rinses, two studies used oral swabs, and in
one study, all subjects had both an oral rinse and an oral swab. Seven studies used HPV
DNA testing, and in one study, patients were tested for HPV-16 E6/E7 mRNA. In the five
studies comprising oropharyngeal cancer patients, the sensitivity of oral HPV testing for
detecting underlying HPV-AOC was 55% (95% CI, 25 to 82) with a specificity of 94% (95%
CI, 85 to 98). Overall sensitivity across all eight eligible studies was 72% (95% CI, 82 to 97)

but with considerable variability in the sensitivity estimates ranging from 93% (95% CI,
81 to 99) to only 12% (95% CI, 7 to 19) (82).
The moderate sensitivity of HPV testing in oral rinses at HPV-AOC diagnosis limits its use
as a screening test for HPV-AOC. However, akin to liquid biopsy, HPV testing in oral rinses
could be used as an adjunct prognostic marker in the tertiary prevention of HPV-AOC, where
the persistence of the HPV in oral rinses throughout treatment may predict HPV-AOC recur-
rences and/or the development of secondary HPV-AOC (Fig. 3). Rettig et al. (83) detected
baseline oral HPV-16 DNA infection in 67/124 (54%) of patients diagnosed with HPV-AOC
with only five patients having persistent HPV-16 infection in oral rinses at 24 months after
treatment. All five patients (100%) with persistent HPV-16 infection (HPV-16 present at base-
line and after therapy) developed recurrent disease, with three with local disease involve-
ment. In contrast, 9 out of 119 (8%) patients without persistent HPV-16 infection developed
recurrent disease (83). Two similar but smaller studies using oral rinses showed comparable
results (84, 85), with a baseline cancer detection rate of HPV-AOC at around 50% and signifi-
cantly higher recurrence rates in patients with persistent oral HPV-16 infection versus those
without, namely, 100% versus 11% (84) and 75% versus 6% (85), respectively. These data
suggest that, although infrequent, persistent HPV-16 infection detected in posttreatment
oral rinses is associated with a poor prognosis of HPV-AOC and represents a potential
marker for long-term tumor surveillance, mainly for the early detection of recurrent HPV-
AOC with local disease involvement.
SUMMARY
Testing for hrHPV using clinically validated molecular HPV assays has dramatically changed
our ability to screen for cervical cancer, gradually replacing traditional morphology-based
screening using the Pap test. In addition to clinician-collected cervical samples and self-
collected cervicovaginal samples, first-void urine is emerging as a credible specimen for
HPV-based cervical cancer screening, triage of HPV screen-positive women, monitoring of
HPV vaccine impact, and HPV testing in groups where a less invasive sample is preferable
(including transgender individuals). Detection of cell-free DNA (including HPV DNA) in blood
samples is slowly transitioning into routine practice, holding great promise for various appli-
cations, including the early detection of HPV-attributable oropharyngeal cancer (HPV-AOC)
and potentially other anogenital cancers, as well as an adjunct prognostic marker in long-
term tumor surveillance. Anti-HPV-16 E6 antibody presence in blood may serve as a poten-
tial screening, diagnostic, and prognostic marker of HPV-AOC, but greater research in this
area is warranted. The moderate sensitivity of HPV testing in oral rinses or swabs at HPV-
AOC diagnosis prevents its use as a screening or diagnostic tool, but HPV testing in oral
rinses represents a promising prognostic tool in the tertiary prevention of HPV-AOC, where
persistence in oral rinses through and after treatment may predict early HPV-AOC recur-
rences and/or the development of secondary HPV-AOC. In addition, HPV testing of oral
rinses will continue to be important for natural history and epidemiological studies.
In conclusion, three specimen types traditionally considered “alternative” for HPV
testing (urine, blood, and oral specimens) are shown to have an increasing clinical, epi-
demiological, and scientific value (Table 1). The HPV community should work together
to identify, report, and optimize all key technical steps (from sample collection to final
result) and to make recommendations for best laboratory and clinical practice. Thus,
we can gain a more accurate insight into the true benefits of application when the
whole testing workflow is optimized and validated.
ACKNOWLEDGMENTS
M.P. is supported by the Horizon 2020 Framework Program for Research and Innovation
of the European Commission, through the RISCC Network (grant no. 847845) and by the
Slovenian Research Agency (grant no. P3-00083). A.V. is supported by the Industrial Research
Fund of the University of Antwerp (IOF-321 SBO, grant no. 44754), and the European Research
Council (URISAMP, grant no. 101040588). The authors would like to express their sincere
gratitude to Manca Krošelj for the initial design of the figures and the subsequent graphic
improvement service.

The institution to which M.P. belongs received research funding, free-of-charge

reagents, and consumables to support research in the last 3 years from Qiagen, Seegene,
Abbott, and Roche, all paid to his employer.
The institution to which K.C. belongs received research funding, reagents, and consumables
to support research in the last 3 years from Cepheid, Euroimmun, GeneFirst, Self-screen, Hiantis,
Seegene, Roche, Abbott, Hologic, and Vaccitech, all paid to her employer. K.C. also attended an
advisory board meeting of Hologic where UK-based travel expenses were supported and is
currently on the advisory board of Vaccitech (with any associated reimbursement paid to
employer).
The institution to which L.A. belongs received funding to support research in the last
3 years from Merck Sharp & Dohme, Roche, GSK, Vitro, Hologic, and Seegene, all paid to
her employer.
The institution to which A.V. belongs received funding to support research and
setting up professional meetings in the last 3 years from Merck, Roche, GSK, Hologic,
Abbott, Novosanis, and Becton, Dickinson and Company, all paid to his employer.
A.V. is the cofounder of Novosanis, a spin-off company of the University of Antwerp,
Belgium, and a subsidiary of OraSure Technologies, Inc., since 2019, and he was a board
member and minority shareholder until January 2019.
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MINIREVIEW

Testing for Human Papillomaviruses in Urine, Blood, and Oral

ABSTRACT Twelve high-risk alpha human papillomavirus (HPV) genotypes cause

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THE BURDEN OF CERVICAL AND OROPHARYNGEAL CANCER AND THE IMPORTANCE

PRINCIPLES OF HPV TESTING—CHOOSING THE RIGHT TEST FOR THE RIGHT

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HPV TESTING IN URINE

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of macroscopically visible differences between ﬁrst-void urine versus subsequent urine

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TESTING FOR HPV IN BLOOD

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TESTING FOR HPV IN ORAL SPECIMENS

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impact of storage time (including at frozen temperatures) on sample quality/target deteri-

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The institution to which M.P. belongs received research funding, free-of-charge

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