European Journal of Obstetrics & Gynecology and Reproductive Biology 216 (2017) 1–11

Contents lists available at ScienceDirect

European Journal of Obstetrics & Gynecology and

Reproductive Biology
journal homepage: www.elsevier.com/locate/ejogrb

Review article

First-void urine: A potential biomarker source for triage of high-risk

human papillomavirus infected women
Severien Van Keera , Jade Pattyna , Wiebren A.A. Tjalmab , Xaveer Van Ostadec ,
Margareta Ievend, Pierre Van Dammea , Alex Vorstersa,*
a
Centre for the Evaluation of Vaccination (CEV), Vaccine & Infectious Disease Institute (VAXINFECTIO), Faculty of Medicine and Health Sciences, University of
Antwerp, Belgium
b
Multidisciplinary Breast Clinic, Unit Gynaecologic Oncology, Department of Obstetrics and Gynaecology, Molecular Imaging, Pathology, Radiotherapy,
Oncology (MIPRO), Faculty of Medicine and Health Sciences, Antwerp University Hospital (UZA)—University of Antwerp, Belgium
c
Proteomics, Proteinscience, Proteomics & Epigenetic Signalling (PPES), Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp,
Belgium
d
Laboratory of Medical Microbiology (LMM), Vaccine & Infectious Disease Institute (VAXINFECTIO), Faculty of Medicine and Health Sciences, University of
Antwerp, Belgium

A R T I C L E I N F O A B S T R A C T

Article history:
Received 20 January 2017 Great interest has been directed towards the use of first-void urine as a liquid biopsy for high-risk human
Received in revised form 18 June 2017 papillomavirus DNA testing. Despite the high correlations established between urinary and cervical
Accepted 24 June 2017 infections, human papillomavirus testing is unable to distinguish between productive and transforming
Available online xxx high-risk infections that have the tendency to progress to cervical cancer. Thus far, investigations have
been primarily confined to the identification of biomarkers for triage of high-risk human papillomavirus-
Key words: positive women in cervicovaginal specimens and tissue biopsies. This paper reviews urinary biomarkers
Human papillomavirus for cervical cancer and triage of high-risk human papillomavirus infections and elaborates on the
Cervical cancer
opportunities and challenges that have emerged regarding the use of first-void urine as a liquid biopsy for
Biomarker
the analysis of both morphological- (conventional cytology and novel immunohistochemical techniques)
First-void urine
Self-sampling and molecular-based (HPV16/18 genotyping, host/viral gene methylation, RNA, and proteins)
biomarkers. A literature search was performed in PubMed and Web of Science for studies investigating
the use of urine as a biomarker source for cervical cancer screening. Five studies were identified reporting
on biomarkers that are still in preclinical exploratory or clinical assay development phases and on
assessments of non-invasive (urine) samples. Although large-scale validation studies are still needed, we
conclude that methylation of both host and viral genes in urine has been proven feasible for use as a
molecular cervical cancer triage and screening biomarker in phase two studies. This is especially
promising and underscores our hypothesis that human papillomavirus DNA and candidate human and
viral biomarkers are washed away with the initial, first-void urine, together with exfoliated cells, debris
and impurities that line the urethra opening. Similar to the limitations of self-collected cervicovaginal
samples, first-void urine will likely not fulfil the high-quality cellularity standards required for
morphological biomarkers. Molecular biomarkers will likely overcome this issue to yield high-
throughput, objective, and reproducible results. When using proper sampling, transport, storage,
preanalytical biomarker concentration techniques, and clinically validated assays, first-void urine is
expected to be a valuable source of molecular biomarkers for cervical cancer screening. Furthermore, as
first-void urine can be easily and non-invasively collected, it is a highly preferred technique among
women and offers the ability to test both primary high-risk human papillomavirus and biomarkers in the
same sample. In addition, the use of first-void urine confers opportunities to reduce loss-to follow-up and
non-adherence to screening subjects.
© 2017 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-
ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Corresponding author at: Campus Drie Eiken-Building R2—Universiteitsplein, 1-2610 Wilrijk, Belgium.
E-mail address: alex.vorsters@uantwerp.be (A. Vorsters).

http://dx.doi.org/10.1016/j.ejogrb.2017.06.036
0301-2115/© 2017 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).
2 S. Van Keer et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 216 (2017) 1–11

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
State of the art: human papillomavirus and cervical cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Interest in human papillomavirus analysis in first-void urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
The need for triage of human papillomavirus infected women . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Biomarkers for cervical cancer screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Urinary biomarkers for cervical cancer screening reported in the literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Opportunities and challenges of first-void urine as a biomarker source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Microscopy-based markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Molecular markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Human papillomavirus DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Host and viral gene DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
RNA-based biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Protein biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Introduction Interest in human papillomavirus analysis in first-void urine

State of the art: human papillomavirus and cervical cancer
With an estimated 527,624 new cancer cases and 265,672
deaths in 2012, cervical cancer remains a significant problem
worldwide [1]. Globally, it is the fourth most common cancer in
women with an age-standardized incidence rate of 14 cases per
100,000. Strikingly, women living in low- and middle-income
countries are most affected by this cancer due to the lack of cervical
cancer prevention measures, representing almost 90% of all
cervical cancer deaths [1]. Human papillomavirus, commonly
known as HPV, is the main aetiological factor of cervical cancer [2].
Although the majority of the population is unaware of the virus,
HPV is the most common sexually transmitted infection, affecting
nearly all sexually active people during their lifetime [3]. Non-
sexual, vertical transmission has also been reported [4]. Fortu-
nately, over 90% of all HPV infections are spontaneously cleared
within two years of acquisition [5]. To date, 200 genotypes of this
virus have been identified, from which at least 40 affect the
anogenital mucosa [6,7]. According to the likelihood of genotypes
to develop cervical cancer, two groups can be distinguished: low-
risk (LR) and high-risk (HR) HPV genotypes. LR-HPV genotypes
mainly cause genital warts, whereas the thirteen genotypes
classified as (probable) HR-HPV
with HPV16 and 18 causing
70% of all cervical cancer cases
can lead to precancerous lesions,
and if left untreated, cervical cancer [8]. Screening based on
primary HR-HPV DNA detection is currently of great interest,
which is more effective and efficient for preventing cervical cancer
and the associated mortality compared to cytology [9,10]. This was
demonstrated based on the higher sensitivity for detecting high-
grade cervical intraepithelial neoplasia (CIN2+) and the lower
cumulative incidence of cervical cancer five years after a negative
HPV test compared to three years after a normal cytology result
[9,11,12]. Nonetheless, challenges remain at the sampling level
regarding better up-take in screening programmes and less
expensive options for follow-up. Additionally, prophylactic HPV
vaccinations will result in a lower prevalence of HR-HPV types [13],
ensuing a drop in the prevalence of true-positive high-grade
precancerous lesions (CIN2+). The consequential decrease in the
‘signal-to-noise’ ratio will thus continue to adversely affect the
sensitivity, specificity, and positive predictive value (PPV) of
cytology to a point when it will no longer be suitable for primary
cervical cancer screening [14].
Recently, great interest has been directed towards the use of
urine as a liquid biopsy for HPV DNA testing because of the high
correlations established between urinary HPV DNA and cervical
infections, the ease of sampling, the lower barrier for women, and
the lower cost of the programme [15–26]. As Papanikolaou
previously indicated (1943), superficial cell layers of a cervix
carcinoma are exfoliated and subsequently mixed with secretions
of the uterus and cervix, which make their way to the vagina and
hence can be recognized in vaginal fluid smears [27]. The theory
behind identifying biomarkers including HPV DNA for cervical
(pre)cancer screening in first-void urine is based on this idea [28],
because we are not interested in the urine itself but instead in the
mucus and debris from exfoliated cells from the genital tract
(particularly the cervix) that line the urethra opening and small
labia and are washed away with the initial urine flow. It hence
follows that the initial flow of urine (first-void) collects most of
this debris, without interfering with the natural history of an HPV
infection by scraping of cervical cells. This finding explains why
the first collected portion of a urine void contains more human
and HPV DNA than the subsequent portion, as was concluded by
the authors of a recent meta-analysis [19]. Our research confirms
these findings, indicating that first-void urine contains signifi-
cantly more HR-HPV (4.8–160 times) and human DNA than the
subsequent fraction [28].
HPV testing of self-collected cervicovaginal samples can be
offered as an alternative strategy for women not attending cervical
cancer screening programmes [29,30] and has been approved for
use in organized, population-based pilot programmes [11].
Recently, it has also been proposed as a primary cervical cancer
screening tool due to its high relative sensitivity and specificity for
CIN2+ compared to clinician-collected samples [31]. Population-
based approaches, such as sending personal invitation letters and
reminders for a scheduled appointment, have proven to be
effective in increasing participation rates [11,32], although barriers
for attending cervical cancer screening have persevered. These
barriers can be diverse, including practical (e.g., lack of time,
intending to go but not getting around to it), emotional (e.g., fear of
pain, embarrassment, fear of the test results/cancer), and cognitive
(e.g., low perceived risk, absence of symptoms) barriers [33,34].
Despite the fact that embarrassment was the most mentioned
barrier, practical barriers such as inconvenience of making an
appointment and not getting around to going for screening, were at
higher odds in a cohort of women overdue for screening, and
 S. Van Keer et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 216 (2017) 1–11 3

therefore, were more predictive of non-attendance to screening
[33]. Both cervicovaginal and urine self-sampling have been
proven effective in increasing participation and screening coverage
of target populations [35–38]. However, compared to the currently
available cervicovaginal self-sampling methods and clinician-
collected smears, (first-void) urine collection is highly preferred
among women [39,40]. First-void urine furthermore allows for
easily repetitive sampling if needed.
The need for triage of human papillomavirus infected women
Despite the high sensitivity of HR-HPV DNA testing, it remains
challenging to identify clinically relevant, i.e., transforming, HR-
HPV infections as only approximately 4% of persistent HR-HPV
infections progress to high-grade lesions [41], among which
approximately 30% can progress to cancer if left untreated [42].
Transforming HPV infections differ from productive infections in
the way that the normal viral life cycle of HPV is halted, followed by
overexpression of the viral oncogenes E6 and E7, which leads to
chromosome instability and subsequent accumulation of
aberrations in host cell genes over time. These aberrations can
be caused by both genetic (e.g., DNA mutations and chromosomal
copy number alterations) and epigenetic alterations (e.g., DNA
methylation and histone modifications) [43]. To avoid over-referral
and over-treatment of women with clinically irrelevant HR-HPV
infections and the associated costs and potential harms caused by
the current treatment methods, there is a need for additional triage
tests that can distinguish productive from transforming HR-HPV
infections. Interesting candidate biomarkers for triage of HR-HPV-
positive women
hereafter referred to as ‘biomarkers’
has been
a topic of great interest, and many potential biomarkers have been
identified to date.
Biomarkers for cervical cancer screening
A biomarker is “a defined characteristic that is measured as an
indicator of normal biological processes, pathogenic processes, or
responses to an exposure or intervention, including therapeutic
interventions” [44]. Ideally, a biomarker should be detected at a
stage early enough to foresee successful treatment and only in the

Fig. 1. Hypothetical one- and two-step triage strategy offering first-void urine self-sampling at home as an alternative to screen non-attendees. Five-yearly primary HR-HPV
testing has been proposed by the European guidelines for quality assurance for cervical cancer screening in organized, population-based cervical cancer screening
programmes [11]. However, it has also been proposed for self-collected cervicovaginal samples for use in organized, population-based pilot programmes for women who do
not attend regular cancer screening programmes despite receiving personal reminders. Nonetheless, due to a lower HPV testing accuracy for use of self-collected versus
clinician-collected samples, one should still be aware that primary HR-HPV testing of self-collected samples is not recommended for national implementation before
successful results have been demonstrated by a pilot study. Currently, a triage of HR-HPV-positive test results is recommended by cytology, followed by repeat testing or
colposcopy [11]. Both microscopy- and molecular-based biomarkers for triage of HR-HPV-positive women were recently reviewed by Ebisch [51] and Luttmer [52]. Discussing
the opportunities and challenges of these biomarkers as urinary biomarker is the main objective of this review. A one-step triage method in which both HR-HPV and
biomarkers can be analysed in the same sample is preferred, as such a method would confer higher compliance to follow-up compared to two-step triage options. HPV:
human papillomavirus.
4 S. Van Keer et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 216 (2017) 1–11

presence of cervical precancerous lesions that will progress to
cervical cancer (CIN2+) [45,46]. Notably, CIN2 is an intermediate,
more regressive condition including both productive and trans-
forming infections
compared to the more histological reproduc-
ible CIN3 diagnosis [47]. The overall impact of a CIN2+ screening
test may therefore be overstated, and hence, reporting CIN2+ and
CIN3+ lesions separately may be more efficient [46]. For a
biomarker to be acceptable, a PPV and negative predictive value
(NPV) of at least 20% and 98%, respectively, should be obtained
[48,49]. Unfortunately, for cervical cancer screening, none of the
biomarkers identified to date offer sufficient prognostic value, and
as such, none serve as an ideal triage method [50–52]. It is
furthermore unlikely that a single biomarker will provide
sufficient sensitivity to detect all high-grade lesions and cancers
(CIN2+) [45,53]. Therefore, a panel of biomarkers is expected to be
necessary for accurate testing with good clinical sensitivity and
specificity. This panel would preferably consist of biomarkers that
are involved in cervical carcinogenesis and that complement each
other concerning, for instance, functionality such that different
aspects of the cancer are targeted for its detection. For example, the
biomarkers could be molecules from different cervical cancer
pathways (cancer- versus immune-related molecules) and from
different biomarker classes (e.g., proteins, (methylated) nucleic
acids).
Recent papers have reviewed the state-of-the-art of triage
markers for HR-HPV infections in cervical samples [14,50–52,54].
Briefly, these biomarkers can be divided in two major groups: (i)
morphological- or microscopy-based biomarkers and (ii) molecu-
lar biomarkers. Conventional cytology, novel immunohistochemi-
cal techniques such as p16INK4a staining and Ki-67/p16 dual stained
cytology, and chromosomal aberrations can be classified under
morphological biomarkers [50–52]. Examples of promising mo-
lecular biomarkers are HPV16/18 genotyping, methylation of host
and viral genes, HPV E6 protein, and E6/E7 mRNA-, and miRNA-
based biomarkers [14,51,52]. Viral load and integration of HPV DNA
into host DNA have been suggested as markers to predict disease
outcome [55,56] but further investigations are needed.
As already mentioned above, none of the biomarkers reported
in the literature to-date meet the criteria as an ideal cervical cancer
screening biomarker for detecting clinically relevant precancerous
lesions. The only available triage strategy that has been sufficiently
evaluated for HR-HPV-positive women, without repeat testing, is
HPV16/18 genotyping in combination with cytology [51,52].
Genotyping of HPV16/18 has all the benefits of HPV DNA-based,
molecular testing, although high colposcopy referrals will endure.
Adding cytology to HPV16/18 genotyping will increase the clinical
value of this triage strategy, resulting in an improved NPV,
although at the cost of the need for a clinician-taken sample for
cytology [51,52]. Their wide evaluation as a triage method makes
them a logical first choice as biomarkers in coming years. However,
with more extensive knowledge on molecular markers from large
population-based prospective studies, cervical cancer screening
may transform to full molecular screening in the future [51].
Thus far, investigations have been primarily confined to the
identification of biomarkers in cervicovaginal specimens and
tissue biopsies. Based on the higher sensitivity of HPV DNA
detection in well-preserved, first-void urine compared to mid-
stream or random urine samples for the presence of cervical HPV
[19,26,40,57], it is expected that biomarkers might also be present
in the first part of the urine void. To the best of our knowledge, no

Fig. 2. Flow chart of the literature search: urinary biomarkers for cervical cancer screening. The search yielded seven and ten hits in PubMed imported into Endnote X7 (‘All
Fields Contain’) and Web Of Science within Endnote X7 (‘Title/Keywords/Abstracts’), respectively. The retrieved records were first screened based on the title and abstract,
followed by full text evaluation. Publications were included that reported the performance of biomarkers (other than HPV itself) analysed in human urine for cervical cancer
screening or, more specifically, triage of high-risk HPV-positive women. After screening for eligibility and addition of manually selected publications, 4 papers and 1 abstract
were included, published between 2003 and 2016. The flow chart was adapted from the PRISMA flow diagram [63].
 S. Van Keer et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 216 (2017) 1–11 5

one has reflected on the opportunities and challenges that have
emerged regarding the use of first-void urine as a liquid biopsy for
triage of HR-HPV-positive women. This paper reviews urinary
biomarkers for cervical cancer screening or for triage of HR-HPV-
positive women and reflects emerging challenges with first-void
urine as a liquid biopsy for analysis of triage biomarkers for HR-
HPV-positive women based on microscopy and molecular assays.
Detection of both HR-HPV DNA and biomarkers in the same, self-
collected first-void urine sample will be an additional asset to
minimize efforts and costs associated with cervical cancer
screening. First-void urine self-sampling will furthermore encour-
age women who are currently reluctant to participate in screening

Table 1
Overview of candidate biomarkers in urine for cervical cancer diagnosis reported in the literature.

Candidate biomarker Phase in Study group Colposcopy/ Collection (i) Pre-analytical urine Outcome Refs
(class) biomarker (n = ) biopsy confirmed method; and processing; and (ii) analysis
a
evaluation (n = ) preanalytical of candidate biomarker
storage
conditions
(time span)
Ratio 16a-hydroxy Phase 1 Case-control No, Pap smear and Early (i) 2/3 ml urine (spiked withNo report of sensitivity, Lee SH
estrone (16a-OH E1)/ study (n = 61) cervicogram; morning internal control) applied to a
specificity, PPV, NPV, or AUC [58]
2-hydroxy estrone (2- cervical disease urine in Serdolit XAD-2 resin column 16a-OH E1/2-OH E1 and
OH E1 (estrogen); ratio (n = 18/61), polyethylene (ii) Gas chromatography- THF/5a-THF proposed as a
5b-tetrahydro-cortisol cervical cancer bottles; mass spectrometry-selected dual biomarker panel for
(THF)/5a-THF (n = 18/61), stored at ion-monitoring (GC/MS/ cervical cancer screening due
(androgen/corticoid) controls (n = 25/
20  C (NA) SIM), normalization to to a significant difference in
(enzyme activity) 61) creatinine concentration concentration between
controls and benign cervical
disease versus cervical
cancers
Collagen I-alpha-I; Phase 1 Case-control Yes, biopsy Spot urine (i) Centrifugation (1,500g, No report of sensitivity, Garbett

collagen XVII-alpha-I study (n = 12) confirmed; CIN2 samples; 15 min, 4 C) ! Amicon specificity, PPV, NPV, or AUC NC [59]
(protein class) (n = 4); cervical stored in Ultra-4 (10,000 NMWCO) of Collagen I-alpha-I increased
cancer (n = 4); aliquots at supernatant ! desalting and in CIN2 > cervical cancer >
controls (n = 4)
80  C (NA) concentration using C18 controls; collagen XVII-
ZipTips alpha-I increased in CIN2 >
(ii) MALDI-TOF(/TOF) MS cervical cancer and is absent
in controls
DAPK1; RARB; TWIST1; Phase 2 Subgroup of Yes, biopsy NA; stored at (i) 5 ml urine HR-HPV detection or Feng QH
and CDH13 women from a confirmed 4 C centrifuged ! cell pellets aberrant methylation of at [61]
(host gene methylation) study (n = 110/129); (processed resuspended in least 1/4 genes: sensitivity of
investigating CIN0 (n = 19), CIN1 within STM ! stored at 84% (ICC) and 64% (CIN2/3),
HPV and CIN (n = 9), CIN2-3/CIS 24 hours)
80  C ! Proteinase K with a specificity of 72% (as
prevalence in (n = 29), ICC digestion (37  C; 1– to CIN0/1) and 81% (as to
West-Africa (n = 72) 2 h) ! DNA isolation of CIN0)
(n = 129) 400 ml urine digest (QIAamp High concordance of HR-HPV
Blood Minicolumn) ! DNA detection or aberrant
concentration to 25 ml methylation of at least 1/4
(Speedvac; RT) genes between paired urine
(ii) Bisulfite conversion of and cervical samples (49–
50–100 ng DNA and 80%) (n = 97/110)
MethyLight (qMSP) analysis
of DAPK1; RARB; TWIST1;
CDH13; U-DNA; M-DNA; and
ACTB
ZNF516; FKBP6; INTS1; Phase 2 Prospective No, paired LBC, NA; flash (i) ccfDNA from 10 ml urine The 4-gene biomarker panel Guerrero-
and HPV16-L1 cohort (n = 40) plasma, and frozen (NA) adsorbed on Q-Sepharose in urinary ccfDNA has Preston R
(host and viral gene serum samples anion-exchange resin, sensitivity of 75%, specificity [62]
methylation) followed by silica-based of 83.3%, PPV of 50%, NPV of
elution with LiCl 93.8%, and AUC of 0.86 for
(ii) Bisulfite conversion and CIN2+ as to NILM/CIN1.
qMSP analysis for ZNF516; Compared to LBC the 4-gene
FKBP6; INTS1; and HPV16-L1. panel has a lower sensitivity,
A target enrichment system better specificity, and similar
was used to sequence the NPV, PPV, and AUC.
HR-HPV epigenome in urine
NA Phase 1 Case-control NA NA Paired cervical scrapes and High accuracy for detecting Rurup R
(host gene methylation) study (n = 53); urine samples were tested cervical cancer (AUC > 0.86 [60]
cervical cancer for HR-HPV DNA and DNA for 4/6 genes)
(n = 29/53), methylation of 6 genes High correlation of DNA
controls (n = 24/ methylation levels between
53) paired urine and cervical
samples (Spearman
correlation = 0.83)
a
: Phase 1: preclinical exploratory studies; Phase 2: clinical assay development for clinical disease and assessment in non-invasive samples; Phase 3: retrospective
longitudinal repository studies; Phase 4: prospective screening studies; Phase 5: prospective intervention study; LBC: liquid-based cytology; NILM: negative for
intraepithelial lesion and malignancy; CIN1-3+: Cervical Intraepithelial Neoplasia grade 1–3+; CIN0: no CIN; CIS: carcinoma in situ; ICC: invasive cervical carcinoma; PPV:
positive predictive value; NPV: negative predictive value; AUC: area under the curve; STM: specimen transport media (Digene); RT: room temperature; ACTB: methylated
b-actin reference gene; U-DNA: unmethylated human sperm DNA control; M-DNA: methylated human sperm DNA control; ccfDNA: circulating cell-free DNA; qMSP:
quantitative methylation-specific PCR; NA: not available.
6 S. Van Keer et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 216 (2017) 1–11

programmes and may increase their compliance for further follow-
up if they are HR-HPV positive (Fig. 1) with minimal efforts by the
screened women.
Urinary biomarkers for cervical cancer screening reported in
the literature
A review of the literature on (bio)markers for cervical cancer
and HPV in urine was performed up to January 3rd 2017 in the two
main databases for health sciences: PubMed (‘All Fields Contain’)
and Web Of Science (‘Title/Keywords/Abstracts’) (Fig. 2). In each
database, the following search terms were used: marker* OR
biomarker*; AND HPV*, human papillomavirus*, human papilloma
virus*, cervical cancer(s), cervical carcinoma*, OR cancer(s) of the
cervix; AND urine. Four papers and one abstract were included
(Table 1). To promote efficiency and consistency in cancer
screening biomarker research, a five-step framework was set up
by the Early Detection Research Network for evaluating the
potential use of a biomarker in cancer screening [45]. Two of the
candidate biomarkers proposed in the articles of our search
reached stage two of the biomarker evaluation, i.e., clinical assay
development [58–60], while the other three only achieved stage
one, i.e., the preclinical exploratory phase [45,46,61,62]. Retro-
spective and large-population-based prospective studies are
required to validate the proposed urinary but also cervicovaginal
sample-based biomarkers, as only few candidate biomarkers have
reached phase three or beyond. None of the studies used first-void
urine samples or a urine conservation medium, or they did not
report such use. For DNA-based biomarkers, use of first-void urine
and immediate mixing with a urine conservation medium are
essential for high biomarker recovery, as is the use of whole urine
for analysis instead of pellet or supernatants [19,26,28,57]. In
contrast, either pellet or cell-free DNA was used in two studies that
investigated DNA methylation biomarkers in urine [61,62]. It must
be further investigated whether using whole urine results in
higher methylation biomarker yields, as is the case for human and
HPV DNA. Searches for patents (worldwide.espacenet.com) and
clinical trials (www.clinicaltrials.gov) using the same search terms
as used for the health science databases yielded 10 and 34 hits,
respectively. Two of the 10 clinical trials actually investigated
biomarkers for cervical (pre)cancer in urine samples
(NCT02714127 and NCT00290459), but no results were available
so far. The patent search resulted in 2 out of 34 hits describing a
urine detection kit for cervical cancer screening (CN104714030 and
CN104198404). Only summaries of the patents were available in
English, providing few details but indicating that the technologies
are based on changes in urine colour and colorimetry.
Opportunities and challenges of first-void urine as a biomarker
source
In the following sections, evidence is discussed that non-
invasive first-void urine self-sampling for cervical cancer screening
is promising and could serve as an alternative method to screen
and triage non-attendees in the future.
Microscopy-based markers
Cytological techniques have a 3–4% higher specificity for
clinically relevant infections compared to primary HR-HPV testing
[12], reducing the number of colposcopy referrals and follow-up
tests. Novel immunohistochemical techniques (e.g., p16INK4a, Ki-
67/p16) have proven to be more sensitive for detecting CIN3+
lesions for triage of HR-HPV-positive women compared to
conventional cytology, without a substantial increase in colposco-
py referrals. Nevertheless, they are not able to distinguish between
productive and transforming HR-HPV infections. Chromosomal
aberrations, and more specifically, loss at 3p and 11q and gains at
3q, have been found to be most frequently altered in cervical
carcinoma [64]; however, they have not been evaluated in large
clinical cohorts of HR-HPV-positive women [51,52].
In our search for candidate biomarkers in urine, two
publications reported the use of urine for analysing morphological
markers for urothelial carcinoma (cancer of the bladder, ureters,

Table 2
Summary of the benefits and drawbacks of microscopy-based/morphological and molecular biomarkers with the aim to use self-collected first-void urine samples for triage
of HR-HPV-positive women.

Microscopy-based/morphological biomarkers
Benefits
Specificity, reduction in colposcopy referrals
Widely evaluated as a triage method
Drawbacks
Sensitivity; necessitates repeat testing; can result in loss to follow-up
Subjective (e.g., affected by prior knowledge of HPV status)
Need for trained cytotechnologist/prone to human error
Suboptimal reproducibility
Suboptimal high-throughput capacity
Not reliable in self-collected samplesa due to need of high quality, cell rich material; necessitates extra clinician-collected cervical sample
Two-step triage; can result in drop out

Molecular biomarkers
Benefits
Objective
Less prone to human error
Reproducibility
High-throughput capacity
Multiplexing format enables small sample volumes and less hands-on time
Possible on the same, self-collecteda (first-void urine) sample
Allows one-step triage; decreases drop out and loss to follow-up

Drawbacks
Necessitates large, population-based retro-, and prospective cohort studies (phase 3–5)b in self-collected (first-void urine) samples
a
Self-sampling yields opportunities to increase screening attendance rates, testing of both HR-HPV DNA and molecular biomarkers in the same sample, and hence
minimizing loss of follow-up.
b
Phase 3: retrospective longitudinal repository studies; Phase 4: prospective screening studies; Phase 5: prospective intervention study [45].
 S. Van Keer et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 216 (2017) 1–11 7

urethra, and renal pelvis) [65,66]. Similar to cervical cytology,
urinary cytology for bladder cancer has a high specificity (ca. 94%)
but is hampered by a low sensitivity (ca. 35%) [67]. Nonetheless,
microscopy-based techniques identifying morphological markers
require cell-rich sample material, which cannot be assured in self-
collected cervicovaginal samples. The available cervical cells are
often of poor morphological quality and diluted due to the large
amount of vaginal cells present in the sample, resulting in a lower
sensitivity [43,51,52]. In addition to this concentration issue,
reproducibility and high-throughput capacity also limit the
applicability of morphological markers as triage markers for HR-
HPV-positive women due to the subjective nature of the method
and need for a trained cytotechnologist, respectively (Table 2).
Molecular-based triage methods, on the other hand, are objective,
highly reproducible, suitable for high-throughput testing, and offer
the opportunity to use self-collected cervicovaginal material
(Table 2).
Molecular markers
Human papillomavirus DNA
The use of first-void urine as a liquid biopsy for HPV DNA testing
is promising. In a recent meta-analysis, the sensitivity and
specificity for detecting any HPV in urine were 87% (95% CI: 78–
92%) and 94% (82–98%), respectively, when results from cervical
samples were used as a reference [19]. However, because of a large
heterogeneity in urine collection and testing methods, the pooled
estimates in this meta-analysis could be incorrectly estimated,
emphasizing the need for standardized methods for human and
HPV DNA detection in urine samples. The use of random or
midstream instead of first-void urine has been identified as a
source of heterogeneity [22,26,40,57,68], resulting in a 22-fold
decrease in accuracy for detecting the presence of cervical HPV
[19]. Ultrafiltration and subsequent DNA extraction of unfractio-
nated, DNA preserved, first-void urine has been proven to yield a
greater amount of HPV DNA-positive samples than paired cervical
samples, using analytically sensitive HPV DNA assays
[16,20,57,69,70]. Based on these data and the literature reported
to date, we propose that optimized HPV DNA detection in urine
should include the following: (i) use of first-void urine captured
with a first-void urine collection device to secure a standardized
urine fraction; (ii) prevention of human/HPV DNA degradation
during extraction and storage by immediately mixing the first-void
urine with a conservation medium that is harmless to humans and
is preferentially prefilled in the vial used for the first-void urine
collection; (iii) processing of a sufficient first-void urine volume;
(iv) recovery of cell-free and cell-associated HPV DNA by
processing whole first-void urine as opposed to only the pellet
or supernatants [26,28,57]; and (v) the use of HPV assays that fulfil
the criteria for primary cervical cancer screening [10], with
optimized assay thresholds for first-void urine (summarized in
Table 3). As we are interested in human and HPV DNA originating
from the cervix, we expect that a larger time interval between two
urinations will positively affect the amount of markers present in
the mucus and cervicovaginal secretions that line the urethra
opening and small labia and are subsequently captured in the first
urine void. However, recent studies indicate no advantage of
testing first-void urine samples taken in the morning versus
samples collected later that day using unfractionated urine
[40,68,71]. The use of human DNA as a first-void urine sample
validity control should be interpreted with caution, since we
observed more rapid degradation of HPV than human DNA [57],
possibly resulting in an underreported HPV prevalence. Therefore,
preference is given to the addition of control DNA to the first-void
urine sample immediately upon collection.
Care should be taken when interpreting urinary HPV DNA
results for primary screening purposes [21]. The clinical validity of
HPV testing using urine samples should be investigated under
optimal conditions. An inferior HPV prevalence was found in the
PaVDaG study where random void urine samples and non-
optimized assay thresholds were used [31]. This is in contrast to
other studies where optimized urine collection, storage, sample
preparation, DNA extraction and amplification methods were used
[16,18,40]. These data make evident that, to-date, less attention has
been paid to optimal urine sampling, storage, and sample
preparation methods compared to DNA extraction and amplifica-
tion methods. These optimizations are, however, of utmost
importance in order to not underestimate infection rates and to
obtain reproducible results in the context of primary HR-HPV-
based screening but also for triage of HR-HPV-positive women
using HPV16/18 genotyping.
Host and viral gene DNA methylation
An important asset of host cell DNA methylation biomarkers is
their ability to distinguish productive from transforming HR-HPV
infections, especially those with a high short-term risk of
progression to cancer. Methylation levels have also been found
to rise in parallel with increased severity and duration of disease
[43,52]. Hereto, host cell DNA methylation has also been proposed
as a potential biomarker for post-treatment follow-up as it enables
differentiation between long-existing and new transforming
lesions [43]. A small set of methylation panels have been evaluated
in self-collected lavage and/or brush-based samples, but only one,
the multiplex qMSP assay based on the bi-marker panel FAM19A4
(Family with sequence similarity 19 (chemokine (C-C motif)-like)
member A4)/mir124-2 (microRNA124-2) has been clinically validat-
ed in a large cohort of HPV-positive women. This assay yields
similar sensitivity and specificity for CIN3+ in both lavage and
brush-based self-collected samples and clinician-taken cervical
smears [72].
In contrast to methylation of human gene promotors resulting
in decreased transcription of tumour suppressor genes due to
hypermethylation, methylation of viral DNA can result in both
increased and decreased viral gene transcription. The exact
mechanism and how viral DNA methylation occurs remains
unclear, but it has been proposed that it is a host defence

Table 3
Key notes for human and HPV DNA detection in urine [10,26,28,57].

Optimized human and HPV DNA detection in urine should include:

- Use of first-void urine; preferentially captured with a first-void urine collection device to secure a standardized urine fraction
- No extensive washing of genitals before urination, as well as collecting the first-void urine sample preceding collection of other cervicovaginal self-samples or clinician-
taken specimens because this could reduce the amount of human/HPV DNA originating from the (exfoliated) cells from the cervix
- Prevention of human/HPV DNA degradation during storage and extraction by immediately mixing the first-void urine with a conservation medium; preferentially
prefilled in the collection vial
- Processing a sufficient amount of first-void urine
- Recovery of both cell-free and cell-associated DNA by processing whole first-void urine rather than only the supernatant or pellet
- Use of validated assays with optimized assay thresholds; for primary HR-HPV screening preferentially HPV assays that fulfil the criteria for primary cervical cancer
screening
8 S. Van Keer et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 216 (2017) 1–11
mechanism to silence viral replication and transcription [43,73].
To-date, only specific CpGs in the late genes L1 and L2 of HPV16, 18,
31, 33, and 45 have shown consistently higher methylation levels
with viral persistence and cell immortalization, increasing the
degree of disease, and may be useful for differentiating trans-
forming from productive infections after sufficiently validated
[50–52,73,74].
Methylation of host and viral genes as urinary biomarkers
include the opportunity for a fully molecular and easily automated
assay using both clinician- and self-collected samples (Table 2).
However, in contrast to host gene methylation, a unique assay
needs to be developed and validated for each HR-HPV type due to
the genetic variation between HPV genotypes. Numerous methyl-
ation markers have been proposed as candidate biomarkers, but
the greatest need is to validate the existing markers in the near
future [73].
In theory, DNA methylation analysis is possible for all biological
sample types containing DNA [73,75,76]. DNA methylation assays
for human and viral genes, however, require high (quality) DNA
content. Therefore, the concentrations of both human and HPV
DNA in first-void urine are expected to represent an important step
in the analysis of methylation biomarkers for cervical precancerous
lesions. As such, similar optimization issues as for human and HPV
DNA detection/genotyping purposes are expected to apply here
(Table 3). By multiplexing for a reference gene and different target
genes in a single PCR reaction, the required amount of sample DNA
is further reduced with improved quality control [75]. Multiplex
quantitative methylation-specific PCR (qMSP) is indeed an
attractive assay that enables the detection of specific DNA
methylation levels in samples containing a limited amount of
methylated DNA in a background of abundant cellular DNA [76,77].
As this is important for self-collected brush- and lavage-based
cervicovaginal samples [75], it is expected to be important for first-
void urine.
When considering DNA methylation markers for other diseases
such as prostate and bladder cancer, urine is a good source of
exfoliated cells from the genitourinary tract [76]. However,
exfoliation of tumour cells depends on tumour characteristics
(e.g., size, stage, grade) and varies among patients, with non-
invasive (tumour) lesions shedding less tumour cells compared to
invasive lesions [77,78]. The same phenomenon is seen in cervical
(pre)cancerous lesions, with better correlations in HPV DNA
presence between urine and cervical samples in more pronounced
cervical lesions [57,79]. The limited amount of DNA in urine indeed
seems challenging upon detection of methylated DNA. Andersson
and colleagues optimized pre-analytic urine processing, demon-
strating that filtration of urine through an 8-mm polycarbonate
filter increases the amount of tumour cells and (potentially
methylated) DNA retained on the filter and, hence, increases the
sensitivity and robustness of the test [78]. For human/HPV DNA,
we have also shown that ultrafiltration of first-void urine increases
the amount of DNA detected in the sample [57]. In contrast,
Guerrero-Preston reported a protocol to extract circulating cell-
free (trans-renal) DNA from urine for the detection of host and viral
methylated genes for cervical cancer diagnosis [62].
RNA-based biomarkers
In contrast to DNA-based methods, in which the absence or
presence of the HPV genome is detected, RNA-based assays are
able to detect changes or differences in gene expression profiles
related to cancer development. Messenger RNA (mRNA) plays an
important role in protein synthesis, whereas its function is
regulated by small non-coding microRNAs (miRNA) by controlling
mRNA stability and its translation into proteins. MiR-20a and miR-
21 have been consistently upregulated, and miR-143, miR-03, and
miR-145 consistently downregulated in studies but have not been
validated in large prospective cohorts [43,51]. This is in contrast to
E6/E7 mRNA, which has been extensively studied, the over-
expression of which has been associated with cellular transforma-
tion and increased disease severity [80]. One assay targeting E6/E7
mRNA from 14 (HR-)HPV types (APTIMA HPV assay, Hologic) has
been fully validated against a standard comparator test according
to the Meijer protocol, showing a similar pooled sensitivity and
superior specificity for CIN2+ in a primary screening setting.
However, these guidelines were developed for HPV DNA-based
assays, and long-term data are not yet available to determine the
risk of CIN3+ after a negative APTIMA HPV test [10].
Liquid biopsy samples have been found to harbour both mRNA
and miRNA [81,82]. Specifically, for urine, both mRNA- and miRNA-
based biomarkers have been described, such as for bladder and
prostate cancer [82–85]. Zika virus RNA is detected at higher levels
in urine compared to blood, and a quantitative reverse transcrip-
tion-PCR (qRT-PCR) assay has been approved by the FDA for this
use. Similar to HPV, Zika virus is sexually transmitted and has been
proposed to actively replicate in the genitourinary tract [86].
Recently, a urinary mRNA biomarker panel for the detection of
bladder cancer confirmed the potential of urine as a liquid biopsy
source for urothelial cell gene expression signatures [84].
Optimization methods similar to those used for DNA (Table 3)
are expected to apply to RNA-based biomarkers as well; for
instance, mRNA has also been shown to degrade due to the
presence of nucleases in urine samples. On the other hand, miRNA
has a high degree of stability in urine [82]. Mixing of the urine with
a buffer containing a chelating agent
inactivating nucleases

might therefore increase the stability of mRNA in urine. The
question regarding whether mRNA and miRNA should be
measured in whole urine, the cell pellet or the supernatant should
be further investigated. For the urinary biomarkers reported to-
date, cell pellets have primarily been investigated for miRNA, and it
was recommended by van de Vrie and colleagues to use midstream
urine when investigating biomarkers for kidney pathology due to
contamination of cells from the lower urinary tract in the first-void
[85]. Because we are interested in biomarkers originating from the
cervix, it is expected that first-void urine rather than midstream
urine will contain more mRNA and miRNA biomarkers associated
with cervical cancer development.
An additional path of growing interest is biomarker detection in
exosomes. These are small membrane vesicles (maximal 100 nm
diameter) secreted by most cell types into the bloodstream, milk,
and female and male urogenital tract body fluids, including
cervicovaginal specimens and urine. Exosomes have the advantage
of being highly stable in bodily fluids, containing high concen-
trations of biological molecules (DNA, proteins/peptides, mRNA,
miRNA, and lipids) protected by their lipid bilayer membrane. In
addition, their ability as protein and RNA biomarker sources has
been described for bladder, renal cell, and prostate cancer [87,88].
Protein biomarkers
Most of the prominent proteomic studies on cervical cancer
have been performed using cell lines, plasma/serum, and cervical
tissue samples. However, a low concordance between studies
exists, and to-date, none of the proposed protein biomarkers have
been sufficiently validated for clinical use. Elevated levels of E6/E7
proteins represent an attractive, disease-specific viral biomarker,
as they are the main mediators of HR-HPV-induced malignant
transformation and, hence, high-grade CIN. A lateral flow-based
assay for detecting E6 protein of HPV16, 18 and 45 has been
developed (OncoE6, Arbor Vita Corporation) and exhibits high
specificity for CIN3+ using clinician-collected cervical samples
from HR-HPV-positive women (93.8% (95% CI: 92.1–95.2%)).
However the sensitivity is limited (54.2% (95% CI: 43.7–64.4%))
[89] possibly because only three HPV genotypes are included. To
 S. Van Keer et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 216 (2017) 1–11
the best of our knowledge, the assay has not been validated using
self-collected samples.
Urine is a suitable material for the identification of protein
biomarkers due its relatively stable composition of proteins and
peptides and its non-invasive, easy method of collection in large
sample volumes [88]. It contains approximately 2000 proteins
originating from different sites [88], i.e., glomerular ultrafiltration
of plasma (systemic), excretion from epithelial cells in the urinary
tract, and shedding of exfoliated epithelial cells from the cervix and
vagina (local) [17]. However, challenges exist regarding urine
processing, including the variability within and between urine
samples of subjects, making it difficult to distinguish disease
biomarkers from normal bio-physiological variations [90–93].
Furthermore, the typical low protein concentration (factor 1000
less compared to, e.g., plasma) and the high concentrations of salts
and other contaminants [88,94] demands additional purification
and concentration steps. Thus far, a standardized protocol for first-
void urine collection and processing upon conservation of proteins
is lacking. A tentative standard protocol has been described for
urine collection and storage (HKUPP; Human Kidney & Urine
Proteome Project; http://www.hkupp.org/), although it is not
based on first-void urine containing cervicovaginal material.
Controversy exists regarding which urine fraction has the least
variable protein concentrations among single sample collections
(e.g., first/second urine sample of the day, random urine) [94,95].
Nevertheless, preanalytical preparation methods are expected to
affect peptide abundances more than biological variations
between pools [94]. For cervicovaginal specimen-derived proteins,
a ratio of the biomarker of interest over total protein concentration
has been proposed for normalization [96]. Despite the proven
necessity of chelating agents to avoid cell-free DNA degradation by
nucleases, the need for protease inhibitors for the proper storage of
urine specimens remains uncertain. In contrast to blood, where
proteases are activated upon collection, proteolytic degradation by
proteases in urine may be already completed by the time of voiding
because urine stagnates for hours in the bladder [90]. Studies have
reported that the absence of protease inhibitors in properly
handled urine samples (stored at
80  C without repeated freeze-
thaws) was not related to differences in the number or identity of
highly abundant proteins for a variable number of years [97,98].
However, we are not interested in proteins excreted in urine but
rather proteins excreted in cervicovaginal material that enter the
first-void urine at the time of voiding. The addition of protease
inhibitors will therefore likely be a requisite for optimal recovery of
the protein biomarkers of interest, especially when the sample is
self-collected at home and transported to the laboratory at room
temperature. Urine preparation methods include, e.g., organic
solvent precipitation, ultrafiltration, ultracentrifugation, dialysis
and lyophilisation, solid phase extraction, and complete evapora-
tion [93,95,97–99], with organic solvent precipitation yielding the
highest recovery rates. Depletion of high abundance proteins could
also increase the concentration of low abundance protein
biomarkers [88] but implicates the risk of removing biomarkers
that are associated with the depleted proteins (e.g., albumin).
Finally, similar to other biomarkers, a panel instead of a single
protein marker will likely increase the accuracy of the assay. Here,
the use of multiplex immunoassays would decrease the required
sample volume and increase the reproducibility in a less expensive,
high-throughput format [100].
Conclusion
There is growing interest in identifying biomarkers from non-
invasive, self-collected samples, which might address both the
non-adherence to screening issue and distinguishing clinically
relevant, transforming HR-HPV infections from productive
9
infections. In contrast to microscopy-based biomarkers, molecu-
lar-based biomarkers offer high-throughput capacity, reproducible
and objective results that are compatible with self-collected
specimens. Nevertheless, for all biomarkers proposed, both
retrospective and large population-based prospective studies are
required for validation in self-collected samples. A limited number
of studies have been reported, especially regarding self-collected
urine, and most available studies are still preclinical exploratory
studies. However, by using proper sampling, transport, storage,
preanalytical biomarker concentration techniques, and clinically
validated assays, first-void urine sampling is expected to be a
valuable method for including non-attending women in cervical
cancer screening programmes. Indeed, high analytical sensitivities
have been reported for primary HPV testing using first-void urine
but, unfortunately, the clinical performance still needs to be
determined. Moreover, the analysis of methylation of both host and
viral genes in urine has been proven feasible for use as a molecular
cervical cancer triage and screening method of urine. This
emphasizes our theory that candidate biomarkers for cervical
cancer are washed away with first-void urine together with
exfoliated cells, debris and impurities that line the urethra
opening.
In conclusion, in the near future, the use of self-sampling
methods that increase compliance with cervical cancer screening
and reduce loss to follow-up will likely increase. We believe that
when appropriately sampled, stored, and processed, first-void
urine is a particularly valuable liquid biopsy source for primary
HPV testing and for analysis of molecular biomarkers via a triage
algorithm to distinguish clinically relevant, transforming infec-
tions from productive infections.
Conflict of interest
P. Van Damme and A. Vorsters are co-founders of a University of
Antwerp spin-off company Novosanis (Belgium).
Acknowledgements
This work was supported by the Industrial Research Fund of the
University of Antwerp (PS ID 32387). S. Van Keer is supported by a
Ph. D. fellowship of the Research Foundation
Flanders (FWO)
(1120816N). None of the sponsors were involved in the writing of
the manuscript. We would like to thank D. Glorie for the design of
the illustration.
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