Abstract
The introduction of self-sampling in cervical cancer screening has
raised the importance of HPV testvalidation on self-collected samples.
This study aimed to evaluate the clinical performance of the OncoPredict
HPV Screening (SCR) assay on self-collected vaginal and first-void urine
(FVU) as part of the VALHUDES framework.
Vaginal (FLOQSwabs) and FVU (Colli-Pee) samples were self-collected by
500 women referred to colposcopy, followed by a clinician-collected
cervical sample prior to colposcopy, which were all tested using
OncoPredict HPV SCR.
OncoPredict HPV SCR demonstrated similar relative clinical sensitivity
to detect cervical intraepithelial neoplasia grade 2 or worse (≥ CIN2)
in urine (ratio: 0.95 [95%CI 0.88-1.02]) and vaginal self-samples
(ratio: 0.96 [95%CI 0.90-1.02]) compared to cervical samples. The
clinical specificity was lower in vaginal but not in urine samples
compared to cervical which improved following cut-off optimization. A
higher cellularity was found in vaginal as compared to cervical and FVU
samples. Moderate to excellent agreement in HPV detection in
self-collected samples and cervical scrapes was demonstrated (Kappa
values: 0.53 to 1.00).
OncoPredict HPV SCR assay demonstrated similar accuracy on
self-collected vaginal and FVU samples compared to cervical samples,
although cut-off adjustment improved clinical specificity when applied
to vaginal samples.
Key words (3 to 6): cervical cancer, self-sampling, diagnostic
accuracy study, Human Papillomavirus (HPV), OncoPredict HPV SCR,
European VALHUDES
Introduction
As proposed in the World Health Organization (WHO) call to action,
screening of 70% of women a key target to support the elimination of
cervical cancer by 2030 (1).
Human Papillomavirus (HPV) testing has been demonstrated to be more
effective than cytology for secondary prevention of cervical cancer
(2,3) and is therefore recommended as a primary
screening tool in current screening algorithms (4).
Additionally, meta-analyses have shown that the clinical accuracy of
PCR-based HPV tests on self-samples is similar to that of cervical
samples (5,6).
In 2021, 48 countries recommended primary HPV-based screening and 17
introduced self-sampling in their national programs or guidelines
(7) as response to WHO Call to Action
(1).
While several HPV tests are currently validated for use in cervical
cancer screening (8), only a few are formally
validated for use on self-collected specimens. The VALidation of HUman
papillomavirus assays and collection Devices for Self-samples and urine
samples (VALHUDES) Framework has defined a standardized protocol to
assess the clinical performance of HPV tests in combination with
self-collection devices (9). Results of a first
installment of VALHUDES demonstrated similar accuracy of first void
urine (FVU) collected with the Colli-Pee device and vaginal specimens
compared to clinician-collected cervical samples using different HPV
assays (10–15). We now report on a second iteration
of VALHUDES undertaken in a different geographic setting and utilizing a
different approach to vaginal sampling.
This present study aimed to evaluate the clinical performance of the
OncoPredict HPV Screening (SCR) assay on vaginal self-samples collected
with FLOQSwab resuspended in 5 ml eNat and FVU with Colli-Pee as
compared to clinician-collected cervical scrapes to detect high-grade
cervical lesions. Secondarily, we investigated the analytical
performance of the assay and evaluated the adequacy of self-collected
samples.
Material and Methods
2.1 Study design
Within the European VALHUDES Framework, 600 women, referred to
colposcopy following a previous cervical abnormality or HPV positivity,
were enrolled between July 2020 and February 2022 in four colposcopy
centers (NHS Lothian, Edinburgh; ASST degli Spedali Civili di Brescia,
Brescia, Italy; European Institute of Oncology IRCCS, Milan, Italy; U.O.
Coordinamento Consultori Familiari, ASSL Sassari - ATS Sardegna,
Sassari, Italy). Exclusion criteria have already been described
(Latsuzbaia et al., submitted).
All women were asked to collect a urine sample followed by a vaginal
specimen. FVU was collected using Colli-Pee FV5000 (Novosanis, Wijnegem,
Belgium). The device captures approximately 13 ml of FVU that are mixed
with 7 ml nucleic acid preservative included within the collection
device. Vaginal self-collection was performed using a FLOQSwab (Copan
Italia Spa, Brescia, Italy). During gynecological examination, a
cervical specimen was collected by a clinician with Cervex-Brush (Rovers
Medical Devices, Oss, The Netherlands) and immediately transferred in 20
ml PreservCyt (Hologic Inc., Bedford, Massachusetts, USA).
All women underwent colposcopy and biopsy was performed if clinically
required. The histological result of the biopsy was used to determine
the disease outcome.
Self-collected vaginal samples were transported dry to the laboratory
together with the 20 ml PreservCyt vial containing cervical samples and
the Colli-Pee tube containing FVU. All specimens were transported to the
laboratories affiliated with the enrolling colposcopy centers. After
arrival in the laboratories, cervical and FVU specimens were shaken for
30 seconds and divided into 1.5 ml aliquots. The dry vaginal swabs were
resuspended in 5 ml of PreservCyt (Hologic Inc., Bedford, Massachusetts,
USA) or 5 ml eNat (Copan Italia Spa, Brescia, Italy). Vaginal samples
were further aliquoted into 0.4 ml volumes. All aliquots were stored at
-20 °C until transferred to MIRRI-IT Biobank of the University of
Milano-Bicocca where they were stored at -80 °C. Results reported in
this manuscript are from the 500 women whose vaginal swabs were
resuspended in eNat.
2.2 HPV testing
Testing of all specimens was performed at the Laboratory Clinical
Microbiology and Virology, School of Medicine and Surgery, University of
Milano-Bicocca (Monza, Italy). Nucleic acid extraction was performed
using a Fluent 480 (Tecan, Männedorf, Switzerland) automated platform
with Quick -DNA/RNA MagBead (Zymo, USA) starting from 400 µl of
sample. Fluent 480 workstation was also used to set-up the real-time PCR
plate of OncoPredict HPV SCR assay (Hiantis, Milan, Italy) according to
manufacturer instructions with 10 µl of mastermix and 5 µl of sample’s
DNA extract. The OncoPredict HPV SCR assay, previously validated for
testing on cervical scrapes in a screening setting
(16), is a partial genotyping assay targeting E6 and
E7 DNA sequences of 13 high-risk Human Papillomavirus (hrHPV) types (HPV
-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and -68).
The test is composed of two separate real-time PCR reactions. A quality
control tube (QC) well allows the assessment of nucleic acid extraction
recovery with an exogenous control gene target added to the sample
before preanalytical processing, additionally the QC tube determines
sample adequacy in terms of human cellularity by the quantification ofC-C Motif Chemokine Receptor 5 (CCR5) gene. A second reaction
well is used to assess the presence of HPV-16, HPV-18 individually and
the 11 other hrHPV types as a pool. Both wells contain an amplification
control to evaluate the potential PCR inhibition. The PCR was carried
out using a CFX384 Touch Real-Time PCR Detection System (Bio-Rad, USA).
All results were considered valid if HPV positive. In the case of HPV
negative result(s) samples were defined as inadequate if i) the
extraction efficiency was below 10%; ii) less than 400 cells/reaction
in cervical samples (16) and 150 cells/reaction in
urine and vaginal samples were detected and iii) there was PCR
inhibition in any of the two reaction wells.
2.3 Statistical analysis
Clinical sensitivity was estimated for cervical intraepithelial
neoplasia grade 2 or worse (≥CIN2) and for cervical intraepithelial
neoplasia grade 3 or worse (≥CIN3). Specificity was estimated for
<CIN2 outcome or accepting negative colposcopy as clinical
endpoint when the gynecologist did not take a biopsy. We used McNemar
tests to evaluate the accuracy differences between index and comparator
tests with statistical significance accepted if p-values <0.05
or when the 95% confidence intervals excluded 1. Cohen’s kappa was
employed to assess HPV test concordance between self- and clinician
taken samples for the entire study population and according to disease
status among specimens and categorized as: poor (0.00–0.19), fair
(0.20–0.39), moderate (0.40–0.59), good (0.60–0.79), and excellent
(0.80–1.00). Mann-Whitney test was used to evaluate differences in
median Ct-values and median number of cells/reaction. All statistical
analyses were conducted using Stata 16.1 (Statacorp, College Station,
TX, USA).
2.4 Ethical Approval
The European VALHUDES study (ClincalTrail.gov: NCT04312737) was
conducted in accordance with the Declaration of Helsinki and approved by
the central Ethics Committee of the Coordinating Centre, ASST degli
Spedali Civili di Brescia, Brescia, Italy (Ethics approval number: NP
3879- Studio WP6-HPVONC) on the 16th of July 2020, subsequently by the
local Ethics Committees of the other participating centres. All women
signed a written informed consent form before to enrolment.
Consorzio Italiano per la Ricerca in Medicina (C.I.R.M.), Milano, Italy,
performed on site and remote monitoring of the study conduction, as
previously described (Latsuzbaia et al., submitted).
Results
3.1 Study population
490 out of the 500 women were included in the study as reported in
Figure 1. The median age of the women included in the study was 37 years
(IQR: 31-47 years, range: 25-64 years), with median age of women with
≥CIN2 lesions being significantly lower of those with <CIN2
result, as previously described (Latsuzbaia et al., submitted). 489
women had colposcopy with the following outcomes: 134 (27.4%) negative,
245 (50.1%) minor colposcopy findings, 104 (21.3%) major colposcopy
and 6 (1.2%) suspicion of cancer. 55% (271/490) of women underwent
biopsy and diagnosis of ≥CIN2 was confirmed in 41.3% (112/271) of
cases. Table 1 reports the characteristics of the study population by
age group and colposcopy center. 28 cervical specimens, 13 vaginal swabs
and 19 FVU samples were excluded from the analysis because they were
inadequate and HPV-negative (Figure 1).