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\begin{document}

\title{Should we use risk selection tests for HPV 16 and/or 18 positive cases:
comparison of p16/Ki67 and cytology}



\author[1]{Karolina Mazurec}%
\author[1]{Martyna  Trzeszcz }%
\author[1]{Maciej Mazurec}%
\author[2]{Joanna Streb}%
\author[3]{Agnieszka Halon}%
\author[2]{Robert  Jach}%
\affil[1]{Corfamed Woman’s Health Center}%
\affil[2]{Uniwersytet Jagiellonski w Krakowie Collegium Medicum}%
\affil[3]{Uniwersytet Medyczny im Piastow Slaskich we Wroclawiu}%


\vspace{-1em}



  
  \date{June 10, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Background}: Major screening abnormalities in pre-colposcopic
stage are tests results that imply direct referral to colposcopy (and/or
expedited treatment) without performing additional high-grade squamous
intraepithelial lesions or worse (HSIL+) risk selection testing.
Currently, both clinically validated HSIL+ risk selection tests, reflex
cytology and reflex p16/Ki67 dual staining (DS), are being compared for
use in primary HPV-based screening to avoid possible overtreatment, but
there is still no sufficient data available for their performance.
\textbf{Methods}: Among 30,066 liquid-based cervical cancer screening
tests results, a group of 332 women was selected with available HRHPV
tests results with 16/18 limited genotyping, liquid-based cytology, DS,
and histology results from standardized colposcopy with biopsy. In HPV
16/18+ cases, three triage approaches were retrospectively analyzed.
Predictive values for detection of HSIL+ were calculated and number of
colposcopies required in each strategy. \textbf{Results}: Both triage
models with DS used (reflex cytology followed by DS, and reflex DS alone
in all cases) had significantly higher PPV for HSIL+ than strategy with
reflex cytology alone (44.2\%/45.7\% vs. 28.3\%; p\textless{}0.0001). In
models with DS, less colposcopies were required (95/92 vs. 152) and less
colposcopies were needed per HSIL+ detection (2,26/2,19 vs. 3,54). Only
1 HSIL+ case was missed in both triage models with DS incorporation.
\textbf{Conclusions}: p16/Ki67 dual-stain may be an effective, alone or
combined with cytology, triage test to detect HSIL+ in patients with
major screening abnormalities in primary HPV-based cervical cancer
screening. Performing cytology as the first triage test improves the
strategy by enabling referrals to expedited treatment in selected cases.%
\end{abstract}%



\sloppy


\textbf{Should we use risk selection tests for HPV 16 and/or 18 positive
cases: comparison of p16/Ki67 and cytology}

Karolina Mazurec\textsuperscript{1}, Martyna
Trzeszcz\textsuperscript{1,2*}, Maciej Mazurec\textsuperscript{1},
Joanna Streb\textsuperscript{3}, Agnieszka Halon\textsuperscript{4},
Robert Jach\textsuperscript{5}

\textsuperscript{1} Corfamed Woman's Health Center, Kluczborska 37,
50-322 Wroclaw, Poland; carlos.mazurec@icloud.com (K.M.);
m.mazurec@corfamed.pl (M.M.)\textsuperscript{2} Division of Pathology
and Clinical Cytology, University Hospital in Wroclaw, Borowska 213,
50-556 Wroclaw, Poland; m.trzeszcz@corfamed.pl (M.T.)\textsuperscript{3}
Department of Oncology, Jagiellonian University Medical College,
Kopernika 50, 31-501 Krakow, Poland; joanna.streb@uj.edu.pl
(J.S.)\textsuperscript{4} Department of Clinical and Experimental
Pathology, Division of Clinical Pathology, Wroclaw Medical University,
Borowska 213, 50-556 Wroclaw, Poland; agnieszka.halon@umed.wroc.pl
(A.H.)\textsuperscript{5} Division of Gynecologic Endocrinology,
Jagiellonian University Medical College, Kopernika 23, 31-501 Krakow,
Poland; jach@cm-uj.krakow.pl (R.J.)

*Corresponding author

\textbf{Correspondence to:} Martyna Trzeszcz, MD, PhD, Division of
Pathology and Clinical Cytology, University Hospital in Wroclaw,
Borowska 213, 50-556 Wroclaw, Poland; m.trzeszcz@corfamed.pl (M.T.);
+48508646805

\textbf{Conflicts of Interest} : Robert Jach has given a lecture
sponsored by the Roche company. The other authors declare no conflict of
interest.

\textbf{Funding} : This research received no external funding.

\textbf{Ethics Statement} : The study was conducted in accordance with
the Declaration of Helsinki and approved by the Ethics Committee of the
Jagiellonian University (opinion ID 118.6120.36.2023).

\textbf{Shortened title} : Should we use triage tests for HPV 16/18+
cases?

\textbf{Abstract}

\textbf{Background} : Major screening abnormalities in pre-colposcopic
stage are tests results that imply direct referral to colposcopy (and/or
expedited treatment) without performing additional high-grade squamous
intraepithelial lesions or worse (HSIL+) risk selection testing.
Currently, both clinically validated HSIL+ risk selection tests, reflex
cytology and reflex p16/Ki67 dual staining (DS), are being compared for
use in primary HPV-based screening to avoid possible overtreatment, but
there is still no sufficient data available for their performance.

\textbf{Methods} : Among 30,066 liquid-based cervical cancer screening
tests results, a group of 332 women was selected with available HRHPV
tests results with 16/18 limited genotyping, liquid-based cytology, DS,
and histology results from standardized colposcopy with biopsy. In HPV
16/18+ cases, three triage approaches were retrospectively analyzed.
Predictive values for detection of HSIL+ were calculated and number of
colposcopies required in each strategy.

\textbf{Results} : Both triage models with DS used (reflex cytology
followed by DS, and reflex DS alone in all cases) had significantly
higher PPV for HSIL+ than strategy with reflex cytology alone
(44.2\%/45.7\% vs. 28.3\%; p\textless{}0.0001). In models with DS, less
colposcopies were required (95/92 vs. 152) and less colposcopies were
needed per HSIL+ detection (2,26/2,19 vs. 3,54). Only 1 HSIL+ case was
missed in both triage models with DS incorporation.

\textbf{Conclusions} : p16/Ki67 dual-stain may be an effective, alone or
combined with cytology, triage test to detect HSIL+ in patients with
major screening abnormalities in primary HPV-based cervical cancer
screening. Performing cytology as the first triage test improves the
strategy by enabling referrals to expedited treatment in selected cases.

\textbf{KEY WORDS:} cervical cancer screening; CIN; high-risk HPV; HPV
16 and 18; p16/Ki67 dual staining; triage

\textbf{Introduction}

Cervical cancer still remains a significant global public health burden,
particularly in low- and middle-income countries where access to regular
screening and preventive interventions is limited {[}1{]}. The
implementation of cervical cancer screening programs has substantially
reduced the incidence and mortality rates of this malignancy in many
high-income countries {[}2{]}. The emergence of 13-14 high-risk types of
human papillomavirus (HRHPV) (family \emph{Papillomaviridae} ,
subfamily\emph{Firstpapillomavirinae} , genus \emph{Alphapapillomavirus}
, species\emph{Alphapapillomavirus} 9) {[}3{]} as a primary etiological
agent in the development of cervical cancer has led to revolutionary
changes in screening strategies {[}4-6{]}. HRHPV testing, with its very
high sensitivity, has gained prominence as a cornerstone in early
detection efforts {[}7-9{]}.

In recent years, advancements in genotyping techniques have further
refined our understanding of HPV's role in cervical carcinogenesis
{[}10{]}. The limited genotyping has opened possibilities to
differentiate between HPV genotypes with varying carcinogenic
propensities. Incorporating this information into clinical
decision-making could enable a targeted approach to colposcopy referral,
risk stratification and minimizing unnecessary interventions for
individuals at lower risk {[}11{]}. Primary HPV-based screening with
genotyping, presents a promising approach to enhance the accuracy of
cervical cancer secondary prevention, specifically in triaging
individuals at highest risk for colposcopy referral {[}12{]}. But
performing a colposcopy in all major screening abnormalities, with
potentially higher risk of histologic high-grade intraepithelial lesions
with a quantification of cervical intraepithelial neoplasia in grade 2
or worse (HSIL/CIN2+) implying a direct referral to colposcopy without
prior triage testing, identified through primary HPV-based screening
with limited genotyping, raises important clinical and resource-related
considerations {[}13{]}. The cost associated with performing
colposcopies in all individuals with major screening abnormalities can
strain healthcare systems and lead to resource allocation challenges.
Furthermore, it may lead to overtreatment, causing unnecessary stress
and discomfort for patients.

The transition to primary HPV-based screening with a limited genotyping
new paradigm needs a critical evaluation of the clinical algorithm
guiding follow-up steps. HPV 16 and/or 18 positive results (HPV 16/18+)
have the highest HSIL/CIN2+ risk among all HRHPV-positive results
{[}14-16{]}. Currently some of recommendations worldwide, including the
2019 American Society for Colposcopy and Cervical Pathology (ASCCP 2019)
Consensus Guidelines, are advising colposcopy for all HPV 16/18+ results
without any triage test {[}14-16{]}. In the ASCCP 2019 Guidelines,
cytology as a triage test for all results is highly valued due to its
importance in referring patients to expedited treatment, defined as
excisional treatment, without preceding confirmatory colposcopy with
biopsy), and not only to colposcopy. It is also recommended that all
cases with HPV 16/18+ ASC-H and HPV 18+ HSIL results should be referred
to colposcopy or expedited treatment and HPV 16+ HSIL patients directly
to expedited treatment {[}14,15{]}. The second examined triage test
approved (e.g. by the U.S. Food and Drug Administration or the Polish
Society of Colposcopy and Cervical Pathology) in selected HPV-positive
cases and in patients with abnormal cotesting results, is p16/Ki67
dual-staining (DS), which is characterized by high sensitivity and
specificity for HSIL/CIN2+ detection {[}17-23{]}.

The landscape of cervical cancer secondary prevention is evolving
rapidly, driven by advances in HRHPV testing and genotyping
technologies. While the use of colposcopy for all major screening
abnormalities detected through primary HPV-based screening with limited
genotyping is the most common management recommended in available
guidelines, it is imperative to critically assess the evidence
supporting this approach for all HPV 16/18+ cases. By evaluating the
potential benefits and limitations demonstrated in different strategy
ramifications, we aim to provide a comprehensive perspective on the need
of colposcopy referral in these patients. The positive predictive value
(PPV) is a valuable determinant of the accuracy of the diagnostic tests.
It depends on the prevalence of the condition in the screened population
and both the sensitivity and the specificity of the test {[}24{]}. PPV
determines the probability that a positive test result correctly
predicts the presence of the disease, so it is more important for
clinicians than sensitivity and specificity {[}25{]}.

This retrospective study is to evaluate performance characteristics of
three different triage models, incorporating cytology alone or DS alone
or in combination, in HPV 16/18 positive patients who had undergone
primary HPV-based cervical cancer screening, for detecting HSIL/CIN2+
cases.

\textbf{Materials and methods}

Study population

The study is a post-hoc analysis including a total of 30,066 cervical
cancer liquid-based screening (LBS) tests results. This dataset
comprises 20,605 results from liquid-based cytology (LBC), 8331 results
from HRHPV with limited HPV 16/18 genotyping, and 1130 results from DS.
These LBS tests were derived from a diverse cohort of women ranging from
15 to 92 years old, with a mean age of 40.9. Data for analysis were
extracted from LBS test results obtained between August 2015 and July
2020. These tests were sourced from opportunistic cervical cancer
screening programs conducted at the Corfamed Woman's Health Center, one
of the largest private outpatient gynecological clinics located in an
urban area of Lower Silesia, Poland. The cervical cancer screening at
the Center employed three distinct models during the study period:
primary cytology with reflex HRHPV, primary cotesting, and primary
cotesting plus (simultaneous cotesting and DS). The reflex HRHPV test
was recommended for cases with minor cytological abnormalities (ASC-US
or LSIL) in the primary cytology screening. DS was conducted for ASC-US
or LSIL cases in both cytology- and cotesting-based models, as well as
for all positive HRHPV test results. The final study cohort consisted of
332 patients aged over 25 years, all of whom had undergone LBS testing
and subsequent colposcopy with biopsy at the Center (Figure 1). This
selected group of patients had no prior HPV status available. A
retrospective analysis of
cytologic-virologic-immunocytochemical-histologic test results was
conducted, and the PPV of cytology and DS as triage tests for specific
major cervical cancer screening abnormalities was evaluated. The study
data were retrieved from the Center's registry, with a detailed
methodology previously described in a series of related studies
{[}26-29{]}. The study received ethics committee approval (ID:
118.6120.36.2023).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Fig. 1. Study group selection -- flowchart. Abbreviations: LBC,
liquid-based cytology; HRHPV, 14 high-risk types human papillomavirus
test; DS, p16/Ki67 dual staining test; HPV 16/18+, human papillomavirus
types 16 and/or 18 positive results; HPV HR12+, human papillomavirus 12
high-risk types other than types 16 and 18 positive results.

Major screening abnormalities were defined in this study as screening
abnormalities with potentially higher HSIL/CIN2+ risk that justifies a
direct referral to colposcopy of HPV 16/18+ patients without prior
triage testing, identified through primary HPV-based screening with
limited genotyping. Management in these cases was according to ASCCP
2019 Risk-Based Management Consensus Guidelines {[}14-16{]}.

HRHPV testing

Detection of HRHPV was performed using the Abbott RealTime High Risk HPV
molecular in vitro PCR test (Abbott Molecular, Des Plaines, IL, USA), in
adherence to the manufacturer's instructions. This test phenotypes 12
types of high-risk HPV DNA (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66,
and 68), as well as genotypes HPV 16 and 18 (limited genotyping). Test
results were classified as HPV 16/18+ if either of these types was
detected, and as HPV HR12+ if one or more of the other twelve non-16 and
non-18 HRHPV types were detected. All screening samples were collected
using the Cervex-Brush device (Rovers Medical Devices, Oss,
Netherlands), and all tests (HRHPV, LBC, and DS) were performed in the
same laboratory.

Liquid-based cytology

Liquid-based SurePath slides were prepared by an external laboratory
using the automatic PrepStain Slide system (Becton Dickinson, Franklin
Lakes, NJ, USA) following the manufacturer's protocol. Residual cervical
samples were stored for three months under specific conditions, enabling
additional tests including DS triage to be performed. This approach
facilitated comprehensive cervical cancer diagnostics from a single
sample collection. A gynecological cytopathologist evaluated all
cytology samples according to the Bethesda 2014 guidelines, being aware
of the HRHPV status of the samples. Quality and control procedures
aligned with benchmarks from US laboratories accredited by the College
of American Pathologists {[}29{]}.

p16/Ki67 dual-stain testing

The CINtec PLUS detection kit (Roche, MTM AG laboratories, Munich,
Germany) was utilized for dual immunocytochemical staining with p16 and
Ki67 proteins. This staining was performed in an automated BenchMark XT
system (Ventana Medical Systems, Inc., Oro Valley, AZ, USA) following
the manufacturer's protocol. DS was conducted using residual cellular
samples from the original SurePath vials stored in the laboratory after
cytology and/or HPV testing. A qualified gynecological pathologist,
specially trained and certified, assessed the DS based on established
definitions {[}30{]}.

Colposcopy protocol

Patients with DS-positive results, ASC-US or LSIL HRHPV-positive
results, or those with cytologic ASC-H or worse, regardless of previous
HRHPV status or with unknown HRHPV status, were referred for a
colposcopy with biopsy. The protocol adhered to Polish recommendations,
with extension of the 2012 ASCCP guidelines and 2015 ASCCP interim
guidelines {[}31-34{]}. The requirements of minimal colposcopy protocol
included endocervical sampling (curettage and brushing), direct biopsies
for all abnormal colposcopic findings or those suggestive for invasion,
or additional random biopsies as per Polish protocols. All histologic
diagnoses of cervical biopsies and endocervical sampling were reviewed
by a gynecological pathologist according to the LAST 2012/WHO 2014
terminology {[}35,36{]} with more details available in a series of
related studies {[}26-29{]}.

Triage models analyzed

Three HSIL/CIN2+ risk triage models for HPV 16/18+ cases in primary
HPV-based cervical cancer screening were compared and analyzed. A reflex
cytology was evaluated in all positive cases in model 1 (M1), in model
1A (M1A) a DS was assessed as the second triage test after NILM, ASC-US
or LSIL results (reflex cytology in all positive cases, as in M1) and in
model 2 (M2) a reflex DS in all HPV 16/18+ cases was analyzed. The
proposed management in the analyzed triage models was presented in
Figure 2.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Fig. 2. Analyzed triage models---flowchart. Abbreviations: HRHPV, 14
high-risk types human papillomavirus test; HPV 16/18+, human
papillomavirus types 16 and/or 18 positive results; M1, Triage model 1;
M1A, Triage model 1A; M2, Triage model 2; LBC, liquid-based cytology;
NILM, negative for intraepithelial lesion or malignancy; ASC-US,
atypical squamous cells of undetermined significance; LSIL, low-grade
squamous intraepithelial lesion; ASC-H, atypical squamous cells-cannot
exclude HSIL; HSIL, high-grade squamous intraepithelial lesion; *HPV 18+
HSIL -- colposcopy or expedited treatment, HPV 16+ HSIL -- expedited
treatment; DS, p16/Ki67 dual staining test; +, positive; -, negative; 1Y
FU, 1-year follow-up; COLPO, colposcopy; COLPO or ExT/ExT, colposcopy or
expedited treatment/expedited treatment.

Statistical analysis

A diagnostic value of the tests and their variations was assessed using
PPV and negative predictive value (NPV), calculated according to
standard definitions. The discrepancies in the diagnostic value between
combinations of screening tests were evaluated using Leisenring et al.
method {[}37{]} with the DTComPair package in R. The statistical
significance was set at p \textless{} 0.05. A licensed PQStat Software
in a 1.6.0 full version (2015 PQStat Statistical Calculation Software,
Poznan, Poland) was used for the statistical analysis.

\textbf{Results}

In 30,066 cervical cancer LBS tests results (20,605 LBC, 8331 HRHPV and
1130 DS) obtained, 443 cases with colposcopy with biopsy performed at
our Center were noted. Among this group, 332 patients were selected as
eligible for the study. Only women over 25 years old were included in
the final study group with age range from 25 to 67 years old, an average
age of 34.7 years and a median age of 33 years old.

All cases in the study group were HRHPV-positive -- 45.8\% HPV 16/18+
(152/332) and 54.2\% HPV HR12+ (180/332). Among HPV 16/18+ cases, 60.5\%
(92/152) were DS-positive and 39.5\% (60/152) were DS-negative. In the
HPV 16/18+ group, 17.1\% of cases had negative cytology (NILM) (26/152),
40.8\% had ASC-US (62/152), 25.0\% had LSIL (38/152), and 17.1\% had
ASC-H+ result (26/152). The histology results in HPV 16/18+ cases were
negative or LSIL/CIN1 in 71.7\% (109/152) of cases and HSIL/CIN2+ in
28.3\% (43/152). The characteristics of the study group are presented in
Table 1.

Tab. 1. Study group characteristics.\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\begin{minipage}[b]{0.47\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.47\columnwidth}\raggedright\strut
Total, No\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
LBS (HRHPV, LBC and DS) + histology results\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
332\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
HPV 16/18+\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
152 (45.8\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
HPV HR12+ -- screening abnormalities evaluated separately {[}27{]}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
180 (54.2\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Results of cytology triage in HPV 16/18+ cases\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
152\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
NILM\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
26 (17.1\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
ASC-US\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
62 (40.8\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
LSIL\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
38 (25.0\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
ASC-H+\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
26 (17.1\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Results of DS triage in HPV 16/18+ cases\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
152\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
DS-positive\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
92 (60.5\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
DS-negative\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
60 (39.8\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Histology results in HPV 16/18+ cases\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
152\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Negative/LSIL/CIN1\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
109 (71.7\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
HSIL/CIN2+\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
43 (28.3\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
HSIL/CIN3+*\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
23 (53.5\%)*\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Age (years)\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Min\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
25\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Max\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
67\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Median\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
33\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Mean\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
34.7\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Abbreviations: LBS, liquid-based screening; HRHPV, 14 high-risk types
human papillomavirus test; HPV 16/18+, human papillomavirus types 16
and/or 18 positive results; HPV HR12+, human papillomavirus 12 high-risk
types other than types 16 and 18 positive results; DS, p16/Ki67 dual
staining test; LBC, liquid-based cytology; NILM, negative for
intraepithelial lesion or malignancy; ASC-US, atypical squamous cells of
undetermined significance; LSIL, low-grade squamous intraepithelial
lesion; ASC-H+, atypical squamous cells-cannot exclude HSIL or worse;
LSIL/CIN1, histologic low-grade squamous intraepithelial lesion;
HSIL/CIN2+, histologic high-grade squamous intraepithelial lesion with a
quantification of cervical intraepithelial neoplasia in grade 2 or
worse; HSIL/CIN3+, histologic high-grade squamous intraepithelial lesion
with a quantification of cervical intraepithelial neoplasia in grade 3
or worse; min, minimum; max, maximum; *, \% of HSIL/CIN2+.

Estimated colposcopy outcomes in different triage strategies for HPV
16/18 positive women in primary HPV-based screening

In the study, triage model M1 required 152 colposcopies, 0 cases were
missed and 43 HSIL/CIN2+ cases were detected, in triage model M1A
95/1/42, and in triage model M2 92/1/42, respectively. The missed
HSIL/CIN2+ cases were caused by no indications for colposcopy according
to the management applied for each of the models. The full results,
including PPV values, for the 3 studied models and all the results sets
are shown in Table 2.

Tab. 2. HSIL/CIN2+ reporting rates and PPVs for detection of HSIL/CIN2+
in HPV 16/18+ cases in the compared triage models.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& HSIL/CIN2+, No & \textless{}HSIL/CIN2, No & Total, No & PPV,
\%\tabularnewline
\midrule
\endhead
Triage of all HPV 16/18+ with cytology alone (M1) & & & &\tabularnewline
NILM & 7 & 19 & 26 & 26.9\tabularnewline
ASC-US & 10 & 52 & 62 & 16.1\tabularnewline
LSIL & 9 & 29 & 38 & 23.7\tabularnewline
ASC-US/LSIL & 19 & 81 & 100 & 19.0\tabularnewline
NILM/ASC-US/LSIL & 26 & 100 & 126 & 20.6\tabularnewline
ASC-H+ & 17 & 9 & 26 & 65.4\tabularnewline
NILM/ASC-US/LSIL/ASC-H+ & 43 & 109 & 152 & 28.3\tabularnewline
Triage of all HPV 16/18+ with cytology and DS (M1A) & & &
&\tabularnewline
NILM DS+ & 7 & 8 & 15 & 46.7\tabularnewline
ASC-US DS+ & 10 & 17 & 27 & 37.0\tabularnewline
LSIL DS+ & 8 & 19 & 27 & 29.6\tabularnewline
ASC-US/LSIL DS+ & 18 & 36 & 54 & 33.3\tabularnewline
NILM/ASC-US/LSIL DS+ & 25 & 44 & 69 & 36.2\tabularnewline
ASC-H+ & 17 & 9 & 26 & 65.4\tabularnewline
NILM/ASC-US/LSIL DS+/ASC-H+ & 42 & 53 & 95 & 44.2\tabularnewline
NILM DS- & (0) & (11) & (11) & -\tabularnewline
ASC-US/LSIL DS- & (1) & (45) & (46) & -\tabularnewline
NILM/ASC-US/LSIL DS- & (1) & (56) & (57) & -\tabularnewline
Triage of all HPV 16/18+ with DS alone (M2) & & & &\tabularnewline
DS+ & 42 & 50 & 92 & 45.7\tabularnewline
DS- & (1) & (59) & (60) & -\tabularnewline
\bottomrule
\end{longtable}

Abbreviations: M1, Triage model 1; M1A, Triage model 1A; M2, Triage
model 2; HPV 16/18+, human papillomavirus types 16 and/or 18 positive
results; NILM, negative for intraepithelial lesion or malignancy;
ASC-US, atypical squamous cells of undetermined significance; LSIL,
low-grade squamous intraepithelial lesion; ASC-H+, atypical squamous
cells-cannot exclude HSIL or worse; DS, p16/Ki67 dual staining test;
HSIL/CIN2+, histologic high-grade squamous intraepithelial lesion with a
quantification of cervical intraepithelial neoplasia in grade 2 or
worse; HSIL/CIN2, histologic high-grade squamous intraepithelial lesion
with a quantification of cervical intraepithelial neoplasia in grade 2;
PPV, positive predictive value; +, positive; -, negative; (No) numbers
in parentheses, potentially missed cases, there was no indications for
colposcopy for these cases.

The number of colposcopies required in triage models with incorporation
of DS, M1A and M2, was around 1,6 times smaller than in model with
cytology alone, M1. The analyzed models were also compared by number of
colposcopies required per HSIL/CIN2+ detection, which was also
approximately two times smaller in M1A and M2 than in M1 (Table 3).

Tab. 3. Number of colposcopies needed to detect HSIL/CIN2+ and number of
missed HSIL/CIN2+ cases in different triage models in primary HPV-based
screening.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& No. of colposcopies needed in each model & No. of colposcopies needed
to detect 1 HSIL/CIN2+ case & No. of missed HSIL/CIN2+
cases\tabularnewline
\midrule
\endhead
Triage model with cytology alone (M1): NILM/ASC-US/LSIL/ASC-H+ & 152 &
3.54 & 0\tabularnewline
Triage model with cytology and DS (M1A): NILM/ASC-US/LSIL DS+/ASC-H+ &
95 & 2.26 & 1\tabularnewline
Triage model with DS alone (M2): DS+ & 92 & 2.19 & 1\tabularnewline
\bottomrule
\end{longtable}

Abbreviations: M1, Triage model 1; M1A, Triage model 1A; M2, Triage
model 2; NILM, negative for intraepithelial lesion or malignancy;
ASC-US, atypical squamous cells of undetermined significance; LSIL,
low-grade squamous intraepithelial lesion; ASC-H+, atypical squamous
cells-cannot exclude HSIL or worse; DS, p16/Ki67 dual staining test;
HSIL/CIN2+, histologic high-grade squamous intraepithelial lesion with a
quantification of cervical intraepithelial neoplasia in grade 2 or
worse; +, positive.

Estimated selected performance of different triage strategies for HPV
16/18 positive women in primary HPV-based screening

The PPV and NPV for HSIL/CIN2+ in M1 were 28.0\% and NA, respectively.
The same parameters in M1A were 44.2\% and 98.3\%, and in M2 45.7\% and
98.3\%, as presented in Table 4.

Tab. 4. Clinical performance with predictive values for detection of
HSIL/CIN2+ in HPV 16/18+ cases in analyzed triage models.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
& HSIL/CIN2+, No. & \textless{}HSIL/CIN2, No. & Total, No. & PPV, \%
(95\% CI) & NPV, \% (95\% CI)\tabularnewline
\midrule
\endhead
Triage model with cytology alone (M1): NILM/ASC-US/LSIL/ASC-H+ & 43 &
109 & 152 & 28.3 (21.3, 36.2) & NA\tabularnewline
Triage model with cytology and DS (M1A): NILM/ASC-US/LSIL DS+/ASC-H+ &
42 & 53 & 95 & 44.2 (34.0, 54.8) & 98.3 (90.6, 99.9)\tabularnewline
Triage model with DS alone (M2): DS+ & 42 & 50 & 92 & 45.7 (35.2, 56.4)
& 98.3 (91.1, 99.9)\tabularnewline
\bottomrule
\end{longtable}

Abbreviations: M1, Triage model 1; M1A, Triage model 1A; M2, Triage
model 2; HPV 16/18, human papillomavirus types 16 and/or 18; NILM,
negative for intraepithelial lesion or malignancy; ASC-US, atypical
squamous cells of undetermined significance; LSIL, low-grade squamous
intraepithelial lesion; ASC-H+, atypical squamous cells-cannot exclude
HSIL or worse; DS, p16/Ki67 dual staining test; HSIL/CIN2+, histologic
high-grade squamous intraepithelial lesion with a quantification of
cervical intraepithelial neoplasia in grade 2 or worse; HSIL/CIN2,
histologic high-grade squamous intraepithelial lesion with a
quantification of cervical intraepithelial neoplasia in grade 2; PPV,
positive predictive value; NPV, negative predictive value; +, positive;
CI, confidence interval.

The difference in PPV between model with cytology triage, M1, and both
models with DS triage, M1A and M2, was statistically significant (28.3\%
vs. 44.2\% -- p \textless{} 0.0001 and 28.3\% vs. 45.7\% - p \textless{}
0.0001), but there was no significant difference in PPV between M1A and
M2. The difference between NPVs in M1A and M2 was also statistically
insignificant. The detailed comparison is presented in Table 5.

Tab. 5. Comparison of predictive values for detection of HSIL/CIN2+ in
selected HPV 16/18+ cases in different triage models.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& M1 (Cytology-alone triage) & M1A (Cytology and DS triage) & \emph{p}
value\tabularnewline
\midrule
\endhead
PPV for HSIL/CIN2+, \% (95\% CI) & 28.3 (21.3, 36.2) & 44.2 (34.0, 54.8)
& \textless{} 0.0001\tabularnewline
& M1 (Cytology-alone triage) & M2 (DS-alone triage) &\tabularnewline
PPV for HSIL/CIN2+, \% (95\% CI) & 28.3 (21.3, 36.2) & 45.7 (35.2, 56.4)
& \textless{} 0.0001\tabularnewline
& M1A (Cytology and DS triage) & M2 (DS-alone triage) &\tabularnewline
PPV for HSIL/CIN2+, \% (95\% CI) & 44.2 (34.0, 54.8) & 45.7 (35.2, 56.4)
& 0.0843\tabularnewline
NPV for HSIL/CIN2+, \% (95\% CI) & 98.3 (90.6, 99.9) & 98.3 (91.1, 99.9)
& 0.38\tabularnewline
\bottomrule
\end{longtable}

Abbreviations: M1, Triage model 1; M1A, Triage model 1A; M2, Triage
model 2; DS, p16/Ki67 dual staining test; HSIL/CIN2+, histologic
high-grade squamous intraepithelial lesion with a quantification of
cervical intraepithelial neoplasia in grade 2 or worse; PPV, positive
predictive value; NPV, negative predictive value; CI, confidence
interval.

\textbf{Discussion}

The landscape of cervical cancer prevention has evolved significantly
with the approach of primary HPV-based screening and limited genotyping.
Our critical examination of the necessity of obligatory colposcopy
referral for all major screening abnormalities detected through this
approach highlights several key points. Overreliance on colposcopy for
all major screening abnormalities detected through primary HPV-based
screening with limited genotyping may lead to overtreatment. In our
population-based non-interventional analysis the PPV for detection of
HSIL/CIN2+ in both triage models with reflex DS (M1A and M2) was
significantly higher than for model with reflex cytology alone (M1),
44.2\% vs. 28.3\% (p \textless{} 0.0001) and 45.7\% vs. 28.3\% (p
\textless{} 0.0001), respectively. Also, both M1A and M2 triage models
were associated with an approximately 40\% reduction in number of
colposcopies required compared to M1 (95/92 vs. 152). The difference in
PPV between triage models with DS incorporation, M1A and M2, was
insignificant and almost imperceptible (44.2\% vs. 45.7\%), as well as
difference in number of colposcopies needed to detect one HSIL/CIN2+
case (2,26 vs. 2,19) and number of missed HSIL/CIN2+ cases (1 case per
each triage model). The highest PPV was achieved in HPV 16/18+
DS-positive cases in M2 (45.7\%). NPVs of both models with DS triage
were very high (98.3\%, no statistically significant difference),
highlighting patients' good protection. All analyzed models had a PPV
levels above 10\%, meeting the criterion for the acceptable triage
strategy in cervical cancer screening {[}38{]}.

The results we obtained for selected combinations of screening tests
results were similar to many other studies, including data presented by
Wright et al {[}39{]}. The PPV in HPV 16/18+ cases with DS triage
published in that paper was slightly lower (35.1\% vs. 45.7\% in our
study) with nearly identical NPV for the same group (96.5\% vs. 98.3\%,
respectively). Also, data presented by FDA was in similar range,
although analyzed separately for HPV 16+ and HPV 18+, both with DS
triage (PPV: 44.0\%/23.9\% and NPV: 93.8/95.3\%, HPV 16+/HPV 18+
respectively) {[}18{]}. Our study showed lower PPV and higher NPV in the
HPV 16/18+ DS-positive cases compared to \selectlanguage{ngerman}Øvestad et al. (PPV: 45.7\% vs.
70.3\%, NPV: 98.3\% vs. 84.4\%) {[}40{]}. The PPV in HPV 16/18+ cases
presented by Naucler et al. was nearly identical to the result in our
analysis (25.1\% vs. 28.3\%) and the value obtained by El-Zein et al.
was around 15\% higher than in our study (43.8\% vs. 28.3\%)
{[}41,42{]}. In turn, PPV for group analyzed in M1A model
(NILM/ASC-US/LSIL DS-positive and ASC-H+ cases), with a triage strategy
using cytology combined with DS, we have not found a reliable comparator
to our research. This is the first study analyzing the following
approach. Due to no full-fledged studies on the subject available, a
complete in detail comparison of all PPV levels obtained in HPV
16/18-positive patients with major screening abnormalities with other
studies was not possible.

In our analysis the highest PPV was obtained in model with DS-alone
triage (M2) with statistically insignificant difference between this
triage model and model with cytology combined with DS (M1A). Also, both
triage models allowed just an effective reduction in number of
colposcopies required in each model comparing to M1 and missed the same
number of HSIL/CIN2+ cases -- only 1 case per model, ensuring high
safety. That confirms the use of DS as a triage test in primary
HPV-based cervical cancer screening strategy for women with selected
major screening abnormalities, allowing accuracy improvement and
reduction in number of invasive procedures (e.g., colposcopy). The
important difference between these two triaging models is the
possibility to refer patients directly to expedited treatment, not only
colposcopy. In M1A due to reflex cytology, for selected screening tests
results colposcopy/expedited treatment or expedited treatment is
recommended. In M2 triage, it does not bring such potential, DS-positive
cases need to be referred to colposcopy and DS-negative cases should go
to 1 year follow-up.

In our study AGC results were excluded from the evaluated models and
were managed based on separate ASCCP 2019 recommendations algorithm
{[}14,15{]}. European Consensus Statement on Expert Colposcopy 2023 also
recommends distinct management for all AGC cases, compared to squamous
epithelial lesions {[}43{]}.

There are several strengths of the study. The non-interventional
analysis of the largest number of LBS results in Poland and Central
Eastern Europe, the insight into the results of screening tests in
private-based opportunistic cervical cancer screening and the wide age
range of participants are some of most important. Joint analysis of all
locally approved cervical cancer screening tests (liquid-based cytology,
HRHPV and DS), which allowed us to compare their accuracy and assess
potential of these tests to be used in a triage strategy for women with
major screening abnormalities in primary HPV-based screening has also
been a strength. Moreover, the study introduces one of the largest
investigations on cytologic-virologic-immunocytochemical-histologic
correlations in cervical cancer screening. A qualified gynecologic
cytopathologist evaluated all LBC and DS. However, the study has
limitations, such as being a retrospective analysis and not all patients
with abnormal screening results had a colposcopy with biopsy at the
Center. The results of colposcopic biopsies carried out outside the
facility were not included in the study due to different colposcopic
protocols and/or histologic terminology and/or lack of p16 stain in
cervical histologic specimens. Our study was conducted in a single
private funds-based Center and the results may not be generalized to
other populations. The sample size was another limitation and could
affect the accuracy of the PPV estimates. To confirm our results further
studies with the multicenter design and greater sample sizes are
required.

\textbf{Conclusions}

In conclusion, the evolution of cervical cancer secondary prevention
strategies calls for a comprehensive assessment of clinical practices.
As we move toward more accurate and personalized approaches, such as
primary HPV-based screening with limited genotyping, the necessity of
obligatory colposcopy referral for all major screening abnormalities
warrants careful consideration. The use of DS as a triage test allows
reduction in number of colposcopy referrals while ensuring safety for
the HPV 16/18-positive patients. The strategy incorporating cytology as
the first triage testing might also improve referrals to expedited
treatment in selected HPV-positive cases.

\textbf{Author Contributions:} Conceptualization, Karolina Mazurec,
Maciej Mazurec and Martyna Trzeszcz; Data curation: Maciej Mazurec and
Karolina Mazurec; Analysis of data: Karolina Mazurec; Interpretation of
data: Karolina Mazurec and Maciej Mazurec; Investigation: Maciej
Mazurec, Martyna Trzeszcz and Karolina Mazurec; Methodology: Karolina
Mazurec, Maciej Mazurec and Martyna Trzeszcz; Writing draft: Karolina
Mazurec, Maciej Mazurec and Martyna Trzeszcz; Draft editing: Karolina
Mazurec, Maciej Mazurec and Martyna Trzeszcz; Supervision and review:
Robert Jach; Review: Agnieszka Halon and Joanna Streb. All authors have
read and agreed to the published version of the manuscript.

\textbf{Data Availability Statement} : The data presented that support
the findings are available from the corresponding author upon reasonable
request.

\textbf{ORCID}

Karolina Mazurec https://orcid.org/0009-0000-9993-103X

Martyna Trzeszcz https://orcid.org/0000-0002-9971-1093

Maciej Mazurec https://orcid.org/0000-0003-2152-157X

Joanna Streb https://orcid.org/0000-0003-2147-7177

Agnieszka Halon https://orcid.org/0000-0003-4240-7899

Robert Jach https://orcid.org/0000-0001-5512-763X

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