ANTICANCER RESEARCH 42: 1-12 (2022)

doi:10.21873/anticanres.15451

Review

Prospects of Improving Early Ovarian Cancer

Diagnosis Using Cervical Cell Swabs
EDYTA BISKUP1, REGITZE SOLLING WILS1, CLAUS HOGDALL2 and ESTRID HOGDALL1

1Department of Pathology, Herlev University Hospital, Herlev, Denmark;

2Department of Gynecology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark

Abstract. Ovarian cancer (OC) has the poorest prognosis diagnosed with advanced stage cancer (stage III-IV
and the highest mortality rate among gynecological according to International Federation of Gynecology and
malignancies, which is largely due to delayed diagnosis. Obstetrics; FIGO) (2). Early diagnosis is crucial for long-
Therefore, an effective detection strategy is a compelling term recovery/curative treatment, as the 5-year survival rate
need. Here, we review the potential use of cervical cell differs dramatically between early stage OC (stage I: 93%)
swabs (Pap specimens, liquid) for early detection of OC. It and late stage OC (stage IV: 23%) (2). An efficient, sensitive
has been shown, that malignant cells exfoliate from the worldwide screening program for OC could alleviate the
ovaries and may be detected in Pap specimens, routinely challenges resulting from late diagnosis and improve the
collected through cervical cancer screening. Using Medical survival of the more than 314,000 women diagnosed with
Subject Headings (MeSH) for searching the PubMed OC yearly (3). On the global scale it means 4 in 100,0000
database we identified eight studies reporting the use of Pap women are diagnosed with OC every year. In Denmark this
specimen in early detection of OC. Six focused on detection number is markedly higher than the world average, reaching
of gene mutations, using gene panels or analysis of TP53 approximately 15 cases per 100,000 (2).
variants. Two studies reported analysis of methylation The widely implemented screening program for cervical
profiles. Seven studies were published in 2018 or later. cancer has proven that a population-based screening can be
Additionally, we found one study without MeSH terms feasible and efficient. Cytological examination of cervical
assigned yet, which postulated using peptide biomarkers smear (Papanicolaou or Pap smear) performed every 3-5
present in Pap-test fluid. In this review we present their main years for women aged 35-64 years has yielded an at least
findings, discuss challenges this approach presents and 80% reduction in the incidence of cervical cancer (4).
include ideas for improved detection. However, the optimal screening strategy for OC is yet to be
found; randomized controlled trials, such as the US Prostate,
Ovarian cancer (OC) is the gynecological malignancy Lung, Colorectal and Ovarian study (PLCO) and the UK
characterized by the poorest prognosis and the highest Collaborative Trial of Ovarian Cancer Screening
mortality rate, which is mainly due to a delay in diagnosis (UKCTOCS), have tested screening strategies consisting of
(1). Symptoms of OC are often discrete and non-specific, a combination of CA-125 serum levels and ultrasonography.
which can mislead both patients and healthcare Sadly, the screening trials did not result in significant
professionals. According to the Danish Gynecological reductions in deaths from OC (5, 6). Advances in genomic
Cancer Database (DGCD) more than half of the patients are profiling and epigenetics technologies have provided new
opportunities for different screening strategies. Studies
focused on detection of mutations and DNA methylation
profiling have explored the possibility for a screening
Correspondence to: Edyta Biskup, Herlev og Gentofte Hospital, program for OC similar to the well-established cervical
Afdeling for Patologi, Borgmester Ib Juuls Vej 1, 2730 Herlev, cancer screening program (7, 8). This approach would ensure
Denmark. Tel: +45 50318853, e-mail: edyta.urszula.biskup@
an accessible screening program with no additional
regionh.dk
procedures and a reasonable cost-benefit balance.
Key Words: Ovarian cancer, Pap test, liquid Pap specimen, cervical The cervical cancer screening program is widely accepted
swab, review. in the population, e.g. in Scandinavian countries the rate of

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participation ranges between 64-81% (9). This means that the provided for 13 cases, and 11 out of these 13 cases (85%)
clinical diagnostic framework is already in place in most were staged as IIIA or higher, indicating that it is primarily
high-resource countries and perhaps can be used to advanced OC that is detected through an abnormal cytology
accommodate OC screening as well, applying material in cervicovaginal samples (14). At earlier OC stages the
already collected, without any additional patient-related quantity of material in the cervical canal originating from the
actions. Briefly, the sample collection proceeds as follows: a ovaries or fallopian tubes may be limited, which imposes
cytobrush used for swabbing the endocervical canal is then major technical requirements to new screening strategies. As
placed in a vial of fixative medium. In addition to the Pap the location of cancerous or precancerous cells to the cervix
smear test, performed to detect early cervical abnormalities, is a prerequisite of cervical screening for OC, it also excludes
the liquid from the vial (hence referred to as liquid Pap a subpopulation of women, who had tubal ligation or
specimen) can be used for diagnostics of human hysterectomy. Menon even questioned whether intrauterine
papillomavirus infection or, potentially, molecular testing for device placement may impede screening using smear, which
cancer (8, 10). The main challenge however is to identify an could potentially exclude a major fraction of women
appropriate analysis of the cervical swab/liquid Pap specimen otherwise eligible for screening (16). Yet, the mechanistic
material that meets the requirements of high specificity and foundation of the detection of OC using cervical smear
sensitivity to detect precancerous cells and/or early-stage OC. samples is complex, as pathogenic mutations were found in
In this review, we outline the current research on the use of Pap smear samples from an HGSC patient who already had
liquid Pap specimen for early diagnosis of OC. Studies tubal ligation (17). Another study investigating the feasibility
applying analyses of mutations and DNA methylation of using cervical cell swabs for endometrial and OC
profiling in cervical cell swabs included in this review were screening suggested that the detection of cancer applying
identified as described in Chapter 3: Search strategy. DNA methylation analysis might not depend on physical
Furthermore, we assess the prerequisites of OC screening relocation of cells from the ovary, tubes or endometria, but
using cervical cell swabs, followed by a discussion of the could be a result of a so-called “field effect” (7). By “field
existing results and the future perspectives of OC screening. effect”, the authors meant that changes in DNA methylation
can be found in normal tissue adjacent to tumor tissues,
Premises for OC Screening exemplified by a study finding changes in DNA methylation
in normal cervical tissue in proximity to cancer tissue (18).
A prerequisite for using cervical cell swabs to detect pre- Most of the studies reviewed in the subsequent sections
cancerous/early stage OC is the transit of OC cells to cervical recognize, however, that material from the ovaries and tubes
canal. The cervix, uterus and fallopian tubes are part of the needs to pass freely to the endocervix and have excluded or
same anatomical unit; the ovaries are not directly connected commented on patients with tubal ligation (17, 19, 20).
to the fallopian tubes, but are in close proximity with the
fimbriae (11). Studies have shown that in many cases OC Search Strategy
does not originate in the ovaries but rather in the fallopian
tubes (12). This precursor lesions in the tubes, named serous The literature search was performed in MEDLINE/PubMed
tubal intraepithelial carcinomas (STIC), are especially found using the following search strategy: [Ovarian Neoplasms
in type II high grade serous carcinoma (HGSC), the most (MesH)] AND [Vaginal Smears(MesH) OR papanicolaou
common type of OC (13). As the fallopian tubes test(MesH)].
communicate directly with the uterus, hence the cervix, it is Inclusion criteria was English language and publication after
reasonable to assume that cells from the tubes may reach the 1990. The search yielded 136 unique results, herein: 1 clinical
cervical canal. The extent of cell shedding from the ovary trial, 18 reviews, 37 case reports (as of August 2021).
towards the uterus and cervical canal remains unknown. In a We focused on a detailed review of studies reporting the
review, several case studies (14) have reported how abnormal possible use of cervical cell swabs in early diagnosis of OC.
cervicovaginal cytology can be indicative of OC, thus Studies (n=8) with relevance for this article are outlined in
supporting the notion of cell shedding from the upper Figure 1.
gynecological tract to the lower regions. Abnormal cytology Since some time may pass between publication of an
in relation to OC encompasses the presence of atypical article and assigning of MeSH terms, we also performed a
glandular cells and/or psammoma bodies, which are small manual search to ensure that we do not miss the most recent
calcifications organized concentrically (14). Psammoma publications. Indeed, a manual search for terms: “Pap
bodies can be found in meningioma, thyroid cancer and test/”Pap specimen” and “ovarian cancer”/”ovarian
gynecological cancers, but may also be an incidental finding carcinoma” resulted in one additional record, namely the
in normal smear samples (15). A review enlisted 24 cases of work by Boylan et al., from January 2021, which had no
OC with abnormal cervicovaginal cytology. Staging was MeSH terms assigned yet (21).

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 Biskup et al: Use of Pap Test in Ovarian Cancer Diagnosis (Review)

Figure 1. Ovarian cancer screening using cervical smear/liquid Pap specimen. A premise of this screening strategy is the presence of material from
a cancerous/precancerous lesion in the smear sample as follows: 1) Precancerous/cancerous cells are shed from the fallopian tube. 2) Cells reach
the uterus and the cervical canal. 3) Cells can be collected from the endocervix with a Pap brush or through intrauterine sampling with a Tao
brush. 4) The material may be subjected to analysis of DNA mutations / methylation patterns or protein profiles. (Figure created with the use of
Biorender/licensed to RSW).

Detection of OC Using Cervical Smears depending on their grade, with type I progressing relatively
slowly (low grade serous tumors, mucinous, endometrioid
Genetic aberrations observed in OC can be divided into and clear cell carcinomas) and type II, characterized by a
sequence-dependent (mutations) and epigenetic (such as DNA more aggressive phenotype (high grade serous ovarian
methylation). Of note, sets of genes affected by either carcinoma, HGSC) (23). Between 2 to 8 driver gene
mechanism may overlap, for example both BRCA1/2 mutations are needed for tumorigenesis, and genetic changes
mutations and silencing by methylation have been reported in the form of point mutations as well as structural mutations
(22). Below, we review studies focused on detecting 1) including copy number variation have been reported in OC
mutations or 2) altered methylation profiles in OC cells, using (24, 25). Mutational profile largely depends on OC
cervical cell swabs as diagnostic material, and hence the histological type (24); [an overview, see Figure 1 in (26)].
potential use of cervical smears in early diagnostics of OC. For example, in HGSC the most frequently mutated genes
In Figure 1 the reviewed studies are categorized according are TP53 (up to 96%) and BRCA1/2 (approximately 20%).
to the primary endpoint analysis, although this division is Otherwise, these neoplasms demonstrate a high degree of
somehow simplistic as the studies often include multiple genomic heterogeneity, due to frequent mutations in repair
analysis, for instance analysis of circulating tumor DNA genes, including homologues recombination (HR) pathway
(ctDNA) in plasma or aneuploidy detection. components (24). On the other hand, clear-cell OC rarely
bears TP53 mutations, but is instead characterized by
DNA Mutations in OC aberrations in KRAS (27), ARID1A (28) and PIK3CA (26).
ARID1A (28) and PIK3AC are also commonly mutated in
Malignant ovarian tumors predominantly (in over 90% cases) endometrioid OC, as well as CTTNB1 (29) and PTEN (30).
derive from epithelium and can be classified into two types, Mutations in KRAS is a hallmark of the mucinous subtype

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(over 60%) (31, 32), whereas low grade serous OC often genetic alterations in one of 18 cancer-related genes selected
bears mutations in KRAS and BRAF genes (33). for the diagnostic panel (AKT1, APC, BRAF, CDKN2A,
Genetic (and epigenetic) changes give rise to different cell CTNNB1, EGFR, FBXW7, FGFR2, KRAS, MAPK1, NRAS,
populations, contributing to tumor heterogeneity. This, on one PIK3CA, PIK3R1, POLE, PPP2R1A, PTEN, RNF43, and
hand, poses a therapeutic challenge, since existence of multiple TP53). In 33% of OC patients genetic alterations detectable
tumor clones often means that therapies are effective only to a by PapSEEK in the tumor tissue were also found in cervical
subset of them, and that the surviving cells may lead to a cell swab. This number was further increased if a so-called
refractory recurrence (34). On the other hand, heterogeneity of Tao brush was used instead of a regular Pap brush. The
the tumor and limitations in tumor tissue sampling may result unquestionable strength of this study was the use of a big
in different mutation profiles between cervical smears and patient cohort, which makes its results more representative.
tumor samples, meaning that the smears are not necessarily Moreover, tumor detection was performed using an 18-gene
representative for the lesions they shed from (25). panel, rather than only one gene (TP53). As mentioned
above, TP53 is commonly mutated in HGSC, but rarely in
DNA Mutations in Cervical Cell other histological types. Therefore, limiting diagnostics only
Swabs in Early Detection of OC to TP53 mutation detection may hamper detection of other
OC types, which albeit less common than HGSC, are by no
We have identified six studies analyzing mutations in means less dangerous. Of note, this study also contains some
cervical cell swabs of OC patients (Figure 1). Three of the new ideas (utilizing Tao brush, detection of circulating tumor
studies focus exclusively on TP53 mutations, whereas others DNA) to further improve sensitivity.
utilize gene panels. The latter typically include cohorts of A more recent work by Jiang and coworkers (25) was
both ovarian and endometrial cancer patients. In two of the inspired by the original study by Kinde et al. (8) and its
studies archive Pap specimens were also analyzed. continuation by Wang et al. (20). Like in the latter study, liquid
The first study by Kinde et al. investigated if cervical cell Pap smear specimen, tumor samples and blood samples were
swabs could be used for detection of OC (8). The purpose of collected from patients diagnosed with epithelial OC or
their work was to verify if tumor cells shed from upper endometrial cancer. First, exome sequencing of 127 cancer
gynecological tract malignancy to the cervical canal. Authors related genes recognized by The Cancer Genomic Atlas
assembled a panel of 12 genes commonly mutated in OC (TCGA) as tumor drivers was performed in OC tumor samples.
(AKT1, APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, Based on that, a panel of eight genes (ARHGAP35, ARID1A,
PIK3CA, PPP2R1A, PTEN and TP53), tested for mutations in BRCA1, EGFR, LRRK2, PIK3CA, RAD21, TP53), potentially
tumor tissue and attempted to find the same mutations in most relevant for OC, was selected for analysis of liquid Pap
corresponding liquid Pap smear specimens from the same smear specimens. Mutations in at least one of these eight genes
patient. In 9 out of 22 patients diagnosed with OC (41%), they were detected in 11 out of 11 liquid Pap smear specimens and
successfully detected the same mutations in the Pap smear showed correlation with tumor tissue. Tumor heterogeneity and
samples as in the tumor tissue. However, 41% seems somewhat limited tissue sampling may explain the fact that only in 5 out
low given that the majority (18/22) of the OC patients presented of 10 tumor samples were found mutations corresponding to
advanced stage OC (FIGO III and IV), where shedding from variants detected in liquid Pap smears specimen.
the site of the cancer could be likely. This number is also much The study by Jiang et al. (25) further corroborates the
lower than for endometrial cancers in the same study, where earlier findings and supports the claim that the DNA shed
100% of the liquid Pap specimens bore mutations identical to from OC cells may be present in liquid Pap smear
those found in corresponding tumor tissues. These findings may specimens. Additionally, an interesting strategy by the
reflect the distance between sampling site and tumor site in OC, authors was to use matched patient leukocytes as a self-
which is why authors suggested uterine sampling for the control. Briefly, the exome sequencing of 127 selected genes
purpose of OC screening (8). was performed both in tumor tissue and in leukocytes in
Another work by the same group followed up on their order to account for natural inter-individual variation.
exploratory study, attempting to increase the sensitivity of Most studies regarding diagnosis of OC using liquid Pap
OC detection in cervical cell swabs (20). Altogether, 245 smear specimens focus generally on two aspects: early
women diagnosed with OC were enrolled, along with healthy detection (studies involving archival material collected when
controls and endometrial cancer patients. Patient material in the patient was still symptom-free) and increased sensitivity.
the form of cervical cell swabs, tumor samples and blood The study by Arildsen and coworkers (35) addressed both
samples (for detection of circulating tumor DNA) were issues, analyzing somatic mutations of TP53 in 15 patients
collected. For tumor detection a parameter dubbed diagnosed with HGSC. For all patients, archive liquid-based
“PapSEEK” score was applied. Samples were regarded as Pap specimens were available (taken 20-95 months before
PapSEEK positive if they either contained aneuploidy or the diagnosis), and additionally for 9 of them diagnostic (i.e.,

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concurrent with diagnosis) liquid Pap samples were should not be ascribed to technical resolution, but simply to
collected. Blood samples were also collected to serve as the absence of neoplastic DNA in liquid Pap specimen.
matching self-controls, similarly to (25). In 6 out of 9 These results put into question possible implementation of
diagnostic samples, somatic TP53 mutations were detected, Pap test in OC diagnosis. It seems, authors claim, that the
giving a ratio of 66%. Interestingly, two stage II A patients sensitivity of the Pap test in the current form is most
had mutated TP53 in liquid Pap samples, emphasizing the probably insufficient for detection of OC (17). Apart from
potential of the method for early detection. Moreover, correlating mutations present in tumor tissue with mutations
mutated TP53 was detected in one of the archive samples, detected in Pap samples, Krimmel and coworkers also
taken 20 months prior to the diagnosis. Detecting OC 20 presented an in-depth analysis of types of TP53 mutations.
months earlier would most probably impact prognosis for the First, they generated an in-silico list of all possible mutations
patient. However, it has been hypothesized that mutations in in TP53 coding region and determined their categories as:
TP53 may occur as early as seven years before the diagnosis. frequent in the cancer database, influencing protein activity,
They were though absent from earlier Pap samples collected pathogenicity, if located in exons 5-8 (DNA binding domain)
for the same patient, which may be explained by precursor and/or if located in hotspots. Then, they related the
lesions not shedding cells until later in the tumorigenic distribution of various categories of mutations as predicted
process (35). In the same study, the authors compared the by the model to 1) their actual distribution, found in the
sensitivity of two detection methods, namely regular digital Universal Mutation Database and 2) to their distribution in
droplet PCR (ddPCR; Bio-Rad) and an ultra-sensitive the cohort used in the study (30 women with or without
ddPCR-based method IBSAFE (currently: SAGAsafe®, HGSC who underwent gynecological surgery due to pelvic
SAGA Diagnostics, Lund, Sweden), which may improve masses suspicious; N=9 with HGSC, N=21 with no
100- to 1,000-fold the detection limit. Indeed, IBSAFE neoplasm or benign neoplasms). This analysis revealed that
performed better, even in samples with as little input as 0.17 indeed, likely pathogenic (affecting the protein activity) or
ng DNA. Directly compared to regular ddPCR, it was able pathogenic mutations appeared in all liquid Pap smear
to detect mutated alleles in three Pap samples as opposed to samples, albeit with higher frequency in liquid Pap samples
two for regular ddPCR. These findings show the importance from women diagnosed with HGSC. Mutations infrequent in
of optimizing highly sensitive and specific methods in early cancer were similarly distributed in both groups. These
non-invasive diagnostics of OC (35). findings suggest that, though a wide spectrum of TP53
A recent study by Paracchini at el. (36) provides further mutations can be seen in benign and malignant conditions
evidence that mutated TP53 variants can be detected in Pap alike, it may be possible to use genetic profiling to infer
smears up to six years prior HGSC diagnosis. Their work about pathogenicity of given mutations and, perhaps, devise
focused exclusively on detection of mutated variants of TP53, a computational tool for risk assessment (17).
in a rather small cohort of 17 patients. Interestingly, the authors
used brush-based and dry stored Pap slides rather than liquid DNA Methylation in OC
Pap tests. The median interval between collection of Pap test
slides and the cancer diagnosis was 14.9 months, with DNA methylation is an epigenetic mechanism where gene
interquartile 3.4-35.9 and maximum 68 months. For three expression is altered by methylation of the cytosine
patients more than one sample was available. In 11 out of 17 nucleotide. Several studies have documented that aberrant
patients (65%), the same TP53 mutation was detected in tumor patterns of methylation play a role in carcinogenesis (38).
samples and in the corresponding Pap smears collected within One of the mechanisms is silencing of tumor suppressor
six months before diagnosis or earlier. Of note, in all but one genes by hypermethylation of their promoter regions (39).
patient mutations found in Pap smears were located in TP53 Activation of oncogenes by hypomethylation of oncogene
gene hotspots. More importantly, however, mutated TP53 promoters have also been observed, although this mechanism
variants were detected is some of the archive samples collected is less investigated compared to hypermethylation (40, 41).
25, 27, 49 and 68 months prior to diagnosis. The TCGA Research Network found 168 genes which were
Krimmel-Morrison et al. also based their detection epigenetically silenced due to elevated promoter methylation
strategy on TP53 alone. They compared genetic profiles of in HGSC as compared to normal fallopian tissue (24). Some
TP53 in liquid Pap specimens with HGSC tumor tissue of the most investigated sites of methylation are the promoter
samples and found tumor-derived mutations only in 3 out of regions of BRCA1, RASSF1A, OPCML and P16INK4a (41).
8 patients (37.8%) (17). For sequencing TP53 from Pap test, A study including OC tumors from 50 women staged I to IV
the authors utilized a highly sensitive method, namely showed hypermethylation of the promoter regions of BRCA1
CRISPR-DS, developed previously by the same group and or RASSF1A genes in 68% of the OC cases and in none of
designed for ultra-accurate sequencing with low DNA input the control cases (42). Hypermethylation of the promotor
(37). Therefore, the relatively low positive ratio (37.8%) region of P16INK4A was also significantly associated with

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OC (43). Moreover, a meta-analysis reported a significant databases of samples from OC patients (tissue and cervical
association between promoter methylation of OPMCL and cell swabs) compared to healthy controls, Wu et al. identified
the risk of OC, and this association was also related to stage a panel of genes on the intersection of all three databases, i.e.,
and poor differentiation of tumor (44). 151 genes highly differently methylated in OC compared to
The use of DNA methylation as a tool for OC methylation levels in samples from healthy individuals (19).
screening/early detection was initially applied on circulating Subsequently, the authors narrowed the selection and designed
cell-free DNA in the plasma or serum of OC patients. a model combining three genes (AMPD3, NRN1, TBX15),
Analyses of the methylation of promoters of different genes, which could predict OC incidence with sensitivity of 81% and
among which are BRCA1, RASSF1A, and OPCML in either a specificity of 84%. This model was constructed based on a
the serum or plasma, have shown results with specificity and so-called training (31 OC, 31 healthy patients) and testing set
sensitivity in the range of 69-90% (45-47). Although a study (21 OC and 21 healthy patients). None of these three genes
conducted in relation to the UKCTOCS trial (48) showed has been previously reported in OC and their role remains
that ctDNA could be found in blood samples from up to two largely unknown. The authors questioned whether these genes
years prior to the diagnosis, the general assumption is that are indeed unique to OC or rather present a common pattern
circulating DNA is mainly present in more advanced disease of aberrant hypermethylation found in cancers, but potentially
and not at precancerous stages (49). This could set the case also in benign conditions.
for using sampled specimen in closer proximity to the site of Wu et al. found no differences in methylation between
the cancer such as cervical cell swab, thus increasing test various stages and grades (18). However, they observed a
sensitivity towards early-stage disease. DNA methylation is small, but significant difference between histological subtypes,
ideal for the purpose of screening/early diagnosis as it is with mucinous OC showing less methylation. A recent review
present early in carcinogenesis. Furthermore, the sites are on epigenetics of OC also presents similar findings, with
stable and tissue-specific and tests may be designed to cover HGSC predominantly displaying a hypomethylated phenotype
a limited key targets, which is practical in large-scale while clear cell OC and endometrioid OC being associated
implementation of test regimens (50, 51). with more hypermethylation events (49). The heterogeneity in
methylation profile according to OC subtype may pose a
DNA Methylation in Cervical Cell challenge in screening and early diagnosis of OC.
Swabs for Early Detection of OC The studies by Chang et al. (7) and Wu et al. (19) do not
match the methylation profile of cervical scrapings with the
The idea of using cervical cell swab and DNA methylation corresponding tumor tissue from the same individual, neither
for OC screening is relatively new and only two studies have they include normal tissue samples. Instead, to assess
presented results with this approach. The first study to concordance in methylation profile, both studies include
investigate the use of DNA methylation as a biomarker in cervical scrapings from age-matched healthy individuals.
cervical cell swabs for OC detection is the study by Chang Demonstrating that a methylation “fingerprint” from OC
and coworkers, where they examined hypermethylation of 14 tumor tissue can be found in corresponding cervical cell
selected genes in patients with OC, endometrial cancer and swab would be a significant proof of concept. However, if
healthy controls (ADRA1D, AJAP1, COL6A2, EDN3, EPO, the test is meant to be used for screening of a generally
HS3ST2, MAGI2, POU4F3, PTGDR, SOX8, SOX17, healthy population, it should be able to reliably identify
ST6GAL2, SYT9, and ZNF614) (7). Using quantitative women in high risk for a malignant pelvic mass. Therefore,
methylation specific PCR (qMSP) they identified two performing validation in OC cervical scrapings versus
hypermethylated genes (POU4F3 and MAGI2) for validation samples collected from healthy individuals as done by (52)
in 30 patients with OC, 30 patients with endometrial cancer and (19), rather than in cervical scrapings versus matching
and 30 healthy controls. For OC, the test yielded a sensitivity tumor tissue seems to be well-grounded.
of 61% and specificity of 62-69%. Both sensitivity and On the other hand, if the test is to be applied in a selected
specificity were higher for the detection of endometrial group of patients with gynecological symptoms, the ability
cancer. The authors recognized that the study was mainly to clearly distinguish between benign and malignant
intended as a proof-of-principle due to the small sample size. condition may be of outmost importance.
Using the same target genes for endometrial cancer and OC In general, the two studies further support the potential
broadens the test coverage but may reduce the specificity of use of cervical cell swabs in the diagnosis of OC. It is yet to
the test. Despite its shortcomings, the study demonstrated the be determined if aberrant DNA methylation is a better
feasibility of testing for aberrant DNA methylation in predictor than the analysis of gene mutations in the cervical
cervical cell swabs and warrants further validation. cell swab as described above (8, 20, 25) or it is better to
The other study on DNA methylation by Wu and coworkers combine both approaches to increase sensitivity and
(2019), focused exclusively on OC (18). Analyzing three large specificity. Additionally, one interesting aspect to pursue

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would be the determination of the extent of “field effect” in four years (2018 and later). Though their results are
OC. If there is a methylation pattern in tissue adjacent to encouraging, there is still room for improvement.
cancerous tissue due to the so-called “field effect”, as Most importantly, sensitivity of the detection is far too
previously demonstrated in cervical cancer (18), analysis of low for screening perspectives. Studies presented in the
methylation could be a promising tool for early OC detection current review estimate the proportion of Pap samples where
since it may not depend exclusively on physical relocation OC-related mutations can be detected to be approximately
of cancer cells to the endocervix. 40% (8, 17, 20). This proportion was higher (ca. 60%) in
some studies focused only on detection of TP53 alterations
Other Markers – Peptides in in HGSC (35, 36). This can be partly explained by cancer
the Early OC Diagnosis cells either not being present in the location where the
sample is being collected or not present in sufficient
Interestingly, a different approach has been suggested by amounts. Some of the ideas for remedying these problems
Boylan et al., who tested if OC can be detected with peptides are presented below.
as disease biomarkers. Using mass spectrometry (MS) the
researchers compared protein profiles of three specimens: Modifications of the Sampling Procedure
primary tumor tissue, a cervical swab and a residual cell-free
fixative from a liquid-based Pap-test of one patient with late Optimization of the sampling procedure was initially
stage HGSC. Indeed, they observed a marked overlap between suggested by Kinde et al. (8) and carried out by Wang and
the profiles (2,293 out of almost 5,000 proteins analyzed were co-workers in the testing of a new screening tool (20). They
detected in all three biospecimens). Some known tumor showed an improvement of the sensitivity of the applied test
markers (Nectin-4, UPAR, FOLR) were absent from the tumor (PapSEEK, a multiplex PCR-based test; see above) from
tissue, but present in Pap test fluid and swab. This, however, 33% to 45% using a Tao brush instead of a conventional Pap
can be explained by these proteins being surface markers, brush. The Tao brush collects material from the endometrial
which can be cleaved and shed into body fluids. cavity instead of the endocervix and may be more efficient
Subsequently, authors compared the protein profiles of the in capturing cancerous cells shed from the ovaries or
patient’s specimens with Normal Pap-test Core Proteome, a fallopian tubes. However, introducing a more invasive
database proposed previously by the same group, consisting method collides with the intention of merely extending the
of 153 proteins identified in samples from healthy cervical smear screening program.
individuals (53), and with literature available up-to-date. Other modifications also aiming to collect the diagnostic
Thus, they suggested several candidates, namely: CA125, material from a closer proximity to the ovaries include
mesothelin, LRG, CD44, folate receptor alpha, UPAR, uterine lavage (55) or use of intravaginal tampons (56).
Nectin-4, Kalikrein-10 and -13, as possible OC biomarkers, The uterine lavage technique [as described in (55)]
all of them present in Pap-test fluid. enabled successful detection of TP53 mutations in up to 80%
This study is undoubtedly interesting, however, rather lavage samples (24 out of 30 patients) of HGSC.
preliminary. First, only one patient was included, and this Interestingly, the cohort included two FIGO stage I patients,
person was diagnosed with already advanced (metastatic) and in both cases the mutated variants were detected.
disease. Second, it must be validated to which extent the Moreover, a mutated TP53 variant was also detected in a
peptide profiles of OC patients are unique and to which patient with occult OC. Briefly, the uterine lavage sample
extent they overlap with healthy controls. More extensive was taken prior to risk-reducing salpingo-oophorectomy
studies are necessary to find out if protein biomarkers can (RRSO) performed due to a germline BRCA mutation.
improve the early diagnosis of OC, alone or in combination Histological analysis performed post RRSO revealed the
with genetic testing. Notably, using proteomics in early presence of small lesions in the intraperitoneal cavity and on
diagnosis of OC was also postulated by Barnabas et al. (54) the ovaries, classifying the patient as stage IIIB. At that
This group, however, used uterine lavage rather than liquid point, patient’s CA-125 and transvaginal ultrasound
Pap specimens. remained inconspicuous, though the tumor-specific variants
were present in the lavage fluid.
Early OC Diagnosis – New Strategies This method has a high diagnostic potential but is not very
and Ideas for Improvement feasible neither comfortable for the patient and bears a
theoretical risk of infection.
Extending the use of cervical cell swabs to diagnosis of OC Another solution, potentially more convenient and less
is a relatively new approach. Apart from the seminal work invasive than lavage, is the use of intravaginal tampons. They
by Kinde and coworkers (8), which was published in 2013, constitute an easily available and broadly accepted hygiene
eight out of nine studies reviewed by us come from the last product. A small pilot study by Eriksen and coworkers (56)

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 ANTICANCER RESEARCH 42: 1-12 (2022)

presents its possible utility. From the original cohort of 33 patients, since it is commonly mutated in HGSC, and HGSC
patients, enrolled with pelvic mass suspicious for malignancy is responsible for about 70% of OC-related deaths (24).
and with planned diagnostic or therapeutic surgery, only 5 Diagnosis of other histological types would most probably
fulfilled all the criteria (confirmed HGSC diagnosis, sufficient require sequencing of cancer-specific gene panels [such as
DNA yield in the tampon and the tumor sample, no tubal those proposed by (20) or (25)]. Sequencing costs keep
ligation). TP53 mutation profiles were compared between decreasing and next generation sequencing becomes more
tumor tissue samples and DNA samples obtained from feasible and sensitive. However, limiting targets of interest
tampons placed in the vagina 8-12 hours before a scheduled would greatly facilitate the diagnostic procedure.
surgery. In 3 out of 5 (60%) participants the tumor and This can be partly addressed by implementation of
vaginal DNA harbored the same TP53 mutations (56). methylation profile analysis along with mutation analysis.
The use of intravaginal tampons presents an attractive way Identifying sites commonly methylated would give a lower
of specimen collection, as it does not require a medical number of variants than in the case of somatic mutations for
professional and can be easily used for serial sampling. the same sequence. In the human genome there are
However, additional studies are necessary to verify the approximately 28.3×106 CpG sites (two levels of magnitude
preliminary findings, especially in the context of early less than there are base pairs) (57), and each of them can
diagnosis. take only one of two states (methylated or not). Identifying
sites in which the methylation status commonly differs
Increasing Sensitivity of Analyses between normal and cancer tissue, between benign and
malignant tumors or between histological types would
The amount of exfoliating OC cells is small, especially in greatly facilitate diagnosis. Further knowledge on
those parts of the gynecologic tract that are anatomically methylation profiles in OC could be obtained through
distant from ovaries, calling for ultra-sensitive detection analysis of samples from large biobanks. That would
methods. It is disputable to which extent cancer cells even hopefully contribute to identification of diagnostic targets in
are present in liquid Pap specimen (17) or in samples cervical cell swabs and help creating a screening strategy
collected from the cervix or uterine cavity by one of the with an optimal coverage of most OC subtypes.
other aforementioned ways (Tao brush, lavage, tampons). An example of a potentially promising diagnostic strategy
However, no matter the sampling strategy, the right tools can is that presented in the study by (58). Using Infinium
improve the detection. It has been for example demonstrated MethylationEPIC BeadChip array the authors identified 84
by Arildsen et al., comparing the use of standard ddPCR loci differently methylated in malignant and benign ovarian
with IBSAFE [see above (35)] and by Maritschnegg et al., diseases. These exploratory results must be further validated
who demonstrated that standard NGS often fails detecting in a larger cohort of cervical cell swabs before any strong
mutated variants, that more sensitive methods (SafeSeqS, conclusion can be drawn. These results may help in the
ddPCR) are more useful (55). Therefore, the progress in identification of new biomarkers for differentiating between
early OC diagnostics should also go in the direction of malignant and benign pelvis disease.
designing highly sensitive detection methods. Optimization of diagnostic algorithms may to some extent
address the issue of insufficient sensitivity of detection. That
Selecting the Most Optimal Targets could include adding other OC-related genes to diagnostic
gene panels, identifying methylation loci unique for tumor
Increased sensitivity of detection can potentially improve or implementing new markers, such as microRNA profiling
early diagnosis; however, the number of mutated variants MS-based proteomics. However, it is also plausible that the
poses a therapeutic challenge. The haploid genome contains low detection rate does not result from shortcomings of the
approximately 3.1×109 base pairs, and, theoretically, each detection tools, but the very biology of OC. If it is evidenced
base can be replaced by one of the other three. Additionally, that indeed only a proportion of OC sheds cells and/or DNA
parts of the genomic sequence can be deleted or moved, in sufficient quantities into the uterine/cervical cavity, the
further expanding the already broad spectrum of variants. Pap test should most probably be supplemented with some
Some of these alterations can result in benign mutations, other type of diagnostic specimen.
while some may be pathogenic.
Screening assays in the whole population, where no tumor Other Types of Diagnostic Specimens –
reference tissue is available, would require sequencing of ctDNA and Liquid Biopsies
cancer-related genes, followed by annotation to the reference
genome, identifying mutations and inferring about their Detection of circulating tumor DNA (ctDNA), i.e., a cancer-
pathogenicity from available databases. Sequencing of TP53 originating component of circulating cell free DNA
alone would allow catching a significant proportion of OC (ccfDNA), present in blood samples, may be considered a

8
 Biskup et al: Use of Pap Test in Ovarian Cancer Diagnosis (Review)

potentially promising approach. Locke et al. postulated that detection methods are utilized. Improvement of the
it could address the problem of tumor heterogeneity and diagnostic algorithms will hopefully further increase
tissue sampling, which make specimens not representative sensitivity and specificity of testing.
for tumor, thus providing a misleading image. ctDNA may
help capturing the true variation of samples and be Conflicts of Interest
representative for broader population of tumor cells.
Moreover, using liquid biopsies and ctDNA could be a The Authors declare no conflicts of interest in relation to this study.
solution when collecting a biopsy is hard or impractical, like
during advanced metastatic disease (50). Authors’ Contributions
The downside of using ctDNA for the diagnostics may be
EB: Conceptualization, Writing - original draft. RSW:
the fact that it is present in very low amounts and does not give Conceptualization; Writing - original draft. CH: Writing - review &
any information about the tumor location or its architecture. editing. EH: EB: Conceptualization, Writing - review & editing.
However, ctDNA can still be considered as a diagnostic tool
for example in conjunction with cervical cell swabs. References
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19, 2017. PMID: 28251877. DOI: 10.1186/s12862-016-0864-0 Accepted October 25, 2021

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