Oral Oncology 67 (2017) 103–108

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Oral Oncology
journal homepage: www.elsevier.com/locate/oraloncology

Sensitivity of tumor surface brushings to detect human papilloma virus

DNA in head and neck cancer
Barbara Kofler a,⇑, Wegene Borena b, Claudia Manzl c, Jozsef Dudas a, Anne-Sophie Wegscheider c,
Pidder Jansen-Dürr d, Volker Schartinger a, Herbert Riechelmann a
a
Department of Otorhinolaryngology, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria
b
Division of Virology, Department of Hygiene, Microbiology, Social Medicine, Medical University of Innsbruck, Peter-Mayr-Strasse 4b, 6020 Innsbruck, Austria
c
Department of Pathology, Medical University of Innsbruck, Müllerstrasse 44, 6020 Innsbruck, Austria
d
Institute for Biomedical Ageing Research, Medical University of Innsbruck, Rennweg 10, 6020 Innsbruck, Austria

a r t i c l e i n f o a b s t r a c t

Article history: Objective: Human papilloma virus (HPV) induced head and neck squamous cell carcinoma (HNSCC) rep-
Received 7 November 2016 resents a distinct tumor subset. We questioned how accurately a brushing from the tumor surface detects
Received in revised form 31 January 2017 HPV in patients with HNSCC.
Accepted 13 February 2017
Materials and methods: Brushings from the tumor surface were compared with HPV DNA isolation from
Available online 23 February 2017
formalin-fixed and paraffin-embedded (FFPE) tumor biopsies, which served as the reference standard. In
both matrices, HPV DNA was detected using a commercially available test kit. In addition, p16 was
Keywords:
assessed in tumor biopsies by immunohistochemistry (IHC). The tumors were considered p16 positive
Brush test
Head and neck cancer
if 70% or more of cancer cells expressed p16.
p16 Results: 93 patients with HNSCC were included. Sensitivity and specificity of the brush test were 83% (95%
Human papillomavirus CI: 67–92%) and 85% (95%CI: 72–93%). Results of p16 IHC were concordant with FFPE samples DNA deter-
minations in 73/93 patients. In 53 patients (57%) the tumor was located in the oropharynx and in 40
patients (43%) the tumor was located in the non-oropharynx region. Sensitivity and specificity of the brush
test in patients with oropharyngeal cancer was higher with 86% (95%CI: 70–95%) and 89% (95%CI: 65–99%).
Conclusion: Superficial brushes from the tumor surface may be used to identify HPV positive HNSCC.
Ó 2017 Elsevier Ltd. All rights reserved.

Introduction the host genome by integration or in a non-integrated episomal

About 85% of adults will have a human papilloma virus (HPV)
infection in their life, but not all infections transform in a carci-
noma, which develops from a persistent HPV induced lesion. In
most cases the infection is transient and eliminated by the immune
system [1]. HPV driven and HPV independent cancer of the head
and neck have a different demographic, clinical, molecular and
genomic entity [2]. The prevalence of this cancer has particularly
increased in North America and Europe. Men younger than
60 years are frequently affected [3]. In 2002, zur Hausen reported
that HPV infects epidermal or mucosal epithelial cells that are able
to proliferate [4]. In head and neck squamous cell carcinoma
(HNSCC) HPV infection has a specific tropism to tonsillar epithe-
lium located in the oropharynx. This is probably due to physiolog-
ically exposed reticulated epithelial cells in tonsillar crypts, which
are susceptible to HPV infection [5]. Virus DNA can interact with
⇑ Corresponding author.
E-mail address: ba.kofler@tirol-kliniken.at (B. Kofler).
state. Integration of the viral genome of high risk (HR) HPV usually
leads to unregulated transcription of the E6 and E7 oncoproteins
[6]. The molecular biology of HPV induced oncogenesis is based
on these two viral oncoproteins, which inhibit key tumor suppres-
sors. The viral oncoprotein E6 induces degradation of p53 and E7
inactivates the retinoblastoma protein (pRb). The inactivation of
pRb through E7 leads to an upregulation of functionally inactive
p16, normally a tumor suppressor protein [7].
The mainstay of HPV diagnostic is the detection of viral nucleic
acid in biopsy specimens [8]. Hybrid Capture IIÒ – a DNA detection
method via signal amplification – has been used as a reference
method for HPV diagnosis [9]. However, in the last few years, a
number of PCR based molecular tests coupled with genotype speci-
fic hybridization have been introduced [10]. In cervical cancer it
seems that the detection of HPV DNA alone cannot distinguish
between a transient infection and oncogenic viral persistence [6].
Adding additional biomarkers such as p16 immunohistochemistry
(IHC) may help to differentiate inactive, non-oncogenic episomal
HPV DNA from oncogenic integrated DNA [11]. HPV DNA combined

http://dx.doi.org/10.1016/j.oraloncology.2017.02.013
1368-8375/Ó 2017 Elsevier Ltd. All rights reserved.

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104 B. Kofler et al. / Oral Oncology 67 (2017) 103–108
with p16 is therefore a frequently used marker to detect oncon-
genic active HPV infection in oropharyngeal squamous cell carci-
noma (OPSCC) [12].
Broglie and coauthors collected liquid-based brush cytology
specimens prospectively during panendoscopy in 51 patients diag-
nosed with OPSCC. From the cell suspension, PCR based HPV DNA
testing and p16 IHC were performed. The accuracy, sensitivity,
specificity, positive predictive value, and negative predictive value
of brush cytology to identify HR HPV DNA positive and p16 positive
OPSCC samples were 88%, 83%, 94%, 95%, and 81%, respectively. The
authors concluded liquid-based brush cytology specimens from
oropharyngeal lesions to be a reliable method to identify patients
with HR HPV OPSCC [13].
Dang and coauthors used oral rinses for HPV detection in 76
patients with OPSCC, 24 patients with non-OPSCC and 110 healthy
persons. In patients with OPSCC and with non-OPSCC HPV was
more likely to be detected than in the healthy control [14]. HPV
oral rinse has also been used as a diagnostic method in a large
cross-sectional prospective cohort based study on the oral rinse
protocol [15] that has been administered in other studies [16,17].
In this study we examined surface swabs (brush test) from clin-
ically suspected upper aerodigestive tract malignancies. We ques-
tioned how PCR based detection of HPV viral genome in the brush
test compares with PCR of formalin-fixed and paraffin-embedded
(FFPE) biopsies from the same lesion. PCR of FFPE biopsies served
as the reference method. Sensitivity and specificity of HPV detec-
tion from the brush test should be estimated.
Methods
In- and exclusion criteria
Patients with clinical suspicion of incident HNSCC treated at the
Department of Otorhinolaryngology – Head & Neck Surgery, Medi-
cal University of Innsbruck were prospectively included between
June 2014 and January 2016. Patients were selected aiming to
obtain a 2:1 ratio of oropharyngeal tumors and tumors from other
sites. Patients were excluded if the histopathological examination
revealed no HNSCC. The study was approved by the ethics commit-
tee of the Medical University of Innsbruck (AN2014-0181 338/4.10).
Specimen harvest and handling
All patients underwent routine examination under anesthesia
and panendoscopy (EUAP) during diagnostic workup. Panen-
doscopy included tracheobronchoscopy, esophagoscopy and laryn-
gopharyngoscopy. Using a cytology brush (digeneÒ HC2 DNA
Collection Device, Qiagen, Hilden, Germany), the surface of the
tumor lesion was vigorously brushed several times. The brushes
were then kept in a sterile container in 0.05% sodium azide and
sent to the Division of Virology, Medical University Innsbruck.
Then tumor biopsies were obtained, fixed in formalin and sent to
the Department of Pathology, Medical University Innsbruck, for
routine histopathological examination and DNA extraction for
HPV examination. An additional tumor biopsy was kept in cell cul-
ture medium and immediately sent to the Laboratory for Molecular
Biology and Oncology, Department of Otorhinolaryngology–Head
& Neck Surgery for p16 immunohistochemistry.
DNA isolation from formalin-fixed and paraffin-embedded (FFPE)
samples
Tumor tissue was fixed in 4% formalin in PBS, paraffin embed-
ded and examined by an experienced pathologist. Areas containing
the highest amount of tumor cells were marked on H&E stained
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slides and macro-dissected for DNA isolation. Genomic DNA was
extracted using EZ1 DNA easy kit (Qiagen, Hilden, Germany) as rec-
ommended by the manufacturer [18]. Briefly, up to three 5 mm
tissue-slices per specimen were used. Samples were lysed at
75 °C for 5 min, followed by a digestion step using proteinase K
solution (600 mAU/ml) at 56 °C overnight. After a centrifugation
step, samples were subsequently processed on the EZ1 workstation
(Qiagen, Hilden, Germany) as recommended. Quality and
concentration of isolated DNA was evaluated by photometric anal-
ysis and specimens were stored at 4 °C until used for HPV
evaluation.
Processing of brush specimens
Nucleic acid extraction was performed within two days of arri-
val of the samples to the laboratory. DNA extraction was per-
formed in a fully automated manner (NucliSensÒ easyMAGÒ,
Biomerieux) [19,20]. A total of 500 ml of the sample was pipetted
into a disposable well. After an initial step of cell lysis, the nucleic
acid component was isolated from the mixture using magnet silica
particles resulting in a total of 110 ml purified DNA extract. Approx-
imately 5-10 ml of this extract was used for HPV DNA detection and
genotyping.
DNA-Amplification and HPV-Genotyping
Real-time PCR was used for the detection of HPV DNA based on
the amplification of the L1 open reading frames (ORF). A PCR for
the house keeping gene beta globin was performed parallel as
internal control for the availability of cellular material. HPV DNA
was considered positive if the fluorescence signal appeared before
the fortieth cycle [8]. In a second step, all HPV positive samples
were further genotyped after an amplification step using reverse
line blot hybridization on nitrocellulose membrane strips contain-
ing genotype specific probes (AmpliQuality HPV-TYPE EXPRESS, AB
AnaliticaÒ, Padova, Italy) [21]. This genotyping kit is able to iden-
tify 40 different HPV types namely, 6, 11, 16, 18, 26, 31, 33, 35,
39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66,
67, 68a/b, 69, 70, 71, 72, 73, 81, 82, 83, 84, 87, 89, and 90 (AB Ana-
liticaÒ, Padova, Italy). Contamination due to carry-over was mini-
mized by using dUTP/UNG system which degrades non-specific
residual RNAs. The detection limit of the test kit is 1000 viral copies
per ml at least for HPV 16 and HPV 6, which is in accordance with
the required detection limit by WHO and international HPV refer-
ence center [22]. Based on International Agency for Research on
Cancer classification, the HPV genotypes were classified as follows
[23,24]: established high risk: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56,
58, 59, probably high risk: 26, 53, 66, 68, 73, 82, established low
risk: 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81.
Immunohistochemistry
Five-micrometer thin paraffin sections were dewaxed and anti-
gens were retrieved in an automated staining system (Ventana,
Discovery, Tucson, AZ, USA). Commercial diagnostic assays were
used for p16 detection (CINtecÒ Histology V-Kit, Roche diagnostics,
Basel, Switzerland). Staining was completed using universal sec-
ondary antibody solution, hematoxilin counterstaining and the
DAB MAP Kit (all Ventana products). Tumor cell areas were evalu-
ated by one experienced observer. Specimens were judged p16
positive if 70% of the cells in tumor areas revealed immunohis-
tochmical reaction products. Staining of fibroblastic stroma cells
was not counted.
 B. Kofler et al. / Oral Oncology 67 (2017) 103–108 105

Data analysis
The frequencies of high risk HPV DNA detection in samples
obtained by brush cytology from HNSCC tumor surfaces and of
high risk HPV DNA detection from FFPE biopsy specimens were
compared. High risk HPV DNA detection from FFPE samples served
as the reference method. For both methods, a binary outcome vari-
able (positive/negative) was obtained. Standard diagnostic accu-
racy measures were calculated [25]. Cohen’s kappa was used as a
measure of concordance and 95% confidence intervals were esti-
mated by bootstrapping. Moreover, the outcomes of HPV DNA
assessments were correlated with the binary outcome of p16
assessment of biopsy specimens and were analyzed using the same
methods. SPSS Statistics 22 software (IBM Corporation, Armonk
NY) was used for data analysis.
Results
Study population
was 0.67 (95% CI 0.52 to 0.81). Sensitivity of the brush test was
83% (95%CI: 67–92%) and specificity was 85% (95%CI: 72–93%).
High risk HPV detection and p16 immunohistochemistry
Of the 93 examined patients with HNSCC, 42 were p16 IHC pos-
itive and 51 were p16 IHC negative. Comparing the results of p16
immunoreactivity with the results of FFPE specimens as reference,
20/93 results were discordant (Table 3). In detail, 9/40 HPV posi-
tive FFPE specimens were p16 IHC negative and 11/53 HPV nega-
tive FFPE specimens were p16 IHC positive. Cohen’s kappa was
0.57 (95% CI = 0.40–0.72), sensitivity was 78% (95% CI = 62–89%)
and specificity was 79% (95% CI = 66–89%).
The 3 diagnostic methods, FFPE biopsies, brush test and p16 IHC
yielded concordant results in 67/93 patients. In 30 patients, all 3
diagnostic methods were HPV positive and in 37, all were HPV neg-
ative. In 8 patients, only p16 IHC was positive. In 6 patients, HPV
DNA was positive in FFPE specimens while the results of the brush
test and p16 IHC were both negative and in 5 patients only the
result of the brush test was HPV positive.

During the study period, 93 patients with HNSCC were

included. Of these, 67 patients were male, 86 patients were first
diagnosed with HNSCC and 5 patients had a second primary cancer
in the head and neck region. The cancer was located at following
sites: oropharynx 53 patients, oral cavity 17 patients, larynx 17
patients and hypopharynx 6 patients. According to the 7th edition
of the UICC staging system 22 patients had stage 1 disease, 8
patients stage 2 disease, 8 patients stage 3 disease, 44 patients
stage 4a, 7 patients UICC stage 4b and 4 patients UICC stage 4c dis-
ease. Patient demographic and disease data are detailed in Table 1.
Detection of HPV DNA from brush test and FFPE biopsies
HPV DNA detection from FFPE biopsies served as the reference
method. In all FFPE specimens sufficient DNA could be extracted.
FFPE biopsies were HPV positive in 40 patients and negative in
53 patients. Also, all brush tests contained sufficient DNA. Brush
tests were HPV DNA positive in 41 patients and HPV DNA negative
in 52 patients (Table 2). Cohen’s kappa measure of concordance
HPV-Genotyping
The most common Genotype was HPV 16, which was found in
37 patients. HPV 16 was positive in 28 patients in both diagnostic
methods (brush test and HPV DNA from FFPE specimens) and in 9
patients in a single diagnostic method (7 patients in HPV DNA from
FFPE specimens/2 patients in brush test). The second most found
high risk virus was HPV 35, which was detected in 4 patients (3
patients in both diagnostic methods/1 patient in HPV DNA from
FFPE specimens). Furthermore, HPV 6 was positive in 3 patients
in one diagnostic method (HPV DNA from FFPE specimens) and
HPV 18 was positive in 2 brush tests. HPV 58 was found in 2
patients, in 1 patient in both diagnostic methods and 1 patient in
a single diagnostic method (HPV DNA from FFPE specimens).
HPV 40, HPV 51, HPV 54, HPV 56, HPV 61, HPV 62, HPV 66, HPV
70, HPV 73 and HPV 90 were positive in 1 patient each diagnosed
by the brush test (Table 4).
Oropharyngeal squamous cell carcinoma

32 of 53 patients with OPSCC were p16 IHC positive, which are

Table 1
60% of this patient collective. Furthermore 28 patients of the p16
Study population.

Gender Table 2
Male 67 Brush test compared with the reference method. Crosstabulation of HPV determina-
Female 26 tions in brushing obtained from the tumor surface (brush test) and DNA isolation
from formalin fixed and paraffin-embedded samples (FFPE). Taking FFPE samples as
Age during initial diagnosis the reference method, the sensitivity of the brush test was 83% and the specificity was
50 9 85%.
51–60 34
61–70 34 Brush test FFPE samples
71–80 12
Negative Positive Total
>80 4
Negative 45 7 52
Location of tumor
Positive 8 33 41
Oropharynx 53
Total 53 40 93
Oral cavity 17
Larynx 17
Hypopharynx 6 Table 3
UICC stage P16 IHC compared with the reference method. Crosstabulation of p16 immunohis-
UICC 1 22 tochemistry (IHC) determinations in tumor samples and DNA isolation from formalin
UICC 2 8 fixed and paraffin-embedded samples (FFPE). Taking FFPE samples as the reference
UICC 3 8 method, the sensitivity of p16 IHC was 78% and the specificity was 79%.
UICC 4a 44
P16 IHC FFPE samples
UICC 4b 7
UICC 4c 4 Negative Positive Total
p16 Negative 42 9 51
Negative (70%) 51 Positive 11 31 42
Positive (70%) 42 Total 53 40 93

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106 B. Kofler et al. / Oral Oncology 67 (2017) 103–108

Table 4 ity and specificity for the brush test in this patient collective was
HPV Genotypes in the brush test and FFPE samples. Crosstabulation of HPV Genotypes 86% (95%CI: 70–95%) and 89% (95%CI: 65–99%).
classified in high risk, probably high risk and low risk and the number of HPV
detection of the different HPV genotypes in HPV brushing from the tumor surface
(brush test) and DNA isolation from formalin fixed and paraffin-embedded samples
(FFPE), as well as only in the brush test and only in FFPE samples. Non-oropharyngeal squamous cell carcinoma
HPV genotypes + in brush + only + only
test and in brush in FFPE In the non-OPSCC group 10 of 40 patients were p16 IHC posi-
FFPE samples test samples tive, which are 25% of this patient collective. HPV DNA in the
High risk: HPV 16, 18, 35, 51, 56, 58 32 4 8 p16 IHC positive non-OPSCC group was found in 5 patients in the
Probably high risk: HPV 66, 73 2 brush test and in 3 patients in FFPE biopsies (Table 6).
Low risk: HPV 6, 11, 40, 54, 61, 70 4 4

Discussion
Table 5
Results of diagnostic methods on patients with OPSCC. Crosstabulation of p16 In this study we questioned if HPV DNA detection from the
immunohistochemistry (IHC) determinations in tumor samples, DNA isolation from brush test obtained from tumor surface in patients with HNSCC
formalin fixed and paraffin-embedded samples (FFPE) and the brush test in patients
yields reasonable results. For HPV DNA detection and genotyping
with oropharyngeal squamous cell carcinoma (OPSCC).
from the brush test, commercially available test kits for real time
P16 IHC FFPE samples Brush test PCR and reverse line blot hybridization were used [26]. The detec-
Negative Positive Negative Positive tion limit of the test kits is below 1000 viral copies per ml. HPV
Negative 14 7 17 4 DNA detection in DNA obtained from formalin-fixed and
Positive 4 28 4 28 paraffin-embedded (FFPE) diagnostic biopsies using the same test
Total 18 35 21 32 kits was used as the reference method. Therefore, this study com-
pares different sampling and DNA extraction methods using iden-
tical detection methods.
FFPE biopsies were high risk HPV positive in 40/93 patients. In
detail, biopsies from the oropharynx were high risk HPV positive in
35/53 patients and biopsies from non-oropharyngeal sites in 5/40
patients (p < 0.001). The brush test from tumor surfaces had a sen-
sitivity of 83% (95%CI: 67–92%) and specificity of 85% (95%CI: 72–
93%) when compared with FFPE specimens derived viral DNA
determination. In patients with OPSCC the brush test could reach
even a higher sensitivity and specificity with 86% (95%CI: 70–
95%) and 89% (95%CI: 65–99%).The brush test may thus serve for
preliminary categorization of HNSCC.
In addition to HPV detection from FFPE specimens and brush
test determinations, p16 IHC was obtained in all patients, 42 were
p16 IHC positive and 51 were p16 IHC negative. In a number of
patients, the 3 diagnostic techniques yielded inconsistent results,
compared with the reference method, 20/93 results were discor-
dant. For 8 patients, of which 3 patients with oropharyngeal can-
cer, 3 patients with oral cancer and 2 patients with laryngeal
Fig. 1. Concordant results of the HPV DNA positive brush test in patients with cancer, p16 IHC was the only positive test result. HPV independent
oropharyngeal squamous cell carcinoma (OPSCC). Venn diagram of HPV positive
p16 overexpression in HNSCC may be due to inactivation of pRb as
results on patients with OPSCC. In the brush test 32 patients are HPV DNA positive,
35 patients are HPV DNA positive in FFPE specimens and 32 patients are p16 IHC a result of gene deletions, point mutations, functional mutations,
positive. The HPV DNA positive brush test reaches 30 concordant results with the or other mechanisms of pRb pathway deregulation [27]. It has been
HPV detection from FFPE specimens and 28 concordant results with the p16 IHC. All reported that the loss of pRb function enables the bypass of p16
3 diagnostic methods obtain 27 concordant results of HPV positivity. mediated cell cycle inhibition and loss of pRb generates an onco-
genic stress that could lead to the activation of p16 expression
Table 6 [28]. Irrespective of the mechanism, presumably any tumor har-
Results of diagnostic methods on patients with non-OPSCC. Crosstabulation of p16 boring high levels of p16 has inactivated pRb in order to facilitate
immunohistochemistry (IHC) determinations in tumor samples, DNA isolation from
tumorigenic proliferation [29]. It is controversially discussed if p16
formalin fixed and paraffin-embedded samples (FFPE) and the brush test in patients
with non-oropharyngeal squamous cell carcinoma (non-OPSCC). is a prognostic indicator on its own [30].
P16 IHC was negative in 14 patients with positive HPV DNA in
P16 IHC FFPE samples Brush test
FFPE specimens or in brush test. In these tumors, episomal, tran-
Negative Positive Negative Positive scriptionally inactive HPV DNA may have been detected by PCR,
Negative 28 2 26 4 but had not resulted in p16 overexpression [31]. In the 6 and 5
Positive 7 3 5 5 samples, which were only HPV positive in FFPE specimens or in
Total 35 5 31 9
the brush test, a contamination or erroneous amplification cannot
be excluded [32],[33]. We assume that some of the 6 FFPE samples
were p16 IHC negative due to episomal HPV localization and the
IHC positive OPSCC group are positive for HPV DNA detection from brush test was negative, because the number of viral copies
the brush test and also 28 of these patients are HPV DNA positive obtained by brushing was too low [32],[34]. Low virus copy num-
in FFPE biopsies (Table 5). The brush test could reach concordant bers in brushings may be due to a low number of brushed cells, tis-
results with the reference method in 30/35 HPV DNA positive FFPE sue necrosis at the tumor surface or by the heterogeneous
specimens and in 28/32 p16 IHC positive patients (Fig. 1). Sensitiv- distribution of the virus with in a tumor tissue [35].

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 B. Kofler et al. / Oral Oncology 67 (2017) 103–108
Using a similar study design, Broglie and coauthors observed a
sensitivity of 100% and a specificity of 74% of brushings obtained
from the tumor surface of 41 patients with oropharyngeal HNSCC
when compared with HPV DNA from the tumor tissues. The
authors observed that false negative cases were associated with a
low cellularity of brush cytology samples [13]. In another recent
study, brush sampling yielded inadequate samples in 16/35
patients with oral or oropharyngeal lesions including various
non-cancerous pathologies. Brushings and DNA from biopsies
yielded consistent HPV detection in 18/19 patients, but were pos-
itive in only 2/19 brush tests with the genotypes HPV 16, and HPV
52 [34].
The most frequently observed HPV genotype in our study was
HPV 16, which was found in 36 patients and is in line with other
reports [36]. The second most common genotype was HPV 35
(n = 4), HPV 6 (n = 3) and HPV 18 (n = 2). Interestingly, in the brush
test a higher variety of genotypes was found (Table 4) than in the
FFPE specimens. This could be caused by touching different and
also healthy tissue during the brushing process. In the 40 non-
OPSCC patient group 5 HPV positive FFPE specimens were found,
in 3 of these patients the brush test was positive for the same
genotype.
The study was based on the Standards for Reporting of Diagnos-
tic Accuracy criteria 2015 [37]. However, there are several limita-
tions. The weakest point is that the patient population is not
representative for HNSCC patients. Initially we included only
patients with oropharyngeal HNSCC. We then realized that we
did not get enough HPV negative samples. Therefore, we started
to include patients with HNSCC at other sites (Table 1). However,
this did not lead to relevant bias. Moreover, several surgeons were
involved in sampling, but all were instructed how to collect sam-
ples. In this study, all brushings were obtained under general anes-
thesia. It is not clear, if similar accuracies may be achieved in
brushing from awake patients.
In conclusion, the brush test from the tumor surface of HNSCC is
a suitable, non-invasive sampling method to detect HPV DNA. It is
useful to obtain quick information about the HPV status in HNSCC
patients, but like HPV DNA detection in FFPE specimens, it is not
sufficient to identify HPV driven HNSCC. In synopsis with p16
determinations, HPV brushings were not inferior to FFPE speci-
mens’ determinations. If it is for calculatory convenience assumed
that oropharyngeal tissues of healthy persons are high risk HPV
free [38] and that the incidence of OPSCC is 10/100,000 and that
all oropharyngeal carcinomas were HPV positive, the positive and
negative predictive values of oropharyngeal brushings in a general
population are close to zero. It is thus questionable, if brushings to
screen for HPV associated carcinomas in a healthy population are
reasonable.
Conflict of interest statement
Authors declare that they have no conflict of interests. The
study has not been submitted to any other journal and is not con-
sidered for publication elsewhere.
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