Polyomavirus JCPyV infrequently detectable in adenoid cystic carcinoma of the oral cavity and the airways

Abstract

Our objective was to assess the presence of three polyomaviruses, namely SV40, JCPyV, and BKPyV, and human papillomaviruses (HPV) in adenoid cystic carcinomas (ACC) of the minor salivary glands (MiSG) in the head and neck region. The study comprised 68 MiSG ACC patients operated during 1974–2012 at the Helsinki University Hospital (Helsinki, Finland). Medical records and 68 histological samples were reviewed. Polyomaviruses were detected with quantitative PCR and the DNA-positive samples were further analyzed for the presence of viral tumor T antigen (T-ag) with immunohistochemistry. HPV genotyping was performed with a Multiplex HPV Genotyping Kit. Only JCPyV DNA was found in ACC samples, being present in 7 (10.3%) out of the 68 samples. The viral load of JCPyV was low varying between 1 to 226 copies/μg DNA. The JCPyV-positive samples originated from trachea (two samples), paranasal sinuses (one), and oral cavity (two). Additionally, JCPyV positivity was found in one lung metastasis of a tracheal tumor and one local disease failure of an oral cavity tumor. Three JCPyV DNA-positive samples showed weak nuclear staining for large T-ag. In conclusion, only JCPyV but not SV40, BKPyV, or HPV was found in ACC from the upper and lower airways. JCPyV copy numbers were low which might support its role as a “hit and run agent” in ACC carcinogenesis.

Full text

ORIGINAL ARTICLE
Polyomavirus JCPyV infrequently detectable in adenoid cystic
carcinoma of the oral cavity and the airways
Hanna Hämetoja
1,2
&Jaana Hagström
2,3
&Caj Haglund
3,4
&Leif Bäck
5
&Antti Mäkitie
5,6,7
&Stina Syrjänen
1
Received: 29 March 2019 / Revised: 13 June 2019 /Accepted: 25 June 2019
#The Author(s) 2019
Abstract
Our objective was to assess the presence of three polyomaviruses, namely SV40, JCPyV, and BKPyV, and human papilloma-
viruses (HPV) in adenoid cystic carcinomas (ACC) of the minor salivary glands (MiSG) in the head and neck region. The study
comprised 68 MiSG ACC patients operated during 1974–2012 at the Helsinki University Hospital (Helsinki, Finland). Medical
records and 68 histological samples were reviewed. Polyomaviruses were detected with quantitative PCR and the DNA-positive
samples were further analyzed for the presence of viral tumor Tantigen (T-ag) with immunohistochemistry. HPV genotyping was
performed with a Multiplex HPV Genotyping Kit. Only JCPyV DNAwas found in ACC samples, being present in 7 (10.3%) out
of the 68 samples. The viral load of JCPyV was low varying between 1 to 226 copies/μg DNA. The JCPyV-positive samples
originated from trachea (two samples), paranasal sinuses (one), and oral cavity (two). Additionally, JCPyV positivity was found
in one lung metastasis of a tracheal tumor and one local disease failure of an oral cavity tumor. Three JCPyV DNA-positive
samples showed weak nuclear staining for large T-ag. In conclusion, only JCPyV but not SV40, BKPyV, or HPV was found in
ACC from the upper and lower airways. JCPyV copy numbers were low which might support its role as a “hit and run agent”in
ACC carcinogenesis.
Keywords Adenoid cystic carcinoma .Polyomavirus .Oral cancer .Oncogenes .Human papillomavirus
Introduction
Adenoid cystic carcinoma (ACC) is a rare malignancy of glan-
dular structures, which most commonly (70%) appears in sal-
ivary glands [1]. Minor glands (MiSGs) are involved more
often than major glands (MaSGs) [1,2]. Etiopathogenesis for
ACC as well as for many other salivary gland tumors is still
unknown. In ACC, genetic studies have found MYB/NFIB
translocations and this fusion oncogene is overexpressed [3].
In addition, studies have provided evidence for Notch-pathway
alterations in 11 to 29% of patients [4]. As the pathogenesis of
ACC remains unknown, even viral background should be con-
sidered. Distinguished viral etiological factors among head and
neck cancers are Epstein-Barr virus (EBV) in nasopharyngeal
carcinoma and human papilloma virus (HPV) 16 in oropha-
ryngeal squamous cell carcinoma [5,6].
Furthermore, head and neck cancer studies have gained
new knowledge on polyomaviruses (HPyVs) and their coin-
fection with other viruses [7–11]. A recent study on oncogenic
DNA viruses in salivary gland tumors increases the interest on
possible role of HPyVs in the pathogenesis of salivary gland
tumors [12].
*Hanna Hämetoja
hanna.hametoja@helsinki.fi
1
Department of Oral Pathology, University of Turku and Turku
University Hospital, Turku, Finland
2
Department of Pathology, University of Helsinki and Helsinki
University Hospital, Helsinki, Finland
3
Research Programs Unit, Translational Cancer Biology Program,
University of Helsinki, Helsinki, Finland
4
Department of Surgery, University of Helsinki and Helsinki
University Hospital, Helsinki, Finland
5
Department of Otorhinolaryngology - Head and Neck Surgery,
University of Helsinki and Helsinki University Hospital,
Helsinki, Finland
6
Division of Ear, Nose and Throat Diseases, Department of Clinical
Sciences, Intervention and Technology, Karolinska Institutet and
Karolinska University Hospital, Stockholm, Sweden
7
Research Programme in Systems Oncology, Faculty of Medicine,
University of Helsinki, Helsinki, Finland
https://doi.org/10.1007/s00428-019-02617-6
Virchows Archiv (2019) 475:609–616
/ Published online: 1 2019
July
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Currently, 13 human HPyVs have been identified [13,14].
Among them, Merkel cell polyomavirus (MCPyV), BK poly-
omavirus (BKPyV), and JC polyomavirus (JCPyV) have been
associated with human cancers [13]. MCPyV is classified as a
grade 2A carcinogen (probable carcinogen) by IARC while
BKPyV and JCPyV are grade 2B carcinogens (possibly carci-
nogenic to humans) [13]. Simian vacuolating virus 40 (SV40)
has an ability to cause cancer in animal models but clinical
consequences to humans are controversial although SV40con-
taminated polio vaccine was used during the years 1955–1963
[13,15,16]. HPyVs are small non-enveloped viruses
consisting of circular double-stranded DNA genome (5.2 kb)
that has two transcriptional units, early and late region [13].
The former encodes large T antigen (T-ag) (90–100 kDA nu-
clear protein) and the latter, small T-ag (17–22 kDa) viral cap-
sidproteinsVP1,VP2,andVP3[13]. Large T-ag acts similarly
as many other known oncoproteins of tumor viruses by inter-
fering with tumor suppressor proteins pRb and p53 [8,11].
Roughly 80–90% of the population are latent carriers of
JCPyV and BKPyV and primary infections are usually mani-
fested subclinically during early childhood [11,15,17]. In
healthy individuals, secretion of JCPyV and BKPyV might
occasionally be detected in urine and saliva [18]. In the case
of immunocompromised patients, however, polyomavirus re-
activation may cause severe complications. These include
polyomavirus-associated nephropathy due to BKPyVreactiva-
tion in kidney transplant recipients and JCPyV-related progres-
sive multifocal leukoencephalopathy [15]. HPVs are widely
studied and the causality of HPV type 16 and squamous cell
carcinoma (SCC) has been classified as grade 1 (IARC vol
100, 2009) [19]. The etiological link between HPV and SCC
has specially been shown in cancers of uterine cervix and oro-
pharynx [6,20]. HPV has also been detected in salivary gland
ACC although the connection between HPV and ACC does
not seem to be strong [21–23]. According to the “hit and run
theory,”different viruses may contribute to tumorigenesis, for
instance, polyomaviruses could augment the oncogenic prop-
erties of HPV during their co-infections [8].
Altogether, studies on HPyVs, HPVs, and salivary gland
tumors are sparse and as far as we know, there are no previous
publications on the presence of polyomaviruses in ACC. The
aim of this study was to assess the prevalence of the three
polyomaviruses, SV40, JCPyV, and BKPyV, and HPV in
ACC samples of minor salivary and mucous glands.
Materials and methods
Patient and tumor characteristics
Tab le 1summarizes the demographic data and tumor charac-
teristics in a retrospective series on 68 patients with MiSG
ACC. The patients were diagnosed and treated at the
Department of Otorhinolaryngology –Head and Neck
Surgery, Helsinki University Hospital (Helsinki, Finland) be-
tween 1974 and 2012. The referral area for this tertiary care
center currently is 1.6 M. The clinical data of this series have
been characterized in our previous study [24] and the Ki67
immunostaining results collected from the same hospital data
were now added. The tumor samples were re-evaluated and
validated according to the diagnostic criteria by the classifica-
tion of the World Health Organization classification (both
2005 and 2017) [3,25]. The present tumor, node, metastasis
(TNM) classification does not include tracheal tumors, and
thus, the TNM classes on six (8.8%) tracheal ACCs are not
given. Additionally, in four cases (5.9%), the TNM class could
not be defined.
Table 1 Characterization of the 68 patients with an adenoid cystic
carcinoma of minor salivary and mucous glands
N%
Sex
Male 29 42.6
Female 39 57.4
Female/male ratio 1.35
Median age, years (range) 58 (24–88)
Tumor site
Oral cavity 41 60.3
Paranasal cavities 6 8.8
Trachea 6 8.8
Nasopharynx 5 7.4
Oropharynx 3 4.4
Ear 4 5.9
Larynx 2 2.9
Esophagus 1 1.5
Tclass
T1 18 26.5
T2 12 17.6
T3 6 8.8
T4 22 32.4
N/A 4 5.9
N class
N0 54 79.4
N1 1 1.5
N2 3 4.4
N/A 4 5.9
Stage
I1623.5
II 12 17.6
III 7 10.3
IV 23 33.8
N/A 4 5.9
N/A, not available
TNM and stage classification for 62 tumors (trachea excluded)
Virchows Arch (2019) 475:609–616
610
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Altogether, 68 paraffin blocks of tumor samples were avail-
able. Samples included 48 samples from primary tumors and
20 disease failures from 15 patients; altogether, we were able
to study samples from 53 patients. In addition, ten normal
salivary gland tissue samples from the same patients were
used as normal controls.
The study comprised MiSG and mucous excreting gland
samples from the head and neck area including trachea and
esophagus. These gland types in the upper gastrointestinal
tract and in the respiratory tract have similar structure and
function. They maintain the overall moisturizing of the muco-
sa. MaSGs on the other hand are activated mainly during
eating when they produce serous saliva. Due to the location
of MiSGs, just beneath the epithelium, different carcinogenic
agents and oncoviruses might have easier access to MiSGs
compared with MaSGs.
Institutional Research Ethics Board approved the study
concept (Dnro 31/13/03/02/2010, 01 February 2010) and
Statistics Finland provided the dates and causes of death.
DNA extraction
Formalin-fixed and paraffin-embedded biopsy samples
DNA was extracted from 5-μm-thick deparaffinized sections
(1 cm
2
in total area) with the high salt method [26]. In brief,
after deparaffinization, the sections were lysed in lysis buffer
(10 mM Tris-HCl, 400 mM NaCl, and 2 mM EDTA, pH 8.2)
with proteinase K (200 μg/mL) overnight at + 37 °C. After
digestion, proteins were precipitated with saturated NaCl and
the DNA with ethanol.
HPV detection
DNA was amplified with primer sets 1 and 2 from the
Multiplex HPV Genotyping Kit® (DiaMex GmbH,
Germany). Primer set 1 contains all HPV primers: nine
biotinylated forward and three reverse primers for ampli-
fying the HPV types under investigation. Primer set 2
(DNA quality control primers) contains primers for the
amplification of a ß-globin gene fragment to verify the
amount and the quality of human genomic sample DNA.
A negative control contained no genomic DNA to confirm
the absence of a contamination in the amplification reac-
tions. Multiplex HPV Genotyping Kit® detects the follow-
ing 24 low risk (LR)- and high risk (HR) -HPV genotypes:
LR-HPV6, 11, 42, 43, 44, and 70 and HR-HPV16, 18, 26,
31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and
82. The labeled hybrids were analyzed with a Luminex
LX-100 analyzer (Bio-Plex 200 System, Bio-Rad
Laboratories, Hercules, USA).
Quantitative detection of SV40, JCPyV, and BKPyV
Presence of SV40, JCPyV and BKPyV DNA in the samples
was detected by quantitative polymerase chain reaction
(qPCR) (Roche, Light Cycler 96) targeting their oncogenic
large T-ag as described by McNees and coworkers, with slight
modification [15]. RNase P was used as a reference gene
(TaqMan® Copy Number Reference Assay RNase, Applied
Biosystems, Foster City, CA, USA) to a relative expression of
the target genes.
The primers and probes were designed as described earlier
[15] and produced by Life Technologies as given in Table 2.
The probes for the target genes SV40, JCPyV, and BKPyV
were labeled with 6-carboxyfluorescein (FAM) and (VIC) was
used for labeling the probe for the reference gene RNase P.
The qPCR reactions were performed in 20 μlvolumeinmicro
titer plate. The following reaction conditions were used:
900 nM of each primer and 100 nM of their analogous probe,
10 μl of TaqMan® Universal Mix II and 300 ng template
DNA. The detection of the reference gene TaqMan® RNase
P (Applied Biosystems) was performed according to the man-
ufacturer’s recommendations. The conditions for all qPCR
reactions were as follows: 2 min at 50 °C, 10 min denaturation
at 95 °C followed by 45 cycles of amplification with 95 °C
denaturation for 15 s, and annealing/extension at 60 °C for
60 s. Amplification data measured as an increase in reporter
fluorescence were collected in real time and analyzed by the
Roche, Light Cycler 96 software.
The linear standard curves for JCPyV and BKPyV were
obtained with a serial dilution of plasmids with amounts rang-
ing from 1.2*10^2ng/μl to 1.2*10^-2 ng/μlforJCPyVand
9.5*10^0 to 9.5*10^-3 ng/μl for BKPyV. The standards for
SV40 detection were constructed with a serial dilution of
COS1 cell line DNA (which contains one copy of
SV40/cell) with an amount range from 5.0*10^4 to
5.0*10^0cells/μl, while the standards for the reference gene
RNase P were acquired with a serial dilution of human pla-
centa DNA extractions (Sigma-Aldrich, Darmstadt, Germany)
ranging from 5.09*10^2 to 5.09*10^-2. Cq values of less than
37 were considered positive. The copy numbers were calcu-
lated as copies in 1 μghumanDNA.
Immunohistochemistry
Immunohistochemistry was performed for JCPyV-positive
samples only as BKPyV and SV40 remained qPCR negative.
For immunohistochemistry, there are 4-μm-thick sections of
formalin-fixed and paraffin-embedded blocks. The slides were
deparaffinized in xylene and rehydrated in a series of ethanol
solutions. Endogenous peroxidase activity was blocked by
incubation of the slides with 3% hydrogen peroxide for
15 min. Epitope retrieval was performed using boiling in
1 mM citrate buffer, pH 6.0 in the microwave for 5 min, twice.
Virchows Arch (2019) 475:609–616 611
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

The primary antibody used was a mouse monoclonal anti-
simian virus T-ag that cross-reacts with JCPyV T-ag (Anti-
SV40 Antibody, clone Pab101, LifeSpan BioSciences Inc.,
Seattle WA, USA) and the dilution of 1:75 was used. The
tissue was incubated with the primary antibody overnight,
followed by detection with Dako REAL Detection System,
Peroxidase/DAB+, Rabbit/Mouse, Dako, Glostrup,
Denmark) and counterstained with hematoxylin.
Results
The presence of JCPyV, BKPyV, SV40, and HPV in ACC
and in normal salivary gland tissue
All 68 samples remained negative for BKPyV, SV40, and
HPV but seven of 68 (10.3%) samples showed JCPyV DNA
positivity. The characterization of the seven patients with
JCPyV-positive ACC is given in Table 3. The viral load of
JCPyV was low in all samples varying from 1 to 226
copies/μg DNA. The locations of JCPyV DNA-positive
ACCs were as follows: two in the trachea, one in the lung
metastasis of a tracheal tumor, one in the paranasal sinuses
(stage IV tumor), two in the oral cavity (stage I and IV tu-
mors), and one in local disease failure of an oral cavity tumor.
All the normal salivary gland control tissues showed negativ-
ity to JCPyV, BKPyV, and SV40.
Patients with JCPyV-positive ACC
Tab le 3shows the details of the seven patients with JCPyV
DNA-positive ACC. The mean age of the patients with
JCPyV DNA-positive tumor was 59 years (range, 43–80)
and female to male ratio was 2.5. In the whole cohort, the
mean age was similar, 58 years (range, 24–88), but the female
and male ratio was smaller, being 1.35. Among the JCPyV-
positive ACCs, the most common growth pattern was cribri-
form (42.8%),and the second was a combination ofcribriform
and tubular (28.6%), followed by tubular (14.3%), and
combination of cribriform and solid (14.3%), which simulates
the pattern of the whole cohort [24]. Neural invasion (perineu-
ral, intraneural, or both) was present in three (42.9%) out of
the JCPyV-positive tumors and in 57.4% in the whole cohort.
Proliferation index of Ki67 immunostaining varied from 4 to
35% (median 30%) among the JCPyV-positive tumors and
from 4 to 80% (median 30%) in the whole cohort.
Immunohistochemistry versus JCPyV DNA positivity
In total, three out of the seven JCPyV DNA-positive tumor
sections showed weak nuclear immunohistochemistry positiv-
ity for large T-ag as presented in Figs. 1and 2.Copynumbers
for two tracheal tumors and one oral cavity tumor were 1.11,
33.95, and 3.25/1 μg DNA, respectively. Immunopositivity
had no association with the clinical outcome of the disease.
Second primary tumors
Of note is that some of the patients with JCPyV DNA-positive
ACC suffered from other malignancies as well. One patient
with a tracheal ACC with thyroid gland invasion was in addi-
tion diagnosed with a papillary microcarcinoma in the thyroid
gland. The patient with an early stage I ACC in oral cavity
died of SCC of the lungs soon after ACC treatment.
Furthermore, the patient with ACC in paranasal sinuses had
recovered from lymphoma 6 years earlier.
Discussion
Our present results show that JCPyV may be found in MiSG
ACC samples byqPCR. However, the prevalence ofJCPyVin
ACC was low, i.e., only 10.3%, and JCPyV presented with
low copy numbers. Three out of the seven JCPyV-positive
tumors were from the oral cavity while the rest were located
in the upper and lower airways. As the number of JCPyV-
positive cases was sparse and the viral loads were low, we
can only speculate the possible viral role as an etiological
Table 2 Primers and probes for
SV40, JCPyV, and BKPyV Name Sequence detection
SV40 primer forward GAT GGC ATT TCT TCT GAG CAA A
SV40 primer reverse GAA TGG GAG CAG TGG TGG AA
JCPyV primer forward TTC TTC ATG GCA AAA CAG GTC TT
JCPyV primer reverse GAA TGG GAA TCC TGG TGG AA
BKPyV primer forward CTT TCT TTT TTT TTT GGG TGG TGT T
BKPyV primer reverse TTG CCA GTG ATG AAG AAG CAA
SV40 T-ag probe 5′-FAM CAG GTT TTC CTC ATTAAA
JCPyV T-ag probe 6 FAM CCA CTT CTC ATT AAA TG
BKPyV T-ag probe 6 FAM AGT GTT GAG AAT CTG C
Adapted from McNees et al. 2005
Virchows Arch (2019) 475:609–616
612
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Table 3 Characterization of the patients with a JCPyV-positive adenoid cystic carcinoma of the minor salivary and mucous glands
Patient Anatomic subsite Age Sex Smoking TNM
classification
Stage Treatment Surgical
margins
Growth
pattern
Proliferation
index Ki67 (%)
Neural
invasion
Disease failure Status Copy number/
1μgDNA
1 Trachea 43 F No Surgery Positive Tubular N/A No Distant
metastasis in
lungs
DOC 1.11
2 Trachea 54 F No Surgery Positive Cribriform
and tubular
35 No NED 33.95
3 Trachea (lung metastasis) 47 F No Surgery N/A Cribriform N/A N/A Distant, local,
locoregional
DOD 7.18
4 Paranasal sinuses 60 M Yes T
4B
N
0
M
0
IVB Oncological Cribriform
and tubular
N/A Yes Local DOC 16.51
5 Oral cavity, gum 80 F N/A T
1
N
0
M
0
I Surgery Negative Cribriform 4 No DOC 225.60
6 Oral cavity, hard palate 59 M N/A T
4B
N
0
M
0
IVB Surgery and
oncological
Positive Cribriform
and solid
30 Yes DOC 3.25
7 Oral cavity, floor of the mouth
(local disease failure)
72 F N/A T
4A
N
0
M
1
IVA Surgery Positive Cribriform N/A Yes Local DOD 15.47
N/A, not available; TNM, tumor, node, metastasis; DOC, dead of other cause; NED, no evidence of disease; DOD, dead of disease
Virchows Arch (2019) 475:609–616 613
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

factor for ACC. A “hit and run hypothesis”has been proposed
for certain oncoviruses. Accordingly, this hypothesis claims
that viruses can mediate cellular transformation through an
initial “hit,”while maintenance of the transformed state is
compatible with the loss (“run”) of viral molecules [27]. The
low copy numbers in our study suggest the role of JCPyV in
hit and run carcinogenesis model rather than as a continuous
driver of the tumor formation. None of the HPyVs were de-
tected in the normal salivary gland control tissue.
We were able to locate large T-ag on nuclei of the tumor
cells by using SV40 antibody, which is cross-reacting with
both JCPyVand BKPyV. qPCR technique is an accurate meth-
od for detecting HPyV DNA, but in the current study, immu-
nohistochemical expression did not correlate well with qPCR
findings, while only three out of the seven JCPyV-positive
samples stained positively. Possible explanation for the nega-
tive staining might be that only few cells in the samples in-
fected with JCPyV are transcriptionally and translationally
active. According to the “hit and run theory,”positive signals
are detected only from sporadic cells (Figs. 1and 2). Another
possibility is that these approximately 7 μm wide cells might
have been cut out from the deeper sections prepared for
immunohistochemistry.
The etiological risk factors for all salivary gland malignan-
cies include exposure to nickel compounds and silica dust,
employment at rubber manufacturing, hairdressers´, and beau-
ty shops, as well as irradiation, EBV, and HIV infection [10,
28,29]. At molecular level, germline BRCA mutations and
genetic variants in DNA double-strand brake repair genes
have been related to the risk for salivary gland cancers includ-
ing ACC [3].
As far as we know, there are no previous studies on the
prevalence of JCPyV or other HPyVs in ACC. As discussed
earlier, 80–90% of population are latent carriers of JCPyVand
BKPyV and HPyVs have been detected in saliva of healthy
individuals, which strengthens the hypothesis of saliva being
the transmission route for HPyVs [11,15,17,18]. Thus, the
reservoir of HPyVs might be in salivary glands or in oral or
oropharyngeal mucosa. An earlier experimental study showed
that the injection of polyomavirus into salivary gland tissue of
mice resulted in tumor formation resembling that of human
pleomorphic adenoma [30]. Further, SV40 DNA has been
identified in pleomorphic adenoma of parotid gland [31].
Overall, studies concerning HPyVs in head and neck can-
cers are rare [7,9,11,32,33]. Kutsuna et al. have linked high
viral JCPyV load to oral SCC of the tongue, but they did not
find any prognostic value for JCPyV [11]. Zheng et al. sug-
gested that JCPyV could have an oncogenic role in the squa-
mous cell carcinogenesis of tongue, pharynx, and larynx [32].
Contradictory to their findings, Polz-Gruszka et al. did not
find JCPyVin any of the 62 oropharyngeal SCC in their series
but found BKPyV in 17.7% of the tumor samples [7]. In these
studies, viral DNA was detected by qPCR [7,11,32]. Some
studies have reported negative HPyV results in head and neck
cancer. Palmieri et al. did not detect SV40, BKPyV, or JCPyV
DNA by qPCR evaluation in oral cavity SCC [9].
HPVs have been studied in ACC but the association does
not seem to be strong [21–23]. However, recently, a new sub-
type of non-keratinizing SCC has been found in the sinonasal
area having features of both ACC and SCC and being usually
HPV type 33 positive [3,34]. This entity is named as HPV-
related multiphenotypic sinonasal carcinoma [3,34]. In our
study, HPV-positive ACCs were not detected.
Further studies are thus needed to understand the possible
role of HPyV in the carcinogenesis of ACC.
Conclusions
Only JCPyV DNA but not SV40, BKPyV, or HPV was found
in ACC from the upper and lower airways. JCPyV copy
Fig. 1 Immunohistochemical staining for large T antigen showing
JCPyV positivity in adenoid cystic carcinoma of the trachea (patient #1
in Table 3)
Fig. 2 Immunohistochemical staining positivity for large T antigen in
adenoid cystic carcinoma of the trachea (patient #2 in Table 3)
Virchows Arch (2019) 475:609–616
614
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

numbers were low which might support its role as a “hit and
run agent”in ACC carcinogenesis.
Acknowledgments The authors thank the Department of Oral Pathology,
Institute of Dentistry, University of Turku for providing the reagents.
Contributions HH and JH contributed to the study concept and design,
data collection, histopathological analysis, interpretation of the results,
and writing. CH contributed to the study concept and provided the labo-
ratory facilities. LB and AM contributed to the study concept and design,
and writing. SS contributed to the study concept and design, data collec-
tion, histopathological analysis, interpretation of the results and writing,
and provided the laboratory facilities for qPCR. All the authors approved
the publication.
Funding Open access funding provided by University of Turku (UTU)
including Turku University Central Hospital. This work was supported by
the Helsinki University Hospital Research Fund (TYH 2018215) and the
Finska Läkaresällskapet.
Compliance with ethical standards
Institutional Research Ethics Board approved the study concept (Dnro 31/
13/03/02/2010, 01 February 2010) and Statistics Finland provided the
dates and causes of death.
Conflict of interest The authors declare that they have no conflict of
interest.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appro-
priate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
References
1. DeAngelis AF, Tsui A, Wiesenfeld D, Chandu A (2011) Outcomes
of patients with adenoid cystic carcinoma of the minor salivary
glands. Int J Oral Maxillofac Surg 40:710–714. https://doi.org/10.
1016/j.ijom.2011.02.010
2. Bjorndal K, Krogdahl A, Therkildsen MH, Charabi B, Kristensen
CA, Andersen E, Schytte S, Primdahl H, Johansen J, Pedersen HB,
Andersen LJ, Godballe C (2015) Salivary adenoid cystic carcinoma
in Denmark 1990-2005: outcome and independent prognostic fac-
tors including the benefit of radiotherapy. Results of the Danish
Head and Neck Cancer Group (DAHANCA). Oral Oncol 51:
1138–1142. https://doi.org/10.1016/j.oraloncology.2015.10.002
3. El-Naggar AK, Chan JKC, Grandis JR, Takata T, Slootweg PJ
(2017) WHO classification of head and neck tumours.
IARCPress, Lyon France 15-17:164–165
4. Stephens PJ, Davies HR, MitaniY, Van Loo P, Shlien A, Tarpey PS,
Papaemmanuil E, Cheverton A, Bignell GR, Butler AP, Gamble J,
Gamble S, Hardy C, Hinton J, Jia M, Jayakumar A, Jones D,
Latimer C, McLaren S, McBride DJ, Menzies A, Mudie L,
Maddison M, Raine K, Nik-Zainal S, O'Meara S, Teague JW,
Varela I, Wedge DC, Whitmore I, Lippman SM, McDermott U,
Stratton MR, Campbell PJ, El-Naggar AK, Futreal PA (2013)
Whole exome sequencing of adenoid cystic carcinoma. J Clin
Invest 123:2965–2968. https://doi.org/10.1172/JCI67201
5. Mesri EA, Feitelson MA, Munger K (2014) Human viral oncogen-
esis: a cancer hallmarks analysis. Cell Host Microbe 15:266–282.
https://doi.org/10.1016/j.chom.2014.02.011
6. Syrjanen S, Rautava J, Syrjanen K (2017) HPV in head and neck
cancer-30 years of history. Recent Results Cancer Res. https://doi.
org/10.1007/978-3-319-43580-0_1
7. Polz-Gruszka D, Morshed K, Jarzynski A, Polz-Dacewicz M
(2015) Prevalence of Polyoma BK virus (BKPyV), Epstein-Barr
virus (EBV) and Human Papilloma virus (HPV) in oropharyngeal
cancer. Pol J Microbiol 64:323–328
8. Guidry JT, Scott RS (2016) The interaction between human papil-
lomavirus and other viruses. Virus Res 231:139–147. https://doi.
org/10.1016/j.virusres.2016.11.002
9. Palmieri A, Carinci F, Martinelli M, Spinelli G, Lo Muzio L, Rubini
C, Scapoli L (2010) Absence of Simian virus 40, BK, and JC
polyomavirus DNA in squamous cell carcinoma limited to the oral
cavity. Head Neck 32(3):375–380. https://doi.org/10.1002/hed.
21197
10. Zheng W, Shu XO, Ji BT, Gao YT (1996) Diet and other risk factors
for cancer of the salivary glands: a population-based case-control
study. Int J Cancer 67:194–198
11. Kutsuna T, Zheng H, Abdel-Aziz HO, Murai Y, Tsuneyama K,
Furuta I, Takano Y (2008) High JC virus load in tongue carcinomas
may be a risk factor for tongue tumorigenesis. Virchows Arch 452:
405–410. https://doi.org/10.1007/s00428-007-0534-0
12. Chen AA, Gheit T, Stellin M, Lupato V, Spinato G, Fuson R,
Menegaldo A, Mckay-Chopin S, Dal Cin E, Tirelli G, Da Mosto
MC, Tommasino M, Boscolo-Rizzo P (2017) Oncogenic DNA
viruses found in salivary gland tumors. Oral Oncol 75:106–110.
https://doi.org/10.1016/j.oraloncology.2017.11.005
13. Burger-Calderon R, Webster-Cyriaque J (2015) Human BK
polyomavirus-the potential for head and neck malignancy and dis-
ease. Cancers (Basel) 7:1244–1270. https://doi.org/10.3390/
cancers7030835
14. Moens U, Rasheed K, Abdulsalam I, Sveinbjornsson B (2015) The
role of Merkel cell polyomavirus and other human polyomaviruses
in emerging hallmarks of cancer. Viruses. 7:1871–1901. https://doi.
org/10.3390/v7041871
15. McNees AL, White ZS, Zanwar P, Vilchez RA, Butel JS (2005)
Specific and quantitative detection of human polyomaviruses BKV,
JCV, and SV40 by real time PCR. J Clin Virol 34:52–62. https://doi.
org/10.1016/j.jcv.2004.12.018
16. Colvin EK, Weir C, Ikin RJ, Hudson AL (2014) SV40 TAg mouse
models of cancer. Semin Cell Dev Biol 27:61–73. https://doi.org/
10.1016/j.semcdb.2014.02.004
17. Burger-Calderon R, Madden V, Hallett RA, Gingerich AD,
Nickeleit V, Webster-Cyriaque J (2014) Replication of oral BK
virus in human salivary gland cells. J Virol 88:559–573. https://
doi.org/10.1128/JVI.02777-13
18. Robaina TF, Mendes GS, Benati FJ, Pena GA, Silva RC, Montes
MA, Janini ME, Camara FP, Santos N (2013) Shedding of poly-
omavirus in the saliva of immunocompetent individuals. J Med
Virol 85:144 –148. https://doi.org/10.1002/jmv.23453
19. http://monographs.iarc.fr/ENG/Monographs/vol100B/mono100B.
pdf. Accessed 01/18 2018
20. Mitra A, MacIntyre DA, Marchesi JR, Lee YS, Bennett PR,
Kyrgiou M (2016) The vaginal microbiota, human papillomavirus
infection and cervical intraepithelial neoplasia: what do we know
and where are we going next? Microbiome. 4:58. https://doi.org/10.
1186/s40168-016-0203-0
21. Huhns M, Simm G, Erbersdobler A, Zimpfer A (2015) HPV infec-
tion, but not EBV or HHV-8 infection, is associated with salivary
gland tumours. Biomed Res Int 2015:1–7. https://doi.org/10.1155/
2015/829349
Virchows Arch (2019) 475:609–616 615
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

22. Qian X, Kaufmann AM, Chen C, Tzamalis G, Hofmann VM,
Keilholz U, Hummel M, Albers AE (2016) Prevalence and associ-
ated survival of high-risk HPV-related adenoid cystic carcinoma of
the salivary glands. Int J Oncol 49:803–811. https://doi.org/10.
3892/ijo.2016.3563
23. Boland JM, McPhail ED, GarciaJJ, Lewis JE, Schembri-Wismayer
DJ (2012) Detection of human papilloma virus and p16 expression
in high-grade adenoid cystic carcinoma of the head and neck. Mod
Pathol 25:529–536. https://doi.org/10.1038/modpathol.2011.186
24. Hametoja H, Hirvonen K, Hagstrom J, Leivo I, Saarilahti K,
Apajalahti S, Haglund C, Makitie A, Back L (2017) Early stage
minor salivary gland adenoid cystic carcinoma has favourable
prognosis. Virchows Arch 471:785–792. https://doi.org/10.1007/
s00428-017-2163-6
25. Barnes L, Eveson WJ, Reichart P, Sidransky D (2005) Pathology
and genetics of head and neck tumours, 4th edn. IARCPress, Lyon
France, pp 221–222
26. Miller SA, Dykes DD, Polesky HF (1988) A simple salting out
procedure for extracting DNA from human nucleated cells.
Nucleic Acids Res 16:1215
27. Nevels M, Tauber B, Spruss T, Wolf H, Dobner T (2001) “Hit-and-
run”transformation by adenovirus oncogenes. J Virol 75:3089–
3094. https://doi.org/10.1128/JVI.75.7.3089-3094.2001
28. Guzzo M, Locati LD, Prott FJ, Gatta G, McGurk M, Licitra L
(2010) Major and minor salivary gland tumors. Crit Rev Oncol
Hematol 74:134–148. https://doi.org/10.1016/j.critrevonc.2009.
10.004
29. Kim D, Hwang YI, Choi S, Park C, Lee N, Kim EA (2013) A case
of tracheal adenoid cystic carcinoma in a worker exposed to rubber
fumes. Ann Occup Environ Med 25:22. https://doi.org/10.1186/
2052-4374-25-22
30. Dawe CJ, Freund R, Mandel G, Ballmer-Hofer K, Talmage DA,
Benjamin TL (1987) Variations in polyoma virus genotype in rela-
tion to tumor induction in mice. Characterization of wild type
strains with widely differing tumor profiles. Am J Pathol 127:
243–261
31. Martinelli M, Martini F, Rinaldi E, Caramanico L, Magri E, Grandi
E, Carinci F, Pastore A, Tognon M (2002) Simian virus 40 se-
quences and expression of the viral large T antigen oncoprotein in
human pleomorphic adenomas of parotid glands. Am J Pathol 161:
1127–1133
32. Zheng Y, Xia P, Zheng HC, Takahashi H, Masuda S, Takano Y
(2010) The screening of viral risk factors in tongue and
pharyngolaryngeal squamous carcinoma. Anticancer Res 30:
1233–1238
33. Mazzoni E, Martini F, Corallini A, Taronna A, Barbanti-Brodano
G, Querzoli P, Magri E, Rotondo JC, Dolcetti R, Vaccher E, Tognon
M (2016) Serologic investigation of undifferentiated nasopharyn-
geal carcinoma and simian virus 40 infection. Head Neck 38:232–
236. https://doi.org/10.1002/hed.23879
34. Bishop JA, Westra WH (2018) Human papillomavirus-related
multiphenotypic sinonasal carcinoma: an emerging tumor type with
a unique microscopic appearance and a paradoxical clinical behav-
iour. Oral Oncol 87:17–20. https://doi.org/10.1016/j.oraloncology.
2018.10.011
Publisher’snoteSpringer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.
Virchows Arch (2019) 475:609–616
616
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com