High prevalence of human papillomaviruses in fresh frozen breast
Journal of Medical Virology 83:2157–2163 (2011)
High Prevalence of Human Papillomaviruses in
Fresh Frozen Breast Cancer Samples
Annika Antonsson,1 Terrence P. Spurr,1 Alice C. Chen,1 Glenn D. Francis,2 Nigel A.J. McMillan,1
Nicholas A. Saunders,1 Michael Law,3 and Ian C. Bennett3*,y
University of Queensland, Diamantina Institute for Cancer, Immunology and Metabolic Medicine,
Princess Alexandra Hospital, Woolloongabba, Brisbane, Queensland, Australia
Pathology Queensland, Princess Alexandra Hospital, Woolloongabba, Brisbane, Queensland, Australia
Breast & Endocrine Unit, Department of Surgery, Princess Alexandra Hospital, Woolloongabba, Brisbane,
While the etiology of breast cancer remains
enigmatic, some recent reports have examined
the role of human papillomavirus (HPV) in
breast carcinogenesis. The purpose of this
study was to determine the prevalence of HPV
in breast cancer tissue using PCR analysis and
sequencing. Fifty-four (54) fresh frozen breast
cancers samples that were removed from a cohort of breast cancer patients were analyzed.
Samples were tested for HPV using comprehensive PCR primers, and in situ hybridization
was performed on parafﬁn embedded tissue
sections. Findings were correlated with clinical
and pathological characteristics. The HPV DNA
prevalence in the breast cancer samples was
50% (27/54) with sequence analysis indicating
all cases to be positive for HPV-18 type. While
HPV patients were slightly younger, no correlation was noted for menopausal status or family
history. HPV positive tumors were smaller with
earlier T staging and demonstrated lesser nodal
involvement compared to HPV negative cancers. In situ hybridization analyses proved negative. The high proportion of HPV positive
breast cancers detected in this series using
fresh frozen tissues cannot be dismissed, however the role of HPV in breast carcinogenesis
remains unclear and may ultimately be ascertained by monitoring future breast cancer incidence amongst women vaccinated against high
risk HPV types.
J. Med. Virol. 83:2157–
2163, 2011. ß 2011 Wiley Periodicals, Inc.
KEY WORDS: breast cancer; human papillomavirus (HPV); polymerase
chain reaction (PCR); in situ
ß 2011 WILEY PERIODICALS, INC.
The papillomaviruses are small, double-stranded
DNA viruses belonging to the Papillomaviridaefamily. So far, more than 110 different HPV types
have been fully characterized, and in addition, several
papillomavirus types have been isolated from a
number of vertebrate species [de Villiers et al., 2004].
Papillomaviruses can be grouped according to tissue
tropism with HPV types found in mucosal lesion being
referred to as mucosal or genital types, while HPV
types found in skin are called skin or cutaneous types.
Mucosal HPV types that are found preferentially in
cervical and other anogenital cancers have been designated high-risk types. These high-risk HPVs have
been identiﬁed as the causative agent in 99.7% or
more of cervical cancers [Walboomers et al., 1999] and
have also been detected in more than 50% of other
anogenital cancers [zur Hausen, 1996]. The most
prevalent high-risk HPV types are HPV-16 and HPV18, which account for 70% of the cancer cases, with
another 10 types making up the other 30%. These
Grant sponsor: Centres for Health Research, Princess Alexandra Hospital Cancer Collaborative Group; Grant sponsor:
Mazda Foundation (to A.A.); Grant sponsor: Prostate Cancer
Foundation of Australia (to A.A.).
There are no conﬂicts of interest.
Ethical approval was obtained through the Princess Alexandra Hospital Ethics Committee.
*Correspondence to: Ian C. Bennett, University of Queensland
Department of Surgery, Princess Alexandra Hospital, Wooloongabba, Brisbane, Australia. E-mail: [email protected]
Accepted 8 August 2011
Published online in Wiley Online Library
HPV types have therefore emerged as one of the most
important identiﬁed risk factors for widespread
human cancer [Parkin and Bray, 2006].
The putative transforming properties of high-risk
HPVs are thought to primarily reside in the E6 and
E7 genes that are consistently expressed in HPVpositive cancers. E6 and E7 are involved in the viral
regulation of cell growth. The HPV E6 gene binds to
the p53 tumor suppressor protein and targets it for
ubiquitin-mediated degradation [Scheffner et al.,
1990] as well as stimulating telomerase activity
[Klingelhutz et al., 1996]. Loss of p53 abrogates the
brake on the cell cycle when DNA damage occurs,
while telomerase activity is required to stop telomere
lengths shortening and cell senescence. The E7 protein targets the active, hypophosphorylated form of
the retinoblastoma protein (pRB) as well as other
members of the RB family [Münger et al., 1989a,b].
This results in the destabilization of pRB/E2F
complexes and the release of active E2F, which
allows transcription of genes required for cell cycle
progression. Therefore, the over-expression of E6
and E7 allows uncontrolled cell growth without checkpoint controls and this sets the cell up for further
mutations, transformation, and the formation of
Apart from cervical and anogenital malignancies,
HPV has also been suggested to play a role in other
cancers including squamous cell carcinomas of the
head and neck and more recently breast cancer.
Breast cancer is the most common invasive cancer diagnosed in females in Australia and is also a leading
cause of cancer death in females [NHMRC National
Breast & Ovarian Cancer Centre, 2009]. Viral infection as an etiological factor in breast cancer genesis
has been raised in a number of studies which have
examined the role of viruses such as murine-mammary tumor virus, simian virus 40, CMV, and Epstein–
Bar virus in breast cancer etiology [Lawson et al.,
2001; Talbot and Crawford, 2004; Tsai et al., 2007].
These studies however have not provided consistent
results with there being quite varied and sometimes
contradictory outcomes. In contrast there have been
some recent, more compelling, results from studies investigating a possible link between HPV and breast
cancer [de Villiers et al., 2005; Kan et al., 2005;
Kroupis et al., 2006]. The suspicion that HPVs could
have a role in human breast cancer is based upon the
in vitro evidence that the most efﬁcient and reproducible model of human mammary epithelial cell immortalization is the expression of high-risk HPV
oncogenes E6 and E7 [Dimri et al., 2005]. HPV-16 has
been identiﬁed in breast tumors in Italian women and
Norwegian women who had previous cervical neoplasia [Hennig et al., 1999b]. HPV-11, -16, and -18 have
been identiﬁed in breast cancer in US and Brazilian
women [Liu et al., 2001; Damin et al., 2004]. Additionally, HPV-33 has been identiﬁed in breast cancer in
Chinese and Japanese women [Yu et al., 1999]. Recently HPV has been identiﬁed in a wide range of
J. Med. Virol. DOI 10.1002/jmv
Antonsson et al.
HPV types in cancer of the breast and nipples [de
Villiers et al., 2005]. Additionally, Kan identiﬁed the
presence of HPV-18 sequences in breast tumors in
Australian women with 48% of the samples being
HPV positive [Kan et al., 2005].
In most of these studies, where relevant analyses
have been performed, no signiﬁcant correlations have
been observed between HPV type and tumor grade,
patient survival, steroid receptor status, HER2, and
p53 expression [Hennig et al., 1999a; Damin et al.,
2004]. However, Kroupis et al.  did show a
tendency for HPV-harboring tumors to be less ER
positive and to display more proliferative features as
well as higher grade. Also, Hennig et al. [1999a]
reported inhibited p53 expression in HPV positive
breast cancer tissue. Previous studies on HPVs in normal breast tissues from healthy women who have had
cosmetic surgery have yielded contradictory results,
one Australian study found no HPV in normal breast
tissue [Kan et al., 2005], while another did ﬁnd HPV
in similar samples [Heng et al., 2009].
However, the studies to date looking at the link
between HPV and breast cancer have been limited in
their methodologies with two deﬁciencies being common; (1) Many of these studies were performed on a
limited number of parafﬁn embedded breast tumors.
(2) The number of HPV types analyzed has been
restricted. To circumvent these problems, this study
examined fresh frozen samples from 54 breast cancer
cases and used a powerful PCR-based approach that
is able to identify up to 150 different HPV types.
MATERIALS AND METHODS
Patients and Samples
This project analyzed 54 breast cancer tissue samples and four healthy breast tissues that were
removed as a part of standard surgical procedures
from patients undergoing surgery at the Wesley
Hospital, Brisbane, Queensland, Australia between
2003 and 2007. Fifty-eight breast tissue biopsies
were obtained from 54 patients, four of which had
matched healthy breast tissue. All tissue samples
were conﬁrmed by histology and the patients’ median
age was 57 years, with a range of 31–88 years.
Data on diagnosis, location, invasive grade, histological type, menopausal status, family history of
breast cancer, cervical cancer diagnosis, and HER2,
estrogen, and progesterone receptors were also
Tissue samples were snap frozen and stored at
808C. Prior to extraction the samples were grinded
in 1.5 ml tubes on dry ice. The tissue samples were
then homogenized in Trizol (Invitrogen, Carlsbad,
CA), and DNA extraction was carried out following
the protocol provided by the manufacturer. The DNA
extracted from the breast tissue specimens was stored
at 208C until analyzed. Furthermore, parafﬁnembedded tissue for in situ hybridization (ISH) was
also available for all patients.
HPV in Breast Cancer
Informed consent was obtained from all patients,
and this project was approved by the Princess Alexandra Hospital Human Research Ethics Committee
Polymerase Chain Reaction (PCR)
The DNA extracted tissue and blood samples were
tested with PCR for HPV DNA with the general
primer-pair FAP59/FAP64 [Forslund et al., 1999], and
the HPV 18 type-speciﬁc primer pairs HPV18FAP59/
HPV18FAP64. New type speciﬁc primers were
designed for HPV-18, using the degenerate FAP
primers as template. The primer sequences for these
HPV-18 type speciﬁc primers were for the forward
primer HPV18FAP59: 50 -TAACTGTGGGTAATCCATATT-30 and for the reverse primer HPV18FAP64: 50 CCAGTATCTACCATATCACCATC-30 . This primer
pair yields an amplicon of 483 bp. The previously
described protocol for FAP59/FAP64 was followed for
the two primer pair sets as described except for the
MgCl2 concentration, which was modiﬁed to 3.5 mM
[Forslund et al., 1999]. Furthermore, all samples were
analyzed for the presence of the human L1 sequence
[Deragon et al., 1990], as a control for human DNA
and as an indirect marker to ensure that a sample did
not contain any PCR-inhibiting substances. Negative
controls (water) were used in each batch of PCR and
the negative controls were added after all the samples
were added to the PCR tubes. None of the negative
controls were positive. Ampliﬁed PCR products were
analyzed by electrophoresis in a 1.5% agarose gel containing TAE buffer and ethidium bromide (Sigma,
Castle Hill, Australia). PCR amplicons were size determined under UV light using the GelDoc software
(Bio-Rad, Sydney, Australia).
Cloning and Sequencing
All positive PCR products were cloned using a
TOPO TA cloning kit (Invitrogen). Samples were
ligated into pCR 2.1 TOPO cloning vector and transformed into TOP10 competent cells (Invitrogen) following the manufacturer’s speciﬁcations. Clones with
inserts were sequenced with both forward and reverse
primers (BigDye1 Terminator v3.1 Cycle Sequencing
Kit; Applied Biosystems, Foster City, CA). The
sequences were analyzed at the Australian Genome
Research Facility Ltd., Brisbane. Sequences obtained
were compared with available sequences in the
GenBank database using the BLAST server (http://
In Situ Hybridization
For the ISH assay, parafﬁn sections from nine of
the strongly HPV positive breast tumors were cut to a
thickness of 5 mm and mounted on Super Frost Plus
slides. One extra section per parafﬁn block was cut
and stained with H&E and used to determine
specimen quality. The high-risk HPV probe,
INFORM1 HPV III Family 16 Probe (B) (Roche
Ventana Medical System, Tucson, Arizona) was used
for the ISH assay. This HPV probe detects 16 different
high-risk (cancer-causing) HPV types (HPV 16, 18, 31,
33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, and 82).
The ISH assay was performed according to the manufacturer’s guidelines using the Discovery1 XT automated system (Roche Ventana Medical System).
Data were analyzed using online statistical software (VassarStats, Vassar College, Poughkeepsie,
NY) and associations between HPV status and various
breast cancer parameters and risk factors were tested
using the Chi-square test.
A total of 54 breast cancer specimens were analyzed
in this study. A summary of the patients’ characteristics and pathology features included in this study can
be found in Table I. The majority of patients were
post-menopausal (59%), mean tumor size was
24.3 mm, and 63% of patients were node negative.
The HPV DNA prevalence in the tissues samples was
50% (27/54; Fig. 1). Sequence analyses of the positive
patient samples indicated that the HPV type present
was HPV-18 in all cases. The DNA sequences of the
HPV-18 positive samples belonged to different strains
of HPV-18. Table II provides a comparison between
HPV positive and HPV negative sub-groups with respect to various patient and tumor characteristics.
The age range of HPV positive patients was 31–70
years of age, with a mean of 50 years. The mean age
of the group as a whole was 57 years and hence, while
the age of the women with a HPV positive breast
cancer tumor was slightly lower than the whole group
recruited, the difference was not signiﬁcant. No significant correlation was found between the HPV positive
and negative sub-groups for menopausal status and
family history data. Similarly for tumor grade, ER
status, PR status, and HER2 status, no difference was
seen between these two subgroups.
However, when tumor size was analyzed, HPV positive cancers tended to be smaller tumors. This was
most markedly demonstrated when analyzed in relation to T staging which showed HPV positive tumors
tending to fall into the smaller T1 and T2 groupings
compared to HPV negative tumors and this was
statistically signiﬁcant (P ¼ 0.03). Additionally when
tumor size was assessed in terms of size being <25 or
>25 mm, 89% of HPV positive cancers were <25 mm
whereas only 63% of HPV negative were <25 mm
(P ¼ 0.05). Similarly, when nodal status was analyzed, HPV positive tumors were more likely to have
less nodal involvement (up to three nodes), compared
to HPV negative tumors.
Comparison of histological cancer types showed
that of the HPV positive tumors 96.3% were of ductal
J. Med. Virol. DOI 10.1002/jmv
Antonsson et al.
TABLE I. Patient Demographic and Pathology Features
Mean tumor size
57.0 years (range 31–88)
It can be argued that the high prevalence of HPV18 could be due to PCR contamination. However, the
same person who has analyzed the breast samples
have been using the same PCR (with the same PCR
reagents and controls) for a skin HPV study and have
never detected HPV-18 in the skin samples [Chen
et al., 2008]. Also, sequencing revealed that several
different strains of HPV-18 were isolated in this
To determine whether the HPV-18 positivity was
localized to the tumor tissue or stromal components,
in situ hybridization analyses were performed on
HPV-18 positive breast cancer tissue slides. No evidence was found of HPV-18 positivity in the breast
tumor tissue or the surrounding stromal/vascular elements (Fig. 2A). In contrast, HPV-18 positive cervical
cancer tissue stained positive for HPV-18 by in situ
hybridization (Fig. 2B).
Three of the four normal breast tissue samples that
were tested were HPV negative, while one of them
was HPV-18 positive. These four normal tissue samples were taken from patients who had breast cancer
and from the same breast. The patient with the HPV18 positive normal tissue had a HPV negative breast
tumor, one of the patients with a HPV negative
normal tissue had a HPV-18 positive breast tumor,
and two patients had both normal tissue and tumor
negative for HPV DNA.
or mixed origin as opposed to lobular types, compared
to HPV negative tumors of which 85.2% were ductal.
No correlation could be made between the two subgroups in relation to a history of cervical cancer as
this information was not reliably recorded. All 54
samples were positive for human L1, suggesting that
the samples did not contain any PCR inhibitory
In this series 50% of fresh frozen breast cancer
tissue samples were found to be positive for HPV,
with HPV-18 being the only HPV type that was
detected. In previous reports worldwide, HPV-16 has
been the most common HPV type detected in breast
cancer samples, followed by HPV-18, but other HPV
types including HPV-6, HPV-11, HPV-16, and HPV-33
have also been recorded [Hennig et al., 1999b; Damin
Fig. 1. Electrophoresis of 26 breast cancer samples tested with PCR using the HPV18FAP59/
HPV18FAP64 primer pair. This PCR yields amplicons of 480 bp. Lanes: Wm, weight marker;
Lane 1–26, breast cancer samples; Lane 27, positive PCR control (HPV-18); Lane 28 negative PCR
control (no DNA).
J. Med. Virol. DOI 10.1002/jmv
HPV in Breast Cancer
TABLE II. Correlation Analysis Between HPV Status and
et al., 2004; Kan et al., 2005; de Villiers et al., 2005;
Heng et al., 2009]. It is interesting that in this current
study performed in Brisbane, Queensland, is very
similar in its results to that obtained in the only two
other Australian studies, including Kan et al. 
who found HPV-18 only in 48% of breast cancer samples from West Australian women (Western Australia
4,000 km from current study) and Heng et al. 
who demonstrated very high HPV prevalence in
breast cancer samples from Sydney women using in
situ PCR techniques, with HPV-18 being the
dominant HPV type (Sydney, New South Wales;
1,000 km from current study). It would seem that
whilst HPV-18 appears to be infecting women from
widely disparate locations in Australia, because of its
relative isolation the HPV proﬁle and prevalence in
Australia is different compared to HPV types identiﬁed in breast cancer from women in Europe, Asia and
the American continents [Damin et al., 2004; de
Villiers et al., 2005; Khan et al., 2008; de Leon et al.,
An unresolved issue is whether the HPV DNA
found in breast cancer samples necessarily reﬂect
Fig. 2. HPV in situ hybridization. Positive cells are stained violet.
A: Breast cancer patient. B: Cervical cancer patient used as a positive control.
active infection and whether HPV is in fact etiologically involved in tumorigenesis, rather than simply
representing fortuitous or chance passage of the virus
through tissues. This controversy is reﬂected in the
recent world literature where reports of the presence
of HPV in malignant tumors of the breast continue to
cause debate with divergent results obtained. Whilst
some studies have reported negative results [de
Cremoux et al., 2008], the majority of recent studies
have indicated positive detection of various HPV
types, most commonly being HPV-16 and HPV-18. de
Villiers et al.  demonstrated that 86% of breast
carcinoma samples harbored papillomavirus sequences, with the most prevalent type being HPV-11. In
another study Damin et al. , reported HPV
DNA to be detected in 25% of breast carcinomas with
the most prevalent type being HPV-16 followed by
HPV-18, but none were detected in benign breast
samples. HPV DNA was detected in 21% of Japanese
breast carcinomas with HPV-16 being the most common type [Khan et al., 2008]. Furthermore, HPV was
not found in any of the benign breast tissue specimens. Breast cancer cases from Mexico have been
found to have a HPV DNA prevalence of 29%, with
J. Med. Virol. DOI 10.1002/jmv
the most common type being HPV-16 followed by
HPV-18 [de Leon et al., 2009]. In all of the above studies PCR analysis was performed on archival parafﬁn
embedded specimens. This present study, which
detected HPV-18 in 50% of cases, was performed on
fresh frozen breast cancer tissue samples which could
explain the higher rate of detection. The use of fresh
frozen samples also minimizes the potential for handling and contamination of the tissue specimens
which likely contributes to the ﬁnding of a speciﬁc
HPV type present within the tumor tissue.
Superﬁcially, it would appear that there is a lack of
consensus on the prevalence and contribution of HPV
to breast cancer formation. Although this current
report has been able to show that 50% of fresh frozen
breast cancer samples were positive for HPV-18, the
level of infection of HPV-18 may be considerably less
than that associated with HPV-caused cervical cancer.
It is unknown whether the low level of HPV-18
presence in the breast cancer tissue is capable of
transforming the cells. This could be supported by the
ﬁnding of one patient with HPV-18 positive normal
tissue but a HPV negative breast cancer, implying a
certain magnitude of infection may be required to
result in a HPV-induced cancer. A recent study has
also showed that HPV-18 can be found in the nucleus
of the breast cancer cells as well as in the normal
breast tissue [Heng et al., 2009]. Ultimately, the contribution of HPV-18 to breast cancer development will
be indicated in patients vaccinated against high-risk
HPV types. Alternatively, the potential contribution
of HPV-18 to breast cancer development may require
sophisticated inducible transgenic mice in which
HPV-18 expression can be controlled in a dose and
Of interest in this study was the fact that HPV positive tumors were statistically more likely to be early
stage tumors, either T1 or T2 lesions or of a size
<2.5 cm. A trend was also noted with respect to nodal
status, with HPV positive tumors being more likely to
be either node negative or have three or less nodes
involved compared to HPV negative tumors. This
could imply that the presence of HPV may at least
have a fundamental role in the growth pattern and
metastatic potential of breast cancers. Other reports
in head and neck and lung cancers have similarly observed that the presence of HPV DNA was correlated
with a better prognosis [Gillison et al., 2000; Iwamasa
et al., 2000].
In this current study, HPV positive cells could not
be identiﬁed with the in situ hybridization technique
in a series of HPV-18 positive breast cancer tissue
slides. In a previous report by de Villiers et al. ,
in situ hybridization was able to positively identify
viral sequences in the breast cancer cells for HPV-6,
HPV-11, and HPV-16. The surrounding stromal
tissues were negative, supporting the case for HPV
having a causative effect. However the explanation
for the negative in situ hybridization result here
remains unclear, but could be reﬂective of a low viral
J. Med. Virol. DOI 10.1002/jmv
Antonsson et al.
load per positive cell or alternatively, as the PCR
ampliﬁcation analysis was performed in fresh frozen
tissues in this study, this may be much more sensitive
than in situ hybridization performed on parafﬁn
embedded tissues. Indeed de Villiers et al. indicated
that the in situ hybridization technique may be less
sensitive and would not necessarily detect a single
viral genome copy per cell; indeed it may be that at
least 10 copies of the viral genome per cell are
required for in situ detection.
The current study showed a tendency for HPV positive carcinomas to be of ductal (96.3%) rather than
lobular type (compared to 85.2% HPV negative
tumors), even thought this was not statistically significant. In de Villiers’ report HPV was found in the
nipple and areola tissues and in major ducts deep to
the nipple, raising the possibility of retrograde transmission of HPV along ducts. It has therefore been postulated that if HPV were involved in carcinoma
transformation that there might be a preponderance
of ductal histological types [Altundag and Baptista,
Only a small number (four) of control tissue samples were included in this study, one of which was
positive. However this should not signiﬁcantly detract
from the ﬁndings, particularly in the context of the
ﬁndings of Heng et al.  whose study did ﬁnd
HPV in a small proportion of control samples but
which is consistent with the expected pathogenic requirement for HPV infection to be present in the
breast tissues for some period before HPV-induced
tumorogenic transformation occurs.
The potential mechanism of transmission of HPV to
the breast remains unclear with opinions being divided between direct contact spread from the female
perineum to the breast or hematological spread.
As already indicated, the presence of HPV has been
demonstrated recently not only in the primary breast
cancer but also in tissue taken from the nipple
regions, which suggests that HPV may enter the
breast by infecting the epithelium of the nipple and
areola. Additionally in two separate studies, HPV-16
was found to be present in breast tumors that occurred in European women with HPV-16 associated
cervical cancer [Hennig et al., 1999b; Widschwendter
et al., 2004], thus raising the possibility that HPV
may be transmitted by hand from the female perineum to the breast. However recent evidence has been
demonstrated for the hematological carriage of HPV
on peripheral blood mononuclear cells which could
provide an alternative pathway for the transmission
of the virus to the breast [Bodaghi et al., 2005; Chen
et al., 2009].
In conclusion, in this study of fresh frozen breast
cancer samples, using a comprehensive PCR screening technique, the presence of HPV-18 only was
detected in 50% of all cases. HPV positive breast cancer cases were noted to be slightly younger, and were
found to be more likely smaller tumors associated
with node negativity, but no other pathological
HPV in Breast Cancer
correlations were noted. While in situ hybridization
studies of corresponding parafﬁn sections were negative, and the etiological role of HPV in breast cancer
remains unclear, the discovery of HPV in such a high
proportion of breast cancers cannot be dismissed. Further investigation of the potential contribution of
HPV-18 to breast cancer development may require research involving sophisticated inducible transgenic
mice in which HPV-18 expression can be controlled in
a dose and time-dependent manner. Ultimately the
role of HPV in breast cancer development will be
ascertained by monitoring the future incidence of the
disease in women administered cervical cancer vaccinations against high-risk HPV types.
The funding bodies played no role in the design or
conduct of the study; the collection, management,
analysis or interpretation of the data; or the preparation, review, or approval of the manuscript. The contents are solely the responsibility of the authors and
do not necessarily represent the ofﬁcial views of the
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