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Infect Dis Obstet Gynecol. 2007; 2007: 60549. Published online 2008 January 2. doi: 10.1155/2007/60549. | PMCID: PMC2233766 |
Human Papillomavirus in Endometrial Adenocarcinomas: Infectious Agent or a Mere “Passenger”? A. Giatromanolaki,1, 2* E. Sivridis,1, 2 D. Papazoglou,3 M. I. Koukourakis,3 and E. Maltezos3 1Department of Pathology, Democritus University of Thrace Medical School, 68100 Alexandroupolis, Greece 2Department of Radiotherapy/Oncology, Democritus University of Thrace Medical School, 68100 Alexandroupolis, Greece 3Second Department of Internal Medicine, Democritus University of Thrace Medical School, 68100 Alexandroupolis, Greece Received June 16, 2007; Accepted November 12, 2007. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Aims. To investigate the possible association
of human papillomavirus (HPV) with endometrial hyperplasias and
neoplasia. Does HPV play any role in the initiation or
prognosis of endometrial adenocarcinomas?
Methods. Twenty-five endometrial adenocarcinomas
of the endometrioid cell type, with and without squamous
differentiation, and twenty-four endometrial hyperplasias of various
forms (simple, complex, and atypical) were analyzed for the
presence of type 16 and 18 HPV by the polymerase chain
reaction (PCR). The results were related to histopathological
features of the tumour, and the patients' age, and
prognosis. Results. Six of 25 endometrial
adenocarcinomas were HPV 16-positive
(  ),
and 5 of 25
(  )
were HPV 18-positive. Simple endometrial hyperplasias was
associated somewhat more commonly with HPV 16 and 18 (2/8 and
1/8 cases, resp.) than hyperplasias progressing to
endometrial adenocarcinomas, namely, atypical endometrial
hyperplasia (1/8 and 0/8 cases, resp.). None of the
positive cases in the series, whether hyperplastic or
neoplastic, demonstrated cytological evidence of HPV
infection. There was no relation between HPV-positive cases
and squamous differentiation, depth of myometrial invasion,
lymphatic involvement, lymphocytic response, patients' age,
or prognosis. Conclusion. It appears that the
presence of HPV in the endometrium, as detected by PCR, does
not play any role in the initiation or prognosis of
endometrial adenocarcinoma. Human papillomavirus
(HPV) infection in the female genital tract has been connected with specific
sites: vulva, vagina, cervix, and specific epithelium—the stratified squamous
epithelium, leading to epithelial cell proliferation and often malignancy. The
infection, most reliably detected by polymerase chain reaction (PCR), is recognized by distinct histological changes in epithelial
cells consisting of multinucleation and koilocytosis [ 1, 2]. Typical examples of
the hyperplastic process include condylomata accuminata, and those of the neoplastic
change include the
intraepithelial neoplasias and the squamous cell carcinomas of the vulva,
vagina, and cervix. In this context, HPV-infected cells have almost equated
with frankly malignant cells and, indeed, the Bethesda system incorporated
koilocytotic atypia and cervical intraepithelial neoplasia grade I (CIN I) into
one category, the low-grade squamous intraepithelial lesions (LSIL), leading
perhaps to excisional cone biopsy [ 3]. A number of reports have
demonstrated the presence of HPV in endometrial adenocarcinomas [ 4– 8], a tissue
close to, but lacking, the stratified
squamous epithelium of the exocervix. Whether such tumours with glandular-type
epithelium may also exhibit morphological evidence of HPV infection is not
clear, as koilocytotic-like changes have hitherto been only reported in the
squamoid component of some endometrial adenocarcinomas [ 9, 10]. There is also
some dispute as to whether the presence of HPV in endometrial tissues
contributes to the development of endometrial neoplasms [ 6, 9, 11– 13]. Thus far,
we only know that HPV is unrelated to prognostic parameters and survival [ 7]; and
there is, of course, little information with regard to the viral presence in endometrial
hyperplasias [ 4, 8]. In view of this paucity of
information, this study was designed to evaluate the presence of HPV in the
endometrium by PCR and its potential role in the genesis of endometrial
adenocarcinoma. The tissues used for this study were drawn from the files of the
Department of Pathology, Democritus University of Thrace Medical School (Alexandroupolis,
Greece). They were all hysterectomy specimens and had been routinely fixed in
10% formol-saline. The original haematoxylin and eosin- (H and E-) stained
sections consisted of 24 endometrial hyperplasias of various forms: simple
(  ), complex (  ), atypical (  ), and 25 endometrial adenocarcinomas:
endometrioid cell type, G1, stage I, with (  ) and without (  ) squamous
differentiation. The endometrioid adenocarcinomas and the various forms of endometrial
hyperplasia were typed along the lines suggested by Buckley and Fox [ 14]. As G1 endometrioid adenocarcinomas were considered, only those composed in
their entirety of glandular elements have no solid components, other than squamous, and no nuclear
atypia, other than low grade [ 15]. The tumour stage was defined according to
FIGO staging system [ 16]. None of the patients in the series had previous
history of vulvar, vaginal, or cervical HPV-related lesion, intraepithelial neoplasia, or carcinoma.
The duration of follow up was 5 years at least. The tissues were
analysed for the presence of type 16 and 18 HPV by PCR amplification (Laboratory of Second
Department of Internal Medicine),
Democritus University of Thrace Medical School (Alexandroupolis, Greece). 2.1. DNA extraction Paraffin sections were cut at 7   m and nonhyperplastic or nonmalignant tissue was
trimmed away from the sample using parallel (H and E-) stained sections as a
guide [ 17]. Normal sections were also cut and served as negative controls. Vigorous
preparations were taken to avoid sample contamination. This was achieved by
cleansing the microtome with 75% ethanol before and after cutting up each
paraffin block, and using sterilized stainless forceps for transferring the
sections. Paraffin wax was removed with xylene,
and the samples were subsequently washed with 100% ethanol (the two steps
repeated twice). DNA was isolated from the resuspended tissue using a
commercially available kit, according to the manufacturer’s instructions
(QIAamp DNA Mini Kit, QIAGEN Inc, Calif, USA). The human p53 and VEGF genes
were used as controls to test the amplification ability of the extracted DNAs. 2.2. Detection of HPV Isolated DNA was subjected to GP5+/GP6+ PCR [ 18].
Briefly, standard PCRs were carried out in 50  L containing 50 mM KCl, 10 mM Tris HCL (pH 8.3), 200  M each dNTP, 3.5  M MgCl 2, 1 U Platinum Taq Polymerase (Gibco, BRL, USA), and 50 pmol each of the GP5+
(5 '-TTTGTTACTGTGGTAGATACTAC-3 ') and GP6+
(5 '-GAAAAATAAACTGTAAATCATATTC-3 ') primers.
Polymerase chain reaction amplification conditions were 96°C (1 minute), 45°C (1.5 minutes) and 72°C
(1 minute) for 40 cycles, followed by final extension at 72°C for 10 minutes. Single PCR was carried out
using type-specific primers to investigate the incidence of both HPV 16 and HPV
18 sequences in GP5+/GP6+ samples
(Cheng et al., 1995). The HPV 16 specific primers were as follows: Forward: 5 '-CCCAGCTGTAATCATGCATGGAGA-3 ' and
Reverse: 3 '-CACACGGGTAATTCAGAAGGT-5 '
generating a 253 bps PCR product. The
HPV 18 specific primers were as follows: Forward: 5 '-CGACAGGAACGACTCCAACGA-3 ' and Reverse:
3 '-TCAATTTAGTAGTTGTAAATGGTCG-5 ' generating a 201 bps PCR product. PCR
amplification was carried out in 25  L final volume containing 50 mM KCl, 10 mM Tris-HCl
(pH8.3), 1.5  M MgCl 2, 200  M each dNTP, and 1 U Platinum Taq Polymerase (Gibco, BRL, US). Polymerase chain reaction
amplification conditions were 96°C (30 seconds), 60°C (30 seconds), and 72°C
(30 seconds) for 35 cycles, followed by final extension at 72°C for 10 minutes. The amplifications were
carried out in a Mastercycler gradient (Eppendorf-Netheler- Hinz GmbH, Hamburg, Germany) thermal cycler and the PCR
products were visualized in ethidium bromide stained agarose gels (2%). In all PCR assays, appropriate
positive controls for HPV 16 (human Caski cell line DNA), HPV 18 (human HeLA
cell line DNA), and cervical squamous cell carcinomas were used and identified.
In addition, the commercially provided positive controls for the identification
of HPV 16 and HPV 18 by Maxim Biotech, Inc (San Francisco, Calif,
USA) were applied successfully. Human lung and liver tissues were tested as
negative controls and were consistently negative. All
reactions were performed in a “blinded” manner by DP. 2.3. Statistics Statistical analysis of the data was performed using Fisher’s exact test
(SPSS, version 11.0.1). Human
papillomavirus (HPV) 16 was a somewhat more common inhabitant than HPV 18 in
endometrial neoplasms and in all forms of endometrial hyperplasia. It was
detected in 24% (versus 20% of HPV 18) of the endometrial adenocarcinomas
studied, and in 16.6% (versus 4% of HPV 18) of the endometrial hyperplasias
( Table 1). Both types of HPV occurred preferentially in simple endometrial
hyperplasia rather than in complex or atypical hyperplasia, and there were
three invasive endometrial neoplasms that were positive for both HPV 16 and 18.
Interestingly, HPV were detected with approximately equal frequency in
endometrial adenocarcinomas with squamous differentiation (1 in 5) and those
without squamous elements (4-5 in 20) ( Table 1). | Table 1HPV detection in
hyperplastic and neoplastic endometrium. |
Detection
of HPV 16/18 DNA sequences in primary endometrial adenocarcinomas by PCR
amplification products in representative specimens are shown in Figures and , respectively. None of the HPV 16 and 18-positive cases, whether hyperplastic or neoplastic,
demonstrated cellular evidence of viral infection, that is, koilocytotic atypia
or multinucleation. There was no statistical correlation between HPV 16/18 and
squamous differentiation, depth of myometrial invasion, lymphatic involvement,
lymphocytic response, patients’ age, or survival (data not shown). The detection of
human papillomavirus (HPV) in the human endometrium, whether hyperplastic or
neoplastic, is fraught with curiosities. HPV was thought as being site- and
tissue-specific, infecting the stratified squamous epithelium of the lower
female genital tract: vulva, vagina, and exocervix [ 19], most commonly in connection with condylomata accuminata,
intraepithelial neoplasias, and invasive carcinomas, and that the infected tissues
suffered the cytopathic effect of multinucleation and koilocytotic atypia.
Still, HPV 16 and 18, as detected by PCR, appear to reside in the endometrium, a simple or
pseudostratified epithelium columnar in type, ciliated, in part, lining glands
or glandular structures, and not
having the characteristic cellular changes of HPV infection [ 1, 2]. Furthermore, the glandular lesions of the
endocervix (adenocarcinoma in situ, invasive adenocarcinoma) may also harbour
HPV 16 and 18 without morphological evidence of multinucleation or koilocytotic
atypia [ 20– 22]. Detecting
HPV DNA sequences in tissues originally fixed in formaldehyde and embedded in paraffin wax may prove
difficult by PCR methods [ 18], and the results obtained are inconsistent, as both specificity and
sensitivity of various HPV PCR primer sets are not unaffected by intermethod
variations [ 23]. This is
reflected in the reported incidence of HPV 16/18 DNA detection in endometrial
adenocarcinomas ranging from 4% to 37.5% [ 4– 8]. Our results fall somewhere in the middle of this
range (24% for HPV 16, and 20% for HPV
18) and are irrespective of the presence or otherwise of squamous
differentiation. Given
that HPV infection precedes the development of cancer [ 24], it is also intriguing that HPV was detected less
frequently in endometrial hyperplasias progressing to adenocarcinomas, namely
atypical hyperplasia, than those not related to such development, namely simple
endometrialhyperplasia, and in carcinoma. This
apparently means that HPV cannot initiate oncogenic events in the endometrium
through the sequence atypical hyperplasia-neoplasia.
Similarly low or even lower, almost negligible, incidence of HPV infection for atypical hyperplasia
were reported earlier [ 4, 8]. Interestingly, the presence
of HPV in endometrial neoplasms was unrelated to histopathological features,
patients’ age, or patients’ survival. This is also the experience of other
investigators [ 8]. It is rather odd, however, that geographical/environmental
conditions may influence the frequency of HPV detection; HPV 16 was detected in
6/47 (13%) endometrioid adenocarcinoma from Japan and 2/38 (5%) from the United
States [ 4]. Others failed to detect HPV DNA in endometrial
carcinomas [ 25– 28], despite
employing relatively large number of cases (66 in one study) and tumours with
squamous cell elements (adenocarcinomas with squamous differentiation, squamous
cell carcinomas) [ 26] or cervical tissues with stage II
endometrial adenocarcinomas [ 28]; they
suggested that the absence of HPV from the malignant endometrium is a hallmark of endometrial, as opposed to endocervical adenocarcinomas
[ 28, 29]. Since HPV 16/18 infection
is, by and large, site- and tissue-specific (vulva, vagina, and cervix stratified squamous epithelium), the endometrium, as indeed the glandular lesions of the endocervix,
may not be a suitable host for HPV replication and maturation. This is further
supported by the absence of relevant epithelial changes, lack of correlation
with histological features or prognosis, and the low incidence rates with
precancerous endometrial lesions. There is, of course, some evidence that
koilocytotic-like changes may occur in the squamoid component of some
endometrial adenocarcinomas with squamous differentiation [ 9, 10] and it is perhaps possible that at this site
HPV positivity is preferentially present [ 30]. Nonetheless, the spread
of HPV in the endometrium is not uncommon [ 4– 8] and several cases in our
material were positive for both HPV 16. We believe, as others do [ 9], that HPV,
originated from the lower genital tract, represents a mere “passenger” in the
endometrium, residing in its simple or pseudostratified columnar epithelium and
having no aetiological or pathogenic role in the development of endometrial
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