Journal of Pathology

J Pathol 2015; 236: 265271

Published online 27 April 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/path.4533

BRIEF DEFINITIVE REPORT

Carcinogenic HPV infection in the cervical squamo-columnar

junction
Jelena Mirkovic,1 Brooke E Howitt,1 Patrick Roncarati,2 Stephanie Demoulin,2 Meggy Suarez-Carmona,2 Pascale
Hubert,2 Frank D McKeon,3 Wa Xian,3 Anita Li,1 Philippe Delvenne,2 Christopher P Crum1,*, and Michael
Herfs2,*,
1
2
3

Department of Pathology, Division of Womens and Perinatal Pathology, Brigham and Womens Hospital, Boston, MA, USA
Laboratory of Experimental Pathology, GIGA-Cancer, University of Lige, Lige, Belgium
Jackson Laboratory for Genomic Medicine, Farmington, CT, USA

*Correspondence to: M Herfs, PhD, Laboratory of Experimental Pathology, GIGA-Cancer, University of Lige, 4000 Lige, Belgium.
E-mail: M.Herfs@ulg.ac.be
Or Christopher P Crum, MD, Division of Womens and Perinatal Pathology, Department of Pathology, Brigham and Womens Hospital, Boston,
MA 02115, USA. E-mail: ccrum@partners.org

CPC and MH contributed equally as senior authors.

Abstract
Recent studies have suggested the involvement of a unique population of cells at the cervical squamo-columnar
junction (SCJ) in the pathogenesis of early (squamous intraepithelial lesion or SIL) and advanced (squamous
cell and adeno-carcinomas) cervical neoplasia. However, there is little evidence to date showing that SCJ cells
harbour carcinogenic HPV or are instrumental in the initial phases of neoplasia. This study was designed to (1)
determine if normal-appearing SCJ cells contained evidence of carcinogenic HPV infection and (2) trace their
transition to early SIL. Sections of cervix from high-risk reproductive age women were selected and SCJ cells
were analysed by using several techniques which increasingly implicated HPV infection: HPV DNA (genotyping
and in situ hybridization)/RNA (PCR), immunostaining for HPV16 E2 (an early marker of HPV infection), p16ink4 ,
Ki67, and HPV L1 protein. In 22 cases with a history of SIL and no evidence of preneoplastic lesion in the excision
specimen, HPV DNA was isolated from eight of ten with visible SCJ cells, six of which were HPV16/18 DNA-positive.
In five of these latter cases, the SCJ cells were positive for p16ink4 and/or HPV E2. Transcriptionally active HPV
infection (E6/E7 mRNAs) was also detected in microdissected SCJ cells. Early squamous atypia associated with
the SCJ cells demonstrated in addition diffuse p16ink4 immunoreactivity, elevated proliferative index, and rare L1
antigen positivity. We present for the first time direct evidence that normal-appearing SCJ cells can be infected by
carcinogenic HPV. They initially express HPV E2 and their progression to SIL is heralded by an expanding metaplastic
progeny with increased proliferation and p16ink4 expression. Whether certain SCJs are more vulnerable than others
to carcinogenic HPV genotypes and what variables determine transition to high-grade SIL remain unresolved, but
the common event appears to be a vulnerable cell at the SCJ.
Copyright 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: HPV; squamo-columnar junction; cervical intraepithelial neoplasia

Received 13 January 2015; Revised 4 March 2015; Accepted 14 March 2015

No conflicts of interest were declared.

Introduction
Human papillomavirus (HPV) infection causes cervical
cancer and its precursor lesions (squamous intraepithelial lesion or SIL), specifically at the squamo-columnar
junction (SCJ) [1,2]. Recent studies of the
gastro-oesophageal and ecto-endocervical junctions
have unearthed residual embryonic cell populations
with both a capacity to differentiate and a vulnerability
to undergo neoplastic transformation [3,4]. The cervical
SCJ cells share an identical immunophenotype [including strong staining for keratin 7 (Krt7)] with over 90%
of high-grade SILs (HSILs) and cervical carcinomas,
Copyright 2015 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk

supporting their role in the initiation of this carcinogenic

sequence [3,5]. The predilection of carcinogenic HPV
infection for these cells provides one explanation why
the number of new cervical cancers yearly worldwide
is nearly 20-fold that of carcinomas in well-developed
lower genital tract squamous epithelium [6].
Despite the proximity of the SCJ to cervical neoplasia
and their shared immunophenotype, there is little information directly linking SCJ cells to HPV infection. This
issue is significant for several reasons. First, the histological link between SCJ infection and SIL development has not been clearly established, apart from limited
immunohistochemical and in situ hybridization studies
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266

that have placed the SCJ cells in close proximity to HSIL

[7]. Second, despite the fact that the concept of latent
HPV infection has been in play for over three decades,
the actual cells harbouring this form of HPV infection
have yet to be revealed.
This study focused on SCJ cells in high-risk
reproductive age women, the main goal being to
determine whether transcriptionally active HPV infection could be observed in these junctional cells.
Herein we provide evidence that the SCJ cells can
be infected by HPV and serve as a nidus for early lesion
development.

Materials and methods

J Mirkovic et al

Immunohistochemical assessment
The procedure is described in detail in the Supplementary materials and methods.

In situ hybridization
As previously described [7], HPV genotypes were
detected by in situ hybridization (ISH) using the Ventana INFORM HPV III family 16 probe (Ventana
Medical Systems, Tucson, AZ, USA) according to
the suppliers recommendations. This kit contains
labelled probes allowing the detection of 12 carcinogenic HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52,
56, 58, and 66). Visualization of hybridized DNA was
performed using Red Counterstain II (Ventana Medical
Systems).

Tissue samples
The protocol was approved by the Ethics Committee
of the University Hospital of Lige (Lige, Belgium)
and by the institutional review board at Brigham and
Womens Hospital (Boston, MA, USA). First, we
searched in our databases (Brigham and Womens
Hospital and University Hospital of Lige) for patients
with a history of SIL who underwent an excision
procedure [loop electrosurgical excision procedure
(LEEP)] during the previous year (July 2013July
2014) but had no evidence of preneoplastic lesion in the
LEEP specimen. Twenty-two patients were selected.
Second, 31 HPV16-positive paraffin-embedded specimens involving the SCJ [10 immature metaplastic
LSILs and 21 classical lesions (7 LSILs and 14
HSILs)] were also obtained. All cases stained positive for Krt7 and were re-examined by experienced
pathologists.

Immunohistochemistry
Immunohistochemical analyses were performed as
previously described [8,9]. Antibodies anti-keratin 7
(Clone RCK 105; Thermo Scientific, Waltham, MA,
USA), anti-Ki67 (Clone SP6; Abcam, Cambridge,
MA, USA), anti-involucrin (Clone SY5; Novocastra,
Newcastle, UK), anti-p16ink4 (Clone JC8; Santa Cruz
Biotechnology, Santa Cruz, CA, USA), anti-HPV
L1 (Clone K1H8; Dako, Glostrup, Denmark), and
anti-HPV16 E2 (kindly provided by Dr S Bellanger,
Institute of Medical Biology, Singapore) were selected.
This latter antibody detects HPV16 E2 in immunohistochemical experiments (Supplementary Figure
1). A cross-reaction with HPV18 E2 was also previously reported by western blots [10] and confirmed
by immunohistochemistry (Supplementary Figure 1).
Signal detection was achieved using the LSAB2 or
Envision kit (Dako) according to the suppliers recommendations. Positive cells were visualized using a
3,3-diaminobenzidine (DAB) substrate and the sections
were counterstained with haematoxylin. Mouse and rabbit control IgG (Santa Cruz Biotechnology) were used
as a negative control.
Copyright 2015 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk

HPV genotyping
The Abbott RealTime High-Risk HPV test (Abbott,
Wiesbaden, Germany) was used to simultaneously genotype HPV16 and 18, and detected 12 other HPV types
(31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68).
Although this assay has been shown to be highly sensitive and specific for the genotyping of HPV16 and
18 in paraffin-embedded samples [11], the collected
results were confirmed by real-time PCR as previously
described [12].

Laser capture microdissection, mRNA extraction,

and amplification
Seven serial sections (6 m thick) of each cervical specimen (immunostained and HPV-typed as
described above) were microdissected using a Leica
LMD7000 system (Leica), and each cell population
(squamous, columnar or SCJ cells) from different
slides but from the same patient was pooled. Total
RNA was extracted using the Nucleospin RNA XS kit
(Macherey-Nagel, Dren, Germany). cDNA library synthesis and amplification were then performed using the
Complete Whole Transcriptome Amplification (WTA2)
Kit (Sigma Aldrich, St Louis, MO, USA), which is
optimized to amplify RNA from paraffin-embedded
and other damaged samples. Before cDNA synthesis, DNase treatment was performed in order to
remove contaminating genomic DNA and to avoid
the detection of HPV DNA by PCR (Supplementary
Figures 2 and 3).

Polymerase chain reaction analysis

HPV16 E6*I, E6*II, E7, and L1 mRNAs as well
as HPV18 E6 and E7 transcripts were detected
in microdissected samples. Primer sequences and
detailed procedure are described in the Supplementary
materials and methods. Samples were loaded on 2%
agarose gels containing ethidium bromide and visualized with a UV transilluminator (Bio-Rad, Hercules,
CA, USA).
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Carcinogenic HPV infection in the cervical SCJ

267

Figure 1. HPV16 E2 immunoreactivity and viral oncogene expression (mRNA) are detected in normal-appearing SCJ cells from high-risk
women. (A) SCJ cells stained for Krt7, HPV16 E2, p16ink4 , and Ki67. Note the absence of p16ink4 immunoreactivity and the positive HPV16 E2
staining (an early marker of HPV infection). HPV DNA was not detected by in situ hybridization (ISH). (B) Target cell populations (squamous,
columnar, and junctional) were detected and microdissected. (C) Total RNA from each cell population was extracted, amplified, and HPV16
E6*I, E6*II, E7, and L1 expression was analysed by PCR. HPV E6 and E7 mRNAs were detected in SCJ cells, suggesting a transcriptionally
active infection in these cells. No HPV oncogene expression or immunohistochemical evidence of HPV infection was observed in squamous
(ectocervix/TZ) or columnar (endocervix) cells immediately adjacent to the SCJ cells. Original magnifications: 40 (microdissection), 100
(immunostaining) or 200 (insets, H&E, ISH).

Results
Detection of HPV in normal-appearing SCJ cells
from high-risk women
The goal was to determine where in the transformation zone HPV could be isolated in women with a
recent history of cervical neoplasia. We focused on the
SCJ, squamous metaplasia, and mature columnar epithelium (Figures 1 and 2). Twenty-two patients with a
prior history of SIL on histological examination but
no evidence of HPV-related lesion in the LEEP specimen were identified. We targeted SCJ cells based on
location (interface of mature squamous and endocervical mucosa), morphology (cuboidal or low columnar
phenotype), and strong Krt7 immunoreactivity. In ten
patients, the SCJ region was visible in serial sections
and contained normal-appearing SCJ cells not associated with reserve or metaplastic cells. HPV DNA was
detected in eight patients. HPV16 and HPV18 were
isolated from five and one of the eight positive cases,
Copyright 2015 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk

respectively. The exact genotype could not be determined in two samples. Similar HPV genotypes were
detected in both the diagnostic biopsies and the excision specimens. To pinpoint the specific cells infected
by HPV, we immunostained for p16ink4 , HPV E2, and
Ki67. In four cases, discrete foci of SCJ cells stained
strongly for both p16ink4 and HPV E2 (Figure 2A and
Supplementary Figure 4). Some rare Ki67-positive cells
were also observed. In one case, HPV E2 immunoreactivity was observed in Krt7-positive SCJ cells without
evidence of p16ink4 expression (Figure 1A). Although
HPV DNA was not detected by ISH (which is known
to have a limited sensitivity when the copy number of
HPV DNA is low [13]), the presence of HPV was confirmed by PCR (Figures 1C and 2C). When total mRNA
was recovered from microdissected SCJ cells and amplified, HPV16 E6*I, E6*II, and E7 were detected in three
of five cases. HPV E6 and E7 transcripts were also
detected in the HPV18-infected specimen (Supplementary Figure 4). Altogether, these results were interpreted
as evidence for a transcriptionally active infection in
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J Mirkovic et al

Figure 2. (A) p16ink4 /HPV16 E2 immunoreactivity in normal-appearing SCJ cells from high-risk women. Some rare Ki67-positive cells were
also observed. HPV DNA was not detected by in situ hybridization (ISH). (B) Target cell populations (squamous, columnar, and junctional)
were detected and microdissected. (C) Total RNA from each cell population was extracted, amplified, and HPV16 E6*I, E6*II, E7, and L1
expression was analysed. HPV E6 and E7 mRNAs were detected in SCJ cells, suggesting a transcriptionally active infection in these cells.
Internal controls included adjacent squamous and columnar epithelia. Original magnifications: 40 (immunostaining and microdissection)
or 200 (insets, H&E, ISH).

these cells. No HPV or immunohistochemical evidence

of HPV infection was detected in the control mature
squamous or endocervical cells immediately adjacent to
the SCJ cells (Figures 1C and 2C). The results obtained
for each HPV-positive cervical specimen tested are summarized in Supplementary Table 1.

HPV infection in both SCJ cells and mild squamous

atypia (also known as early CIN)
A high percentage of HSILs express SCJ-related
biomarkers [3,5]. To clarify the histological link
between SCJ infection and SIL development, cervical
biopsies with SCJ cells accompanied by an underlying squamous atypia were identified (Figure 3A).
Significantly, the patches of p16ink4 /HPV E2-positive
cells were associated with an increased proliferation.
Both HPV DNA (ISH) and mRNAs (PCR) were also
detected (Figures 3A and 3B). However, epithelial
differentiation/stratification seemed insufficient to produce virions, as suggested by the absence of HPV L1
expression in early SIL (Figure 3B).
Copyright 2015 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk

SCJ cells appear to give rise to reserve cells capable

of squamous differentiation, seen during embryogenesis
and transformation zone development [7]. We observed
cuboidal cells on the surface of mild metaplastic atypias,
suggesting early SIL development. All analysed lesions
stained positive for keratin 7, p16ink4 , HPV E2, and Ki67
(Figure 3C). Interestingly, focal apical L1 immunoreactivity was also detected in five of ten cases (Supplementary Figure 5) and confirmed at the mRNA level
(Figure 3D). However, L1 expression was weak compared with that observed in mature ectocervical/TZ
SIL (Supplementary Figure 5) and this result was not
interpreted as evidence confirming virion production.
Based on morphology and Krt7 positivity, we identified remaining SCJ cells in direct continuity with
atypical epithelium in 10/21 (47.6%) SILs seen near
the SCJ. In contrast to the strong and diffuse (basal
or full thickness) p16ink4 immunostaining observed
in all (pre)neoplastic lesions, no contiguous SCJ
cells expressed this marker. However, in three cases,
these normal-appearing SCJ cells displayed HPV E2
immunoreactivity in keeping with HPV16 infection
(Figure 4A).
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Carcinogenic HPV infection in the cervical SCJ

269

Figure 3. Characterization of the early steps of cervical neoplasia initiated within the SCJ. Early cervical neoplasia stained for Krt7, HPV16
E2, p16ink4 , and Ki67. The patches of p16ink4 /HPV E2-positive cells were significantly associated with increased proliferation. HPV DNA was
detected by in situ hybridization (ISH) in both early SIL (A) and immature metaplastic lesions (C). After microdissection, mRNA extraction,
and amplification, HPV16 E6*I, E6*II, E7, and L1 expression was analysed (B, D). Note the L1 mRNA expression in immature metaplastic
SIL. Adjacent squamous and columnar epithelia do not express viral oncogenes and do not display immunohistochemical evidence of HPV
infection. Original magnifications: 100 [immunostaining (AC), H&E (C), ISH (C)] or 200 [insets, H&E (A) and ISH (A)].

Discussion
We have previously shown that cells with an SCJ
immunophenotype can be found on the surface of HSIL
present at or near the SCJ and share both HPV DNA
(ISH) and p16ink4 immunoreactivity with these lesions
[7]. Moreover, we showed that most lesions overexpressing SCJ-related biomarkers were HPV16-positive, irrespective of their lesion grade [5]. In a subsequent study
published recently, a spectrum of immature metaplastic lesions near the SCJ ranging from bland-appearing
metaplasia to HSIL were highly associated with HPV16
or 18 and stained positive for Krt7 [14]. The implication from these studies was that SCJ infection by HPV
initiated this progressive sequence from immature metaplasia through HSIL. The current study is the first to
focus specifically on the SCJ as an initial site of infection
and a potential reservoir of HPV nucleic acids prior to
the development of SIL.
Copyright 2015 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk

We selected several techniques that were intended

to highlight early and late HPV infection: HPV DNA
(ISH), HPV mRNAs (PCR), and immunohistochemical detection of HPV16 E2, p16ink4 , and HPV L1,
with isolated positivity for HPV E2 signifying the earliest phase of infection as shown previously [10]. Predictably, the earliest sign of infection in normal SCJ
cells was manifested by positive E2 immunoreactivity and HPV detection (by PCR) (Figures 1 and 2).
p16ink4 staining was negative or variable and proliferative activity (Ki67 staining) was absent or weak.
In contrast, when a squamous lesion developed, HPV
DNA (ISH) and diffuse p16ink4 staining were also
detected, accompanied by an increase in proliferative
index (Figure 3). An intriguing finding was the presence
of L1 mRNA signal in these latter immature lesions
accompanied by rare immunostaining suggesting L1
protein. It is impossible to confirm that virions were
present without ultrastructural confirmation. However,
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J Mirkovic et al

Figure 4. (A) Some remaining SCJ cells in direct continuity with atypical epithelium are infected by HPV. Note the absence of both p16ink4
and HPV L1 immunoreactivity in SCJ cells and the positive HPV16 E2 staining. (B) Schematic illustration of the HPV infection sequence in
the cervical SCJ. SCJ cells are infected, initially display expression of HPV E2, and terminate with proliferation that signifies the earliest
morphological evidence of SIL. Original magnifications: 40 (H&E and Krt7) or 100 (p16, HPV E2, and L1).

the presence of these signals is of interest given the

rarity of immunohistochemical evidence of L1 antigen detection in HPV16-associated lesions in the lower
genital tract [15].
Figure 4B proposes a model for an HPV infection
sequence in the SCJ, whereby the SCJ cells are infected
and initially display HPV E2 expression, followed
by metaplastic outgrowth that signifies the earliest
morphological evidence of SIL. This model is supported by the observations in this study, but several
fundamental questions remain. The first is whether the
SCJ is the universal site of latent HPV. This would
seem unlikely given the geographic range of HPV
infection in the lower genital tract. However, given its
exposure level as a cuboidal surface population, the
SCJ could be viewed as a preferred site of initial HPV
Copyright 2015 Pathological Society of Great Britain and Ireland.
Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk

infection. One study showed that ablation of this area

by cryotherapy reduced the detection rate of cervical
HPV by 50% [16]. It would be important to know
if this reduction was SCJ-related and specifically a
preferential reduction in HPV16 infection. Second and
third questions would be what the trigger is that initiates lesion development in the infected SCJ cells and
whether the HPV16-positive immature proliferations
that ensue are destined to become persistent high-grade
SIL as implied in some reports [5,14]. Given the high
percentage of CIN2s that disappear spontaneously,
it is highly likely that certain patient-specific factors
favour the progression of a subset of these lesions to a
persistent CIN3 [17]. This raises the additional question
of whether certain individuals are uniquely prone to
developing HSIL through some facet of SCJ biology or
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Carcinogenic HPV infection in the cervical SCJ

immune surveillance. Addressing these questions could

improve our understanding of the characteristics that
place such a small number of women at the greatest risk.
It would also provide further insight into the possible
effects of pre-emptive SCJ ablation on the subsequent
acquisition or persistence of HPV infection.

Acknowledgments
We thank our clinical colleagues at the University of
Lige and Brigham and Womens Hospital for their
cooperation. This work was supported in part by a grant
from the National Cancer Institute (5R21CA173190-02
to CPC), by the Belgian Fund for Medical Scientific
Research (FNRS), by the Centre Anti-Cancereux prs
lUniversit de Lige, by the Faculty of Medicine of the
University of Lige, and by the Fonds Lon Frdricq.
We thank Estelle Dortu for her technical assistance. We
are also grateful to Dr Sophie Bellanger (Institute of
Medical Biology, Singapore) for kindly providing us
with the anti-HPV E2 antibody.

Author contributions
JM, CPC, and MH designed the study. PR, MSC, and
MH performed experiments. JM, BH, AL, PD, and CPC
collected the tissue samples. JM, CPC, and PD reviewed
all cases. PH, FM, WX, CPC, and MH interpreted the
data. SD and MH generated figures. CPC and MH wrote
the manuscript. All authors had final approval of the
submitted manuscript.

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SUPPORTING INFORMATION ON THE INTERNET

The following supporting information may be found in the online version of this article:
Supplementary materials and methods.
Figure S1. Anti-HPV E2 antibody efficiently detects E2 protein of both HPV16 and HPV18 in immunohistochemical experiments.
Figure S2. Control experiments for the detection of HPV16 E6*I, E6*II, E7, and L1 mRNAs.
Figure S3. Control experiments for the detection of HPV18 E6 and E7 transcripts.
Figure S4. (A) p16ink4 /HPV E2 immunoreactivity in normal-appearing SCJ cells from HPV18-infected women. (B) Target cell populations (squamous,
columnar, and junctional) were detected and microdissected.
Figure S5. Focal apical L1 immunoreactivity was detected in immature metaplastic lesions (A). However, L1 expression was weak compared with
that observed in mature ectocervical/TZ SIL (B).
Table S1. Characteristics of each HPV-positive cervical specimen tested (no evidence of CIN in the LEEP specimen).

Copyright 2015 Pathological Society of Great Britain and Ireland.

Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk

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