1. C L I N I C A L A N D LA B O R A T O R Y I N V E S T I G A T I O N S DOI 10.1111/j.1365-2133.2005.06562.x
Human papillomavirus-associated plantar epidermoid cyst
related to epidermoid metaplasia of the eccrine duct
epithelium: a combined histological,
immunohistochemical, DNA–DNA in situ hybridization and
three-dimensional reconstruction analysis
K. Egawa, N. Egawa* and Y. Honda
Departments of Dermatology and Surgical Pathology, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860-0811, Japan
*Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan
Summary
Correspondence Background We recently proposed that certain palmoplantar epidermoid cysts may
Kiyofumi Egawa. be related to eccrine ducts and that human papillomavirus (HPV) 60 may play a
E-mail: kuroibo@kaiju.medic.kumamoto-u.ac.jp role in their pathomechanism. However, the origin of palmoplantar epidermoid
cysts is still controversial.
Accepted for publication
9 October 2004
Objectives To examine the contribution of eccrine ducts and HPV 60 in the devel-
opment of epidermoid cysts.
Key words: Methods Five epidermoid cysts and four ridged warts that had developed on the
eccrine duct, epidermoid cyst, human soles of a patient were studied histologically, immunohistochemically and by
papillomavirus 60, inclusion cyst, metaplasia DNA–DNA in situ hybridization. Using serial sections obtained from its entire
body, a three-dimensional reconstruction (3DR) analysis was performed on the
Conflicts of interest:
None declared. smallest cyst to analyse the relationship between the epidermoid cyst, eccrine
duct and the overlying epidermis.
Results Histological and DNA–DNA in situ hybridization analyses demonstrated
both homogeneous intracytoplasmic inclusion bodies pathognomonic for HPV
60 infection and HPV 60 DNA sequences not only in all of the epidermoid cysts
and ridged warts but also in the acrosyringeal portion of an eccrine duct, with
the dermal portion of which the smallest cyst had been revealed to connect by
3DR analysis. However, immunohistochemical analyses using antibodies against
human carcinoembryonic antigen (CEA), involucrin and several cytokeratins
(CKs) revealed that the immunoreactivity of the cyst was not identical to
that of the eccrine dermal duct but was identical to that of suprabasal layers of
the epidermis.
Conclusions It was clearly demonstrated that an HPV 60-associated epidermoid cyst
with immunoreactivities for CEA, involucrin and CKs which were identical to
those of the epidermis connected with the eccrine dermal duct, supporting the
idea that certain palmoplantar epidermoid cysts may develop following the epi-
dermoid metaplasia of eccrine ducts with HPV 60 infection.
Epidermoid cysts of palmoplantar location (palmoplantar epi- epidermoid cysts, with or without infection by human papil-
dermoid cysts) have been described as being caused by lomavirus (HPV) 60,5 and therefore proposed the alternative
implantation of epidermal fragments into the dermis as a pathogenic hypothesis that certain palmoplantar epidermoid
result of a penetrating or blunt injury.1 However, this assump- cysts may develop from eccrine ducts and that HPV 60 may
tion has been questioned, and the necessity of a search for a play a role in this development. However, on the basis of the
more common aetiology has been stressed.2 previous results,3–5 it is still likely that such epidermoid cysts
We previously observed3,4 that eccrine sweat ducts were can develop as a result of the implantation of epidermal frag-
frequently found in and ⁄or connecting with palmoplantar ments that contain eccrine ducts with or without HPV 60
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2. 962 HPV-associated eccrine epidermoid cyst, K. Egawa et al.
infection from injuries as they do occasionally develop after
injuries3,6 and are mainly located beneath the weight-bearing
areas of the foot, i.e. the balls and heels.7
Indeed, Abe et al.8 and Ohnishi and Watanabe9 questioned
the hypothesis of an ‘eccrine duct origin of the epidermoid
cyst’ on the basis of the immunoreactivities of several cyto-
keratins (CKs) which were not identical to those of sweat
glands and ducts but were identical to those of suprabasal
layers of the epidermis in plantar epidermoid cysts with HPV
infection.
However, Egawa et al.10 proposed the idea that such a CK
profile in HPV-associated palmoplantar epidermoid cysts might
be a reflection of the epidermoid metaplasia of the eccrine
duct epithelium in the process of cyst formation in the eccrine
ducts, as it was revealed that the CK profile of cholesteatomas,
which are histologically defined as epidermoid cysts develop-
ing in the middle ear,11 cannot be used as a variable to decide
whether the origin of epidermoid cysts is epidermal or meta-
plastic.12 Thus, the origin of palmoplantar epidermoid cysts is
still controversial.
Three-dimensional reconstruction (3DR) analysis from serial
sections is now a powerful tool for increasing our comprehen-
sion of the relationships among structural components, for the
indication of the connections between components, and for
revealing the distribution of components.13–15 In the present
study, using serial sections obtained from the entire body of
an epidermoid cyst, we attempted 3DR analysis to demon- Fig 1. Three subcutaneous nodules (solid arrows) and two ridged
strate the fine structure of an HPV 60-associated epidermoid warts (open arrows) on the left sole.
cyst of eccrine duct origin, in combination with histological,
immunohistochemical and DNA–DNA in situ hybridization
analyses. excised specimens were fixed in 10% formalin, and proc-
essed with an automatic processor.
Materials and methods
Histopathology
Patient
Four-micrometre thick sections were obtained using a micro-
A 21-year-old Japanese woman presented to Kumamoto Uni- tome from all of the biopsy specimens, stained with haema-
versity Hospital in April 1995, with several subcutaneous nod- toxylin and eosin (H&E), and examined microscopically
ules and warts on the soles of her feet. She first noticed (Figs 2 and 3). From the smallest cyst, gross serial sections
several miliary-sized asymptomatic nodules on her soles were obtained from its entire body, and all sections were
3 months before presentation and 1 year after having had sev- numbered in the order of sectioning, from one side to the
eral plantar warts treated with cryotherapy, although she could other. Fifty serial sections were obtained from each of the
not say whether or not the nodules developed in the same other cysts. Every alternate section was stained with H&E for
positions as the treated plantar warts. The nodules had gradu- histological analysis (Fig. 3), whereas the remaining sections
ally enlarged and had come to cause tenderness during the were used for further examinations such as immunohisto-
previous 3 months. chemical and DNA–DNA in situ hybridization analyses.
At the time of her first visit, examination showed three
subcutaneous nodules of 5–8 mm diameter and two small
Processing of histological and immunohistochemical data
flat warty lesions with clinical features of ridged warts16,17
for computer analysis
on the left sole (Fig. 1) and one nodule of 7 mm diameter
on the right sole. Her health was otherwise good. After Using photomicrographs of histopathological and immunohis-
informed consent was obtained, all the lesions were tochemically stained sections (Fig. 4a), we made montages by
removed successfully by scalpel under local anaesthesia. tracing the outlines of the histological ductal structures expres-
However, about 6 months later, a further subcutaneous sing carcinoembryonic antigen (CEA) in serial sections in red,
nodule of similar size appeared on the left sole, with two and those of the cyst wall and the overlying epidermis in blue
ridged warts. They were also removed by scalpel. The (Fig. 4b).
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3. HPV-associated eccrine epidermoid cyst, K. Egawa et al. 963
(Fig. 4d). The three-dimensional images were rotated along
various axes to analyse the relationship between the cyst,
eccrine ducts and the overlying epidermis.
Immunohistochemistry
An immunohistochemical procedure for human CEA,3,4,15,18
CKs8,9,19 and involucrin20 was performed on paraffin-embed-
ded sections by the avidin–biotin–peroxidase complex (ABC)
method,21 utilizing a Vectastain ABC kit (Vector Laboratories,
Burlingame, CA, U.S.A.). A polyclonal antibody to human CEA
(Dako, Glostrup, Denmark) and monoclonal antibodies to var-
ious CKs (OV-TL 12 ⁄30, RCK108 and AE1 ⁄AE3, Dako; LL002,
Biomeda, Foster City, CA, U.S.A.; and CAM5.2, Becton Dickin-
son, San Jose, CA, U.S.A.) and involucrin (Novocastra, New-
Fig 2. Histological features of a ridged wart. Vacuolar structures are castle-upon-Tyne, U.K.) were used as primary antibodies
seen in the thickened horny layer and homogeneous intracytoplasmic (Table 1). In the case of CKs, the procedure was performed
inclusion bodies are seen in the granular and upper spinous cell layers with or without proteinase K predigestion or autoclave heating
of the acanthotic epidermis. A tiny keratinous cyst is seen just beneath for antigen retrieval as described previously.22 Negative con-
the acanthotic epidermis (arrow). trols for immunostaining were performed by substituting the
primary antibodies with phosphate-buffered saline or nonim-
mune mouse or rabbit IgG.
Three-dimensional reconstruction analysis
An image scanner (IX-4025; Canon, Tokyo, Japan) and a per-
DNA–DNA in situ hybridization
sonal computer (Apple Macintosh 8500) were used in the
3DR imaging. Montages of the serial sections obtained from HPV 60 DNA sequences were detected in formalin-fixed, paraf-
the entire body of the epidermoid cyst were displayed on a fin-embedded tissue sections using the DNA–DNA in situ hybrid-
television monitor (Multiscan 17s; Sony, Tokyo, Japan). The ization method as described previously.23,24 Hybridization was
outlines of the cysts and the eccrine ducts were digitized and performed at 37 °C for 16 h using digoxigenin-labelled HPV
stored on the computer to make a wire-frame of these in vivo 60 complete genome DNA which we had previously cloned
structures (Fig. 4c). Based on the wire-frame, three-dimen- from a plantar epidermoid cyst.3 The hybridization mixture
sional images of the structures were reconstructed using an consisted of 10% dextran sulphate, 2 · saline sodium citrate
image analysis program (Specular Infini-D; Specular, Amherst, (0Æ3% sodium chloride, 0Æ03 mol L)1 sodium citrate, pH 7Æ0),
MA, U.S.A.) and were displayed on the television monitor 400 lg mL)1 sheared herring sperm DNA (Sigma, St Louis,
Fig 3. Representative histological pictures
selected from the serial sections obtained
from the entire body of an epidermoid
cyst. Section numbers appear at the lower
left corner of each picture. Varying
histological features coming from a
hypertrophied eccrine dermal duct (no.
200, inset) connecting the apical portion
of the cyst with an acrosyringium in the
overlying epidermis are seen in the serial
sections. The ecrine duct, appearing as a
ductal structure in the apical portion of
the cyst wall (nos 180–190), goes
upwards through the dermis to the
acrosyringium (nos 200–210) and
connects with it in the overlying
epidermis (no. 220). The acrosyringium is
extremely dilated in the horny layer (no.
230).
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4. 964 HPV-associated eccrine epidermoid cyst, K. Egawa et al.
Fig 4. Computer-based, three-dimensional
reconstruction (3DR) analysis of a human
papillomavirus 60-associated plantar
epidermoid cyst. (a) Histological section
(the same as Figure 3, no.190). (b)
Tracing of the outline of the histological
ductal structures expressing
carcinoembryonic antigen (red) and of the
cyst wall and overlying epidermis (blue).
(c) A wire-frame of the epidermoid cyst
generated by computer using a montage
of tracings obtained from all the serial
sections. (d) 3DR analysis visualizing the
fine structure of the epidermoid cyst
connecting with the eccrine dermal duct.
Table 1 Immunohistochemical staining in a human papillomavirus (HPV) 60-associated plantar epidermoid cyst
Normal eccrine gland
Secretory Eccrine
Antibody Specificity portion dermal duct Epidermis Cyst wall HD
CEA +++ +++ – – ++ (IML)
AE1 ⁄ AE3 CKs 10, 14–16, 19 ⁄ 1–8 +++ +++ +++ +++ +++
LL002 CK14 – ++ ++ (LL) ++ (OL) + (OL)
OV-TL 12 ⁄ 30 CK7 +++ – – – –
RCK108 CK19 + – – – –
CAM5.2 CKs 8, 18 +++ – – – –
Involucrin – – +++ (UL) +++ (IL) ++ (IL)
HD, hypertrophied eccrine dermal duct with which the HPV 60-associated epidermoid cyst connected; CEA, carcinoembryonic antigen;
CK, cytokeratin; IML, innermost layers; LL, lower layers; OL, outer layers; UL, upper layers; IL, inner layers; –, negative; +, weak; ++,
moderate; +++, strong.
MO, U.S.A.), 50% formamide and 2 lg mL)1 of probe DNA. (Figs 3 and 5c). The characteristic histological feature com-
The digoxigenin-labelled probe was detected by the streptavi- monly seen in all the ridged warts and the epidermoid cysts
din-biotinylated polyalkaline phosphatase detection system was the presence of eosinophilic homogeneous intracyto-
(DNA detection system; BRL, Gaithersburg, MD, U.S.A.). The plasmic inclusion bodies pathognomonic for HPV 60
phaeochromes used were nitroblue tetrazolium and 5-bromo- infection3–7,16,17,24–27 in the granular and upper spinous cell
4-chloro-3-indolylphosphate (Fig. 5). layers of the acanthotic epidermis of the ridged warts or in
the inner cell layers of the cyst wall (Figs 2 and 5c). Vacuolar
structures, also pathognomonic for HPV 60 infection,4,7 were
Results
also seen both in the horny layer of the ridged warts and in
keratinous material within the cyst cavity (Figs 2 and 5c).
Histology
In the serial sections obtained from the entire body of the
In the ridged warts, the epidermis was acanthotic with hyper- smallest cyst (Fig. 3), ductal structures coming from an eccrine
keratosis and partial hypergranulosis. In one lesion, a tiny dermal duct were seen between the apical portion of the cyst
keratinous cyst was seen just beneath the acanthotic epidermis and an acrosyringium in the overlying epidermis in the dermis
(Fig. 2). Epidermoid cysts were lined by stratified squamous that connects the two histological components (Fig. 3, nos.
epithelium, showing keratinization of the epidermoid type 190–220). The eccrine dermal duct was hypertrophied with
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5. HPV-associated eccrine epidermoid cyst, K. Egawa et al. 965
duct but not the secretory portion. In contrast, OV-TL 12 ⁄30
(against CK7), RCK108 (against CK19) and CAM5.2 (against
CKs 8 and 18) stained only the secretory portion. A monoclo-
nal antibody against involucrin did not stain any portions of
the normal eccrine sweat gland.
Epidermis of the sole
CEA was expressed only in the acrosyringeal epithelium in the
epidermis. LL002 stained the lower cell layers; AE1 ⁄AE3
stained whole cell layers; OV-TL 12 ⁄30, RCK108 and CAM5.2
did not stain any cell layers; involucrin was expressed in the
upper cell layers of the epidermis.
Plantar epidermoid cyst
CEA was not expressed in the cyst wall but was expressed in
the acrosyringeal structures in the cyst cavity. LL002 stained
the outer cell layers; AE1 ⁄AE3 stained whole cell layers; OV-
TL 12 ⁄30, RCK108 and CAM5.2 did not stain any cell layers;
involucrin was expressed in the inner cell layers of the cyst
wall.
Hypertrophied eccrine dermal duct with stratified
squamous epithelium with which an epidermoid cyst
connected
Fig 5. Histological localization of human papillomavirus (HPV)
60 DNA sequences. A high-power view of the same section The innermost cell layers of the stratified epithelium of the
(a,c; haematoxylin and eosin) and a serial section (b,d; DNA–DNA hypertrophied eccrine dermal duct expressed CEA. Expression
in situ hybridization) of Figure 3, no. 230. Homogeneous of involucrin was also noted in the inner cell layers; LL002
intracytoplasmic inclusion bodies (a,c) and HPV 60 DNA sequences stained the outer cell layers; AE1 ⁄AE3 stained whole cell
(b,d) are seen in the acrosyringeal epithelium (a,b) as well as in the
layers; OV-TL 12 ⁄30, RCK108 and CAM5.2 did not stain any
cyst wall (c,d).
cell layers of the duct.
The results are summarized in Table 1.
stratified squamous epithelium (Fig. 3, no. 200, inset); the
acrosyringium was extremely dilated in the horny layer
DNA–DNA in situ hybridization
(Fig. 3, no. 230; Fig. 5a); and the homogeneous intracytoplas-
mic inclusion bodies were seen in the acrosyringeal epithelium HPV 60 DNA sequences were detected in all the epidermoid
(Fig. 5a) as well as in the epidermoid cyst (Fig. 5b). cysts and ridged warts. In the smallest epidermoid cyst ana-
Ductal structures suggestive of eccrine ducts were observed lysed with 3DR, the DNA sequences were identified both in
in three of the other four epidermoid cysts, using 50 serial the acrosyringeal portion of the eccrine duct (Fig. 5b) and in
sections obtained from each cyst. the cyst connecting with its dermal portion (Fig. 5d).
Three-dimensional reconstruction analysis Discussion
The analysis clearly demonstrated that the smallest epidermoid Implantation of epidermal fragments into the dermis has been
cyst connected with the eccrine dermal duct (Fig. 4d). thought to be the main cause of palmoplantar epidermoid
cysts.1 Although we have proposed an alternative pathogenic
hypothesis that certain palmoplantar epidermoid cysts may
Immunohistochemical observations
develop from eccrine ducts and that HPV (especially HPV 60)
may play a role in this development,3,4,10,17 there has been a
Normal eccrine sweat gland
lack of direct evidence to support this hypothesis.
A polyclonal antibody against CEA and a monoclonal antibody HPV 60 was initially identified25,26 in and isolated5 from
AE1 ⁄AE3 (against CKs 10, 14–16, 19 ⁄1–8) stained all the plantar epidermoid cysts with homogeneous intracytoplasmic
secretory, dermal duct and acrosyringeal portions of the ec- inclusion bodies, and was also subsequently found in flat
crine sweat gland. LL002 (against CK14) stained the dermal lesions retaining the appearance of dermal ridges on their
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6. 966 HPV-associated eccrine epidermoid cyst, K. Egawa et al.
surface (ridged warts).16,17 Thus, the clinical morphology of appearance of CKs characteristic of stratified and cornifying
HPV 60-induced lesions also remains unclear. In the present epithelia, indicating a true change in the differentiation of the
study, using 3DR analysis, it was clearly demonstrated that an middle ear epithelium.12 Interestingly, HPV has also been
HPV 60-associated plantar epidermoid cyst connected with the identified in cholesteatoma.31
dermal portion of an eccrine duct. HPV 60 DNA sequences Although the epidermoid cyst analysed with 3DR in this
were identified not only in the epidermoid cyst but also in study connected with an eccrine dermal duct, the immunore-
the acrosyringeal portion of the eccrine duct by DNA–DNA activities for CEA, involucrin and several CKs of the cyst wall
in situ hybridization. All the other plantar epidermoid cysts and were not identical to those of normal eccrine dermal ducts
ridged warts that developed simultaneously on the same but were identical to those of the suprabasal layers of the
patient also harboured HPV 60 DNA sequences, suggesting a epidermis, as observed by Abe et al.,8 Ohnishi and Watanabe9
close association between the lesions and HPV 60 infection. A and Yokogawa et al.32 This suggests that the immunoreactivi-
tiny keratinous cyst, which might be a precursor of an HPV ties of the palmoplanatar epidermoid cysts may also be an
60-associated plantar epidermoid cyst, was also seen just indication of the metaplastic origin of the cysts from eccrine
beneath the acanthotic epidermis of a ridged wart. ducts. In the present case, the eccrine dermal duct with
In a previous report, two plantar epidermoid cysts were which the epidermoid cyst connected was hypertrophied
attached to the epidermis and open to the surface in a patient with stratified squamous epithelium and expressed both CEA
with multiple plantar epidermoid cysts harbouring both HPV and involucrin, suggesting a transitional form of the squa-
60 DNA sequences and eccrine ducts,3 suggesting that the mous metaplasia of the eccrine duct epithelium.
cysts developed from the acrosyringeal or uppermost portions However, as Epstein and Kligman have suggested that kera-
of eccrine ducts. As HPV 60 was frequently identified in tinizing cysts may develop from any part of the epithelial sys-
palmoplantar epidermoid cysts concomitantly with eccrine tem,33 further studies are still required to determine whether
ducts,3,4 we assumed that HPV 60 had a high affinity for the cases in which neither HPV infection nor an association
eccrine duct epithelia and played a role in cyst formation.17 with eccrine ducts has been found in ordinary histological sec-
Supporting this idea, recent studies28,29 have suggested that tions reflect a false-negative result or are an indication of epi-
papillomaviruses, including HPVs, may target epidermal stem dermal inclusion cysts resulting from the implantation of
cells, which may exist in association with eccrine ducts in the epidermal fragments.
human palmoplantar skin.29
In the context of the previous findings and the results of
Acknowledgments
the present case, we speculate that HPV 60 initially infects the
upper parts of the eccrine duct, such as the acrosyringeal epi- We thank Ms Chiemi Shiotsu and Ms Yoshiko Sonoda for tech-
thelium, where ridged warts develop, and then migrates into nical assistance and Gyohei Egawa, Haruki Egawa and Motoe
various parts of the dermal portions of the eccrine duct, where Egawa for help in drawing montages of histological pictures.
the virus-associated epidermoid cysts develop.
It has been revealed that HPV 60-associated palmoplantar
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