Journal of Medical Virology 61:289–297 (2000)

Human Papillomavirus Infection and

Non-Melanoma Skin Cancer in Immunosuppressed
and Immunocompetent Individuals
Catherine A. Harwood,1* T. Surentheran,2, Jane M. McGregor,1,3 Patricia J. Spink,2 Irene M. Leigh,1
Judith Breuer,2 and Charlotte M. Proby1
1
Department of Academic Dermatology, Royal Hospitals NHS Trust, London, United Kingdom
2
Department of Virology, Royal Hospitals NHS Trust, London, United Kingdom
3
Department of Photobiology, St John’s Institute of Dermatology, London, United Kingdom

The role of human papillomavirus (HPV) in ano-
genital carcinogenesis is established firmly, but
a similar role in non-melanoma skin cancer re-
mains speculative. Certain immunosuppressed
individuals have an increased incidence of both
viral warts and non-melanoma skin cancer, that
has prompted the suggestion that HPV may play
a pathogenic role. Differences in the techniques
used to detect HPV DNA in skin, however, have
led to discrepancies in the prevalence and spec-
trum of HPV types reported in these malignan-
cies. This study describes the use of a compre-
hensive degenerate PCR technique to compare
the HPV status of 148 Non-melanoma skin can-
cers from immunosuppressed and immunocom-
petent individuals. HPV DNA was detected in 37/
44 (84.1%) squamous cell carcinomas, 18/24
(75%) basal cell carcinomas and 15/17 (88.2%)
premalignant skin lesions from the immunosup-
pressed group compared with 6/22 (27.2%)
squamous cell carcinomas, 11/30 (36.7%) basal
cell carcinomas and 6/11 (54.4%) premalignan-
cies in the immunocompetent group. Epidermo-
dysplasia verruciformis HPV types prevailed in
all lesion types from both groups of patients. In
immunosuppressed individuals, cutaneous HPV
types were also identified at high frequency, and
co-detection of multiple HPV types within single
tumours was commonly observed. This study
represents the largest and most comprehensive
analysis of the HPV status of non-melanoma skin
cancers yet undertaken; whereas there are
clearly significant differences in non-melanoma
skin cancers from immunosuppressed and im-
munocompetent populations, we provide evi-
dence that the prevalence and spectrum of HPV
types does not differ in squamous cell carcino-
mas, basal cell carcinomas or premalignancies
within the two populations. These data have im-
portant implications for future investigation of
the role of HPV in cutaneous carcinogenesis at a
functional level. J. Med. Virol. 61:289–297, 2000.
© 2000 Wiley-Liss, Inc.
KEY WORDS: degenerate PCR; viral carcino-
genesis; renal transplant recipi-
ents; epidermodysplasia verru-
ciformis
INTRODUCTION
Non-melanoma skin cancers are the most prevalent
malignancies in fair-skinned populations world-wide
[Ko et al., 1994]. Epidemiological and molecular data
implicate ultraviolet radiation as the most important
etiological factor, but other agents including the im-
mune response, genetic predisposition and viral infec-
tion may also be involved. A number of viruses have
been proposed in the development of non-melanoma
skin cancer, but the most plausible evidence to date is
that for human papillomavirus (HPV) [Proby et al.,
1996; zur Hausen, 1996].
HPV is recognised increasingly as an important hu-
man carcinogen [zur Hausen, 1996]. The best estab-
lished association with human malignancy is that of
high-risk mucosal HPV types and anogenital cancer. A
role for HPV in cutaneous malignancy is also impli-
cated in the rare inherited condition epidermodyspla-
sia verruciformis that is characterised by a predisposi-
Abbreviations: HPV, human papillomavirus; PCR, polymerase
chain reaction.
Grant sponsor: Joint Research Board of St. Bartholomew’s;
Grant sponsor: Research Advisory Committee of St. Bartho-
lomew’s; Grant sponsor: Royal London School of Medicine and
Dentistry, Queen Mary and Westfield College.
*Correspondence to: Dr Catherine A. Harwood, Centre for Cu-
taneous Research, St. Bartholomew’s and the Royal London
School of Medicine and Dentistry, Queen Mary and Westfield
College, 2, Newark Street, London E1 2AT, UK. E-mail:
caharwood@doctors.org.uk
Accepted 15 November 1999

© 2000 WILEY-LISS, INC.

290
tion to HPV infection and the development of
cutaneous squamous cell carcinomas on sun-exposed
sites [Majewski and Jablonska, 1995]. Cellular tumour
suppressor proteins p53 and pRB are important targets
for the viral oncoproteins E6 and E7 respectively in
anogenital cancer, but the oncogenic mechanisms of
HPV in epidermodysplasia verruciformis are uncer-
tain, and there seems to be a crucial additional require-
ment for ultraviolet radiation [Majewski and Jablon-
ska, 1995].
An association between virus warts and skin cancer
has also been proposed in renal transplant recipients
[Walder et al., 1971; Boyle et al., 1984]. Renal trans-
plant recipients have a marked increased susceptibility
to both viral warts and non-melanoma skin cancer; the
incidence of squamous cell carcinomas in particular is
increased 50–100-fold, with a reversal in the normal
squamous cell carcinomas to basal cell carcinomas ra-
tio. Viral warts and cutaneous squamous cell carcino-
mas co-localise on sun-exposed sites and ultraviolet
light seems to be an important factor in the develop-
ment of both warts and cancers [Glover et al., 1993]. In
addition, clinical and histological features of trans-
plant squamous cell carcinomas indirectly support the
progression of virus warts through increasingly dys-
plastic squamous lesions to invasive squamous cell car-
cinomas [Blessing et al., 1989].
Until recently it has been technically difficult to de-
tect the 80 or more characterised HPV types in skin
cancers, considerably hindering research in this area.
In early studies, detection of HPV DNA varied both in
overall prevalence from 0–64% and in the HPV types
detected [reviewed in Proby et al., 1996]. These dis-
crepancies largely reflect the detection methods used;
DNA-hybridisation based techniques were generally
employed using a limited number of HPV probes that
were not informative for the majority of HPV types. As
a consequence, the true prevalence of HPV in cutane-
ous lesions was underestimated. Where polymerase
chain reaction (PCR) was employed, early methods
used type-specific primers capable of detecting only a
limited range of HPV types [Soler et al., 1993; Stark et
al., 1994; Arends et al., 1997]. This was frequently com-
pounded by the use of paraffin-embedded material that
yields sub-optimal results compared with fresh frozen
tissue [Dyall-Smith et al., 1991; Smith et al., 1993; Fer-
randiz et al., 1997]. More recently, studies using a de-
generate PCR approach have consistently demon-
strated a high prevalence (65–81%) of HPV DNA in
transplant tumours [Berkhout et al., 1995; de Jong-
Tieben et al., 1995; Shamanin et al., 1996; de Villiers et
al., 1997]. Even in these studies, however, the spec-
trum of HPV types detected has differed considerably,
reflecting the differing sensitivity and specificity pro-
files of the primer sets used.
Our group has now established a degenerate PCR
technique that includes nested primer pairs for muco-
sal and cutaneous as well as epidermodysplasia verru-
ciformis-HPVs to create a comprehensive, specific and
highly sensitive methodology for detection and geno-
Harwood et al.
typing of all known HPVs [Harwood et al., 1998; Storey
et al., 1998; Harwood et al., 1999]. We have used this
approach to compare HPV DNA detection in malignant
and premalignant non-melanoma skin cancers in im-
munosuppressed and immunocompetent individuals.
MATERIALS AND METHODS
Patients
Immunosuppressed individuals. Patients com-
prised 32 immunosuppressed renal transplant recipi-
ents (26 male, 6 female). The mean age was 58.2 years
(40–72 years) and duration of transplant was 12.3
years (range 2–24 years). All renal transplant recipi-
ents were receiving systemic immunosuppression with
prednisolone, azathioprine or cyclosporin. In addition,
lesions from 4 patients (2 male, 2 female) immunosup-
pressed for other reasons were included (haematologi-
cal malignancy, n ⳱ 3; long-term azathioprine and
prednisolone for ulcerative colitis, n ⳱ 1). Details of
skin type and cumulative sun exposure were obtained.
Immunocompetent individuals. Lesions from 51
immunocompetent individuals were analysed. These
patients included 34 males and 17 females, mean age
73.4 years (range 28–92 years). Careful assessment of
patient records was made to exclude any possible
sources of endogenous or iatrogenic immunosuppres-
sion.
Tissue Samples
Specimens from the immunosuppressed group com-
prised 44 squamous cell carcinomas, 24 basal cell car-
cinomas, and 17 premalignant lesions (carcinoma in
situ or actinic keratoses). From the immunocompetent
group, 22 squamous cell carcinomas, 30 basal cell car-
cinomas and 11 premalignancies were analysed. All le-
sions were histologically confirmed, and squamous cell
carcinomas classified as well-, moderately- or poorly
differentiated. The tissue analysed was collected at the
time of surgery, immediately snap frozen and stored at
−70°C. Care was taken to avoid potential cross-
contamination between specimens at the collection
stage by using separate surgical equipment if more
than one biopsy was taken, and freezing each specimen
in individual cryotubes in separate canisters of liquid
nitrogen. Six large squamous cell carcinomas were sub-
divided into 2 to 4 sections, and each section analysed
independently.
DNA Extraction
Samples were minced finely with sterile scalpels on a
petri dish, washed twice in PBS and resuspended in
lysis buffer containing proteinase K to a concentration
of 0.1 mg/ml. After lysis overnight at 37°C, samples
were pelleted and the supernatant removed to a fresh
tube. DNA was then extracted by a standard phenol-
chloroform-isoamyl alcohol technique followed by etha-
nol precipitation [Maniatis et al., 1989]. Specific
precautions were taken to prevent and monitor cross-
contamination during the DNA extraction process. All
procedures were carried out under conditions of strict
HPV and Skin Cancer
pre- and post-PCR separation [Kwok and Higuchi,
1989]. Lesions were extracted in 27 separate proce-
dures. Samples from immunocompetent patients were
extracted simultaneously with samples from immuno-
suppressed patients. Multiple samples from individual
patients were usually extracted in at least 2 different
batches. HPV negative skin (as defined by the fact that
the sample was negative with our methodology) was
used as a negative tissue control in 17 extraction pro-
cedures. Buffer extraction controls that did not contain
tissue were placed in the middle or at the end of an
extraction procedure. Although the identity of the
samples was not masked, they were coded after extrac-
tion, and the coding only revealed after completion of
PCR and sequencing. The coding was known to one
investigator only.
PCR Primers
We have established previously a panel of 9 degen-
erate primer pairs and 1 single round of primer pairs
located within the highly conserved L1 (major capsid
protein) open reading frame (ORF) of the HPV genome
for detection of cutaneous, mucosal and epidermodys-
plasia verruciformis-HPV types [Harwood et al., 1998].
One modification was made to this panel of primers for
the purposes of this study in that the nested primer
pair previously used for detection of mucosal HPV
types was replaced by a semi-nested version compris-
ing MY11 and GP6 [Snijders et al., 1990]; this gave a
larger PCR fragment, increasing the length of sequence
available for HPV genotyping from approximately 150
bases to 200 bases [Harwood et al., 1999]. We have
confirmed previously the capacity of this primer panel
to detect and identify a broad spectrum of HPV types
and also to detect the presence of mixtures of two or
more HPV types within single lesions [Harwood et al.,
1999].
PCR Amplifications
PCR amplifications were carried out exactly as de-
scribed previously [Harwood et al., 1999]. All PCR re-
actions were performed on a Perkin-Elmer 480 ther-
mal-cycler and 100–200 ng of cellular DNA was used as
template in each first round PCR reaction. For each set
of reactions, negative controls for reagents (water only)
and genomic DNA (human placental DNA, Sigma)
were included and processed in the same way as the
lesional samples throughout all PCR steps, as were the
negative controls for DNA extraction. None of the nega-
tive controls was positive for HPV. HPV plasmid (0.01
pg) containing the genotype of HPV-2, -4, -5, or -6
served as positive controls. Before amplification with
the HPV primers, samples were amplified with beta-
globin primers PC04 and GH20 to confirm adequate
preservation of DNA [Resnick et al., 1990].
Sequence Analysis
Amplified PCR products that appeared as a visible
band after ethidium bromide staining were purified af-
ter separation in a 2% agarose low melting point gel
291
(QIAquick Gel Extraction Kit, QIAGEN, Germany) and
sequenced directly by fluorescent dideoxynucleotide
chain termination cycle sequencing on a Perkin-Elmer
2400 thermal cycler (ABI Prism Dye Terminator Cycle
Sequencing Ready Reaction Kit, Perkin-Elmer) with
both forward and reverse primers. The products were
analysed on a Perkin-Elmer 377 ABI Prism automated
sequencer.
The nucleotide sequences obtained were edited using
the Sequence Navigator computer software (Macin-
tosh). Sequences of 140 bases or more with fewer than
5% unidentified bases were processed. The forward and
reverse complement sequences were aligned and ho-
mology of the consensus sequence was compared with
those of known HPV types available through the Gen-
Bank database (National Center for Biotechnology In-
formation, National Institutes of Health, Bethesda,
MD) using the GCG Blast programme. In accordance
with established guidelines, a nucleotide sequence was
regarded as an HPV type if it shared over 90% homol-
ogy with a known type, and a related type if the se-
quence homology was less than 90% [Chan et al., 1995].
Confirmation of Results Obtained in Clinical
Specimens With L1 Degenerate Primers Using
E6 Type-Specific Primers
Type-specific primers for the E6 ORF of HPVs -10,
-23, -24, and -27 were designed using E6 nucleotide
sequence data (Los Alamos National Laboratory HPV
sequence Database, 1997) as described previously
[Harwood et al., 1999]. The HPV types were randomly
chosen from amongst those that had occurred more
than once in the series of clinical specimens. Using the
E6 ORF primers, PCR amplification of several repre-
sentative clinical samples was used as additional con-
firmation of the results obtained with the degenerate
nested L1 primers. Amplification reactions were car-
ried out in 50 ␮l of reaction mixture as described above.
Forty cycles of PCR were performed (95°C for 1 min,
55°C for 1 min and 72°C for 1 min) followed by exten-
sion at 72°C for 5 min. In plasmid titration experi-
ments, each E6 primer set detected its relevant target
plasmid to a sensitivity of at least 0.01 fg.
Statistical Analysis
Statistical analysis was performed using the Chi
squared test with Yates correction where appropriate.
RESULTS
Clinical Features
It is well recognised that non-melanoma skin cancers
occur 10–20 years earlier in Renal transplant recipi-
ents compared to the immunocompetent population
[Glover et al., 1993], and it was therefore not possible
to undertake an age-matched comparison of renal
transplant recipients and immunocompetent patients.
There were, however, no significant differences in gen-
der, skin type or annual cumulative sun exposure be-
tween the 2 groups (data not shown).
292
TABLE I. Comparison of HPV DNA Prevalence in Non-Melanoma Skin Cancers From Immunocompetent and
Immunosuppressed Individuals†
SCC BCC
Positive Positive
Immune lesions lesions
status Patients Lesions (%) Patients Lesions (%)
RTR 14 40 33 (82.5)* 15 24 18 (75)**
IS 4 4 4 (100 — — — —
IC 20 22 6 (27)* 28 30 11 (37)**
†
RTR, renal transplant recipient; IS immunosuppressed individuals excluding renal transplant recipients (see text); IC, immunocompetent
individuals; SCC, squamous cell carcinoma; BCC, basal cell carcinoma; CIS, premalignant lesions (carcinoma in situ and actinic keratoses).
*P < 0.001.
**P < 0.01.
***P < 0.05.
Non-Melanoma Skin Cancers From Renal
Transplant Recipients
Squamous cell carcinomas. HPV DNA was
found in 33/40 (82.5%) of transplant squamous cell car-
cinomas (Table I). Epidermodysplasia verruciformis-
HPV types predominated, being present in 29/33
(87.9%) HPV positive lesions. Cutaneous HPV types
were found in 21/33 (63.6%) and mucosal types in 5/33
(15.2%). Mixed infections in which two or more HPV
types were co-detected, occurred in 21/33 (63.6%) of
HPV positive lesions. The majority of mixed infections
contained between 2 and 4 distinct types, usually as
combinations of cutaneous and epidermodysplasia ver-
ruciformis-types.
Analysis of lesions according to HPV groups and
clusters is summarised in Table II Overall, no indi-
vidual HPV type emerged in specific association with
squamous cell carcinomas. Of the cutaneous group, 7
distinct types were identified (HPV -10, -27, -2, -1, -3,
-77, -28). Twenty-one recognised epidermodysplasia
verruciformis-HPV types or HPV sequences with a
GenBank database accession number were identified
(HPV-5, -14, -19, -20, -22, -23, -24, -25, -36, -37, -38, -49,
-75, -RTRX1, -RTRX2, -RTRX5, RTRX32, -vs20-4,
-vs42-1,-vs73-1,-vs92-1, Z95963). Three epidermodys-
plasia verruciformis-related types with less than 90%
sequence homology to sequences registered in the Gen-
Bank database were also found (5-related, 23-related
and 24-related). Just 3 mucosal types were identified
(HPV-11,-16, and -66) in 5 lesions.
All 4 squamous cell carcinomas from immunosup-
pressed non-renal transplant recipients were observed
to harbour HPV DNA. In each case a single infection
with an epidermodysplasia verruciformis-HPV type
was found.
Subdivision of squamous cell carcinomas. Six
squamous cell carcinomas were subdivided and each
portion analysed separately. The results are included
in Table III. In 4 of the 5 HPV DNA positive squamous
cell carcinomas, at least one HPV type was common to
two or more portions of the lesion analysed (e.g., HPV-
36 in Patient B, lesion 4; HPV-19 in Patient B, lesion
5). In some lesions, however, different portions har-
boured additional or differing HPV types, suggesting
possible spatial separation of HPV types within lesions
Harwood et al.
CIS Total
Positive Positive
lesions lesions
Patients Lesions (%) Lesions (%)
9 17 15 (88)*** 81 66 (81.5)*
— — 4 4 (100)
8 11 6 (54.5)*** 63 23 (36.5)*
harbouring multiple HPV types (e.g., Patient D, Lesion
4). In one lesion (Patient C, lesion 10), HPV DNA was
not detected in either of the two sections analysed.
Basal cell carcinomas. Twenty-four basal cell
carcinomas from transplant recipients were analysed
of which 18 (75%) contained HPV DNA. Of these HPV
positive lesions, 72.2% contained epidermodysplasia
verruciformis-types, 50% cutaneous HPV types and
11.1% mucosal types. The prevalence of mixed infec-
tions and the HPV types detected were broadly similar
to those found in squamous cell carcinomas (Table II).
Premalignant lesions (carcinoma in situ and
actinic keratoses). HPV DNA was detected in 15/17
(88%) of premalignancies and the spectrum of types
detected was again similar to that observed in both
basal cell carcinomas and squamous cell carcinomas.
Multiple lesions. In patients from whom multiple
lesions were analysed (Table III), clustering of certain
HPV types was found, with these HPV types occurring
in both benign and malignant lesions located on differ-
ent anatomical sites, removed on different occasions
(up to 5 years apart), and analysed independently. For
example, Patient A harboured HPV RTRX5 in 2 basal
cell carcinomas from different sites as well as in a squa-
mous cell carcinomas and a viral wart. Similarly, HPV-
19 and -27 were identified in carcinoma in situ, squa-
mous cell carcinomas and viral warts from Patient B.
This trend also extended to HPV negative lesions; 7
HPV negative squamous cell carcinomas occurred in
just 3 individuals, of whom Patient C had 4 HPV nega-
tive lesions.
Non-Melanoma Skin Cancers From
Immunocompetent Individuals
There were several notable differences between le-
sions from immunocompetent compared with immuno-
suppressed patients. The overall prevalence of HPV
DNA in squamous cell carcinomas, basal cell carcino-
mas and carcinoma in situ was significantly lower in
immunocompetent individuals than in Renal trans-
plant recipients (P < 0.001, P < 0.01 and P < 0.05 re-
spectively). The spectrum of types detected also dif-
fered; epidermodysplasia verruciformis-HPV types
were still most often detected in these lesions, but cu-
 TABLE II. Spectrum of HPV Types Detected in HPV Positive Non-Melanoma Skin Cancers From Immunosuppressed and Immunocompetent Individuals*
HPV Types Detected in HPV Positive Lesions (%)a
No. HPV b
Immune positive Cutaneous EVb
status Lesion lesions A2 A4 E Totald a1 a2 b1 b2 Other Totald Mucosalb Mixedc
RTR SCC 33 14 9 3 21 3 8 3 7 11 29 5 21
(41.4) (27.3) (9.1) (63.6) (9.1) (24.2) (9.1) (21.2) (33.3) (87.9) (15.2) (63.6)
BCC 18 5 4 1 9 1 4 0 1 12 13 2 10
(27.8) (22.2) (5.6) (50) (5.6) (22.2) (5.6) (66.7) (72.2) (11.1) (55.6)
CIS 15 7 5 0 11 3 7 1 1 2 12 2 8
(46.7) (33.3) (73.3) (20) (46.7) (6.7) (6.7) (13.3) (80) (13.3) (53.3)
IS SCC 4 0 0 0 0 1 0 0 1 2 4 0 0
(25) (25) (50) (100)
IC SCC 6 0 0 0 0 0 1 0 0 5 6 0 0
(16.7) (83.3) (100)
BCC 11 1 0 0 1 0 0 2 0 10 10 1 3
(9.1) (9.1) (18.2) (90.1) (90.1) (9.1) (27.3)
CIS 6 2 0 0 2 1 1 0 0 1 3 1 0
(33.3) (33.3) (16.7) (16.7) (16.7) (50) (16.7)
*RTR, renal transplant recipient; IS immunosuppressed (non RTR); IC, immunocompetent; SCC, squamous cell carcinoma; BCC, basal cell carcinoma; CIS, carcinoma in situ; EV, epider-
modysplasia verruciformis.
a
The percentage prevalence of each HPV type is calculated with respect to the total HPV positive lesions for that tumour, rather than as a percentage of all lesions examined.
b
HPV types: These have been grouped according to the phylogenetic classification described in Los Alamos National Laboratory HPV sequence Database, 1977. The HPV types detected within
these groups included the following: Cutaneous: A2 ⳱ HPV group A2 (HPV −3, −10, −28, −77); A4 ⳱ HPV group A4 (HPV −2, −27, −57); E ⳱ HPV group E (HPV −1, −41). EV: a1 ⳱ EV HPV
cluster a1 (HPV −5, −8, −12, −36); a2 ⳱ EV HPV cluster a2 (HPV −14, −19, −20, −21, −25); b1 ⳱ EV HPV group b1 (HPV −9, −15, −37, −vs92-1); b2 ⳱ EV HPV group b2 (HPV −22, −23, −38,
−RTRX1, −VS42); Other ⳱ other EV HPV types detected which have not yet been classified (HPV −24, −49, −75) or have not yet been fully characterised but are registered in the GenBank
database (−RTRX2, −RTRX4, −RTRX5, −RTRX7, −RTRX9, −VS20-4, −RTRX32, −PSOX1, −IA16 and HPVs L1 with accession numbers Z95963, Z95969, AF027135, L383888, AF019978), or
which are putatively novel types (15-related, RTRX9-related, 5-related, 23-related, 24-related). Mucosal: 4 types were detected (HPV −6, −11, −16, and −66).
c
This term includes those lesions in which 2 or more distinct HPV genotypes were detected.
d
Note that these figures are not additive due to the presence of mixed infection.
294 Harwood et al.

TABLE III. HPV Typing of Multiple Lesions From Selected Renal Transplant Recipients
HPV typing
Patient Lesion Site EV Cutaneous Mucosal
A 1. CIS Nose 20 27 —
2. CIS Ear 5, 14, VS92-1 27 11
3. BCC Wrist 14, RTRX5, VS20-4 27 11
4. BCC Neck RTRX5 10, 27 —
5. BCC Nose 14,RTRX4, AF027135 — —
6. SCC Ear RTRX5, VS20-4 2 —
7. VW Wrist RTRX5, Z95963 3, 57 —
B 1. CIS Hand RTRX9 — —
2. CIS Hand 19 27 —
3. CIS Finger 19 3 —
4. SCC Handa
(i) 36 27 —
(ii) 36 10 16
5. SCC Handa
(i) 19 27 —
(ii) 19 — 66
6. SCC Hand 19 — —
7. SCC Finger 37 — —
8. SCC Cheek 14, 19 — 11
9. VW Foot 36 27 —
10. VW Neck 14, 19, 38 — —
11. VW Chest 19, 36 — —
C 1. CIS Forehead — 77 —
2. CIS Back 5-related — —
3. CIS Arm — 77 —
4. BCC Cheek — — —
5. BCC Scapula — — —
6. SCC Chest 5 27, 77 —
7. SCC Chest — — —
8. SCC Ear — — —
9. SCC Hand — 27 —
10. SCC Shouldera
(i) — — —
(ii) — — —
11. SCC Temple — — —
12. VW Forehead 24, RTRX9 27, 77 —
13. VW Chest RTRX9 27 —
D 1. CIS Arm 5 28 —
2. SCC Forearma
(i) RTRX5 10 —
(ii) 23, 49 10 —
3. SCC Leg RTRX5 — —
4. SCC Thigha
(i) RTRX1 — —
(ii) 5 — —
5. SCC Forearm 5, 23 10 —
6. VW Hand RTRX5 2, 10 —
E 1. SCC Foreheada
(i) 24, VS42-1 — 11
(ii) 25 — —
(iii) — — 11
(iv) — — 11
2. SCC Nose 24, VS42-1, RTRX5 — —
3. SCC Nose 24, 38 77 —
4. VW Ear 25, 38 — 11
5. VW Hand 25 28 11
F 1. SCC R. Ear — — —
2. SCC L. Ear RTRX32 — —
3. SCC Scalp RTRX32 — —
4. SCC Forehead RTRX32, VS92-1 — —
G 1. SCC R. Ear — 1, 27 —
2. SCC Forearm VS 73-1 27 —
3. SCC Hand 22 10 —
4. VW Thumb RTRX1 1, 77 —
a
These lesions were subdivided and the resulting samples were analysed independently (see text). Benign viral warts (VW) for these patients
had previously been analysed [Harwood et al., 1999] and the HPV types detected are shown here for comparison with malignant and
premalignant lesions.
HPV and Skin Cancer
taneous types were uncommon (p<0.001) and mixed in-
fection with multiple HPV types was found in only 3/23
(13%) of positive lesions compared with 39/72 (54.17%)
of positive renal transplant recipient lesions (P <
0.001).
Confirmation of Results
Five samples that were found to harbour HPV-27
using the degenerate L1 ORF primers and 5 samples
that were HPV-27 negative were amplified with the
HPV-27 E6 type specific primers and in all cases the
results confirmed the L1 primer results. Similarly, the
presence of HPV-23, -24, and -10 was confirmed in the
lesions tested.
DISCUSSION
This is the largest series to date comparing the HPV
status of non-melanoma skin cancers from immuno-
competent and immunosuppressed individuals. It em-
ploys a comprehensive and sensitive degenerate PCR
methodology designed to detect all known HPV types
with approximately equal sensitivity, and it has previ-
ously been rigorously validated against other method-
ologies [Harwood et al., 1999]. A high prevalence of
HPV DNA was detected in lesions from immunosup-
pressed patients (70/85, 82.35%), with no significant
differences in either HPV prevalence or types between
squamous cell carcinomas, basal cell carcinomas and
premalignant lesions. In immunocompetent patients
the overall HPV DNA prevalence of 23/63 (36.5%) was
significantly lower (P < 0.001) with only epidermodys-
plasia verruciformis-types found in immunocompetent
squamous cell carcinomas, in contrast to the additional
cutaneous or mucosal types found in transplant squa-
mous cell carcinomas, often as mixed infection. thus
epidermodysplasia verruciformis-HPV types predomi-
nated in all tumours but co-detection of multiple HPV
types within single lesions was almost entirely con-
fined to renal transplant recipients (P < 0.001).
Comparison With Previous Data on HPV Status
in IS Versus IC Lesions
Our data highlight potentially important differences
in both the HPV prevalence and the spectrum of HPV
types present in Non-melanoma skin cancers from im-
munocompetent and immunosuppressed patients. Fur-
thermore, they indicate that within each population,
the HPV status of individual non-melanoma skin can-
cer types is similar. This is the first study to examine
equivalent numbers of each non-melanoma skin cancer
type from both immunocompetent and immunosup-
pressed individuals using a sufficiently comprehensive
detection technique to allow conclusions to be drawn.
Whilst the overall prevalence of HPV DNA in skin tu-
mours is similar to that reported by other investigators
using degenerate PCR [de Jong-Teiben et al., 1995;
Berkhout et al., 1995; Shamanin et al., 1996; de Villiers
et al., 1997], the spectrum of types that we have ob-
295
served has shown certain differences (see below). This
is largely a reflection of the PCR primers used; our
methodology has incorporated certain features of a
number of previous approaches and has thereby al-
lowed detection of a more comprehensive range of HPV
types. For example, in post-transplant non-melanoma
skin cancer, the nested PCR method developed by
Berkhout et al. [1995] detected only epidermodysplasia
verruciformis or epidermodysplasia verruciformis-
related viruses in 49/61 (80%) squamous cell carcino-
mas, 4/8 (50%) basal cell carcinomas, 14/15 (93%) ac-
tinic keratoses, 2/5 (40%) in situ carcinomas [Berkhout
et al., 1995]. In a separate study, Shamanin et al.
[1996] identified HPV in 13/20 (65%) squamous cell
carcinomas and 3/5(60%) basal cell carcinomas, but
only 20% of the HPV types were epidermodysplasia
verruciformis-associated; the majority consisted of ei-
ther high risk mucosal types (HPV-16,-51,-54,-56,-
61,and -69) or cutaneous types (HPV-41 and -60). Con-
firmation that these discrepancies are likely to reflect
the differing profiles of the PCR primers employed was
provided by a recent study [de Villiers et al., 1997] in
which differences were found in HPV types detected in
lesions analysed by 2 different methodologies [that of
Shamanin et al., 1996 and Berkhout et al., 1995].
There have been fewer studies assessing the HPV
status of non-melanoma skin cancer in immunocompe-
tent patients. The most comprehensive study using de-
generate PCR is that reported by Shamanin et al.
[1996] in which the HPV status identified in non-
melanoma skin cancers from renal transplant recipi-
ents was compared with that from 36 non-melanoma
skin cancers from immunocompetent patients; HPV
DNA was detected in 8/25 (32%) squamous cell carci-
nomas and 4/11 (36%) basal cell carcinomas. A broad
spectrum of HPV types was found including epidermo-
dysplasia verruciformis-associated types (HPV-8 , -9,
-23, and -25), common cutaneous types (HPV-4 and -7),
and mucosal types (HPV-6b, -32,-34, -42, and -51). As
indicated above, however, this methodology may be in-
sufficiently comprehensive in the detection of epider-
modysplasia verruciformis-HPV types in particular [de
Villiers et al., 1997]. This is further confirmed by our
findings in a larger series of immunocompetent non-
melanoma skin cancers of predominantly epidermodys-
plasia verruciformis-types but also some cutaneous
and mucosal types.
Detection of Multiple HPV Types
In addition to differences in the spectrum of HPV
types harboured by immunocompetent and immuno-
suppressed lesions, the frequent observation in our
study of mixed infections in transplant tumours (39/66,
59.1%) contrasts with the findings in cancers from im-
munocompetent patients (3/23, 13.04%), and this dif-
ference is significant (P < 0.001). This might represent
co-infection of single cells by multiple HPV types, or a
mixture of cells infected by single virus types. In one
report in which several genital HPV types were de-
296
tected within a single anal wart from a renal trans-
plant recipient, localisation by in situ hybridisation
suggested that each HPV type maintained regional
separation within the lesion [Christensen et al., 1997].
Unger et al. [1997] also reported the presence of mul-
tiple HPV types clustered in geographically distinct ar-
eas within anogenital warts in immunosuppressed HIV
positive patients. In contrast, using double fluores-
cence hybridisation HPV-1 and-63 were co-detected
within nuclei from a plantar wart from an immunocom-
petent patient, although only the cytopathogenic effect
of HPV-63 was evident in the infected cells [Egawa et
al., 1993]. Our data from 6 squamous cell carcinomas
divided into 2 or more portions for the purposes of HPV
genotyping provides some evidence in support of re-
gional separation of individual HPV types within le-
sions. Evaluation by in situ hybridisation of cutaneous
malignancies from transplant recipients will be neces-
sary to clarify the spatial localisation of multiple HPV
genotypes within single lesions.
Significance of HPV DNA to the Development of
Non-Melanoma Skin Cancer
One line of evidence often considered to provide sup-
port for the possibility that HPV may play a role in the
development of non-melanoma skin cancer is the ob-
servation that the squamous cell carcinoma to basal
cell carcinoma ratio is reversed in transplant recipients
[Glover et al., 1993]. Our finding of a high prevalence of
HPV DNA in both squamous cell carcinomas and basal
cell carcinomas in transplant recipients may indicate
that the role of HPV differs in the two tumour types,
leading to preferential development of squamous cell
carcinomas. The mechanisms by which HPV may exert
such a role in carcinogenesis have yet to be established,
but one possible model is that the viruses have a pro-
moter effect, acting in conjunction with specific tumour
initiators or other promoters, the most important of
which is ultraviolet radiation. In such a capacity it
might be envisaged that a diverse spectrum of HPV
types could be involved in promoting carcinogenesis,
explaining why no specific HPV type has emerged as
strongly associated with non-melanoma skin cancer.
Although the prevalence of HPV DNA in lesions from
immunocompetent individuals was significantly lower
in our study, HPV may nonetheless also be relevant to
the development of skin cancer in the context of a nor-
mal immune system. In these circumstances, the copy
number of the virus in the majority of lesions may be
lower than is currently detectable by PCR, or the virus
may be pathogenic in certain susceptible individuals
only. Indeed, further complexity is likely to arise from
the interaction of HPV with other host factors; our
group have, for example, recently identified an associa-
tion between a p53 polymorphism and increased sus-
ceptibility to HPV-induced cancer, including non-
melanoma skin cancer[Storey et al., 1998].
Alternatively, HPV may be simply a coincidental cu-
Harwood et al.
taneous “passenger.” Indeed, despite the strong asso-
ciation between HPV and non-melanoma skin cancer
that is emerging, HPV DNA is also detected in normal
skin. In one study, 16% of normal skin samples from
individuals with non-melanoma skin cancer were
found to contain HPV [Stark et al., 1994], and we have
also reported previously the presence of HPV DNA in
4/12 samples of normal skin from PUVA-treated pa-
tients with non-melanoma skin cancer [Harwood et al.,
1998]. Furthermore, the majority of hair follicles from
immunosuppressed patients were found to harbour
HPV DNA [Boxman et al., 1997]. Astori et al. [1998]
found epidermodysplasia verruciformis-associated
HPV types in 50% of 6 normal peri-lesional skin
samples, adjacent to actinic keratoses, basal cell carci-
nomas and benign nevi. Seven of 20 (35%) of normal
skin samples taken at the time of cosmetic surgery
were also positive for HPV DNA in this study. Most
recently one group found HPV DNA in 8/42 (19%) of
skin from healthy volunteers [Weissenborn et al.,
1999]. Finally, a high prevalence of HPV DNA in the
proliferative but benign skin condition psoriasis has
also been reported [Favre et al., 1998; Weissenborn et
al., 1999]. Our results should therefore be interpreted
in the context of these findings. Thus, it is possible that
immunosuppression and ultraviolet light may both
play a causal role in increasing susceptibility to cuta-
neous viral infection (or activation of latent HPV infec-
tion) and skin cancer independently, and this may ex-
plain the increased detection of HPV DNA in these
malignancies. Another plausible explanation is that
the replication of HPV generally or certain HPV types
in particular is preferentially enhanced by a hyperpro-
liferative (pre)malignant epidermis in the permissive
milieu of systemic immunosuppression.
In summary, our study demonstrates for the first
time an equivalent high prevalence, broad spectrum
and frequent multiple infections with HPV in both
squamous cell carcinomas, basal cell carcinomas and
premalignant lesions from immunosuppressed indi-
viduals. By comparison, the spectrum and prevalence
of HPV in malignancies from immunocompetent pa-
tients is significantly lower, but is also equivalent be-
tween different lesion types. Many important questions
remain concerning the relevance of these findings to
carcinogenesis. In contrast with anogenital cancer in
which epidemiological, molecular and functional data
fulfil the World Health Organisation criteria for viral
carcinogenesis, these criteria have yet to be met in non-
melanoma skin cancer [IARC, 1995]. In particular,
where multiple infection is present, the spatial locali-
sation, viral load, physical state and transcriptional
activity of the viruses present in tumours and normal
skin will need to be established as this may have a
critical bearing on any role of HPV in cutaneous carci-
nogenesis. Nonetheless, this study provides the broad-
est basis to date from which to start addressing the
functional role of HPV in skin carcinogenesis.
HPV and Skin Cancer
ACKNOWLEDGMENTS
CAH is supported by a Medical Research Council
(U.K) Clinical Training Fellowship. TS is supported by
a Joint Research Board grant and PJS by a Research
Advisory Committee grant from St Bartholomew’s and
the Royal London School of Medicine and Dentistry,
Queen Mary and Westfield College.
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