Novel Human Papillomavirus (HPV) DNA Sequences fromRecurrent Cutaneous and Mucosal Lesions of a Stoma-Carrier

Ulrike Wieland, Gerd E. Gross,* Ansgar Hofmann,† Nib Sohendra,‡ Hans Peter Berlien,§ and Herbert PfisterInstitute of Virology, University of Cologne, Cologne, Germany; *Department of Dermatology, University of Rostock, Rostock, Germany; †Orpegen Pharma,Heidelberg, Germany; ‡Endoscopic Surgery, Department of Surgery, University of Hamburg, Germany; §Department of Laser-Medicine, Hospital Berlin-Neukoelln, Germany

Recent studies have demonstrated a high prevalence ofhuman papillomavirus (HPV) types originally believedto be restricted to patients with epidermodysplasiaverruciformis (EV) in benign and malignant skin tumorsof the general population. Other groups detected typicalmucosal HPV in skin tumors. We have investigatedrecurrent leukoplakial cutaneous and mucosal lesionslocated around the ileostoma of a woman with ulcerativecolitis for the presence of HPV. Cutaneous, muco-cutaneous, and mucosal ileostoma-biopsies wereanalyzed by three different polymerase chain reactionprotocols for genital, cutaneous, and cutaneous EV-associated HPV types. Polymerase chain reactionproducts were cloned, sequenced, and submitted tophylogenetic analyses. HPV-DNA sequences of the EV-

Human papillomaviruses (HPV) infect epithelial cells ofmucous membranes and of the skin and can lead to alarge variety of benign and malignant epithelial tumors(for review see zur Hausen, 1994; IARC, 1995;Wieland and Pfister, 1997). Today, 80 HPV types and

numerous novel HPV-DNA fragments have been identified (Myerset al, 1996). HPV types are genotypes. A new type has to differ in itsL1 gene DNA sequence by at least 10% from every other known HPVtype. HPV subtypes and variants are defined as having viral DNAsequence similarities between 90 and 98% or above 98%, respectively(Wheeler and Icenogle, 1995; van Ranst et al, 1996). Papillomavirusesare phylogenetically classified into groups A–E. Human papil-lomaviruses belong to group A (mainly genital HPV), B [epidermodys-plasia verruciformis (EV)-HPV], or E (cutaneous PV) (Chan et al,1995; Myers et al, 1996). HPV types found in the skin as HPV1–4,10, 26–29 are mainly associated with benign neoplasms (Myers et al,1996; Gross et al, 1997). Recently, known and putatively novel HPVtypes of the EV-HPV group B1 have been found in benign andmalignant skin tumors of immunocompromised (transplant recipients)and immunocompetent patients (Tieben et al, 1994; Berkhout et al,1995; de Jong-Tieben et al, 1995; Hopfl et al, 1997). Others havefound cutaneous HPV (groups B2 and E) or typical mucosal genitalHPV types (group A) in benign and malignant skin tumors (Euvrard

Manuscript received November 8, 1997; revised February 24, 1998; acceptedfor publication March 27, 1998.

Reprint requests to: Dr. Ulrike Wieland, Institut fuer Virologie, UniversitaetKoeln, Fuerst-Pueckler-Strasse 56, D-50935 Koeln, Germany.

Abbreviations: EV, epidermodysplasia verruciformis; nt, nucleotide.

0022-202X/98/$10.50 · Copyright © 1998 by The Society for Investigative Dermatology, Inc.

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HPV group could be detected in all biopsies, whereasgenital/mucosal or cutaneous HPV types were not found.HPV types detected comprised HPV20, HPV23, HPV38,and four putatively novel HPV types that belong todifferent clusters of the EV-HPV group B1. DifferentHPV types prevailed in cutaneous, mucocutaneous, andmucosal lesions and the number of HPV sequences foundper lesion varied between one and three. Our data showthe association of recurrent lesions around a stoma andat the ileum with known and novel EV-HPV types. Theseresults emphasize the plurality of HPV and yield datafor the possible transmission of cutaneous HPV tomucosal areas of the intestine. Key words: intestine/leuko-plakia/polymerase chain reaction. J Invest Dermatol 111:164–168, 1998

et al, 1993; Soler et al, 1993; Shamanin et al, 1994, 1996; Stark et al,1994; for review see Pfister and Ter Schegget, 1997). In this study wedescribe the association of known and putatively novel EV-HPV typeswith benign recurrent cutaneous, mucocutaneous, and mucosal lesionsaround an ileostoma.

MATERIALS AND METHODS

Clinical data and histology A 55 y old woman presented in 1995 withrecurrent and increasing papillomatous lesions around her ileostoma. Thepatient’s primary disease was ulcerative colitis since 1975. She had had acolectomy and establishment of an ileostoma with a Kock’s pocket in her rightlower abdominal quadrant in 1982. Since 1990 she had had several smallrevisions of the stoma because of the formation of leukoplakia-like lesions atthe skin-mucosa transition. In June 1995 her clinical findings were grayish-white, papillomatous plaques on the upper circumference of the ileostoma witha sharp outline and with a downward extension of 7–10 mm into the ileum(Fig 1). In the skin area surrounding the ileostoma no irritation was present.An endoscopic examination of the ileum in July 1995 only revealed minute,superficial ulcera at the terminal ileum just behind the Kock’s pocket (Fig 2).Biopsies of the affected skin (biopsy A) and of the skin-mucosa transition (Band C) were taken; an intestinal biopsy (D) of the ulcerous ileal mucosa wasobtained during endoscopy.

The histologic diagnosis of biopsies A-C was akanthosis without signs ofkoilocytosis. The skin biopsy showed epidermal hyperplasia, hyperortho- andparakeratosis, hypergranulosis, and papillomatosis (Fig 3). Biopsy D showed anacute ulcerous inflammation and detached intestinal epithelial cells. Neithersigns of malignancy nor signs of koilocytosis were detectable.

The patient was locally treated with three cycles of podophyllotoxin solutionin August 1995. The therapy had no effect. In December 1995 the externalparts of the papillomatous lesions were removed by a CO2 laser swift lase. Fourweeks later the lesions recurred. The patient was then referred to a medicallaser center in Berlin, Germany. Direct and endoscopically controlled Nd-YAGlaser were without persistent effect. The same was true for the use of toluidine

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Figure 1. Peristomal lesion. Greyish-white, papillomatous leukoplakia-likeplaque on the upper circumference of the ileostoma.

Figure 2. Ulcerations of the intestinal mucosa. Endoscopy showed minutesuperficial ulcerations (arrows) of the terminal ileum adjacent to the stoma.

photodynamic therapy alone and combined with CO2 laser evaporation (threesessions between 1996 and July 1997). A second biopsy from the leukoplakia-like lesion located within the ileostoma showed a benign polyploid mucosalhyperplasia.

Sample preparation and polymerase chain reaction (PCR) Formalin-fixed paraffin-embedded tissues were processed with the QIAamp Tissue Kit(Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Tenmicroliters of purified total cellular DNA were employed in each PCR reaction(25 ng DNA per µl as determined by ethidium bromide fluorescent quantitation;Sambrook et al, 1989). Each sample was analyzed with three different PCRprotocols suitable for the detection of genital (GP5 1/6 1 PCR), (muco)cutane-ous/EV (CPIIG/IIS/CPI PCR), and EV-HPV-types (CP65–70 PCR), respect-ively. PCR were performed as described previously (Tieben et al, 1993;Berkhout et al, 1995; de Roda Husman et al, 1995). An enzyme mixwith proofreading activity (Expand High Fidelity PCR System, Boehringer,Mannheim, Germany) was used. β-globin PCR was performed with all biopsiesto demonstrate that the samples contained adequate DNA and were free ofsubstances inhibitory to PCR (Saiki et al, 1985). To avoid PCR contamination,the suggestions of Kwok (1990) were diligently considered. Five negativecontrols (water instead of patient samples and HPV-negative paraffin embeddedtissues) were included in each PCR run; 0.1 pg full-length HPV8, HPV1a,and HPV16 plasmid served as positive controls (Danos et al, 1980; Seedorf et al,1986; Steger et al, 1990).

Cloning and sequencing of PCR products CP65–70 positive PCRproducts were cloned into pUC19 (Boehringer). Internal PCR was repeatedwith CP66/CP69 primers (Berkhout et al, 1995) that carried BamH1 andEcoR1 sites at their 59 ends, respectively. The internal PCR product (374–389 bp) was purified (QIAquick spin PCR purification kit, Quiagen), digested(BamH1/EcoR1), and cloned into dephosphorylated BamH1/EcoR1-digestedpUC19 using T4 DNA ligase (GibcoBRL, Eggenstein, Germany) and CaCl2transformation of Escherichia coli DH5α (Clontech, Paloalto, CA) (Sambrook

Figure 3. Leukoplakia-like HPV-positive lesion. (a) Histology of theleukoplakia-like plaque (biopsy A) revealed epidermal hyperplasia withhyperortho- and parakeratosis, hypergranulosis, and papillomatosis (hematoxylinand eosin; scale bar, 250 µm). (b) Magnification from (a); scale bar, 25 µm.

et al, 1989). Both strands of those clones that were shown to carry a BamH1/EcoR1 insert were sequenced using reagents and conditions supplied with theT7 sequencing kit (Pharmacia Biotech, Freiburg, Germany). Some of theCP66/69 PCR products had an internal EcoR1 site and thus the clonedsequences were shorter than the expected size of µ380 bp (Table I).

Sequence analyses, alignments, and phylogenetic analyses Sequenceanalyses, alignments, and phylogenetic analyses were performed as describedpreviously (Wieland et al, 1994, 1997). MacVector 6.0 and AssemblyLIGN 1.0(Oxford Molecular Group PLC, U.K., 1996) were used for translation ofnucleotide sequences and alignments. Phylogenetic analyses were performedwith the ‘‘Heidelberg Unix Sequence Analysis Resources’’ (HUSAR, GermanCancer Research Center, Heidelberg, Germany). Unrooted phylogenetic treeswere calculated according to the ‘‘neighbor joining method’’ (Saitou and Nei,1987). Bootstrapping was employed on all data sets to evaluate confidencelevels for the groupings of a tree (Felsenstein, 1985). Over 100 published HPVL1 sequences served as reference sequences for the alignments and phylogeneticanalyses (Myers et al, 1996).

RESULTS

Detection of HPV DNA in four peristomal lesions All biopsieswere tested by three different PCR protocols for genital (GP51/61and CPII/I PCR), cutaneous (CPII/I PCR), and EV- (CPII/I andCP65–70 PCR) HPV types (Tieben et al, 1993; de Roda Husman

166 WIELAND ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table I. HPV typing

Biopsy Clone(s) HPV typea Closest relative % Homology (DNA) HPV groupb

A A1–A5 S-A HPV17 76.6% (258 of 337 nt) B1skinB B1, B4 S-B HPV15 82.8% (159 of 192 nt) B1 b1transition B2, B5 S-B/X2 HPV24 64.1% (66 of 103 nt) B1skin/mucosa B3, B6 HPV23 HPV23 97.9% (185 of 189 nt) B1 b2

reference typeC C1–C5 HPV38 HPV38 99.0% (190 of 192 nt) B1 b2transition reference typeskin/mucosaD D1 HPV23 HPV23 97.4% (184 of 189 nt) B1 b2ileal D2–D5 HPV20 HPV20 100% (201 of 201 nt) B1 a2mucosa D6 S-D HPV38 82.6% (281 of 340 nt) B1 b2

aHPV typing was performed by dideoxy chain termination sequencing of cloned HPV L1 fragments and alignment with 150 known HPV L1 reference sequences.bHPV groups according to Chan et al (1995) and Myers et al (1996) (see text for details).

et al, 1995; Berkhout et al, 1995). HPV-specific sequences could notbe detected in any of the biopsies when repeatedly tested with GP51/61 or CPII/I PCR; however, HPV-specific DNA fragments of theexpected length (374–389 bp) were readily detectable in all fourbiopsies with CP65–70 PCR. Of each biopsy, the internal PCRproduct generated with primers CP66/69 was cloned into the vectorpUC19 as described in Materials and Methods. Five or six clones wereobtained from each sample (Table I).

Identification of novel EV-HPV sequences HPV typing wasperformed by sequencing of cloned HPV L1 fragments and comparisonof the sequences located between the primers with 150 known HPVL1 sequences (Myers et al, 1996). In the four biopsies we found sevendifferent HPV L1 sequences: HPV20, HPV23, HPV38, a subtype ofthe putatively new HPV type RTRX2 (S-B/X2), and three furthersequences representing putatively novel HPV types (Table I). Thelatter were named S-A, S-B, and S-D. RTRX2 is a new HPV L1sequence [276 nucleotides (nt)] recently described by Berkhout et al(1995). Seventy-two nucleotides of S-B/X2 overlapped with RTRX2and in this overlap S-B/X2 and RTRX2 were 95.8% identical (69 of72 nt), thus probably representing subtypes of the same HPV type.The closest relatives of S-A, S-B, and S-D were HPV17, HPV15, andHPV38, respectively (Table I, Fig 4). The nucleotide sequences ofS-A, S-B, and S-D differed by more than 10% from their closestrelatives (23.4%, 17.2%, 17.4%, respectively), and thus representputatively new HPV types. At the amino acid level the differences totheir closest relatives were 20.5% (23 of 112 amino acids) for S-A,15.6% (10 of 64 amino acids) for S-B, and 18.6% (21 of 113 aminoacids) for S-D (Fig 4). 159 nt of S-B overlapped with a putatively novelHPV sequence named X29 (R. Berkhout, University of Amsterdam, theNetherlands, personal communication). S-B and X29 were almostidentical (158 of 159 nt, 99.4%). Each of the new HPV sequenceswere only found in one of the four biopsies, respectively. The numberof HPV sequences found per lesion varied between one and three.Biopsies A and C carried only one HPV sequence each: identical S-Aand HPV38 sequences were found in all clones of these lesions,respectively. Three different HPV sequences were found in biopsy B(S-B, S-B/X2, HPV23) and in biopsy D (HPV20, HPV23, S-D).HPV23 was the only HPV type that occurred in more than one biopsy,namely in biopsies B and D (Table I).

Phylogenetic analyses of the novel HPV L1 sequences and of 26reference sequences showed that S-A, S-B, and S-D clustered withHPV L1 sequences of the HPV group B1 (Fig 5). According to theLos Alamos National Laboratory HPV sequence database (Myers et al,1996), the phylogenetic tree of group B1 forms two major brancheseach of which is subdivided in two minor branches, resulting in theclusters a1, a2, b1, and b2, besides some isolated branches (comparewith legend to Fig 5). In the tree shown in Fig 5, S-B, S-B/X2, andS-D were located next to the HPV types that were identified as theirclosest relatives by sequence alignments (Table I) and their positionin the tree was corroborated by bootstrap values above 800. S-B andS-D were assigned to the B1 clusters b1 and b2, respectively. RTRX2

has previously been classified as an isolated type within the group B1(Myers et al, 1996). The position of S-A in the tree was not stable(224 of 1000 and 240 of 1000 bootstrap replicates) and thus it was notassigned to any cluster.

DISCUSSION

In this study we have investigated recurrent skin and mucosal leuko-plakia-like lesions of a stoma-carrier that were refractory to therapy.By cloning and sequencing of PCR products, we have found sevendifferent HPV L1 DNA sequences in four biopsies of the leukoplakiallesions around the ileostoma and of the adjacent ileal mucosa. PCR-derived HPV L1 sequence fragments are suitable for HPV typing(Bernard et al, 1994; Tachezy et al, 1994). Comparison of the detectedsequences with 150 known HPV L1 sequences showed that three ofthe seven L1 DNA sequences corresponded to the known HPV types20, 23, and 38 that belong to the HPV group B1 (Chan et al, 1995;Myers et al, 1996). Four of the detected L1 DNA sequences differedby more than 10% from any other known HPV sequence and thusrepresent putatively novel HPV types. They were provisionally namedS-A, S-B, S-B/X2, and S-D. Their complete genome needs to becloned before they can definitively be recognized as new HPV types.S-B/X2 is probably a subtype of the L1 sequence RTRX2 that wasrecently found in a squamous cell carcinoma (SCC) (Berkhout et al,1995). Phylogenetic analyses confirmed that S-A, S-B, and S-Dbelonged to the HPV group B1 (Fig 5). Group B1 comprises HPVtypes associated with the rare skin disease EV and several new HPVL1 sequences (Myers et al, 1996). These new HPV B1 sequences aswell as established EV-HPV types were recently detected in benign(papillomas, warts, keratoacanthomas), premalignant (actinic keratoses,Bowen’s disease), and malignant (Bowen’s carcinomas, basal cell carcin-omas, SCC) skin tumors (Tieben et al, 1994; Berkhout et al, 1995; deJong-Tieben et al, 1995; Shamanin et al, 1996; Hopfl et al, 1997).Leukoplakial lesions around a stoma as in the patient investigated here,have, to our knowledge, not yet been described before. ConcerningHPV and the digestive tract, HPV types 2, 6, 11, 16, and 18 havebeen found in benign and (pre)malignant oral leukoplakias, but therole of HPV in oral leukoplakia is not yet clear (Gassenmeier andHornstein, 1988; Wen et al, 1997). Some studies have associated HPVtypes 6, 11, 16, and 18 with oral and esophageal cancer and onenovel HPV B1 sequence named ‘‘Togawa’’ was recently found in anesophageal SCC (IARC, 1995; Togawa and Rustgi, 1995; Myers et al,1996). Although we used PCR protocols suitable for the detection ofmucosal and typical cutaneous HPV, we only found HPV group B1sequences in the patient investigated here. The PCR protocol thatyielded positive results in this study was specifically designed to detectEV-HPV types at low copy numbers, but it is also suitable for theamplification of some mucosal HPV types as HPV6, HPV11, HPV13,HPV16, HPV18, HPV31, and HPV33 (Berkhout et al, 1995). This isto our knowledge, the first detection of EV-HPV types in an intestinallesion. It has to be considered, however, that the intestinal mucosadescribed here differed from normal intestinal mucosa: due to the

VOL. 111, NO. 1 JULY 1998 NEW HPV SEQUENCES IN RECURRENT LEUKOPLAKIAL LESIONS 167

Figure 4. Novel HPV L1 sequences. DNA and, above, deduced amino acidsequences of the new HPV L1 sequences S-A, S-B, S-B/X2, and S-D comparedwith their closest relatives, respectively (for details see text and Table I). (a) S-A (337 nt) and HPV17 (nt 6752–7091; Delius and Hofman, 1994); (b) S-B(192 nt) and HPV15 (nt 6731–6922; Delius and Hofman, 1994); (c) S-B/X2(103 nt) and HPV24 (nt 6753–6855; Delius, DKFZ, Heidelberg, Germany,unpublished, 1995); (d) S-D (340 nt) and HPV38 (nt 6686–7026; Delius,unpublished, 1995). Conserved positions are shown by points, and gaps areindicated by dashes. GenBank accession numbers are AF012460–AF012461.

stoma surgery it had direct contact to the skin. Two of the lesionsinvestigated by us contained only one HPV type each, and the twoother lesions carried three different HPV types each (Table I). Thesefindings are in accordance with previous studies of HPV-associated

Figure 5. Phlyogenetic analysis of the novel HPV L1 gene sequences.The phylogenetic unrooted tree was calculated according to the neighborjoining method of Saitou and Nei (1987) (for details see Materials and Methods).The four novel HPV L1 sequences, S-A, S-B, S-B/X2, and S-D, and 26 HPVL1 group B1 reference sequences (Myers et al, 1996) of the correspondinglengths were employed in the analysis. Bootstrap values above 500 are shown.The scale bar represents percentage divergence along the branches. L1 sequencesfound in the investigated patient are shown in bold letters. The branch of S-B/X2 was shortened (dotted line) because the differences to RTRX2 were over-represented due the reduced length of S-B/X2. Of the group B1 referencesequences shown in the tree (from right to left), HPV19, HPV25, HPV20,HPV14, and HPV21 belong to cluster a2; HPV5, HPV36, HPV47, HPV12,ICPX1, and HPV8 belong to cluster a1; RTRX4, RTRX5, HPV24, andRTRX2 are not classified; HPV17, HPV37, RTRX3, HPV9, and HPV15belong to cluster b1; and RTRX1, HPV23, HPV 22, HPV 38 belong to clusterb2; RTRX6 and HPV49 are not classified (Myers et al, 1996) (compare with text).

skin lesions. Berkhout et al (1995) detected more than one HPV typein over 30% of SCC biopsies. Similarily, Shamanin et al (1996) foundup to four different HPV sequences in SCC and basal cell carcinomas,and up to two different HPV sequences in common warts of renaltransplant recipients. The four biopsies investigated here were shownto carry different HPV types, respectively. The only type that wasfound in two of the biopsies was HPV23. HPV23 occurred at thetransition skin/mucosa and in the ileal mucosa (Table I). Because thepatient had had the habit of regularly flushing her ileostoma, HPVparticles or virus-containing cells could have been carried from theskin surrounding the stoma to the intestinal mucosa. The two otherHPV sequences found in the intestinal lesion (HPV20 and S-D) didnot occur in any of the other biopsies, however. The detection ofHPV-DNA by PCR does not necessarily prove that the patient’speristomal lesions were caused by an HPV infection; however, thehistology of biopsies A–C, the clinical similarity with leukoplakias, andthe strong tendency to reoccur after therapy, which is typical for manyHPV-induced lesions, make a causal connection between the peristomalpathology and the detected HPV types likely.

We would like to thank Silke von Ahlfen for excellent technical assistance. This workwas supported by the ‘‘Bundesministerium fur Bildung, Wissenschaft, Forschung undTechnologie,’’ grant number 01 KS 9502.

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