Novel Human Papillomavirus (HPV) DNA Sequences from Recurrent Cutaneous and Mucosal Lesions of a Stoma-Carrier
1998; Elsevier BV; Volume: 111; Issue: 1 Linguagem: Inglês
10.1046/j.1523-1747.1998.00256.x
ISSN1523-1747
AutoresUlrike Wieland, Gerd Gross, Ansgar Hofmann, Nib Sohendra, Hans Peter Berlien, Herbert Pfister,
Tópico(s)Nonmelanoma Skin Cancer Studies
ResumoRecent studies have demonstrated a high prevalence of human papillomavirus (HPV) types originally believed to be restricted to patients with epidermodysplasia verruciformis (EV) in benign and malignant skin tumors of the general population. Other groups detected typical mucosal HPV in skin tumors. We have investigated recurrent leukoplakial cutaneous and mucosal lesions located around the ileostoma of a woman with ulcerative colitis for the presence of HPV. Cutaneous, mucocutaneous, and mucosal ileostoma-biopsies were analyzed by three different polymerase chain reaction protocols for genital, cutaneous, and cutaneous EV-associated HPV types. Polymerase chain reaction products were cloned, sequenced, and submitted to phylogenetic analyses. HPV-DNA sequences of the EV-HPV group could be detected in all biopsies, whereas genital/mucosal or cutaneous HPV types were not found. HPV types detected comprised HPV20, HPV23, HPV38, and four putatively novel HPV types that belong to different clusters of the EV-HPV group B1. Different HPV types prevailed in cutaneous, mucocutaneous, and mucosal lesions and the number of HPV sequences found per lesion varied between one and three. Our data show the association of recurrent lesions around a stoma and at the ileum with known and novel EV-HPV types. These results emphasize the plurality of HPV and yield data for the possible transmission of cutaneous HPV to mucosal areas of the intestine. Recent studies have demonstrated a high prevalence of human papillomavirus (HPV) types originally believed to be restricted to patients with epidermodysplasia verruciformis (EV) in benign and malignant skin tumors of the general population. Other groups detected typical mucosal HPV in skin tumors. We have investigated recurrent leukoplakial cutaneous and mucosal lesions located around the ileostoma of a woman with ulcerative colitis for the presence of HPV. Cutaneous, mucocutaneous, and mucosal ileostoma-biopsies were analyzed by three different polymerase chain reaction protocols for genital, cutaneous, and cutaneous EV-associated HPV types. Polymerase chain reaction products were cloned, sequenced, and submitted to phylogenetic analyses. HPV-DNA sequences of the EV-HPV group could be detected in all biopsies, whereas genital/mucosal or cutaneous HPV types were not found. HPV types detected comprised HPV20, HPV23, HPV38, and four putatively novel HPV types that belong to different clusters of the EV-HPV group B1. Different HPV types prevailed in cutaneous, mucocutaneous, and mucosal lesions and the number of HPV sequences found per lesion varied between one and three. Our data show the association of recurrent lesions around a stoma and at the ileum with known and novel EV-HPV types. These results emphasize the plurality of HPV and yield data for the possible transmission of cutaneous HPV to mucosal areas of the intestine. epidermodysplasia verruciformis nucleotide Human papillomaviruses (HPV) infect epithelial cells of mucous membranes and of the skin and can lead to a large variety of benign and malignant epithelial tumors (for review see zur Hausen, 1994zur Hausen H. Human Pathogenic Papillomaviruses. Springer, Berlin1994Crossref Google Scholar;IARC, 1995IARC IARC Monographs on the Evaluation of Carcinogenic Risks to Humans – Human Papillomaviruses. World Health Organization, Lyon, France1995Google Scholar;Wieland and Pfister, 1997Wieland U. Pfister H. Papillomaviruses in human pathology: epidemiology, pathogenensis, and oncogenic role.in: Gross G.E. Barrasso R. Human Papillomavirus Infection – A Clinical Atlas. Ullstein Mosby, Berlin1997: 1-18Google Scholar). Today, 80 HPV types and numerous novel HPV-DNA fragments have been identified (Myers et al., 1996Myers G. Baker C. Wheeler C. Halpern A. McBride A. Doorbar J. Human Papillomaviruses. Los Alamos National Laboratory, Los Alamos1996Google Scholar). HPV types are genotypes. A new type has to differ in its L1 gene DNA sequence by at least 10% from every other known HPV type. HPV subtypes and variants are defined as having viral DNA sequence similarities between 90 and 98% or above 98%, respectively (Wheeler and Icenogle, 1995Wheeler C. Icenogle J. A closer look at papillomavirus variants.Human Papillomaviruses 1995. Los Alamos National Laboratory, Los Alamos1995: III41-46Google Scholar;van Ranst et al., 1996van Ranst M. Trachezy R. Burk R.D. Human papillomaviruses: a neverending story?.in: Lacey C. Papillomavirus Reviews: Current Research on Papillomaviruses 1996. Leeds University Press, Leeds, U.K.1996: 1-19Google Scholar). Papillomaviruses are phylogenetically classified into groups A–E. Human papillomaviruses belong to group A (mainly genital HPV), B [epidermodysplasia verruciformis (EV)-HPV], or E (cutaneous PV) (Chan et al., 1995Chan S.Y. Delius H. Halpern A.L. Bernard H.U. Analysis of genomic sequences of 95 papillomavirus types: uniting typing, phylogeny, and taxonomy.J Virol. 1995; 69: 3074-3083Crossref PubMed Google Scholar;Myers et al., 1996Myers G. Baker C. Wheeler C. Halpern A. McBride A. Doorbar J. Human Papillomaviruses. Los Alamos National Laboratory, Los Alamos1996Google Scholar). HPV types found in the skin as HPV1–4, 10, 26–29 are mainly associated with benign neoplasms (Myers et al., 1996Myers G. Baker C. Wheeler C. Halpern A. McBride A. Doorbar J. Human Papillomaviruses. Los Alamos National Laboratory, Los Alamos1996Google Scholar;Gross et al., 1997Gross G.E. Jablonska S. Hügel H. Skin diagnosis.in: Gross G.E. Barrasso R. Humanpapillomavirus Infection. Ullstein Mosby, Berlin1997: 65-123Google Scholar). Recently, known and putatively novel HPV types of the EV-HPV group B1 have been found in benign and malignant skin tumors of immunocompromised (transplant recipients) and immunocompetent patients (Tieben et al., 1994Tieben L.M. Berkhout Rjm Smits H.L. et al.Detection of epidermodysplasia verruciformis-like human papillomavirus types in malignant and premalignant skin lesions of renal transplant recipients.Br J Dermatol. 1994; 131: 226-230Crossref PubMed Scopus (85) Google Scholar;Berkhout Rjm et al., 1995Berkhout Rjm Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar;de Jong-tieben et al., 1995de Jong-tieben L.M. Berkhout Rjm Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. van der Woude F.J. ter Schegget J. High frequency of detection of epidermodysplasia verruciformis-associated human papillomavirus DNA in biopsies from malignant and premalignant skin lesions from renal transplant recipients.J Invest Dermatol. 1995; 105: 367-371Crossref PubMed Scopus (138) Google Scholar;Höpfl et al., 1997Höpfl R. Bens G. Wieland U. Petter A. Zelger B. Fritsch P. Pfister H. Human papillomavirus DNA in non-melanoma skin cancers of a renal transplant recipient: detection of a new sequence related to epidermodysplasia verruciformis associated types.J Invest Dermatol. 1997; 108: 53-56Abstract Full Text PDF PubMed Scopus (38) Google Scholar). Others have found cutaneous HPV (groups B2 and E) or typical mucosal genital HPV types (group A) in benign and malignant skin tumors (Euvrard et al., 1993Euvrard S. Chardonnet Y. Pouteil-noble C. Kanitakis J. Chignol M.C. Thivolet J. Tourraine J.L. Association of skin malignancies with various and multiple carcinogenic and noncarcinogenic human papillomaviruses in renal transplant recipients.Cancer. 1993; 72: 2198-2206Crossref PubMed Scopus (113) Google Scholar;Soler et al., 1993Soler C. Chardonnet Y. Allibert P. Euvrard S. Schmitt D. Mandrand B. Detection of mucosal human papillomavirus types 6/11 in cutaneous lesions from transplant recipients.J Invest Dermatol. 1993; 101: 286-291Abstract Full Text PDF PubMed Google Scholar;Shamanin et al., 1994Shamanin V. Glover M. Rausch C. Proby C. Leigh I.M. zur Hausen H. Specific types of human papillomavirus found in benign proliferations and carcinomas of the skin of immunosuppressed patients.Cancer Res. 1994; 54: 4610-4613PubMed Google Scholar,Shamanin et al., 1996Shamanin V. zur Hausen H. Lavergne D. et al.Human papillomavirus infections in nonmelanoma skin cancers from renal transplant recipients and nonimmunosuppressed patients.J Natl Cancer Inst. 1996; 88: 802-811Crossref PubMed Scopus (281) Google Scholar;Stark et al., 1994Stark L.A. Arends M.J. McLaren K.M. Benton E.C. Shahidullah H. Hunter Jaa Bird C.C. Prevalence of human papillomavirus DNA in cutaneous neoplasms from renal allograft recipients supports a possible viral role in tumour promotion.Br J Cancer. 1994; 69: 222-229Crossref PubMed Scopus (89) Google Scholar; for review see Pfister and Ter Schegget, 1997Pfister H. Ter Schegget J. Role of HPV in cutaneous premalignant and malignant tumors.Clinics Dermatol. 1997; 15: 335-347Abstract Full Text PDF PubMed Scopus (110) Google Scholar). In this study we describe the association of known and putatively novel EV-HPV types with benign recurrent cutaneous, mucocutaneous, and mucosal lesions around an ileostoma. A 55 y old woman presented in 1995 with recurrent and increasing papillomatous lesions around her ileostoma. The patient's primary disease was ulcerative colitis since 1975. She had had a colectomy and establishment of an ileostoma with a Kock's pocket in her right lower abdominal quadrant in 1982. Since 1990 she had had several small revisions of the stoma because of the formation of leukoplakia-like lesions at the skin-mucosa transition. In June 1995 her clinical findings were grayish-white, papillomatous plaques on the upper circumference of the ileostoma with a sharp outline and with a downward extension of 7–10 mm into the ileum (Figure 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 (Figure 2). Biopsies of the affected skin (biopsy A) and of the skin-mucosa transition (B and C) were taken; an intestinal biopsy (D) of the ulcerous ileal mucosa was obtained during endoscopy.Figure 2Ulcerations of the intestinal mucosa. Endoscopy showed minute superficial ulcerations (arrows) of the terminal ileum adjacent to the stoma.View Large Image Figure ViewerDownload (PPT) The histologic diagnosis of biopsies A-C was akanthosis without signs of koilocytosis. The skin biopsy showed epidermal hyperplasia, hyperortho- and parakeratosis, hypergranulosis, and papillomatosis (Figure 3). Biopsy D showed an acute ulcerous inflammation and detached intestinal epithelial cells. Neither signs of malignancy nor signs of koilocytosis were detectable. The patient was locally treated with three cycles of podophyllotoxin solution in August 1995. The therapy had no effect. In December 1995 the external parts of the papillomatous lesions were removed by a CO2 laser swift lase. Four weeks later the lesions recurred. The patient was then referred to a medical laser center in Berlin, Germany. Direct and endoscopically controlled Nd-YAG laser were without persistent effect. The same was true for the use of toluidine photodynamic therapy alone and combined with CO2 laser evaporation (three sessions between 1996 and July 1997). A second biopsy from the leukoplakia-like lesion located within the ileostoma showed a benign polyploid mucosal hyperplasia. Formalin-fixed paraffin-embedded tissues were processed with the QIAamp Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Ten microliters 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., 1989Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning – A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1989Google Scholar). Each sample was analyzed with three different PCR protocols suitable for the detection of genital (GP5 +/6 + PCR), (muco)cutaneous/EV (CPIIG/IIS/CPI PCR), and EV-HPV-types (CP65–70 PCR), respectively. PCR were performed as described previously (Tieben et al., 1993Tieben L.M. ter Schegget J. Minnaar R.P. et al.Detection of cutaneous and genital HPV types in clinical samples by PCR using consensus primers.J Virol Meth. 1993; 42: 265-280Crossref PubMed Scopus (160) Google Scholar;Berkhout Rjm et al., 1995Berkhout Rjm Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar;de Roda Husman et al., 1995de Roda Husman A.M. Walboomers Jmm van den Brule A.J.C. Meijer Cjlm Snijders PjF The use of general primers GP5 and GP6 elongated at their 3′ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR.J Gen Virol. 1995; 76: 1057-1062Crossref PubMed Scopus (1090) Google Scholar). An enzyme mix with proofreading activity (Expand High Fidelity PCR System, Boehringer, Mannheim, Germany) was used. β-globin PCR was performed with all biopsies to demonstrate that the samples contained adequate DNA and were free of substances inhibitory to PCR (Saiki et al., 1985Saiki R.K. Scharf S. Faloona F. Mullis K.B. Horn G.T. Erlich H.A. Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.Science. 1985; 230: 1350-1354Crossref PubMed Scopus (6510) Google Scholar). To avoid PCR contamination, the suggestions ofKwok, 1990Kwok S. Procedures to minimize PCR-product carry-over.in: Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols – A Guide to Methods and Applications. Academic Press, San Diego1990: 142-145Crossref Google Scholar were diligently considered. Five negative controls (water instead of patient samples and HPV-negative paraffin embedded tissues) were included in each PCR run; 0.1 pg full-length HPV8, HPV1a, and HPV16 plasmid served as positive controls (Danos et al., 1980Danos O. Katinka M. Yaniv M. Molecular cloning, refined physical map and heterogeneity of methylation sites of papilloma virus type 1a DNA.Europ J Biochem. 1980; 109: 457-461Crossref PubMed Scopus (39) Google Scholar;Seedorf et al., 1986Seedorf K. Krämmer G. Dürst M. Suhai S. Röwekamp W.G. Human papillomavirus type 16 DNA sequence.Virology. 1986; 145: 181-185Crossref Scopus (553) Google Scholar;Steger et al., 1990Steger G. Olszewsky M. Stockfleth E. Pfister H. Prevalence of antibodies to human papillomavirus type 8 in human sera.J Virol. 1990; 64: 4399-4406PubMed Google Scholar). CP65–70 positive PCR products were cloned into pUC19 (Boehringer). Internal PCR was repeated with CP66/CP69 primers (Berkhout Rjm et al., 1995Berkhout Rjm Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar) that carried BamH1 and EcoR1 sites at their 5′ 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-digested pUC19 using T4 DNA ligase (GibcoBRL, Eggenstein, Germany) and CaCl2 transformation of Escherichia coli DH5α (Clontech, Paloalto, CA) (Sambrook et al., 1989Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning – A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1989Google Scholar). Both strands of those clones that were shown to carry a BamH1/EcoR1 insert were sequenced using reagents and conditions supplied with the T7 sequencing kit (Pharmacia Biotech, Freiburg, Germany). Some of the CP66/69 PCR products had an internal EcoR1 site and thus the cloned sequences were shorter than the expected size of ≈380 bp (Table 1).Table IHPV typingBiopsyClone(s)HPV typeaHPV typing was performed by dideoxy chain termination sequencing of cloned HPV L1 fragments and alignment with 150 known HPV L1 reference sequences.Closest relative% Homology (DNA)HPV groupbHPV groups according to Chan et al (1995) and Myers et al (1996) (see text for details).AA1—A5S-AHPV1776.6% (258 of 337 nt)B1skinBB1, B4S-BHPV1582.8% (159 of 192 nt)B1 b1transitionB2, B5S-B/X2HPV2464.1% (66 of 103 nt)B1skin/mucosaB3, B6HPV23HPV2397.9% (185 of 189 nt)B1 b2CC1—C5HPV38reference type HPV3899.0% (190 of 192 nt)B1 b2transitionskin/mucosaDD1HPV23reference type HPV2397.4% (184 of 189 nt)B1 b2ilealD2—D5HPV20HPV20100% (201 of 201 nt)B1 a2mucosaD6S-DHPV3882.6% (281 of 340 nt)B1 b2a HPV typing was performed by dideoxy chain termination sequencing of cloned HPV L1 fragments and alignment with 150 known HPV L1 reference sequences.b HPV groups according to Chan et al., 1995Chan S.Y. Delius H. Halpern A.L. Bernard H.U. Analysis of genomic sequences of 95 papillomavirus types: uniting typing, phylogeny, and taxonomy.J Virol. 1995; 69: 3074-3083Crossref PubMed Google Scholar, Myers et al., 1996Myers G. Baker C. Wheeler C. Halpern A. McBride A. Doorbar J. Human Papillomaviruses. Los Alamos National Laboratory, Los Alamos1996Google Scholar (see text for details). Open table in a new tab Sequence analyses, alignments, and phylogenetic analyses were performed as described previously (Wieland et al., 1994Wieland U. Hartmann J. Suhr H. Salzberger B. Eggers H.J. Kühn J. In vivo genetic variability of the HIV-1 vif gene.Virology. 1994; 203: 43-51Crossref PubMed Scopus (45) Google Scholar,Wieland et al., 1997Wieland U. Seelhoff A. Hofmann A. Kühn J.E. Eggers H.J. Mugyenyi P. Schwander S. Diversity of the vif gene of human immundeficiency virus type 1 in Uganda.J Gen Virol. 1997; 78: 393-400Crossref PubMed Scopus (19) Google Scholar). MacVector 6.0 and AssemblyLIGN 1.0 (Oxford Molecular Group PLC, U.K. 1996) were used for translation of nucleotide sequences and alignments. Phylogenetic analyses were performed with the “Heidelberg Unix Sequence Analysis Resources” (HUSAR, German Cancer Research Center, Heidelberg, Germany). Unrooted phylogenetic trees were calculated according to the “neighbor joining method” (Saitou and Nei, 1987Saitou N. Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees.Mol Biol Evol. 1987; 4: 406-425PubMed Google Scholar). Bootstrapping was employed on all data sets to evaluate confidence levels for the groupings of a tree (Felsenstein, 1985Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap.Evolution. 1985; 39: 783-791Crossref PubMed Google Scholar). Over 100 published HPV L1 sequences served as reference sequences for the alignments and phylogenetic analyses (Myers et al., 1996Myers G. Baker C. Wheeler C. Halpern A. McBride A. Doorbar J. Human Papillomaviruses. Los Alamos National Laboratory, Los Alamos1996Google Scholar). All biopsies were tested by three different PCR protocols for genital (GP5+/6+ and CPII/I PCR), cutaneous (CPII/I PCR), and EV- (CPII/I and CP65–70 PCR) HPV types (Tieben et al., 1993Tieben L.M. ter Schegget J. Minnaar R.P. et al.Detection of cutaneous and genital HPV types in clinical samples by PCR using consensus primers.J Virol Meth. 1993; 42: 265-280Crossref PubMed Scopus (160) Google Scholar;de Roda Husman et al., 1995de Roda Husman A.M. Walboomers Jmm van den Brule A.J.C. Meijer Cjlm Snijders PjF The use of general primers GP5 and GP6 elongated at their 3′ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR.J Gen Virol. 1995; 76: 1057-1062Crossref PubMed Scopus (1090) Google Scholar;Berkhout Rjm et al., 1995Berkhout Rjm Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar). HPV-specific sequences could not be detected in any of the biopsies when repeatedly tested with GP5+/6+ or CPII/I PCR; however, HPV-specific DNA fragments of the expected length (374–389 bp) were readily detectable in all four biopsies with CP65–70 PCR. Of each biopsy, the internal PCR product generated with primers CP66/69 was cloned into the vector pUC19 as described in Materials and Methods. Five or six clones were obtained from each sample (Table 1). HPV typing was performed by sequencing of cloned HPV L1 fragments and comparison of the sequences located between the primers with 150 known HPV L1 sequences (Myers et al., 1996Myers G. Baker C. Wheeler C. Halpern A. McBride A. Doorbar J. Human Papillomaviruses. Los Alamos National Laboratory, Los Alamos1996Google Scholar). In the four biopsies we found seven different HPV L1 sequences: HPV20, HPV23, HPV38, a subtype of the putatively new HPV type RTRX2 (S-B/X2), and three further sequences representing putatively novel HPV types (Table 1). The latter were named S-A, S-B, and S-D. RTRX2 is a new HPV L1 sequence [276 nucleotides (nt)] recently described byBerkhout Rjm et al., 1995Berkhout Rjm Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar. Seventy-two nucleotides of S-B/X2 overlapped with RTRX2 and in this overlap S-B/X2 and RTRX2 were 95.8% identical (69 of 72 nt), thus probably representing subtypes of the same HPV type. The closest relatives of S-A, S-B, and S-D were HPV17, HPV15, and HPV38, respectively (Table 1, Figure 4). The nucleotide sequences of S-A, S-B, and S-D differed by more than 10% from their closest relatives (23.4%, 17.2%, 17.4%, respectively), and thus represent putatively new HPV types. At the amino acid level the differences to their 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 amino acids) for S-D (Figure 4). 159 nt of S-B overlapped with a putatively novel HPV sequence named X29 (R. Berkhout, University of Amsterdam, the Netherlands, personal communication). S-B and X29 were almost identical (158 of 159 nt, 99.4%). Each of the new HPV sequences were only found in one of the four biopsies, respectively. The number of HPV sequences found per lesion varied between one and three. Biopsies A and C carried only one HPV sequence each: identical S-A and 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 1). Phylogenetic analyses of the novel HPV L1 sequences and of 26 reference sequences showed that S-A, S-B, and S-D clustered with HPV L1 sequences of the HPV group B1 (Figure 5). According to the Los Alamos National Laboratory HPV sequence database (Myers et al., 1996Myers G. Baker C. Wheeler C. Halpern A. McBride A. Doorbar J. Human Papillomaviruses. Los Alamos National Laboratory, Los Alamos1996Google Scholar), the phylogenetic tree of group B1 forms two major branches each of which is subdivided in two minor branches, resulting in the clusters a1, a2, b1, and b2, besides some isolated branches (compare with legend to Figure 5). In the tree shown in Figure 5, S-B, S-B/X2, and S-D were located next to the HPV types that were identified as their closest relatives by sequence alignments (Table 1) and their position in the tree was corroborated by bootstrap values above 800. S-B and S-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., 1996Myers G. Baker C. Wheeler C. Halpern A. McBride A. Doorbar J. Human Papillomaviruses. Los Alamos National Laboratory, Los Alamos1996Google Scholar). The position of S-A in the tree was not stable (224 of 1000 and 240 of 1000 bootstrap replicates) and thus it was not assigned to any cluster. In this study we have investigated recurrent skin and mucosal leukoplakia-like lesions of a stoma-carrier that were refractory to therapy. By cloning and sequencing of PCR products, we have found seven different HPV L1 DNA sequences in four biopsies of the leukoplakial lesions around the ileostoma and of the adjacent ileal mucosa. PCR-derived HPV L1 sequence fragments are suitable for HPV typing (Bernard et al., 1994Bernard H.U. Chan S.Y. Manos M.M. et al.Identification and assessment of known and novel human papillomaviruses by polymerase chain reaction amplification, restriction fragment length polymorphisms, nucleotide sequence, and phylogenetic algorithms.J Infect Dis. 1994; 170: 1077-1085Crossref PubMed Scopus (443) Google Scholar;Tachezy et al., 1994Tachezy R. van Ranst M.A. Cruz Y. Burk R.D. Analysis of short novel human papillomavirus sequences.Biochem Biophys Res Comm. 1994; 204: 820-827Crossref PubMed Scopus (41) Google Scholar). Comparison of the detected sequences with 150 known HPV L1 sequences showed that three of the seven L1 DNA sequences corresponded to the known HPV types 20, 23, and 38 that belong to the HPV group B1 (Chan et al., 1995Chan S.Y. Delius H. Halpern A.L. Bernard H.U. Analysis of genomic sequences of 95 papillomavirus types: uniting typing, phylogeny, and taxonomy.J Virol. 1995; 69: 3074-3083Crossref PubMed Google Scholar;Myers et al., 1996Myers G. Baker C. Wheeler C. Halpern A. McBride A. Doorbar J. Human Papillomaviruses. Los Alamos National Laboratory, Los Alamos1996Google Scholar). Four of the detected L1 DNA sequences differed by more than 10% from any other known HPV sequence and thus represent putatively novel HPV types. They were provisionally named S-A, S-B, S-B/X2, and S-D. Their complete genome needs to be cloned before they can definitively be recognized as new HPV types. S-B/X2 is probably a subtype of the L1 sequence RTRX2 that was recently found in a squamous cell carcinoma (SCC) (Berkhout Rjm et al., 1995Berkhout Rjm Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar). Phylogenetic analyses confirmed that S-A, S-B, and S-D belonged to the HPV group B1 (Figure 5). Group B1 comprises HPV types associated with the rare skin disease EV and several new HPV L1 sequences (Myers et al., 1996Myers G. Baker C. Wheeler C. Halpern A. McBride A. Doorbar J. Human Papillomaviruses. Los Alamos National Laboratory, Los Alamos1996Google Scholar). These new HPV B1 sequences as well 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 carcinomas, SCC) skin tumors (Tieben et al., 1994Tieben L.M. Berkhout Rjm Smits H.L. et al.Detection of epidermodysplasia verruciformis-like human papillomavirus types in malignant and premalignant skin lesions of renal transplant recipients.Br J Dermatol. 1994; 131: 226-230Crossref PubMed Scopus (85) Google Scholar;Berkhout Rjm et al., 1995Berkhout Rjm Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar;de Jong-tieben et al., 1995de Jong-tieben L.M. Berkhout Rjm Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. van der Woude F.J. ter Schegget J. High frequency of detection of epidermodysplasia verruciformis-associated human papillomavirus DNA in biopsies from malignant and premalignant skin lesions from renal transplant recipients.J Invest Dermatol. 1995; 105: 367-371Crossref PubMed Scopus (138) Google Scholar;Shamanin et al., 1996Shamanin V. zur Hausen H. Lavergne D. et al.Human papillomavirus infections in nonmelanoma skin cancers from renal transplant recipients and nonimmunosuppressed patients.J Natl Cancer Inst. 1996; 88: 802-811Crossref PubMed Scopus (281) Google Scholar;Höpfl et al., 1997Höpfl R. Bens G. Wieland U. Petter A. Zelger B. Fritsch P. Pfister H. Human papillomavirus DNA in non-melanoma skin cancers of a renal transplant recipient: detection of a new sequence related to epidermodysplasia verruciformis associated types.J Invest Dermatol. 1997; 108: 53-56Abstract Full Text PDF PubMed Scopus (38) Google Scholar). Leukoplakial lesions around a stoma as in the patient investigated here, have, to our knowledge, not yet been described before. Concerning HPV and the digestive tract, HPV types 2, 6, 11, 16, and 18 have been found in benign and (pre)malignant oral leukoplakias, but the role of HPV in oral leukoplakia is not yet clear (Gassenmeier and Hornstein, 1988Gassenmeier A. Hornstein O.P. Presence of human papillomavirus DNA in benign and precancerous oral leukoplakias and squamous cell carcinomas.Dermatologica. 1988; 176: 224Crossref Scopus (47) Google Scholar;Wen et al., 1997Wen S. Tsuji T. Li X. Mizugaki Y. Hayatsu Y. Shinozaki F. Detection and analysis of human papillomavirus 16 and 18 homologous DNA sequences in oral lesions.Anticancer Res. 1997; 17: 307-312PubMed Google Scholar). Some studies have associated HPV types 6, 11, 16, and 18 with oral and esophageal cancer and one novel HPV B1 sequence named “Togawa” was recently found in an esophageal SCC (IARC, 1995IARC IARC Monographs on the Evaluation of Carcinogenic Risks to Humans – Human Papillomaviruses. World Health Organization, Lyon, France1995Google Scholar;Togawa and Rustgi, 1995Togawa K. Rustgi A. A novel human papillomavirus sequence based on L1 general primers.Virus Res. 1995; 36: 293-297Crossref PubMed Scopus (15) Google Scholar;Myers et al., 1996Myers G. Baker C. Wheeler C. Halpern A. McBride A. Doorbar J. Human Papillomaviruses. Los Alamos National Laboratory, Los Alamos1996Google Scholar). Although we used PCR protocols suitable for the detection of mucosal and typical cutaneous HPV, we only found HPV group B1 sequences in the patient investigated here. The PCR protocol that yielded positive results in this study was specifically designed to detect EV-HPV types at low copy numbers, but it is also suitable for the amplification of some mucosal HPV types as HPV6, HPV11, HPV13, HPV16, HPV18, HPV31, and HPV33 (Berkhout Rjm et al., 1995Berkhout Rjm Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar). This is to our knowledge, the first detection of EV-HPV types in an intestinal lesion. It has to be considered, however, that the intestinal mucosa described here differed from normal intestinal mucosa: due to the stoma surgery it had direct contact to the skin. Two of the lesions investigated by us contained only one HPV type each, and the two other lesions carried three different HPV types each (Table 1). These findings are in accordance with previous studies of HPV-associated skin lesions.Berkhout Rjm et al., 1995Berkhout Rjm Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar detected more than one HPV type in over 30% of SCC biopsies. Similarily,Shamanin et al., 1996Shamanin V. zur Hausen H. Lavergne D. et al.Human papillomavirus infections in nonmelanoma skin cancers from renal transplant recipients and nonimmunosuppressed patients.J Natl Cancer Inst. 1996; 88: 802-811Crossref PubMed Scopus (281) Google Scholar found up to four different HPV sequences in SCC and basal cell carcinomas, and up to two different HPV sequences in common warts of renal transplant recipients. The four biopsies investigated here were shown to carry different HPV types, respectively. The only type that was found in two of the biopsies was HPV23. HPV23 occurred at the transition skin/mucosa and in the ileal mucosa (Table 1). Because the patient had had the habit of regularly flushing her ileostoma, HPV particles or virus-containing cells could have been carried from the skin surrounding the stoma to the intestinal mucosa. The two other HPV sequences found in the intestinal lesion (HPV20 and S-D) did not occur in any of the other biopsies, however. The detection of HPV-DNA by PCR does not necessarily prove that the patient's peristomal lesions were caused by an HPV infection; however, the histology of biopsies A–C, the clinical similarity with leukoplakias, and the strong tendency to reoccur after therapy, which is typical for many HPV-induced lesions, make a causal connection between the peristomal pathology and the detected HPV types likely. We would like to thank Silke von Ahlfen for excellent technical assistance. This work was supported by the “Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie,” grant number 01 KS 9502.
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