Human papilloma virus in erosive oral lichen pianus
M. Jonteii', S. Watts', M. Wallstrom', L. Levin' and K. Sloberg^ Departments of EndodontologylOral Diagnosis^ and Oral Surgery', University of Gothenburg, Sweden, and Dental Research Center', University of North Carolina at Chapel Hill, NC, USA
Jontell M, Watts S, Wallstrom M, Levin L, Sloberg K: Human papillomavirus in erosive oral lichen pianus. J Oral Pathol Med 1990; 19: 273-7. Several types of human papilloma viruses (HPV) have been associated with benign and malignant squamous cell tumours of mucosal epithelium. To identify HPV in erosive oral lichen pianus (OLPe), considered as a premalignant lesion, tissues from 20 patients were examined by Southern blot hybridization with "Plabeled HPV DNA probes. Type 11 was found in 6 of the lesions while HPV types 6, 16 and 18 were not detected in any of the tissues examined. Using a type-specific polymerase chain reaction (PCR) assay for HPV-6, 11, 16 and 18, HPV-11 was detected in 8 of the samples (all of those positive by Southern blot), and, in addition, HPV-6 was found in 5 samples and HPV-16 in 3 samples. Overall, by the more sensitive PCR assay, 65% of samples were positive for HPV DNA, The finding of HPV DNA in many of the samples using two different techniques indicates a high prevalence of HPV in the OLPe afflicted oral mucosa. However, the role of HPV in the pathogenesis of OLPe has yet to be determined.
Erosive oral lichen pianus (OLPe) is usually resistant to permanent cure. The chronic erosive lesion, which consists of an atrophic mucosa with ulcers, demands regular examinations since OLPe is considered as a premalignant condition, A transformation into oral carcinoma over 5 years has been suggested in approximately 1% ofthe cases (1), The association between certain HPV and the development of various types of benign and malignant genital tumors is a well-recognized phenomenon (2), In recent years, HPV has also been identified in oral squamous cell carcinomas which suggests that HPV may be involved in oral carcinogenesis as well (3-7), Significant correlation has been established between the presence of HPV antigens and the degree of dyspiasia of premalignant disorders such as oral leukoplakias (5, 8, 9), In two recent studies, the association between HPV and oral lichen pianus has been examined (10, 11), HPV was identified in 8 out of 10 lesions (11), However, no figures were provided separately for OLPe, The present study was undertaken to examine the presence of HPV in OLPe and to determine the different DNA types. Material and methods
Patients - Patients with OLPe (17 women, mean age = 59 yr, range = 36-81
Key words: human papilloma virus: lichen pianus, erosive: oral mucosa, PCR Southern blot hybridization.
Accepted for publication March 15, 1990,
yr; 3 men, mean age = 55, range = 30-77 was immediately transferred to a yr) were randomly selected from records — 70°C freezer and stored at this temof the Departments of Endodontology/ perature until further processing. Identification and typitig of HPV Oral Diagnosis and Oral Surgery, University of Gothenburg, Requirements DNA by Southern blottittg - Total cellufor inclusion were presentation with bi- lar DNA was extracted from the bioplateral erosive lesions of the buccal mu- sies by sodium dodecyl sulfate (SDS)cosa, as well as additional OLPe lesions pronase treatment, phenol-chloroform on the dorsum of the tongue or in the extraction, and ethanol precipitation. gingiva. Special attention was given to Digestion with RNAse was followed by exclude lichenoid reactions, topographi- repeated extraction and precipitation, A cally related to buccal amalgam fillings. portion of the cellular DNA was digestLesions of lupus erythematosus and be- ed with restriction endonuclease Bam nign mucous membrane pemphigoid HI which cleaves HPV 6, 11, 16 and 18 were excluded by immunohistochem- DNAs 1-2 times. The DNA was subjected to electrophoresis in 1,0% agaristry. Tissue processing and morphologic ex- ose gels in tris-acetate-EDTA buffer amittation - Biopsies were taken with a with appropriate controls of plasmidscalpel under local anesthesia (Xylo- cloned HPV DNAs (obtained from Dr, cain-epinephrin, Astra, Sweden), The HARALD ZUR HAUSEN, Heidelberg, specimens were divided in two parts, FRG), The DNA was denatured and one for histopathologic examination to transferred from the gel to "Geneverify the clinical diagnosis and the Screen Plus" hybridization membranes Other part for identification and typing (New England Nuclear) by the method of HPV DNA, The part of the tissue of Southern (12), The membranes were used for histopathologic analysis was hybridized under stringent conditions subjected to fixation in 5% buffered (Tn-23°C) of 50% formamide, 0,2 M parafonnaldehyde. The staining proce- NaCl, 20 niM sodium phosphate, pH dure of the specimens was performed 6,8, 5X Denhardt's solution, 0,1% SDS, in hematoxylin and eosiri according to 250 ixg/inl depurinated salmon sperm routine methods. The sections were ex- DNA, 10% dextran sulfate at 41"C to amined in a Olympus BH2 microscope mixtures of nick-translated '-P-labeled and the microphotographs were taken probes of HPV DNA types 6/11 and 16/ with an Olympus 0M2 camera using a 18 (>lxlO'' cpm/|ig), Episomal viral Agfa Pan 25 film. The portion of the DNA was identified by autoradiogratissue subjected to HPV DNA analysis phy with a sensitivity of less than 5 pg
274
JONTELL et al.
kbp 23.1 9.4-
12 3 4 5 6 7 8 _ .•iti.i^r.;
6.6-
4.3-
Fig, L Southern blot hybridization of total cellular DNA from OLPe samples hybridized to HPV-11-type-specific probe (nucleotides 3900-4557) from the E5b subgenomic region. Dashed lines indicate migration positions of Hind Ill-digested bacteriophage lambda DNA molecular weight markers, 23.1, 9,4, 6.6, 4,3, 2.3, and 2,0 kilobase pairs in size as denoted. Arrows indicate migration positions of episomal HPV DNA form I (covalently-closed circular) as well as partially-digested form II (nicked circular) and form III (cleaved linear) viral DNAs, Approximately 10 (ig of each sample DNA digested with Bam HI restriction endonuclease was electrophoresed in each lane. Lane I; HPV-6-containing genital condyloma acuminatum, lane 2; HPV-11-containing laryngeal papilloma, lane 3; sample HA, lane 4; sample BC, lane 5; sample BD, lane 6; sample EB, lane 7; samples ES, lane 8; sample LP, lane 9; sample LH,
or 0,5 viral genome equivalent/cell. Additionally, multi-cut restriction endonuclease digestion with Pst I or Hinc II was performed or, alternatively, hybridization with type-specific subgenomic probes for specific HPV types was used for further type identification. Type-specific detection of HPV DNAs by ampliftcation assay - For polymerase chain reaction amplification (13, 14), unique oligonucleotide pairs (20-mers)
corresponding to the DNA sequence of HPV 6, 11, 16 and 18 were chosen from the E6 genomic region in order to amplify corresponding type-specific fragments of 166 bp (nucleotides 139-304), 138 bp (nucleotides 139-276), 398 bp (nucleotides 110-507), and 438 bp (nucleotides 105-542), respectively. Amplification reactions in 50 |,il volume contained approximately 1 ng sample DNA, 2 U Taq polymerase, 200 |iM
Table 1, HPV analysis of erosive oral lichen pianus lesions ofthe buccal mucosa.
Sample
Sex
Age
HA UW
F F
55 37
ES
F
71
EB
F
LK
M
EL
F
IS
F
LP LH BD BC LO IA IB RN RM GE EBl SC AF
F F F F F F
61 30 73 63 73 49 53 46 77 41 81 57 36 60 59 11 66
F M F F F M F
Southern blot hybridization 6/11 16/18
PCR Assay Risk
11
16
18
-l-a -hb -l-a -l-a
Resuits
+b -t-b — _ — _ — —
-t-
—
_ _
each deoxynucleotide triphosphate, 0.5 (iM each oligonucleotide primer in 10 mM Tris-HCl, pH 8,3, 50 mM KCl, 1,5 mM MgClj, 0,01% (w/v) gelatin. Amplifications were performed for 40 cycles using 1 min at 95°C for denaturation, 30 s at 64°C for annealing, and 20 s at 72°C for extension, HPV-6 and 11 primers were used in a single reaction as these primers had been shown not to yield interference in control samples. Separate reactions were performed with HPV 16 and 18 primer pairs due to interference when primer pairs were combined in reaction mixtures (WATTS et al., personal communication). In sensitivity tests, this amplification procedure detected less than 5 fg of each HPV DNA, One-third of the pooled reaction products for each sample were denatured with sodium hydroxide and applied using a dot blot manifold to hybridization membranes in quadruplicate. Type-specific oligonucleotides corresponding to HPV-6, 11, 16 or 18 sequences within each amplified fragment were radiolabeled and hybridized to dot blots in 5X SSPE (lXis 0,18 M sodium chloride, 0.01 M sodium phosphate, pH 6,8, 0,001 M disodium ethylenediamine tetraacetate), 5X Denhardt's solution, 0,5% SDS for 16 h at 17-19°C below the oligonucleotide Tj, Dot blots were washed at room temperature and 37°C in 2X SSPE-0,1% SDS prior to overnight autoradiographic exposure. Under these conditions, no crossreaction occurred between typespecific internal oligonucleotide hybridizations to control HPV-6, 11, 16 and 18 DNAs. The sensitivity of dot blot detection was less than 1 ng HPV plasmid DNA per dot,
-f-
-
S*
c
* Cigarette smoking (C) ** Snuff (S), All positive samples were identified as containing HPV-11 DNA by either: a) Hybridization to a HPV-11 type-specific probe encompassing the E5b open reading frame region (nucl. 3900-4557). b) Pst I and Hinc U restriction endonuclease cleavage patterns.
Overall, 30% of OLPe lesions examined (6 out of 20) were found to contain HPV DNA hybridizing to the 6/11 probe (Table 1), By digestion with Hinc II and/ or Pst I restriction endonuclease or by hybridization to a HPV-11-specific probe, the specific HPV was determined to be Type 11 (Fig, 1). None of the samples hybridized to HPV 16 or 18, With our development of polymerase chain reaction assay for type-specific E6 subgenomic regions of HPV-6, 11, 16 and 18, we decided to reassay the OLPe samples by the new, more sensitive technique to verify Southern blot positivity. Although possible contamination of biopsy samples previously obtained could not be ruled out, we expected the assay
HPV in erosive oral liehen plantis to be worthwhile since disposable plasticware and careful techniques were used in sample processing. Although no samples were positive by hybridization for HPV-18 amplification, positive samples were found for HPV-6, HPV-11, and HPV-16. Eight samples were positive for HPV-11, including all of those determined to be positive by Southern blot analysis. Five samples were positive for the closelyrelated HPV-6 and three samples for HPV-16. Two samples, specifically EBl and SC were positive for multiple HPV types. Overall, 13 of 20 samples (65%) were positive for some HPV type, predominantly HPV-11, All samples showed histopathologic characteristics of OLPe, i,e, an atrophic epithelium with no or mild dyspiasia, a subepithelial infiltrate of lymphocytes and basal liquefaction (Fig, 2A, B). The histopathologic examination of the HPV positive specimens showed no morphologic characteristics of HPV infection such as koilocytosis (15), Koilocytosis is suggested to be a cytopathic effect of HPV (16), characterized as a perinuclear clear zone adjacent to a pyknotic, irregularly-shaped nucleus within the spinous cells of the squamous epithelium (17), The demographic data of the patients with lesions containing HPV were not apparently different from the patients with lesions negative for HPV. The prevalence of smoking habits, medication and history of various types of diseases
were similar in the two groups. Finally, no differences were observed in the clinical appearance of the HPV negative and positive OLPe lesions. Discussion Of the various types of oral lichen pianus, OLPe is the form which has lost the most tissue integrity. Although the normal pathways for activation of the defence mechanisms are thereby seriously affected, OLPe is rarely associated with malignant transformation. However, malignancy-associated HPV-16 was recently identified in a single case of oral lichen pianus. To evaluate a possible association of HPV and oral lichen pianus, further studies were requested (10), The presence of HPV-16 or 18 DNA, particularly in an integrated state in the cellular chromosome, seems to be related to the development of cervical mahgnancy while HPV types 6 and 11 are predominantly found in condylomata acuminata, laryngeal papillomas and mild cervical dysplasias (2), In some cases, HPV-11 and 16 in particular have been found in squamous cell carcinomas of the buccal mucosa, tongue, larynx (5, 7, 18-20), In a few cases of vulvar and laryngeal carcinoma, rearranged HPV6 or 11 episomal DNAs have been associated with malignancy (21, 22), Our limited investigation of the samples by Pst I and Hinc II restriction endonuclease digestion did not indicate any major
Fig. 2. Typieal oral liehen pianus lesions with basal liquefaction and dense infiltrate mononuelear cells, HPV was detected in A) but not in B), HE, x 100,
275
genome rearrangements but minor alterations would not have been detectable in our analysis. To evaluate the association of various types of HPV with OLPe, it will be necessary to examine a larger number of lesions. The specimens in the present study represent tissues from atrophic and erosive OLPe lesions. The prevalence of HPV in these lesions was found to be different from what was observed by Southern blot hybridization where 87% of the reticular type of OLP contained HPV (11). This difference might be explained by a higher degree of keratosis in the reticular type of lesion, a hypothesis supported by the concept that viral replication may require keratinization. A 1000-fold increase in the sensitivity of virus genome detection by the PCR assay versus the Southern blot assay can account for the detection of additional positive samples as well as additional HPV DNA types in the same samples by the former technique in our study. The malignant transformation as well as growth control and natural history of HPV infections have been related to the immune status ofthe host (23). Since local immunosuppression by corticosteroids is a valuable drug and widely used in attempt to keep OLPe under control, it is worth remembering that long-term treatment with corticosteroids might contribute to malignant progression of OLPe. Steroids are known to decrease the density of Class II antigen expressing Langerhans cells (24) which thereby open avenues to viral antigens and an increased risk of malignant transformation (25, 26), In addition, a transcriptional activation element induceable by glucocorticoids has been identified in the regulatory regions of HPV-6, 11,16 and 18 genomes which in treated patients with virus could lead to over-expression of HPV genes associated with cell culture transformation (27), These observations emphasize the necessity of a regular and careful examination of OLPe patients treated with steroids. Observations indicating that T-lymphocytes dominate the histopathologic picture of OLPe lesions support the hypothesis that cell-mediated immunity is primarily involved in the pathogenesis of the disorder. Although it is still unknown to what tissue structures the lymphocytes are directed, their activity is probably indirectly responsible for the disruption ofthe epithelial hning. There is no evidence in OLPe for primary changes in the epithelium, as for exam-
276
JONTELL et al.
pie an increased prevalence of epithelial dyspiasia, which would make this disorder per se to be more predisposed for neoplastic development than other
chronic ulcers of the oral mucosa. Nevertheless, due to the widespread breakdown of the weakened epithelium, this tissue is constantly exposed to various
a b o d e
insults and recovery which might contribute to a malignant transformation. In addition, trauma is thought to play an essential role in the introduction of HPV into the basal cell layer of the epithelium in the initiation of the infection. Thus, HPV may represent one of the factors which could be responsible, in combination with other carcinogens like tobacco and alcohol, for the development of squamous cell carcinoma in OLPe (6, 23), A large number of various forms of oral mucosal lesions have been observed to contain different types of HPV (10), The existential association between HPV and these lesions has provided a basis for suggestions of the etiological role of HPV. However, some studies of healthy oral tissues indicate that even the normal oral mucosa carries HPV (11), Further evidence is required from epidemiologicai studies in order to uncover an etiological role for HPVs in the pathogenesis of different oral mucosal lesions. Acknowledgments - This study was supported by a grant DE 08214 from the US Public Health Service,
References 1, HOLMSTRUP P, THORN J J, RINDUM J,
Fig. 3. Dot-blot hybridization detection of HPV-11-specific amplified sequences from erosive lichen pianus DNA samples. Denatured amplifieation products from erosive lichen pianus samples as well as control plasmid DNAs (unamplified) and amplified HPV-containing control tissue DNAs were hybridized to a^^P-labeled HPV-11-specific oligonucleotide representing sequences within the amplified product (nucleotides 261-280). Row 6 in columns «-
M. Jonteii', S. Watts', M. Wallstrom', L. Levin' and K. Sloberg^ Departments of EndodontologylOral Diagnosis^ and Oral Surgery', University of Gothenburg, Sweden, and Dental Research Center', University of North Carolina at Chapel Hill, NC, USA
Jontell M, Watts S, Wallstrom M, Levin L, Sloberg K: Human papillomavirus in erosive oral lichen pianus. J Oral Pathol Med 1990; 19: 273-7. Several types of human papilloma viruses (HPV) have been associated with benign and malignant squamous cell tumours of mucosal epithelium. To identify HPV in erosive oral lichen pianus (OLPe), considered as a premalignant lesion, tissues from 20 patients were examined by Southern blot hybridization with "Plabeled HPV DNA probes. Type 11 was found in 6 of the lesions while HPV types 6, 16 and 18 were not detected in any of the tissues examined. Using a type-specific polymerase chain reaction (PCR) assay for HPV-6, 11, 16 and 18, HPV-11 was detected in 8 of the samples (all of those positive by Southern blot), and, in addition, HPV-6 was found in 5 samples and HPV-16 in 3 samples. Overall, by the more sensitive PCR assay, 65% of samples were positive for HPV DNA, The finding of HPV DNA in many of the samples using two different techniques indicates a high prevalence of HPV in the OLPe afflicted oral mucosa. However, the role of HPV in the pathogenesis of OLPe has yet to be determined.
Erosive oral lichen pianus (OLPe) is usually resistant to permanent cure. The chronic erosive lesion, which consists of an atrophic mucosa with ulcers, demands regular examinations since OLPe is considered as a premalignant condition, A transformation into oral carcinoma over 5 years has been suggested in approximately 1% ofthe cases (1), The association between certain HPV and the development of various types of benign and malignant genital tumors is a well-recognized phenomenon (2), In recent years, HPV has also been identified in oral squamous cell carcinomas which suggests that HPV may be involved in oral carcinogenesis as well (3-7), Significant correlation has been established between the presence of HPV antigens and the degree of dyspiasia of premalignant disorders such as oral leukoplakias (5, 8, 9), In two recent studies, the association between HPV and oral lichen pianus has been examined (10, 11), HPV was identified in 8 out of 10 lesions (11), However, no figures were provided separately for OLPe, The present study was undertaken to examine the presence of HPV in OLPe and to determine the different DNA types. Material and methods
Patients - Patients with OLPe (17 women, mean age = 59 yr, range = 36-81
Key words: human papilloma virus: lichen pianus, erosive: oral mucosa, PCR Southern blot hybridization.
Accepted for publication March 15, 1990,
yr; 3 men, mean age = 55, range = 30-77 was immediately transferred to a yr) were randomly selected from records — 70°C freezer and stored at this temof the Departments of Endodontology/ perature until further processing. Identification and typitig of HPV Oral Diagnosis and Oral Surgery, University of Gothenburg, Requirements DNA by Southern blottittg - Total cellufor inclusion were presentation with bi- lar DNA was extracted from the bioplateral erosive lesions of the buccal mu- sies by sodium dodecyl sulfate (SDS)cosa, as well as additional OLPe lesions pronase treatment, phenol-chloroform on the dorsum of the tongue or in the extraction, and ethanol precipitation. gingiva. Special attention was given to Digestion with RNAse was followed by exclude lichenoid reactions, topographi- repeated extraction and precipitation, A cally related to buccal amalgam fillings. portion of the cellular DNA was digestLesions of lupus erythematosus and be- ed with restriction endonuclease Bam nign mucous membrane pemphigoid HI which cleaves HPV 6, 11, 16 and 18 were excluded by immunohistochem- DNAs 1-2 times. The DNA was subjected to electrophoresis in 1,0% agaristry. Tissue processing and morphologic ex- ose gels in tris-acetate-EDTA buffer amittation - Biopsies were taken with a with appropriate controls of plasmidscalpel under local anesthesia (Xylo- cloned HPV DNAs (obtained from Dr, cain-epinephrin, Astra, Sweden), The HARALD ZUR HAUSEN, Heidelberg, specimens were divided in two parts, FRG), The DNA was denatured and one for histopathologic examination to transferred from the gel to "Geneverify the clinical diagnosis and the Screen Plus" hybridization membranes Other part for identification and typing (New England Nuclear) by the method of HPV DNA, The part of the tissue of Southern (12), The membranes were used for histopathologic analysis was hybridized under stringent conditions subjected to fixation in 5% buffered (Tn-23°C) of 50% formamide, 0,2 M parafonnaldehyde. The staining proce- NaCl, 20 niM sodium phosphate, pH dure of the specimens was performed 6,8, 5X Denhardt's solution, 0,1% SDS, in hematoxylin and eosiri according to 250 ixg/inl depurinated salmon sperm routine methods. The sections were ex- DNA, 10% dextran sulfate at 41"C to amined in a Olympus BH2 microscope mixtures of nick-translated '-P-labeled and the microphotographs were taken probes of HPV DNA types 6/11 and 16/ with an Olympus 0M2 camera using a 18 (>lxlO'' cpm/|ig), Episomal viral Agfa Pan 25 film. The portion of the DNA was identified by autoradiogratissue subjected to HPV DNA analysis phy with a sensitivity of less than 5 pg
274
JONTELL et al.
kbp 23.1 9.4-
12 3 4 5 6 7 8 _ .•iti.i^r.;
6.6-
4.3-
Fig, L Southern blot hybridization of total cellular DNA from OLPe samples hybridized to HPV-11-type-specific probe (nucleotides 3900-4557) from the E5b subgenomic region. Dashed lines indicate migration positions of Hind Ill-digested bacteriophage lambda DNA molecular weight markers, 23.1, 9,4, 6.6, 4,3, 2.3, and 2,0 kilobase pairs in size as denoted. Arrows indicate migration positions of episomal HPV DNA form I (covalently-closed circular) as well as partially-digested form II (nicked circular) and form III (cleaved linear) viral DNAs, Approximately 10 (ig of each sample DNA digested with Bam HI restriction endonuclease was electrophoresed in each lane. Lane I; HPV-6-containing genital condyloma acuminatum, lane 2; HPV-11-containing laryngeal papilloma, lane 3; sample HA, lane 4; sample BC, lane 5; sample BD, lane 6; sample EB, lane 7; samples ES, lane 8; sample LP, lane 9; sample LH,
or 0,5 viral genome equivalent/cell. Additionally, multi-cut restriction endonuclease digestion with Pst I or Hinc II was performed or, alternatively, hybridization with type-specific subgenomic probes for specific HPV types was used for further type identification. Type-specific detection of HPV DNAs by ampliftcation assay - For polymerase chain reaction amplification (13, 14), unique oligonucleotide pairs (20-mers)
corresponding to the DNA sequence of HPV 6, 11, 16 and 18 were chosen from the E6 genomic region in order to amplify corresponding type-specific fragments of 166 bp (nucleotides 139-304), 138 bp (nucleotides 139-276), 398 bp (nucleotides 110-507), and 438 bp (nucleotides 105-542), respectively. Amplification reactions in 50 |,il volume contained approximately 1 ng sample DNA, 2 U Taq polymerase, 200 |iM
Table 1, HPV analysis of erosive oral lichen pianus lesions ofthe buccal mucosa.
Sample
Sex
Age
HA UW
F F
55 37
ES
F
71
EB
F
LK
M
EL
F
IS
F
LP LH BD BC LO IA IB RN RM GE EBl SC AF
F F F F F F
61 30 73 63 73 49 53 46 77 41 81 57 36 60 59 11 66
F M F F F M F
Southern blot hybridization 6/11 16/18
PCR Assay Risk
11
16
18
-l-a -hb -l-a -l-a
Resuits
+b -t-b — _ — _ — —
-t-
—
_ _
each deoxynucleotide triphosphate, 0.5 (iM each oligonucleotide primer in 10 mM Tris-HCl, pH 8,3, 50 mM KCl, 1,5 mM MgClj, 0,01% (w/v) gelatin. Amplifications were performed for 40 cycles using 1 min at 95°C for denaturation, 30 s at 64°C for annealing, and 20 s at 72°C for extension, HPV-6 and 11 primers were used in a single reaction as these primers had been shown not to yield interference in control samples. Separate reactions were performed with HPV 16 and 18 primer pairs due to interference when primer pairs were combined in reaction mixtures (WATTS et al., personal communication). In sensitivity tests, this amplification procedure detected less than 5 fg of each HPV DNA, One-third of the pooled reaction products for each sample were denatured with sodium hydroxide and applied using a dot blot manifold to hybridization membranes in quadruplicate. Type-specific oligonucleotides corresponding to HPV-6, 11, 16 or 18 sequences within each amplified fragment were radiolabeled and hybridized to dot blots in 5X SSPE (lXis 0,18 M sodium chloride, 0.01 M sodium phosphate, pH 6,8, 0,001 M disodium ethylenediamine tetraacetate), 5X Denhardt's solution, 0,5% SDS for 16 h at 17-19°C below the oligonucleotide Tj, Dot blots were washed at room temperature and 37°C in 2X SSPE-0,1% SDS prior to overnight autoradiographic exposure. Under these conditions, no crossreaction occurred between typespecific internal oligonucleotide hybridizations to control HPV-6, 11, 16 and 18 DNAs. The sensitivity of dot blot detection was less than 1 ng HPV plasmid DNA per dot,
-f-
-
S*
c
* Cigarette smoking (C) ** Snuff (S), All positive samples were identified as containing HPV-11 DNA by either: a) Hybridization to a HPV-11 type-specific probe encompassing the E5b open reading frame region (nucl. 3900-4557). b) Pst I and Hinc U restriction endonuclease cleavage patterns.
Overall, 30% of OLPe lesions examined (6 out of 20) were found to contain HPV DNA hybridizing to the 6/11 probe (Table 1), By digestion with Hinc II and/ or Pst I restriction endonuclease or by hybridization to a HPV-11-specific probe, the specific HPV was determined to be Type 11 (Fig, 1). None of the samples hybridized to HPV 16 or 18, With our development of polymerase chain reaction assay for type-specific E6 subgenomic regions of HPV-6, 11, 16 and 18, we decided to reassay the OLPe samples by the new, more sensitive technique to verify Southern blot positivity. Although possible contamination of biopsy samples previously obtained could not be ruled out, we expected the assay
HPV in erosive oral liehen plantis to be worthwhile since disposable plasticware and careful techniques were used in sample processing. Although no samples were positive by hybridization for HPV-18 amplification, positive samples were found for HPV-6, HPV-11, and HPV-16. Eight samples were positive for HPV-11, including all of those determined to be positive by Southern blot analysis. Five samples were positive for the closelyrelated HPV-6 and three samples for HPV-16. Two samples, specifically EBl and SC were positive for multiple HPV types. Overall, 13 of 20 samples (65%) were positive for some HPV type, predominantly HPV-11, All samples showed histopathologic characteristics of OLPe, i,e, an atrophic epithelium with no or mild dyspiasia, a subepithelial infiltrate of lymphocytes and basal liquefaction (Fig, 2A, B). The histopathologic examination of the HPV positive specimens showed no morphologic characteristics of HPV infection such as koilocytosis (15), Koilocytosis is suggested to be a cytopathic effect of HPV (16), characterized as a perinuclear clear zone adjacent to a pyknotic, irregularly-shaped nucleus within the spinous cells of the squamous epithelium (17), The demographic data of the patients with lesions containing HPV were not apparently different from the patients with lesions negative for HPV. The prevalence of smoking habits, medication and history of various types of diseases
were similar in the two groups. Finally, no differences were observed in the clinical appearance of the HPV negative and positive OLPe lesions. Discussion Of the various types of oral lichen pianus, OLPe is the form which has lost the most tissue integrity. Although the normal pathways for activation of the defence mechanisms are thereby seriously affected, OLPe is rarely associated with malignant transformation. However, malignancy-associated HPV-16 was recently identified in a single case of oral lichen pianus. To evaluate a possible association of HPV and oral lichen pianus, further studies were requested (10), The presence of HPV-16 or 18 DNA, particularly in an integrated state in the cellular chromosome, seems to be related to the development of cervical mahgnancy while HPV types 6 and 11 are predominantly found in condylomata acuminata, laryngeal papillomas and mild cervical dysplasias (2), In some cases, HPV-11 and 16 in particular have been found in squamous cell carcinomas of the buccal mucosa, tongue, larynx (5, 7, 18-20), In a few cases of vulvar and laryngeal carcinoma, rearranged HPV6 or 11 episomal DNAs have been associated with malignancy (21, 22), Our limited investigation of the samples by Pst I and Hinc II restriction endonuclease digestion did not indicate any major
Fig. 2. Typieal oral liehen pianus lesions with basal liquefaction and dense infiltrate mononuelear cells, HPV was detected in A) but not in B), HE, x 100,
275
genome rearrangements but minor alterations would not have been detectable in our analysis. To evaluate the association of various types of HPV with OLPe, it will be necessary to examine a larger number of lesions. The specimens in the present study represent tissues from atrophic and erosive OLPe lesions. The prevalence of HPV in these lesions was found to be different from what was observed by Southern blot hybridization where 87% of the reticular type of OLP contained HPV (11). This difference might be explained by a higher degree of keratosis in the reticular type of lesion, a hypothesis supported by the concept that viral replication may require keratinization. A 1000-fold increase in the sensitivity of virus genome detection by the PCR assay versus the Southern blot assay can account for the detection of additional positive samples as well as additional HPV DNA types in the same samples by the former technique in our study. The malignant transformation as well as growth control and natural history of HPV infections have been related to the immune status ofthe host (23). Since local immunosuppression by corticosteroids is a valuable drug and widely used in attempt to keep OLPe under control, it is worth remembering that long-term treatment with corticosteroids might contribute to malignant progression of OLPe. Steroids are known to decrease the density of Class II antigen expressing Langerhans cells (24) which thereby open avenues to viral antigens and an increased risk of malignant transformation (25, 26), In addition, a transcriptional activation element induceable by glucocorticoids has been identified in the regulatory regions of HPV-6, 11,16 and 18 genomes which in treated patients with virus could lead to over-expression of HPV genes associated with cell culture transformation (27), These observations emphasize the necessity of a regular and careful examination of OLPe patients treated with steroids. Observations indicating that T-lymphocytes dominate the histopathologic picture of OLPe lesions support the hypothesis that cell-mediated immunity is primarily involved in the pathogenesis of the disorder. Although it is still unknown to what tissue structures the lymphocytes are directed, their activity is probably indirectly responsible for the disruption ofthe epithelial hning. There is no evidence in OLPe for primary changes in the epithelium, as for exam-
276
JONTELL et al.
pie an increased prevalence of epithelial dyspiasia, which would make this disorder per se to be more predisposed for neoplastic development than other
chronic ulcers of the oral mucosa. Nevertheless, due to the widespread breakdown of the weakened epithelium, this tissue is constantly exposed to various
a b o d e
insults and recovery which might contribute to a malignant transformation. In addition, trauma is thought to play an essential role in the introduction of HPV into the basal cell layer of the epithelium in the initiation of the infection. Thus, HPV may represent one of the factors which could be responsible, in combination with other carcinogens like tobacco and alcohol, for the development of squamous cell carcinoma in OLPe (6, 23), A large number of various forms of oral mucosal lesions have been observed to contain different types of HPV (10), The existential association between HPV and these lesions has provided a basis for suggestions of the etiological role of HPV. However, some studies of healthy oral tissues indicate that even the normal oral mucosa carries HPV (11), Further evidence is required from epidemiologicai studies in order to uncover an etiological role for HPVs in the pathogenesis of different oral mucosal lesions. Acknowledgments - This study was supported by a grant DE 08214 from the US Public Health Service,
References 1, HOLMSTRUP P, THORN J J, RINDUM J,
Fig. 3. Dot-blot hybridization detection of HPV-11-specific amplified sequences from erosive lichen pianus DNA samples. Denatured amplifieation products from erosive lichen pianus samples as well as control plasmid DNAs (unamplified) and amplified HPV-containing control tissue DNAs were hybridized to a^^P-labeled HPV-11-specific oligonucleotide representing sequences within the amplified product (nucleotides 261-280). Row 6 in columns «-
