| Abstract|| |
Background: Papillomatosis is a known histopathologic pattern usually seen in human papillomavirus (HPV) infection and verruca vulgaris is the typical example. This pattern is also detected in some other benign cutaneous lesions such as nevus sebaceous (NS), seborrheic keratosis (SK), trichilemmoma (TL) and inverted follicular keratosis (IFK). The association between papillomatous lesions and HPV infection is questionable. Objective: The objective of this study was to investigate the presence of HPV deoxyribonucleic acid (DNA) in non-genital benign papillomatous skin lesions (NS, SK, TL and IFK) by polymerase chain reaction (PCR). Materials and Methods: A total of 100 specimens of non-genital NS, SK, TL and IFK were retrieved from archives of Dermatopathology Department of Razi Hospital, between 2003 and 2010. The conventional PCR using consensus GP5+/GP6+ primer and hydroxymethylbilane synthase gene as inner control was performed. Results: PCR for HPV DNA revealed no positive results in any of 28 seborrheic keratosis (SK), 28 nevus sebaceous (NS), 28 inverted follicular keratosis (IFK) and 13 trichilemmoma (TL) studied specimens. Conclusion: Papillomatosis is usually a characteristic pattern of HPV infection. However, we found no association between HPV infection and non-genital benign papillomatous lesions.
Keywords: Human papillomavirus, inverted follicular keratosis, nevus sebaceous, papillomatosis, polymerase chain reaction, seborrheic keratosis, trichilemmoma
|How to cite this article:|
Kambiz KH, Kaveh D, Maede D, Hossein A, Nessa A, Ziba R, Alireza G. Human papillomavirus deoxyribonucleic acid may not be detected in non-genital benign papillomatous skin lesions by polymerase chain reaction. Indian J Dermatol 2014;59:334-8
|How to cite this URL:|
Kambiz KH, Kaveh D, Maede D, Hossein A, Nessa A, Ziba R, Alireza G. Human papillomavirus deoxyribonucleic acid may not be detected in non-genital benign papillomatous skin lesions by polymerase chain reaction. Indian J Dermatol [serial online] 2014 [cited 2021 Jun 15];59:334-8. Available from: https://www.e-ijd.org/text.asp?2014/59/4/334/135475
What was known?
Some of the benign papillomatous lesions show histhopathological pattern that mimics human papillomavirus (HPV)-induced cutaneous lesions, so it is reasonable to investigate HPV infection in them.
| Introduction|| |
Papillomatosis is a minor histopathologic pattern of irregular wavy projections of epidermal surface and underlying dermal papilla, under the light microscopy. The prototypes are verruca vulgaris, seborrheic keratosis (SK) and acrokeratosis verruciformis.  In some papillomatoses such as verruca vulgaris and bowenoid papulosis cythopathologic effect of HPV can be detected.  These changes include parakeratosis above the epidermal projection, acanthosis, vacuolated koilocytes and coarse granular layer.  While other focal epidermal proliferations that don't present viral cytopathic effect might show basaliod cells, atypical keratinocytes or elongation of dermal papilla.  The association between these kinds of lesions and HPV infection is questionable.
HPVs are a heterogeneous group of small double strand deoxyribonucleic acid (DNA) viruses. The pathogenesis of HPV initiates as it reaches the basal layer through minor erosions of the epithelium. HPV DNA integrates into the host genome of epithelial cells. However, it can remain in extra-chromosomal states, induce epithelial proliferation and cause differentiation arrest resulting in verrucous or papillomatous lesions.  The virus actually delays nuclear condensation in differentiating keratinocytes forming the koilocyte, known as the pathognomonic cell of HPV infection. 
Viral gene expression is closely linked to the differentiation level of the infected epithelial cells. The El and E2 genes are transcribed in the basal cell layer and are responsible for viral genome replication. The E5, 6, 7 genes remove a brake on supra-basal cell cycling, induce epithelial proliferation and epithelial differentiation arrest. Vegetative viral growth occurs in differentiation epithelial compartment. Late viral proteins L1, L2 and E4 are made for assembly of high copy number of the viral genome into virions in the upper terminally differentiated layers of the stratum spinosum and granulosum. 
More than 100 types of HPV have been detected. After isolation of HPV from verruca in 1949, it has been detected in various benign and malignant proliferation of mucocutaneous epithelium such as respiratory papillomatosis, cervical and anogenital squamous cell carcinoma and some cancers of the head and neck.  Some of these lesions exhibit HPV type-specific clinicopathologic features.  Using highly sensitive polymerase chain reaction (PCR), widespread distribution of HPV was detected even in normal skin. 
Some of the benign papillomatous lesions such as nevus sebaceous (NS), SK, trichilemmoma (TL) and inverted follicular keratosis (IFK) show papillomatous epidermal hyperplasia, that mimic HPV-induced cutaneous lesions. It seems reasonable to investigate HPV infection in these types of lesions in our patient's population. Although few studies reported HPV association with some of these lesions, this association was not confirmed by other authors and in different patient populations.
In this study, we investigated HPV infection in NS, SK, TL and IFK in our immunocompetent patients, by conventional PCR.
| Materials and Methods|| |
The formalin-fixed, paraffin-embedded blocks of benign papillomatous lesions of NS, SK, TL and IFK were retrieved from archives of Dermatopathology Department of Razi Hospital, Tehran University of Medical Sciences, between 2003 and 2010. All specimens were selected from non-immunocompromised patients. Minimum of three verrucae vulgaris were included as positive controls.
For each specimen, one section was taken for H and E staining. The specimens were reviewed by a board-certified Dermatopathologist and the primary diagnosis was confirmed.
Then, other two 5 μm sections were used for extraction of DNA for HPV testing. To ensure the availability of DNA in these specimens homosapiens hydroxymethylbilane synthase (HMBS) gene was checked in extracted DNA (inner control). HMBS exists in all nucleated human cells,  and therefore, only samples containing HMBS were included.
After extracted DNA content was confirmed and normalized, amplification using GP5+/6+ consensus primer (Bioneer Corp., Korea). Stages of PCR process have been summarized in [Table 1]. The GP5+/6+ primer, which was available on our center, amplifies a 150 base pair region of highly conserved L1 region of many HPV types. Some studies believe that this primer is sufficiently sensitive to detect many mucosal, cutaneous and epidermodysplasia verruciformis (EV) HPV types.  Theoretically, HPV consensus primers, amplifies a highly conserved L1 region for HPV capsid protein of many types of HPV 1A, 2, 5, 6, 8, 11, 13, 16/18, 26, 27, 30, 31/33, 35, 39, 40, 41, 42, 44, 45, 47, 48, 51, 52, 53, 54, 55, 57,58, 59 as well as uncharacterized types.  However, specific HPV genotypes such as HPV may not be readily detected by the consensus primers. 
For all specimens positive and negative controls were processed simultaneously. To eliminate the potential contaminate, all stages were performed under appropriate hood.
The PCR products were analyzed using electrophoresis on agarose gels and stained with ethidium bromide that binds organic bases. The appearance of the 150 base-pair band, similar to positive control for L1 gene is considered as a positive result.
| Results|| |
Our archive consisted of 28 NS, 28 SK, 13 TL and 28 IFK specimens. Three verrucae plantares positive controls were included. Conventional PCR and gel electrophoresis were used to find HPV DNA in the paraffin blocks. Investigation for HPV DNA by PCR revealed no positive results in any of NS, SK, TL and IFK specimens, but the results were positive in verrucae plantares specimens. [Figure 1] and [Figure 2] show the negative result of PCR in NS, SK, IFK, TL (the positive result in [Figure 1] corresponds to verruca plantares). Results have been summarized in [Table 2].
|Figure 1: Gel electrophoresis (7 is the positive control, 8 is a negative control, 6 is the block of 88-775 (wart) 1,5,9 are seborrheic keratosis (negative result), 2,10,11 are nevus sebaceous (negative result), 3,4 are inverted follicular keratosis (negative result), 12 is trichilemmoma (negative result))|
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|Figure 2: Gel electrophoresis (1 is a negative control, 2 is a positive control, 3,4,10 are inverted follicular keratosis negative result, 5,6,7,13 are seborrheic keratosis (negative result), 8,11,12 are nevus sebaceous (negative result) 9 is trichilemmoma (negative result))|
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| Discussion|| |
Papillomatosis is one of the major cytopathic effects of HPV infection, but HPV DNA is not necessarily associated with all papillomatous lesions. Some studies that sought the association between HPV and NS, SK, TL and IFK are discussed separately as follows.
NS of jadassohn is a complex hamartoma, involving not only the pilosebaceous follicle, but also the epidermis and other adnexal structures.  It shows basaloid epidermal hyperplasia and papillomatosis as well as dilated apocrine glands in the dermis. 
There was only one study that investigated the association of NS and HPV; however, they got different results from ours. Carlson et al. studied 28 NS without secondary tumors (NWOT) and 32 NS with secondary tumors (NSWT) for HPV DNA using nested PCR and HPV 16 was the most prevalent HPV type in 27% of pure NS lesions. 
Higher frequency of EV, genital-mucosal, co-infection and HPV 16 DNA was detected in NSWT compared to NWOT.  Higher frequency of HPV co-infection in NSWT can lead to higher load of HPV DNA and increase the risk of HPV even unknown types detection in their NS specimens. They found at least one HPV DNA and also some novel EV HPV genotypes in 82% of all specimens, but nearly each type of HPV was seen only once in isolated specimen, except for HPV 16 and HPV 38 (in secondary tumors).
In our study, none of the NS specimens was complicated with secondary tumor and no HPV DNA was detected among these 28 NS. The different results might be because of difference in technics and the possibility of over detection of HPV DNA by nested PCR and or presence of specific HPV genotypes not readily detected by the consensus primers. 
SK (senile wart, basal cell papillomas) are common, often multiple, benign tumors, which usually appear in middle life. They are composed of basaloid cells with a varying admixture of squamoid cells. 
Preliminary studies reported high prevalence of HPV associated with SK in genital areas. SK in anogenital area often resembles condylomata acuminata clinically and histopathologically. To put an end to this dilemma, Li and Ackerman proposed that "SK," which contains HPV are actually condylomata acuminate. 
Most studies that investigated non-genital SK found evidence of HPV infection only in minority or none of the cases. Several methods like electron microscopy,  PCR for HPV DNA,  in situ PCR for type specific HPV  (in situ PCR enables visualization of the cellular localization of HPV DNA and is more accurate and sensitive methods comparing to in situ hybridization  ) were used.
However, some studies reported positive results. Applying nested PCR with consensus primers revealed more positive results for EV HPV DNA in non-genital SK compared to healthy controls.  In nested PCR double genome amplification is used and the significant difference in positive results from two steps PCR genome amplification, strongly suggested a very low copy number for EV HPV DNA.  Gushi et al. study detected mucosal HPV-18, 81, 6 in most of their samples.  In their studies, the increased age of patients and healthy controls and number of lesions were associated with increased/added HPV detection.  Therefore, colonization of HPV as commensal in the elderly with co-infection of multi H of multi HPV has been suggested. 
Surprisingly, there was no overlap between accused HPV types detected in these studies, except for HPV 6. , One possible explanation would be that venereal transmission and genital infection with HPV (HPV 6, 11) can increases the risk of total body skin exposure to these HPVs and also other HPV types as well. And the affected site could be the source of such differences in virus detection.  However, none of those studies mentioned any history of genital SK in their patients. Co-infections with multiple HPV types make the role of a distinct HPV in the pathogenesis of SK questionable. 
Our results did not reveal HPV DNA in any of the 28 non-genital SK. Although, positive results of three verrucae plantares, positive controls, rules out technical defects. The GP5+/GP6+ primer was applied only if the specimens contained the human genome (positive for HMBS). As a result all old specimens lacking genome were excluded.
Whether TL are true benign neoplasms or virus-induced and the hair follicle would be the source for both EV-HPV reservoir and TL origin remains a subject of controversy. 
Some previous studies didn't detect any evidence for papillomavirus infection applying immunoperoxidase HPV techniques,  in situ hybridization for HPV 1, 2, low and high risk genital HPV types,  and PCR. 
However, Rohwedder et al. could detect potentially novel EV-associated HPV in TL cases, by modification of PCR conditions. They reduced annealing temperature from 55°C to 50°C. 
HPV sequences, especially the EV-associated types have been detected as commensals, especially in immunocompromised people. Unfortunately the healthy controls were not included for comparison and method modification might cause over-detection of HPV DNA.
As TL is a rare follicular involved lesion, the total of 13 specimens was included in our study. Our findings did not show HPV DNA in any of those lesions. However, we used the common PCR conditions and GP5+/6+ primers detecting L1 fragments that are naturally conserved among HPV types. However as we applied the common 60c annealing temperature our results might get different from the latter study.
Although IFK seemed to be an endophytic variant of irritated SK  and same hypothesis for HPV association with IFK could be presumed, no previous study has detected HPV within these lesions.
Our results confirm the previous studies of negative results that applied ISH for HPV types 1, 2 and GM-types,  and IHC staining of polyvalent HPV antibodies,  to detect any association with HPV.
Limitations of this study
There are some limitations that should be noted. First of all, we tested our samples with conventional PCR. The consensus primer that we used was GP5+/6+ from the HPV L1 gene. Deletion of L1 gene in HPV would cause false negative results. Therefore, it is better that this study be confirmed by nested PCR or HPV mRNA testing and using other primers. Second, for better evaluation we should test more samples, but some of these tumors are extremely rare.
| Conclusions|| |
Although papillomatosis is usually a characteristic pattern of HPV infection, several studies did not detect HPV in papillomatous lesions and the role of HPV in papillomatous lesions remains controversial.
In general, controvert results could be caused by the use of large variability in different studies such as HPV subtypes studied, the technique of PCR or ISH and the sample type (fresh, frozen or paraffin, etc.) and also different geographical populations.
Although positives results of multiple HPV types in both healthy skin and lesional tissue might be owing to the contamination; however, HPV infection may be commensal and over detection of HPV DNA in some studies may have resulted from applying double DNA amplification in nests or type-specific PCR. However, co-infections with multiple HPV types make the role of a distinct HPV in the pathogenesis of papillomatous lesions questionable.
Interestingly, HPV DNA is not detected invariably in all condyloma acuminata, even in those that display koliocytes.  Furthermore, it has been proposed that HPV can initiate and promote some skin lesions to a point of no return and subsequently, become undetectable or possibly be removed by the immune system. 
In this study, we observed no association between HPV infection and non-genital benign papillomatous skin lesions of NS, SK, TL and IFK lesions in our population of immunocompetent Iranian patients. However, number of samples might not be high enough to refuse the possible association between HPV and papillomatosis. It is also possible that the PCR technique used in this study is sensitive for detecting mucosal HPV; however, cannot efficiently detect the cutaneous types.
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What is new?
The number of researches about the role of HPV in benign papillomatos non-genital skin lesions is limited. In this research, we investigate the role of HPV in four of these lesions (nevus sebaceous (NS), seborrheic keratosis (SK), Trichilemmoma (TL), inverted follicular) by PCR methods.
[Figure 1], [Figure 2]
[Table 1], [Table 2]