No Evidence of Human Papillomaviruses in Non-genital Seborrheic Keratosis

Abstract

Background:

Seborrheic keratosis (SK) is a benign epidermal tumor of unknown etiology. Because of its wart-like morphology, Human papillomaviruses (HPVs) have been suggested as a possible causative agent. Viral involvement, however, has not been confirmed yet despite research and the association between HPVs and seborrheic keratosis has not been studied among Iranian population by PCR.

Objectives:

The aim of this case-control study was to evaluate the presence of HPVs DNA in non-genital SK by PCR.

Materials and Methods:

Fifty biopsy specimens obtained from patients with non-genital SK and 50 controls were analyzed using polymerase chain reaction (PCR).

Results:

No HPVs DNA was detected by PCR within the tissue extracts from paraffin-embedded SK samples, while one of the controls was HPVs DNA positive. The age range of the patients was 20 to 82 yrs (mean = 52). Twenty-eight patients (56%) were males and 22 patients (44%) were females. The most common anatomic site was the face. Histopathologic changes due to viral infection such as koilocytosis (10%), dyskeratosis (66%), mitosis (28%), and parakeratosis (88%) were evident within the lesions. The most common histologic type was acanthotic type.

Conclusion:

Our results showed that there is no association between HPVs and seborrheic keratosis in investigated subjects.

Introduction

What was known?

Seborrheic keratosis (SK) is a benign epidermal tumor of unknown etiology. Because of its wart-like morphology and their histological similarity to warts HPVs have been suggested as a possible causative agent.

Seborrheic keratoses (SKs) are the most common human benign neoplasm, often seen in the elderly people.[1] Although the etiology and pathogenesis of seborrheic keratosis are still not well understood, new insights have been gained in recent years; however, the nature of seborrheic keratoses is still disputed. Due to their histological similarity to warts (hyperkeratinization, acanthosis, papillomatosis in the epidermis), SKs have been assayed for the presence of human papillomaviruses (HPVs). Zhao et al., showed HPVs in four of 89 seborrheic keratoses by light microscopy and electron microscopy.[2] In addition, HPVs have been detected in a small number of studies.[3,4] It has also been found in the seborrheic keratoses of patients who have epidermodysplasia verruciformis.[5] Furthermore, the DNA of epidermodysplasia-verruciformis associated HPVs have been detected in non-genital seborrheic keratoses.[6] The present study was conducted to evaluate the presence of HPVs in tissue samples of confirmed seborrheic keratoses.

Materials and Methods

Sample collection

Confirmed cases of non-genital SK referred to Pathology Department of Imam Reza Hospital, Mashhad, Iran, from 2003 to 2010 were selected for this study. Skin biopsies removed for cosmetic reasons were used as control specimens.

The exclusion criteria for cases and controls were inadequacy of tissue, not fulfilling the criteria for definite diagnosis of SK, those with unmatched clinical records and negative samples in internal control PCR.

Clinical records were surveyed for all patients and some data including age, gender and site of the lesion were considered. One hundred and six cases of non-genital seborrheic keratosis were collected. Then, their histological sections were reviewed by a dermatopathologist under the light microscopy. After revision of the sections and assurance of consistency, 50 seborrheic keratoses with a definite diagnosis of seborrheic keratosis and adequate amount of tissue were entered in our project. After proper age and sex matching, 50 case and 50 control samples were selected and their formalin-fixed and paraffin-embedded blocks were retrieved from the archive.

DNA preparation

Fifteen μm thick paraffin-embedded tissue sections were prepared (with a sterilized microtome) and 1-3 sections were considered for DNA extraction using commertial kit according to manufacturer's recommendations (TaKaRa, Kyoto, Japan).

Detection of HPV DNA

HPVs genome was detected by PCR. The primers Gp6+ (5′-GAAAAATAAACTGTAAATCATATTC-3′) and Gp5+ (5′-TTTGTTACTGTGGTAGATACTAC-3′) were used for amplification of a 142 bp fragment of HPVs L1 gene as described previously.[7] TA vector containing HPV L1 fragment was used as a positive control and distilled water was used as template in negative control tube. All samples were amplified in 15 μ1 of reaction mixture containing 1.5 μl of 10X PCR buffer, 1.5 mM MgCl₂, 0.3 μl dNTP (Fermentas Inc., Germany), 2.5 u Taq DNA polymerase (Cinagen, Iran), 0.3 μl of 10 pmol primersee 1 μl DNA (150 ng/μl). A 268 bp fragment of human β-globin gene was amplified by PCR using GH20 and PCO4 primers as an internal control.

The amplification program was the denaturation step 5 min in 94°C, 35 cycles of 94°C for 30 s, 55°C for 45 s, 72°C for 45 s, and a final 72°C elongation step for 5 min. The PCR product was visualized on a 2% agarose gel by Green-Viewer staining.

Simultaneously, all the histopathological sections of our 50 case subjects were evaluated histopathologically for changes regarding viral infections and to determine the type of lesion.

Statistical analysis

Data management and analysis was performed using SPSS 16 and statistical tests of Chi-square and Fisher exact test.

Results

The baseline characteristics for our case and control groups are as follows:

Out of 5485 skin biopsies, 106 were seborrheic keratosis (1.93%). A total of 100 biopsies, comprising 50 cases and their 50 age and sex matched controls were included in the study. For each set, the mean and standard deviation of age was 52 ± 1 years and ranged from 20 to 82 years. Each group consisted of 28 (56%) males and 22 (44%) females. The anatomic sites of non-genital seborrheic keratoses were the face, the scalp, the forehead, the extremities, the chest and the back.

In histopathologic evaluation of the samples for changes regarding viral infection, 32 (64%) had parakeratosis overlying papillary projections, 44 (88%) parakeratosis, 33 (66%) dyskeratotic cell, 18 (36%) hemorrhagic parakeratosis, 14 (28%) mitosis, 20 (40%) coarse keratohyalin granules and 5 (10%) koilocytosis. Frequency of SK types in non-genital SK was the acanthotic, irritated, hyperkeratotic, inverted follicular keratosis and the reticulated type, respectively.

Hundred samples comprising 50 cases and 50 controls submitted for detection of HPVs DNA by PCR were evaluated for the presence of β-globin DNA, [Figure 1]. The positive rates of the case and control groups for the 268 bp β-globin PCR; were respectively 32% and 30%, indicating sufficient DNA for gene amplification in these samples.

Figure 1.

The products of β-globin PCR in the case and control subjects. Lanes M is DNA size marker: Lanes 16 and 17 are positive and negative controls, respectively; Lanes 2, 3, 5, 6, 9, 10, 11, 13 and 15 are positive for β-globin PCR

After exclusion of β-globin-negative samples (to exclude false negativity), 31 subjects (16 cases, 15 controls) were included for the HPVs PCR using GP5+/GP6+ primer sets [Figure 2]. The HPVs DNA was not detected in any of the 16 case subjects. Of our 15 control subjects, one was positive for HPVs DNA by PCR. According to Fisher exact test, the difference between the two groups is considered statistically insignificant (P = 0.48).

Figure 2.

HPVs PCR results: The lane number 9 shows HPVs PCR positive result. The lanes 22 and 23 are positive controls and the lanes 20 and 21 are negative controls for HPVs PCR amplification. Lane M is DNA size marker

Discussion

The present study could not demonstrate HPVs genome in the non-genital SK tissue samples. This result is similar to the results of the following studies, which reported the absence of HPVs genome: Lee et al., reported that HPV types 6/11, 31, and 33 were not found in non-genital SK.[8] Also, 28 (96%) cases of non-genital SK did not contain HPVs DNA sequences in Zhu's study.[9] It is interesting to note that HPVs detection by IHC in all of the 31 inverted follicular keratoses (IFK) samples performed by Asadi-Amoli revealed negative result.[10] Moreover, in a case-control study conducted by Shih, negative PCR result was obtained in an exophytic nodule diagnosed as IFK.[11]

On the other hand, in contrast to our findings, eight reports suggested a possible role of HPVs in non-genital SK. Zhao et al., showed HPVs in four of 89 seborrheic keratoses by light microscopy and electron microscopy.[2] Gushi et al., detected HPVs in 95 of the 104 cases by PCR and Southern blot analysis.[4] Tsambaos et al., who used in situ hybridation, showed HPVs in 34 of 173 SK cases vs. none of 173 normal skin.[12] Li et al., showed epidermodysplasia verruciformis (EV) -associated HPVs DNA in 42 of 55 (76%) non-genital SK biopsies vs. only 13 of 48 (27%) healthy controls (P < 0.005).[6] Jeong et al. detected EV-associated HPVs DNA in 15 of 40 non-genital SK (37.5%) compared with 1 of 40 cutaneous squamous cell carcinomas (SCC) samples and 3 of 40 healthy controls.[3] Roncalli de Oliveira et al. suggested that HPVs infection may promote the occurrence of SK-like lesions in EV patients.[5] Jacyk et al. indicated the presence of DNA related to the HPV-5 group of papillomaviruses in seborrheic keratoses in three patients by using reverse blot hybridization.[13] In 2004, Bertoli et al., detected HPVs in lesions of a patient with dermatosis papulosa nigra.[14]

The negativity of HPVs PCR results could be due to one of the following reasons: Absence of association between HPVs and non-genital SK, and technical errors. We used very sensitive and specific methods to detect the tissue HPVs. We believe that our negative results are not due to technical errors for the following reasons: (1) PCR method used in this study is more sensitive than in situ hybridization; (2) TA vector containing HPV L1 fragment was used as a positive control in all HPVs PCRs and it showed the expected fragment size using DNA size marker. Also, the negative control (in which we added sterile purified water instead of sample DNA) was negative by the HPVs PCR. (we took great care during the experiments to prevent cross-contamination; all negative controls returned negative results); (3) Moreover, as short DNA fragments are useful as a template for PCR,[15] GP5+/6+ primer sets were chosen to increase the success rate. (The most widely used PCR methods for the detection of HPV types are based on MY09/11 (yielding a 450 bp fragment) and GP5+/6+ (yielding a 142 bp fragment) primer sets. All these primers, target the L1 region of the HPV genome. The HPVs PCR using GP5+/6+ primer sets seems to be more sensitive, more reliable and reproducible when compared to MY09/11.

As mentioned earlier, the quality of each DNA sample was verified by amplification of portions of ubiquitous human β-globin gene using a GH20/PCO4 primer pair that yielded a 268 bp fragment. Successful amplification of the β-globin gene fragment indicated the integrity of the DNA samples and the absence of PCR inhibitors. PCR amplifications that targeted portions of the HPV L1 region were then performed on all samples, using GP5+/GP6+ (yielding a 142 bp fragment).

A question that comes to mind is that why so many β-globin negative samples were reported?

In general, an adequate amount of tissue samples and undertaking a procedure to produce high-quality extracted DNA are important factors for obtaining successful results from PCR. DNA can be extracted from paraffin-embedded tissues, but this is usually unsuitable for most molecular techniques. This unsuitability could result from; (a) The fragmentation of nucleic acids:[16] (b) The degradation of DNA; and (c) The absence of a detectable amount of target DNA in the small tissue samples or biopsy specimens.[17] The fragmentation of nucleic acids is due to formalin fixation and the degradation of DNA is because of: The type of fixative solution used, the storage conditions, tissue thickness and the time required for fixation to complete, and The long time between surgical tissue removal and fixation.[18]

The degradation of DNA and the fragmentation of nucleic acids could not have been prevented because our study was a retrospective study and we used archival formalin-fixed paraffin-embedded tissues. However, all of the histological sections with adequate amount of tissue were chosen to assure the presence of enough human cells and therefore a detectable amount of target DNA. It can be concluded that regarding measures taken for gathering those histological sections with adequate amount of tissue and the optical density results that showed quantity of the isolated DNA, absence of human cells could not have been the culprit behind β-globin negative results and Probably the degradation of DNA and the fragmentation of nucleic acids were responsible. (Isolated DNA was quantitated by using UV absorbance measurements, taking into account an optical density conversion factor at 260 nm of 1 at 50 μg of DNA/ml).

Presence of inhibitors in the PCR reaction mixture could be another reason for the negative results but as the positive control showed DNA bands of the correct molecular weights, the role of these inhibitors in producing negative results is rejected. Moreover, as we used traditional kit for extracting DNA from paraffin-embedded tissues, probably the presence of inhibitors in purified DNA is not possible.

Decreased β-globin amplification rate from archival tissues stored for variable time periods was another possibility for the negative results. However, Park showed no decrease in the amplification rate with longer periods of storage.[19] Moreover, as the HPVs positive control subject in our study was one of the oldest, the role of longer periods of storage is ruled out.

Our results is the same as other studies which relate the disease to middle and old age and prove higher incidence in men and also in mentioning the face as the most common site for non-genital SK, indicating the possible role of UV light exposure.[3,6,10]

The results of this study indicate that koilocytosis, parakeratosis overlying papillary projections, hemorrhagic parakeratosis and coarse keratohyalin granules can be present in non-HPV associated SK. An implication of this is the possibility that these changes are not merely HPVs related. Moreover, out of 16 case subjects which were included for the GP PCR, only 2 (12.5%) had koilocytosis. Asadi's study yeilded 5 (16.1%) koilocytosis in the study subjects.[10] Therefore, it can be suggested that in this study, perinuclear vacuolization as was used by Zhu[9] is a better term compared to koilocytosis.

Conclusions

Thus, our findings can be in favor of the concept of less important role and low frequency of HPVs infection in non-genital SK in Iran. Obviously, other mechanisms like sun exposure are involved in formation of SK.

Several limitations to this study need to be acknowledged: Since some studies have reported some difficulties in reproducing PCR results with formalin-fixed paraffin-embedded tissues,[20] it is possible that if fresh tissues had been examined, HPVs positive cases might have been higher. Amplification step could be hampered by formalin. As we used archival formalin-fixed paraffin-embedded tissues, it was not possible to examine the patients for the presence of other HPVs related lesions to rule out surface contamination with HPVs.

Another study should be conducted comparing the association of HPVs DNA with SK in paraffin-embedded and fresh tissues. Other methods like real-time PCRs using specific primers and probes or microarrays which are more sensitive than the conventional PCRs are recommended for HPVs DNA detection in the samples. Forslund reported high prevalence of cutaneous HPVs DNA on the top of SKs but not in “stripped” biopsies of the lesion.[21] On the other hand, numerous studies performed in Eastern countries reported the presence of HPVs in SK.[3,4,5,6,7,13] It can be proposed that the positive results in these studies is due to the contamination of the lesion surface. To investigate this issue, patients should be tested for HPVs DNA in swab samples collected on top of SK and in biopsies of the same lesion, obtained after stripping with tape to remove superficial layers.

What is new?

Our findings can be in favor of the concept of less important role and low frequency of HPVs infection in non-genital SK in Iran.