Human papillomavirus-associated balanoposthitis – a marker for penile intraepithelial neoplasia?

Abstract

The purpose of this study was to analyse, among men treated with diathermy, whether there was a difference in balanoposthitis between men with histopathologically benign human papillomavirus–associated lesions and those with penile intraepithelial neoplasia. Data were derived from patient material from a previously published study. Two clinically identical lesions from the same genital site were collected for analysis with routine histopathology and with nested PCR. In total, 292 men were included, of which 47 (16%) had penile intraepithelial neoplasia. Of those with penile intraepithelial neoplasia, 19/47 (40%) reported problems consistent with balanoposthitis, compared with only 15/245 (6%) patients with benign lesions (p < 0.0001). A statistical difference in percentage distribution was also seen regarding morphology (p = 0.001) and location (p = 0.0003) of the lesions among the men having benign lesions with and without balanoposthitis. It is not possible to take biopsies from patients with genital warts, but this study suggests that one probably should be more observant for genital dysplasia among those men with warts with a history of balanoposthitis than those with no such history.

Keywords

Balanoposthitis balanitis condyloma genital warts human papillomavirus HPV nested PCR penile intraepithelial neoplasia PIN penile cancer sexually transmitted infection

Introduction

Genital human papillomavirus (HPV) infection can present diverse clinical conditions. Lesions can demonstrate acuminate, papular, macular or seborrhoeic keratosis-like characteristics. Symptoms are also variable; some patients are undisturbed by the warts, some are cosmetically disturbed and others experience symptoms such as itching.¹ About 40 HPV types are considered to be mucosal and are further classified into low- and high-risk types. The classic low-risk HPV types are HPV 6 and 11, probably accounting for up to 90% of genital warts.² High-risk types, such as HPV 16 and 18, have been described as important for development of genital dysplasia, predominantly studied on the cervix uteri (cervical intraepithelial neoplasia, CIN). However, HPV-associated dysplastic lesions on the vulva (VIN), penis (PIN) and anus (AIN) have also been described.³

Balanitis is an inflammatory response of the glans penis, while posthitis is an inflammation of the inner aspect of the foreskin. In uncircumcised men, usually both the glans and the foreskin are involved, thus “balanoposthitis”. Balanoposthitis represents a complex clinical entity. Some forms are associated with microbiological agents like candida and bacteria. Other kinds of balanoposthitis, such as plasma cell, circinate and non-specific balanoposthitis, are of unknown etiology.⁴ A large proportion of the male population will probably experience brief courses of balanoposthitis during their life-time. The most common form of balanoposthitis is the non-specific type. The prevalence of balanoposthitis in the male population is unknown, but it is no doubt that this disease is a common reason for men to attend an STI clinic.⁴ Other causes of genital skin problems are the genital dermatoses such as lichen planus and lichen sclerosus, which present clinically with symptoms similar to balanoposthitis.

HPV-associated balanoposthitis has previously been described.⁵ The condition manifests as inflammation of the penile skin prior to appearance of visible HPV-associated lesions or simultaneously with the lesions and is clinically a form of non-specific balanoposthitis. Symptoms include burning, redness, tenderness, itching and dyspareunia. Clinical indications include erythema, fissures and oedema. Treatment of the condition is difficult. Local steroid creams, sometimes combined with antifungal and antibacterial components, are recommended as symptomatic therapy. The HPV-associated lesions are often treated by surgery, sometimes by using penoscopy in order to visualize the lesions.⁶ However, the condition might be long-lasting and in some cases cure is not accomplished until the HPV infection is completely eradicated. The purpose of this study was to determine whether incidence of balanoposthitis differs between men with histopathologically benign and dysplastic lesions. Baseline data of the HPV-associated lesions in men with PIN and in those with benign lesions has previously been described in two separate papers,^7,8 in which the aspect of balanoposthitis has not been illuminated.

Methods

Patients

Between 2004 and 2007, male patients attending the STI clinic at Karolinska Hospital, with clinical genital HPV infections planned for surgical treatment, were asked to participate in the study, as already described.^7,8 Diathermy was the surgical method used for all patients. The men were seen mainly by one doctor (AW). All men exhibited multiple lesions (those with solitary lesions were excluded). Two clinically identical lesions from the same genital site were collected with punch biopsy or by scissor excision. One sample was put in formalin for routine histopathological preparation and evaluation, and the other was frozen at −70℃ for PCR analysis. The macroscopic morphology of the lesions was classified into acuminate, papular, macular and seborrhoeic keratosis-like. Colour and location were also recorded. Data on genital symptoms and/or signs in agreement with balanoposthitis (such as itching, redness and fissures) at the first visit were retrospectively evaluated in the patient files. Sexual orientation was asked for and circumcision status was noted. The men with PIN were offered an HIV test.

Histopathological evaluation

Biopsies were prepared according to the routine at the histopathological laboratory according to standard routines and stained with haematoxylin-eosin. The lesions were categorized as benign or as dysplastic (PIN) according to established criteria as previously described.^7,8 Several sections were made from each biopsy, for thorough analysis and observation. In order to estimate histopathological signs of balanoposthitis, all biopsies from the men with PIN were reviewed, and the dermal inflammatory infiltrates were graded as light, moderate and heavy.

DNA extraction

Frozen biopsy samples were prepared. DNA was extracted using the high-salt method.⁹ The samples were lysed with proteinase K at 37℃ overnight. The proteins were then precipitated with saturated 6M NaCl, followed by centrifugation. After centrifugation, the supernatant was removed and DNA was precipitated with absolute ethanol. The DNA pellet was dissolved in sterile water and stored at −20℃.

PCR

HPV DNA was detected with nested PCR using MY09/MY11 as external primers and GP05 + bio-GP06+ as internal primers, targeting the L1 open-reading frame of the HPV genome. PCR was conducted in a 50-µl reaction containing PCR buffer, 20 pmol of each primer, 200 µM each of deoxynucleotide triphosphate and 1.25 U AmpliTaq Gold^R DNA polymerase (Perkin Elmer, New Jersey, USA) and 300 ng of DNA. Amplification started with initial denaturation at 95℃ for 10 min followed by 30 cycles of denaturation at 95℃ for 30 s, annealing at 55℃ for 55 s and elongation at 72℃ for 60 s. One µl of the PCR product was taken for the second PCR and biotinylation with the internal primer pair of GP05 + bio-GP06+. The cycle consisted of denaturation at 95℃ for 60 s, annealing at 40℃ for 60 s and extension at 72℃ for 90 s. A total of 40 cycles were performed. Good laboratory practice was adhered to during DNA extraction and PCR experiments.

HPV genotyping

After HPV amplification, the PCR products were HPV-genotyped with hybridization to fluorescence-labeled polystyrene bead microarrays (Luminex suspension array technology) using the Multimetrix kit (Progen Biotechnik GmbH, Heidelberg, Germany). The assay can detect the following 24 HPV types: low-risk HPV: 6, 11, 42, 43, 44 and 70; high-risk HPV: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82. At the final step, the beads were washed three times with 100 µl blocking buffer and finally re-suspended in 100 µl blocking buffer for 5 min on a shaker. They were then analyzed for internal bead colour and R-phycoerythrin reporter fluorescence on a Luminex 100 analyzer.¹⁰ The median reporter fluorescence intensity (MFI) of at least 100 beads was computed for each bead set in the sample. The cutoff value was defined for each HPV probe individually as follows: 1.5 times the background MFI + 5MFI.

Statistical analyses

Fisher’s exact test was used to investigate whether there was a difference in the proportion of incident balanoposthitis between subjects with PIN and those with benign lesions and to evaluate whether the percentage distribution of the clinical parameters (morphology, colour and location) differed for groups with or without a history of balanoposthitis among those with PIN and benign lesions. Fisher’s exact test was also used to investigate whether the percentage distribution of inflammatory infiltrates (mild, moderate and heavy) differed between men with PIN with and without a history of balanoposthitis. For this parameter the Cochran-Armitage trend test was used in order to see differences in severity of the inflammatory infiltrates between these men with and without balanoposthitis.

Results

A total of 303 men were included in the original study, which previously has been described^7,8; in 11 men with benign lesions, patient files were lacking, and these men were excluded from the analysis. Of 292 men included in this study, 47 (16%) had PIN. Of the men with PIN, 19/47 (40%) had problems consistent with balanoposthitis, compared to only 15/245 (6%) of patients with benign lesions (p < 0.0001). In the group of men with benign lesions, 7/245 men also had a history of genital skin problems (five cases with lichen sclerosus and two men with circinate balanitis), which were not included in the group of patients with HPV-associated balanoposthitis. All of the men with a history of balanoposthitis were heterosexual, and only 1/34 was circumcised (one man with PIN II), who due to the lack of a foreskin had balanitis (only involvement of the glans). HIV tests were performed in 17/19 of the men with PIN, and all were HIV negative.

Table 1 describes the morphology and location of the lesions as well as dysplastic grade for those subjects with PIN. We found no significant difference for the various parameters between the men with or without balanoposthitis; however there was a non-significant trend for more balanoposthitis with advanced grades of PIN. Table 2 describes the same parameters for the men with benign lesions. A significant difference in percentage distribution for the morphology was seen (p = 0.001). Macular lesions were the most common lesions for the men with balanoposthitis, and acuminate warts were most common in the group without this condition. There was also a significant difference in percentage distribution for location (p = 0.0003). The foreskin was the most common location for men with balanoposthitis, whereas the penile shaft and the pubis region were most common for those without balanoposthitis. However, of the men with balanoposthitis, 7/19 with PIN and 2/15 with benign lesions had their HPV-associated lesions on other genital parts than the foreskin and the glans.

Table 1.

Balanoposthitis among men with PIN. Morphology, location of lesions and PIN grade (n = 47).

Balanoposthitis (n = 19)	Yes	No
Morphology of lesions:
Acuminate (n = 6)	1	5
Papular (n = 13)	4	9
Macular (n = 27)	13	14
Seborrhoeic keratosis-like (n = 1)	1	0
Location of lesions:
Foreskin (n = 23)	11	12
Glans (n = 3)	1	2
Penile shaft (n = 16)	5	11
Pubis (n = 5)	2	3
PIN grade:
PIN I (n = 19)	5	14
PIN II (n = 13)	6	7
PIN III (n = 15)	8	7

PIN: penile intraepithelial neoplasia.

Table 2.

Balanoposthitis among men with histopathologically benign lesions. Morphology and location of lesions (n = 245).

Balanoposthitis (n = 15)	Yes	No
Morphology of lesions:
Acuminate (n = 106)	4	102
Papular (n = 83)	2	81
Macular (n = 41)	9	32
Seborrhoeic keratosis-like (n = 15)	0	15
Location of lesions:
Foreskin (n = 60)	12	48
Glans (n = 5)	1	4
Penile shaft (n = 90)	2	88
Pubis (n = 70)	0	70
Perianal (n = 11)	0	11
Scrotum (n = 5)	0	5
Groin (n = 2)	0	2
Perineum (n = 1)	0	1
Location unknown (n = 1)	0	1

In Table 3, the HPV type distribution is shown. HPV 16 and HPV 6 were the most common HPV types in PIN and in benign lesions, respectively. In two and three cases of PIN and benign lesions, respectively, there was a mix of benign and malignant HPV types. Table 4 demonstrates the histopathological evaluation of men with PIN, by grade and balanoposthitis status. No significant differences were found.

Table 3.

Patients with HPV-associated balanoposthitis. HPV types in PIN (n = 19) and in benign lesions (n = 15).

HPV type(s)	PIN lesions (and PIN grade)	Benign lesions
6	4 (1 PIN I, 2 PIN II, 1 PIN III)	7
11	1
16	6 (1 PIN I, 1 PIN II, 4 PIN III)	1
31	1 (PIN II)
33	1 (PIN II)
45	1 (PIN III)
70	1 (PIN III)
73	1 (PIN I)
6,16,66	1
6,16,52,56,73	1
16,42,58	1 (PIN I)
42,53	1 (PIN I)	1
HPV negative	2 (1 PIN II, 1 PIN III)	2
HPV test not done	1

HPV: human papillomavirus; PIN: penile intraepithelial neoplasia.

Table 4.

Histopathological evaluation of inflammatory infiltrates in PIN patients (n = 47) with and without balanoposthitis.

	Balanoposthitis
	Yes (n = 19)	No (n = 28)
Inflammatory infiltrate
Light	1 (1 PIN III)	6 (3 PIN I, 2 PIN II, 1 PIN III)
Moderate	7 (2 PIN I, 1 PIN II, 4 PIN III)	11 (6 PIN I, 3 PIN II; 2 PIN III)
Heavy	11 (3 PIN I, 4 PIN II, 4 PIN III)	11 (5 PIN I, 3 PIN II, 3 PIN III)

PIN: penile intraepithelial neoplasia.

Discussion

In this study, we analysed how many men with genital warts who at their first visit to the STI clinic at Karolinska Hospital had problems with balanoposthitis. Patient files from a group of patients with genital warts who required surgical treatment (diathermy) were retrospectively recorded. Some other data about these men have already been published.^7,8

This study has some limitations. Balanoposthitis is usually fluctuating, the signs and symptoms come and go, thus the number of cases may be underestimated. Lack of accurate recall is another limitation, thus genital symptoms may be under-reported. In such cases, the skin problems were probably very mild, self-healing and did not affect the patient. Generally, at the consultation, enough time is given for the patients to ventilate their concerns and to ask questions, so we believe that the risk of under-reporting is limited. Compared to our previous studies,^5,6 the description of lesions is less complete. However, as the main purpose of this study was not the aspect of balanoposthitis; we have no penoscopic evaluation and no grading of the inflammatory changes by naked eye examination. The inflammatory infiltrates from the biopsies in the PIN patients with and without balanoposthitis have been graded. No significant differences were found between the two groups although there was a tendency whereby heavy inflammatory infiltrates were more commonly found among patients with balanoposthitis (occurring in 11/19 men with balanoposthitis compared to 11/28 without balanoposthitis). HPV infection itself always shows a varying degree of dermal inflammation, which might be associated with regression of lesions,^11,12 and therefore the presence of inflammatory cells also among patients without balanoposthitis was expected. The collection of samples might be another limitation. Instead of splitting one lesion into two parts, two different samples from two clinically identical lesions were taken from each patient, one for routine histopathology and one for HPV typing. The lesions are sometimes quite small and might be difficult to prepare; sectioning for histopathology is difficult for small lesions. By taking two clinically identical lesions from the same site, we have reduced the risk of taking lesions induced by different HPV types, a possibility that unfortunately cannot be fully excluded. In cases of mixed lesions in the individual patient, we chose the type of lesions that were most common.

HPV-associated balanoposthitis is, in our experience, an important clinical problem that might be long-lasting. It is associated with redness, itching and fissures that cause dyspareunia, and some men are unable to have sexual intercourse for long periods of time.^5,13 Balanoposthitis might also be the first symptom of a genital HPV infection and is sometimes the reason for HPV-infected men to attend the clinic, usually due to problems affecting their sexual life. Some men, especially those with flat lesions, might not have noticed the HPV lesions themselves. Of our patients with PIN, 19/47 had balanoposthitis. There was a tendency that the condition seemed to be more common with advanced PIN grades, but the difference was not statistically significant. In contrast, only 15/245 patients with benign genital warts had balanoposthitis. The seven men with previously diagnosed genital dermatoses (lichen sclerosus and circinate balanitis) were not included in the group of men with HPV balanoposthitis, but it cannot be fully excluded that they also suffered from this since the symptoms might be similar. However, inclusion of these subjects would not abrogate the significance of the difference between men with PIN and those with benign lesions. As expected, balanoposthitis was more common if the HPV lesions were located on the foreskin. However, some men with lesions on other parts of the penis also experienced problems with redness and fissures of the foreskin. In our previous study, the mean duration of balanoposthitis was 11 months (range 0–48).⁵ Since exploring this aspect of balanoposthitis was not the main purpose when we started this study, we do not have data for the duration of genital skin problems among these men.

Balanoposthitis in association with HPV infection is probably due to an HPV-induced lymphocyte inflammatory response in the dermis, usually representing a good prognostic marker for immunologically-induced elimination of latent virus as has previously been shown for skin warts.^11,12 It is currently unclear why there was a significant difference between balanoposthitis in subjects with PIN and those with benign lesions. It is feasible that the oncogenic viruses tend to induce a stronger inflammatory response than the benign HPV types. On the other hand, avoiding rather than inhibiting the immune response is considered the key mechanism for HPV persistence.¹⁴ The role of inflammation in HPV infection is complex since it involves mechanisms capable of preventing infection, clearing ongoing infection as well as leading to persistence and progression.¹⁵ To our knowledge, no studies exist that from an immunological point of view can explain our findings. To further elucidate the role of balanoposthitis in relation to genital HPV infection, prospective studies are needed.

Ethics approval

Ethics approval was obtained by the Ethics committee of the Karolinska Institute. Patient consent was also approved.

Contribution

AW initiated the study, saw the patients and took the biopsies. M-AH performed the histopathology and SS did the PCR tests.

Footnotes

Conflict of interest

The authors declare no conflict of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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