Verrucous pilar cysts infected with beta human papillomavirus

Abstract

Epidermoid cysts with histologic features of human papillomavirus (HPV) infection have been previously reported and are commonly termed verrucous cysts. We report a series of eight histologically distinct verrucous pilar cysts, distinguished from traditional verrucous epidermoid cysts by trichilemmal keratinization, as well as two verrucous hybrid pilar-epidermoid cysts. These lesions contain characteristic stratified epithelial linings with abrupt transitions to compact eosinophilic keratin, as well as areas of papillomatosis, coarse intracytoplasmic keratohyalin granules, and vacuolar structures suggestive of HPV-induced cytopathic change. HPV-24, a β genus HPV species, was identified by degenerate PCR in DNA extracted from two of the lesions, and the presence of β-HPV E4 protein was confirmed by immunohistochemistry. HPV-60, the HPV species most commonly reported in verrucous epidermoid cysts, was not detected. Verrucous pilar cysts represent histologically and potentially etiologically distinct lesions which may be underrecognized.

Introduction

Plantar and non-plantar epidermoid cysts with histologic features of human papillomavirus (HPV) infection have been reported and are commonly termed verrucous cysts.¹ Here, we report several examples of distinct pilar verrucous cysts distinguished from traditional epidermoid verrucous cysts by trichilemmal keratinization. Two of the pilar and hybrid pilar-epidermoid verrucous cysts demonstrated evidence of infection with HPV-24 by both PCR and immunohistochemistry.

Case Reports

The database of recent cases from a large commercial dermatopathology laboratory was queried for “verrucous cyst” using a natural language search, and the identified cases were reviewed. This review identified eight cysts with stratified epithelial linings and abrupt transitions to compact keratin consistent with pilar cysts. Areas of the cyst linings additionally demonstrated papillomatosis, coarse intracytoplasmic keratohyalin granules, and vacuolar structures suggestive of HPV-induced cytopathic change (Figure 1A, D–F). In addition, two cysts with histologic evidence of HPV-induced cytopathic change and areas of both abrupt transition to compact keratin and stratified squamous lining with a granular layer transitioning to loose keratin consistent with hybrid pilar-epidermoid cysts were identified (Figure 1B and C).

Figure 1. Verrucous pilar cysts.

A-F, H&E stained biopsy sections demonstrating abrupt transitions from the stratified epithelial lining to compact keratin consistent with pilar cysts, along with papillomatosis, coarse intracytoplasmic keratohyalin granules, and vacuolar structures consistent with HPV-induced cytopathic change. B and C, Some cysts also demonstrated areas of stratified squamous lining transitioning to loose keratin consistent with hybrid pilar-epidermoid cysts. A-C, 40× original magnification. D-F, 100× original magnification.

The identified cases were evenly split between men and women, with patient age ranging from 19 to 70 years-old, and a wide range of non-acral anatomical sites (Table 1). All but one were initially reported with a diagnosis of verrucous cyst. Combined or hybrid pilar features were reported for three of the lesions. In-situ hybridization for common low- and high-risk HPV types (6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, and 68) was negative, as was PCR amplification of extracted DNA with HPV-60-specific primers.² However, PCR with degenerate primers targeting the L1 gene in a broad spectrum of HPV types was positive in two cases (Figure 2).³ Sequencing of the amplicons demonstrated 93% to 96% identity to HPV-24. Immunohistochemistry with monoclonal antibodies against a conserved epitope in the β-HPV E4 protein (Doorbar, unpublished) was performed on all cases. In the two cases positive for HPV-24 by PCR, as well as one additional hybrid cyst which was negative by PCR, strong cytoplasmic staining was apparent in dyskeratotic and vacuolated cells within the cyst cavity, as expected for E4 (Figure 3A–C). Staining was absent in the other cases (Figure 3D).

Table 1.

Verrucous pilar and hybrid pilar-epidermoid cysts

Case	Cyst Type	Age	Sex	Location	Initial Diagnosis	Clinical Impression	PCR	IHC
1	Hybrid	57	Female	Left mid back	Combined epidermoid and verrucous cyst	r/o BCC vs. EIC	-	β-HPV E4
2	Pilar	30	Male	Left upper forehead	Verrucous cyst	r/o cyst	-	-
3	Pilar	52	Male	Left distal pretibial region	Verrucous cyst	NUB vs. DN vs. DF	-	-
4	Pilar	52	Male	Right lateral back	Combined pilar and verrucous cyst	Inflammatory papule r/o cyst	HPV-24	β-HPV E4
5	Pilar	70	Female	Posterior scalp	Verrucous cyst	NUB vs. BCC	-	-
6	Hybrid	62	Female	Right preauricular area	Hybrid epidermoid and pilar cyst	r/o EIC	HPV-24	β-HPV E4
7	Pilar	57	Male	Right under arm	Ruptured combined pilar and verrucous cyst with granulomatous dermatitis	Sebaceous cyst	-	-
8	Pilar	66	Male	Left mid back	Verrucous cyst Superimposed prurigo changes The lesion extends to the deep margin	r/o ISK	-	-
9	Pilar	57	Female	Right thigh	Verrucous cyst, ruptured	EIC vs. DF vs. DFSP vs. MCC vs. other	-	-
10	Pilar	19	Female	Right distal pretibial region	Verrucous cyst	NUB vs. cyst	-	-

Abbreviations: BCC, basal cell carcinoma; DF, dermatofibroma; DFSP, dermatofibrosarcoma protuberans; DN, dysplastic nevus; EIC, epidermal inclusion cyst; HPV, human papillomavirus; IHC, immunohistochemistry; ISK, inflamed seborrheic keratosis; MCC, Merkel cell carcinoma; NUB, neoplasm of uncertain behavior; PCR, polymerase chain reaction.

Figure 2. Detection of human papillomavirus DNA in verrucous pilar cysts by polymerase chain reaction.

DNA was isolated from formalin-fixed paraffin-imbedded tissue using Maxwell 16 FFPE Plus LEV purification kits (Promega, Madison, WI). Amplification was performed by polymerase chain reaction using degenerate primers targeting the L1 gene of a broad subset of human papillomavirus (HPV) species.³ PCR products were run on an agarose gel with ethidium bromide. The predicted amplicon size is 480 base pairs. P, control Pap smear with high-risk HPV infection; C, control condyloma acuminatum; VV, control verruca vulgaris; VP, control verruca plantaris; 1–10, cases; X, gel loading error.

Figure 3. Detection of human papillomavirus E4 protein in verrucous pilar cysts by immunohistochemistry.

A-C, Monoclonal antibodies against a conserved epitope in the β-HPV E4 protein highlights intracytoplasmic inclusions in dyskeratotic and vacuolated cells within the cyst cavity. D, Example of negative staining with the same antibody. A and D, 100× original magnification. B and C, 400× original magnification.

Discussion

As far as we are aware, only a single HPV-infected pilar cyst has been previously reported, and the specific HPV type infecting the cyst was not identified.⁴ In contrast, most verrucous cysts reported to date have demonstrated epidermoid features, including an attenuated stratified squamous epithelial lining with a granular layer and loose keratin in the cyst lumen (Table 2).^1,5–8 Nevertheless, given the relative ease with which we were able to identify additional pilar cysts with verrucous features, we suspect this phenomenon may be more common than the paucity of reports would suggest. The majority of cases reported here were initially diagnosed as verrucous cysts, without distinction between epidermoid and pilar features. This may be in part because cytopathic changes induced by HPV infection can complicate identification of cyst types. For example, in conjunction with verrucous projections in these pilar cysts, there is often hypergranulosis, a feature more typical of epidermoid cysts. However, careful examination of the lining cells and the presence of thick eosinophilic compact keratin in the cyst lumen should allow for the correct diagnosis.

Table 2.

Reports of verrucous cysts

Reference	Age/Sex	Site	Histology	HPV Type	Detection Method	Additional Notes
Ashida M et al28	50/M	Extremities	Verrucae, some with cystic changes	3, 27, 60	IHC, Southern blot
Egawa K et al11	2–84	Palmoplantar surface	Epidermoid cysts	60	IHC, ISH	47/119 positive
Egawa K et al12	23/M	Right sole	Epidermoid cyst	57	PCR, ISH
Egawa K et al5	8–61	Plantar surface	Epidermoid cysts	n.d.	IHC, EM	11/14 positive
Elston DM et al7	-	Scalp	Epidermoid cyst	n.d.	IHC
Honda A et al29	50/M	Hand, arm, leg	Plantar cyst	60	IHC, ISH, Southern blot
Kashima M et al14	21/F	Plantar surfaces	Epidermoid cyst	57	ISH
Kato N30	-	Plantar surface	Epidermoid cyst	60	IHC	2 cases
Kawasa M et al10	-	Plantar surfaces	Epidermoid cysts	60	ISH	6/10 cases positive
Kawase et al15	31/M	Scrotum	Epidermoid cyst	6, 11	IHC, ISH
Kim H et al13	28/M	Right flank	Epidermoid cyst	59	IHC, PCR
Kitasato H et al31	-	Plantar surface	Epidermoid cysts	57	PCR, Southern blot	3/18 cases positive
Lee S et al2	-	Palmoplanatar and non-palmoplantar	Epidermoid cysts	60	PCR with sequencing	9/63 cases positive
Meyer LM et al1	29–59	Neck, eyebrow, forearm, upper back	Epidermoid cysts	n.d.	PCR	5 cases
Misago4	52/F	Right thigh	Pilar cyst	n.d.	IHC
Park HS et al32	-	Palmoplantar surfaces	Epidermoid cysts	60	PCR, IHC	4/13 cases positive
Pusiol T et al16	86/F	Perianal	Invasive squamous cell carcinoma arising from a verrucous cyst	16	IHC, ISH, PCR
Ramagosa R33	75/M	Chest	Epidermoid cyst	6, 8	PCR
Rios-Buceta LM et al6	53/F	Right plantar foot	Epidermoid cyst	n.d.	IHC
Sato Y et al8	16/F	Plantar surface	Epidermoid cyst	n.d.	IHC
Soyer HP et al18	37–82	Face, extremities	Epidermoid cysts	n.d.	PCR	5 cases
Terada T34	41/M	Neck	Epidermoid cyst with features of seborrheic keratosis	n.d.	IHC	SK like area was positive for HPV
This study	19–66	Various	Pilar (8) and Pilar-epidermoid hybrid (2) cysts	24	PCR with sequencing, ISH, IHC	2/10 positive by PCR 3/10 positive by IHC

Abbreviations: EM, electron microscopy; HPV, human papillomavirus; IHC, immunohistochemistry; ISH, in-situ hybridization; PCR, polymerase chain reaction; SK, seborrheic keratosis

A substantial fraction of reported verrucous epidermoid cysts have occurred at pressure points on the plantar surface,^5,6,8 although HPV-infected epidermoid cysts of other locations have been reported as well.^1,7 Verrucous epidermoid cysts are usually solitary and sometimes co-occur with non-infected cysts.⁷ When a specific HPV type is identified, HPV-60 is most common, especially in plantar cysts.^9–11 Other HPV types reported to infect cysts include HPV-57 and HPV-59.^12–14 HPV-6 and HPV-11, which typically infect genital and mucosal surfaces, were detected in one scrotal epidermoid cyst.¹⁵ In general, cysts have not been found to contain high-risk HPV types, although squamous cell carcinoma arising from a HPV-16-infected cyst has been reported.¹⁶ In many reports of verrucous epidermoid cysts, no specific HPV type was identified, particularly for lesions on non-plantar skin.^1,4,7,17,18

Two of the verrucous pilar cysts presented here demonstrated evidence of infection with HPV-24, a β genus HPV. HPV-24 is among the HPV species associated with epidermodysplasia verruciformis (EV), a genetic or acquired skin disease characterized by multiple pre-malignant verrucous papules and macules caused by chronic HPV infection.¹⁹ Genetic EV typically results from loss of function mutations in EVER1, EVER2, or CIB1,^20,21 while acquired EV may result from iatrogenic immunosuppression in organ transplant patients, HIV infection, or other medical conditions leading to compromised immune function. In fact, ruptured epidermoid cysts infected with HPV-24, as well as EV-associated HPV-20 and HPV-80, have been reported in one patient with idiopathic CD4 lymphopenia and chronic Mycobacterium avium intracellulare infection.²² In contrast, β HPV species typically produce asymptomatic infection in immunocompetent patients.²³ None of the patients in this series had a known history of immunosuppression. Furthermore, none of the lesions demonstrated the characteristic histological features of EV, including large keratinocytes with blue-grey cytoplasm and mild cytologic atypia.

In the majority of the verrucous pilar cysts in this series, HPV DNA was not detected by ISH or PCR. However, these methods are limited by their lack of universal sensitivity across all HPV types. In particular, the ISH cocktails used are designed to detect HPV species associated with condyloma acuminata or lesions at high risk for malignant transformation, not HPV species associated with common warts. While the PCR primer sets used are predicted to have broader sensitivity based on their degenerate sequences, their sensitivity spectrum in practice is not completely defined. Several prior reports failed to directly detect either HPV DNA or protein in at least some histologically characteristic verrucous epidermoid or plantar cysts.^2,10,11,17 Given the number of HPV species and the inherent limitations of the available detection methods for HPV, the histologically verrucous pilar cysts negative for HPV DNA by ISH and PCR in this series were most likely infected with HPV types not targeted by the probes and primers used.

This report identifies verrucous pilar cysts histologically distinct from verrucous epidermoid cysts reported previously. Unlike verrucous plantar cysts, HPV-60 was not found in any of the verrucous pilar cysts. Given the absence of follicles, pilar cysts do not occur on plantar surfaces, suggesting distinct etiologies for verrucous pilar cyst and verrucous plantar cyst formation in addition to infection with different HPV species. Verrucous epidermoid cysts have been reported on non-plantar locations, but given the relatively small number of reports in which specific HPV types have been identified, potential differences in HPV infection patterns between verrucous pilar and non-plantar epidermoid cysts remain unknown. It is possible that distinct HPV types isolated from pilar and epidermoid cysts reflect the topographic infection preferences of specific HPV types, as has been reported for bacteria and fungi.²⁴ Specifically, while HPV-24 has been identified as a commensal resident of hair follicles,²⁵ HPV-57 and −60 have been identified in non-follicular epithelia from glabrous skin.^26,27 It remains unclear whether HPV infection of the follicular epithelium drives the formation of verrucous pilar cysts or whether verrucous pilar cysts represent secondary or passenger HPV infection of existing cyst epithelia. Nevertheless, verrucous pilar cysts clearly represent histologically and potentially etiologically distinct lesions which are likely significantly underreported.