Recent advances in cutaneous HPV infection

More than 200 types of human papillomavirus (HPV) have been reported to date and have been associated with various dermatological diseases. Among dermatological diseases, viral verrucae are the most commonly reported to be associated with HPV. Epidermodysplasia verruciformis (EV) consists of three types: typical EV is an autosomal recessive genetic disorder with TMC6/TMC8 gene mutations, atypical EV develops due to various gene mutations that cause immunodeficiency, and acquired EV develops due to acquired immunodeficiency. Generalized verrucosis differs from EV in that it involves numerous verrucous nodules (mainly on the limbs), histopathologically no blue cells as seen in EV, and infection with cutaneous α‐HPVs as well as β‐HPVs. HPV‐induced skin malignancies include squamous cell carcinoma (SCC) caused by β‐HPV (especially HPV types 5 and 8) in EV patients, organ transplant recipients, and healthy individuals, and SCC of the vulva and nail unit caused by mucosal high‐risk HPV infection. Carcinogenesis of β‐HPV is associated with sunlight. Mucosal high‐risk HPV‐associated carcinomas may also be sexually transmitted. We focused on Bowen's disease of the nail, which has been the subject of our research for a long time and has recently come to the fore in the field of dermatology.


| INTRODUC TI ON
Human papillomavirus (HPV) is a well-known causative virus of cervical cancer, with more than 200 types reported thus far, and the number is still increasing (International HPV Reference Center, http://www.hpvce nter.se/). It is a double-stranded circular DNA virus consisting of E1, E2, E4, E5, E6, E7, L1, and L2 genes and infects the skin basal cells through minute wounds.
HPV types correlate very well with the clinical presentation of skin disease. We herein review the HPVs associated with skin tumors, especially generalized warts, and the mechanism underlying skin cancer development caused by HPVs.

| HPV detection methods
For HPV typing, the author basically extracts DNA from paraffin blocks using Promega's Maxwell 16 Tissue DNA Purification Kit (Promega Corporation) and subjects it to polymerase chain reaction (PCR). HPV is difficult to detect with a single primer, and the author presently uses the SK1/2 primer for cutaneous warts 1 and L1C1/L1C2 primers for malignant tumors. 2 GP5+/GP6+ 3 and CP primers 4 are used for both benign and malignant skin tumors.
PCR products are used for typing by direct sequencing. HPV Monoclonal Antibody (K1H8) (Thermo Fisher Scientific) is used for HPV immunostaining. This antibody is against HPV type 1, and according to the author's experience, both benign and malignant types can be detected. HPV RNA expression can be confirmed by RNA in situ hybridization (RNAscope; Advanced Cell Diagnostics, Inc.). HPV virus particles can be observed by electron microscopy using paraffin-embedded section combined with immunostaining and in situ hybridization.

| PV and animal experiments
Animal models of HPV have been difficult to develop due to its species specificity. However, with the recent publication of PVs that infect rodents, promising animal models are now available. PVs infecting rodents, such as MnPV1 and McPV2 detected in African multimammate rats, called Mastomys; MaPV1 in Syrian golden hamsters; MmiPV in European harvest mice; RnPV1 in Norway rats; and EdPV1 in North American porcupines have been reported and used in research. 5 The recently discovered MmuPV1 is a PV found in the mouth and perinasal papillomas of mice in India and has been analyzed in detail. 6 This virus, originally called MusPV, is now known as MmuPV1 and specifically infects mouse skin and has a structure similar to carcinogenic β-HPV. 7 Further analyses using these animal models are expected to be performed in the future.

| Viral warts and HPV
Among dermatological diseases, warts are the most commonly reported to be associated with HPV. Typical diseases and HPVs detected in them are summarized below.

| Verruca vulgaris
Verruca vulgaris is a keratotic nodule that occurs mainly on the hands and feet, especially on the soles of the feet, where it can develop into mosaic warts, and is often difficult to treat. Those that proliferate externally on the face are called filiform warts. Verruca vulgaris is caused by infection with HPV types 2a/27/57, among others.

| Myrmecia
Myrmecia is an HPV type 1a infection that occurs on the soles of the feet and other parts of the body in children and may be accompanied by pain.

| Flat warts
Flat warts (HPV types 3/10) are common on the face, dorsum of the hands, and forearms. They are flat, raised, normal skin-colored to brownish nodules with a tendency to fuse. Rarely, atypical eruptions may occur in immunosuppressed or other conditions, and a biopsy may be necessary.

| Condyloma acuminatum
Condyloma acuminatum is a sexually transmitted infection of the vulva, in which HPV types 6/11 is detected. Histopathologically, characteristic koilocytosis is seen. Seborrheic keratosis and Bowenoid papulosis (HPV type 16 and others) must be differentiated. A biopsy is sometimes required.

| Epidermodysplasia verruciformis and β -HPV infection
Epidermodysplasia verruciformis is an autosomal recessive genetic disorder that can be divided into three types: typical EV, caused by mutations in EVER1 and EVER2 proteins; atypical EV, caused by various genetic mutations that result in immunodeficiency; and acquired EV, caused by an acquired immunodeficiency state. Recently, a new mutation in typical EV, CIB1, was discovered. 8 The TMC6 gene, TMC8 gene, and CIB1 gene encode EVER1, EVER2, and CIB1 proteins, respectively, and are thought to form a complex. 8 Atypical EV presents with skin lesions similar to those of typical EV but are associated with a variety of infections from early childhood, and all have congenital immunodeficiency of the T-cell lineage. 9 Genetic mutations associated with atypical EV have been identified as RHOH, 10 STK4, 11 CORO1A, 12 DCLRE1C, 13 DOCK8, 14,15 RASGRP1, 16 LCK, 17 and TPP2. 18 Acquired EV is divided into HIV-associated 19 and iatrogenic EV. 20 Epidermodysplasia verruciformis presents with a characteristic skin rash, tinea versicolor, flat wart-like skin rash, seborrheic keratosis-like skin rash due to persistent β-HPV infection, and squamous cell carcinoma (SCC) and other lesions appear in the exposed areas after 20-30 years old. As mentioned above, many EVs are caused by mutations in the TMC6, TMC8, and CIB1 genes. Unlike HPV types 2a/27/57, which cause verruca vulgaris, and HPV types 16/18, which cause malignant tumors, the infecting HPV is a β-HPV lacking E5.
Regarding the mechanisms by which β-HPV escapes the control of the EVER1/EVER2/CIB1 complex in EVs, in healthy individuals, the EVER1/EVER2/CIB1 complex restricts transcription of β-HPV and does not cause clinical symptoms. E5 and E8, which are expressed by other cutaneous HPVs (α2, α4, γ, and μHPVs), can antagonize the EVER1/EVER2/CIB1 complex. However, the general absence of HPV lesions may be influenced by additional regulatory factors: in EV patients, the absence of the EVER1/EVER2/CIB1 complex results in transcription of β-HPV, which leads to the development of EV lesions. There may be limiting factors for cutaneous HPVs other than β-HPV that are regulated similarly to healthy individuals. 8,21 However, of interest in this model is the presence of an as-yet-unidentified EVER1/EVER2/CIB1 complex-independent factor that specifically limits persistent infection by α, γ, and μHPVs.

| Generalized verrucosis with genetic mutations
In recent years, more than 20 verrucous lesions distributed in multiple anatomic sites, including the fingers, have been described as generalized verrucosis (GV), a cutaneous manifestation of systemic immunodeficiency with a background of hereditary or acquired immunodeficiency conditions. 22 Clinical manifestations (verrucous type, skin rash distribution), other symptoms, the family history, and the personal history (blood disorders, organ transplantation) are important in diagnosing GV. In addition to the usual blood tests (blood counts, immunoglobulins), HIV infection should be examined. 22 If HIV infection is seen, there is a possibility of acquired EV. A useful algorithm for diagnosing GV by genetic mutations has also been reported. 23 Generalized verrucosis is a disease characterized by numerous generalized warts on the extremities, including the hands and feet, and the absence of blue cells histopathologically, as seen in EV.
Although there are many diseases that can cause GV, the authors experienced GV due to GATA2 deficiency ( Figure 1). 24 Clinically, the patient had numerous verruca vulgaris-like nodules on the extremities, including the hands and feet. HPV type 2 and several other HPVs were detected in multiple verrucae. As the concepts of GV and EV become more widespread, more cases of hereditary diseases are likely to be found in patients with multiple warts, such as in our patient.
F I G U R E 1 Epidermodysplasia verruciformis (EV) and generalized verrucosis (GV). EV is classified as typical EV, atypical EV, and acquired EV. EV is characterized by flat skin rash and pathologically β-HPV infected keratinocytes, "blue cells." GV differs from EV in having more verrucous nodules being pathologically similar to normal verrucae. In GV, β-HPV is detected in addition to α-HPV, such as HPV type 2. AIDS, acquired immunodeficiency syndrome; HPV, human papillomavirus.

| Treatment of GV
Treatment options for warts vary widely. According to UK guidelines, the most highly recommended method of treating hand warts involves salicylic acid and liquid nitrogen therapy, followed by localized bleomycin injection, local immunotherapy, topical 5-FU, and LASER. 25 The same is true for regular refractory warts, but the associated genetic mutations make radical treatment of cases difficult.
There are many reports concerning the treatment of GV and other refractory warts, but most of these are case reports of small case series. The aforementioned British guidelines for the treatment of warts in immunocompromised patients suggest that surgical techniques and tissue destruction, along with salicylic acid, are effective in reducing the volume of warts. Caution should be exercised when treating GVs with the expectation of an immune response, especially since GV with genetic mutations is associated with immune abnormalities. In our experience with GATA2 mutation cases, liquid nitrogen therapy caused the warts to expand, so it is possible that the virus spread by destroying the tissue ( Figure 1). 24 Cidofovir, local immunotherapy, imiquimod, diazepam, and retinoids have also been reported to be effective in treating refractory warts with immunodeficiency. 25 In addition, the efficacy of the HPV vaccine has been reported as well. However, it should be noted that the HPV vaccine is prophylactic, and its mechanism of efficacy is unknown. 26 In addition, a case of GATA2 mutation was reported in which hematopoietic stem cell transplantation was effective, 27 suggesting a close relationship between GV and host immunity.

| New analyses of hereditary refractory generalized warts and their characteristic HPV gene expression patterns
At present, in the diagnosis of hereditary diseases with generalized warts, genetic analyses and HPV gene expression can be simultaneously examined by whole-transcriptome sequencing using a single biopsy sample. 28 Interestingly, even though the infecting virus is the usual HPV type 2 infection, the mode of infection differs between healthy subjects and those with Treeman syndrome (CD28 deficiency). In Tree-man syndrome, no capsid protein is detected (no viral particles are produced), and the E6 and E7 gene expression is high only in the basal layer, suggesting a latent infection state. 29 In future studies of refractory generalized warts with genetic mutations, such an analysis that approaches the disease from both the viral protein and genetic aspects may be useful.

| β -HPV AND NON -MEL ANOMA S KIN C AN CER
Among β-HPVs, HPV types 5 and 8 in EV patients have been identified as carcinogenic (Group 2B) by the International Agency for Research on Cancer. β-HPV infection (HPV types 5 and 8) has been identified in non-melanoma skin cancer in EV. However, β-HPV is less detectable in SCC than in precancerous conditions. The "hit and run theory" has been proposed as to why β-HPV triggers but does not maintain carcinogenesis. 30 Normally, keratinocytes stop the cell cycle and repair DNA when exposed to sunlight in order to inhibit carcinogenesis. If repair still proves difficult, apoptosis is induced.
β-HPV infection, such as HPV5 and HPV8, suppresses these fac- Epidermodysplasia verruciformis has been reported in Japan, although it is rare. We encountered several cases of EV. The first case had a tinea versicolor-like rash since childhood, and trichilemmal carcinoma appeared as an adult (Figures 1 and 2). 34 In the second patient, a brownish skin rash occurred in her teens, with multiple SCCs and basal cell carcinoma on the face and trunk, and a mutation in the TMC8 gene was identified. HPV type 5 was detected in the skin rash. 35

| MUCOSAL HI G H -RIS K HPV AND NON -MEL ANOMA S KIN C AN CER
Genus α mucosal high-risk HPVs are the causative viruses of cervical, penile, vaginal, anal, and oropharyngeal cancers. More than 600 000 people are diagnosed with HPV-associated cancers annually. 36 However, few studies have been conducted on skin tumors.

| Carcinogenesis due to mucosal high-risk HPV compared to skin cancer due to β -HPV
The life cycle of mucosal high-risk HPVs is reviewed here, including an example of cervical cancer. Mucosal high-risk HPV infects discrete cell populations at the squamocolumnar junction (SCJ), and the HPV genome is maintained at a low copy number as an episome. As the infected basal cells replicate and migrate to the spinous layer, viral E6 and E7 expression re-enters the differentiating cells into the cell cycle and initiates epithelial cell proliferation. As the cells move into the upper layers, replication of the viral genome occurs, and the F I G U R E 2 β-HPV or mucosal high-risk HPVs-associated non-melanoma skin cancer. β-HPV-associated skin cancers include EV (EV)associated SCC. Although β-HPV-associated SCC in healthy individuals has been suggested, it is often undetectable in practice, and the "hit and run theory" has been proposed as the reason for this. Bowen's disease of the nail and SCC are known to be associated with mucosal high-risk HPV. In particular, Bowen's disease of the nail is classified into two types: Periungual type and subungual/longitudinal melanonychia type. In addition, there are many reports of mucosal high-risk HPV infection in genital SCC. As a carcinogenic mechanism, ultraviolet light is important in the case of β-HPV-associated carcinomas. For mucosal high-risk HPV, the anatomical site of infection is important, and it is thought to have a sexually transmitted aspect. EV, epidermodysplasia verruciformis; HPV, human papillomavirus; HR, high-risk; SCC, squamous cell carcinoma.

| Clinical features of nail SCC/Bowen's disease
Bowen's disease of the nail apparatus is known to be caused by mucosal high-risk HPV infection. 38,46 The author reported that mucosal highrisk HPV type 56 is detected in Bowen's disease of the nail, especially in the type that presents with longitudinal melanonychia. 40,47 Although similar reports have been accumulated both in Japan and overseas, the fact that Bowen's disease of the nail is associated with mucosal highrisk HPV is not widely known in fields other than dermatology.
The author analyzed 136 previously reported cases of HPVassociated SCC/Bowen's disease of the nail apparatus. 48 Patients were between 12 and 85 years old (mean age 52.2 years old) with a male-to-female ratio of 2.5:1. Patients with Bowen's disease tended to be younger than those with SCC, with an average age of 50 vs.
56 years old. There were 53 cases of invasive carcinoma and 83 of intraepithelial carcinoma. Multiple lesions were seen in 11 cases (8%), suggesting that an immunocompromised status may also play a role in disease development. The affected digits in the previously reported cases were one to three on the right and three on the left, with fewer than five on both the left and right. A small number of cases involving toes were also seen. This characteristic distribution strongly suggests that nail SCC/Bowen's disease is a sexually transmitted disease.

| Nail SCC/Bowen's disease in the context of mucosal high-risk HPV-associated visceral cancer
Mucosal high-risk HPV-associated visceral cancers include cervical, oral, oropharyngeal, anal, penile, and vulvar cancers. We proposed the term "nail apparatus intraepithelial neoplasia (NIN)." 48

| Distribution of mucosal high-risk HPV infection around the nail apparatus
A noteworthy point is that, in all cases in which the clinical picture was confirmed, continuity from the proximal and lateral nail fold was observed, strongly suggesting HPV infection of the nail matrix. These results suggest that the pathogenesis differs depending on the HPV type.
We examined the distribution of HPV infection using anti-HPV antibody in tissue from a case of HPV type 56-positive Bowen's disease. 48

| CON CLUS ION
Various HPV infections and testing methods in dermatological diseases have been described. In addition to viral carcinogenesis, it has been pointed out that β-HPV is also involved in the pathogenesis of many SCCs. It is important to examine the relationship between β-HPV and sunlight, which has been pointed out in the past, through comprehensive genetic analyses. Although mucosal high-risk HPV has not been implicated in many skin cancers, its characteristic in-