Acquired Epidermodysplasia Verruciformis With <scp>HPV5 RNA</scp> Expression Using <scp>RNA</scp> Chromogenic In Situ Hybridization

A skin disorder, phenotypically resembling epidermodysplasia verruciformis (EV) but affecting patients with chronic acquired immunosuppression, is referred commonly to as acquired epidermodysplasia verruciformis (AEV). Typically occurring in the context of HIV infection, immunosuppressive therapy, or hematological neoplasia, AEV presents clinically with EV-like skin lesions, including flat wart-like papules and dyspigmented macules [1, 2]. Patients with EV have germline mutations in EVER1/TMC6 or EVER2/TMC8 genes and are prone to infections with beta-genus human papillomaviruses (HPV), notably HPV5 and HPV8 [1]. This can lead to the development of characteristic lesions and cutaneous carcinogenesis, with squamous malignancy manifesting in up to 50% of patients later in life [3]. The pathogenesis of AEV, along with its potential for progression to squamous malignancy, remains poorly understood. We present a case of AEV that stained positive for HPV5 RNA using RNA chromogenic in situ hybridization (CISH). This sample came from a 27-year-old female with a medical history including perinatal HIV, chronic bronchiectasis, cerebral palsy, left-sided paraplegia, and HIV-related encephalopathy. She sought dermatological care for diffuse wart-like lesions present since her teenage years. She noted occasional itching and bleeding upon scratching. Her recent CD4 count was at 567 with an HIV viral load of 87 copies/mL. At the time, she was undergoing treatment involving daily emtricitabine-tenofovir and atazanavir. Upon physical examination, numerous sharply demarcated hypopigmented macules, ranging from 2 to 4 mm, were observed on the face (Figure 1A), neck, and upper thighs. Additional scattered lesions appeared elsewhere on her body. A shave biopsy of a lesion on the right superior shoulder was performed, considering possible diagnoses such as flat warts, post-inflammatory hypopigmentation, and idiopathic guttate hypomelanosis. Pathological evaluation revealed a well-defined zone of acanthosis, epidermal dysmaturation (Figure 1B), and viral cytopathic changes with pale vesicular nuclei, discernible nuclei, and increased blue-gray cytoplasm (Figure 1C). The RNAscope (ACDBio, Newark, CA) HPV5 assay was positive, demonstrating viral RNA expression in the mid and upper portions of the affected epidermis (see Figure 1D). Although the patient did not develop additional lesions, the existing ones did not respond to topical treatments with tretinoin or salicylic acid cream. The patient eventually succumbed to a lung infection. We identified six other cases of AEV from chronically immunosuppressed patients. However, staining with HPV5 RNA CISH in these cases was negative. In situ hybridization for HPV5 (ACDBio, Newark, CA) was performed using the RNAScope 2.5 LSx Reagent Kit-RED (ACDBio, Neward, CA) according to default manufacture protocols. In total, seven formalin-fixed-paraffin-embedded (FFPE) samples were stained using an automated staining system (Leica Bond RX, Leica Biosystems, Deer Park, IL). After cover-slipping, whole-slide scans were acquired by imaging at 0.5 μm/px using the PhenoImager HT (Akoya Biosciences). This study introduces the utility of RNA CISH in FFPE samples of AEV, which has not yet been previously documented in routine clinical skin biopsies. In contrast to PCR and sequencing based techniques, which cannot completely rule out contamination from the common skin flora hosting beta HPV viruses, RNA CISH circumvents this issue more effectively. The method enables the detection of viral RNA within the context of epidermal histology, facilitating the examination of RNA expression patterns at the single-cell level across different epidermal compartments and at various evolutionary stages of HPV-driven lesions. In our patient, the observed pattern of HPV5 expression in the upper epidermis closely aligns with documented murine models and, more broadly, adheres to the biological characteristics of HPVs [4, 5]. However, the fact remains that only one out of seven patients in the study exhibited positive staining for HPV5. This raises the question of infection rate, which appears to be lower than expected based on previous PCR studies in AEV and EV-related lesions [1, 6, 7]. This incongruity implies varying forms of immune dysfunction that could lead to inadequate clearance of specific beta-HPV infections. Another explanation is that detection rates in PCR studies may be artificially conflated by the possibility of skin flora contamination. Nevertheless, our modest staining positivity rate demonstrates that our study is limited by the sole testing of HPV5 and small sample size. Recent studies suggest that in addition to HPV5, more than 20 other beta-HPV types may be able to generate an EDV-like histopathologic phenotype including but not limited to HPV8, 14, 20, 25, and so on [3, 8, 9]. Furthermore, pathogenesis and implicated HPVs may differ between inherited and acquired forms of EV [10]. Given the finite success of HPV5 RNA CISH, future studies could assess the utility of RNA CISH for detection other beta HPVs, with the hope for eventual development of a “pan-beta HPV” probe for the most commonly implicated pathogens in AEV. Although the current practicality of RNA CISH in AEV remains limited, the advantages of in situ colocalization of HPV allows for it to be a useful adjunctive test for disease diagnosis and characterization. Considering these factors, we view RNA CISH studies as potentially invaluable for future clinical use and investigations into the biology of beta-HPVs in AEV. Consent for the publication of all patient photographs and medical information was provided by the authors at the time of article submission to the journal, stating that all patients gave consent for their photographs and medical information to be published in print and online and with the understanding that this information may be publicly available. The authors declare no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request.