ABSTRACT

Background

Extramammary Paget’s disease (EMPD) is a rare intraepithelial adenocarcinoma that mainly affects the anogenital and axillary regions. Although its etiology has not been fully elucidated, there is evidence that androgen receptors (AR) are expressed in most cases of EMPD. However, the role of androgen signaling in the pathogenesis of EMPD remains unclear.

Objective

To evaluate the role of androgen signaling in tumor growth of AR-positive EMPD.

Methods

Patient-derived organoids were established and cultured from two AR-positive EMPD patients: one man and one woman. Cultured organoids were treated with androgen agonists and/or antagonists, then subjected to analysis of changes in organoid proliferation, as well as changes in androgen signaling pathway-specific genes.

Results

Organoid cultures were established from each EMPD sample. These organoids were immunohistologically and genetically identical to the original tumor. For each organoid sample, viable cell number increased in response to androgen exposure. The mRNA level of Fkbp5, a known AR target gene, increased in a concentration-dependent manner in organoids exposed to the synthetic androgen R1881. Conversely, the AR inhibitor darolutamide suppressed the viable cell number in a concentration-dependent manner. The mRNA expression levels of MKI67 and Fkbp5 were also suppressed by darolutamide.

Conclusion

Our results indicate that androgen signaling is a key pathway involved in the growth of AR-positive EMPD. Therefore, androgen signaling inhibition may be a novel treatment option for EMPD patients who require systemic therapy.

Introduction

Extramammary Paget’s disease (EMPD), a rare intraepithelial adenocarcinoma, predominantly occurs in the genital and axillary regions. Histopathological analysis indicates that it is characterized by intraepidermal proliferation of large glycogen-rich Paget cells [1]. Generally, Paget cell growth is slow; most cases are diagnosed as in situlesions and are curable with surgical resection. However, the risk of metastasis significantly increases when Paget cells invade the dermis, and the prognosis for advanced EMPD is very poor [2], [3], [4].

Although the pathogenesis of EMPD has not been fully elucidated, signaling through androgen hormone receptors (AR) is associated with EMPD development. There is evidence that 54–90 % of EMPD cases are AR-positive [5], [6], [7]. Additionally, the expression levels of AR and androgen-producing enzymes (including 5α-reductase and 17β-hydroxysteroid dehydrogenase type 5) are higher in invasive cases than in in situcases [7], [8].

Androgens (e.g., testosterone and dihydrotestosterone) are hormones essential for the development of male reproductive organs and secondary sexual characteristics. These hormones act through the AR, a ligand-dependent nuclear transcription factor. Androgens activate the AR by binding to ligand-binding domains in the cytoplasm. The activated AR translocates into the nucleus, where the AR-ligand complex dimerizes and binds to other co-activators. This complex then binds to promoter regions in an array of target genes with diverse functions. These target genes include secreted proteins, fusion genes, growth stimulators, PI3K modulation, transcription factors, metabolic enzymes, cell cycle regulators, and glucuronidases [9], [10].

AR expression has been demonstrated in various types of cancer, including prostate, bladder, kidney, and lung. Furthermore, AR signaling pathway activation reportedly promotes tumorigenesis and metastasis in several types of cancer [11]. Notably, 90 % of prostate cancers grow in an androgen-dependent manner, and anti-androgen therapy is now regarded as first-line therapy. This includes androgen deprivation therapy with luteinizing hormone-releasing hormone analog (LH-RHa), or combined androgen blockade therapy involving LH-RHa with an AR inhibitor (e.g., darolutamide) [12]. Furthermore, a phase II trial of combined androgen blockade therapy (a combination of bicalutamide and leuprorelin) showed a high overall response rate of 41.7 % in patients with AR-positive salivary gland cancer [13].

Because the AR signaling pathway has an important role in multiple clinically relevant malignancies, we hypothesized that it also could serve as an important target in EMPD. However, because of its rarity, effective models for the analysis of EMPD pathogenesis have not been established. Here, we established stable EMPD organoid cultures from two AR-positive EMPD patients, then used these cultures to examine the effects of androgen pathway stimulation or inhibition on EMPD.

Section snippets

Patients

This study protocol was approved by the ethics committees of Keio University School of Medicine (Approval No. 2019–0279 and 2020–0356). Two EMPD cases treated at Keio University Hospital in 2020 (EMPD#1 and EMPD#2) were included in this study. EMPD#1 was an 80-year-old female patient with a primary perineal lesion, and EMPD#2 was a 55-year-old male patient with a primary groin lesion (Fig. 1). EMPD #1 is an untreated patient and EMPD #2 has been treated with capecitabine at the referring

Establishment of AR-positive EMPD organoids

EMPD#1 was generated from an 80-year-old female patient who had EMPD affecting the vulva, along with ilio-inguinal lymph node metastasis. She had received no previous treatment. Organoids were established from ulcerative nodules of the primary lesion on the right labia majora at the time of palliative resection (Fig. 1a). Pathological analysis revealed that the original EMPD tissue exhibited sheet-like proliferation of tumor cells, with deep subcutaneous tissue infiltration. Immunohistochemical

Discussion

Monophasic tumor cell culture is an established approach for analysis of malignant cells. However, this approach cannot analyze tumor tissue in a three-dimensional manner; it also cannot analyze relationships between the tumor and surrounding stromal cells [15]. To overcome these limitations, three-dimensional culture systems using spheroids and patient-derived tumor xenografts have been established for multiple tumor types [16], [17], [18]. Nevertheless, in rare tumors such as EMPD, these