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A multicenter study on TROP2 as a potential targeted therapy for extramammary Paget disease in Japan

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Introduction

Extramammary Paget disease (EMPD), a rare form of skin cancer that typically affects older individuals, is known to particularly affect areas with apocrine sweat glands1,2,3. The pathogenesis and risk factors of EMPD remain incompletely understood, despite intensive efforts to genetically analyze this condition4,5. Studies have revealed the slow-growing nature of EMPD tumors, with them remaining as in situ lesions for long periods, resulting in a favorable prognosis6,7,8,9,10,11,12. In a clinical context, the diagnosis of EMPD can sometimes be difficult because of the resemblance to various inflammatory skin diseases1. An additional complication is dermal invasion, which occurs in a significant proportion of cases and can increase the risk of metastasis1,7,8,11. Against this background, the prompt surgical removal of EMPD lesions is recommended to minimize their potential impact on overall health and well-being6. It is also important to distinguish EMPD from mammary Paget disease, a specific type of breast cancer that extends to the epidermis, and secondary EMPD, a rare type of visceral cancer that extends to the epidermis or mucosa, given the difference in management strategy for these latter two types compared with that for primary EMPD1,2. The differentiation of these tumors can be facilitated by immunohistochemical staining panels, including CK7, GCDFP15, CK20, and CDX21,2. Meanwhile, EMPD lesions tend to have an unclear tumor border and hidden skip lesions, making complete tumor removal difficult13. Various methods are available to achieve complete removal, such as Mohs micrographic surgery, wide local excision with or without mapping biopsies, and photodynamic diagnosis-guided surgery14,15,16,17,18,19. Nonetheless, even with these approaches, anatomical constraints can prevent complete removal in some cases13. Although conventional chemotherapy drugs such as docetaxel, paclitaxel, cisplatin, TS-1, and 5-fluorouracil are used to treat metastatic EMPD, they are generally associated with a poor prognosis20,21,22. There is thus an urgent need for alternative treatments.

Trophoblast Cell Surface Antigen 2 (TROP2) is a 36-kDa transmembrane glycoprotein that can be found in various normal epithelial tissues, including in the skin23,24, esophagus, and lung, as well as in several types of tumors like breast25, prostate26, ovarian27, pancreatic28, gastric29, and colon cancers30. This protein plays a crucial role in tumor cell proliferation, apoptosis, migration, invasion, and metastasis31. It is commonly thought that high TROP2 expression contributes to cancer progression, however, various reports have suggested that TROP2 acts as both promoter and suppressor of cancers32. As a promoter, TROP2 promotes proliferation and epithelial-to-mesenchymal transition (EMT) of cancer cells through modulating several signaling pathways25,26,27,28,29,30. In some cancers such as SCC and cholangiocarcinoma, however, loss of TROP2 promotes proliferation and EMT through affecting its downstream molecules, suggesting that it acts as tumor suppressor in the steady state33,34. Even though, overexpression of TROP2 is commonly associated with a poor prognosis in many cancer types, making it a promising target for anticancer therapy30. An antibody–drug conjugate (ADC) called sacituzumab govitecan, which targets TROP2, has recently been approved for metastatic breast cancer and metastatic urothelial carcinoma, highlighting the potential of anti-TROP2 agents in cancer treatment25,35. However, the role of TROP2 in EMPD remains poorly understood. Previous studies suggested the potential for TROP2-targeted therapy for EMPD based on the overexpression of TROP2 in primary lesions23, but no functional study was possible due to the lack of an EMPD cell line. We recently overcame this obstacle by establishing the first EMPD cell line, KS-EMPD-136. In the current study, we collected primary and metastatic paired samples of EMPD from multiple Japanese institutions to investigate the expression and functional significance of TROP2 in EMPD using these paired samples and the KS-EMPD-1 cells. The findings revealed that TROP2 was highly expressed in EMPD tissues, regardless of their primary or metastatic nature, and that it played a regulatory role in the proliferation and migration of EMPD cells in vitro. These findings illuminate the role of TROP2 in EMPD development and suggest its potential as a therapeutic target for this cancer.

Results

Patients

The demographic data of all 54 patients are shown in Table 1. Overall, 40 of the patients (74.1%) were male and 14 (25.9%) were female, with a mean age of 72.0 years. In terms of ethnicity, only one patient (1.9%) was Chinese, while the rest were Japanese (98.1%). The primary tumors were predominantly found in the genital area (n = 48, 88.9%), followed by anogenital (n = 4, 7.4%), perianal (n = 1, 1.9%), and axillary areas (n = 1, 1.9%). The majority of primary tumors were thick, with a mean thickness of 7.0 mm. As for the metastatic lesions, most were regional lymph nodes (n = 53, 98.1%), while one metastasis was found in the urinary bladder. The H-scores of the primary and metastatic tumors were high, with means of 218.7 and 231.5, respectively.

Effect of TROP2 knockdown on proliferation of KS-EMPD-1 cells

To investigate the impact of TROP2 on the proliferation of EMPD cells, KS-EMPD-1 cells were transfected with TROP2-targeted siRNA and evaluated for viable cell number. Knockdown efficiency of TROP2 was confirmed by qRT-PCR and western blotting. TROP2 expression was significantly downregulated by siRNA at both mRNA and protein levels, with approximately 80% knockdown efficiency (Fig. 2A, B, Supplementary Fig. S1, S2A, S2B). Inhibition of TROP2 significantly reduced the number of viable cells compared with that under control conditions (Fig. 2C). The expression of genes that may affect viable cell number (i.e., cell proliferation-, survival-, and apoptosis-related genes) was also analyzed in TROP2-knockdown samples (Fig. 2D, Supplementary Fig. S2C). The cell proliferation-related gene CCND1 was slightly downregulated in TROP2-knockdown samples, while C-MYC was slightly upregulated. In terms of cell survival-related genes, MCL1 and BCL-XL were slightly upregulated, but BCL2 was downregulated in TROP2-knockdown samples. Finally, the apoptosis-related genes BAX and BAK1 were both significantly upregulated by TROP2 knockdown.

Effect of TROP2 knockdown on migration and invasion of KS-EMPD-1 cells

TROP2 reportedly regulates the process of cell migration37,38. To investigate its effect on the migration of KS-EMPD-1 cells, the expression of genes encoding MMPs and cell migration- and EMT-related factors was analyzed. MMPs, except for MMP2, were significantly upregulated by the inhibition of TROP2 (Fig. 3A, Supplementary Fig. S3A). Regarding cell migration- and EMT-related genes, CDH1 (encoding E-cadherin) was significantly upregulated and VIM was significantly downregulated by TROP2 inhibition (Fig. 3B, Supplementary Fig. S3B). Meanwhile, SNAI1 and ZEB1 were significantly upregulated on days 1 and 2, but their levels later decreased. There was no clear change of TWIST1 upon TROP2 inhibition, while ZEB2 was upregulated overall, with its level peaking on day 2 (Fig. 3B). Protein expression of cell migration- and EMT-related molecules whose mRNA expression was significantly changed at 1 day after siRNA transfection was further investigated. Protein expression of vimentin and SNAIL was not detected in this cell line. Protein expression of ZEB1 was significantly upregulated by TROP2 knockdown, whereas no significant changes were observed regarding E-cadherin and ZEB2 (Supplementary Fig. S4, S5). Invasion and migration were further analyzed using functional assays. TROP2 knockdown did not affect the invasion of KS-EMPD-1 cells in in vitro invasion assays (Fig. 3C). Migration assay showed that TROP2 inhibition did not affect migration of the cells. However, when the proliferation of the cells was inhibited by mitomycin (MMC), knockdown of TROP2 significantly inhibited the migration of the cells (Fig. 3D). Knockdown of TROP2 decreased the viability of the cells, but subsequent treatment with MMC canceled the effect (Supplementary Fig. S4).

TROP2-targeted ADC prevents proliferation of KS-EMPD-1 cells

To evaluate the effect of TROP2-targeted ADC on EMPD, KS-EMPD-1 cells were treated with sacituzumab (anti-TROP2 antibody) or sacituzumab govitecan (TROP2-targeted ADC). Compared with the findings under the sacituzumab-treated control conditions, sacituzumab govitecan treatment significantly decreased the number of viable cells even at the lowest concentration (0.1 μg/mL) in a dose-dependent manner (Fig. 4). IC50 of sacituzumab govitecan was calculated as 11.54 ± 5.48 μg/mL, a concentration lower than that in the patients’ plasma (80.0 μg/mL39).

Discussion

In most cases of EMPD, the tumor grows slowly. However, in advanced cases, EMPD can be aggressive. No completely effective treatments are available for such cases, which often have a poor outcome. Chemotherapy and radiation therapy have conventionally been used for unresectable EMPD tumors, but their effectiveness is limited20,22. An anti-PD-1 antibody, nivolumab, has recently been approved in Japan for treating unresectable epithelial skin malignancies including EMPD40. Nevertheless, the response rate of nivolumab for this disease is not high, underscoring the need for novel therapeutic agents for EMPD. ADC is a new class of targeted therapeutic agent, which consists of a monoclonal antibody linked to a cytotoxic agent (payload) via a linker. When the ADC binds to a specific cell surface antigen, it enters the target cell, releases its payload, and ultimately kills the cell41,42,43. ADCs were initially used to treat hematological malignancies, but their use has been expanded to include solid cancers. The US Food and Drug Administration (FDA) has approved several ADCs for the treatment of solid cancers, and many other ADCs targeting various molecules are currently under development. Interestingly, target molecules of FDA-approved ADCs, such as NECTIN444,45,46,47,48,49,50, HER351, TROP223,24, and HER252,53,54. are widely expressed in various skin cancers. We previously reported the strong and diffuse expression of TROP2 in primary lesions of EMPD. In the current study, we collected paired EMPD tissues from multiple institutions to compare TROP2 expression between primary and metastatic lesions, and performed in vitro experiments to address the functional roles of TROP2 in EMPD.

In the current study, we made several important findings. First, most of the EMPD tissues, regardless of their primary or metastatic status, showed strong and diffuse expression of TROP2 protein in immunohistochemical analysis. Second, the KS-EMPD-1 cells treated with a TROP2-targeted ADC, sacituzumab govitecan, showed significantly reduced viability compared with those treated with sacituzumab (anti-TROP2 antibody) alone. Third, knockdown of TROP2 in KS-EMPD-1 reduced cell viability and cell migration, but did not change cell invasion. These findings suggest that a TROP2-targeted ADC such as sacituzumab govitecan may be a promising treatment for unresectable, primary and metastatic EMPD.

Regarding the first finding, comparisons of TROP2 expression in primary and metastatic lesions were also reported in other tumors. For example, using tissues from triple-negative breast cancer patients, TROP2 expression was compared among three patient groups: those with only a primary tumor with or without neoadjuvant chemotherapy and those with a metastatic tumor. Similar to our results in EMPD, primary and metastatic triple-negative breast cancer lesions highly expressed TROP2 and there was no significant difference in TROP2 H-score between the primary and metastatic lesions55. Although TROP2 H-score was a prognostic factor of metastatic triple-negative breast cancer, the predictive value of TROP2 for targeted therapy in this particular disease is still unclear. Against this background, there is also the same need to deeply understand the significance of TROP2 expression in EMPD.

Sacituzumab govitecan is an FDA-approved TROP2-targeted ADC used for metastatic triple-negative breast cancer. Its efficacy in other cancers, such as colorectal, pancreatic, ovarian, and gastric cancers, as well as esophageal adenocarcinoma, renal cell cancer, and squamous cell carcinoma, has also been evaluated56. To date, no clinical trials assessing the efficacy of TROP2-targeted ADC for treating EMPD have been performed; however, our results clearly showed that EMPD cells are highly sensitive to sacituzumab govitecan in vitro, suggesting the potential application of this ADC to EMPD. When considering the application of sacituzumab govitecan to EMPD, attention needs to be paid to adverse effects since TROP2 is also highly expressed in normal skin tissue such as in epidermal keratinocytes, hair follicles, and sweat glands24.

With regard to the in vitro analyses performed here, the efficacy of TROP2 inhibition is unclear; reductions in both viability and migration were observed upon TROP2 silencing in KS-EMPD-1 cells, but the underlying mechanisms influencing these functions are rather complicated. Regarding cell viability, the levels of proliferation-related or survival-related factors were not consistently reduced in PCR, although the apoptosis-related factors BAX and BAK1 were slightly upregulated. As for the wound healing assays, KS-EMPD-1 cells treated with MMC to inhibit their proliferation also showed significantly reduced migration. Considering that the knockdown of TROP2 upregulated CDH1 and downregulated VIM, the expression pattern opposite to what occurs in EMT, inhibition of migration observed in TROP2-knockdown cells is a consistent result with the gene expression pattern57,58. However, there were no significant changes at protein levels. In addition, TROP2 knockdown significantly upregulated mRNA and protein expression of ZEB1, a marker of EMT induction58. These results make it difficult to evaluate the role of TROP2 in EMT of EMPD cells. At present, there is no report explaining the relationship between TROP2 and EMT in EMPD. In other types of cancer, however, TROP2 is suggested to induce EMT and eventually promote metastasis through affecting several signaling pathways such as AKT/β-catenin and PI3K pathways38,59. In EMPD, activation of C-X-C motif chemokine receptor 4-stromal cell-derived factor-1 (SDF-1) axis is suggested to promote lymphatic metastasis through EMT-related features60 and it is also reported that expression of SDF-1 and TROP2 could be possible diagnostic and prognostic markers in papillary thyroid carcinoma61. Thus, future analyses looking at these factors would be beneficial to evaluate the relationship between TROP2 and EMT in EMPD. According to these functional analyses, although TROP2 plays some roles in EMPD biology, the impacts of TROP2 inhibition are not so strong as to independently exert anticancer effects. An ADC that targets TROP2 would be a better strategy for unresectable EMPD.

It should be noted that our study has some limitations. First, we were only able to use one cell line, KS-EMPD-1, as it is the only cell line available for EMPD. Second, we could not perform in vivo assays due to the lack of access to disease models. Although a few disease models have been established, including patient-derived xenograft models62,63 and cancer tissue-oriented spheroids64, they cannot currently be used by the wider research community. Unfortunately, KS-EMPD-1 does not have the ability to grow in mice, which limits our ability to establish a disease model. We hope to overcome this limitation in future studies to confirm the effects of sacituzumab govitecan in vivo.

In summary, we found strong and diffuse TROP2 expression in primary and metastatic EMPD. Sacituzumab govitecan successfully reduced the number of viable cells of EMPD in a dose-dependent manner. TROP2-targeted ADC, such as sacituzumab govitecan, should be a promising treatment for unresectable EMPD.

Methods

Patients and ethical approval

This retrospective research was conducted in accordance with the tenets of the Declaration of Helsinki. The experimental protocols, permission to perform the present study, and a waiver for informed consent were approved by the Ethics Committee of Kyushu University Hospital (Approval number: 23257–00, approved on November 7, 2023). Obtaining informed consent from each patient was not possible due to the retrospective nature of this study. Patients were given a full opportunity to decline participation through opt-out. Permission to perform the present study was also obtained from each participating institution’s ethics committee. Patients were diagnosed and treated in each institution and a total of 108 patient samples (54 patients) were examined. Secondary EMPD were carefully excluded from this study through preoperative imaging, endoscopy, and immunohistochemistry panels for excised tumors.

Immunohistochemistry

Formalin-fixed, paraffin-embedded EMPD tissues were sliced into 4- to 5-μm-thick sections for immunohistochemical staining. The staining procedure for TROP2 was carried out using the methods described in our previous reports23,44,65. The primary antibody used was rabbit anti-human TROP2 (1:1,000, ab214488; Abcam, Cambridge, UK), while the secondary antibody used was N-Histofine Simple Stain AP MULTI (pre-diluted, 414,261; Nichirei Biosciences, Tokyo, Japan). The sections were further incubated with FastRed II (415,261; Nichirei Biosciences) and counterstained by hematoxylin to stain the nucleus. For CK7, the antigen was retrieved by incubation with a protease (715,231; Nichirei Biosciences). Then, we used mouse anti-human CK7 (prediluted by the supplier, 713,481; Nichirei Biosciences) as the primary antibody, followed by N-Histofine Simple Stain MAX-PO MULTI (724,152; Nichirei Biosciences) as the secondary antibody. The chromogenic substrate used was 3,3′-diaminobenzidine tetrahydrochloride (725,191; Nichirei Biosciences). The stained sections were visualized and images were captured using a Nikon ECLIPSE 80i microscope (Nikon, Tokyo, Japan). The samples were observed under the microscope after staining. In these staining methods, positive signals of TROP2 and CK7 were detected as red and brown, respectively.

We evaluated the immunohistochemical results using a semiquantitative approach, H-score66. Briefly, the score was calculated as the percentage of positive cells (0%–100%) multiplied by the staining intensity (0–3 +). The intensity was graded as no staining (0), weakly positive (1 +), moderately positive (2 +), and strongly positive (3 +). The final H-score could thus range from 0 to 300.

Cell culture

An EMPD cell line, KS-EMPD-136, was cultured in EBM2 medium (C-22111; Takara Bio Inc., Tokyo, Japan) supplemented with 10 ng/mL Heregulin β1 (100–03; PeproTech, Cranbury, NJ) and antibiotic–antimycotic (15,240–062; Thermo Fisher Scientific, Waltham, MA). The medium was refreshed every 2–3 days and cells were passaged at 80% confluence by trypsinization.

siRNA transfection

Cells were seeded into 96-well plates (5,000 cells/well), 12-well plates (1.5 × 105 cells/well), or 6-well plates (2.0 × 105 cells/well) and incubated for 24 h at 37 °C in 5% CO2. To knock down TROP2, cells were transfected with negative control siRNA or TROP2 siRNA using Lipofectamine RNAiMAX (13,778,150; Thermo Fisher Scientific). Briefly, Silencer Negative Control No. 1 siRNA (AM4635; Thermo Fisher Scientific) and TROP2 siRNA (s8364; Thermo Fisher Scientific) were diluted with RNase-free water and further mixed with Opti-MEM I reduced serum medium (31,985–070; Thermo Fisher Scientific) and Lipofectamine RNAiMAX. The final siRNA concentration was set to 10 nM. The mixture was incubated for 20 min at room temperature and added to the cells. The cells were then incubated for 1–4 days at 37 °C in 5% CO2 and used for the subsequent experiments. Knockdown efficiency of TROP2 was confirmed at the mRNA and protein levels by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting, respectively.

RNA extraction and qRT-PCR

RNA was extracted from cells using the RNeasy Mini Kit (74,104; Qiagen, Hilden, Germany). The extracted RNA was converted to cDNA using a PrimeScript RT Reagent Kit (RR037A; Takara Bio Inc., Tokyo, Japan). Subsequent quantitative PCR was performed using TBGreen Premix Ex Taq (RR420; Takara Bio Inc.). The PCR conditions were as follows: 95 °C for 30 s and 40 cycles of 95 °C for 5 s and 60 °C for 20 s. β-Actin (ACTB) was used as an internal control gene. The sequences of the primers used are listed in Supplementary Table S1. The expression of each gene relative to that under the control conditions was calculated using the comparative Ct method.

Protein extraction and western blotting

To extract protein, cells were lysed with M-PER mammalian protein extraction reagent (78,501; Thermo Fisher Scientific) supplemented with 1% protease inhibitor cocktail (P8340; Sigma-Aldrich, St. Louis, MO). Cell lysates were incubated for 5 min at 4 °C with rotation, further centrifuged at 14,000 rpm for 10 min at 4 °C, and the supernatant was collected. Protein samples were then mixed with a 2 × sample buffer (30,566–22; Nacalai Tesque Inc., Kyoto, Japan) and heated for 5 min at 96 °C. Using the protein samples, western blotting was performed as reported previously51,65. The primary antibodies used were rabbit anti-human TROP2 (1:1,000, ab214488; Abcam) and rabbit anti-human β-actin (1:2,000, 4970; Cell Signaling Technology, Danvers, MA), mouse anti-human E-cadherin (1:5,000, 610,181; BD Biosciences, Franklin Lakes, NJ), rabbit anti-human ZEB1 (1:1,000, ab155249; Abam), rabbit anti-human ZEB2 (1:1,000, ab138222; Abcam), and rabbit anti-human SNAIL (1:1,000, 3879; Cell Signaling Technology). Goat anti-rabbit IgG horseradish peroxidase (7074; Cell Signaling Technology) was used as a secondary antibody at a dilution of 1:10,000. The immunological bands were then treated with SuperSignal West Pico Chemiluminescent Substrate (34,580; Thermo Fisher Scientific) and imaged using the ChemiDoc XRS Plus System (Bio-Rad Laboratories Inc., Hercules, CA). The intensity of the bands was measured using ImageJ software (National Institutes of Health, Bethesda, MD).

Proliferation assay

Cells were seeded at a density of 5,000 cells/well into 96-well plates and incubated for 24 h at 37 °C in 5% CO2. Cells were then transfected with siRNAs as described above and incubated for 1–4 days at 37 °C in 5% CO2. Each day, 10 μL of Cell Counting Kit-8 solution (CCK-8, 343–07,623; Dojindo, Kumamoto, Japan) was added to each well and incubated for 2 h at 37 °C. After this incubation, absorbance at 450 nm was measured using an iMark microplate reader (Bio-Rad Laboratories Inc.).

Migration assay

Cells were seeded at a density of 2 × 104 cells/well into a 96-well ImageLock tissue culture microplate (Essen Bioscience, Ann Arbor, MI) pre-coated with type I collagen (Nitta Gelatin Inc., Osaka, Japan) and incubated for 24 h at 37 °C in 5% CO2. Cells were then transfected with negative control or TROP2 siRNA as described above. After 48 h of transfection, the cell monolayers were scratched using a wound-maker (Essen Bioscience). The IncuCyte Cell Imaging System (Essen Bioscience) automatically captured images of the scratched area every 2 h for a total of 24 h. The IncuCyte software was used to analyze the wound area relative to that at 0 h. To prevent cell proliferation, the cells were treated with mitomycin C (5 μg/mL, 10,107,409,001; Roche Diagnostics GmbH, Basel, Switzerland) 2 h before scratching.

Invasion assay

Cells were seeded at a density of 2 × 105 cells/well into a 6-well plate and transfected with negative control or TROP2 siRNA as described above. After the incubation for 48 h at 37 °C in 5% CO2, cell invasion assay was performed using a CytoSelect 24-well cell invasion assay (CBA-110; Cell Biolabs, Inc., San Diego, CA) following the manufacturer’s instructions. Briefly, siRNA-transfected cells were harvested by trypsinization and suspended in medium without any supplements. Cell suspensions were then added into cell culture inserts and the inserts were transferred into wells containing the complete medium. Cells were further incubated for 24 h at 37 °C in 5% CO2. After this incubation, noninvasive cells were removed using a swab and the culture inserts were stained and observed under a microscope. To quantify the invading cells, stained cells were further incubated with extraction solution for 10 min at room temperature and the absorbance of the resultant solution at 570 nm was measured using an iMark microplate reader.

Treatment of the KS-EMPD-1 cells with TROP2-targeted ADC

Cells were seeded into 96-well plates (5,000 cells/well) and incubated for 24 h at 37 °C in 5% CO2. To investigate the effect of TROP2-targeted ADC, cells were treated with various concentrations (final concentrations: 0.1–100 μg/mL) of sacituzumab (A2031; Selleck Chemicals, Houston, TX) or sacituzumab govitecan (E2841; Selleck Chemicals) at 37 °C in 5% CO2. PBS was used as a vehicle control. At 72 h of incubation, viable cells were quantified using CCK-8 solution as described above. Absorbance at 450 nm was measured using an iMark microplate reader and cell viability relative to that of PBS-treated control cells was calculated. IC50 of sacituzumab govitecan was calculated with GraphPad Prism 7 software (GraphPad Software, San Diego, CA).

Statistical analysis

Experiments were independently repeated at least three times. Significance analyses were performed with GraphPad Prism 7 software. The normality of the data distribution was analyzed by Shapiro–Wilk test. The significance of differences between two independent groups was examined by Student’s unpaired two-tailed t tests. When the data were not normally distributed, Mann–Whitney U test was used. A p value of less than 0.05 was considered statistically significant. Wilcoxon matched-pairs signed rank test was used for the comparison of TROP2 expression in paired clinical samples.

Data availability

All data generated or analyzed during this study are included in the main text and its supplementary information files.

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Acknowledgements

We thank the patients who participated in this research and provided EMPD tissue. We also thank Ms. Mieko Ogawa for her technical support in the immunohistochemistry. The research collaborators in this multi-institutional study were recruited with support from the Japan Dermatopathology Society.