Whole genome sequencing of HER2-positive metastatic extramammary Paget's disease: a case report.

BACKGROUND: Extramammary Paget's disease (EMPD) is a rare cancer that occurs within the epithelium of the skin, arising predominantly in areas with high apocrine gland concentration such as the vulva, scrotum, penis and perianal regions. Here, we aim to integrate clinicopathological data with genomic analysis of aggressive, rapidly-progressing de novo metastatic EMPD responding to HER2-directed treatment in combination with other agents, to attain a more comprehensive understanding of the disease landscape. METHODS: Immunohistochemical staining on the scrotal wall tumor and bone marrow metastasis demonstrated HER2 overexpression. Whole genome sequencing of the tumor and matched blood was performed. RESULTS: Notable copy number gains (log2FC > 0.9) on chromosomes 7 and 8 were detected (n = 81), with 92.6% of these unique genes specifically located on chromosome 8. Prominent cancer-associated genes include ZNF703, HOOK3, DDHD2, LSM1, NSD3, ADAM9, BRF2, KAT6A and FGFR1. Interestingly, ERBB2 gene did not exhibit high copy number gain (log2FC = 0.4) although 90% of tumor cells stained HER2-positive. Enrichment in pathways associated with transforming growth factor-beta (TGFß) (FDR = 0.0376, Enrichment Ratio = 8.12) and fibroblast growth factor receptor (FGFR1) signaling (FDR = 0.0082, Enrichment Ratio = 2.3) was detected. Amplicon structure analysis revealed that this was a simple-linear amplification event. CONCLUSION: Whole genome sequencing revealed the underlying copy number variation landscape in HER2-positive metastatic EMPD. The presence of alternative signalling pathways and genetic variants suggests potential interactions with HER2 signalling, which possibly contributed to the HER2 overexpression and observed response to HER2-directed therapy combined with other agents in a comprehensive treatment regimen.

Whole genome methylation sequencing reveals epigenetic landscape and abnormal expression of FABP5 in extramammary Paget's disease.

BACKGROUND: Extramammary Paget's disease (EMPD) is a rare cutaneous malignant tumor with a high recurrence rate after surgery. However, the genetic and epigenetic alterations underlying its pathogenesis remain unknown. DNA methylation is an important epigenetic modification involved in many biological processes. METHODS: In this study, enzymatic methyl-sequencing (EM-seq) technique was used to investigate the landscape of genome-wide DNA methylation from three pairs of tumor tissues and adjacent tissues of patients with EMPD. Additionally, we conducted histopathological examinations to assess the expression of fatty acid-binding protein 5 (FABP5) in another three paired samples from EMPD patients. RESULTS: The cluster analysis showed the good quality of the samples. A differential methylation region (DMR) heat map was used to quantitatively characterize genome-wide methylation differences between tumors and controls. Global DNA methylation level is lower in EMPD tissue compared to matched controls, indicating that DNA methylation discriminates between tumor and normal skin. And the top hypomethylation gene on the promoter region in tumor tissues was FABP5 on chromosome 8 with 38.44% decreased median methylation. We next identified the expression of FABP5 in paired tumors and adjacent tissues in three additional patients with EMPD. Immunofluorescence results showed FABP5 highly expressed in tumor tissues and co-located with CK7, CK20 and EMA. GO and KEGG enrichment analysis showed DMR genes on promoter are mainly enriched in the calcium ion transport, GTPase mediated signal transduction, Rap1 signaling pathway and GnRH signaling pathway. CONCLUSION: Taken together, our findings provide the first description of the whole genome methylation map of EMPD and identify FABP5 as a pathogenic target of EMPD.

KS-EMPD-1: a novel cell line of primary extramammary Paget's disease.

Extramammary Paget's disease (EMPD) is a rare skin cancer that mainly occurs in apocrine sweat gland-rich areas in elderly people. The prognosis of metastatic EMPD is unfavorable because of the lack of fully effective systemic therapies. However, the difficulty in establishing a model of EMPD has hampered basic research for exploring its pathogenesis and optimal treatments. Here, we established for the first time an EMPD cell line (named KS-EMPD-1) from a primary tumor on the left inguinal region of an 86-year-old Japanese male. The cells were successfully maintained for more than 1 year, with a doubling time of 31.2 ± 0.471 h. KS-EMPD-1 exhibited constant growth, spheroid formation, and invasiveness, and was confirmed to be identical to the original tumor by short tandem repeat analyses, whole exome sequencing, and immunohistochemistry (CK7+CK20-GCDFP15+). Western blotting of the cells revealed the protein expression of HER2, NECTIN4, and TROP2, which have recently attracted attention as potential therapeutic targets for EMPD. KS-EMPD-1 was highly sensitive to docetaxel and paclitaxel on chemosensitivity test. The KS-EMPD-1 cell line is a promising resource for basic and preclinical research on EMPD to better define the tumor characteristics and treatment strategy of this rare cancer.

The utility of p63, CK7, and CAM5.2 staining in differentiating pagetoid intraepidermal carcinomas.

BACKGROUND: Extramammary Paget disease (EMPD), pagetoid squamous cell carcinoma in situ (PSCCIS), and Paget disease of the breast (PD) are intraepidermal carcinomas with overlapping histopathologic features. CK7 and CAM5.2 stains are frequently utilized to distinguish PSCCIS from EMPD and PD. However, some cases of PSCCIS can stain positively for CAM5.2 and CK7, indicating a potential pitfall with these stains. p63 has been shown to distinguish PSCCIS from EMPD. We assessed p63 staining in PD and compared it to p63 staining of PSCCIS and EMPD. METHODS: A retrospective search for 15 examples each of PSCCIS, EMPD, and PD with remaining tissue in the paraffin block was performed. The diagnosis was confirmed by a board-certified dermatopathologist and immunostaining for p63, CK7, and CAM5.2 was performed. Staining >55% was scored as positive. Staining <55% was scored as negative and an approximate percentage of positive cells was recorded. RESULTS: Diffuse nuclear expression for p63 was detected in 100% (15/15) of PSCCIS cases, 0% (0/15) of PD cases, and 0% (0/15) of EMPD cases. CK7 and CAM5.2 stains were positive in 100% of PD. CAM5.2 was positive in 100% of EMPD and CK7 was positive in 93% of EMPD. CAM5.2 was positive in 0% of PSCCIS biopsy specimens, but partial staining was seen in 20%. CK7 was positive in 13%, but partial staining was seen in 47%. CONCLUSIONS: p63 immunostaining is a highly sensitive and specific method for differentiating between PSCCIS and PD or EMPD. While CAM5.2 and CK7 are also useful ancillary stains in this differential diagnosis, false-positive and false-negative staining occurs with these two markers.

Immunohistochemical and clinicopathological study regarding nardilysin on extramammary Paget's disease.

It has been reported that nardilysin (NRDC), a metalloendopeptidase which regulates various growth factors and cytokines, is associated with malignancies in a conflicting manner, in which it promoted gastric, hepatocellular, and colorectal cancers and suppressed pancreatic ductal adenocarcinoma. However, it has not been investigated how NRDC is associated with cutaneous malignancies for now. Immunohistochemical staining has revealed that NRDC expression is observed in all extramammary Paget's disease (EMPD) cases. Notably, other cutaneous malignancies including basal cell carcinoma, squamous cell carcinoma, and eccrine porocarcinoma, did not show increased NRDC expression in immunohistochemistry. EMPD typically presents several types of lesions including nodules, and positive staining of NRDC on EMPD was observed regardless of the type of lesions. Examination using samples taken from nodular lesions showed that some cases showed heterogenous NRDC expression within each lesion. We also found that NRDC staining was weaker in the marginal parts of EMPD lesion than in the central parts in several cases, and tumor cells tend to be distributed beyond the macroscopic skin lesions in these cases. It was speculated that decreased NRDC expression in the marginal zones of the skin lesions may be associated with the ability of tumor cells to produce the cutaneous manifestation of EMPD. This study suggests that NRDC may be associated with EMPD like other malignancies reported previously.

Immunohistochemical expression of TRPS1 in mammary Paget disease, extramammary Paget disease, and their close histopathologic mimics.

BACKGROUND: Trichorhinophalangeal syndrome type 1 (TPRS1) expression has been found to be highly sensitive and specific for breast carcinomas. The frequency of TRPS1 expression in cutaneous neoplasms such as mammary Paget disease (MPD) and extramammary PD (EMPD) is currently unknown. We assessed the utility of TRPS1 immunohistochemistry (IHC) in the evaluation of MPD, EMPD, and their histopathologic mimics, squamous cell carcinoma in situ (SCCIS) and melanoma in situ (MIS). METHODS: Twenty-four MPDs, 19 EMPDs, 13 SCCISs, and 9 MISs were subjected to immunohistochemical analysis using anti-TRPS1 antibody. The intensity (none, 0; weak, 1+ ; moderate, 2+ ; strong, 3+ ) and proportion (<1%, absent; 1%-25%, focal; 26%-75%, patchy; >75%, diffuse) of TRPS1 expression were recorded. Relevant clinical data were documented. RESULTS: TPRS1 expression was present in 100% (24/24) of MPDs, with 88% (21/24) of MPDs exhibiting strong, diffuse immunoreactivity. Sixty-eight percent (13/19) of EMPDs showed TRPS1 expression. Intriguingly, EMPDs lacking TRPS1 expression were consistently of perianal origin. TRPS1 expression was seen in 92% (12/13) of SCCISs but was absent in all MISs. CONCLUSIONS: TRPS1 may be useful to distinguish MPDs/EMPDs from MISs, but its utility is limited in distinguishing them from other pagetoid intraepidermal neoplasms such as SCCISs.

Paget cells of the esophagus: A clinicopathologic and immunohistochemical study of 10 cases.

OBJECTIVES: Extramammary Paget's disease (EMPD) of the esophagus is very rare and the clinicopathologic features are not well characterized. DESIGN: We analyzed 10 cases with reported presence of Paget or Pagetoid tumor cells in the esophageal specimens collected between 2005 and 2018 at our institution. RESULTS: The cohort included 7 males and 3 females with a median age of 67 years. Histologically, 7 cases were secondary Pagetoid spread of tumor cells directly from an underlying invasive adenocarcinoma (pADC) located at the distal esophagus, all CK7 + with variable intracytoplasmic mucin. The clinical course of those secondary cases was dependent on the underlying malignancies. Only 3 cases were primary, including 2 Pagetoid squamous cell carcinoma in-situ (pSqCCis) and 1 Pagetoid adenocarcinoma in-situ (pADCis) with focalstromal invasion. The primary cases showed similar clinical and endoscopic presentations. Immunohistochemically, the singly dispersed Paget or Pagetoid tumor cells frequently showed loss of E-cadherin and gain of vimentin expression. Seven cases, including 5 pADC, 1 pSqCCis and the pADCis showed aberrant p53 expression. Four patients, all pADC, died of disease at a median follow-up of 10 months, while the others were alive. CONCLUSIONS: Paget or Pagetoid tumor cells in the esophagus can be primary or secondary to an invasive carcinoma. The clinical outcomes depend on the underlying malignancy and extensiveness of disease. Frequent p53 aberrancies and epithelium-mesenchymal transition likely play a role in the pathogenesis.

Transcription factor activating enhancer-binding protein 2B expression correlates with invasiveness and prognosis of extramammary Paget's disease.

BACKGROUND: Extramammary Paget's disease (EMPD) is a slowly advancing malignancy that sometimes progresses to the invasion of the dermis, systemic metastases, and death. Although there have been reports that dermal invasion is associated with poor prognosis, no molecular markers of this invasion have been identified thus far. The aim of this study was to identify key molecules for predicting the risk of EMPD dermis invasion. METHOD: We performed microarray screening for three cases of in-situ EMPDs, three cases of invasive EMPDs, and three cases of normal epidermis. We identified a molecule that exhibited a stepwise increase in expression. Further, we analyzed 47 cases of EMPD using immunohistochemical staining (IHC) and examined the correlated clinicopathological findings, including prognosis. RESULT: We examined molecules that showed stepwise differences with invasion. We focused on transcription factor activating enhancer-binding protein 2 B (TFAP2B). Of the 47 EMPD patients, 38 (80.9 %) and 9 (19.1 %) had low and high TFAP2B expression, respectively. TFAP2B expression was significantly correlated with invasion into the dermis, mass formation, and preoperative lymph node metastasis (p = 0.001, 0.042, and 0.033, respectively). The cumulative postoperative recurrence-free rate in the TFAP2B-high expression group was significantly lower than that in the TFAP2B-low expression group (P < 0.001). In univariate analysis of recurrence-free survival, TFAP2B expression was found to be a significant factor (p = 0.006). CONCLUSION: The expression of TFAP2B, which was comprehensively found by microarray screening, may correlate with the invasiveness of EMPD and may be an unfavorable prognostic factor.

Autophagic factors in Paget disease.

BACKGROUND: Despite autophagy being a principal mechanism of tumor progression, its role has been never studied in Paget disease, a difficult to treat intraepithelial neoplasia affecting mainly the breast and the vulvar regions. MATERIAL AND METHODS: Twenty seven cases (17 extramammary and 10 mammary) of Paget disease were immunohistochemically studied for the expression of the two principal autophagic factors, LC3B and p62. RESULTS: The majority of Paget diseases showed strong cytoplasmic expression of p62 in contrast to nearby keratinocytes which presented nuclear-only p62 staining. LC3B was negative or only mildly positive in neoplastic cells. No difference was seen between mammary and extramammary cases. CONCLUSION: The immunohistochemical autophagic profile of Paget disease suggests a down-regulated autophagic process which, thus, may be implicated in the invasive potential of these cells.

Cyclin-dependent kinase 4/6 inhibitors suppress tumor growth in extramammary Paget's disease.

Extramammary Paget's disease (EMPD) is a rare adnexal neoplasm commonly seen in the genital areas among the senior population. The prognosis of advanced EMPD is not favorable; thus, the development of potential treatments has long been sought. Cyclin-dependent kinase (CDK) 4/6 inhibitors such as abemaciclib and palbociclib have been proven effective against metastatic breast cancer; however, no studies have addressed CDK4/6 inhibitors as an EMPD treatment. We herein examine the efficacy of CDK4/6 inhibitors against an EMPD patient-derived xenograft (PDX) model. Abemaciclib (50 mg/kg/day) or palbociclib (120 mg/kg/day) was given orally to tumor-bearing NOD/Scid mice over a 3-week period. We also investigated the protein expression levels of CDK4/6 and cyclin D1 through immunohistochemical staining using EMPD clinical samples. Treatment with abemaciclib or palbociclib as a single agent was found to significantly suppress tumor growth in EMPD-PDX. The Ki-67-positive ratio of the treated EMPD-PDX tumors was significantly lower than that of the nontreated tumors. Clinically, the expression levels of CDK4 and cyclin D1 were significantly higher in the EMPD tumor cells than in the normal epidermis. Our results suggest that CDK4/6 inhibitors could be novel and potent therapeutics for the treatment of EMPD.

Trop2 Expression in Extramammary Paget's Disease and Normal Skin.

Extramammary Paget's disease (EMPD) is a rare skin cancer arising in the apocrine gland-rich areas. Most EMPD tumors are dormant, but metastatic lesions are associated with poor outcomes owing to the lack of effective systemic therapies. Trophoblast cell surface antigen 2 (Trop2), a surface glycoprotein, has drawn attention as a potential therapeutic target for solid tumors. Sacituzumab govitecan, an antibody-drug conjugate of Trop2, has recently entered clinical use for the treatment of various solid cancers. However, little is known about the role of Trop2 in EMPD. In this study, we immunohistochemically examined Trop2 expression in 116 EMPD tissue samples and 10 normal skin tissues. In normal skin, Trop2 was expressed in the epidermal keratinocytes, inner root sheaths, and infundibulum/isthmus epithelium of hair follicles, eccrine/apocrine glands, and sebaceous glands. Most EMPD tissues exhibited homogeneous and strong Trop2 expression, and high Trop2 expression was significantly associated with worse disease-free survival (p = 0.0343). These results suggest the potential use of Trop2-targeted therapy for EMPD and improve our understanding of the skin-related adverse effects of current Trop2-targeted therapies such as sacituzumab govitecan.

Integrative bioinformatics approaches to map key biological markers and therapeutic drugs in Extramammary Paget's disease of the scrotum.

Extramammary Paget's disease (EMPD) is an intra-epidermal adenocarcinoma. Till now, the mechanisms underlying the pathogenesis of scrotal EMPD is poorly known. This present study aims to explore the knowledge of molecular mechanism of scrotal EMPD by identifying the hub genes and candidate drugs using integrated bioinformatics approaches. Firstly, the microarray datasets (GSE117285) were downloaded from the GEO database and then analyzed using GEO2R in order to obtain differentially expressed genes (DEGs). Moreover, hub genes were identified on the basis of their degree of connectivity using Cytohubba plugin of cytoscape tool. Finally, GEPIA and DGIdb were used for the survival analysis and selection of therapeutic candidates, respectively. A total of 786 DEGs were identified, of which 10 genes were considered as hub genes on the basis of the highest degree of connectivity. After the survival analysis of ten hub genes, a total of 5 genes were found to be altered in EMPD patients. Furthermore, 14 drugs of CHEK1, CCNA2, and CDK1 were found to have therapeutic potential against EMPD. This study updates the information and yields a new perspective in the context of understanding the pathogenesis of EMPD. In future, hub genes and candidate drugs might be capable of improving the personalized detection and therapies for EMPD.

Overexpression of Janus kinase 2 protein in extramammary Paget's disease.

Extramammary Paget's disease is a rare malignant tumor of the skin that occurs primarily in the genitocrural region. Although the prognosis of extramammary Paget's disease with distant metastasis is poor, an effective therapy has not been established. Because Janus kinase 2 has attracted attention as a therapeutic target in several cancers, we investigated the expression of the Janus kinase 2 protein and the relationship between its level of expression and clinical significance in 53 patients with extramammary Paget's disease in our hospital. Immunohistochemistry showed that most extramammary Paget's disease tissues were positive for Janus kinase 2 (50/53, 94.3%), and the immunostaining intensity of Janus kinase 2 was correlated with the degree of invasiveness, lymph node metastasis and distant metastasis. Based on these findings, Janus kinase 2 may be a promising therapeutic target in extramammary Paget's disease.

Characteristic Clinicopathological Features of Secondary Extramammary Paget Disease With Underlying Anorectal Adenocarcinoma: Evenly Circumferential Perianal Distribution, Fibroepithelioma of Pinkus-like Changes, and Subepidermal Mucin Deposits Without Invasive Tumor Cells.

ABSTRACT: This study aimed to identify the clinical and histopathological characteristics of secondary extramammary Paget disease (EMPD) with underlying anorectal adenocarcinoma so as to differentiate it from primary cutaneous EMPD. Seventeen and 8 cases of primary and secondary EMPD with anorectal adenocarcinoma, respectively, were retrieved from the pathology archive and the clinical and histopathological features reviewed. The tumor samples from 21 cases were totally resected specimens, whereas 3 and 1 of secondary and primary cases were punch biopsied, respectively. All 8 (100%) cases of secondary EMPD presented evenly distributed perianal lesions. By contrast, 4 of 17 (23.5%) primary EMPD cases had perianal skin lesions and displayed an uneven, asymmetrical distribution around the anus. Fibroepithelioma of Pinkus-like changes and subepidermal mucin deposits with no or few invasive tumor cells were observed in 6 (75%) and 3 (37.5%) of the 8 secondary EMPD cases, respectively, although 3 secondary case samples were small biopsy specimens. Both the histopathological changes were not observed in any of the 17 primary EMPD cases. Evenly circumferential perianal distribution, fibroepithelioma of Pinkus-like changes, and subepidermal mucin deposits without invasive tumor cells were characteristic to cases of secondary EMPD with anorectal adenocarcinoma. These clinicopathological features could be used to differentiate between secondary and primary EMPD.

Unbiased Detection of Driver Mutations in Extramammary Paget Disease.

PURPOSE: Extramammary Paget disease (EMPD) is an uncommon skin malignancy whose genetic alterations are poorly characterized. Previous reports identified mutations in chromatin remodeling genes and PIK3CA. In order to unambiguously determine driver mutations in EMPD, we analyzed 87 EMPD samples using exome sequencing in combination with targeted sequencing. EXPERIMENTAL DESIGN: First, we analyzed 37 EMPD samples that were surgically resected using whole-exome sequencing. Based on several in silico analysis, we built a custom capture panel of putative driver genes and analyzed 50 additional formalin-fixed, paraffin-embedded samples using target sequencing. ERBB2 expression was evaluated by HER2 immunohisotochemistry. Select samples were further analyzed by fluorescence in situ hybridization. RESULTS: A median of 92 mutations/sample was identified in exome analysis. A union of driver detection algorithms identified ERBB2, ERBB3, KMT2C, TP53, PIK3CA, NUP93, AFDN, and CUX1 as likely driver mutations. Copy-number alteration analysis showed regions spanning CDKN2A as recurrently deleted, and ERBB2 as recurrently amplified. ERBB2, ERBB3, and FGFR1 amplification/mutation showed tendency toward mutual exclusivity. Copy-number alteration load was associated with likelihood to recur. Mutational signatures were dominated by aging and APOBEC activation and lacked evidence of ultraviolet radiation. HER2 IHC/fluorescence in situ analysis validated ERBB2 amplification but was underpowered to detect mutations. Tumor heterogeneity in terms of ERBB2 amplification status was observed in some cases. CONCLUSIONS: Our comprehensive, unbiased analysis shows EMPD is characterized by alterations involving the PI3K-AKT pathway. EMPD is distinct from other skin cancers in both molecular pathways altered and etiology behind mutagenesis.

PD-L1 Expression in Extramammary Paget Disease: A Case Series.

ABSTRACT: The PD-1/PD-L1 pathway plays a critical role in the physiologic inhibition and modulation of the immune response in normal tissue. Many tumors evade immune detection and response by upregulating PD-L1 expression. Humanized monoclonal PD-1 and PD-L1 antibodies have proven as both tolerable and effective treatment in many neoplasms. Extramammary Paget disease (EMPD) is a deformative and debilitating cutaneous malignancy in which definitive treatment options are limited with high recurrence rates after surgical excision. To the best of our knowledge, there is little published information regarding EMPD and PD-L1 expression. We evaluated 18 EMPD surgical pathology cases for tumor cell and tumor-associated inflammatory (TAI) cell PD-L1 expression. We identified PD-L1 tumor cell expression in 3 (17%) of the cases: 2 of 4 invasive cases (50%) and 1 of 14 (7%) noninvasive cases. One invasive case had lymph nodal metastasis with PD-L1 tumor cell expression. The host inflammatory response intensity and PD-L1 expression were variable in cases negative for tumor cell PD-L1 expression; however, a marked inflammatory response and TAI PD-L1 expression were present in all cases positive for tumor cell PD-L1 expression. In conclusion, 1 in 14 (7%) in situ EMPD cases showed tumor cell PD-L1 expression and 2 of 4 invasive cases (50%) showed tumor cell PD-L1 expression. TAI cells were more often positive (83%) than tumor cells (17%) for PD-L1 expression.