Pathogenesis Of Extramammary Paget's Disease Research Articles

Overexpression of heat shock protein 90 through the downregulation of miR-9-5p in extramammary Paget's disease.

Heat shock protein 90 (HSP90) facilitates diverse cellular processes by interacting process with more than 200 client proteins. Overexpression of HSP90 contributes to the pathogenesis of various malignant tumors, and HSP90 inhibitors attenuate the progression of malignant tumors in vitro/vivo. Numerous clinical trials have used HSP90 inhibitors to treat several cancers, and pimitespib (an HSP90 inhibitor) is covered by insurance for advanced gastrointestinal stromal tumor in Japan. In this study, we investigated the expression pattern of HSP90 and analyzed its clinical significance in extramammary Paget's disease (EMPD). All 77 EMPD tissues investigated were positive for HSP90 expression. The immunoreactivity of HSP90 in fetal cases due to EMPD tended to be highly stained. Although there was no significant difference in HSP90 mRNA levels between 24 paired lesional and nonlesional tissues, microRNA-inhibiting HSP90 levels in tumor tissues were significantly decreased compared with those in normal tissues. Thus, HSP90 may play an important role in the pathogenesis of EMPD and may be a novel therapeutic target for EMPD.

Detection of MSH2 Gene Methylation in Extramammary Paget's Disease by Methylation-Sensitive High-Resolution Melting Analysis.

Background Extramammary Paget's disease (EMPD) is a rare skin tumor. Hypermethylation in the MSH2 promoter resulting in the downregulation of its protein expression shows a high detection rate in EMPD tumor tissue, which indicates that the methylation of MSH2 may play an important role in the pathogenesis of EMPD. Objective This study aims to establish a rapid analysis strategy based on the methylation-sensitive high-resolution melting curve (MS-HRM) to detect the methylation level of the MSH2 promoter. Methods With the use of universal methylated human DNA products, we established the MS-HRM standard curve to quantitatively detect the methylation level of the MSH2 promoter. Then, all 57 EMPD tumor DNA samples were analyzed. Pyrosequencing assay was also carried out to test the accuracy and efficacy of MS-HRM. Besides, a total of 54 human normal and other cancerous tissues were included in this study to test the reliability and versatility of the MS-HRM standard curve. Results In this study, by using the established MS-HRM, we found that 96.5% (55/57) EMPD tumor samples had varying methylation levels in the MSH2 promoter ranging from 0% to 30%. Then, the methylation data were compared to the results obtained from pyrosequencing, which showed a high correlation between these two techniques by Pearson's correlation (r = 0.9425) and Bland–Altman plots (mean difference = −0.1069) indicating that the methylation levels analyzed by MS-HRM were consistent with DNA pyrosequencing. Furthermore, in 23 normal and 31 other cancerous tissue samples, there were two colorectal cancer (CRC) tissues that tested MSH2 methylation positive (1% and 5%) which confirmed that our established MS-HRM can be widely applied to various types of samples. Conclusion MS-HRM standard curve can be used for the detection of the methylation level of MSH2 in EMPD tumor samples and other cancerous tissues potentially, which presents a promising candidate as a quantitative assay to analyze MSH2 promoter methylation in routine pathological procedure.

Genetic Analysis of Mismatch Repair Genes Alterations in Extramammary Paget Disease.

Extramammary Paget disease (EMPD) is a rare cutaneous malignant neoplasm. The familial occurrence of EMPD and the high risk of concomitant secondary tumors in EMPD patients have gained much attention. These findings highlight the importance of genetic alterations in the tumorigenesis of this skin cancer. Genetic tests and functional analysis of mismatch repair (MMR) genes were performed in EMPD. The results showed that 8 of 20 cases with germline MMR genes mutations and 5 of them exhibited microsatellite instability (MSI). Immunohistochemical staining showed that the tumor tissues from 20 patients had the normal expression of MLH1 but 5 cases had the reduced expression of MSH2. There is a nearly significant correlation between MSI and germline mutations. In 172 cases, rates of germline and somatic mutations were 34.3% and 13.4%, respectively. The mutations of MLH1 V384D (15.7%), R217C (4.1%), and I219V (5.2%) were common in this cancer. In addition, the yeast 2-hybrid and immunoprecipitation assays exhibited reduced interaction between MLH1 and PMS2 in MLH1 V384D and R217C but not I219V. Moreover, MLH1 V384D and R217C had impaired MMR activity compared with the wild-type and I219V mutation by an in vitro MMR assay. The germline mutations in MMR genes are involved in the pathogenesis of EMPD and partially explain the genetic abnormalities for this disease.

Oncogenic mutations in extramammary Paget's disease and their clinical relevance

Extramammary Paget's disease (EMPD) is a rare cutaneous malignant neoplasm. The genetic alterations underlying its pathogenesis have less been described. Therefore, we analyzed the possible mutations in the KRAS, HRAS, NRAS, BRAF, ARAF, RAF1, PIK3CA, AKT1, CTNNB1 and APC genes as well as methylation and expression of CDH1 in 144 EMPD cases and 42 matched normal skin tissues. A distinct mutation profile was identified in EMPDs with 27 (19%) cases mutant for RAS and RAF genes and 50 (35%) cases harboring oncogenic mutations in PIK3CA and AKT1. Moreover, a mutually exclusive pattern was observed in the genetic variants in these two signaling pathways. No mutation was detected in CTNNB1 and APC genes. High prevalence of low expression and hypermethylation of CDH1 gene was detected in 33 and 48% of the EMPD cases, respectively. Furthermore, PIK3CA and AKT1 mutations were significantly correlated with CDH1 hypermethylation which could explain why the majority of EMPD cases with mutant PIK3CA and AKT1 were invasive. Our study demonstrates that genetic variants associated with constitutive activation of RAS/RAF and PI3K/AKT pathways are involved in the pathogenesis of EMPD. This may represent novel therapeutic targets for this skin cancer.

PI3K, Rac1 and pPAK1 are overexpressed in extramammary Paget's disease

Phosphatidylinositol 3-kinase (PI3K), Ras-related C3 botulinum toxin substrate 1 (Rac1) and P21-activated protein kinase 1 (PAK1) appear to play important roles in the pathogenesis of several tumors, but their expressions in extramammary Paget's disease (EMPD) have not been investigated yet. To investigate the potential contribution of the PI3K, Rac1 and PAK1 to the development of EMPD. Thirty-five paraffin-embedded EMPD specimens were subjected to immunohistochemical staining for PI3K (85α), Rac1 and pPAK1. All the 35 primary EMPD specimens, including 20 non-invasive EMPD, 13 invasive EMPD and 2 metastatic lymph nodes, showed cytoplasm overexpression of PI3K (85α), Rac1 and pPAK1. The expression (% positive cells) of PI3K(85α), Rac1 and pPAK1 (90.1 ± 8.6, 91.4 ± 9.5 and 89.6 ± 10.8% ) in EMPD were significantly higher than in apocrine glands of normal skin ( 20.1 ± 11.9, 29.8 ± 8.9, 41.1 ± 13.4%), and the expression in invasive EMPD with lymph node metastasis (98.2 ± 1.7, 98.8 ± 0.7 and 98.4 ± 0.9%) are significantly higher than in invasive EMPD without lymph node metastasis (94.1 ± 2.6, 96.5 ± 1.7 and 95.3 ± 1.1%) and non-invasive EMPD (85.2 ± 8.4, 87.1 ± 9.9 and 83.1 ± 10.6%). There were significant positive correlations of the expression levels between PI3K (85α) and Rac1, as well as between Rac1 and pPAK1 in EMPD. These results indicate that PI3K, Rac1 and PAK1 may play important roles in the pathogenesis of EMPD.

Hematopoietic Progenitor Kinase 1, Mitogen-Activated Protein/Extracellular Signal-Related Protein Kinase Kinase Kinase 1, and phosphoMitogen-Activated Protein Kinase Kinase 4 are Overexpressed in Extramammary Paget Disease

The c-Jun amino-terminal kinase (JNK) pathway seems to play important roles in the pathogenesis of several tumors, but its significance in extramammary Paget disease (EMPD) has not been investigated yet. The purpose of the study was to investigate the potential contribution of the JNK-associated molecules, such as hematopoietic progenitor kinase 1 (HPK1), mitogen-activated protein/extracellular signal-related protein kinase kinase kinase1 (MEKK1), transforming growth factor-β activated kinase 1 (TAK1), and phosphomitogen-activated protein kinase kinase 4 (p-MKK4) to the development of EMPD. Thirty-five paraffin-embedded EMPD specimens were subjected to immunohistochemical staining for HPK1, MEKK1, TAK1, and p-MKK4. All the 35 EMPD, including 13 dermal invasive EMPD and 2 lymph node metastasis, showed cytoplasmic overexpression of HPK1, MEKK1, and p-MKK4. The expression (%positive cells) of HPK1, MEKK1, and p-MKK4 in EMPD (92.3% ± 8.6%, 92.9% ± 8.6%, and 92.7% ± 7.4%, respectively) were significantly higher than in normal eccrine sweat gland cells (51.6% ± 10.4%, 44.7% ± 11.7%, 0% ± 0%). In addition, the expression of HPK1-, MEKK1-, and p-MKK4 in invasive EMPD was significantly higher than in noninvasive EMPD. Meanwhile, the expression of TAK1 was basically low and no significantly different between EMPD and normal controls. In conclusion, these results indicate that JNK pathway may play a role in the pathogenesis of EMPD.

Multiple novel apoptotic pathways are upregulated in extramammary paget’s disease

<h3>Aim</h3> To investigate the expression of the novel anti-apoptotic markers GRP78 and MUC13 in extramammary Paget's disease (EMPD). <h3>Method</h3> Twelve archival cases of EMPD, accessioned from 2001 until 2010 were assessed for expression of bcl-2, GRP78 and MUC13 using standard immunohistochemical and immunofluor-escent techniques. <h3>Results</h3> Twelve cases of EMPD were examined, four of which included an invasive component. Eight of twelve cases (67%) were positive for bcl2. Eight of eleven cases (72%), and four of eight cases (50%) with available tissue were positive for GRP78 and MUC13, respectively. <h3>Discussion</h3> Bcl2 is a well known regulator of apoptosis. GRP78 overexpression has been shown to significantly increase resistance to apoptosis <i>in vitro</i> and <i>in vivo.</i> MUC13 is overexpressed in a range of epithelial malignancies, and has poorly defined anti-apoptotic properties, as well as a role in proliferation activity via the p38MAPK signalling pathway, and anti-adhesive roles promoting cell motility. Our study has demonstrated an overexpression of the anti-apoptotic markers GRP78, MUC13, and bcl2 in EMPD. Overexpression of these anti-apoptotic markers is a novel finding in this disease and likely to be important in the pathogenesis of EMPD.

Nerve growth factor, brain‐derived neurotrophic factor and their high‐affinity receptors are overexpressed in extramammary Paget's disease

Neurotrophin (NT) systems appear to play important roles in the pathogenesis of several tumors, but their expression in extramammary Paget's disease (EPD) has not been investigated. Thirty-four paraffin-embedded EPD specimens (32 primary EPD and 2 metastatic to lymph nodes) were subject to immunohistochemical staining for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT3, NT4, their high-affinity receptors (TrkA, TrkB and TrkC) and the common low-affinity receptor, p75 NT receptor (p75). All 34 EPD specimens, including 2 metastatic to lymph nodes, showed cytoplasmic overexpression of NGF, BDNF, TrkA and TrkB. The expression (% positive cells) of NGF, BDNF, NT3, NT4, TrkA and TrkB (81.6 ± 14.9, 86.0 ± 10.4, 89.6 ± 14.9, 87.8 ± 17.9, 83 ± 14.4 and 86.2 ± 11.7%) in EPD was significantly higher than in normal skin (21.6 ± 6.5, 27.6 ± 4.5, 19.7 ± 10.1, 8.2 ± 10.0, 25.0 ± 5.3 and 25.4 ± 6.4%), and the expression of these factors in invasive EPD was significantly higher than in noninvasive EPD. Interestingly, Paget cells were negative for p75 and TrkC in all the 34 EPD specimens. These results suggest that overexpression of NGF, BDNF and their high-affinity receptors (TrkA and TrkB) might play a role in the pathogenesis of EPD.

Immunohistochemical analysis of the mammalian target of rapamycin signalling pathway in extramammary Paget’s disease

We have previously observed that persistent activation of the serine/threonine kinase, protein kinase B (AKT) is a frequent event in extramammary Paget's disease (EMPD). AKT promotes cell proliferation by its ability to coordinate mitogenic signalling with energy- and nutrient-sensing pathways that control protein synthesis through the atypical serine/threonine kinase, mammalian target of rapamycin (mTOR). CDK2, a member of the serine/threonine kinase family of cyclin-dependent kinases, is a key regulator of G(1)-S cell cycle progression, and has recently been shown to be one of the targets of AKT. The AKT-mTOR-p70 ribosomal protein S6 kinase (p70S6K) pathway has been described in some human malignancies, but not in EMPD. To investigate the immunohistochemical staining of the AKT-mTOR-p70S6K pathway in EMPD and to evaluate the relationships among the components. Samples of primary EMPD tissue were subjected to immunohistological staining with phosphorylated (p)-AKT, p-mTOR, p-4E-binding protein 1 (p-4EBP1), p-p70S6K/S6K1, p-ribosomal protein S6 (p-S6) and CDK2. Ten normal skin samples served as a control. Of the 32 EMPD tissue samples, 29, 27, 26, 29, 26 and 32 samples were positive for p-AKT, p-mTOR, p-4EBP1, p-p70S6K/S6K1, p-S6 and CDK2 staining, respectively. All these cell signalling molecules showed higher positivity in invasive EMPD than in EMPD in situ. There were significant correlations between p-AKT, p-mTOR, p-4EBP1, p-p70S6K/S6K1 and p-S6 and CDK2. The activation of the AKT-mTOR-p70S6K pathway may play an important role in the pathogenesis of EMPD. The high expression of the components of the pathway was highly correlated with CDK2 expression, suggesting that the AKT/mTOR pathway may induce the malignant transition through CDK2 in EMPD. The AKT-mTOR-p70S6K pathway might be a potential therapeutic target in EMPD.

Differential expression of two new members of the p53 family, p63 and p73, in extramammary Paget’s disease

The proteins p53, p63 and p73 are known to be overexpressed and to play important roles in the pathogenesis of many tumours, but the expression of p63 and p73 has not previously been investigated in extramammary Paget's disease (EMPD). To investigate the potential contribution of p53, p63 and p73 in the pathogenesis of EMPD. In total, 35 paraffin wax-embedded tissue samples from patients with EMPD were examined using immunohistochemical staining for p53, p63 and p73. All of the 35 EMPD specimens, including all 6 invasive EMPD and 2 metastatic lymph-node specimens, showed nuclear overexpression of both p53 and p73. The expression levels (percentage of positive cells) of p53 and p73 (90.66 +/- 12.53% and 80.20 +/- 13.07%) in EMPD were significantly higher than those of normal skin. There was a significant correlation between the expression levels of p53 and p73 in EMPD. In 29 of 35 EMPD specimens, there was no nuclear expression of p63, and weak or moderate staining was found in only 6 specimens. The expression level of p63 in EMPD was significantly less than that in normal skin. Our study shows that the concordant overexpression of p53 and p73 and the decreased expression of p63 may play a pivotal role in the pathogenesis of EMPD. The decreased expression of p63 may play a more important role in the pathogenesis of EMPD than the overexpression of p53 and p73.

Expression of insulin-like growth factor-1 receptor, p-AKT and p-ERK1/2 protein in extramammary Paget's disease

The insulin-like growth factor-1 (IGF-1) receptor (R)-induced phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK)/ERK signal transduction cascade, which have critical roles in prevention of apoptosis and regulation of cell cycle progression, plays an important role in tumorigenesis. The expression of IGF-1R, AKT and ERK1/2 has been described in some human malignancies, but not in extramammary Paget's disease (EMPD). To study the expression of IGF-1R, p-AKT and p-ERK1/2 in EMPD and to evaluate the relationships among them. Thirty-six tissue samples of 34 patients with primary EMPD were subjected to immunohistochemical staining for IGF-1R, p-AKT and p-ERK1/2. Of thirty-six EMPD tissue samples, 34, 34 and 28 were positive for IGF-IR, p-AKT and p-ERK1/2 expression, respectively; 27, 23 and 17 of the 36 specimens stained positive for IGF-IR, p-AKT and p-ERK1/2 in more than half of Paget's cells, respectively. There were significant correlations between the IGF-1R and p-AKT expression as well as between IGF-1R and p-ERK1/2 expression. Taken together, these results indicate that IGF-1R is overexpressed, and AKT and ERK1/2 are frequently phosphorylated in EMPD. Our study shows that the expression of IGF-1R and the induction of p-AKT and the p-ERK1/2 pathway may play an important role in the pathogenesis of EMPD. The IGF-IR system might be a potential therapeutic target in EMPD.

Expression of phosphorylated Stat3, cyclin D1 and Bcl-xL in extramammary Paget disease

Stat3 (Signal transducer and activator of transcription-3) is an oncogene that plays a critical role in regulating fundamental processes associated with malignant transformation and cell survival. It participates in oncogenesis through upregulation of genes encoding apoptosis inhibitors (Bcl-xL) and cell cycle regulators (cyclin D1). The expression of Stat3, Bcl-xL and cyclin D1 protein has not been investigated in extramammary Paget disease (EMPD). To study the expression of phosphorylated Stat3 (p-Stat3), Bcl-xL and cyclin D1 protein in EMPD and to evaluate the relationships among them. Thirty-six tissue samples from 34 patients with primary EMPD were subjected to immunohistochemical staining for p-Stat3, cyclin D1 and Bcl-xL. Thirty-five of 36 specimens were clearly positive for p-Stat3 in EMPD, while 30 of 36 and 32 of 36 were positive for cyclin D1 and Bcl-xL expression, respectively. In all of four invasive EMPD specimens, strong and frequent expression of these three molecules was evident; moreover, two invasive EMPD specimens with lymph nodal metastasis showed very strong nuclear and membranous p-Stat3 staining. Two metastatic lymph node specimens showed very strong nuclear and local membrane p-Stat3 staining. There were significant correlations between p-Stat3 and cyclin D1 expression and between p-Stat3 and Bcl-xL expression. Our study shows that the expression of p-Stat3, cyclin D1 and Bcl-xL may play a pivotal role in the pathogenesis of EMPD.

HER-2/neu expression in Paget disease of the vulva and the female breast

Objectives. Paget disease of the vulva is a rare lesion that accounts for <1% of vulva neoplasms. A 12% prevalence of invasive Paget carcinoma and a 4% prevalence of associated adenocarcinomas are described. Furthermore, a high recurrence rate of 30% after surgical therapy is observed. This study aims to search for therapeutic strategies for recurrent Paget disease, which are less mutilating and less aggressive than reexcision, x-ray therapy, or chemotherapy. Trastuzumab (Herceptin™) is a recombinant monoclonal antibody against HER-2/neu, approved by the U.S. FDA for the treatment of patients with HER-2/neu-positive metastatic breast carcinomas. The results of recent studies indicate that HER-2/neu oncoprotein may play a role in the pathogenesis of extramammary Paget disease. Methods. Using HercepTest™, we analyzed HER-2/neu overexpression in seven noninvasive Paget lesions, two invasive lesions, and one Paget disease of the vulva with underlying adenocarcinoma. In addition, we investigated five mammary Paget diseases. Results. Overexpression of HER-2/neu oncoprotein labeling exclusively the membranes of Paget cells was demonstrated in 8 out of 10 cases. One noninvasive and one with underlying adenocarcinoma stained negatively. Overexpression of HER-2/neu was demonstrated in all five cases of mammary Paget disease. Conclusion. Using HercepTest™ as a standardized detection system, overexpression of HER-2/neu can be demonstrated in a majority of both noninvasive and invasive Paget disease of the vulva. The use of Trastuzumab should be considered for the treatment of patients with recurrent Paget disease of the vulva with overexpression of HER-2/neu.

Overexpression of transforming growth factor-beta3 immunohistochemical staining in extramammary Paget's disease, but downregulated expression in Bowen's disease.

The role of transforming growth factor-beta (TGF-beta) in carcinogenesis is complex, with some reports indicating a tumor inhibition role and others indicating a tumor promotion role. In particular, TGF-beta3 is thought to play a key role in controlling epithelial homeostasis. Immunopositive p53 has been demonstrated in a variety of human malignant tumors and its role in oncogenesis and tumor progression is thought to be important. Extramammary Paget's disease (EPD) and Bowen's disease are skin cancers of unknown histogenesis. To clarify the role of TGF-beta3 and p53 in EPD and Bowen's disease and to better understand the origin of these disorders. Specimens were obtained from 12 patients with EPD and 12 patients with Bowen's disease seen at our clinic between 1993 and 2000. TGF-beta3 and p53 immunohistochemical staining was performed. In three of the 12 EPD patients and five of the 12 Bowen's disease patients, positive p53 staining was detected. In contrast, TGF-beta3 overexpression was detected in all EPD patients, whereas downregulated TGF-beta3 expression was detected in all Bowen's disease patients. The present data suggest different roles for TGF-beta3 in abnormal epidermal cells in EPD and Bowen's disease. Thus, TGF-beta3 expression may be modulated differently via a p53-dependent or -independent pathway in the pathogenesis of EPD and Bowen's disease. Moreover, high TGF-beta expression appears to be a useful indicator of tumor activity in EPD.

Extramammary Paget disease is characterized by the consistent lack of estrogen and progesterone receptors but frequently expresses androgen receptor.

Extramammary Paget disease (EPD) is an uncommon cutaneous malignant neoplasm that arises in areas rich in apocrine glands (perineum, vulva, and axilla). Apocrine gland origin or apocrine differentiation of cells of EPD has been suggested. Estrongen, progesterone, and androgen hormone receptors have been reported to exhibit a characteristic pattern of expression in mammary apocrine type carcinomas; however, their expression in EPD has not been elucidated fully. By using immunohistochemical methods, we studied the expression of steroid receptors in EPD on formalin-fixed paraffin-embedded tissue samples from 28 patients with EPD without associated visceral malignant neoplasms or adnexal carcinoma. Androgen receptor (AR) was identified in 15 of 28 cases. The proportion of AR-positive cells varied from 1% to more than 75%; 8 cases expressed AR in more than 10% of cells. Strong AR expression also was seen in the invasive carcinoma arising from 1 case of EPD. All cases lacked immunohistochemically detectable estrogen and progesterone receptors. The immunophenotype characteristic of apocrine carcinomas (AR-positive, estrogen receptor-negative, progesterone receptor-negative) was seen in a substantial proportion of EPD cases. Results suggest that AR expression is a factor in pathogenesis of EPD. This may be important for the therapy of recurrent or invasive disease.