Assessment of the methods used to detect HER2-positive advanced extramammary Paget's disease.

The human epidermal growth factor receptor 2 (HER2) is recognized as an oncogene as well as a therapeutic target in various cancers. Certain patients with advanced extramammary Paget's disease (EMPD) have also been reported to express HER2, which is therefore considered a therapeutic target for EMPD. However, an accurate methodology to determine HER2-positive EMPD has not been established. To assess the optimal methods for detection of HER2-positive EMPD, 73 EMPD samples were analyzed by immunohistochemical (IHC) staining, fluorescence in situ hybridization (FISH), and the HER2 testing algorithm for breast cancer of the American Society of Clinical Oncology/College of American Pathologists, which combined the results of IHC staining and FISH. The results showed discordance in the rate of positive IHC staining and FISH results. While 68.6% (24/35) of the metastatic samples showed equivocal or positive IHC staining, only 37.1% (13/35) were positive by FISH. To assess the accuracy of these methods, the degree of HER2 expression detected by each method was correlated with the staining profiles of activated downstream signaling pathways involving phosphorylated p44/42 MAPK (Thr202/Tyr204) (p-ERK1/2) and phosphorylated AKT (Ser473) (p-AKT). Among 16 lymph node metastasis samples, all HER2-positive samples as determined by the testing algorithm stained positively for both p-ERK1/2 and p-AKT. On the other hand, 10-14.3% of the samples determined by FISH or IHC showed negative staining for p-ERK1/2 and p-AKT. The results showed that combining the results of IHC and FISH according to the HER2 testing algorithm is a useful method for accurately evaluating HER2-positive EMPD.

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

BACKGROUND: 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. OBJECTIVES: To investigate the potential contribution of the PI3K, Rac1 and PAK1 to the development of EMPD. METHODS: Thirty-five paraffin-embedded EMPD specimens were subjected to immunohistochemical staining for PI3K (85α), Rac1 and pPAK1. RESULTS: 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. CONCLUSIONS: These results indicate that PI3K, Rac1 and PAK1 may play important roles in the pathogenesis of EMPD.

The expression of HER-2 in extramammary Paget's disease.

Extramammary Paget's disease (EMPD) is a rare intraepidermal adenocarcinoma. The common sites of EMPD involvement are the vulva, perineal, perianal, scrotal and penile skin. Several studies have shown that HER-2/neu, also known as c-erbB-2, is amplified and overexpressed in many cancers. In this study, we investigated the expression and clinical significance of HER-2 in Japanese patients with EMPD. Keratinocytes in epidermis were slightly positive for HER-2. As for EMPD, 19 of 31 EMPD were positive for HER-2 (61%). There is significant correlation between the presence of invasion and strong positivity (3+) for HER-2 (p < 0.02). Furthermore, there is significant correlation between the presence of lymph node metastasis and strong positivity (3+) for HER-2 (p < 0.02). These results suggest that patients with EMPD strongly positive for HER-2 may have high risk for lymph node metastasis and should be followed up carefully. The observed overexpression of HER-2 in EMPD presents a potential therapeutic target for adjuvant treatment of this disease. Treatment with trastuzumab is well established in breast cancer with HER-2 overexpression and is recommended by several consensus statements. The results of the present study indicate that targeting therapies for HER-2, such as trastuzumab, may be used for EMPD particularly in patients with invasive and/or metastatic 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-ß 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.

HER-2/neu expression in extramammary Paget disease: a clinicopathologic and immunohistochemistry study of 47 cases with and without underlying malignancy.

Extramammary Paget disease (EMPD) is an infrequent skin cancer sometimes representing a secondary event caused by extension of an underlying carcinoma. Her-2/neu overexpression in breast cancer is correlated with a more aggressive behavior, but anti-Her-2/neu therapy improves survival in these patients. We investigated Her-2/neu expression by immunohistochemistry in cases of EMPD with and without underlying malignancy to try to correlate with tumor recurrence, progression and possible targeted therapy. Forty-seven cases were analyzed (6 from the scrotum, 7 perianal region, 1 axilla and 33 vulva). Two cases had invasive EMPD (one from vulva and one from scrotum). The overall Her-2/neu expression was 31.9%. Of the noninvasive EMPD of the vulva (32 cases), Her-2/neu was shown in 38%. The case of invasive vulvar EMPD was negative. All six scrotal EMPD lacked Her2/neu expression. Her-2/neu was expressed in two of seven perianal cases (33.3%). The EMPD on the axilla (one case) was negative. Eighteen cases had recurrence, and of these, 44.4% expressed Her-2/neu in the initial lesion. A high proportion of EMPD showed Her-2/neu expression (31.9%), indicating that these patients may benefit from targeted therapy. The proportion of positive cases was higher in lesions that had recurred at last follow up (44.4%), suggesting a more aggressive behavior.

BD ProEx C immunostaining in extramammary Paget's disease and perineal melanoma.

The differential diagnosis of perineal biopsies can include squamous intraepithelial lesions, extramammary Paget's disease, and melanoma. Less frequently two of these lesions coexist. BD ProEx C is a recently developed immunoassay that targets expression of two genes shown to be associated with cervical cancer. Immunostaining for ProEx C has been validated in cervical cytology and positive staining has also been shown to be strongly associated with human papilloma virus (HPV)-induced cervical and anal intraepithelial neoplasia in biopsies. We observed positive staining for ProEx C in Paget cells in all of 26 cases of Paget's disease irrespective of tissue site (extramammary, mammary) and in melanoma cells in all of 12 cases of primary perineal melanoma with immunostaining in >50% of malignant cells in 73% of Paget disease cases and 43% of perineal melanoma cases. Positive staining was heterogeneous and exclusively nuclear in all cases. In situ hybridization was negative for low-risk and high-risk HPV subtypes in all Paget and melanoma cases that were tested. Currently neither of these lesions is known to be HPV related although according to the literature the possibility of a role for HPV in melanoma is still unsettled. Relevant literature is reviewed.

HER-2/neu targeting for recurrent vulvar Paget's disease A case report and literature review.

BACKGROUND: The treatment of Paget's disease of the vulva particularly for recurrences can be challenging. Overexpression of the HER-2/neu protein has been found in about 30% of vulvar Paget's cases therefore presenting a potential therapeutic target. CASE: We report the case of a 52-year-old patient with persistent Paget's disease of the vulva despite eight surgical excisions over a 15-year period. Immunohistochemistry demonstrated overexpression of the HER-2/neu protein in the vulva resection specimen. Treatment with Trastuzumab resulted in a significant regression of her disease and resolution of symptoms. CONCLUSION: Based on our case report, therapeutic targeting of HER-2/neu for patients with Paget's disease of the vulva using for example Trastuzumab is a potentially effective, alternative approach, and warrants further investigation.

Fatty acid synthase expression in Paget's disease of the vulva.

We explored the immunohistochemical expression of fatty acid synthase (FAS) in Paget's disease of the vulva (PDV) and its association with clinico-pathological features. FAS is a recently discovered molecule involved in energy supply of normal cells. FAS is also overexpressed in neoplastic tissues because of their increased necessity of energy. Specimens from 20 patients with PDV were immunohistochemically evaluated; increased FAS expression was observed in 7 of 8 patients with invasive PDV (87%), in 3 of 4 patients with microinvasive PDV (75%), and in 1 of 8 patients with noninvasive PDV (12%). Statistical analysis revealed that increased FAS expression was associated with invasive PDV (p = 0.04). To our knowledge, this association of FAS in PDV is the first to be reported in literature. These observations reveal that FAS is a reliable marker of aggressiveness in PDV. The knowledge of FAS statistical association in invasive PDV is an important finding that may stratify these patients in different prognostic groups and determine therapeutic approaches for patient care.

Cyclooxygenase-2 is a possible target of treatment approach in conjunction with photodynamic therapy for various disorders in skin and oral cavity.

BACKGROUND: Anti-cancer effects of cyclooxygenase (COX)-2 inhibitors have been reported, but not fully investigated in skin and oral diseases. 5-aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) for treating those patients with skin and oral lesions is a highly sophisticated procedure, but the incidence of disease recurrence after treatment is rather significant. OBJECTIVE: To confirm that COX-2 could be a molecular target in adjunctive therapy to ALA-based PDT, we investigated (i) COX-2 expression in various skin and oral diseases, and (ii) the inhibitory effects on cellular growth of COX-2 selective inhibitor (nimesulide), ALA-based PDT and their combination on human oral squamous cell carcinoma (SCC) cell lines. METHODS: A total of 129 biopsy samples from the skin and oral mucosal lesions were tested immunohistochemically for COX-2 expression. Then the in vitro effects of nimesulide, ALA-based PDT, and their combination were determined on two SCC cell lines, HSC-2 and HSC-4. Three different methods (MTT assay, double-staining for annexin V and propidium iodide, caspase-3/CPP32 fluorometric protease assay) were applied for evaluation of their inhibitory effects on these two cell lines. RESULTS: Among the skin diseases, a considerable number of COX-2 high expressers were found in actinic keratosis (15 of 25, 60%), Bowen's disease (13 of 17, 76%) and extramammary Paget's disease (15 of 15, 100%). In contrast, only one of 33 (3%) basal cell carcinoma tumours was a COX-2 high expresser. Among the oral mucosal biopsies, the proportion of COX-2 high expressers increased gradually from hyperplasia (one of six, 17%) through mild dysplasia (five of eight, 63%) and moderate dysplasia (20 of 23, 87%) to severe dysplasia (two of two, 100%). Nimesulide had an inhibitory effect in vitro on HSC-2 (proven to be a COX-2 high expresser), but not on HSC-4 (a COX-2 non-expresser). While ALA-based PDT showed an inhibitory effect on both HSC-2 and HSC-4, most importantly the combination of nimesulide and ALA-based PDT demonstrated a significant synergistic effect on the cellular growth inhibition of only HSC-2, but not of HSC-4. CONCLUSIONS: Our study strongly suggests that COX-2 can be one of the molecular targets in treating various skin and oral diseases. The results from our in vitro experiments also prompt us to develop a new protocol with a combination of COX-2 selective inhibitor and ALA-based PDT for more effective treatment of those diseases.

Expression of cyclooxygenase-2 (COX-2) in non-neoplastic and neoplastic vulvar epithelial lesions.

OBJECTIVE: Cyclooxygenase-2 (COX-2) overexpression has been associated with parameters of tumor aggressiveness and unfavourable clinical outcome in several solid tumors. We investigated by immunohistochemistry the expression of COX-2 in normal vulvar tissue, non-neoplastic vulvar epithelial lesions, vulvar intraepithelial neoplasia (VIN) and invasive vulvar cancer (IVC). METHODS: The expression pattern of COX-2 was studied in normal vulvar tissue, in six cases of lichen sclerosus (LS), seven cases of squamous cell hyperplasia (SCH), 20 VIN, 2 Paget's disease and 36 IVC. The relationship between COX-2 expression and clinicopathologic parameters in IVC patients has been also addressed. Sections were incubated with normal rabbit serum for 15 min, then with rabbit polyclonal antiserum against human COX-2 (Cayman, Ann Arbor, MI, USA). The results were reported as mean +/- standard error (SE) of COX-2 integrated density values (IDV). RESULTS: Higher levels of tumor/stroma COX-2 IDV ratio were found in stages III-IV (mean +/- SE = 3.5 +/- 0.8) than stages I-II disease (mean +/- SE = 1.4 +/- 0.3) (P value = 0.04). In the subgroup of stage I cases, tumor/stroma COX-2 IDV values were higher in cases with > 1 mm stromal invasion (T1b) than cases with <== 1 mm stromal invasion (T1a) (mean +/- SE = 1.6 +/- 0.3 vs. mean +/- SE = 0.6 +/- 0.1) (P = 0.033). Moreover, we observed higher tumor/stroma COX-2 IDV in cases with metastatic lymph node involvement than cases without lymph node involvement (mean +/- SE = 3.5 +/- 0.8 vs. mean +/- SE = 1.3+/-0.4) (P = 0.037). CONCLUSION: This study suggests that COX-2 overexpression may contribute to vulvar tumorigenesis and progression. Moreover, the correlation of tumor/stroma COX-2 IDV ratio with tumor extension and metastatic lymph node involvement, which represent the major prognostic parameters in this neoplasia, implies that tumor/stroma COX-2 IDV ratio could have a prognostic role in vulvar cancer.

The role of vascular endothelial growth factor-A (VEGF-A) and platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) in Paget's disease of the vulva and breast.

BACKGROUND: Paget's disease of the vulva and the breast are uncommon conditions. The pathogenesis underlying these diseases is still unclear. Vascular endothelial growth factor-A (VEGF-A), a potent angiogenic factor, has been demonstrated in a variety of tumour cell types and is thought to be involved in tumour expansion. Platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) has also been shown to stimulate angiogenesis. MATERIALS AND METHODS: Fifty-four cases of Paget's disease of the vulva, including 10 with an associated invasive adenocarcinoma, and 38 cases of Paget's disease of the breast, including 26 with available associated ductal carcinoma in situ (DCIS) and/or invasive carcinoma of the breast, were evaluated immunohistochemically for the expression of VEGF-A and PD-ECGF/TP. RESULTS: VEGF-A was not expressed in Paget's disease of the vulva or breast. PD-ECGF/TP was expressed in 22 out of 54 (41%) cases of Paget's disease of the vulva. Four of the cases associated with invasive disease (40%) expressed PD-ECGF/TP. Twenty-one out of 38 (55%) cases of Paget's disease of the breast were positive for PD-ECGF/TP. CONCLUSION: Our data suggest that PD-ECGF/TP may have a role to play in the pathogenesis of Paget's disease of the vulva and the breast. The role of VEGF-A in Paget's disease of the vulva and the breast remains to be fully elucidated.

Immunohistochemical study of inducible nitric oxide synthase in skin cancers.

BACKGROUND: Nitric oxide (NO) is synthesized from the amino acid L-arginine by NO synthase (NOS). Experimental evidence suggests that increased express of inducible NOS (iNOS), which is an NOS isoform and calcium independent, is related to various pathological processes, such as inflammation and cancer. METHODS: In this study, we used immunohistochemistry to investigate iNOS expression in a series of basal cell carcinomas (BCC), Bowen's disease, squamous cell carcinomas (SCC), extramammary Paget's disease (EPD) and metastatic tumors of the skin. RESULTS: Only 1 of 16 BCC cases was positive for iNOS and the intensity of staining was weak. In most of the 10 cases of Bowen's disease, iNOS was weakly expressed and there was a wide range in the percentage of positive tumor cells. Twelve of the 16 cases of SCC were positive for iNOS and the extent of positivity was greater than in Bowen's disease. Two of the 7 cases of EPD were positive for iNOS, and 12 of the 15 cases of metastatic cancer were positive. Well-differentiated adenocarcinomas were diffusely positive, whereas poorly-differentiated ones showed strong and heterogeneous staining. CONCLUSIONS: These results indicated that the expression of iNOS may reflect the proliferation of tumor cells and that a heterogeneous distribution of iNOS may correlate with a wide variety of biological behavior of tumor cells.

Human collagenase-3 is expressed in malignant squamous epithelium of the skin.

Co-expression of several members of the matrix metalloproteinase (MMP) family is a characteristic of human carcinomas. To investigate the role of the recently cloned collagenase-3 (MMP-13) in epidermal tumors, we studied samples representing malignant (basal and squamous cell carcinoma, Paget's disease), pre-malignant (Bowen's disease, solar keratosis), and benign (keratoacanthoma, seborrheic keratosis, linear epidermal nevus) tumors. Basal cell carcinomas expressed collagenase-3 mRNA in focal areas of keratinized cells, the squamous differentiation of which was confirmed by positive immunostaining for involucrin. Apoptosis was observed in central parts of these foci. In squamous cell carcinomas, collagenase-3 expression was detected at the epithelial tumor front and less frequently in the surrounding stromal cells. Collagenase-3 mRNA co-localized with immunostaining for laminin-5, an adhesion molecule suggested to participate in the migration of tumor cells. The pre-malignant and benign tumors were mostly negative for collagenase-3. Stromelysin-1, a potential activator of latent collagenases, was frequently expressed by stromal cells surrounding the malignant tumors, and the two MMPs occasionally co-localized in keratotic foci. Our results demonstrate that in basal cell carcinomas, expression of collagenase-3 is associated with terminal differentiation of epithelial cells. Furthermore, the gene is activated during skin carcinogenesis, and we suggest a role for collagenase-3 in degradation of the extracellular matrix associated with malignant epithelial growth.

Expression of glutathione S-transferase-pi in malignant skin tumors.

GST-pi has been known to be markedly increased in human (pre) neoplasms of several organs. In this paper, the significance of immunohistochemical detection of GST-pi in human malignant tumors of the skin was studied. In specimens from 40 patients with various skin cancers, malignant melanoma, Paget's disease and undifferentiated squamous cell carcinoma showed strong reactivity in GST-pi staining. The reactions were negative or weak in Bowen's disease, basal cell epithelioma and solar keratosis. In normal melanocytes, eccrine, apocrine, and breast gland cells stained positively but not in keratinocytes, sebaceus gland and fibroblasts. While immunohistochemical detection of GST-pi in the skin was not specific for malignancies, it contributed to aid the distinction of squamous cell carcinoma from other keratinocytic tumors. GST-pi might provide potentially useful information on chemosensitivity of skin cancer, and might serve as a biomarker of disease activity.

Immunohistochemical demonstration of peptidylarginine deiminase in human sweat glands.

Human skin is known to contain protein-bound citrulline. This is the product of enzymatic deimination of arginine residues catalyzed by peptidylarginine deiminase. We probed frozen sections of human skin with a rabbit antiserum raised to rat skeletal muscle peptidylarginine deiminase using the avidin-biotin-peroxidase complex technique. This led us to interesting findings. No staining was observed in epidermis, inner root sheaths of hair follicles, sebaceous glands, and hair erector muscle. However, we noticed specific staining of the cytoplasm of secretory and myoepithelial cells of both eccrine and apocrine sweat glands. The procedure also stained neoplastic cells present in specimens dissected from extramammary Paget's disease. The data mean that peptidylarginine deiminase may be used as a new marker in the classification of skin neoplasms showing sweat gland differentiation. Possible localization of multiple types of peptidylarginine deiminases in human skin is discussed.

Fosfatasa alcalina:valoración sérica en bioquímica clínica

Efectos de la edad y sexo en la proporción de fosfatasa alcalina en la sangre. Métodos de análisis. Preparación del paciente y recolección de la muestra. Constancia de la actividad