Frequent PIK3CA-activating mutations in hidradenoma papilliferums.

Hidradenoma papilliferum (HP) is a benign epithelial tumor most commonly seen in the vulva. It is proposed to be derived from the anogenital mammary-like glands and is histologically very similar to the mammary intraductal papilloma (IP). Approximately 60% of mammary IPs have activating mutations in either PIK3CA or AKT1, with each gene accounting for 30% of cases. In this study, we screened the mutation statuses of PIK3CA, AKT1, RAS, and BRAF in 30 HPs. The results showed that activating mutations in either PIK3CA or AKT1 were identified in 20 tumors (67%); 19 tumors had PIK3CA mutations (63%; 13 in exon 20 and 6 in exon 9), and 1 had an AKT1 E17K mutation (3%). BRAF V600E mutation was found in an HP that also had a PIK3CA H1047R mutation. No RAS mutation was found. The mutation status was not correlated with the degree of epithelial cell hyperplasia. We conclude that although there might be site-related variations in the mutation frequencies of PIK3CA and AKT1 genes, HP is histologically and also genetically very similar to the mammary IP, suggesting that HP can be viewed as the extramammary counterpart of mammary IP.

MMPs and angiogenesis affect the metastatic potential of a human vulvar leiomyosarcoma cell line.

Gynaecological leiomyosarcoma (gLMS) represent a heterogeneous group of soft tissue sarcoma, characterized by rare incidence, high aggressiveness and propensity to infiltrate secondary organs, poor prognosis and lethality, because of the lack of biological mechanisms that underlying their progression and effective pharmaceutical treatments. This study was focused on some of the aspects of progression and dissemination of a subtype of gLMS namely vulvar LMS (vLMS). We therefore used a vulvar LMS-derived cell line namely SK-LMS-1, coupled with in vitro and in vivo assays. We observed that SK-LMS-1 cells have a strong invasive capacity in vitro, through the activity of matrix metalloproteinases 2 and 9, while in vivo these cells induce a strong angiogenic response and disseminate to the chick embryo liver. Therefore, we postulate that metalloproteinases are involved in the spreading behaviour of SK-LMS-1. Further investigations are necessary to better understand the molecular and cellular machinery involved in the progression of this malignancy.

Comprehensive evaluation of caspase-14 in vulvar neoplasia: an opportunity for treatment with black raspberry extract.

OBJECTIVE: The aim of this study is to determine the expression of caspase-14, a key protein in maturation of squamous epithelia, in archival malignant and premalignant vulvar squamous lesions and examine in-vitro effects of a black raspberry extract (BRB-E) on a vulvar squamous cell carcinoma (VSCC) cell line. METHODS: VSCC cell cultures were exposed to different BRB-E concentrations and used to create cell blocks. Immunohistochemistry for caspase-14 was performed on cell block sections, whole tissue sections, and a tissue microarray consisting of normal vulvar skin, lichen sclerosus (LS), classic and differentiated vulvar intraepithelial neoplasia (cVIN and dVIN respectively), and VSCC. RESULTS: LS demonstrated abnormal full thickness (5/11) or absent (1/11) caspase-14 staining. dVIN often showed markedly reduced expression (4/7), and cVIN occasionally demonstrated either absent or reduced caspase-14 (6/22). VSCC predominantly had absent or markedly reduced caspase-14 (26/28). VSCC cell cultures demonstrated a significant increase in caspase-14 (p=0.013) after BRB-E treatment: 7.3% (±2.0%) of untreated cells showed caspase-14 positivity, while 21.3% (±8.9%), 21.7% (±4.8%), and 22.6% (±5.3%) of cells were positive for caspase-14 after treatment with 200, 400, and 800 µg/mL BRB-E, respectively. Pair-wise comparisons between the treatment groups and the control demonstrated significant differences between no treatment with BRB-E and each of these treatment concentrations (Dunnett's adjusted p-values: 0.024, 0.021, and 0.014, respectively). CONCLUSIONS: Caspase-14 is frequently decreased in premalignant and malignant vulvar squamous lesions, and is upregulated in VSCC cell culture by BRB-E. BRB-E should be further explored and may ultimately be incorporated in topical preparations.

mRNA sequencing of novel cell lines from human papillomavirus type-16 related vulval intraepithelial neoplasia: consequences of expression of HPV16 E4 and E5.

Vulval intraepithelial neoplasia is a precursor of vulval cancer and is commonly caused by infection with Human Papillomavirus (HPV). Development of topical treatments for vulval intraepithelial neoplasia requires appropriate in vitro models. This study evaluated the feasibility of primary culture of vulval intraepithelial neoplasia biopsy tissue to produce cell lines for use as in vitro models. A potentially immortal cell line was produced which gave rise to three monoclonal lines. These lines were characterized for HPV genomic integration and for viral gene expression using ligation-mediated PCR and quantitative PCR. Distinct patterns of viral integration and gene expression were observed among the three lines. Integration and expression data were validated using deep sequencing of mRNA. Gene ontology analyses of these data also demonstrated that expression of the HPV16 E4 and E5 proteins resulted in substantial changes in the composition of the cell membrane and extracellular space, associated with alterations in cell adhesion and differentiation. These data illustrate the diverse patterns of HPV gene expression potentially present within a single lesion. The derived cell lines provide useful models to investigate the biology of vulval intraepithelial neoplasia and the interactions between different HPV gene products and potential therapeutic agents.

Aldehyde dehydrogenase-1 predicts favorable prognosis in patients with vulvar squamous cell carcinoma.

BACKGROUNDS: Aldehyde dehydrogenase-1 (ALDH1) has been considered as a potential cancer stem cell marker in different types of cancer. In the present study, we investigated the expression of ALDH1 in vulvar squamous cell carcinoma, and evaluated its correlation with clinicopathological factors in patients suffering from this disease. MATERIALS AND METHODS: One hundred and fifty-four patients with vulvar squamous cell carcinoma, together with their verified histopathological and complete clinical data in Norway were included in the study. All paraffin-embedded samples of the primary vulvar carcinoma were recruited. The presence of ALDH1 was detected by immunohistochemistry and compared against commonly recognized prognostic factors. RESULTS: By immunohistochemical staining, the expression of ALDH1 was observed in 10/154 (6.5%) vulvar squamous cell carcinomas, while being extensively expressed in the suprabasal cells in normal vulvar epithelia from patients with benign gynecological disease and non-malignant epithelia adjacent to the tumor cells. In addition, ALDH1 was highly expressed in stromal fibroblasts, blood vessels and keratinized pearl of the carcinoma in all the samples. Patients with ALDH1-positive tumors had a significantly longer disease-specific survival (p=0.042). CONCLUSION: Contrary to the characteristics of cancer stem cells shown in other types of cancer with positive expression of ALDH1, the positive expression of ALDH1 in patients with vulvar squamous cell carcinoma indicates a significantly better prognosis. Furthermore, there is a trend that the expression of ALDH1 is associated with better histological differentiation.

Evolution of the predictive markers amphiregulin and epiregulin mRNAs during long-term cetuximab treatment of KRAS wild-type tumor cells.

Molecular mechanisms other than activating KRAS mutations should underlie the occurrence of weaker versus stronger responses to cetuximab (CTX) in EGFR-dependent carcinomas with either an intact KRAS signaling or in which KRAS mutations do not predict CTX efficacy. We hypothesized that KRAS wild-type (WT) tumor cell-line models chronically adapted to grow in the presence of CTX could be interrogated to establish if the positive predictive value of the mRNAs coding for the EGFR ligands amphiregulin (AR) and epiregulin (EPI) could be significantly altered during and/or after treatment with CTX. Gene expression analyses using real-time (kinetic) RT-PCR were performed to monitor the transcriptional evolution of EGFR ligands EGF, TGFα, AR, BTC, EPI, NRG and HB-EGF in experimental modes induced to exhibit acquired resistance to the mono-HER1 inhibitor CTX, the mono-HER2 inhibitor trastuzumab (Tzb) or the dual HER1/HER2 inhibitor lapatinib (LPT). Gene expression signatures for EGFR ligands distinctively related to the occurrence of unresponsiveness to CTX, Tzb or LPT, with minimal overlap between them. CTX's molecular functioning largely depended on the overproduction of the mRNAs coding for the EGFR ligands AR and EPI. Thus, a dramatic down-regulation of AR/EPI mRNA expression occurred upon loss of CTX efficacy in EGFR-positive tumor cells with an intact regulation of RAS signaling. Unlike KRAS mutations, which are informative of unresponsiveness to CTX solely in mCRC, our hypothesis-generating data suggest that expression status of AR and EPI mRNAs might be evaluated as dynamic predictors of response in KRAS WT patients receiving any CTX-based therapy.

Expression pattern of class I histone deacetylases in vulvar intraepithelial neoplasia and vulvar cancer: a tissue microarray study.

BACKGROUND: Epigenetic regulation is an important mechanism leading to cancer initiation and promotion. Histone acetylation by histone deacetylases (HDACs) represents an important part of it. The development of HDAC inhibitors has identified the utility of HDACs as a therapeutic target. Little is known about the epigenetic regulation of vulvar intraepithelial neoplasia (VIN) and vulvar squamous cell cancer (VSCC). In this study, the expression of class I HDACs (HDAC 1, 2 and 3) was compared in a series of VIN and VSCC tissues. METHODS: A tissue micro array (TMA) with specimens from 106 patients with high-grade VIN and 59 patients with vulvar cancer was constructed. The expression of HDACs 1, 2 and 3 were analyzed with immunohistochemistry (IHC). The nuclear expression pattern was evaluated in terms of intensity and percentage of stained nuclei and was compared between vulvar preinvasive lesions and vulvar cancer. RESULTS: HDAC 2 expression was significantly higher in VIN than in VSCC (p < 0.001, Fisher's test). Also, 88.7% (n = 94/106) of VIN samples and only 54.5% (n = 31/57) of VSCC samples were scored at the maximum level. Conversely, HDAC 3 expression was significantly higher in VSCC (93%, 53/57) compared to VIN (73.6%, 78/106, p = 0.003), whereas only a small difference in the expression of HDAC 1 was found between these two entities of vulvar neoplasia. CONCLUSIONS: These results suggest that epigenetic regulation plays a considerable role in the transformation of VIN to invasive vulvar neoplasia.

A bioanalytical investigation on the exquisitely strong in vitro potency of the EGFR-DNA targeting type II combi-molecule ZR2003 and its mitigated in vivo antitumour activity.

ZR2003 is a type II of combi-molecule designed to target DNA and the epidermal growth factor receptor (EGFR) without requirement for hydrolysis. In human tumour cell lines cultured as monolayers, it showed 6.5-35 fold greater activity than Iressa. Further evaluation in 3D organ-like multilayer aggregates showed that it could block proliferation at submicromolar level. However, despite the superior potency of ZR2003 over Iressa in vitro, its activity xenograft models was not significantly different from that of Iressa. To rationalize these results, we determined the tumour concentration of both ZR2003 and Iressa in vivo and more importantly in vitro in multicellular aggregates. The results showed that in A431 and 4T1 xenografts, the level of ZR2003 absorbed in the tumours were consistently 2-fold less than those generated by Iressa. Likewise, in the multicellular aggregates model, the penetration of ZR2003 was consistently lower than Iressa. In serum containing media, the level of extractable or free ZR2003 was also inferior to those of Iressa. The results from this bioanalytical study, suggest that the discrepancy between the in vitro and in vivo potency of ZR2003 when compared with Iressa, may be imputed to its significantly lower tumour concentration.

Expression of indoleamine 2,3-dioxygenase predicts shorter survival in patients with vulvar squamous cell carcinoma (vSCC) not influencing on the recruitment of FOXP3-expressing regulatory T cells in cancer nests.

OBJECTIVE: Regulatory T cells (Tregs), and the enzyme indoleamine 2,3-dioxygenase (IDO), have potential regulatory properties for immune escape in cancer. Inhibitors of IDO are available and could potentially be used in vulvar cancer if IDO was proved to drive progression of the disease. The aim of this study was to evaluate the expression of factor forkhead boxP3 (FOXP3), a marker of Tregs, and IDO in vulvar squamous cell carcinoma (vSCC), and to verify their prognostic significance. METHODS: 76 primary tumors and 35 lymph node metastases derived from 76 patients with full clinical history were analyzed. The intratumoral infiltration of Tregs and IDO expression within cancer were evaluated by immunohistochemistry. RESULTS: The number of Tregs in primary tumor and in corresponding lymph node metastasis was significantly correlated. Intensity of Treg infiltrates in the primary and metastatic sites was not correlated to IDO expression and had no influence on the overall patient survival. High IDO expression was associated with significantly worse overall survival among vSCC patients and was found to be an independent prognostic factor similarly to the tumor grade and patient's age. CONCLUSIONS: The degree of intratumoral Treg infiltrates is an individual feature and remains stable throughout the course of the disease without impact on the patient's survival. IDO expression predicts shorter survival of vSCC patients. If immunologic tolerance of the tumor is promoted by the overexpression of IDO it will not influence the number of intratumoral Tregs. IDO expression seems to be an independent prognostic factor in patients with vSCC.

Serum carbonic anhydrase IX and its prognostic relevance in vulvar cancer.

INTRODUCTION: Therapeutic options in advanced or recurrent vulvar cancer are limited. The identification of new prognostic factors and markers for therapy stratification is therefore highly desirable. Carbonic anhydrase IX (CAIX) is up-regulated in various solid tumors and a promising new target. We therefore determined CAIX serum concentration and its prognostic relevance in correlation to intratumoral CAIX expression in patients with primary vulvar cancer. METHODS: Thirty-one serum samples of patients with primary vulvar cancer were prospectively collected before surgery and analyzed for CAIX by enzyme-linked immunosorbent assay. In addition, intratumoral CAIX expression was determined by immunohistochemistry and correlation with serum CAIX and clinicopathological factors, and outcome was analyzed. RESULTS: Preoperative serum concentration of CAIX ranged between 56 and 879 pg/mL (median, 147 pg/mL; mean, 237.29) and was significantly higher in patients with high intratumoral expression (median, 269 pg/mL vs 126 pg/mL, P = 0.03). High serum CAIX was not associated with any of the analyzed clinicopathological parameters. However, disease-free survival was shorter in patients with high preoperative serum CAIX (above median; P = 0.012). By immunohistochemistry, 26% of the tumors showed a moderate or strong expression of CAIX, whereas 74% showed weak or no expression. High intratumoral expression of CAIX was also associated with unfavorable disease-free survival (P = 0.043). CONCLUSIONS: Carbonic anhydrase IX serum concentration is higher in patients with high intratumoral expression, and elevated preoperative serum values are associated with unfavorable prognosis. Serum CAIX might therefore be an easily assessable marker to stratify patients for adjuvant therapy and potentially monitor response. Carbonic anhydrase IX is differentially expressed in vulvar cancer and potentially associated with negative outcome.

Overexpression of CDC25B, CDC25C and phospho-CDC25C (Ser216) in vulvar squamous cell carcinomas are associated with malignant features and aggressive cancer phenotypes.

BACKGROUND: CDC25 phosphatases are important regulators of the cell cycle. Their abnormal expression detected in a number of tumors implies that their dysregulation is involved in malignant transformation. However, the role of CDC25s in vulvar cancer is still unknown. To shed light on their roles in the pathogenesis and to clarify their prognostic values, expression of CDC25A, CDC25B and CDC25C in a large series of vulvar squamous cell carcinomas were examined. METHODS: Expression of CDC25A, CDC25B, CDC25C and phosphorylated (phospho)-CDC25C (Ser216) were examined in 300 vulvar carcinomas using immunohistochemistry. Western blot analysis was utilized to demonstrate CDC25s expression in vulvar cancer cell lines. Kinase and phosphatase assays were performed to exclude cross reactivity among CDC25s isoform antibodies. RESULTS: High nuclear CDC25A and CDC25B expression were observed in 51% and 16% of the vulvar carcinomas, respectively, whereas high cytoplasmic CDC25C expression was seen in 63% of the cases. In cytoplasm, nucleus and cytoplasm/nucleus high phospho-CDC25C (Ser216) expression was identified in 50%, 70% and 77% of the carcinomas, respectively. High expression of CDC25s correlated significantly with malignant features, including poor differentiation and infiltration of vessel for CDC25B, high FIGO stage, presence of lymph node metastases, large tumor diameter, poor differentiation for CDC25C and high FIGO stage, large tumor diameter, deep invasion and poor differentiation for phospho-CDC25C (Ser216). In univariate analysis, high expression of phospho-CDC25C (Ser216) was correlated with poor disease-specific survival (p = 0.04). However, such an association was annulled in multivariate analysis. CONCLUSIONS: Our results suggest that CDC25C and phospho-CDC25C (Ser216) play a crucial role and CDC25B a minor role in the pathogenesis and/or progression of vulvar carcinomas. CDC25B, CDC25C and phospho-CDC25C (Ser216) were associated with malignant features and aggressive cancer phenotypes. However, the CDC25s isoforms were not independently correlated to prognosis.

Increased matrix metalloproteinases 2 and 9 and tissue inhibitor of matrix metalloproteinase 2 expression is associated with progression from vulvar intraepithelial neoplasia to invasive carcinoma.

OBJECTIVE: The expression of matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) and tissue inhibitors of matrix metalloproteinases 1 (TIMP-1) and 2 (TIMP-2) in vulvar intraepithelial neoplasia (VIN I-III) and in vulvar invasive carcinoma were evaluated. DESIGN: A retrospective study. SETTING: Oulu University Hospital, Finland. SAMPLE: The study population consisted of 68 patients with vulvar neoplasia (13 VIN I, 5 VIN II, 6 VIN III and 44 squamous cell carcinomas). METHODS: Paraffin-embedded tissue samples were examined by immunohistochemistry. MAIN OUTCOME MEASURES: MMP-2, MMP-9, TIMP-1 and TIMP-2 expression in VIN compared to vulvar carcinoma. RESULTS: In VIN I-III MMP-2 expression was positive in 13%, MMP-9 in 13%, TIMP-1 in 50% and TIMP-2 in 17% of patients. The positive expressions in patients with vulvar carcinoma were 52% for MMP-2, 36% for MMP-9, 41% for TIMP-1 and 78% for TIMP-2. CONCLUSIONS: We conclude that over-expression of MMP-2, MMP-9 and TIMP-2 may be associated with the progression from VIN to invasive vulvar squamous cell carcinoma.

Assessment of promising protein markers for vulva cancer.

OBJECTIVES: To validate the results of a previous study with the tissue microarray technology showing that cyclooxygenase 2 (COX-2) overexpression and absent caspase 3 expression are associated with poor disease-specific survival in univariate analysis. METHODS: The study group comprised 80 consecutive patients with vulva cancer treated in the period from 1999 to 2003 in a university hospital. A tissue microarray with 3 tumor tissue cores per patient was constructed and stained with antibodies against COX-2, caspase 3, epidermal growth factor receptor, p16 INK4, cyclin D1, and Ki-67. The impact of the expression of these protein markers and selected clinicopathologic variables on disease-specific as well as disease-free survival was measured. Cox proportional hazard model was used for both univariate and multivariate analyses. RESULTS: In multivariate analysis, lymph node metastases and strong COX-2 expression were related to disease-free (hazard ratio [HR], 8.33, 95% confidence interval [CI], 2.97-23.36; P < 0.001; and HR, 6.42; 95% CI, 2.33-17.72; P < 0.001) and disease-specific survival (HR, 6.04; 95% CI, 2.12-17.19; P = 0.001; and HR, 5.11; 95% CI, 1.82-14.36; P = 0.002). In the present series, no association was found between caspase 3 expression and survival. CONCLUSION: The prognostic significance of COX-2 overexpression was confirmed. In contrast, in the present series, no relation was found between caspase 3 expression and survival.

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.

Decreased survival in EGFR gene amplified vulvar carcinoma.

OBJECTIVE: We undertook an extensive molecular characterization of the epidermal growth factor receptor (EGFR) gene in vulvar squamous cell carcinomas to investigate EGFR mutation and/or genomic amplification and its association with EGFR protein expression, high-risk human papillomavirus (HPV) status and clinical outcome. METHODS: A cohort of 51 vulvar cancer patients distributed across all FIGO stages was selected for immunohistochemistry (IHC) and fluorescence in situ hybridization. EGFR expression and gene amplification were correlated with high-risk HPV status, EGFR mutational status and clinical prognostic variables. Fisher's exact tests, Kaplan-Meier survival estimates and a Cox proportional-hazards model were utilized. RESULTS: EGFR gene amplification and chromosome 7 high polysomy were observed in 12% and 6% of cases, respectively. IHC of malignant tissue with 3+ staining demonstrated 100% sensitivity and 79% specificity to detect EGFR gene amplification, yielding a 39% positive predictive value. Decreased survival (p<0.025) was observed in patients with gene amplification, and was associated with a more statistically robust 3.3 hazard ratio (p<0.005) in the Cox proportional-hazards model that controlled for age at diagnosis, stage and lymph node metastasis. Univariate analysis confirmed that EGFR gene amplification was associated with the absence of high-risk HPV (p<0.001). Common activating EGFR gene mutations were not identified. CONCLUSION: A subset of patients with vulvar squamous cell carcinoma was identified with EGFR gene amplification that was HPV-independent and associated with poor prognosis. Given the association of EGFR amplification with response to targeted therapy in other tumor types, these patients may be candidates for therapeutic strategies that target the EGFR pathway.

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.

The role of angiogenesis and COX-2 expression in the evolution of vulvar lichen sclerosus to squamous cell carcinoma of the vulva.

OBJECTIVES: We aimed to determine whether premalignant changes in vulvar lichen sclerosus (LS) could be identified by analysing markers of angiogenesis and the expression of the enzyme cyclooxygenase-2 (COX-2). METHODS: Eight cases of histologically diagnosed vulvar LS, which showed an evolution to carcinoma of the vulva histologically documented, were compared to 10 cases of vulvar LS, for which follow-up information was available for at least 9 years, and to 10 cases of LS adjacent to squamous cell carcinoma (SCC) of the vulva. The microvessel density (MVD), and the expression of vascular endothelial growth factor (VEGF) and of COX-2 were analysed. RESULTS: Difference of MVD between unchanged LS cases and LS cases evolving to SCC and LS adjacent to SCC cases was statistically significant (P=0.008, Wilcoxon Mann-Whitney test). Difference of VEGF and COX-2 expression between unchanged LS cases and LS cases evolving to SCC and LS adjacent to SCC cases were statistically significant (P=0.007 and P=0.01, respectively; Fisher's exact test). CONCLUSIONS: Our study addresses the possibility that immunohistochemical studies may add information to permit the identification of LS as a precursor lesion that has a greater potential to evolve into SCC. These data may identify characteristics of vulvar LS disclosing alterations that indicate the further development to cancer; therefore, it may allow the identification of a group of LS patients who need a careful follow-up and adjunctive biopsies.

Increased cyclooxygenase-2 expression associated with inflammatory cellular infiltration in elderly patients with vulvar cancer.

As the relationship between inflammation and carcinogenesis grows stronger, the role of cyclooxygenase-2 (COX-2) and epidermal growth factor (EGFR) has been highlighted in the pathogenesis and progression of human cancer. In view of the fact that vulvar cancer is characterized by precancerous inflammatory changes in elderly patients, the expressions of COX-2 and EGFR are expected to show different patterns of distribution according to age and other prognostic factors. To verify whether there was a relationship between their expression and clinicopathologic parameters in vulvar cancer, we investigated the inflammatory cellular infiltration and the expression of COX-2 and EGFR by immunohistochemical analysis. Eleven of 19 samples (57.8%) were stained positive for COX-2, and 17 (89.4%) for EGFR. The portion of inflammatory cellular infiltration in adjacent normal tissue was also higher in the older age group, and showed a strong correlation with COX-2 positivity (P = 0.002). Furthermore, COX-2 expression was significantly more frequent in patients over 60 years of age compared to those under 50 years (P = 0.009). COX-2 expression was noted to be high in moderate and well-differentiated cases, whereas, poorly differentiated carcinoma was negative for COX-2 expression (P = 0.023). However, EGFR expression was not differently distributed on the basis of stage, age, tumor grading, or presence of lymph node metastasis. Our article suggests that vulvar cancer in elderly patients may be associated with inflammation, and thus with increased COX-2 expression. In light of these findings, a clinical trial designed to assess the addition of COX-2 targeted therapy to conventional treatment in vulvar cancer would be helpful for consideration of additional treatment options and possibly avoiding the serious surgical morbidity in elderly patients.

Cyclooxygenase 2 expression in cervical intraepithelial neoplasia and vulvar cancer.

OBJECTIVE: The purpose of this study is to describe the expression of cyclooxygenase (COX) 2 cervical intraepithelial neoplasia (CIN) and vulvar cancer specimens. MATERIALS AND METHODS: Archived tissues from 21 cases including 6 cases negative for CIN (no CIN), 71 low-grade (CIN 1) diseases, 8 high-grade (CIN 2, 3) diseases, and 14 vulvar cancer cases were examined. Immunohistochemistry was evaluated in COX-2 expression in tissue using an isoform-specific COX-2 polyclonal antibody. Specimens were assigned an immunohistochemical score for intensity of staining and the percent of cells stained. The slides were scored by 2 independent pathologists and compared across histological categories. RESULTS: A greater proportion of cells were stained in specimens with high-grade CIN (p = 0.01). Staining intensity was not statistically different among the 3 groups. Higher scores were found for vulvar cancer as compared with normal vulva (p = 0.01). CONCLUSIONS: The increase in COX-2 in cervical cancer precursors may provide a potential target for prevention studies.

Cyclooxygenase-2 (COX-2) immunostaining does not correlate with the degree of vulvar neoplasia.

OBJECTIVE: The cyclooxygenase-2 (COX-2) enzyme is up-regulated in inflammatory and neoplastic conditions. In the last decade, its biological role has been investigated in various pre-invasive and invasive cancers with the hope that it can serve as a target for cancer prevention and treatment. METHODS: We evaluated the expression of COX-2 in vulvar biopsies to determine its relationship to the degree of dysplasia. COX-2 expression was studied by immunohistochemistry in 62 consecutive vulvar biopsies divided into four diagnostic groups. Group 1 included inflamed vulva (n = 14); group 2, vulvar intraepithelial neoplasia (VIN) I and VIN II (n = 20); group 3, VIN III and carcinoma in situ (n = 18); and group 4, invasive squamous cell carcinoma (n = 10). Representative sections were immunostained using polyclonal anti-COX-2 antibodies at concentration 1:25 without pretreatment. Immunostaining was scored according to the proportion of positive epithelial cells in the vulvar mucosa as 0 (no positive cells), 1(< 5% positive), 2 (6-50% positive), or 3 (> 50% positive). RESULTS: Mean immunostaining scores were 1.6, 1.4, 0.7, and 1.2 for groups 1, 2, 3, and 4, respectively. Scores were different between the groups (chi(2) = 9.908, P = 0.019) as shown by Cochran-Mantel-Haenszel statistical analysis (modified ridit scores), but did not correlate with age or the degree of dysplasia. The strongest staining for COX-2 was in the inflammatory group. CONCLUSION: COX-2 staining in inflamed, dysplastic, and malignant vulvar epithelium is variable but, as shown in this study, does not correlate with the degree of vulvar dysplasia or malignancy.