The EphA2 receptor tyrosine kinase is frequently overexpressed in invasive breast cancer cells. Moreover, these malignant cells have unstable cell-cell contacts, which preclude EphA2 from interacting with its ligand, EphrinA1, which is anchored to the membrane of adjacent cells. This defect is important because ligand binding causes EphA2 to transmit signals that negatively regulate tumor cell growth and survival, whereas the absence of ligand binding favors these same behaviors. In our present study, human adenoviral type 5 (HAd) vectors were engineered to express secreted-forms of EphrinA1. These vectors were used to infect MDA-MB-231 human breast cancer cells, or MCF-10A human breast epithelial cells providing matched controls. Infection with HAd-EphrinA1-Fc (HAd vector expressing extracellular domain of human EphrinA1 attached to Fc portion of human IgG 1 heavy chain) caused increased EphA2 activation and turnover and consequently decreased tumor cell viability in soft agar assays. Consistent with this observation, infection of MDA-MB-231 cells with HAd-EphrinA1-Fc prevented tumor formation in xenograft models. Furthermore, therapeutic modeling via intratumoral inoculation revealed that HAd-EphrinA1-Fc significantly inhibited subsequent tumor growth as compared to matched controls. These results suggest that targeting of EphA2 with adenoviral vectors may have therapeutic value.

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Aim To determine whether BAP1 gene and protein expression associates with different prognostic parameters in uveal melanoma and whether BAP1 expression correctly identifies patients as being at risk for metastases, following enucleation of the primary tumour. Methods Thirty cases of uveal melanoma obtained by enucleation between 1999 and 2004 were analysed for a variety of prognostic markers, including histological characteristics, chromosome aberrations obtained by fluorescence in situ hybridisation (FISH) and single nucleotide polymorphism (SNP) analysis and gene expression profiling. These parameters were compared with BAP1 gene expression and BAP1 immunostaining. Results The presence of monosomy of chromosome 3 as identified by the different chromosome 3 tests showed significantly increased HRs (FISH on isolated nuclei cut-off 30%: HR 11.6, p=0.002; SNP analysis: HR 20.3, p=0.004) for death due to metastasis. The gene expression profile class 2, based on the 15-gene expression profile, similarly provided a significantly increased HR for a poor outcome (HR 8.5, p=0.005). Lower BAP1 gene expression and negative BAP1 immunostaining (50% of 28 tumours were immunonegative) were both associated with these markers for prognostication: FISH cut-off 30% monosomy 3 (BAP1 gene expression: p=0.037; BAP1 immunostaining: p=0.001), SNP-monosomy 3 (BAP1 gene expression: p=0.008; BAP1 immunostaining: p=0.002) and class 2 profile (BAP1 gene expression: p<0.001; BAP1 immunostaining: p=0.001) and were themselves associated with an increased risk of death due to metastasis (BAP1 gene expression dichotomised: HR 8.7, p=0.006; BAP1 immunostaining: HR 4.0, p=0.010). Conclusions Loss of BAP1 expression associated well with all of the methods currently used for prognostication and was itself predictive of death due to metastasis in uveal melanoma after enucleation, thereby emphasising the importance of further research on the role of BAP1 in uveal melanoma.

ABBREVIATIONS RASSF1ARAS association domain family protein 1A RARβ retionic acid receptor β MSP methylation specific PCR ERα estrogen receptor α PRB progesterone receptor B ACKNOWLEDGEMENTSWe are grateful to Department of Biotechnology, India, for providing the financial support to conduct this study. S.M. is grateful to University Grant Commission for award of Junior Research Fellowship. The authors thank Dr. Cecile Rochette Egly for the kind gift of RARβ monoclonal antibody. Research PaperDetection of RASSF1A and RARβ Hypermethylation in Serum DNA from Breast Cancer Patients ABSTRACTBreast cancer is fast emerging as the leading cancer amongst females, especially in young females in metropolitan cities in India. The epigenetic alterations involved in the onset and progression of breast cancer may serve as biomarkers for early detection and prognosis of the disease. Furthermore, using body fluids such as serum offers a noninvasive method to procure multiple samples for such analyses. In this study, we examined methylation status of two normally unmethylated but biologically significant cancer genes, RAS association domain family protein 1A (RASSF1A) and retionic acid receptor β (RARβ) by methylation specific PCR (MSP) in invasive ductal carcinomas of the breast and paired serum DNA. RASSF1A was found to be methylated in 17 of 20 (85%) breast tumors; while sera from 15 of 20 (75%) of the patients showed concordant methylated RASSF1A, with a sensitivity of 88%. RARβ was methylated in 2/20 (10%) breast tumors. A gene unmethylated in the tumor DNA was always found to be unmethylated in the matched serum DNA for both RASSF1A and RARβ genes; hence specificity was 100%. Immunohistochemical analysis of RARβ protein in 15 breast carcinoma patients harboring unmethylated RARβ in tumors and serum DNA showed the expression of RARβ protein in tumors and paired normal breast tissues, confirming the MSP findings, suggesting that RARβ promoter is functional in these cases. This study underscores the potential utility of DNA methylation based screening of serum, a readily accessible body fluid, as a surrogate marker for early detection of breast cancer.

Distinction of primary skin adnexal carcinomas from cutaneous metastasis of adenocarcinomas is challenging. In this study, we evaluated podoplanin immunoreactivity in a series of primary skin adnexal tumors and adenocarcinomas metastatic to skin using a D2-40 antibody. The initial test series were composed of a total of 93 cases including 32 primary skin adnexal carcinomas, 46 benign primary adnexal tumors, and 15 cutaneous metastatic adenocarcinomas. We found that variable D2-40 reactivity was seen in all of the primary cutaneous carcinomas including sebaceous carcinomas (10/10), squamous cell carcinomas (10/10), porocarcinomas (4/4), trichilemmal carcinomas (4/4), skin adnexal carcinomas not otherwise specified (4/4), and in the majority of benign skin adnexal tumors. In contrast, no podoplanin immunoreactivity was seen in any of the 15 (0/15) cutaneous metastases. To confirm the initial findings and to further explore the utility of podoplanin reactivity in the distinction of these tumors, we also examined a test set of 35 unknown cases, including 21 adenocarcinomas metastatic to skin and 14 primary adnexal tumors, in a blinded fashion. In this test set of cases, podoplanin was negative in 22 cases and positive in 13 cases. Of the 22 podoplanin negative cases, 20 were proven to be metastatic adenocarcinoma. Of the 13 D2-40 positive cases, 12 were proven to be primary adnexal tumors. Our results suggest that podoplanin can be a useful tool to distinguish primary skin adnexal carcinomas form adenocarcinomas metastatic to skin with high sensitivity (94.5%) and specificity (97.2%).