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Ruppen I.,Tumor Markers Group | Grau L.,Tumor Markers Group | Orenes-Pinero E.,Tumor Markers Group | Ashman K.,Spanish National Cancer Research Center | And 4 more authors.
Molecular and Cellular Proteomics | Year: 2010

KiSS-1 is a metastasis suppressor gene reported to be involved in the progression of several solid neoplasias. The loss of KiSS-1 gene expression has been shown to be inversely correlated with increasing tumor stage, distant metastases, and poor overall survival in bladder tumors. To identify the molecular pathways associated with the metastasis suppressor role of KiSS-1 in bladder cancer, we carried out a proteomics analysis of bladder cancer cells (EJ138) transiently transfected with a vector encompassing the full-length KiSS-1 gene using an iTRAQ (isobaric tags for relative and absolute quantitation) approach. Protein extracts collected after 24- and 48-h transfection were fractionated and cleaved with trypsin, and the resulting peptides were labeled with iTRAQ reagents. The labeled peptides were separated by strong cation exchange and reversed phase LC and analyzed by MALDI-TOF/TOF MS. Three software packages were utilized for data analysis: ProteinPilot for identification and quantification of differentially expressed proteins, Protein Center for gene ontology analysis, and Ingenuity Pathways Analysis to provide insight into biological networks. Comparative analysis among transfected, mock, and empty vector-exposed cells identified 1529 proteins with high confidence (>99%) showing high correlation rates among replicates (70%). The involvement of the identified proteins in biological networks served to characterize molecular pathways associated with KiSS-1 expression and to select critical candidates for verification analyses by Western blot using independent transfected replicates. As part of complementary clinical validation strategies, immunohistochemical analyses of proteins regulated by KiSS-1, such as Filamin A, were performed on bladder tumors spotted onto tissue microarrays (n = 280). In summary, our study not only served to uncover molecular mechanisms associated with the metastasis suppressor role of KiSS-1 in bladder cancer but also to reveal the biomarker role of Filamin A in bladder cancer progression and clinical outcome. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Cubilla A.L.,Institute Patologia e Investigacion | Lloveras B.,Institute cataladOncologia | Lloveras B.,Hospital Of Mar | Alejo M.,Johns Hopkins University | And 16 more authors.
American Journal of Surgical Pathology | Year: 2011

Background: One third to one half of penile squamous cell carcinomas (SCCs) are related to human papillomavirus (HPV) infection. Viral detection is usually carried out by polymerase chain reaction (PCR) or other molecular methods. In this study, we evaluated p16 immunohistochemical expression, which is simpler and less costly, as a potential marker of high-risk HPV (HR-HPV) infection in penile SCC. DESIGN AND METHODS: We pathologically classified 202 invasive penile carcinomas and performed HPV genotyping by short PCR fragment (SPF)10 PCR and p16 immunohistochemistry. We also evaluated HPV and p16 according to the histologic subtypes of penile SCC. Tumors depicting continuous p16 immunostain in all neoplastic cells were considered positive. HPV and p16 status were compared using classifier performances and concordance indexes. RESULTS: Evidence of HPV (low-risk and high-risk genotypes) was found in 63 cases (31%) by PCR. Fifty-three p16-positive cases were identified (26%). Overexpression of p16 had a sensitivity of 67% and a specificity of 91% for defining the HPV status. Concordance indexes between p16 and HPV status were high (≥78%) in general cases and in all histologic subtypes of penile SCC. The stain was useful in the differential diagnosis of basaloid and low-grade warty carcinomas. Low-risk HPV genotypes were found in 5 tumors, 4 of which were p16 negative. Basaloid and nonbasaloid high-grade (grade 3) SCCs were more likely to be HR-HPV positive when compared with grades 1 to 2 tumors (P<0.000001 and 0.0417, respectively). Conclusions: p16 overexpression was found to be a reliable marker for HR-HPV and a helpful tool in the differential diagnosis of low-grade verruciform and high-grade solid penile tumors. SCC variants depicting basaloid features were more likely to be HPV and p16 positive than low-grade, keratinizing lesions. We also observed a tendency toward HPV positivity in high-grade nonbasaloid tumors. Our results indicated a concordance between HPV and p16 status and this observation may have diagnostic and prognostic implications. Copyright © 2011 by Lippincott Williams & Wilkins.

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