title: Methods to identify molecular expression of mTOR pathway: a rationale approach to stratify patients affected by clear cell renal cell carcinoma for more likely response to mTOR inhibitors.
abstract: Since target therapy with mTOR inhibitors plays an important role in the current management of clear cell renal cell carcinoma (RCC), there is an increasing demand for predictive biomarkers, which may help to select patients that are most likely to benefit from personalized treatment. When dealing with formalin-fixed paraffin-embedded (FFPE) cancer tissue specimens, several techniques may be used to identify potential molecular markers, yielding different outcome in terms of accuracy. We sought to investigate and compare the capability of three main techniques to detect molecules performing an active function in mTOR pathway in RCC. Immunohistochemistry (IHC), Western blot (WB) and immunofluorescence (IF) analyses were performed on FFPE RCC tissue specimens from 16 patients by using the following mTOR pathway-related: mTOR (Ser235/236), phospho-mTOR (p-mTOR/Ser2448), phospho-p70S6k (p-p70S6k/Thr389), both monoclonal and polyclonal, phospho-S6Rb (p-S6Rb) and phospho-4EBP1 (p-4EBP1/Thr37/46). No single molecule was simultaneously revealed by all three techniques. Only p-p70S6k was detected by two methods (IHC and IF) using a monoclonal antibody. The other molecules were detected exclusively by one technique, as follows: p-mTOR and polyclonal p-p70S6K by IHC, p70S6K, p-S6Rb and p-4EBP1 by WB, and, finally, mTOR by IF. We found significant differences in detecting mTOR pathway-related active biomarkers by using three common techniques such as IHC, WB and IF on RCC samples. Such results have important implications in terms of predictive biomarker testing, and need to be related to clinical end-points such as responsiveness to targeted drugs by prospective studies.
authors.affiliation: Departments of Pathology and Diagnostic, University of Verona Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Fiorini
authors.affiliation: Medical Oncology, Azienda Ospedaliera Universitaria Integrata di Verona AOUI, University of Verona Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Massari
authors.affiliation: Departments of Pathology and Diagnostic, University of Verona Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Pedron
authors.affiliation: Departments of Pathology and Diagnostic, University of Verona Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Sanavio
authors.affiliation: Medical Oncology, Azienda Ospedaliera Universitaria Integrata di Verona AOUI, University of Verona Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Ciccarese
authors.affiliation: Urologic Clinic, Department of Oncological and Surgical Sciences, University of Verona Italy.
authors.forename: Antonio Benito
authors.initials: AB
authors.lastname: Porcaro
authors.affiliation: Urologic Clinic, Department of Oncological and Surgical Sciences, University of Verona Italy.
authors.forename: Walter
authors.initials: W
authors.lastname: Artibani
authors.affiliation: Department of Medicine, Università di Verona Verona, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Bertoldo
authors.affiliation: Departments of Pathology and Diagnostic, University of Verona Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Zampini
authors.affiliation: Medical Oncology, Azienda Ospedaliera Universitaria Integrata di Verona AOUI, University of Verona Italy.
authors.forename: Teodoro
authors.initials: T
authors.lastname: Sava
authors.affiliation: Departments of Pathology and Diagnostic, University of Verona Italy.
authors.forename: Miriam
authors.initials: M
authors.lastname: Ficial
authors.affiliation: Departments of Pathology and Diagnostic, University of Verona Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Caliò
authors.affiliation: Departments of Pathology and Diagnostic, University of Verona Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Chilosi
authors.affiliation: Anatomic Pathology, Card. G. Panico Hospital Tricase (LE), Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: D'Amuri
authors.affiliation: Pathology Unit, Department of Clinical and Experimental Medicine, University of Foggia Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Sanguedolce
authors.affiliation: Medical Oncology, Azienda Ospedaliera Universitaria Integrata di Verona AOUI, University of Verona Italy.
authors.forename: Giampaolo
authors.initials: G
authors.lastname: Tortora
authors.affiliation: Departments of Pathology and Diagnostic, University of Verona Italy ; ARC-Net Research Center, Università di Verona and Azienda Ospedaliera Universitaria Integrata di Verona AOUI Verona, Italy.
authors.forename: Aldo
authors.initials: A
authors.lastname: Scarpa
authors.affiliation: Department of Pathology and Molecular Medicine, Wellington School of Medicine, University of Otago Wellington, New Zealand.
authors.forename: Brett
authors.initials: B
authors.lastname: Delahunt
authors.affiliation: Medical Oncology, IRCSS Ospedale San Matteo Pavia, Italy.
authors.forename: Camillo
authors.initials: C
authors.lastname: Porta
authors.affiliation: Departments of Pathology and Diagnostic, University of Verona Italy.
authors.forename: Guido
authors.initials: G
authors.lastname: Martignoni
authors.affiliation: Departments of Pathology and Diagnostic, University of Verona Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Brunelli
keywords: Western blot; clear cell renal cell carcinoma; immunofluorescence analysis; immunohistochemical; mTOR; methods; p-mTOR; p-p70S6k