The whole saliva sample was collected for a 5-minute
period using a cotton wool swab inserted in the mouth (Salivette®, Sarstedt AG & Co., Nümbrecht, Oberbergischer Kreis, Germany). The saliva sample was subsequently diluted (1:1) in a PBS solution containing protease inhibitors (0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA, and 0.01 mg/mL aprotinin A) and 0.05% Tween-20 and was stored at -20°C until analysis. Sections of formalin-fixed, paraffin-embedded incisional biopsy specimens of the tumor were evaluated by H&E staining and used for immunohistochemistry. The histological grade of malignancy was performed employing two parameters of a recognized grading system: degree ATM Kinase Inhibitor price of keratinization and nuclear pleomorphism [11]. ELISA Salivary protein levels were measured by sandwich ELISA, in accordance with the procedures recommended by the manufacturers. The following kits were used: Epidermal Growth Factor Receptor (CBA 018) and c-erbB2/c-neu Rapid Format ELISA kit (QIA10), both from Calbiochem® (Darmstadt, Hessen, Germany) and Human EGF (DuoSet, R&D Systems, Minneapolis, EPZ-6438 manufacturer MN, USA). The total protein content in the saliva was determined using the Bradford method [12] (Sigma, Saint Louis, MO, USA) according to the BSA standard (Fermentas Life Sciences, Vilnius, Lithuania). The total protein content was
used to normalize the EGF, EGFR, and Her-2 values for each sample. Immunohistochemistry Cobimetinib manufacturer (IHC) IHC reactions for the detection of EGFR and Her-2 antigens were performed using the monoclonal antibodies clone 31G7 (Zymed Laboratories Inc., San Francisco, CA, USA) and clone CB11 (Novocastra Laboratories, Newcastle upon Tyne, UK), respectively. Sections
of oral mucosa and breast carcinoma were used as EGFR and Her-2 AR-13324 positive controls, respectively. Evaluation of IHC EGFR expression was evaluated on the basis of extent and intensity of immunolabeling in tumor cell membranes, classified on a four-point scale: 0 (no labeling, or labeling in < 10% of tumor cells); 1 (weak labeling, homogeneous or patchy, in > 10% of the tumor cells); 2 (moderate labeling, homogeneous or patchy, in > 10% of the tumor cells); 3 (intense labeling, homogeneous or patchy, in > 10% of the tumor cells). These scores were subsequently grouped into two categories: negative (0 or 1) and positive labeling (2 or 3) [13]. The Her-2 protein immunoexpression was analyzed using the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines for Her-2 testing in breast cancer (0, no staining or membrane staining is observed in < 10% of the tumor cells; 1+, faint/barely perceivable membrane staining is detected in > 10% of the tumor cells, and only part of the membrane is stained; 2+, weak to moderate complete membrane staining is observed in > 10% of the tumor cells; 3+, strong complete membrane staining is observed in > 30% of the tumor cells).