Koos Boeve

148 Chapter 8 Technical validation of OSCC-methylation markers For the validation, saliva from in total 10 OSCC patients were collected: seven males and three females with a median age of 63 years and with pT1-2 (n = 7) and pT3-4 (n = 3) staged tumors. For methylation status in the original tumor tissue, six fresh frozen (FF) tumor biopsies and nine formalin fixed paraffin embedded (FFPE) tumor resection tissues were available for DNA isolation. Saliva samples were collected from healthy (non-cancer) controls. Five patients were planned to undergo benign corrective jaw surgery (median age 45 years, significant younger than the OSCC patients p = 0.050) and five patients were scheduled to receive dental implants (median age 67 years, age-matched with the OSCC patients). Characteristics of the patients and controls are summarized in Table 1. All patients and controls had no prior history of HNSCC or immunological diseases such as Sjögren’s syndrome and no apparent infections in the oral cavity during saliva collection. Saliva was collected preoperatively on the day of surgery between 07:00 and 10:00 AM to exclude variation due to circadian rhythm. Patients and controls had at least 90 min without stimulation of the salivary glands by drinking, smoking or eating. Patients and controls deposited 2 ml whole saliva into a 15 ml falcon tube without a time limit. Samples were recoded for lab processing. Ethics approval and consent to participate Written approval and informed consent of all twenty patients and controls included in the validation study was obtained. Because of the non-invasive character of saliva sample collection, this research was not a clinical study with human subjects as meant in the Medical Research Involving Human Subjects Act as was concluded by the local Medical Ethics Review Board of the University Medical Center Groningen (M12.116657) and no further approval was required. DNA isolation Saliva DNA integrity was preserved by adding 2.5 ml of 1 tablet Roche Complete mini Protease Inhibitor Cocktail (pro. #. 04693159001) dissolved in 10 ml filtered (4°C) PBS. The saliva PBS mixture was equally divided in three 1.5 ml Eppendorf Tubes and centrifuged at 14000 rpm for 10 min at 4°C. The pellets were incubated in 600 µl 1% SDS-proteinase K. Both the pellet and the supernatant were separately stored at -80°C. Tumor DNA was isolated as follows. Approximately eight 10 µm thick sections were cut from the FFPE blocks. For quality control, the first and last section (3 µm thick) were HE- stained to check for tumor load. A dedicated head and neck pathologist marked areas with >60% neoplastic cells. The 10 µm FFPE sections were deparaffinized using xylene and