Koos Boeve

153 Methylation biomarkers for the detection of tumor DNA in saliva technical validation. The design and technical validation of C11orf85 , KCNA5 and SIPA1 QMSP was optimal for further analysis. In addition, based on literature search we included EDNRB [19] , HOXA9 [24] , NID2 [24] and TIMP3 [20] , as these genes were reported to be associated with OSCC in saliva. Technical validation of OSCC-specific methylation markers to detect tumor cells in saliva from patients with OSCC To select methylation markers with high sensitivity and high specificity, we collected a total of 2 ml of saliva from 10 patients with OSCC and from 10 non-cancer controls (five orthognathic and five dental implant patients) referred to as controls in this study (Table 1). Median amount of isolated DNA from saliva was 64 µg (range: 6 to 140 µg) among the OSCC patients, 32 µg (range: 16 to 75 µg) among the orthognathic patients and 32 µg (range: 20 to 57 µg) among the dental implant patients (Table 1). There were no significant differences in DNA yield from the pellets between the OSCC, orthognathic and dental implant patients. QMSP analysis of the seven selected methylation markers on bisulfite-treated DNA from saliva cells from 10 OSCC patients and 10 controls, revealed significant differences in methylation levels of EDNRB (p = 0.016) and KCNA5 (p < 0.001) (Figure 2). In fact, methylation of C11orf85 , HOXA9, NID2 and SIPA1 was detected in all controls and methylation of EDNRB in 50% of the controls (not associated with age) (Figure 2). Five control patients were significantly younger than the OSCC patients (Table 1). Comparing QMSP data from saliva from OSCC with either controls of similar or younger age, revealed a difference for only KCNA5 methylation (both OSCC-patients vs younger or older controls p = 0.001) and EDNRB methylation (only OSCC vs younger controls p = 0.003) (data not shown). Age-matched analysis did not affect the results of the other methylation markers. One explanation for the fact that not all markers were hypermethylated in saliva cells of OSCC patients compared to saliva cells of controls could be that the original tumor is not methylated for each of these methylation markers. To evaluate the effect on the sensitivity and NPV of detecting tumor cells in saliva in patients with methylated tumor tissues, the methylation status of these seven markers was tested in available tumor tissues of these same 10 OSCC patients. Methylation of four markers ( EDNRB, C11orf85, KCNA5 and SIPA1) was detected in all 10 tumor tissues (Supplementary data 2). Methylation was detected in nine ( HOXA9 ) and seven ( NID2 and TIMP3 ) of the 10 tumor tissues (Supplementary data 2). When performing the analysis with OSCC cases showing methylation of tumor tissue of HOXA9, NID2 or TIMP3, no differences were found in methylation levels in saliva between OSCC cases and controls (data not shown).