Body fluids such as saliva are potential resources for development of biomarkers for detection, diagnosis, and prognosis of HNSCC. Aberrant promoter hypermethylation has been recently proposed as a means for detection of HNSCC in salivary rinses. Recently, our group published the utility of evaluating the promoter region methylation status of various genes as a tool for detection of HNSCC. In our study, seven genes, comprised of DAPK
, were identified as part of a panel that could distinguish salivary rinses from HNSCC patients and healthy controls 
. With a pilot cohort of 61 HNSCC patients, we also found that the detection of these markers in pretreatment salivary rinse is a likely prognostic indicator for local recurrence and poor survival 
Salivary rinses used for promoter hypermethylation assay in the literature have been collected either with or without an exfoliating brush 
. An exfoliating brush could be used to include cells from deep epithelial layers in the oral cavity and oropharynx. It also allows for a broad sampling of epithelial cells from multiple sites in the upper aerodigestive tract. Although both salivary rinses collected with and without an exfoliating brush has been reported in detection of promoter hypermethylation, the clinical significance of exfoliating brush use in salivary rinse collection for detection of promoter hypermethylation is unknown. There has been no direct study of the correlation of promoter hypermethylation between salivary rinses collected with and without an exfoliating brush.
In this study, we first determined the promoter methylation pattern of seven individual genes, including P16, CCNA1, DCC, TIMP3, MGMT, DAPK, and MINT31, in 57 paired salivary rinses collected with or without an exfoliating brush from patients with HNSCC, and then evaluated the concordance of promoter hypermethylation between salivary rinses collected without brush and those with brush. As shown in , the clinical and pathological characteristics of these 57 patients with HNSCC appeared comparable to the patient cohort we previously published, and were broadly representative of standard clinical practice. To circumvent the possible confounding factors that may be involved in salivary rinse collection, we have collected each pair of salivary rinses (with and without an exfoliating brush) sequentially during one visit to the physician's office. Quantitative methylation-specific PCR was used to detect the promoter hymermethylation in salivary rinse sample DNA. This real-time PCR methodology allows a more objective, robust, and rapid assessment of promoter methylation status. Give the sensitivity of the QMSP technique used to detect the presence of methylated alleles in a background of normal at a threshold of 1/1,000 to 1/10,000, this strategy allowed us to define methylated genes that were highly specific for tumor, and rarely or never present in any of the aerodigestive sites that shed cells in salivary rinses.
We reported that promoter hypermethylation frequencies of P16
, and MINT31
could be detected in salivary rinses collected without an exfoliating brush at levels comparable to those in salivary rinses collected with brush. We showed that the promoter hypermethylation frequencies of these studied genes in salivary rinses collected with and without an exfoliating brush were between 8.8% and 31.6% and between 5.2% and 38.6%, respectively 
Our study also demonstrated a concordance of gene promoter methylation between salivary rinses collected with an exfoliating brush and without brush from patients with HNSCC. In our study, Spearman rank analysis showed a strong correlation for promoter hypermethylation of P16, CCNA1, DCC, MGMT, DAPK, and MINT31, although a weak correlation for TIMP3 promoter hypermethylation was found (The reason why TIMP3 was poorly concordant was unknown.). We also found a strong agreement for promoter hypermethylation of these markers (). Meanwhile, as revealed by Cohen's kappa statistic, we found moderate agreements of promoter methylation at CCNA1, DCC and DAPK and substantial agreements of promoter methylation at P16, MGMT and MINT31. It should be noted that the kappa statistic also depends on the underlying methylation prevalence that may lead may lead to smaller kappa even the percent agreement is higher. In addition, the techniques we used for paired salivary rinse collection may attenuate the concordances of gene promoter methylation to some extent, making it seem like the techniques are less agreeable than they actually are. We don't exclude the possibility that the initial salivary rinses without brushing capture much of the loose epithelial and tumor cells whereas the rinses with brushing capture fewer tumor cells, since there was already a prior rinse. As an additional point, it remain to be investigated whether detection of methylation markers from saliva more than once will increase the total percentage of the positive cases, no matter using brush or not.
The concordance of promoter hypermethylation between salivary rinses collected with and without an exfoliating brush may have biological implications. To date, the mechanism leading to the presence of gene promoter hypermethylation in salivary rinse is not well understood. It is likely that 1) aggressive tumors may undergo increase rate of mechanical dissociation or shedding into salivary rinses. Those tumor with a higher burden of epigenetic alteration would be more frequently detected in salivary rinses; 2) salivary rinse tumor DNA with epigenetic alterations may also originate from cells that have left the primary site and have invaded the circulatory system but are still not capable of metastasis to new organ; 3) premalignant clonal patches expanded will beyond primary tumor location, resulting a large surface area of epigenetically altered cells to shed into the saliva 
. Previous studies hypothesized that salivary rinses collected without an exfoliating brush may not have enough oropharyngeal cells to meet cutoffs for positive biomarker findings in case. Brushing, which are site specific, may help overcome these obstacles 
. However, based on our current study, at least for study of gene promoter methylation in salivary rinses, detection of gene promoter hypermethylation between salivary rinses collected with and without an exfoliating brush is concordant, suggesting that an exfoliating brush is not necessary to be used for salivary rinse collection.
The concordance of promoter hypermethylation between salivary rinses collected with and without an exfoliating brush have potentially important clinical implications. In comparison to collection of salivary rinses with an exfoliating brush for development of epigenetic biomarkers for epigenetic study, there are numerous potential advantages for the collection of salivary rinses without using an exfoliating brush. For instance, the method is non-invasive (the sample is relatively easy and painless to acquire), it has the potential for low cost (no expenses of exfoliating brush), and lends itself to easy administration (it is likely patients can perform this collection by themselves). Thus, the collection of salivary rinses without using an exfoliating brush could be an efficient, cost effective and reliable method for obtaining material for detection of HNSCC related markers.
In summary, the present study compared the detection of similar promoter hypermethylation frequencies of seven individual genes between salivary rinses collected with and without an exfoliating brush from patients with HNSCC. Moreover, our study for the first time demonstrated a strong concordance of gene promoter hypermethylation between salivary rinses collected with and without brush. This study suggests that use of an exfoliating brush may not be necessary for salivary rinse collection for the detection of promoter hypermethylation biomarkers of HNSCC detection.