HPV and Methylation Indicators in Paired Tumor and Saliva in HNSCC

Human papilloma virus type 16 (HPV16) is a causative agent for some head and neck squamous cell carcinoma (HNSCC) and an independent risk factor for oropharyngeal SCC. The goal of this study was to examine HPV16 associated gene methylation in paired saliva and tumor DNA with assessment of the sensitivity, specificity, positive predictive, and negative predictive value for saliva HPV as a test for HNSCC. HPV16 status was determined by quantitative PCR (qPCR) in 35 primary HNSCC paired tumor and saliva specimens. Tumor cut points ≥0.03 and ≥0.1 and saliva cut points >0 and ≥0.001 were used to classify results as HPV positive or negative. Aberrant methylation was determined by the methylation-specific multiplex ligation probe amplification (MS-MLPA) assay. The frequency of promoter hypermethylation in tumor samples was 66% (23/35) versus 17% (6/35) in saliva. Two of 35 paired tumor and saliva samples had commonly methylated genes. HPV and methylation were correlated for IGSF4 (p=0.01) in tumor samples (cut point ≥0.03) and for ESR1 in saliva samples (cut point >0). Although the sensitivity of HPV detection in saliva was significantly reduced when saliva cut points were increased from >0 to ≥0.001, the specificity and positive predictive values were 100% at saliva cut point of ≥0.001, regardless of tumor cut points. Within clearly defined parameters, HPV detection in saliva DNA shows promise as a non invasive approach for tumor HPV status. Methylated genes detected in saliva may be useful in early detection and as potential predictive markers of HNSCC. Further confirmation and validation in larger cohorts is required.


Introduction
Accurate and reliable stratification of head and neck squamous cell carcinoma (HNSCC) for prediction of outcomes has been challenging, mainly because of the numerous anatomic sites and sub-sites from which tumors can arise.HNSCC affect more than 500,000 people worldwide each year, accounting for 5% of all malignancies, and a gradually increasing rate over the last three decades (Brockstein et al., 2004).In the United States, approximately 40,250 cases of oral cavity and pharynx HNSCC are expected in 2012 with an estimated 7,850 deaths (American Cancer Society [ACS], 2012).
While it is becoming more firmly established that HPV positive HNSCC have better survival outcomes than HPV negative HNSCC (Gillison et al., 2000;SR Schwartz, Yueh, McDougall, Daling, & SM Schwartz, 2001;Weinberger et al., 2006;Ang et al., 2010;Worsham et al., 2013b), believed to be because of better response to chemo radiation (Fakhry et al., 2008), the underlying mechanism for these improved prognosis outcomes remains underexplored.Epigenetic events of promoter hypermethylation represent important tumor-specific markers occurring early in tumor progression.Our group has demonstrated using high-throughput methods, the contribution of both genetic (Worsham et al., 2003;Worsham et al., 2006b) and epigenetic events (Maruya et al., 2004), often working together (Worsham et al., 2006a), in the development and progression of HNSCC.A recent study of over 27,000 CpGs , while supporting differential DNA methylation in HPV-associated HNSCC also indicted epigenetic modulation of signal transduction pathways (Worsham, Chen, Ghanem, Stephen, & Divine, 2013a).Also, studies are beginning to establish a mechanistic role for promoter methylation with improved survival outcomes in HPV positive HNSCC.Gubanova et al. (2012) showed that promoter hypermethylation and concordant low SMG-1 expression correlated not only with HPV positive status and improved patient survival, but also enhanced response to radio therapy in HPV-positive HNSCC cell lines.
Epigenetic alterations identified in saliva and cancerous lesions with biomarker potential would have high clinical significance in risk assessment and early detection.The goal of this study was to examine the utility of saliva as a non-invasive approach to detect high risk HPV and associated aberrantly methylated genes in paired saliva and tumor HNSCC.

Cohort
The study cohort consisted of 35 HNSCC with saliva and tumor tissue from the same patient.Sites in the head and neck included oral cavity (OC), oropharynx (OP), hypopharynx (HP), larynx and other site (consisting of nasopharynx, paranasal sinuses, neck mass, head and neck skin).Patients diagnosed within the Henry Ford Health System with both saliva and tumor tissue collected between 2006 and 2007 were included in the study.Risk factor information was obtained from patient questionnaires and electronic medical records.
This study was approved by the Henry Ford Health System Institutional Review Board committee and informed consent was obtained from all enrolled subjects.

DNA Extraction and HPV Detection
Two milliliters of saliva were collected from each study subject in Oragene DNA kits (DNA Genotek Inc, Ontario, Canada), and saliva DNA was extracted according to the manufacturer's instructions.

Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) Assay
Archival tissue and saliva DNA were interrogated for methylation status using the multi-gene methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay.MS-MLPA (Worsham et al., 2006a;Chen et al., 2007), a modification of the conventional MLPA assay (Schouten et al., 2002) allows for the simultaneous detection of changes in methylation status as well as copy number changes of approximately 41 different DNA sequences in a single reaction requiring only 20ng of human DNA.
Briefly, the MS-MLPA panel in the presence of HhaI detects aberrant promoter hypermethylation by taking advantage of a HhaI site in the gene probes of interest.The control gene probes, without a HhaI site, serve as undigested controls.A normal control DNA sample will generate 41 individual peaks for all probes in the absence of HhaI and 15 separate peaks in the presence of HhaI (Stephen et al. 2010).Normal controls for methylation assays are run using DNA from paraffin-embdedded squamous epithelium from individuals with no evidence of cancer.

Gene Probe Panels
The 41 gene probe panel (ME001B, www.mlpa.com)used in this cohort interrogates 38 unique genes implicated in cancer (24 tumor suppressor genes) for methylation status in two separate reactions (one in the absence of the methyl-sensitive enzyme HhaI and one in the presence of the HhaI enzyme).There are two probes each for MLH1, RASSF1 and BRCA2, and a normal control DNA sample will generate 41 individual peaks in the absence of HhaI and 15 individual peaks in the presence of HhaI.

Statistical Analysis
Fisher's exact tests were used to examine the association between HPV and each gene as well as age, gender, smoking, race, site and stage in tumor and saliva samples.Wilcoxon Mann-Whitney tests were used to compare continuous variables between groups.The sensitivity, specificity, positive predictive value, and negative predictive value of saliva results were calculated using 2x2 tables.Statistical significance was set at p<0.05.All data were analyzed using SAS 9.2.

HPV
In this study HPV positivity cut points for saliva were set at >0 or ≥0.001 and for tumor at ≥0.03 or ≥0.1.The criteria for cut points of >0 or ≥0.001 for saliva and ≥0.1 for tumor were set by Zhao et al. (2005) to measure sensitivity and specificity of HPV detection in matched tumor and saliva specimens.The tumor cut point of ≥0.03 was previously determined by our group (Stephen et al., 2012).HPV data was not available for 4 of 35 tumor samples.Their corresponding saliva samples were HPV negative at cut point ≥0.001, but at cut point >0, one saliva sample was HPV positive.For saliva, 12/35 samples at cut point >0 and 3/35 samples at cut point ≥0.001 were HPV positive.Of the 31 tumor samples with HPV data, 10 samples at cut point ≥0.03 and 8 at cut point ≥0.1 were HPV positive.Of the 35 cases, at tumor cut point of ≥0.03 and saliva cut point of >0, seven were positive for HPV in the saliva and tumor from the same patient, decreasing to 6 matched pairs when the tumor HPV cut point was raised to ≥0.1.However, when the saliva cut point was raised to ≥0.001, this decreased to three cases for both tumor and saliva, regardless of tumor cut point.Of the 35 matched cases, 17 had agreement for lack of HPV in saliva and tumor regardless of cut points.This was increased to 21 matched cases at saliva cut point of ≥0.001.
There were a few discordances between the saliva and tumor samples but only at saliva cut point of >0, with 4 and 5 discordant cases at tumor cut point ≥0.03 and ≥0.1, respectively.The latter is reflected in the sensitivity, specificity, positive predictive value, and negative predictive value for saliva HPV.At saliva HPV cut point of ≥0.001, specificity and positive predictive values were 100% regardless of tumor cut points; however, sensitivity was low (Table 1).The sensitivity of HPV detection in saliva at tumor cut point ≥0.03 went from 70% to 30% at saliva cut points >0 and ≥0.001, respectively.Similarly at tumor cut point ≥0.1, the sensitivity decreased from 75% to 37.5% at saliva cut points >0 and ≥0.001, respectively (Table 1).Older individuals (over 61 years) with HNSCC were more likely to be HPV negative (p=0.009 at a tumor HPV cut point of ≥0.03 (Table 2), whereas oropharyngeal tumors were more likely to be HPV positive (p=0.015) at a saliva cut point of ≥0.001 (Table 3).S -Saliva, AA -African American, CA -Caucasian American, HP -Hypopharynx, OC -Oral cavity, OP -Oropharynx a Site differs significantly between the saliva HPV groups.All 3 of the HPV positive patients have OP sites.

Methylation and HPV
In the tumor samples, at HPV cut point of ≥0.03, methylation of RARB (p=0.04),CDKN2B (p=0.027) and IGSF4 (p=0.027) was associated with being HPV positive; only IGSF4 (p=0.01) was associated with HPV positive status at tumor cut point ≥0.1.In saliva samples, methylation of ESR1 correlated with HPV positive status (p=0.034)but only at cut point >0.There was no association between HPV status and methylation in saliva at cut point ≥0.001.

Discussion
Alterations in gene sequences, expression levels and protein structure or function have been associated with every type of cancer.These molecular markers can be useful in detecting cancer, determining prognosis and monitoring disease progression or therapeutic response.DNA-based tumor markers usually have distinct characteristics rendering them attractive targets for molecular diagnosis of cancer in body fluids (Goessl, Muller, Straub, & Miller, 2002).Body fluids used for early detection of cancer include sputum for lung cancer (Palmisano et al., 2000;Belinsky et al., 2006), urine for urologic tumors (Hoque et al., 2004), and breast fluid (Lee et al., 2004).Currently for head and neck cancers there is no routine early detection test even though these cancers are often curable if found early.There is, however, evidence that oral examination screenings for early detection of high-risk populations for HNSCC may be cost effective (Speight et al., 2006).HPV detection in saliva DNA presents an added advantage in screening for HNSCC as it is readily available and noninvasive.Also, its feasibility as a screening tool for high-risk individuals for HNSCC is supported by recent studies (Zhao et al., 2005;Chen et al., 2013).
A study from our group examining saliva from HNSCC patients and normal controls demonstrated that saliva gene-based algorithms can differentiate patients from normal controls (Sethi, Benninger, Lu, Havard, & Worsham, 2009).In particular a gain in copy number of PMAIP1 alone or in combination with PTPN1 in saliva can differentiate HNSCC cases from normal controls with a high degree of sensitivity and specificity.This suggests that saliva can represent tumor-associated genetic alterations, making it a useful screening tool for detection and monitoring of head and neck cancers.A study by Carvalho et al. (2011) confirmed the presence of a panel of known hypermethylated genes in HNSCC in the saliva of HNSCC patients.They also found that gene hypermethylation is associated with local recurrence and poorer overall survival.In a follow up study, TIMP3 methylation alone was associated with local recurrence-free survival in HNSCC, indicating its possible use as a salivary rinse biomarker for HNSCC recurrence (Sun et al., 2012).We found TIMP3 to be frequently methylated in saliva (3/6) and tumor (7/23) samples.TIMP3 was also methylated in the paired saliva and tumor of one case.
Other studies on salivary rinses by Rettori et al.(2013) and Kaur et al.(2010) have found that different ethnic cohorts from different geographic regions also have very similar gene hypermethylation patterns.This suggests that detection of methylated genes in salivary rinses could constitute a specific and robust marker that could be applied across geographic populations.
In our current study the frequency of promoter methylation was greater in the tumor samples when compared to saliva (66% vs 17%).We found that for 10 cases the primary tumor and corresponding saliva were concordant for lack of methylation.Of the 23 cases in which the primary tumor was methylated only 4 (17%) presented with methylation of 1 or more genes in the corresponding saliva.This may be due to dilution effects of normal (and presumed nonmethylated) genomes present in saliva from normal mucosa (Carvalho et al., 2008).It is also possible that the methylation rate of each specific gene could vary because of differences in the cohort composition among different studies, specifically tumor localization (Rettori et al., 2013).Also, different methods of specimen collection may affect the quantity of altered cells in saliva.Our saliva samples were obtained as spit saliva.However, it has been shown that hypermethylation patterns in saliva collected with or without exfoliating brush do not differ (Sun et al., 2012).
Saliva specimens in 2 cases displayed gene methylation lacking in the corresponding primary tumor.Some reasons for this might include 1) spurious results in saliva, 2) methylated genes comprising only a minor tumor clone without or insufficient representation in the tumor DNA, or 3) methylation unrelated to the tumor.
Several studies have shown the presence of HPV16 in saliva of HNSCC patients (Smith et al., 2004;Zhao et al., 2005).A recent study from our group found that the overall prevalence of HPV in saliva was significantly different between control subjects, screening subjects without head and neck cancer, and patients with head and neck cancer (Chen et al., 2013).At cut point >0, each group had an overall HPV prevalence of over 5%, with a higher prevalence of 30.8% in the HNC patient group.At cut point >0.001, the prevalence was 0% in the control, 1.2% in the screening, and 16.7% in the HNC group.HPV prevalence in the HNC group at both cut points was different across sites (<0.001) and significantly higher in the oropharynx (OP) than larynx or site as other after Hochberg's adjustment (Chen et al., 2013), and support the findings of our current study that non-OP sites are more likely to be HPV negative than OP sites.This corresponds to studies of tumor tissue where the frequency of HPV infection is significantly higher in OP than in other sites of the head and neck including HP, larynx, and OC (Kreimer et al., 2005;Dayyani et al., 2010;Stephen et al., 2013).Chuang et al. (2008) correlated the presence of HPV16 in surveillance salivary rinses with disease progression in HNSCC.Similar to pap smear screening performed for early detection of cervical cancers (Sankaranarayanan, Budukh, & Rajkumar, 2001), HPV detection in saliva DNA could help with early detection of HNSCC, especially in OPSCC, with a distinct advantage of a non-invasive approach.
In our current study, we found HPV in 32% (10/31) and 26% (8/31) of our tumor samples at cut point ≥0.03 and ≥0.1, respectively.In the saliva, HPV was present in 34% (12/35) and 9% (3/35) at cut point >0 and ≥0.001, respectively.As saliva cut points for HPV increased from >0 to ≥0.001, the sensitivity was significantly reduced regardless of tumor cut points (70% to 30% at tumor cut point ≥0.03 and 75% to 37.5% at tumor cut point of ≥0.1).A limitation of this study was the relatively small samples size of only 35 paired cases.Regardless, at saliva cut point >0 and tumor cut point ≥0.1, positive predictive values (PPV) and negative predictive values (NPV) in our study were comparable to the Zhao et al. study (2005) of 92 matched saliva and tumor cases (PPV of 54.6% in our study vs 53.3% and NPV of 90% in our study vs 80.6%).
Promoter hypermethylation in HNSCC has been reported for numerous tumor suppressor genes.In this study, IGSF4, ESR1, CDKN2B and RARB methylation were significantly associated with HPV positive status at various tumor and saliva cut points.In the tumor samples, at HPV cut point of ≥0.03, methylation of RARB (p=0.04),CDKN2B (p=0.027) and IGSF4 (p=0.027) was associated with being HPV positive; only IGSF4 (p=0.01) was associated with HPV positive status at tumor cut point ≥0.1.In saliva samples, methylation of ESR1 correlated with HPV positive status (p=0.034)but only at cut point >0.There was no association between HPV status and methylation in saliva at cut point ≥0.001.IGSF4 is a novel immunoglobulin-like intercellular adhesion molecule first characterized as a tumor suppressor of non-small cell lung cancer and termed TSLC1 (Murakami et al., 1998;Kuramochi et al., 2001), in which silencing was primarily achieved by allelic loss and promoter methylation.IGSF4 is located at the region of 11q23.2 and spans more than 300 kilobases (Murakami et al., 1998).Promoter hypermethylation of IGSF4 has been reported in nasopharyngeal carcinomas (Hui et al., 2003).
In HNSCC, promoter hypermethylation of IGSF4 is a primary as well as a disease progression event, indicating complete abrogation of tumor suppressor function (Worsham et al., 2006).IGSF4 hypermethylation is also a highly frequent event in cervical cancers where epigenetic silencing of IGSF4 has been implicated in the progression from high-risk HPV-containing, high-grade CIN lesions to invasive cervical cancer (Steenbergen et al., 2004).Furthermore, IGSF4 silencing was accompanied by complete loss or significant decrease of IGSF4 mRNA expression in these cell lines.In esophageal squamous cell carcinoma (ESCC), loss of IGSF4 protein expression as a consequence of promoter hypermethylation, a late stage event in ESCC carcinogenesis, has been implicated in invasion and metastasis and aggressive tumor behavior through the disruption of cell-cell interactions (Ito et al., 2003).Additionally, IGSF4 may offer a promising new therapeutic target in ESCC because expression can be restored by a demethylating agent (Ito et al., 2003).
ESR1, located at 6q25.1, is important for hormone binding, DNA binding, and activation of transcription (Ponglikitmongkol, Green, & Chambon, 1988).ESR1 has metastasis-suppressor properties in breast cancer cells (Garcia, Derocq, Freiss, Rochefort, 1992), suggesting a tumor-suppressor role for ESR1 (Issa et al., 1994).Methylation of CpG sites in the ESR1 promoter, with concordant loss or downregulation of ESR1 expression, is the primary mechanism in prostate cancer (Li et al., 2000).ESR1 is methylated in Barrett's metaplastic and dysplastic samples as well as in some adenocarcinoma samples, suggesting that DNA hypermethylation is an early epigenetic event in the progression of esophageal adenocarcinomas (EAC) (Eads et al., 2000).A previous study by our group on laryngeal squamous cell carcinomas found that aberrant methylation of ESR1 was an independent predictor of late stage diagnosis (Stephen et al., 2010).ESR1 also exhibits age-dependent methylation in colon mucosa (Issa et al., 1994), the cardiovascular system (Post et al., 1999), ulcerative colitis (Issa et al., 2001), and prostate cancer, suggesting that ESR1 may be involved in age-dependent increase in cancer incidence.
Cyclin-dependent kinase inhibitor 2B (CDKN2B), also known as p15, inhibits CDK4 and regulates cell growth by controlling cell cycle G1 progression.Inactivation of CDKN2B gene at the genomic and epigenetic level is a frequent event in human oral SCCs (Yeh et al., 2003) and in HNSCC (Shintani et al., 2001;Worsham et al., 2003;Worsham et al., 2006).In precancerous oral tissues and lesions of the head and neck (Shintani et al., 2001;Worsham et al., 2006;Chen et al., 2007;Stephen et al., 2007a;Stephen et al., 2007b), aberrant methylation of CDKN2B has been implicated as an early event in the pathogenesis of these lesions.Preferential methylation of CDKN2B has been shown to be a useful tumor marker in undifferentiated nasopharyngeal carcinoma (Wong et al., 2003).As a consistent target of aberrant promoter hypermethylation, CDKN2B may serve as a useful biomarker and a potential therapeutic target for gene reactivation studies and in disease monitoring for progression.
The alteration of RARB via DNA hypermethylation has several implications in HNSCC.Decreased expression of RARB has been associated with increased keratinizing squamous differentiation in HNSCC cells (Wan et al., 1999).More importantly, in our primary HNSCC cohort (Chen et al., 2007), RARB silencing by promoter hypermethylation in early and late stage tumors suggest that DNA methylation of RARB is an early epigenetic event.Also, it has been demonstrated that the demethylating agent 5-aza-CdR can restore RARB inducibility by retinoid ATRA (9-cis-RA) in most cell lines (Youssef et al., 2004).

Conclusion
HPV detection in saliva DNA shows promise as a non invasive approach for tumor HPV status within clearly defined parameters.Given the limited sample size of this study, for clinical relevance, the results will require confirmation and validation in larger cohorts.Pursuing the feasibility of detecting HPV and methylation markers in saliva DNA is relevant to not only early detection of HNSCC but also in disease monitoring in response to treatment as well as predictors of HNSCC especially in high risk individuals.Also, unlike genes that are inactivated by nucleotide sequence variation, genes silenced by epigenetic mechanisms are still intact and thus retain the potential to be reactivated by environmental or medical intervention (Olden, Issac, & Roberts, 2011).

Table 1 .
Sensitivity and specificity

Table 3 .
Saliva HPV and demographics at saliva cut point >0.001