ORIGINAL ARTICLE

Evaluation of the methylation profile of exfoliated cell samples from patients with headand neck squamous cell carcinoma

Ana Luiza Bomfim Longo, MSc,1 Marianna M. Rettori, PhD,1 Ana Carolina de Carvalho, MSc,1 Luiz Paulo Kowalski, PhD,2 Andre Lopes Carvalho, PhD,3

Andre Luiz Vettore, PhD1*

1Laborat�orio de Biologia Molecular do Cancer, Departamento de Ciencias Biol�ogicas, Universidade Federal de S~ao Paulo, S~ao Paulo, Brazil, 2Departamento de Cirurgia de Cabecae Pescoco, Hospital A.C. Camargo, S~ao Paulo, Brazil, 3Departamento de Cirurgia de Cabeca e Pescoco Hospital do Cancer de Barretos, Barretos, Brazil.

Accepted 5 April 2013

Published online 18 April 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.23345

ABSTRACT: Background. Silencing of tumor suppressor genes plays avital role in head and neck carcinogenesis. The purposes of this studywere to determine the methylation profile of exfoliated tumors cells col-lected from patients with head and neck squamous cell carcinoma(HNSCC) and to evaluate its prognostic significance.Methods. The methylation profile and level of a 20-gene panel wereevaluated by quantitative methylation-specific polymerase chain reaction(qMSP) in exfoliated tumor cell samples from 96 patients with HNSCC.Results. CCNA1 (60.4%), DCC (54.2%), and TIMP3 (35.4%) were fre-quently methylated in these samples. Patients with exfoliated tumors

cells positive for DCC methylation showed a trend toward a lower localrecurrence-free survival.Conclusion. These findings indicate that a low invasive method could beused to access the methylation profile of exfoliated cells from patientswith HNSCC. Moreover, our data provide evidence that hypermethylationof DCC could be useful as prognostic indicator for this malignancy.VC 2013 Wiley Periodicals, Inc. Head Neck 36: 631–637, 2014

KEY WORDS: head and neck squamous cell carcinoma (HNSCC),methylation, exfoliated cell, qMSP

INTRODUCTIONHead and neck squamous cell carcinoma (HNSCC) isone of the most common cancers worldwide, withapproximately 650,000 new cases per year.1 Over pastyears, diagnosis and management of patients withHNSCC have improved through combined efforts insurgery, radiotherapy, and chemotherapy, but long-termsurvival rates have improved only marginally, and the 5-year overall survival (OS) rate is around 50%.2 Latediagnosis and frequent locoregional recurrences are themajor causes for the poor prognosis.2,3 Clinically effec-tive biomarkers for the identification of patients at highrisk for recurrence that may benefit from adjuvant

therapy or intensive surveillance to detect recurrence atan earlier stage are being pursued. New methods forearly recurrence diagnosis based on molecular character-istics of the tumors could provide valuable opportunitiesfor improvement of survival rates.

There is increasing evidence that, in addition to geneticalterations, epigenetic processes play a major role in car-cinogenesis. One important epigenetic phenomenon is themethylation of genomic DNA occurring almost exclu-sively at cytosine residues, that become methylated to5-methylcytosine in the dinucleotide CpG.4 DNA methyl-ation is associated with several changes in chromatinstructure, including the regulation of histone methylationand acetylation and the recruitment of proteins to themethylated sites. This usually leads to the obstruction ofthe promoter, hindering gene transcription, and subse-quent gene silencing.5 Genes involved in the cell cyclecontrol, DNA repair, apoptosis, cell adhesion, and signaltransduction have already been described as inactivatedby aberrant promoter methylation (or hypermethylation)in different human cancers.6–8 Moreover, each tumor typeseems to have its own distinct pattern of methylation9,10

and it was previously reported that aberrant methylationis a frequent event in HNSCC.11

HNSCC detection is currently based on clinical exami-nation of the upper aerodigestive tract and histologicanalysis of suspicious areas. In the last years, the hyper-methylation pattern of saliva samples from patients withHNSCC has been evaluated as noninvasive tests for earlycancer diagnosis.11,12 DNA hypermethylation can be

*Corresponding author: A. L. Vettore, Laborat�orio de Biologia Molecular doCancer, UNIFESP, Rua Pedro de Toledo, 669 – 11o andar, S~ao Paulo, SP, Brazil04039-032. E-mail: andre.vettore@gmail.com

Contract grant sponsors: Fundac~ao de Amparo �a Pesquisa do Estado deS~ao Paulo – FAPESP grants (2008/58460-9 and 2005/02580-8) A.L.B.L.and A.C.C. were recipients of scholarships from Fundac~ao de Amparo �aPesquisa do Estado de S~ao Paulo – FAPESP (07/56471-0 and 07/56245-0). M.M.R was the recipient of a scholarship from Coordenac~aode Aperfeicoamento de Pessoal de N�ıvel Superior – CAPES. A.L.C. hasa National Counsel of Technological and Scientific Development – CNPqscholarship (313181/2009-8). A.L.V. has a National Counsel of Techno-logical and Scientific Development – CNPq scholarship (302360/2008-5).

Additional Supporting Information may be found in the online version ofthis article.

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measured in tissue samples using a real-time quantitativemethylation-specific polymerase chain reaction (qMSP)approach. The ability to quantify methylation allows thedelineation of clinically meaningful threshold values ofmethylation to improve sensitivity and specificity in thedetection of tumor-specific signals.13–16

The feasibility of cancer detection in body fluids alsoopens the potential for surveillance after treatment. Mo-lecular detection techniques have the potential to predicttumor recurrence before clinical symptoms or physicalexamination changes. Salivary DNA promoter hypermeth-ylation analysis has been found to be an efficient molecu-lar tool for early diagnosis of HNSCC and several geneshave already been identified as hypermethylated in sali-vary rinse samples (X).11,12,17–21 In light of these consid-erations, biomarkers based on the methylation profileevaluation in exfoliated mucosa cell samples could repre-sent a powerful class of minimally invasive markers forHNSCC detection. For this reason, in this study, we char-acterized the promoter methylation status of 20 genes intumor cells collected with an exfoliating brush frompatients with HNSCC at diagnosis and after the last cura-tive treatment. We also evaluated its prognosis signifi-cance as well as its value as biomarker for early detectionof HNSCC recurrence.

MATERIALS AND METHODS

Tissue samples

This prospective cohort study involved 200 consecutivepatients with HNSCC treated between 2006 and 2008 atHospital A.C. Camargo, S~ao Paulo, Brazil. The studyincluded patients not previously treated, over 18 years ofage, treated with curative intent, and presenting withtumors at the oral cavity or oropharynx. Of those partici-pants initially enrolled in the screening study, 51 wereexcluded for not filling the inclusion criteria (previouslyuntreated patients with HNSCC, over 18 years of age,treated with curative intent, primary tumors), and theother 47 were excluded for tumors being localized in thelarynx. For 6 samples, we were not able to obtain goodexfoliated tumor cell samples. The exfoliated tumor cellswere prospectively collected at diagnosis (pretreatment, n5 96) and 7 to 15 days after the last curative treatment(posttreatment, n 5 87). For the control population, 79exfoliated epithelial cell samples from healthy accompa-nying patients (68% men; median age, 46.3; and 38%smokers) were collected.

Because of the scarcity of DNA quantity after bisulfitetreatment of many samples and the large number of genesselected, it would be virtually impossible to evaluate allpossible candidate genes in all samples. Thus, a step-by-step selection was conducted, and a set of “best” geneswas evaluated in the expanded cases or control cohorts ofsamples. The first step was to verify the methylation sta-tus of all the target genes in a small group of controlsamples and the ones with genes showing low specificitywere excluded. Next, the methylation profile of theremaining genes was analyzed in an evaluation set con-sisting of tumor samples and genes presenting low sensi-tivity were also discarded. By the end, genes that could

better distinguish HNSCC from control samples wereselected to be tested in the expanded cohorts.

The experimental protocol was approved by the EthicsCommittees of the Hospital A. C. Camargo, Hospital doCancer de Barretos and Universidade Federal de S~aoPaulo. Clinicopathological information was collectedfrom the patients’ medical records. Smoking was definedas use of tobacco, chewable or smoked, for at least 1 yearcontinuously. Alcohol use was defined as intake of morethan 2 alcoholic drinks per day for at least 1 yearcontinuously.

Exfoliated tumor cells were obtained by brushing thetumor surface with a gynecological exfoliating brush(Kolplast, S~ao Paulo, Brazil). The brush was gently agi-tated to release the obtained material into 2 mL of normalsaline solution. After centrifugation, the supernatant wasdiscarded and DNA was isolated from the pellet.

DNA extraction

Genomic DNA from exfoliated cell samples wasextracted by digestion with 50 mg/mL proteinase K (Invi-trogen, Carlsbad, CA) in the presence of 1% sodium do-decyl sulfate at 48�C for 48 hours, followed by phenol/chloroform extraction and ethanol precipitation.

Bisulfite treatment

Sodium-bisulfite conversion of 2 lg of genomic DNAwas performed, as previously described.22 In brief,genomic DNA was denatured, diluted in freshly preparedbisulfite solution, and incubated for 3 hours at 70�C inthe dark. Bisulfite-modified DNA was desalted, desulfo-nated, and precipitated. The bisulfite-modified genomicDNA was dissolved in water and stored at -80�C.

Target gene selection

A total of 20 genes were selected for the examination ofmethylation abnormalities. All the genes evaluated in thisstudy present tumor suppressor activities and their silenc-ing could contribute to the tumorigenic process. Amongthese genes are CCNA1, CCND2, CDKN2A, CDKN2B,p14ARF, and SOCS1, which are involved in cell cycle con-trol, CDH1 in cell adhesion, ESR1, APC, and TIMP3 insignal transduction processes, MLH1 in DNA repair,MT1G in cell–cell signaling processes, and SFRP1 andHIN1 in cell differentiation and proliferation. The functionof AIM1, DCC, HIC1, and UCHL1 are not yet well under-stood. MINT1 and MINT31 are chromosomal regions fre-quently described as methylated in human cancers. It couldbe shown that these genes are affected by aberrant pro-moter methylation in association with transcription silenc-ing in different types of human malignancies.

Quantitative methylation-specific polymerase chainreaction analyses

The qMSP analysis were conducted, as previouslydescribed.23 Basically, 30 ng of bisulfite-modified DNAwas used as a template in fluorogenic qMSP assays car-ried out in a final volume of 20 lL in the ABI PRISM

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7500 Sequence Detection System (Applied Biosystems,Foster City, CA). Each sample was run in triplicate andthe reaction mixture contained 3 lL of bisulfite-modifiedDNA, 1.2 lM of forward and reverse primers, 200 nmol/L of the probe, 0.5U of platinum Taq polymerase (Invi-trogen, Carlsbad, CA), 200 lmol/L dNTPs, 16.6 nmol/Lammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L mag-nesium chloride (2.5 mmol/L for CDKN2A), 10 mmol/Lmercaptoethanol, 0.1% dimethyl sulfoxide, and 1X ROXdye (Invitrogen). Polymerase chain reaction was con-ducted with the following conditions: 95�C for 2 minutes,followed by 45 cycles at 95�C for 15 seconds, and 60�Cfor 1 minute. Each plate included serial dilutions (30–0.0003 ng) of a positive control (leukocyte DNA methyl-ated in vitro) allowing the construction of calibrationcurves. Primer and probe sequences are provided in Sup-plementary Table S1, online only. The relative DNAmethylation was determined as a ratio of methylation-spe-cific polymerase chain reaction–amplified gene to ACTBand then multiplied by 100 for easier tabulation (meanvalue of triplicates of gene of interest divided by themean value of ACTB triplicates 3 100). Each sample wasanalyzed in triplicate and mean value and SD were calcu-lated. When SD of the triplicate was greater than 0.3, thesample was reanalyzed. A cutoff value of �0.1% wasused to score HNSCC exfoliated cell samples as positiveones for the genes AIM1, CDKN2A, SFRP1, TIMP3,CCND2, ESR1, MINT1, p14ARF, and CDH1, and a cutoffvalue of �1% was adopted for the genes MINT31,MLH1, SOCS1, HIN1, DCC, MT1G, and UCHL1. Thesecutoff values were chosen for being clinically relevantand also to exclude very low-level background readingsthat can occur in certain individual for certain genes.24

For the other 4 genes (CCNA1, CDKN2B, APC, andHIC1), no cutoff value was adopted.

Statistical analysis

Statistical analysis was performed using the softwareSPSS 19.0 for Windows. Categorical variables were com-pared using Pearson’s chi-square test or Fisher exact test,as appropriate. Quantitative variables were comparedusing unpaired t test. Survival was estimated from thefirst day of treatment until the last follow-up or date ofdeath. Survival curves were calculated using the Kaplan–Meier method and compared by log-rank test. Resultswere calculated with 95% confidence intervals. For allcomparisons, we considered statistical significance whenp < .05.

RESULTS

Patient characteristics and clinical predictors

Ninety-six patients with HNSCC were included in thisstudy (Table 1). They were mainly men (78.1%), withages ranging from 20 to 90 (mean, 58.9; median, 57.0).Alcohol or tobacco consumption (current or past) wasfound in 79.2% and 81.3% of the cases, respectively. Pri-mary tumor sites included the oral cavity (76%) and oro-pharynx (24%). Based on the TNM staging system,54.2% of patients had neoplastic cells in their lymphnodes, whereas 58.3% were T3/T4. Surgery followed byradiotherapy was adopted as the curative treatment in

52.1% of the cases, whereas surgery alone (28.1%) andradiotherapy alone (19.8%) were also used. The medianfollow-up period for these patients was 31 months andrecurrences occurred in 42.7% of the cases.

Prevalence of aberrant methylation in head and necksquamous cell carcinoma exfoliated cell samples

The first step was to verify the methylation status ofthe 20 target genes in 25 exfoliated epithelial cell samplescollected from healthy individuals. This analysis showedthat APC, CCND2, CDH1, ESR1, HIC1, HIN1, MINT1,MT1G, CDKN2B, SFRP1, and UCHL1 were frequentlymethylated in control samples and, for this reason, thesegenes were set aside. The remaining 9 genes were identi-fied as rarely methylated in control samples and theirmethylation profile was analyzed in 42 exfoliated tumorcell samples collected at diagnosis (pretreatment) frompatients with HNSCC. This analysis revealed that hyper-methylation of AIM1 (15.2%), CDKN2A (0%), MINT31(8.7%), MLH1 (8.3%), p14ARF (5.0%), and SOCS1

TABLE 1. Demographic and clinical characteristics of patients with headand neck squamous cell carcinoma included in the study (n 5 96).

Characteristic No. of patients %

Age>60 y 55 57.3�60 y 41 42.7

SexMale 75 78.1Female 21 21.9

Tobacco consumptionYes 78 81.3No 18 18.7

Alcohol consumptionYes 76 79.2No 20 20.8

Tumor siteOral cavity 73 76.0Oropharynx 23 24.0

T classificationT1/T2 40 41.7T3/T4 56 58.3

N classificationcN0 44 45.8cN1 52 54.2

First curative treatmentSurgery 1 radiotherapy 50 52.1Surgery 27 28.1Radiotherapy 19 19.8

RecurrenceYes 41 42.7No 55 57.3

Recurrence siteLocal 16 16.7Regional (neck) 5 5.2Locoregional 4 4.2Local 1 distant 5 5.2Regional 1 distant 1 1.0Distant 10 10.4

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(11.6%) are rare events in HNSCC exfoliated cells har-vested at diagnosis. On the other hand, CCNA1 (60.4%),DCC (54.2%), and TIMP3 (35.4%) are frequently methyl-ated in these samples.

Therefore, these 3 genes (CCNA1, DCC, and TIMP3)that could better distinguish HNSCC exfoliated cells fromcontrol samples were selected to be tested in theexpanded cohort of HNSCC exfoliated cell samples col-lected at diagnosis (n 5 96) and control subjects (n 580). By the end, CCNA1 was methylated in 60.4% (58 of96) of all pretreatment samples examined, DCC in 54.2%(52 of 96), and TIMP3 in 35.4% (34 of 96; Table 2). Thisanalysis also confirmed that methylation of these 3 geneswas highly associated with HNSCC cases (specificity>91%; Table 2). In this way, we were able to define a 3-gene panel whose methylation in exfoliated tumor cellswas highly associated with HNSCC cases. We alsoobserved that 82.3% of the pretreatment HNSCC exfoli-ated cell samples showed hypermethylation of at least 1of these 3 genes.

Based on the above results, the methylation profile ofthis 3-gene panel was evaluated in exfoliated cell samplescollected from patients with HNSCC just after the last cu-rative treatment (posttreatment). This analysis showed aremarkable decrease in the methylation frequency ofCCNA1 (8.0%), DCC (9.2%), and TIMP3 (2.3%) in thesesamples (Table 2).

Correlation between aberrant methylation and patientcharacteristics

The methylation patterns of CCNA1, DCC, and TIMP3in the pretreatment HNSCC exfoliated cell samples andof HIC1 in the pretreatment plasma samples were ana-lyzed for potential correlations with clinical characteris-tics of patients with HNSCC including age, sex, tobaccoand alcohol consumption, tumor site, vascular emboliza-tion, perineural infiltration, and OS. This analysis showedthat the hypermethylation of CCNA1 in HNSCC exfoli-ated cells was correlated with patients aged >60 years (p5 .022; Supplementary Table S2, online only). No associ-ations were observed between the other clinical featuresand hypermethylation status of any of the target genestested.

The OS at 3 years for the patients with HNSCCincluded in this study was 60%. As expected, advanced Tclassification (p 5 .004) and lymph node metastasis at di-agnosis (p 5 .001) were significantly associated withreduced OS (Table 3).

TABLE 2. Comparison of hypermethylation detection on head and neck squamous cell carcinoma exfoliated cells samples and normalexfoliated cell controls.

Genes Control no. (%) Pretreatment no. (%) After-treatment no. (%) Specificity, % (95% CI) Sensitivity, % (95% CI)

CCNA1 6 (7.6) 58 (60.4) 7 (8.0) 92.4 (85.5–97.0) 60.4 (50.0–70.3)DCC 7 (8.9) 52 (54.2) 8 (9.2) 91.1 (82.9–95.6) 54.2 (43.7–64.4)TIMP3 7 (8.9) 34 (35.4) 2 (2.3) 91.1 (82.9–95.6) 35.4 (25.9–45.8)

Abbreviation: CI, confidence interval.

Promoter hypermethylation frequency for the 3 genes selected in control samples (n 5 80), pretreatment (n 5 96), and posttreatment exfoliated tumor cell samples (n 5 87).

TABLE 3. Local control and overall survival rates according to the clinicalvariables.

Variables3-yearOS (%) p value

3-year localcontrol (%) p value

Sex .321 .746Male 60.2 62.9Female 67.7 61.4

Tobacco consumption .465 .207No 71.4 83.3Yes 59.1 60.8

Alcohol consumption .023 .034No 84.7 83.2Yes 52.9 55.8

Tumor site .539 .651Oral cavity 58.5 56.8Oropharynx 56.1 54.5

T classification .004 .062T1/T2 78.6 73.9T3/T4 47.8 56.2

N classification .001 .744Negative 74.0 61.9Positive 46.0 63.6

M classification .004 .398M0 65.7 62.9Mx 30.0 60.0

Vascular embolization .469 .544Negative 62.3 62.7Positive 55.6 55.6

Perineural invasion .062 .405Negative 69.8 64.7Positive 40.9 54.4

Margins .529 .392Negative 64.5 64.5Positive 45.0 50.0

Lymph node involvement .567 .448Negative 58.1 68.3Positive 54.1 57.8

Second primary tumor .543 .833Negative 62.1 62.0Positive 50.0 66.7

Curative treatment .509 .678Surgery 60.6 53.5Radiotherapy 53.3 71.4Surgery 1 radiotherapy 67.4 67.5Surgery 1 radiotherapy

1 chemotherapy50.0 62.5

Radiotherapy 1chemotherapy

57.1 59.1

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Analyses of OS and local recurrence-free survival werenot able to identify significant associations with the hy-permethylation profile of the investigated genes in theexfoliated cell samples collected from patients withHNSCC. However, although the difference in local recur-rence–free survival between patients with and withoutDCC hypermethylation in the pretreatment HNSCC exfo-liated cell samples was not significant, a trend toward aworse prognosis was noticed for the patients with methyl-ated DCC when compared with patients with HNSCCpresenting unmethylated DCC (p 5 .081; Figure 1).

DISCUSSIONSeveral clinicopathological parameters have been impli-

cated in the determination of HNSCC prognosis, recur-rence, and survival. Despite the evolution in management,the OS of HNSCC is around 50%.2 Detection of oral can-cer in the early asymptomatic stage could dramaticallyimprove the cure rates and patients’ quality of life byminimizing extensive and debilitating treatments. There-fore, novel diagnostic approaches based on the molecularcharacteristics of the tumors25 and biomarker discovery isan important area of ongoing research in HNSCC.

The ideal biomarker must be accessible using noninva-sive protocols, inexpensive to quantify, specific to the dis-ease of interest, and provide a reliable early indication ofdisease before clinical symptoms appear. Biomarkers arebeing evaluated for risk stratification, chemoprevention,screening, diagnosis and classification, prognosis, predic-tion of treatment, therapy tracking, and posttreatment sur-veillance.26 Instead of analyzing the tumor samplesthemselves, the molecular composition of a tumor can beindirectly characterized by analyzing body fluid samples(plasma, saliva, urine, bronchial lavage, and sputum),thereby avoiding the need of invasive procedures.

Promoter hypermethylation is an essential mechanismfor epigenetic inactivation of key genes in the develop-ment of HNSCC. In recent years, several studies havereported methylation profile analyses in body fluids as

saliva and plasma from patients with HNSCC.11,12,18

King et al27 (2002) compared the DNA yield, quality, andassociated costs of buccal cell DNA collected using cyto-brushes (buccal exfoliated cells) or mouthwash and con-cluded that the collection of buccal cells with cytobrushesis cost-effective in large-scale studies and yields sufficientquantity and quality of DNA for genotyping.

In light of these observations, we used qMSP to exam-ine the methylation status of 20 target genes in HNSCCexfoliated cell samples collected at diagnosis and just af-ter the last curative treatment from patients with HNSCC.

To our knowledge, this is the first study examining themethylation profile of exfoliated cell samples harvestedfrom patients with HNSCC with cytobrushes. Exfoliatingtechniques have the advantage of being minimally inva-sive and of not requiring local anesthetic. The use of anexfoliating brush allows sampling of the full thickness ofstratified squamous epithelium of the oral mucosa.28 Thisanalysis allowed the identification of a 3-gene panel(CCNA1, DCC, and TIMP3) that showed high specificity(>90%) and sensitivity (>80%).

The protein encoded by CCNA1 belongs to the highlyconserved cyclin family, whose members are character-ized by a dramatic periodicity in protein abundancethrough the cell cycle. Cyclins function as regulators ofCDK kinases. CCNA1 was found to bind to importantcell cycle regulators, such as Rb family proteins, tran-scription factor E2F-1, and the p21 family proteins.29,30

Previous analyses demonstrated that the CCNA1 promoterwas methylated in kidney, colon, spleen, testis, and smallintestine, but not in brain, liver, pancreas, or heart.31

Tissue inhibitor of metalloproteinases 3 (TIMP3)belongs to a family of genes that inhibit matrix metallo-proteinases. Numerous studies have indicated that TIMPsinhibit cellular invasion, tumorigenesis, metastasis, andangiogenesis,32 and others described hypermethylation ofTIMP3 in a variety of tumor types. Esteller et al9 (2001)analyzed promoter hypermethylation changes of 12 genes,including TIMP3, in DNA from over 600 primary tumorsamples and observed hypermethylation of TIMP3 in co-lon, breast, lung, brain, kidney, and liver. Ninomiya etal33 (2008) analyzed TIMP3 hypermethylation in esopha-geal squamous cell carcinoma samples and found a posi-tive association between TIMP3 hypermethylation andearlier recurrence and poor survival. Hoque et al34 (2008)analyzing TIMP3 methylation in 175 urine sediment frompatients with bladder cancer found that TIMP3 methyla-tion was associated with an almost 2.5-fold increase inthe risk of death even after adjusting for the presence ofmetastasis. Although hypermethylation of TIMP3 is fre-quently described in various tumor types, there are fewreports in HNSCC.20,35,36 Righini et al20 (2007) found ahigh frequency of TIMP3 hypermethylation in tumor andsaliva samples of patients with HNSCC, whereas Car-valho et al11 (2008) analyzed the methylation profile of21 genes in HNSCC saliva samples taken at diagnosisfrom individuals of the American population and foundTIMP3 frequently methylated. In our study, TIMP3 wasfrequently hypermethylated in HNSCC exfoliated cellscollected at diagnosis.

The deleted in colon cancer (DCC) gene is a putativetumor-suppressor gene that encodes a transmembrane

FIGURE 1. Local recurrence-free rate according to DCC hyper-methylation pattern in pretreatment head and neck squamouscell carcinoma (HNSCC) exfoliated cell samples (p 5 .081)

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HEAD & NECK—DOI 10.1002/HED MAY 2014 635

protein with structural similarity to neural cell adhesionbranch of the immunoglobulin superfamily.37 Functionalstudies show that, in addition to DCC being a tumor sup-pressor gene, its protein product may participate in cell–cell and cell–matrix interactions and is involved in bothepithelial and neuronal-cell differentiation.38,39 Mountingevidence suggests that DCC may be inactivated in severalsolid tumors arising outside the colon and rectum, includ-ing epithelial tumors of the stomach, pancreas, head andneck, and breast.40–43 Reestablishment of DCC expressionhas been shown to suppress tumorigenicity.44 DCC pro-moter region has been found to be hypermethylated inseveral malignancies including epithelial tumors of thelung, stomach, colon, esophageal, head and neck, and inhematologic disorders as multiple myeloma and follicularlymphoma.6,45–47 We also found DCC often hypermethy-lated in the HNSCC exfoliated cell samples collected atdiagnosis.

Our results suggested that detection of CCNA1, DCC,and TIMP3 hypermethylation is a common and specificevent in HNSCC exfoliated cells. Furthermore, the meth-ylation frequency of these genes became a rare event inthe exfoliated tumor cell samples collected after the treat-ment, suggesting that the therapy approaches applied inthe patients were effective and the tumors were removedin the majority of the HNSCC cases evaluated. However,we trust that more analyses are needed to confirm if theaberrant methylation of these genes could be useful asmolecular markers for the surveillance of patients withHNSCC.

In brief, the current study represents the first evaluationof the methylation profile of HNSCC exfoliated cell sam-ples harvested with an exfoliating brush. We identified 3genes frequently hypermethylated in these samples col-lected at diagnosis and demonstrated that the aberrantmethylation of these genes is scarce in samples collectedafter treatment. Although no significant association wasfound between aberrant methylation of any of the investi-gated genes and OS in patients with HNSCC, weobserved a trend toward poor prognosis for patients whohad HNSCC with DCC hypermethylation. Of note, alarger study cohort needs to be evaluated in support ofour findings.

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