RESEARCH ARTICLE

p16CDKN2A SNP rs11515 was not associated with headand neck carcinoma

Ugo Borges Pinheiro & Carlos Alberto de Carvalho Fraga & Danilo Cangussu Mendes &Luciano Marques-Silva & Lucyana Conceição Farias & Marcela Gonçalves de Souza &

Mariana Batista Soares & Kimberly Marie Jones & Sérgio Henrique Souza Santos &

Alfredo Maurício Batista de Paula & Gustavo Velásquez-Meléndez &

André Luiz Guimarães

Received: 26 November 2013 /Accepted: 28 February 2014 /Published online: 15 March 2014# International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract Head and neck squamous cell carcinoma (HNSCC)is considered a serious public health problem in many coun-tries. Recently, genetic variations have been considered asimportant factors to cancer susceptibility and prognosis.More specifically, genetic polymorphisms have been associ-ated with the development and prognosis of HNSCC. Thepurpose of the current study was to investigate an associationamong p16CDKN2A gene polymorphism at rs11515, age, andHNSCC aggressiveness. PCR–RFLP analysis was used toinvestigate the p16CDKN2A gene in 96 patients with HNSCCand in 100 individuals without HNSCC. A case group wascategorized by age in younger (<60 years) and older(≥60 years) patients. Differences between the case and controlgroups were determined using Fisher and chi-squared tests.Time of survival was calculated from the date of diagnosis tothe date of last follow-up visit or to the date of death using theKaplan–Meier estimator and comparing this to the log-rank

test. Statistical significance was set at p<0.05. In the presentstudy, no association was established between HNSCC andrs11515 polymorphism, as indicated in a previous study. Wefound that HNSCC individuals with large-sized tumors andwith metastatic disease presented worse overall survival, con-sistent with fundamental concepts that establish the effects oftumor size and lymph node metastasis to HNSCC outcomes.This study identified that there is no difference in the distri-bution of rs11515 between the control and HNSCC groups. Inaddition, no differences between rs11515 genotypes and clin-icopathological parameters were observed.

Keywords Suppressor gene .CDKN2A . Polymorphism .

Cancer . Genetic

Introduction

Head and neck squamous cell carcinoma (HNSCC) representsthe sixth most prevalent type of cancer worldwide and hasbeen considered a serious and growing public health problemin many countries [1, 2]. The oral cavity, oropharynx, hypo-pharynx, and larynx are the most common sites of the diseasein the head and neck region and appear to have similar riskfactors [2]. Potential carcinogenic agents, such as tobaccosmoke and alcohol, are the most common risk factors [3];however, HNSCC etiology is complex due to its multigenicnature [4]. Classically, the development of HNSCC has beendescribed in patients above the age of 60 [1]. However, somestudies have shown an increased incidence of HNSCC inyounger patients [5–7]. Currently, there is no consensus ofwhether or not HNSCC in younger patients has a worseprognosis than in older patients because the traditional riskfactors seem to actively contribute to the etiopathogenesis of

U. B. Pinheiro : C. A. de Carvalho Fraga :D. C. Mendes :L. Marques-Silva : L. C. Farias :M. G. de Souza :M. B. Soares :A. M. B. de Paula :A. L. Guimarães (*)Department of Dentistry, Universidade Estadual de Montes Claros,Hospital Universitário Clemente de Faria, Avenida CulaMangabeira,562, Montes Claros, Minas Gerais 39400-000, Brazile-mail: andreluizguimaraes@gmail.com

K. M. JonesProgram in Health Sciences, Universidade Estadual de MontesClaros, Montes Claros, Brazil

S. H. S. SantosDepartment Pharmacology, Universidade Federal de Minas Gerais,Belo Horizonte, Brazil

G. Velásquez-Meléndez (*)Department of Nursing, Universidade Federal de Minas Gerais, BeloHorizonte, Brazile-mail: jguveme@gmail.com

Tumor Biol. (2014) 35:6113–6118DOI 10.1007/s13277-014-1809-0

the disease in both groups [5, 6]. Moreover, while somestudies show a better prognosis for younger patients [8–10],other studies have shown an increased risk of death in youngerpatients [11, 12].

Recently, it was demonstrated that genetic variationscould be associated with HNSCC susceptibility [13, 14],prognosis, and survival [15–18]. p16CDKN2A is a poly-morphic gene, and an adjacent polymorphism was iden-tified recently in the 3′ untranslated region of exon 3[19, 20]. rs11515 polymorphism has been associatedwith low expression of p53 and with tumor aggressive-ness. This study explores whether or not this adjacentpolymorphic variant may have some functional rele-vance [19, 21–23]. Therefore, the aim of this study isto assess whether or not there is an association betweenthe p16CDKN2A gene polymorphism rs11515, patient age,and HNSCC aggressiveness, considering that a betterunderstanding of the relationship between genetic var-iations and disease process may contribute to moreaccurate risk assessments, prognosis, and treatment ofHNSCC.

Methods

Patients

The present analysis was based on a retrospective study de-sign. Ninety-six patients with HNSCC, who underwent diag-nosis and treatment from 1997 to 2011, were recruited fromthe databases of several surgical clinics that treat the head andneck in Montes Claros, Brazil. The study group was dividedinto two categories, one with 50 patients under the age of 60and the other with 46 patients who were 60 years old or older.These groups were matched for tumor node metastasis (TNM)staging and smoking history. As part of inclusion criteria, weused only samples with sufficient material available for DNAextraction. These cases underwent surgical resection withpostoperative radiotherapy. Treatment of the patientsconsisted of standard radiotherapy using the conventionaltwo-dimensional technique delivered with megavoltage(Cobalt-60 or 6 MV linear accelerator). Total doses rangedfrom 50 to 70 Gy in daily fractions of 2 Gy, five times a week.Patients with preoperative treatment as well as those withother histological types were excluded.

A control group of 100 randomly chosen individuals with-out HNSCC were also enrolled in the study. Sample sizecalculation of the control group was performed to estimatethe number of people by considering the size of the localpopulation (confidence level 95 %, standard error <9 %, andevent prevalence 50.0 %). Both case and control groups camefrom the same geographical region. The control group wasage-matched.

Ethics statement

Information on age, sex, tobacco smoking, alcohol use, med-ical history, family cancer history, tumor site, TNM clinicalstaging, and survival were obtained from medical charts. Thestudy was approved by the relevant institutional review board(State University of Montes Claros, process number 1187/08).

Clinical data

Tobacco use was investigated using a process described in aprevious study [6]. Briefly, ex-drinkers and ex-smokers weresubjects who had abstained from any type of drinking orsmoking for at least 1 year. Only those who had never smokedwere considered nonsmokers. Smokers and ex-smokers weregrouped combined. Physical description of skin color was notused because, in Brazil, it is a poor predictor of genomicancestry [24].

All patients were staged according to the UICC TNMClassification of Malignant Tumors (1997) [25, 26]. Lesionsof HNSCC were classified according to the primary site asdescribed in the International Classification of Diseases(ICD-10) for Oncology. The anatomical sites reviewed in thisstudy included both anterior and posterior sites. Anterior sitesincluded 43 (44.8 %) in the mouth and perioral region (C00,C01, C02, C04, C05, C06.0, C06.2) and 28 (29.2 %) in theoropharynx (C09–C10). The posterior sites were 25 (26.0 %)in the hypopharynx–larynx region (C12, C13, C32). Patientswith a diagnosis of carcinoma in situ or multiple head andneck carcinomas were excluded from the study. All patientswere asked about the occurrence of cancer in first-degreerelatives. The term cancer was defined using the WHO defi-nition of “an uncontrolled growth and spread of cells that mayaffect almost any tissue of the body.”

DNA isolation and p16CDKN2A genotyping

DNAwas isolated from ten 10-μm-thick tissue sections fromeach block of HNSCC specimens, using the DNeasy TissueKit (Qiagen, Chatsworth, CA) as described before [15]. Thetube was processed according to the manufacturer’s protocol,using xylene treatment to remove paraffin as outlined in theDNeasy user manual [36]. Briefly, the paraffin was removedby vortexing and 10 min of incubation with 1.2 mL of xylene,followed by two washes with pure (200 proof) ethanol. Theair-dried pellet was then incubated with 20 μL of proteinase Kand 180 μL of ATL lysis buffer (from the DNeasy kit) for 16 hin a heat block at 56 °C. The lysed emulsion was furtherpurified with the DNeasy spin-column kit. DNA was finallyrecovered in a single elution step with 100 μL of AE solutionfrom the kit.

The rs11515 of the p16CDKN2A gene was assessed by re-striction fragment length polymorphism (RFLP). This

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polymerase chain reaction for p16CDKN2Awas carried out in atotal volume of 25 μL containing approximately 100 ng ofgenomic DNA as template, 0.5 μL of each of the F primer 5′-GCCTGTTTTCTT TCTGCCCTCTG-3′ and the R primer 5′-CGAAAGCGGGGTGGGTTGT-3′ (20 pmol/μL), 2.5 μL ofdNTP mix (25 mM) (AMRESCO, Ohio, CA), 2.5 μL of 10×PCR buffer, 1.25μL of magnesium chloride (50 mM), and 2.5units of Platinum Taq DNA polymerase (Invitrogen LifeTechnologies, Carlsbad, CA, USA). The 144-bp PCR productfrom the p16CDKN2A gene was digested by HaeIII restrictionendonuclease (Sigma-Aldrich, St. Louis, MO) that recognizesa restriction site. Thus, the wild-type C allele has two bands(109 and 35 bp), while the G allele has only one band(144 bp). For this procedure, 10 μL of amplified DNA wasdigested with 2.5 U of HaeIII for 16 h at 37 °C. PCR andrestriction reactions were performed in a thermocycler(Eppendorf AG, Hamburg, Germany). DNA sequencing wascarried out to confirm the p16CDKN2A genotyping by PCR–RFLP.

Electrophoresis

The PCR products for polymorphism and digested fragmentswere verified by 10 % polyacrylamide gel electrophoresis at200 V of constant voltage for 1.0 h and stained with silvernitrate. Electrophoresis results were estimated using a 100-bpladder.

Statistical analysis

The statistical significance of the differences between the caseand control group distributions for alleles, genotypes, andclinicopathological features was determined using the Fisheror chi-squared test. Time of survival was calculated from thedate of diagnosis to the date of the last follow-up visit or to thedate of death. The records of each patient were reviewed,considering the same parameters, for 2–3,737 days. The timeof survival was calculated using the Kaplan–Meier estimatorand compared to results of the log-rank test. All analyses wereperformed using SPSS 18.0 (SPSS Inc., Chicago), and statis-tical significance was set at p<0.05.

Results

Association between p16CDKN2A polymorphism and HNSCC

To determine whether the presence of either of the singlenucleotide polymorphisms (SNP) of the p16CDKN2A genewas associated with HNSCC, chi-squared tests were per-formed comparing HNSCC and control groups. The genotypefrequencies of rs11515 polymorphisms of the p16CDKN2A genein both groups are presented in Table 1. No differences

between the control and HNSCC groups were observed. Thedistribution of genotypes was similar to the data from aCentral South African population and an African-Americanpopulation (NCBI dbSNP database).

Association between p16CDKN2A polymorphismand clinicopathological parameters in HNSCC patients

The genotype distributions of p16CDKN2A polymorphism ac-cording to age, molecular features, and clinicopathologicalparameters are summarized in Table 2. Thirteen patients fromeach group presented TNM I/II. No differences betweenp16CDKN2A genotypes and clinicopathological parameterswere observed.

Table 1 Genotype frequencies of the RS11515 polymorphisms of thep16 gene in HNSCC patients and the control group

Gene variant/genotype Controls n (%) HNSCC n (%) p value

RS11515

GG 07 (7) 07 (7.3)

CG 26 (26) 15 (15.6) 0.200

CC 67 (67) 74 (77.1)

All values were calculated using the χ2 test

Table 2 p16 genotype and its association with and clinicopathologicalfeatures in the case group

Variables Number (%) p value

GG CG CC

Age*

Young 2 (28.6) 9 (60.0) 39 (52.7) 0.379Older 5 (71.4) 6 (40.0) 35 (47.3)

Gender*

Male 6 (85.7) 14 (93.3) 68 (91.9) 0.825Female 1 (14.3) 1 (6.7) 6 (8.1)

Family history of any cancer*

Absent 6 (85.7) 10 (66.7) 41 (55.4) 0.243Present 1 (14.3) 5 (33.3) 33 (44.6)

Smoking status*

Never 6 (85.7) 15 (100) 70 (94.6) 0.368Ever 1 (14.3) 0 (0.0) 4 (5.4)

Anatomic sites*

Anterior 2 (28.6) 8 (53.3) 33 (44.6) 0.552Posterior 5 (71.4) 7 (46.7) 41 (55.4)

TNM clinical staging*

I/II 1 (14.3) 6 (40.0) 19 (25.7) 0.383III/IV 6 (85.7) 9 (60.0) 55 (74.3)

Locoregional metastasis*

Absent 0 (0.0) 8 (53.3) 27 (36.5) 0.053Present 7 (100) 7 (46.7) 47 (63.5)

*p<0.05 (significant, analyzed by Fisher’s or χ2 test)

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Molecular findings and the relative survival rate of HNSCCpatients

Once again, the time of survival was calculated from the dateof diagnosis to the date of the last follow-up visit or to date ofdeath. The records of each patient were reviewed, consideringthe same parameters. All deaths were caused by locoregionaland/or metastatic disease. For the purposes of analysis, pa-tients who died without evidence of recurrencewere excluded.The time of survival is displayed by means of the Kaplan–Meier method. In the present study, the mean survival of allHNSCC patients was 1,124.24 days from diagnosis. Factorswhich impacted on survival were differently distributedamong the groups. We observed no association betweenp16CDKN2A genotypes and time of survival. Considering clin-icopathological parameters, TNM staging impacted onsurvival.

Discussion

A background of genetic and environmental factors modulatesthe biological behavior of cancer. Among them, DNA poly-morphisms and their cooperative interaction with related ex-ogenous factors have been identified as pivotal modulators ofthe biological behavior of malignancy [27–32]. Thep16CDKN2A tumor suppressor gene is one of the key regulatorsof the cell cycle and is often inactivated in solid tumors [33].In vitro evidence using functional assay has reported thatp16CDKN2A is growth inhibitory and arrests cells in the G0–G1 phase of the cell cycle. The mechanism of action ofp16CDKN2A appears to be through the inhibition of CDK4and CDK6, leading to a blockage of pRB phosphorylation.A significant number of primary tumors have also demon-strated methylation of the p16CDKN2A, leading to inactivation[21]. It has been demonstrated by our group that p16CDKN2A

methylation is a common event in HNSCC development.Also, p16CDKN2A methylation has an impact in HNSCC sur-vival [5]. Moreover, since p16CDKN2A methylation has alsobeen found in precursor lesions of HNSCC [28], this marker isan interesting candidate for early detection and risk assess-ment. The SNP rs11515 is located in exon 3 of p16CDKN2A

gene. Although not leading to an amino acid sequence change,it has been associated with low expression of p53, tumorsusceptibility, a more aggressive course of malignant melano-ma [19], and a shortened tumor-specific survival rate in blad-der carcinomas [22, 34, 35].

The p16CDKN2A is a tumor suppressor gene that encodes fora cyclin-dependent kinase inhibitor that plays an importantrole in the regulation of the G1/S phase cell cycle checkpoint.The inactivation of this gene was observed in many tumortypes [41–45] by point mutation, homozygous deletion, andmethylation of the promoter region [46, 47]. In recent years,

several studies have demonstrated that genetic polymor-phisms are associated with susceptibility to, or protectionfrom, cancer development. Although the rs11515 G allele isassociated with p16 INK4a and p14 ARF inactivation, its rolein HNSCC has not been established. The rs11515 G allele as arisk factor has been most extensively characterized for mela-noma [12, 13, 30], having an increased frequency in patientsfrom families with a high risk of melanoma [12]. The G alleleoccurred more often in patients with tumors that have nodefined telomere maintenance mechanism. It was also a prog-nostic marker for poorer survival in glioblastoma multiforme.In the present study, we hypothesized that the rs11515 G allelemay be associated with worse prognosis for HNSCC patients.In the present study, no association was established betweenHNSCC susceptibility and rs11515 polymorphism, as indicat-ed in a previous study [23]. Previously, we demonstrated thatmethylation on the CDKN2A gene was present in 76 % of thepatients. The effect of the SNP rs11515 might be influencedby complex molecular mechanisms. It was demonstrated thatthe binding of MiR 601 to CDKN2A was predicted to beaffected by the rs11515 [18]. Considering these facts, it canbe speculated that different epigenetic and genetic pathwaysmay affect cancer evolution and prognosis.

HNSCC patients frequently exhibit a worse overall surviv-al with about 60 % of all patients reaching 5 years of survival.We found that HNSCC individuals with large-sized tumorsand with metastatic disease presented worse overall survival.These findings are consistent with fundamental conceptsthat establish the contribution of tumor size and lymphnode metastasis factors to the HNSCC outcome. Aprevious study showed that the rs11515 G allele isstrongly associated with susceptibility to some types ofhuman cancer, including individuals with bladder carcinomaand glioblastoma multiforme. By using bivariate tests, weobserved that neither allelic nor genotypes had an impact onthe overall survival using multivariate statistical tests. Futurestudies are needed to foster a better understanding of theparticipation of rs11515 polymorphism in the outcome ofpatients with HNSCC.

This study concluded that there are no differences in thedistribution of rs11515 between the control and HNSCCgroups. In addition, no differences between rs11515 geno-types and clinicopathological parameters were observed.These findings may contribute to a better understanding ofthe relationships between disease process and genetic factorsand eventually lead to more accurate diagnosis, treatment, andprognosis in HNSCC patients.

Acknowledgments This study was supported by grants from theConselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior(CAPES), and Fundação de Amparo a Pesquisa do Estado de MinasGerais (FAPEMIG). Dr. Guimarães, Dr. Santos, and Dr. de Paula areresearch fellows of CNPq.

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Conflicts of interests None.

Ethical statement All authors abide by the Association for MedicalEthics’ ethical rules of disclosure.

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