.T Clin Pathol 1997;50:509-512

Deletion and methylation of the tumoursuppressor gene p 16/CDKN2 in primary headand neck squamous cell carcinoma

M Victoria Gonzailez, Marta F Pello, Carlos L6pez-Larrea, Carlos Suairez,Maria J Menendez, Eliecer Coto

AbstractAims-To study the homozygous deletionand methylation status of the 5' CpGisland of the p16 and p15 genes (9p2l) in aset of primary advanced head and necksquamous cell carcinomas (SCC) and totest whether inactivation ofthese genes bythese mechanisms contributes to headand neck SCC development.Methods-DNA was extracted from freshtumours. Homozygous deletion was deter-mined by the polymerase chain reaction(PCR) followed by hybridisation with thecorresponding probe, radioactively la-belled by the random priiing method.Methylation status ofthe CpG island ofthe5' region of these genes was assessed bydigestion with the appropiate restrictionenzymes followed by PCR and subsequenthybridisation with the correspondingprobe. The presence of point mutationswas determined by PCR-SSCP (singlestrand conformation polymorphism).Results-The p16 and p15 genes werehomozygously deleted in 20% and 10% ofthe tumours, respectively. No point muta-tions were found at p16 and p15. The 5'CpG island at the p16 gene was methylatedin 20% of the cases.Conclusions-The tumour suppressorgene p16 is inactivated through homo-zygous deletion or methylation in a sig-nificant proportion of cases of head andneck SCC.( Clin Pathol 1997;50:509-512)

Laboratorio deGeneticaMolecular-Servicio deInmunologia, HospitalCentral de Asturias,33006 Oviedo, SpainM V GonzalezC L6pez-LarreaM J MenendezE Coto

Servicio deOtorrinolaringologiaM F PelloC Suarez

Correspondence to:Dr Coto.

Accepted for publication8 April 1997

Keywords: head and neck squamous cell carcinoma;tumour suppressor genes; homozygous deletion; methy-lation; CpG island; single strand conformation; poly-morphism (p 16, p 15)

Head and neck squamous cell carcinoma(SCC) is a common type of cancer stronglyassociated with tobacco and alcoholconsumption.' Several genetic alterations havebeen reported to contribute to head and neckSCC development. Mutations in the oncogeneHa-Ras and overexpression of cyclin D havebeen described.2" Loss of heterozygosity stud-ies identified several chromosome regions thatare frequently deleted.5 Loss of heterozygosityand mutations at the p53 gene are frequent inhead and neck SCC, as well as loss ofheterozygosity at 9p21."8 However, the p16gene, a candidate tumour suppressor gene from

the 9p21 region, has a very low rate of pointmutations."The tumour suppressor gene p1 6/INK4A

encodes a protein that belongs to the INK4family of cdk inhibitors. It binds specifically tocyclin dependent kinase (CDK) 4/6 andprevents it from forming a complex with cyclinDI, thus blocking the entrance into the Glphase of the cell cycle.'" The cyclin D1/CDK4complex phosphorylates the retinoblastomaprotein (pRB), thus releasing cells from thegrowth inhibitory effect of unphosphorylatedpRB. The lack of p16 or the expression of amutated form of the p16 protein results inabnormal cell cycling and growth, the featuresthat characterise tumour development." Therole of p16 as a tumour suppressor gene inhead and neck SCC has recently been demon-strated by transfection of p16 into head andneck SCC cell lines that lacked p16 function,resulting in cell cycle arrest.'2

In accordance with its central role in cellgrowth inhibition, the p16 gene is frequentlymutated in most human cancers, following thetwo hits model for the inactivation of tumoursuppressor genes-one allele is deleted and theother has a point mutation or is deleted. Celllines and primary tumours from most humancancer types show a high frequency of loss ofheterozygosity at chromosome 9p2l, the re-gion where the p16 locus is located. However,they differ in the nature of the secondmutation-either a point mutation, resulting ina stop codon, or an amino acid change or asecond deletion, resulting in the homozygousdeletion of the p16 gene.'3"-" More recently,several authors have established an alternativemechanism for p 16 inactivation through methy-lation of a 5' CpG island, resulting in the trans-criptional silencing of the p1 6 gene.20 21 Analy-sis of several head and neck SCC derived celllines and primary tumours indicated thathomozygous deletion and transcriptional si-lencing through methylation of the 5' CpGisland would be a major mechanism for p16inactivation in this type of cancer.22 23The p1 5/INK4B gene is related to p16/

INK4A and is located 28 kilobases centromericto the p 16 gene on 9p2 1. Transforming growthfactor 3 (TGF,B) induces the expression ofp1 5,which in turn inhibits the cell cycle, thus medi-ating the inhibitory function of TGFf3.24We describe the homozygous deletion and

methylation of p 1 6/INK4A in a significantnumber of primary head and neck SCC,

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p16 p15

Telomere -

El E2 E3

p16-1 p16-2

El

p15-1 p1 5-2

E2

Figure 1 Map of the p16 andp15 genes, indicating (arrows) the primers usamplification of exon 1 ofp16 andplSfor the assessment of homozygous delemethylation status of both genes. 0 HpaII sites; *SmaI site.

indicating that inactivation of this tipressor gene through these two gen4nisms would be a basic event in theof this type of cancer.

MethodsPATIENTS AND DNA EXTRACTIONTumour samples were collectedpatients. The fresh tumour tissue wasected. Specimens containing moreneoplastic cells were placed in lyzcontaining 1% sodium dodecyl sulpand proteinase K for 24 hours atDNA was obtained by phenol-extraction and ethanol precipitatior

HOMOZYGOUS DELETION OF THE P16 GENEA 340 base pair sequence encompassing thefirst exon ofthe p16/INK4A gene was amplifiedwith primers p16-1: 5'GAAGAAAGAGGAG-GGGCTGGCTGGTCACCA3' and p 16-2:5'GCGCTACCTGATTCCAATTCCCCTG-CAAAC3'. Reactions consisted of 100 ng ofgenomic DNA from tumour samples, 20 pmolof each primer, 200 jM of each dNTP,2.5 mM MgCl,, 1 x reaction buffer and oneunit of Taq polymerase in a total volume of20 gl. PCR conditions consisted of 20 cycles of30 seconds at 95°C, one minute at 62°C(annealing) and one minute at 72°C (exten-sion). The reactions (5 gl each) were electro-phoresed overnight on a 1.5% agarose gel andblotted on to a nylon membrane. Hybridisationwas at 42°C with a formamide hybridisationsolution containing the p16 (first exon) probe

Table 1 Homozygous deletion and methylation of the 5' CpG island in the pl6 and pl5genes in 20 cases ofhead and neck squamous cell carcinoma

Case LOH 9p2* pl6 delt pl6 met4 pl5 delt pl5 met,1 NI +23 NI + +4 -5 NI + +6 +78 + +9 NI10 + +11 NI - - -

12 + - - - -

13 + - - - -

14 + - - - -

15 + -+16 + -

17 + - +18 + -

19 + -

20 NI +

*LOH 9p2 1, + allelic loss; -heterozygosity retained; NI not informative.tpl 5/p16 met, + methylation of the 5' CpG island; -no methylation.tp 1 5/p 16 del, + deletion of the first exon; -no deletion.

radioactively labelled by the random primingmethod. After three washes of 30 minutes at650C with 0.1 x SSC/0.5% SDS, membranes

Centromere were autoradiographed at -800C for 2-12hours.To test the quality of the DNA, an 1125 base

pair sequence corresponding to nucleotides0.3 kb 755-1880 ofthe N-acetyl transferase-2 (NAT-2)

gene was amplified with primers 5'GGGGAT-edin the CATGGACATTTGAAG3' and 5'CTTCC-tion and CAAGATAATCACAGGCC3' (EMBL acces-

sion number X14672). Southern blot analysis,umour sup- including hybridisation with a NAT-2 radiola-etic mecha- belled probe, was performed as described for theprogression analyis of the p16 gene.

HOMOZYGOUS DELETION OF THE P15 GENEThe first exon of p1 5/INK4B was amplifiedwith primers p15-1: 5'GCAGCGTGGGAAA-

from 20 GAAGGGAAG3' and p15-2: 5'GCCCTGG-s microdis GGCCCCAGCTA3', yielding a fragment ofe microdis- 260 base pairs. Annealing temperature wassis solution 65"C and PCR was performed under the same,hate (SDS) conditions as the amplification of the p1 6 gene.37°Ca and The reactions were electrophoresed (10 glchloroform each), blotted on to a nylon membrane and-chloroform hybridised with a p 15 exon 1 probe.

METHYLATION ASSAYWe examined the methylation status of twoHpaII sites (CCGG) at the 5' CpG island ofthe p 16 and p 15 genes. One SmaI site(CCCGGG), coincident with one HpaII siteon the 5' CpG region of p16 was also studied(fig 1). Tumour DNA (1 jg) was digested with20 units of HpaII and 20 units of SmaI(Boehringer Mannheim, Germany). The di-gested DNA (50 ng) was subjected to 20 cyclesof PCR with the primers used for the analysisof homozygous deletion of the p16 gene. Theamount of amplified DNA was quantified afterSouthern blot analysis of 10 pl of the PCRproduct with a p1 6 exon 1 probe, as describedabove. The same protocol was followed for theexon 1 ofp 15, amplified with the same primersthat were used for the homozygous deletion ofp15.

PCR-SSCP (SINGLE STRAND CONFORMATIONPOLYMORPHISM)Tumour DNA was amplified with primers forp16 exon 1 and p15 exon 1. SSCP analysis wasadapted from the original method of Orita etat5 and PCR was performed as previouslydescribed.8 Formamide denaturing loadingbuffer (100 pl) was added to the reaction mix-ture and heated to 98°C for five minutes afterwhich 5 gl was immediately loaded on to a 6%polyacrylamide gel containing 10% glycerol.Electrophoresis was carried out at 10 W for 12hours.

ResultsHOMOZYGOUS DELETION OF THE P16 AND P15GENESTumour DNA samples from 20 patients weresubjected to PCR for the assessment ofhomozygous deletion (table 1). PCR per-formed over 25-30 cycles rendered an amplifi-cation product in all samples checked. How-

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pl6ICDKN2 in primary head and neck squamous cell carcinomla

1 2 3 4 5 6 7 8 9 gene that encodes the inhibitor of cyclinD/CDK4 complexes. This gene, pl6/INK4A,

_ _ _ ___isthe target of the 9p2l loss of heterozygosityobserved in many human tumours.Y 2 The

_ . -~ - - p1 5/INK4B gene maps to the same region, 28kilobases centromeric to p16.23 However, p15mutations are a rare event in human

18 26 28tumours. 2 Some cases harboured p16homozygous deletion that did not involve15.29 Moreover, in the rare cases where p15

was deleted, this deletion also included the first-- So exon of p 16.30

We analysed the p16 and p15 genes in a setof 20 cases of primary advanced head and neckSCC. According to Knudson's two hits model

p for tumour suppressor genes, p16 would beinactivated in human tumours by allele lossand point mutation. Previous studies usingtumour cell lines showed a high incidence ofp16 mutations. However, this was not consist-

___ !W-1; ent with the results obtained from fresh tumoursamples. p16 is frequently mutated in heredi-tary melanoma and pancreatic carcinoma butnot in many other human cancers.'3 16 31 We didnot find any evidence of p16 mutation in thefirst and second exons of p16 in the 20 headand neck SCC studied. Some authors havedescribed the presence of p16 mutations in10% of head and neck SCC, while others have

(A) Amplification of a fragment of the NAT-2 gene (nucleotides 755 to 1880). found a much lower incidence (one in 29lification of exon 1-p15 of tumour DNA samples (lane 9, tumour showing exon f a

mozygous deletion). (C) Amplification of exon 1-p15 of tumour DNA samples cases).y digested with HpaII (lane 1, tumour harbouring methylation of the p15 5'CpG We used PCR to identify the cases displaying`D) Amplification of exon 1-p16 of tumour samples (lanes 3, 7, and 9, cases homozygous deletions, another known mech-womozygous deletion). (F) Amplification of exon 1-p16 of tumour DNA 2vy digested with HpaII (lanes 1 and 2, cases showing methylation of the p16 5' anism of p16 inactivation.2 We failed tond). amplify the first exon of p16 in four cases and

the first exon of p15 in two cases, both alsoever, when fewer than 20 cycles were used shwn p16 omzgu deti.Teesome~~~~~~~~~sape.io iedaypout showing p 16 homozygous deletion. These

some samples did not yield any product, tmuswudntepesayp1 rtiindicating that this was the ideal number of anmould ouldnot express anype o6 protelncycles to minimise the contaminating effect of cycle, allowing it to progress in some situatonsnon-tumour cells. All cases showed amplifica-tion with the primers for the NAT-2 gene (fig that would otherwise lead to a cell cycle arrest.

2).Thiswastake as a control forthequaliy of Reed et al have reported a higher incidence2). lThis was taken as a control for the quality of (6% ohmzyusdltn. 1 TeyueDNA. Four of 20 cases (20%o) did not amplify (67%/) of homozygous deletion.32 They usedthe Fir of hesp1 0n (fig n2) Tmh ,lf microsatellite analysis for the assessment of this

gene first exon wahomozygously de in alteration. The different experimental ap-two cairstesx( ) tat werezyglso deletedfor th proach might account for the discrepancy inpw16 gene (see fi 2). the results, since the presence of contaminatingDNA from normal tissue could have made us

METHYLATION STATUS OF THE PI6 AND PI5 GENES

All the samples were digested with HpaII andSmaI and subjected to amplification of the firstexon of the p16 and p15 genes. Hybridisationwith the corresponding probes and subsequentautoradiography showed the methylation ofp16 and p15 in four cases (20%) and one case

(5%), respectively (table 1, fig 2).

POINT MUTATIONS IN THE P16 AND PI5 GENES

The 20 cases were subjected to PCR-SSCPanalysis for the detection of point mutations inthe first exon of the p1 6 and p1 5 genes. Noneof them showed any band shift. We had previ-ously studied the second exon with the same

approach and no abnormalities were found.8

DiscussionAllelic loss of the chromosome 9p2i region isone of the most frequent genetic alterations inhead and neck SCC.7' This region contains the

miss some cases harbouring homozygous dele-tions. However, this is unlikely, as we reducedthe number of PCR cycles to 20 to minimisethis masking effect.

Hypermethylation of the 5' CpG island ofthe p16 gene correlates with gene silencing.This hypermethylation is frequently present inmany types of cancers.'3 Our results indicatethat p16 was inactivated by this mechanism in20% of the cases studied. The p16 gene is themajor target of inactivation by methylation inhead and neck SCC as the incidence of p15methylation is much lower. Thus, p 15 methyla-tion would not be a specific event contributingto progression of the disease. This is in linewith the finding that hypermethylation of p16and p15 is a selective phenomenon dependingon the tissue of origin. Thus, in most epithelialderived tumours (lung, head and neck SCC,breast, prostate, and colon) methylation wouldaffect p1 6 exclusively, while p1 5 would be the

A

B

D

E

Figure 2(B) Amp1-p15 ho,previousl1island). (showing /previouslCpG islai

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main target of inactivation in gliomas andleukaemias.3

In 40% of our patients the p16 gene wasinactivated either by homozygous deletion ormethylation. The importance of p16 altera-tions in the development of head and neckSCC indicates that this tumour suppressorgene could be a major target of antitumourtreatments. In accordance with this, it has beenseen that transfection of head and neck SCClines with the p 16 gene has reverted thetumour phenotype.'2

This work was supported by Grant FISS 94/1421 and 96/1418(EC). MVG is the recipient of a fellowship of the Fundacion defomento en Asturias de la Investigacion Cientifica aplicada y laTecnologia.

1 Decker J, Goldstein JC. Risk factors in head and neck can-cer. N Engl _r Med 1982;306:1151-5.

2 Nufiez F, Dominguez 0, Coto E, Suarez-Nieto C, Perez P,Lopez- Larrea C. Analysis of ras oncogene mutations inhuman squamous cell carcinomas of the head and neck.Surg Oncol 1992;1:405-11.

3 Saranath D, Panchal RG, Nair R, Mehta AR, Sanghavi V,Sumegi J. Oncogene amplification in squamous cellcarcinoma of the oral cavity. Jpn .7 Cancer Res 1989;80:430-7.

4 Wang X, Pavelic ZP, Li Y-Q, Wang L, Gleich L, Radack K,et al. Gene amplification and overexpression of the cyclinGI gene in head and neck squamous cell carcinoma. _7 ClinPathol: Mol Pathol 1995;48:M256-9.

5 Nawroz H, van der Riet P, Hruban RH, Koch W, RuppertJM, Sidransky D. Allelotype of head and neck squamouscell carcinoma. Cancer Res 1994;54:1152-5.

6 Boyle JO, Hakim J, Koch W, van der Riet P, Hruban RH,Roa RA, et al. The incidence of p53 mutations increaseswith progression of head and neck cancer. Cancer Res1994;53:4477-80.

7 Van der Riet P, Nawroz H, Hruban RH, Corio R, Tokino K,Koch W, et al. Frequent loss of chromosome 9p2l-22 earlyin head and neck cancer progression. Cancer Res 1994;54:1156-8.

8 Gonzalez MV, Pello MF, L6pez-Larrea C, Suarez C,Menendez MJ, Coto E. Loss of heterozygosity andmutation analysis of the p 16 (9p21) and p53 (17p 13) genesin squamous cell carcinoma of the head and neck. ClinCancer Res 1994;1:1043-9.

9 Zhang S-Y, Klein-Szanto AJP, Sauter ER, Shafarenko M,Mitsunaga S, Nobori T, et al. Higher frequency ofalterations in the p1 6/CDKN2 gene in squamous cell car-cinoma cell lines than in primary tumours of the head andneck. Cancer Res 1994;54:5050-3.

10 Serrano M, Hannon GJ, Beach D. A new regulatory motif incell-cycle control causing specific inhibition of cyclinD/cdk4. Nature 1993;366:704-7.

11 Serrano M, Gomez-Lahoz E, DePinho RA, Beach D,Bar-Sagi D. Inhibition of Ras-induced proliferation andcellular transformation by p 16NKS Science 1995;267:24952.

12 Liggett WH, Sewell DA, Rocco J, Ahrendt SA, Koch W,Sidransky D. p 15 and p 16 are potent growth suppressors ofhead and neck squamous carcinoma cell in vitro. Cancer Res1996;56:4119-23.

13 Caldas C, Hahn SA, Da Costa LT, Redstone MS, SchutteM, Seymour AB, et al. Frequent somatic mutations andhomozygous deletions of the p 16 (MTS 1) gene in pancre-atic adenocarcinoma. Nlat Genet 1994;8:27-32.

14 Mori T, Miura K, Aoki T, Nishihira T, Mori S, Nakamura YFrequent somatic mutations ofthe MTS1/CDK4 (multipletumour suppressor/cyclin dependent kinase 4 inhibitor)gene in esophageal cell carcinoma. Cancer Res 1994;54:3396-97.

15 Okamoto A, Demetrick DJ, Spillare EA, Hagiwara K, Hus-sain SP, Bennett WP, et al. Mutations and alteredexpression of p1 61N4 in human cancer. Proc Natl Acad SciUSA 1994;91:11045-9.

16 Spruck C, Gonzalez-Zulueta M, Shibata A, Simoneau A,Lin M, Gonzales F, et al. p 16 gene in uncultured tumours.Nature 1994;370:183-4.

17 Cairns P, Mao L, Merlo A, Lee DJ, Schwab D, Eby Y, et al.Rates of p16 (MTS1) mutations in primary tumours with9p loss. Science 1994;265:415-7.

18 Jen J, Harper JW, Bigner SH, Bigner DD, Papadopoulos N,Markowitz S, et al. Deletion of p 16 and p 15 genes in braintumours. Cancer Res 1994;54:6353-8.

19 Gonzalez MV, Artimez ML, Rodrigo L, L6pez-Larrea C,Menendez MJ, Alvarez V, et al. Mutation analysis of thep53, APC, and p16 genes in the Barrett's oesophagus, dys-plasia, and adenocarcinoma. JClin Pathol 1997;50:212-17.

20 Herman JG, Merlo A, Mao L, Lapidus RG, Issa J-PJ,Davidson NE, et al. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNAmethylation in all common human cancers. Cancer Res1995;55:4525-30.

21 Gonzalez-Zulueta M, Bender CM, Yang AS, Nguyen TD,Beart RW, van Torout JM, et al. Methylation of the 5' CpGisland of the p1 6/CDKN2 tumour suppressor gene in nor-mal and transformed human tissues correlates with genesilencing. Cancer Res 1995;5:4531-5.

22 Cairns P, Polascik TJ, Eby Y, Tokino K, Califano J, Merlo A,et al. Frequency of homozygous deletion at p16/CDKN2 inprimary human tumours. Nat Genet 1995;11:210-2.

23 Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, BurgerPC, et al. 5' CpG island methylation is associated withtranscriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat Med 1995;1:686-92.

24 Hannon GJ, Beach D. p 15IKNB is a potential effector ofTGFinduced cell cycle arrest. Nature 1994;371:257-61.

25 Orita M, Suzuki Y, Sekiya T, Hayashi K. Rapid and sensitivedetection of point mutations and DNA polymorphismsusing the polymerase chain reaction. Genomics 1989;5:874-9.

26 Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, HarshmanK, Tavtigian SV, et al. A cell cycle regulator potentiallyinvolved in the genesis ofmany tumour types. Science 1994;264:436-40.

27 Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, CarsonDA. Deletions of the cyclin-dependent kinase-4 inhibitorgene in multiple human cancers. Nature 1994;368:753-6.

28 Stone S, Dayananth P, Jiang P, Weaver-Feldhaus JM,Tavtigian SV, Cannon-Albright L, et al. Genomic structure,expression and mutational analysis of the p15 (MTS2)gene. Oncogene 1995;11:987-91.

29 Otsuki T, Clark HM, Wellman A, Jaffe ES, Raffeld M.Involvement of CDKN2 (p16INK"\/MTS1) and p151NK4B/MTS2 in human leukemias and lymphomas. Canicer Res1995;55:1436-40.

30 Stone S, Jiang P, Dayananth P, Tavtigian SV, Katcher H,Parry D, et al. Complex structure and regulation of the p 16(MTS1) locus. Cancer Res 1995;55:2988-94.

31 Hussussian CJ, Struewing JP, Goldstein AM, Higgins PAT,Dracopoli NC. Germline p16 mutations in familialmelanoma. Nat Genet 1994;8:15-21.

32 Reed AL, Califano J, Cairns P, Westra WH, Jones RM, KochW, et al. High frequency of p 16 (CDKN2iMTS- 1 /INK4A)inactivation in head and neck squamous cell carcinoma.Cancer Res 1996;56:3630-3.

33 Herman JG, Jen J, Merlo A, Baylin SB. Hypermethylation-associated inactivation indicates a tumour suppressor rolefor p 15 NK4B. Cancer Res 1996;56:722-7.

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