loss of heterozygosity and mutation analysis of the (9p2l ......d9s 157 d9s 171 l134t l13 p53 :ac/gt...

Vol. 1, 1043-1049, September 1995 Clinical Cancer Research 1043

Loss of Heterozygosity and Mutation Analysis of the p16 (9p2l)

and p53 (l7pl3) Genes in Squamous Cell Carcinoma of the

Head and Neck1

Maria Victoria Gonzalez,2 Marta F. Pello,

Carlos L#{243}pez-Larrea, Carlos Su#{225}rez,

MarIa J. Men#{233}ndez, and Eliecer Coto3

Laboratorio de Gen#{233}tica Molecular [M. V. G., M. J. M., E. C.],

Servicio de Otorrmnolaringologla [M. F. P., C. S.], and Servicio de

InmunologIa [C. L. L.], Hospital Central Universitario de Asturias,33006 Oviedo, Spain

ABSTRACT

We analyzed ablelic loss at the p13 gene (l7pl3) and atchromosome region 9p21 in 35 primary head and neck

squamous cell carcinomas. Loss of heterozygosity (LOH) at

p53 and 9p2l was found in 50 and 75% of informative cases,respectively. LOH at the p53 gene did not increase signifi-cantly with tumor stage, but was more frequent in moder-ately and poorly differentiated tumors than in well-differ-

entiated tumors. LOH plus mutation or homozygousdeletion of p53 was limited to advanced stage and poorlydifferentiated tumors. Allelic loss at 9p2l is frequent inearly stage head and neck squamous cell carcinoma and is

not significantly associated with LOH at p5.3. The second

exon of the p16/MTSI/CDKN2 gene was found to be ho-

mozygoushy deleted in 1 of 19 cases showing LOH at 9p2l,

but direct sequencing did not show mutations in theremaining 18 cases. This suggests that p16 plays a limited

role in the development of head and neck squamous cellcarcinoma.

INTRODUCTION

HNSCC4 is one of the most common types of cancer in

the world. More than 10,000 Americans die each year from

HNSCC (1). Its 5-year survival rate has not changed in recent

years, and is still one of the lowest among the prevalent

cancers. HNSCC is strongly associated with tobacco and

alcohol consumption (2).

Little is known about the genetic events involved in its

progression, although some genes have been reported to con-

tribute significantly (3). Mutations have been found in the

oncogene Ha-ras (4). Overexpression of cyclin D has been

reported to be associated with aggressive behavior of HNSCC

Received 2/17/95; revised 4/4/95; accepted 5/4/95.

1 This work was supported by Grant FISS 94/1421 (E. C.).2 Recipient of a fellowship of the FiCYT.

3 To whom requests for reprints should be addressed.4 The abbreviations used are: HNSCC, head and neck squamous cellcarcinoma; TSG, tumor suppressor gene; LOH, loss of heterozygosity;

SSCP, single-strand conformation polymorphism; cdk4, cychin-depen-dent kinase 4; VTR, variable tandem repeat(s).

(5-7). AJlebotyping using DNA microsatellite markers represen-

tative of all human chromosomes have found regions of com-

mon LOH (8, 9), an event associated with the inactivation of

TSGs. To date, the highest mutation rate for any gene in the

HNSCC is that of p53 (chromosome l7pl3; Refs. 10-13),

which is also mutated in most human cancers (14). Mutations of

the p.53 gene in HNSCC resembles those found in the squamous

cell lung carcinoma. p.53 is known to be a TSG (15) that encodes

a nuclear protein of 53,000, which acts as a transcription factor

(16-19) through sequence-specific DNA binding (20, 21). Mu-

tations in p53 abrogate its transactivating activity, leading to

uncontrolled cell growth (22). Approximately 90% of the p53

mutations are located in exons 5-9. These exons encode the

surface of the protein, and amino acids at this region are directly

involved in DNA binding (23).

Recent studies have described the presence of homozygous

deletions in multiple tumor-derived cell lines (24) and in pri-

mary melanoma (25, 26), pancreatic carcinoma (27), and esoph-

ageal squamous cell carcinoma (28) on another TSG, p16/

MTSJ/CDKN2, mapped to chromosome 9p2l. The protein binds

to cdk-4, preventing it from forming a complex with cyclin D.

The cyclin D/cdk-4 complex allows the cell cycle to progress

through G1, thus permitting cell division. If cdk-4 is overex-

pressed or expressed at the wrong moment, then pitS would

exert its role as a TSG by binding cdk-4 and inhibiting cell

growth (29).

TSGs are generally inactivated by the boss of one copy and

inactivation ofthe remaining copy by point mutation or deletion.

In an attempt to determine the robe of p53 and p16, we have

analyzed several microsatellite markers that are intragenic or

closely linked to these genes, and searched for mutations at

these genes in 35 unselected HNSCCs.

MATERIALS AND METHODS

Patients and DNA Extraction. Paired tumor and blood

samples were collected from 35 patients in the operating

room immediately after operation. For tumor DNA extrac-

tion, the fresh tumor tissue was isolated by microdissection,

and specimens containing more than 70% neoplastic cells

were placed at 37#{176}Cfor 24 h in lysis solution containing SDS

and proteinase K. DNA was obtained after phenol-chloro-

form extraction and ethanol precipitation (30). DNA from

WBCs was obtained in accordance with a previously de-

scribed protocol (31).

The 35 samples were from the pharynx (20 samples),

larynx (13 samples), and mouth floor (2 samples). Histologi-

cally, 13 were well-differentiated, 12 were moderately differen-

tiated, and 10 were poorly differentiated tumors. In reference to

the tumor stage, 1 neoplasm was stage I, 9 were stage II, 15 were

stage III, and 10 were stage IV tumors.

Research. on July 11, 2021. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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D9s 157 D9S 171

L134T L13

P53 :AC/GT

J�.p53: VTR

1044 Mutation Analysis of the p53 and p16/CDKN2 Genes in HNSCC

L25T L26T L117T L136T

L118T L122T

Fig. I LOH for microsatellites p53-AC/GT and p53-VTR, both intragenic to the p53 gene and D9S171 and D9S157. Arrows, deleted alleles,

characterized by a significant reduction of the signal in tumor (7) DNA relative to normal (L) DNA.

Microsatellite LOH Analysis. Microsatellite markers,

D9S157 and D95171, were analyzed to determine the LOH at

chromosome region 9p2i. One dinucleotide repeat and one VTR

polymorphism, both of which are intragenic to p53, were also

analyzed. Primers for these polymorphic markers were as pre-

viousby described (32-34). PCR was performed in 15-pA reac-

tion volumes containing 100 ng genomic DNA, 10 pmol of each

primer, 200 p.M each dATP, dGTP, and dTTP, 100 p.M dCTP, 2

mM MgC1, 0.10 p.Ci [32P]dCTP, 1 X Taq buffer, and 1 unit Taq

polymerase (Promega). Conditions consisted of an initial dena-

turing step of S mm at 98#{176}C,followed by 30 cycles of 1 mm at

98#{176}Cand 1 mm at 55#{176}Cand a final extension of S mm at 75#{176}C.

Taq polymerase was added after the initial denaturing step. PCR

products were mixed with 1 volume of denaturing loading

buffer (95% formamide, 1% xylene cyanol), heated at 98#{176}Cfor

S mm, and electrophoresed on a 6% denaturing polyacrylamide

gel (45% urea, 5.7% acrylamide, 0.3% bis-acrylamide). After

electrophoresis the gels were vacuum dried and autoradio-

graphed at -80#{176}Cfor 2 to 24 h.

After visual inspection of bands of the two matched sam-

ples, LOH was defined as a reduction of at least 50% in the

allelic intensity in tumor DNA.

PCR Amplification of p16 and p53 Genes. GenomicDNA from primary tumors was amplified for exons S and 6 and

7-9 ofp53 with the primers and under the conditions previously

described (35, 36). Exon 2 of pitS was amplified with primers

42F (GGAAATFGGAAACFGGAAGC) and 551R (TCT

GAGC1TFGGAAGCTCT) as described previously (24). Reac-

tions consisted of 200 ng genomic DNA, 50 pmol of each

primer, 0.2 mM each dNTP, 2 mrvi MgC1, 1 X reaction buffer, and

1 unit Taq polymerase (Promega) in a final volume of 50 p.1.

Amplification conditions were as described for microsatellite

LOH analysis, with an extension step of 1 mm at 72#{176}C.Aliquots

of each reaction mixture were electrophoresed on a 1 % agarose



1 2�4567M

A

C

B

Clinical Cancer Research 1045

gel, and the gels were ethidium bromide stained and photo-

graphed.

Multiplex PCR consisted of the same reaction conditions

and contained two sets of primer pairs, permitting the simulta-

neous amplification of two gene sequences.

SSCP Analysis of p53 and p16. Primers and annealing

temperatures for amplification of exons 5-8 of p53 have been

described previously (35, 36). Exon 2 of p16 was split into two

fragments. The first half ofpl6 was amplified with primers 42F

(see above) and 16.6 (CCAGGTCCACGGGCAGA, annealing

temperature 55#{176}C),and the second half with primers 551R (see

above) and 16.3 (ACTCTCACCCGACCCGT, annealing tem-

perature 52#{176}C).SSCP analysis was adapted from the original

method of Orita et a!. (37). PCR was done as described for

microsatellite analysis. Formamide-denaturing loading buffer

(100 p.1) was added to the reaction mixture and heated at 98#{176}C

for S mm. Five p.1 were immediately loaded on a 6% polyacryl-

amide gel containing 10% glycerol. Electrophoresis was at 10 W

for 12 h. Tumors showing band shifts were reamphified to

confirm the presence of shifts.

Direct Sequencing of the p16 and p53 Genes. PCRproducts were purified from agarose gels with the Sephaglas

Band Prep kit (Pharmacia). Subsequently, the double-stranded

DNA was directly sequenced by the dideoxy method with

35S-labeled ATP using the Sequenase Version 2.0 kit following

the manufacturer’s protocol (USB). PCR primers and other

internal oligonucleotides derived from the p53 and p16 genes

were used as sequencing primers. Sequenase reaction products

were separated on a 6% polyacrylamide sequencing gel and

autoradiographed. Mutations were confirmed by sequencing

both strands from two different amplifications.

RESULTS

Deletion and Mutation Analysis ofp53 (l’lpl3) and p16

(9p2l) Genes. To determine the relative incidence of LOH at

l’7pl3 and 9p2l, we compared the microsatellite patterns of

DNA samples from 35 HNSCCs with their normal tissue (lym-

phocytes) counterparts (Fig. 1). Two l7pl3 microsatellites were

studied, both of them intragenic to the p53 gene. Thus, LOH at

this region is due to the loss of one of the p53 alleles. A total of

32 cases were informative for at least one of the two p53

markers, and LOH was found in the tumor tissue of 14 patients.

A very faint band was obtained for the tumor DNA alleles in

two cases. In these two cases a PCR product was not obtained

for exons S and 6 and 7 to 9, suggesting that deletions have

eliminated both p53 alleles (Fig. 2). To determine the mutational

status of the remaining allele, we amplified exons 5-9 in the 14

cases showing LOH. SSCP and direct-sequencing analysis

showed the presence of at least one mutation in 4 cases (Fig. 3).

Three were missense mutations (codon 210, AAC:Asn-*AGC:

5cr; codon 154, GGC:Gly-�GTC:Val; codon 173, GTG:

Val-*ATG:Met) and one was a stop mutation at codon 196

(CGA:Arg-�TGA:stop) (Fig. 4).

The chromosomab region 9p2i is frequently deleted in

HNSCC and other tumors, and homozygous deletion for mark-

ers mapped to this region has been found in bladder, melanoma,

and lung cancers. We analyzed 2 microsatellites (D9S171 and

D9S157) at 9p2l in the 35 HNSCC tumors. A total of 31 cases

12345

- � - - �- -

,�53(L1-9)

._p16(E2)

\�53(E�-6)

�53

Fig. 2 Amplification of sequences of the p.53 and pitS genes. A, mul-

tiplex PCR (p16-exon 2 + p53-exons 5 and 6) of normal (Lane 1) and

tumor (Lane 2) DNAs from case 51. Multiplex PCR (p16-exon 2 +

p53-exons 7-9) of normal (Lane 3) and tumor (Lane 4) DNAs from the

same case. Lanes 5-7, amplification of p16-exon 2, p53-exons S and 6,

and p53-exons 7-9, respectively, in a control DNA. M, marker. B,multiplex PCR of pitS (exon 2) and p53 (exons S and 6) in five cases,

two ofthem showing homozygous deletion ofp53. C, multiplex PCR of

p16 (exon 2) and p53 (exons 7-9) of seven cases showing LOH at 9p21.Lane 8, homozygous deletion of pitS. Lanes 1, 4, and 6, reduced

amplification of pitS.

were informative for at least one of the markers, and LOH was

found in 19 of them (Fig. 1).

A gene at 9p2l with the properties of a TSG has been

recently described (29). This gene, p16/MTSI/CDKN2, is fre-

quently mutated in human cancer cell lines (24), and somatic

mutations have been demonstrated in primary melanomas and

esophageal and pancreatic carcinomas. To determine the muta-

tional status of the p16 gene in the HNSCC, we amplified the

second exon, where previous studies have located most pitS

mutations. We failed to obtain a PCR product in one of the

tumors that showed LOH at 9p2l (Fig. 2). This case gave a

normal size p53 product (exons S and 6 and 7-9), but we also

failed to amplify two regions encompassing the second exon of

p16 in a SSCP experiment (Fig. 3). These data suggest the

homozygous deletion of a 9p2l region that included the p16

locus. In four cases a p16 PCR product was obtained, but with



Lr,

I-.U,

I-- - #{182}�:� � �kTT�

p16 - E253 . E 8


p53�E5

Fig. 3 SSCP analysis of exons 5 and 8 ofp53, and exon 2 (first half, amplified with primers 42F and 16.6) ofpl6. *, tumors showing band shifts.

Tumor 25 shows homozygous deletion of p53. Tumor 51 shows homozygous deletion of p16.

a reduced yield (Fig. 2). We suspect the homozygous deletion of

this gene in these tumors, with the amplification observed cor-

responding to ‘ ‘contaminating’ ‘ nontumor cells. The presence of

normal tissue DNA in these cases was indicated by the fact that

a significant reduction in the autoradiographic intensity of the

deleted allele was obtained instead of the total absence of a

signal that characterizes the LOH in the tumor free of normal

‘ ‘contaminant’ ‘ tissue. SSCP and direct-sequencing analysis of

the cases showing LOH at 9p2l failed to detect any difference

relative to the normal p16 sequence (Fig. 3).

A significant difference was obtained for p53 LOH in

tumor grade (x2 - 6.8, P < 0.025, using x2 test for indepen-

dence). LOH at this locus is more frequent in poorly and

moderately differentiated tumors than in well-differentiated tu-

mors. When considering the tumor stage, LOH at both regions

occurred at the earliest stages (I and II) at a frequency that did

not differ significantly with respect to the advanced stages (III

and IV). In 10 cases we found LOH at p53 and 9p2l but no

significant association between them was found, suggesting that

they are independent genetic events in HNSCC.

DISCUSSION

We have analyzed the role of mutations at the p53 and pitS

genes in the progression of HNSCC. These two loci map at

regions showing a high frequency of LOH (8, 9, 38). Several

authors have previously described mutations at the p53 gene in

the HNSCC (10-13, 39-42). Mutations at this gene have also

been found in normal epithelia from smokers, which supports

the hypothesis of field cancerization in the development of

HNSCC (40). Chung et a!. (41) found p53 mutations in 47% and

37% of the patients with stage I or II tumors and stage III or IV

tumors, respectively. Boyle et al. (42) described a frequency of

19% in noninvasive lesions, increasing to 43% in invasive

carcinomas. We have found p53 LOH in 50% of our tumors, a

frequency similar to that described by other authors. LOH at the

p53 gene is not limited to advanced stages: it occurs in 33% of

the early stage (I/Il) tumors and in 55% of the advanced stage

(III/IV) tumors. Our work suggests that p53 LOH is an early

event that does not increase significantly with tumor progres-

sion. However, a second mutation, either deletion or point

mutation, would be more frequent in advanced stages.

LOH at the p53 gene is significantly associated with mod-

erately and poorly differentiated tumors. Several authors have

described that moderately and poorly differentiated tumors are

frequently aneuploid (43-45), and the loss of the wild-type p53

protein has been correlated with aneuploidy in several cancer

types. Our work suggests that the hypothesis of p53 as a guard-

ian that controls the genome stability, previously suggested for

other cancers (46, 47), could also apply to HNSCC.

LOH at 9p2l occurs at a high frequency in several human

cancers, including HNSCC (38). This genomic region contains

the p16 gene that encodes an inhibitor of the cell cycle, thus

having the property of a TSG. Initial studies demonstrated a

high frequency of pitS mutations, either microdeletions and

point mutations, in most human cancer cell lines (24, 48). The

name of multiple tumor suppressor 1 (MTS-1) was then given to

this gene. Analysis of tissue from melanomas, pancreatic ade-

nocarcinomas, and squamous esophageal carcinomas demon-

strated the presence of p16 mutations at a high frequency

(25-28). However, recent studies on primary carcinomas of

bladder, lung, and head and neck found gene mutations in a

limited number of cases (49, 50). All ofthese tumors show LOH

at 9p2l. Our study did not find any point mutation at p16 in

those cases showing LOH at 9p2l. However, homozygous de-

letion of p]6 was found in one case, suggesting a robe for this

gene in the development of this tumor. We suspect the homozy-

gous deletion of p16 in three additional HNSCC cases that

showed a reduced amount of the amplified product, as deter-

mined by the reduced ethidium bromide staining and autoradio-

graphic (SSCP) signals of the PCR products. Several tumors

contained a significant amount of contaminating non-neopbastic

tissue (infiltrating cells and normal mucosa). The presence of

DNA from nontumor tissue contaminating the DNA from neo-

plastic tissue is also a problem faced by all of the previous




ACGT

210

/C

A

ACGT

173

G�A

ACGT

154C

G

AGCT

196

C-T

Fig. 4 Mutations in the p.53 gene in four tumors showing LOH at this gene. Missense mutations occurred at codons 210, 154, and 173 in three cases.In one case, the nucleotide change at codon 196 was a non-sense mutation.

studies on HNSCC and other cancer types, as demonstrated by

the fact that in most of the cases that showed LOH at any

polymorphic marker the deleted allele is characterized by a

reduced signal rather than by the total absence of the band. This

general problem makes it difficult to identify homozygous de-

letion affecting any locus. In our study, homozygous deletions at

the p53 and p16 genes could not be confirmed because of

insufficient DNA for Southern blot analysis.

An alternative interpretation of LOH could be allelic im-

balance, whereby a chromosome region is amplified, increasing

the yield of PCR product from the allele amplified. However,

duplication/amplification would not account for the alterations

seen on chromosomes 9p and l7p. Previous works have tested

the p53 and p16 genes with Southern blot analysis, and no

amplification of these genes was described (49).

In a recent report, the homozygous deletion ofpl6 has been

described in 19% of 31 bladder primary tumors, while no point

mutations at the second exon of p16 were found in the same set

of tumors (50). It is possible that some mutations affecting pitS

on exons other than the second exon (e.g., mutations at exons 1

and 3, or at the regulatory sequences) have not been detected.

Recently, Zhang et a!. (51) analyzed the pi6/CDKN2 gene in 68




primary tumors of the head and neck. None of these tumors

showed homozygous deletion, and seven had misssense or

nonsene base changes (51). These along with our own work,

showing a reduced frequency of homozygous deletion or base

changes of p16 in the primary tumors, suggest that this gene is

not the only target of 9p21 LOH in the HNSCC. Alternatively,

loss of one copy could affect cell growth and tumor develop-

ment.

LOH at 9p2l was not found to be significantly associated

with tumor stage or tumor grade in the HNSCC. It occurs in

early stages (I/Il) at a higher frequency (75%) than LOH at the

p53 gene (33%). Interestingly, the case showing homozygous

deletion ofpl6 was an early (I) stage tumor, while the two cases

with homozygous p53 deletion were advanced (III) stage tu-

mors. A high frequency of boss of chromosome 9p in preinva-

sive lesions, suggesting that this is an early event in HNSCC

progression, has been described previously (38).

Cancer progression requires several genetic events, either

mutation at proto-oncogenes or tumor suppressor genes. It has

been suggested that HNSCC arises following 6-10 independent

genetic events (52). Our work suggests that loss of the pitS gene

through homozygous deletion, although at a low frequency,

could contribute to HNSCC development.

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1995;1:1043-1049. Clin Cancer Res M V González, M F Pello, C López-Larrea, et al. and neck.and p53 (17p13) genes in squamous cell carcinoma of the head Loss of heterozygosity and mutation analysis of the p16 (9p21)

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