Loss of Heterozygosity and Mutation Analysis of the (9p2l ......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
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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;
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
1048 Mutation Analysis of the p53 and p16/CDKN2 Genes in HNSCC
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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