(CANCER RESEARCH 5.1. 2128-21.12. May I. 1993]
p53 Mutation Is a Common Genetic Event in Ovarian Carcinoma1
Ben J. Milner,2 Lindsey A. Allan, Diana M. Eccles, Henry C. Kitchener, Robert C. F. Leonard, Kevin F. Kelly,
David E. Parkin, and Neva E. Haites
Department i>jMolecular anil Cell Biology ¡Medica/ Genetics), University- of Aberdeen. Medical Schon! Buildings, Foresterhill. Aberdeen ABV 2ZD ¡B.J. M.. L A. A., K. F. K.,
N. E. H.I: Imperial Cancer Research fund Medical Oncology Unit. Western General Hospital. Edinburgh EH4 2XU ID. M. E., R. C. E L.¡:and Department of Obstetrics and
Gynaecology. Aberdeen Knyal Infirmary. Foresterhill. Aberdeen AB9 2ZB ¡H.C. K.. D. E. P./. Scollanti
MATERIALS
ABSTRACT
Using the single-strand
conformational
polymorphism
technique,
we
have screened 66 malignant ovarian tumors forp53 mutation in exons 5 to
8. Thirty-four of the tumors demonstrated a single-strand conformational
polymorphism band shift in this region of the gene, including 6 in exon 5,
7 in exon 6, 12 in exon 7, and 10 in exon 8 (one of the tumors showed a shift
for exons 7 and 8). All of the single-strand conformational polymorphism
shifts have been further characterized by DNA sequencing, and 31 of 35
have been shown to represent genuine DNA alterations. These include 27
point mutations (23 missense, 2 nonsense, and 2 silent mutations), 3 dele
tions (a 2-base pair deletion introducing, by frameshift, a stop codon
further downstream; a 3-base pair deletion; and an unusual 6-base pair
deletion made up of separate 2-base pair and 4-base pair deletions), and a
4-base pair insertion (introducing a stop codon downstream). In total, 29
of the 66 (44% ) carcinomas analyzed had mutations affecting the primary
sequence of the p53 protein.
p53 mutation was found in tumors of all International Federation of
Gynecologists and Obstetricians stages, suggesting that it might be an
earlier genetic event in the progression of epithelial ovarian tumors than
previously thought. A significantly greater number of p53 mutations were
seen in high-grade serous carcinomas than in those of endometrioid and
ruminons types (0.02
I"
0.01). Analysis of the distribution of point
AND METHODS
Clinical Materials. Fresh tumor tissue was collected following surgical
debulking and stored frozen at -20°C, with peripheral blood samples from the
same patients. A total of 66 ovarian carcinomas were analyzed. 37 of which
were collected at the Western General Hospital in Edinburgh, and 29 of which
were collected at Aberdeen Royal Infirmary. A variety of borderline and benign
ovarian tumors of epithelial origin (15 of each) were also collected for analysis.
DNA Extraction. Blood and tumor DNA extraction was carried out by
standard methods (17).
SSCP Analysis. Polymerase chain reaction was carried out tor p53 exons
5-8 on 100 ng of tumor DNA using 5 pmol of the following primer pairs:
exon 5, 5'-TTCCTCTTCCTACAGTACTC-3'
5 '-GCCCCAGCTGCTCACC ATCG-3 ' ;
exon 6, 5'-GGCCTCTGATTCCTCACTGATT-3'
5 ' -AG AG ACCCCAGTTGCAAACC-3
';
exon 7, 5'-CTTGCCACAGGTCTCCCCAA-3'
5 ' -AGGGGTC AGCGGC AAGCAG A-3 ' ;
exon 8. .V-TGCTTCTCTTTTCCTATCCTGA-3'
5 ' -CGCTTCTTGTCCTGCTTGCT-3
'.
mutations showed no preference for any particular mutation type.
The following program was used: 30 cycles of 94°C,2 min; 55°C.2 min; 72°C,
3 min; incorporating [a-'2P]CTP. Point mutations in the tumor DNA were
INTRODUCTION
demonstrated by running tumor and normal control DNA polymerase chain
reaction products, in adjacent lanes, on 5% nondenaturing acrylamide gel
containing 0. 5, or 10% glycerol at 20°Cusing either 0.5X or I.Ox Tris borate
It is becoming increasingly clear that the p53 tumor suppressor gene
plays a central role in the maintenance of normal cell growth and
differentiation ( 1). Inacti vation of the p53 gene by mutation is, to date,
the most commonly detected genetic lesion in human cancer (2, 3). It
is conceivable that the loss of normal function of this gene is involved
in the etiology of the whole spectrum of tumor types, from the more
common cancers of colon (4), breast (5), and lung (6) to the less
common cancers such as the soft tissue sarcomas (7. 8).
Ovarian cancer can undoubtedly be included as being affected by
loss of normally functioning p53 with mutations already reponed by
a number of groups (9-13). We report the detection of p53 mutations
in exons 5-8 of the p53 gene, previously defined as containing the
"hot spot" regions of mutation in human cancer (14). using SSCP1
analysis and direct DNA sequencing in a large group of ovarian
carcinomas. In lung (2). colorectal (2), and hepatocellular carcinomas
(15. 16), a specific mutation type has been found to predominate.
Statistical analysis was therefore carried out on the distribution of
mutation type in ovarian carcinoma, using both our results alone and
those previously published (9. 10, 12. 13) as a whole.
Received 11/9/92: accepted 2/23/93.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by the Scottish Hospitals Endowments Research Trust (B. J. M.J and the
EDTA buffer (18). The appearance of a band shift in the tumor DNA when
compared with the normal banding pattern for the particular stretch of DNA is
characteristic of a change in the secondary structure of the mutant single
strands caused by the altered primary sequence (19).
Direct DNA Sequencing. Polymerase chain reaction was carried out using
primers hybridizing externally to the regions amplified for SSCP analysis in a
second independent reaction. Five pmol each of the following primer pairs
were used:
exons 5 and 6. 5'-TGTTCACTTGTGCCCTGACT-3'
5'-GGAGGGCCACTGACAACCA-3';
exon 7,
5'-CAGGTCTCCCCAAGGCGCACTGGCC-3'
5 ' -TGTGC AGGGTGGC AAGTGGC-3 ' ;
exon 8,
5'-TGGGAGTAGATGGAGCCTGG-3'
5'-AGGAAAGAGGCAAGGAAAGG-3',
with the same program as for SSCP analysis. The antisense primer was biotinylated at the 5' end to facilitate the purification of single-stranded template
using streptavidin-conjugaled
magnetic beads (Dynabeads
M-280 Streptavi-
din; Dynal UK, Ltd.). The sequencing reactions were carried out using the T7
sequencing kit (27-1682-01; Pharmacia LKB) with [a-"S]ATP and the sense
primer for each exon (exon 6 was sequenced using an additional internal sense
primer 5'-TGGTTGCCCAGGGTCCCCAG-3')
and were run on a 6» dena
turing acrylamide gel (20).
Statistical Analysis. A simple \~ test was used to analyze the distribution
of point mutations.
RESULTS
Medical Research Council (L. A. A.).
: To whom requests for reprints should be addressed, at Department of Molecular and
Cell Biology (Medical Genetics). University of Aberdeen. Medical School Buildings.
Foresterhill. Aberdeen AB9 2ZD. Scotland.
' The abbreviation used is: SSCP. single-strand conformational polymorphism.
p53 Mutation Detection by SSCP Analysis. Of the 66 ovarian
carcinomas analyzed for p53 exons 5 to 8, 34 demonstrated confor
mational band shifts (those shown in Table 1; see also Fig. 1). These
2128
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/>5.Ã-Mutation in Ovarian Carcinoma
Table 1 Ovarian tumors with p53 SSCP .v/if/r.vand characterized
mutatuw.\
analysisTGC
detected by SSCP
CodeS3IS55S21G3G24G34S49G20G59GI7SSG41S41S16S61S43G26G15S45S60GI9G12FISIIG60G56GIIG2IS53G43045SI5S24G28S46PathologySerous
GGCGTG
-»
141Codon
adenocarcinomaSerous
5Exon
ATGCGC
-»
adenixarcinomaSerous
5Exon
173Codon
CACCGC
->
adenocarcinomaSerous
5Exon
175Codon
CACCAT
->
adenixarcinomaSerous
5Exon
175Codon
CGTCGC
->
179Codon
ademxarcinomaSerous
5Exon
CCCATC
-»
ademxarcinomaEndometrioid
5Exon
181Codon
GLYVAL
->
METARG
->
HISARG
-»
HISHIS
->
ARC,ARG
->
PROILE
->
adenocarcinomaSerous
adenocarcinomaEndometrioid
adenocarcinomaSerous
adenocarcinomaBorderline
mucinousSerous
adcmxarcinomaMixed
tumorSerornucinous
Mullerian
adenocarcinomaSerous
6Exon
6Exon
6Exon
6Exon
6Exon
6Exon
6Exon
6Exon
195Codon
208Codon
213Codon
213Codon
213Codon
213Codons
15Codon 2 13-2
220Codon
ACCGAC
-»
GTCCGA
-»
CGGCGA
-»
CGGCGA
->
CGGCGA
->
TGACGA.
->
ATTTAT
CAT. ACT -»
TGT2-hase
-»
adenocarcinomaSerous
7Exon
229Codon
ademxarcinomaSerous
adenocarcinomaSerous
ademxarcinomaSerous
ademxarcinomaEndometrioid
7Exon
7Exon
7Exon
7Exon
8Exon
236Codon
236Codon
242Codon
241,
244No
mutationCodon
deletion.frameshift
pair
eodonTAC —¿
stop at
TGCTAC
-»
TGCTCC,
->
TGCGGC
TGC ->
GACGGC
-»
CYSTYR
->
CYSSER.
-»
CYSGLY
CYS -»
ASPGLY
-»
adentxarcinomaSerous
ademxarcinomaSerous
ademxarcinomaSerous
adentxarcinomaSerous
adenocarcinomaSerous
adentxarcinomaF.tidornetrioid
adentxarcinomaSerous
7Exon
7Exon
7Exon
7Exon
7Exon
7Exon
7Exon
245Codon
245Codon
248Codon
259Codon
259Codon
259N't)
mutationCodon
AGCGGC
-»
AGCCGG
-»
TGGGAC
-»
GTCGAC
->
GTCGAC
->
TACGGA
-»
SERGLY
->
SERARG
-»
TRPASP
-»
VALASP
-»
VALASP
->
TYRGLY
->
ademxarcinomaSerous
adentxarcinomaMucinous
adentxarcinomaSerous
adentxarcinomaSerous
adentxarcinomaSerous
adentxarcinomaUndifterentialed
8Exon
8Exon
8Exon
8Exon
8Exon
8Exon
266Codon
266Codon
273Codon
273Codon
274Codon
276Codon
GTAGGA
->
TGACGT
-»
TGTCGT
->
TGTGTT->CTT4-base
->
insert.frameshift
pair
ctxlonGAC —¿
stop at
-> GGCCYS
carcinomaEndometrioid
8Exon
281No
mutationNo
adentxarcinomaAdentxarcinoma,
8Exon
no specific typeFIGO"stageIIIIIIIIIIIIIIIIIIcIVmnIMinmIKinininHeIIIIIIIIIIcIblaIIIIIIIIIIIIIIIIIIIIIlaIIIExon
8pS3Codon
mutationmutation
' International Federation of Gynecologists and Obstetricians.
were fairly well distributed between the four exons tested. Tumor S45
showed a shift for exons 7 and 8. A shift for exon 6 was detected in
one of the 15 borderline ovarian tumors tested (see Table 1), but no
such band shifts were found in the DNA of any of the benign ovarian
cysts.
Characterization of SSCP Band Shifts by Direct Sequencing.
Table 1 lists, in primary sequence order, the mutations which were
characterized using direct DNA sequencing (examples of which are
shown in Fig. 2). It was confirmed that four of the SSCP shifts did not
represent genuine DNA mutations (3 in exon 8 and one in exon 7). In
all, 32 sequence alterations were found. 3 of which (including that in
the borderline tumor) were silent. The remaining 29 mutations are
described in detail in Table I. Of the 25 point mutations identified. 23
were missense alterations, and 2 resulted in the insertion of a stop
eodon. Three deletions were found, one of which resulted in the loss
of one amino acid from the region encoded by exon 7 (Fig. 2, tumor
G15). The most striking deletion was that causing the net removal of
a 6-base pair region apparently as the result of the deletion of two
smaller units. The other 2-base pair deletion (Fig. 2. tumor S6I), as
well as a 4-base pair insertion, are frameshift mutations which would
VALGLY
-»
STOPARG
->
CYSARG
->
CYSVAL
->
LEUs
->
304/305ASP
-»GLY
sequencing the constitutive DNA from the same individuals, we could
not exclude the possibility of their being natural polymorphisms.
There is growing evidence that this silent mutation is, in fact, a
relatively common natural polymorphism (21, 22). Table 2 shows the
distribution of point mutation types identified in this study, including
Table 2 )c analysis of the distribution of p5i point mutation type
resultsGC^AT
TG
~*G
C^G
C^A
T-A
result in the premature formation of a stop eodon and ultimately a
truncated protein. One can usually identify a deletion or insertion from
the SSCP results when all of the major bands shift together (Figs. 1
and 2. tumors G15 and S61).
Statistical Analysis. A simple \~ test was use<l to analyze the
distribution of point mutations. The three silent mutations detected in
exon 6, eodon 213, were not used in this analysis, since without
THRASP
-»
VALARG
-»
ARGARG
->
ARGARG
-»
ARGARG
->
STOPARG,
->
-»ILETYR
HIS, SER
CYS238TYR-»
CC
GT
AC
GT
Our
results"Observed23II95435512
10Transition
6Transversion
2Transversion
3Transversion
1Transversion
THA
ATotalsX2ObservedTransition
325Expected'111.992.802.305.581.291.04258.978
(0.5 > P > 0.1)All
(0.05 >Expected''26.386.165.0612.272.842.2
P > 0.02)
" All results inelude those already published 19. 10. 12. 13).
'' The expected distribution of mutations was calculated based on a comprehensive
review of p5J mutations in all cancer types (2).
2129
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p53 Mutation
in Ovarian Carcinoma
Fig. I. Examples of SSCP band shifts for p53 exons 6 and 7. Lanes C, control DNA taken from pe
ripheral blood. Normal (N) and aberrant (A ) banding
patterns are shown.
TUMORS:
C
BAND PATTERN:
N
C S43 G12 G15
S11 S15 S16
N
N
A
N
DISCUSSION
Mutation of the p53 gene was identified in 44% of the invasive
ovarian carcinomas analyzed. It would seem that with such a high
frequency of mutations detected in these tumors, the SSCP technique
is a sensitive one. Michaud et al. (23) have reported SSCP detection
of 20 of 20 known mutations, and to date, in our own laboratory, we
A
C G
A
A
A
N
A
N
EXON7
EXON7
EXON6
their distribution among 30 previously published mutations (9, 10. 12.
13). In comparison with a compilation of over 300 point mutations
reported in a variety of different cancers (2), the distribution of mu
tation types was not significantly altered when we took our results
alone (0.5 > P > 0.1). However, when we looked at all 55 known
ovarian cancer point mutations as a whole, a significant overall de
viation from the expected mutation distribution was observed (0.05 >
P > 0.02). Table 3 shows that a significantly higher rate of p53
mutation is found in serous carcinomas than in endometrioid and
mucinous types (0.02 > P > 0.01). The frequency of p53 mutation
does not seem to correlate significantly with a later stage of disease
(Table 4; 0.5 > P > 0.1).
A
S11 S31 S61 C
have not found any p53 mutations by DNA sequencing analysis in
tumors with no observable SSCP band shift (data not shown). The
appearance of four false positives does suggest that the SSCP tech
nique lacks specificity. However, the SSCP technique is clearly useful
as a screening method in the selection of tumors likely to possess p53
mutations, providing the more diagnostic technique of DNA sequenc
ing is used for confirmation purposes.
Such a high proportion of tumors with mutations in exons 5 to 8 of
the p53 gene has yet to be reported in ovarian cancer using the
SSCP/direct sequencing approach. Mazars et al. (10) reported p53
mutation in 11 of 30 (36%) carcinomas, and Okamoto et al. (11) found
SSCP shifts in 9 of 31 (29%) cases, although not all of these were
verified by sequencing. It is interesting that such a high frequency of
p53 mutation does not appear to be found in other common cancers,
such as those of breast (33% of carcinomas; Ref. 24) and colon (25%
of carcinomas; Ref. 25).
Analysis of the distribution of point mutations has in some cancers
revealed a preference for a particular nucleotide substitution, whether
it be a transition (purine to purine or pyrimidine to pyrimidine) or a
transversion (purine to pyrimidine or vice versa). In colorectal carci-
T
A
C
G
T
A C
G
T
c
A
C
C
Fig. 2. Sequencing data for three of the tumors
shown to have a band shift in Fig. I. In each case
a wild-type sequence can be seen that is due to the
presence of contaminating normal tissue in the tu
mor samples.
A
•¿
=T]del
TUMOR
S16
TUMOR
G15
TUMOR
S61
2130
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p53
Mutation
in Ovarian Carcinoma
Table 3 r analysis0 of the correlation between P53 mutation and histopalhology
ro]e
jn epithelial
ovarian
tumor
progression,
although
it is Still
not
clear at what stage in tumor development p53 mutation occurs.
Mutation positive''
negativeTotalsSerous24
Mutation
"r
.
(56%)''
1943Endometrioid2(15%)
II13Mucinous1
(17%)
56Totals27
ACKNOWLEDGMENTS
3562
The authors would like to thank Consultant Pathologists Ian D. Miller.
Department of Pathology, Aberdeen Royal Infirmary, and Alastair M. Lessells,
= 8.577 (0.02 > P > 0.01).
Mutation positive, those tumors in which a mutation has been confirmed bv sequenc-
_
, .
~
...
„¿
. ..
pathological review of cases used in the study.
Table 4 ^ analysis" of the correlation between p53 mutation and FIGO stage
REFERENCES
Stage"Mutation
positive'Mutation
. . _,..
.
,
r
..
Pathology Department, Western General Hospital. Edinburgh, tor the htsto-
' Numbers in parentheses, percentages of tumors with pSJ mutation.
(20%)''1213113 (38%)58III21 (55%)1738IV1(25%)34Total283765
negativeTotal13
" x2 = 6.188 (0.5 >/>>0.1).
''FIGO stage I: growth limited to ovaries; II. growth limited to pelvis; III: growth
limited to peritoneum; IV: metastasis outwith the peritoneum.
' Mutation positive, those tumors in which a mutation has been confirmed by sequenc
ing
Numbers in parentheses, percentages ot tumors with p53 mutation.
noma, a significantly increased proportion of p53 mutations are GC to
AT transitions, whereas in lung carcinomas GC to TA transversions
are preferred (2). A high proportion of p53 mutations in hepatocarcinomas are also GC to TA transversions, occurring predominantly in
codon 249 of the gene (15, 16). This transversion is thought to occur
as a direct result of exposure to the environmental carcinogens benzo(a)pyrene (in cigarette smoke) and aflatoxin B,. in lung cancer and
hepatocellular carcinoma, respectively (26-29). In an attempt to iden
tify a bias for a particular mutation type which might have similar
etiological implications in ovarian cancer, we carried out a statistical
analysis on the distribution of point mutations detected in our tumors.
No significant frequency imbalance was apparent between the possi
ble mutation types based on the results of this study alone (Table 2).
However, using the 30 additional published ovarian cancer point
mutations in the analysis, a significant overall deviation from the
expected mutation distribution was observed. Despite the significance
of this result, it is not possible to identify one predominant transition
or transversion type, as has been the case for colorectal (2). lung (2),
and hepatocellular carcinomas ( 15, 16). where particular carcinogens
were implicated. Clearly, the accumulation of more mutation data may
help to resolve this matter.
In colon cancer, in particular, p53 mutation seems to occur during
the later stages of tumorigenesis (30). Mazars' work on ovarian cancer
also suggested that p53 mutation could be a late event, based on the
heterogeneous appearance of mutations within certain tumors. How
ever, in agreement with Okamoto, we have shown that tumors of all
four International Federation of Gynecologists and Obstetricians
stages can have p53 mutation (Table 4), indicating that this genetic
event may occur earlier in carcinoma progression, perhaps while the
tumor is still limited to the pelvic region.
A correlation which we did find was one between the histológica!
classification, a parameter which is apparently independent of Inter
national Federation of Gynecologists and Obstetricians staging, and
p53 mutation (Table 3). The serous adenocarcinomas, of which most
were poorly differentiated, had a significantly higher incidence of p53
mutations than those of the mucinous and endometrioid types (0.02 >
P > 0.01 ). This suggestion is supported by the studies on chromosome
17 loci in ovarian cancer (31), the alÃ-eleloss of which was more
common in serous and high-grade tumors.
In conclusion, the high level of p53 mutations detected in ovarian
carcinomas shows that mutation of the gene is likely to play a major
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p53 Mutation Is a Common Genetic Event in Ovarian Carcinoma
Ben J. Milner, Lindsey A. Allan, Diana M. Eccles, et al.
Cancer Res 1993;53:2128-2132.
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