Oncogene (1997) 15, 2951 ± 2958
 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00
TP53 mutational pattern in Spanish and Polish non-small cell lung cancer
patients: null mutations are associated with poor prognosis
Josep Maria de Anta1, Ewa Jassem2, Rafael Rosell3, Marta MartõÂ nez-Roca1, Jacek Jassem2,
Eva Martõ nez-LoÂpez1, Mariano MonzoÂ1, Jose Javier SaÂnchez-HernaÂndez4, Isabel Moreno3
and Montserrat SaÂnchez-CeÂspedes1
1
Laboratory of Molecular Biology of Cancer and 3Medical Oncology Service, Universitat AutoÁnoma de Barcelona, Hospital
Universitari Germans Trias i Pujol, Box 72, Ctra. Canyet s/n, 08916 Badalona, Barcelona, Catalonia, Spain; 2Department of
Oncology and Radiotherapy, Gdansk School of Medicine, 7 Debinki St., 80-211, Gdansk, Poland; 4Department of Preventive
Medicine (Statistics), Universidad AutoÂnoma de Madrid, Avda. del Arzobispo Morcillo s/n, 28029, Madrid, Spain
Inactivation of TP53 tumor suppressor gene is the most
frequent molecular alteration in NSCLC, involving up to
60% of cases. Furthermore, TP53 mutational spectrum
is related to the type of mutagen exposure, as well as
racial and/or diet di€erences. Nearly 95% of TP53
perturbations a€ect codons included within exons 5 ± 8
which encode for almost the entire DNA-binding domain.
In this study we addressed the possible prognostic value
of the molecular alterations identi®ed in exons 5 ± 8 of
the TP53 gene in DNAs from 151 paran-embedded
NSCLC sections corresponding to 59 Spanish and 92
Polish stage I-IIIA resected patients. PCR/single-strand
conformation polymorphism (SSCP) analysis revealed
that the occurrence of TP53 exon 5 ± 8 mutations was
17/59 (29%) in the Spanish cohort and 17/92 (18%) in
the Polish group. However, when DNA sequencing
analysis was performed, these frequencies were reduced
because of the presence of SSCP-false positive, intronic
and silent mutations and polymorphisms. Fifteen of the
59 Spanish NSCLC tumors (25%) harbored TP53
mutations a€ecting exons 5 ± 8 coding sequences,
whereas only 12 of 92 Polish neoplasms (13%) contained
alterations in the central hydrophobic region of p53. Our
results indicate that the occurrence of TP53 mutations
a€ecting exon 5 ± 8 coding sequences in some European
NSCLC populations may be lower than previously
reported, and that the TP53 mutational patterns of
these cohorts di€er somewhat. The Spanish NSCLC
patients contained missense mutations (9/59, 15%) and a
relatively high percentage of null mutations (5/59, 8%)
while the Polish patients mostly harbored missense
mutations (9/92, 10%) and only one tumor contained a
null type (1/92, 1%). Moreover, most TP53 missense
mutations in the Spanish group were located outside the
conserved regions, whereas the same mutations in the
Polish group a€ected conserved amino acids. Furthermore, the Polish patients harbored a high percentage of
G?A transitions (most of them at non-CpG sites), while
G?T transversions were predominant in the Spanish
group. Our ®ndings suggest that there may be di€erent
racial or exogenous factors in these two populations
which may help to explain both the distinct TP53
mutational pattern and the lower frequency obtained in
the Polish group. The presence of missense mutations did
not confer a worse clinical outcome in these subsets of
NSCLC patients. However, patients whose tumors
Correspondence: R Rosell
Received 12 June 1997; revised 1 August 1997; accepted 5 August
1997
contained null TP53 gene mutations had a 5 month
median disease-free survival time in contrast with 42
months in those patients without mutations (P=0.008).
These ®ndings suggest that loss of p53 function may
enhance tumor progression in NSCLC patients independently of whether dominant negative TP53 missense
mutations are present.
Keywords: NSCLC; TP53 null mutations; prognosis;
TP53 mutational pro®le
Introduction
Lung cancer (LC) is the leading cause of cancer death
in the US, and a similar trend is seen in Europe
(Bon®ll et al., 1996). Non-small cell lung cancer
(NSCLC) is the most common type of LC, representing 80% of cases and consisting of adenocarcinoma,
squamous cell carcinoma, large-cell carcinoma, and
some rare histological types. Although tumor extent is
one of the most signi®cant prognostic parameter
considered, the molecular pathogenesis of NSCLC
must be understood in order to discover other
potential, independent factors in¯uencing outcome.
Inactivation of TP53 tumor suppressor gene emerge
as the most frequent molecular disturbance in human
neoplasms, although frequency and type of mutation
di€er substantially among cancers. Particularly in
NSCLC, the TP53 mutational frequency is considerably variable among di€erent populations and studies,
ranging from 20 ± 60% (Kishimoto et al., 1992; Lee et
al., 1994; Ryberg et al., 1994; Takagi et al., 1995).
The TP53 tumor suppressor gene encodes for a 53
KD DNA sequence-speci®c transcription factor with
multiple functions: gene transcription, DNA synthesis
and repair, apoptosis and angiogenesis (Harris, 1996;
Ko and Prives, 1996). In the cellular context, the
biological behavior of p53 protein can be modulated by
binding to cellular and oncoviral proteins as well as by
alterations in TP53 sequence. Therefore, it has been
suggested that detection of TP53 abnormalities may
have diagnostic, prognostic, and therapeutic implications (Harris and Hollstein, 1993). In NSCLC
particularly, the clinical value of TP53 alterations as
a prognostic tool is still a controversial issue. Some
studies indicate that p53 immunohistochemistry is
indicative of a poor prognosis (Quinlan et al., 1992;
Carbone et al., 1994), while others demonstrate the
TP53 mutational pattern in Spanish and Polish NSCLC patients
JM de Anta et al
2952
contrary (Lee et al., 1995). Similarly, the potential role
of TP53 mutations is also debatable. Carbone et al.
(1994) did not associate these perturbations with major
aggressiveness in NSCLC patients, while other
investigators observed the opposite e€ect (Horio et
al., 1993; Mitsudomi et al., 1993). However, these
investigations did not analyse the TP53 mutational
pattern, and the interpretation of the single use of
screening methods without DNA sequencing in these
studies is dicult. Although there are tumors that have
lost one TP53 allele or contain a mutation that
abrogates p53 function (null phenotype), most TP53
perturbations found in human neoplasms are of
missense type. These mutations normally behave as
dominant negative oncogenes, requiring only one
mutated TP53 copy to inhibit cell proliferation by
complexing the wild-type form (Farmer et al., 1992;
Kern et al., 1992). Nearly 90 ± 95% of TP53 mutations
reported in human cancers have been detected in exons
5 ± 8 and their intervening introns, the latter resulting
in deleted exons and the formation of truncated
proteins (Hollstein et al., 1991; Caron de Fromentel
and Soussi, 1992; Kishimoto et al., 1992; Mitsudomi et
al., 1992). Most of this region (exons 5 ± 8) includes the
sequence speci®c DNA binding domain of the protein,
where the vast majority of alterations are present.
Remaining mutations are outside the central core of
the protein, a€ecting the transactivation domain
(amino terminal region) and the oligomerization and
DNA-damage binding domains (carboxy terminal
region). Surprisingly, most of them are of null type
generating a non-functional protein and few cases
contain missense mutations (Hartmann et al., 1995).
Unlike RB and APC tumor suppressor genes which are
inactivated by null mutations, most TP53 mutations
are of missense type. These mutations normally
stabilize p53 protein, leading to its accumulation,
which can then be detected by immunohistochemistry.
Nuclear overexpression of p53 protein has been
routinely used as a simple method of screening tumor
samples for the presence of TP53 alterations, although
this presence is not always associated with a mutational
change (Wynford-Thomas, 1992; Dix et al., 1994).
A large body of evidence indicates that several
factors, including endogenous metabolites and exogenous carcinogen compounds can induce DNA damage
(Zarbl et al., 1985). The nature and the location of the
mutational change within the TP53 gene di€er greatly
among tumors of di€erent histological type which may
be a re¯ection of some DNA genotoxic agents
(Greenblatt et al., 1994). The TP53 mutational spectra
of cancer patients belonging to di€erent populations,
or in groups of patients exposed to particular DNA
mutagens has been studied by several investigators
(VaÈhaÈkangas et al., 1992; Takeshima et al., 1993;
Thorlacius et al., 1993; Saitoh et al., 1994; Shiao et al.,
1995; Takagi et al., 1995; Taylor et al., 1996; Liloglou
et al., 1997). However, as far as we know, there are no
studies comparing the TP53 mutational pattern in
di€erent European NSCLC patient population groups,
as well as investigations demonstrating a correlation
between certain TP53 mutations and prognosis in
NSCLC. We were therefore prompted to examine a
surgical series of 59 Spanish and 92 Polish NSCLC
patients (stage I, II and III, according to TNM
classi®cation) for the presence of molecular alterations
in this gene in order to determine: ®rst, the occurrence
of TP53 alterations a€ecting exon 5 ± 8 coding
sequences in both populations and their relationship
to clinical parameters and outcome; second, whether
TP53 mutational clustering occurred; and third,
whether di€erences in the patterns of mutations
existed in NSCLC patients from geographically
distinct European populations (Spain and Poland).
Results
TP53 mutational frequency
Paran embedded tumors from one hundred ®fty-one
completely resected NSCLC patients who did not
receive adjuvant treatment were evaluated. Median
age at diagnosis was 61.6 years (range 33 ± 82); 95%
were smokers. Squamous cell carcinoma was the most
common histological subtype (64%), followed by
adenocarcinoma (30%) and large cell carcinoma (5%)
(Table 1). These NSCLC specimens were screened by
PCR/SSCP for the presence of TP53 mutations in
exons 5 ± 8. Seventeen of 59 (29%) Spanish and 17 of
92 (18%) Polish samples displayed mobility-shifts in
the PCR/SSCP autoradiograms. All tumor DNA with
suspicious migratory bands on SSCP were analysed by
direct sequencing to assess TP53 genotypes in exons
5 ± 8. In the Polish group of patients, two samples were
SSCP-false positive, containing in fact wild-type alleles.
Furthermore, three showed point mutations at introns
6 and 7 that did not alter splice sites, leading to a
Table 1 TP53 mutational pattern in NSCLC patients in relation to clinicopathological factors and country
None (%)
Median age (year)
Sex
Male
Female
Histology
SCC
AC
LCC
Stage
I
II
III
61
Barcelona, Spain
Missense (%)
60
Null (%)
64
None (%)
59
Gdansk, Poland
Missense (%)
55
Null (%)
71
43 (96)
2 (4)
9 (100)
0 (0)
5 (100)
0 (0)
67 (82)
15 (18)
8 (89)
1 (11)
1 (100)
0 (0)
29 (78)
12 (70)
4 (80)
5 (14)
3 (18)
1 (20)
3 (8)
2 (12)
0 (0)
53 (88)
26 (90)
3 (100)
7 (12)
2 (7)
0 (0)
0 (0)
1 (3)
0 (0)
22 (81)
8 (73)
15 (72)
4 (15)
3 (27)
2 (9)
1 (4)
0 (0)
4 (19)
44 (92)
12 (75)
26 (93)
4 (8)
4 (25)
1 (4)
0 (0)
0 (0)
1 (3)
SCC, squamous cell carcinoma; AC, adenocarcinoma; LCC, large cell carcinoma
TP53 mutational pattern in Spanish and Polish NSCLC patients
JM de Anta et al
decrease in the total number of TP53 mutations to
13% in these patients. Furthermore, two tumors in the
Spanish group harbored polymorphisms at codon 213
(CGA?CGG) which did not alter the encoded
arginine (samples 7 N1 and 12 N1). Overall, the
percentage of gene alteration was 25%. There were no
di€erences between clinicopathological factors and
TP53 status (Table 1).
TP53 mutational pattern
When both series were examined separately, some
di€erences in the nature of TP53 mutations surfaced.
In Spanish NSCLC specimens, nine (15%) were
missense, one (2%) silent and ®ve (8%) null
mutations. Of these changes, one (2%) was nonsense
type (Figure 1) and four (7%) frameshift mutations,
two (3%) deletions and two (3%) insertions. Conversely, all TP53 mutations in the Polish cohort were
single-base substitutions, nine (10%) missense, two
(2%) silent and one (1%) nonsense mutations (Table
2).
There were some di€erences in the nature of the
TP53 missense mutations in both groups of patients.
Although TP53 mutations in Spanish patients were
relatively dispersed, 22% of missense changes a€ected
codon 151, whereas a further 22% involved codon 196.
Moreover, there was a paucity of missense changes
a€ecting conserved amino acids in the Spanish group
of NSCLC patients, with only one mutation a€ecting
codon 176 at domain III (Table 3). Therefore, eight
mutations a€ected non-conserved amino acids (three
mutations were located between domains II and III
(amino acids 142 ± 171), four between regions III and
IV (amino acids 181 ± 234) and one beyond domain V
(starting amino acid 286) (Figure 2). Conversely, most
TP53 missense mutations in Polish NSCLC specimens
were identi®ed in domains IV and V (8/9, 89%),
whereas 1/9 (11%) of those gene alterations involved a
non-conserved amino acid (Table 4). In the Polish
patients, TP53 mutational clustering at codons 245 and
282 occurred, 44% of missense mutations a€ected
codon 245 and 22% codon 282. Single-nucleotide
substitutions at codons 245 and 248 (5/9, 56%) are
hotspots in human cancer, but only one (patient 80,
codon 248) (1/9, 11%) has previously been considered
a hotspot in LC (Greenblatt et al., 1994) (Table 4).
The nature of any nucleotide perturbation was also
monitored. DNA sequencing analysis revealed that the
Spanish group of NSCLC patients contained a slight
predominance of transversions (55%) versus transitions
(45%) (Table 5). The most frequent type of transversions was G?T (36%), whereas G?A was the most
common transition (27%), all of them located at nonTable 3
Patient
29
48
13
5
15
53
7
16
35
5
N0
N0
N1
N2
N2
N0
N1
N1
N0
N0
Patient
11
14
3
50
13
N2
N0
N2
N0
N2
Exon
5
5
5
5
5
6
6
6
7
8
TP53 mutations in Spanish NSCLC patients
Missense and silent mutations
Nucleotide
Amino acid
Codon
change
substitution
151
176
166
151
154
193
196
196
242
291
Exon
Codon
5
7
7
8
8
135
239
239
212
293
CCC ± ACC
TGC ± TTC
TCA ± TTA
CCC ± TCC
GGC ± GTC
CAT ± CGT
CGA ± CCA
CGA ± CCA
TGC ± TGT
AAG ± GAG
Pro ± Thr
Cys ± Phe
Ser ± Leu
Pro ± Ser
Gly ± Val
His ± Arg
Arg ± Pro
Arg ± Pro
Cys ± Cys
Lys ± Glu
Null mutations
Nucleotide
change
TGC ± TGA
(+1) A frameshift insertion
(+1) A frameshift insertion
(71) T frameshift deletion
(71) G frameshift deletion
Conserved
domain
outside
III
outside
outside
outside
outside
outside
outside
±
outside
Structural
change
Cys ± Stop
ter AA 263
ter AA 263
ter AA 246
ter AA 344
Figure 1 Automated DNA cycle sequencing of two samples
containing TP53 mutations. (a) Tumor 14 N0 contains a 1 bp
single-nucleotide frameshift insertion at codon 239. (b) Tumor 11
N2 shows a nonsense mutation at codon 135
Table 2
No. tumors
No. mutations
Missense
Null
Nonsense
Deletion fs
Insertion fs
Splice junction
Silent
TP53 mutations in NSCLC
Barcelona, Spain (%)
Gdansk, Poland (%)
59
15
9
5
1
2
2
0
1
92
12
9
1
1
0
0
0
2
(25)
(15)
(8)
(2)
(3)
(3)
(0)
(2)
(13)
(10)
(1)
(1)
(0)
(0)
(0)
(2)
Figure 2 p53 functional and evolutionary conserved domains.
Codons included in exon 5 through 8 and in the evolutionary
conserved regions are depicted
2953
TP53 mutational pattern in Spanish and Polish NSCLC patients
JM de Anta et al
2954
CpG sites (Table 5). Conversely, G?T transversions
were observed at a lower frequency in Polish tumors
(27%). These patients exhibited an abundance of G?A
transitions (46%), mostly at non-CpG nucleotides
(40%) (Table 5).
TP53 status and clinical outcome
When we searched for correlations between certain
TP53 genotypes (missense, null, versus wild type) and
clinical outcome in the whole group of patients, the
presence of TP53 null mutations were associated with a
worse disease-free survival. Median disease-free survival time for patients with null mutations was 5 months
Table 4 TP53 mutations in Polish NSCLC patients
Patient
10
76
11
12
14
80
91
21
39
65
72
Exon
5
5
7
7
7
7
7
8
8
8
8
Missense and silent mutations
Nucleotide
Amino acid
Codon
change
substitution
154
141
245
245
245
248
245
282
286
282
297
GGC ± GGT
TGC ± TGT
GGC ± GTC
GGC ± GAC
GGC ± GAC
CGG ± CAG
GGC ± GTC
CGG ± GGG
GAA ± AAA
CGG ± GGG
CAC ± AAC
Conserved
domain
Gly ± Gly
Cys ± Cys
Gly ± Val
Gly ± Asp
Gly ± Asp
Arg ± Gln
Gly ± Val
Arg ± Gly
Glu ± Lys
Arg ± Gly
Leu ± Asn
±
±
IV
IV
IV
IV
IV
V
V
V
outside
Null mutations
Patient Exon
15
5
Patient
17
42
93
Codon
Nucleotide change
Structural
change
166
TCA ± TGA
Ser ± Stop
Intronic mutations
Intron
Nucleotide change
6
6
7
compared to 42 months in those patients without
mutations (P=0.008) (Figure 3). The clinical behavior
of patients containing null mutations also di€ered from
those harboring missense mutations (5 months versus
24 months of median disease-free survival time),
although this di€erence did not reach statistical value.
In those patients harboring missense perturbations, we
were unable to demonstrate di€erences in prognosis
between those containing mutations a€ecting conserved
amino acids and those including nucleotide substitutions at non-conserved residues (data not shown).
Furthermore, there were no di€erences in overall
survival among the group of patients without
mutations and those with missense mutations (median
survival time 38 months and 45 months, respectively).
Looking for stronger predictive factors, and using
multivariate analysis, stage IIIA was the only
signi®cant independent factor in¯uencing survival
(hazard ratio 1.7; P50.001).
Figure 3 Disease-free survival curve of 151 European NSCLC
patients according to TP53 mutations identi®ed in exons 5 ± 8 by
DNA sequencing obtained by the Kaplan Meier method. Median
disease-free survival was 42 months for 127 patients without
mutation in contrast to 5 months for six patients containing null
mutations (P=0.008). The median disease-free survival for the 18
patients with missense mutations was 24 months
Nucleotide position
13964
13846
14133
G±C
G±T
G±A
Table 5 TP53 mutational spectra in di€erent NSCLC populations
Study
Barcelona,
Spain
Gdansk,
Poland
Tokyo,
Japan (1)
Los Angeles,
USA (2)
Taipei,
Taiwan (3)
Helsinki,
Finland (4)
Bergen
and Oslo,
Norway (5)
Pittsburgh,
USA (6)
Hong Kong,
China (7)
USA (8)
Merseyside,
UK (9)
% Transitions
%
CpG
non-CpG
mutations G:C-A:T G:C-A:T G:C-A:T A:T-G:C
% Transversions
Total
Other
25
0
27
27
18
Total
45
G:C-C:G G:C-T:A A:T-C:G A:T-T:A
18
36
0
0
55
7
13
6
40
46
0
46
27
27
0
0
54
0
56
15
21
36
15
51
11
30
6
2
49
10
60
5
5
9
14
23
14
55
5
5
77
5
44
25
31
56
6
62
19
6
0
13
38
0
45
9
17
26
14
40
14
34
11
0
60
6
31
17
17
33
17
50
13
33
3
0
50
4
31
10
20
30
0
30
40
30
0
0
70
0
27
12
19
31
0
31
38
19
12
0
69
4
60
28
7
18
16
46
23
64
19
9
42
73
15
9
32
18
3
0
8
0
58
27
12
11
(1) Kishimoto et al., 1992; (2) Miller et al., 1992; (3) Lee et al., 1994; (4) RidanpaÈaÈ et al., 1994; (5) Ryberg et al., 1994; (6) Law et al., 1995; (7)
Takagi et al., 1995; (8) Casey et al., 1996; (9) Liloglou et al., 1997. 1 and 8 analysed mutations outside exons 5 ± 8. (2) SCLC were also included
TP53 mutational pattern in Spanish and Polish NSCLC patients
JM de Anta et al
Discussion
Tumor suppressor genes are inactivated by multiple
mechanisms. Ablation of genes like RB, APC,
BRCA1 and BRCA2 is generally produced by
nucleotide mutations which lead to the synthesis of
truncated proteins that are normally unfunctional
(null mutations). However, most TP53 alterations,
around 83% in NSCLC, behave as dominantnegative missense mutations. These alterations
primarily involve amino acid residues contained in
the central hydrophobic region of the protein (mostly
included in exons 5 ± 8) which mediate the DNAspeci®c transactivating function of p53. Many
researchers have focused their investigations on the
potential value of p53 immunohistochemistry (IHC)
as a prognostic tool (Quinlan et al., 1992; Navone et
al., 1993; Carbone et al., 1994; Harpole et al., 1995;
Lee et al., 1995; Passlick et al., 1995; Nishio et al.,
1996). The utility of IHC as a screening method is
dubious, as null mutations (nonsense, frameshift and
splice junction mutations) result in the production of
shortened polypeptides and most are not recognized
by speci®c antibodies because of antigenic site loss
(Greenblatt et al., 1994). Therefore, the use of PCRbased methods becomes compulsory if we are to
ascertain the possible prognostic value of null
mutations.
We used PCR/SSCP analysis (Orita et al., 1989)
followed by DNA sequencing to detect TP53
mutations. Fifteen (25%) of 59 Spanish cases
contained mutations in exons 5 through 8 of TP53
gene, while such alterations were identi®ed in 12
(13%) of 92 Polish tumors (Table 2 and 5). Although
much higher frequencies have been described in the
literature (Table 5), the relatively low incidence in the
Spanish group is not so surprising if we compare it to
previous reports as shown in Tables 5 and 6 (Ryberg
et al., 1994; Law et al., 1995; Takagi et al., 1995;
Liloglou et al., 1997). Only one of these studies
included non-smoking NSCLC patients (Takagi et al.,
1995), and, as re¯ected in Table 6, the TP53
mutational occurrence seen in the Spanish cohort is
not signi®cantly di€erent from the frequencies
reported by most investigators. We have no explanation for the low number of mutations found in the
Polish cohort, but considering that both groups were
equally determined, it may either re¯ect a random
e€ect, or even that other non TP53 mutation
mechanisms, may play a part in NSCLC progression. In this context, Miller et al. (1992) found that
30% of lung tumors examined harbored TP53 allelic
losses without detectable point mutations at exons 4
through 8. Factors, other than TP53 gene mutations,
may account for a greater part of NSCLC oncogenesis
in cohorts with lower frequencies of TP53 mutation;
the polymorphic VNTR region ¯anking the H-RAS1
gene (Ryberg et al., 1994) and point mutations in the
K-RAS proto-oncogene (Rosell et al., 1993, 1996) are
two examples. Kishimoto et al. (1992) and Casey et al.
(1996) found higher frequencies, partly because they
analysed exons 2 through 11, and one of these studies
included perturbations found in SCLC patients in
whom the occurrence of TP53 alterations is higher
(Takahashi et al., 1991). In the present study, the
frequency of missense TP53 mutations was greater
than the prevalence of null mutations (Table 2).
However, when the Polish population was observed,
there was a scarcity of perturbations that lead to p53
ablation, since only one sample contained a nonsense
mutation (1%). Nevertheless, null mutations made up
33% of the total number of mutations in the Spanish
NSCLC tumors in contrast to lower frequencies
documented by other investigators (Kishimoto et al.,
1992; Lee et al., 1994; Ryberg et al., 1994; Takagi et
al., 1995; Casey et al., 1996).
Most of the DNA changes in the Spanish group
were G?T transversions, 50% on the non-transcribed
Table 6 Signi®cance levels (P) of pairwise comparisons of the TP53 mutational frequencies in di€erent NSCLC populations using the Fisher
Exact Test (2611 Table)
Barcelona,
Spain
Barcelona,
6
Spain
Gdansk,
0.06
Poland
Tokyo,
50.001
Japan (1)
Los Angeles,
50.001
USA (2)
Taipei,
0.07
Taiwan (3)
Helsinki,
0.01
Finland (4)
Bergen and Oslo,
0.5
Norway (5)
Pittsburgh,
0.5
USA (6)
Hong Kong,
0.8
China (7)
USA (8)
50.001
Merseyside,
0.8
UK (9)
Gdansk,
Poland
Tokyo,
Japan
Los
Angeles,
USA
Taipei,
Taiwan
Helsinki,
Finland
Bergen
and Oslo, Pittsburgh,
Norway
USA
Hong
Kong,
China
USA
Merseyside,
UK
6
50.001
6
6
50.001
6
50.001
0.7
6
50.001
0.3
0.2
6
50.001
0.09
0.2
1
6
0.002
0.002
0.002
0.2
0.03
6
0.03
0.02
0.02
0.3
0.2
1
6
0.03
0.001
0.001
0.08
50.001
0.6
0.8
0.3
50.001
1
50.001
0.1
0.2
0.02
0.06
50.001
0.8
50.001
0.03
0.006
0.08
6
50.001
1
(1) Kishimoto et al., 1992; (2) Miller et al., 1992; (3) Lee et al., 1994; (4) RidanpaÈaÈ et al., 1994; (5) Ryberg et al., 1994; (6) Law et al., 1995; (7)
Takagi et al., 1995; (8) Casey et al., 1996; (9) Liloglou et al., 1997. 1 and 8 analysed mutations outside exons 5 ± 8. (2) SCLC were also included
2955
TP53 mutational pattern in Spanish and Polish NSCLC patients
JM de Anta et al
2956
strand (Tables 3), which are compatible with exposure
to benzo(a)pyrene and to elevated levels of oxygen free
radicals in the blood of cigarette smokers (Reid and
Loeb, 1992). The Polish samples contained more
transversions than transitions. However, changes
related to the exposure to di€erent carcinogenic
burden such as G?A transitions at non-CpG sites
(40%), were the most frequent substitution in Polish
patients, followed by G?T (27%) and G?C (27%)
transversions (Table 5). This preponderance of nonCpG G?A transitions agree with the results obtained
by Liloglou et al. (1997) in smoking NSCLC patients
from Merseyside, UK, an industrialised region with a
similar climate to that of Gdansk.
The Polish tumor samples contained a high number
of missense substitutions (75% of the total mutations),
with most altering conserved amino acids belonging to
domains IV and V. However, none of these mutations
were located at codon 157, the most frequently
described mutated site in LC. Forty-six percent of
mutations were identi®ed at codons 245, 248 and 282,
all of which are commonly mutated in human cancer.
Amino acids 245 and 282 participate in the stabilization of the DNA binding surface of the protein, and
some mutants a€ecting these codons alter p53
transcriptional activity and growth suppression
(Greenblatt et al., 1994). Nevertheless, none of the
mutants changing amino acids 245 and 282 were the
substitutions most commonly reported in LC (Greenblatt et al., 1994). Amino acid 248, also a hotspot in
LC, directly interacts with DNA, and it is assumed
that missense changes at this position may a€ect the
DNA-speci®c associated transcription of p53. Nevertheless, most TP53 missense mutations found in those
tumors probably do not confer a growth advantage.
The fact that there was no di€erence in survival
between the group of patients without mutations and
those with missense mutations is not surprising if we
keep in mind the fact that a signi®cant number of these
mutants retain latent ability to bind DNA and still
form tetramers. These data may help to explain why
missense mutations found in this study did not result in
major aggressiveness in this set of NSCLC patients.
Moreover, no mutations were detected at residue Arg
273 which is probably involved in the allosteric
regulation of the p53 tetramer. Interestingly, Japanese
researchers found an association between mutations at
the 273 hotspot and poor survival in NSCLC (Huang
et al., 1997).
Most TP53 missense mutations in the Spanish
cohort were located outside the conserved regions
(Table 3). This is not so novel, since other authors have
found a similar trend in breast cancer in which some
55% of mutations were located outside domains II ± V
(Sjogren et al., 1996). Most TP53 hotspot codons are
located within domains II ± V. However, there are some
residues which are also relative hotspots lying outside
these evolutionary conserved regions (151 ± 152, 157 ±
158, 163, 194 (Lys4Arg), 195 (Ile4Thr), 205, 220 and
266). Two of eight Spanish patients (25%) had
mutations outside the conserved domains which
a€ected one of these codons (amino acid 151,
Pro4Thr, Pro4Ser). The potential growth advantage
of these perturbations in unknown, but considering
that most mutations (67%) in the Spanish group
changed no relevant amino acids, it seems plausible
that these alterations may not a€ect the growth
inhibitory pathway mediated by p53 in these NSCLC
patients. Although, patients with null mutations had a
poor clinical outcome, suggesting that p53 loss may
confer a greater aggressiveness. However, the prognostic value of certain TP53 alterations in NSCLC and
other cancers has yet to be completely elucidated.
Many studies have used the IHC approach to ascertain
the prognostic value of p53 accumulation, albeit with
contradictory results, since IHC analysis is unable to
detect most null mutations. Most investigators have
only screened TP53 mutations a€ecting the central core
of the protein and is believed that the combination of
IHC plus DNA sequencing of exons 2 through 11 is
mandatory in order to detect all p53 perturbations
(Casey et al., 1996). Functional assays in yeast to
characterize the status of TP53 in tumors have recently
been described (Inga et al., 1997; Lomax et al., 1997).
By using DNA sequencing methods, some authors
have shown that null mutations as well as missense
mutations a€ecting amino acids of the DNA-binding
domain are equally associated with poor prognosis in
breast cancer patients (Saitoh et al., 1994). Our results
prompted us to conclude that: (a) the low percentage
of TP53 mutations a€ecting exons 5 ± 8 coding
sequences in our study suggest that other mechanisms
including TP53 allelic losses, alterations and/or
aberrant expression of genes involved in the p53
mediated growth suppression pathway may exist in
certain populations of NSCLC patients; (b) either
ethnic disparities or di€erent carcinogen burden may
explain to some extent the distinct mutational pro®les
of the two populations studied: G?A transitions at
non-CpG sites were predominant in the Polish patients,
and G?T transversions in the Spanish cohort; (c)
missense mutations which do not perturb essential
amino acids or generate certain hotspot mutants seem
to be irrelevant in NSCLC prognosis; and (d) TP53
null mutations are linked to poor outcome in NSCLC.
We believe that mechanisms other than TP53 missense
and null mutations in the sequence-speci®c DNA
binding region warrant investigation.
Materials and methods
Patients and DNA isolation
One hundred and ®fty-one patients surgically-resected with
NSCLC, stages I to IIIa, according to the international
TNM classi®cation (Mountain, 1986) were studied. All 151
patients for whom follow-up and tumor samples were
available had undergone thoracotomy and resection
between 1986 and 1992 as treatment of their disease.
Data on LC recurrences and cause of death were obtained.
Tumors were collected from 59 Spanish and 92 Polish
NSCLC patients, the latter obtained through Gdansk
School of Medicine.
Three to ®ve 5 mm tumor paran embedded sections were
cut, microdissected from the surrounding normal tissue and
collected in an eppendorf tube. To remove paran, 1 ml of
xylene was added, centrifuged and rinsed with ethanol. After
centrifugation, DNA from sections was extracted in a lysis
bu€er (10 mM Tris-HCl pH 8.5, 2.5 mM MgCl2, 50 mM KCl,
0.5% Tween 20 containing 250 mg/ml of proteinase K) at
568C for 24 h. Then samples were centrifuged to clean them
up. Supernatants were recovered, incubated at 958C for
10 min to inactivate proteinase K and used for ampli®cation.
TP53 mutational pattern in Spanish and Polish NSCLC patients
JM de Anta et al
PCR/SSCP (single-strand conformation polymorphism) and
DNA sequencing
To analyse TP53 mutations (exons 5 ± 8), 1 ± 5 ml of the
isolated genomic DNA sample were used as a template and
ampli®ed in 10 m M Tris HCl (pH 9.0 at 258C), 50 mM KCl,
0.1% Triton X-100, 1.5 mM MgCl2, 200 mM of each dNTP
and 0.5 mM of each amplimer (Navone et al., 1993) in a
50 ml volume. 1 ml of a 1/100 dilution of the PCR was used
as a template for a 20 ml nested PCR reaction. DNA
ampli®cation was performed in 10 mM Tris HCl (pH 9.0 at
258C), 50 mMKCl, 0.1% Triton X-100, 1.5 mM MgCl2,
200 mM dATP, 200 mM dTTP, 200 mM dGTP, 150 mM
dCTP, 0.5 mCi (50 mM) of [a32P]-dCTP (Amersham Inc.,
UK), 0.5 mM of nested TP53 primers (Navone et al., 1993)
in a 9600 Perkin-Elmer Cetus DNA thermal cycler. The
thermal pro®le used was 948C for 1 min, 156(948C for
20 s, 568C for 20 s and 728C for 20 s), and 748C for 4 min.
Two to four ml of the nested PCR reaction were mixed with
an equal volume of denaturing solution (98% formamide,
10 mM EDTA pH 8.0, 0.02% xylene cyanol, 0.02%
bromophenol blue). The DNA mixture was denatured at
958C for 5 min, chilled on ice and loaded onto a nondenaturing 6% polyacrylamide ± 16TBE gel containing
10% glycerol. Electrophoresis was performed at 4 W for
16 h at 48C, or alternatively at room temperature and
cooled with a fan. The gel was dried and ®nally exposed to
an autoradiographic ®lm (Kodak OAT, USA) using
intensifying screens at 7808C for several hours and
developed. Each experiment was repeated at least twice
for each sample.
To determine the nature of TP53 mutations detected by
SSCP technique, PCR products obtained from ampli®ed
genomic DNA positive for SSCP were analysed by automated
DNA cycle sequencing. Genomic DNAs were reampli®ed
using speci®c primers for each exon and, PCR products were
directly sequenced using the appropriate ¯uorescent nested
primer. For cycle sequencing, primers and primer dimers
contained in the PCR ampli®ed product were previously
removed by using S-300 HR Sephacryl microcolumns
(Pharmacia Biotech, Uppsala, Sweden). Puri®ed PCR
products were used as a templates in a cycle sequencing
reaction using a ¯uorescent nested primer (Pharmacia
Biotech). Brie¯y, a cycle sequencing reaction for each
nucleotide was performed using 500 ng of PCR template,
3 pmoles of a ¯uorescent sequencing primer, dNTPs with the
corresponding dideoxynucleotide, and 1.25 U Taq DNA
polymerase in a sequencing bu€er containing MgCl2. First,
DNA was denatured at 958C for 2 min and then 30 cycles of
ampli®cation (958C 30 s, 56 ± 658C 30 s, 728C 40 s) were
performed followed by a soak pro®le at 48C. Then 4 ml of
stop solution containing formamide and dextran blue was
added, and the mixture was previously denatured at 958C for
3 min before loading into a prewarmed denaturing 6%
polyacrylamide-8 M urea gel on an ALF express DNA
sequencer. Samples were run at 40 W for 2 or 3 h and the
sequencing data obtained were compared with the wild-type
TP53 sequence. Independent PCR products obtained from
each SSCP-positive genomic DNA sample were analysed at
least twice to con®rm mutations.
Statistical analysis
For the purpose of statistical analysis the TP53 abnormalities were classi®ed into three major groups: no
mutations, missense mutations and null mutations. The
relationship among alterations of the TP53 gene and
clinicopathological features was tested by the chi-square
and Fisher exact tests. Also di€erences in TP53 mutation
frequency and pattern were examined with the same tests.
To analyse the relationship between TP53 mutations and
survival time of the patients, Kaplan ± Meier estimations as
well as Log ± Rank and Tarone ± Ware tests were
performed and mean survival times were computed
according to Kaplan and Meier. The multivariate analyses
were performed with the stepwise proportional hazards
regression model for censored survival data (Cox's model).
Acknowledgements
This work was supported by grant 95/0177 from the Fondo
de Investigaciones Sanitarias de la Seguridad Social and by
a grant from Bristol-Myers Squibb, Madrid, Spain. We
thank Ms. Maura O'Sullivan-Brown for assistance on the
English version of the manuscript.
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