Mutations in the p53 Tumor Suppressor Gene: Clues to Cancer

Etiology and Molecular Pathogenesis

M. S. Greenblatt, W. P. Bennett, M. Hollstein, et al.

Cancer Res 1994;54:4855-4878. Published online September 1, 1994.

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[CANCERRESEARCH54, 4855-4878, Septemberi5, 1994J

Perspectives in Cancer Research

Mutations in the p53 Tumor Suppressor Gene: Clues to Cancer Etiology

and Molecular Pathogenesist
M. S. Greenblatt, W. P. Bennett, M. Hollstein, and C. C. Harris2
Laboratory of Human Carcinogenesis, Division of Cancer Etiology, National Cancer institute, Bethesda, Maryland 20892 (M. S. G., W. P. B., C. C. HI, and Division of
Toxicology, German Cancer Research Center, D-69120 Heidelburg, Germany [M. H.]

I. Introduction

The p53 tumor suppressor gene has come to the forefront of cancer Ref. 10). Important sources of the spontaneous generation of point
research because it is commonly mutated in human cancer and the mutations in human cells include deamination of cytosine and 5-meth
spectrum of p53 mutations in these cancers is providing clues to the ylcytosine (1 1), DNA polymerase infidelity (12), depurination (13),
etiology and molecular pathogenesis of neoplasia (1—3).Detection of and oxidative damage from free radicals generated by biological
p53 abnormalities may have diagnostic, prognostic, and therapeutic processes (14, 15).
implications (4). The most well-studied endogenous mechanism of DNA damage at
The 15-year history of p53 investigations is a paradigm in cancer this time is the phenomenon of deamination of 5-methylcytosine.
research, illustrating the convergence of previously parallel lines of Methylation of DNA is one epigenetic mechanism involved in regu
basic, clinical, and epidemiological investigation and the rapid trans lating gene expression. Cytosine and 5-methylcytosine residues can
fer of research findings from the laboratory to the clinic. p53 is clearly spontaneously deaminate to uracil and thymine, respectively, which if
a component in biochemical pathways central to human carcinogen not repaired will result in G:C—>A:T transitions. These mutations
esis; p53 protein alterations due to missense mutations and loss of p53 occur most frequently at CpG dinucleotides (a cytosine followed by a
protein by nonsense or frameshift mutations provide a selective ad guanine), which are frequently methylated (9, 11, 16).
vantage for clonal expansion of preneoplastic and neoplastic cells (5). DNA replication in humans is performed by multicomponent protein
The potential for a missense mutation to cause loss of tumor suppres machines that include several DNA polymerases, which vary in charac
sor function and gain of oncogenic activity, i.e., to transform cells by teristics such as exonuclease activity and fidelity of replication. Error
two mechanisms, is one explanation for the commonality of p53 rates vary from 112,000 for DNA polymerase-@3,which repairs only short
mutations in human cancer. Recent studies investigating the mecha gaps, to 1/50,000- 1/1,000,000 for other polymerases which contain
nisms underlying the biological activity of p53 indicate that the exonuclease activity (17). The most common base substitutions observed
protein is involved in gene transcription, DNA synthesis and repair, are G:C—*
A:T and A:T—@ G:C transitions and G:C—*
T:A transversions.
genomic plasticity, and programmed cell death (1—6).These complex Imbalances in deoxynucleoside triphosphate pools (18), mutations in
biochemical processes are performed by multicomponent protein ma DNA polymerase-a (19), and slippage of DNA polymerase at nucleotide
chines; therefore, it is not surprising that the p53 protein forms repeats (Ref. 8; section II.B.2) are examples of mechanisms contributing
complexes with other cellular proteins (Fig. 1) and that some viral to infidelity of DNA synthesis. Considering the multiplicity of proteins
oncoproteins alter the functions of these machines by binding to p53 involved in DNA synthesis, DNA repair, mitosis, and the cell cycle, the
and perturbing its interaction with other cellular protein components. number of potential gene targets which could interfere with replication
In this Perspective, we will focus on the origin of p.53 mutations, fidelity in somatic cells is large.
the mutational spectrum of p.53 in human cancers, and the hypotheses Abnormalities in some of these processes could result in the mu
generated by the analysis of p53 mutations in premalignant and tator phenotype (20, 21), which could increase the probabilities of
malignant cells. The interpretation ofp53 mutations in human cancers both neoplastic transformation and the generation of increasingly
is based on observations of the patterns of DNA damage induced by malignant subclones during tumor progression (22, 23). If such
chemical and physical mutagens in model systems. In this Introduc mutations occur in germ cells and are nonlethal, they could lead to
tion, we will review these data, which provide the background for heritable syndromes of increased cancer risk. The hypothesis that
many of the inferences drawn from p53 mutational analysis. inherited genomic instability may be associated with increased cancer
risk is supported by studies of two diseases. Children with Bloom's
A. Origins of Mutation syndrome have an increased risk of childhood cancers, and their cells
show an increase in cytogenetic abnormalities (24). Germline muta
Both exogenous carcinogens and endogenous biological processes
are known to cause mutations (7—9).Classes of DNA damage include
tions in two DNA mismatch repair genes, hMSH2 (25, 26) and
hMLHJ (27, 28), recently have been identified as the genetic abnor
deletion, insertion, and base substitution, either transition (change of
malities responsible for HNPCC.3 Mutations of these genes are
a pyrimidine to another pyrimidine or a purine to another purine) or
transversion (change of a pynmidine to a purine or vice versa;
3 The abbreviations used are: HNPCC, hereditary nonpolyposis colorectal carcinoma;
aprt, adenine phosphoribosyl transferase; dhfr, dihydrofolate reductase; hprt, hypoxan
Received 4/27/94; accepted 7/20/94. thine-guanine phosphoribosyl transferase; CHO, Chinese hamster ovary; BPDE, 75,8R-
Thecostsof publicationof thisarticleweredefrayedin partby the paymentof page dthydroxy-9R,1OS-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene; BP, benzo(a)pyrene; IHC,
charges. This article must therefore be hereby marked advertisement in accordance with immunohistochemistry; SSCP, single-stranded conformation polymorphism; PCR, polymer
18 U.S.C. Section 1734 solely to indicate this fact. sac chain reaction; XP-C, xeroderma pigmentosum, complementation group C; PAH, poly
i Due to space limitations and the breadth of this topic, comprehensive reviews which cyclic aromatic hydrocarbon; AFB, aflatoxin B@; HCC, hepatocellular carcinoma; HBV,
are the source of additional citations will be cited frequently. hepatitisB virus; SCL@ small cell lung carcinoma;NSCLC, ron-small cell lung carcinoma
2 To whom requests for reprints should be addressed, at Laboratory of Human Carcino NPC, nasopharyngeal carcinoma EBV, Epstein-Barr virus; HPV, human papilloma virus;
genesis, Building 37, Room 20)5, National Cancer Institute, Bethesda, MD 20892. CAT, chloramphemcol acetyltransferase; LFS, Li-Fraumeni Syndrome; MP, mean pairwise.
4855

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Copyright © 1994 American Association for Cancer Research
 p53 MUTATIONS AND CANCER

flG.1 FIG. 2A
Tr.lnsdc:' @atic@n
[ Mutations and -@Oligom.rlzat$on and

@
4.― Dvrnairi Sequence
DNA Binding
Specific
Domain
NucI@r Localization
Domains
MUTATIONAL
SPECTRUM
OF
110 273 ALLHUMANCANCER
14O@
120
100 1 24•
2% 8%
@ I 235 282
@ I 17%
4O@
20 .kli .a

.4 as
.4@V @P $V@ LARGE T-Ag ______
ISBIS H$P70 HSX 543
@ 1 1.4 EISMK £RC@.0
I IIYNPA NPA@ 17%
15@41 MOM2

SKINCANCERS
@ 2B SQUAMOUS CELL BASAL CELL MURINE (UV-INDUCED)
3% 2% 7%
1O%@ @j1% 13%

HEADANDNECKCANCERS
2E 2F 2G
NASOPHARYNX ORAL PHARYNX/LARYNX
6% 10/
0 10%

29%

LEGENDFORFiGURE2 A-O
U G:CtoC:G•
G:CtoT:A
•
G:CtoA:T
•
A:TtoT:A•A:Tto
G:C
@ U A:T
toC:G U-Non-CpG
•
CC:TT
toThAA Othir
Fig. 1. Schematic of the p53 molecule. Verticallines, distribution of mutations; horizontal bars, regions of protein binding. The p53 protein consists of 393 amino acids with functional
domains, evolutionarily conserved domains, and regions designated as mutational hotspots. Functional domains include the transactivation region (amino acids 20-42, orange area),
sequence-specific DNA binding region (amino acids 100—293),nuclear localization sequence (amino acids 316—325,dark green area), and oligomenzation region (amino acids 319—360,light
green area). Cellular or oncoviral proteins bind to specific areas of the p53 protein. Evolutionarily conserved domains (amino acids 17—29,97—292,and 324—352;rose-colored area) were
determined using the MACAW program as described in the text. Seven mutational hotspot regions within the large conserved domain are identified: amino acids 130—142,151—164,171—181,
193-200, 213-223, 234-258, and 270-286. Functional domains and protein binding sites were compiled from references in text and from X. Wang and C. Harris(unpublished data). Vertical
lines above the schematic, missense mutations;lines below schematic, nonmissense mutations. The majority of missense mutations are in the conserved hydrophobic midregion, while
nonmissensemutations(nonsense,frameshift,splicing,and silent mutations)are distributedthroughoutthe protein, determinedprimarilyby sequencecontext, as described in the text.
Fig. 2. A-G. spectrum of p53 mutations in various tumor types. (n) the number of mutations for which exact base change or deletion was confirmed by DNA sequencing. A, the mutational
spectrumofp53 in all reportedtumorsand cell lines(n = 2567) in the database.B, squamous(n 32 induding Bowen's disease)and (C)basal cell (n 52)carcinomas ofthe skin in humans
and (D) UV light-inducedmurine skin tumors (n = 30) exhibit frequent G:C—÷A:T transitions and occasional tandem mutations at dipynmidine sites (usually CC:GG—@1T:AA),
both
characteristicof UV-induceddamage.Squamouscell carcinomashave been linkedwith occupationalexposureto PAHa,and one-fourthcontainG:C-@T:A transversions,which are consistent
with the mutations caused by these carcinogens in model systems. The mutational spectra differ by site of origin of head and neck squamous carcinomas [(E) nasopharynz n 17; (F) oral,
n 69; (G) pharvnxilar,nx. n 42J. 4856

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Copyright © 1994 American Association for Cancer Research
 p53 MIJFATIONS AND CANCER

FIG.2H-0 2H ADENOCARCINOMA 21 SQUAMOUS

LUNG
CANCERS 14%
BYCELL
TYPE 45%

8%

2J SMALL CELL 2K LARGECELL

4%

5%

13%

LUNG
CANCERS
a SMOKERS 2M NON-SMOKERS
6% 6%
6%
/0

11%
@ I 3%

8%

COLONSOMATICMUTATIONS LI-FRAUMENI
GERMUNEMUTATiONS
2N

8%

5%

15%

Fig. 2. (continued) H-O, spectrumof p53 mutationsin various tumortype&The mutationalspectra in lung cancers differ among histological types [(H) adenocarcinonia n 61; (1) sqisimous
n = 75; (J) 5771011cell (SfLC), n = 92; (K) large @dL n 27]. Menocarcinomasdisplaya lowermutationprevalence, a kwer percentage of G:C—@TA tranavetsions, aixi a higherpercentage
of
G:C-÷Altaiultians.The mutational specus in lungeancervaiy byanddnghistccy[(L)nnokers n = 169;(M)noiwnoke,@ n = 161.Themostcommon mutations in(N)coloncarcinoma(n = 324)
and (0) LFS (germline p53 mutations, n = 47) are GC-*A@T transitions at @pG dinucIeOtideS,which may be the result of spontaneous deamination of 5-inethylcytosine.
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Copyright © 1994 American Association for Cancer Research
 p53 MUTATIONS AND CANCER

associated with deletions and insertions in microsatellite sequences of
somatic cells and a genetic predisposition to tumors of the colon and
other tissues (29). Somatic mutations in !ZMSH2 have also been
observed. Therefore, the mutator phenotype can be both a predispos
ing inherited factor initiating carcinogenesis or a secondary
change produced during this multistage process.
A well-studied mechanism by which chemical
their ultimate carcinogenic metabolites cause mutations is by forming
covalent adducts with the nucleotides in DNA, increasing the proba
bility of errors during DNA replication (30). Some small carcinogen
DNA adducts, such as 06-methylguanine resulting from alkylating
agents, may cause DNA polymerase to misread the base pairing due
to the altered hydrogen bonding properties of a base which contains an
additional methyl or ethyl group. Laboratory studies have established
that the most common mutations caused by alkylating agents are
G:C—+A:T transitions, consistent with 06-methylguanine
with thymine (31). Bulky carcinogen-DNA adducts may render the
bases unreadable and stall the replication machinery. DNA poly
merases may fill these noninstructive sites preferentially with adenine.
If this occurs opposite to a guanine (a common target for bulky
carcinogens), a thymine will pair with the adenine in the next round
of DNA synthesis, resulting in a G:C—*T:A transversion (32). Studies
using prokaryotic and eukaryotic cells and site-specific mutagenesis
assays (13, 33) have shown that some carcinogenic
“fingerprint―
(1, 34, 35); specific types and locations of DNA adducts
have been linked with the mutational spectrum of an agent. These
patterns have been studied in eukaryotes using both exogenous genes
introduced into the appropriate host cell by a shuttle vector (33, 36)
and endogenous genetic loci (34) such as apri, dhfr, and hprt.
B. Mutations in Model Systems
The carcinogen-induced mutational spectra in prokaryotes and in
target genes in eukaryotes can be compared to spontaneous mutational
spectra, although uncertainty exists in the extrapolation of in vitro data
to human carcinogenesis. Confounding variables which could affect
experimental systems include differences in the genes and cell types
studied, interspecies variability of DNA repair, and the potential
mutagenicity of transfection and shuttle vectors. The interpretation of
mutational spectra is complicated, and observed mutations may not
somatic reflect all events at the DNA level. The DNA sequence can influence
both carcinogen-DNA adduct formation and DNA repair, and some
carcinogens and mutations may be either more readily detected due to clonal expansion
or less readily detected due to inhibition of the mutant cell population.
1. Mutational Spectra in Housekeeping Genes
The majority of spontaneous mutations in the model systems used
are single base substitutions. Studies in prokaryotes indicate that
carcinogen-induced mutations occur at nucleotides which are targets
for carcinogen-DNA interactions and alterations (37). Differences
mispairing
have been observed between the spontaneous mutational spectra
of the endogenous aprt gene of CHO cells and both bacterial genes
and exogenous genes carried by shuttle vectors in mammalian cells
(38). This may reflect either cell-specific or gene-specific biological
properties.
A comparison of the spontaneous mutational spectrum at the aprt
locus in CHO cells with the spectra induced by UV radiation, ionizing
radiation, or BPDE illustrates carcinogen-specific damage (Table 1).
agents produce a
The mutational spectrum of UV light is consistent with the mutagen
esis models of the promutagenic cyclobutane and pyrimidine-pyrimi
done (6—4)photoproducts. Ionizing radiation often causes chromo
somal abnormalities, deletions, and losses (39, 40). BPDE, which
primarily binds to the 2-amino group of deoxyguanine, produces
predominantly the G:C—*T:A transversions predicted of a bulky car
cinogen-DNA adduct; a similar mutational spectrum for BPDE has
been observed in human cell assays (41). The hprt locus allows
comparison of mutational spectra obtained by exposing cultured cells
to carcinogens with those observed in human donor lymphocytes
exposed in vivo to environmental carcinogens (42). A recent com
prehensive database has been developed which contains more than

modeli'Gene Table ISpontaneous and induced mutational spectra: in vitro and anima!
[Ref.]G:C-@A:TG:C-'T:AG:C—@C:GA:T—*G:CA:T-@T:AA:T—@C:GCC:GG—'TT:AADe1C@dCHO
and Mutagen (n)―
aprt gene
Spontaneous (30) [281]
UV light (31) [282] 52 6 6 3 10 6 6 3 3
Ionizing radiation (16) [39] 19 6 6 6 13 19 0 31 0
BPDE (21) [283]71
5Human 513 623 140 07 90 00 06 50
aprt gene
Spontaneous (74) [43]
UV light (55) [43] 47 13 7 15 9 7 NR 2 0
BPDE (102) [43] 14 69 13 2 1 1 NR 1 0
MNNG (103)(43] 63 13 2 10 7 5 NR 0 1
0Animal
ENU (81) [43]28 3112 98 520 178 258 9NR NR15 50
p53 gene@
Mouse skin
UV light (33) [44, 451
BP (7) [46] 14 71 14 0 0 0 0 0 0
Butadiene (10)e 10 40 10 30 0 0 0 10 0
DMBA (18) [46, 48] 22 17 11 11 17 6 0 11 6
0Rat
MCA (7) [284]42 2918 570 140 018 00 021 00 00
esophagus
[285]90000000100Rat
NMBA (10)
liver
choline-deficient diet (9) 11 101 101 10
[286]5501
a Values, percentagesof each class of mutation;MNNG, N-methyl-N'-nitro-N-nitrosoguanidine;ENU, N-ethyl-N'-nitrosourea;DMBA, dimethylbenzanthracene;MCA, methyl
cholanthrene; NMBA, nitrosomethylbenzylamine; CHO< Chinese Hamster Ovary; Ref., reference no; NR, not reported.
h Number of mutations sequenced.
C Deletions.

d Insertions.
e R. Wiseman, personal communication.
I Examples of mutation prevalence may be found in Refs. 287 and 288.

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Copyright © 1994 American Association for Cancer Research
 p53
1000 human hprt mutations (43). The mutational spectra found at
the aprt locus of CHO cells and the hprt locus of human cells are
detailed in Table 1. The most striking similarities are the high
percentages of G:C—'T:A transversions at both loci in cells ex
posed to BPDE and high percentages of G:C—*A:T transitions in
UV-exposed cells, consistent with models predicted for these
agents. The largest difference between the two systems is in the
spontaneous mutational spectrum. In the aprt locus, G:C— A:T
transitions account for 71% of the 30 spontaneous alterations,
whereas in the hprt locus, only 28% of the 74 mutations are
G:C—t@
A:T (P = 0.001, Fisher's exact test). Thus, cell-specific
gene-specific features may affect spontaneous mutation patterns.
2. Mutational Spectra in Animal Models
Animal models provide opportunities for mutational spectrum stud
ies that cannot be performed with human tumors. For example, dose
response experiments can define the mutational spectra of carcinogens
in vertebrate neoplasms. The spectrum of carcinogen-induced muta
tions in cancer-related and housekeeping genes can be compared in
rodent and human cells, assessing the relevance of these model
systems to human carcinogenesis. Furthermore, transgenic
can provide mechanistic insights into the interplay between environ
mental carcinogens and host susceptibility factors such as carcinogen
metabolism and alterations of oncogenes and tumor suppressor genes.
The frequency of p53 alterations in murine primary tumors is
generally lower than in human tumors. p53 mutations have been found
in tumors of the breast (42%), lung (8%), skin (30%), and other tissues
(Table 1). Alterations generally have not been found in colon or liver
tumors. The low prevalences in lung and colon tumors are surprising
for several reasons: (a) the gene is highly conserved between mice
and humans; (b) p.53 mutation frequency is high in human lung and
colon cancers; (c) the morphological features of lung and colon
adenocarcinomas are similar in humans and mice; and (d) lung and
colon tumors from both species frequently contain ras mutations. The
mutation database in laboratory animals is still limited (Table 1), and
more studies of different carcinogens and dose regimens are needed.
The most well-studied system is skin carcinogenesis in the mouse.
Several carcinogens have been examined in rat, hamster, and nonhu
man primate models, but mutation rates are generally too low to
generate meaningful spectra.
Uv exposure efficiently induces squamous skin carcinomas in
several strains of inbred mice (44, 45). In these studies, as in human
tumors, there is a striking preference for single base transitions and
tandem mutations at dipyrimidine sites (30 of 33; 91% of mutations).
BP, a carcinogen found in tobacco smoke and burning fossil
induces papillomas and squamous carcinomas in Sencar mice when
applied topically. p53 mutations were identified in 1 of 8 papillo
mas and 6 of 12 carcinomas (46) with 5 G:C—@ T:A transversions,
1 G:C—t@C:G transversion, and 1 G:C—@. A:T transition (Table 1).
These data provide the first characterization of the p53 mutation
spectrum of BP-induced tumors in vertebrates. Skin tumors in
duced by 7,12-dimethylbenz(a)anthracene provide a contrasting
mutation spectrum. Eighteen p53 mutations have been reported
(46—48), and the mutation pattern is notable for a high frequency
(40%) of mutations at A:T base pairs and a low frequency of
G:C—―
T:A transversions. This is consistent with the mutagenic
specificity of 7,12-dimethylbenz(a)anthracene (48).
These mutational spectra of aprt, dhfr, and hprt genes and animal
models serve as a foundation with which to compare the spectra
observed in cancer-related genes in humans. Differences in spontane
ous mutation patterns may reflect differences in inherent characteris
tics of various cell types (e.g., DNA repair capacities), whereas
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Copyright © 1994 American Association for Cancer Research
MUTATIONS AND CANCER
induced mutation patterns that override the spontaneous profiles are
more likely to reflect mutagen-DNA interactions.
II. p53 Mutational Spectrum Analysis in Human Cancers
One of the goals of carcinogenesis research is to identify the precise
molecular alterations responsible for neoplastic transformation, ab
normal differentiation, and growth control. Molecular epidemiology
has developed to attempt to integrate traditional epidemiological
investigation of cancer risk factors with the substantial expansion of
or knowledge of the molecular mechanisms of cellular processes (49).
Mutational spectrum analysis, the study of the types and locations
of DNA alterations, describes the often characteristic patterns of DNA
changes caused by endogenous and exogenous mutagens. Alterations
of cancer-related genes found in tumors not only represent the inter
actions of carcinogens with DNA and cellular DNA repair processes
but also reflect the selection of those mutations which provide pre
malignant and malignant cells with a growth advantage. Study of the
frequency, timing, and mutational spectra of abnormalities of p53 and
other cancer-related genes can thus generate and test a variety of
hypotheses in carcinogenesis. These include questions regarding car
animals cinogen-DNA interactions, functions of the affected gene products,
mechanisms of carcinogenesis in specific organs or tissues, and fea
tures of general cell biology such as DNA replication and repair.
The information derived from this type of mutational spectrum
analysis is most relevant when it addresses alterations of genes vital to
carcinogenesis. A burgeoning literature (reviewed in Refs. 1, 2, 5, and
6) attests to the important role of p53 protein in normal cell function
and neoplastic transformation. Mutations of p53 are the most common
genetic abnormality yet found in human cancers. The prevalence of
p53 mutations varies among tumor types, ranging from 0 to 60% in
major cancers (Table 2), and is over 80% in some histological sub
types. All classes of mutations occur in the p53 gene (Fig. 2A);
three-fourths of substitutions occur at G:C base pairs.
The p53 gene is well-suited to mutational spectrum analysis for
several reasons. First, since p53 mutations are common in many
human cancers, a sizable database has accrued whose analysis can
yield statistically valid conclusions (50). Its modest size (1 1 exons,
393 amino acids) permits study of the entire coding region, and it is
highly conserved in vertebrates, allowing extrapolation of data from
animal models (51). Point mutations which alter p53 function are
distributed over a large region of the molecule, especially in the
hydrophobic midportion (1), where many base substitutions alter p53
conformation and sequence-specific transactivation activity (sec.
II.C.1.); thus, correlations between distinct mutants and functional
fuels, changes are possible. Frameshift and nonsense mutations which trun
cate the protein can be located outside of these regions, so evaluation
of the entire DNA sequence yields relevant data. This situation differs
from that of the ras oncogenes. Transforming mutations of ras occur
primarily in three codons and are thus limited to a few sequence
motifs which identify a critical functional domain (52). The diversity
of p53 mutational events permits more extensive inferences regarding
mechanisms of DNA damage and mutation.
For this Perspective, we compiled 2567 mutations in human carci
nomas or cell lines from over 300 papers published or in press through
January 1, 1994, as identified by searches of Medline and Current
Contents in January, 1994. Only mutations confirmed by DNA
sequencing were entered into the database. Selected additional reports
were added to illuminate specific discussions. An updated version of
the database is available from the European Molecular Biology Lab
oratory Data Library through e-mail; details of its compilation
have been reported separately (50). Here we analyze these data with
attention to: (a) questions regarding carcinogenesis which have been
4859
 p53 MLflA11ONS AND CANCER

cancers'@Tumor Table 2 Preyalence and spectra ofp53 mutations in human

type p.53
(n)―Prey. Mut.c %G:C-a A:T %G:C—aT:A %G:C-+ C:G %A:T—'G:C %A:T—*T:A %A:T-@ C:G %Del/Ins/ OtherCpG W HOTSPOTS1
All tumors 37 41 17 8 11 6 4 13 24 •175
lb H
(2567) •245G> S,D,C,v
•248lb W,Q
249 R> S,M
•273
lb H,C
Lung 56 24 40 9 7 5 3 11 9 157 V> F
(897) •24@
lb W,L,Q
249 R> M,S
*273 R> L,H,C
Colon 50 63 9 3 11 4 1 8 47 ‘175@>H
(960) •245G> S,D,V
•248lb W,Q
p273 lb H,C
p282 lb W
Esophagus 45 38 16 3 15 14 3 12 18 None
(279)
Ovary 44 42 9 12 18 3 6 9 23 @273@>C,H
(386)
Pancreas 44 41 13 6 17 8 5 10 21 @273@>H
(170)
Skin 44 41 12 6 4 4 2 31 17 @248R>W
(220) 278 P> S,F
Gastric 41 47 6 3 17 5 6 16 35 None
(314)
Headandneck 37 31 18 11 14 5 3 19 13 @@4flbQWJ@
(524)
Bladder 34 37 14 21 9 3 2 14 16 280R>T
(308)
Sarcoma 31 47 14 6 7 6 1 19 21 None
(339)
Prostate 30 44 13 6 16 0 6 16 28 None
(87)
Hepatocellular 29 20 37 8 11 12 3 9 9 249 lb S,M,W
(716)
Brain 25 50 11 5 14 1 3 15 30 *175@>H
(456) •248
R> Q,W
Adrenal 23
(31)
Breast 22 36 13 8 11 7 6 16 23 @175R>H
(1536) •248R> Q,W
*273 R> H,C
Endometrium 22 51 10 4 14 0 6 14 37 •24@B.> W,Q
(224)
Mesothelioma 22
(23)
Renal 19
(102)
Thyroid 13 48 9 13 9 0 4 17 28 *248R>Q,G,W
(299) 273 lb.H
Hematological 12 49 7 7 12 4 5 15 31 •175P.> H
(1916) •24@lb. Q,W
Carcinoid 11
(61)
Melanoma 9
(70)
Parathyroid 8
(13)
Cervix 7 35 35 10 0 0 10 10 25 ‘273@>C
(350)
Neuroblastoma 1
(212)
Wilms 0
(41)
Testes 0
(40)
Pituitary 0
(27)
Pheochromocytoma 0
(47)
a Mutationalspectrum of a tumor type was calculated only if 20 mutationswere reported.
b n, the number of tumors of each cell type evaluated for p53 mutation by PCR-based techniques as compiled in the p53 mutation database (50).
C Prevalence of p53 mutations in cancers from each organ/tissue, as detected by PCR-based techniques, screening at least exons 5—8. Premalignant lesions such as adenomas or

dysplastic lesions are not included in prevalence data. Prevalence in all cancers is a crude estimate derived from statistics on worldwide cancer incidence and the prevalence of p53
mutations for each type as calculated from the reports in the mutation database (see text).
d Del, deletion; Ins, insertions.
@ e of mutationswhich are G:C—@
A:T transitionsat CpG dinucleotides.
I Hotspots for the entire database are defined as codons at which at least 50 mutations have been reported. Hotspots for individual tumor types include codons at which at least 10
mutations, or 10% of all mutations for that tumor, have been reported. Letters refer to amino acid change, using standard one letter code. Amino acid substitutions which occur more
than once are listed in descending order of frequency at each codon.
*, CpG site.

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 p53 MIThATIONS AND CANCER

addressed by mutation analysis, and conclusions reached to date; (b)
how mutational spectrum analysis and in vitro experiments provide
complementary information on carcinogenesis; and (c) new hypoth
eses generated from the accumulated information to be specifically
addressed by future studies of p53 mutations.
A. Analytical/Technical Considerations
1. Methods ofAnalysis for p53 Abnormalities
The methods of analysis for mutations can bias conclusions regard
ing the prevalence and nature of p53 mutations. A few caveats
concerning techniques and experimental design are warranted before
examining the data in detail.
Solid tumors are heterogeneous, consisting of tumor cells mixed
with nonneoplastic stromal cells and necrotic debris. Since tumors
undergo clonal evolution, different sites within a malignancy may also
vary in genotype and phenotype (21); a false negative result
arise from sampling error. Microscopic assessment is important to
ensure that tissue samples contain adequate amounts of the desired
histological features and to avoid analysis of necrotic debris. Samples
may be enriched for tumor content by several methods, such as
manual microdissection of slides (removing only the tumor tissue for
study) and cell sorting using DNA flow cytometry (53, 54).
DNA sequencing is required for precise identification of mutations,
but an altered p53 gene or protein can be detected by other immuno
logical or chemical tests (55—58).Because DNA sequencing is time
and labor intensive, studies evaluating human cancers for p53 muta
tions often use screening tests, performing DNA sequencing only on
tumors which are likely to contain mutations. The sensitivity and
specificity of these techniques are critical factors which could intro
duce bias into p53 mutation prevalence data.
The wild-type p53 protein usually resides in the cell nucleus, but
has a very short half-life and is present in such small quantities that it
cannot be detected by routine IHC. Missense mutations often increase
the half-life and quantity of the p53 protein, allowing its detection by
IHC (58). Many investigators have used IHC as a screening test prior
to DNA sequencing, and staining is often considered a surrogate
marker for gene mutation even in the absence of confirmatory DNA
studies. In the 84 studies we identified which report IHC and sequenc
ing in the same tumor sets, positive staining was found in 44% of
tumors by IHC, while 36% contained mutations. However, the sen
sitivity of IHC in these studies was only 75% (range, 36—100%),and
the positive predictive value was only 63% (range, 8—100%), with
considerable variation among tumor types. Thus, the status of the p53
protein by IHC should not be equated with wild-type or mutant
genotype.
Discrepancies between IHC and sequencing results may be due to
false negative and positive results from technical aspects of sample
preparation and analysis or the result of biological phenomena respon
sible for the discordance (reviewed in Ref. 58). Mutations which
result in deletion or truncation of the protein (nonsense and frame
shift) do not cause protein accumulation. Therefore, IHC will be less
sensitive in detecting mutations in tumor types with high proportions
of these nonmissense mutations. Some tumors, including melanoma
(59, 60) and testicular carcinoma (61, 62), are frequently immuno
positive for p53 in the absence of mutation. Potential mechanisms for
positive IHC due to accumulation and staining of nonmutant p53
protein include stabilization of p53 protein by binding to viral or
cellular proteins and DNA damage from chemical or physical geno
toxic agents (58). Another hypothesis is that mutations are occurring
in a gene downstream of p53, such as WAF1/Cipi, a Mr 21,000
protein which is transactivated by the p53 protein and is an inhibitor warranted when mutations are not found in exons 5—8.The tissue
of cyclin-dependent kinases (63, 64). Interruption of the p53 pathway
4861
could interfere with a feedback loop which regulates expression of
wild-type p53. This can be tested by searching for mutations in genes
downstream in the p53 biochemical pathway in the cancers in which
staining is frequently detected but which rarely contain p53 mutations.
Other methods of screening tumors to identify those likely to
contain mutations involve detection of altered electrophoretic mobil
ity patterns of DNA sequences containing point mutations. The most
common techniques, SSCP (65—67),denaturant gradient gel electro
phoresis, and constant denaturant gel electrophoresis (68, 69), have a
sensitivity and specificity of approximately 90% in detecting muta
tions confirmed by DNA sequencing (67, 69, 70). Sequencing should
be done to confirm that positive results represent mutations and not
technical artifacts or germline polymorphisms, which have been
described at codons 72 and 213 (71, 72).
PCR-based techniques, including SSCP, constant denaturant gel
electrophoresis, and DNA sequencing, are subject to false negative
might and false positive results. The polymerases used in PCR to amplify
specific gene sequences are subject to error rates from 1/10,000 to
more than 1/500 base pairs, depending on reaction conditions (73).
Large deletions might not be detected if the missing segment includes
primer sites, and the wild-type gene of stromal tissue is amplified.
Contamination of a PCR reaction with other airborne PCR products
may also lead to false positive or negative sequencing results (74).
Therefore, it is imperative that all mutations found by direct sequenc
ing of PCR products be confirmed by sequencing of a second, mdc
pendent PCR product. This practice has not been followed uniformly;
hence, some mutations in the database may represent false positives.
There also is considerable inconsistency among papers in the extent of
analysis. Mutation prevalence data are included in this report only if
a minimum of exons 5—8was sequenced. Due to underdetection,
mutation prevalence rates are likely underestimated in the database,
although reported rates for some tumors were probably inflated due to
preselection of samples by screening tests. Thus, absolute mutation
prevalence rates are difficult to assess, given the heterogeneity of
techniques in cited reports.
2. Extent ofp53 Gene Analysis
The p53 gene consists of 11 exons; exons 2—1
1 code for the protein
of 393 amino acids. Early studies noted that most p53 mutations
occurred in the regions of the gene which are highly conserved
through evolution and presumably of functional importance, primarily
in exons 5—8(codons 126—306) (55). Based on these preliminary
data, most investigators have confined their analyses to exons 5—8
(and sometimes exon 4). Partly because of this bias in searching, 95%
of the reported mutations have been found in exons 5—8and their
intervening introns (mutations in introns which affect splicing of the
next exon have been classified as mutations of the deleted exon). We
have identified 50 studies in which sequencing of the entire coding
region of p53 was reported. Of the 560 mutations reported in these
papers, 87% were in exons 5—8,and most of the others were in exons
4 (8%) and 10 (4%). The distribution varied among tumor types.
Mutations outside exons 5—8were most frequent in bladder (28%)
and hepatocellular (24%) carcinomas and least frequent in head and
neck and hematopoietic malignancies (2% each). Over 70% of these
mutations generated stop codons or frameshifts; therefore, they likely
would be missed by IHC screening.
Thus, evaluation of only exons 5—8is likely to underestimate the
prevalence of p53 mutations, especially nonmissense mutations, by
over 20% in some tumors. Analysis of exons 4 and 10 is therefore
specific differences in the prevalence of mutations in various exons

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 p53
are clues that detailed p.53 mutational spectrum analysis can reveal
subtle patterns of genetic damage.
Another potential source of underestimating the prevalence of p.53
mutations might be the presence of mutations in unevaluated DNA
sequences, such as its 2 promoters or its 10 introns. Although the
intron bases adjacent to exons, which are responsible for splicing, are
often examined, complete evaluation of introns is not common. Re
verse transcription-PCR, a technique using reverse transcriptase to
amplify mRNA and sequence the resulting complementary DNA,
cannot evaluate introns, which have been removed by splicing. Mu
tations in intronic regulatory regions may be prevalent and undetected by
screening with IHC (if protein does not accumulate) and PCR-based
techniques (if primers do not encompass the relevant intron sequence).
3. Epidemiokgkal Considerations
Specific features of populations evaluated for p53 mutations may
also create bias in the conclusions. For some cancer types, differences
in frequency or spectrum have been detected between groups
differ in ethnicity or nationality, possibly due to specific carcinogen
exposures or inherited features in these populations (sec. II.D.3.). If
atypical subgroups are overrepresented, features of the mutational
spectrum of other groups may be obscured.
In order to yield valid conclusions, molecular epidemiology must
adhere to standard epidemiological principles of statistical analysis:
i.e., selection of appropriate control and study groups, assessment for
potential confounding variables (which may be different for each
tumor type), and adequate sample sizes to minimize type I errors (e.g.,
the premature assignment of relationships between putative carcino
gens and cancers) and provide the statistical power to detect important
differences and avoid type II errors (the erroneous conclusion that no
difference exists). Although these admonitions may seem elemental,
the literature contains many examples of preliminary conclusions
based on small sample sizes, which often pass into general opinion
without sufficient critique. The database provides a powerful tool to
highlight the areas in which data are inadequate and focus future endeav
ors.
B. Patterns of Mutation: Insights into DNA Replication
and Repair
1. DNA Repair and Strand Bias of Mutations
Since all organisms are constantly exposed to exogenous and en
dogenous mutagens, sophisticated systems of DNA repair to maintain
the integrity of the genome have evolved. DNA repair may be defined
as a cellular response which restores the normal nucleotide sequence
and stereochemistry of DNA following damage. DNA damage often
causes a physical block to transcription; therefore, repair is generally
faster in actively transcribed genes and is closely linked to other
cellular pathways which control gene expression (75). Several re
cently described genes code for enzymes exhibiting both DNA repair
and gene transcription activities, suggesting a potential molecular
mechanism for control of cellular proliferation until damage is cor
rected (76, 77). In addition, the p53 gene is repaired faster than the
dhfr gene, and much more efficiently than the inactive genomic
regions (78). In vitro studies and observations in repair-deficient organ
isms indicate that repair is important in determining mutation patterns.
Importantly, repair is faster on the transcribed (template) strand of
DNA than on the nontranscribed (coding) strand (75). Experiments in
bacteria, yeast, and mammalian cells and in vivo mouse skin carcino
genesis models have shown that mutations induced by exogenous
carcinogens preferentially occur on the nontranscribed strand of genes
such as dhfr, hprt (79), and p53 (46, 75). That is, the mutations
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MUTATIONS AND CANCER
induced on the nontranscribed (coding) strand by UV light or BP are
much more likely to be CC-+ U than GG—AA and G—@ T than
C—@
A, respectively. This difference is thought to be an evolutionary
adaptation which ensures that structure and function of essential
proteins with short half-lives are maintained, increasing the likelihood
of cell survival. Repair of the nontranscribed strand can occur later
and still preserve the fidelity ofthe gene in the daughter cells. If repair
of the nontranscribed strand does not take place, then daughter cells
might arise with mutations that are lethal or that confer a survival
advantage on the mutant clone.
The hypothesis that deficient DNA repair can affect mutation
patterns in humans was tested by measuring the repair of UV-induced
cyclobutane pyrimidine dimers in the individual DNA strands of p53
in a normal, repair-proficient human fibroblast strain and in fibro
blasts from a patient with the repair-deficient disorder XP-C (78).
Selective repair of the transcribed DNA strand of p53 is observed in
both normal and XP-C fibroblasts, and the strand bias of repair is
more pronounced in XP-C cells. Since mutations may occur due to
which replication errors at the sites of unrepaired DNA damage, these results
predict that the majority of p.53 mutations in skin cancers, especially
those from patients with XP-C, would occur on the nontranscribed
strand. Indeed, 100% of tandem mutations in skin cancers from XP-C
patients and four of seven from non-XP patients exhibit such a strand
bias (80, 81). More data in non-XP cancers are needed. Tumors from
XP-Cpatientsare muchmorelikelythan sporadiccancersto contain
tandem pyrimidine transitions (58 versus 11%), suggesting a differ
ential ability to repair this type of DNA damage. This sequence of studies
is an example of testing in the laboratory of a hypothesis generated by
analysis of the p53 mutation spectrum and then predicting the result of
further molecular epidemiological investigation of human tumors.
Some tumor types have been linked by epidemiological and labo
ratory evidence to specific carcinogens with known mutational spec
tra, such as lung cancer and exposure to cigarette smoke (section
II.D.5.C). Ninety-one percent of G:C—+T:A transversions in lung
cancer occur on the nontranscribed DNA strand (i.e., are G—+
T rather
than C—+
A), supporting the hypothesis that DNA repair is an impor
tant determinant of mutation specificity of cancer-related genes in
human cancer. The hypothesis may be extended to postulate that a
nontranscribed strand bias in the mutation pattern of a specific base
substitution or tumor type implies that an exogenous carcinogen may
be responsible. Evaluating the entire p53 database for strand bias may
therefore provide clues to suspected and unrecognized carcinogens.
Four patterns of base substitution exhibit strand bias: G—+
T (87%
on the nontranscribed strand) and T—* G (67%) transversions, and
A—p
G (70%) and nonCpG site G-+ A (65%) transitions (Table 3).
G:C—*
A:T transitions occurring at CpG dinucleotides are reported
separately from other G:C—@
A:T transitions because of differences in
mechanism discussed in section I. Bulky carcinogens such as BP,
other PAHs (82), and AFB (82—84)have been associated with tran
sitions and transversions at both G:C and A:T base pairs (85). The
relative contributions of these and other agents to human somatic
mutations are unknown.
In support of the hypothesis that tumors which are associated with
environmental carcinogens should contain a high proportion of base
substitutions with bias toward the nontranscribed strand, Table 3 lists
all tumor types in descending frequency of the prevalence of the base
substitution types which exhibit strand bias (G—* T, non-CpG site
G—+A, A-t' G, and T—t'G). Among the tumors which most frequently
contain these types of mutations are tobacco-associated cancers of the
lung (most frequent), cervix, esophagus, head and neck (fourth, fifth,
and sixth most frequent, respectively), and hepatocellular carcinoma
(second most frequent), which is associated with dietary AFB expo
sure in certain geographic regions (section II.D.5.B). Most of these
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 p53 MUTATIONS AND CANCER

Table 3DNA strandbias by tumor%

G-+ATumorTotal
T—Gtype % of% G—TonNon-CpG on% A-@G%
at strand G—aA nontran nontran
strandAlltumors50171711487657067(2567)Lung6640157395529164(328)HCC61381011389777571(224)Pancreas56132117510069730(63)Cervix55351001086100NAe50(20)Esophagus53161915
(n)°Muts bias site―% G—'D% non-CpG% A—@G%T—*Gnontran strand―nontran strandon strandon

Skin401222527742'@20100(110)Gastric4061117671693857(121)Thyroid39917941003810050(46)Colon36915II268646650(324)

a ,@ number of mutations detected in benign and malignant tumors at each site. These numbers differ from values obtained from Table 2 by multiplying the total number
tested X prevalence rates because: (a) some tumors contain multiple mutations; (b) mutations in nonmalignant precursors are included here but not in Table 2 prevalence data; and
(c) only sequenced mutations are included here, whereas mutations determined by other techniques may be included in prevalence statistics.
b Percentage of p53 mutations that are one of the indicated base substitutions associated with strand bias.
C Percentage of p53 mutations of indicated base substitution on both strands.

d Percentage of indicated base substitution on nontranscribed strand.

e NA, not applicable.
@ I light-induced transitions at tandem pyrimidine sites are a cause of C—*
T transitions with nontranscnbed strand bias in experimental models. Therefore, in skin cancer, C—'T
transitions are considered to be carcinogen-induced nontranscribed strand mutations.

substitutions exhibit marked strand bias. When strand bias is not
apparent, it may reflect a mechanism of mutation which does not
exhibit strand bias [such as non-bulky DNA adducts of alkylating
agents, which cause mainly G:C—A:T transitions due to mispairing
(31, 86)]. Alternatively, multiple carcinogens could be involved, and
the bias patterns could negate each other (e.g., in esophageal and lung
cancer, one tobacco-associated carcinogen might cause G—A and
another, C—p
T transitions). In vitro studies which establish the mu
tational spectrum of a mutagen can be complementary to analyses of
clinical specimens for strand bias in base substitutions and can im
plicate potential tumor-specific carcinogens (section II.D.5). In the
lower half of Table 3 (i.e., tumors which exhibit few mutations with
strand bias) are cancers which have not been strongly associated with
environmental carcinogens (87), supporting the nontranscribed strand
bias-exogenous carcinogen hypothesis.
2. Deletions and Insertions: DNA Replication
Hypothesis
Previous analyses of short deletions and insertions in p53 (88) and
other genes associated with genetic disease (89) have concluded that
the DNA sequence context is the most important factor determining
these events. Almost all short deletions and insertions occur at mono
tonic runs of two or more identical bases or at repeats of 2- to 8-base
pair DNA motifs, either in tandem or separated by a short intervening
sequence. Several mechanisms are probably involved (90). The most
well-studied is called slipped mispairing, a misalignment of the tem
plate DNA strands during replication that leads to either deletion, if
the nucleotides excluded from pairing are on the template strand, or
insertion, if they are on the primer strand. When direct repeat se
quences mispair with a complementary motif nearby, the intervening
oligonucleotide sequence may form a loop between the two repeat
motifs and be deleted (88, 89). More lengthy runs and sequence
repeats are more likely to generate frameshift mutations. In vitro
research which detects errors in replication of reporter genes has
helped quantify this phenomenon (reviewed in Ref. 17).
The database contains 285 deletions or insertions of 30 base pairs,
and the Mispairing
including 98 single base pair deletions, 40 single base pair insertions,
and 217 total events of @6 base pairs. Almost all (265; 90%) are at
monotonic runs and short direct repeats, supporting the prior obser
vations that mispairing is the primary mechanism of short deletions
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 p53 MUTATIONSAND CANCER
and insertions in human genes. Both deletions and insertions increase
in frequency as the number of repeated bases in a run increases. The
region of p53 which most frequently contains deletions or insertions
is codons 151—159.Twenty-seven (9% of all deletions and insertions
and 1% of all p53 mutations) have been reported at this G:C-rich
sequence with multiple runs and direct repeats, confirming in vitro
observations regarding sequence motifs which predispose to mispair
ing and other mechanisms of frameshift mutations.
Much remains unknown regarding the cellular phenomena which
influence the risk of mispairing. Factors which affect DNA polymer
ase function, e.g., binding to polymerase accessory proteins, are areas
of interest for mechanistic study (89). Some bulky chemical mutagens
induce frameshift mutations in prokaryotic models through mecha
nisms related to adduct-induced deformation of the DNA helix. These
adduct-induced mutations occur more frequently in monotonic or
tandem base runs and at G:C-rich sequences (82, 90, 91). The con
tribution of chemical carcinogens to frameshift mutations of p53 in
eukaryotic cells is potentially significant. Tumor-related differences in
deletion prevalence may reflect organ-specific carcinogens or tissue
specific properties that regulate replication fidelity.
It is uncertain whether the recently described human mismatch
repair genes hMSH2 and hMLHJ (25—28), which are responsible for
causing mutations at tandem repeat DNA sequences (microsateilites)
in families with FINPCC (section I.A; Refs. 92 and 93), play a role in
p53 deletions and insertions. Most events in colon and other cancers
occur at consecutive base runs, but 40% are at short tandem repeats.
None of the seven deletions at the most mutable tandem repeat
sequence in the p53 gene (GTG GTG GTG at codons 216—218)are
found in colon cancers. Future studies might include evaluation of
HNPCC tumors for slippage mutations at repeat sequences, assess
mont of the frequency of microsatellite changes in cancers which
exhibit frequent deletions and insertions, and evaluation of mutations
in hMLHJ, hMSH2, and other mismatch repair genes.
C. Determinationof Functionsand Propertiesof p53
Mutational spectrum analysis complements in vitro studies of the
relationship between p53 structure and function and suggests many
experiments which can test the hypothesis that some mutants of p53
alter its function and confer upon cells a growth advantage. In other
tumor suppressor genes (e.g., RB and APC), the most common ab
normalities found are deletions or nonsense mutations which elimi
nate protein expression, indicating that loss of their function promotes
tumorigenesis (94, 95). The presence of mutated p53 protein in tumors
suggests that cells acquire a selective growth advantage from not only
loss but also missense alteration of p53, frequently accompanied by
loss or mutation of the second allele. Analysis of all p53 mutations in
human cancers reveals a nonrandom distribution within the p53 mol
ecule, implying that specific mutant proteins possess different growth
stimulatory properties (Fig. 1). Identification of these locations has
focused research of p53 structure and function toward specific regions
of the protein, such as exons 5—8.What is the functional importance of
these regions? Are mutations more frequent here because of the gain and
loss of specific functions? Are the types of p53 mutations seen in other
regions of the molecule different than those in the conserved regions?
1. Structure-Function Relationships ofps3 Protein
Homologies have been noted between p53 domains and other
proteins, providing clues to both structure and function. The confor
mations of wild-type and some mutant p53 proteins and their rela
tionship to the critical functions of sequence-specific DNA binding
and transactivation have been examined using full-length and trun
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Copyright © 1994 American Association for Cancer Research
cated proteins. Some of these inferences have been corroborated by
recent evidence describing the nuclear magnetic resonance analysis
and the crystal structure of several p53 domains (96, 97).
A schematic of the p53 molecule, indicating the functional regions
most frequently evaluated, is shown in Fig. 1. The NH2-terminal of
p53 contains an acidic domain similar to those found in other fran
scription factors (5). Chimeric proteins containing amino acids 20—42
fused to the DNA binding site of Gal4 will transactivate reporter
genes linked to Gal4-binding DNA sequences (98). Interestingly, this
region also is responsible for binding to cellular protein Mdm2, and
direct inhibition of transactivation is the likely mechanism by which
Mdni2 interferes with p53 function (99—102).A proline-rich linking
region between amino acids 60 and 92 begins the hydrophobic mid
section of the protein. Between amino acids 100 and 295 lies the
critical region (delineated by studies of truncated and chimeric pro
teins) which determines p53 conformation, specific binding to DNA
consensus sequences, and sequence-specific transcriptional activity
and which may be essential for growth suppression (103). This region
also binds to SV4O T-antigen (104). The recently described crystal
structure of the DNA-binding domain identifies several motifs which
directly contact DNA, including nine amino acids which abut the p53
consensus binding sequence (97). The positively charged carboxy
terminus contains the basic nuclear localization sequence (amino
acids 316—325)and oligomerization domain (amino acids 319—360;
Refs. 5, 96, 105, and 106). Oligomerization into dimers or tetramers
appears to be required for DNA binding and transactivation function
(97, 107, 108). The observation that the nonsense mutation
CGC—@
TGA at codon 342 has been reported 10 times in human
tumors supports the experimental evidence that truncation of the last
50 amino acids does lead to a selection advantage in vivo.
Many mutations in the hydrophobic midregion change the confor
mation of the molecule, perhaps by partial denaturation. This exposes
an epitope between amino acids 213—217,which is the target for
mAb24O, a monoclonal antibody that binds specifically to conforma
tionally altered or denatured p53 (97). This antibody has been used as
a marker of altered p53 conformation in immunoprecipitation and
immunoblotting studies. The importance of conformation in p53 func
tion has been demonstrated using truncated and mutant p53 molecules
(including temperature-sensitive mutants whose conformation
changes from wild-type at 32°Cto mutant at 37°C)and by modulating
wild-type conformation by oxidation. Molecules whose conformation
is altered by mutation or oxidation (109) lose sequence-specific DNA
binding and transactivation activity; temperature-sensitive mutants
exhibit this loss only at 37°C,when they exist in mutant conformation
(110, 111). Chimeric proteins, with the central p53 conformation
region linked to foreign nuclear localization and transactivation re
gions, maintain sufficient p53 transactivating properties to sup
press growth of lung and colon carcinoma cells (103, 112), indicating
that the amino and carboxy termini contribute generic functions but not
specificity.
Humans and laboratory animals which develop tumors carrying p53
mutations may also develop antibodies against the protein, and this
phenomenon is now being investigated as a possible marker for
diagnosis and prognosis. Anti-p53 antibodies occur in patients whose
tumors contain missense mutations, but they are usually directed
against epitopes in the amino and carboxy termini and not the
conformation-determining midregion, suggesting that the native pro
tein can be immunogenic. Antibodies have been detected in the serum
of patients with carcinomas of the bladder (28%), lung (15%), breast
(12%), esophagus (16%), liver (25%), pancreas (19%), and lympho
mas (5%) and at least seven other types of cancer, as well as in
patients with precursor lesions such as Barrett's esophagus (5%; Refs.
113—118). In several anecdotal cases, the antibodies have been de

tected in serum samples in high-risk patients (Barrett's esophagus,
tobacco smokers, and occupational vinyl chloride exposure) several
years before the clinical detection of cancer.4 Hypotheses regarding
anti-p53 antibodies include: (a) does the immune response affect
carcinogenesis, e.g., does presence of anti-p53 antibody affect rates of
progression to malignancy of preneoplastic lesions? (b) can the de
tection of anti-p53 antibodies be used as a screening test in patients at is shorter than the span identified by in vitro and evolutionary studies
high risk for specific cancers (e.g., smokers, Barrett's esophagus, and
occupational carcinogens)? and (c) can the presence of serum anti-p53
antibodies identify individuals most responsive to immunotherapy?
2. Distribution of Mutations and the Relationship to p53
Conservation through Evolution
The clonal selection theory of carcinogenesis posits a growth ad
vantage for clones containing mutant p53; therefore, sites where
missense mutations frequently occur presumably identify amino acids
whose replacement leads to loss of p53 suppressor function and
perhaps gain of oncogenic function. Previous reviews have noted that
major mutational “hotspots―
occur in four of five domains labeled I—V
(I, amino acids 13—19; II, amino acids 117—142; III, amino
171—181;IV, amino acids 234—258;and V, amino acids 270—286),
which are highly conserved in evolution and are presumed to contain
amino acids essential for p53 function (Fig. 1; Ref. 51). Of the 393
amino acids in human p53, 96 (24%) are conserved in 8 species for
which the p53 sequence is reported. By comparing all hotspot sites to
the pattern of AA conservation in all species, we can address the
hypothesis that common mutations affect conserved amino acids
important to p53 function.
Fig. 1 demonstrates the distribution of missense and nonmissense
mutations in the database. Missense mutations are the most common
type (79% of mutations) in the conserved midregion but are uncom
mon (23%) in the amino and carboxy termini, where nonmissense
mutations (nonsense, frameshift, splicing, and silent mutations) pre
dominate. The rarity of missense mutations in the amino and carboxy
termini suggests that in these regions the integrity of a single amino
acid residue is not essential to function or conformation. Of note, no
mutations have been reported in conserved domain I. Nonconserved
residues within exons 5—8also show a low proportion of missense
mutations, supporting the hypothesis that substitutions of noncon
served amino acids do not provide cells with a positive selection
advantage. The most commonly mutated amino acids in the DNA
binding region are close to the protein-DNA interface (97). The
distribution of deletions and insertions in p53 appears to be related
primarily to DNA sequence repeats (Fig. 1; section II.B.2) and not to
characteristics of the protein; as predicted, they occur frequently at
both conserved and nonconserved codons.
We analyzed patterns of evolutionary conservation using the Mul
tiple Alignment Construction & Analysis Workbench (MACAW)
program (119). This method identifies the most highly conserved
portion as amino acids 97—292(MP-scores, 359.4 and 471.2 for two
contiguous conserved regions). This area corresponds almost exactly
to the region which determines sequence-specific DNA binding and
transactivation (section II.C.1). When lower stringency for sequence
similarity is allowed, secondary conserved blocks are noted between
amino acids 17—29(MP-score, 40.4), corresponding to the transacti
vation region identified through Gal4 binding experiments, and amino
acids 324—352(MP-score 114.3), encompassing the oligomerization
domain. Higher stringencies fail to identify subregions within amino
acids 97—292,including domains II—V,that are significantly more
homologous than the sequence as a whole. Detailed analysis of the
4 G. Trivers and T. Soussi, personal communication.
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p53 MUTATION5AND CANCER
patterns of mutation clusters in conserved domains Il—Vand else
where adds to the evolutionary data and provides clues to amino acids
of greater and lesser importance.
The predominance of missense over nonmissense mutations begins
and ends abruptly at codons 130 and 286 (Fig. 1), suggesting that
these are the boundaries of the conformation-determining region. This
as important for DNA binding specificity (amino acids 93—293)and
suggests that changes to amino acids 93—130may not alter p53
conformation significantly. This region contains three short (3-sheets
and an unstructured loop, of which only one amino acid (Lys 120)
interacts with DNA (97). Forty-four codons are relative hotspots for
missense mutation, arbitrarily defined as more than 12 mutations
reported in the database (>0.5% of all mutations); 40 of the 44 hotspot
amino acids (91%) are conserved in all 8 species. This represents only
42% of the 96 conserved AM, suggesting that mutations at many
other conserved sites confer little or no growth advantage; this hy
pothesis can be tested by functional assays of mutants created by
site-directed mutagenesis.
Some amino acids in the previously identified hotspot domains are
acids
affected much more frequently than others, and within each domain
are some highly conserved amino acids which are rarely mutated. For
example, only 3 of the 25 amino acids in domain II (132 Lys, 135 Cys,
and 141 Cys) are relative hotspots. All three of these residues begin or
end (3-strands, although other amino acids which flank (3-strands are
not hotspots (97). Of the six hotspot codons which contain 23% of
reported p53 mutations (Fig. 1), five contain CpG dinucleotides
(codons 175, 245, 248, 273, and 282 but not codon 249), suggesting
a shared mechanism (deamination of 5-methylcytosine) for the prey
alent G:C—@ A:T transitions reported at these sites. The resulting
mutants at some of these codons have been shown to alter p53 transcrip
tional activity and growth suppression (section II.D.5.F). Only two of
these amino acids (Arg 248 and 273) directly contact DNA, but all appear
to be Critical in stabilizing the protein-DNA interface (97).
Some relative hotspots for missense mutations occur outside the
previously recognized conserved domains, marking three unrecog
nized regions of potential functional significance (Fig. 1). Between
domains II and III lies a stretch of 14 amino acids, 151—164,of which
one-half are conserved and at which 167 missense mutations (and 33
nonmissense mutations) have been reported. This region forms a
(3-strand which is not adjacent to DNA (97). Its DNA sequence is rich
in G:C base pairs, often repetitive. No codons in this span are mutated
as frequently as the six hotspots noted above. Despite the fact that five
CpG sites occur here (section II.C.2), G:C—t'A:T transitions are rare.
This suggests that these sites may not be methylated or that the AA
substitutions resulting from deamination (three of which are silent)
offer little growth advantage, perhaps because they are not adjacent to
DNA (97). These hypotheses also are testable using in vitro mutagen
esis, evaluating the mutants for transactivation, growth suppression,
and other properties, as has been done for other CpG site mutations
(section II.D.5.F).
Two other highly mutated areas lie between domains III and IV.
Amino acids 193—200are almost completely conserved; missense
mutations are common at the first three codons (His-Leu-Ile). Codons
213—223also define a region of high conservation, with missense
mutational hotspots at codons 216 (Val) and 220 (Tyr). Two other
conserved moieties which are frequently altered are 205 Tyr and 266
Gly. Of these mutational hotspots, five contain a single favored
mutant: 157 G:C—@
T:A, Val>Phe; 194 G:C—*
T:A, Lys>Arg; 195
T:A—*C:G, Ile>Thr; 205 A:G-+ G:C, Tyr>Cys; and 220 A:T-+ G:C,
Tyr>Cys. Codon 157 mutations, especially G:C—*T:A transversion,
are especially frequent in lung cancer, perhaps indicating a tobacco
4865
 p53
specific carcinogen (section II.D.5.C) or a growth advantage
to bronchial cells.
Taken together, these specificities do not exclude that certain re
gions of DNA may be susceptible to specific mutagenic events,
only favored mutants providing a selection advantage. The finding
that tyrosine residues are affected at several hotspots suggests that
they are important to p53 function. Potential reasons include contri
bution to p53 conformation or modulation of phosphorylation
although a mechanism has not yet been demonstrated.
Some other specific residues with potential importance are serines
9, 15, 37, 315, and 392, targets for phosphorylation by DNA-activated
protein kinase, p34cdc kinase, and casein kinase II. In vitro mutagen
esis studies creating alanine residues which are not phosphorylated
have implicated serine 15 in human p53 as playing a role in suppres
sor function; conflicting results have been found by altering serine
392 (120—122). That no mutations of these amino acids have been
reported is inconsistent with a strong selective advantage and argues
against the hypothesis that these residues contribute significantly to
suppressor function.
Our conclusions regarding evolutionary aspects of p53 are: (a) the
most highly conserved region corresponds to the area of p53 respon
sible for sequence-specific DNA binding functions (amino acids 97—
292); (b) the transactivation, nuclear localization, and oligomerization
domains are only moderately conserved, provide generic functions
which are not p53-specific, and are not significantly functionally
altered by single amino acid substitutions; and (c) based on mutational
spectrum analysis, essential conserved residues and favored mutants
occur at previously described domains Il—Vand three additional
regions (1) codons 151—164,especially codons 151—152(Pro-Pro),
157—158(Val-Arg), and 163 (Tyr); (2) codons 193—195,especially
the codon 194 Lys>Arg and 195 Ile>Thr mutants; and (3) codons
213—220,especially the Tyr>Cys mutant at codon 220. Two other
residues which are both conserved and frequently mutated are codon
205, another tyrosine which often mutates to a cysteine, and glycine at
codon266.A searchof the SwissProt,PIR International,andtranslated
Genbank files found no other reported sequences homologous to these
three regions; therefore, their specific properties remain obscure.
D. Clues to Carcinogenesis
1. p53 in the Context of Multistep Carcinogenesis
Is alteration of function of the p53 pathway a prerequisite for
neoplastic transformation? The frequency of p53 mutations argues for
a critical role, but for most tumor types mutations have been found in
only 20—50% of cases, with a maximum of 81% in oral mucosa
squamous cell, 70% in small cell lung cancers, and 68% in anaplastic/
undifferentiated thyroid cancer. Current techniques are unlikely to
miss more than 15—20% of coding sequence abnormalities.
frequent and important to functional abrogation are mutations in the
p53 promoter or unevaluated introns, epigenetic inactivation
and/or alteration of genes or proteins downstream in the p53 pathway?
Is p53 only one of several pathways whose disruption is sufficient for
neoplastic transformation? Support for this concept comes from the
observations that both p53 and ras mutations are common events but
often occur independently (67, 123—127),and either may be associ
ated with aggressive tumor behavior (4, 128, 129). Does neoplastic
transformation require a critical mass of genetic injury, in which p53
is a frequent but not essential casualty? In this scenario, loss of p53
genomic stabilization properties would predispose a cell to an accel
eration in the rate of genetic damage and greatly increase the likeli
hood of neoplastic transformation and/or malignant progression. Re
search which attempts to unify molecular and cellular theories of
carcinogenesis should address these and other questions. Evaluation
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MUTATIONS AND CANCER
peculiar of mutation patterns of p53 and other genes in clinical tumors can be
an important part of this process.
Some cancers are thought to arise from a single cell, while for
with others a “field
defect―of the entire mucosal surface is thought to result
in multiple clones of transformed cells and multiple primary neo
plasms (130). Field cancerization is thought to occur in epithelia of the
upper aerodigestive tract, bladder, and ulcerative colitis-associated
status, colon (131—133).Careful identification of the stages of this process
and molecular analysis at each stage can help illuminate the processes
of clonal evolution in specific tumors. The multiplicity of p53 muta
tional events indicates that identical mutations are unlikely to occur
independently, and specific mutants may be used as clonal markers.
For example, the field cancerization hypothesis for head and neck
squamous carcinoma is supported by molecular analyses which have
found discordant mutations among nonneoplastic respiratory mucosa,
primary cancers, and second upper aerodigestive primary lesions,
suggesting that p53 is an important early target for mutations which
can occur at multiple mucosal sites (132, 133).
2. Timing ofps3 Mutations in Human Cancer
Several human cancers develop through multiple, morphologically
defined stages, such as the adenoma-carcinoma sequence in the colon
and the dysplasia progression in the oral and bronchial mucosae. The
anatomically defined stages provide milestones for marking the tim
ing of p53 mutation, and there is evidence that some tumor types
undergo early and others late p53 mutation. The issue has both
mechanistic and practical implications, including the use of p53 as a
potential marker for early diagnosis and as an intermediate biomarker
for chemoprevention studies. This section reviews current data assess
ing early and late alterations in human tumors. Early is defined here
as more than one morphological stage before invasion of the basement
membrane, which is the traditional hallmark of malignancy. Late
means just before, or after, crossing the basement membrane. Alter
ations in the p53 pathway (mutation or epigenetic inactivation) are
thought to be generally early events in cancers of the lung, esophagus,
head and neck, breast, cervix, and stomach and generally late events
in cancers of the brain, thyroid, liver, and ovary (134—139).Cancers
of the colon, bladder, liver, and perhaps other organs may develop
through multiple pathways; p53 alterations may be early events in
some pathways and late events in others for these tumor types. Some
of the better characterized examples are discussed here.
Squamous cell carcinomas account for 25% of lung cancers and the
large majority of cancers of the upper aerodigestive tract. These
tumors usually progress through multiple, histologically recognizable
stages of dysplasia (usually subdivided as mild, moderate, and se
vere), carcinoma in situ, and transgression of the basement membrane,
How which marks the beginning of invasive cancer. Despite the small size
of preneoplastic lesions, which frequently measure less than 100 p@m,
of p53, some genetic studies have been done, including allelic deletion, SSCP,
and sequence analysis. However, the bulk of the data come from IHC
using p53 protein accumulation as a surrogate marker for missense
mutation (section II.A.1).
Genetic and cytogenetic analyses of preinvasive bronchial lesions
have shown that p53 mutations can precede invasion. Three reports
(140—142) document missense mutations, p53 allelic deletion, and
chromosome l'7p deletion in dysplastic mucosa associated with car
cinoma. However, composite IHC data show p53 protein accumula
tion in 0% of normal bronchial mucosae, 7% of squamous metapla
sias, 25% of mild dysplasias, 32% of moderate dysplasias, 69% of
severe dysplasias, 57% of carcinomas in situ, 70% of microinvasive
carcinomas, and 76% of fully invasive lung carcinomas (140—144).
These results support a multistage model for squamous lung carcinoma in
4866
 p53 MUTATIONS
which p53 mutation occurs in 25% of the earliest neoplastic lesions, and
the large majority of mutations occur before invasion develops.
The timing of p53 alterations in the oral cavity, oropharynx, and
larynx is addressed by at least 10 published reports, usually using IHC
as a marker for putative missense mutation in small progenitor lesions.
Several groups have reported p53 alterations (jositive IHC or muta
tion) at or before the stage of severe dysplasia, including some
histologically normal mucosa (132, 145—149).Future studies could
contribute to this area by providing additional histological detail to
IHC studies, further molecular data, or correlating additional factors
(e.g., DNA content and other genetic changes) with p53 alterations.
The timing of p53 mutation in squamous neoplasms in the esophagus
appears similar to those in the oropharynx (150—152).
Sporadic colorectal cancers generally develop through an adenoma
carcinoma sequence which has been extensively analyzed for alter
ations in oncogenes and tumor suppressor genes (reviewed in Ref.
153). Allelic deletion and p53 point mutation rarely occur until the
late adenoma/carcinoma boundary (154; reviewed in Ref. 153). In
contrast, in ulcerative colitis, an inflammatory condition predisposing
to colorectal cancer in non-polypoid, non-adenomatous, dysplastic
mucosa, p53 mutation and allelic deletion are found in the earliest
recognized dysplastic lesions (131, 155, 156). It is notable that several
IHC studies find protein accumulation at earlier stages of sporadic
colon cancer, suggesting a nonmutational mechanism of protein
accumulation during incipient neoplasia (157, 158).
3. p53 Genotype and Cancer Phenotype
The conventional method for describing the phenotype and predict
ing the clinical behavior of a cancer has been histological grading as
assessed by light microscopy of tumor cells. IHC, which identifies
specific cellular antigens, and other specialized staining techniques
can yield more detailed phenotypes and sometimes clinically useful
information, but these traditional measures are merely crude descrip
tions of essential tumor behaviors. Correlation of phenotypes, such as
histological grade or proliferative rate, with precise molecular events,
such as alterations of p53 or other genes (e.g., metastasis-related gene
nm23, RB, and ras), promises to expand our understanding of tumor
behavior and perhaps foster new paradigms of risk assessment, preven
tion, early detection, determination of prognosis, and antineoplastic ther
apies.
The relationship of p53 mutations to tumor grade and stage has
been evaluated in many tumor types. Associations of p53 mutation or
positive IHC staining with higher grade and more advanced stage are
frequent, although sample sizes are often too small to draw statisti
cally valid conclusions from individual studies. A relationship be
tween p53 mutation and advanced tumor stage has been noted for
cancers of the endometrium (159), cervix (159), ovary (138, 139,
160), liver (136, 137), prostate (161), and bladder (162, 163), indi
cating that for these tumors p53 mutation may be a late event con
tributing to tumor progression, but not for lung (67, 164, 165), head
and neck (132, 149, 166), and breast (134, 135), in which mutations
are known to occur in early stage tumors and precursor lesions.
Studies of brain neoplasms are archetypes for establishing the role
of p53 in tumor progression and clonal dedifferentiation. Low grade
gliomas, which are indolent and often curable by resection, may
transform to high grade, aggressive, rapidly lethal glioblastomas. Two
studies evaluating both low grade tumors and recurrent high grade
tumors document the acquisition of p53 mutations in five of six
tumors after transformation from low to high grade, although low
grade tumors may also contain p53 mutations (167, 168). The p53
mutational spectra of low grade astrocytomas versus high grade
glioblastomas are similar, supporting the concept that the high grade
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AND CANCER
tumors evolve from the low grade precursors and are subject to the
same mutagens. As 45% of mutations are G:C—@ A:T transitions at
CpG dinucleotides plus short deletions, the mutagens responsible for
progressive genetic damage in these tumors may be endogenous
cellular processes (section II.D.5.F).
Recent studies have correlated p53 mutation with metastasis and
other important but less well-studied tumor phenotypes, such as
angiogenesis (169). The regulation of specific genes which contribute
to these behaviors [such as MDR-1 (170) and thrombospondin (171)]
is one area in which the known transcriptional activating function of
p53 could contribute to acquisition of the metastatic phenotype. Since
p53 regulates genomic stability, and prevents cell cycle entry in
response to DNA damage, loss of p53 function would be expected to
correlate with aneuploidy and increased proliferative rates, two fea
tures often associated with tumor progression and poor prognosis.
Preliminary studies have noted a statistically significant association
between aneuploidy and p53 mutation for ovarian (172), colorectal
(173), and gastric (54) cancers and a positive trend in esophageal
cancer (53). Other properties which are being studied for a relation
ship to p53 function include radiosensitivity and hormone responsive
ness (161, 174—176).
These data support a critical role for p53 in tumor progression. The
results to date of p53 mutation analysis indicate that the details of this
process are complex and probably mutant- and tissue-specific. Spe
cific p53 functions potentially involved include the pathways of gene
transactivation, cell cycle checkpoint control, and programmed cell
death and suggest unbounded opportunities for in vitro testing. Future
research might address p53 mutation and mechanisms of tumor inva
sion, metastatic properties such as angiogenesis, and drug resistance.
Such studies could clarify questions generated by clinicopathological
observations, which are often inconclusive due to unavoidable
confounding variables or sample size limitations.
4. Immortalization and Neoplastic Transformation:
In Vitro Features
Much in vitro carcinogenesis research uses cultured human cells
derived from normal or malignant tissues (177). Tumor cell lines often
differ from the corresponding in vivo normal tissues in properties such
as protein or marker expression, response to growth modulators, and
oncogene abnormalities. p53 mutations are found twice as frequently
in all cell lines compared with all clinical tumor specimens (53% vs
26%); 15 of 20 tumor types have an excess of mutations in cell lines.
Potential explanations for this observation include: (a) mutations
might be underdetected in clinical specimens due to technical reasons;
(b) mutations could arise frequently during the process of establishing
a cell line and, therefore, reflect in vitro mutagenic events which are
not relevant to in vivo carcinogenesis; (c) clones with mutated p53
which comprise a minority of the in situ tumor could have an in vitro
growth advantage over clones without such mutations. According to
this hypothesis, the mutations would reflect in vivo mutagens, but p53
mutation would not be an important early event. The genetic insta
bility triggered by loss of p53 function would lead eventually to clonal
evolution and tumor progression.
Mutational spectrum analysis supports the hypothesis that cell line
mutations do reflect in vivo mutagens. Distribution of mutation sites
and percentages of each type of base substitution, frameshift muta
tions, and CpG site mutations are similar in clinical tumors and cell
lines. Experience in the establishment of cell lines from cervical
(178—180),thyroid (181, 182), nasopharyngeal (183, 184), and other
cancers strongly suggests that cells with abnormal p53 function have
a selective growth advantage in vitro. This advantage also may exist
in athymic nude mice, as suggested by the higher prevalence of p53
 p53 MUTATIONS AND CANCER

mutations in xenografts than clinical specimens of head and neck
carcinomas (183, 185). This hypothesis warrants further investigation
into the features of in vitro cell growth and what properties of
wild-type p53 may be inhibitory.
5. Carcinogens and Specific Organ Carcinogenesis
The narrow mutational spectra exhibited by some mutagens has
popularized the idea that each agent might leave a specific identifying
“fingerprint―
of site and type of DNA damage (35). It is probably
more realistic to expect that carcinogens will produce mutation pat
terns which are characteristic and instructive but not as unique as
fingerprints. Table 2 contains the mutational spectra of all cancers for
which more than 20 mutations were reported at the time of analysis of
the database, in descending order of mutation prevalence.
A) Skin Carcinoma and UV Light. A clear example of the value
of mutational spectrum analysis in identifying carcinogen-specific
mutations is seen in skin carcinomas, the most common type of human
cancer, in which the role of UV light as the major carcinogen is
unquestioned. Exposure to UV light increases the risk of both basal
cell and squamous cell carcinoma, as well as the less common but
more lethal melanoma (186, 187). This physical mutagen produces
distinctive pyrimidine dimers that, if unrepaired, can produce tandem
mutations, most characteristically CC—tTT transitions. Tandem
dipyrimidine mutations are infrequently caused by mutagens other
than UV light (188, 189) and have rarely been observed in noncuta
neous malignancies, i.e., 10 in over 2400 tumors with p53 mutation.
Thus, the observation that these tandem mutations are common in
squamous cell (9%) and basal cell (12%) carcinomas of the skin (Fig.
2, B-D) directly incriminates both exposure to UV light as the cause
of damage to the p53 gene and the loss of its tumor-suppressor
function in the development ofthe cancers (190). The detection of rare
cells with p53 mutations at dipynmidine sites in sun-exposed nonma
lignant skin indicates that UV light initiated these mutations at the
earliest stage of skin carcinogenesis (191).
Mutations at dipyrimidine sites in skin carcinomas show a nonran
dom distribution among sites within the p.53 gene. Of the 30 CC:GG
dinucleotides (18 on the nontranscribed strand) in conserved domains,
transitions are common (5 or more) at only five (codons 151—152, HBV infection and p53 mutations in the same tumors (208—216).
247—248,178—179,278, and 281—282);frequency of mutations also
varies at other dipyrimidine sites (CT:GA, TT:AA, and TC:AG).
Recent work shows that rates of cyclobutane dimer repair vary among
codons within p53 (192). Comparison of the frequency of transition
mutations to rates of repair shows that 94% of C:G—@ T:A or CC: areas; 54% prevalence in high AFB exposure areas, where almost all
GG—TT:AA transitions occur at sites where more than 10% of cases were HBV positive). The combination of these exposures may
cyclobutane pyrimidine dimers remain 24 h after UV treatment,
suggesting that slow repair of these dimers is responsible for some of
the site specificity of UV-induced mutations. The dipyrimidine sites
which are frequently mutated in XP-C patients were not evaluated in 211, and 216).
this study. It remains unknown whether variations in site-specific
DNA repair in XP patients follow the same patterns as normal
individuals and whether these patterns explain the different dipyrimi
dine mutation site specificity seen in XP-C. Mutant selection due to and AFB (217, 218). This nested case-control analysis shows statis
functional changes of specific amino acid substitutions also is likely to tically significant associations among the presence of AFB and its
play a role in determining this specificity. To further define the
mechanisms responsible for site specificity of mutations, study of the
functional consequences (e.g., transactivation) of the commonly
found mutants in skin and other cell types would be of interest.
Whereas p53 mutations are common in basal cell carcinoma and
squamous cell carcinoma, they are infrequent in melanomas (Table 2).
Since sunlight also is a risk factor for melanomas (reviewed in Ref.
193), the rarity of p53 mutations is surprising, although the frequent
accumulation of p53 in the nuclei of these tumor cells (59, 186, 194)
supports a role for the p53 biological pathway in tumorigenesis.
Evaluation in melanomas of other genes and proteins which interact
with and stabilize p53 (e.g., Mdm2) or act downstream in the p53
pathway [e.g., WAF1/Cipi (63)] would be informative. The p1611――
gene, which like WAF1/Cipi encodes an inhibitor of cyclin-depend
ent kinases, is another candidate tumor suppressor gene involved in
melanomas and other cancers (122, 195, 196).
B) Hepatocellular Carcinoma, Aflatoxin, and Hepatitis. The
viral-chemical etiology and multistage pathogenesis of HCC are ar
chetypal of human carcinogenesis. It is estimated that 75—90%of
HCC cases are attributable to HBV (197). The relative risk of HCC is
elevated in viral hepatitis carriers with chronic active hepatitis (198).
These findings suggest that cell proliferation and/or inflammatory
response associated with chronic active hepatitis are the critical fac
tors responsible for the increased probability of neoplastic transfor
mation of the precursor cells of HCC.
HBVDNAintegratesintoHCCcellsat randomsitesin thegenome.
It contains the X gene, which codes for a protein (HBX) that modu
lates the transactivation of many cellular genes and is a candidate viral
oncoprotein (199). Transgenic mice containing HBX gene in their
germline have an increased frequency of HCC (200). HBX protein
binds with p53 in vitro and in vivo (201), inhibits p53 sequence
specific DNA binding and transactivation activities, partially disrupts
p53 oligomerization, and prevents p53 binding to transcription-repair
coupling factor ERCC3 (202). These mechanisms of epigenetic p53
inactivation could account for the transactivation properties of HBX.
Correlation of p53 staining and mutation data with HBX protein
expression in HCCS could help assess the interactions between HBV
and p53.
AFB also is considered to be a significant etiological factor in
certaingeographicareas (e.g., southernAfricaand Asia),wherethis
mycotoxin is consumed in food contaminated by Aspergillus flavis
(reviewed in Ref. 203). Epidemiological studies conducted in the
1970s and 1980s provided statistical evidence that dietary consump
tion of AFB was positively correlated with incidence of HCC and
suggested a synergistic interaction with alcoholic beverage consump
tion (204) and chronic active viral hepatitis (205—207).
Many investigators have reported data on the presence of chronic
Their results indicate that HBV infection alone does not influence the
rate of p53 mutation and that AFB exposure is the most important
influence on mutation prevalence (28 versus 29% mutation prevalence
in HBV-positive versus HBV-negative cases in low AFB exposure
be synergistic in causing mutation, but data are insufficient for defm
itive conclusions (28 of 49 AFB positivefHBV-positive cases versus 1
of S AFB positive/HBV-negative cases contain mutations; Refs. 208,
The results from a recent prospective cohort study of 18,244 people
provide convincing evidence that AFB has an etiological role in
hepatocellular carcinogenesis and indicate a synergy between HBV
metabolites in urine specimens, serum HBV surface antigen positiv
ity, and HCC risk. The finding of the promutagenic AFB-N7-guanine
adduct in the urine is further evidence that AFB has been activated to
its electrophilic ultimate carcinogenic metabolite, AFB 8,9-oxide.
Human hepatocytes in vitro can enzymatically activate AFB to its
8,9-oxide (219); the interindividual variation in forming the AFB-N7-
guanine adduct may be 10-fold or greater (220). Since several iso
forms of cytochrome P450 enzymes can activate AFB (221), the
interpretation of pharmacokinetics data and its use in risk assessment
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 p53 MUTATIONS AND CANCER

will be complicated. The mutational spectrum of AFB (222, 223) has
been established in experimental systems; G:C—t@
T:A transversions
are the most common base substitutions (224—226). The high fre
quency of AGG—'AGT transversions on the nontranscribed strand at
p53 codon 249 in HCCs from areas of China and Mozambique with
a high HCC incidence could be due to the high mutability of the third
base of codon 249 by AFB and/or a selective growth advantage
hepatocyte clones carrying this specific 249@ mutant in liver
chronically infected by HBV.
The preferential mutability hypothesis has been tested by Aguilar et
a!. (227), who found that in a human liver cell line exposed to AFB,
the third base in codon 249 is preferentially but not exclusively
mutated compared to immediately adjacent codons, suggesting that
both preferential mutability and clonal selection are involved in hu
man hepatocellular carcinogenesis. This highly sensitive and specific
genotypic mutation assay also can be used to determine the p53
mutation load in nontumorous liver tissue from donors living in
geographic areas of high HCC incidence in which urinary AFB and/or
macromolecular-AFB adducts have been detected. A pilot study using
a genotypic mutation assay has compared the load of codon 249scr
(AGG->AGT) mutant cells in nontumorous liver tissues from HCC
patients from Qidong, People's Republic of China, urban Thailand,
and the United States (228). The loads of codon 249@Tmutations were
elevated in specimens from Qidong, slightly elevated in one biopsy
from Thailand, but did not exceed background levels in specimens
from the United States. These results indicate a positive association
between loads of codon 249ser mutation and dietary exposure to AFB.
They also suggest that, in some populations, p53 mutation occurs
early in hepatocarcinogenesis, and clonal expansion of these mutant
cells occurs in the nonmalignant tissue. A number of hypotheses could
account for a selection advantage for these mutant cells, including
altered responses to growth factors or resistance to programmed cell
death induced by hepatitis viral infection. These and other hypotheses can
be tested by in vitro models using human hepatocytes (219) and clinico
pathological studies, such as correlation of aflatoxin-DNA adducts with
codon249G:C—*
T:Atransversionsin individualpatients.Thegenotypic
mutation assay also might be applied to other tumor-specific hotspots.
C) Tobacco:Lungand OtherCarcinomas.Tobaccosmokingis
the leading preventable cause of cancer mortality, accounting for 40%
of cancer deaths in men and 20% in women. Cigarette smoke is a
complex mixture with thousands of constituents including tumor
initiators, promoters, complete carcinogens, and cocarcinogens (229).
The contributions of many of these agents to carcinogenesis in the
lung and other organs are not known. Some of the compounds found
in tobacco smoke, most notably N-nitrosamines and PAHs, have been
purified, tested in model systems, and found to be in vitro carcinogens
which induce characteristic DNA abnormalities (section I.B). This
finding has encouraged mutational spectrum analysis in tobacco
associated cancers to test the hypothesis that patterns of mutation can
implicate specific agents in tobacco responsible for inducing human
cancers. Compounds of interest include N-nitrosamines, PAHs (e.g.,
BP), active oxygen species, aldehydes, and metals. The p53 muta
tional spectra in tobacco-associated cancers show surprising intertu
mor variation, suggesting that many different tobacco-related carcin
ogens may be germane to human cancers.
1) Lung Carcinoma. Cigarette smoking is now thought to be
responsible for 90% of lung carcinomas in men and 78% in women
(230). A large body of epidemiological evidence has confirmed a
dose-response relationship proportional to duration and amount of
smoking (231, 232). How does mutational spectrum analysis further
define this association?
p53 mutations are common in lung cancer, with the highest prey
alence (70%) in SCLC and the lowest (33%) in adenocarcinomas. The
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prevalent mutation is G:C—@'T:A transversion with a predominance of
guanine residues on the nontranscribed DNA strand (Fig. 2, H-K), and
the frequency of transitions at CpG sites (9%) is lower than in almost
all other cancers. This spectrum is consistent with data for several
different types of chemical carcinogens found in tobacco smoke. The
highly mutagenic metabolites of PAHs such as BP preferentially
of attack deoxyguanines and lead to mutations (Table 1), and one of the
quantitatively minor adducts from the tobacco-specific N-nitrosamine
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone also leads to
G:C—@
T:A transversions (233).
Analysis of p53 mutations in the database in relation to smoking
history yields statistically significant conclusions consistent with the
experimental data. The frequency of p53 mutation and of G:C—@ T:A
transversion on the nontranscribed DNA strand are positively cone
lated with lifetime cigarette consumption (234). Compared with non
smokers, smokers have significantly lower frequencies of all transi
tions (35 versus 69%; P < 0.05), G:C—*A:T transitions (21 versus
69%; P < 0.001), and higher rates of G:C—*T:A transversions with a
DNA nontranscribed strand bias (29 versus 0%; P < 0.01; Fig. 2, L-M;
Ref. 235). These observations are consistent with the model of pref
erential repair of carcinogen-induced damage on the transcribed DNA
strand (75, 236). Cigarette smoke and some of its components have
been shown in vitro (229, 237) to increase mutation frequency and
cause defects in DNA repair, which may contribute to the p53 muta
tion spectrum. These results also have implications in the current
controversies concerning the relative risk of lung cancer in nonsmok
ers exposed to environmental (passive) tobacco smoke. Molecular
analysis would contribute important data to the debate by determining
whether critical mutations in lung cancers from these individuals
show a spectrum similar to smokers (a dose-response increase in
G:C— T:A transversions with exposure to environmental tobacco
smoke), although a large sample set may be required to detect changes
in the spectrum.
Evidence exists of potential differences in pathways of carcinogenesis
among histological types of lung cancer. In vitro studies of lung cancer
differentiation support a pluripotent cell of origin common to all types, or
multiple cells which can differentiate by variable, often overlapping
pathways (238—240).Sufficient data exist to analyze the mutational
spectra of squamous cell, large cell, adenocarcinoma, and SCLC (Fig. 2,
H-K). p53 mutations are found in 70% of SCLC and 47% of NSCLC,
including 65% of squamous and 60% of large cell, but only 33% of
adenocarcinomas (140, 234, 241—246),a prevalence which more closely
approximates that of other adenocarcinomas than of other lung cancer
histologies. The mutational spectra are notable for an excess of
A:T—@
G:C mutations in SCLC (11 versus 6%) and an excess of deletions
and insertions in NSCLC (14 versus 4%). Subdividing NSCLC types
reveals that the prevalence of G:C—@ T:A transversions in large cell,
squamous, and small cell carcinomas is similar (43—49%),but they are
less common in adenocarcinoma. This finding is consistent with the
weaker association of adenocarcinoma with cigarette smoking (247—
249), since G:C—÷T:A transversions are thought to result from bulky
molecules found in tobacco smoke. In adenocarcinomas, higher propor
tions of G:C—@A:T (non-CpG site) and G:C—@ C:G mutations are seen,
suggesting the presence of other carcinogens. Since adenocarcinomas are
the most common cell type in women and squamous cell is less frequent,
analysis of mutational spectrum by gender (controlling for cell type and
smoking history) may provide clues to different carcinogenesis pathways,
hormonal influences, or differing susceptibilities to tobacco-associated
carcinogens.
2) Other Tobacco-associated Cancers. Epidemiological studies
have linked cigarette smoking to other cancers with attributable risks
ranging from 20 to 90% (250). Mutational spectrum analysis of some
of these tumors should reflect the effects of relevant tobacco-associ

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 p53 MUTATIONS AND CANCER

ated carcinogens. Despite the similarity of risk factors, the mutational
spectra of these tumors differ from each other and differ significantly
from the lung cancer spectrum. This may be due to different constit
uents of tobacco or nontobacco-related carcinogens.
Head and Neck and Esophageal Carcinomas: The combination of
tobacco use and drinking alcoholic beverages is responsible for ap
proximately 75% of esophageal and 90% of head and neck cancers,
most of them squamous cell (250). Two lines of evidence derived
from the database implicate environmental carcinogens
neck carcinogenesis: (a) over one-half of all mutations in head and
neck cancers are substitutions at base pairs which usually demonstrate
nontranscribed strand bias (Table 3; section II.B.1), and 80% of these
occur on the nontranscribed strand; and (b) CpG transitions (sugges
tive of endogenous events; section II.D.5.F) are uncommon (13%).
Compared with lung cancers, G:C—*T:A transversions are less com
mon (18 versus 40%), while A:T—*G:C (14%) and non-CpG
G:C—* A:T (18%) transitions are prominent, as are deletions and
insertions (19%). Of 18 deletions and insertions, 12 can be localized
to repetitive DNA sequences, consistent with the hypothesis that
mispairing during replication causes frameshifts (section II.B.2), and
two are large deletions at splice site codons, suggesting that the actual
DNA alteration is an intronic point mutation. Chemical carcinogens
such as PAHs in tobacco smoke may contribute to these frameshift
mutations (section II.B.2).
Subdivision of head and neck carcinomas by anatomic region of
origin shows differing patterns of p53 mutations among
primary mucosal sites (Fig. 2, E-G). In laryngeal
primaries, the mutation prevalence is relatively low (34%), but the
spectrum resembles that of lung cancer. G:C—@' T:A transversions,
primarily on the nontranscribed strand, are the most common mutation
(34%), and non-CpG G:C—@ A:T transitions also are common. Pri
mary tumors in the oral cavity have a high mutation prevalence
and exhibit a spectrum in which non-CpG site G:C—*A:T transitions,
A:T—*G:C transitions, deletions, and insertions predominate. Thus, in
this class of cancers, subgroups which are often grouped together
when addressing carcinogenesis and therapy demonstrate mutational
differences, which could be due to differences in carcinogen
(e.g.,solubletobacco-specific
N-nitrosamines
in oralcancers
versus smoking
tobacco combustion products in laryngeal primaries) or variation in
carcinogen activation or DNA repair between distinct
These hypotheses can be tested in the laboratory and by molecular
epidemiological studies of mutational spectra of well-characterized
tumor types in populations with well-defined exposures.
The epidemiology of primary NPC differs from other head and
neck sites; these tumors are associated with dietary N-nitrosamines,
EBV, and perhaps genetic factors (183). Clinical NPCS rarely contain
mutated p53 (4%). One hypothesis for this low frequency of mutation
could be the epigenetic inactivation of p53 by EBV proteins (251).
The spectrum of the few p53 mutations in NPC tumors and lines is
unusual, reflecting a lack of mutations known to be associated with
tobacco-related carcinogens or N-nitrosamines (G:C—*T:A, non-CpG
G:C—*A:T, and A:T—@ G:C substitutions). G:C—*
C:G transversions,
which comprise only 3% of p53 mutations at other head and neck sites
and 7% in all cancers, are common (5 of 17; 29% of mutations;
@ P 0.00001 versus other head and neck sites), and four of five are on
the nontranscribed strand, suggesting the action of a chemical carcin
ogen. Further molecular epidemiological studies are indicated to gen
erate a statistically meaningful database.
The spectrum in esophageal squamous cell carcinomas also shows
less frequent G:C—*T:A transversions than in lung carcinomas
versus 40%), with corresponding increases in G:C—@' A:T (non-CpG
site 22 versus 16%) and A:T—@G:C (14 versus 7%) transitions, as well
as A:T—*T:A transversions (15 versus 5%). As discussed (section I.B;
Table 1), PAHs, N-nitrosamines, and alkylating agents are among the
compounds which cause these substitutions in experimental systems.
Risk factors for squamous cell carcinoma in addition to tobacco smoking
are alcoholic beverage consumption, and in China, dietary N-nitrosamine
ingestion (which could account for non-CpG site G:C—* A:T transitions;
Ref. 252). Future studies could address the carcinogenicity and muta
tional spectra of these suspected carcinogens in cultured human esopha
geal and upper aerodigestive cells or in animal models.
in head and As in the lung, adenocarcinoma of the esophagus is increasing in
frequency relative to squamous cell; the last decade has seen a 3-fold
increase in the incidence of adenocarcinoma and a 23% decrease in
squamous cell carcinoma (253, 254). Adenocarcinoma of the esoph
agus resembles carcinoma of the gastric cardia and usually arises
within the intestinal metaplasia of Barrett's esophagus. Chronic gas
tric reflux is considered a risk factor for this condition, but risk factors
for neoplastic transformation are unknown. Few studies of the muta
tions in esophageal adenocarcinomas or Barrett's esophagus have
been performed, but they suggest differences in mutagens from squa
mous carcinoma. Of the mutations reported, 6 of 9 (67%) have been
G:C—* A:T transitions at CpG sites, compared with a 14% incidence
of CpG site mutations in squamous cell esophageal cancer. Evaluation
of more adenocarcinomas will be important to generate statistically
valid comparisons with squamous carcinomas of the esophagus and
with gastric cardia cancers.
Bladder Carcinoma: One-half of bladder carcinomas in men and
the various one-third in women are attributable to smoking (255). A significant
and pharyngeal association has been found in superficial bladder cancers between p53
nuclear staining by IHC and the number of cigarettes smoked (256).
The p53 mutational spectrum in bladder cancer is unusual with a high
frequency of G:C—@ C:G transversions (21 versus 7% for all tumors)
with a nontranscribed DNA strand bias. G:C—@ A:T transitions at both
(81%) CpG and non-CpG sites are the most common mutation, with non
transcribed DNA strand bias at non-CpG sites. One study has found a
hotspot G:C—@C:G (Tyr>Lys) mutation at codon 280, but this has not
been confirmed. Two reports have compared the spectra in a total of 34
smokers and 23 nonsmokers (163, 257) with similar spectra found in the
exposure two groups. Further epidemiological studies, including quantitative
histories, are warranted to evaluate the data for more subtle
patterns.
mucosal sites. D) Radiation. 222Radon is a colorless, odorless, inert gas, a natu
rally occurring decay product of @8uraniumwhose decay emits a-par
tide radiation. Conclusive epidemiological evidence implicates occu
pational exposure to radon in uranium miners as a cause of lung
cancer (reviewed in Refs. 258 and 259). Early epidemiological studies
of domestic radon and lung cancer risk exposure suggest that the
dose-response and relative risk for residential radon exposure is the
same as for occupational exposure (260—262). Radon exposure and
cigarette smoking increase risk synergistically, and analysis of human
tumors may reflect interactions between radon and tobacco carcino
gens (258, 259, 262).
Two studies report the molecular analysis of the p.53 gene in lung
cancers from uranium miners (140, 263). Missense p53 mutations
were found frequently in both studies. A mutational hotspot (16 of 30
mutations) at codon 249, AGG—@A@ Arg—+Met, was observed in
miners from the Colorado Plateau who were exposed to high amounts
of radon. Three of the codon 249met mutations occurred in lung
cancers from never smokers, implicating a non-tobacco-associated
carcinogen. Since these miners also were heavily exposed to other
carcinogens found in the mines, the hypothesis that radon was respon
(16 sible for the specific base substitution requires further testing.
Other sources of ionizing radiation which have been associated
with the development of hematological and solid malignancies in
dude therapeutic radiation, atomic bomb explosions, and the radio
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 p53 MUTATIONS AND CANCER
logical contrast agent Thorotrast. Mutational spectrum analysis of
cancersin these patientscouldindicateradiation-inducedevents.p53
mutations in lung cancers from nine nonsmoking atomic bomb sur
vivors from Hiroshima, Japan, were compared to eight nonexposed
controls; no differences in prevalence or mutational spectrum were
noted in this small study (235). The study of larger numbers and a
greater variety of radiation-related cancers would contribute more
statistically meaningful data to the issue of radiation-induced mu
tagenesis in human cancer.
E) Cervical Carcinoma and Human Papifioma Virus. Epidemi
ological studies established the link between cervical carcinoma and
HPV types 16 and 18, and the molecularbasis for this association
emerging from laboratory research features the p53 pathway. HPV
types 16 and 18 both produce E6 protein, which complexes
cellular protein AP6 and degrades p53 in the cytoplasm (264, 265), an
example of epigenetic p53 inactivation. Mutation data would support
this hypothesis if p53 mutations were found in HPV-negative more
frequently than HPV-positive cancers, and IHC would be expected to
demonstrate no p53 staining in HPV-positive cases and nuclear stain
ing in HPV-negative cases with missense mutations. Early reports
found that mutation frequency was 100% in HPV-negative and 0% in
HPV-positive tumors and cell lines (179, 266, 267), but this dramatic
difference has not been validated in further studies of clinical tumors
(159, 178, 268—271). These later studies confirmed the rarity of
mutations in HPV-positive tumors (4%) and lines (7%); mutations
have been found in 7 of 39 of HPV-negative carcinomas now reported
(18%; P 0.0007 versus HPV-positive tumors).
These data indicate that dysfunction of the p53 pathway contributes
to cervical carcinogenesis, but unless mutations or other alterations in
the p53 pathway are demonstrated in all HPV-negative tumors, it
cannot be considered essential. Future studies might address the low
rate of mutations in HPV-negative tumors by searching for other p53
pathway abnormalities, such as occult mutations, downstream gene
abnormalities, or other epigenetic mechanisms of inactivation.
F) Endogenous Sources of Mutation. As discussed in section I.A,
not all mutations are caused by exogenous carcinogens. Normal
cellular events which are mutagenic include deamination of 5-meth
ylcytosine at CpG dinucleotides, DNA polymerase infidelity due to
mispairing at repeat sequences or other mechanisms, and oxygen
radical damage (reviewed in section l.A. and Refs. 14 and 272).
Studies of constitutive cellular genes to determine baseline mutation
rates in human and other mammalian cells have found rates of
between 10 ‘° and i0@ mutations/gene/cell generation (10, 85).
However, these may underestimate the true mutation rate because of
insensitivity of the assays to some classes of mutations (e.g.,
hemoglobin variants with point mutations were detectable) or failure
of an assay's cell culture conditions to select clones with mutations
but no growth advantage (10, 85). This background level of mutations
implies that populations will always exhibit a baseline rate of cancer,
even in the absence of carcinogen exposure. The rates of endogenous
mutations may also be affected by exogenous events; future studies on
the effects of carcinogens and cocarcinogens on deamination and
deletion are warranted. Mutational spectrum analysis may be able to
define these baseline mutational frequencies by identifying the pro
portions of mutations in cancer-related genes which are caused by
endogenous processes.
1) CpG Deamination. It cannot be proven that an individual
G:C—@ A:T transition found at a CpG dinucleotide in a tumor results
from deamination of 5-methylcytosine (as opposed to mispairing of an
O°-methylguanine adduct with thymine, for example). However, the
in vitro rates of spontaneous deamination, the clustering of spontane
ous G:C—+ A:T mutations in experimental systems at sites where
cytosine is methylated, and the large database of observed mutations
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Copyright © 1994 American Association for Cancer Research
suggests that this is the usual mechanism by which G:C—'A:T tran
sitions arise at CpG sites in human tumors. Examination of the p53
database for associated features such as amino acid selection and
strand bias of CpG transitions may yield further insights into this
process in tumor cells.
The p53 coding region contains 39 CpG dinucleotides (i.e., 78 base
pairs) which are potential sites for G:C—@A:T transition from deamina
tion. Transitions at these sites account for 24% of all p53 mutations
described, with large variations among tumors. Seven sites (codons 175,
196, 213, 245, 248, 273, and 282) account for 90% of CpG transitions, or
21% of all p53 mutations. These mutations occur equally on both DNA
strands; strand bias in recognition and repair of G:T mismatches is thus
unlikely to contribute to the selection of mutants.
with Some mechanisms by which cells with some CpG transitions ac
quire a selective growth advantage have been studied. At codons 248
and 273, both C:G—@ T:A and G:C—*A:T transitions commonly occur,
implying that both amino acid changes (Arg>Trp and >Gln at 248,
Arg>Cys and >His at 273) result in a selection advantage. At codons
175 and 282, only one of the two mutations is selected (175, Arg>His
but not >Cys; 282, Arg>Trp but not >Gln). Soussi and colleagues
have constructed all potential transition mutants at CpG hotspot
codons 175, 248, and 273 and assessed conformation and in vitro
transcriptional activation in the sarcoma cell line SAOS, which con
tains no endogenous p53 protein.5 Transactivation, which is thought
to contribute to p53 tumor suppressor function, was assessed by
transcriptional assays using the reporter gene CAT linked to an up
stream p53 binding region. Wild-type and mutant p53 constructs
cotransfected into target cells with the reporter constructs were com
pared in their transcriptional activating abilities. The favored Arg>His
mutant at codon 175 assumes the mutant conformation and transac
tivation properties, whereas the rare Arg>Cys mutant retains wild
type conformation and activity. Interestingly, both mutants at codons
248 (Arg>Trp and >Gln) and 273 (Arg>Cys and >His) maintain
apparent wild-type conformation by antibody analysis but acquire
mutant transactivation function. These two amino acids are in direct
contact with DNA (97). This evidence supports the studies (section
II.C.1) which connect p53 transactivation activity with growth sup
pression, but they imply that conformational changes as detected by
these methods are not necessary for modulation of this function (at
least in SAOS sarcoma cells). Future studies could address other
features of CpG site mutants which could eliminate p53 suppressor
activity. It is possible that the more infrequent mutants are function
ally silent, and their presence in cancer cells does not contribute to the
only cancer phenotype.
Thus, observational and experimental analysis supports the hypoth
esis that selection advantage due to altered transactivation and perhaps
other functions is responsible for the hotspot mutations at CpG
dinucleotides. Evidence argues against strand bias in mismatch repair
of CpG transitions.
2) Cancers with High Rates of CpG Mutations, Deletions, and
Insertions. The reasons discussed above argue strongly that deami
nation of 5-methylcytosine, an endogenous mechanism of mutation, is
primarily responsible for G:C—@ A:T transitions at CpG dinucleotides
(273). Similarly, deletions and insertions may result from misalign
ment due to polymerase infidelity during replication, another endog
enous mechanism. Transitions and frameshifts also may be induced by
carcinogen-DNA adducts and other mechanisms (section II.B.1.A).
We hypothesize, however, that the sum of CpG transitions, deletions,
and insertions, and potentially of other mutations which may eventu
ally be linked specifically to endogenous processes, may be used as a
5 K. Ory and T. Soussi, personal communication.
4871
 p53 MUTATIONSAND CANCER
Fig. 3. The mutational spectrum in hepatocellular Legend:
carcinomas varies by geographic origin in Asia (Qi
dong, n = 34; other mainland China, n = 11; Tai
wan, n = 26; and Japan, n = 123).
crude indicator of the proportion of mutations which are an inevitable
consequence of biological processes.
Transitions at CpG sites, deletions, and insertions comprise 37% of all
reported p53 mutations but 45% of mutations of brain, colon (Fig. 2N),
stomach, endometrial, thyroid, and hematological cancers. This suggests
that endogenous cellular processes may contribute to a significant frac
tion of these tumors. The mutational spectrum of LFS implicates 5-meth
ylcytosine deamination as the primary event in the formation of p53
germline mutations; 53% of germline mutations are G:C—+ A:T transi
tions at CpG sites (Fig. 20). The second most frequent class of mutations
(13%) are deletions and insertions, mostly of a single base pair at repeat
sequences, a pattern which is consistent with the slipped mispairing
mechanism. Thus, two-thirds of LFS cases can be attributed to endoge
nous-type mutational events, compared with slightly more than one-third
of p53 somatic mutations found in human cancers. Mutations known or
suspected to be associated with carcinogen exposure (A:T—t'G:C and
non-CpG site G:C—* A:T transitions, G:C—@
T:A transversions, and tan
dem mutations) represent only 23% of LFS mutations versus 46% of
somatic mutations (Fig. 2, A and 0).
Comparison to germline mutations of other genes associated with
cancer or other diseases supports the concept that these putative
mechanisms of endogenous mutations are important sources of germ
line mutation, but mutation patterns are not consistent and probably
reflect specific functional characteristics of the gene and protein. For
example, germline mutations in the APC gene cause familial adenom
atous polyposis, a syndrome in which multiple colon polyps, adeno
mas, and eventually carcinomas develop in all affected
Germline and somatic mutations in APC result in truncation or com
plete loss of the protein in 96% of cases and in an altered protein in
only 4% (274). Thus, frameshift mutations are prominent (48% of
mutations), and CpG site G:C—+ A:T transitions, although the most
frequent base substitution, comprise only 14% of all mutations. Sim
ilar to p53 germline mutations, however, three-quarters ofAPC germ
line mutations are consistent with the endogenous processes of deami
nation and slipped mispairing. The spectrum of mutations in the factor
IX gene, which causes hemophilia B, contains similarities and differ
ences from the spectra in the tumor suppressor genes (273, 275).
Approximately two-thirds are missense substitutions, less than 10%
are deletions and insertions, and the remaining one-quarter are splice
site alterations and promoter mutations. Transitions at CpG sites are
common (approximately 36%), but other base substitutions are more
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Copyright © 1994 American Association for Cancer Research
@,J-
JAPAN
prevalent in factor IX mutations than in p53 germline mutations.
Expansion of the data sets on germline mutations in other genes such
as HPRT, APC, DCC, hMLHJ, and hMSH2 may suggest patterns
which are now inapparent.
3) Mutational Spectrum Differences among Populations. Al
though the focus of mutational spectrum analysis in HCC has been on
the role of codon 249@ mutations and AFB exposure, deeper scrutiny
of the data produces additional hypotheses. Since the spectrum in
HCC is particularly dependent on a population's environmental cx
posure, subclassification of the data by geographic region may reveal
patterns hidden by the AFB-related tumors. Fig. 3 depicts the muta
tional spectra of tumors from Qidong, other regions of mainland
China, Taiwan, and Japan. While tumors from Qidong contain an
overwhelming preponderance of G:C—@T:A transversions (95%) due
to association with AFB, Japanese and Taiwanese tumors harbor this
type in only 26% of cases each. That both of these latter groups
exhibit some codon 249@1mutations suggests that AFB may be a risk
factor even in populations not normally considered to be at high risk
for dietary contamination. The remainder of the spectrum differs
between the groups, however, and suggests other potential carcino
gens. In Taiwanese patients, the most frequent base change for all
HCCs is A:T—@ T:A transversion (41%); the strand bias (all 11 mu
tations are A—@ T) suggests that an exogenous carcinogen is respon
sible. For Japanese patients, G:C—*A:T (at CpG and non-CpG sites)
and A:T—'G:C transitions (also with nontranscribed strand bias) are
both common, suggesting environmental carcinogens; deletions and
persons. insertions, linked to both endogenous and exogenous mutational
mechanisms, also are frequent. The identification of relevant carcin
ogens with these spectra will be important. Only nine mutations have
been reported in HCC from Western countries; five are G:C—+ A:T
transitions, three of which are at CpG sites. Thus, no pattern suggesting
carcinogen exposure is yet discernible.
Environmental exposures or inherited characteristics which vary
among populations may be important determinants of mutational
spectra in other tumors as well. Sufficient data are available to
evaluate lung, gastric, and breast cancers for differences in spectrum
between Japanese and Western patients. Compared to Western pa
tients, Japanese lung tumors show a higher frequency of G:C—@ A:T
transitions (29 versus 20%, P 0.13; most are at non-CpG sites) and
a lower frequency of G:C—+ T:A transversions (30 versus 43%,
P = 0.01), both with a nontranscribed strand bias. These differences
4872
 p53 MUTATIONS AND CANCER
are not found in SCLC but are seen in both adenocarcinomas
(G:C—s A:T transitions: 48 versus 20%, P = 0.02; G:C—+T:A trans
versions: 14 versus 27%, P = 0.21) and non-adeno NSCLC (24 versus
20%, P = 0.004; 29 versus 55%, P 0.0003, respectively). Deletions
and insertions are more frequent in Japanese tumors (18 versus 10%,
P = 0.22). These results suggest differences between Western
Japanese individuals, either in exogenous carcinogens or in cancer
susceptibility factors such as germline polymorphisms in genes
involved in carcinogen metabolism (49).
Most of the gastric tumors analyzed have been from Japan, where
incidence rates are much higher than in Western countries. Epidemi
ological studies in Japan have found an association between gastric
cancer and dietary salt and N-nitrosamines (276). Comparison of the
mutational spectra reveals that endogenous-type mutations are seen in
72% of Western and 55% of Japanese tumors. More Western tumors
must be analyzed to provide a more statistically valid spectrum. The
major type of p53 mutation seen in Japanese but not Western tumors
is A:T—@ G:C transition (20 of 69 versus 1 of 18, P = 0.03), with
strand bias favoring T—@ C on the nontranscribed strand, the opposite
of the usual A:T—*G:C strand bias. Perhaps this represents the con
sequences of alkylthymine adducts from N-nitrosamines (277—279)in
the Japanese diet.
No specific exogenous carcinogens have been conclusively linked
with development of human breast carcinoma. Although only 25% of
breast cancers contain p53 mutations, 15% of these are G:C—@' T:A
transversions, 16% are G:C—'A:T transitions at non-CpG sites, and
11% are A:T—@' G:C transitions, with nontranscribed strand bias of 89,
63, and 75%, respectively, suggesting that exogenous bulky carcino
gens may play a role. The responsible carcinogens may vary among
populations. Two reports of breast cancers from Japanese women
(135, 280) indicate a mutational spectrum different from tumors in
Western women. G:C—*T:A transversions are uncommon in Japanese
cases (1 of 48 versus 25 of 175, P = 0.02), while A:T—@ G:C
transitions are more common (8 of 48 versus 10 of 175, P = 0.01).
Thus, A:T—*G:C transitions with nontranscribed strand bias are
prominent in the spectra of three different types of tumors in Japanese
patients (HCC, gastric, and breast). Whether this represents the effect
of a regional environmental agent(s) or population differences in
carcinogen metabolism or DNA repair is an area for future investigation.
Transitions at CpG sites, deletions, and insertions are common in both
populations, suggesting a similar contribution of endogenous mutations.
These analyses suggest that molecular studies with careful identi
fication of geographic and ethnic features of populations can make a
significant contribution to carcinogenesis research. Future research
should evaluate statistically valid sample sizes in order to address
specific hypotheses of differences in carcinogenesis among popula
tions. For example, studies of Japanese populations in different geo
graphic regions (Japan, Hawaii, and mainland United States) have
demonstrated differences in cancer rates. Molecular studies on these
cancers should be done to search for changes in mutational spectra,
which would suggest that environmental carcinogens differ among
geographic regions for several tumor types.
4)p53 Mutation and Prognosis. Since tumors with p53 mutations
often behave more aggressively, the value of p53 as a prognostic factor
has been addressed. Studies have implicated p53 protein expression as an
independent prognostic factor in carcinomas of the breast, stomach,
colon/rectum, bladder, and NSCLC (reviewed in Ref. 129).
Future studies might integrate hypotheses of carcinogenesis into
studies evaluating clinical outcomes. Since p53 mutation may corre
late with other indicators of poor prognosis, such as aneuploidy, gene
amplification, and high proliferative fraction (section II.D.3), it will
be important to adhere to careful epidemiological methodologies to
assure that p53 status adds information to established prognostic
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Copyright © 1994 American Association for Cancer Research
markers. This can be accomplished by performing prospective studies
in conjunction with large clinical trials to ensure statistical power,
facilitate multivariate analyses to control for other established prog
nostic variables, and assess the responses to therapies in relation to
p53 status. The ability to perform IHC and PCR-based diagnostic
and studies from archived tissue means that some of these studies could be
accomplished on materials from completed clinical trials which may
already have reached their end points, expediting the answers to some
questions. Comparison of multiple molecular markers in the same
tumors can help to determine the relative importance to carcinogen
esis of abnormalities in various cellular pathways.
Such studies can address both specific and general hypotheses regard
ing carcinogenesis, for example: that tumors with p53 mutations might be
more resistant to therapy-induced apoptosis; that tumors with mutations
are more likely to be associated with amplification ofother cancer-related
genes which influence therapy; whether all cancers have abnormalities of
the p53 pathway, or of a specific combination of pathways; the relation
ship of response to therapies to molecularly determined tumor pheno
types and genotypes; identification of subsets of patients with favorable
and unfavorable prognoses that should be analyzed separately and cx
cluded from standard treatment protocols; and whether innovative ther
apies (e.g., antisense oligonucleotides and gene therapy) can be tailored
to specific molecular features of a tumor to improve the probability of
successful outcome.
ifi. Conclusions
p53 mutational spectrum analysis contributes to a variety of mech
anistic questions in carcinogenesis, including integration of molecular
and cellular hypotheses. Examples which are of ongoing interest
include: what is the timing and sequence of the genetic changes
contributing to neoplastic transformation, progression, and metasta
sis? Does cancer always arise from a single transformed clone, or can
multiple transforming events occur in some tissues? How do carcin
ogen-host interactions, such as individual differences in carcinogen
metabolism and DNA repair, influence an individual's risk of devel
oping cancer? Do different histological types of cancer contain char
acteristic mutations reflecting a pattern of mutagen exposure, and do
cancers of similar histological types in different organs share these
patterns? Are abnormalities of the p53 pathway critical for all tumors?
In cancers with low rates of p53 mutation, is p53 inactivated by
epigenetic events, or is its function interrupted by changes in other
genes which participate in p53-mediated pathways?
The emerging field of molecular epidemiology promises important
contributions to cancer research, but full realization of its potential
will require careful adherence to the tenets of both laboratory science
and epidemiology. This will require increased interaction between
laboratory researchers, clinicians, and epidemiologists in order to
design studies which can adequately address important hypotheses.
For example, published reports of the molecular features of cancers
(and other diseases) should include important epidemiological data
such as gender, age, ethnicity, geographic origin, and, when appro
priate, associated medical conditions, tobacco smoking, and occupa
tional or other exposures. Large scale clinical trials which include data
on the relevant variables can provide tumor tissue for molecular study.
This may be accomplished using archived materials from past studies;
future trials could collect and preserve tissue specifically for molec
ular analyses. Attention to specimen handling at all times would
minimize tissue degradation and maximize the reliability of laboratory
findings.
Mutational spectrum analysis and molecular epidemiology can be
applied in innovative ways to both generate and address basic and
practical hypotheses including the areas of chemical, physical, and bio
4873
 p53 MUTATIONS
logical carcinogens, risk assessment, host factors, control of cell and
tissue behaviors, mechanisms of subcellular communication, protein
function, DNA replication, transcription, translation, and evolution.
Acknowledgments
We appreciate discussion and comments from our colleagues, including
Ettore Appella, Mark Boguski, Kathleen Forrester, Stephen Friend, S. Perwez
Hussain, David Lane, W. Edward Mercer, Ruggero Montesano, Moshe
Oren, Jennifer Pietenpol, Varda Rotter, Thierry Soussi, Glen Trivers, Bert
Vogelstein, Xin Wang, and Stuart Yuspa. The expert graphic and editorial
assistance of Dorothea Dudek and the assistance of Peter Shields with
statistical analyses are also appreciated.
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