[CANCER RESEARCH 54, 4750-4755,
September 1. 1994]
p53 Mutations and Microsatellite Instability in Sporadic Gastric Cancer:
When Guardians Fail1
John G. Strickler, Jian Zheng, Qiuping Shu, Lawrence J. Burgart, Steven R. Alberts, and Darryl Shibata2
L J. B.I
and Oncolog\ ¡S.R. A.j, Mayo Clinic, Rochester, Minnesota 55905, ami Department
Departments of Anatomical Pathology [J. G. S., L.
BJ aiu
Southern California School of Medicine, Los Angeles. Californiaia 90033 [J. Z. Q. S., D. SJ
ABSTRACT
Genetic instability may underlie the etiology of multistep gastric carcinogenesis. The altered microsatellites observed in tumors with the ubiq
uitous somatic mutation i ISM i phenotype may represent the expression
of such instability. Similarly, p53 mutations may allow the accumulation
of genetic alterations caused by multiple mechanisms. In 40 sporadic
gastric adenocarcinomas,
nine tumors (22.5%) with p53 mutations in
exons 5-8, and six tumors (15%) with the USM+ phenotype, were de
tected. None of the tumors had both alterations. The tumors with p53
mutations were predominantly in the proximal stomach whereas the
I SM + tumors were predominantly in the distal stomach. The mutantp53
alÃ-eleswere homogeneously distributed throughout the primary tumors,
but usually absent from adjacent normal or dysplastic epithelium, indi
cating that p53 mutations are typically acquired before the bulk of clonal
expansion. The loss of mutant p53 alÃ-elesduring progression was also
rarely observed in metastatic foci. Altered microsatellites were homoge
neously present in the I SM+ primary and metastatic tumors and one
synchronous tubular adenoma, but were not detected in adjacent normal
and metaplastic epithelium. These findings also demonstrate that the
USIVI+ phenotype is expressed before the bulk of clonal expansion. In
most (5 of 6) l SM+ tumors, the sizes of the altered microsatellites
differed between regions, indicating that the instability usually persists
during clonal expansion. These Findings indicate that both /'.•>.!
mutations
and the I'SM + phenotype are present prior to the bulk of tumor growth
and therefore may contribute
malignant transformation.
to, rather than be a late consequence
of,
arrest may allow the repair of DNA damage, or trigger apoptosis (38).
Losses of wild type p53 alÃ-elesare also associated with gene ampli
fication (39).
Instability would be expected to be most effective at promoting
malignant transformation if it were acquired or expressed during early
tumor progression (17). Indeed, germ-line mutations of p53 and
hMSH2 are present in hereditary cancer families (40) and HNPCC
(24, 25). In order to determine possible interactions and when these
alterations are acquired or expressed, sporadic gastric cancers were
screened for the RER+ phenotype and p53 mutations, and their
topographical distributions were analyzed.
INTRODUCTION
Specimens.
phenotype (16, 17). The specific types and timing of multiple mutations in
gastric cancers are poorly characterized as the frequencies of these alterations
are typically less than 50% (18).
The presence of a "mutator" phenotype is thought to be critical for
multistep carcinogenesis since the normal mutation rate may be in
sufficient to account for the accumulation of multiple mutations (19).
Mutations in the p53 tumor suppressor gene and the loci responsible
for the USM+ phenotype may contribute to some aspects of increased
genetic instability. The USM+ phenotype is manifest by widespread
alterations in the sizes of simple repeat sequences secondary to defects
Received 3/30/94; accepted 6/28/94.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
' This work was supported in part by Grant CA-58704 from the NIH.
2 To whom requests for reprints should be addressed, at 12(K) N. State St., No. 736,
Los Angeles, CA 90033.
1The abbreviations used are: MS, microsatellitcs; USM, ubiquitous somatic mutations;
HNPCC, hereditary nonpolyposis coloréela!cancer; GE, gastrocsophageal;
heterozygosity; PCR, polymerase chain reaction.
LOH, loss of
University of
in DNA repair after replication (20-27). USM+ cell lines demon
strate markedly increased mutation rates in MS (26, 27). Although the
majority of MS alterations are likely to be neutral or selection inde
pendent, the instability may increase the probability that critical
oncogenic mutations occur. Recent studies have demonstrated that the
USM+ phenotype is present in 18 to 38% of gastric adenocarcinomas,
especially in poorly differentiated tumors (16, 17, 28).
p53 is a transcription factor with many diverse roles (29, 30). One
apparent role is to ensure genomic integrity by preventing the expan
sion of cells with genetic alterations caused by diverse mechanisms
(31-34). p53 promotes the expression of WAFI/Cipl which inhibits
the cell cycle by binding to cyclin-dependent kinases (35-37). This G,
MATERIALS
Gastric cancer is one of the most common tumors in the world. Its
earliest oncogenic alterations may occur decades before transforma
tion (1). The histological progression associated with the intestinal
type of gastric cancer have been well documented, with apparent
evolution through a sequence of superficial gastritis, chronic atrophie
gastritis, intestinal metaplasia, and dysplasia (2, 3). Similarly, the genetic
evolution of cancer involves the accumulation of multiple mutations (4).
In gastric cancer, altered loci include p53 (5-10), APC (11, 12), c-K-ra.v
(13-15), and multiple MS3 in cancers with a replication error (RER) or USM
of Pathology,
AND METHODS
DNA was extracted from 40 formalin-fixed, paraffin-embed
ded sporadic gastric cancers from the Mayo Clinic (41). Regions of at least
50% tumor cells and normal cells were isolated with microdissection. All
tumors were considered primary gastric adenocarcinomas, although the exact
origins were uncertain for the 15 proximal tumors arising in the cardia or the
gastroesophageal (GE) junction. Only 4 of the 15 proximal tumors had evi
dence of Barrett's epithelium. The remaining 25 distal tumors were present in
the body or antrum of the stomach. The adenocarcinomas were classified as
either of the intestinal or diffuse types (42). Clinical data were collected from
review of medical records.
Detection of p53 Mutations. The specimens were screened forp5.i muta
tions in exons 5-8 by using PCR and single-strand conformation polymor
phism (43). Bands of abnormal mobility were reamplified, cloned (TA Cloning
Kit, Invitrogcn), and sequenced. The mutations were verified by hybridization
of a mutation-specific 17-mer probe back to the DNA amplified from the
primary specimen.
Detection of USM+ Tumors. Pairs of tumor and adjacent normal tissue
were amplified at three MS loci consisting of CA repeat sequences [Mfd27
(44), Mfd41 (45), Mfd47 (46)], and their sizes were compared (27). Tumors
with MS sizes different from their normal tissues in at least one-half of the
tested loci were designated as USM+ and were analyzed as below. For one
case, the Mfd57 locus (47) was also analyzed.
Topographical Distributions of Altered MS and Mutant p53 AlÃ-eles.
The topographical distributions of the mutant p53 alÃ-elesor altered MS were
determined with selective UV radiation fractionation (41, 48). Multiple blocks
of normal, tumor, and métastaseswere analyzed. From each stained micro
scopic section, 8—20different cell groups (100-300 cells) consisting of histologically defined phenotypes were isolated and amplified for 40-50 PCR
cycles. Normal tissues were not contaminated with tumor and tumor pheno
types were at least 70% pure. The r>53 PCR products were analyzed with
mutant and wild type specific oligomer probes (43). The MS from different
topographical regions were analyzed as previously described (27).
4750
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P.Õ3AND THE USM+
PHENOTYPE
RESULTS
IN GASTRIC
CANCER
Table 2 p53 mutation spectrum
The 40 sporadic gastric cancers were screened for the USM+
phenotype and p53 mutations in exons 5-8. The USM+ phenotype
was detected in six (15%) tumors and p53 mutations in nine (22.5%)
tumors (Table 1). p53 mutations were also detected in two other
tumors, but could not be confirmed by hybridization of corresponding
mutation-specific oligomer probes back to PCR products from the
Case123456789Codon282276136135282261273175179MutationCGG
TipGCC
to TOO Arg to
GlyCAA
to GGC Ala to
StopTOC
to TAA Gin to
TipCGG
to TGG Cys to
TipTCCA'GG
to TGG Arg to
TCCA(TCCA)GGCGT
to
HisCGC
to CAT Arg to
HisCAT
to CAC Arg to
to AAT His to AsnTypemC
" P, proximal stomach; D, distal stomach.
primary specimens or multiple tumor regions obtained by selective
UV radiation fractionation. None of the tumors had both a USM+
phenotype and p53 mutation, or more than one p53 mutation. All of
the USM+ and p53 mutant tumors were of the intestinal type, while
none of three diffuse tumors showed the USM+ phenotype or p53
mutations.
Most p53 mutations (67%) were present in the proximal stomach.
All six proximal /;5J-positive cases were located at the GE junction
and did not arise from Barrett's metaplasia, as determined by routine
transitionG
transversionG
or C
transitionG
or C
transversionmC
or C
transition4-base
insertionmC
pair
transitionmC
transitionG
or C transversionLocation"PPPDDDPPP
PHENOTYPE
i
È
histológica! examination. Of the eight proximal adenocarcinomas in
which p53 mutations were not identified, four appeared to arise from
Barrett's esophagus and four were at the GE junction. All of the
tumors with p53 mutations were of advanced stage with metastasis to
lymph nodes.
In contrast to the /?5^-positive cases, most (83%) USM+ tumors
(except Case 15) were located in the distal stomach. Three USM+
cases were metastatic to lymph nodes. The three node-negative
USM+ cases included two with invasion limited to the submucosa
and one invading through the muscularis propria to the serosa. All six
of the USM+ cases are currently alive with no evidence of recurrent
neoplasm (16-30 months follow-up).
The nine p53 mutations included one nonsense and seven missense
mutations (Table 2). A single tumor had a 4 base pair duplication of
the adjacent sequence. All of the point mutations occurred at G or C
bases, with one-half consistent with spontaneous deamination at CpG
sites. This spectrum of p53 changes is similar to that of other studies
(8, 9). The topographical distributions of the mutant p53 alÃ-eleswere
determined for all positive cases (Fig. 1). Only the distributions of the
mutant alÃ-elesand not LOH are reported here since the large numbers
MUTATION
NOT DETECTED
LESION
ABSENT
MUTATION
DETECTED
LESION NOT
TESTED
Fig. 1. Topographical distributions of p53 mutations. The mutant alÃ-eleswere associ
ated with specific histológica! phenotypes. The p53 mutations were homogeneously
present in the primary gastric cancers, suggesting that they were acquired prior to the bulk
of clona] expansion. p53 mutations were absent from most examples of dysplasia except
for Case 1. Intestinal metaplasia was not adjacent to these cancers. In Cases 8 and 9, the
p53 mutations present in the primary lesions were absent in some of the métastases.
of amplification cycles prevent reliable quantitation. The p53 muta
tions were absent from normal epithelium and stroma, but were
homogeneously present in all positive primary tumors. Most dysplasTable 1 Characteristics of gastric cancers
tic lesions (4 of 5 possible cases) adjacent to the positive tumors did
and/)5.i
not contain the mutant alÃ-ele.The exception was Case 1 (Fig. 2).
mutation
mutation
+668.5153360334232p53
cases4065.61525832373132712281821USM
detected961.1630990271836USMnot detected2566.681752(12238177181213
p53 mutations were present in the majority of metastatic lesions
(5 of 6 possible cases) with predominately homogeneous metastatic
studiedAv
No.
ageSiteProximalDistalMétastasesAbsentPrésentTypeIntestinalDiffuseGradeLowHighMetaplasiaPresentAbsentFamily
topographical distributions. In Case 8, however, the p53 mutation was
present in 12 but absent from 2 lymph node metastasis (Fig. 3). There
were no morphological differences between the métastaseswith or
without mutant p53 alÃ-eles.The small (3 mm) solitary lymph node
metastasis in Case 9 also lacked the p53 mutation present in the
primary lesion.
The topographic distributions of altered MS were determined for
the six USM+ tumors (Fig. 4). Altered MS were not detected in
adjacent normal, hyperplastic, or metaplastic epithelium, although
nonclonal MS changes in individual cells may escape detection. In
Case 13, one altered MS locus was detected in a small (0.5 cm)
tubular adenoma distinct from the primary tumor (Fig. 5). In the
primary tumors and their métastases,altered MS from at least one
locus could be detected throughout the carcinomas. The patterns were
complex as the same or different-sized MS additions or deletions
could be detected from adjacent tumor cell groups (Fig. 6). The
history"PositiveNegativeAll
exception was Case 13 in which intratumor heterogeneity of the
altered MS loci was not detected. In Case 10, the primary tumor had
a homogeneous distribution of the same altered MS, but its métastases
" The family history was considered positive if any cancer was noted in parents or
had different-sized MS. These intratumor differences suggest that MS
siblings.
4751
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pS3 AND THE USM+
PHENOTYPE
IN GASTRIC'
CANCER
A B C D
MUTANT
WILD
TYPE
Fig. 2. l h spiisi K gastric epithelium positive for pSJ mutation in Case 1. a, the dysplasia was identified microscopically (H & E stain of a conventional microscope section, X 40).
b, then small protective ink dots were placed on the dysplasia (left dot), normal gastric mucosa (right upper dot), and deeper cancer (right lower dot) (H & E stain of section used
for selective UV radiation fractionation and PCR. The lack of a coverslip and the relatively lower resolution of the video camera system accounts for the poorer image), c. the p53
mutation was present in the dysplasia and deeper cancer (A, B) but absent in the normal epithelium (C, D).
alterations usually continue to accumulate during macroscopic tumor
expansion.
The p53 mutations were not germ line since they were absent from
normal tissues. The mutations responsible for the MS instabilities are
unknown for the USM+ tumors. However, the family histories were
unremarkable for cancer or HNPCC (49) when compared with USMnegative cases (Table 1), except for Case 13 in which both parents had
colorectal cancer and a brother had pancreatic cancer. Although this
gastric cancer presented at 74 years of age, it may represent a late
manifestation of a germ-line mutator mutation. The average age of the
USM+ patients was 68.5 years, which was slightly greater than the
average for all cases (65.6 years).
dence that both p53 mutations and the USM+ phenotype may fre
quently (23) or infrequently (20, 51) coexist.
In this study, p53 mutations and the USM+ phenotype were not
observed in the same tumors. Marked regional specificity was noted
as tumors with p53 mutations were located predominately in the
proximal stomach, whereas the USM+ tumors were predominately in
the distal stomach. Regional specificity is also present for sporadic
USM + colorectal tumors which predominately arise in the proximal
colon (20, 21, 23). This distribution is also consistent with studies
which have demonstrated a high incidence of p53 mutations in ade-
DISCUSSION
many adenocarcinomas at the GE junction (52). Interestingly, the
epidemiology differs between proximal and distal gastric cancers (53).
Most adenocarcinomas of the GE junction exhibit aneuploidy and
LOH at multiple loci (54, 55), and p53 mutations are much more
common in aneuploid compared to diploid gastric tumors (7). One
other study also failed to detect p53 mutations among four USM+ (as
defined in this study) gastric cancers (17). Aneuploidy or LOH are
relatively rare in USM+ colorectal and endometrial tumors (21, 23,
51, 56).
The strategy used in this study utilizes the topographical distribu
tions of mutations to provide information on when they were acquired
relative to the bulk of tumor growth. This strategy requires both the
mutation and its clonal expansion, since mutations in individual cells
Genetic instability may underlie the etiology of many tumors as
multiple mutations are thought necessary for malignant transforma
tion (19). The altered MS present in USM+ tumors provide evidence
of widespread instability or a mutator phenotype (20, 21, 23). This
mutator phenotype has been best described for colorectal cancers
arising in families with HNPCC. The hMSH2 and hMLHl genes have
been linked to most of these familial tumors (24, 25, 50). A defect in
DNA repair after replication is responsible for the multiple MS
alterations observed in USM+ tumors (24, 26, 27). Similarly, p53
mutations are associated with defects in G, arrest after genetic dam
age, which may allow the genetic changes generated by multiple
mechanisms to accumulate (31). In colorectal cancer, there is evi
nocarcinomas which arise at the GE junction or are associated with
Barrett's metaplasia (10). It is difficult to distinguish between a
gastric, or an esophageal origin arising from Barrett's esophagus, for
4752
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pSJ AND THE USM+
PHENOTYPE
IN GASTRIC
CANCER
would be difficult to detect. Therefore definitive conclusions on the
p53 mutations or USM+ phenotypes are limited to whether they were
acquired prior to, or during the bulk of clonal expansion of the
individual cancers.
The homogeneous topographical tumor distributions of the p53
mutations and the lack of the same mutations in adjacent normal
A B C D
1 •••
2
3
4
5
6
A
B
C
D
••••
•••
•• •
••
•«
Fig. 5. Dysplastie epithelium of the tubular adenoma with altered MS from Case 13.
H & E, x 100.
ü
LU
MUTANT
WILD TYPE
Fig. 3. Absence of the mutant p53 alÃ-elein métastasesof Case 8. The p53 mutation was
homogeneously present in the primary tumor (Al-6, Bl-6, Cl-3) and one small
metastasis in a lymph node (D2). The p53 mutation was absent in normal lymphocytes
(C4, 5) and one lymph node metastasis (Co, Dl). A no dot control (D3) verified the
inactivation of the unprotected regions of the tissue section. A composite tumor section
(/)•/),normal genomic control (D5), and a water blank (Do) are also presented. Consistent
demonstration of LOH was not possible due to the high number of amplification cycles,
although in this example loss of the normal alÃ-eleappeared to have occurred in one
metastasis (D2) but not in the primary tumor.
LLJOOOOOOOO
^occcroccrcrircra:
S<LLJLLJLLILLILlJLlJLiJLLI
er oc er
O Q z)
2
2
W
•ÌLL.CLCLCLCLCLCLCLCL
CCCCLJJLULLILIJLLJUJLIJLIJ
OIDLLILULULULULULULU
2C/3QQQQQQQQ
CASE 10
ADENOMA
CASE
13
Mfd47
_t
.Mfd27
Fig. 6. Example of the altered MS distributed in Case 15. The altered MS differed
between adjacent tumor regions suggesting that MS alterations continued to accumulate
during clonal expansion.
METAPLASIA OR HYPERPLASIA
Fig. 4. Graphic representation of the relative proportions of MS sizes present in each
USM+ tumor. Normal and tumor regions were isolated and then amplified at different MS
loci. The results represent at least two different isolations and analysis. As the exact cells
could not he isolated from the duplicate sections and not every MS was amplified from the
same fractions, the results between MS loci cannot be directly compared. Each horizontal
line represents the sizes and relative proportions of the altered MS present in the tumors.
Altered MS were not detected in nonmalignant tissue, but were detected throughout all
tumors and a tubular adenoma. At least one altered MS was present in every tumor region.
For example, in Case 11, a loss of two CA repeats at the Mfd27 locus was present in all
tested tumor regions, but was absent from normal mucosa. In contrast, at the Mfd47 locus,
two CA repeat additions and deletions were present throughout one block of the primary
tumor, but in the other block, some regions exhibited only two CA repeat deletions,
whereas other regions exhibited only two CA repeat additions. At the Mfd41 locus, two,
three, and four CA repeat additions and two CA repeat deletions were present. An
example of the altered MS in Case 15 is presented in Fig. 6.
tissues suggest that they were acquired somatically before the bulk of
clonal expansion, and therefore at or before malignant transformation.
Definitive evidence of p53 mutation before malignant transformation
was observed in one case in which the identical point mutation was
present in adjacent dysplasia, although most examples of dysplasia
adjacent to the cancers with ¡>53
mutations lacked the same mutations.
Other studies have also provided evidence that p53 mutations can
occur in the preneoplastic lesions of gastric adenomas, intestinal
metaplasia (57, 58), and Barrett's esophagus (10, 55).
When the USM+ phenotype is acquired is more difficult to deter
mine as the underlying mutations are unknown. Although the hMSH2
and liMLHI genes have been linked to most USM+ colorectal cancers
in HNPCC, other loci are also implicated (22, 50). As yet, the
mutations responsible for the majority of sporadic USM+ tumors
have not been characterized. The high average age of presentation of
4753
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pSJ AND THE USM+
PHENOTYPE
ACKNOWLEDGMENTS
The technical assistance of Susan Leong and the secretarial assistance of
Kelly Hain were greatly appreciated.
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p53 Mutations and Microsatellite Instability in Sporadic Gastric
Cancer: When Guardians Fail
John G. Strickler, Jian Zheng, Qiuping Shu, et al.
Cancer Res 1994;54:4750-4755.
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