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119
Differences in Patterns of TP53 and KRAS2 Mutations
in a Large Series of Endometrial Carcinomas with or
without Microsatellite Instability
Elizabeth M. Swisher, M.D.1
Stacia Peiffer-Schneider, Ph.D.1
David G. Mutch, M.D.1
Thomas J. Herzog, M.D.1
Janet S. Rader, M.D.1
Alaa Elbendary, M.D.1
Paul J. Goodfellow, Ph.D.2
1
Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis,
Missouri.
2
Department of Obstetrics and Gynecology and
Department of Surgery, St. Louis, Missouri.
Presented at the Society for Gynecologic Oncologists’ 29th Annual Meeting, Orlando, Florida, February 7–11, 1998.
Supported in part by Grant CA71754. The assembly and characterization of the endometrial carcinoma specimens investigated was supported by a
grant from the American Cancer Society (RPG-96042-MGO).
The authors thank Kathryn Trinkaus, Ph.D., and
the Biostatistics Department of the Washington
University Cancer Center for statistical assistance
in evaluation of the significance of the difference in
the number of doubly mutant tumors.
Address for reprints: Washington UniversitySchool
of Medicine, Department of Obstetrics and Gynecology, Box 8064, 1 Barnes-Jewish Hospital Plaza,
St. Louis, MO 63110.
Received April 1, 1998; accepted May 26, 1998.
© 1999 American Cancer Society
BACKGROUND. The correlation between tumor microsatellite instability (MSI) and
the accumulation of mutations in KRAS2 and TP53 is uncertain. The authors
evaluated the TP53 and KRAS2 genes for mutations in sporadic endometrial carcinomas with microsatellite instability (MSI) and matched MSI negative controls to
determine whether defective DNA mismatch repair impacts the patterns of mutations in two genes known to be involved in endometrial tumorigenesis.
METHODS. Twenty-five MSI positive endometrial tumors were matched for prognostic factors with 25 MSI negative tumors. Mutations in codon 12 and 13 of KRAS2
were assessed using a polymerase chain reaction (PCR) restriction assay. Mutations in codon 61 of KRAS2 and exons 5– 8 of TP53 were evaluated using PCR
amplification and single strand conformation variant (SSCV) analysis. All variants
were subjected to direct DNA sequencing.
RESULTS. KRAS2 and TP53 mutations were identified with equal frequency in the
MSI positive and MSI negative groups. For TP53, the authors identified 5 mutations
(20%) in the MSI positive specimens compared with 8 (32%) in the MSI negative
group. For KRAS2, there were identified 8 mutations (32%) in the MSI positive
specimens compared with 7 (28%) in the MSI negative tumors. The mutational
spectra evident from sequence analysis of TP53 and KRAS2 variants were similar
between MSI negative and MSI positive tumors. MSI negative tumors were more
likely to have mutations in both KRAS2 and TP53 than MSI positive tumors, which
were rarely mutant in both genes (P ⫽ 0.046).
CONCLUSIONS. Although the overall frequency of mutations in TP53 and KRAS2 is
similar, MSI positive tumors are less likely to have mutations in both genes than
MSI negative sporadic endometrial carcinomas. MSI positive and MSI negative
endometrial carcinomas may arise through distinct genetic pathways. Cancer
1999;85:119 –26. © 1999 American Cancer Society.
KEYWORDS: endometrial carcinoma, microsatellite instability, TP53, KRAS2, mismatch repair, replication error.
T
he genetic events that lead to the formation of endometrial carcinoma remain elusive. Like colorectal carcinoma, endometrial carcinoma often arises from a well-defined precancerous lesion (atypical
endometrial hyperplasia). Although a genetic model for colorectal
tumorigenesis is well established, only a few genes that have important roles in endometrial tumorigenesis are known. Tumor behavior
and risk factors vary for different histologies of endometrial carcinoma. Thus, there may be more than one oncogenic pathway in
endometrial tumorigenesis.
A high proportion of sporadic endometrial carcinomas (15–25%)
are characterized by instability at simple DNA repeat sequences
120
CANCER January 1, 1999 / Volume 85 / Number 1
called microsatellites.1–3 A defect in the DNA mismatch repair system leads to a failure to correct
slipped-strand mispairing during replication, resulting
in polyclonal replication errors or microsatellite instability (MSI).4,5 MSI positive cell lines with defective
DNA mismatch repair accumulate mutations at a
much higher rate than MSI negative cell lines. Thus,
MSI positive tumors have been said to have a mutator
phenotype. It is presumed that this mutator phenotype will confer an oncogenic advantage to MSI positive tumor cells, a consequence of more rapid accumulation of mutations in genes that are important in
tumorigenesis.
Hereditary nonpolyposis colorectal carcinoma
(HNPCC) is a familial cancer syndrome in which tumors frequently exhibit the MSI phenotype. Colorectal
carcinoma and endometrial carcinoma are the first
and second most common cancers in HNPCC. These
patients carry germline mutations in 1 of 4 DNA mismatch repair genes (MSH2, MLH1, PMS1, and
PMS2).6 –10 Many MSI positive sporadic colorectal carcinomas have somatic mutations in MSH2 or
MLH1.11,12 On the other hand, the genetic basis for
defective mismatch repair for the majority of MSI positive sporadic endometrial carcinomas is unknown.13
The genetic targets of defective mismatch repair
in MSI positive tumors is poorly defined. MSI positive
colorectal carcinomas have an increased incidence of
frameshift mutations in the tumor growth factor
(TGF)-␤2 receptor gene and in the BAX gene, which
are important in the TP53-directed apoptotic pathway.14 –17 Recent reports by Tashiro et al. and Teng et
al. found a higher rate of mutation of the PTEN gene in
MSI positive endometrial carcinomas compared with
MSI negative endometrial carcinomas.18,19 Some, but
not all, studies have shown MSI positive colorectal
carcinomas to have a lower rate of TP53 and KRAS2
mutations than MSI negative colorectal carcinomas.20 –22
We hypothesized that sporadic MSI positive endometrial carcinomas arise from genetically distinct
pathways more often than MSI negative endometrial
tumors, and this difference would be reflected in the
pattern of mutations in genes commonly involved in
endometrial tumorigenesis. Two of the genes most
frequently implicated in endometrial carcinoma are
the tumor suppressor gene TP53 and the protooncogene KRAS2, which are mutated in 10 –30% of cases.23–27 We performed a mutation analysis for these
two genes in MSI negative and MSI positive tumors.
We matched the MSI positive and MSI negative specimens for prognostic factors known to be important in
endometrial carcinoma and which might impact the
frequency of mutation in these two genes. Using DNA
sequence analysis, we sought to determine whether
the specific types of mutations differed between MSI
positive and MSI negative tumors.
MATERIALS AND METHODS
Specimens
Tumor samples were obtained at the time of total
abdominal hysterectomy and bilateral salpingo-oophorectomy for primary endometrial carcinoma at
Washington University Medical Center from 1993 to
the present. The study protocol had been approved by
the institutional review board at Washington University, and all patients gave informed consent prior to
participation. Tissue specimens were frozen at -70°C
and DNA was prepared using proteinase K and phenol
extraction. High neoplastic cellularity for the tumor
tissues used as the source of DNA was histologically
confirmed by one pathologist. Normal DNA was extracted from peripheral blood as previously described.28 For a few cases, normal myometrium was
used as a source of normal DNA in lieu of blood
samples. Polymerase chain reaction (PCR)– based
analysis for microsatellite instability was performed as
previously described, using 7 simple sequence repeat
markers (FABP3, D2S123, D3S1029, D5S346, D10S212,
D17S261, and D18S55)2 along with the BAT-26 and
MLH1(TA)nTn markers.13 Tumors were classified as
MSI positive if they exhibited instability at two or
more markers. Characteristics of the MSI positive
specimens have been previously described.13
Clinical and pathologic information was used to
match 25 MSI positive specimens with 25 MSI negative endometrial tumors that served as controls. The
known prognostic factors in endometrial carcinoma of
grade, histology, surgical stage, and patient race were
matched between groups. Surgical stage was defined
according to the International Federation of Obstetrics and Gynecology (FIGO) 1988 criteria minus information on cytology.29 Age was matched between
groups as closely as possible after other prognostic
factors were matched.
TP53 Mutation Analysis
Exons 5– 8 of the TP53 gene were amplified from
genomic tumor DNA using primers derived from the
flanking intronic sequence of each exon. Primer sequences, reaction conditions, and PCR conditions
have been detailed previously.30 Twenty-seven rounds
of PCR cycling were performed, incorporating
␣32PdCTP (Amersham, Arlington Heights, IL). Each
amplimer was denatured at 94°C for 2 minutes, chilled
on ice, and electrophoresed through nondenaturing
gels of 0.5⫻ MDE (J. T. Baker) with and without 5%
glycerol at room temperature for 14 –20 hours at
TP53 and KRAS2 in Endometrial Carcinomas with Microsatellite Instability/Swisher et al.
4-5 W. Gels were dried and products were visualized
by autoradiography at -70°C. PCR amplification and
single-strand conformation variant (SSCV) analysis
was repeated on all variants and their corresponding
normal cellular DNA to verify the somatic nature of
the variation.
KRAS2 Mutation Analysis
KRAS2 codons 12 and 13 were evaluated by PCR amplification and restriction digestion as previously described.31 Briefly, the two assays use mutagenic primers, which introduce a new restriction site next to the
target codon. Any missense mutation in the codon
causes loss of the introduced restriction site. For
codon 12, mutagenic primers introduce a restriction
site for BstN I in wild-type alleles, resulting in a diagnostic 114 base pair fragment. For codon 13, mutagenic primers insert a Hae III restriction site, resulting
in an 83 base pair fragment. The mutant allele is
identified as a restriction fragment of different size by
electrophoresis through a 6% polyacrylamide gel. Gels
were stained with ethidium bromide, and products
were visualized under ultraviolet illumination. All
codon 12 and 13 KRAS2 mutants identified in this
assay were then subjected to PCR amplification and
SSCV analysis of exon 1 using primers and PCR conditions previously described.32 Variant bands were cut
out from the SSCV gel, eluted, and reamplified. Direct
DNA sequencing was performed following PCR purification as described below.
KRAS2 codon 61 mutations were identified by PCR
amplification and SSCV analysis of exon 2. Primers for
exon 2 amplification were 5´-cccttctcaggattcctaca-3´
and 5´-agaaagccctccccagtcct-3´. PCR reactions were
performed in 10 ␮L containing 20 ng DNA, 0.07 mM
each deoxy-NTP, 0.2 mM of each primer, 0.5 units Taq
polymerase, 0.1 mCi ␣32PdCTP (Amersham), 10 mM
Tris HCl (pH 8.5), 1.5 mM MgCl2, and 100 mM KCl.
Twenty-seven rounds of PCR cycling were performed,
with each cycle consisting of 30 seconds of denaturing
at 94°C, 30 seconds of annealing at 59°C, and 30 seconds of extension at 72°C. SSCV analysis was performed in identical fashion to that described for TP53.
All variants and the corresponding normal cellular
DNA were subjected to repeat PCR amplification and
SSCV analysis. Variants present in normal as well as
tumor DNA were assumed to be polymorphisms, as
KRAS2 germline mutations have not been described.
These variants were not subject to sequence analysis.
Only variants present in tumor samples and not found
in corresponding normal DNA were sequenced.
121
Variant Analysis
Variant TP53 and KRAS2 bands were excised from the
dried SSCV gels. DNA was eluted, reamplified, and
electrophoresed on a 1% low-melt agarose gel or purified with a PCR product purification kit (Qiagen,
Chatsworth, CA). Templates were sequenced directly
using the dideoxy chain termination method with radiolabeled primers as previously described,33 or using
the fmol Sequencing Kit (Promega, Madison, WI). In
cases of previously undescribed TP53 mutations, variant sequences were confirmed by sequencing the
complementary strand or by restriction digest when
appropriate.
Statistics
The statistical significance of differences between the
MSI positive and MSI negative control groups was
evaluated with the McNemar chi-square test for
paired observations.
RESULTS
Table 1 describes the clinical features of the specimens investigated and summarizes the mutation data
for the 25 paired MSI negative and MSI positive tumors. The race of the patient was matched exactly; 3
patients in each group were African American (12%)
and the remaining patients were white. The median
age (66 years) was the same in each group. International Federation of Gynecology and Obstetrics (FIGO)
surgical stage was matched exactly between specimens, except for Case 1033, which was a Stage IB
tumor (a tumor invading less than one-half the thickness of the myometrium) with only 1 mm invasion
and was matched with a Stage IA tumor (with no
invasion). The breakdown of the 25 MSI positive specimens by surgical stage is as follows: 5 Stage IA, 11
Stage IB, 6 Stage IC (invading greater than or equal to
one-half the myometrial thickness without serosal
penetration), 1 Stage IIA (involving endocervical mucosa), and 2 IIIC (involving pelvic or para-aortic lymph
nodes). Nine tumors were ⱖStage IC (36%). In each
group, all tumors were adenocarcinomas with endometrioid histology. MSI positive tumors were matched
to MSI negative endometrioid carcinomas of the same
grade, without regard to the presence of squamous
elements. Grade was matched nearly exactly. Of 25
tumors in each group, 7 (28%) were FIGO Grade 1, 15
(60%) were FIGO Grade 2, and 3 (12%) were FIGO
Grade 3.
KRAS2 and TP53 mutations were identified in
equal frequency in the MSI positive and MSI negative
groups (Table 2). Of 25 specimens, 12 (48%) of the MSI
positive and 10 (40%) of the MSI negative tumors had
2
3
2
3
3
1
2
2
2
2
2
2
2
2
1
2
2
1
1
2
1
1
1
2
2
37
82
1027
1033
1044
1061
1062
1064
1070
1078
1085
1086
1087
1089
1094
1096
1100
1102
1110
1112
1125
1128
1133
1141
1145
IA
IC
IB
IB*
IIIC
IB
IC
IB
IB
IIIC
IB
IIA
IC
IB
IB
IB
IC
IA
IB
IC
IB
IA
IA
IC
IA
Tumor
stage
244
144
273
181
274
5
8
5
8
Codon
7
Exon
cag(gln) to tag(stop)
insert A before cat
cgc(arg) to cac(his)
gtt(val) to ggt(gly)
ggc(gly) to gtc(val)
Nucleotide change
GAT
GTT
CAC
GAT
AGC
GGA
12
61
12
13
12
GTT
GTT
Nucleotide
12
12
12
Codon
69
1042
1117
1029
1111
1093
1072
1129
1058
1119
1028
38
1066
92
1065
1043
1059
1045
1126
1049
1081
1109
1046
1054
169
Specimen
I.D.
2
3
2
3
3
1
2
2
2
2
2
2
2
2
1
2
2
1
1
2
1
1
1
2
2
Tumor
grade
Tumor
stage
Exon
IA
IC
IB
IA*
IIIC
IB
IC
IB
IB
IIIC
IB
IIA
IC
IB
IB
IB
IC
IA
IB
IC
IB
IA
IA
IC
IA
5
138
280
173
272
8
8
5
splice
248
192
266
5
7
6
8
Codon
gcc(ala) to gtc(val)
aga(arg) to aaa(lys)
gtg(val) to ggg(gly)
gtg(val) to ggg(gly)
intron 4 del A of AG
cgg(arg) to tgg(trp)
cag(gln) to tag(stop)
gga(gly) to gaa(glu)
Nucleotide change
TP53 mutation
KRAS2 mutation
TP53 mutation
12
12
13
12
12
61
12
Codon
GTT
GTT
GAC
TGT
GTT
CAC
GAT
Nucleotide
KRAS2 mutation
MSI: microsatellite instability.
a
Each MSI positive tumor is matched with the MSI negative tumor to its right. Grade and stage for MSI negative specimens are identical to those given for their matched MSI positive specimens. The exception is 1033, with 1 mm invasion (Stage 1B*), which was matched with a
Stage 1A* tumor 1029. Wild-type KRAS2 codons 12, 13, and 61 are GGT, GGC, and CAA, respectively.
Tumor
grade
Specimen
I.D.
MSI negative tumors
MSI positive tumors
TABLE 1
Clinical Features and Mutation Data for MSI Positive Endometrial Tumors and Their Matched MSI Negative Controlsa
122
CANCER January 1, 1999 / Volume 85 / Number 1
TP53 and KRAS2 in Endometrial Carcinomas with Microsatellite Instability/Swisher et al.
TABLE 2
Mutations in MSI Positive and MSI Negative Endometrial Tumors
No. (%) of cases
Gene
Mutation
type
MSI positive
MSI negative
TP53
TP53
TP53
TP53
KRAS2
KRAS2
KRAS2
Transition
Transversion
Frameshift
Total
Transition
Transversion
Total
2
2
1
5 (20%)
5
3
8 (36%)
5
2
1
8 (32%)
5
2
7 (32%)
MSI: microsatellite instability.
a KRAS2 and/or TP53 mutation. For TP53, we identified 5 mutations (20%) in the MSI positive specimens
compared with 8 (36%) in the MSI negative group. For
KRAS2, we identified 8 mutations (36%) in the MSI
positive specimens compared with 7 (32%) in the MSI
negative tumors. Although the overall frequency of
mutations in both KRAS2 and TP53 were similar, the
distribution of these mutations varied between the
two groups (Table 1). In the MSI positive group, significantly more tumors (5, 20%) were found to have
mutations in both KRAS2 and TP53 than in the MSI
negative group (1, 5%, P⫽ 0.046). Representative mutation analysis and DNA sequence analysis for TP53
and KRAS2 variants are demonstrated in Figures 1 and
2, respectively.
The presence of a TP53 mutation was significantly
associated with higher grade tumors. In 14 Grade 1
tumors, 1 TP53 mutation was identified (7.1%). In
contrast, among 6 Grade 3 tumors, 4 TP53 mutations
were identified (67%, P ⫽ 0.01, two-tailed). There was
a trend toward higher frequency of KRAS2 mutations
in higher grade tumors; 2 of 14 Grade 1 and 3 of 6
Grade 3 tumors exhibited KRAS2 mutations (P ⫽ 0.13).
TP53 and KRAS2 mutation status did not correlate
with surgical stage.
The TP53 and KRAS2 mutation spectra variants
were similar in MSI negative and MSI positive tumors
(Table 2). Two of 13 TP53 mutations (15%) were insertion or deletion (frameshift) mutations, with 1 occurring in each group. The percentages of transition
and transversion mutations were likewise equally distributed between the two groups for both KRAS2 and
TP53. The fraction of transition mutations occurring at
CpG dinucleotides was also equal for both TP53 and
KRAS2 between MSI positive and MSI negative tumors.
Of the 15 KRAS2 mutations identified, 2 occurred
in codon 13 and 2 occurred in codon 61. One KRAS2
codon 13 and one codon 61 mutation were found in
123
each group. The number of TP53 mutations identified
in each exon examined was also similar between MSI
negative and MSI positive tumors.
DISCUSSION
MSI is diagnostic for a mutator phenotype attributable
to defective DNA mismatch repair. Mutation rates are
increased for MSI positive cells and are presumed to
confer an oncogenic advantage.4,5 The MSI phenotype
has been noted in atypical hyperplasia adjacent to MSI
positive endometrial carcinoma, and as such appears
to be an early event in endometrial carcinoma development.34 Because MSI appears to be an early event in
the development of a subset of endometrial carcinomas, the mutator phenotype is likely to influence the
entire cascade of genetic events that contribute to
tumor formation and progression. The genes that accumulate mutations in tumor cells with the mutator
phenotype are, for the most part, unknown. MSI positive cancers could accumulate mutations in genes
known to be involved in endometrial tumorigenesis at
an increased frequency, and/or accumulate mutations
in genes not involved in MSI negative tumors. We
hypothesized that the MSI phenotype would influence
the mutation spectra of genes known to be involved in
endometrial tumorigenesis, in particular TP53 and
KRAS2.
We observed high mutation rates in both KRAS2
and TP53 in this study, but we did not detect a difference in either the frequency or the types of mutations
in these genes. There was, however, a statistically significant difference in the proportion of tumors that
were found to have mutations in both KRAS2 and
TP53 (P ⫽ 0.046). Because TP53 mutations are more
frequent in higher grade and stage endometrial carcinomas as well as in certain histologic types, we
matched our MSI negative and MSI positive groups for
these features in an effort to control for differences in
mutation rates that could be attributed to these factors. The difference noted in this study in the distribution of KRAS2 and TP53 mutations may not have
been evident if we had not closely matched our tumors for many prognostic features. Caduff et al. reported the results of a study of 10 MSI positive endometrial carcinomas investigated for TP53 mutations
by immunohistochemistry and for mutations in codon
12 of KRAS2 using a PCR amplification and restriction
digest assay.39 They found no difference in mutation
rates in MSI positive tumors compared with a large
group of unmatched MSI negative tumors. In our
study, which relied on different mutation detection
methods and matched MSI positive and negative tumors, we were able to detect a difference in the pattern of KRAS2 and TP53 mutations in both MSI posi-
124
CANCER January 1, 1999 / Volume 85 / Number 1
FIGURE 1.
Representative SSCV and
DNA sequence analysis for exon 8 of
TP53 is shown. Arrows indicate variant
conformers. N: normal (wild-type) DNA
pattern; M: tumor specimens containing
variant conformers representing TP53
mutations. DNA sequence analysis demonstrates the wild-type sequence AGA at
codon 280 (arginine). Tumor 1058 demonstrates a G-to-A transition, resulting in
the sequence AAA at codon 280 (lysine).
FIGURE 2. Detection of KRAS2 mutations is shown. Arrows indicate restriction fragments or conformational variants associated with mutations. (A)
Polymerase chain reaction amplification
and restriction analysis to detect codon
12 and 13 mutations is shown. N: normal (wild-type) pattern; M: tumor specimen heterozygous for a codon 12 or 13
mutation. (B) Single strand conformation
variant analysis and DNA sequence
analysis (of the noncoding DNA strand)
to detect KRAS2 codon 61 mutations is
shown. N: normal cellular DNA; T: tumor
DNA from specimen 1087. The wild-type
sequence for codon 61 is CAA (glutamine). The conformational variant
from tumor 1087 has a CAC (histidine) at
codon 61 (GTG on the noncoding
strand).
tive and MSI negative tumors. The fact that more MSI
negative tumors have mutations in both genes,
whereas MSI positive tumors tend to have mutations
in only one of these genes, supports the hypothesis
that endometrial tumors with defective mismatch repair arise via a different genetic pathway or pathways
than endometrial tumors with intact mismatch repair.
Several groups have compared KRAS2 and TP53
mutation frequency between MSI positive and MSI
negative colorectal carcinomas. Methods for the detection of MSI and for mutation analysis have differed
among studies.5 Some authors have reported an inverse correlation between the MSI phenotype and
KRAS2 and/or TP53 mutation rates.20 –22 Other groups
found no difference in KRAS2 and TP53 mutation rates
between MSI positive and MSI negative colorectal carcinomas.35–37 Konishi et al. noted an inverse correlation with MSI phenotype and TP53 and KRAS2 mutations in tumors from Japanese HNPCC patients.
However, this correlation did not hold for sporadic
MSI positive colorectal carcinomas.38 It is difficult to
determine whether inherited and sporadic tumors
with defective DNA mismatch repair differ in how they
accumulate mutations. Not all studies specified
whether the MSI positive colorectal carcinomas were
sporadic or familial. In studies in which only sporadic
TP53 and KRAS2 in Endometrial Carcinomas with Microsatellite Instability/Swisher et al.
colorectal carcinomas were investigated, there was no
association between the frequency of TP53 and KRAS2
mutation and the MSI phenotype.36 –38 Thus, sporadic
MSI positive colorectal carcinomas with somatic mutations in DNA mismatch repair genes may progress
through different oncogenic pathways than HNPCC
tumors with constitutional mutations in mismatch repair genes. If inherited and sporadic colorectal carcinomas differ in how they accumulate mutations, it
might explain some of the discordant results regarding
TP53 and KRAS2 mutation frequency and MSI phenotype in colorectal carcinomas.
The observed frequency of TP53 and KRAS2 mutations in this study (26% and 30%, respectively) is
high. Other studies have noted TP53 and KRAS2 mutations in 9 –30% of endometrial carcinomas.23–27,40
The high mutation frequency that we observed is consistent with the nature of the tumors we investigated
and the sensitive mutation detection methods used.
TP53 mutation is correlated with higher grade in our
study, as has been noted previously by other investigators.23,24 Poorly differentiated endometrial carcinomas are more likely to have defective mismatch repair,3 and, as expected, our series includes a high
percentage of Grade 2 and 3 tumors. We believe that
the composition of our tumor panel explains the high
incidence of TP53 mutations in this study.
Most studies of KRAS2 in endometrial carcinoma
have been restricted to analysis of codon 12.27,32,39,41
Greater than one-fourth of the mutations (4 of 15) that
we observed occurred in codons 13 and 61, increasing
our total KRAS2 mutation rate from 22% to 30%. To
the best of our knowledge, KRAS2 mutations in codon
61 have not previously been reported in endometrial
carcinoma. Other investigators studying endometrial
carcinoma have used dot blot oligohybridization or
direct DNA sequencing to identify codon 61 mutations.26,42 The PCR and SSCV methods we used may be
more sensitive in identifying codon 61 mutations. One
codon 61 mutation was found in the MSI positive and
1 in the MSI negative tumors. The uncommon codon
13 and 61 mutations in KRAS2 seem to be distributed
equally between the cancers with intact and those
with defective mismatch repair.
Only a few genetic targets of defective mismatch
repair in MSI positive cancers have been identified.
Colon carcinomas from HNPCC patients have a very
high rate of mutation in the TGF-␤ type II receptor
gene.14 –16 The majority of these mutations involve
insertion or deletion in a string of 10 adenosine residues, a typical target of defective mismatch repair. The
recently discovered PTEN tumor suppressor gene has
been shown to be mutated in a very high percentage
of MSI positive endometrial carcinomas (12 of 14,
125
85%), in contrast to MSI negative endometrial carcinomas (4 of 12, 33%).18 PTEN mutations in MSI positive endometrial carcinomas include both insertion
and deletion mutations in repeats as well as single
base substitutions.18,19 It is unclear why this gene
seems to be a particular target for mutation in endometrial carcinomas with defective mismatch repair
and whether this predilection for PTEN mutations applies to other MSI positive human cancers.
Endometrial carcinomas with MSI are less likely
than MSI negative tumors to have mutations in both
TP53 and KRAS2. This difference in the mutation pattern of these two genes supports the hypothesis that at
least a subset of endometrial tumors with defective
DNA mismatch repair arise via a different oncogenic
pathway than tumors without MSI. An improved understanding of the genetic targets of defective mismatch repair will clarify how the MSI phenotype contributes to oncogenesis in endometrial carcinoma.
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