Monitoring of endometrial K-ras mutation in
The age of the patients at the surgery ranged from 27 to 71 years with a
mean of 53.6 years (Table 1). They were treated with 20 mg of TAM daily for 24to 106 months with a mean of 52.4 months (dose of TAM ranged from 14.4 g to
tamoxifen-treated patients with breast cancer.
63.6 g). There is no significant difference in duration of TAM use, and total doseof TAM with or without K-ras mutations. Six patients underwent chemotherapy,
and 7 patients underwent adjuvant radiotherapy to prevent the local recurrenceof breast cancer. None of the patients underwent radiotherapy for to induce
cessation of castration of the ovarian function. One patient underwent TAM
Hiroshi Tsujioka1, Toru Hachisuga2, Miyoko Fukuoka1, Taeko Ueda1, Shinji Horiuchi1, Kyoko Shirota1, Toshiyuki
treatment in combination with gonadotrophin-releasing hormone analog
Yoshizato1, Makoto Emoto1, Tatsuhiko Kawarabayashi1, Masahide Kuroki3, and Shingo Miyamoto1
(GnRHa). There are no patients who developed atypical endometrial hyperplasia
1Department of Obstetrics and Gynecology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
or endometrial carcinoma during monitoring period. There is no significant
, 2Department of
difference in height, bodyweight, body Mass Index and gravidity of patients with
Obstetrics and Gynecology, University of Occupational and Environmental Health, Kitakyushu, Japan, 3Department of
or without K-ras mutations.
Biochemistry, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
Mutations in codon 12 of K-ras were observed initially in 13 (46.4 %) of 28
patients. In these 13 patients with K-ras mutations, all patients were
Aim: A high incidence of endometrial K-ras mutations has been reported in Tamoxifen (TAM)-treated patients with breast cancer. We examined the changes in the
postmenopausal-amenorrheic status, and no endometrial K-ras mutation was
frequency of endometrial K-ras mutations after the cessation of TAM treatment.
found in 6 premenopausal patients. The postmenopausal-amenorrheic status is
Method: DNA was extracted from fresh cytological or polypectomy samples of the endometrium in 28 patients who had undergone TAM treatment of breast cancer.
significantly frequent in patients with K-ras mutations (p<0.05). When it passed
Mutations were detected by an enriched polymerase chain reaction-enzyme-linked minisequence assay (Sumitomo Metal Industry, Inc, Tokyo, Japan). K-ras codon 12
more than 2 years after cessation of TAM treatment, repeated examinations did
mutations were monitored in these 28 patients.
not show mutations in any of the 13 patients in whom they were initially
-2year cessation +2year
Results: An initial examination detected endometrial K-ras mutations in 13 of the 28 patients. However, repeated examinations performed after cessation of TAM
detected (Table 2). Shown in Figure 1, PCR-enzyme-linked minisequence assay
Figure 2. The Enriched enriched polymerase chain reaction (PCR)-enzyme-linked
treatment did not detect endometrial K-ras mutations in any of these 13 patients. No endometrial K-ras mutation has been detected in repeated examinations
of case 1 and 2 revealed that no endometrial K-ras mutation has been detected
minisequence assay, showing K-ras codon 12 mutations. In Case 1: , mutations were
performed for these patients for more than 2 years since the cessation of TAM treatment. In addition, the 15 patients who did not have endometrial K-ras mutations
at 2 years after cessation of TAM treatment (Figure 2). In contrast, the 15
GAT (3+) in lane 1, GAT (±) in lane 2, and GTT (2+) in lane 3, and only GGT (wild
in the initial examination did not demonstrate them in repeat examinations.
patients who did not have endometrial K-ras mutations in the initial
type) was shown in lanes 4 and 5. In Case 2: , mutations were GCT (+) and GAT (3+)
Conclusion: The cessation of TAM treatment may reduce the risk of developing endometrial cancers through K-ras mutations.
examination did not demonstrated them in repeat examinations (Table 2).
in lane 6, GCT (3+) in lane 7, and GCT (±) in lane 8, and only GCT (wild type) wasshown in lane 9. BACK: background
Tamoxifen (TAM) is a nonsteroidal triphenylethylene derivative that has proven to be effective in the adjuvant treatment of breast cancer by increasing both the
TAM-related polyps obtained from other 31 patients were evaluated for Ki-67
disease-free and overall survival. TAM is generally well tolerated and it has been believed to have few adverse effects. However, several large epidemiological studies
index and apoptosis index. Immunohistochemical staining for apoptotic body are
have confirmed that the relative risk of endometrial cancers is 2- to 3-fold that of controls, and the risk increases with the duration and cumulative dose of TAM
shown in Figure 3 and clinicopathological features are summarized in table 3.
There is no significant difference in Ki-67 index with or without K-ras mutations.
K-ras mutations are considered to correlate with the phenotypic progression from atypical hyperplasia to endometrial cancer. A mutation in codon 12 of the K-ras
Apoptosis index is significantly higher in polyps with K-ras mutations.
gene has been identified in from 4.5% to 23% cases of atypical endometrial hyperplasia and in 11% to 26% cases of endometrioid carcinomas of the endometrium. Thepresence of K-ras codon 12 mutations has been reported in 27% to 76% of the TAM-treated endometria. Prasad et al found that 5 (17%) of the 29 TAM-associatedendometrial cancers showed K-ras mutations and also stated that the frequencies of K-ras mutations were similar as in sporadic endometrial cancer. Thus, it is
Table 2. Monitoring of the K-
ras mutations in the endometriums of the tamoxifen-treated patients
considered that the frequencies of K-ras mutations in TAM-associated endometrial cancers are lower than those of all TAM-treated patients. This fact suggested that
Case 1: K-ras mutation(+)
Case 2: K-ras mutation(+)
there exist the presence of repair systems and/or checkpoint-mediated fail-safe mechanisms to deal with K-ras mutations in humans. The significance of K-ras
mutations in the development of TAM-related endometrial cancers remains unclear. The purpose of the present study was to investigate the changes in K-ras
mutations after the cessation of TAM treatment.
GCT(1+) and GAT(3+)
Materials and Methods
2. Papanicolaou staining
Since April 1993, gynecologic examinations of patients who received adjuvant
hormonal treatment after undergoing surgery for breast cancer have been performed
1. Endometrial cell
at the outpatient gynecological clinic of Fukuoka University Hospital. Since March
2000, endometrial polypectomy specimens of patients receiving TAM treatment have
Case 3: K-ras mutation(+)
Case 4: K-ras mutation(-)
been examined for the presence of K-ras mutations. Eight frozen polypectomy
specimens were available for analysis and mutations was were found in 5 specimens
(62.5%). Between February 2003 and September 2004, endometrial cytological
samples of 86 patients receiving TAM treatment were screened for the presence of K-
ras mutations. K-ras mutations was were found in 35 (40.6%) of these 86 patients.
After an initial analysis of endometrial K-ras mutations, patients were repeatedly
examined every 6 months. During the monitoring of endometrial K-ras mutations, 28
patients stopped undergoing TAM treatment. These 28 patients were monitored
3. Wash in buffer
periodically before and after the cessation of TAM treatment, whereas 5 patients who
Figure 1. Fresh endometrial cytological samples were obtained using an
Figure 3. Apoptotic body expression in TAM-related
started monitoring around the cessation were included. We analyzed these 28 patients
endocyte device (Laboratorie CCD, Paris, France). The cellular material was
categorized into 2 types: one was smeared onto glass slides and processed for
to clarify the influence of TAM cessation to endometrial K-ras mutations.
routine Papanicolaou staining, whereas the other was used for an analysis of K-
significantly higher in polyps with K-ras mutation (case 1,
2, and 3) than polyps without K-ras mutation (case 4).
The endometrial polyps were confirmed by a hysteroscopy. Fresh endometrial
cytological samples were obtained using an endocyte device (Laboratorie CCD, Paris,
Table 1. Clinical date of the tamoxifen-treated patient
France). The cellular material was categorized into 2 types: one was smeared onto
Table 3. Clinicopathological features of TAM-related endometrial polyps.
glass slides and processed for routine Papanicolaou staining, whereas the other was
used for an analysis of K-ras mutations (Figure 1).
Detection of a mutation in codon 12 of the K-ras gene
DNA was extracted from cytological specimens using the standard phenol and
P; endometrial polypectomy specimen
chloroform method. Mutations in codon 12 of the K-ras gene were analyzed by an
Apoptosis index (%)
enriched polymerase chain reaction (PCR)-enzyme-linked minisequence assay
(Sumitomo Metal Industry, Inc., Tokyo, Japan). This assay was based on the
enrichment of the mutant K-ras gene as previously described, followed by the
incorporation of a biotin-labeled nucleotide specific for mutant gene, which was then
The formation of DNA adducts is a key event in the multistage process of carcinogenesis. Unrepaired or inefficiently repaired DNA adducts may cause
quantified enzymatically using chromogenic substance. The K-ras gene amplified by
mispairing during DNA replication, resulting in mutations. Specific mutations in crucial gene encoding proteins for cell cycle control and growth might trigger
PCR was captured by the probes that were designed to detect the K-ras codon 12 wild
tumorigenesis. Although some investigators have failed to find any evidence of TAM-DNA adducts in the endometriums, Shibutani et al detected them in 50% of the
type (GGT) and six 6 mutants (GAT, GCT, GTT, AGT, CGT, and TGT), which were
endometrial samples obtained from TAM-treated women. The site-specific N2-deoxyguanosinyl tamoxifen adducts display a high miscoding and mutagenic potential
finally subsequently measured using a microtiter plate reader to detect their the
and primarily generate G-to-T transversions in mammalian cells. The presence of alpha-acetoxytamoxifen-induced DNA damage causes a dose-related increase in
presence and quantity. The results of a semiquantitative analysis were scored as (3+),
mutation frequency. The codons 12 and 14 of the K-ras gene are reported to be hotspots for carcinogen-DNA
(2+), (1+), (±), and (-) according to the percentage of mutant ras genes. ; these scores
adduct formation in human bronchial epithelial cells. The DNA adducts that formed at codon 12 of the K-ras gene were poorly repaired in comparison to those at
represent greater than 20%, 2% to 20%, 0.2 to 2%, less than 0.2%, or 0% (not detected)
other codons, including codon 14. A high incidence of mutations in codon 12 of the K-ras gene was also found in TAM-related endometrial polyps.
of the mutant, respectively, according to the manufacturer's instructions.
Mammalian cells have developed numerous repair systems to deal with various types of DNA damage and maintain genomic integrity. Bulky DNA adducts, such
The oligonucleotide primers were as follows: upstream for the first and second PCR;
as those formed by TAM, are typically removed by the nucleotide excision repair system (NER). One study using cells proficient and deficient in NER showed that it
5'-TAAACTTGTGGTAGTTGGAACT-3', downstream for the first PCR; 5'-
plays a significant role in the removal of TAM-DNA adducts. The reported absence of endometrial K-ras mutations in more than half of all patients who receive with
GTTGGATCATATTCGTACAC-3' and downstream for the second PCR; 5'-
TAM treatment indicates that the level of TAM-DNA adducts in uterine samples may depends on the individual variability in either the extent of metabolic
CAAATGATCTGAATTAGCTG-3'. One microliter of a 10-fold dilution of the first PCR
activation or capacity of NER. In the present study, no endometrial K-ras mutation was detected in premenopausal patients. It suggested that menstruation
product was digested with 2.5U of BsrI restriction enzyme (New England Biolabs,
probably plays an important role in the removal of K-ras mutations of the endometrium. We also revealed that endometrial K-ras mutations were detected initially
Beverly, MA). The details of this method have been described in previous studies.
in 13 (46.4 %) of 28 TAM-treated patients, and no endometrial K-ras mutations has been detected in any of these 13 patients when it passed more than two years
This study (No. 158) was approved by the ethics committee of Fukuoka University
after cessation of TAM treatment. These 13 patients were postmenopausal-amenorrheic and this fact suggested that there exist the removal mechanism of K-ras
School of Medicine. Informed content was obtained from each patient.
mutations in humans other than menstruation. This event is supported by the presence of various repair systems and/or checkpoint-mediated failsafe mechanisms,
such as apoptosis or cellular senescence, in response to the immediate induction of K-ras mutations in humans. These findings suggest the potential usefulness of
Statistical analysis was performed using Dr. SPSS II 11.0.1 J (SPSS Japan, Tokyo,
novel molecular targeted therapies for the treatment of precancerous lesions of the endometrium. The cessation of TAM treatment may reduce the risk of developing
Japan). The student's t-test and the chi-square test were used to assess the association
endometrial cancers through K-ras mutations.
between categorical variables. Statistical significance was set at a value of P<0.05.
post: postmenopausal patient, ame; TAM and GnRHa related amenorrheic patient, pre; premenopausal patient, C; cyclophosphamide, E; epirubicin, M; Methotrexate, F; 5-fluorouracil
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SIRT1 Regulates Circadian Clock GeneExpression through PER2 Deacetylation Gad Asher,David Gatfield,Markus Hans Reinke,Charna Florian Kreppel,Raul Mostoslavsky,Frederick W. Alt,and Ueli 1Department of Molecular Biology, Sciences III, University of Geneva, 30, Quai Ernest Ansermet, CH-1211 Geneva-4, Switzerland2Division of Gene Therapy, University of Ulm, Helmholtzstrasse 8/1, D-89081 Ulm, Germany3Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02114, USA4Howard Hughes Medical Institute, Children's Hospital, Center for Blood Research, and Department of Genetics,Harvard University Medical School, Boston, MA 02115, USA*Correspondence: DOI 10.1016/j.cell.2008.06.050