Frequency of K-ras Mutation in Endometrioid Endometrial Adenocarcinoma
Papitchaya Watcharanuruk1*, Supaporn Suwiwat2, Siriwong Lattakanjanang3
1
Department of Biomedical Sciences, Faculty of medicine, Prince of Songkla University,
Songkhla 90110, Thailand
2
Department of Pathology, Faculty of medicine, Prince of Songkla University, Songkhla
90110, Thailand
*e-mail: papitchaya_59@hotmail.com
Abstract
K-ras oncogene plays an important role in the mechanism of carcinogenesis in many
human cancers. Previous studies have shown that K-ras mutation is found in 10% to 30% of
endometrial carcinomas. However, the K-ras mutations in Thai patients with endometrial
carcinoma have not yet been investigated. Our purpose was to examine the frequency of Kras gene mutation in endometrioid adenocarcinoma and investigate the correlation with
clinicpathological variables. Genomic DNA was extracted from formalin-fixed paraffinembedded tissue sections. Polymerase chain reaction amplification for K-ras codon 12 and
13 were performed, followed by sequencing. K-ras mutations were detected in 10 of 62
(16.1%) endometrioid adenocarcinomas. Six mutations were found at codon 12 (9.7%), and
two mutations at codon 13 (3.2%). Interestingly, we also detected 2 samples with a double
point mutations, codon 12 and codon 6 (1.6%), and codon 12 and codon 7 (1.6%). No
significant relationship was seen between the presence of K-ras mutations and
clinicopathological variables such as age, grade, and depth of myometrial invasion. Our
findings suggest that mutation of K-ras gene may be associated with pathogenesis of
endometrioid adenocarcinoma in subgroup patients but with no evidence of prognostic value.
Keywords: K-ras mutation, endometrioid adenocarcinoma
Introduction
Endometrial carcinoma is the third common malignant tumor of the female genital
system in Thailand with the incidence rate of 2.8/100,000/years [1]. Recently, the incidence
in Thai women younger than 40 years of age who have obesity seems to be increasing [2, 3].
Endometrioid adenocarcinoma is the major type of endometrial carcinoma, approximately
80% of all cases. This type is commonly associated with high levels of estrogen. In addition,
it is related to the history of obesity, diabetes, hypertension, nulliparity, and chronic
anovulation [4, 5]. The pathogenensis of endometrioid adenocarcinoma also involves a wide
range of molecular alterations, including microsatellite instability, PTEN mutation, K-ras (cKirsten-ras/ Kirsten Rat Sarcoma virus) mutation, and β-catenin mutation [6, 7].
K-ras is a proto-oncogene and it encodes a 21 kDa small monomeric guanine
nucleotide-binding protein (G-protein). The G-protein has been implicated mainly in
regulating cell growth and differentiation. Mutations of K-ras oncogene result in constitutive
activation of the signal transduction pathway and subsequently upregulated proliferation and
impaired differentiation [8]. Previously published studies have shown that K-ras gene
mutation is found in 10% to 30% of endometrial carcinomas [9, 11-14]. K-ras mutation has
been reported in association with submicroscopic myometrial invasion and depth of
myometrial invasion in endometrial cancer [10]. In addition, K-ras mutations have been
identified in endometrial hyperplasia and complex atypical hyperplasia, suggesting that K-ras
mutation may be an early event in the carcinogenesis of endometrium [11, 12]. However, Kras mutations in uterine adenocarcinoma in Thai patients have not yet been investigated. In
this study, we examine the frequency of K-ras mutations in Thai patients with endometrioid
adenocarcinoma and investigate the correlation between the presence of K-ras mutations and
clinicopathogical variables including age, grade, and depth of myometrial invasion.
Methodology
Patients and tissue samples
Formaldehyde-fixed, paraffin-embedded tissue from cases of endometrioid
adenocarcinoma of the uterus were collected for this study. The samples were obtained from
62 cases treated at Songklanagarind University Hospital during 2011-2012, including
hysterectomy specimens in all cases. This study was approved by the Hospital Ethics
Committee.
DNA extraction and PCR analysis
DNA was extracted from 5-μm from paraffin section using QIAamp Tissue Kit
(Qiagen) following the manufacturer’s protocol. The PCR for K-ras exon2 was performed
using primer K-ras forward: 5’-GGTACTGGTGGAGTATTTGAT-3’, and K-ras reverse:5’ACTCATGAAAATGGTCAG AG-3’. To determine that all samples originally contained
DNA of sufficient quality and quantity, samples were co-amplified for the presence of an
internal standard, beta globin. The beta-globin primer were PC04:5’-CAACTTCATCCACG
TTCACC-3’, and GH20:5’-GAAGAGCCAAGGACAGGTAC-3’. The amplification reaction
was performed with initial denaturation at 94oC for 4 min, followed by 40 cycles of
denaturation 94oC for 45 sec, annealing at 51 oC (K-ras), 55 oC (beta-globin) for 45 sec, and
extension at 72 oC for 1 min, and with final extension at 72 oC for 7 min. The 291 bp of K-ras
product and the 268 bp of beta-globin product were run on a 2% agarose gel electrophoresis
and stained with ethidium bromide for size verification.
Direct sequencing
The PCR products were purified using Purelink PCR Purification kit (Invitrogen)
according to the manufacturer’s instruction. The purified PCR products were use as template
in cycle sequencing with the Big Dye Terminator v1.1 kit (Applied Biosystem). The
sequencing reactions were ethanol precipitated and run on a ABI PRISM 310 DNA sequencer
(Applied Biosystem).
Statistical analysis
The relationship between K-ras mutation and clinicopathological variables was
determined using Fisher’s Exact test. Statistical significance was set at P < 0.05.
Results
K-ras mutations were analyzed in 62 endometrioid adenocarcinomas and the
incidence of K-ras mutations was 16.1% (10 of 62, Table1). Of the 10 mutations, 60% (6 of
10) were found in codon 12, 20% (2 of 10) in codon 13, and 20% (2 of 10) in codon 12 with
codon 6 and codon 12 with codon 7, subsequently. No significant relationship was seen
between the presence of K-ras point mutation and clinicopathological variables, including
age, grade, and depth of myometrial invasion as shown in Table 2.
Table 1. Type of K-ras point mutations and amino acid change
Mutation
Hotspot codon mutations
codon 12
Nucleotide change
Amino acid change
K-ras mutation
GGT > TGT
GGT > GAT
GGT > AGT
GGT > GTT
Gly12Cys (G12C)
Gly12Asp (G12D)
Gly12Ser (G12S)
Gly12Val (G12V)
2
2
1
1
GGC > TGC
GGC > GAC
Gly13Cys (G12C)
Gly13Asp (G12D)
1
1
GGT > TGT
TTT > CTT
Gly12Cys (G12C)
Phe6Leu (F6L)
codon 13
codon 12 and codon 6
1
codon 12 and codon 7
1
GGT > GAT
GTG > ATG
Gly12Asp (G12D)
Val7Met (V7M)
Table 2. Clinicopathologic variables related to presence of K-ras mutation.
Data
Total
Age (years)
<50
50-60
>60
Number of cases
62
Cases of K-ras mutation
10
p value
12
31
19
3
5
2
NS
Histological grade
G1
G2
G3
33
16
13
6
3
1
NS
Myometrial invasion (%)
none
<50%
≥50%
5
32
25
0
6
4
NS
NS= No Significant
Discussion and Conclusion
The results from this study show that 16.1% (10 of 62) endometrioid
adenocarcinomas demonstrated K-ras mutations. The frequency of K-ras mutation in
endometrioid adenocarcinomas in our study is similar to other published reports, ranging
from 10% to 30% [11-14]. K-ras mutational analysis in this study revealed that each affected
case contained a single point mutation, either within codons 12 (G12C, G12D, G12S, and
G12V) or 13 (G13C and G13D). These data types of K-ras gene mutations are consistent
with the previous reports [15]. Most of the K-ras mutations found in human tumors involve
these codon 12 and 13, coding for glycines located in proximity of the catalytic site of RAS.
Any mutation resulting in the incorporation of a different amino acid at these positions leads
to a reduction of the intrinsic GTPase activity of RAS [16]. It is interesting that our data
showed a double point mutation of K-ras mutation (2 of 62) at codon 12 and 6 (G12C, F6L),
and codon 12 and 7 (G12D, V7M).
In summary, our findings demonstrate the contributions of K-ras mutation both a
single point mutation within codon 12 or 13 and a double point mutation to pathogenesis of
endometrioid adenocarcinoma and indicate no correlation with clinicopathological variables
such as age, grade, and depth of myometrial invasion.
References
1. Khuhaprema T, Srivatanakul P, Sriplung H, Wiangnon S, Sumitsawan Y, Attasara P, editors. Cancer in
Thailand Vol.V 2001-2003. Bangkok 2010.
2. Hanprasertpong J, Sakoprakraikij S, Geater A. Endometrial cancer in Thai women aged 45 years or
younger. Asian Pac J Can Prev. 2008; 9:58-62.
3. Manchana T, Khemapech N. Endometrial Adenocarcinoma in Young Thai Women. Asia Pacific J Cancer
Prev. 2008; 9:283-6.
4. Dahlgren E, Friberg LG, Johansson S, Lindström B, Odén A, Samsioe G, et al. Endometrial carcinoma;
ovarian dysfunction—a risk factor in young women. Eur J Obstet Gynecol Reprod Biol. 1991; 41:143-50
5. Doll A, Abala M, Rigaua M, Monge M, Gonzaleza M, Demajoa S, et al. Novel molecular profiles of
endometrial cancer-new light through old windows. J Steroid Biochem Mol Biol. 2008; 108:221-9.
6. Soliman PT, Oh JC, Schmeler KM, Sun CC, Slomovitz BM, Gershenson DM, et al. Risk factors for young
premenopausal women with endometrial cancer. Obstet Gynecol. 2005; 105:575-80.
7. Bansal N, Yendluri V, Wenham RM. The molecular biology of endometrial cancer and the implications for
pathogenesis, classification, and targeted therapies. Cancer Control. 2009; 16:8-13.
8. Kiaris H, Spandidos D. Mutations of ras genes in human tumors (review). Int J Oncol. 1995; 7:413-21.
9. Lax SF. Molecular genetic pathways in various types of endometrial carcinoma: from a phenotypical to a
molecular-based classification. Virchows Archiv. 2004; 444:213-223.
10. Alexander-Sefre F, Salvesen HB, Ryan A, Singh N, Akslen LA, MacDonald N, et al. Molecular assessment
11.
12.
13.
14.
15.
16.
of depth of myometrial invasion in stage I endometrial cancer: a model based on K-ras mutation analysis.
Gynecol Oncol. 2003; 91:218-25.
Sasaki H, Nishii H, Takahashi H, Tada A, Furusato M, Terashima Y, et al. Mutation of the Ki-ras
protooncogene in human endometrial hyperplasia and carcinoma. Cancer Res. 1993; 53:1906-10.
Dobrzycka B, Terlikowski SJ, Mazurek A, Kowalczuk O, Niklińska W, Chyczewski L, et al. Mutations of
the KRAS oncogene in endometrial hyperplasia and carcinoma. Folia Histochem Cytobiol. 2009; 41:14350.
Duggan BD, Felix JC, Muderspach LI, Tsao JL, Shibata DK. Early mutational activation of the c-Ki-ras
oncogene in endometrial carcinoma. Cancer Res. 1994; 54:1604-7.
Lax S, Kendall B, Tashiro H, Slebos RJ, Hedrick L. The frequency of p53, K-ras mutations, and
microsatellite instability differs in uterine endometrioid and serous carcinoma: evidence of distinct
molecular genetic pathways. Cancer. 2000; 88:814-24.
Xiong J, He M, Jackson C, Ou JJ, Sung CJ, Breese V, et al. Endometrial carcinomas with significant
mucinous differentiation associated with higher frequency of K-ras mutations: A morphologic and
molecular correlation study. Int J Gynecol Cancer. 2013; 23:1231-6.
Scheffzek K, Ahmadian M, Wittinghofer A. GTPase-activating proteins: helping hands to complement an
active site. Trends Biochem Sci. 1998; 23:257-62.
Acknowledgements: This work was supported by a grant from faculty of medicine, Prince of
Songkla University.