RAS genes are the most frequently mutated oncogenes detected in human cancer. In this study we analyzed the presence of mutations at codon 12 of the KRAS gene in 78 women with ovarian tumor, including 64 invasive ovarian cancers and 14 borderline ovarian tumors, using an RFLP-PCR technique and we evaluated whether such alterations were associated with the selected clinicopathological parameters of the patients. KRAS codon 12 gene mutations were found in 6,2% of ovarian cancer tissue and in 14,3% of the borderline ovarian tumor. KRAS mutations were found with a significantly higher frequency in mucinous and borderline tumors compared to serous tumors (p&lt;0,01). Mutation frequency was correlated with the histological type of tumor, but not with stage, grade or patients age

Bozena Dobrzycka

Lech Chyczewski

Marek Kulikowski

Oksana Kowalczuk

Sławomir J Terlikowski

Wiesława Niklińska

Folia Histochemica et Cytobiologica

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©Polish Histochemical et Cytochemical SocietyFolia Histochem Cytobiol. 2009:47(2): 221 (221-224) doi: 10.2478/v10042-009-0039-6IntroductionOvarian cancer is the sixth most frequent female can-cer type and the sixth most frequent cause of deathfrom cancer among women in Poland. Approximately2700 new cases appear every year, constituting an age-standardized incidence rate of 14,4 per 100 000, whichis one of the highest incidence rates observed world-wide [1].Most human malignancies are the end result of anaccumulation of mutations within tumor-suppressorgenes and oncogenes as well as of the dysregulation ofspecific genes resulting in the antiapoptotic proteinseliminations [2]. Molecular studies have identifiedseveral genetic alterations such as p53, KRAS, c-erB-2,PTEN, HER-2/neu and BRCA1 mutations in ovariantumors [3,4].The RAS family of oncogenes is constituted ofthree principal members -KRAS, HRAS and NRAS – allof which have been implicated in the development ofhuman malignancies. The KRAS oncogene resides onchromosome 12p12 and encodes a 21-kD protein(p21RAS) involved in the MAP-kinase signal trans-duction pathway, modulating cellular proliferation anddifferentiation. Mutations of the KRAS oncogene resultin constitutive activation of this signal transductionpathway and consequently unregulated proliferationand impaired differentiation [5,6]. Activation of RAS oncogenes also occurs in ovari-an tumors. Some studies have shown that KRAS muta-tions are more frequent in mucinous than in nonmuci-nous neoplasm [7-9], whereas other studies have notrevealed correlation with histological type [10]. All thereported studies are based on a relatively small numberof patients and therefore, the results remain a subjectof debate.In this study, we analyzed the presence of muta-tions at codon 12 of the KRAS gene in 78 ovariantumors by using a restriction fragment length poly-morphism-polymerase chain reaction technique(RFLP-PCR) and we evaluated whether such alter-ations correlated with the selected clinicopathologicalparameters of the patients.Materials and methodsIn this study, we analyzed the presence of mutations at codon 12 ofthe KRAS gene in 78 women with ovarian tumor, including 64invasive ovarian cancers and 14 borderline ovarian tumors. Alltumors were staged according to the criteria of the InternationalCorrespondence: B. Dobrzycka, Dept. of Gynecological andObstetrical Nursing, Medical University of Bia³ystok, Warszawska 15, 15-062 Bia³ystok, Poland; tel.: (+4885) 748 88 63,fax.: (+4885) 74 88 860, e-mail: bdobrzycka@gmail.comFOLIA HISTOCHEMICAET CYTOBIOLOGICAVol. 47, No. 2, 2009pp. 221-224Mutations in the KRAS gene in ovarian tumorsBo¿ena Dobrzycka1, S³awomir J. Terlikowski1, Oksana Kowalczuk2, Wies³awa Nikliñska3, Lech Chyczewski2, Marek Kulikowski11Department of Gynecological and Obstetrical Nursing, 2Department of Clinical Molecular Biology,3Department of Histology and Embryology, Medical University of Bia³ystok, PolandAbstract: RAS genes are the most frequently mutated oncogenes detected in human cancer. In this study we analyzed thepresence of mutations at codon 12 of the KRAS gene in 78 women with ovarian tumor, including 64 invasive ovarian can-cers and 14 borderline ovarian tumors, using an RFLP-PCR technique and we evaluated whether such alterations were asso-ciated with the selected clinicopathological parameters of the patients. KRAS codon 12 gene mutations were found in 6,2%of ovarian cancer tissue and in 14,3% of the borderline ovarian tumor. KRAS mutations were found with a significantly high-er frequency in mucinous and borderline tumors compared to serous tumors (p<0,01). Mutation frequency was correlatedwith the histological type of tumor, but not with stage, grade or patients age. Key words: ovarian tumor, KRAS, point mutation, molecular detectionFederation of Gynecology and Obstetrics (FIGO). The median ageof the patients was 58 (range, 34-75) years. The protocol was pre-viously approved by the Bioethical Committee of the MedicalUniversity of Bia³ystok (R-I-003/206/2004). Serial paraffin sections were stained with H&E for light micro-scopic study. Clinicopathological information was obtained frommedical charts. Histopathological examination was performedaccording to the WHO classification. Tissues were immediatelyfrozen in liquid nitrogen after operation and stored at -80°C untiluse. We microscopically confirmed that the tumor specimens con-sisted mainly of carcinoma tissue (80%). DNA extraction was per-formed with a "GeneElute Mammalian Genomic DNA MiniprepKit" (Sigma), according to the manufacturer's instructions. BeforeDNA extraction, tumor samples (30 – 50 mg) were minced by theuse of a sterile scalpel and then digested for overnight at 37°C in180 μl of tissue lysis buffer from the kit, containing 20 μl of pro-teinase K solution (10 mg/ml). Isolated DNA was stored at -20°Cbefore further assays. The detection of KRAS mutations at codon 12 was performedby PCR-RFLP method and the results of the detection were veri-fied by direct sequencing of PCR products. DNA amplification wasperformed in 20 μl reaction mixture containing 10 – 100 ng ofgenomic DNA isolated from tissue, 1.5 mM of MgCl2, 0,2 mM ofdNTPs (Sigma), 0.2  M of each of the primers K1 and DD5P and1,0 U of Taq DNA polymerase (Sigma) in 1× PCR buffer suppliedby the polymerase manufacturer. The K1 upstream primer (5'-ACT GAA TAT AAA CTT GTG GTA GTT GGA CCT -3') wasimmediately upstream of K-RAS codon 12 and introduced a G to Csubstitution at the first position of the codon 11 of K-RAS in orderto create a BstOI restriction site (5'-CCT/AGG-3') within the aboveamplified fragment which overlapped the first two nucleotides ofcodon 12 and was lost when codon 12 mutations took place. Thedownstream primer DD5P was as follows: 5'-TCA TGAAAA TGGTCA GAG AA-3'. After the initial DNA denaturation at 95°C for 3 min, PCR wascarried out for 40 cycles (94°C for 15 s, 56°C for 15 s, 72°C for 15 s)followed by terminal extension of PCR products at 72°C for 7 min-utes. PCR products were then digested with a restriction endonu-clease BstOI. For this purpose, five-microliter aliquots of the post-PCR reaction mixture were digested with 10 U of the restrictionendonuclease BstOI (PROMEGA) in the appropriate reactionbuffer (supplied by the enzyme manufacturer) in the final volumeof 10 μl at 60°C for 3 h. The additional aliquot of 5 U of theenzyme was added to the reaction mixture after the first hour of thedigestion. The enzyme recognized the sequence 5'-CCTGG-3',which was present in codon 12 KRAS wild type PCR products, butwas absent from the mutant ones. As a result, only the wild typemolecules were digested into two fragments – 160 bp and 29 bplong. The digestion products were then electrophoresed on a 6%native polyacrylamide gel, stained with ethidium bromide and pho-tographed on a ultraviolet light transilluminator with the use ofUVI-KS 400i/Image PC system. The non-restricted PCR productswere 189 bp long, whereas the wild type codon 12 products, beingrestricted inside codon 12 sequence, were 160 bp long. All mutations were then confirmed by direct sequencing of thePCR products. For this purpose, antisense strain of PCR productswere sequenced with the antisense primer DD5P, an ABI PRISMBigDye Terminator v.3.1 Cycle Sequencing Ready Reaction(Applied Biosystems) and an automatic ABI PRISM 377 DNAsequencer (Applied Biosystems). A wild-type control DNA sample(without KRAS codon 12 mutation) and a known mutation samplewere included in all the experiments. All experiments were dupli-cated precisely. For statistical analysis, the χ2 test was performed;p<0.01 was considered significant.ResultsThe age of the patients ranged from 34 to 75 years(mean, 58). KRAS gene mutations at codon 12 in tumortissue were detected in 4 of 64 (6.2%) cases with pri-mary invasive ovarian carcinomas and in 2 of 14(14,3%) cases with a borderline ovarian tumor (Fig. 1).We detected KRAS gene mutation in 1 of 36 serousadenocarcinomas (2.7%), 3 of 13 mucinous adenocar-cinomas (23%) and 2 of 14 borderline ovarian tumors(14.3%). No mutations were detected in undifferentiat-ed carcinomas, papillary adenocarcinomas, endometri-oid adenocarcinomas, nonepithelial carcinomas and inclear cell neoplasms (Table 1, 2). Overall, a KRAS codon 12 point mutation wasdetected in approximately 10% (8/78) of the examinedtumors and did not correlate with the malignant poten-tials (e.g. stage and grade) and patients' age (Table 1).There was a tendency towards a higher incidence ofKRAS mutations in the mucinous tumors – 23% (3/13)than in the borderline and serous tumors – 14.3%(2/14) and 2.7% (1/36), respectively. These statisticaldifferences are significant (p<0.01). 222 B. Dobrzycka et al.©Polish Histochemical et Cytochemical SocietyFolia Histochem Cytobiol. 2009:47(2): 222 (221-224) doi: 10.2478/v10042-009-0039-6Fig. 1. Examples of mutation analysis of KRAS gene. (A)  KRAS codon 12 restriction fragment length polymorphism analysis. (B) Asequencing electropherograms with KRAS codon 12 mutants.  (←) mutation in KRAS gene, (*) a mismatch base in artificial forwardprimer, SM, size marker; WT, wild type, M, mutant.DiscussionEpithelial ovarian tumors are a complex clinical, diag-nostic and therapeutic challenge because of the diffi-culty of early detection, lack of known precursorlesions and high mortality rates. Advanced-stage dis-ease display aggressive chemoresistant behavior withpatients demonstrating a shorter median survival rate[11,12]. Extensive studies have been performed on humansamples assessing the presence of activated forms ofKRAS in numerous malignant neoplasms, e.g.: pan-creas (60%), biliary tract (32%), large intestine (32%),small intestine (20%), gastrointestinal tract (site inde-terminate) (19%), thymus (15%) and endometrium(14%) [13].In epithelial ovarian cancers, the incidence ofKRAS point mutations is between 15% and 39%[13,14]. In our study KRAS mutation frequencies seemto be highly related to tumor histology. There was atendency towards a higher incidence of KRAS muta-tion in the mucinous tumors (23%) than in the border-line and serous tumors (14.3% and 2.7%), respective-ly. In general, KRAS mutations occur more frequentlyin mucinous tumors, including borderline malignan-cies, than in nonmucinous tumors such as serous car-cinomas. Thus, point mutations in the KRAS geneseem to be more commonly associated with mucinouscarcinomas [15]. Our earlier study showed that mucinous borderlinetumors are clearly distinct from their serous counter-parts [16,17]. In this study we found that more muci-nous ovarian tumors have KRAS mutations than bor-derline tumors and other invasive cancers. Thus, bothmucinous ovarian cancer and borderline ovarian tumorare basically different from other ovarian cancers sincethe former are initiated by specific mutations (e.g.KRAS ) while the latter are initiated by different mech-anisms [18].A few previous studies have determined the prog-nostic role of KRAS alteration in ovarian cancer [19].Several studies found no correlation between KRASgene mutations and survival [10,20], while very fewconcluded that there is a significant negative relation-ship using multivariate analysis [21].We found 3/13 of the investigated mucinous ovari-an cancer tissues (23%) to have a mutation in KRAScodon 12, which was the highest frequency detected ina subgroup of patients in this study, and significantlyhigher than the frequency of 1/36 for serous adenocar-cinomas (2.7%). All the two borderline ovarian tumors(2/14) found to have a mutation in KRAS codon 12were mucinous (14.3%). Thus, KRAS mutations fre-quency in our study was correlated with the histologi-cal type of tumor but not with other clinicopathologi-cal parameters such as grade, stage or patients age. 223Mutations in the KRAS gene in ovarian tumors©Polish Histochemical et Cytochemical SocietyFolia Histochem Cytobiol. 2009:47(2): 223 (221-224) doi: 10.2478/v10042-009-0039-6Table 1. Distribution of KRAS mutations according to clinico-pathological characteristics of the examined ovarian lesions.Table 2. KRAS gene mutations in the examined ovarian lesions.Acknowledgments: This work was supported by the MedicalUniversity of Bia³ystok, grant No. 3-90987P. References[ 1] Wojciechowska U, Dikowska J, Tarkowski W, et al. Nowot-wory z³oœliwe w Polsce w 2004 roku. Centrum OnkologiiInstytut im. Marii Sk³odowskiej-Curie, Warszawa 2004.[ 2] Blum HE. Molecular therapy and prevention of liver diseases.Adv Med Sci. 2007;52:29-36.[ 3] Kar R, Sen S, Singh A, et al. Role of Apoptotic Regulators inHuman Epithelial Ovarian Cancer. Cancer Biol Ther. 2007;22:6(7). [ 4] Kolasa IK, Rembiszewska A, Janiec-Jankowska A, et al.PTEN mutation, expression and LOH at its locus in ovariancarcinomas. Relation to TP53, K-RAS and BRCA1 muta-tions. Gynecol Oncol. 2006;103(2):692-697.[ 5] Keller JW, Haigis KM, Franklin JL, et al. Oncogenic K-RASsubverts the antiapoptotic role of N-RAS and alters modula-tion of the N-RAS: gelsolin complex. Oncogene. 2007;26(21):3051-3059.[ 6] Patra SK. Ras regulation of DNA-methylation and cancer.Exp Cell Res. 2008;314(6):1193-1201.[ 7] Garrett AP, Lee KR, Colitti CR, et al. K-RAS mutation maybe an early event in mucinous ovarian tumorigenesis. Int JGynecol Pathol. 2001;20(3):244-251.[ 8] Hø gdall EV, H?gdall CK, Blaakaer J, et al. K-RAS alterationsin Danish ovarian tumour patients. From the Danish "Malo-va" Ovarian Cancer study. Gynecol Oncol. 2003;89(1):31-36. [ 9] Semczuk A, Postawski K, Przadka D, et al. K-RAS gene pointmutations and p21ras immunostaining in human ovariantumors. Eur J Gynaecol Oncol. 2004;25(4):484-488.[10] Varras MN, Sourvinos G, Diakomanolis E, et al. Detectionand clinical correlations of ras gene mutations in human ovar-ian tumors. Oncology. 1999;56(2):89-96.[11] Bhoola S, Hoskins WJ. Diagnosis and management of epithe-lial ovarian cancer. Obstet Gynecol. 2006;107(6):1399-1410. [12] Chien JR, Aletti G, Bell DA, et al. Molecular pathogenesisand therapeutic targets in epithelial ovarian cancer. J CellBiochem. 2007;102(5):1117-1129. [13] Karnoub AE, Weinberg RA. Ras oncogenes: split personali-ties. Nat Rev Mol Cell Biol. 2008;9(7):517-531.[14] Tammela J, Odunsi K. Gene expression and prognostic sig-nificance in ovarian cancer. Minerva Ginecol. 2004; 56(6):495-502.[15] Mayr D, Hirschmann A, Löhrs U, et al. KRAS and BRAFmutations in ovarian tumors: a comprehensive study of inva-sive carcinomas, borderline tumors and extraovarianimplants. Gynecol Oncol. 2006;103(3):883-887.[16] Terlikowski S, Sulkowski S, Lenczewski A, et al. Study ofborderline and invasive mucinous ovarian tumors using Ki-67(MIB 1) antibodies and nucleolar organizer region (NOR)staining. Arch Gynecol Obstet. 1999;263:29-33. [17] Terlikowski S, Lenczewski A, Famulski W, et al. Expressionof nucleolar organizer regions (NORs) in ovarian epithelialtumors. Folia Histochem Cytobiol. 2001;39(2):161-162.[18] Fabjani G, Kriegshaeuser G, Schuetz A, et al. Biochip for K-RAS mutation screening in ovarian cancer. Clin Chem. 2005;51(4):784-787.[19] Singer G, Shih IeM, Truskinovsky A, et al. Mutational analy-sis of K-RAS segregates ovarian serous carcinomas into twotypes: invasive MPSC (low-grade tumor) and conventionalserous carcinoma (high-grade tumor). Int J Gynecol Pathol.2003;22(1):37-41.[20] Cuatrecasas M, Erill N, Musulen E, Costa I, Matias-Guiu X,Prat J. K-RAS mutations in nonmucinous ovarian epithelialtumors: a molecular analysis and clinicopathologic study of144 patients. Cancer. 1998;82(6):1088-1095. [21] Scambia G, Masciullo V, Benedetti Panici P, et al. Prognosticsignificance of ras/p21 alterations in human ovarian cancer.Br J Cancer. 1997;75(10):1547-1553.Submitted: 15 December, 2008Accepted after reviews: 21 February, 2009224 B. Dobrzycka et al.©Polish Histochemical et Cytochemical SocietyFolia Histochem Cytobiol. 2009:47(2): 224 (221-224) doi: 10.2478/v10042-009-0039-6

Mutations in the KRAS gene in ovarian tumors.

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Mutations in the KRAS gene in ovarian tumors.

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