Microsatellite In sta bil ity (MSI) Anal y sis in Pa tients with Endometrial Can cer*

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1 Pol J Pathol 2005, 56, 1, PL ISSN Gra yna Dec 1, Hanna Romanowicz-Makowska 2, Beata Smolarz 2, Andrzej Kulig 2 Microsatellite In sta bil ity (MSI) Anal y sis in Pa tients with Endometrial Can cer* 1 De part ment of Gy ne col ogy and Ob stet rics, Med i cal Uni ver sity, ódÿ, 2 Laboratory of Molecular Genetics, Department of Pathology, Institute of Polish Mother s Memorial Hospital, ódÿ Microsatellite in sta bil ity (MSI) seems to be im por - tant for the de vel op ment of var i ous hu man can cers in - clud ing spo radic endometrial can cer. The aim of this study was eval u a tion of microsatellite in sta bil ity in 20 postmenopausal women with endometrial adeno carci - noma in DNA sam ples ob tained from can cer tis sue and blood of the same pa tients. Con trol DNA was ob tained from nor mal endometrial tis sue (n=25). MSI was stud - ied at five loci con tain ing sin gle- or dinucleotide re peat sequences and mapping to different chromosomal loca - tions: BAT-25 (at lo cus 4q12), BAT-26 (2p16), D2S123 (2p16-p21), D5S346 (5q21-q22) and D17S250 (17q11.2-q12). No dif fer ences in the MSI fre quen cies be - tween blood and can cer tis sue ob tained from pa tients were de tected. The microsatellite in sta bil ity sta tus was sig nif i cantly higher in endometrial can cer tis sue [5/20 (25%)] as com pared to con trol [3/25 (12%)] (p<0.05). There were no sig nif i cant dif fer ences be tween MSI pres - ence in the sub groups as signed to the histological grades (p>0.05). The re sults sug gest that the microsatellite in - sta bil ity seems to be im por tant in the de vel op ment of spo radic endometrial can cer. Introduction Microsatellite se quences are short re peat nu cle o tide se - quences dis sem i nated in whole genome in nor mal con di tions. In Eucaryota ge nome there are re peated se quences con sist ing of one, two, three and four nu cleo tides. Over 90% of the till this mo ment stud ied microsatellite sequences from mono - nucleotides to tetranucleotides show poly mor phism. Small de le tions or ex pan sions in tu mor DNA, man i fested as shifts in allelic electrophoresis mobility characterize microsatellite in - stability (MSI). Genetic instability is considered to be responsible for a rapid accumulation of somatic mutations in various tu mor sup pres sor genes and onco genes, thus play ing an im - por tant role in the ini ti a tion and pro gres sion of ma lig nant tu - mors [16, 17]. Pre vi ous stud ies have in di cated that MSI seems to be im por tant in the de vel op ment of var i ous hu man can cers [2, 25, 33] in clud ing spo radic endometrial can cer (EC) [7, 9, 21, 31]. Endometrial can cer is one of the most com mon ma lig - nant neoplasms, which ap pear in uter ine body [26]. About 80% of the cases are di ag nosed af ter meno pause. The high - est in ci dence es ti mated in years is mov ing to 6 and 7 de cade of life at pres ent [4]. Endometrial can cer is fourth the most com mon fe male car ci noma [29]. An nu ally 150,000 new cases of this can cer are noted world wide. Ev ery year 65 new cases of endometrial can cer in age group years are di ag nosed among ev ery 100,000 women. Some risk fac tors have been iden ti fied, re lated to re pro - duc tion (such as early age at men ar che, late age at meno pause and nulliparity) or more immediately estrogen-related (i.e. con di tions such as the polycystic ovar ian syn drome) [4, 30]. Prognostic factors important in the evaluation of EC are: tu mor size, tu mor grade, pres ence or ab sence and depth of myometrial invasion, myometrial lymphatic/vascular space invasion, steroid hormone receptor status, ploidy and proliferative indices. How ever, these fac tors pres ent an in com - plete picture of the tumor biology. Therefore, investigation of other prognostic factors is of special clinical relevance, particu larly in view of the un ex pect edly pro gres sive course of the dis ease and fre quent re lapses in some cases. MSI is char ac ter ized by small in ser tions or de le tions within short tan dem re peats in tu mor DNA when com pared to the cor re spond ing nor mal DNA. MSI was firstly dem on - strated in pa tient with he red i tary nonpolyposis colorectal carcinoma (HNPCC), an inherited cancer syndrome that * This work was sup ported by the grant No. 6P05E12220 from the State Com mit tee for Sci en tific Re search (KBN). 15

2 G. Dec et al pre dis poses also to endometrial can cer, which rep re sents the sec ond most com mon ma lig nancy in HNPCC fam i lies [14, 24]. Later stud ies have also dem on strated that MSI is pres - ent in about 20% of spo radic colorectal tu mors [20]. Endo - metrial can cer is one of the most com mon extracolonic tu mors as so ci ated with HNPCC, and MSI has been re ported to be pres ent in 9 45% of sporadic cases [5, 6, 12, 19, 27]. In the pres ent study MSI sta tus in women with spo - radic endometrial can cer was in ves ti gated. Material and Methods Endometrial can cer sam ples Twenty pa tients with histologically-proven di ag no sis of endometrial adenocarcinoma were in cluded in the study (mean age ±SD 63.75±4.72 years). Endometrial can cer was classified as sporadic after evaluation of patient s family his tory in or der to ex clude the pres ence of the HNPCC syn drome. Fam ily his tory for can cer was eval u ated by ques - tion naire in ter views in endometrial can cer pa tients at tend - ing the De part ment of Ob stet rics and Gy ne col ogy of the Medical University in ódÿ after the initial surgical treat - ment. Tu mor tis sues and blood were ob tained from post - menopausal women with endometrial adenocarcinoma treated at Department of Ob stet rics and Gy ne col ogy be - tween 2002 and Tis sues were frozen immediately and stored at -70 C. All tu mors were staged ac cord ing to the cri - te ria of the In ter na tional Fed er a tion of Gy ne col ogy and Ob - stet rics (FIGO). There were 7 tumors of I stage, 6 of II stage and 7 of III stage in to tal. DNA from nor mal endometrial tis - sue (n=25) served as a con trol. DNA isolation DNA was ex tracted from fresh ma te rial us ing com - mer cially avail able QIAmp Kit (Qiagen GmbH, Hilden, Ger many) DNA pu ri fi ca tion kit according to man u fac - turer s in struc tion. Microsatellite analysis Tu mor DNA and cor re spond ing nor mal DNA were an a lyzed us ing a panel of five microsatellite mark ers for mononucleotide and dinucleotide re peat se quences: BAT25 (at lo cus 4q12), BAT26 (2p16), D2S123 (2p16-p21), D5S346 (5q21-q22) and D17S250 (17q11.2-q12) [3]. All primer se quences were as re ported in Ge nome Da ta Base (GDB, at: The PCR was car ried out in a Perkin-Elmer/Gene Amp, PCR Sys tem 2400 ther mal cycler. The ther mal cy - cling con di tions were 60s at 94 C, 60s at 60 C, 60s at 72 C, re peated for 30 step cy cles. PCR am pli fi ca tion was per formed in a fi nal vol ume of 25 µl. The re ac tion mix ture con tained 5 ng of genomic DNA, 0.2 µmol of each ap pro - pri ate primer (ARK Sci en tific GmbH Biosystems, Darmstad, Ger many), 2.5 mm MgCl 2, 1 mm dntps and 1 unit of Taq Poly mer ase (Qiagen GmbH, Hilden, Ger - many). PCR prod ucts were frac tion ated by de na tur ing elec tro pho re sis in a 6% polyacrylamide gel (PAGE) and vi su al ized by sil ver stain ing. Statistical analysis For statistical analysis, the χ 2 test was per formed, p<0.05 was con sid ered sig nif i cant. Results A sam ple was clas si fied as MSI-high (MSI-H) if two or more mark ers showed in sta bil ity, MS-sta ble (MSS) if no in sta bil ity was noted, and MSI-low (MSI-L) if a sin gle marker re vealed novel bands com pared with the cor re spon - d ing normal DNA. TABLE 1 Num ber of endometrial can cer pa tients and con trol with the pres ence or ab sence of microsatellite in sta bil ity (MSI) MSI status Endometrial cancer patients (n=20) Control (n=25) Number Frequency Number Frequency MSI * * MSI-H MSI-L MSS * p<0.05 as com pared to con trols 16

3 MSI in endometrial cancer Fig. 1. Num ber of endometrial can cer pa tients (n=20) pre sent ing or not microsatellite in sta bil ity in re la tion to tu mor grade. MSI was de ter mined in 20 endometrial car ci noma tis - sues and blood from pa tients and in 25 con trol sam ples. There were no dif fer ences in MSI pres ence be tween blood and can cer tis sue ob tained from the same endometrial can - cer patients. Five out of 20 (25%) tu mors tested were found to be MSI pos i tive, 4 MSI-high and 1 MSI-low (Ta ble 1). It can be seen from the Ta ble 1 that there were sig nif i cant dif fer - ences in MSI fre quency be tween women with endometrial can cer and con trol. The fre quency of MSI pres ence in can - cer sam ples was higher than in nor mal sam ples (p<0.05). A de pend ency of the MSI fre quency dis tri bu tion on the tu mor grade eval u ated ac cord ing to FIGO cri te ria in women with endometrial can cer is dis played in Fig ure 1. The histological anal y sis of tu mor grade showed a lack of as so ci a tion (p>0.05) be tween tu mor grade and microsa - tellite instability presence. Discussion Endometrial tumorigenesis is still poorly un der stood. The de vel op ment of endometrial can cer is as so ci ated with an ac - cumulation of specific genetic alterations [22]. The activation of onco genes, the loss or in ac ti va tion of repressor genes and im paired mis match-re pair func tion are known to be in volved in the de vel op ment of tu mors. It is well known, that MSI plays an im por tant role in the pathogenesis and dis ease pro gres sion of endometrial can cer [1, 11, 15, 23, 18]. Many stud ies have doc u mented that MSI may be a marker of a ten dency for rep li ca tion er rors in hu man can - cers [13, 23, 31]. De fects in DNA mis match-re pair genes such as MLH1, MSH2 and MSH6 lead to rep li ca tion er - rors re vealed as in sta bil ity in microsatellite mark ers. Sev - eral stud ies have in di cated that loss of MLH1 ex pres sion can ac count for a large pro por tion of spo radic endometrial tu mors [8, 10, 28]. In the ma jor ity of endometrial tu mors with high MSI the loss of MLH1 ex pres sion was found, whereas loss of ex pres sion was less fre quent in tu mors with in ter me di ate MSI. Thus, patho log i cal ex pres sion of MLH1 does not seem to ac count for all tu mors with a MSI-pos i tive phe no type, in di cat ing that other mis match re pair genes might also be in volved [28]. In the light of sub stan tial ev i dence that the pro gres - sion of endometrial can cer can be as so ci ated with microsatellite in sta bil ity, it seems rea son able to check a possible cor re la tion be tween MSI and clin i cal sta tus of endometrial can cer pa tients. The pa tients in cluded in the study re ceived no che mo ther apy or hor mone ther apy. In this work con ducted on 20 endo metrial car ci noma pa tients we find a cor re la tion be tween MSI and oc cur rence of can - cer. Twenty-five per cent of endometrial can cer pa tients pre sented MSI, while only 3 nor mal sam ples from 25 healthy in di vid u als (12%) showed this pat tern. 17

4 G. Dec et al How ever, the histological anal y sis of tu mor grade sho - wed a lack of cor re la tion be tween tu mor grade and the num ber of patients presenting MSI. Re cent stud ies have sug gested that ge netic al ter ations, in clud ing p53 mu ta tions, loss of heterozygosity, and K-ras, PTEN and Her2/neu gene am pli fi ca tion, can be de tected in endometrial can cer [17]. Our find ings pro vide ad di tional evidence that genetic alterations, including MSI, may occur as rel a tively early events in the de vel op ment of endometrial cancer. The im pli ca tions of MSI in DNA from pa tients with endometrial can cer may be as so ci ated with dif fer ent tumo - rigenic path ways. The ge nome-wide MSI may be cor re lated to the ex is tence of pathogenetic mech a nisms in duc ing pro - gressive accumulation of sequence errors and providing a selective advantage during malignant evolution. Our study im plies that it is pos si ble that the MSI pro cess may be in volved in the appearance and/or pro gres sion of endometrial can cer. Fur ther stud ies, con ducted on a larger pop u la tion, are re quired to clar ify this point. Ref er ences 1. Arzimanoglou II, Gilbert F, Bar ber HRK: Microsatellite in sta bil ity in hu man solid tu mors. Can cer 1998, 82, Benachenhou N, Guiral S, Gorska-Flipot I, Labuda D, Sinnett D: Fre quent loss of heterozygosity at the DNA mis match-re pair loci hmlh1 and hmsh3 in spo radic breast can cer. Br J Can cer 1999, 79, Boland CR, Thibodeau SN, Ham il ton SR: Na tional Can cer In sti tute Work shop on Microsatellite In sta bil ity for can cer de tec tion and fa mil - ial pre dis po si tion: de vel op ment of in ter na tional cri te ria for the de ter - mi na tion of microsatellite in sta bil ity in colorectal can cer. Can cer Res 1998, 58, Bremond A, Bataillard A, Thomas L: Can cer of the endometrium. 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5 MSI in endometrial cancer tion and gene si lenc ing is the pri mary cause of microsatellite in sta bil ity in spo radic endometrial can cers. Hum Mol Genet 1999, 8, Spaczyñski M: Onkologia Ginekologiczna. Ur ban &Part ner, Wroc³aw Stefansson I, Akslen LA, Mac Don ald N, Ryan A, Das S, Jacobs IJ, Salvesen HB: Loss of hmsh2 and hmsh6 ex pres sion is fre quent in spo radic endometrial car ci no mas with microsatellite in sta bil ity: a pop - u la tion-based study. Clin Can cer Res 2002, 8, Sturzeneker R, Bevilacqua RA, Haddad LA, Simpson AJ, Pena SD: Microsatellite in sta bil ity in tu mors as a model to study the pro cess of microsatellite mu ta tions. Hum Mol Genet 2000, 9, Thibodeau SN, Bren G, Schaid D: Microsatellite in sta bil ity in can cer of the prox i mal co lon. Sci ence 1993, 260, Address for correspondence and reprint requests to: Beata Smolarz M.D. Laboratory of Molecular Genetics Department of Pa thol ogy In sti tute of Pol ish Mother s Me mo rial Hos pi tal Rzgowska 281/289, ódÿ Phone: smolbea@wp.pl 19