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1、论著 Study of microsatellite instability in epithelial ovarian tumors Yuan LU1,Xi-shi LIU 1 ,Yue-xiang WANG2,Hou-yan SONG2,Nanbert ZHONG3, 4 (1. Department of Gynecology, The Hospital of Obstetrics and Gynecology,Fudan University,Shanghai 200011, China; 2. The Key Laboratory of Molecular Medicine,Mini
2、stry of Education,Fudan University; 3. Peking University Center of Medical Genetics; Department of Medical Genetics, School of Basic Medical Sciences,Peking University, Beijing 100083, China; 4. Department of Human Genetics,New York State Institute for Basic Research in Developmental Disabilities,St
3、at- en Island,NY 10314,USA) ABSTRACT Objective:To evaluate the frequency of MSI in epithelial ovarian tumors and its relation- ship with clinicopathologic features. Methods:Ninety fresh specimens of epithelial ovarian tumors,in- cluding 74 primary and 16 secondary tumors, were collected. Microsatell
4、ite analysis was carried out using 5 mono- and dinucleotide markers from the National Cancer Institute Consensus Panel by fluorescence-la- beled polymerase chain reaction. Results:Of 90 epithelial ovarian tumors analyzed,18 demonstrated a high level of microsatellite instability(MSI-H) ,30 demonstra
5、ted a low level of microsatellite instability (MSI-L) ,and the remaining 42 exhibited microsatellite stability(MSS) . Frequency of microsatellite in- stability(MSI)at loci BAT-25 was higher than that at any other loci. No correlation was found between MSI level and patient age,tumor type,tumor diffe
6、rentiation(P 0. 05) . But the microsatellite instabili- ty-high phenotype correlates with clinical stage. It tended to occur more frequently in early-stage tumors (P =0. 03) . Conclusion:The frequent MSI in epithelial ovarian tumors suggests that it is an early event to involve in the development of
7、 epithelial ovarian tumors. KEY WORDS Ovarian neoplasms; DNA, Satellite; Polymerase chain reaction Ovarian carcinoma is one of the most lethal canc- ers of women in China , in which 90% are epithelial o- varian tumors 1. Since the identification of the associ- ation of microsatellite instability( MS
8、I) with colon cancer in 1993 by three independent groups,it has been shown to play a significant role in several other cancers,leading to the identification of an important genetic pathway in the development of cancer 2. MSI is identified when alleles of novel sizes are detected in microsatellite se
9、quences derived from cancer DNA that are not present in normal tissues of the same individu- al. The discovery of MSI in colorectal cancers and its linkage to HNPCC (hereditary nonpolyposis colorectal cancer syndrome)in 1993 has resulted in many ad- vances related to detection of cancer susceptibili
10、ty,re- sponse to chemotherapy,and clinical outcome 3. The role and incidence of MSI in ovarian cancer are matters of great controversy. In some studies,how- ever,MSI frequencies have ranged from 0 to 53% in sporadic ovarian cancers 4, 5. These studies varied greatly in terms of the type and number o
11、f loci studied, the criteria used for defining MSI,and the histological subtypes of ovarian cancer analyzed,however. There were no commonly accepted criteria for the number, type,and identity of microsatellites to be used in MSI testing until 1997,when the NCI(National Cancer In- stitute)Workshop on
12、 HNPCC proposed specific mark- ers for MSI assessment in HNPCC,consisting of 2 mononucleotide repeats(BAT26 and BAT25)and 3 dinucleotiderepeats( D5S346,D2S123and D17S250) ,collectively known as the NCI panel 6. This standard panel was subsequently adapted for use in assessing ovarian cancer. The rol
13、e and incidence of MSI in epithelial ovari- an tumor remains unknown. This study was conducted to evaluate the frequency of MSI in epithelial ovarian tumors and its relationship with clinicopathologic fea- tures. It may help us understand the molecular events involved in the development of ovarian t
14、umors. 1 Patients and Methods 1. 1 Tissue samples and clinical information Matched pairs of fresh tumor tissue samples and peripheral blood samples were obtained from 90 women with ovarian epithelial cancer. Peripheral blood were obtained from patients as a source of normal DNA. These patients were
15、collected from The Obstetrics and Gynecology Hospital and Oncology Hospital of Fudan University. Clinical information was obtained from pa- thology reports and/ or patient charts.Tumor stages were assigned according to the classification system of the International Federation of Gynecology and Obste
16、t- rics ( FIGO) .Histological diagnosis was assigned based on the World Health Organization(WHO)clas- sification. Involvement of human subjects was followed the federal regulation and approved by Institution Re- view Board. Of the 90 patients,the average age is(50. 33 11. 82)years; 74 were primary c
17、ancers, 16 were recur- rent cancers; 20 had stage disease, 7 had stage , 44 had stage ,and 5 had stage ;9 were high grade , 23 were middle grade , 39 were low grade ; 44 Fund project (基金项目) :Supported by the National Natural Sciences Foundation of China (30571952)and Special Fund for Promotion of Ed
18、uca- tion, Ministry of Education P. R. C (05JC14012)国家自然科学基金和教育部教育振兴行动计划特殊专项 ( “九八五” 工程) Corresponding author s e-mail, lxsdoc hotmail. com 26 北京大学学报 (医学版) JOURNAL OF PEKING UNIVERSITY (HEALTH SCIENCES) Vol. 38 No. 1 Feb. 2006 were serous adenocarcinoma, 5 were mucous adenocar- cinoma,13 were endome
19、triod cancer,11 were clear- cell carcinoma,and 17 were the other epithelial ovari- an tumors,respectively. 1. 2 Research methods 1. 2. 1 DNA extraction Fresh tumor tissue samples and peripheral blood samples were digested by protein- ase K overnight. DNA isolation and preparation were according to m
20、olecular cloning:a laboratory manual. DNA was diluted to 50 mg/ L. 1. 2. 2 Microsatellite loci and primer The five mark- ers ( BAT25, BAT26, D5S346,D2S123, and D17S250)recommended by the NCI were evaluated, in which BAT25 and BAT26 are mononucleotide repeat sequences and D5S346, D2S123,and D17S250 a
21、re di- nucleotide repeat sequences.The primer sequences were obtained from genebank. Oligonucleotide forward primers were synthesized and fluorescently 5 labeled with FAM or HEX by genecane biotechical company. Table 1 presents five microsatellite loci primer se- quences and amplication size. Table
22、1 Five microsatellite loci primer sequences and amplication size Sequences Amplication size Bat-25 F: 5-TCGCCTCCAAGAATGTAAGT-3 R: 5-TCTGCATTTTAACTATGGCTC-3 120 bp Bat-26 F: 5-TGACTACTTTTGACTTCAGCC-3 R: 5-AACCATTCAACATTTTTAACCC 3 116 bp D2S123 F: 5-AAACAGGATGCCTGCCTTTA-3 R: 5- GGACTTTCCACCTATGGGAC-3
23、197 - 227 bp D17S250 F: 5-GGAAGAATCAAATAGACAAT-3 R: 5-GCTGGCCATATATATATTTAAACC-3 153 bp D5S346 F: 5-ACTCACTCTAGTGATAAATCGGG-3 R: 5-AGCAGATAAGACAGTATTACTAGTT-3 96 - 122 bp F, forward; R, reverse. 1. 2. 3 PCR(polymerase chain reactions) Multiple polymerase chain reactions(PCRs)were carried out in a 15
24、 L mixture containing approximately 100 ng DNA, 1. 5 L 10 PCR buffer,1. 5 L 5 mol/ L of each PCR primer(Genecane Biotechical Company) , 2. 5 mmol/ L GeneAmp dNTP mix 4 uL,AmpliTaq Gold DNA Polymerase 0. 5 uL,25 mmol/ L MgCl2 3uL,Sterlie D. I. H2O 2. 5 uL. PCR amplification was conducted in a GeneAmp
25、 PCR System Thermo Cy- cler 9700. Samples were denatured at 95 for 12 mi- nutes and then subjected to 10 cycles consisting of de- naturation(94 for 15 seconds) ,annealing(55 for 15 seconds) ,and extension(72 for 30 sec- onds) ,and then to 30 cycles consisting of denaturation (89 for 15 seconds) ,ann
26、ealing(55 for 15 sec- onds) ,and extension(72 for 30 seconds)and a fi- nal extension step at 72 for 10 minutes,then stored at 4 . The PCR amplication were loaded on the ABI PRISM 3700 Genetic Analyzer(Applied Biosystems) . The data were collected automatically and analyzed u- sing the GeneScan 3. 7
27、and Genotyper Analysis soft- ware(Applied Biosystems) ,which automatically de- termined the actual size of the PCR products and the a- mount of fluorescent signal. 1. 2. 4 MSI analysis MSI was indicated by the pres- ence of novel peaks in the tumor tissue that were not seen in normal control tissue
28、from the same patient or by a difference in microsatellite lengths in the 2 sam- ples. Tumors exhibiting MSI at 2 or more markers were defined as MSI-high(MSI-H) . Tumors showing insta- bility at only 1 marker were defined as MSI-low(MSI- L) . Tumors in which no markers exhibited MSI were considered
29、 to exhibit microsatellite stability(MSS) . 1. 2. 5 Statistical analysis x2or Fisher s exact test was used as appropriate to determine differences be- tween variables using SPSS 11. 5. P 0. 05) . But the MSI-H phenotype corre- 36卢 媛, 等 上皮性卵巢癌中微卫星不稳定的研究 late with clinical stage,it tended to occur mor
30、e fre- quently in early-stage tumors(P = 0. 03) . The detail is present in Table 4. Table 2 The frequence of MSI at every loci LociMSIPercent BAT-253235.5% (32/90) BAT-2644.4% (4/90) D2S1231516.5% (15/90) D5S3461718.8% (17/90) D17S2501213.3% (12/90) Table 3 MSI of various number loci N(Positive loci
31、)nPercent 04246.6% (42/90) 13033.3% (30/90) 21213.3% (12/90) 333.3% (3/90) 433.3 (3/90) Table 4 Association between MSI status and clinicopathologic variables in 90 epithelial ovarian tumors n (%) MSSMSI-LMSI-HTotalP Stage 4 (20%)7 (35%)9 (45%)200.003 4 (57%)2 (29%)1 (14%)7 25 (58%) 15 (35%) 3 (7%)4
32、3 0 (0%)2 (50%)2 (50%)4 Grade High3 (38%)4 (50%)1 (12%)80.905 Middle14 (44%) 12 (38%) 6 (18%)32 Low21 (42%) 19 (38%) 10 (20%)50 Type Serous20 (48%) 13 (31%) 9 (21%)420.194 Mucous1 (20%)2 (40%)2 (40%)5 Endometriod 8 (61%)5 (39%)0 (0%)13 Clear cell7 (64%)0 (0%)4 (36%)11 Others6 (32%)9 (47%)4 (21%)19 P
33、rimary33 (44%) 25 (34%) 16 (22%)740.529 Recurrence9 (56%)5 (31%)2 (13%)16 3 Discussion Microsatellites are widely used for linkage analysis and studies on loss of heterozygosity and are generally highly polymorphic. Assessment of MMR defects has become an important tool in tumor molecular pathology
34、and clinical practice. MSI has been detected in many solid malignancies although the definition of instability applied has been variable. It is most commonly found in sporadic malignancies that also occur in the HNPCC syndrome such as colorectal, stomach, endometrial and ovarian cancer. MSI may impa
35、rt a favorable prognosis in colorectal,gastric,pancreatic and probably oesoph- ageal cancers but a poor prognosis in non small cell lung cancer. In clinical studies colorectal cancers dem- onstrating MSI respond better to chemotherapy while in vitro studies using MSI positive cell lines show resist-
36、 ance to radiotherapy and chemotherapy 7. MSI-H and MSI-L/ MSS phenotypes appear to characterize 2 differ- ent pathways of carcinogenesis. Patients who present with MSI-H carcinomas have a better prognosis than those with MSI-L/ MSS tumors.The status of MMR may be important in predicting tumor respo
37、nse to clini- cal therapy 6. Several lines of indirect evidence suggest that MSI might play a role in the genesis of ovarian carcinoma. Ovarian carcinoma and endometrial carcinomas are two common exocolonic tumors occurring in patients with HNPCC,which is characterized by a high MSI fre- quency. MSI
38、 occurs in approximately 20% of endome- trial carcinomas,a tumor closely related to ovarian car- cinoma 8. Nevertheless,there is no general agreement about the frequency of MSI in sporadic ovarian carcinomas. Some studies have reported very low frequencies, whereas others have reported high frequenc
39、ies(as high as 53%) 9 -13. In addition,several studies have sug- gested an association between MSI and certain histolog- ical types of ovarian carcinoma. For example,Fujita et al 14demonstrated that the incidence of MSI was signif- icantly higher in the endometrioid subtype of ovarian tumor( 50%) th
40、an in other histological subtypes (8%) . Ohwada et al.9found MSI happened more frequently in mucinous adenocarinoma(38%)than in serous carcinoma(13%) . King et al 15. found a high frequency of MSI in endometrioid carcinoma(33. 3%) and a relatively lower frequency in invasive serous car- cinoma(8%) .
41、 Tangir et al.16and Haas et al.13re- ported that MSI-H occurred with very low frequency in patients with serous adenocarcinoma(6. 5% and 0, respectively)but relatively often in epithelial border- line ovarian tumors(27. 8% and 30%) .Shih et al.17did not find any MSI in 31 ovarian neoplasms of low ma
42、lignant potential. Arzimanoglou et al.18,how- ever,demonstrated that MSI inovarian cancer is not definitely associated with a specific histopathologic sub- type. In most of these studies,unfortunately,different kinds of microsatellite markers were used and MSI was assessed with the demonstration of
43、instability at only one locus,which is no longer considered as an ade- quate criterion for MSI positivity. Most of these studies were done before the development of a standard panel of microsatellite marker sand standardized criteria for determining MSI. This may at least partly explain the controve
44、rsial frequency of MSI observed in these stud- ies. Until now,the NCI markers and criteria have been used in some studies of MSI in ovarian cancer. Sood et al.19found MSI-H in 12% of invasive ovarian tumors,and Gras et al 20 reported that MSI-H was limited to endometrioid and clear cell carcinomas (
45、12. 5%) . Liu J et al.21demonstrated that 20% of endometrioid carcinomas were MSI-H. Kathy et al.22 found MSI-H in 6 of 42(14. 3%)ovarian clear cell carcinomas using the new-standard NCI markers and criteria. Dellas A et al.23concluded MSI-H in carci- nomas was significantly associated with poor dif
46、ferentia- tion( P = 0. 03)and higher clinical stage( P = 46 北京大学学报 (医学版) JOURNAL OF PEKING UNIVERSITY (HEALTH SCIENCES) Vol. 38 No. 1 Feb. 2006 0. 03) . In the current study,we found MSI-H in 18 of 90 (20%)ovarian epithelial carcinomas using the new- standard NCI markers and criteria. The incidence
47、of MSI-H tended to occur more frequently in poor differ- entiation and mucous adenocarcinoma and clear cell carcinoma. But it showed no correlation with patient age,tumor differentiation,different histological types or other clinicopathologic features. the MSI-H pheno- type was significantly associa
48、ted with clinical stage,it tended to occur more frequently in early-stage tumors (P =0. 03) . In conclusion,our study suggests that MSI-H has a role in the pathogenesis of epithelial ovarian cancers. Microsatellite instability is a relatively common event in ovarian carcinoma. It is an early event a
49、nd it is in- volved in the development of epithelial ovarian tumors. References 1Greenlee RT,Murray T,Bolden S,et al. Cancer statistics J . CA Cancer J Clin, 50: 7 -33. 2 Thibodeau SN,Bren G,Schaid DJ. Microsatellite instability in cancer of the proximal colon J . Science, 1993, 260: 816 -819. 3Sood AK. Holmes R,Hendrix MJC,et al. Application of the Na- tional Cancer Institute International Criteria for determination of microsatellite instability in ovarian cancer J . C
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