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• W1996860520 abstract "Gastric cancer (GC) remains a leading cause of cancer mortality worldwide. Genetic factors are implicated, including DNA mismatch repair (MMR) deficiency manifested as tumor microsatellite instability (MSI). However, a standardized panel of markers and a definition of low-versus-high level MSI in GC are lacking. We examined a population-based cohort of early onset (≤50 yrs) gastric cancer. We identified 211 cases of early onset gastric cancer in Central-East Ontario from 1989 to 1993, with archival material available for 139 cases. Testing included a six-mononucleotide marker panel and a three-MMR immunohistochemical panel. Overall, 30% (41 of 139) of GC were MSI+, with allelic shifts at one to eight markers. An unexpected discordance between the BAT-25, BAT-26, and BAT-40 markers was observed in the MSI+ cases. Six cases showing multiple loci instability (≥3 markers MSI+/MSI-high) demonstrated MMR protein deficiency. Three novel hMLH1 mutations (two germline frameshift and one somatic nonsense) were also found. The only significant clinicopathological associations were increased tumor size in MSI+ cases (P = 0.04) and Lauren histotype (P = 0.006) and tumor grade (P = 0.007) in MSI-high cases. Tumor size, location, depth, nodal status, and Ming subtype were significant prognostic variables. Therefore, we propose a new definition of high-level MSI based on unifying characteristics of instability of more than or equal to three of six mononucleotide markers and loss of MMR protein expression. Gastric cancer (GC) remains a leading cause of cancer mortality worldwide. Genetic factors are implicated, including DNA mismatch repair (MMR) deficiency manifested as tumor microsatellite instability (MSI). However, a standardized panel of markers and a definition of low-versus-high level MSI in GC are lacking. We examined a population-based cohort of early onset (≤50 yrs) gastric cancer. We identified 211 cases of early onset gastric cancer in Central-East Ontario from 1989 to 1993, with archival material available for 139 cases. Testing included a six-mononucleotide marker panel and a three-MMR immunohistochemical panel. Overall, 30% (41 of 139) of GC were MSI+, with allelic shifts at one to eight markers. An unexpected discordance between the BAT-25, BAT-26, and BAT-40 markers was observed in the MSI+ cases. Six cases showing multiple loci instability (≥3 markers MSI+/MSI-high) demonstrated MMR protein deficiency. Three novel hMLH1 mutations (two germline frameshift and one somatic nonsense) were also found. The only significant clinicopathological associations were increased tumor size in MSI+ cases (P = 0.04) and Lauren histotype (P = 0.006) and tumor grade (P = 0.007) in MSI-high cases. Tumor size, location, depth, nodal status, and Ming subtype were significant prognostic variables. Therefore, we propose a new definition of high-level MSI based on unifying characteristics of instability of more than or equal to three of six mononucleotide markers and loss of MMR protein expression. Gastric carcinoma (GC) is a leading cause of cancer mortality worldwide. Genetic pathways involved in its development are not clearly delineated, although several genetic alterations are implicated including DNA mismatch repair (MMR) deficiency, manifesting as the microsatellite instability (MSI) phenotype.1Bevan S Houlston RS Genetic predisposition to gastric cancer.Q J Med. 1999; 92: 5-10Crossref Scopus (41) Google Scholar GC is also a manifestation of inherited cancer predisposition syndromes, including hereditary nonpolyposis colorectal cancer syndrome (HNPCC; MIM 114500) due to germline mutations in MMR genes hMSH2,2Leach FS Nicolaides NC Papadopoulos N Liu B Jen J Parsons R Peltomaki P Sistonen P Aaltonen LA Nystrom-Lahti M Guan XY Zhang J Meltzer PS Yu JW Kao FT Chen DJ Cerosaletti KM Fournier REK Todd S Lewis T Leach RJ Naylor SL Weissenbach J Mecklin JP Järvinen H Petersen GM Hamilton SR Green J Jass J Watson P Lynch HT Trent JM de la Chapelle A Kinzler KW Vogelstein B Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer.Cell. 1993; 75: 1215-1225Abstract Full Text PDF PubMed Scopus (2089) Google Scholar, 3Wang Q Lasset C Desseigne F Saurin JC Maugard C Navarro C Ruano E Descos L Trillet-Lenoir V Bosset JF Puisieux A Prevalence of germline mutations of hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 genes in 75 French kindreds with nonpolyposis colorectal cancer.Hum Genet. 1999; 105: 79-85Crossref PubMed Scopus (101) Google Scholar hMLH1,3Wang Q Lasset C Desseigne F Saurin JC Maugard C Navarro C Ruano E Descos L Trillet-Lenoir V Bosset JF Puisieux A Prevalence of germline mutations of hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 genes in 75 French kindreds with nonpolyposis colorectal cancer.Hum Genet. 1999; 105: 79-85Crossref PubMed Scopus (101) Google Scholar, 4Papadopoulos N Nicolaides NC Wei YF Ruben SM Carter KC Rosen CA Haseltine WA Fleischmann RD Fraser CM Adams MD Venter JC Hamilton SR Petersen GM Watson P Lynch HT Peltomaki P Mecklin JP de la Chapelle A Kinzler KW Vogelstein B Mutation of a mutL homolog in hereditary colon cancer.Science. 1994; 263: 1625-1629Crossref PubMed Scopus (1761) Google Scholar hMSH6,5Akiyama Y Sato H Yamada T Nagasaki H Tsuchiya A Abe R Yuasa Y Germ-line mutation of the hMSH6/GTBP gene in an atypical hereditary nonpolyposis colorectal cancer kindred.Cancer Res. 1997; 57: 3920-3923PubMed Google Scholar hPMS2,6Nicolaides NC Papadopoulos N Liu B Wei YF Carter KC Ruben SM Rosen CA Haseltine WA Fleischmann RD Fraser CM Adams MD Venter JC Dunlop MG Hamilton SR Petersen GM de la Chapelle A Vogelstein B Kinzler KW Mutations of two PMS homologues in hereditary nonpolyposis colon cancer.Nature. 1994; 371: 75-80Crossref PubMed Scopus (1433) Google Scholar and hMLH3.7Wu Y Berends MJ Sijmons RH Mensink RG Verlind E Kooi KA van der Sluis T Kempinga C van der Zee AG Hollema H Buys CH Kleibeuker JH Hofstra RM A role for MLH3 in hereditary nonpolyposis colorectal cancer.Nat Genet. 2001; 29: 137-138Crossref PubMed Scopus (146) Google Scholar hMSH2 and hMLH1 mutations account for most cases of colorectal cancer (CRC) exhibiting MSI as currently listed in mutation databases (eg, in the database of the International Collaborative Group on HNPCC [http://www.insight-group.org/] and in the human gene mutation database [http://archive.uwcm.ac.uk/uwcm/mg/hgmd0.html]). The human MMR system repairs DNA replication errors or physico-chemical induced damage. Microsatellite regions are susceptible to mutation due to slippage of DNA polymerase during DNA replication. Failure to excise these errors may lead to frameshift mutations in target genes such as TGFβRII, IGFIIR, E2F-4, and BAX.8Nakachi A Miyazato H Shimoji H Hiroyasu S Isa T Shiraishi M Muto Y Microsatellite instability in patients with gastric remnant cancer.Gastric Cancer. 1999; 2: 210-214Crossref PubMed Google Scholar, 9Loeb LA Microsatellite instability: marker of a mutator phenotype in cancer.Cancer Res. 1994; 54: 5059-5063PubMed Google Scholar, 10Loeb LA A mutator phenotype in cancer.Cancer Res. 2001; 61: 3230-3239PubMed Google Scholar, 11Chung YJ Song JM Lee JY Jung YT Seo EJ Choi SW Rhyu MG Microsatellite instability-associated mutations associate preferentially with the intestinal type of primary gastric carcinomas in a high-risk population.Cancer Res. 1996; 56: 4662-4665PubMed Google Scholar, 12Chung YJ Park SW Song JM Lee KY Seo EJ Choi SW Rhyu MG Evidence of genetic progression in human gastric carcinomas with microsatellite instability.Oncogene. 1997; 15: 1719-1726Crossref PubMed Scopus (74) Google Scholar, 13Oliveira C Seruca R Seixas M Sobrinho-Simoes M The clinicopathological features of gastric carcinomas with microsatellite instability may be mediated by mutations of different “target genes”: a study of the TGFbeta RII, IGFII R, and BAX genes.Am J Pathol. 1998; 153: 1211-1219Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar Carriers of MMR germline mutations have a fourfold increased risk of GC and a high risk of early onset CRC (early onset, CRC presenting at ≤50 years).14Watson P Lynch HT Extracolonic cancer in hereditary nonpolyposis colorectal cancer.Cancer. 1993; 71: 677-685Crossref PubMed Scopus (673) Google Scholar, 15Lynch HT Smyrk T Hereditary nonpolyposis colorectal cancer (Lynch syndrome): an updated review.Cancer. 1996; 78: 1149-1167Crossref PubMed Scopus (470) Google ScholarUnlike MSI testing in CRC,16Boland CR Thibodeau SN Hamilton SR Sidransky D Eshleman JR Burt RW Meltzer SJ Rodriguez-Bigas MA Fodde R Ranzani GN Srivastava S A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257PubMed Google Scholar, 17Boland CR Thibodeau SN Hamilton SR Sidransky D Eshleman JR Burt RW Meltzer SJ Rodriguez-Bigas MA Fodde R Ranzani GN Srivastava S Cancer Res. 1998; 58 (Perucho M: Correspondence. Cancer Res 1999, 59:249-256. In response to:): 5248-5257PubMed Google Scholar, 18Umar A Boland CR Terdiman JP Syngal S de la Chapelle A Ruschoff J Fishel R Lindor NM Burgart LJ Hamelin R Hamilton SR Hiatt RA Jass J Lindblom A Lynch HT Peltomaki P Ramsey SD Rodriguez-Bigas MA Vasen HF Hawk ET Barrett JC Freedman AN Srivastava S Revised Bethesda guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability.J Natl Cancer Inst. 2004; 96: 261-268Crossref PubMed Scopus (2430) Google Scholar there is no consensus on MSI testing in GC. Thus widely variable results on the frequency and definition of MSI in sporadic GC have been reported depending on the type (mono-, di-, tri-, tetra-, or pentanucleotide) and number of microsatellites used. For instance, low-frequency MSI (MSI-L; allelic shifts in one locus) in sporadic GC in Japan varies from 16 to 39%. High-frequency MSI (MSI-H; variously defined as ≥30 to 40% of loci with allelic shifts and/or BAT-26 locus instability alone) is reported in about 5 to 10% of these cancers.19Renault B Calistri D Buonsanti G Nanni O Amadori D Ranzani GN Microsatellite instability and mutations of p53 and TGF-beta RII genes in gastric cancer.Hum Genet. 1996; 98: 601-607Crossref PubMed Scopus (86) Google Scholar, 20Han HJ Yanagisawa A Kato Y Park JG Nakamura Y Genetic instability in pancreatic cancer and poorly differentiated type of gastric cancer.Cancer Res. 1993; 53: 5087-5089PubMed Google Scholar, 21Rhyu MG Park WS Meltzer SJ Microsatellite instability occurs frequently in human gastric carcinoma.Oncogene. 1994; 9: 29-32PubMed Google Scholar This is similar to Western populations with 24 to 84% showing MSI-L and 2 to 15% MSI-H.22Hayden JD Cawkwell L Quirke P Dixon MF Goldstone AR Sue-Ling H Johnston D Martin IG Prognostic significance of microsatellite instability in patients with gastric carcinoma.Eur J Cancer. 1997; 33: 2342-2346Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 23Theuer CP Campbell BS Peel DJ Lin F Carpenter P Ziogas A Butler JA Microsatellite instability in Japanese vs European American patients with gastric cancer.Arch Surg. 2002; 137 (discussion, 965–966): 960-965Crossref PubMed Scopus (38) Google Scholar The National Cancer Institute (NCI) developed guidelines for MSI testing in CRC using a panel of five microsatellite loci containing two mononucleotide repeats (BAT-25 and BAT-26) and three dinucleotide repeats (D2S123, D5S346, and D17S250) and defined MSI-L and MSI-H as instability in one and two or more markers, respectively. MSI analysis using BAT-25 and BAT-26 are usually sufficient to establish MSI-H in CRC,24Hoang JM Cottu PH Thuille B Salmon RJ Thomas G Hamelin R BAT-26, an indicator of the replication error phenotype in colorectal cancers and cell lines.Cancer Res. 1997; 57: 300-303PubMed Google Scholar, 25Zhou XP Hoang JM Li YJ Seruca R Carneiro F Sobrinho-Simoes M Lothe RA Gleeson CM Russell SE Muzeau F Flejou JF Hoang-Xuan K Lidereau R Thomas G Hamelin R Determination of the replication error phenotype in human tumors without the requirement for matching normal DNA by analysis of mononucleotide repeat microsatellites.Genes Chromosomes Cancer. 1998; 21: 101-107Crossref PubMed Scopus (227) Google Scholar however, Suraweera et al26Suraweera N Duval A Reperant M Vaury C Furlan D Leroy K Seruca R Iacopetta B Hamelin R Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR.Gastroenterology. 2002; 123: 1804-1811Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar proposed the addition of the quasimonomorphic mononucleotide markers NR-21, NR-22, and NR-24 that have a lower frequency of polymorphisms in African and Caucasian populations and are useful complements to BAT markers. Although the NCI criteria are effective in identifying MSI-H in CRC, they should be applied with caution for other malignancies. Therefore, the optimal set of markers to diagnose MSI in GC may be different and needs to be studied.Despite the nonuniformity of MSI testing in GC, a number of investigators have reported clinicopathological associations and prognostic significance of MSI-H in sporadic GC. Some suggest that MSI positive (MSI+) tumors occur predominantly in the gastric antrum, whereas others demonstrate an even distribution throughout the stomach.27Lin JT Wu MS Shun CT Lee WJ Wang JT Wang TH Sheu JC Microsatellite instability in gastric carcinoma with special references to histopathology and cancer stages.Eur J Cancer. 1995; 31A: 1879-1882Abstract Full Text PDF PubMed Scopus (52) Google Scholar Some authors report a higher frequency of MSI-H in GC with an intestinal/atypical appearance and in poorly differentiated subgroups.20Han HJ Yanagisawa A Kato Y Park JG Nakamura Y Genetic instability in pancreatic cancer and poorly differentiated type of gastric cancer.Cancer Res. 1993; 53: 5087-5089PubMed Google Scholar, 22Hayden JD Cawkwell L Quirke P Dixon MF Goldstone AR Sue-Ling H Johnston D Martin IG Prognostic significance of microsatellite instability in patients with gastric carcinoma.Eur J Cancer. 1997; 33: 2342-2346Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 27Lin JT Wu MS Shun CT Lee WJ Wang JT Wang TH Sheu JC Microsatellite instability in gastric carcinoma with special references to histopathology and cancer stages.Eur J Cancer. 1995; 31A: 1879-1882Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 28Strickler JG Zheng J Shu Q Burgart LJ Alberts SR Shibata D p53 mutations and microsatellite instability in sporadic gastric cancer: when guardians fail.Cancer Res. 1994; 54: 4750-4755PubMed Google Scholar Patients with MSI-H GC have also been shown to have a lower incidence of lymph node metastasis and an improved long-term survival (64 to 88%) compared with patients with microsatellite stable (MSS; defined as the absence of allelic shifts in tumor versus normal DNA) tumors (39 to 53%).13Oliveira C Seruca R Seixas M Sobrinho-Simoes M The clinicopathological features of gastric carcinomas with microsatellite instability may be mediated by mutations of different “target genes”: a study of the TGFbeta RII, IGFII R, and BAX genes.Am J Pathol. 1998; 153: 1211-1219Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 28Strickler JG Zheng J Shu Q Burgart LJ Alberts SR Shibata D p53 mutations and microsatellite instability in sporadic gastric cancer: when guardians fail.Cancer Res. 1994; 54: 4750-4755PubMed Google Scholar Of note, there are few studies specific to MSI in early onset gastric cancer (EOGC), and they have been limited to single-institution or multicenter collaborations showing variable MSI definitions, frequencies, and associations.29Hayden JD Cawkwell L Sue-Ling H Johnston D Dixon MF Quirke P Martin IG Assessment of microsatellite alterations in young patients with gastric adenocarcinoma.Cancer. 1997; 79: 684-687Crossref PubMed Scopus (30) Google Scholar, 30Shiao YH Bovo D Guido M Capella C Cassaro M Busatto G Russo V Sidoni A Parenti AR Rugge M Microsatellite instability and/or loss of heterozygosity in young gastric cancer patients in Italy.Int J Cancer. 1999; 82: 59-62Crossref PubMed Scopus (20) Google Scholar, 31Hayden JD Cawkwell L Dixon MF Pardal F Murgatroyd H Gray S Quirke P Martin IG A comparison of microsatellite instability in early onset gastric carcinomas from relatively low and high incidence European populations.Int J Cancer. 2000; 85: 189-191Crossref PubMed Google Scholar, 32Lim S Lee HS Kim HS Kim YI Kim WH Alteration of E-cadherin-mediated adhesion protein is common, but microsatellite instability is uncommon in young age gastric cancers.Histopathology. 2003; 42: 128-136Crossref PubMed Scopus (50) Google Scholar, 33Carvalho R Milne AN van Rees BP Caspers E Cirnes L Figueiredo C Offerhaus GJ Weterman MA Early-onset gastric carcinomas display molecular characteristics distinct from gastric carcinomas occurring at a later age.J Pathol. 2004; 204: 75-83Crossref PubMed Scopus (51) Google Scholar EOGC has a more aggressive clinical course than older cases, and the characterization of molecular alterations responsible in this cohort can be important in the understanding and future management of these patients.To clarify the role of MSI in EOGC, we used a well-defined population-based cohort of EOGC (≤50 years) and a hexaplex panel of quasimonomorphic mononucleotide markers (BAT-25, BAT-26, BAT-40, NR-21, NR-22, and NR-24) and two dinucleotide markers (D5S346 and D17S250) to determine MSI status. Five commonly used histological classifications were used to characterize the specific GC subtype. The objectives of this study were 1) to determine the frequency of MSI and MSI-H in EOGC; 2) to examine the loss of MMR protein expression (hMLH1, hMSH2, and hMSH6) in MSI+ cases using immunohistochemistry (IHC), methylation, and mutational analyses; and 3) to characterize genotype-phenotype correlations between MSI status and clinicopathological variables.Materials and MethodsPatients, Tissue Collection, Pathology Review, and DNA ExtractionUsing the Ontario Cancer Registry, 211 GC cases ≤50 years diagnosed between 1988 and 1993 in Central-East Ontario (population ∼ 4.8 million) were identified. Research Ethics Board of Mount Sinai Hospital, Toronto, Canada approved all study protocols. Formalin-fixed paraffin embedded tissue (resections or biopsies) and pathology reports were obtained from 35 hospitals in which these patients were treated. Patient identifiers were removed, and study numbers were assigned. Clinical information (age and gender), gross pathology (Borrmann type,34Borrmann R Geshwulste des Magens und Duodenums. Springer-Verlag, Berlin1926Google Scholar tumor size, and tumor location), and lymph node involvement were obtained from pathology reports and/or clinical records where available. Histopathological features (tumor grade, tumor depth, and histological subtype) were assigned.35Sobin L Wittekind C UICC, International Union against Cancer: TNM Classification of Malignant Tumours. Wiley-Liss, New York2002Google Scholar Two pathologists (JB and RR) jointly assessed and classified cases according to five classification systems: Carneiro,36Carneiro F Seixas M Sobrinho-Simoes M New elements for an updated classification of the carcinomas of the stomach.Pathol Res Pract. 1995; 191: 571-584Crossref PubMed Scopus (104) Google Scholar Goseki,37Goseki N Takizawa T Koike M Differences in the mode of the extension of gastric cancer classified by histological type: new histological classification of gastric carcinoma.Gut. 1992; 33: 606-612Crossref PubMed Scopus (128) Google Scholar Lauren,38Lauren P The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An attempt at a histo-clinical classification.Acta Pathol Microbiol Scand. 1965; 64: 31-49Crossref PubMed Scopus (4976) Google Scholar Ming,39Ming SC Gastric carcinoma: a pathobiological classification.Cancer. 1977; 39: 2475-2485Crossref PubMed Scopus (467) Google Scholar and World Health Organization.40Watanabe H Jass JR Sobin LH Histological Typing of Oesophageal and Gastric Tumours. Springer-Verlag, Berlin1990Crossref Google Scholar Matched pairs of formalin-fixed paraffin embedded normal and tumor tissue were obtained where possible (n = 138 of 139). Hematoxylin and eosin (H&E) slides were used as reference to microdissect unstained 7- to 10-μm slides. Laser capture microdissection (Arcturus, Mountain View, CA) was used for cases with low tumor cellularity (<70%) and volume (biopsies, n = 35). Normal and tumor DNA was extracted using QIAamp DNA minikit (Qiagen Inc., Mississauga, ON, Canada) following the manufacturer's instructions.MSI AnalysisFive markers endorsed by the NCI consensus panel16Boland CR Thibodeau SN Hamilton SR Sidransky D Eshleman JR Burt RW Meltzer SJ Rodriguez-Bigas MA Fodde R Ranzani GN Srivastava S A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257PubMed Google Scholar, 18Umar A Boland CR Terdiman JP Syngal S de la Chapelle A Ruschoff J Fishel R Lindor NM Burgart LJ Hamelin R Hamilton SR Hiatt RA Jass J Lindblom A Lynch HT Peltomaki P Ramsey SD Rodriguez-Bigas MA Vasen HF Hawk ET Barrett JC Freedman AN Srivastava S Revised Bethesda guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability.J Natl Cancer Inst. 2004; 96: 261-268Crossref PubMed Scopus (2430) Google Scholar of microsatellite markers to define MSI in CRC (three mononucleotide markers: BAT-25, BAT-26, and BAT-40; two dinucleotide markers: D5S346 and D17S250) and three described by Suraweera et al26Suraweera N Duval A Reperant M Vaury C Furlan D Leroy K Seruca R Iacopetta B Hamelin R Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR.Gastroenterology. 2002; 123: 1804-1811Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar in CRC and GC (NR-21, NR-22, and NR-24) were used (Supplemental Table S1; http://jmd.amjpathol.org/). Oligonucleotides were radioactively labeled using T4 PNK (New England Biolabs, Ipswich, MA). Polymerase chain reactions (PCRs) were performed in a 15-μl volume containing a minimum of 20 ng of DNA; 0.4–0.6 mmol/L forward, reverse, and γ-33P-labeled reverse primers (Canadian Life Technologies, Burlington, ON, Canada); 2.5 mmol/L MgCl2; 166 μmol/L dNTPs; and 0.5 U Platinum TaqDNA polymerase (Invitrogen Canada Inc., Burlington, ON, Canada). Thirty-five cycles of 94°C for 30 s, 56 to 58°C for 30 s, and 72°C for 40 seconds were performed with an initial denaturation step of 94°C for 4 minutes and final extension step of 72°C for 10 minutes using the Perkin Elmer GeneAmp PCR system 9600 (PerkinElmer Life and Analytical Science Inc., Boston, MA). PCR products were diluted 1:1 with a loading buffer (98% formamide, 0.1% xylene cyanol, 0.1% bromophenol blue, and 10 mmol/L ethylenediaminetetraacetic acid [pH 8.0]), denatured for 4 minutes at 94°C, and rapidly cooled on ice before gel loading. From each case, 3.5 μl was electrophoresed on 7% denaturing polyacrylamide gels for 2 to 3 hours at 60 to 70W, dried, and visualized by autoradiography. Three independent evaluators (JB, ND, and SG) read the autoradiograms. Cases demonstrating MSI were confirmed by a repeat PCR and electrophoresis. The results of the repeat PCR were consistent in all cases.Definition of MSI and Characterization of MSI-H CasesMicrosatellite positive (MSI+) cases were initially defined as cases showing instability in at least one marker. Tumors in which allelic shifts were not detected in any of the markers were defined as MSS. In the absence of standardized criterion for defining MSI-H status in GC, MSI-H was defined as multiple loci instability in more than or equal to three of six to eight (38 to 50%) markers tested, and MSI-L was defined as one to two loci instability.IHC for hMLH1, hMSH2, and hMSH6Monoclonal antibodies (Abs) against human hMLH1 (clone G168728; BD Biosciences, Mississauga, ON, Canada), hMSH2 (clone FE11; Oncogene Research Products, Mississauga, ON, Canada), and hMSH6 (clone 44; BD Biosciences) were used. Sections (4 μm) were deparaffinized and dehydrated. Endogenous peroxidase activity was quenched in 3% H2O2 for 15 minutes. Sections were washed in water, pretreated using heat-induced epitope retrieval in 0.01 mol/L citrate buffer, pH 6.0, and cooled for 10 minutes. Sections were placed in 0.05 mol/L Tris-buffered saline, pH 7.6, where all intervening washes were carried and all incubations done in RT. Nonspecific Ig-binding sites were blocked using 20% protein block with avidin for 15 minutes. Primary Ab incubations (hMLH1, 1:40; hMSH2, 1:100; and hMSH6, 1:300) were done for 60 minutes. Secondary Ab (1:200) (biotinylated horse anti-mouse; Vector Laboratories, Burlington, ON, Canada) was applied for 30 minutes. The Vectastain Elite avidin and biotinylated horseradish peroxidase complex (Vector Laboratories) was applied for 30 minutes. Sections were incubated for 10 minutes in a chromogen mixture containing 3,3′-diaminobenzidinetetrahydrochloride (Sigma, Diagnostics, Mississauga, ON, Canada) in 0.05 mol/L Tris-buffered saline and 0.03% H2O2. A final wash and counterstain in Mayer's hematoxylin was done before final dehydration steps and mounting in permount (Fisher Scientific Ltd., Ottawa, ON, Canada).The normal IHC staining pattern for hMLH1, hMSH2, and hMSH6 MMR proteins is nuclear. Notably, lymphocytes, normal gastric epithelium, and smooth muscle served as internal positive controls for each case. Pathologists (JB, RR, and AP) scored IHC slides using a semiquantitative system. A case was designated “immuno-negative” when complete loss or reduction (minimum 95% to maximum 100% loss) in tumor nuclei staining and corresponding intact gastric mucosae/lymphocyte staining was observed for any MMR proteins. Cases with at least 10% tumor nuclei staining were scored “intact,” and cases with crush artifact or absent internal control staining were scored “equivocal.”Hypermethylation of hMLH1 PromoterhMLH1 immunonegative cases were analyzed for hMLH1 methylation using methylation-specific PCR (MSP) for the 5′ CpG promoter of hMLH1. DNA from normal and tumor from cases of interest were chemically modified using the CpGenome DNA Modification kit (Chemicon International, Temecula, CA) as per the manufacturer's instructions. Two sets of oligonucleotides, one specific for the methylated and the other for the unmethylated promoter, were used to amplify bisulfite-modified normal and tumor DNA as previously described.41Herman JG Umar A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1686) Google Scholar Cell lines SW48 and HCT116 were used as positive and negative controls, respectively. Cases showing PCR amplification of tumor DNA using the methylation-specific primer set were scored hMLH1 methylation positive.hMLH1 Mutational AnalysesGermline (normal) and tumor DNA from hMLH1 immunonegative cases were analyzed for mutations in all 19 exons and intron-exon boundaries of hMLH1 (oligonucleotide sequences, PCR, and denaturing high performance liquid chromatography [d-HPLC] conditions are available on request). Each amplicon was electrophoresed on 2% agarose for confirmation. Denaturing high performance liquid chromatography was used to screen 18 of 19 exons for mutations/variants (Transgenomics Inc., Omaha, NE), except for exon 12, which was sequenced.42Liu W Smith DI Rechtzigel KJ Thibodeau SN James CD Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations.Nucleic Acids Res. 1998; 26: 1396-1400Crossref PubMed Scopus (229) Google Scholar, 43Kuklin A Munson K Gjerde D Haefele R Taylor P Detection of single-nucleotide polymorphisms with the WAVE DNA fragment analysis system.Genet Test. 1997; 1: 201-206Crossref PubMed Scopus (133) Google Scholar Chromatograms generated from normal DNA of noncancer patients were used as normal controls for each exon. Samples showing any variations in peak morphology relative to the normal chromatogram were sequenced using Thermosequenase Radiolabeled Terminator Cycle Sequencing kit (Amersham Biosciences Corp., Piscataway, NJ) as per the manufacturer's instructions. Results were obtained after autoradiography. Variations and mutations were confirmed by a second manual sequencing reaction from a separate amplification from the original DNA extraction at least twice in both forward and reverse directions.Statistical AnalysesClinicopathological parameters were obtained for most cases. Age, gender, overall survival, cause of death, tumor grade, Carneiro, and World Health Organization histotypes were available for all cases. Tumor site/region (proximal, middle, or distal), Goseki, Lauren, and Ming histotypes were known for 130, 123, 135, and 101 cases, respectively. Analyses of tumor size, Borrmann type, tumor depth of invasion, and nodal status were restricted to resection specimens only (n = 104). Tumor size was available for 100 of 104 resections. Three resections did not have Borrmann morphology, and one resection did not have nodal status specified. All resections had complete tumor depth data. Testing for associations between MSI and clinicopathological variables was performed using Pearson's χ2 test or Fisher's exact test for categorical variables and Student's t-test for continuou" @default.
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• W1996860520 date "2005-10-01" @default.
• W1996860520 modified "2023-10-17" @default.
• W1996860520 title "Tumor Microsatellite Instability in Early Onset Gastric Cancer" @default.
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