Immunohistochemical Analysis Reveals High Frequency of PMS2 Defects in Colorectal Cancer
2005; Elsevier BV; Volume: 128; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2005.01.056
ISSN1528-0012
AutoresKaspar Truninger, Mirco Menigatti, Judith Luz, Anna Russell, Ritva Haider, Jan‐Olaf Gebbers, Fridolin Bannwart, Hueseyin Yurtsever, Joerg Neuweiler, Hans‐Martin Riehle, Maria Sofia Cattaruzza, Karl Heinimann, Primo Schär, Josef Jiricny, Giancarlo Marra,
Tópico(s)Colorectal Cancer Screening and Detection
ResumoBackground & Aims: Germline mutations in the DNA mismatch repair (MMR) genes MSH2, MSH6, or MLH1 predispose to colorectal cancer (CRC) with an autosomal dominant inheritance pattern. The protein encoded by PMS2 is also essential for MMR; however, alterations in this gene have been documented only in extremely rare cases. We addressed this unexpected finding by analyzing a large series of CRCs. Methods: Expression of MSH2, MSH6, MLH1, and PMS2 was studied by immunohistochemistry in 1048 unselected, consecutive CRCs. Where absence of MMR proteins was detected, microsatellite instability and cytosine methylation of the respective gene promoter were analyzed. The DNA of patients presenting with PMS2-deficient cancers was examined for germline and somatic alterations in the PMS2 gene. Results: An aberrant pattern of MMR protein expression was detected in 13.2% of CRCs. Loss of expression of MSH2, MSH6, or MLH1 was found in 1.4%, 0.5%, and 9.8%, respectively. PMS2 deficiency accompanied by microsatellite instability was found in 16 cases (1.5%) with a weak family history of cancer. The PMS2 promoter was not hypermethylated in these cases. Despite interference of the PMS2 pseudogenes, we identified several heterozygous germline mutations in the PMS2 gene. Conclusions: PMS2 defects account for a small but significant proportion of CRCs and for a substantial fraction of tumors with microsatellite instability. However, the penetrance of heterozygous germline mutations in PMS2 is considerably lower than that of mutations in other MMR genes. The possible underlying causes of this unorthodox inheritance pattern are discussed. Background & Aims: Germline mutations in the DNA mismatch repair (MMR) genes MSH2, MSH6, or MLH1 predispose to colorectal cancer (CRC) with an autosomal dominant inheritance pattern. The protein encoded by PMS2 is also essential for MMR; however, alterations in this gene have been documented only in extremely rare cases. We addressed this unexpected finding by analyzing a large series of CRCs. Methods: Expression of MSH2, MSH6, MLH1, and PMS2 was studied by immunohistochemistry in 1048 unselected, consecutive CRCs. Where absence of MMR proteins was detected, microsatellite instability and cytosine methylation of the respective gene promoter were analyzed. The DNA of patients presenting with PMS2-deficient cancers was examined for germline and somatic alterations in the PMS2 gene. Results: An aberrant pattern of MMR protein expression was detected in 13.2% of CRCs. Loss of expression of MSH2, MSH6, or MLH1 was found in 1.4%, 0.5%, and 9.8%, respectively. PMS2 deficiency accompanied by microsatellite instability was found in 16 cases (1.5%) with a weak family history of cancer. The PMS2 promoter was not hypermethylated in these cases. Despite interference of the PMS2 pseudogenes, we identified several heterozygous germline mutations in the PMS2 gene. Conclusions: PMS2 defects account for a small but significant proportion of CRCs and for a substantial fraction of tumors with microsatellite instability. However, the penetrance of heterozygous germline mutations in PMS2 is considerably lower than that of mutations in other MMR genes. The possible underlying causes of this unorthodox inheritance pattern are discussed. Repair of mismatches, non—Watson-Crick base pairs arising during DNA replication or recombination, requires the mismatch repair (MMR) proteins MSH2, MSH3, MSH6, MLH1, and PMS2 and several factors involved in DNA replication.1Jiricny J. Marra G. DNA repair defects in colon cancer.Curr Opin Genet Dev. 2003; 13: 61-69Crossref PubMed Scopus (74) Google Scholar Most base-base mismatches (G/T, G/G, and so on) are recognized by the MSH2/MSH6 heterodimer, whereas small insertion/deletion loops arising in mononucleotide and dinucleotide repeats (the so-called microsatellites) through strand misalignments can be bound either by the MSH2/MSH6 or the MSH2/MSH3 heterodimer. The partial redundancy of MSH3 and MSH6 in the repair of insertion/deletion loops has a profound effect on microsatellite instability (MSI), one of the key phenotypic traits of MMR-deficient tumors.2Ionov Y. Peinado M.A. Malkhosyan S. Shibata D. Perucho M. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis.Nature. 1993; 363: 558-561Crossref PubMed Scopus (2410) Google Scholar, 3Honchel R. Halling K.C. Thibodeau S.N. Genomic instability in neoplasia.Semin Cell Biol. 1995; 6: 45-52Crossref PubMed Scopus (80) Google Scholar Thus, whereas MSH2-deficient tumors invariably display MSI because both heterodimers are defective, the degree of MSI in MSH6-deficient tumors can vary4Berends M.J. Wu Y. Sijmons R.H. Mensink R.G. van der Sluis T. Hordijk-Hos J.M. de Vries E.G. Hollema H. Karrenbeld A. Buys C.H. van der Zee A.G. Hofstra R.M. Kleibeuker J.H. Molecular and clinical characteristics of MSH6 variants an analysis of 25 index carriers of a germline variant.Am J Hum Genet. 2002; 70: 26-37Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 5Plaschke J. Kruger S. Pistorius S. Theissig F. Saeger H.D. Schackert H.K. Involvement of hMSH6 in the development of hereditary and sporadic colorectal cancer revealed by immunostaining is based on germline mutations, but rarely on somatic inactivation.Int J Cancer. 2002; 97: 643-648Crossref PubMed Scopus (72) Google Scholar, 6Hendriks Y.M. Wagner A. Morreau H. Menko F. Stormorken A. Quehenberger F. Sandkuijl L. Moller P. Genuardi M. Van Houwelingen H. Tops C. Van Puijenbroek M. Verkuijlen P. Kenter G. Van Mil A. Meijers-Heijboer H. Tan G.B. Breuning M.H. Fodde R. Wijnen J.T. Brocker-Vriends A.H. Vasen H. Cancer risk in hereditary nonpolyposis colorectal cancer due to MSH6 mutations impact on counseling and surveillance.Gastroenterology. 2004; 127: 17-25Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar because the MSH2/MSH3 heterodimer partially compensates for the loss of MSH2/MSH6 in insertion/deletion loop repair.7Genschel J. Littman S.J. Drummond J.T. Modrich P. Isolation of MutSbeta from human cells and comparison of the mismatch repair specificities of MutSbeta and MutSalpha.J Biol Chem. 1998; 273: 19895-19901Crossref PubMed Scopus (334) Google Scholar, 8Chang D.K. Ricciardiello L. Goel A. Chang C.L. Boland C.R. Steady-state regulation of the human DNA mismatch repair system.J Biol Chem. 2000; 275: 18424-18431Crossref PubMed Scopus (161) Google Scholar The converse is not true, however, because the MSH2/MSH6 heterodimer compensates for the absence of MSH2/MSH3 in insertion/deletion loop repair. Correspondingly, primary alterations in the MSH3 gene have not been found in tumors with MSI. Like their biological roles, the biochemical characteristics of the 3 MSH polypeptides also differ, inasmuch as MSH2 deficiency leads to the proteolytic degradation of both MSH3 and MSH6, whereas MSH2 is largely stable in the absence of one of its cognate partners.1Jiricny J. Marra G. DNA repair defects in colon cancer.Curr Opin Genet Dev. 2003; 13: 61-69Crossref PubMed Scopus (74) Google Scholar This means that cells with destabilized MSH2 will also appear as MSH3 and MSH6 negative in immunohistochemistry, whereas cells with mutated MSH3 or MSH6 will be seen to lack solely the affected polypeptide. The MLH1/PMS2 heterodimer was suggested to act as a matchmaker between the mismatch recognition complex and the downstream MMR factors.1Jiricny J. Marra G. DNA repair defects in colon cancer.Curr Opin Genet Dev. 2003; 13: 61-69Crossref PubMed Scopus (74) Google Scholar Interestingly, as in the case of the MSH proteins, the stabilities of the MLH1 and PMS2 polypeptides are different. Thus, when MLH1 is not expressed, or when it is mutated such that it is either destabilized or it cannot interact with PMS2, the latter protein is degraded. In contrast, MLH1 remains stable in the absence of PMS2,8Chang D.K. Ricciardiello L. Goel A. Chang C.L. Boland C.R. Steady-state regulation of the human DNA mismatch repair system.J Biol Chem. 2000; 275: 18424-18431Crossref PubMed Scopus (161) Google Scholar, 9Räschle M. Marra G. Nyström-Lahti M. Schär P. Jiricny J. Identification of hMutLbeta, a heterodimer of hMLH1 and hPMS1.J Biol Chem. 1999; 274: 32368-32375Crossref PubMed Scopus (148) Google Scholar possibly through interactions with other MutL homologues such as PMS1 or MLH3. MSI analyses of tumor DNA suggested that 10%-20% of colorectal cancers (CRC) may be associated with MMR system malfunctions.10Grady W.M. Markowitz S.D. Genetic and epigenetic alterations in colon cancer.Annu Rev Genomics Hum Genet. 2002; 3: 101-128Crossref PubMed Scopus (246) Google Scholar In sporadic (ie, nonfamilial) CRCs, the MMR defect is associated with the silencing of MLH1 transcription by cytosine methylation.11Herman J.G. Umar A. Polyak K. Graff J.R. Ahuja N. Issa J.P. Markowitz S. Willson J.K. Hamilton S.R. Kinzler K.W. Kane M.F. Kolodner R.D. Vogelstein B. Kunkel T.A. Baylin S.B. Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci U S A. 1998; 95: 6870-6875Crossref PubMed Scopus (1698) Google Scholar In families affected by hereditary nonpolyposis colon cancer (HNPCC, also called Lynch syndrome), MMR malfunction has been linked primarily with heterozygous germline mutations in MSH2 or MLH1. Due to the partial functional redundancy of MSH3 and MSH6, germline alterations in MSH6 are less frequent and are associated with colon and endometrial cancers in families that often do not fulfill Amsterdam criteria for diagnosis of HNPCC12Vasen H.F. Watson P. Mecklin J.P. Lynch H.T. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC.Gastroenterology. 1999; 116: 1453-1456Abstract Full Text Full Text PDF PubMed Scopus (2089) Google Scholar, 13Wijnen J. de Leeuw W. Vasen H. van der Klift H. Moller P. Stormorken A. Meijers-Heijboer H. Lindhout D. Menko F. Vossen S. Moslein G. Tops C. Brocker-Vriends A. Wu Y. Hofstra R. Sijmons R. Cornelisse C. Morreau H. Fodde R. Familial endometrial cancer in female carriers of MSH6 germline mutations.Nat Genet. 1999; 23: 142-144Crossref PubMed Scopus (366) Google Scholar (sometimes referred to as “HNPCC-like” or “attenuated HNPCC”). Nevertheless, the inheritance pattern in families carrying MSH6 germline mutations is autosomal dominant, as in families with germline alterations in MSH2 or MLH1. Germline mutations in the PMS2 gene have been reported in 6 subjects presenting with a severe childhood cancer syndrome characterized by CRC and brain tumors in the first 2 decades of life (3 cases were diagnosed as Turcot’s syndrome).14Nicolaides N.C. Papadopoulos N. Liu B. Wei Y.F. Carter K.C. Ruben S.M. Rosen C.A. Haseltine W.A. Fleischmann R.D. Fraser C.M. et al.Mutations of two PMS homologues in hereditary nonpolyposis colon cancer.Nature. 1994; 371: 75-80Crossref PubMed Scopus (1446) Google Scholar, 15Hamilton S.R. Liu B. Parsons R.E. Papadopoulos N. Jen J. Powell S.M. Krush A.J. Berk T. Cohen Z. Tetu B. et al.The molecular basis of Turcot’s syndrome.N Engl J Med. 1995; 332: 839-847Crossref PubMed Scopus (915) Google Scholar, 16Miyaki M. Nishio J. Konishi M. Kikuchi-Yanoshita R. Tanaka K. Muraoka M. Nagato M. Chong J.M. Koike M. Terada T. Kawahara Y. Fukutome A. Tomiyama J. Chuganji Y. Momoi M. Utsunomiya J. Drastic genetic instability of tumors and normal tissues in Turcot syndrome.Oncogene. 1997; 15: 2877-2881Crossref PubMed Scopus (76) Google Scholar, 17De Rosa M. Fasano C. Panariello L. Scarano M.I. Belli G. Iannelli A. Ciciliano F. Izzo P. Evidence for a recessive inheritance of Turcot’s syndrome caused by compound heterozygous mutations within the PMS2 gene.Oncogene. 2000; 19: 1719-1723Crossref PubMed Scopus (117) Google Scholar, 18Trimbath J.D. Petersen G.M. Erdman S.H. Ferre M. Luce M.C. Giardiello F.M. Cafe-au-lait spots and early onset colorectal neoplasia a variant of HNPCC?.Fam Cancer. 2001; 1: 101-105Crossref PubMed Google Scholar, 19De Vos M. Hayward B.E. Picton S. Sheridan E. Bonthron D.T. Novel PMS2 pseudogenes can conceal recessive mutations causing a distinctive childhood cancer syndrome.Am J Hum Genet. 2004; 74: 954-964Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar A careful mutational analysis in these cases showed that the germline alterations were biallelic, suggesting a recessive inheritance pattern reminiscent of similar syndromes observed in children found to be compound heterozygotes or true homozygotes for mutations in MSH2 or MLH1.20Gallinger S. Aronson M. Shayan K. Ratcliffe E.M. Gerstle J.T. Parkin P.C. Rothenmund H. Croitoru M. Baumann E. Durie P.R. Weksberg R. Pollett A. Riddell R.H. Ngan B.Y. Cutz E. Lagarde A.E. Chan H.S. Gastrointestinal cancers and neurofibromatosis type 1 features in children with a germline homozygous MLH1 mutation.Gastroenterology. 2004; 126: 576-585Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 21Wang Q. Lasset C. Desseigne F. Frappaz D. Bergeron C. Navarro C. Ruano E. Puisieux A. Neurofibromatosis and early onset of cancers in hMLH1-deficient children.Cancer Res. 1999; 59: 294-297PubMed Google Scholar, 22Ricciardone M.D. Ozcelik T. Cevher B. Ozdag H. Tuncer M. Gurgey A. Uzunalimoglu O. Cetinkaya H. Tanyeli A. Erken E. Ozturk M. Human MLH1 deficiency predisposes to hematological malignancy and neurofibromatosis type 1.Cancer Res. 1999; 59: 290-293PubMed Google Scholar, 23Vilkki S. Tsao J.L. Loukola A. Poyhonen M. Vierimaa O. Herva R. Aaltonen L.A. Shibata D. Extensive somatic microsatellite mutations in normal human tissue.Cancer Res. 2001; 61: 4541-4544PubMed Google Scholar, 24Whiteside D. McLeod R. Graham G. Steckley J.L. Booth K. Somerville M.J. Andrew S.E. A homozygous germ-line mutation in the human MSH2 gene predisposes to hematological malignancy and multiple cafe-au-lait spots.Cancer Res. 2002; 62: 359-362PubMed Google Scholar, 25Bougeard G. Charbonnier F. Moerman A. Martin C. Ruchoux M.M. Drouot N. Frebourg T. Early onset brain tumor and lymphoma in MSH2-deficient children.Am J Hum Genet. 2003; 72: 213-216Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar To date, heterozygous germline mutations in PMS2 have not been reported in HNPCC families as defined by Amsterdam criteria. This finding is contrary to expectations, because PMS2 is essential for MMR. MLH1 can interact with other partners, notably PMS19Räschle M. Marra G. Nyström-Lahti M. Schär P. Jiricny J. Identification of hMutLbeta, a heterodimer of hMLH1 and hPMS1.J Biol Chem. 1999; 274: 32368-32375Crossref PubMed Scopus (148) Google Scholar and MLH326Lipkin S.M. Wang V. Jacoby R. Banerjee-Basu S. Baxevanis A.D. Lynch H.T. Elliott R.M. Collins F.S. MLH3 a DNA mismatch repair gene associated with mammalian microsatellite instability.Nat Genet. 2000; 24: 27-35Crossref PubMed Scopus (265) Google Scholar (Cannavó et al, manuscript in preparation); however, neither heterodimer could be shown to play a major role in human MMR. Correspondingly, cell lines not expressing PMS2 display MSI and their extracts are MMR deficient to an extent similar to that observed in cells mutated in MLH1 or MSH2.9Räschle M. Marra G. Nyström-Lahti M. Schär P. Jiricny J. Identification of hMutLbeta, a heterodimer of hMLH1 and hPMS1.J Biol Chem. 1999; 274: 32368-32375Crossref PubMed Scopus (148) Google Scholar, 27Ma A.H. Xia L. Littman S.J. Swinler S. Lader G. Polinkovsky A. Olechnowicz J. Kasturi L. Lutterbaugh J. Modrich P. Veigl M.L. Markowitz S.D. Sedwick W.D. Somatic mutation of hPMS2 as a possible cause of sporadic human colon cancer with microsatellite instability.Oncogene. 2000; 19: 2249-2256Crossref PubMed Scopus (27) Google Scholar, 28Risinger J.I. Umar A. Glaab W.E. Tindall K.R. Kunkel T.A. Barrett J.C. Single gene complementation of the hPMS2 defect in HEC-1-A endometrial carcinoma cells.Cancer Res. 1998; 58: 2978-2981PubMed Google Scholar It might therefore be anticipated that, similarly to MSH2, MLH1, and MSH6, germline mutations in a single PMS2 allele would predispose to CRC. We addressed this curious finding by analyzing the expression of PMS2 along with that of its heterodimeric partner MLH1 and with that of MSH2 and MSH6 in a large series of unselected CRCs. This approach, supported by the analysis of the PMS2 gene, allowed us to identify patients affected by cancers with a primary defect of PMS2 (ie, not secondary to an MLH1 defect) and to describe their phenotype. Between January 2000 and June 2002, 1048 consecutive CRCs were collected from patients who underwent surgery at 5 Swiss hospitals: Triemli Hospital Zurich (F.B.), University Hospital Zurich (H.-M.R.), Cantonal Hospital of Lucerne (J.-O.G.), Cantonal Hospital of Aarau (H.Y.), and Cantonal Hospital of St Gallen (J.N.). No tumors were excluded from this study. In the first instance, the expression of MSH2, MSH6, MLH1, and PMS2 was investigated by immunohistochemistry. In tumors with aberrant patterns of MMR protein expression, MSI analysis was performed using the mononucleotide marker BAT26. We then analyzed the methylation status of the MLH1 or PMS2 gene promoters in tumors not expressing MLH1 or PMS2, respectively. Finally, for patients presenting with PMS2-deficient tumors, blood and tumor DNA were analyzed for the presence of germline and somatic mutations and for the loss of heterozygosity (LOH) at the PMS2 locus. Information about the family history of cancer was obtained from a questionnaire sent to the family doctors and to the patients. In our study, cases fulfilling the Amsterdam criteria II (or revised Amsterdam criteria)12Vasen H.F. Watson P. Mecklin J.P. Lynch H.T. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC.Gastroenterology. 1999; 116: 1453-1456Abstract Full Text Full Text PDF PubMed Scopus (2089) Google Scholar for diagnosis of HNPCC are designated “AC,” whereas cases not complying with AC but fulfilling the revised Bethesda guidelines for MSI testing (Table 2 of Umar et al29Umar A. Boland C.R. Terdiman J.P. Syngal S. de la Chapelle A. Ruschoff J. Fishel R. Lindor N.M. Burgart L.J. Hamelin R. Hamilton S.R. Hiatt R.A. Jass J. Lindblom A. Lynch H.T. Peltomäki P. Ramsey S.D. Rodriguez-Bigas M.A. Vasen H.F. Hawk E.T. Barrett J.C. Freedman A.N. 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 (2487) Google Scholar; guidelines for the identification of tumors that should be tested for MSI) are designated “BG.” Cases fulfilling neither AC nor BG are termed “sporadic.”Table 2Characteristics of Patients With MMR-Deficient TumorsAll casesCRCs not expressingMLH1PMS2MSH2MSH6TotalAC or BGaPercentage refers to the number of patients whose family pedigree was available (MSH2, 11; MSH6, 4; MLH1, 96; PMS2, 16; for details see Supplementary Table 2). Sporadic CRCs were separated from those fulfilling the AC for HNPCC diagnosis or BG for MSI testing (for definitions, see Patients and Methods).SporadicaPercentage refers to the number of patients whose family pedigree was available (MSH2, 11; MSH6, 4; MLH1, 96; PMS2, 16; for details see Supplementary Table 2). Sporadic CRCs were separated from those fulfilling the AC for HNPCC diagnosis or BG for MSI testing (for definitions, see Patients and Methods).TotalAC or BGaPercentage refers to the number of patients whose family pedigree was available (MSH2, 11; MSH6, 4; MLH1, 96; PMS2, 16; for details see Supplementary Table 2). Sporadic CRCs were separated from those fulfilling the AC for HNPCC diagnosis or BG for MSI testing (for definitions, see Patients and Methods).SporadicaPercentage refers to the number of patients whose family pedigree was available (MSH2, 11; MSH6, 4; MLH1, 96; PMS2, 16; for details see Supplementary Table 2). Sporadic CRCs were separated from those fulfilling the AC for HNPCC diagnosis or BG for MSI testing (for definitions, see Patients and Methods).TotalAC or BGaPercentage refers to the number of patients whose family pedigree was available (MSH2, 11; MSH6, 4; MLH1, 96; PMS2, 16; for details see Supplementary Table 2). Sporadic CRCs were separated from those fulfilling the AC for HNPCC diagnosis or BG for MSI testing (for definitions, see Patients and Methods).SporadicaPercentage refers to the number of patients whose family pedigree was available (MSH2, 11; MSH6, 4; MLH1, 96; PMS2, 16; for details see Supplementary Table 2). Sporadic CRCs were separated from those fulfilling the AC for HNPCC diagnosis or BG for MSI testing (for definitions, see Patients and Methods).TotalAC or BGaPercentage refers to the number of patients whose family pedigree was available (MSH2, 11; MSH6, 4; MLH1, 96; PMS2, 16; for details see Supplementary Table 2). Sporadic CRCs were separated from those fulfilling the AC for HNPCC diagnosis or BG for MSI testing (for definitions, see Patients and Methods).SporadicaPercentage refers to the number of patients whose family pedigree was available (MSH2, 11; MSH6, 4; MLH1, 96; PMS2, 16; for details see Supplementary Table 2). Sporadic CRCs were separated from those fulfilling the AC for HNPCC diagnosis or BG for MSI testing (for definitions, see Patients and Methods).Patients, no. (%)139103 (74)28 (29)68 (71)16 (11.5)12 (75)4 (25)15 (10.8)9 (82)2 (18)5 (3.6)3 (75)1 (25)Age at presentation, y Mean (±SD)69.9 (14.9)72.6 (14.1)57.9 (15.7)78.5 (8.2)62.8 (14.7)59.4 (15.4)73.0 (5.0)62.5 (17.2)52 (12.7)69.0 (7.1)60.4 (5.1)62.3 (6.1)58 Median7476577861.55774.56453695861 Range27–9427–9427–9152–9442–8242–8266–7734–8734–7164–7457–6957–69Sex, no. (%) Male62 (45)39 (38)13 (46)25 (37)14 (88)10 (83)4 (100)7 (47)5 (56)1 (50)2 (40)01 (100) Female77 (55)64 (62)15 (54)43 (63)2 (12)2 (17)08 (53)4 (44)1 (50)3 (60)3 (100)0Site of tumor, no. (%) Cecum35 (25)31 (30)6 (21)23 (34)1 (6)1 (8)03 (20)1 (11)1 (50)000 Ascending colon50 (36)35 (34)6 (21)28 (41)7 (44)3 (25)4 (100)6 (40)4 (44)02 (40)2 (67)0 Hepatic flexure9 (6)8 (8)2 (7)6 (9)1 (6)1 (8)0000000 Transverse colon8 (6)7 (7)1 (4)6 (9)1 (6)1 (8)0000000 Splenic flexure2 (1)0002 (13)2 (17)0000000 Descending colon7 (5)4 (4)3 (11)02 (13)2 (17)01 (7)1 (11)0000 Sigmoid colon14 (10)11 (11)5 (18)3 (4)1 (6)1 (8)01 (7)1 (11)01 (20)1 (33)0 Rectum14 (10)7 (7)5 (18)2 (3)1 (6)1 (8)04 (27)2 (22)1 (50)2 (40)01 (100) Proximal to splenic flexure102 (73)81 (79)15 (54)63 (93)10 (63)6 (50)4 (100)9 (60)5 (56)1 (50)2 (40)2 (67)0 Distal to splenic flexure37 (27)22 (21)13 (46)5 (7)6 (37)6 (50)06 (40)4 (44)1 (50)3 (60)1 (33)1 (100)Pathologic classification of the primary tumor, no. (%)bTNM and tumor grade classification according to Sobin LH, Wittekind CH. TNM classification of malignant tumours. 6th ed. New York: Wiley-Liss, 2002. Only T and N stages are reported because they are histologically confirmed in all cases. T17 (5)5 (5)3 (11)1 (2)2 (13)1 (8)1 (25)000000 T217 (12)13 (13)3 (11)9 (13)1 (6)1 (8)03 (20)2 (22)0000 T392 (66)68 (66)18 (64)45 (66)10 (63)8 (67)2 (50)10 (67)6 (67)2 (100)4 (80)2 (67)1 (100) T423 (17)17 (16)4 (14)13 (19)3 (19)2 (17)1 (25)2 (13)1 (11)01 (20)1 (33)0Pathologic classification of the regional lymph nodes, no. (%)bTNM and tumor grade classification according to Sobin LH, Wittekind CH. TNM classification of malignant tumours. 6th ed. New York: Wiley-Liss, 2002. Only T and N stages are reported because they are histologically confirmed in all cases. N094 (68)67 (65)18 (64)44 (65)14 (88)10 (83)4 (100)11 (73)6 (67)2 (100)2 (40)1 (33)1 (100) N122 (16)20 (19)5 (18)13 (19)1 (6)1 (8)00001 (20)00 N223 (17)16 (15)5 (18)11 (16)1 (6)1 (8)04 (27)3 (33)02 (40)2 (67)0 No lymph node involvement94 (68)67 (65)18 (64)44 (65)14 (88)10 (83)4 (100)11 (73)6 (67)2 (100)2 (40)1 (33)1 (100) Lymph node involvement45 (32)36 (35)10 (36)24 (35)2 (12)2 (17)04 (27)3 (33)03 (60)2 (67)0Tumor grade, no. (%) Well differentiated (G1)2 (1)2 (2)1 (4)1 (1)000000000 Moderately differentiated (G2)80 (58)55 (53)22 (79)29 (43)12 (75)8 (67)4 (100)9 (60)6 (67)2 (100)4 (80)2 (67)1 (100) Poorly differentiated (G3)57 (41)46 (45)5 (18)38 (56)4 (25)4 (33)06 (40)3 (33)01 (20)1 (33)0MSIcMSI as detected by BAT26 analysis, except for PMS2 cases where dinucleotide repeats flanking PMS2 on chromosome 7 were also investigated. Unstable127 (92)dIn one case, DNA was not suitable for analysis.99 (96)27 (96)66 (97)15 (100)dIn one case, DNA was not suitable for analysis.11 (100)4 (100)12 (80)7 (78)2 (100)1 (20)1 (33)0 Stable11 (8)4 (4)1 (4)2 (3)0003 (20)2 (22)04 (80)2 (67)1 (100)Promoter methylationeOnly MLH1 or PMS2 promoter were analyzed in MLH1-deficient or PMS2-deficient CRCs, respectively. Present84 (82)14 (50)65 (96)000 Absent19 (18)14 (50)3 (4)15 (100)dIn one case, DNA was not suitable for analysis.11 (100)4 (100)a Percentage refers to the number of patients whose family pedigree was available (MSH2, 11; MSH6, 4; MLH1, 96; PMS2, 16; for details see Supplementary Table 2). Sporadic CRCs were separated from those fulfilling the AC for HNPCC diagnosis or BG for MSI testing (for definitions, see Patients and Methods).b TNM and tumor grade classification according to Sobin LH, Wittekind CH. TNM classification of malignant tumours. 6th ed. New York: Wiley-Liss, 2002. Only T and N stages are reported because they are histologically confirmed in all cases.c MSI as detected by BAT26 analysis, except for PMS2 cases where dinucleotide repeats flanking PMS2 on chromosome 7 were also investigated.d In one case, DNA was not suitable for analysis.e Only MLH1 or PMS2 promoter were analyzed in MLH1-deficient or PMS2-deficient CRCs, respectively. Open table in a new tab The study protocol was approved by the institutional review boards. Tumors were fixed in buffered formalin and embedded in paraffin. Four-micrometer sections were mounted on glass slides coated with organosilane (DakoCytomation, Glostrup, Denmark), deparaffinized, and rehydrated. Antigen retrieval was accomplished by heating the sections in a pressure cooker at 120°C for 2 minutes in 10 mmol/L citrate-buffered solution (pH 6.0). DakoCytomation peroxidase blocking reagent and goat serum were sequentially used to suppress nonspecific staining due to endogenous peroxidase activity and unspecific binding of antibodies, respectively. Incubations with primary monoclonal antibodies were performed as follows: anti-hMSH2, 24 hours at 4°C with 1 μg/mL antibody NA26 (Oncogene Research, Darmstadt, Germany); anti-hMSH6, 2 hours at room temperature with 4 μg/mL antibody G70220 (Transduction Laboratories, Basel, Switzerland); anti-hMLH1, 1 hour at room temperature with 1.2 μg/mL antibody 13271A (PharMingen, Basel, Switzerland); anti-hPMS2, 24 hours at 4°C with 3 μg/mL antibody 65861A (PharMingen, Basel, Switzerland). After washing, anti-mouse secondary antibodies conjugated to peroxidase-labeled polymer (Dako EnVision+ kit) were applied for 30 minutes at room temperature, and the peroxidase activity was developed by incubation with 3,3′-diaminobenzidine chromogen solution (Dako). The sections were then counterstained with hematoxylin. Lack of protein expression was clearly evident as absence of nuclear staining in tumor cells despite nuclear staining in proliferating cells in normal crypts and stroma. DNA was extracted from histologic sections and blood with QIAamp DNA and DNeasy, whereas RNeasy and Omniscript/Sensiscript RT kits (Qiagen, Basel, Switzerland) were used for RNA isolation and complementary DNA synthesis. MSI analysis was performed by analyzing the mononucleotide repeat BAT26 and, for PMS2-deficient tumors, several dinucleotide repeats on chromosome 7p22. LOH analysis was performed using 8 microsatellite markers flanking PMS2 on chromosome 7p22. LOH was scored at any informative marker if the area under one allelic peak in tumor DNA was reduced by >50% relative to the other allele after correcting for the relative peak areas in DNA from normal tissue. Search for germline mutations was performed on genomic DNA and complementary DNA by sequencing all 15 PMS2 exons and their intron-exon boundaries. Primers described by Nicolaides et al30Nicolaides N.C. Carter K.C. Shell B.K. Papadopoulos N. Vogelstein B. Kinzler K.W. Genomic organization of the human PMS2 gene family.Genomics. 1995; 30: 195-206Crossref PubMed Scopus (104) Google Scholar were used except for exons 1, 3, 5, and 10–14, for which new primers were designed to allow a more specific amplification and to avoid coamplification of PMS2 pseudogenes. All primers and reaction conditions are reported in Supplementary Table 1 (see supplemental material online available at:http://www.imcr.unizh.ch/research/researchdetail.StaffId=11).Table 1Characteristics of Patients and CRCs in Relation to Tumor MMR StatusCharacteristicAll casesMMR-proficient CRCMMR-deficient CRCDifference (95% CI)PaComparison between MMR-proficient and -deficient CRCs (t and χ2 tests). Logistic regression: the odds ratio was 1.6 (95% CI, 1.1–2.4) for women versus men, 7.1 (95% CI, 4.7–10.9) for proximal versus distal colon, 3.1 (95% CI, 2.0–4.8) for no lymph node involvement, and 3.4 (95% CI, 2.2–5.2) for poorly versus well plus moderately differentiated tumors.Patients, no. (%)1048 (100)909 (86.7)139 (13.2)Age at presentation, y Mean (±SD)69.7 (11.9)69.7 (11.4)69.9 (14.9).82 Median717174 Range23–9423–9327–94Sex, no. (%) Male609 (58.1)547 (60.2)62 (44.6)15.6 (6.8–24.3)<.001 Female439 (41.9)362 (39.8)77 (55.4)Site of tumor, no. (%) Cecum127 (12.1)92 (10.1)35 (25.2)<.00005 Ascending colon134 (12.8)84 (9.2)50 (36.0) Hepatic flexure34 (3.2)25 (2.8)9 (6.5) Transverse colon48 (4.6)40 (4.4)8 (5.8) Splenic flexure21 (2.0)19 (2.1)2 (1.4) Descending colon36 (3.4)29 (3.2)7 (5.0) Sigmoid colon343 (32.7)329 (36.2)14 (10.1) Rectum305 (29.1)291 (
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