Immunohistochemistry versus Microsatellite Instability Testing for Screening Colorectal Cancer Patients at Risk for Hereditary Nonpolyposis Colorectal Cancer Syndrome
2008; Elsevier BV; Volume: 10; Issue: 4 Linguagem: Inglês
10.2353/jmoldx.2008.080062
ISSN1943-7811
Autores Tópico(s)Colorectal Cancer Treatments and Studies
ResumoGermline mutations in the mismatch repair genes mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2), MSH6, and postmeiotic segregation increased 2 (PMS2) lead to the development of hereditary nonpolyposis colorectal cancer (HNPCC). Diagnosis of HNPCC relies on the compilation of a thorough family history of cancer, documentation of pathological findings, tumor testing for microsatellite instability (MSI) and immunohistochemistry (IHC), and germline mutation analysis of the suspected genes. As a hallmark of HNPCC, microsatellite instability is widely accepted as a primary method for identifying individuals at risk for HNPCC. It serves as an excellent, easy-to-evaluate marker of mismatch repair deficiency. Recent improvements in MSI testing have significantly enhanced the accuracy and reduced its cost. Proficiency testing for MSI is available, and laboratory-to-laboratory reproducibility of such testing can be easily evaluated. In addition, the combination of microsatellite instability testing, MLH1 promoter methylation analysis, and BRAF (V600E) mutation analysis can distinguish a sporadic colorectal cancer from one associated with HNPCC, helping to avoid costly molecular genetic testing for germline mutations in mismatch repair genes. In this article, we discuss the development of MSI markers used for HNPCC screening and focus on the advantages and disadvantages of MSI testing in screening for HNPCC patients. We conclude that MSI is as sensitive and specific as IHC, given its excellent reproducibility and its potential capability to indicate mutations not be detected by IHC. MSI has been used and will continue to prevail as the primary screening tool for identifying HNPCC patients. Germline mutations in the mismatch repair genes mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2), MSH6, and postmeiotic segregation increased 2 (PMS2) lead to the development of hereditary nonpolyposis colorectal cancer (HNPCC). Diagnosis of HNPCC relies on the compilation of a thorough family history of cancer, documentation of pathological findings, tumor testing for microsatellite instability (MSI) and immunohistochemistry (IHC), and germline mutation analysis of the suspected genes. As a hallmark of HNPCC, microsatellite instability is widely accepted as a primary method for identifying individuals at risk for HNPCC. It serves as an excellent, easy-to-evaluate marker of mismatch repair deficiency. Recent improvements in MSI testing have significantly enhanced the accuracy and reduced its cost. Proficiency testing for MSI is available, and laboratory-to-laboratory reproducibility of such testing can be easily evaluated. In addition, the combination of microsatellite instability testing, MLH1 promoter methylation analysis, and BRAF (V600E) mutation analysis can distinguish a sporadic colorectal cancer from one associated with HNPCC, helping to avoid costly molecular genetic testing for germline mutations in mismatch repair genes. In this article, we discuss the development of MSI markers used for HNPCC screening and focus on the advantages and disadvantages of MSI testing in screening for HNPCC patients. We conclude that MSI is as sensitive and specific as IHC, given its excellent reproducibility and its potential capability to indicate mutations not be detected by IHC. MSI has been used and will continue to prevail as the primary screening tool for identifying HNPCC patients. The diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) at the molecular level relies on the presence of a deleterious germline mutation in one of the mismatch repair (MMR) genes. Because cancer morbidity and mortality can be dramatically reduced by colonoscopic screening of individuals with the HNPCC syndrome and by prophylactic surgeries, molecular screening of colorectal cancer patients for HNPCC is now feasible.1Aaltonen LA Salovaara R Kristo P Canzian F Hemminki A Peltomaki P Chadwick RB Kaariainen H Eskelinen M Jarvinen H Mecklin JP de la Chapelle A Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease.N Engl J Med. 1998; 338: 1481-1487Crossref PubMed Scopus (981) Google Scholar2Hampel H Frankel WL Martin E Arnold M Khanduja K Kuebler P Nakagawa H Sotamaa K Prior TW Westman J Panescu J Fix D Lockman J Comeras I de la Chapelle A Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer).N Engl J Med. 2005; 352: 1851-1860Crossref PubMed Scopus (1096) Google Scholar3Salovaara R Loukola A Kristo P Kaariainen H Ahtola H Eskelinen M Harkonen N Julkunen R Kangas E Ojala S Tulikoura J Valkamo E Jarvinen H Mecklin JP Aaltonen LA de la Chapelle A Population-based molecular detection of hereditary nonpolyposis colorectal cancer.J Clin Oncol. 2000; 18: 2193-2200Crossref PubMed Scopus (445) Google Scholar4Schmeler KM Lynch HT Chen LM Munsell MF Soliman PT Clark MB Daniels MS White KG Boyd-Rogers SG Conrad PG Yang KY Rubin MM Sun CC Slomovitz BM Gershenson DM Lu KH Prophylactic surgery to reduce the risk of gynecologic cancers in the Lynch syndrome.N Engl J Med. 2006; 354: 261-269Crossref PubMed Scopus (606) Google Scholar The challenge is to establish a strategy that is able to screen effectively for HNPCC. Microsatellite instability (MSI) in colorectal cancer was discovered in 1993 and was subsequently found to be present in colon cancer tissue from most HNPCC patients.5Aaltonen LA Peltomaki P Leach FS Sistonen P Pylkkanen L Mecklin JP Jarvinen H Powell SM Jen J Hamilton SR et al.Clues to the pathogenesis of familial colorectal cancer.Science. 1993; 260: 812-816Crossref PubMed Scopus (2559) Google Scholar6Ionov Y Peinado MA 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 (2390) Google Scholar7Peltomaki P Aaltonen LA Sistonen P Pylkkanen L Mecklin JP Jarvinen H Green JS Jass JR Weber JL Leach FS et al.Genetic mapping of a locus predisposing to human colorectal cancer.Science. 1993; 260: 810-812Crossref PubMed Scopus (802) Google Scholar8Thibodeau SN Bren G Schaid D Microsatellite instability in cancer of the proximal colon.Science. 1993; 260: 816-819Crossref PubMed Scopus (2785) Google ScholarGenotyping for microsatellite instability was initially used to screen for HNPCC,1Aaltonen LA Salovaara R Kristo P Canzian F Hemminki A Peltomaki P Chadwick RB Kaariainen H Eskelinen M Jarvinen H Mecklin JP de la Chapelle A Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease.N Engl J Med. 1998; 338: 1481-1487Crossref PubMed Scopus (981) Google Scholar,3Salovaara R Loukola A Kristo P Kaariainen H Ahtola H Eskelinen M Harkonen N Julkunen R Kangas E Ojala S Tulikoura J Valkamo E Jarvinen H Mecklin JP Aaltonen LA de la Chapelle A Population-based molecular detection of hereditary nonpolyposis colorectal cancer.J Clin Oncol. 2000; 18: 2193-2200Crossref PubMed Scopus (445) Google Scholar while immunohistochemistry (IHC) analysis of the MMR proteins has been more recently proposed as an alternative method for screening HNPCC.2Hampel H Frankel WL Martin E Arnold M Khanduja K Kuebler P Nakagawa H Sotamaa K Prior TW Westman J Panescu J Fix D Lockman J Comeras I de la Chapelle A Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer).N Engl J Med. 2005; 352: 1851-1860Crossref PubMed Scopus (1096) Google Scholar Two recent studies have indicated that microsatellite instability testing and immunohistochemistry are both highly effective strategies for selecting patients for molecular genetic testing (germline mutation analysis).2Hampel H Frankel WL Martin E Arnold M Khanduja K Kuebler P Nakagawa H Sotamaa K Prior TW Westman J Panescu J Fix D Lockman J Comeras I de la Chapelle A Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer).N Engl J Med. 2005; 352: 1851-1860Crossref PubMed Scopus (1096) Google Scholar,9Pinol V Castells A Andreu M Castellvi-Bel S Alenda C Llor X Xicola RM Rodriguez-Moranta F Paya A Jover R Bessa X Accuracy of revised Bethesda guidelines, microsatellite instability, and immunohistochemistry for the identification of patients with hereditary nonpolyposis colorectal cancer.JAMA. 2005; 293: 1986-1994Crossref PubMed Scopus (447) Google Scholar However, it is unclear which approach should be used as the primary method for screening HNPCC. Here, we summarize both the early and more recent literature data on the use of MSI, discuss the molecular basis of microsatellite instability in MMR-deficient tumors, and outline the advantages and limitations of this methodology. Our analysis indicates that given several merits of MSI that IHC does not have (see Advantages of MSI, below), MSI is an excellent, easy to use marker for identifying HNPCC. Therefore, it is important that clinicians are aware of the pros and cons of these two tests as both are widely used in screening HNPCC cases.Literature ReviewMicrosatellite InstabilityMicrosatellites are short, tandemly repeated DNA sequences of 1 to 6 bases scattered throughout the human genome;10Tautz D Hypervariability of simple sequences as a general source for polymorphic DNA markers.Nucleic Acids Res. 1989; 17: 6463-6471Crossref PubMed Scopus (1660) Google Scholar,11Weber JL May PE Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction.Am J Hum Genet. 1989; 44: 388-396PubMed Google Scholar they can be affected by a form of genomic instability called microsatellite instability.5Aaltonen LA Peltomaki P Leach FS Sistonen P Pylkkanen L Mecklin JP Jarvinen H Powell SM Jen J Hamilton SR et al.Clues to the pathogenesis of familial colorectal cancer.Science. 1993; 260: 812-816Crossref PubMed Scopus (2559) Google Scholar,6Ionov Y Peinado MA 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 (2390) Google Scholar,8Thibodeau SN Bren G Schaid D Microsatellite instability in cancer of the proximal colon.Science. 1993; 260: 816-819Crossref PubMed Scopus (2785) Google Scholar,12Peltomaki P Lothe RA Aaltonen LA Pylkkanen L Nystrom-Lahti M Seruca R David L Holm R Ryberg D Haugen A et al.Microsatellite instability is associated with tumors that characterize the hereditary non-polyposis colorectal carcinoma syndrome.Cancer Res. 1993; 53: 5853-5855PubMed Google Scholar MSI is a change in length of a microsatellite allele due to either insertion or deletion of repeating units during DNA replication and failure of the DNA mismatch repair system to correct these errors. MSI analysis has been used as a screening method to identify HNPCC patients and a subgroup of colorectal cancer patients for further genetic testing.The DNA Mismatch Repair System and HNPCCDNA MMR is an effective post-replication mechanism. Most errors that occur during DNA replication are immediately corrected by the 3′ to 5′ exonuclease activity of DNA polymerase. It is estimated that 99.9% of the mutations that escape the proofreading activity of DNA polymerase (DNA polymerase slippage) are repaired by the DNA MMR system, particularly single-bp mismatches and “loop outs” of unpaired bases.13Vogelstein B Kinzler KW The Genetic Basis of Human Cancer. McGraw-Hill, Medical Pub Division, New York2002Google Scholar The replication machinery slips more frequently on repetitive sequences than on non-repetitive sequences, so microsatellite instability occurs in the repetitive sequences in MMR-deficient cells. The causes of MMR defects are: i) germline mutations in any one of the five DNA MMR genes—mutS homolog 2 (MSH2), mutL homolog 1 (MLH1), MSH6, and, infrequently, postmeiotic segregation increased 2 or 1 (PMS2 or PMS1), causing HNPCC14Lynch HT de la Chapelle A Hereditary colorectal cancer.N Engl J Med. 2003; 348: 919-932Crossref PubMed Scopus (1624) Google Scholar; and ii) somatic inactivation of MLH1 caused by promoter hypermethylation in approximately 15% of sporadic colorectal cancer.5Aaltonen LA Peltomaki P Leach FS Sistonen P Pylkkanen L Mecklin JP Jarvinen H Powell SM Jen J Hamilton SR et al.Clues to the pathogenesis of familial colorectal cancer.Science. 1993; 260: 812-816Crossref PubMed Scopus (2559) Google Scholar,8Thibodeau SN Bren G Schaid D Microsatellite instability in cancer of the proximal colon.Science. 1993; 260: 816-819Crossref PubMed Scopus (2785) Google Scholar,15Herman 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 ScholarIn MMR-deficient cells, genes that contain a microsatellite in their coding regions are more prone to frameshift mutations. Mutations in key genes that regulate cell growth and apoptosis ultimately lead to dysregulated cell proliferation and/or cell death, which further speeds the evolution of colorectal cancer.16Umar A Risinger JI Hawk ET Barrett JC Testing guidelines for hereditary non-polyposis colorectal cancer.Nat Rev Cancer. 2004; 4: 153-158Crossref PubMed Scopus (146) Google Scholar One example is the well studied frameshift mutations in the TGF-βRII gene, which commonly occurs in colorectal cancer but not in endometrial cancer. In most colorectal cancers, the polyadenine tract mutations affect both alleles of TGF-βRII, suggesting that TGF-βRII functions as a tumor suppressor during colorectal cancer development and is a critical target of inactivation in mismatch repair-deficient tumors.17Kuismanen SA Moisio AL Schweizer P Truninger K Salovaara R Arola J Butzow R Jiricny J Nystrom-Lahti M Peltomaki P Endometrial and colorectal tumors from patients with hereditary nonpolyposis colon cancer display different patterns of microsatellite instability.Am J Pathol. 2002; 160: 1953-1958Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar18Myeroff LL Parsons R Kim SJ Hedrick L Cho KR Orth K Mathis M Kinzler KW Lutterbaugh J Park K et al.A transforming growth factor beta receptor type II gene mutation common in colon and gastric but rare in endometrial cancers with microsatellite instability.Cancer Res. 1995; 55: 5545-5547PubMed Google Scholar19Parsons R Myeroff LL Liu B Willson JK Markowitz SD Kinzler KW Vogelstein B Microsatellite instability and mutations of the transforming growth factor beta type II receptor gene in colorectal cancer.Cancer Res. 1995; 55: 5548-5550PubMed Google Scholar Similar frameshift mutations in coding microsatellites also occur in other genes involved in growth control and apoptosis (TCF4, IGFIIR, BAX, and RIZ), as well as in genes involved in DNA mismatch repair itself (MSH6, MSH3, and MSH2).14Lynch HT de la Chapelle A Hereditary colorectal cancer.N Engl J Med. 2003; 348: 919-932Crossref PubMed Scopus (1624) Google ScholarMSI as a Marker for HNPCC ScreeningThe original (1997) Bethesda guidelines20Boland 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,21Rodriguez-Bigas MA Boland CR Hamilton SR Henson DE Jass JR Khan PM Lynch H Perucho M Smyrk T Sobin L Srivastava S A National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome: meeting highlights and Bethesda guidelines.J Natl Cancer Inst. 1997; 89: 1758-1762Crossref PubMed Scopus (930) Google Scholar proposed a panel of five microsatellite markers for the uniform analysis of MSI in HNPCC. This panel, which is referred to as the Bethesda panel, included two mononucleotide (BAT-25 and BAT-26) and three dinucleotide (D5S346, D2S123, and D17S250) repeats. Samples with instability in two or more of these markers are defined as MSI-High (MSI-H), whereas those with one unstable marker are designated as MSI-Low (MSI-L). Samples with no detectable alterations are MSI-stable (MSS). Because mononucleotide markers appear to be more sensitive than dinucleotide markers for the detection of MSI-H, limitations in the original panel resulting from inclusion of dinucleotide repeats were addressed at a 2002 National Cancer Institute workshop, and revised recommendations for MSI detection were proposed. The revision mainly recommends testing a secondary panel of mononucleotide markers, such as BAT-40, to exclude MSI-L in cases in which only the dinucleotide repeats are mutated.22Umar 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 According to the revised Bethesda guidelines, strategies based on MSI testing were effective in identifying MLH1/MSH2 mutation carriers (sensitivity 81.8% and specificity 98.0%).9Pinol V Castells A Andreu M Castellvi-Bel S Alenda C Llor X Xicola RM Rodriguez-Moranta F Paya A Jover R Bessa X Accuracy of revised Bethesda guidelines, microsatellite instability, and immunohistochemistry for the identification of patients with hereditary nonpolyposis colorectal cancer.JAMA. 2005; 293: 1986-1994Crossref PubMed Scopus (447) Google ScholarAdvantages of MSIMicrosatellite Instability Serves as an Excellent, Easy-to-Evaluate Marker of MMR Deficiency, and Recent Improvements in MSI Testing Significantly Enhance Accuracy and Reduce CostA hallmark of tumors in HNPCC is microsatellite instability. Typically half or more of all microsatellites have mutations (contraction or elongation) in the tumor cells; therefore, microsatellite instability serves as an excellent, easy-to-evaluate marker of mismatch repair deficiency. Since both HNPCC and MSI are caused by MMR defects, MSI can be used as a surrogate marker of HNPCC and has been widely accepted as a primary method for identifying individuals at risk for HNPCC.As mentioned under Literature Review, a recent follow-up NCI workshop recognized the limitations of the original Bethesda panel20Boland 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,21Rodriguez-Bigas MA Boland CR Hamilton SR Henson DE Jass JR Khan PM Lynch H Perucho M Smyrk T Sobin L Srivastava S A National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome: meeting highlights and Bethesda guidelines.J Natl Cancer Inst. 1997; 89: 1758-1762Crossref PubMed Scopus (930) Google Scholar due to the inclusion of dinucleotide repeats, which are less sensitive and less specific than mononucleotide repeats for identification of cancers with MMR deficiency.22Umar 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 To improve the accuracy of MSI testing using the Bethesda panel of MSI markers, a panel of five mononucleotide markers was developed and incorporated into a multiplex fluorescence assay: the Promega (Madison, WI) MSI Analysis System.23Bacher JW Flanagan LA Smalley RL Nassif NA Burgart LJ Halberg RB Megid WM Thibodeau SN Development of a fluorescent multiplex assay for detection of MSI-High tumors.Dis Markers. 2004; 20: 237-250Crossref PubMed Scopus (189) Google Scholar These mononucleotide repeat markers are quasi-monomorphic; that is, almost all individuals are homozygous for the same common allele for a given marker. The use of monomorphic markers simplifies data interpretation. The added pentanucleotide repeat markers ensure that the tumor and matching normal specimen are from the same individual.23Bacher JW Flanagan LA Smalley RL Nassif NA Burgart LJ Halberg RB Megid WM Thibodeau SN Development of a fluorescent multiplex assay for detection of MSI-High tumors.Dis Markers. 2004; 20: 237-250Crossref PubMed Scopus (189) Google Scholar,24Suraweera 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 The Promega system can help resolve cases of MSI-L into either MSI-H or MSS.25Murphy KM Zhang S Geiger T Hafez MJ Bacher J Berg KD Eshleman JR Comparison of the microsatellite instability analysis system and the Bethesda panel for the determination of microsatellite instability in colorectal cancers.J Mol Diagn. 2006; 8: 305-311Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar The representative electropherograms of the Promega MSI Analysis System are shown in Figure 1. The microsatellite markers included in the Bethesda panel20Boland 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 Scholar21Rodriguez-Bigas MA Boland CR Hamilton SR Henson DE Jass JR Khan PM Lynch H Perucho M Smyrk T Sobin L Srivastava S A National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome: meeting highlights and Bethesda guidelines.J Natl Cancer Inst. 1997; 89: 1758-1762Crossref PubMed Scopus (930) Google Scholar22Umar 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 and the Promega MSI Analysis System,25Murphy KM Zhang S Geiger T Hafez MJ Bacher J Berg KD Eshleman JR Comparison of the microsatellite instability analysis system and the Bethesda panel for the determination of microsatellite instability in colorectal cancers.J Mol Diagn. 2006; 8: 305-311Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar,26Baker K Raut P Jass JR Microsatellite unstable colorectal cancer cell lines with truncating TGFbetaRII mutations remain sensitive to endogenous TGFbeta.J Pathol. 2007; 213: 257-265Crossref PubMed Scopus (15) Google Scholar as well as other commonly used MSI markers, are summarized in Table 1.Table 1The Loci Information for Commonly Used MSI MarkersMicrosatellite markerRepeat typeChromosomal location (gene near marker/GenBank number)Repeat motif*Non-repetitive nucleotides are indicated as dots.24Suraweera 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 Scholar52Dietmaier W Wallinger S Bocker T Kullmann F Fishel R Ruschoff J Diagnostic microsatellite instability: definition and correlation with mismatch repair protein expression.Cancer Res. 1997; 57: 4749-4756PubMed Google ScholarBethesda panel20Boland 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 Scholar21Rodriguez-Bigas MA Boland CR Hamilton SR Henson DE Jass JR Khan PM Lynch H Perucho M Smyrk T Sobin L Srivastava S A National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome: meeting highlights and Bethesda guidelines.J Natl Cancer Inst. 1997; 89: 1758-1762Crossref PubMed Scopus (930) Google Scholar22Umar 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 ScholarPromega kit24Suraweera 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 Scholar25Murphy KM Zhang S Geiger T Hafez MJ Bacher J Berg KD Eshleman JR Comparison of the microsatellite instability analysis system and the Bethesda panel for the determination of microsatellite instability in colorectal cancers.J Mol Diagn. 2006; 8: 305-311Abstract Full Text Full Text PDF PubMed Scopus (191) Google ScholarBAT-25Mononucleotide4q12 (c-kit, intron 16)TTTT.T.TTTT.(T)7.A(T)25XXBAT-26Mononucleotide2p16.3-p21 (hMSH2 gene, intron 5)(T)5 . . . . . . (A)26XXNR-21Mononucleotide14q11.2 (SLC7A8, 5′UTR)(T)21XNR-24Mononucleotide2q11.2 (ZNF2, 3′UTR)(T)24XMONO-27Mononucleotide2p21 (MAP4K3, intron 13)(A)27XBAT-40Mononucleotide1p13.1 (HSD3B2)TTTT.TT . . (T)7 . . . . . . . . . . TTTT.(T)40D2S123Dinucleotide2p16 (MSH2)(CA)13TA(CA)15(T/GA)7XD5S346Dinucleotide5q21/22 (APC)(CA)26XD17S250Dinucleotide17q11.2-q12 (BRCA1)(TA)7 . . . . . . . . . . . . . . .X . . . . . . . . . . (CA)24Penta CPentanucleotide21q22.3 (AL138752)(AAAAG)3–15XPenta DPentanucleotide9p12-13.3 (AC003656)(AAAAG)2–17XMYCL1Complex1p34.3 (MYCL1)GAAAA(GAAA)2TAAA(A/G)10 GAAAGA(GAAA)14 GAAA (GAAAA)8GAAAAA(GAAAA)2* Non-repetitive nucleotides are indicated as dots. Open table in a new tab Since the Promega MSI Analysis System utilizes a multiplex fluorescence assay, PCR for all five mononucleotide markers and two pentanucleotide nucleotide markers can be performed in a single reaction. The size of the amplified products can be easily visualized using capillary electrophoresis. The cost of MSI testing is significantly reduced. In addition, in situations where the availability of representative tumor sections is limited, MSI testing can be done on one tumor section, whereas at least four tumor sections are required for IHC.Proficiency Testing for MSI Is Available and the Reproducibility of MSI Testing is Close to 100%The most common type of quality control in which clinical laboratories participate is proficiency testing, in which testing is performed along with routine laboratory work. Proficiency testing is a method of externally validating the accuracy of laboratory performance by testing samples and comparing results of all participating laboratories. To comply with Clinical Laboratory Improvement Amendments of 1988, laboratories performing moderate and high complexity tests must be enrolled in regulatory proficiency testing for their particular specialties/subspecialties of testing. A convenient way to accomplish this is by subscribing to a proficiency testing program that monitors those analytes. In the United States, proficiency testing for MSI is provided by the College of American Pathologists (CAP).In the most recent CAP Proficiency Survey for microsatellite instability testing (2007 MSI-B), 71 laboratories were enrolled in this survey. The participant summary provided evidence that mononucleotide markers have higher specificity (ie, a lower false positive rate) for instability than dinucleotide markers. CAP also provided a detailed summary on several clinically important issues such as the number and types of markers used, methods used to perform the assay, and definition of MSI-H and MSI-L phenotypes. This information is valuable to clinical laboratories that are currently offering this test as well as to those that are planning to launch this test. Thirty-four of the 53 laboratories reported performing IHC together with MSI, whereas 19 of 53 laboratories did not perform IHC testing. With two recent CAP Proficiency Surveys combined (2007 MSI-A and MSI-B), 101 of 103 laboratories (98%) reported the same results, indicating that the reproducibility of MSI testing can be evaluated and, more importantly, that it is satisfactorily high.In terms of quality control and interpretation of MSI testing in clinical settings, studies from a six-center consortium indicated that optimal PCR quality is essential to getting interpretable results. In most c
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