Heterogeneous Staining for Mismatch Repair Proteins during Population-Based Prescreening for Hereditary Nonpolyposis Colorectal Cancer
2007; Elsevier BV; Volume: 9; Issue: 4 Linguagem: Inglês
10.2353/jmoldx.2007.060162
ISSN1943-7811
AutoresNatasha Watson, Fabienne Grieu, M. J. Morris, Jennet Harvey, Colin J.R. Stewart, Lyn Schofield, Jack Goldblatt, Barry Iacopetta,
Tópico(s)Colorectal Cancer Treatments and Studies
ResumoThe aim of this study was to determine the frequency of microsatellite instability (MSI+) in tumors from a population-based series of young colorectal cancer patients and its correlation with the loss of expression of mismatch repair (MMR) proteins. The BAT-26 mononucleotide repeat was used to screen for MSI+ in all colorectal cancers diagnosed in Western Australia throughout a 5-year period in patients <60 years of age. MSI+ was found in 75 of 1003 (7.5%) cases, of which six contained a concomitant mutation in BRAF and were therefore excluded from further investigations as possible hereditary nonpolyposis colorectal cancer. Immunohistochemistry was used to evaluate expression of the four major MMR proteins (MLH1, MSH2, MSH6, and PMS2) in the remaining 69 MSI+ tumors. Complete loss of MLH1 and PMS2 expression or of MSH2 and MSH6 expression was found in 35 (51%) and 17 (25%) cases, respectively, whereas other patterns of complete loss were observed in eight cases (12%). Eight tumors (12%) were initially recorded as showing normal expression, but on review seven were reclassified as having abnormal staining because of heterogeneous patterns of MMR loss. Three of these seven cases had previously been found to have germline mutations. Because of possible misinterpretation of heterogeneous immunohistochemistry staining for MMR protein loss, MSI testing is recommended as the initial screen for population-based detection of hereditary nonpolyposis colorectal cancer. The aim of this study was to determine the frequency of microsatellite instability (MSI+) in tumors from a population-based series of young colorectal cancer patients and its correlation with the loss of expression of mismatch repair (MMR) proteins. The BAT-26 mononucleotide repeat was used to screen for MSI+ in all colorectal cancers diagnosed in Western Australia throughout a 5-year period in patients <60 years of age. MSI+ was found in 75 of 1003 (7.5%) cases, of which six contained a concomitant mutation in BRAF and were therefore excluded from further investigations as possible hereditary nonpolyposis colorectal cancer. Immunohistochemistry was used to evaluate expression of the four major MMR proteins (MLH1, MSH2, MSH6, and PMS2) in the remaining 69 MSI+ tumors. Complete loss of MLH1 and PMS2 expression or of MSH2 and MSH6 expression was found in 35 (51%) and 17 (25%) cases, respectively, whereas other patterns of complete loss were observed in eight cases (12%). Eight tumors (12%) were initially recorded as showing normal expression, but on review seven were reclassified as having abnormal staining because of heterogeneous patterns of MMR loss. Three of these seven cases had previously been found to have germline mutations. Because of possible misinterpretation of heterogeneous immunohistochemistry staining for MMR protein loss, MSI testing is recommended as the initial screen for population-based detection of hereditary nonpolyposis colorectal cancer. The microsatellite instability (MSI+) phenotype in tumor DNA, also referred to as MSI-high, is a consequence of defects in the DNA mismatch repair system.1Ionov 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 (2396) Google Scholar MSI+ occurs in ∼10% of sporadic colorectal carcinomas (CRCs) and in almost all tumors associated with hereditary nonpolyposis colorectal cancer (HNPCC; Lynch syndrome). The majority of sporadic MSI+ CRCs arise in the proximal colon of older patients and are associated with acquired, methylation-induced transcriptional silencing of MLH1 gene expression.2Herman 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 (1691) Google Scholar Germline mutations in MLH1 and MSH2 and to a lesser extent MSH6 and PMS2 account for almost all MSI+ CRCs associated with the HNPCC syndrome.3Mitchell RJ Farrington SM Dunlop MG Campbell H Mismatch repair genes hMLH1 and hMSH2 and colorectal cancer: a HuGE review.Am J Epidemiol. 2002; 156: 885-902Crossref PubMed Scopus (119) Google Scholar The identification of potential inherited mismatch repair (MMR) gene mutation carriers is currently the major clinical application for MSI screening. Accurate diagnosis of this syndrome is of great importance for family risk management because regular colonoscopy has been demonstrated to improve the survival of mutation carriers.4Järvinen HJ Aarnio M Mustonen H Aktan-Collan K Aaltonen LA Peltomaki P De La Chapelle A Mecklin JP Controlled 15-year trial on screening for colorectal cancer in families with hereditary nonpolyposis colorectal cancer.Gastroenterology. 2000; 118: 829-834Abstract Full Text Full Text PDF PubMed Scopus (1094) Google Scholar There is widespread concern, however, that most mutation carriers in the population are not being identified.5Lynch HT Riley BD Weissman SM Coronel SM Kinarsky Y Lynch JF Shaw TG Rubinstein WS Hereditary nonpolyposis colorectal carcinoma (HNPCC) and HNPCC-like families: problems in diagnosis, surveillance, and management.Cancer. 2004; 100: 53-64Crossref PubMed Scopus (73) Google Scholar,6Terdiman J It is time to get serious about diagnosing Lynch syndrome (hereditary nonpolyposis colorectal cancer) with defective DNA mismatch repair in the general population.Gastroenterology. 2005; 129: 741-744Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Several reasons are likely to account for this, including the failure of clinicians to carefully document family cancer histories and to refer patients for genetic evaluation. For patients who are referred to family cancer clinics, tumors are usually tested for MSI in the first instance using polymerase chain reaction (PCR)-based methods. MSI testing is not available, however, in the large majority of routine pathology service laboratories, and these rely on immunohistochemistry (IHC) technique to detect loss of MMR protein expression as a surrogate marker for the presence of MSI. Whether or not all or clinicopathological subsets of CRCs should be routinely tested for MMR defects to assist with the detection of HNPCC is still a matter of conjecture. There is also considerable debate as to whether MSI or IHC is the technically superior screening approach.7Chapusot C Martin L Puig PL Ponnelle T Cheynel N Bouvier AM Rageot D Roignot P Rat P Faivre J Piard F What is the best way to assess microsatellite instability status in colorectal cancer? Study on a population base of 462 colorectal cancers.Am J Surg Pathol. 2004; 28: 1553-1559Crossref PubMed Scopus (38) Google Scholar8Ward RL Turner J Williams R Pekarsky B Packham D Velickovic M Meagher A O'Connor T Hawkins NJ Routine testing for mismatch repair deficiency in sporadic colorectal cancer is justified.J Pathol. 2005; 207: 377-384Crossref PubMed Scopus (47) Google Scholar9Jass JR Re: Ward et al. Routine testing for mismatch repair deficiency in sporadic colorectal cancer is justified (letter to the editor). J Pathol 2005, 207:377–384.J Pathol. 2006; 208: 590-591Crossref PubMed Scopus (4) Google Scholar10Mangold E Pagenstecher C Friedl W Fischer HP Merkelbach-Bruse S Ohlendorf M Friedrichs N Aretz S Buettner R Propping P Mathiak M Tumors from MSH2 mutation carriers show loss of MSH2 expression but many tumors from MLH1 mutation carriers exhibit weak positive MLH1 staining.J Pathol. 2005; 207: 385-395Crossref PubMed Scopus (88) Google Scholar11Shia J Klimstra DS Nafa K Offit K Guillem JG Markowitz AJ Gerald WL Ellis NA Value of immunohistochemical detection of DNA mismatch repair proteins in predicting germ-line mutation in hereditary colorectal neoplasms.Am J Surg Pathol. 2005; 29: 96-104Crossref PubMed Scopus (128) Google Scholar12Southey MC Jenkins MA Mead L Whitty J Trivett M Tesoriero AA Smith LD Jennings K Grubb G Royce SG Walsh MD Barker MA Young JP Jass JR St. John DJ Macrae FA Giles GG Hopper JL Use of molecular tumor characteristics to prioritize mismatch repair gene testing in early-onset colorectal cancer.J Clin Oncol. 2005; 23: 6524-6532Crossref PubMed Scopus (182) Google Scholar13Evans GD Lalloo F Mak T Speake D Hill J Is it time to abandon microsatellite instability as a pre-screen for selecting families for mutation testing for mismatch repair genes?.J Clin Oncol. 2006; 24: 1960-1962Crossref PubMed Scopus (10) Google Scholar With the aim of improving the rate of HNPCC detection in the state of Western Australia, our laboratory is conducting a population-wide study that uses MSI as the prescreen test followed by IHC for positive cases. All CRC patients <60 years of age and diagnosed in this state throughout a 5-year period were tested for MSI. The coexistence of a BRAF oncogene mutation in tumors found to be MSI+ was used as a criterion to exclude cases that were sporadic in origin.14Deng G Bell I Crawley S Gum J Terdiman JP Allen BA Truta B Sleisenger MH Kim YS BRAF mutation is frequently present in sporadic colorectal cancer with methylated hMLH1, but not hereditary nonpolyposis colorectal cancer.Clin Cancer Res. 2004; 10: 191-195Crossref PubMed Scopus (332) Google Scholar The remaining MSI+/wild-type BRAF cases were further evaluated for loss of MMR protein expression and subsequent germline testing for consenting individuals. All CRC cases diagnosed in the state of Western Australia from 2000 to 2006 inclusive were identified by electronic searches of the public and private pathology service provider databases. Only patients <60 years of age at diagnosis were selected for the study. This age cutoff was chosen as a compromise between the feasibility of screening for MSI+ in large sample numbers while at the same time maximizing the capture of HNPCC cases and reducing sporadic cases. An additional 24 CRC patients <30 years of age and diagnosed in Western Australia during the period from 1993 to 1999 were also included in the study as potential high-risk cases for HNPCC. Archival tissue blocks obtained from surgical resection or biopsies were selected to contain maximal tumor content. All cases were screened for MSI and BRAF mutation. No information on family history of cancer was available at the time of MSI+ screening or IHC. Ethics approval for the project was obtained from the Human Research Ethics Committees of each hospital and from the Confidentiality of Health Information Committee. The ethical issues involved in the phenotypic screening (MSI and IHC) of archival tumor tissues without patient consent are described elsewhere.15Zeps N Iacopetta B Schofield L George J Goldblatt J Waiver of individual patient consent in research: when do potential benefits to the community outweigh private rights?.Med J Aust. 2007; 186: 88-90PubMed Google Scholar The MSI status for all tumors was determined using fluorescent-single strand conformation polymorphism (F-SSCP) to detect deletions in the BAT-26 mononucleotide repeat as described previously.16Iacopetta B Grieu F Routine detection of the replication error phenotype in clinical tumor specimens using fluorescence-SSCP.Biotechniques. 2000; 28: 566-570PubMed Google Scholar F-SSCP was also used to screen for the common V600E point mutation in the BRAF oncogene.17Li WQ Kawakami K Ruszkiewicz A Bennett G Moore J Iacopetta B BRAF mutations are associated with distinctive clinical, pathological and molecular features of colorectal cancer independently of microsatellite instability status.Mol Cancer. 2006; 5: 2Crossref PubMed Scopus (149) Google Scholar Several 10-μm sections cut from formalin-fixed, paraffin-embedded tumor blocks were digested at 50°C with proteinase K for at least 48 hours before heat inactivation (95°C, 10 minutes) of the enzyme. Primer sequences and PCR conditions for the amplification of BAT-26 and BRAF were the same as described previously.16Iacopetta B Grieu F Routine detection of the replication error phenotype in clinical tumor specimens using fluorescence-SSCP.Biotechniques. 2000; 28: 566-570PubMed Google Scholar,17Li WQ Kawakami K Ruszkiewicz A Bennett G Moore J Iacopetta B BRAF mutations are associated with distinctive clinical, pathological and molecular features of colorectal cancer independently of microsatellite instability status.Mol Cancer. 2006; 5: 2Crossref PubMed Scopus (149) Google Scholar IHC was performed on tumors from 2000 to 2004 found to be MSI+ and BRAF wild type and on all tumors diagnosed in 2005 or 2006. Tissue sections of 4-μm thickness were cut from the same tumor blocks used for MSI+ screening and placed onto silane-coated slides. After dewaxing and rehydration, they were stained for MLH1, PMS2, MSH2, and MSH6 using commercially available antibodies (clones G168-15, A16-4, G219-1129 and 44, respectively; BD PharMingen) at the recommended dilutions and using standard IHC methods. Antigen retrieval was performed using Target retrieval solution (DAKO, Botany, NSW, Australia) and a Decloaker pressure cooker (BioCare Medical, Stafford, QLD, Australia) at 121°C for 20 minutes. The detection system used was the Mach3 kit (BioCare Medical) as recommended by the supplier. Lymphocytes and normal colonic epithelium located adjacent to tumor cells served as internal controls for positive MMR protein expression. Cases were initially scored by a pathologist (J.H.) as positive for expression (MMR normal) if nuclear staining was present in any of the malignant cells. Cases were initially scored as negative for expression (loss of MMR expression) if all tumor cells showed complete loss of staining while the adjacent normal cells showed nuclear staining. A total of 1059 tumors from CRC patients <60 years of age and diagnosed from 2000 to 2004 were screened for MSI+ using the BAT-26 marker alone, of which 1003 gave a result. Representative F-SSCP results are shown in Figure 1. Deletion of the BAT-26 allele was found in 75 of 1003 (7.5%) cases. The frequency of MSI+ in different age groups was 22.2% (≤29 years), 14.7% (30 to 39 years), 10.6% (40 to 49 years), and 4.4% (50 to 59 years). Mutations in BRAF (V600E) were found in 6 of 75 (8%) of the MSI+ tumors, and all were in patients 54 to 59 years of age. Based on previous observations,14Deng G Bell I Crawley S Gum J Terdiman JP Allen BA Truta B Sleisenger MH Kim YS BRAF mutation is frequently present in sporadic colorectal cancer with methylated hMLH1, but not hereditary nonpolyposis colorectal cancer.Clin Cancer Res. 2004; 10: 191-195Crossref PubMed Scopus (332) Google Scholar these were excluded from additional follow-up as possible HNPCC. The remaining 69 MSI+/BRAF wild-type tumors were further investigated by IHC for expression of the major MMR proteins. All cases showed appropriate positive staining of normal colonic epithelial cells and lymphocytes with all four antisera tested. In MSI+ tumors, loss of MLH1 expression usually occurs in conjunction with PMS2 loss, whereas MSH2 loss is usually accompanied by MSH6 loss. These common patterns of loss were observed in 35 (51%) and 17 (25%) cases, respectively (Table 1). Tumors in this category showed complete loss of staining for the two relevant proteins in all malignant cells. Another eight tumors showed complete loss of expression for MSH6 alone (three cases); PMS2 alone (two cases); MSH2 alone (one case); PMS2 and MSH6 (one case); or MLH1, PMS2, and MSH6 (one case). There were insufficient tumor cells in one case to allow accurate evaluation of staining. The remaining eight tumors were initially assessed as showing normal expression for all four MMR proteins, although a degree of staining heterogeneity was noted in some cases.Table 1Patterns of MMR Protein Expression in 69 MSI+ Tumors from Young CRC PatientsPattern of loss (n)*One case contained insufficient tumor tissue for proper evaluation by IHC.MLH1†−, loss of expression in all tumor cells; +, expression in all tumor cells; H, heterogeneous loss of expression in tumor cells; U, unsatisfactory IHC result.PMS2MSH2MSH6Common (52) 35−−++ 17++−−Other (8) 3+++− 2+−++ 1+−+− 1++−H 1−−+−Heterogeneous (7) 2++HH 2HH++ 1+H++ 1UH++ 1+++HNo loss (1) 1++++* One case contained insufficient tumor tissue for proper evaluation by IHC.† −, loss of expression in all tumor cells; +, expression in all tumor cells; H, heterogeneous loss of expression in tumor cells; U, unsatisfactory IHC result. Open table in a new tab The single Familial Cancer Program serving the entire state of Western Australia (population, 2 million) was notified of the 69 young CRC patients (<60 years) with MSI+/BRAF wild-type tumors identified in this study. Eighteen (26%) were already known to this service as germline MMR gene mutation carriers (12 MSH2, five MLH1, one MSH6) in either the proband (n = 13) or members of their immediate family (n = 5). Three mutation carriers were among the eight tumors initially reported as showing normal MMR protein expression: one case each for MLH, MSH2, and MSH6. This prompted additional review of all eight MSI+ cases showing apparently normal expression. Heterogeneous staining patterns were observed for seven cases and included examples from each of the four MMR proteins (Table 2 and Figure 2). Two patterns of staining heterogeneity were observed. In two of the tumors there were confluent areas of staining loss involving multiple adjacent gland profiles (zonal loss; Figure 2A), whereas five tumors showed intraglandular variation in staining with strongly immunoreactive cells admixed with unstained cells (focal loss; Figure 2B). In some cases, the neoplastic cells toward the periphery of tumor aggregates or the advancing tumor margin appeared more consistently immunoreactive, whereas central glands were often negative. However, there was no clear correlation between the distribution of staining and morphological features associated with the MSI phenotype such as mucinous differentiation or presence of tumor-infiltrating lymphocytes. The remaining case was a biopsy sample of an in situ tumor that showed apparently normal expression of all four MMR proteins. This case had previously been diagnosed at 18 years of age with a small bowel tumor, and his family has a known MSH2 germline mutation.Table 2Heterogeneous Staining Patterns for MMR Proteins in MSI+ CRCCaseMLH1PMS2MSH2MSH61079*Patient lost to follow-up.Moderate intensity in 25% cells, heterogeneity within glandsModerate intensity in 25% cells, heterogeneity within glandsStrong uniform stainingVariable staining intensity909†Known MSH6 germline mutation.Strong uniform stainingStrong uniform stainingVariable staining intensity with areas of focal lossFocal areas with loss of staining308*Patient lost to follow-up.Strong uniform stainingStrong uniform stainingMostly strongly positive, <10% of glands showed heterogeneityMostly strongly positive, <10% of glands showed heterogeneity500‡Known MSH2 germline mutation.Mostly strongly positiveStrong uniform stainingHeterogeneous staining both zonal and within individual glandsHeterogeneous staining both zonal and within individual glands857§Known MLH1 germline mutation.Weak nuclear signal in ∼25% of cells, heterogeneity within glandsWeak-moderate signal in ∼25% of cells, heterogeneity within glandsStrong uniform stainingStrong uniform staining727¶Germline mutation test result pending.Focal areas of reduced intensity in <1% of glandsComplete loss of staining in ∼10% of glands; heterogeneity in <10%Strong uniform stainingFocal areas of complete loss in <1% of glands184∥Patient deceased before blood sample could be obtained for testing.Unsatisfactory stainAreas of focal lossStrong uniform stainingStrong uniform staining* Patient lost to follow-up.† Known MSH6 germline mutation.‡ Known MSH2 germline mutation.§ Known MLH1 germline mutation.¶ Germline mutation test result pending.∥ Patient deceased before blood sample could be obtained for testing. Open table in a new tab A second CRC cohort from <60-year-old patients who were diagnosed in 2005 or 2006 (n = 208) was evaluated simultaneously for MSI and IHC to evaluate the degree of concordance between the two techniques. Both analyses were performed blinded to the results of the other test. Using BAT-26 alone, 20 of 208 (9.6%) tumors were found to be MSI+ (Table 3). The same number of tumors showed loss of MMR expression using the criteria of complete loss of expression in tumor cells in the presence of staining of adjacent nonmalignant cells. Discordant results were observed for only 2 of 208 (1%) cases. These comprised one MSI− tumor that showed complete loss of MSH6 expression and one MSI+ tumor with clonal loss of both MSH2 and MSH6. The latter case can therefore be considered equivalent to the seven MSI+ tumors in the previous cohort (2000 to 2004 diagnosis period) that showed heterogeneous staining patterns. Importantly, among the remaining 187 MSI− tumors that did not show complete loss of expression, 140 (75%) displayed heterogeneous or clonal patterns of loss for at least one of the four MMR proteins.Table 3Concordance between MSI and IHC in CRC from Young Patients (<60 Years)MMR expression (n = 208)MSI−MSI+No loss*Defined as any expression for all four MMR proteins. Includes cases showing weak, clonal, or heterogeneous staining, or variable staining intensity.1871†Clonal losses of both MSH2 and MSH6.Complete loss‡Defined as complete loss of at least one MMR protein.1§Complete loss of MSH6 expression only.19* Defined as any expression for all four MMR proteins. Includes cases showing weak, clonal, or heterogeneous staining, or variable staining intensity.† Clonal losses of both MSH2 and MSH6.‡ Defined as complete loss of at least one MMR protein.§ Complete loss of MSH6 expression only. Open table in a new tab Most estimates of the proportion of CRCs that are attributable to HNPCC range from 1 to 3%18de Leon MP Pedroni M Benatti P Percesepe A Di Gregorio C Foroni M Rossi G Genuardi M Neri G Leonardi F Viel A Capozzi E Boiocchi M Roncucci L Hereditary colorectal cancer in the general population: from cancer registration to molecular diagnosis.Gut. 1999; 45: 32-38Crossref PubMed Scopus (57) Google Scholar19Salovaara 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 (446) Google Scholar20Samowitz WS Curtin K Lin HH Robertson MA Schaffer D Nichols M Gruenthal K Leppert MF Slattery ML The colon cancer burden of genetically defined hereditary nonpolyposis colon cancer.Gastroenterology. 2001; 121: 830-838Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar21Katballe N Christensen M Wikman FP Orntoft TF Laurberg S Frequency of hereditary non-polyposis colorectal cancer in Danish colorectal cancer patients.Gut. 2002; 50: 43-51Crossref PubMed Scopus (84) Google Scholar22Cunningham JM Kim CY Christensen ER Tester DJ Parc Y Burgart LJ Halling KC McDonnell SK Schaid DJ Walsh Vockley C Kubly V Nelson H Michels VV Thibodeau SN The frequency of hereditary defective mismatch repair in a prospective series of unselected colorectal carcinomas.Am J Hum Genet. 2001; 69: 780-790Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar23Hampel 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 (1105) Google Scholar with frequencies in unselected patients <50 years of age and <45 years of age estimated at 14 and 17%, respectively.12Southey MC Jenkins MA Mead L Whitty J Trivett M Tesoriero AA Smith LD Jennings K Grubb G Royce SG Walsh MD Barker MA Young JP Jass JR St. John DJ Macrae FA Giles GG Hopper JL Use of molecular tumor characteristics to prioritize mismatch repair gene testing in early-onset colorectal cancer.J Clin Oncol. 2005; 23: 6524-6532Crossref PubMed Scopus (182) Google Scholar,21Katballe N Christensen M Wikman FP Orntoft TF Laurberg S Frequency of hereditary non-polyposis colorectal cancer in Danish colorectal cancer patients.Gut. 2002; 50: 43-51Crossref PubMed Scopus (84) Google Scholar Identification of pathogenic mutations in MMR genes provides a definitive genetic test that can assist with the management of affected families. Because mutation screening is expensive, clinical guidelines based mainly on age and family history of cancer have been proposed to help with the prioritization of individuals for genetic testing (Amsterdam and Bethesda criteria). Although this approach to the detection of HNPCC has relatively good specificity, it suffers from poor sensitivity with the result that most MMR gene mutations in the community remain undiagnosed. The reasons for this have been outlined elsewhere.5Lynch HT Riley BD Weissman SM Coronel SM Kinarsky Y Lynch JF Shaw TG Rubinstein WS Hereditary nonpolyposis colorectal carcinoma (HNPCC) and HNPCC-like families: problems in diagnosis, surveillance, and management.Cancer. 2004; 100: 53-64Crossref PubMed Scopus (73) Google Scholar,6Terdiman J It is time to get serious about diagnosing Lynch syndrome (hereditary nonpolyposis colorectal cancer) with defective DNA mismatch repair in the general population.Gastroenterology. 2005; 129: 741-744Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar With the aim of increasing the detection rate for HNPCC in the Western Australian population, our group is trying the use of MSI as the initial test to select young CRC patients that are to be offered MMR genetic testing. During the course of this work, it became apparent that a relatively high incidence of errors can occur in the interpretation of IHC results for MMR protein expression because of heterogeneity of staining patterns. This presents as a major difficulty for population-based HNPCC detection programs that use IHC as the initial screening test in the absence of supporting data from MSI testing. Because routine pathology laboratories are at the front line of population-based screening for HNPCC and are almost totally reliant on the IHC technique, we propose that serious consideration be given to alternative strategies that are based on MSI analysis. In a process that was totally blinded to both family history and germline mutation status, more than 1000 consecutive CRCs from relatively young patients (<60 years) were evaluated for MSI+. Approximately 7.5% tested positive and, because of their young age, were therefore considered strong candidates for HNPCC. BRAF mutation testing was useful for excluding a small proportion of these MSI+ cases (6 of 75, 8%) as being acquired rather than hereditary in origin.14Deng G Bell I Crawley S Gum J Terdiman JP Allen BA Truta B Sleisenger MH Kim YS BRAF mutation is frequently present in sporadic colorectal cancer with methylated hMLH1, but not hereditary nonpolyposis colorectal cancer.Clin Cancer Res. 2004; 10: 191-195Crossref PubMed Scopus (332) Google Scholar The presence of BRAF mutation serves as a surrogate marker for methylation of the MLH1 promoter. All six MSI+ patients with BRAF mutation were 54 to 59 years of age, suggesting this marker is unlikely to be of value for the exclusion of sporadic MSI+ cases in patients below this age group. Patients <60 years of age comprise ∼25% of nonselected CRC cohorts, implying that ∼1.8% of all CRCs fall into the high-risk category for HNPCC as defined by the criteria of young age, MSI+, and wild-type BRAF. Although the germline mutation status is not yet known for all of the high-risk cases, this value is within the range of 1 to 3% cited above for the frequency of HNPCC observed in other population-based studies.18de Leon MP Pedroni M Benatti P Percesepe A Di Gregorio C Foroni M Rossi G Genuardi M Neri G Leonardi F Viel A Capozzi E Boiocchi M Roncucci L Hereditary colorectal cancer in the general population: from cancer registration to molecular diagnosis.Gut. 1999; 45: 32-38Crossref PubMed Scopus (57) Google Scholar19Salovaara 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 (446) Google Scholar20Samowitz WS Curtin K Lin HH Robertson MA Schaffer D Nichols M Gruenthal K Leppert MF Slattery ML The colon cancer burden of genetically defined hereditary nonpolyposis colon cancer.Gastroenterology. 2001; 121: 830-838Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar21Katballe N Christensen M Wikman FP Orntoft TF Laurberg S Frequency of hereditary non-polyposis colorectal cancer in Danish colorectal cancer patients.Gut. 2002; 50: 43-51Crossref PubMed Scopus (84) Google Scholar22Cunningham JM Kim CY Christensen ER Tester DJ Parc Y Burgart LJ Halling KC McDonnell SK Schaid DJ Walsh Vockley C Kubly V Nelson H Michels VV Thibodeau SN The frequency of hereditary defective mismatch repair in a prospective series of unselected colorectal carcinomas.Am J Hum Genet. 2001; 69: 780-790Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar23Hampel 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
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