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Molecular Biology and Genetics |
Dana Farber Cancer Institute [S. S. W., G. T., M. L., J. G., S. S., E. F.], Brigham and Women’s Hospital [G. T., M. L., S. S.], Harvard Medical School, Boston, Massachusetts 02115, and Ludwig Institute of Cancer Research [S. S. W., J. S., R. D. K.], Department of Medicine [R. D. K.] and Cancer Center [R. D. K.], University of California San Diego School of Medicine, La Jolla, California 92903
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Predicting families with a high likelihood of having a disease-causing mutation is of great importance, because mutation detection in a number of genes is time consuming and costly. Therefore, efforts have been made to find the most sensitive clinical criteria, based on family history, to be used to select families for mutation detection analyses. Selection of samples using a clinical or molecular phenotype could ideally reduce the sample number for mutation screening. Studies of families fulfilling the Amsterdam criteria, which are the most restrictive criteria for HNPCC, have identified germ-line MSH2 and MLH1 mutations with a relatively high sensitivity (
60%) and specificity (70%; Refs. 13, 14, 15
). Analysis of HNPCC cases identified by less strict criteria such as the Modified Amsterdam (16)
and Bethesda criteria (2)
, which include extracolonic tumors, led to an increased sensitivity and a decreased specificity for the identification of germ-line MSH2 and MLH1 mutations. The Bethesda criteria, which are the least restrictive clinical criteria, predicted germ-line MSH2 and MLH1 mutations with an even higher sensitivity (94%) and a lower specificity (50%; Ref. 15
). Early-onset colorectal cancer or cases with a family history of endometrial cancer have also been shown to be independent predictors of germ-line mutations (17
, 18)
. These studies indicate that the restrictive criteria identify HNPCC cases with the highest likelihood of having a germ-line mutation but exclude many cases that have a germ-line mutation. In contrast, the less restrictive criteria identify almost all cases with a germ-line mutation but include many cases without a germ-line mutation. In a clinical setting where genetic testing is desirable but expensive, the established clinical criteria for HNPCC have significant disadvantages as a starting point for identifying individuals for testing.
MSI, also termed the replication error phenotype, in CRC tumors from early-onset cases and cases with a family history, is a strong predictor of germ-line mutations in MMR genes such as MSH2, MLH1, and PMS2 but not MSH6 (18, 19, 20, 21, 22) . MSI can be detected in 90% of tumors with a germ-line MMR defect (23) . This genomic instability is characterized by small deletions or insertions within simple repeat sequences, usually mono- or dinucleotide repeat sequences, in tumor DNA compared with corresponding DNA from normal tissue or blood (24) . The altered size of the repeat sequences is the result of frameshift errors during DNA replication combined with the failure to repair these errors because of a DNA MMR defect (3) . The loss of expression of MSH2 and MLH1 in tumors has also been suggested to be of value in predicting a germ-line mutation in MMR genes (25, 26, 27) . In this method, IHC analysis of tumors using MLH1 and MSH2 antibodies has been used to detect loss of expression of MSH2 or MLH1. Neither MSI nor IHC can be used to definitively diagnose HNPCC among unselected CRC cases. This is because 15% or more of sporadic CRC cases have been found to be microsatellite unstable (28, 29, 30, 31) . MSI in the majority of these cases is associated with methylation of the MLH1 promoter and silencing of the MLH1 gene (30 , 32 , 33) . Thus, a significant proportion of unselected CRC cases will be sporadic, MSI positive cases that do not express MLH1 but do not have a germ-line MLH1 mutation in contrast to familial CRC cases with germ-line mutations in MLH1 or MSH2 (34 , 35) .
We have studied previously the ability of the different clinical criteria for HNPCC to predict a germ-line mutation in MLH1 and MSH2 using 70 cases of familial colorectal cancer (15 , 36) . The families were selected by meeting at least one of several established HNPCC criteria including the Amsterdam, Modified Amsterdam, Bethesda, or HNPCC-like criteria (15) . This study indicated that those clinical criteria, which identify most of the HNPCC cases having a germ-line MMR defect, also include many individuals who do not have such defects. In the present study, we have determined the tumor MSI status for 48 of 70 families used in our previous study and tested 24 of these individuals for MSH2 and MLH1 expression using IHC to determine whether these tests could improve the selection criteria for identifying individuals for genetic testing. The results presented here demonstrate that MSI testing using the NCI 5-marker set identified all HNPCC cases where a germ-line MSH2 or MLH1 mutation was observed by DNA sequencing or where loss of expression of one of these two genes was observed. In contrast, five germ-line MSH2 or MLH1 mutations were observed that resulted in a MSI-H phenotype where both MSH2 and MLH1 proteins were still expressed in the tumors, suggesting that the IHC analysis may be a less useful indicator than MSI analysis of the presence of a germ-line mutation in either MSH2 or MLH1 in suspected HNPCC cases.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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are a subset of the 70 families published for the sequencing of MLH1 and MSH2 (15
, 36)
and represent all of the cases for which a tumor sample was available. The families were classified according to various criteria: Amsterdam I, Modified Amsterdam, Bethesda criteria 2,3,4,7, and HNPCC-like. Five families met the HNPCC-like criteria only, 5 families met the Modified Amsterdam criteria only, whereas all others fulfilled the Amsterdam and/or Bethesda criteria, and in some cases also the Modified Amsterdam criteria.
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. For most exons, one of the primers had an 18 base M13 (-21) forward sequence at its 5'-end, and the other primer had an 18 base M13 (-28) reverse sequence at its 5' end. Several of the exons had only one M13-tailed primer listed because unambiguous sequence could only be generated from one end of the exon. For some of the exons, three primers are listed: in those cases, the noncoding strand primer without an M13 tail at its 5' end was used routinely, and the noncoding strand primer with an M13 tail was only used if the PCR product required sequencing of its noncoding strand. Genomic DNA was isolated from blood samples as described previously. PCR was performed in 25–50 µl reactions containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.1 mM each dATP, dCTP, dGTP, TTP, 0.2 µM of each primer, 5 ng/µl DNA, and 0.05 unit/µl Taq polymerase (Taq Platinum; Life Technologies, Inc., Gaithersburg, MD). Thermocycling conditions were: 94°C, 4 min, followed by 11 cycles, each with a denaturing step at 94°C for 20 s, an extension step at 72°C for 20 s, and with a 20-s annealing step that decreased 1°C/cycle (beginning at 60°C in the first cycle and decreasing to 50°C in the eleventh cycle), and the eleventh cycle was then repeated 24 times for a total of 35 cycles of PCR. Finally, the reactions were incubated for 6 min at 72°C, followed by storage at 4°C.
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Tumor Analysis.
DNA for tumor analysis was extracted from microdissected tissue samples as follows. Ten µm paraffin sections were cut using a new disposable blade and collected on glass slides. Slides were baked at 55°C in an oven for 3 h. Paraffin sections were deparaffinized with xylene, washed with ethanol, and rehydrated in deionized water. Lesional tissue was visualized under the microscope, microdissected with a 30-gauge 1/2-inch sterile needle, and collected in a sterile tube containing 20 µl of digesting buffer (10 mM Tris, 1 mM EDTA, 1% Tween 20 and 200 µg/ml proteinase K). From the same slides, normal (nontumor) tissue was microdissected using a new 30-gauge 1/2-inch sterile needle and put in a separate sterile tube containing 50 µl of digesting buffer. The digestion was performed at 37°C for 36 h. The samples were then heated at 94°C for 5 min to inactivate the proteinase K and centrifuged, and the supernatant was used as template for PCR amplification and subsequent MSI analysis.
Analysis of microsatellite sequences for instability was performed using a PE/ABI377 DNA sequencer. PCR reactions were performed in a PE/ABI 9600 thermocycler, and PE/ABI GeneScan software was used for data interpretation. The microsatellite loci referred to as the 5-marker set were BAT25, BAT26, D2S123, APC, and Mfd15, and the five additional microsatellite loci analyzed were BAT40, MYCL, D18S69, D18S58, and D10S197 (29 , 37) . Primer sequences were as described, and both fluorescently labeled and unlabeled primers were obtained from Life Technologies, Inc. PCR was performed in 10-µl reactions, using 50–80 ng of DNA isolated from microdissected tissue samples, 0.2 µM each primer (from a 2 µM stock concentration), 1x PE/ABI Buffer 2, 2.5 mM MgCl2, 250 µM each of the 4 deoxynucleotide triphosphates (from a 2.5 mM stock; Boehringer Mannheim), and 0.25 unit of PE/ABI Amplitaq Gold. The thermocycling conditions were 1 cycle of 10 min 95°C, followed by 11 touchdown cycles consisting of 10 s 98°C; 30 s 60°C; 60°C decreasing 1°C/cycle; 1 min 70°C, followed by 37 cycles of 10 s 98°C; 1 min 58°C; 1 min 70°C, 10 min 99°C, followed by 1 cycle of 6 min 72°C. Samples were prepared for analysis by pooling 1 µl of each of up to five reactions, precipitating them with ethanol, drying the pellet, and suspending it in 1.5 µl of H2O. The resulting suspension was then mixed with 2.5 µl of formamide, 0.4 µl of blue dextran/EDTA and 0.6 µl GS350 (Tamra; PE/ABI). One µl of the final mixture was heated at 95°C for 2.20 min, placed on ice, and then loaded onto an acrylamide gel (FMC BioProducts, Rockland, ME) on a PE/ABI377 sequencer.
IHC was performed exactly as described previously (32) . The antibodies used for these studies were anti-MSH2 FE11 (Oncogene Research products, Cambridge, MA) and anti-MLH1 G168-728, (PharMingen, San Diego, CA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. One case (no. 1448) containing a MLH1 splice site mutation also had the MSH2 G322D missense change thought to be a polymorphism,4
and one case (no. 1755) containing a MLH1 splice site mutation also contained the MLH1 V716M missense change; we did not have appropriate material to allow us to determine whether the two MLH1 changes were associated with the same or different alleles.
MSI Analysis of Tumors.
Tumors from 48 cases were examined for MSI using both the NCI-recommended 5-marker test where MSI-H is defined as two or more markers of five showing instability and the NCI recommended 10-marker set, where MSI-H is defined as 4 or more markers of 10 showing instability (Table 1
; Refs. 29
, 37
). This study design also allowed evaluation of the BAT26 single microsatellite test for MSI (38)
. Using the 5-marker test, the MSI-H phenotype was found in 28 (58%) of the 48 families tested. These included all 14 cases having a germ-line MSH2 or MLH1 mutation and all 5 cases in which loss of expression of either MSH2 or MLH1 was observed in the absence of a detected germ-line mutation. Thus, classifying tumors as MSI-H using the 5-marker test had a 100% sensitivity for detecting cases where a definitive MSH2 or MLH1 defect (germ-line mutation or loss of expression) could be demonstrated. The 10-marker test was slightly less effective for MSI analysis. Tumors from 26 cases were MSI-H using the 10-marker test, of which 25 were classified as MSI-H and 1 was classified MSI-L by the 5-marker test. However, only 12 of 14 cases with a germ-line mutation and all 5 cases where loss of expression without a germ-line mutation were MSI-H using the 10-marker test. When BAT26 was analyzed, it was found to be unstable in 23 of 28 (82%) cases that were MSI-H by the 5-marker test, it was unstable in 11 of the 14 (79%) cases with a germ-line MSH2 or MLH1 mutation, it was unstable in 15 of the 19 (79%) cases with either a germ-line MSH2 or MLH1 mutation or loss of expression of MSH2 or MLH1, and it was unstable in 2 of the 7 cases that were MSI-L by the 5-marker test. Analysis of the proportion of microsatellite markers found to be unstable in the tumors classified as MSI-H by the 5-marker criteria revealed that MLH1 mutant cases had a significantly higher proportion of unstable mononucleotide repeat markers compared with MSH2 mutant cases (Table 3)
. This difference was independent of whether all three mononucleotide repeats were analyzed or whether only BAT26 was analyzed.
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. Selected examples of IHC analysis of cases expressing MLH1 or showing loss of expression of either MSH2 or MLH1 are presented in Fig. 1
. There were 4 examples of loss of MSH2 expression and 1 example of loss of MLH1 expression in MSI-H cases where no germ-line mutation in MSH2 or MLH1 was present. In addition, there was 1 example of MSH2 expression in a case with a germ-line MSH2 mutation and there were 4 examples of MLH1 expression in cases where a germ-line mutations in MLH1 was found. The MSH2 mutant case that expressed MSH2 contained a single codon deletion (N596del) and would be predicted to express a full-length protein. Two of the MLH1 mutant cases that expressed MLH1 had protein-truncating mutations late in the gene (exon 16, K604X and exon 19, c.2104–2105delAG) and might be expected to express large fragments of MLH1. The remaining two MLH1 mutant cases that expressed MLH1 had splice site mutations (c.677G
A and IVS7-AG) that might be predicted to result in skipping of exon 8; because this would not result in an in-frame deletion, these mutations would be predicted to be more deleterious to MLH1 expression.
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. These 9 cases were either Modified Amsterdam or HNPCC-like, and none of them had a germ-line mutation in either MSH2 or MLH1. The value of these clinical criteria for predicting the presence of germ-line mutations in MSH2 and MLH1 has been evaluated previously using the 70-sample set (15)
. Because this study used only 48 of the 70 families included in the first analysis, the sensitivity and specificity of each clinical criteria for predicting germ-line mutations in MSH2 and MLH1 was recalculated for comparison and found to be similar to that reported previously. Using the 48 families, the Amsterdam criteria showed the highest specificity (68%) for detecting germ-line mutations but a low sensitivity (57%), thus missing many mutations. In contrast, all of the samples with identified germ-line mutations met at least one of the Bethesda criteria. However, the specificity of the Bethesda criteria was only 29%, indicating that most of the samples meeting these latter criteria did not contain a germ-line mutation that could be detected by sequence analysis. It should be noted that of the 5 cases in which loss of expression of either MSH2 or MLH1 was observed in the absence of a detected germ-line mutation, 1 of the 5 did not meet the Bethesda criteria. The MSI status of the cases was also evaluated relative to the clinical characteristics of the cases. Eighty-six % (24 of 28) of the MSI-H families met at least one of the Bethesda criteria, and the specificity of the Bethesda criteria for identifying MSI-H cases was 63%. In contrast, 54% of the MSI-H cases met the Amsterdam criteria, and the specificity of the Amsterdam criteria for identifying MSI-H cases was 80%. Thus, similar to the situation with germ-line mutation detection, the Bethesda criteria identify most but not all MSI-H cases at the expense of including many cases that are not MSI-H, whereas the Amsterdam criteria identify a smaller proportion of the MSI-H cases but include a much smaller proportion of cases that are not MSI-H. Similar results on the relationship between MSI status and clinical criteria have also been reported in two recent studies (39 , 40) .
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(a) Dye terminator sequencing of PCR-amplified exons using a capillary sequencer is adequate for detecting heterozygous germ-line mutations in MSH2 and MLH1.
(b) The Bethesda criteria identify all cases having a germ-line MSH2 or MLH1 mutation but included many cases that did not have such defects, as well as including many cases that were not MSI-H and were unlikely to have MMR defects.
(c) All of the tumors from cases having a germ-line defect in MSH2 or MLH1 or showing loss of expression of one of these genes in a tumor sample were found to be MSI-H using the NCI-recommended 5-marker test. In contrast, analyses with just the BAT26 mononucleotide repeat was inadequate to detect all of the MMR-defective cases or all of the cases found to be MSI-H by the 5-marker test. Similarly, the 10-marker test did not detect all of the MMR-defective cases.
(d) A combination of the Bethesda criteria and MSI-H defined the smallest number of cases that included all of the germ-line MSH2 or MLH1 mutations found but excluded one case where loss of MSH2 expression in the absence of a germ-line mutation was seen.
(e) IHC analysis was of little use in helping to define those cases where a germ-line mutation was found because the tumors from some cases containing a pathogenic mutation nonetheless express protein that is detected by IHC.
We have described an improved set of PCR primers that allow amplification and sequencing of each exon of MSH2 and MLH1 without the need for nested PCR. The results obtained here showed that dye terminator sequencing on either a PE/ABI377 or PE/ABI3700 sequencer was capable of detecting all heterozygous mutations that could be detected using dye primer sequencing. Dye primer sequencing is well suited for mutation detection in heterozygotes because of the uniformity of sequencing chromatogram peak heights obtained but suffers from the disadvantage of being tedious because of the need to perform four sequencing reactions/sample. The ability to use dye terminator sequencing offers a significant advantage because it is less tedious to perform, and using a capillary sequencer, it is less expensive because it is possible to work with smaller sequencing reaction volumes.
Previous studies have shown that the majority of tumors from HNPCC patients show MSI, and similarly, a smaller proportion of sporadic cancers show MSI (18 , 19 , 23 , 24 , 29) . An NCI-sponsored workshop has recommended two standard sets of microsatellite markers for use in MSI analysis (29 , 37) . These markers were recommended based on the observation that they each showed a high degree of instability in a subset of primarily sporadic tumors in which a high proportion of markers were unstable (29) . However, these markers have not been well validated using samples in which the nature of the MMR defects has been established. The results presented here show that the NCI 5-marker test detected 100% of the samples shown to have either a germ-line mutation in MSH2 or MLH1 or loss of expression in the absence of a detected mutation. To our knowledge, this is the first study to actually evaluate whether the NCI 5-marker test can actually detect all HNPCC cases having a germ-line mutation in a MMR gene and demonstrate this is indeed true. An unexpected feature of the data was that the MSH2-defective tumors showed a lower level on mononucleotide repeat instability compared with the MLH1-defective tumors. The functional basis for this is unclear and could represent a mechanistic difference between MSH2 and MLH1 defects, a difference in the nature of inactivation of the second allele in the tumors, or it could be attributable to statistical variation in a small sample set. Some studies have suggested that mononucleotide repeat instability and, in particular instability of BAT26, is sufficient for detection of MSI (28 , 38) . However, in the present study of HNPCC cases, such criteria would have missed a significant number of cases that proved to have a defect in MSH2. Similarly, a recent study of sporadic colon tumors identified several cases where BAT26 was stable, but expression of either MSH2 or MLH1 was absent (41) . Interestingly, the studies that have reported successful use of BAT26 have primarily analyzed sporadic tumors, or sporadic and HNPCC tumors from Finland, and in each of these sample sets MLH1 is the gene that is most often defective (28 , 30 , 33 , 38 , 42) . Similar to this, all of the cases with a MLH1 germ-line mutation analyzed here showed BAT26 instability.
IHC analysis of MMR gene expression has proven useful in the detection of MMR defects, particularly in the case of sporadic tumors where loss of MLH1 expression is often seen (29
, 30
, 32, 33, 34, 35)
. In the present study, IHC analysis was not useful in predicting MSH2 or MLH1 gene defects. This is because there were 5 cases where significant pathogenic germ-line mutations, including protein-truncating mutations, were found but nonetheless the tumors produced presumably nonfunctional proteins that were detected by IHC. This is not surprising, however, because in at least 3 of the cases, the nature of the mutation predicted that a full length or almost full-length, but nonfunctional, protein would be produced. In the other two cases, it is possible that the mutation resulted in the production of a stable protein fragment that was detected by IHC; it is also possible that the wild type allele was still present in the tumor, and the protein fragment produced caused a dominant-negative effect. It is also reasonable to expect the presence of proteins that would be detected by IHC in cases where a germ-line missense mutation was present. Indeed, other studies have also observed that IHC can detect expression of MSH2 or MLH1 in tumors from cases where an MSH2 or MLH1 mutation was present, although such expression was only observed in cases with missense mutations (43, 44, 45)
, a result that might be expected. Our observation of MSH2 and MLH1 protein expression in a significant proportion of HNPCC cases containing a germ-line MMR defect including cases containing protein-truncating mutations indicates that IHC is unlikely to be a definitive primary test for detecting the presence of germ-line mutations in MMR genes in HNPCC cases. We observed a much higher proportion of MSH-H cases that expressed both MSH2 and MLH1 even when a germ-line MSH2 or MLH1 mutation was present than observed in a recent large scale study of unselected CRC cases, where 8% of MSI-H cases expressed both MSH2 and MLH1 (46)
. The difference between these two studies is that our study focused on suspected HNPCC cases where germ-line mutations would be found, whereas the recent large-scale study included a high proportion of sporadic CRC cases, which are primarily attributable to silencing of MLH1 and hence loss of expression of MLH1 (29
, 30
, 32, 33, 34, 35)
. In the analysis of suspected HNPCC cases, IHC seems more useful in the analysis of MSI-H cases where no germ-line MSH2 or MLH1 mutations were found than as a primary screen for the presence of MMR defects. Five such cases were identified in which MSH2 (4 cases) or MLH1 (1 case) expression was absent, and these would be candidates for analysis for the presence of other types of MSH2 or MLH1 defects, such as deletion mutations (6
, 9, 10, 11)
.
A critical question regarding the molecular analysis of CRC cases suspected of being HNPCC is what strategy should be used to analyze such cases for germ-line defects in MSH2 and MLH1, the major HNPCC genes (6)
,4
so as to minimize the work and expense involved while maximizing the fraction of defects detected. Our studies as well as those of others indicate that the Bethesda criteria identify a greater proportion of CRC cases having a germ-line MMR defect than other clinical criteria. Thus, the Bethesda criteria perform very well in achieving the intended goal to help guide which CRC families should undergo molecular evaluation for HNPCC. The data presented here indicate that a second step of MSI analysis using the NCI 5-marker test would yield the smallest number of cases (24 from a total of 48) where all of the MSH2 and MLH1 germ-line mutations were present. Not only is MSI analysis the only molecular method that detected all cases containing a germ-line MMR defect in MSH2 and MLH1, in so much as MSI analysis is significantly less expensive than DNA sequence analysis, MSI analysis is also a cost-effective second step. In our study, IHC analysis of MSI-H cases was not definitive for identifying cases with germ-line MMR defects in MSH2 and MLH1. Because our study indicates that MSI-H cases that express MSH2 and MLH1 proteins would still need to be analyzed for germ-line mutations in both MSH2 and MLH1, if IHC analysis were performed after MSI analysis, it would only reduce the overall amount of DNA sequence analysis required by 20%. In contrast, if IHC analysis is performed on only those cases that are MSI-H where DNA sequencing did not identify a germ-line mutation in MSH2 or MLH1, IHC analysis provided an indication of whether MSH2 or MLH1 contained some sort of as yet detected germ-line defect in 5 of 7 of MSI-H, mutation-negative cases where definitive IHC was possible. This would be extremely useful in guiding additional analysis, such as the use of conversion analysis or analysis for deletion mutations (6
, 9, 10, 11)
. In contrast, if conversion and deletion mutation analysis were applied at earlier stages in the analysis, such as before MSI testing or before sequence analysis, these methods would only detect mutations in a small proportion of cases and would not significantly improve the efficiency of mutation detection by DNA sequencing.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a grant from the Starr Foundation. R. D. K. is an inventor on both issued patents and pending patent applications covering some of the molecular diagnostic procedures discussed in this study. 
2 To whom requests for reprints should be addressed, at Ludwig Institute for Cancer Research, UCSD School of Medicine-CMME3080, 9500 Gilman Drive, La Jolla, CA 92093-0660. Phone: (858) 534-7804; Fax: (858) 534-7750; E-mail: rkolodner{at}ucsd.edu.
3 The abbreviations used are: HNPCC, hereditary nonpolyposis colon cancer; CRC, colorectal cancer; NCI, National Cancer Institute.
4 Internet address for database: http://www.nfdht.nl/database/mdbchoice.htm; MMR, mismatch repair; MSI, microsatellite instability; IHC, immunohistochemistry.
Received 12/11/01. Accepted 4/23/02.
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REFERENCES |
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X. Llor, E. Pons, R. M. Xicola, A. Castells, C. Alenda, V. Pinol, M. Andreu, S. Castellvi-Bel, A. Paya, R. Jover, et al. Differential Features of Colorectal Cancers Fulfilling Amsterdam Criteria without Involvement of the Mutator Pathway Clin. Cancer Res., October 15, 2005; 11(20): 7304 - 7310. [Abstract] [Full Text] [PDF]
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M. C. Southey, M. A. Jenkins, L. Mead, J. Whitty, M. Trivett, A. A. Tesoriero, L. D. Smith, K. Jennings, G. Grubb, S. G. Royce, et al. Use of Molecular Tumor Characteristics to Prioritize Mismatch Repair Gene Testing in Early-Onset Colorectal Cancer J. Clin. Oncol., September 20, 2005; 23(27): 6524 - 6532. [Abstract] [Full Text] [PDF]
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 S. Chen, P. Watson, and G. Parmigiani Accuracy of MSI testing in predicting germline mutations of MSH2 and MLH1: a case study in Bayesian meta-analysis of diagnostic tests without a gold standard Biostat., July 1, 2005; 6(3): 450 - 464. [Abstract] [Full Text] [PDF]
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 V. Pinol, A. Castells, M. Andreu, S. Castellvi-Bel, C. Alenda, X. Llor, R. M. Xicola, F. Rodriguez-Moranta, A. Paya, R. Jover, et al. Accuracy of Revised Bethesda Guidelines, Microsatellite Instability, and Immunohistochemistry for the Identification of Patients With Hereditary Nonpolyposis Colorectal Cancer JAMA, April 27, 2005; 293(16): 1986 - 1994. [Abstract] [Full Text] [PDF]
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 S. Oda, Y. Maehara, Y. Ikeda, E. Oki, A. Egashira, Y. Okamura, I. Takahashi, Y. Kakeji, Y. Sumiyoshi, K. Miyashita, et al. Two modes of microsatellite instability in human cancer: differential connection of defective DNA mismatch repair to dinucleotide repeat instability Nucleic Acids Res., March 18, 2005; 33(5): 1628 - 1636. [Abstract] [Full Text] [PDF]
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W Kievit, J H F M de Bruin, E M M Adang, J L Severens, J H Kleibeuker, R H Sijmons, T J Ruers, F M Nagengast, H F A Vasen, J H J M van Krieken, et al. Cost effectiveness of a new strategy to identify HNPCC patients Gut, January 1, 2005; 54(1): 97 - 102. [Abstract] [Full Text] [PDF]
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L.R. Lipton, V. Johnson, C. Cummings, S. Fisher, P. Risby, A.T. Eftekhar Sadat, T. Cranston, L. Izatt, P. Sasieni, S.V. Hodgson, et al. Refining the Amsterdam Criteria and Bethesda Guidelines: Testing Algorithms for the Prediction of Mismatch Repair Mutation Status in the Familial Cancer Clinic J. Clin. Oncol., December 15, 2004; 22(24): 4934 - 4943. [Abstract] [Full Text] [PDF]
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A. Muller, G. Giuffre, T. B. Edmonston, M. Mathiak, B. Roggendorf, E. Heinmoller, T. Brodegger, G. Tuccari, E. Mangold, R. Buettner, et al. Challenges and Pitfalls in HNPCC Screening by Microsatellite Analysis and Immunohistochemistry J. Mol. Diagn., November 1, 2004; 6(4): 308 - 315. [Abstract] [Full Text] [PDF]
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 B Perez-Ordonez, N N Huynh, K W Berean, and R C K Jordan Expression of mismatch repair proteins, {beta} catenin, and E cadherin in intestinal-type sinonasal adenocarcinoma J. Clin. Pathol., October 1, 2004; 57(10): 1080 - 1083. [Abstract] [Full Text] [PDF]
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R L Ward and E Mokany Germline testing of mismatch repair genes is not aided by prescreening tumours for allelic loss Gut, March 1, 2004; 53(3): 471 - 472. [Full Text] [PDF]
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 A. Umar, C. R. Boland, J. P. Terdiman, S. Syngal, A. d. l. Chapelle, J. Ruschoff, R. Fishel, N. M. Lindor, L. J. Burgart, R. Hamelin, et al. Revised Bethesda Guidelines for Hereditary Nonpolyposis Colorectal Cancer (Lynch Syndrome) and Microsatellite Instability J Natl Cancer Inst, February 18, 2004; 96(4): 261 - 268. [Abstract] [Full Text] [PDF]
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A. E. de Jong, M. van Puijenbroek, Y. Hendriks, C. Tops, J. Wijnen, M. G. E. M. Ausems, H. Meijers-Heijboer, A. Wagner, T. A. M. van Os, A. H. J. T. Brocker-Vriends, et al. Microsatellite Instability, Immunohistochemistry, and Additional PMS2 Staining in Suspected Hereditary Nonpolyposis Colorectal Cancer Clin. Cancer Res., February 1, 2004; 10(3): 972 - 980. [Abstract] [Full Text] [PDF]
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 B. Diergaarde, H. Braam, G. N. P. van Muijen, M. J. L. Ligtenberg, F. J. Kok, and E. Kampman Dietary Factors and Microsatellite Instability in Sporadic Colon Carcinomas Cancer Epidemiol. Biomarkers Prev., November 1, 2003; 12(11): 1130 - 1136. [Abstract] [Full Text] [PDF]
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E. Renkonen, Y. Zhang, H. Lohi, R. Salovaara, W. M. Abdel-Rahman, M. Nilbert, K. Aittomaki, H. J. Jarvinen, J.-P. Mecklin, A. Lindblom, et al. Altered Expression of MLH1, MSH2, and MSH6 in Predisposition to Hereditary Nonpolyposis Colorectal Cancer J. Clin. Oncol., October 1, 2003; 21(19): 3629 - 3637. [Abstract] [Full Text] [PDF]
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 Y. Hendriks, P. Franken, J. W. Dierssen, W. de Leeuw, J. Wijnen, E. Dreef, C. Tops, M. Breuning, A. Brocker-Vriends, H. Vasen, et al. Conventional and Tissue Microarray Immunohistochemical Expression Analysis of Mismatch Repair in Hereditary Colorectal Tumors Am. J. Pathol., February 1, 2003; 162(2): 469 - 477. [Abstract] [Full Text] [PDF]
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I. Gazzoli, M. Loda, J. Garber, S. Syngal, and R. D. Kolodner A Hereditary Nonpolyposis Colorectal Carcinoma Case Associated with Hypermethylation of the MLH1 Gene in Normal Tissue and Loss of Heterozygosity of the Unmethylated Allele in the Resulting Microsatellite Instability-High Tumor Cancer Res., July 15, 2002; 62(14): 3925 - 3928. [Abstract] [Full Text] [PDF]
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AA frameshift mutation in MSH2 exon 4. H, tumor tissue not expressing MSH2 from case 4103 containing the c.704–705