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Clinical Chemistry 54: 1844-1854, 2008. First published September 4, 2008; 10.1373/clinchem.2008.108902
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(Clinical Chemistry. 2008;54:1844-1854.)
© 2008 American Association for Clinical Chemistry, Inc.

Cancer Diagnostics

Multiplex SNaPshot Genotyping for Detecting Loss of Heterozygosity in the Mismatch-Repair Genes MLH1 and MSH2 in Microsatellite-Unstable Tumors

Maria Bujalkova1, Katarina Zavodna1, Tomas Krivulcik1, Denisa Ilencikova2, Brigitte Wolf3, Michal Kovac1,4, Judith Karner-Hanusch3, Karl Heinimann4, Giancarlo Marra5, Josef Jiricny5 and Zdena Bartosova1,a

1 Laboratory of Cancer Genetics, Cancer Research Institute of Slovak Academy of Sciences, Bratislava, Slovakia; 2 National Cancer Institute, Bratislava, Slovakia; 3 Research Laboratories, Department of Surgery, Medical University of Vienna, Vienna, Austria; 4 Division of Medical Genetics UKBB, Research Group Human Genetics, Department of Biomedicine, University of Basel, Basel, Switzerland; 5 Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland.

aAddress correspondence to this author at: Laboratory of Cancer Genetics, Cancer Research Institute of Slovak Academy of Sciences, Vlarska 7, 833 91 Bratislava, Slovakia. Fax 421–2-59327250; e-mail zdena.bartosova{at}savba.sk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: In the workup of patients with suspected hereditary nonpolyposis colorectal cancer (HNPCC), detection of loss of heterozygosity (LOH) could help pinpoint the mismatch-repair (MMR) gene carrying the germline mutation, but analysis of microsatellite markers has proved unreliable for this purpose. We developed a simple, low-cost method based on single-nucleotide polymorphism (SNP) genotyping and capillary electrophoresis for the assessment of LOH at 2 MMR loci simultaneously.

Methods: We used the Applied Biosystems SNaPshot® Multiplex Kit with meticulously selected primers to assess 14 common SNPs in MLH1 [mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli)] and MSH2 [mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)] and optimized the protocol for DNA isolated from peripheral blood and fresh/frozen or archival microsatellite-unstable tumors from patients with confirmed (n = 42) or suspected (n = 25) HNPCC. The 42 tumors from patients with confirmed MLH1 or MSH2 germline mutations were used to validate the method’s diagnostic accuracy against results obtained with DNA sequencing or multiplex ligation-dependent probe amplification.

Results: The SNaPshot assay provided better detection of certain SNPs than DNA sequencing. The MLH1 and MSH2 SNP marker sets were informative in 82% and 76% of the 67 cases analyzed, respectively. The new assay displayed 100% specificity for detecting LOH and predicted the location of the germline mutation in 40% of the cases (54% of those involving MLH1, 22% in MSH2).

Conclusions: Our SNP-based method for detecting LOH in MLH1 and MSH2 is simple to perform with instruments available in most clinical genetics laboratories. It can be a valuable addition to protocols now used to guide mutational screening of patients with suspected HNPCC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The DNA mismatch-repair (MMR)1 genes MLH12 [mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli); MIM 120436] and MSH2 [mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli); MIM 120435] are best known for their roles in the correction of errors (base/base mismatches, insertion/deletion loops) arising during DNA replication, although they also participate in homologous recombination, cell cycle checkpoint signaling, and somatic hypermutation and class-switch recombinations involving the immunoglobulin genes (1). Germline mutation of MMR genes (MLH1 or MSH2 in most cases) leads to the hereditary nonpolyposis colon cancer (HNPCC) syndrome (MIM 114500), which predisposes affected individuals to the development of cancers of the colorectum, endometrium, and several other organs according to a mechanism following the so-called microsatellite instability (MSI) pathway. The more than 1000 MLH1 and MSH2 variants that have been described (2) include pathogenic nonsense or frameshift alterations that lead to loss of protein expression, missense mutations that can functionally impair the encoded protein, and single-nucleotide polymorphisms (SNPs). The role played by these SNPs is not fully clear, although there is evidence to suggest that they contribute to the development of various types of cancer (3)(4)(5)(6)(7), including those of the colorectum (8)(9) and endometrium (10).

In the well-known 2-hit hypothesis of tumorigenesis, the heterozygous germline mutation of an MMR gene represents the "first hit" in HNPCC, but the loss of the MMR function and the MSI associated with it are observed only after somatic inactivation of the wild-type allele. This "second hit" is believed to be caused in many HNPCC cases by a loss of heterozygosity (LOH). For this reason, LOH assays could be a potentially useful addition to the prescreening protocols used to orient germline testing in individuals with suspected HNPCC. The algorithms currently used to identify HNPCC patients are based largely on various combinations of microsatellite genotyping and immunohistochemistry (IHC) assessment of MMR proteinexpression patterns. No consensus exists on the order in which these 2 methods should be used, or even on the need to perform both types of testing (11)(12). Although the most accurate results are generally acknowledged to be obtained with a combination of MSI testing and IHC analysis, this approach may not always be feasible or advisable, depending on the laboratory and/or the characteristics of the case at hand (13)(14)(15)(16)(17). Thus far, the value of LOH assessments in these algorithms has proved to be limited, but the assays in question have been based on the analysis of microsatellite markers (18), which are often unstable in MMR-deficient tumors and thus uninformative for the study of LOH events. LOH can also be detected by multiplex ligation-dependent probe amplification (MLPA) or by DNA sequencing, but MLPA analysis identifies only LOHs that involve changes in exon dosages, and sequencing of DNA extracted from archival tumor tissues is often unsuccessful. In addition, neither of these mutation-detection methods can predict the mutated MMR gene, because their use in LOH assessment is limited to cases in which the germline mutation is already known.

In their recent study of the mechanisms underlying MMR gene inactivation in HNPCC tumors, Ollikainen et al. (19) used MALDI-TOF mass spectrometry to quantitatively assess LOH at the sites of germline mutations and in 2 intragenic SNPs in MLH1. High-throughput SNP-genotyping strategies of this type are extremely attractive in research settings but require robotics and other instrumentation that are still beyond the reach of many clinical genetics laboratories. To further explore the potential of intragenic SNPs as markers of LOH at MMR loci, we used a medium-throughput method based on single-nucleotide primer extension and capillary electrophoresis for SNP genotyping at MLH1 and MSH2 simultaneously. We evaluated the method’s performance for detecting LOH and for predicting the gene harboring germline changes in HNPCC patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The study protocol received institutional review board approval, and written informed consent was obtained from all individuals whose DNA was analyzed. Unless otherwise stated, commercial products were used according to the manufacturers’ instructions.

dna samples
We analyzed genomic DNA from peripheral blood leukocytes (PBLs) of 20 healthy volunteers of Caucasian origin and DNA from PBL and tumor tissue samples from 67 cancer patients (also of Caucasian origin). Patients 1–42 were known carriers of pathogenic germline mutations in MLH1 or MSH2 (20)(21)(22)(23)(24)(25)(26). Patients 43–67 were suspected HNPCC probands. Although IHC analysis had demonstrated loss of MLH1 or MSH2 protein expression in most of these patients’ tumors, DNA sequencing and MLPA analyses had failed to detect germline mutations in any of the major MMR genes. All 67 tumors (66 colorectal cancers, 1 urothelial tumor) had displayed MSI. We used standard phenol-chloroform extraction to isolate genomic DNA from most PBL and tumor samples [fresh, frozen, or formalin-fixed and paraffin-embedded (FFPE) samples]. Alternatively, we used the Qiagen QIAamp DNA Mini Kit for some PBL and fresh/frozen tumor tissue samples and the RecoverAllTM Total Nucleic Acid Isolation Kit (Ambion/Applied Biosystems) for some FFPE tumor samples.

snp selection and multiplex pcr amplification of targets
Table 1 summarizes the characteristics of the 14 SNP markers (7 in MLH1, 7 in MSH2) used in our assay. Targets containing these SNPs were amplified in different reactions, depending on the nature of the template DNA (Fig. 1 , Table 1 ). When DNA from PBLs or fresh/frozen tumors was used, 7 MLH1 and 4 MSH2 fragments (length, 250–535 bp) were amplified in an undecaplex reaction (Fig. 1A ). In contrast, DNA from FFPE tissues was subjected to 3 quadruplex reactions, yielding a total of 12 amplicons (each <250 bp; Fig. 1B , Table 1 ). All reactions were carried out in 25-µL volumes containing 12.5 µL of Multiplex PCR Master Mix (Qiagen), a mixture of primers (each at a final concentration of 0.07–3 µmol/L), and up to 100 ng of template DNA. Primer sequences and PCR conditions are provided in Supplemental Table 1 and the Supplemental Data Text, respectively, in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol54/issue11.


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  		Table 1. Characteristics of the SNP markers used in this study.


Figure 1
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  		Figure 1. Multiplex PCRs of MLH1 and MSH2 targets containing SNPs.

Reaction products were resolved on a 30 g/L agarose gel stained with ethidium bromide. (A), DNA from PBLs and fresh/frozen tumor tissue was amplified in an undecaplex PCR. Amplicons 1 and 10 include 2 and 3 MSH2 SNPs, respectively. Genes and amplicon numbers are shown on the left. (B), Three quadruplexes (Q1, Q2, Q3) from amplification of DNA extracted from FFPE tissues. Amplicon numbers (white labels) correspond to those in (A). MSH2 fragment 10 was divided into amplicons 10a and 10b (see Table 1Up ). N, nontumor (PBL) DNA; T, tumor DNA; M, size marker; Pro, promoter.

single-base extension of snp-specific primers
We evaluated 35 SNP-specific oligonucleotide primers designed with the ABI PRISM® SNaPshot® Multiplex Kit (Applied Biosystems). To select the 14 primers for multiplex SNaPshot genotyping of the 14 MLH1 and MSH2 SNPs (Table 2 ), we used a meticulous, systematic assessment protocol, which included empirical testing in a SNaPshot reaction without a template to exclude self-priming. Each primer consisted of a specific sequence (18–27 nucleotides long) complementary to the analyzed region plus a poly(GACT) or poly(A) tail, which was added to ensure spatial resolution of the extension products during capillary electrophoresis (final length, 18–82 nucleotides). Oligonucleotides exceeding 35 nucleotides were purified by HPLC (Generi Biotech).


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  		Table 2. Characteristics of the SNP-specific oligonucleotide primers used in the multiplex SNaPshot assay.

SNaPshot reactions were carried out in a 10-µL final volume containing SNaPshot Multiplex Ready Reaction Mix (5 µL), primer mix (final concentrations, 0.02–0.6 µmol/L), and templates (4 µL) consisting of the multiplex PCR products described above, which had been purified with the QIAquick PCR Purification Kit (Qiagen). Quadruplex PCR products amplified from archival DNA were pooled and purified on the same column before the SNaPshot reaction. The cycling program included 25 cycles of 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 30 s. Extension products were purified by a 15-min incubation with 1 U of shrimp alkaline phosphatase (Promega) at 37 °C and a subsequent 15-min incubation at 80 °C to inactivate the enzyme. The purified products (0.5 µL) were mixed with 9 µL of formamide and 0.5 µL of GeneScan-120 LIZ Size Standard (Applied Biosystems) and separated by capillary electrophoresis (ABI PRISM 310 Genetic Analyzer; Applied Biosystems). The results were analyzed with GeneMapper 3.0 software (Applied Biosystems).

dna sequencing and mlpa
We used AmpliTaq Gold® PCR Master Mix (Applied Biosystems) to amplify fragments for DNA sequencing (primer sequences and cycling conditions available upon request). We used the BigDye® Terminator v3.1Cycle Sequencing Kit (Applied Biosystems) as described previously to sequence purified PCR products (21).

MLPA assays for LOH were carried out with the SALSA MLPA Kit P003 MLH1/MSH2 (MRC-Holland). We used an ABI 310 Genetic Analyzer for sequencing and MLPA reactions and analyzed the results with the Sequencing Analysis 5.2 and GeneMapper 3.0 programs, respectively (Applied Biosystems). We used Microsoft Excel sheet data generated according to the MLPA manufacturer’s recommendations to determine MLH1/MSH2 exon dosage in DNAs from nontumor and tumor samples.

definitions of loh
SNaPshot.
For each SNP marker, we expressed the relative proportions of alleles 1 and 2 in nontumor and tumor samples as the ratio of the heights of the corresponding peaks. An LOH index was calculated by dividing the ratio of allele 2 to allele 1 in the nontumor DNA by the corresponding ratio in the tumor DNA. LOH positivity was defined as an LOH index of <0.5 (reflecting a substantial loss of allele 1 in the tumor sample) or >1.5 (indicative of substantial loss of allele 2).

DNA sequencing.
When point mutations were detectable as double peaks in the reference DNA, the loss or marked reduction of one of the peaks in the tumor DNA was considered indicative of LOH. In cases characterized by small deletions and/or insertions that shifted the entire sequence downstream from the mutation, LOH positivity was defined as the absence of this shift or a marked reduction of one allele in the mixed pattern of peaks.

MLPA analysis.
Tumor-derived DNA is inevitably contaminated by DNA from nontumor tissue within and/or adjacent to the tumor. We therefore defined the homozygous loss of an exon as a decrease in the exon-peak area in tumor DNA of at least 40%, compared with the corresponding peak in the reference DNA containing a heterozygous deletion. Such losses were considered indicative of LOH only when the SD was <20%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
mlh1 and msh2 snp genotypes based on multiplex snApSHOT analysis vs dna sequencing
We used PBL DNA from 20 healthy volunteers to compare the results of our SNP-genotyping method with those obtained from DNA sequencing, the current gold standard for genotyping. The genotypes obtained with the 2 methods were concordant for all but 2 SNPs, which are located in MSH2 intron 1 (c.211+9C/G and c.211+98T/C); both SNPs are included in MSH2 amplicon 1 (see Fig. 1AUp ). In 11 cases, SNaPshot analysis revealed heterozygosity for both of these polymorphisms, whereas sequencing analysis of the same DNAs showed homozygosity at both markers (c.211+9C/C and c.211+98T/T). When we resequenced the SNP at position +9 by means of an amplicon generated with a different primer pair (27), the locus displayed heterozygosity (+9C/G) in all 11 cases.

frequency of heterozygosity and informativeness of the mlh1 and msh2 snp markers
To estimate the frequency of heterozygosity at the selected SNP markers, we used the SNaPshot-based method to analyze genomic DNA from PBL samples from the 67 unrelated patients with MSI tumors. High heterozygosity rates (>20%) were observed for all 14 markers (33%–61% for those in MLH1, 21%–52% for MSH2; Table 1Up ). The MLH1 and MSH2 marker sets were informative (i.e., at least one of the 7 markers was heterozygous) in 82% and 76% of the samples, respectively, and both sets were informative in 63% of the samples.

multiplex snApSHOT-based detection of loh at mlh1 and msh2
Fig. 2 shows representative SNaPshot electrophoretograms documenting LOH at MLH1 and MSH2. The new method revealed LOH at the mutated locus in 17 (40%) of the 42 patients with known germline MMR gene mutations [13 (54%) of the 24 patients with known MLH1 mutations and 4 (22%) of the 18 patients with MSH2 mutations; Table 3 ]. To evaluate the reliability of our LOH-detection method, we reanalyzed tumor DNAs from the 42 patients with known mutations by either DNA sequencing (in the 34 cases characterized by single-base substitutions, small deletions, or insertions) or MLPA (in 8 patients with large germline rearrangements). Table 3 shows that the reference method analysis, which was confined to the heterozygous gene region known to harbor a germline mutation, confirmed all 13 of the LOH events involving MLH1 detected with the SNP-based method and 2 of the 4 events detected in MSH2. The other 2 LOH events at MSH2 (cases 27 and 29) could not be confirmed or excluded because the quality of the tumor DNA was inadequate for MLPA analysis. Of the 9 cases that were uninformative in the SNP-based assays, 6 exhibited LOH in reference assays (cases 7, 25, 34, 40–42), 2 others (cases 18 and 31) were LOH negative in the reference assay, and sequencing failed in the ninth (case 16) because of poor-quality DNA. In the 16 SNP-informative cases without SNaPshot-detectable LOH, the absence of LOH was confirmed by reference methods in 13 cases; the other 3 samples (cases 6, 26, and 32) could not be sequenced because of the low quality of the DNA.


Figure 2
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  		Figure 2. Use of 14 SNPs and the multiplex SNaPshot method to detect LOH in MLH1 and MSH2.

Capillary electrophoresis of SNaPshot products was performed with an ABI 310 Genetic Analyzer, and SNP profiles of nontumor and tumor DNA were analyzed with GeneMapper 3.0 software as described in Materials and Methods. For each SNP marker, the relative proportions of alleles 1 and 2 in nontumor and tumor samples were expressed as the ratio of the heights of the corresponding peaks. An LOH index was calculated by dividing the ratio of allele 2 to allele 1 in nontumor DNA by the ratio of allele 2 to allele 1 in tumor DNA. LOH positivity was defined as an index of <0.5 (reflecting substantial loss of allele 1 in the tumor sample) or >1.5 (indicative of substantial loss of allele 2). (A), An example of LOH in the MLH1 gene (patient no. 4 displaying LOH at 7 informative SNP markers of the MLH1 gene). (B), LOH in MSH2 (patient no. 67 showing LOH at 6 informative SNP markers of the MSH2 gene). Note: In contrast to fluorescence capillary DNA sequencing, the C nucleotide appears as a black peak, and the G nucleotide is blue. *, reverse extension primer.


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  		Table 3. LOH at MLH1 and MSH2 as determined by SNaPshot and reference methods.1

When we compared SNaPshot-based detection of LOH with that based on sequencing and/or MLPA and excluded the 6 cases in which neither reference analysis could be carried out (see Supplemental Table 2 in the online Data Supplement), the new method exhibited 100% specificity and 71% sensitivity in the combined analysis of MLH1 and MSH2. It displayed excellent sensitivity (93%) for detecting LOH involving MLH1. The low sensitivity (29%) that emerged in the assessment of MSH2 is largely because 2 of the 4 cases of SNaPshot-based LOH detection had to be excluded from the validation analysis because the tumor DNAs could not be analyzed with MLPA. Table 3Up shows that SNaPshot-based detection of LOH correctly predicted the gene harboring the germline mutation in 17 (40%) of the 42 cases analyzed [13 (54%) of 24 cases involving MLH1; 4 (22%) of 18 cases in MSH2].


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have developed a simple, rapid, and low-cost method for medium-throughput detection of LOH in the MLH1 and MSH2 genes that is based on the simultaneous analysis of multiple SNPs. It involves single-nucleotide primer extension in multiplex SNaPshot reactions followed by capillary electrophoresis and can be used in any laboratory equipped with genetic analyzers.

Applied Biosystems claims that its SNaPshot Multiplex Kit can be used to analyze up to 10 SNPs simultaneously, but with meticulous primer selection we succeeded in analyzing all 14 MLH1 and MSH2 SNPs in a single reaction. Interestingly, when we compared SNaPshot genotyping of PBL DNA with SNP genotyping based on DNA sequencing, the new method detected 2 SNPs in MSH2 intron 1 (c.211+9C>G and c.211+98T>C) that were missed by the reference method. The failure of DNA sequencing at this locus can probably be linked to the secondary structure of this region, which contains 3 GC-rich motifs that might form a hairpin. This hairpin is likely to be more stable when the template strand contains +9G rather than +9C. This fact may lead to preferential amplification of the +9C allele during the PCR and overrepresentation of this template in the sequencing mix. Like polymerases used in the PCR, the sequencing enzyme is also more likely to preferentially copy the +9C allele. Consequently, the G peak would not be present in the electrophoretogram, and the patient would appear to be homozygous for the +9C allele. Unlike DNA sequencing, the SNaPshot reaction is based on primer annealing immediately adjacent to the SNP position and subsequent extension by a single nucleotide. The chemistry of this reaction is much less susceptible to secondary-structure effects, and both nucleotides will be detected at position +9, even in the presence of unequal amounts of the 2 allele-specific amplicons. The same secondary-structure problem might affect the SNP at position +98, which is close to +9. SNaPshot is also better than DNA sequencing for detecting KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) mutations in DNA isolated from tumor samples containing admixtures of nontumor cells (28). Therefore, regardless of whether the DNA is constitutional or tumoral, SNaPshot surpasses sequencing in terms of its ability to detect single-nucleotide changes in the presence of unequal amounts of allele-specific amplicons.

SNP-based approaches are more reliable for LOH detection than the analysis of microsatellite markers, which are often unstable in MMR-deficient tumors and which are usually located several mega–base pairs from the gene of interest. MALDI-TOF mass spectrometric analysis of 2 intragenic SNP markers has recently been used to investigate the role of LOH in MLH1 inactivation in colorectal and endometrial cancers (19). This approach proved to be very promising, although the authors noted that a higher number of polymorphisms would have improved the rates of heterozygosity. Our SNaPshot-based method was developed around 14 SNPs, each of which presents a heterozygosity frequency of >20% in the general population. These choices ensured high overall rates of heterozygosity for the MLH1 and MSH2 SNP sets (82% and 76%, respectively, based on an analysis of 67 samples). Compared with the results obtained with DNA sequencing or MLPA analysis of the known mutant locus in 36 tumors, the new method displayed 100% specificity and an overall sensitivity of 71% for detecting LOH at MLH1 or MSH2. The overall sensitivity might well have been higher if we had not had to exclude 2 of the 4 cases of MSH2 LOH from our validation analysis because of technical failure of the reference-method analyses (DNA sequencing and MLPA analysis are both difficult to perform with DNA extracted from tumor samples).

Compared with MALDI-TOF mass spectrometry, SNaPshot-based analysis of intragenic SNPs is associated with substantially lower equipment costs: all that is needed is an automated DNA sequencer. Therefore, our method is especially useful in clinical laboratories involved in the diagnostic workup of patients with suspected HNPCC, where sequencers are already being used for mutation scanning. Assessment of the tumor’s MSI status and/or IHC studies of MMR protein expression patterns are widely used as first-line strategies for selecting cases in which germline testing for MLH1/MSH2 mutations is indicated. Previous attempts to improve the predictive value of these prescreening protocols with the addition of LOH studies have been unsuccessful (18), but the detection of allelic loss in that study was based on the examination of microsatellite markers, which are (as we have seen) unreliable for this purpose.

Our experience suggests that SNP-based assessment of LOH might have several potential roles in this setting (Fig. 3 ). For example, in many laboratories, suspected HNPCC tumors initially undergo PCR-based MSI testing (gray area in Fig. 3 ), ideally with the new MSI multiplex typing systems based on quasi-monomorphic markers, which can be used without reference to matching nontumor DNA. These kits are much more reliable and cost far less per patient, i.e., approximately US $50 vs US $300 for older methods based on uniplex analyses of a large panel of markers, including dinucleotides, in both nontumor and tumoral tissues (29)(30)(31). Tumors found to be PCR-MSI positive are then subjected to IHC analysis of MMR protein expression. SNaPshot-based LOH analysis of MSI-positive tumors could be a cost-effective addition to this diagnostic algorithm. This analysis can be performed rapidly on the same DNA samples used for MSI testing, and interpretation of the results is straightforward and objective. Although its ability to predict germline mutations of MLH1 or MSH2 is limited, it is highly specific, and the cost per patient (approximately US $30 for simultaneous analysis of both genes) is considerably lower than for IHC (roughly US $50 for each protein). Therefore, by revealing the presence of MLH1 or MSH2 LOH in an MSI-positive tumor, our method could eliminate further need for IHC analysis and appreciably lower the cost of prescreening in a nonnegligible percentage of cases. This approach could be especially useful when technical difficulties arise in the execution and/or interpretation of IHC assays (32)(33)(34)(35)(36)(37). The final results of IHC analyses are influenced by several factors, including those related to preparing the FFPE sample (type of fixative, fixation times, temperatures reached during the embedding procedure). The results of SNP-based LOH detection could also be helpful when normal immunostaining for all 4 MMR proteins is observed despite the tumor’s MSI positivity and a family history that is strongly suggestive of HNPCC (Fig. 3 , right). In one study (38), normal MMR protein expression was observed in 8% of MSI-positive tumors, and the rates that have been reported by other groups are only slightly lower (29). Results of this type (admittedly uncommon) can and do occur in the presence of some missense mutations, and LOH detection may identify the mutated gene in some of these cases.


Figure 3
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  		Figure 3. Potential roles of the SNaPshot-based assay for LOH at MLH1/MSH2 in the diagnostic workup of patients with suspected HNPCC.

For detailed description, see Discussion.

The SNP-genotyping method for MLH1 and MSH2 genes that we have described appears to be a promising, cost-effective adjunct to prescreening protocols used to guide germline testing for MMR defects in patients with colorectal cancers. Its specific roles in this setting can be determined only from data obtained in a large prospective study, which is currently being undertaken by our group. In the meantime, it is also important to recall that this novel assay also has a number of potential roles in research focusing on disease associations with MLH1 and MSH2 SNPs and SNP haplotypes, and it can also be readily adapted to the genetic analysis of other loci exhibiting a high frequency of polymorphisms in the general population.


   Acknowledgments
 
Author Contributions: Each author confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) Significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors’ Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. The following authors reported a potential conflict of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: This work was supported primarily by the European Commission (EU contract QLG1-CT-2000–01230) and in part by the Slovak Government (2003SP51–028–0800/028–0801), the Ministry of Health of the Slovak Republic (2006/26-NOU-02), the VEGA Grant Agency (2/5131/25), the Swiss National Science Foundation (310000–108434), and the Swiss Cancer League/Oncosuisse (01358–03–2003). K. Zavodna was a fellow of the European Social Fund Project (13120200038).

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We thank Ritva Haider for technical assistance. Marian Everett Kent provided very valuable editorial assistance.


   Footnotes
 
1 Nonstandard abbreviations: MMR, mismatch repair; HNPCC, hereditary nonpolyposis colorectal cancer; MSI, microsatellite instability; SNP, single-nucleotide polymorphism; LOH, loss of heterozygosity; IHC, immunohistochemistry; MLPA, multiplex ligation-dependent probe amplification; PBL, peripheral blood leukocyte; FFPE, formalin-fixed, paraffin embedded.

2 Human genes: MLH1, mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli); MSH2, mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli); KRAS, v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; MSH6, mutS homolog 6 (E. coli); PMS2, PMS2 postmeiotic segregation increased 2 (S. cerevisiae).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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