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Clinical Chemistry 49: 552-561, 2003; 10.1373/49.4.552
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(Clinical Chemistry. 2003;49:552-561.)
© 2003 American Association for Clinical Chemistry, Inc.

Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry-based Detection of Microsatellite Instabilities in Coding DNA Sequences: A Novel Approach to Identify DNA-Mismatch Repair-deficient Cancer Cells

Thomas Bonk1,2, Andreas Humeny1,2, Johannes Gebert2,2, Christian Sutter2, Magnus von Knebel Doeberitz2 and Cord-Michael Becker1,a

1 Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander Universität Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany.

2 Abteilung für Molekulare Pathologie, Pathologisches Institut, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 220, D-69120 Heidelberg, Germany.

aAuthor for correspondence. Fax 49-9131-85-22485; e-mail cmb{at}biochem.uni-erlangen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Inherited defects in the DNA mismatch repair system lead to increased loss or gain of repeat units in microsatellites, commonly referred to as microsatellite instability (MSI). MSIs in coding regions of critical genes contribute to the pathogenesis of DNA-mismatch repair-deficient cancers, particularly those associated with the hereditary nonpolyposis colorectal cancer syndrome (HNPCC). MSI typing is therefore increasingly used to guide the molecular diagnosis of HNPCC.

Methods: We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to identify MSIs in mononucleotide repeats within the coding sequences of genes relevant to the pathogenesis of MSI+ neoplastic lesions. After a primer extension reaction of PCR products encompassing the microsatellites, the molecular masses of the extension products were determined by MALDI-TOF-MS.

Results: MSIs were detected by MALDI-TOF-MS in the GART, AC1, TGFBR2, MSH3, and MSH6 genes in neoplastic tissues and MSI+ colorectal cancer cell lines but not in MSI- control tissues. The analysis of peak-integral ratios in a single spectrum of the peaks representing insertions or deletions compared with the full-length microsatellites allowed relative quantification of MSIs. MALDI-TOF-MS-based genotyping results were confirmed by conventional DNA sequencing and electrophoresis.

Conclusions: Because of its reliability, short run times, and low costs, this semiquantitative procedure represents an effective alternative, in particular for diagnostic high-throughput typing of MSIs in neoplastic lesions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cancer cells are characterized by multiple genetic alterations and an overall genomic instability that abrogate normal growth and differentiation control. In colorectal and other epithelial cancers, two major types of genetic instabilities have been identified: (a) chromosomal instability (1), which refers to gross changes in chromosome structure and number; and (b) microsatellite instability (MSI)1 (2)(3), which represents small somatic alterations at the nucleotide level recognized as length variations of short repetitive sequences termed microsatellites. In contrast to chromosomal instability tumors, MSI colorectal cancers are usually diploid or near diploid, show a low frequency of loss of heterozygosity, and exhibit a reduced occurrence of APC, ras, and p53 mutations (1)(2)(4), suggesting distinct but not mutually exclusive molecular pathogenic pathways (5).

MSI is a hallmark of hereditary nonpolyposis colorectal cancer (HNPCC) but is also found in ~15% of sporadic tumors of the colorectum and other organs (3)(6). Already present in preneoplastic lesions, MSI represents an early event that contributes to tumor development and progression. MSI colorectal cancers display a variety of distinct clinicohistopathologic features, including predilection for the proximal colon, mucinous or undifferentiated histology, and prominent lymphocytic infiltration, and in comparison with microsatellite stable tumors have a favorable prognosis (7). Recent clinical evidence suggests that patients with MSI colorectal cancers who receive adjuvant chemotherapy have an excellent prognosis (8).

At the molecular level, MSI is a consequence of mutational inactivation or epigenetic silencing of DNA-mismatch repair genes [for a review, see Ref. (9)]. In DNA-mismatch repair-deficient cells, microsatellites are particularly susceptible to DNA polymerase slippage and strand misalignment during DNA replication (10)(11)(12). These replication-dependent events create insertion-deletion loops, leading to gains or losses of short repeat units within microsatellites. If these genetic alterations affect microsatellites located in noncoding or nonregulatory sequences, no functional consequences are expected. However, length variations in coding microsatellites (cMS) that produce frameshift mutations inevitably lead to truncated proteins if the respective gene is expressed. Several cMS-containing genes that encode proteins involved in the regulation of cell growth (TGFBRII and IGF2R) (13)(14), programmed cell death (BAX and caspase-5) (15)(16), DNA-mismatch repair (hMSH3 and hMSH6) (17), transcription (TCF4)(18), or protein translocation (SEC63) (19) are frequently mutated in MSI cancers. Biallelic inactivation and variable frequencies of instability among cMS of identical type and length strongly suggest that frameshift mutations in some of these genes are selected for and contribute to MSI carcinogenesis. Recently, it has been shown that MSI cancer cells are prime targets for cytotoxic T cells directed against frameshift-induced neopeptides (20). This might explain at least in part the strong lymphocytic infiltration and favorable prognosis observed for MSI+ tumors (21).

As a sensitive surrogate marker for mismatch repair deficiency, particularly in neoplastic lesions of potential HNPCC patients, MSI classification therefore is increasingly used in diagnostic molecular pathology.

Apart from its mere diagnostic value, efficient screening for MSIs of the large number of cMS-containing candidate genes within the human genome (18)(19)(22) provides an immediate clue to the functional relevance of mutations for the pathogenesis of MSI+ cancers (18)(19)(22). Methods commonly used for the detection of MSIs, including chromatography, electrophoresis, and DNA sequencing, are time-consuming and expensive and suffer from low sensitivity in the detection of quantitative differences. In this situation, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based genotyping offers a promising technical alternative (23)(24)(25)(26)(27)(28)(29). Here we describe a novel method for enhanced genotyping of MSIs that affect mononucleotide repeats with prospects for high throughput by use of MALDI-TOF-MS for semiquantitative analysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patient samples
Blood and tissue samples were obtained from HNPCC patients attending the Department of Surgery of the University of Heidelberg. Written informed consent was obtained. Chromosomal leukocyte DNA was extracted from peripheral EDTA blood with use of the RapidPrepTM Macro Genomic DNA Isolation Kit (Pharmacia) according to the manufacturer’s protocol. DNA preparation from matched healthy/cancer tissues and cancer cell lines as well as conventional MSI classification has been described previously (19)(30).

pcr for maldi-tof-ms-based genotyping
PCR reactions (10 µL) contained 12 pmol of both forward and reverse primers (Table 1 ; MWG Biotech); 50 ng of DNA template; 1 U of PAN Taq Polymerase in supplied NH4+ buffer (PAN) supplemented by 2.0 mM (AC1, TGFBR2, MSH6), 2.2 mM (GART), or 2.5 mM (MSH3) MgCl2, respectively; and 2 nM of each deoxynucleotide triphosphate. PCR conditions were 96 °C for 2 min; 35 cycles at 96 °C for 60 s and 54 °C (MSH3), 56 °C (AC1), 58 °C (MSH6), 59 °C (TGFBR2), or 61 °C (GART), respectively, for 30 s; and finally 72 °C for 3 min.


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  		Table 1. Sequences of the primers for PCR and extension reactions of the microsatellites investigated.

primer extension reactions for maldi-tof-ms-based genotyping
Purified PCR products (for GART, 82 bp, nt 2370–2451; for AC1, 86 bp, nt 21–106; for TGFBRII, 102 bp, nt 304–405; for MSH3, 154 bp, nt 1003–1146 plus 4 nucleotides of intron 6 and 6 nucleotides of intron 7; for MSH6, 94 bp, nt 3207–3300) were used as templates for the primer extension reaction (10 µL). The extension mixture contained 12 pmol of extension primer (Table 1Up ), 1–2 U of Thermosequenase, 1x reaction buffer (Pharmacia), and 2 nmol of assay-specific deoxynucleotide triphosphates and dideoxynucleotide triphosphates (for GART, dTTP and ddCTP; for AC1, dATP and ddTTP; for TGFBRII and MSH3, dATP and ddGTP; for MSH6, dCTP and ddTTP). Extension reactions were performed at 96 °C for 2 min; 40 cycles at 94 °C for 30 s and 49 °C (MSH3, MSH6), 55 °C (TGFBR2), 56 °C (AC1), or 60 °C (GART), respectively, for 30 s; and finally 72 °C for 3 min.

sample purification for maldi-tof-ms-based genotyping
Both PCR and primer extension products were purified with magnetic beads (GenoPure) as described by the supplier (Bruker Daltonik GmbH).

maldi-tof-ms-based internal quantification of msi
Two synthetic oligonucleotides, GARTwt (5'-AGC CAC TCT GGC CTT TTT TTT TTC-3') and GART-1 (5'-AGC CAC TCT GGC CTT TTT TTT TC-3'), which corresponded to the reaction products of the wild-type microsatellite (wt) and the one-nucleotide-deleted microsatellite (-1) of the GART primer extension assay were mixed in defined molar ratios (1:9, 1:4, 3:7, 2:3, and 1:1). After MALDI-TOF-MS analysis, the signal-integral ratios (SIRs) of the peaks representing both oligonucleotides were determined with the oligonucleotide GARTwt serving as an internal standard. Ten individual experiments were performed for each molar ratio.

maldi-tof-ms
Aliquots of the purified samples were spotted onto matrix crystals of 3-hydroxypicolinic acid on a stainless steel target and air dried. Mass determinations were performed on a BiflexTM III MALDI-TOF mass spectrometer (Bruker Daltonik GmbH) equipped with a nitrogen laser ({lambda} = 337 nm) and delayed extraction. Laser-desorbed positive ions were analyzed after acceleration by 20 kV in the linear mode. External calibration was performed with a standard oligonucleotide mixture. Usually, 30 individual spectra were averaged to produce a mass spectrum. SIRs of deletion and insertion peaks were determined with use of the peak integral of the wt alleles as an internal reference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
correlation of allele amount and sir
MSI is generally recognized by length variations in PCR-amplified short repetitive sequences in tumor DNA compared with DNA from nonneoplastic cells. However, PCR-based amplification of such repeats frequently leads to artifactual smaller- or larger-sized amplimers because of DNA polymerase slippage, thus requiring that artifactual amplimers be distinguished from true unstable alleles of identical size. As a first step toward semiquantitative MSI analysis by MALDI-TOF-MS, we mixed two synthetic oligonucleotides representing the 24- and 23-bp primer extension products of the wt and the -1 deletion alleles, respectively, of an A10 repeat [phosphoribosylglycinamide formyltransferase gene (GART); MIM 138440] in defined molar ratios. The peak ratios for the wt and -1 alleles revealed a linear correlation between the molar ratios of oligonucleotides and observed intraspectral SIRs (Fig. 1 ). By this approach, it can be ruled out that different ion yields lead to different signal intensities. These data demonstrate that significant changes in the relative amounts of differently sized microsatellite alleles are directly reflected by the SIR pattern.



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  		Figure 1. MALDI-TOF-MS analysis of SIRs of synthetic oligonucleotides representing the genotypes of the microsatellite in the GART gene.

The synthetic oligonucleotides GARTwt and GART-1A, representing the wt allele and the deletion of one A in the microsatellite located in exon 17 of the GART gene, as shown in Fig. 2Up , were analyzed by MALDI-TOF-MS. The oligonucleotides were mixed in defined molar ratios ranging from 1:9 to 1:1, referring to the amount of wt allele oligonucleotide. After the generation of MALDI-TOF-MS spectra, the SIRs were determined for both peaks, representing the GARTwt and GART-1A oligonucleotides. Ten individual experiments were performed for each molar ratio.

coding microsatellite length variations in colorectal tumors and cell lines
On the basis of the linear correlation of SIRs, we examined the primer extension product profiles and SIR values of five microsatellites in 10 blood samples, 15 colorectal cancer cell lines, and 12 matched colorectal healthy/tumor tissues. The MSI status of these samples had previously been determined by chromatographic methods. Molecular weight analysis of extension products in these samples required external calibration with use of a standard oligonucleotide mixture. For calculation of SIR values, peak integrals of the wt extension products served as an internal standard (isSIR). The microsatellites subjected to this analysis are located in the coding region of the genes encoding transforming growth factor ß receptor type II (TGFBR2; MIM 190182), DNA-mismatch repair proteins (MSH3; MIM 600887; MSH6, MIM 600678), the trifunctional protein GART, and a protein of unknown function (AC1; Table 1Up ). They were chosen as target sequences based on the following criteria: (a) these microsatellites comprise short mononucleotide repeats of different types and lengths; and (b) their patterns of stable and unstable alleles in colorectal tumors and cell lines have previously been characterized.

We first examined the mass spectra and isSIR values of primer extension products for all five microsatellites in blood lymphocytes of healthy donors. Representative MALDI-TOF-MS spectra are depicted in Figs. 2–6 . All microsatellites analyzed showed characteristic peaks of the predicted molecular masses for both the unextended primer and for the extension product comprising the wt repeat. In addition, peaks of lower signal intensities exhibiting molecular masses that corresponded to shorter-sized primer extension products were observed in these control samples. These additional peaks most likely represent PCR artifacts attributable to DNA polymerase slippage. To quantify the relative amounts of these differently sized microsatellite primer extension products, we determined their SIRs. In mismatch repair-proficient control blood samples, isSIR values for GART, AC1, TGFBRII, MSH3, and MSH6 microsatellite primer extension products representing one or two nucleotide deletions (-1/wt; -2/wt) were in the range of 0.02–0.5. In contrast, isSIR values for one or two nucleotide insertions (+1/wt; +2/wt) remained undetectable (Tables 2 and 3 ).



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  		Figure 2. MALDI-TOF-MS-based genotyping of MSIs in GART exon 17.

(A), schematic primer extension assay for genotyping MSIs in GART. The scheme depicts the genomic sequences of the GART wt allele (nt 2407–2440), including the microsatellite 10-A repeat (nt 2411–2420), according to numbering starting with the A in the start codon (GenBank accession no. X54199). GART sequences containing the deletion and insertion of one A (nt 2410–2420 and nt 2412–2420), respectively, are shown below the wt genomic sequence. The microsatellite is shown in bold. (B), MALDI-TOF-MS analysis of MSIs in GART. MALDI-TOF-MS spectra of control DNA, DNA of patients B184 (control tissue and cancer tissue) and B218 (control tissue and cancer tissue), and DNA of the KM12 cell line. The remaining primer (6026 Da) served for internal mass calibration. The molecular mass of 7211 Da represents the wt allele, whereas the molecular masses of 6907 and 7215 Da indicate the deletion and insertion of one A in the microsatellite, respectively.



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  		Figure 3. MALDI-TOF-MS-based genotyping of MSIs in AC1 exon 1.

(A), schematic primer extension assay for genotyping the MSIs in AC1. The scheme depicts the genomic sequences of the AC1 wt allele (nt 56–88), including the microsatellite 10-T repeat (nt 59–68), according to numbering starting with the A in the start codon (GenBank accession no. D82070). AC1 sequences with the deletion and insertion of one T (nt 55–88 and nt 57–88), respectively, are shown below the wt genomic sequence. The microsatellite is shown in bold. (B), MALDI-TOF-MS analysis of MSIs in the AC1 gene. MALDI-TOF-MS spectra of control DNA, DNA of patient B245 (control tissue and cancer tissue), and DNA from cell lines KM12, HCT116, and SW48. Unextended primer (6152 Da) was used for internal mass calibration. The molecular mass of 8318 Da represents the wt allele, whereas the molecular mass of 8005 Da indicates the deletion of one T in the microsatellite.



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  		Figure 4. MALDI-TOF-MS-based genotyping of MSIs in TGFBR2 exon 3.

(A), schematic primer extension assay for genotyping MSIs in the TGFBR2. The scheme depicts genomic sequences of the TGFBR2 wt allele (nt 357–389), including the microsatellite 10-A repeat (nt 374–383), according to numbering starting with the A in the start codon (GenBank accession no. D50683). The TGFBR2 sequences with the deletion and insertion of one T (nt 357–390 and nt 357–388), respectively, are shown below the wt genomic sequence. The microsatellite is shown in bold. (B), MALDI-TOF-MS analysis of MSIs in TGFBR2. MALDI-TOF-MS spectra of control DNA and DNA of patients B210 (control tissue and cancer tissue) and B245 (control tissue and cancer tissue). Remaining unextended primer (6223 Da) was used for internal mass calibration. The molecular mass of 8414 Da represents the wt allele, whereas the molecular mass of 8101 Da indicates the deletion of one A in the microsatellite.



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  		Figure 5. MALDI-TOF-MS-based genotyping of MSIs in MSH3 exon 7.

(A), schematic primer extension assay for genotyping the MSIs in MSH3. The scheme depicts genomic sequences of the MSH3 wt allele (nt 1096–1128), including the microsatellite 8-A repeat (nt 1114–1121), according to numbering starting with the A in the start codon (GenBank accession no. J04810). The MSH3 sequences with the deletion and insertion of one T (nt 1096–1129 and nt 1096–1127), respectively, are shown below the wt genomic sequence. The microsatellite is shown in bold. (B), MALDI-TOF-MS analysis of MSIs in MSH3. MALDI-TOF-MS spectra of control DNA, DNA of patient B174 (control tissue and cancer tissue), and DNA of cell lines KM12 and HCT116. Remaining unextended primer (6651 Da) was used for internal mass calibration. The molecular mass of 8126 Da represents the wt allele, whereas the molecular mass of 7813 Da indicates the deletion of one A in the microsatellite.



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  		Figure 6. MALDI-TOF-MS-based genotyping of MSIs in MSH6 exon 5.

(A), schematic primer extension assay for genotyping the MSIs in MSH6. The scheme depicts genomic sequences of the MSH6 wt allele (nt 3237–3269), including the microsatellite 8-C repeat (nt 3254–3261), according to numbering starting with the A in the start codon (GenBank accession no. U54777). The MSH6 sequences with the deletion and insertion of one T (nt 3237–3270 and nt 3237–3268), respectively, are shown below the wt genomic sequence. The microsatellite is shown in bold. (B), MALDI-TOF-MS analysis of MSIs in MSH6. MALDI-TOF-MS spectra of control DNA, DNA of patient B174 (control tissue and cancer tissue), and DNA of cell lines LS180, KM12, and HCT116. Remaining unextended primer (4852 Da) was used for internal mass calibration. The molecular mass of 6296 Da represents the wt allele, whereas the molecular masses of 6007, 6585, and 6874 Da indicate the deletion of one A and insertion of one or two As in the microsatellite, respectively.


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  		Table 2. Results of MALDI-TOF-MS-based genotyping of MSIs in GART, AC1, and TGFBR2.1


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  		Table 3. Results of MALDI-TOF-MS-based genotyping of MSIs in MSH3 and MSH6.1

We next investigated microsatellite primer extension products in 15 colorectal cancer cell lines and in 12 primary colorectal tumors and adjacent healthy mucosa. The microsatellite peak pattern of healthy mucosa samples resembled that of control blood samples (Figs. 2–6Up Up Up Up Up ). The isSIR values of extension products reflecting nucleotide deletions or insertions never exceeded 0.54 or were absent, respectively (Tables 2Up and 3Up ). In the cancer tissues of six HNPCC patients (B174, B180, B184, B210, B218, and B245) and in six tumor cell lines (KM12, SW48, HCT116, LS174T, LS180, and LoVo; Tables 2Up and 3Up ), however, the isSIR values of primer extension products were significantly increased. Differences ranged from 2-fold differences for the TGFBRII microsatellite in B184 healthy vs cancer tissues up to 33-fold differences for the MSH6 microsatellite in the LS174T cell line vs blood samples. Among these samples, increased isSIR values were easily detectable for extension products representing nucleotide insertions because of the particularly low (zero) integrals of wt peaks in control samples (GART in KM12 and SW48, TGFBRII in B245, and MSH6 in B184, KM12, SW48, and HCT116; Tables 2Up and 3Up ). In some cell lines, the absence of extension products representing the wt repeat length precluded the calculation of isSIR values of deletion mutant extension products for three microsatellites (AC1 in SW48, TGFBRII in SW48 and HCT116, and MSH3 in HCT116). In the absence of wt extension products, prominent microsatellite mutant peaks could be unambiguously identified by comparison with the blood or mucosa peak pattern [AC1 in SW48 (Fig. 3Up ) and MSH3 in HCT116 (Fig. 5Up )]. Overall, 31 true unstable microsatellite alleles showing increased isSIR values were identified in the cancer tissues and cell lines analyzed. These included 4 alterations in GART (-1 and +1 frameshifts), 4 alterations in AC1 (-1 frameshift), 11 alterations in TGFBRII (-1, -2, and +1 frameshifts), 5 alterations in MSH3 (-1 and -2 frameshifts), and 7 alterations in MSH6 (+1 and +2 frameshifts). All MALDI-TOF-MS-based results were subsequently confirmed by DNA sequencing and showed a 100% concordance with the microsatellite allele pattern obtained previously by conventional methods. These data demonstrate the suitability of MALDI-TOF-MS for reliable MSI typing of tumor cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In molecular pathology, the analysis of MSIs in affected genes serves as a diagnostic tool, especially for screening neoplastic lesions for a genetic predisposition to HNPCC (19)(31)(32). Usually, electrophoretic and chromatographic methods are used for this purpose. However, these methods are hampered by limitations ranging from insufficient sensitivity and molecular resolution to long time exposures; they therefore appear unsuitable for high-throughput analysis. In contrast, MALDI-TOF-MS-based genotyping offers high molecular resolution, accuracy, and reproducibility combined with excellent sensitivity as shown previously for detection of single-nucleotide polymorphisms (23)(24)(25)(27)(28) and genotyping of dinucleotide tandem repeats with use of ribozyme-cleaved RNA transcripts (33). In addition, the high-throughput option of MALDI-TOF-MS genotyping (29) may be fully exploited by automated post-data processing. After external calibration with a standard oligonucleotide mixture, the accuracy of mass determination is ensured by internal calibration using the theoretical masses of the primers or the extension products generated. Finally, the spectra are interpreted, the quality of each spectrum is determined. and the observed genotypes are saved electronically (34).

In the present study, we applied these features of MALDI-TOF-MS to the analysis of MSIs. Using DNA from healthy tissue and peripheral blood as a control, we were able to identify unstable microsatellite alleles in primary tumors and cell lines also subjected to conventional MSI typing. In MALDI-TOF-MS-based MSI analysis, quantitative assessment of extension products by isSIR is critical. This approach ensured the reproducibility of the isSIR values obtained; interassay variation was ~5%. Still, the wt isSIR must be established for each individual MSI assay because the lengths and nucleotide patterns of repeats as well as their flanking sequences appear to affect the isSIR observed. For example, the theoretical probability for DNA slippage increases with microsatellite length. Moreover, assay conditions that modulate the isSIR, including temperature and Mg2+ concentration, must be optimized. Overall, a significant increase in isSIR was a reliable criterion of MSI and allowed true unstable alleles to be distinguished from aberrant extension products arising from DNA polymerase slippage.

Our strategy of MALDI-TOF-MS-based MSI classification depends on the generation of short primer extension products; thus, it is restricted to microsatellites containing 8–10 mononucleotide repeats. To ensure selective annealing of the extension primer to the target DNA sequence, the primer needs to be complementary to either one of the nonrepetitive sequences flanking the microsatellite. Depending on the flanking sequence of the microsatellites, reverse or forward extension primers were selected that yielded optimal melting temperatures, high specificity, and reduced formation of secondary structures [e.g., primer dimers, and loops (25)(27)(28)]. On the other hand, in MALDI-TOF-MS analysis, signal intensities of oligonucleotides decrease parallel to increases in molecular mass (28). Experience has shown that the molecular masses of single-strand extension products should be 4–10 kDa. Given that the molecular masses of single nucleotides are in the range of ~300 Da, appropriate extension products should be composed of 13–33 nucleotides. As evident from these basic criteria of primer design, microsatellites with up to 15 repeat units should be analyzable by this approach. These requirements are contrasted, however, by the reference marker panel for MSI classification recommended by the ICG/NCI (BAT25, BAT26, D2S123, D5S346, and D17S250), which includes microsatellites exceeding this repeat size limit (7). Still, the microsatellite panel used in this study (GART, AC1, TGFBRII, MSH3, and MSH6) was sufficient to define a high degree of MSI, i.e., at least 40% of markers show instability in three primary tumors (B184, B218, and B245) and five cell lines (KM12, SW48, HCT116, LS174T, and LoVo). Nevertheless, a more generalized application of this technology in the future will require the characterization of additional MALDI-TOF-MS-compatible microsatellites and comparison of their MSI scoring values with the reference marker panel. The most immediate use of MALDI-TOF-MS-based MSI typing may be envisioned for instability in coding microsatellites: In the human genome, these coding repeats exist in large numbers, producing frameshift mutations at these repeats as a consequence of MSIs (19). Thus, high-throughput MSI screening of coding microsatellite candidate sequences should facilitate the identification of real target genes involved in MSI carcinogenesis. Moreover, coding microsatellite frameshift mutations often generate truncated proteins with unique frameshift C-terminal sequences. Determination of coding region MSI and frameshift mutation patterns in tumors by MALDI-TOF-MS might help in the design of specific frameshift peptide-based therapeutic vaccines for affected patients (20).

In summary, MALDI-TOF-MS-based genotyping of mononucleotide MSIs offers a cost-effective, high-throughput alternative to conventional electrophoretic and chromatographic methods. Because of its high molecular resolution, excellent accuracy, reproducibility, and automation properties, this method offers the potential to replace other methods in molecular medicine (23)(24)(25)(26)(27)(28)(29). Although the feasibility of our semiquantitative approach has been demonstrated on HNPCC neoplasms and colorectal cancer cell lines, it may easily be adapted to MSI classification of cancers in other organs.


   Acknowledgments
 
The skillful technical assistance of Claudia Sass is gratefully acknowledged. We thank Bruker Daltonik GmbH (Bremen, Germany) for providing the Biflex III MALDI-TOF mass spectrometer. We are grateful to Dr. Markus Kostrzewa (Bruker Saxonia Analytik GmbH, Leipzig, Germany) for helpful discussions. This work was supported in part by grants from Deutsche Forschungsgemeinschaft, German-Israeli-Foundation of Scientific Research and Development, Fonds der Chemischen Industrie (to C.M.B.), and the Deutsche Krebshilfe (to M.v.K.D. and J.G.).


   Footnotes
 
1 Nonstandard abbreviations: MSI, microsatellite instability; HNPCC, hereditary nonpolyposis colorectal cancer; cMS, coding microsatellite; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; SIR, signal-integral ratio; wt, wild type; and isSIR, signal-integral ratio internal standard.

2 These authors contributed equally to this work.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lengauer C, Kinzler KW, Vogelstein B. Genetic instability in colorectal cancers. Nature 1997;386:623-627.[CrossRef][Medline] [Order article via Infotrieve]
  2. Ionov 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;363:558-561.[CrossRef][Medline] [Order article via Infotrieve]
  3. Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon. Science 1993;260:816-819.[Abstract/Free Full Text]
  4. Konishi M, Kikuchi-Yanoshita R, Tanaka K, Muraoka M, Onda A, Okumura Y, et al. Molecular nature of colon tumors in hereditary nonpolyposis colon cancer, familial polyposis, and sporadic colon cancer. Gastroenterology 1996;111:307-317.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  5. Perucho M, Peinado MA, Ionov Y, Casares S, Malkhosyan S, Stanbridge E. Defects in replication fidelity of simple repeated sequences reveal a new mutator mechanism for oncogenesis. Cold Spring Harb Symp Quant Biol 1994;59:339-348.[Abstract/Free Full Text]
  6. Aaltonen LA, Peltomaki P, Mecklin JP, Jarvinen H, Jass JR, Green JS, et al. Replication errors in benign and malignant tumors from hereditary nonpolyposis colorectal cancer patients. Cancer Res 1994;54:1645-1648.[Abstract/Free Full Text]
  7. Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RW, et al. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 1998;58:5248-5257.[Abstract/Free Full Text]
  8. Hemminki A, Mecklin JP, Jarvinen H, Aaltonen LA, Joensuu H. Microsatellite instability is a favorable prognostic indicator in patients with colorectal cancer receiving chemotherapy. Gastroenterology 2000;119:921-928.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  9. Peltomaki P. DNA mismatch repair and cancer. Mutat Res 2001;488:77-85.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  10. Hancock JM. The contribution of slippage-like processes to genome evolution. J Mol Evol 1995;41:1038-1047.[ISI][Medline] [Order article via Infotrieve]
  11. Karthikeyan G, Chary KV, Rao BJ. Fold-back structures at the distal end influence DNA slippage at the proximal end during mononucleotide repeat expansions. Nucleic Acids Res 1999;27:3851-3858.[Abstract/Free Full Text]
  12. Hartenstine MJ, Goodman MF, Petruska J. Base stacking and even/odd behavior of hairpin loops in DNA triplet repeat slippage and expansion with DNA polymerase. J Biol Chem 2000;275:18382-18390.[Abstract/Free Full Text]
  13. Markowitz S, Wang J, Myeroff L, Parsons R, Sun L, Lutterbaugh J, et al. Inactivation of the type II TGF-ß receptor in colon cancer cells with microsatellite instability. Science 1995;268:1336-1338.[Abstract/Free Full Text]
  14. Souza RF, Appel R, Yin J, Wang S, Smolinski KN, Abraham JM, et al. Microsatellite instability in the insulin-like growth factor II receptor gene in gastrointestinal tumours. Nat Genet 1996;14:255-257.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  15. Rampino N, Yamamoto H, Ionov Y, Li Y, Sawai H, Reed JC, et al. Somatic frameshift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype. Science 1997;275:967-969.[Abstract/Free Full Text]
  16. Schwartz S, Jr, Yamamoto H, Navarro M, Maestro M, Reventos J, Perucho M. Frameshift mutations at mononucleotide repeats in caspase-5 and other target genes in endometrial and gastrointestinal cancer of the microsatellite mutator phenotype. Cancer Res 1999;59:2995-3002.[Abstract/Free Full Text]
  17. Malkhosyan S, McCarty A, Sawai H, Perucho M. Differences in the spectrum of spontaneous mutations in the hprt gene between tumor cells of the microsatellite mutator phenotype. Mutat Res 1996;316:249-259.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  18. Duval A, Gayet J, Zhou XP, Iacopetta B, Thomas G, Hamelin R. Frequent frameshift mutations of the TCF-4 gene in colorectal cancers with microsatellite instability. Cancer Res 1999;59:4213-4215.[Abstract/Free Full Text]
  19. Woerner SM, Gebert J, Yuan YP, Sutter C, Ridder R, Bork P, et al. Systematic identification of genes with coding microsatellites mutated in DNA mismatch repair-deficient cancer cells. Int J Cancer 2001;93:12-19.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  20. Linnebacher M, Gebert J, Rudy W, Woerner S, Yuan YP, Bork P, et al. Frameshift peptide-derived T-cell epitopes: a source of novel tumor-specific antigens. Int J Cancer 2001;93:6-11.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  21. Dolcetti R, Viel A, Doglioni C, Russo A, Guidoboni M, Capozzi E, et al. High prevalence of activated intraepithelial cytotoxic T lymphocytes and increased neoplastic cell apoptosis in colorectal carcinomas with microsatellite instability. Am J Pathol 1999;154:1805-1813.[Abstract/Free Full Text]
  22. Mori Y, Yin J, Rashid A, Leggett BA, Young J, Simms L, et al. Instabilotyping: comprehensive identification of frameshift mutations caused by coding region microsatellite instability. Cancer Res 2001;61:6046-6049.[Abstract/Free Full Text]
  23. Schiebel K, Meder J, Rump A, Rosenthal A, Winkelmann M, Fischer C, et al. Elevated DNA sequence diversity in the genomic region of the phosphatase PPP2R3L gene in the human pseudoautosomal region. Cytogenet Cell Genet 2000;91:224-230.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  24. Bonk T, Humeny A. MALDI-TOF-MS analysis of protein and DNA. Neuroscientist 2001;7:6-12.[Abstract]
  25. Humeny A, Bonk T, Berkholz A, Wildt L, Becker C-M. Genotyping of thrombotic risk factors by MALDI-TOF mass spectrometry. Clin Biochem 2001;34:531-536.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  26. Bonk T, Humeny A, Sutter C, Gebert J, von Knebel-Döberitz M, Becker C-M. Molecular diagnosis of familial adenomatous polyposis (FAP): genotyping of adenomatous polyposis coli (APC) alleles by MALDI-TOF mass spectrometry. Clin Biochem 2002;35:87-92.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  27. Humeny A, Schiebel K, Seeber S, Becker C-M. Identification of polymorphisms within the bovine prion protein gene (Prnp) by DNA sequencing and genotyping by MALDI-TOF-MS. Neurogenetics 2002;4:59-60.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  28. Humeny A, Bonk T, Becker K, Jafari-Boroujerdi M, Stefani U, Reuter K, et al. A novel recessive hyperekplexia allele GLRA1 (S231R): genotyping by MALDI-TOF mass spectrometry and functional characterisation as a determinant of cellular glycine receptor trafficking. Eur J Hum Genet 2002;10:188-196.[CrossRef][Medline] [Order article via Infotrieve]
  29. Nissum M, Preuss D, Harig A, Lieberwirth U, Betz C, Neumann S, et al. High-throughput genetic screening using matrix-assisted laser desorption/ionization mass spectrometry. Psychiatr Genet 2002;12:109-117.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  30. Sutter C, Gebert J, Bischoff P, Herfarth C, von Knebel Doeberitz M. Molecular screening of potential HNPCC patients using a multiplex microsatellite PCR system. Mol Cell Probes 1999;13:157-165.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  31. Shibata D, Aaltonen LA. Genetic predisposition and somatic diversification in tumor development and progression. Adv Cancer Res 2001;80:83-114.[ISI][Medline] [Order article via Infotrieve]
  32. Loukola A, Eklin K, Laiho P, Salovaara R, Kristo P, Jarvinen H, et al. Microsatellite marker analysis in screening for hereditary nonpolyposis colorectal cancer (HNPCC). Cancer Res 2001;61:4545-4549.[Abstract/Free Full Text]
  33. Krebs S, Seichter D, Forster M. Genotyping of dinucleotide tandem repeats by MALDI mass spectrometry of ribozyme-cleaved RNA transcripts. Nat Biotechnol 2001;19:877-880.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  34. Pusch W, Kraeuter KO, Froehlich T, Stalgies Y, Kostrzewa M. Genotools SNP manager: a new software for automated high-throughput MALDI-TOF mass spectrometry SNP genotyping. Biotechniques 2001;30:210-215.[ISI][Medline] [Order article via Infotrieve]



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