[CANCER RESEARCH 59, 3899–3903, August 15, 1999]

Advances in Brief

Quantitative Analysis of Aberrant p16 Methylation Using Real-Time QuantitativeMethylation-specific Polymerase Chain Reaction1

Y. M. Dennis Lo,2 Ivy H. N. Wong, Jun Zhang, Mark S. C. Tein, Margaret H. L. Ng, and N. Magnus HjelmDepartments of Chemical Pathology [Y. M. D. L., J. Z., M. S. C. T., N. M. H.] and Anatomical and Cellular Pathology [I. H. N. W., M. H. L. N.], The Chinese University of HongKong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region

Abstract

We have developed a quantitative method for methylation analysis ofthe p16 gene based on real-time methylation-specific PCR (MSP). Real-time MSP is sensitive enough to detect down to 10 genome equivalents ofthe methylatedp16sequence. Application of real-time MSP to DNA fromtumor-derived cell lines revealed complete concordance with conventionalMSP analysis. Quantitative data generated by real-time MSP were ex-pressed as the methylation index, which was defined as the percentage ofbisulfite-converted DNA that consisted of methylated target sequences.The methylation index was shown to be inversely correlated withp16genetranscription during demethylation treatment of cell lines with 5-aza-2*-deoxycytidine. The application of real-time MSP to bone marrow aspi-rates from patients with multiple myeloma revealed complete concord-ance with conventional MSP analysis. Real-time quantitative MSP mayhave applications in elucidating diverse biological processes involvingDNA methylation and may become a valuable diagnostic tool for detectingtumor-associated epigenetic changes in cancer patients.

Introduction

The role of DNA methylation in tumorigenesis has attracted con-siderable attention recently (1). The detection of aberrant DNA meth-ylation in tumors has implications for the understanding of the fun-damental mechanisms of oncogenesis (1, 2) and may form the basisfor new molecular assays for cancer detection and monitoring (3–6).Established methods for methylation analysis include methylation-sensitive restriction enzyme treatment followed by Southern blotting(7) and PCR (8), bisulfite sequencing (9), restriction site creation bybisulfite modification (10), and MSP3 (11). The need for quantitativedata has prompted some investigators to develop quantitative methodsfor methylation analysis (12, 13). However, these quantitative toolsfor methylation analysis generally require the use of gel electrophore-sis and radioisotopes. The advent of real-time quantitative PCR (14,15) has allowed the performance of nonisotopic, rapid, and highlyaccurate quantitative amplification analysis via the continuous opticalmonitoring of a fluorogenic PCR. In this study, we investigated thepossibility of quantitative methylation analysis using real-time MSP.

Materials and Methods

Cell Lines. The tumor-derived cell lines HS-Sultan, ARH-77, IM-9, RPMI8226, NCI-H929, U266 B1, HeLa, and Raji were obtained from the AmericanType Culture Collection (Manassas, VA) and cultured using previously estab-lished conditions (7). DNA from cultured cell lines was extracted as describedpreviously (7).

Patient Materials. Eight patients with multiple myeloma were recruitedwith informed consent from subjects investigated at the Department of Ana-tomical and Cellular Pathology, Prince of Wales Hospital, Hong Kong. DNAfrom bone marrow aspirates was extracted as described previously (7). Thestudy was approved by the Ethics Committee of The Chinese University ofHong Kong.

Demethylation Treatment. Cell lines were treated with 1–3mM 5-aza-29-deoxycytidine (Sigma Chemical Co., St. Louis, MO) in RPMI 1640 supple-mented with 10 or 15% fetal bovine serum, as described previously (7), andcultured for 10 days.

Bisulfite Conversion of DNA Samples.Bisulfite conversion of DNAsamples was carried out essentially as described (11) and was based on theprinciple that treatment of DNA with bisulfite would result in the conversionof unmethylated cytosine residues into uracil. Methylated cytosine residues, onthe other hand, would remain unchanged. Thus, the DNA sequences ofmethylated and unmethylated genomic regions following bisulfite conversionwould be different and distinguishable by sequence-specific PCR primers.

Bisulfite conversion was carried out using reagents provided in a CpGe-nome DNA Modification Kit (Intergen, New York, NY). Onemg of DNA wastreated with sodium bisulfite following the manufacturers’ recommendations.Following conversion, the bisulfite-converted DNA was resuspended in a totalvolume of 25ml.

Conventional MSP. Conventional MSP was carried out as described pre-viously (6, 11). The sense and antisense primers for the bisulfite-convertedmethylated sequence were p16MF (59-TTA TTA GAG GGT GGG GCG GATCGC-39) and p16MR (59-GAC CCC GAA CCG CGA CCG TAA 39), respec-tively (product size, 150 bp; Ref. 11). The sense and antisense primers for thebisulfite-converted unmethylated sequence were p16U-5 (59-TTA TTA GAGGGT GGG GTG GAT TGT-39) and p16U-3 (59-CAA CCC CAA ACC ACAACC ATA A-39), respectively (product size, 151 bp; Ref. 11). The sense andantisense primers for the unconverted wild-type sequence were p16WF (59-CAG AGG GTG GGG CGG ACC GC-39) and p16WR (59-CGG GCC GCGGCC GTG G-39), respectively (product size, 140 bp; Ref. 11). Thirty-fivecycles of PCR were carried out using reagents supplied in a GeneAmp DNAAmplification Kit using AmpliTaq Gold as the polymerase (Perkin-Elmer,Foster City, CA) as described previously (6). PCR products were analyzed byagarose gel electrophoresis and ethidium bromide staining.

Real-Time Quantitative MSP. Real-time quantitative PCR is based on thecontinuous optical monitoring of the progress of a fluorogenic PCR (14, 15).In this system, apart from the two amplification primers as in conventionalPCR, a dual-labeled fluorogenic hybridization probe is also included (14). Onefluorescent dye serves as a reporter (FAM), and its emission spectra isquenched by a second fluorescent dye (TAMRA). During the extension phaseof PCR, the 59to 39 exonuclease activity of the Taq DNA polymerase (16)cleaves the reporter from the probe, thus releasing it from the quencher,resulting in an increase in fluorescent emission at 518 nm.

Three real-time MSP systems were developed for the detection and quan-titation of the bisulfite-converted methylated version of thep16 gene (thep16M system; system 1), the bisulfite-converted unmethylated version of the

Received 5/21/99; accepted 7/2/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by grants from the Hong Kong Research Grants Council and theDirect Grants Scheme from the Chinese University of Hong Kong. Y. M. D. L. is arecipient of the Industrial Support Fund. M. S. C. T. was supported by the PathologicalSociety of Great Britain and Ireland, a Zonchonis Special Enterprise Award, and aJohnson-Ewart Smart Fund Award from the University of Manchester (Manchester,United Kingdom).

2 To whom requests for reprints should be addressed, at the Department of ChemicalPathology, The Chinese University of Hong Kong, Room 38023, 1/F, Clinical SciencesBuilding, Prince of Wales Hospital, 30-32 Ngan Shing Street, Shatin, New Territories,Hong Kong Special Administrative Region. E-mail: loym@cuhk.edu.hk.

3 The abbreviations used are: MSP, methylation-specific PCR; FAM, 6-carboxyfluo-rescein; TAMRA, 6-carboxy-tetramethylrhodamine; RT-PCR, reverse transcriptase PCR;CV, coefficient of variation; Ms-SNuPE, methylation-sensitive single-nucleotide primerextension; COBRA, combined bisulfite restriction analysis.

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p16gene (the p16U system; system 2), and the unconverted wild-type versionof the p16 gene (the p16W system; system 3). For system 1, the primersp16MF and p16MR (see above; Ref. 11) were used in conjunction with afluorogenic probe p16MT [59-(FAM)-AGT AGT ATG GAG TCG GCG GCGGG-(TAMRA)-39]. For system 2, the primers p16UF and p16UR (see above;Ref. 11) were used in conjunction with a fluorogenic probe p16UT [59-(FAM)-AGG TAG TGG GTG GTG GGG AGT AGT ATG GAG TTG-(TAMRA)-39].For system 3, the primers p16W-5 (59-GTG GGG CGG ACC GC 39) and

p16W-3 (59-GCC GCG GCC GTGG-39) were used in conjunction with afluorogenic probe, p16W-T [59-(FAM)-AGC AGC ATG GAG CCG GCGG-(TAMRA)-39]. The fluorogenic probes contained a 39-blocking phosphategroup to prevent probe extension during PCR.

Fluorogenic PCRs were set up in a reaction volume of 50ml using com-ponents (except fluorogenic probes and amplification primers) supplied in aTaqMan PCR Core Reagent Kit (Perkin-Elmer, Foster City, CA). Fluorogenicprobes were custom-synthesized by PE Applied Biosystems. PCR primerswere synthesized by Life Technologies (Gaithersburg, MD). Each reactioncontained 5ml of 103 buffer A; 300 nM each amplification primer; 25 nM

corresponding fluorogenic probe; 200mM each dATP, dCTP, and dGTP; 400mM dUTP; and 1.25 units of AmpliTaq Gold. The concentrations of MgCl2

were at 2 mM for the p16M real-time MSP, 1.75 mM for the p16U real-timeMSP, and 2 mM for the p16W real-time MSP. DMSO (Merck, Darmstadt,Germany) was added at final concentrations of 5% for both the p16M andp16U real-time MSPs and 10% for the p16W real-time MSP. Fiveml of theconverted DNA were used per real-time MSP. DNA amplifications werecarried out in a 96-well reaction plate format in a PE Applied Biosystems 7700Sequence Detector (Perkin-Elmer).

Thermal cycling was initiated with a first denaturation step of 10 min at95°C. The subsequent thermal profile for the p16M and p16U real-time MSPswas 95°C for 15 s, 55°C for 30 s, and 72°C for 1 min. For the p16W real-timeMSP, the corresponding thermal profile was 95°C for 30 s, 50°C for 1 min, and72°C for 1 min. Data obtained following 40 cycles of amplification wereanalyzed. Multiple negative water blanks were included in every analysis.

A calibration curve was run in parallel with each analysis. A humanplasmacytoma cell line, HS-Sultan (ATCC CRL-1484), previously shown tohavep16methylation by methylation-sensitive restriction enzymes and South-ern blotting techniques (7) as well as by conventional MSP (6), was used forconstructing the calibration curve for the p16M real-time MSP. Serial dilutionsof HS-Sultan DNA were made in water. Similarly, peripheral blood DNA froma healthy individual previously shown to be negative for methylatedp16sequences was used for constructing the calibration curve for the p16W (priorto bisulfite conversion) and p16U (following bisulfite conversion) real-timeMSP systems. A conversion factor of 6.6 pg of DNA per diploid cell was usedfor expressing quantitative results in genome equivalents (17). One genomeequivalent was defined as the amount of a particular target sequence in a singlereference cell.

Amplification data, collected by the 7700 Sequence Detector and stored ina Macintosh computer (Apple Computer, Cupertino, CA), were then analyzedusing the Sequence Detection System software (Version 1.6.3) developed byPE Applied Biosystems.

The methylation index (%) in a sample was calculated using the followingequation:

Methylation index 5M

M 1 U3 100%

whereM is the quantity of methylatedp16 sequences measured by the p16Mreal-time MSP following bisulfite conversion andU is the quantity of un-methylatedp16 sequences measured by the p16U real-time MSP followingbisulfite conversion.

The completeness of bisulfite conversion was estimated by calculating thefractional concentration of converted DNA that remained in the wild-typesequence (%W) using the following equation:

%W 5W

W 1 M 1 U3 100%

whereW is the quantity of wild-typep16 sequences measured by the p16Wreal-time MSP following bisulfite conversion,M is the quantity of methylatedp16 sequences measured by the p16M real-time MSP following bisulfiteconversion, andU is the quantity of unmethylatedp16sequences measured bythe p16U real-time MSP following bisulfite conversion.

This calculation was performed for every real-time MSP analysis. The meanfractional concentration of p16W sequences following bisulfite treatment ofcell line DNA was 2.9% (range, 0.02–8.2%), showing that the modificationstep was largely complete.

Fig. 1. Detection of aberrant promoter methylation of thep16 gene by real-timequantitative MSP.A, amplification plot of fluorescence intensity against PCR cycle.Curves, particular input quantities (inset) of HS-Sultan DNA.X axis, the cycle number ofa quantitative PCR.Y axis,DRn (Delta Rn), which is the fluorescence intensity over thebackground (14).B, plot ofCT (Ct) against the input target quantity (common logarithmicscale). The input target quantity was expressed as genome-equivalents of HS-Sultan DNA.The correlation coefficient was 0.995.C, correlation of the measured and the actual inputmethylation indices of artificial mixtures of DNA containing methylated and unmethyl-atedp16 sequences.

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RT-PCR. Cell lines were washed in PBS and pelleted by centrifugation.The cell pellet was resuspended in 0.5 ml of guanidinium thiocyanate solution[4 M guanidinium thiocyanate, 0.5% sarkosyl, 25 mM sodium citrate (pH 7),and 0.1M 2-mercaptoethanol]. Total RNA was then extracted by a single-stepmethod as described previously (18). Twomg of total RNA were reverse-transcribed and amplified using primers for thep16 gene using conditionsreported previously (7). RT-PCR forb2-microglobulin transcripts was per-formed to check for the integrity of the RNA samples (7).

Results

Development of Real-Time Quantitative MSP.To determinethe dynamic range of real-time quantitative MSP, we preparedserial dilutions of HS-Sultan DNA containing methylatedp16sequences (6, 7); the samples were then bisulfite-converted andsubjected to analysis by the p16M real-time quantitative MSP. Fig.1A shows that the amplification curve shifted to the right as theinput target quantity was reduced. This was expected because

reactions with fewer target molecules required more amplificationcycles to produce a certain quantity of reporter molecules thanreactions with more target molecules. The system was sensitiveenough to detect down to 10 genome equivalents of methylatedp16sequence. Using wild-type and bisulfite-converted DNA from anormal individual, we showed the detection limits of the p16U andp16W real-time MSP systems to be 10 genome equivalents and 1genome equivalent, respectively.

Fig. 1B shows a plot of the threshold cycle (CT) of the p16Mreal-time MSP against the input target quantity, with the latter plottedon a common logarithmic scale. TheCT was set at 10 SDs above themean baseline fluorescence calculated from cycles 1 to 15 and wasinversely proportional to the starting target copy number (logarithmicscale) used for amplification (14). The linearity of the graph demon-strates the large dynamic range and accuracy of real-time quantitativePCR. Similar results were obtained for the p16U and p16W systems,

Fig. 2. Amplification plots of real-time MSP analysis ofcell line DNA. Red, p16M system;green, p16U system.Xaxis, cycle number of a quantitative PCR.Y axis, DRn(Delta Rn), which is the fluorescence intensity over thebackground (14).

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using bisulfite-converted and unconverted DNA, respectively, from anormal individual.

The reproducibility of bisulfite conversion followed by real-timeMSP was tested by performing six replicate bisulfite conversions ofHS-Sultan DNA (100 pg), followed by real-time quantitative p16MMSP analysis. The CV of theCT values of these replicate analyseswas 5.9%. The corresponding CV for the p16U system was deter-mined using multiple bisulfite conversions of DNA from a normalindividual and was calculated to be 5.6%. The analytical CV for thep16W system was 3.6%.

Measurement of Methylation Index in Artificial Mixtures ofMethylated and Unmethylated p16 Sequences.To validate themeasurement of the methylation index, we prepared artificial mixturesof varying proportions of bisulfite-converted HS-Sultan DNA andDNA from a normal individual and subjected them to the p16M andp16U real-time MSP systems. Fig. 1Cshows a plot of the observedagainst the actual input methylation indices. The correlation coeffi-cient was 0.983.

Real-Time Quantitative MSP on Cell Lines. DNA samples ex-tracted from eight cell lines (ARH-77, HS-Sultan, IM-9, RPMI 8226,NCI-H929, U266 B1, HeLa, and Raji) were subjected to real-timeMSP analysis using the p16M and p16U systems. The amplificationplots of these real-time MSPs are shown in Fig. 2. Only methylatedp16 sequences were detected in DNA from HS-Sultan, NCI-H929,Raji, RPMI 8226, and U266 B1. Only unmethylatedp16 sequenceswere detected in DNA from HeLa. A mixture of methylated andunmethylatedp16 alleles was detected for IM-9 and ARH-77. Con-ventional gel-based MSP was also carried out for DNA extracted fromthese cell lines and the results were completely concordant (results notshown).

The calculated methylation indices for these cell lines were asfollows: HS-Sultan, 100%; HeLa, 0%; IM-9, 0.4%; ARH-77, 78%;NCI-H929, 100%; Raji, 100%; RPMI 8226, 100%; and U266 B1,100%. RT-PCR analysis indicated that the five cell lines with amethylation index of 100% (HS-Sultan, NCI-H929, Raji, RPMI 8226,and U266 B1) did not have detectable level ofp16 transcription(results not shown). Positive RT-PCR signals, however, were ob-served for HeLa, IM-9 and ARH-77, with a signal intensity that wasinversely correlated with the methylation index (results not shown).

Correlation of Methylation Index with Transcriptional Activa-tion following Demethylating Treatment. The correlation betweenthe methylation index and the transcriptional status of thep16 gene

was studied using the cell lines HeLa, Raji, RPMI 8226, HS-Sultan,and NCI-H929. Expression of thep16 gene was monitored by RT-PCR following demethylation treatment using different concentra-tions of 5-aza-29-deoxycytidine (Fig. 3). The corresponding methyl-ation indices are shown in Fig. 3 under the corresponding lanes of thegel. A reduction in the methylation index was observed with increas-ing concentrations of 5-aza-29-deoxycytidine, indicating increasingdemethylation. The methylation index was negatively correlated withp16 transcription, withp16 mRNA detectable when the methylationindex was#85% (Fig. 3).

Quantitative MSP Analysis of Bone Marrow Aspirates fromMultiple Myeloma Patients. DNA from bone marrow aspirates from8 patients with multiple myeloma was analyzed with both conven-tional MSP and real-time quantitative MSP. Of the eight cases,methylatedp16 sequences were detected by both techniques in five.No methylatedp16 sequences were detected in the remaining threecases. As a control, unmethylatedp16 sequences were found inbisulfite-converted DNA in all eight cases. The median methylationindex in the five cases with detectable aberrantp16 methylation was0.32% (interquartile range, 0.06–0.37%).

Discussion

In this study, we have developed a novel method for quantitativemethylation analysis using real-time MSP. This method has the com-bined advantages of MSP (high specificity and sensitivity; Ref. 11)and real-time PCR (rapidity, large dynamic range, and anticontami-nation properties; Refs. 14 and 15).

In the first part of our study, we tested the real-time MSP systemsusing cell lines that were well-characterized with regard to the meth-ylation status of thep16promoter region (7). The data obtained usingthe real-time MSP systems were completely concordant with conven-tional MSP results and also with data obtained using methylation-sensitive restriction enzymes and Southern blotting (7). The real-timesystems, however, were much more rapid and were able to generateresults in a convenient 96-well format that was amenable to large-scale analysis.

Quantitative results generated by real-time MSP are dependent ona number of parameters, as follows: the efficiency of the bisulfiteconversion step, including DNA loss during the process and thecompleteness of bisulfite modification (parameter 1); the copy num-ber of methylated and unmethylated target molecules (parameter 2);and the methylation density of the primer and probe binding sites(parameter 3). Variability in parameter 1 is compensated by the use ofthe methylation index, which essentially compares methylated andunmethylated sequences that have been successfully converted. Thedependence of quantitative MSP on parameter 2 is clearly seen in Fig.1, B andC, where MSP results accurately reflect the number of inputtarget molecules. The effect of parameter 3 can be seen for the celllines HS-Sultan, RPMI 8226, U266 B1, NCI-H929, ARH-77, andIM-9, for which the methylation density has been extensively studied(7). These previous data indicate that HS-Sultan, RPMI 8226, U266B1, and NCI-H929 are more heavily methylated than ARH-77, which,in turn, is more heavily methylated than IM-9 (7). These results areconsistent with the methylation indices measured by real-time MSP,as follows: HS-Sultan, RPMI 8226, U266 B1, and NCI-H929, 100%;ARH-77, 79%; and IM-9, 0.4%.

The dependence of the methylation index on both parameters 2 and3 suggests numerous applications of real-time MSP. For example,real-time MSP may be used for the monitoring of changes in meth-ylation density during neoplastic progression (19). Another example isthe use of this technology in the quantitation of tumor-derived DNAin the plasma or serum of cancer patients (5, 6). However, in many of

Fig. 3. Correlation of the methylation index withp16 expression. RT-PCR productswere electrophoresed in an ethidium bromide-stained agarose gel.Arrow, position of thep16RT-PCR product. The names of the cell lines analyzed are shown:Lanes He, HeLa;Lanes Ra, Raji;Lanes RPMI, RPMI 8226;Lanes HS, HS-Sultan;Lanes NCI, NCI-H929;Lanes M, molecular weight marker (1-kb ladder from Life Technologies, Inc.). Thenumbers(top) denote the concentrations (mM) of 5-aza-29-deoxycytidine added. Themethylation indices for each cell line under particular conditions are shown at thebottom.

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the potential applications of quantitative MSP, it is likely that one ofthese parameters may be the predominant one that is of interest. Thesystem can be modified to be more dependent on either parameter 2or 3 by the alteration in primer or probe sequences.

Existing quantitative methods for methylation analysis include Ms-SNuPE (12) and COBRA (13). Both of these methods require post-PCRprocessing, gel electrophoresis, and the handling of radioisotopes. CO-BRA, in addition, requires the use of restriction enzymes (13). Real-timeMSP, on the other hand, does not require any post-PCR processing, isnonisotopic, and does not need restriction enzyme treatment. It is likely,however, that, for certain applications, Ms-SNuPE or COBRA may beused in conjunction with real-time MSP. For example, Ms-SNuPE can beused for the initial detailed elucidation of the methylation status ofindividual CpG sites. This information can then be used to designreal-time MSP systems that can subsequently be used for large-scalesample analysis.

The biological relevance of the methylation index, as measured byreal-time MSP, was illustrated by correlating the methylation indexwith p16 mRNA expression. Thus, the cell lines HS-Sultan, Raji,RPMI 8226, U266 B1, and NCI-H929, which all had a methylationindex of 100%, had no detectablep16 expression by RT-PCR analy-sis. In contrast, the cell lines HeLa, IM-9, and ARH-77, with meth-ylation indices of 0, 0.4, and 78%, all showedp16expression, with alevel of transcription that was inversely correlated with the methyla-tion index.

To further demonstrate the biological implication of methylationindex measurement, we applied real-time MSP to cell lines that hadbeen treated with the demethylating agent, 5-aza-29-deoxycytidine. Asexpected, treatment with the demethylating agent resulted in a reduc-tion in the methylation index (Fig. 3). Our data once again showed aninverse relationship betweenp16gene expression and the methylationindex. The highest methylation index observed in this study that wascompatible withp16 transcription was 85% (Fig. 3). We envisionreal-time MSP having a possible future application in the evaluationof novel demethylating agents, which may have potential clinicalapplication in cancer treatment.

Recently, there has been considerable interest in the use of meth-ylation analysis in the clinical detection of tumors, such as the analysisof aberrantp16 methylation in sputum (3) or bronchoalveolar lavagefluid (4) for lung cancer diagnosis and methylation analysis in theplasma/serum of lung (5) and liver cancer (6) patients. Real-time MSPwill provide a quantitative dimension for this type of analysis and mayallow one to follow the progress of patients and to assess the effect oftreatment. In addition, the suitability of real-time PCR to large-scaleapplication, due to its rapidity and resistance to carryover contamina-tion, may also catalyze the widespread use of methylation analysis inclinical practice. The establishment of real-time quantitative MSP alsoprovides a valuable tool for investigators to study the various techni-cal parameters affecting MSP, ranging from bisulfite conversion andprimer/probe design to DNA amplification conditions. This develop-ment will be very useful for optimizing protocols, which will allowthe reliable use of MSP to different clinical specimen types.

Because aberrant promoter methylation of thep16 gene has beendescribed in numerous cancers (2), the real-time MSP systems describedhere can be applied to many malignancies. Furthermore, the principlesinvolved in the development of the currentp16MSP systems can also beused to develop similar real-time quantitative systems for aberrant meth-ylation affecting other tumor suppressor genes. In addition, becausereal-time PCR has the ability to accommodate multiple fluorescent labels(20), it is possible that real-time MSP assays for multiple genes can becombined in a time-efficient multiplex format.

In this study, we have demonstrated the potential clinical utility ofreal-time MSP by applying the technique to bone marrow aspirates

from patients with multiple myeloma. Aberrant promoter methylationof thep16gene has been reported in this malignancy (21). The resultsobtained using conventional and real-time MSP were completelyconcordant. In addition, real-time MSP was able to provide an addi-tional quantitative parameter, namely, the methylation index, for thesesamples, which might be useful for the follow-up of these patients.

In addition to detecting aberrant methylation in cancer, real-timequantitative MSP may also have application for studying other bio-logical processes involving DNA methylation. For example, quanti-tative MSP would be a useful tool in the monitoring of methylationpatterns of imprinted genes during development and in the quantita-tive analysis of clonality by the measurement of X-inactivation pat-terns.

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REAL-TIME QUANTITATIVE METHYLATION-SPECIFIC PCR

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1999;59:3899-3903. Cancer Res   Y. M. Dennis Lo, Ivy H. N. Wong, Jun Zhang, et al.   Reaction Real-Time Quantitative Methylation-specific Polymerase Chain

Methylation Usingp16Quantitative Analysis of Aberrant

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