The Journal of Molecular Diagnostics, Vol. 13, No. 5, September 2011

Copyright © 2011 American Society for Investigative Pathology

and the Association for Molecular Pathology.

Published by Elsevier Inc. All rights reserved.

DOI: 10.1016/j.jmoldx.2011.05.009

Genomic Changes in Gliomas Detected UsingSingle Nucleotide Polymorphism Array inFormalin-Fixed, Paraffin-Embedded Tissue

Superior Results Compared with Microsatellite Analysis

Shuko Harada,* Lindsay B. Henderson,†

James R. Eshleman,* Christopher D. Gocke,*Peter Burger,* Constance A. Griffin,*† andDenise A.S. Batista*†‡

From the Department of Pathology,* Johns Hopkins Medical

Institutions, Baltimore; the McKusick-Nathans Institute of Genetic

Medicine,† Johns Hopkins University School of Medicine,

Baltimore; and the Cytogenetics Laboratory,‡ Kennedy Krieger

Institute, Baltimore, Maryland.

Deletion or loss of heterozygosity (LOH) in chromo-somes 1p and 19q in oligodendrogliomas (ODGs)have diagnostic, prognostic, and therapeutic implica-tions. Current clinical assays are limited because theprobes or primers interrogate only limited genomicsegments. We investigated the use of single nucleotidepolymorphism (SNP) arrays for identifying genomicchanges in gliomas from FFPE tissues. DNA was ex-tracted from FFPE tissues of 30 brain tumor cases (15ODGs and 15 non-ODGs) and assayed on the Illuminaarray with 300,000 markers. SNP results were com-pared with standard short tandem repeat (STR) assaysof chromosomes 1p and 19q. Fifteen ODGs had LOHby STR and deletion by array on both 1p and 19q. Tennon-ODGs had no evidence of LOH on 1p and 19q bySTR, seven of which had no abnormalities for thesechromosomes; three had partial deletions by SNP ar-ray. Five non-ODG cases had partial LOH or deletionby both assays. No major discordance was found be-tween SNP array and STR results. Advantages of SNParrays include no need for an accompanying normalsample, the ability to find small segmental deletions,the potential to distinguish between deletions andcopy neutral LOH, and whole-genome screening toallow discovery of new, significant loci. Assessmentof genomic changes in routine glioma specimens us-ing SNP arrays is feasible and has great potential as anaccurate clinical diagnostic test. (J Mol Diagn 2011, 13:

541–548; DOI: 10.1016/j.jmoldx.2011.05.009)

Gliomas are the most common primary tumors of thecentral nervous system in adults. The current WorldHealth Organization classification and grading system isbased on histologic features and distinguishes astrocy-tomas, oligodendrogliomas (ODGs), and oligoastrocyto-mas or mixed gliomas.1 Treatment and prognosis arelargely dependent on the diagnosis. However, classifica-tion is sometimes difficult using a histopathological ap-proach alone.

Considerable evidence suggests that acquired non-random genetic alterations could influence the diagnosis,disease progression, and response to conventional treat-ments of ODGs. Specifically, co-deletion of the short armof chromosome 1 (1p) and the long arm of chromosome19 (19q) correlates with the histologic diagnosis of clas-sic ODG, increased chemosensitivity compared with anastrocytoma of a similar grade, and improved overallsurvival.2 Other markers that are used in the evaluation ofadult malignant gliomas include methylation of the O-6methylguanine-DNA methyltransferase (MGMT) genepromoter, alterations in the epidermal growth factor re-ceptor (EGFR) pathway, and isocitrate dehydrogenase 1(IDH1) gene mutations.2 These markers are not yet testedas a standard of care, but assessment of 1p and 19qstatus is widely implemented in the neuro-oncologicalmanagement of patients with ODGs and anaplasticODGs.

Currently, deletion or loss of heterozygosity (LOH) ofchromosome arms 1p and 19q are tested either by fluo-rescence in situ hybridization (FISH) or by short tandemrepeat (STR, microsatellite) analysis.3 Both assays arelimited in that the probes or primers interrogate only smallportions of the chromosomes, and thus small deletionscan be missed or misinterpreted as involving the wholearm. Additionally, STR loci are not always informative;that is, some may be germline homozygous. In this situ-

Accepted for publication May 10, 2011.

Supplemental material for this article can be found at http://jmd.amjpathol.org or at doi: 10.1016/j.jmoldx.2011.05.009.

Address reprint requests to Denise A.S. Batista, Ph.D., Johns HopkinsUniversity, 600 N. Wolfe St., Park Bldg. SB 202, Cytogenetics Laboratory,

Baltimore, MD 21287. E-mail: dbatist1@jhmi.edu.

541

ndrogli

542 Harada et alJMD September 2011, Vol. 13, No. 5

ation, STR analysis of normal tissue from the same patient(eg, peripheral blood, buccal swab, or uninvolved braintissue) confirm the noninformativeness of the loci but donot resolve the underlying question of LOH. Also, normaltissue often is not readily available for comparison, espe-cially for consult cases. Furthermore, FISH can detectdeletions but cannot detect copy neutral (CN)-LOH, andSTR analysis cannot distinguish CN-LOH from deletions.

High-resolution array-based comparative genomic hy-bridization (aCGH) and single nucleotide polymorphism(SNP) array provide an alternative method for character-izing chromosomal alterations in ODGs and other glialtumors. Array-based CGH has been shown to be a usefulmethod for detecting deletions of 1p/19q in gliomas withthe added advantage of providing information regardingother genomic changes.4,5 A genome-wide associationstudy used SNP array to identify five susceptibility loci forglioma.6 In addition, a recent meta-analysis of genome-wide CGH data of 467 cases demonstrated World HealthOrganization grade-specific aberration profiles in astro-cytoma.7 A gain of chromosome 7 with a hot spot at 7q32was seen frequently even in low-grade astrocytomas.Analysis of pediatric low-grade astrocytomas using IlluminaHumanHap550K SNP arrays identified duplication of 7q34.8

In contrast, gains on chromosomes 7, 8q, 19q, and 20 andlosses on 9p, 10, 18q, and Xp were reported to be associ-ated with shorter overall survival in persons with oligoden-droglial tumors.9 Thus array-based technology has provedto be a powerful tool for research and has great potential tobecome a valuable clinical diagnostic application.

The results of the aforementioned studies were obtainedusing DNA isolated from fresh frozen tumor tissues. How-ever, in usual pathology practice, it is not easy to obtainfrozen tissues for array-based assays. In contrast, formalin-fixed, paraffin-embedded (FFPE) tissues are almost alwaysavailable. Oosting et al10 used Illumina BeadArray platformon FFPE colorectal tumor tissues and showed nearly iden-tical patterns of genomic change to fresh frozen tissues.Recently, successful array-based molecular karyotypingusing Affymetrix GeneChip Mapping on FFPE solid tumorsamples has been reported.11,12 However, few studieshave evaluated the performance of array-based assays onFFPE samples for brain tumors. Johnson et al13 character-ized a series of 15 matched FFPE and frozen astrocytomas

Table 1. Patients and Brain Tumor Characteristics

Variable ODG

No. of cases 15Age (mean � SEM) 48.33 � 4.47Sex 9 male, 6 femaleDiagnosis, N (%) ODG, 9 (30%)

Anaplastic ODG, 5 (16.7%)Oligosarcoma � ODG, 1 (3.3%

WHO grade 2, No. (%) 9 (30%)WHO grade 3, No. (%) 6 (70%)WHO grade 4, No. (%) NA

AC, astrocytoma; NA, not available; NS, not significant; ODG, oligode

using a customized CGH array; however, aCGH fails to

detect CN-LOH, a common mechanism of LOH in solidtumors.14 In contrast, SNP arrays allow one to determineboth the copy number status and the genotype, therebydetecting regional gains/losses and CN-LOH.

In this study, we investigated the use of SNP arrays toidentify genomic changes in gliomas using DNA ex-tracted from FFPE tissues.

Materials and Methods

Tumor Samples and DNA Extraction

This study was covered under Institutional Review Boardapproval and comprised FFPE tissue samples from 30brain tumor cases (15 ODGs and 15 non-ODGs) that hadbeen submitted for routine clinical analysis of 1p/19q lossvia our existing STR assay. All samples were obtained in2010. The mean age of the patients analyzed was 42.7years (range, 14 to 82 years). Detailed demographics aresummarized in Table 1.

H&E-stained slides from FFPE tumor samples werereviewed by a pathologist (P.B.), and tumor tissue wasselected for analysis. Corresponding tissue from two tofive unstained, 10-�m-thick tissue sections was removedusing Pinpoint reagents (ZymoResearch, Orange, CA) aspreviously described.3,15 Genomic DNA was extractedfrom the sample with use of a QIAmp DNA Kit (Qiagen,Valencia, CA) and quantified by OD at 260 nm using theNanoDrop ND-1000 (NanoDrop Technologies, Inc., Wil-mington, DE).

DNA Quality Assessment

DNA qualities were determined with use of a BioScoreScreening and Amplification Kit (Enzo Life Sciences,Plymouth Meeting, PA). The BioScore assay is an isother-mal whole-genome amplification method that can gener-ate more than 10 �g of DNA from 100 ng of high-qualitygenomic DNA. In brief, 100 ng of DNA was incubatedwith primers, nucleotide mix, and enzyme at 37°C. After 1hour, 5 �L of Stop Buffer was added to the mixture. Theresulting amplified DNA was purified with use of aQIAquick PCR Purification Kit (Qiagen), and yields weremeasured by NanoDrop (NanoDrop Technologies, Inc.)

Non-ODG P value

15 NS37.7 � 3.35 0.053

7 male, 8 female NSFibrillary AC, 3 (10%) NAAC, 2 (6.7%)Pineal parenchymal tumor of intermediate

differentiation, 1 (3.3%)Anaplastic AC, 7 (23.3%)Glioblastoma, 2 (6.7%)

5 (16.7%) NS8 (56.6%) NS2 (6.7%) NA

oma; WHO, World Health Organization.

)

and calculated. Depending on the DNA yield, DNA qual-

SNP Array of Paraffin-Embedded Gliomas 543JMD September 2011, Vol. 13, No. 5

ity was classified as poor, intermediate, good, or excel-lent (�1 �g, 1 to 3 �g, 3 to 10 �g, or �10 �g, respec-tively, as per the manufacturer’s protocol). This assaywas performed only to assess the quality of the DNA andnot to generate DNA for the SNP array. However, in someinstances SNP array was performed on paired speci-mens (nonamplified DNA and DNA amplified by Bio-Score) to assess performance.

STR (Microsatellite) Analysis of 1p/19q LOH

STR assay of chromosomes 1p and 19q was performedby multiplex PCR of five STR loci on chromosome 1(D1S199, D1S186, D1S162, D1S312, and D1S226) andthree loci on chromosome 19q (D19S918, D19S112, andD19S206), as previously described.3 Briefly, fluorescent-labeled PCR products were detected by capillary elec-trophoresis with use of the ABI 3130 Genetic Analyzerand GeneMapper software version 4 (Applied Biosys-tems, Carlsbad, CA). Interpretation was made by themolecular pathologist (C.D.G., J.R.E.) based on the ratioof the peak height (intensity) of the longer allele to theshorter allele at heterozygous loci.3

Illumina Infinium II SNP Array

DNA samples extracted from FFPE tissue (200 ng opti-mal) were run on the Illumina Infinium II SNP array with300K markers (HumanCytoSNP-12, Illumina Inc., San Di-ego, CA) without any optimization or change in the man-ufacturer’s regular protocol. For 14 cases, DNA afterwhole-genome amplification using a BioScore Screeningand Amplification Kit (Enzo Life Sciences) also was run inparallel with DNA not subject to pre-amplification. For onecase, a matched specimen from peripheral blood alsowas assayed by SNP array. The B allele frequency (BAF)and Log R ratio (LRR) data were analyzed using IlluminaKaryoStudio software version 2.0 and CNV (copy numbervariation) partition V2.4.4.0. We excluded known benigncopy number changes from our analysis. Moreover, onlychanges larger than a few megabases were considered.BAF represents the frequency of B alleles at a given SNP.LRR is the ratio between the observed and the expectedprobe intensity, thus indicating copy number. Deviation ofLRR (Log R Dev) was used to assess noisiness of the SNParray data. SNP array results were compared with our stan-dard clinical STR assay of chromosomes 1p and 19q. Ab-normalities of other chromosomes also were analyzed, andthe results were compared with pathological diagnoses.

Statistical Analysis

The �2 test and paired Student’s t-test were used toevaluate statistical significance.

Results

We analyzed SNP array data from 30 FFPE glioma spec-imens (Table 1). The quality of DNA extracted from FFPE

tissues was assessed with use of the BioScore Screening

and Amplification Kit (Enzo Life Sciences). The detailedresults are shown in Table 2. The majority of cases (90%)had excellent14 or good-quality13 DNA; three cases hadonly intermediate-quality DNA. No poor-quality cases ofDNA were found. The excellent and good-quality caseshad lower SNP array noise and lower Log R Dev com-pared with those with intermediate-quality DNA [0.34 �0.026 (mean � SEM) versus 0.45 � 0.029 versus 0.96 �0.07, respectively, P � 0.0001; see also SupplementalTable S1 at http://jmd.amjpathol.org]. Overall, DNA qual-ities correlated well with Illumina SNP array quality deter-mined by Log R Dev. The whole genome pre-amplifiedDNA samples had significantly higher deviation com-pared with matched non-pre-amplified samples (1.05 �0.33, n � 14, versus 0.44 � 0.20, n � 14; P � 0.0001). Insome cases we could not obtain the amount of DNArequired for testing per the manufacturer’s protocol andthe input on the SNP array was less than optimal, beingas low as 76 ng (optimal DNA input is 200 ng) (Table 2).The amount of DNA did not appear to affect the quality ofthe SNP array results, because there was no correlationbetween DNA input and Log R Dev. These results sug-gest that, for the Illumina platform, the quality of the DNAis a more important factor for successful SNP array anal-ysis than is DNA quantity. Nevertheless, even for the mi-croarrays with higher Log R Dev, all data were interpretablewith respect to 1p and 19q (Table 2 and Figure 1).

All 15 ODG cases showed LOH of 1p and 19q by STRtesting. By SNP array analysis, all of these cases alsoshowed abnormalities of these two regions: 13 had de-letion of both whole chromosome arms (1p and 19q)(cases 1 to 13), and one case had partial deletion ofthese chromosome arms that included all of the STR loci(1p12 to 1pter and 19q13.11 to 19qter) (case 14, Figure1A). For the last case (case 15), although it was clearlyabnormal, the SNP plots could not be unequivocally in-terpreted as deletion or CN-LOH because of the wavi-ness of the LRR. The LRR smoothed plot was close tocopy number two (ratio 0) for part of chromosome 1p butmoved slightly to the left (deletion) for 19q and part of 1q,whereas BAF was very well separated for both entirearms. Therefore interpretation of deletion or CN-LOH wasnot entirely clear. Of note, this case had several otherchromosomes with minimally shifted LRR in the presenceof a separated BAF, thereby posing a challenge to dis-cerning between deletion and CN-LOH (see Supplemen-tal Table S2 at http://jmd.amjpathol.org).

Ten non-ODG cases showed no evidence of whole-arm 1p/19q LOH by STR. For seven of these cases, theSNP array results were concordant, that is, no abnor-malities were found on 1p and 19q (cases 16 to 22). Incontrast, three cases had partial deletions of 1p and/or19q by SNP array. In two of these discordant cases(cases 23 and 24), the deletions were small and lo-cated outside of the STR loci. In the third case (case25), STR analysis showed no evidence of LOH at themost proximal 19q locus and homozygosity at the othertwo 19q loci, which was interpreted as no evidence ofLOH overall. However, for this patient, the SNP arrayshowed partial deletion of 19q, including a segment

that spans the two distal STR loci read as homozygous

ality de

544 Harada et alJMD September 2011, Vol. 13, No. 5

(19q13.32 to 19qter) (Table 2 and Figure 1C). Thiscase shows that SNP array analysis could help avoidambiguous calls.

STR analysis of the five remaining non-ODG casesshowed normal results for the loci at 1p for all caseswith the exception of one case that had LOH for thedistal three loci at 1p (case 30). For 19q, all five casesshowed LOH by STR. Array data showed that threecases (cases 26 to 28) had partial deletion of 19q andone (case 30) had partial deletion of both 1p and 19q,which was consistent with the STR results. One casethat had LOH at 19q only by STR had a small deletion

Table 2. Summary of Diagnosis, DNA and Array Parameters, SN

Caseno. Diagnosis DNA quality

DNA input(ng) L

1 ODG Excellent 2002 ODG Excellent 2003 ODG Good 1804 ODG Good 1845 ODG Good 766 ODG Good 2007 ODG Good 2008 ODG Good 2009 ODG Excellent 120

10 Anaplastic ODG Intermediate 18011 Anaplastic ODG Excellent 15612 Anaplastic ODG Good 17613 Anaplastic ODG Good 20014 Anaplastic ODG Good 20015 ODG �

oligosarcomaExcellent 116

16 Fibrillary AC Excellent 172

17 Fibrillary AC Good 200

18 Fibrillary AC Intermediate 200

19 AC Excellent 200

20 AC Intermediate 200

21 Pinealparenchymaltumor

Excellent 148

22 Anaplastic AC Excellent 200

23 Anaplastic AC Excellent 200

24 Anaplastic AC Excellent 200

25 Anaplastic AC Good 200

26 Anaplastic AC Excellent 160

27 Anaplastic AC Good 190

28 Glioblastoma Excellent 152

29 Glioblastoma Excellent 200

30 Anaplastic AC Good 192

AC, astrocytoma; del, deletion; dup, duplication; No abnl, no abnorm

on 1p detected by array and duplication of entire chro-

mosome 19 (case 29; Figure 1D). Retrospectively, theallelic imbalance we saw on STR analysis in this casewas consistent with trisomy, as the peak height ratio ofthe allele in two STR sites on chromosome 19q was 1.3(one locus was homozygous). Overall, no major discor-dance was seen between SNP array and STR results(Table 3), with the added advantage that SNP arraycan identify additional segmental deletions and dupli-cations and distinguish between deletions causingLOH and CN-LOH.

We next analyzed other chromosomal abnormalitiesdetected by SNP array (see Supplemental Table S2 at

, and STR Results for Chromosomes 1p and 19q

ev

SNP array results STR results

1p 19q 1p 19q

del 1p del 19q LOH LOHdel 1p del 19q LOH LOHdel 1p del 19q LOH LOHdel 1p del 19q LOH LOHdel 1p del 19q LOH LOHdel 1p del 19q LOH LOHdel 1p del 19q LOH LOHdel 1p del 19q LOH LOHdel 1p del 19q LOH LOHdel 1p del 19q LOH LOHdel 1p del 19q LOH LOHdel 1p del 19q LOH LOHdel 1p del 19q LOH LOHdel p12¡pter del q13.11¡qter LOH LOHdel/CN-LOH 1p del/CN-LOH 19q LOH LOH

No abnl No abnl NoLOH

NoLOH

No abnl No abnl NoLOH

NoLOH

No abnl No abnl NoLOH

NoLOH

No abnl No abnl NoLOH

NoLOH

No abnl No abnl NoLOH

NoLOH

No abnl No abnl NoLOH

NoLOH

No abnl No abnl NoLOH

NoLOH

No abnl del q13.41¡qter NoLOH

NoLOH

del p31.1p31.1 No abnl NoLOH

NoLOH

No abnl del q13.32¡qter NoLOH

NoLOH

No abnl del q13.31¡qter NoLOH

LOH

No abnl del cen¡q13.42 NoLOH

LOH

No abnl delq13.31¡q13.32;

NoLOH

LOH

del q13.32¡qterdel p35.1¡p36.11

dup entirechromosome

NoLOH

LOH

del p32.1¡pter, dupp13.3p13.3

del p12¡qter DistalLOH

LOH

tected; ODG, oligodendroglioma.

P array

og R D

0.210.450.520.550.390.570.520.500.340.980.340.350.390.410.35

0.44

0.60

1.07

0.51

0.83

0.27

0.22

0.25

0.33

0.38

0.37

0.43

0.46

0.25

0.24

http://jmd.amjpathol.org). Three cases with high Log R

SNP Array of Paraffin-Embedded Gliomas 545JMD September 2011, Vol. 13, No. 5

Dev were excluded from this analysis (cases 10, 18, and20). In general, grade 3 and 4 lesions had significantlymore abnormal chromosomes [5.17 � 0.60 in grade 2 (n �12), 9.46 � 1.28 in grade 3 (n � 13), and 9.50 � 3.50 ingrade 4 (n � 2); P � 0.007 for grade 2 versus 3 and P �0.039 for grade 2 versus 4]. The number of abnormalchromosomes in grade 2 ODGs and non-ODGs was similar[5.22 � 0.80 (n � 9) and 5.00 � 0.58 (n � 3), respectively],whereas grade 3 non-ODG had more abnormal chromo-somes than did grade 3 ODG [10.63 � 1.66 (n � 8) and 7.6 �1.94 (n � 5), respectively], although this finding was notstatistically significant (P � 0.269).

Table 3. Concordance for 1p/19q Results Between STR and SNP

SNP array results No LOH

No LOH 7Partial deletion/LOH* 3 (cases 23–25)Deletion/LOH both arms 0Total 10

*Partial deletion/LOH means that only a segment of the chromosome†One case (case 29) with 19 LOH by STR had additional deletions in‡

Array revealed del 1p12àpter and 19q13.11àqter in this case (case 14).§One case (case 15) had deletion/CN-LOH.

Distinct differences and some similarities in distributionof abnormal chromosomes were noted between the twotumor groups analyzed (Figure 2 and Table 4; see alsoSupplemental Table S2 at http://jmd.amjpathol.org). Asexpected, the chromosome 1 and 19 abnormalities wereseen more commonly in ODG (100% in ODGs versus38.5% in non-ODGs for chromosome 1 abnormalities;100% in ODGs versus 53.8% in non-ODGs for chromo-some 19). Chromosome 15 abnormalities were onlyfound in ODGs where 5 of 14 cases had a commondeleted region between bands q13.1 and q22.31. Alter-natively, only non-ODG cases had abnormalities of chro-

Figure 1. Examples of SNP array for chromo-somes 1 and 19. SNP array data with IlluminaHumanCytoSNP-12 illustrated with KaryoStudiosoftware; red data show smoothed signal intensityvalues (LRR) (Log base 2 ratio of observed andexpected intensities; LogR 0, copy number two)and blue data points represent the BAF of eachindividual SNP (B-allele frequency of 0 equals noB-allele; 1 equals only B-alleles present). For illus-tration purposes, the locations of markers used forthe STR assay were shown by the ideograms in A.A: Case 14: SNP array showed deletion of most ofbut not the entire arms of chromosomes 1p and19q, whereas STR called LOH on both arms. B:Case 15: SNP array showed abnormal 1p and 19q,as indicated by the separation of BAF. However,the wavy and minimally shifted LRR made it diffi-cult to distinguish between deletion and LOH. C:Case 25: SNP array showed deletion of the distalportion of chromosome 19q, whereas STR couldnot be interpreted clearly because of homozygos-ity. The array also showed loss of distal 1q(q43àqter) and LOH of 19p (p12àpter). D: Case 29:SNP array revealed small complex deletion on 1pand duplication of entire chromosome 19, includ-ing q arm, whereas STR results for 1p and 19qwere normal and showed LOH, respectively.

STR results

artial LOH orOH of one arm LOH both arms Total

0 0 7(cases 26–30) 1‡(case 14) 9

0 14§ 145 15 30

a change and not the entire arm.duplication in 19q by SNP array.

Array

PL

5†

showed1p and

546 Harada et alJMD September 2011, Vol. 13, No. 5

mosome 10, with 4 of 13 cases showing deletion or LOHfrom band q25.1 to the end of the long arm. Duplicationof a portion of the long arms of chromosomes 7 (partic-ularly q35 to q36.1) and 8 (q22.3 to q24.21) was seenmore frequently in non-ODG (6 of 13 verus 1 of 14 inODG, for both chromosomes). Deletion or LOH of 9p, inparticular from p21.2 to p21.3, was observed frequentlyin both groups of gliomas (8 of 13 non-ODGs and 6 of 14ODGs). Loss of the entire chromosome 14 was a relativelyfrequent finding and was seen in 4 of 14 ODG cases, but innon-ODG cases the observed changes affecting this chro-mosome were smaller in size and typically non-overlapping,and thus not showing a pattern. Deletion or LOH of 17p wasseen in approximately 80% of non-ODG cases (10 of 13cases), whereas only one ODG case had LOH of this re-gion. The type of abnormality, whether gain or loss or CN-LOH, seemed to be specific to certain chromosomes. Ex-amples of this observation include duplications onchromosomes 7q and 8q, in contrast to deletions on chro-mosome 13 and 14 and CN-LOH on chromosome 17. Ex-cluding 1p and 19q, more than half of the chromosomeabnormalities seen in ODG were due to whole chromosomechanges, whereas in non-ODG about 85% of the chromo-some changes were segmental abnormalities.

Discussion

In current clinical practice, assessment of 1p and 19q sta-tus is widely implemented in the neuro-oncological man-agement of patients with ODGs and anaplastic ODGs. Anaccurate test to assess 1p and 19q status is needed. Be-cause of the limitation of the probes or primers that interro-gate only small portions of the chromosomes, current as-says may not be comprehensive enough in some cases,especially since the genes that are being targeted by theLOH are currently unknown. Array-based CGH and SNParray are powerful tools and have been used extensively inthe diagnosis of constitutional chromosome abnormalities inpersons with developmental delay. These techniques arestarting to make their way into the realm of acquired abnor-malities found in neoplasia. In this study, we demonstratedthat assessment of genomic changes in gliomas using SNParray in FFPE samples is feasible and has great potential asan accurate clinical diagnostic test.

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

ODGNon-ODG

Case

sw

ith a

bnor

mal

ity(%

)

Chromosome #

Figure 2. Frequency (%) of abnormal cases for each chromosome.

The quality of DNA extracted from FFPE samples is animportant factor for a successful and accurate micro-array analysis. Routine extraction using the Qiagen QIAmpDNA Kit yielded good-quality DNA in 90% of our cases asassessed by the BioScore Screening and AmplificationKit (Enzo Life Sciences), which accurately predicted ar-ray performance. When the quality was “intermediate,” asobserved in three of our cases, the SNP array resultswere noisier, as indicated by a higher SD (Log R Dev).This higher deviation can hinder accurate analysis ofchromosomal changes, in particular small-sized abnor-malities, and therefore these three specimens could notbe fully analyzed. However, the SNP arrays from theseDNA samples still yielded data of sufficient quality foranalysis of whole arm changes on chromosomes 1p and19q. The amount of DNA recommended by the manufac-turer (Illumina Inc.) for input onto the Illumina SNP array is200 ng (at 50 ng/�L). Nevertheless, the SNP array hadan acceptable performance with as low as 76 ng DNA,and this suboptimal amount did not appear to affect thequality of the data, in particular the BAF, in the limitednumber of cases that we studied. These results arerather surprising and this factor is an important con-sideration because the amount of DNA extracted fromFFPE samples may be very low, especially from smallbiopsy specimens.

A few attempts to use copy number arrays with FFPEtissue have been made previously. Mohapatra et al16,17

used aCGH to analyze FFPE brain tumors. However, theirmethod included optimization with a two-step labelingprocedure using an amine-modified nucleotide for gen-eration of aCGH probes. Our method did not require anymodifications; therefore, the FFPE samples can be runsimultaneously with samples from fresh tissues such asperipheral blood or bone marrow. This point is particu-larly important because the Illumina micro-array we usedis packaged in a 12-sample-per-chip format.

Some limitations of the SNP array analysis on FFPEsamples were noted. Because of noise and waviness onLRR lines, it was difficult in some cases to distinguish

Table 4. Summary of Most Frequent ChromosomeAbnormalities Detected by SNP Array, ExcludingChromosomes 1 and 19

Chromosome andminimum shared

region Abnormality ODG Non-ODG

4p16.1pter Loss/LOH 5/14 0/135q32q33.1 Loss/LOH 1/14 4/137q35q36.1 Gain 1/14 6/138q22.3q24.21 Gain 1/14 6/139p21.2p21.3 Loss/LOH 6/14 8/13*10q25.1qter Loss/LOH 0/14 4/1313q12.11q13.2 Loss 2/14 8/1314 whole chromosome Loss 4/14 0/13†

15q13.1q22.31 Loss 5/14 0/1317p13.1p13.2 Loss/LOH 1/14 10/1318q22.3qter Loss/LOH 5/14 1/1320p12.1pter Gain 0/14 4/13

*Two of these cases had homozygous deletion.†Only small losses not affecting the whole chromosome

between deletion in a small percentage of cells and CN-

SNP Array of Paraffin-Embedded Gliomas 547JMD September 2011, Vol. 13, No. 5

LOH. One particular case (case 15) had an LRR close tozero while clearly showing four tracks for the BAF. Thissituation could indicate CN-LOH, but because of the wav-iness of the LRR we could not unambiguously interpret itand preferred to call it deletion/CN-LOH. In the nearfuture, modification of DNA extraction methods and ad-ditional optimization steps may further improve the qualityof DNA, which should result in higher performance of theSNP arrays.

In our analyses of SNP arrays we detected all of thechanges that were identified by our routine clinical testusing microsatellites (STRs), with the added advantagethat the arrays provided information for the entire arm ofchromosomes 1p and 19q instead of just the segmentscovered by the microsatellites. This information allowedus to diagnose loss of the entire 1p and 19q arms in 14 of15 ODG cases examined, but only partial loss of 1p and19q in one case (case 14).

The proposed mechanism of concurrent 1p and 19qchromosome loss in ODG is reciprocal whole-arm ex-change at the centromere forming two derivative chro-mosomes: der(1;19)(p10;q10) and der(1;19)(q10;p10).The latter derivative chromosome containing the long armof chromosome 1 and the short arm of chromosome 19 issubsequently retained, while subsequent loss of the for-mer derivative chromosome results in simultaneous lossof 1p and 19q.18 In fact, Vogazianou et al19 recentlysuggested that the breakpoints of t(1;19) translocationmay be in the pericentromeric regions of 1p and 19q, notdisrupting any known protein coding genes. However,different mechanisms may be involved in the case, withonly partial loss of 1p and 19q and with the centromeresand pericentromeric regions retained.

In addition, the SNP array detected partial deletionsand duplications on chromosomes 1 and 19 that were notidentified by STR analysis. Interestingly, one case hadtrisomy of chromosome 19 by SNP array, and it wasinterpreted as LOH by STR analysis (case 29). Both de-letion and duplication result in allelic imbalance, whichcould not be distinguished by the STR analysis, becausethis method only detects allelic imbalance. Retrospec-tively, the allelic imbalance we saw on STR analysis in thiscase was consistent with trisomy because the peakheight ratio of the allele in two STR sites on chromosome19q was 1.3 (the remaining one locus was homozygous).This case was diagnosed as glioblastoma and had ad-ditional duplications in chromosomes 7 and 20.

Three out of 10 non-ODG cases (30%) that did nothave evidence of LOH by STR analysis had partial dele-tions of either 1p or 19q. One out of five cases (20%) withLOH of some of the loci interrogated by STR was found tohave additional deletions within 1p or 19q. In these sce-narios, SNP array has the ability to accurately identifysmall or partial deletions. The clinical significance ofthese small or partial deletions is not currently under-stood. However, collecting such data with clinical corre-lation would be crucial for the discovery of genomic le-sions responsible for tumorigenesis and progression.

High-resolution genomic copy number profiling of glio-mas has been performed recently either by aCGH or SNP

array. In glioblastoma or astrocytic tumors, chromosome

7 gain, 9p deletion, and/or chromosome 10 loss are re-ported to be a characteristic pattern.20 In oligodendro-gliomas, combined 1p/19q and 19q loss were signifi-cantly associated with longer survival, and gains on 7,8q, 19q, and 20 and losses on 9p, 10, 18q, and Xq wereassociated with shorter survival.9 Loss of 1p and 19qcorrelates inversely with TP53 mutations (chromosome17p13.1), 10q deletions, and amplification of EGFR (chro-mosome 7p12).2 Others have shown a significantly short-ened survival time in patients with a glioblastoma with13q14 (RB1) deletion or 17p13.1 (TP53) deletion/LOH.21

Our study also identified frequent gains on chromosomes7 and 8q, losses on 13q, and LOH on 17p in non-ODG,especially in grade 3 and 4 tumors, whereas thesechanges were less frequent in ODG (Table 4).

In summary, we have demonstrated that assessmentof genomic changes in gliomas using Illumina SNP mi-croarrays on FFPE samples is feasible and has greatpotential as an accurate clinical diagnostic test. Smalldeletions or LOH that could be missed by the currentassays are detectable using the SNP array, which has thepotential to also distinguish between simple deletion andCN-LOH. The identification of CN-LOH as a mechanismof loss highlights the importance of using a strategy thatutilizes informative markers such as SNPs or microsatel-lites. At the same time, SNP arrays allow the identificationof additional chromosome abnormalities. The number ofcases in this study is relatively small, but a pattern ofchromosomal abnormalities starts to emerge, and ourstudy shows the potential of SNP array analysis in iden-tifying crucial loci in gliomas. The clinical significance ofthese small deletions, CN-LOH, and abnormalities onother chromosomes has yet to be determined and willrequire further studies.

Acknowledgments

We thank Katie Beierl and Amanda Stafford (MolecularDiagnostic Laboratory, Johns Hopkins University, Balti-more, MD), Janet Biscoe, Victoria Stinnett, Linjie Wo, andElizabeth Wohler (Cytogenetics Laboratory, KennedyKrieger Institute, Baltimore, MD) for their technical exper-tise, and Dr. Athanasios Tsiatis for helpful discussions.

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