Loss of heterozygosity (LOH) is a com-mon contributor to tumorigenesis, lead-ing to the loss of a wild-type allele andthe unmasking of a recessive mutation.Scans of genomic copy number (CN)can reveal LOH due to hemizygous dele-tions, but LOH can also occur independ-ently of CN change, where one chromo-some or chromosomal region has beenduplicated and its homologue has beendeleted.When LOH occurs without CNchange, it is commonly termed copy-neutral LOH.

The Affymetrix® Genome-Wide HumanSNPArray 6.0 provides industry-leadingCN detection with more than 1.8 millionmarkers, including more than 900,000SNPs for LOH identification.The abilityof Affymetrix SNP arrays to combine CNand LOH detection for the identificationof copy-neutral LOH is changing the par-adigm for analyzing chromosomalchanges in different cancer types.Without LOH detection, traditional bacte-rial artificial chromosome (BAC) or com-parative genomic hybridization (CGH)arrays from other commercial providersonly provide half of the picture.

This Application Note describes the fre-quency and relevance of copy-neutralLOH in a variety of cancer samples, andpresents a review of recent publicationsin which Affymetrix SNP arrays wereused to simultaneously study CN andLOH.These publications identify signifi-cant and common chromosomal aberra-tions that cannot be identified whenexamining CN alone.

Introduction

Cancer samples can exhibit chaoticgenomes. By definition, cancer results froman accumulation of genetic alterations thatlead a cell population from initiationthrough promotion and then progression.These genetic alterations include subtlechanges, such as small gains and losses andnucleotide substitutions, and more conspic-uous alterations, such as changes in chro-mosomal copy number (CN), translocationsand high-level amplifications. These are thecause and effect of impairments in cell cycleregulation leading to errors in replication,recombination and cell division.

Cancer research on the DNA level has his-torically emphasized CN profiling throughcytogenetic techniques and focused molecu-lar analyses, such as microsatellite PCR. Yetsignificant changes in the genome can occurwithout changes in chromosomal CN, andthe scope of molecular analyses has notoffered a feasible approach to genome-wideobservations of these events.

Copy-neutral loss of heterozygosity(LOH) represents one example of a genom-ic abnormality in which no net change inCN occurs, yet the abnormality can con-tribute to tumorigenesis. Copy-neutralLOH can occur due to duplication of onechromosome segment along with loss ofthe corresponding homologous region, sothat the cell retains two copies derivedfrom one parental source and no copiesderived from the other parental source. Theacquired homozygosity can contribute totumorigenesis by activating potential

oncogenes, unmasking mutated tumor sup-pressor genes or contributing to patho-genicity as a result of altered gene expres-sion due to imprinting.

Copy-neutral LOH events cannot bedetected when scanning cancer genomes forCN alone, but they can be detected whenviewing CN in parallel with LOH or viewingallele-specific CN. Although the majority ofcancer genome screens have focused on CNalone, recent studies combining CN andLOH detection in a single experiment, usingAffymetrix Genome-Wide Human SNPArrays, have revealed a growing list of cancertypes that present frequent and recurringcopy-neutral LOH.

This Application Note presents a collec-tion of recent articles demonstrating theimportance and relevance of copy-neutralLOH in cancer.

Publications

LOH AND FOLLICULAR LYMPHOMA

Follicular lymphoma (FL) is a common typeof non-Hodgkin’s lymphoma that originatesfrom B-cell lymphocytes and is mostly exclu-sive to adults. The majority of FL cases pres-ent a t(14;18) translocation resulting in theconstitutive over-expression of an altered Bcl-2 that blocks apoptosis. While knowledge ofgenomic changes beyond this translocationhas been limited, and prognosis based ongenomics is not yet available for this patientcohort, a few CN abnormalities have beenassociated with the disease, including del6q,del1p32-36, +7, +12 and +X.

Copy-neutral Loss ofHeterozygosity in Cancer

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Application Note

Affymetrix SNP Arrays

Charles W. Ross1 and colleagues usedAffymetrix SNP arrays2 to simultaneouslydetect CN and genotype in 46 FL sampleswith paired CD3+ T cell controls. This wasthe first time that researchers have examinedFL samples simultaneously for CN and LOHon a genome-wide level. A number of regionswere consistently affected by gains and dele-tions, as previously seen, but this experimentalso identified a number of regions presentingLOH where CN was unaffected.

On chromosome 1p, for example, aregion containing p73 and CASP9 exhibit-ed a high frequency of LOH with and with-out deletion (Figure 1). In total, LOH atchromosome 1 was observed in 50 percentof the samples, becoming the second mostfrequent genetic lesion ever described inFL. The majority of these LOH events wereexamples of copy-neutral LOH, with 15samples exhibiting no CN change andeight samples harboring deletions. Bydetecting CN alone, only the eight events,17 percent of all samples, would have beendetected, and the significance of thisregion would have been underestimated.Interestingly, chromosome 1p LOH wasthe only genetic lesion significantly over-represented in FL grade 2 as compared withgrade 1 samples, suggesting the potentialdifferential importance of LOH, includingcopy-neutral LOH, between these grades.

Chromosome 6p also exhibited a highrecurrence of copy-neutral LOH, with 30percent of FL samples presenting LOH inthe affected area. Eighty-six percent of theseevents were without CN change, indicatinga prevalence of 6p copy-neutral LOH.Interestingly, copy-neutral LOH at this siteco-occurred with LOH at chromosome 1p.

The authors comment, “Copy-neutralLOH is not detectable using either con-ventional cytogenetics or array-CGH and,therefore, has not been previouslydescribed in FL.” The novelty of thesefindings, therefore, is rooted in the factthat adequate means to detect these typesof changes had simply not been applied tothis disease type.

ALLELIC IMBALANCE AND MYELOPROLIFER-

ATIVE DISEASE

Myeloproliferative disease (MPD) is charac-terized by excess production of cells in thebone marrow. This group of diseases isdivided into four main groups: chronicmyelogenous leukemia (CML), which con-tains the Philadelphia chromosome, andthree diseases without this translocation.These include Polycythemia vera (PV),Essential thrombocytosis (ET), andMyelofibrosis (MF). A clonal mutation ofJAK2 tyrosine kinase (V617F) occurs withhigh frequency in patients with PV, ET andMF, suggesting a common pathogenesis forthe diseases that is negative for thePhiladelphia chromosome.

Go Yamamoto3 and coauthors usedAffymetrix SNP arrays4 to detect CN andLOH across a sampling of MPD cases. TheMPD samples were characterized by high

tumor heterogeneity, where CN or LOHevents may only affect a minor populationwithin the whole tumor. Because genotyp-ing, by definition, assigns a single genotypeacross the whole tumor sample, it was noteffective at detecting LOH in minor popula-tions of these MPD samples.

Instead, the authors took advantage ofallele-specific CN detection, which pro-vides a CN value for separate alleles at agiven SNP. When 100 percent of cells haveLOH, allele-specific CN values would be“0 and 2” for the two SNP alleles. Duringretention of heterozygosity, allele-specificCN values would be “1 and 1” for het-erozygous SNPs. The utility of allele-spe-cific CN in detecting LOH of mixed sam-ples is that CN values can fall betweenintegers, such that a tumor in which 50percent of the cells exhibited LOH woulddisplay allele-specific CN values of “0.5and 1.5.”

AFFYMETR IX ® PRODUCT FAM I LY > ARRAYSAFFYMETR IX ® PRODUCT FAM I LY > ARRAYS

Figure 1: Each row represents one sample; the x-axis represents chromosome position. (A)LOH is indicated by blue for 46 samples, along a stretch of chromosome 1p. (B) Copy numberis indicated on a scale from red (gain) to blue (loss) for 58 samples. CN for 46 of these samplesis aligned horizontally with LOH predictions from the same sample. Red arrows = samples pre-senting copy-neutral LOH; black arrows = samples presenting LOH with CN loss.

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In Figure 2, Yamamoto, et al. demonstrateboth the sensitivity of allele-specific CN tomixed populations and the significance ofcopy-neutral LOH events identified by thismethod. In Figures 2A and 2B, the same pri-mary acute myeloid leukemia (AML) speci-men is studied using paired (Figure 2A) orunpaired (Figure 2B) references. It is clear thatthe whole of chromosome 17 remains diploid,but copy-neutral LOH of a portion of thetumor sample is detected on chromosome17p. Because the allele-specific CN values(green and red lines) do not reach 0 and 2, butinstead fall somewhere closer to 0.8 and 1.2, itwas determined that the copy-neutral LOHevent occurred in approximately 20 percent ofthe tumor population.

Also of interest: in the pairwise compari-son (Figure 2A), less of the populationappears to display the duplication than theproportion displaying the deletion (i.e., thered line deviates from CN = 1 less than thegreen line). This is explained by the fact thata residual tumor component contains thegain but not the loss in the reference sample,which was the bone marrow sample in com-plete remission (Figure 2C).

The JAK2 gene is located on chromosome9p, a region with a high frequency of copy-neutral LOH or gain in MPD cases shown tobe JAK2mutation-positive (Figure 2D, E, F).In Figures 2D and 2F, copy-neutral LOH isdetected that encompasses the JAK2 muta-tion, and in Figure 2E, this region displays aduplication. In these examples, only the gainbut not the copy-neutral LOH events couldhave been detected without the aid of allele-specific CN. In addition, this work con-tributed to the understanding of diseasemechanism, as the authors comment that thiswork demonstrated “how strongly and effi-ciently a genetic change (point mutation)works to fix the next alteration (mitoticrecombination) in the tumor population dur-ing clonal evolution in human cancer.”

In total, the authors detected a minorcopy-neutral LOH subpopulation in 63percent of the MPD cases that were JAK2mutation positive. Interestingly, the preva-

lence of copy-neutral LOH differedbetween MPD diseases, with chromosome9p copy-neutral LOH present in 100 per-cent of PV cases and 90 percent of IMFcases, but only 27 percent of ET cases.

COPY-NEUTRAL LOH IN NEUROBLASTOMAS

Neuroblastoma comprises 6 to 10 percent ofall childhood cancers and results in 15 percent

of cancer deaths in children. The causeremains unknown, but it usually begins in theadrenal gland, or may stem from the neck,chest or spinal cord. For most patients the dis-ease has spread by the time of diagnosis.

Rani E. George5 and colleagues comparedprimary neuroblastoma samples with pairedblood from 22 children to detect somatic CNand LOH events across the genome6. LOH

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Figure 2: Allelic imbalance in AML samples (A-C) and MPD samples (D-F) were detected.For each image, the top panel of blue dots represents raw and unsmoothed CN values across thechromosome. The second panel showing a dark blue line is smoothed CN. Below the cytoband,green notches represent heterozygous SNP genotype calls and pink notches represent conflictinggenotype calls between paired tumor and normal samples. On the bottom panel, red and green linesrepresent smoothed allele-specific CN for each of two SNP alleles. The blue bar at the bottom indi-cates regions of LOH while the pink bar at the bottom indicates a gain. (A) AML sample comparedto paired normal displays copy-neutral LOH on chromosome 17p in a portion of the tumor population.(B)The same AML sample displays the same copy-neutral LOH but in an unpaired analysis to anony-mous reference samples. (C) Residual tumor component is identified in the bonemarrow sample thathad been used as the paired reference in part A. (D - F) Allelic imbalance is detected in JAK2 muta-tion-positive MPD samples. (D) 9p copy-neutral LOH was detected in approximately 20 percent ofthe population. (E) Allelic imbalance due to a duplication of 9p was detected. (F) Two discrete popu-lations displaying copy-neutral LOH were detected in a single sample, such that the majority of cellsexhibiting copy-neutral LOH had a small region affected (pink arrows) whereas copy-neutral LOHextended further down chromosome 9 for a minority of affected cells (green arrows).

was common and found to be recurring in anumber of regions, including chromosomes11q, 3p and 1p. The majority of these LOHregions were associated with a reduction inCN, indicating that homozygosity arosedue to a hemizygous deletion. Additionally,one of 15 samples exhibiting LOH on 11qlacked an associated CN change.

Chromosome 11p also displayed frequentLOH, but in this region neither deletionsnor gains were detected. Instead, for the fourneuroblastoma samples exhibiting LOH on11p, all remained diploid across the chro-mosome arm, indicating that frequent 11pLOH always occurred in the form of copy-neutral LOH for these samples (Figure 3).

The authors state that the recurringcopy-neutral LOH regions on chromo-

some 11p “may result in gain of functionof the genes in this region, as for example,IGF2, which is known to induce neurob-lastoma cell proliferation. Alternately, thetargeted gene within this region on theduplicated allele may also contain inacti-vating mutations or be suppressed by epi-genetic mechanisms, resulting in loss of

function. Further studies are required todetermine which gene or genes are mutat-ed and how the mutations that led totheir inactivation were selected for in thefirst place.” It is interesting to note thatthe mechanism of LOH for 11p and 11qis evidently different, because the com-mon 11q LOH occurred with deletionwhile the common 11p LOH consistentlyrepresented acquired copy-neutral LOH(Figure 3).

ADDITIONAL PUBLICATIONS

JUVENILE MYELOMONOCYTIC LEUKEMIA7

Juvenile myelomonocytic leukemia(JMML) cells are generally affected byderegulation of the RAS pathway throughseveral possible mechanisms. In approxi-mately 11 percent of cases, RAS hyperac-tivity is caused by inactivation of the NF1tumor suppressor gene, which resides onchromosome 17q. In Flotho, et al. (Oncogene,2007), an assortment of JMML samples wasstudied to detect common regions of allelicgains and losses.

In addition to revealing numerous spo-radic aberrations that had not been previous-ly identified for this cancer type, these exper-iments uncovered large regions of copy-neu-tral LOH on chromosome 17q in 80 percentof patient samples with the NF-1 mutation.Sequence analysis confirmed that the inacti-vating NF1 mutation was present on bothalleles in all of these cases. In contrast, copy-neutral LOH was not detected in any of thesamples lacking this mutation. The authorswrite that “these findings underscore thatisodisomy is not a coincidental observation inthe leukemic genome of patients with NF-1who develop JMML.”

CUTANEOUS SQUAMOUS CELL CARCINOMAS8

Little is known about the genomic causesof cutaneous squamous cell carcinoma(SCC), the second most commonly diag-nosed cancer type in fair-skinned popula-tions. In this study, Purdie, et al. (Genes,Chromosomes & Cancer, 2007) identifiedrecurring patterns of chromosomal alter-ations including loss, gain and LOH. Themost frequent aberration detected was thatof LOH on chromosome 9p (observed in 81percent of SCC samples).

Although copy-neutral LOH has neverbefore been detected in SCC samples, theSNP arrays revealed copy-neutral LOH at9p in 23 percent of these samples. In fact,the researchers found that copy-neutralLOH can be common across SCC samples,

AFFYMETR IX ® PRODUCT FAM I LY > ARRAYSAFFYMETR IX ® PRODUCT FAM I LY > ARRAYS

Figure 3: A total of 22 neuroblastoma tumors were characterized for both LOH (left) andCN (right) on a single SNP array. Blue = LOH; yellow = heterozygosity retained. On chromo-some 11p, four samples displayed LOH without change in CN. In contrast, on chromosome 11q,15 samples displayed LOH, 14 of which also showed an accompanying hemizygous deletion.

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“Sequence analysis confirmed that the inactivat-

ing NF1 mutation was present on both alleles in

all cases of copy-neutral LOH.”

with recurring acquired copy-neutral LOHdetected on 2q, 7q, 8p, 9p, 9q, 13, 17q and18q. The underestimation of copy-neutral

LOH frequency in SCC is a result of theabsence of previous analyses matching CNwith LOH in these sample types.

COLORECTAL CANCER CELL LINES9

In this study, Melcher, et al. studied col-orectal cancer cell lines were studied usingboth spectral karyotyping (SKY) andAffymetrix SNP arrays (Cytogenetic GenomeResearch, 2007). Both methodologies suc-cessfully identified complex chromosomalaberrations, but the SNP arrays revealedadditional and frequent copy-neutral alter-ations representing copy-neutral LOH.

Genes involved in early-acting tumor sup-pression, such as APC, CDKN2A, MLH1and MSH2, were present in recurring copy-

neutral LOH regions. In contrast, genesinvolved later in the adenoma to carcinomatransition were not affected by copy-neutralLOH These results are consistent with themodel, stating that inactivation of tumor sup-pressor genes through LOH can contribute tothe early stages of disease.

MOLECULAR ALLELOKARYOTYPING10

In a study by Kawamata, et al. (Blood,2007), molecular allelokaryotyping—eval-uating chromosomal abnormalities areevaluated with specificity to separate SNPalleles across the genotype—was performed

across a large cohort of 399 pediatric acutelymphoblastic leukemia (ALL) samples.Whole and/or partial chromosome copy-neutral LOH was observed in approximate-ly 25 percent of cases, with chromosome 9the most commonly affected chromosome.

This is the first study to indicate thatcopy-neutral LOH across chromosome 9 or9p is common in pediatric ALL. WhileJAK2 mutations, located on 9p, are com-mon in myeloproliferative disorders (MPD),this mutation is rare in ALL. Also, thesecases were negative for the commonlyknown JAK2 mutations, suggesting thatanother unidentified region serves as a basisof mechanism for the 9p copy-neutral LOHeffect in ALL.

An interesting aside: most whole-chro-mosomal copy-neutral LOH cases were con-centrated in hyperdiploid ALL samples(HD-ALL), whereas copy-neutral LOH thatoccurred across only part of the chromo-somes was generally detected in non-HD-ALL cases, probably as a result of mitoticrecombination rather than mis-segregation.

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“Copy-neutral LOH rendered tumors homozy-

gous for pre-existing mutations in genes includ-

ing CDKN2A and TP53.”

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FOLLICULAR LYMPHOMA11

Transformation to a more aggressive lym-phoma (t-FL) is common in patients withfollicular lymphoma (FL), but the mecha-nism of this transformation remains to bedefined. Fitzgibbon, et al. (Leukemia, 2007)assessed the contribution of acquired copy-neutral LOH to the transition of FL to t-FLand identified recurring regions of copy-neu-tral LOH. Sixty-five percent of LOH eventsoccurred without CN change, and theseexamples of copy-neutral LOH were presentin 88 percent of tumor samples, locating to16 different chromosomes. Sequence analysisindicated that copy-neutral LOH renderedtumors homozygous for pre-existing muta-tions in genes including CDKN2A andTP53. These results confirmed previousobservations by this group that mitoticrecombination follows mutation in thesesamples, resulting in the unmasking of themutated gene version.

UPD IN COLORECTAL CANCER12

In this analysis of CN and LOH across ade-nocarcinoma samples (Andersen, et al.,Carcinogenesis, 2007), half of the LOHevents detected occurred with deletion,whereas the remaining half representedcopy-neutral LOH, indicating copy-neutralLOH. These copy-neutral LOH regionswere focused in specific locations, includ-ing 8q, 13q and 20q. Transcriptionalanalysis of adenocarcinoma samples com-pared to normal mucosa confirmedincreased gene expression associated withCN gains, decreased expression in regionsof CN loss and no effect on gene expressionin chromosome regions displaying copy-neutral LOH. This implies that these copy-neutral LOH regions influence tumorigen-esis not through altered gene expressiondue to imprinting, but through a change inthe allelic representations of the genes.

Conclusion

This Application Note represents nine exam-ples of peer-reviewed publications high-lighting the prevalence and significance of

copy-neutral LOH. Consistently, it wasdetermined that copy-neutral LOH wascommon and recurring in a wide range ofcancer types. These LOH events sometimesaffected known genes and mutations or atother times suggested regions that maycontain novel somatic events contributingto cancer development.

In all cases, the approach of combininggenome-wide CN with genome-wide LOHdetection was necessary to understand thefull spectrum of gross chromosomal changescontributing to disease. Affymetrix SNParrays provide the ability to determine bothwhole-genome SNP genotype and CN in asingle experiment. The SNP Array 6.0combines more than 900,000 SNPs forLOH identification with an almost equalnumber of additional non-polymorphicprobes empirically selected for dose-dependence CN prediction. The result is afull 1.8 million markers for CN detection,enabling discovery of small CN changeswith high confidence and precise fine map-ping of CN breakpoints. The combinedhigh coverage of SNPs and CN probes forcombined CN and LOH detection in a sin-gle array makes the SNP Array 6.0 the idealsolution for cancer genome studies.

REFERENCES

1. Ross C. W., et al. Comprehensive Analysisof Copy Number and Allele Status IdentifiesMultiple Chromosome Defects UnderlyingFollicular Lymphoma Pathogenesis. ClinicalCancer Research 13(16) (2007).

2. Using the GeneChip® Human Mapping50K Array Xba.

3. Yamamoto G., et al. Highly SensitiveMethod for Genome-wide Detection ofAllelic Composition in Nonpaired, PrimaryTumor Specimens by Use of AffymetrixS ing l e -Nuc l eo t id e -Po l ymorph i smGenotyping Microarrays. American Journalof Human Genetics 81:114-126 (2007).

4. Using GeneChip® Human Mapping 50Kand 250K Arrays.

5. George R. E., et al. Genome-WideAnalysis of Neuroblastomas using High-Density Single Nucleotide PolymorphismArrays. PLoS ONE 2(2): e255 (2007).

6. Using GeneChip® Human Mapping 10Kand 250K Arrays.

7. Flotho C., et al. Genome-wide single-nucleotide polymorphism analysis in juve-nile myelomonocytic leukemia identifiesuniparental disomy surrounding the NF1locus in cases associated with neurofibro-matosis but not in cases with mutant RAS orPTPN11. Oncogene 1-6 (2007).

8. Purdie K. J., et al. Allelic Imbalances andMicrodeletions Affecting the PTPRD Genein Cutaneous Squamous Cell CarcinomasDetected Using Single NucleotidePolymorphism Microarray Analysis. Genes,Chromosomes & Cancer 46:661-9 (2007).

9. Melcher R., et al. SNP-Array genotypingand spectral karyotyping reveal uniparentaldisomy as early mutational event in MSS-and MSI-colorectal cancer cell lines.Cytogenetic Genome Research 118:214-21(2007).

10. Kawamata N., et al. Molecularallelokaryotyping of pediatric acute lym-phoblastic leukemias by high resolution sin-gle nucleotide polymorphism oligonu-cleotide genomic microarray. Blood (2007).

11. Fitzgibbon J., et al. Genome-wide detec-tion of recurring sites of uniparental disomyin follicular and transformed follicular lym-phoma. Leukemia 1-7 (2007).

12. Andersen C. L., et al. Frequent occur-rence of uniparental disomy in colorectalcancer. Carcinogenesis 28(1):38-48 (2007).

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NOTES

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Part No. 702615 Rev. 1

©2008 Affymetrix, Inc. All rights reserved. Affymetrix®, ®, GeneChip®, HuSNP®, GenFlex®, Flying Objective™, CustomExpress®, CustomSeq®, NetAffx®, Tools To Take You As Far As Your Vision®, The Way Ahead™, Powered byAffymetrix™, GeneChip-compatible™, and Command Console® are trademarks of Affymetrix, Inc. All other trademarks are the property of their respective owners. Array products may be covered by one or more of the following patentsand/or sold under license from Oxford Gene Technology: U.S. Patent Nos. 5,445,934; 5,700,637; 5,744,305; 5,945,334; 6,054,270; 6,140,044; 6,261,776; 6,291,183; 6,346,413; 6,399,365; 6,420,169; 6,551,817; 6,610,482; 6,733,977; and EP619 321; 373 203 and other U.S. or foreign patents.

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