Regions of acquired uniparental disomy at diagnosis of follicular lymphoma are associated with both...

doi:10.1182/blood-2008-08-174953Prepublished online January 13, 2009;

Andrew Lister and Jude FitzgibbonA. Johnson, Elias Campo, Wing C. Chan, Randy D. Gascoyne, Bryan D. Young, Louis M. Staudt, T.Gribben, Andreas Rosenwald, German Ott, Lisa M. Rimsza, Harald Holte, Jean-Baptiste Cazier, Nathalie Derville O'Shea, Ciaran O'Riain, Manu Gupta, Rachel Waters, Youwen Yang, David Wrench, John transformationlymphoma are associated with both overall survival and risk of Regions of acquired uniparental disomy at diagnosis of follicular

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1

Regions of acquired Uniparental Disomy at diagnosis of Follicular Lymphoma

are associated with both overall survival and risk of transformation

Derville O’Shea1, Ciarán O’Riain1, Manu Gupta1, Rachel Waters2, Youwen Yang1, David

Wrench1, John Gribben1, Andreas Rosenwald3, German Ott3, 4, Lisa M. Rimsza5, Harald Holte6,

Jean-Baptiste Cazier1,7, Nathalie A. Johnson8, Elias Campo9, Wing C. Chan10, Randy D.

Gascoyne1, Bryan D Young1, Louis M. Staudt11, T. Andrew Lister1 and Jude Fitzgibbon1.

1Centre for Medical Oncology, Barts and the London School of Medicine, London, UK; 2Centre

for Statistics in Medicine, Oxford University, Oxford, UK; 3Institute of Pathology, University of

Würzburg; 4Department of Clinical Pathology, Robert-Bosch-Krankenhaus, Stuttgart, Germany;

5Department of Pathology, and Arizona Cancer Center, University of Arizona, Tucson, AZ

85724; 6Department of Oncology, Cancer Clinic Norwgian Radium Hospital, Rikshospitalet,

Oslo, Norway; 7Bioinformatics and Biostatistics, Cancer Research UK, London WC2A 3PX, UK

8Department of Pathology and Division of Medical Oncology, British Columbia Cancer Agency,

Vancouver, British Columbia, Canada; 9Department of Pathology and Hospital Clinic, Institut

d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Spain;

10Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha,

NE, USA. 11Metabolism Branch, Center for Cancer Research, National Cancer Institute, National

Institutes of Health, Bethesda, MD, USA.

Short title: aUPD at diagnosis in Follicular Lymphoma

Blood First Edition Paper, prepublished online January 13, 2009; DOI 10.1182/blood-2008-08-174953

Copyright © 2009 American Society of Hematology




2

Corresponding author: Derville O’Shea, Centre for Medical Oncology, Barts and the London

School of Medicine, Charterhouse Square, London EC1M6BQ, UK,

Phone: 00442078826009;

Fax 00442078826004;

email: [email protected]




3

Abstract

Acquired homozygosity in the form of segmental acquired uniparental disomy (aUPD) has been

described in follicular lymphoma (FL) and is usually due to mitotic recombination. SNP array

analysis was performed using the Affymetrix 10K 2.0 Gene-chip array on DNA from 185

diagnostic FL patients to assess the prognostic relevance of aUPD. Genetic abnormalities were

detected in 118/182 (65%) patients. Number of abnormalities was predictive of outcome; > 3

abnormalities was associated with inferior overall survival (OS) (p<0.03). Sites of recurrent aUPD

were detected on 6p (n=25), 16p (n=22), 12q (n=17), 1p36 (n=14), 10q (n=8) and 6q (n=8). On

multivariate analysis aUPD 1p36 correlated with shorter OS (p=0.05). aUPD 16p was predictive of

transformation (p=0.03) and correlated with poorer progression free survival (p=0.02). aUPD is

frequent at diagnosis of FL and impacts on probability of disease transformation and clinical

outcome.




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Introduction

Follicular Lymphoma (FL) is the most common indolent lymphoma and has marked

heterogeneity in outcome,1-3 with frequent transformation to aggressive lymphoma impacting

dramatically on overall survival (OS).4,5 The genomic hallmark of FL is t(14;18)(q32;q21) with

many secondary abnormalities described, 6,7 although few have confirmed prognostic value 6,8,9

and the target genes remain largely unidentified.

Single nucleotide polymorphism (SNP) arrays allow detection of copy neutral loss of

heterozygosity (LOH) undetectable by previous methodology, termed acquired uniparental

disomy (aUPD). We and others have described aUPD in several hematological malignancies10,11

including FL.12,13 It is due to mitotic recombination or non-disjunction and may render a cell

homozygous for a pre-existing abnormality leading to clonal selection. Both FL series profiled to

date have shown frequent, non-random regions of aUPD; however, its prognostic implication has

not been addressed. We aimed to clarify the incidence of aUPD in 185 diagnostic FL patients

and correlate this with clinical outcome.

Patients, materials and methods

Patient Information

Tumor extracted DNA from 185 untreated patients with FL presenting between 1974 and 2001

was obtained through the Lymphoma/Leukemia Molecular Profiling Project (LLMPP). Clinical

data was available in 169/182 cases (93%) cases (Table S1), transformation data was available




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for 141 cases and 39 patients transformed to aggressive lymphoma. Research Ethics Committee

approval was obtained from the London School of Medicine before initiation of the study.

10K GeneChip assay

SNP array genotyping was performed using the 10K 2.0 GeneChip (Affymetrix Inc., Santa

Clara, CA) as previously described.14

Signal intensity data were analyzed by GeneChip DNA analysis software and GTYPE was used

for calling genotypes. GOLF,an in-house software was used for copy number (CN) and UPD

estimation, (http://bioinformatics.cancerresearchuk.org/~cazier01/). In the absence of germ-line

controls the definition of aUPD was based on runs of consecutive homozygous markers defined as

maximum two heterozygous calls in 50 consecutive SNPs. This was described by Gupta et al and

briefly this stringent criterion is based on 10K 2.0 data available from 90 independent germ-line

samples from the CORGI study consortium16 giving a false positive rate of 3.3%.15 CN was

determined based on log2 ratio of signal intensity from the lymphoma sample versus the pooled

signal intensity of 10 unrelated control DNA samples. A ratio below 0.75 and above 1.25 on at

least three consecutive SNPs was defined as a loss and a gain respectively. The data was also

analyzed using Partek Genomics Suite (Partek Incorporated, St Louis, MO). Sex chromosomes

were excluded from analysis. SNP and gene annotations used NCBI genome build 35.

Mutation analysis

Mutation screening was performed for the coding region of ID4, BRD2, HLA DQB1, SOCS1,

CDK4, DYRK2, PTEN and specific exons of NFKB2 (Table S2) (protocols available on request).




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Statistical analysis

Fisher’s exact test and Mann-Whitney U test were used to determine association with clinical

characteristics. OS was defined as the time from diagnosis to death, or for patients remaining

alive, the time from diagnosis to last contact. Progression free survival (PFS) was defined as the

time from diagnosis to first clinical progression, transformation or death from any cause, or for

patients remaining alive and disease free, the time from diagnosis to last contact. Transformation

to aggressive lymphoma was defined histologically or by clinical criteria. Kaplan-Meier survival

estimates were obtained and the logrank test used to compare differences between the groups

with UPD versus those without. Multivariate Cox regression was used to determine if regions of

recurrent abnormality remained independently predictive of PFS and OS after adjusting for the

International Prognostic Index (IPI). Statistical significance was set at p<0.05.

Results and Discussion

This study of 185 diagnostic FL cases addresses the clinical impact of aUPD using the well

characterized LLMPP cases.17 It confirms earlier reports that aUPD is frequent at diagnosis of FL,

occurs non-randomly at recurring chromosomal locations 12,13 and now shows that aUPD is

clinically important. SNP genotype call rates were > 90% for 182/185 cases analyzed.

Abnormalities including CN and copy neutral changes were detected in 118/182 (65%) cases

(Figure 1). The number of abnormalities ranged from range 0-9 (median 1) with median LOH size

of 48 Mb (range 3.4 -241 Mb). The 20 cases with > 3 abnormalities at diagnosis had inferior OS

(p=0.03) compared to those with ≤ 3 abnormalities (n=149) (Figure 2A). aUPD was seen in 96/118

(81%) abnormal cases. Sites of recurrent aUPD were detected on chromosome 6p (n=25), 16p




7

(n=22), 1p (n=16), 12q (n=17), 10q (n=8) and 6q (n=8). aUPD6p was observed in 25/182 (14%)

cases (Table S3) and as shown previously,13 aUPD6p was associated with aUPD1p (p=0.002).

aUPD16p occurred in 22/182 (12%) of cases (Table S4) and 10/22 cases transformed to aggressive

lymphoma (p<0.03).

aUPD16p correlated with poorer PFS (p=0.01) (Figure 2B) but not OS and remained significant

after adjusting for IPI (Hazard ratio (HR) 1.80 (CI 1.09-2.98), p=0.02). The association of

aUPD16p with transformation (p=0.03) represents a potential new risk factor for this event with a

trend seen towards shorter time to transformation with aUPD16p (HR 1.94 (CI 0.92-4.13) p=0.08).

Loss of 1p36 has been previously associated with poorer OS in FL.6 aUPD1p was present in

16/182 (9%) cases and analysis showed that patients with aUPD1p36 (n=14) had inferior OS

(p=0.01) (Figure 2C) and PFS (logrank p=0.05). On multivariate analysis aUPD1p36 correlated

with poorer OS after adjusting for IPI (HR 2.10 (CI 1.00-4.43), p=0.05) but not for PFS (HR 1.70

(CI 0.90-3.18) p=0.10). aUPD12q was present in 17/182 (9%) cases, 10q abnormalities, (loss

(n=9) and aUPD (n=8)) were seen in 17/182 (9%) cases and chromosome 6q abnormalities were

identified in 21/182 (11%) cases (loss (n=13) and aUPD (n=8)). Loss of 6q and inferior outcome

has been shown previously in FL;9 no significant associations were seen here for OS, PFS or

transformation with 6p, 6q, 10q or 12q abnormalities. Recurrent regions of gain were identified on

chromosome 2p (involving REL), chromosome 7, 8q, 12q (involving MDM2) and 18q, areas of

gain previously described in FL.18-20 No statistically significant associations were seen here with

clinical outcome. The regions of aUPD, copy number loss and gain were verified using Partek

Genomic Suite.

The recurring regions of abnormality seen here demonstrated considerable overlap with previous

analysis using cell-sorted FL samples, but the frequency of abnormalities was higher13 suggesting

that in FL purified tumor populations are preferred for accurate genotyping as DNA from non-

tumor cells will interfere with the detection of abnormalities. It is probable that some of the




8

“normal” cases in this study may have low malignant cell content, below the threshold of detection

of this array. Identification of gene lesions made homozygous has proven difficult in FL, in this

series none of the target genes screened were mutated (Table S1) and it is probable that other

selective mechanisms are important.21 The regions identified here were invariably large pointing

towards clonal events but this may be an underestimate and comparison of tumor and germ-line

DNA is required to distinguish smaller somatic UPD from inherited events.22 This study focuses

on recurrent regions of LOH making it less likely to detect a rarely reported constitutional

abnormality.23 The use of higher resolution platforms may identify cases with more localized

aberrations suitable for further characterization by next-generation sequencing.

Authorship

Contribution: D.O. designed the study, analyzed data and wrote the paper; D.O., C.O., Y.Y., and

D.W. performed research; M.G., R.W., J.G., B.D.Y., and J.C. analyzed data; A.R., G.O., L.R.,

H.H., N.J., E.C., W.C.C. and R.D.G. collected data; L.M.S., T.A.L. and J.F. designed the study

and wrote the paper.

Conflict of interest disclosure: The authors declare no competing financial interests.

Acknowledgments The work was supported by grants from Cancer Research UK, the Medical Research Council and

the NIH SPEC grant 5UO1CA114778. D.O. is supported by a Medical Research Council

Clinical Research Fellow grant. C.O. is a Cancer Research UK Barts-Cambridge Molecular

Pathology Clinical Research Fellow.




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18. Schwaenen C, Viardot A, Berger H, et al. Microarray-based genomic profiling reveals novel genomic aberrations in follicular lymphoma which associate with patient survival and gene expression status. Genes Chromosomes Cancer. 2009;48:39-54. 19. Cheung KJ, Shah SP, Steidl C, et al. Genome-wide profiling of follicular lymphoma by array comparative genomic hybridization reveals prognostically significant DNA copy number imbalances. Blood. 2008. 20. Viardot A, Barth TF, Moller P, Dohner H, Bentz M. Cytogenetic evolution of follicular lymphoma. Semin Cancer Biol. 2003;13:183-190. 21. Milani L, Gupta M, Andersen M, et al. Allelic imbalance in gene expression as a guide to cis-acting regulatory single nucleotide polymorphisms in cancer cells. Nucleic Acids Res. 2007;35:e34. 22. Gondek LP, Tiu R, O'Keefe CL, Sekeres MA, Theil KS, Maciejewski JP. Chromosomal lesions and uniparental disomy detected by SNP arrays in MDS, MDS/MPD, and MDS-derived AML. Blood. 2008;111:1534-1542. 23. O'Keefe CL, Gondek LP, Tiu R, et al. Identification of chromosomal abnormalities in healthy bone marrow using 250K SNP arrays. Blood 2006;108:Abstract [2076]. 24. Neri A, Fracchiolla NS, Migliazza A, Trecca D, Lombardi L. The involvement of the candidate proto-oncogene NFKB2/lyt-10 in lymphoid malignancies. Leuk Lymphoma. 1996;23:43-48.




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Figure 1 : Global view of LOH including aUPDs, losses and gains in 182 FL samples across the 22 autosomes using GOLF. Aberrations (gain in red, loss in green and aUPD in blue) for individual samples are shown to the right of the chromosome.




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Figure 2: Survival (a) Overall survival by number of abnormalities (b) Progression free survival by aUPD 16p (c) Overall survival by aUPD 1p36

0 10 20 300

20

40

60

80

100

Logrank p < 0.03

</= 3 abnormalities (n=149)

> 3 abnormalities (n=20)

Time (years)

Aliv

e (%

)

0 5 10 15 20 250

20

40

60

80

100

Logrank p = 0.01

a UPD 16p (n=21)

Non 16p UPD (n=148)

Time (years)

Pro

gre

ssio

n (%

)

0 10 20 300

20

40

60

80

100Logrank p = 0.01

1p36 UPD (n=11)

Non 1p36 UPD (n=158)

Time (years)

Aliv

e (%

)




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