Human Cancer Biology

Comprehensive Analysis of Copy Number and Allele Status Identifies Multiple Defects Underlying Follicular Lymphoma Pathogenesis Charles W. Ross,2 Peter D. Ouillette,1Chris M. Saddler,1Kerby A. Shedden,3 andSamiN.Malek1

Abstract Purpose: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin’s lym- phoma in the Western world. The clinical course is variable and only in part explained by known tumor-intrinsic or -extrinsic factors. FL carries the hallmarkchromosomal translocation t(14;18), deregulating the expression of Bcl-2, but this is not sufficient to explain either FL biology or clin- ical behavior. Experimental Design: We have employed high-density genomic profiling technology using the Affymetrix 50K-XbaI oligonucleotide single nucleotide polymorphism ^ chip platform to interro- gate the genomes of 58 fluorescence-activated cell ^ sorted (FACS) FL specimens for chromo- somal copy number changes and 46 specimens for loss of heterozygosity (LOH). Results: We report (a) previously unknown high-frequency copy-neutral LOH (uniparental dis- omy) in FL on chromosomes1p(f50%)and6p(f30%);(b) that del6q is complex, as reported, with at least two regions of minimal common loss at 6q13-15 and 6q23-24, and that in addition, f8% of FL specimens contain a homozygous deletion at 6q23.3-24.1that spans the negative NFnBregulatorA20 and the p53 apoptosis effector PERP ;(c) that combined analysis of chro- mosome17p for LOH, copy number, and p53 mutations shows that most p53 mutationsinFLdo not involve del17p. Finally, we map high-frequency LOH with and without copy loss on chromo- somes 9p, 10q, and 16p and genomic gains on 2p15-16 and 8q24.22-24.3. Conclusions: This comprehensive description of the pathologic anatomy of the FL genome uncovers novel genetic lesions and should aid with identification of relevant to FL biology and clinical behavior.

Follicular lymphoma (FL) is the most common indolent B cell The importance of tumor-extrinsic factors (infiltrating immune lymphoma and remains incurable with current therapies. FL cells) on FL biology and outcome was recently shown using has a varied clinical course. Genetically, FL is characterized by array-based expression analysis and tissue array–based immu- the hallmark balanced chromosomal translocation t(14;18), nohistochemistry (infiltrating macrophage content;refs. 4–6). leading to deregulated expression of Bcl-2 (1, 2). Transloca- Tumor-intrinsic factors, including genetic markers, for FL tion(14;18) is found in about 85% of FL cases, thus predicting biology and outcome have been less well defined, in large part the existence of additional tumor-intrinsic and -extrinsic factors due to an incomplete view of the pathologic anatomy of the FL that are important in FL biology and clinical behavior (3). genome (7, 8). Unlike other hematologic neoplasms, like acute myelogenous leukemia or chronic lymphocytic leukemia (CLL; refs. 9, 10), where genomic information is prominently used for patient risk stratification, genome-based prognostication is Authors’ Affiliations: 1Division of Hematology and Oncology, Department of Internal Medicine, Departments of 2Pathology, and 3Statistics, University of not yet available for FL patients. Michigan, Ann Arbor, Michigan Genomic changes in FL have been difficult to study Received 2/21/07; revised 4/23/07; accepted 5/18/07. comprehensively because FL cells do not grow well ex vivo. Grant support: Leukemia and Lymphoma Society of America Special Fellow Nonetheless, over the years, recurrent abnormalities have been Award and a Leukemia Research Foundation New Investigator Award. This research is supported (in part) by the NIH through the University of Michigan’s described using either conventional karyotyping or comparative Cancer Center Support Grant (5 P30 CA46592). genomic hybridization (CGH;refs. 7, 8, 11–15): del6q, The costs of publication of this article were defrayed in part by the payment of page del1p32-36, +7, +12, +X and der(18)t(14;18), as well as others. charges. This article must therefore be hereby marked advertisement in accordance Essentially, nothing is known about mutations or gene with 18 U.S.C. Section 1734 solely to indicate this fact. deregulations as part of or secondary to these changes. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Because cancer is fundamentally a genetic disease, the C.W. Ross and P.D. Ouillette contributed equally to this work. discovery of genes mutated in various forms of cancer has Requests for reprints: Sami N. Malek, Division of Hematology and Oncology, advanced our understanding of the specific pathogenetic Department of Internal Medicine, University of Michigan, 1500 E. Medical Center mechanisms that operate in various cancer cell types (16). Drive, Ann Arbor, MI 48109-0936. Phone: 734-763-2194; Fax: 734-647-9654; E-mail: [email protected]. Many cancers are heterogeneous and represent multiple F 2007 American Association for Cancer Research. biologically and clinically distinct subtypes. For most cancer doi:10.1158/1078-0432.CCR-07-0456 types, sporadic information on recurrent genetic changes has

www.aacrjournals.org 4777 Clin Cancer Res 2007;13(16) August 15, 2007 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Human Cancer Biology accumulated, but a genome-wide, high-resolution, comprehen- informative for LOH analysis, resulting in an effective median and sive view of all copy number changes or loss of heterozygosity mean inter-SNP distance for LOH searches of about 50 and 140 kb, (LOH) is emerging for just a few (17–20). respectively. Given the recent development of high-density technologies Genomic DNA sequencing. Primers to amplify exons and adjacent p53, IRF4, Blimp1, ID4 p73 to measure genome-wide changes in gene or genome copy intronic sequences of human , and and primers to amplify selected genomic regions containing informative number or allele identities in DNA extracted from tumor cells, SNPs were designed using the primer 3 program.4 Primer sequences can we reasoned that it would prove promising to interrogate the FL be found in Supplementary Table S3. PCR products were generated genomes of a large number of samples using single nucleotide using DNA templates from Qiagen Repli-g kit-amplified, fluorescence- polymorphism (SNP) chip technology (21). SNP chip technol- activated cell–sorted (FACS) CD20+ or CD3+ cells. Amplifications were ogy has the advantage of allowing for simultaneous LOH done using Taq polymerase. PCR amplicons were prepared for direct detection and copy number estimation, permitting the detec- sequencing with internal nested sequencing primers using the tion of copy-neutral LOH (22, 23). exonuclease/shrimp alkaline phosphatase method (USB). In this study, we describe an unbiased, high-density, whole- Data analysis. SNP chip data outputs from the Affymetrix genome view of LOH and copy number changes in FL. These Chromosome Copy Number Tool software were used to perform data may serve as a reference for the detection of FL-associated LOH analysis. dChipSNP, a freely available software package (26), was gene mutations and development of genomics-based clinical used to visualize copy number changes by analyzing Affymetrix.CEL files and GDAS software output data. LOH display was done using a assays in the future. Using highly purified FL preparations and software program developed for these studies in our laboratory (LOH paired T cell–derived DNA, we were able to detect novel FL- tool). The LOH tool is a Java-based genome browser that displays LOH associated genetic changes, including formerly unknown high- for entire across multiple patient samples simultaneous- frequency copy-neutral LOH at 1p and 6p, and have established ly. An executive copy of this software and instructions for use are an overview of the pathologic FL genome. available on disc by obtaining an academic license from the University of Michigan. In total, 58 patient samples (out of a total of 78 screened samples) could be analyzed for copy changes (with sufficient CD20+ Materials and Methods cell-derived DNA available) and 46 for LOH (with sufficient paired CD3+ T cell DNA available) for this study. The remaining samples did + + Source material and Internal Review Board-Medicine approval. Source not yield sufficient DNA from either CD20 cells, CD3 cells, or both material for this study was cryopreserved cell suspensions of variable for this analysis. viability representing low grade FL from 58 adult patients. The 58 sam- Mutation Surveyor (SoftGenetics LLC) software was used to compare ples had been collected between 1990 and 2005 and processed by the experimental DNA sequences from FL samples against GenBank- clinical flow cytometry laboratory at the University of Michigan. Patient derived cDNA sequences or genomic sequences, complemented by specimen sources included 52 lymph node biopsies, 4 bone marrow visual inspection of sequence chromatograms. aspirates, and 2 peripheral blood samples. Patients ranged in age from Quantitative real-time PCR analysis of genomic copy number changes. 35 to 79 years (median age 55 years), with a male-to-female ratio of 1:1. Primers and TaqMan-based probes for Q-PCR applications were pur- Subclassification of the FL by the WHO criteria was verified by a chased from Applied Biosystems (Primers-on-demand). Primer/probe hematopathologist (C.W.R.) using histopathologic, cytologic, and mixtures included Hs00751717_s1 (PERP, chr.6q), Hs00250581_s1 immunophenotypic features (24, 25). Thirty-two of the specimens (BCL11A, chr.2p), Hs01851142_s1 (RAG2, chr.11), Hs01112790_s1 were classified as FL, grade 1, and the remainder were grade 2. The (NHLRC1, chr.6p), Hs01937396_s1 (ZCCHC5, chr. X), Hs00369747_s1 research protocol was approved by the University of Michigan Medical (ACTRT2, chr.1p), Hs00272432_s1 (RSC1A1, chr.1p), and Hs002- Center Institutional Review Board (2005-0338). 52427_s1 (C8orf17, chr.8q). Triplicate amplification reactions included  Flow cytometry. Aliquots of cryopreserved, postdiagnostic FL cells primers/probes, TaqMan 2 Universal PCR Master Mix, No AmpErase were washed and recovered by centrifugation and stained with UNG, and 10 ng of unamplified genomic template DNA extracted from + phycoerythrin-conjugated anti-CD20, FITC-conjugated anti-CD3, and CD20 -sorted cells in a 20-AL reaction volume. Reactions were done on an APC-conjugated anti-CD45 (eBioscience). Propidium iodide was added ABI 7900HT machine. The ability of the assay to discriminate one from two after the final washing to a concentration of 1 Ag/mL to discriminate dead genomic allele copies was confirmed using the X chromosome probe set in + + - + - + C cells, and viable CD45 /CD20 /CD3 and CD45 /CD20 /CD3 cells were most reactions and comparing t values with the gender of individual sorted on a high-speed FACS Aria (Becton Dickinson) flow cytometer. cases. Standardization of relative copy number estimates for genomic C C Preparation of sample DNA and hybridization to Affymetrix 50k-XbaI- regions of interest was done using the D T method with the t values for mapping arrays. Sorted CD20+ or CD3+ cells were digested overnight the RAG2 locus as reference. in 100 mmol/L Tris (pH, 8.0), 50 mmol/L EDTA, 50 mmol/L NaCl, 0.5% SDS, and 100 Ag/mL of proteinase K (Invitrogen) at 56jC. DNA was extracted using phenol-chloroform and precipitated using ammo- Results nium acetate, ethanol, and glycogen. About 250 Ag of DNA was digested for 10 h at 37jC, and the samples were subsequently processed The 50K-SNP chip assay can detect single copy gains or according to the manufacturer’s (Affymetrix) instructions. Hybridiza- losses. We initially tested the ability of the 50K-SNP chip assay tion of labeled DNA fragments to the 50k-XbaI-mapping arrays was to detect single copy gains and losses in a cohort of CLL done in an Affymetrix Hybridization Oven 640 for 16 h at 48jC with patients for which clinical information was available. Using rotation at 60 rpm. Washing and staining was done on an Affymetrix data for the X chromosome for 76 CLL patients, we showed Fluidics Station 450. Scanning was done using an Affymetrix Scanner 100% sensitivity and specificity to identify males and females 3000 7G with autoloader. Data files were archived and transferred using (one versus two copies of the X chromosome;data not shown). CAB (windows cabinet) files. Next, we proceeded to analyze a collection of FACS-sorted FL Selected technical characteristics of the 50k-XbaI SNP chips used in specimens. We reasoned that FACS sorting of cells would result this study are a total combined number of f58,000 SNPs, a median intermarker distance of about 16 kb, a mean intermarker distance of about 47 kb, and an average heterozygosity of 0.29;therefore, on average, about 16,800 SNPs per individual FL genome will be 4 http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

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Fig. 1. The SNP chip assay can detect single copy differences in the FL genome. Copy number estimates for each SNP position were calculated using dChipSNPand displayed for all SNP positions for all patients for the indicated chromosomes. Blue, copy losses; red, copy gains.The physical position of SNPs is not linear along the displayed portions of the chromosome. A, chromosome X. Left, LOH analysis; right, copy number analysis.Text files generated through the use of theAffymetrix program Chromosome Copy NumberTool for all patients were imported into the LOH tool, and all individual positions of LOH between CD3+ cell-derived DNA and paired tumor CD20+ cell-derived DNA graphed as a blue tickmarkacross the length of the chromosomes. A, right, black asterisk, female patients; red asterisk, male patients. Red arrow, patients with discordant results between gender and copy number analysis. Blackarrow, patients with LOH.The estimated copy numbers for all SNP posi tionsforFL2are displayed along the entire chromosome below the X chromosome display; red line, 2N state. B, copy number analysis for SNP rs10487741in theTcell receptor g variable region locus. Top,TRGV locus schematically depicted. The estimated copy numbers for all SNP positions for FL 25 are depicted below the display; red line, 2N state. C, copy number display of SNP positions in the E light-chain locus on chromosome 22. Top, Ig-E-locus is schematically depicted. Blackarrows, cases exhibiting locus deletions. The estimated copy numbers for all SNP positions for FL 51are depicted below the display; red line, 2N state. in very highly pure FL cells and would allow for the detection of locus relative to the position of the informative SNPs of homozygous losses and single copy gains. interest. As predicted, copy number estimates for SNP In Fig. 1A, we have displayed data using the X chromosome rs10487741 uniformly showed relative gains in CD20+ FL cells analysis (LOH analysis, left;copy number analysis, right). (strong red signal) as compared with the a/h TCR-positive and Female patients (asterisks) were identified as 2N, and female TRGV-rearranged CD3+ T cells that were used as the copy patients with a loss of one X chromosome, as detected by LOH number comparator for this analysis. (black double arrows), were identified as 1N. Only 4:56 (7%, As a final semiquantitative assessment to show the ability of red single arrows) of informative samples were not, demon- the assay to detect mono- and biallelic deletions, we analyzed the strating the expected X chromosome copy number, but instead E light-chain locus on chromosome 22. In Fig. 1C, we have showed 2N and 3N estimates. Analysis of X chromosome copy displayed a screenshot of copy number estimates for all samples number estimates for the paired normal T cell DNA in three of for the IgV-E locus and flanking SNP positions. Above the the four cases gave expected results (no. 41 lacked T cell data), screenshot is a schema depicting the physical boundaries of this supporting true copy gains in the tumor cells as described (11). locus relative to the copy number estimates. As expected, We next proceeded to test the assay’s ability to detect multiple samples (black arrows) showed visible deletions (blue) amplifications using data from the analysis of the T cell of various lengths at this locus, presumably reflecting the mono- receptor g variable region (TRGV) on chromosome 7 for all or biallelic genomic losses following IgV-E rearrangements. samples (Fig. 1B). SNP rs10487741 is embedded in the TRGV In summary, these data in aggregate and Q-PCR data region and is expected to be deleted in T cells as part of T cell presented in Supplementary Table S2 provide confidence that antigen receptor rearrangements that occur during T cell single-copy losses or gains can be detected at high apparent ontogeny. In Fig. 1B, we show copy number estimates at TRGV sensitivities and specificities using the assay conditions and a schema depicting the physical boundaries of the TRGV employed in this study.

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Detection of high-frequency copy-neutral LOH at chromosome co-occurred with copy-neutral LOH at 6p (log odds ratio, 1.9; 1p in FL. Having confirmed that we could reliably detect copy 95% confidence interval, 0.6-3.1). number changes and LOH in these samples, we proceeded to Detection of novel high-frequency copy-neutral LOH at scan the FL genome for novel changes. chromosome 6p in FL. Focusing our attention on chromo- Through comparison of LOH and copy number estimates, we some 6p, we detected novel high-frequency LOH in 14:46 detected a very high frequency of copy-neutral LOH on (30%) of cases at 6p that was copy neutral in all but two cases chromosome 1p (see Fig. 2, red arrows). Additionally, LOH (Fig. 3A). Combined Q-PCR analyses and genomic SNP with copy loss was detected at 1p (see Fig. 2, black arrows). sequencing confirmed these findings (Supplementary Tables Combined, the frequency of these lesions was 23:46 (50%), the S1 and S2). second most frequent genetic lesion ever described in FL. Scanning of genes located in these areas with relevance to B Confirmatory allele copy number measurements by Q-PCR on cell or cancer physiology identified IRF4 and ID4 as potential randomly selected cases using primers/probes embedded in 1p candidates for this abnormality, but sequence analysis of all (within ACTRT2), within RAG2 (a reference gene present in coding exons failed to identify mutations in these genes. two copies in all FL genomes) and on chromosome X (see High-density mapping of del(6q) and identification of A20 and Materials and Methods and Supplementary Table S2), com- PERP as possible del(6q) candidate genes. Loss of material from bined with sequence analysis of informative SNPs in tumor and chromosome 6q has been described in non-Hodgkin lympho- paired normal DNA (PCR-based LOH analysis, Supplementary ma (NHL), and a number of nonoverlapping minimally Table S1), confirmed uniparental disomy in these cases. deleted regions have been proposed by various investigators Attempting further subclassification using anatomic criteria, over the years (28–30). Various del6q have been associated we could identify a small telomeric lesion of about 8.5 Mb in with negative prognosis in NHL and FL;nothing is known length that was part of all lesions (1p type I) and a second type about specific gene aberrations on 6q in FL (15, 31). of lesion of about 25 Mb in length (1p type II). Both lesions are We analyzed chromosome 6q by LOH and copy number fully contained in cytoband 1p36, deletion of which confers analysis in all FL samples and detected a total of 22:58 (38%) poor outcome in patients with neuroblastoma (27). positive samples (see Fig. 3A and B). Chromosome 1p lesions of types I and II harbor the p73 We readily detected at least two nonoverlapping regions of locus, and we proceeded to sequence all coding exons of p73 in loss at frequencies of 16:58 (28%) at 6q13-15 (MDR1: physical 16 FLs. We did not detect mutations, and due to the lack of boundaries, 73.69-88.07 Mb) and 13:58 (22%) at 6q23.2-24.2 fresh FL tissue, we were not able to measure the influence of the (MDR2: physical boundaries, 132.45-138.31 Mb). Additional LOH/copy loss at p73 on p73 expression. large, recurrent, contiguous regions of loss both between MDR1 Finally, 1p LOH was the only genetic lesion significantly and MDR2 and telomeric to MDR2 could be identified, overrepresented in FL grade 2, as compared with grade 1 lesions supporting prior reports that multiple genes of importance (log odds ratio, 1.37;95% confidence interval, 0.28-2.46) and may reside on 6q.

Fig. 2. Detection of high-frequency copy-neutral LOH on chromosome 1p36 in FL. A, LOH display for chromosome 1p. Vertical solid lines, 10-Mb intervals. Roman numerals and arrows, subtypes of the 1p36 region. The approximate positions of the genes p73 and CASP9 are indicated. B, copy number display for chromosome 1. The estimated copy numbers for all SNP positions for FL 60 are depicted below the chromosome 1display; red line, 2N state. Red arrows, FL patients with copy-neutral LOH on chromosome 1p; blackarrows, LOH with copy loss.

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Fig. 3. Detection of high-frequency copy-neutral LOH on chromosome 6p in FL and high-resolution mapping of del6q in FL. A, LOH display for .Vertical solid lines, 10-Mb intervals.Vertical red lines, minimal deleted regions. B, copy number display for chromosome 6. The approximate positions of various genes are indicated. The estimated copy numbers for all SNP positions for FL 26 are depicted below the chromosome 6 display; red line, 2N state. Red arrows, FL patients with copy-neutral LOH; blackarrows, samples with LOH and copy loss. Red dotted lines, boundaries of minimally deleted regions (MDR1and MDR2). C, copy number estimates for a subregion on chromosome 6q23.3-24.1. Each row represents one patient. Arrows, patients with homozygous deletions.The approximate positions of two genes, A20 and PERP, are indicated.The estimated copy numbers for all SNP positions for FL 62 are depicted below the chromosome 6 display; red line, 2N state.

Scanning the regions of loss for copy estimates indicative of expression studies were hampered by the lack of access to fresh biallelic deletions revealed five cases with presumed homozygous FL tissue. deletion at 6q23.3-24.1 within MDR2 (Fig. 3C). Mean copy Blimp1, found mutated or underexpressed in subsets of diffuse number estimates for these cases were 0.34 F 0.2 (mean F SD). large B cell lymphoma (34, 35) and located outside of MDR1 or In comparison, average copy number estimates for the entire FL MDR2 in FL, was not found to be mutated in our FL cohort. cohort for all of chromosomes 1, 2, 4, 15, 19, and 20 were 2.01 Detection of genetic changes in FL on chromosomes 16p, 9p, and F 0.19, respectively. Delineation of a minimal homozygously 10q. Completing the genome-wide survey for LOH and copy deleted region uncovered two genes, A20, a well-studied NFnB number changes, we identified three additional regions of interest: regulator (32), and PERP, a p53 apoptosis effector (33). chromosome 16p LOH (Supplementary Fig. S1) in 12:46 (26%) Confirmatory copy number Q-PCR analysis using primers/ of cases (mostly copy neutral), chromosome 9p LOH (Supple- probe with PERP confirmed progressively fewer copy number mentary Fig. S2) in 9:46 (20%) of cases (copy neutral or with copy estimates in reference cases, candidate monoallelic deleted loss) spanning the CDKN2A and CDKN2B gene loci, and cases, and candidate homozygously deleted cases (Supplemen- chromosome 10q LOH and copy loss (Supplementary Fig. S2), tary Table S2). in which the PTEN locus was included in 7:46 (15%) of cases. Sequencing of all coding exons for both genes, A20 and FL-associated 10q lesions co-occurred with 6q-MDR2 lesions PERP,inf50 FL cases failed to uncover mutations, but (log odds ratio, 2.4, 95% confidence interval, 0.7-4.1).

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Most p53 mutations in FL are not associated with structural Finally, p53 mutations co-occurred with 6q-MDR2 lesions, changes at 17p. CLL patients with del17p by fluorescence 8q24 gains, and 16p LOH (log odds ratios, 2.3, 2.3, and 2.0;95% in situ hybridization have a very poor prognosis, with an ave- confidence intervals, 0.5-4.2, 0.5-4.2 and 0.2-3.8, respectively). rage survival of about 2.5 years (9). Given the importance of The non-Hodgkin’s lymphoma-associated amplification on this cytogenetic abnormality in CLL, we proceeded to analyze chromosome 2p15-16 invariably includes BCL11A and the 17p regions in our FL cohort. REL. Hodgkin’s lymphoma and NHL, including FL, are In Fig. 4A, we have displayed LOH calls on 17p. Three pa- known to harbor a genomic amplification on chromosome tients showed LOH of about 7 to 18 Mb length at this chromo- 2p15-16 in 10% to 40% of samples (36, 37). In Fig. 5A and B, somal position, a frequency of 3:58, or about 5%. Unlike the we display copy number estimates for chromosome 2p15-16 situation in CLL, where del17p invariably deletes one p53 allele, for all 58 samples. As can be seen, 9:58 (16%) of samples one of the three FL cases (case 65) did not involve the p53 showed an amplification of varying length, and a minimally locus. In Fig. 4B, we display matching copy number data and, in amplified region could be derived spanning f600 kb (physical Fig. 4C, a p53 mutation analysis. position, 60.5-61.1 Mb on chr.2). These copy gains were It is commonly assumed that the del17p indicates that p53 is confirmed by Q-PCR with primers/probe for BCL11A (Supple- mutated on the retained allele. We decided to test this mentary Table S2). Interestingly, even this relatively small relationship directly and found 6:58 (10%) patients with consensus region contained both BCL11A and REL, providing mutations in p53 (exons 5-9), including 4 patients with support to the hypothesis that both genes may play a missense mutations and 2 patients with frameshifts. pathogenetic role in FL (38). Interestingly, del17p did not predict for mutated p53, as only Delineation of a minimal region of amplification on chromo- 1:6 cases (FL 67) had both a del17p involving the p53 gene and some 8q. A genome-wide review of copy number data showed a p53 mutation on the retained allele. Most p53 mutations frequent low-level amplifications of all or parts of chromo- occurred without notable chromosomal alterations at 17p. some 8 in 10:58 (17%) of the specimens (see Fig. 6). Further

Fig. 4. Comparative analysis of chromosome 17pLOH, copy number estimates, and p53 mutation analysis. A, LOH display for chromosome 17p.Vertical solid lines, 10-Mb intervals.Vertical arrow, location of the p53 gene at 17p13.1. B, copy number display for chromosome 17p.Vertical arrow, location of the p53 gene at 17p13.1.The estimated copy numbers for all SNP positions for FL 67 are shown below the chromosome17pdisplay; red line, 2N state. C, p53 mutation analysis for exons 5 to 9. Column 1, sample numbers. Column 2, nucleotide and amino acid residue for that position in the cDNA NM_000546 or genomic DNA (same result); column 3, nucleotide and amino acid residue for that position in FACS-sorted CD20+ cells. Red, p53 mutations without 17pLOH or copy loss; green, 17pLOH and copy loss without p53 mutations; purple, 17p LOH without p53 mutations; black, 17pLOH and copy loss with p53 mutations; blue, 17p copy loss and p53 mutation data not available.

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Fig. 5. The lymphoma-associated amplification on chromosome 2p15-16 invariably includes BCL11A and REL. A, genomic copy number display for parts of chromosome 2. The physical positions of BCL11A and REL are indicated. Arrows, samples with amplifications.The estimated copy numbers for all SNP positions for FL 53 are displayed below the chromosome 2 display; red line, 2N state. B, genomic copy number display for a subregion of chromosome 2p15-16, with a schema of the BCL11A and REL loci in relation to individual SNP positions. analysis narrowed a region of minimal amplification to This analysis suggests (a) that chromosome 18 may harbor 8q24.22-8q24.23 (physical boundaries 134.43-telomere at an additional, amplified gene with relevance in FL biology f144 Mb), thereby excluding the Myc locus, which has located on der(18)t(14;18) other than Bcl-2;and ( b) that a previously been suspected as being the primary target of 8q significant fraction of additional FL cases harbor trisomy 18, amplifications. These copy gains were confirmed by Q-PCR suggesting a mechanism to explain increased Bcl-2 expression with primers/probe for C8orf17 (Supplementary Table S2). independent of t(14;18) (43). Scanning of 8q24.22-8q24.23 identified a minimum of four genes that have previously been implicated in tumor Discussion biology, including the potassium channel KCNK9 (39), the NFnB-activating NIBP (40), the protein tyrosine In this study we are presenting high-density genomic kinase PTK2/FAK (41), and the protein tyrosine phosphatase profiling data, including LOH and copy number changes, for PTP4A3 (42). a series of FL specimens. These data have implications for basic Amplifications of der(18)t(14;18) do not involve Bcl-2. The as well as clinical research in FL, are novel in nature, and have genetic hallmark lesion of FL is the translocation t(14;18), not been reported for this important malignancy. juxtaposing the Bcl-2 gene and the immunoglobulin heavy- We have described novel, high-frequency regions of LOH and chain locus enhancer elements. Fine mapping of the break- copy loss as well as copy-neutral LOH (uniparental disomy) on points showed that the part of chromosome 18 that did not multiple chromosomes in the FL genome. Copy-neutral LOH is contain Bcl-2 was present in three copies as der(18)t(14;18) not detectable using either conventional cytogenetics or array- (11), as previously reported (43), whereas the remainder of this CGH and, therefore, has not been previously described in FL. chromosome was disomic (Supplementary Fig. S3A and B). The occurrence of alterations at 1p36 in about half of all FL Trisomy 18 (black arrows) was detected as well. may be of clinical significance because a similar region has been

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Fig. 6. Identification of a minimally amplified region on chromosome 8q24.22-23 in FL that excludes Myc.The boundaries of a minimal amplified region on chromosome 8q24.22-24.3 are indicated. The estimated copy numbers for all SNP positions for FL 67 are displayed below the chromosome 8 display; red line, 2N state.

shown to confer poor prognosis for children with neuroblas- patients with various cancers (48, 49). Both genes are attractive toma (27). It certainly makes analysis of the impact of this targets for future studies on well-preserved FL tissue. lesion on FL outcome a research priority. The data presented on the well-known amplification on Chromosome 6p LOH spans the IRF4 locus. This may be chromosome 2p15-16 show that BCL11A and REL are always significant to FL pathogenesis because others have recently coamplified. BCL11A is a Krueppel zinc finger transcription shown that the transcriptional regulator IRF4 is necessary for B factor essential for pre–B cell development, with restricted cell germinal center exit (44). expression in tissues;it should be investigated further for the Deletion 6q has been reported to confer negative prognostic role it assumes in malignant B cell physiology (9, 50). impact on patients with NHL, but a critical gene has not been The analysis of a minimally amplified region on chromo- identified in any of the studied regions. some 8q24 excludes Myc as the critically relevant gene and Analysis of a novel homozygously deleted region at 6q23.3- suggests a short list of genes that all have proven roles in cancer 24.1 identifies A20 and PERP as two novel genes of potential cell properties or cell proliferation. interest in FL biology. Combined analysis of chromosome arm 17p for LOH, copy A20 is a negative regulator of NFnB and Toll-like receptor loss, and p53 mutations shows that most p53 mutations in FL signaling (32). Studies in multiple cellular and murine systems are not associated with chromosomal alterations at 17p, and suggest that reduction in A20 function results in sustained that the development of clinically useful tests based on NFnB signaling, a molecular feature of many NHL subtypes genomic profiling should include p53 sequence analysis. (45). A20 knock-out animals develop a lethal inflammatory In summary, this large genomic profiling study extends syndrome and die shortly after birth, precluding possible knowledge about the FL genome and suggests basic and clinical observation of tumor proneness (46). research implications. The high-resolution mapping data PERP, a gene induced by p53, executes part of the p53 highlight novel chromosomal regions of potential importance apoptosis program. Radiation-induced apoptosis in thymocytes to FL biology and candidate genes worthy of further analysis. or neuronal cells is impaired in PERP-/- mice (47). Down- The clinical importance of these findings awaits testing of regulation of PERP has been implicated in the poor outcome of various genetic FL subgroups for prognostic power.

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Charles W. Ross, Peter D. Ouillette, Chris M. Saddler, et al.

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