Genome-Wide Detection of Recurring Sites of Uniparental Disomy in Follicular and Transformed Follicular Lymphoma

Genome-wide detection of recurring sites of uniparental disomy in follicular and transformed follicular lymphoma

J Fitzgibbon1, S Iqbal1, A Davies1, D O’Shea1, E Carlotti1, T Chaplin1, J Matthews1, M Raghavan1, A Norton2, TA Lister1 and BD Young1

1Cancer Research UK, Centre for Medical Oncology, Barts and the London School of Medicine and Dentistry, London, UK and 2Department of Histopathology, St Bartholomew’s Hospital, London, UK

Single-nucleotide polymorphism (SNP) array analysis was biopsy samples has been used to determine the critical copy performed using the 10K GeneChip array on a series of 26 number changes in these tumours.10–13 More recently, array- paired follicular lymphoma (FL) and transformed-FL (t-FL) biopsies and the lymphoma cell lines SCI-1, DoHH2 and based high-density genotype analysis has become available. The RL2261. Regions of acquired homozygosity were detected in application of this approach to other malignancies has not only 43/52 (83%) primary specimens with a mean of 1.7 and 3.0 revealed copy number changes but has also uncovered the aberrations in the FL and t-FL, respectively. A notable feature presence of large regions of homozygosity in the absence of copy was the occurrence of recurring sites of acquired uniparental number change.14–18 Such regions, which have been termed disomy (aUDP) on 6p, 9p, 12q and 17p in cell lines and primary acquired uniparental disomy (aUPD) or isodisomy, appear to be samples. Homozygosity of 9p and 17p arose predominantly in due to mitotic recombination between the two chromosomal t-FL and in three cases rendered the cell homozygous for a pre- 19 existing mutation of either CDKN2A or TP53. These data homologues and are undetectable by CGH array technology. suggest that mutation precedes mitotic recombination, which These events can result in the selection of daughter cells made leads to the removal of the remaining wild-type allele. In all, 18 homozygous for a pre-existing mutation.20–24 We have used cases exhibited abnormalities in both FL and t-FL samples. In single nucleotide polymorphism (SNP) array technology to 10 cases blocks of homozygosity were detected in FL that were investigate regions of loss of heterozygosity in the absence of absent in the subsequent t-FL sample. These differences support the notion that FL and t-FL may arise in a proportion copy number change in order to establish the contribution of of patients by divergence from a common malignant ancestor these abnormalities to the evolution of FL and t-FL. cell rather than by clonal evolution from an antecedent FL. Leukemia (2007) 21, 1514–1520; doi:10.1038/sj.leu.2404696; published online 10 May 2007 Materials and methods Keywords: single-nucleotide polymorphisms; uniparental disomy; follicular lymphoma; transformation Tissue samples Fifty-two tumour specimens derived from 26 patients with FL that subsequently transformed to diffuse large B-cell lymphoma, referred to as t-FL, were identified from the tissue archive at St Bartholomew’s Hospital, London. These patients formed part Introduction of a tumour panel of 29 patients reported previously and the patient numbering has been maintained.25 Patients 3, 14 and 29 Follicular lymphoma (FL) is the second most common B-cell were excluded from analysis as DNA was limited. In the malignancy representing one-quarter of non-Hodgkin’s lympho- remaining 26 patients (median 45 years (22–70)), transformation mas (NHL). Although the disease has a relatively long median occurred a median of 3.8 years (range 1–14.6 to years) after 1,2 survival of 8–10 years and patients have initially a high rate of presentation, with patients having received a median of 3 (0–13) response, the illness follows a fluctuating course of progression therapies before transformation. Median follow-up from diag- punctuated by remissions of variable duration. For as many as nosis for all patients at the time of analysis was 16 years (range half of patients, transformation to a more aggressive lymphoma 2–21 years). The median age at transformation was 52 (range may occur, usually diffuse large B-cell lymphoma transformed- 27–73) years. Presentation lymph nodes were analysed in 10/26 FL (t-FL), and this event is often associated with a particularly patients (Patients 6, 7, 9, 12, 17, 19, 20 and 23–25). The 3–6 poor response to treatment. Although up to 85% of FL cases t(14;18)(q32;q21) cell lines DoHH2, RL2261 and SCI-1 were are characterized by the t(14;18)(q32;q21) translocation, clin- also included for analysis. Local Research Ethics Committee ical heterogeneity is reflected in a complex accumulation of approval was obtained before initiation of the study. secondary genetic events within these tumours,7–9 many of which are present in only a subset of specimens.9 The mechanism of transformation and the relationship 10K 2.0 GeneChip assay between the two disease stages remain obscure. Comparative Genomic DNA was extracted from snap-frozen lymph nodes by genomic hybridization (CGH) analysis of sequential FL and t-FL conventional methods. DNA probes were prepared using the GeneChip mapping assay protocol (Affymetrix Inc., Santa Clara, Correspondence: Dr J Fitzgibbon, Cancer Research UK Department of CA, USA) with the modification that PCR products were purified Medical Oncology, Barts and the London School of Medicine and using the Ultrafree-MC filtration column (Millipore, Billerica, Dentistry, Charterhouse Square, London EC1M 6BQ, UK. E-mail: jude.fi[email protected] MA, USA). Signal intensity data were analysed by the GeneChip Received 30 September 2006; revised 27 February 2007; accepted 12 DNA analysis software, which uses a model algorithm to March 2007; published online 10 May 2007 generate SNP calls. Regions of alteration were defined by the aUPD in follicular lymphoma J Fitzgibbon et al 1515 presence of 496% homozygosity in at least 50 contiguous SNPs Results using a custom designed software, genome oriented laboratory file (GOLF). Information on GOLF is available on request from Pattern of abnormalities in paired FL and t-FL biopsies Professor Bryan D Young, (email: [email protected]) SNP genotype arrays were used to analyse homozygosity and software package and guidelines for use of the GOLF patterns in sequential samples obtained from 26 patients who Programme are accessible at http://www.cancer.qmul.ac.uk/ had at least one episode of FL and t-FL. For most of these paired staff/young.html. The copy number ratio was calculated against samples, germline DNA was unavailable. It was therefore a combination profile of six germline DNA samples (Supple- necessary to establish a criterion for the detection of abnormal mentary Information). runs of homozygosity. The presence of 496% homozygosity in at least 50 contiguous SNPs was found to detect no abnormalities in DNA of 23 remission bone marrows (data not shown) and was therefore adopted. As the mean physical distance Mutation analysis between individual SNPs on the 10K chip system is 258 kb these Aliquots of DNA were diluted to a final concentration of 50– parameters will bias detection to long runs of homozygosity. 100 mg/ml. Primers and precise amplification conditions were Using this criterion, blocks of homozygosity representing derived either from previously published studies or cDNA/ acquired regions of UPD (black), deletion (green) and amplifica- genomic comparison, and are available on request. Selective tion (red) were detected in 43/52 (83%) lymph node biopsies screening of patient DNA was performed as determined by its with an average 1.7 and 3.0 aberrations detected in FL and t-FL, corresponding homozygosity profile. Mutation analysis was respectively (Figure 1). carried out by PCR-direct sequence analysis of selected regions Overall, the number of FLs exhibiting abnormalities (20/26: of protein tyrosine phosphatase, non-receptor type 11 (PTPN11) 77%) was lower than that observed for t-FL (23/26: 88%) with (exons 3 and 13);26 histone H4 (HIST1H4D), Splicing factor, 18 patients having detectable abnormalities in both episodes of arginine/serine-rich 3 (SFRS3), cyclin-dependent kinase inhibi- disease. The pattern of aberration detected in these patients was tor 1A (CDKN1A/p21WAF1/CIP1),27 cyclin D3 (CCND3) and consistent with two distinct mechanisms of transformation. t-FL cyclin-dependent kinase inhibitor 2A (CDKN2A) were all appeared to arise by a process of direct evolution in eight screened in their entirety. Tumour protein 53 (TP53) screening patients where the aberrations detected in FL were maintained of this series has been published previously.25 PCR products at transformation. Figure 2a shows a genomic profile obtained were sequenced directly by use of an ABI 377 DNA sequencer from patient 9 who exhibited a block of homozygosity on 12q in (PE Applied Biosystems, Foster City, CA, USA). Unincorporated both FL and t-FL samples with the acquisition of several primers were removed by ultrafiltration and sequencing data additional abnormalities within the t-FL sample. The other 10 analysed using DNAStar (DNAStar Inc., Madison, WI, USA). patients have a more complex pattern with the detection of

Figure 1 A summary of the regions of homozygosity (regions with 496% homozygous calls in a minimum region of 50 contiguous SNPs) in paired FL-tFL biopsies and three cell lines (DoHH2, RL2261, SCI-1) for each chromosome in the samples analysed using the 10 K 2.0 SNP array (see also Supplementary data). Black: represents regions of homozygosity without signiﬁcant chromosome copy number changes (signal intensity ratio compared between 0.75 and 1.25); green, regions of homozygosity with relative chromosome copy number loss (signal intensity ratio, o0.75); red, regions of homozygosity with relative copy number gains (signal intensity ratio41.25). Presentation FL samples were analysed in 10/ 26 cases. Remission BM samples were analysed in six cases (7, 8, 16, 18, 20 and 21). Patient 8 contains a germline region of homozygosity of 11p. t(14;18) status is known for 22/26 cases. t(14;18) breakpoint status was as follows: patients 1–2, 4–6, 9–12, 17–20, 22, 24, 26 and 28 (MBR), 8, 16 and 25 (mcr) and 21 and 27 (atypical). The status of four patients (7, 13, 15 and 23) was not determined. Note: when a number of events colocalize to the same chromosome the automated generated homozygosity image can appear to merge these events together, for example, t-FL patient 22; a region of aUPD (black) and ampliﬁcation (red) on chromosome 11 appear to occur simultaneously. Patient numbering is as reported previously.25

Leukemia aUPD in follicular lymphoma J Fitzgibbon et al 1516

Figure 2 A genome-wide map (1–22, X) showing blocks of homozygosity for FL (top) and t-FL (bottom) in patients 9 and 21. Black: represents regions of homozygosity without signiﬁcant chromosome copy number changes (signal intensity ratio compared between 0.75 and 1.25); green; regions of homozygosity with relative chromosome copy number loss (signal intensity ratio, o0.75); red; regions of homozygosity with relative copy number gains (signal intensity ratio41.25).

abnormalities within the FL that were absent at transformation. this was a feature of the patient’s germline DNA. The exis- Figure 2b shows the genomic proﬁle obtained for case 21 that tence of large regions of germline homozygosity are rare and harbours aberration on chromosomes 1, 2, 3, 4 and 13 within are more commonly associated with particular recessive the FL sample that were absent at transformation. The detection disorders.28 of regions of homozygosity on 2p and 6p common to both FL and t-FL in this patient suggests that these lymphomas evolved separately from an ancestor B cell. Eight pairs had at least one Recurrent sites of homozygosity biopsy in which no abnormalities were detectable (Figure 1). Seventy-eight of the 120 homozygous events (65%) detected These cases were not included for direct comparison as we are represented regions of aUPD. Such abnormalities were found in unable to discriminate between samples having no abnormality 23/26 (88%) tumour pairs involving 16 different chromosomes and those where the existence of contaminating normal DNA (range 1–6). Regions of recurring aUPD (n43) were localized to within the sample may have prevented the detection of changes chromosomes 6, 9, 12 and 17. The size of these regions was within these tumours. variable, included whole chromosomes (6) or chromosomal SNP genotype analysis was also performed on remission arms (6p, 9p, 12q, 17p) and usually ran from a point on the bone marrow DNA available in six cases (7, 8, 16, 18, 20 chromosome to the telomere. and 21). In all but one case the observed homozygosities aUPD6p-tel was detected in four patients (12, 17, 21 and 26), were absent from the remission sample demonstrating that where the block of homozygosity was present in both FL and the these were somatically acquired. A single 11 Mb region of corresponding t-FL biopsy. aUPD arose in two further cases homozygosity on 11p seen in both FL and t-FL of patient 8 was (5 and 15) from an apparent loss and duplication of the whole also present in the remission bone marrow indicating that chromosome 6 (Figure 3a). Patient 2 showed a region of

Leukemia aUPD in follicular lymphoma J Fitzgibbon et al 1517

Figure 3 (a) Schematic representation of SNP calls on chromosome 6 in FL and t-FL biopsies. Six paired FL (on the left) and t-FL (on the right) are shown for patients 5, 12, 15, 17, 21 and 26 where aUPD runs from a point on the chromosome to the telomere (12, 17, 21, 26) or involves the entire chromosome (5, 15). Heterozygous calls are indicated in red (designated AB) and the homozygous calls (designated AA or BB) are in blue. In each case the copy number ratio when compared to a bank of normal DNAs was one, indicating aUPD. (b) Schematic representation of the regions of aUPD 6p. aUPD in tumours specimens 2, 5, 12, 15, 17, 21 and 26 are shown in orange and cell line SCI-1, in green. FL biopsy obtained from patient 2 contains a B9 Mb region of aUPD on 6p24.3-p22.3 ﬂanked by regions of no calls (dotted lines). Mitotic recombination events cluster to a B10 Mb region (purple shading). The location of the genes included for mutation analysis is indicated.

interstitial aUPD localizing to an approximately 9 Mb region of aUPD of lymphoma cell lines 6p. There was an increase in the number of no calls for the SNPs The t(14;18)-bearing cell lines SCI-1, DoHH2 and RL2261 were residing on either side of this region. This no call profile suggests subjected to similar genotype analysis. It was notable that these that normal contaminating cells may be masking the detection cell lines also contained regions of aUPD similar to those seen of aUPD6p-tel in this sample. Overall, the boundaries of mitotic in primary tumour material: SCI-1 (6p, 12q and 17p); DoHH2 recombination on 6p all locate within an approximate 10 Mb (9p and 12q) and RL2261 (12q and 17p) (Figure 1). Additionally, (34–44 Mbs) region of p21 (Figure 3b). the investigation of copy number data identified a small Homozygosity of 9p occurred in six patients and included homozygous deletion on 9p in DoHH2 (Figure 4). three cases each with a deletion (8, 9 and 27) or aUPD (10, 18 and 28) of 9p. These alterations were present in three of the FL specimens and in all six corresponding t-FL. Patient 8 had a Mutation studies deletion in the FL biopsy and a region of aUPD in the t-FL Mutation analysis was performed for genes previously found to sample. Regions of aUPD12q were detected in five patients (1, be deregulated in haematological malignancies and locating to 5, 9, 13 and 19) with a further two patients with amplification of sites of homozygosity on 6p (HISTY1H4D, SFRS3, CDKN1A and 12q (15, 22). aUPD12q was detected in FL and t-FL biopsies CCND3), 9p (CDKN2A), 12q (PTPN11) and 17p (TP53). No with the site of mitotic recombination arising within a 13 Mb mutation was detected in the coding region of genes localizing region of 12cen-q13 (33–46 Mbs) in three cases (9, 13 and 19) to 6p or 12q. Patient 27 had a heterozygous missense mutation (Figure 1). Finally, all five cases identified with homozygosity of in CDKN2A (385 T4G: Y129D) (data not shown). Homo- 17p (4, 8, 9, 10 and 22) had arisen in cases of t-FL. zygosity of 17p was observed in five patients and was restricted

Leukemia aUPD in follicular lymphoma J Fitzgibbon et al 1518 these tumours as seen on 6p in patient 2. The possibility however that these biopsies do not harbour regions of homozygosity can not be excluded completely since 10% of t(14;18) þ ve FL do not harbour secondary cytogenetic abnormalities.9 Normal cell contamination will have an even greater effect on mutation analysis particular when attempting to discriminate between heterozygous and homozygous gene mutation. In contrast with aUPD, where 50% of DNA from the normal contaminating cells will contribute to the aUPD Figure 4 Chromosome 9 copy number ratio (log2 ratio) at each SNP profile, all DNA arising from the normal cell pool will contribute locus for the cell line DoHH2. SNPs are plotted (dots) as per wild-type sequence. This provides an explanation for the chromosomal location from p arm (left) to q arm (right). DoHH2 exhibits both aUPD9p and a homozygous deletion of SNPs spanning presence of heterozygous mutation for patients 9 and 27 in the CDKN2A locus (located between the vertical lines). TP53 and CDKN2A, respectively where homozygous mutations would be expected based on their aUPD profiles. We cannot however rule out the possibility that mutation in CDKN2A or TP53 arise following mitotic recombination and are not there- to t-FL. TP53 mutation status of these biopsies were included as fore the basis of daughter selection and the acquisition of aUPD. part of a previous study.25 Two of these patients, 4 (13344 T4C: To search for underlying genetic lesions made homozygous in I195T) and 22 (13129_13130 ins C frameshift) showed FL we analysed four regions of recurrent aUPD on chromosomes homozygous mutation in their t-FL biopsy. Case 9 was 6, 9, 12 and 17. These chromosomal regions have all been heterozygous for a TP53 mutation (14057 G4T: G244C) that implicated previously in the pathogenesis of FL.9 As our study in was also detected in the corresponding FL biopsy sample. There leukemia indicated that mitotic recombination and the resultant were no mutations observed in samples obtained from patients UPD has a role in rendering a leukemic cell homozygous for a 8 or 10. pre-existing sequence mutation we have restricted our initial studies in lymphoma to the search for alterations in the coding region of selective genes. Mutation of CDKN2A (9p) and TP53 Discussion (17p) are established secondary events in transformation7,25,35,36 and homozygous mutation of these genes was identified in three High-density array-based SNP analysis provides an efficient cases (DoHH2: CDKN2A; patients 4 and 22: TP53). This means to determine sites of aUPD within the cancer genome. confirmed our previous observations in AML that mutation The acquisition of aUPD is nonrandom, varies within and precedes mitotic recombination which represent a second hit between cancers,15,29,30 and in leukemia is frequently asso- resulting in the removal of the remaining wild-type allele.24 The ciated with homozygous gene mutation.20–24 In FL the muta- absence of mutation in other samples with aUPD 9p or 17p may tional status of genes is less established and the secondary reflect the existence of either additional mutational targets on events more complex. We therefore sought to determine these chromosomes or alternative mutational mechanisms, for whether aUPD also arose in these tumours and how these example, methylation of CDKN2A.35 The finding of a single events could contribute to the clinical evolution of the disease. mutation (patient 27) within the patient panel is in agreement Regions of aUPD were detected in the majority of samples with previous studies showing only rare mutation of CDKN2A and located to 16 different chromosomes. This figure may well in FL.36 be an under-representation of the actual frequency of aUPD, as The occurrence of 6p and 12q abnormalities in both FL the criterion set to distinguish between aUPD and the normal (diagnostic and relapse samples) and t-FL samples suggests that occurrence of homozygosity within the genome in this study is these aberrations may represent early events in the development biased towards long regions of homozygosity that encompass a of the disease. The region of 6p is highly gene rich and includes minimum of 50 contiguous SNPs. As the mean separation several genes (HIST1H4D,37 HLA-DRA,38 HLA-DQB1,38 between adjacent SNPs on the 10K system is approximately BRD2,39 SFRS3,40 CDKN1A,27 CCND341,42) subject to dere- 258 kb, sizeable regions of aUPD are very likely to go gulation in haematological malignancy. As the number of undetected. This inherent bias for abnormalities spanning large independent samples harbouring aUPD6p is limited and in the distances may explain the high frequency of aUPD events absence of matching germline material to map with confidence involving whole chromosome arms, the absence of events on the precise recombination boundaries, genes mapping within or the shorter chromosomes and their preponderance over changes distal of this mitotic recombination cluster region were included in DNA copy number that typically locate to discrete for mutation analysis. No mutations have been identified in the chromosomal regions. genes tested (Figure 3b); it seems likely that the selective basis of The occurrence of normal cells within the tumour is a aUPD in lymphoma will prove more difficult to resolve than in recognized and prognostic marker in FL31 with the amount of AML where the gene targets are better established. We are also infiltrate varying between biopsy with a mean of approximately unable to rule out the possibility that epigenetic changes or shifts 30% (Randy Gascoyne, personal communication). This is likely in tumour allelotype could be responsible for daughter selection to lead to a masking and an under reporting of acquired following mitotic recombination. We expect that the accumula- homozygosity events. In a series of diluting experiments using tion of additional cell line (for example, see www.sanger.ac.uk/ both the Illumina and Affymetrix platforms, where tumour and genetics/CGP/CopyNumberMapping/) and biopsy material with its corresponding normal cells were mixed at differing propor- aUPD6p, and the use of higher density arrays will assist in tions, approximately 70% tumour content was defined as a defining minimal boundaries of aUPD and narrow attention to a lower limit for the detection of aUPD.32–34 The presence of non- smaller number of genes. This is also the case for aUPD12q tumour cells therefore is the most likely explanation for the where the region of aUPD includes a large portion of the long absence of acquired homozygosities in 23% of the biopsies arm of the chromosome. Silencing mediator of retinoic acid and tested in this study and/or the occurrence of no calls within thyroid hormone receptor (SMRT) has been proposed as the

Leukemia aUPD in follicular lymphoma J Fitzgibbon et al 1519 candidate gene in this region43 as it resides within a recurrent pathways of clonal evolution in follicular lymphomas. Genes breakpoint region on 12q24 region in transformed NHL. Since Chromosomes Cancer 2004; 39: 195–204. SMRT is deleted in only one copy of 12q in the DoHH2 cell line 10 Martinez-Climent JA, Alizadeh AA, Segraves R, Blesa D, Rubio- it seems unlikely to act as the selective basis of aUPD on this Moscardo F, Albertson DG et al. Transformation of follicular lymphoma to diffuse large cell lymphoma is associated with a chromosome. heterogeneous set of DNA copy number and gene expression Eighteen of the 26 patients exhibited abnormalities in both FL alterations. Blood 2003; 101: 3109–3117. and t-FL samples. SNP genotype analysis detected abnormalities 11 Goff LK, Neat MJ, Crawley CR, Jones L, Jones E, Lister TA et al. The in 10 of these FL tumours, which were absent in the use of real-time quantitative polymerase chain reaction and corresponding transformed episodes of the disease. It is possible comparative genomic hybridization to identify amplification of therefore that FL and t-FL may arise independently from a B-cell the REL gene in follicular lymphoma. Br J Haematol 2000; 111: 44 618–625. precursor and not through the emergence of a more 12 Nagy M, Balazs M, Adam Z, Petko Z, Timar B, Szereday Z et al. aggressive, subclone of cells from within the FL pool. This Genetic instability is associated with histological transformation of hypothesis, based initially on the clonal relationship of Ig VHDJH follicle center lymphoma. Leukemia 2000; 14: 2142–2148. sequence variability in a case of FL and t-FL,44 represents an 13 Hough RE, Goepel JR, Alcock HE, Hancock BW, Lorigan PC, important new concept to emerge in this field and could have Hammond DW. Copy number gain at 12q12–14 may be important significant clinical and therapeutic relevance. On the basis of in the transformation from follicular lymphoma to diffuse large B the natural history of FL transformation one can speculate that cell lymphoma. Br J Cancer 2001; 84: 499–503. 14 Calhoun ES, Hucl T, Gallmeier E, West KM, Arking DE, Maitra A the events giving rise to FL from this proposed B-cell precursor et al. Identifying allelic loss and homozygous deletions in may be more easily acquired than for the onset of t-FL. There is pancreatic cancer without matched normals using high-density however very little data to ascertain the frequency of de novo single-nucleotide polymorphism arrays. Cancer Res 2006; 66: DLBCL recurring with FL for a direct comparison to be made. 7920–7928. The use of SNP genotype analysis provides a further means of 15 Raghavan M, Lillington DM, Skoulakis S, Debernardi S, Chaplin T, visualizing the genomic changes responsible for cancer devel- Foot NJ et al. Genome-wide single nucleotide polymorphism analysis reveals frequent partial uniparental disomy due to somatic opment and sites of UPD that go undetected by previous recombination in acute myeloid leukemias. Cancer Res 2005; 65: methods. Although the clinical heterogeneity of FL is still 375–378. reflected in a complex accumulation of genetic events the 16 Gaasenbeek M, Howarth K, Rowan AJ, Gorman PA, Jones A, current study has identified mitotic recombination as a key event Chaplin T et al. Combined array-comparative genomic hybridiza- in the origin and evolution of this disease. tion and single-nucleotide polymorphism-loss of heterozygosity analysis reveals complex changes and multiple forms of chromosomal instability in colorectal cancers. Cancer Res 2006; 66: Acknowledgements 3471–3479. 17 Midorikawa Y, Yamamoto S, Ishikawa S, Kamimura N, Igarashi H, Sugimura H et al. Molecular karyotyping of human hepato- We thank Randy Gascoyne for sharing his unpublished flow data cellular carcinoma using single-nucleotide polymorphism arrays. in FL, Susan Weiss for assistance in preparing the article, Iram Oncogene 2006; 25: 5581–5590. Gull and Victoria Miller for sample collection and storage, and 18 Nielaender I, Martin-Subero JI, Wagner F, Martinez-Climent JA, Jean-Baptiste Cazier and Manu Gupta for helpful comments on Siebert R. Partial uniparental disomy: a recurrent genetic mechan- the article. Derville O’Shea is a MRC training fellow and ism alternative to chromosomal deletion in malignant lymphoma. Emanuela Carlotti is a Bart’s and the Royal London Charitable Leukemia 2006; 20: 904–905. 19 Robinson WP. Mechanisms leading to uniparental disomy and Foundation fellow. The work was also supported by the Cancer their clinical consequences. Bioessays 2000; 22: 452–459. Research UK and Kay Kendall Leukaemia Fund. 20 James C, Ugo V, Casadevall N, Constantinescu SN, Vainchenker W. A JAK2 mutation in myeloproliferative disorders. Pathogenesis and therapeutic and scientific prospects. Trends Mol Med 2005; References 11: 546–554. 21 Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR 1 Horning SJ. Natural history of and therapy for the indolent non- et al. A gain-of-function mutation of JAK2 in myeloproliferative Hodgkin’s lymphomas. Semin Oncol 1993; 20: 75–88. disorders. N Engl J Med 2005; 352: 1779–1790. 2 Gallagher CJ, Gregory WM, Jones AE, Stansfeld AG, Richards MA, 22 Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S Dhaliwal HS et al. Follicular lymphoma: prognostic factors for et al. Acquired mutation of the tyrosine kinase JAK2 in human response and survival. J Clin Oncol 1986; 4: 1470–1480. myeloproliferative disorders. Lancet 2005; 365: 1054–1061. 3 Cullen MH, Lister TA, Brearley RI, Shand WS, Stansfeld AG. 23 Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ Histological transformation of non-Hodgkin’s lymphoma: a pro- et al. Activating mutation in the tyrosine kinase JAK2 in spective study. Cancer 1979; 44: 645–651. polycythemia vera, essential thrombocythemia, and myeloid 4 Hubbard SM, Chabner BA, DeVita VT, Simon R, Berard CW, Jones metaplasia with myelofibrosis. Cancer Cell 2005; 7: 387–397. RB et al. Histologic progression in non-Hodgkin’s lymphoma. 24 Fitzgibbon J, Smith LL, Raghavan M, Smith ML, Debernardi S, Blood 1982; 59: 258–264. Skoulakis S et al. Association between acquired uniparental 5 Acker B, Hoppe RT, Colby TV, Cox RS, Kaplan HS, Rosenberg SA. disomy and homozygous gene mutation in acute myeloid Histologic conversion in the non-Hodgkin’s lymphomas. J Clin leukemias. Cancer Res 2005; 65: 9152–9154. Oncol 1983; 1: 11–16. 25 Davies AJ, Lee AM, Taylor C, Clear AJ, Goff LK, Iqbal S et al. A 6 Bastion Y, Sebban C, Berger F, Felman P, Salles G, Dumontet C limited role for TP53 mutation in the transformation of follicular et al. Incidence, predictive factors, and outcome of lymphoma lymphoma to diffuse large B-cell lymphoma. Leukemia 2005; 19: transformation in follicular lymphoma patients. J Clin Oncol 1997; 1459–1465. 15: 1587–1594. 26 Tartaglia M, Kalidas K, Shaw A, Song X, Musat DL, van der Burgt I 7 Lossos IS. Higher-grade transformation of follicular lymphoma – a et al. PTPN11 mutations in Noonan syndrome: molecular continuous enigma. Leukemia 2005; 19: 1331–1333. spectrum, genotype–phenotype correlation, and phenotypic hetero- 8 Lossos IS, Levy R. Higher grade transformation of follicular geneity. Am J Hum Genet 2002; 70: 1555–1563. lymphoma: phenotypic tumor progression associated with diverse 27 Galmarini CM, Bouchet BP, Audoynaud C, Lamblot C, Falette N, genetic lesions. Semin Cancer Biol 2003; 13: 191–202. Bertholon J et al. A p21/WAF1 mutation favors the appearance of 9 Hoglund M, Sehn L, Connors JM, Gascoyne RD, Siebert R, Sall T drug resistance to paclitaxel in human noncancerous epithelial et al. Identification of cytogenetic subgroups and karyotypic mammary cells. Int J Cancer 2006; 119: 60–66.

Leukemia aUPD in follicular lymphoma J Fitzgibbon et al 1520 28 Engel E. A fascination with chromosome rescue in uniparental involving p16 and p15 are associated with histologic progression disomy: Mendelian recessive outlaws and imprinting copyrights in follicle center lymphoma. Blood 1998; 91: 4677–4685. infringements. Eur J Hum Genet 2006; 14: 1158–1169. 37 Kurata M, Maesako Y, Ueda C, Nishikori M, Akasaka T, Uchiyama T 29 Murthy SK, DiFrancesco LM, Ogilvie RT, Demetrick DJ. Loss of et al. Characterization of t(3;6)(q27;p21) breakpoints in B-cell non- heterozygosity associated with uniparental disomy in breast Hodgkin’s lymphoma and construction of the histone H4/BCL6 carcinoma. Mod Pathol 2002; 15: 1241–1250. fusion gene, leading to altered expression of Bcl-6. Cancer Res 30 Teh MT, Blaydon D, Chaplin T, Foot NJ, Skoulakis S, Raghavan M 2002; 62: 6224–6230. et al. Genomewide single nucleotide polymorphism microarray 38 Jordanova ES, Philippo K, Giphart MJ, Schuuring E, Kluin PM. mapping in basal cell carcinomas unveils uniparental disomy as a Mutations in the HLA class II genes leading to loss of expression of key somatic event. Cancer Res 2005; 65: 8597–8603. HLA-DR and HLA-DQ in diffuse large B-cell lymphoma. 31 Dave SS, Wright G, Tan B, Rosenwald A, Gascoyne RD, Chan WC Immunogenetics 2003; 55: 203–209. et al. Prediction of survival in follicular lymphoma based on 39 Greenwald RJ, Tumang JR, Sinha A, Currier N, Cardiff RD, molecular features of tumor-infiltrating immune cells. N Engl J Rothstein E et al. mu-BRD2 transgenic mice develop B-cell Med 2004; 351: 2159–2169. lymphoma and leukemia. Blood 2004; 103: 1475–1484. 32 Lindblad-Toh K, Tanenbaum DM, Daly MJ, Winchester E, Lui WO, 40 Chen W, Itoyama T, Chaganti RS. Splicing factor SRP20 is a novel Villapakkam A et al. Loss-of-heterozygosity analysis of small-cell partner of BCL6 in a t(3;6)(q27;p21) translocation in transformed lung carcinomas using single-nucleotide polymorphism arrays. follicular lymphoma. Genes Chromosomes Cancer 2001; 32: Nat Biotechnol 2000; 18: 1001–1005. 281–284. 33 Zhao X, Li C, Paez JG, Chin K, Janne PA, Chen TH et al. An 41 Shaughnessy J, Gabrea A, Qi Y, Brents L, Zhan F, Tian E et al. integrated view of copy number and allelic alterations in the Cyclin D3 at 6p21 is dysregulated by recurrent chromosomal cancer genome using single nucleotide polymorphism arrays. translocations to immunoglobulin loci in multiple myeloma. Blood Cancer Res 2004; 64: 3060–3071. 2001; 98: 217–223. 34 Peiffer DA, Le JM, Steemers FJ, Chang W, Jenniges T, Garcia F 42 Smith ML, Arch R, Smith LL, Bainton N, Neat M, Taylor C et al. et al. High-resolution genomic profiling of chromosomal aberra- Development of a human acute myeloid leukaemia screening tions using Infinium whole-genome genotyping. Genome Res panel and consequent identification of novel gene mutation in 2006; 16: 1136–1148. FLT3 and CCND3. Br J Haematol 2005; 128: 318–323. 35 Pinyol M, Cobo F, Bea S, Jares P, Nayach I, Fernandez PL et al. 43 Song L, Zlobin A, Ghoshal P, Zhang Q, Houde C, Weijzen S et al. p16(INK4a) gene inactivation by deletions, mutations, and hyper- Alteration of SMRT tumor suppressor function in transformed non- methylation is associated with transformed and aggressive variants Hodgkin lymphomas. Cancer Res 2005; 65: 4554–4561. of non-Hodgkin’s lymphomas. Blood 1998; 91: 2977–2984. 44 Matolcsy A, Schattner EJ, Knowles DM, Casali P. Clonal evolution 36 Elenitoba-Johnson KS, Gascoyne RD, Lim MS, Chhanabai M, Jaffe of B cells in transformation from low- to high-grade lymphoma. ES, Raffeld M. Homozygous deletions at chromosome 9p21 Eur J Immunol 1999; 29: 1253–1264.

Supplementary Information accompanies the paper on the National Centre for Biotechnology Information website (http:// www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE7425)

Leukemia