BCL2 Mutations in Diffuse Large B-Cell Lymphoma

ORIGINAL ARTICLE BCL2 mutations in diffuse large B-cell lymphoma

JM Schuetz1,6, NA Johnson2,6, RD Morin1, DW Scott3,KTan3, S Ben-Nierah3, M Boyle3, GW Slack3, MA Marra1, JM Connors4, AR Brooks-Wilson1,5,7 and RD Gascoyne3,7

BCL2 is deregulated in diffuse large B-cell lymphoma (DLBCL) by the t(14;18) translocation, gene amplification and/or nuclear factor-kB signaling. RNA-seq data have recently shown that BCL2 is the most highly mutated gene in germinal center B-cell (GCB) DLBCL. We have sequenced BCL2 in 298 primary DLBCL biopsies, 131 additional non-Hodgkin lymphoma biopsies, 24 DLBCL cell lines and 51 germline DNAs. We found frequent BCL2 mutations in follicular lymphoma (FL) and GCB DLBCL, but low levels of BCL2 mutations in activated B-cell DLBCL, mantle cell lymphoma, small lymphocytic leukemia and peripheral T-cell lymphoma. We found no BCL2 mutations in GC centroblasts. Many mutations were non-synonymous; they were preferentially located in the flexible loop domain, with few in BCL2-homology domains. An elevated transition/transversions ratio supports that the mutations result from somatic hypermutation. BCL2 translocations correlate with, and are likely important in acquisition of, additional BCL2 mutations in GCB DLBCL and FL. DLBCL mutations were not independently associated with survival. Although previous studies of BCL2 mutations in FL have reported mutations to result in pseudo-negative BCL2 protein expression, we find this rare in de-novo DLBCL.

Leukemia (2012) 26, 1383--1390; doi:10.1038/leu.2011.378 Keywords: BCL2; lymphoma; DLBCL; t(14;18); RNA-seq; somatic hypermutation

INTRODUCTION BCL2 protein overexpression in DLBCL has been associated with 11 Diffuse large B-cell lymphomas (DLBCLs) represent the most poor prognosis. The addition of rituximab to chemotherapy may 12 common subtype of non-Hodgkin lymphoma (NHL).1 Gene overcome the impact of BCL2 expression on prognosis; however, expression profiling classifies DLBCL into at least two molecular BCL2 expression remains relevant when analysis is restricted to 7,13,14 groups, including the germinal center B-cell (GCB) and activated specific molecular subtypes of DLBCL. Indeed, drugs that B-cell (ABC) subtypes.2 GCB DLBCLs are characterized by the target BCL2 are being investigated in clinical trials, thus BCL2 overexpression of genes active in normal germinal center B cells, mutations may be clinically important in the future. such as BCL6, CD10 and CD38; while ABC DLBCLs overexpress The BCL2 family of proteins comprises members with both pro- genes normally induced during B-cell activation, such as MUM1, and anti-apoptotic roles. All have at least one of the four c-FLIP and BCL2.2 structurally conserved motifs known as BCL2-homology (BH) 15 BCL2, discovered because of its involvement in t(14;18) in domains. BCL2, BCL-XL and other anti-apoptotic members follicular lymphoma (FL),3 has a central role in the inhibition of contain all four BH domains, while pro-apoptotic members apoptosis.4 BCL2 is normally transiently expressed during B-cell (for example, BAX and BAK) either contain BH1, BH2 and BH3, or maturation. The t(14;18) translocation causes constitutive over- BH3 alone (the so-called ‘BH3-only’ class). The BH3 domain of expression of BCL2 by juxtaposing it to immunoglobulin heavy pro-apoptotic members inserts into a hydrophobic groove domain chain gene enhancer elements. This translocation is found in formed by the BH1, BH2 and BH3 domains of anti-apoptotic B20% of DLBCL,5 most often in the GCB subtype of DLBCL.6 Other members, a key interaction for the negative regulatory role of 16 mechanisms of BCL2 deregulation, more often observed in BCL2 family members. 17 ABC DLBCLs, include amplification of the BCL2 gene or its Using the transcriptome sequencing method RNA-seq in 89 transcriptional upregulation through constitutive activation of DLBCL samples, we recently identified genomic changes that drive 18,19 the nuclear factor-kB pathway.7 Saito et al.8 reported that the BCL2 the pathogenesis of adult DLBCL. The most commonly 19 promoter can also be aberrantly hypermutated in DLBCL, which mutated gene, mutated in 33/89 transcriptomes (37%) was BCL2. may prevent its inhibition by MIZ1 and BCL6.8 They found a high The aims of the current study were to determine the frequency number of mutations in BCL2 and suggested that the mutations and distribution of BCL2 mutations in DLBCL in a large number described were the result of somatic hypermutation (SHM). of cases, assess the relationship between gene mutation and Mutations in BCL2 have been shown to cause false-negative BCL2 protein expression or BCL2 translocation, and determine the protein expression results in immunohistochemistry assays in impact of mutations on immunohistochemical detection of FL;9,10 however, no large study has so far investigated whether this protein expression and clinical outcome. We also tested whether occurs in DLBCL. BCL2 mutations are specific to DLBCL by sequencing the BCL2

1Canada’s Michael Smith Genome Sciences Center, BC Cancer Agency, University of British Columbia, Vancouver, British Columbia, Canada; 2Division of Hematology and Oncology, Jewish General Hospital, Montreal, Que´bec, Canada; 3Department of Pathology, Centre for Lymphoid Cancer, BC Cancer Agency, Vancouver, British Columbia, Canada; 4Division of Medical Oncology, Centre for Lymphoid Cancer, BC Cancer Agency, Vancouver, British Columbia, Canada and 5Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada. Correspondence: Dr RD Gascoyne, Department of Pathology and Experimental Therapeutics, BC Cancer Research Centre, Room 5-113, 675 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada. E-mail: [email protected] 6These authors contributed equally to this work. 7These authors contributed equally to this work. Received 12 October 2011; revised 30 November 2011; accepted 1 December 2011; accepted article preview online 22 December 2011; advance online publication, 13 January 2012 BCL2 mutations in DLBCL JM Schuetz et al 1384 gene in other lymphoma subtypes with and without BCL2 Immunohistochemistry translocations. Re-sequencing BCL2 in 298 DLBCL tumors revealed Standard immunohistochemical methods were used for all antibodies. that it is highly mutated in this NHL subtype, showing that BCL2 protein expression was assessed in 262 DLBCL samples. Whole alteration of BCL2 in DLBCL can occur not only by the established sections of FFPET (fixed paraffin-embedded tissue) were stained with mechanisms of translocation or amplification, but also, frequently, antibody clone 124 (Dako, Burlington, ON, Canada), which targets through specific point mutations. amino acids 41--54. In all, 135 (52%) of these samples were also included in tissue microarray that was stained with antibody clone 124 and clone E17 (Epitomics, Burlingame, CA, USA), which targets amino acids 50--90 (96/135 samples were previously reported20,21). Samples with MATERIALS AND METHODS 50% or more malignant cells expressing BCL2 were defined as BCL2 Samples and patients positive. In all, 512 samples were used: 429 primary tumor biopsies, 24 DLBCL cell In all, 69 DLBCL cases were stained with CD10, BCL6 and MUM1 to þ lines, 51 germline DNA samples and 8 tonsil CD77 centroblast samples. determine cell of origin subtype by Hans criteria.22 Gene expression data Primary tumor samples consisted of 298 DLBCL (Table 1), 25 MCLs (mantle for cell of origin molecular subtyping were available for an additional cell lymphomas), 25 peripheral T-cell lymphomas (PTCLs), 30 SLLs (small 132 DLBCL cases, totaling 201/262 cases with cell of origin information lymphocytic lymphomas), 26 FLs and 25 PMBCLs (primary mediastinal B- (Table 1). Immunohistochemical stains were independently evaluated cell lymphomas). All samples were taken before treatment and diagnosed by three hematopathologists and samples with discrepant results were according to the World Health Organization 2008 criteria by expert reconciled by consensus review using a mutliheaded microscope. hematopathologists.1 Cell lines and their respective sources are listed in Supplementary File 1. In a subset of 51 of the above individuals (25 patients with DLBCL and 26 patients with FL), normal peripheral blood Cytogenetic analysis lymphocytes were used as a source of germline DNA. In addition, CD77 þ In 175 cases, the presence or absence of a BCL2 translocation could be centroblasts purified from pediatric and adult tonsils using Miltenyi confirmed (Table 1) by FISH (fluorescence in situ hybridization) (n ¼ 164), magnetic bead separation according to the manufacturer’s protocol using a commercial Vysis dual color break-apart probe (Abbott Molecular, (Miltenyi, Auburn, CA, USA) were used as a source of normal germinal Abbott Park, IL, USA) applied to FFPET, or by G-banded karyotype center B cells. Patients with DLBCL were treated with curative intent with (n ¼ 11).23 For samples in which non-synonymous BCL2 mutations were cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) with detected, FFPET were retrieved for FISH analysis. Samples containing 45% (n ¼ 212) or without (n ¼ 86) rituximab (R) (Table 1). Ethical approval for of cells displaying break-apart signals were considered positive for the this study, conducted in accordance with the Declaration of Helsinki, was presence of a translocation. The break-apart probe identifies BCL2 granted by the University of British Columbia/British Columbia Cancer translocations, and while t(14;18) is most common, translocations Agency Clinical Research Ethics Board. with (a) IGK (k light chain, 2p11.2); (b) IGL (l light chain, 22q11.2) and

Table 1. Clinical characteristics of samples subjected to Sanger sequencing, categorized according to the presence of coding mutations in BCL2

Clinical characteristics No mutation, Synonymous mutation, Non-synonymous P-value n ¼ 194 (%) n ¼ 65 (%) mutation, n ¼ 39 (%)

IPI 0--1 58 (34) 18 (35) 15 (43) 0.88 2--3 76 (45) 22 (42) 14 (40) 4--5 35 (21) 12 (23) 6 (17) Data not available 25 (À)13(À)4(À)

Primary therapy R-CHOP 141 (73) 43 (66) 28 (72) 0.61 CHOP 53 (27) 22 (34) 11 (28)

BCL2 protein (n ¼ 262)a Positive 99 (60) 41 (68) 34 (92) 0.0007 Negative 59 (36) 13 (22) 3 (8) Discrepant 7 (4) 6 (10) 0 (0) Data not available 29 (À)5(À)2(À)

Cell of origin (n ¼ 201) GCB 34 (27) 35 (76) 26 (84) o0.00001 Non-GCBb 90 (73) 11 (24) 5 (16) Data not available 70 (À)19(À)8(À)

BCL2 translocation (n ¼ 175) Translocation 14 (13) 26 (67) 23 (79) o0.00001 No translocation 93 (87) 13 (33) 6 (21) Data not available 87 (À)26(À)10(À) Abbreviations: ABC, activated B-cell; CHOP, cyclophosphamide, doxorubicin, vincristine, prednisone regimen; DLBCL, diffuse large B-cell lymphoma; GCB, germinal center B cell; IPI, international prognostic index; R-CHOP, CHOP with rituximab. aBCL2 protein expression detection by clone 124 was performed in 262 samples, of which 135 (52%) samples were also stained with clone E17. The calculation of the P-value does not include the discrepant cases. BCL2 translocation status was determined in 175 cases. Some cases do not have data for all variables. The percentages in parentheses reﬂect the number of cases/ total cases with available data within each group; for example, 99 cases were BCL2 protein positive and had no mutations, representing 99/(99+59+7) within the group of samples without mutations. bNon-GCB includes activated B-cell (ABC) type and tumors not classiﬁable by gene expression, or non-GCB by Hans criteria.22

Leukemia (2012) 1383 --1390 & 2012 Macmillan Publishers Limited BCL2 mutations in DLBCL JM Schuetz et al 1385 (c) complex variant translocations involving chromosomes 14, 18 and one (OS) was determined from the date of diagnosis to the date of death of any or two other chromosomes24 would also be detected using this assay. cause. The median follow-up in patients alive at last follow-up was for a median 5.7 years (0.1--15.9 years). OS and PFS were calculated using the Transcriptome sequence data Kaplan--Meier method and the log rank test was used to compare survival 20 2 RNA-seq data included 31 DLBCL libraries (each RNA-seq library is curves between groups. The w test and Fisher’s exact test were used to constructed from a single tumor sample) described by Morin et al.18 and test for association between variables; P-values of o0.05 were considered 58 additional DLBCL libraries.19 Briefly, paired end reads, obtained by significant. Unless otherwise specified, P-values reported are calculated sequencing both ends of each DNA fragment of an RNA-seq library, were from Fisher’s exact test. These statistical analyses were performed using aligned to the human reference genome (hg18) and exon junction SPSS software, version 14 (SPSS Inc., Chicago, IL, USA). sequences (to allow correct alignment of reads spanning exons) using the Burrows-Wheeler Aligner.25 Single nucleotide variants were identified with SNVMix software (http://compbio.bccrc.ca/software/snvmix/).26 Known RESULTS polymorphisms, including those found in dbSNP (build 130) and any Frequent somatic BCL2 mutations in DLBCL and FL variants in published genomes, were removed, resulting in a list of The median depth of coverage of BCL2 for all RNA-seq libraries candidate mutations in the genome, including in BCL2. was 58-fold. The BCL2 gene was identified as the most mutated expressed gene in DLBCL transcriptomes19 (Figure 1), with 33 of Sanger sequencing 89 (37%) DLBCL samples containing at least one candidate For all samples described in ‘samples and patients,’ genomic DNA was mutation in the coding region, 26 of which had at least one non- extracted using the QIAGEN ALL PREP kit (Qiagen, Hilden, Germany). DNA synonymous mutation. On average, 5.53 mutations affected the was quantified using PicoGreen (Invitrogen, Burlington, ON, Canada) in a BCL2 coding region in the 26 mutated DLBCL transcriptomes. BCL2 Victor2 fluorescence plate reader (Perkin-Elmer, Waltham, MA, USA). was mutated twice as often as the next most commonly mutated Four BCL2 amplicons were sequenced, with primers designed using gene, PIM1 (Table 2), and 5.6 times more than EZH2. There were far Primer3(ref. 27) to cover all exons and 1.4 kb upstream of the BCL2 more mutations in BCL2 than in other genes known to be targets transcriptional start site. Supplementary File 2 lists each amplicon’s primers of SHM, such as PIM1 or BCL6,29 which were only mutated in 25/89 and PCR conditions. --21M13F (TGTAAAACGACGGCCAGT) and M13R and 8/89 samples, respectively (Table 2). Of known SHM targets, (CAGGAAACAGCTATGAC) extensions were added to 50 ends of forward only BCL2 had significantly more mutations in GCB samples and reverse PCR primers, respectively, for uniform sequencing conditions. (P ¼ 0.004; Table 2). Of the 10 other BCL2 family genes consistently PCR products were purified using AmPure magnetic beads (Agencourt expressed (with expression defined as mean RPKM17X20), the Bioscience, Beverly, MA, USA) and eluted in 30 ml Tris EDTA (10 mM Tris, average number of samples with a candidate mutation was 1; only 0.1 mM EDTA pH 8.0) according to the manufacturer’s instructions. In all, two other genes in the family, MCL1 and BCL2L11, had candidate 2 ml of purified product was cycle sequenced using Big Dye Terminator Mix mutations in more than two samples. V.3 at 1/24 chemistry in total volume of 4 ml (Applied Biosystems, Foster The high frequency of BCL2 mutations was then determined in a City, CA, USA). Both forward and reverse directions were sequenced. larger set of NHL tumors (of DLBCL, MCL, SLL, FL, PMBCL and PTCL Mutation Surveyor v3.24 (Soft Genetics, State College, PA, USA) was used subtypes) by Sanger sequencing. All three exons and 1.4 kb to assemble against build hg18 and examine the sequences. upstream of the BCL2 transcriptional start site were successfully bi-directionally sequenced in 97% of samples. After exclusion In-silico functional assessment of BCL2 mutations of known polymorphic variants (rs1801018, rs1473418 and 28 rs2279115), 594 heterozygous mutations were detected in PolyPhen, a tool for in-silico classification of amino-acid changes as 104 of the 298 (35%) primary DLBCL samples (Figure 2 and ‘benign’, ‘possibly damaging’ or ‘probably damaging’, was used to predict Supplementary File 3). This high mutation rate is only sixfold lower possible deleterious effects of non-synonymous mutations on BCL2 than that of the rearranged IGH gene (9 Â 10À4 mutations/bp for protein. ‘Damaging’ variants affect annotated active sites or post- BCL2, compared with 5.8 Â 10À3 mutations/bp for IGH30). Of these, translational modification sites, or have predicted effects on the three- 127/594 were single nucleotide substitutions in the coding region dimensional structure of the protein. of BCL2, of which 60/594 (10%) were non-synonymous, predicting amino-acid substitutions or truncation of the BCL2 protein in 38 Statistical analysis samples. Of note, some samples contained multiple non-synon- Progression-free survival (PFS) was determined from the date of diagnosis ymous mutations (n ¼ 4 in 2 samples, n ¼ 3 in 3 samples, n ¼ 2in9 to the date of relapse, progression or death of any cause. Overall survival samples, n ¼ 1 in the remaining 24 samples). In addition, 115

Figure 1. Mutation frequency in 28 commonly mutated genes in B-cell NHL. The bar height is the total number of non-synonymous candidate mutations in each gene detected by RNA-seq in 89 cases. These 28 genes include 26 described by Morin et al. as signiﬁcantly mutated, and two genes (CARD11 and TNFAIP3) also known to be targets of mutation in DLBCL.

& 2012 Macmillan Publishers Limited Leukemia (2012) 1383 --1390 BCL2 mutations in DLBCL JM Schuetz et al 1386 Table 2. Candidate mutations in known targets of somatic hypermutation identiﬁed by RNA-seq in DLBCL cases

Gene Any mutation Total mutations S NS NS GCB NS ABC NS U Tn Tv Tn/Tv ratio P-value

MYC 1 1 0 1 1 0 0 1 0 NA 0.333 PAX5 6 6 0 6 2 3 1 3 3 1 0.407 BCL6 8 9 2 6 4 2 0 8 1 8 9.65E--04 PIM1 25 65 17 17 8 7 2 42 23 1.83 4.24E--07 BCL2 33 123 28 26 23 2 1 85 38 2.24 7.31E--16 Abbreviations: ABC, activated B-cell; DLBCL, diffuse large B-cell lymphoma; GCB, germinal center B cell. RHOH, a known somatic hypermutation target, was not mutated in our samples. No truncating mutations were observed in any of these genes. Any mutation: number of DLBCL cases with any type of mutation; total mutations: total number of mutations in gene among all DLBCL cases (some cases have more than one mutation); S: number of DLBCL cases with synonymous mutations; NS: number of DLBCL cases with non-synonymous mutations; NS GCB: number of GCB-type DLBCL cases with non-synonymous mutations in gene; NS ABC: number of ABC-type DLBCL cases with non-synonymous mutations; NS U: unclassiﬁed DLBCL cases with non-synonymous mutations; Tn: number of transitions; Tv: number of transversions; Tn/Tv ratio: ratio of transitions to transversions for all mutations; P-value that Tn/Tv is signiﬁcantly different than expected (0.333) in a one-tailed binomial exact test. RNA-seq data are also described in Morin et al.

positions had mutations in more than one case (296/594 generated, one would expect a 1:2 transition/transversion ratio. mutations were in these recurrent positions). The most frequently Indeed, in DLBCL samples, the transition/transversion ratio was observed non-synonymous mutations occurred at amino acids inverted (343/241, 1.42:1), thus demonstrating a clear enrichment 59 and 60 (9/38 samples), in the flexible loop domain (FLD). for transitions (P ¼ 4.07 Â 10À18) supporting previous reports8 that Heterozygous insertions/deletions (indels) were detected in 9/104 BCL2 mutations likely occurred as a result of SHM. The transition/ samples; none was found in the coding region. transversion ratio was similar for data obtained by RNA-seq and Mutations in BCL2 were also frequently detected in FL, but not Sanger methodologies. Consistent with SHM, we observed SLL, PTCL, PMBCL or MCL (Figure 2). Sequencing BCL2 in 26 FL ‘clustering’ of mutations (see Figure 2) at promoter sites. In our samples revealed 139 mutations, including 5 indels in introns. large set of cases, we observed that this clustering of mutations is In contrast, only 11 heterozygous mutations were detected in most pronounced near P1 (the more 50 promoter) in GCB tumors. the other 105 samples comprising SLL, PTCL, PMBCL and MCL. Clustering is apparent near both promoters in FL and, interest- Sequencing of matched constitutional DNA (from 26 FL and 25 ingly, the few mutations seen in MCL, PTCL or SLL are close to P2. DLBCL cases) and 8 normal centroblasts revealed, respectively, The large number of mutations close to the promoters, with a tail- one heterozygous synonymous variant and four additional off in the distribution with increasing distance from the promoter, variants (two being the common SNPs (single nucleotide is characteristic of SHM. Transitions were most enriched in the GCB polymorphisms) rs2279115 and rs1801018, while the other two samples (ratio of 1.48:1) compared with the ABC cases (ratio of were synonymous variants), implying that 499% of sequence 0.63:1), where there was no evidence of enrichment for transitions. variations observed in GCB-type DLBCL and FL samples are The high transition/transversion ratio in BCL2 translocation- somatic mutations specific to the tumor. Matched tumor DNA for positive cases (P ¼ 2.50 Â 10À13) compared with BCL2 transloca- the 25 DLBCL cases had an average of 5.7 mutations per sample tion-negative cases (P ¼ 0.07) supports the previous report of the (range: 0--28), while matched tumor DNA for 26 the FL cases had BCL2 translocation in placing BCL2 at high risk of acquiring 5.4 mutations per sample on average (range: 0--19), with only 4/26 mutations through SHM.32 These data strongly suggest that FL tumor DNA having no mutations. presence of BCL2 translocation, rather than deregulation of BCL2 In all, 23/24 DLBCL cell lines also had mutations (Supplementary through other mechanisms such as transcriptional upregulation File 4), with an average of 11 BCL2 mutations per cell line (range: seen in ABC subtypes of DLBCL or deregulation of miR 15--16 in 1--79). Compared with tumor samples, cell lines had many CLL, has a pivotal role in the acquisition of BCL2 mutations in GCB homozygous mutations, with 42% of mutations homozygous. DLBCL and FL. However, 17% of DLBCL cases without a detectable BCL2 translocation contained mutations of BCL2, indicating that other mechanisms may also induce mutations in BCL2. These BCL2 mutations are associated with GCB phenotype and presence other mechanisms appear to be tied to BCL2 expression level: in of BCL2 translocation the RNA-seq data BCL2 mRNA levels where higher in BCL2 BCL2 mutations were significantly enriched in GCB DLBCL mutated cases than in cases without BCL2 mutations (Supple- and highly associated with the presence of BCL2 translocation mentary File 5), when comparing only cases with no evidence for (see Figure 2 and Table 1). Cell of origin molecular subtype BCL2 translocation (t-test P ¼ 0.016). Furthermore, BCL2 protein information was available for 201 DLBCL samples (95 GCB and 106 status was available in eight cases with BCL2 mutations and no ‘non-GCB’, which included 44 cases of ABC and 22 cases BCL2 translocation. Although the numbers in this subset analysis unclassifiable as defined by gene expression profiling and 40 are too small to be statistically significant, 6/8 cases were positive cases of non-GCB defined by the Hans immunohistochemical for BCL2 protein, indicating BCL2 is indeed expressed in samples 22 algorithm ). Mutations were associated with GCB vs non-GCB without translocation. The lack of BCL2 mutations in germline tumors (Po0.00001) (Table 1). In addition, only 7/44 ABC samples DNA and normal centroblasts is consistent with previous reports33 (16%) had any mutation in BCL2 (synonymous or non-synon- that, unlike BCL6, BCL2 is not a target of SHM in normal tissue. ymous) (range: 0--6), while 61/95 GCB samples (64%) had at least one mutation (range: 0--51, P ¼ 7.34 Â 10À8). Two of the seven ABC samples with BCL2 mutations also had a BCL2 translocation by Non-synonymous mutations are enriched in the FLD, between the FISH. Translocations of BCL2 were detected in 25/26 FL (96%) and BH1 and BH3 domains and at the amino terminus in 61/172 DLBCL (35%) (Table 1), and were significantly associated Non-synonymous mutations in BCL2 were significantly enriched in with the presence of BCL2 mutations (Po0.00001). three areas corresponding to the amino terminus, the FLD and If these mutations occurred as a result of SHM, one would amino acids 108--135 located between the BH1 and BH3 domains expect a high transition/transversion ratio from activation-induced of the protein, compared with all other domains (Figure 3). All deaminase activity, which removes amino groups from cytosines, mutations, their predicted amino-acid changes and, where creating C4T changes.31 If the mutations were randomly applicable, their dbSNP identifiers, are provided in Supplementary

Leukemia (2012) 1383 --1390 & 2012 Macmillan Publishers Limited BCL2 mutations in DLBCL JM Schuetz et al 1387 22 GCB 20 68% n =95 18 16 14 12 10 8 6 number of mutations number 4 2 Figure 3. Non-synonymous mutations found in the BCL2 protein. 0 Functional domains of the protein are indicated in different colors

0 and are named on the ﬁgure; mutations in a given domain are 500 1500 2000 1000 shown in the same color. Mutations predicted by PolyPhen to

190000 192000 194000 be damaging to BCL2 function are indicated as triangles. 14 FL TM, transmembrane domain. 12 96% n =26 10 8 6 BCL2 mutations are not a common cause of pseudo-negative 4 BCL2 protein expression in DLBCL 2 number of mutations number The large sample size in this study allowed us to determine that 0 ‘pseudo-negative’ BCL2 protein expression due to BCL2 mutations 0

500 is not frequent in de-novo DLBCL. Both synonymous and 1000 1500 2000

190000 192000 194000 non-synonymous BCL2 mutations were associated with BCL2

14 ABC protein expression (P ¼ 0.0007). In 135 cases, both monoclonal 12 6% antibodies 124 and E17 could be compared. Only 13/135 cases 10 n =44 (10%) were discordant for staining with clones 124 and E17 8 (Table 1). Of nine cases negative for clone 124 and positive for E17, 6 none had non-synonymous mutations that could interfere with 4 the binding site for clone 124. Four cases were considered 2 number of mutations number 0 negative for clone E17 and positive for clone 124 but no non-

0 synonymous mutations were detected in these cases. Variability in 500 1500 1000 2000 tumor content (percent cells positive) may account for these 190000 192000 194000 discrepancies. Thus, although ‘pseudo-negative’ BCL2 protein 14 MCL / PTCL /SLL expression from BCL2 mutations has been reported in FL before 0% 12 n =80 and after histological transformation to DLBCL, this is a rare event 10 in de-novo DLBCL.9 8 6 4 BCL2 mutations do not confer a more favorable OS 2 number of mutations number 0 Patients with BCL2 mutant DLBCL do not have better clinical

0 outcome compared to patients with BCL2 wild-type DLBCL, 500 1000 1500 2000 suggesting that BCL2 mutations do not result in loss of 190000 192000 194000 anti-apoptotic function or improved chemosensitivity. In this P1 P2 study, BCL2 protein expression was associated with inferior survival in CHOP-treated (P ¼ 0.003) but not R-CHOP-treated patients (P ¼ 0.807), consistent with previous publications.12 We SAMPLE not translated EXON % t(14;18) translated focused on prognostic effects of BCL2 mutations and conducted number of samples SEQUENCED AMPLICON comparisons when sufficient numbers of samples with and PROMOTER without each variable were available. The presence of a BCL2 translocation or non-synonymous mutation was not significantly Figure 2. Mutations found in NHL tumors by Sanger re-sequencing. associated with OS or PFS in CHOP or R-CHOP-treated patients (all The number of mutations at each position is shown on the y axes; P-values40.05). We also tested within the GCB subtype and within distance along the BCL2 gene is indicated on the x axis. A model of the BCL2 gene is shown beneath the x axis of the top left cases that had a BCL2 translocation, and within cases with BCL2 figure, 50 -- 3 0 from left to right. non-synonymous mutations located within the FLD (in the presence or absence of a BCL2 translocation). No impact was File 3. Only 9/60 non-synonymous mutations were detected in found on OS or PFS. In a Cox regression model that included the BH domains, which are important for inhibiting apoptosis. International Prognostic Index, use of rituximab, presence of BCL2 In contrast, 18 non-synonymous mutations were found in sites expression and presence of mutations within the FLD, the effect of that would affect the BCL2’s putative p53-binding domain34 BCL2 mutations was also not statistically significant. In some (Figure 3), including an annotated caspase cleavage site at D34. analyses, the number of cases included was too small to make firm D34 mutants have reduced ability to induce apoptosis,35,36 conclusions about the prognostic significance of BCL2 mutations. including p53-mediated apoptosis.34 8/51 non-redundant, non- However, overall, BCL2 mutations do not seem to have an impact synonymous mutations were predicted to be ‘probably damaging’ on patient outcomes. by PolyPhen, while an additional 15/51 were predicted to be Given that novel BH3 mimetics, for example, ABT-737, are ‘possibly damaging’ (Figure 3). For instance, non-synonymous currently being evaluated in patients with lymphoid malignancies, mutations at R146 may impair the interaction between BCL2 and it is important to determine if BCL2 mutations may be a potential BAX.37 One variant introduced an early stop codon, which would cause of resistance to these agents. Of the 24 cell lines included in result in a form of BCL2 with no transmembrane domain. this study, we found non-synonymous mutations in the FLD of

& 2012 Macmillan Publishers Limited Leukemia (2012) 1383 --1390 BCL2 mutations in DLBCL JM Schuetz et al 1388 BCL2 in three cell lines that have been previously evaluated for cleavage signal at D34 (that we found mutated in DLBCL patients), responses to ABT-737.38 These included KARPAS 422 (with the which renders BCL2 pro-apoptotic.35,36 The FLD also has important mutations G47D and P59L), OCI-LY1 (with F49L mutation) and roles in paclitaxel-induced apoptosis,43 cell-cycle progression,44 --46 SU-DHL6 (with I48IF mutation). All three are very sensitive to and interaction with Nur77, a cell death-related protein.37 The ABT-737; thus mutations in the FLD may not interfere with the transmembrane domain of BCL2 is required for Nur77 to trigger binding of BH3 mimetics. Further work would be necessary to cell death; interestingly, we found a truncating mutation in one show whether these or other mutations impair the binding of BH3 sample, which would then lack this domain. mimetics to BCL2. Almost half of the observed non-synonymous mutations were predicted by PolyPhen to affect protein function. Although 28/51 unique non-synonymous mutations were marked ‘benign’ by DISCUSSION PolyPhen, this does not preclude a functional role. For example, The high frequency and non-random pattern of BCL2 mutations non-synonymous mutations at residue A43 were scored as that we and others8 have observed supports the notion that ‘benign’ by PolyPhen; but previous work47 has shown that these are noteworthy events in DLBCL. BCL2 mutations have been changes at this position provide resistance to autoimmunity via previously described in DLBCL and FL, but their frequency, reduction of T cells. compared with other genes mutated in the DLBCL genome, had The most common mutations observed in this study (amino not been established. Furthermore, previous studies have acids 59 and 60) are located in a regulatory binding site for p53.34 combined cell line data in analysis with primary patient samples, BCL-XL binds p53 through its own region homologous to the FLD making results hard to interpret. of BCL2, as well as through the loop between a3 and a4.48 The With novel sequencing technologies, we demonstrated that the helices a3 and a4 are located between BH3 and BH1 domains, and number of BCL2 mutations is at least threefold higher than that of we observed many non-synonymous mutations between these the second most mutated gene, PIM1, and only sixfold lower than domains of BCL2, with four cases containing non-synonymous that published for rearranged IGH.30 We show that BCL2 is mutations between the two helices (amino acids 117--123). mutated at a higher rate than all known targets of SHM29 except Phosphorylation of BCL2 affects several BCL2 functions, IGH in GCB DLBCL. Our data support other reports that BCL2 depending on cellular context, including survival,49 autophagy50 mutations are associated with the presence of a BCL2 transloca- and cell-cycle control.51 It would therefore be interesting to tion and likely occur as a consequence of aberrant SHM,8,32,39 --41 further study the functional effect of non-phosphomimetic suggesting that the BCL2 locus may have properties that hinder mutations at S87, such as S87R reported here. DNA repair machinery when occupied by other proteins in a GCB- Another novel aspect of this study is that it addresses the type specific manner. However, 6/39 (15%) of the cases with non- potential clinical ramifications of BCL2 mutations in DLBCL. Most synonymous mutations in this study occurred in the absence of a clinical laboratories use BCL2 antibody clone 124 from Dako BCL2 translocation, suggesting that rearrangement is not a (Abbott) to test for BCL2 protein expression. Mutations in the requirement for BCL2 mutations. FLD have been reported to result in ‘pseudo-negative’ or ‘false- The high transition/transversion ratio suggests that aberrant negative’ BCL2 expression,10 where BCL2 could be detected with SMH is a predominant feature of the GCB-type DLBCL but, unlike an alternative anti-BCL2 clone E17,9 suggesting that the E17 clone previous work,8 we do not find evidence for SHM in ABC-type is more accurate for determining BCL2 protein status. However, DLBCL or normal germinal center B cells. This could be due to the previous studies were done using selected cases and with larger number of ABC DLBCL samples in our study. Other targets relatively small sample sizes. We find that ‘pseudo-negative’ of aberrant (and normal) SHM have been described, including BCL2 protein expression with clone 124 is rare in de-novo DLBCL. BCL6, PAX5, MYC and RHOH.29 These have all been suggested as We therefore do not support recent arguments, suggesting that uniquely hypermutated in DLBCL and not in other NHLs. Our pathology laboratories change antibodies.9 transcriptome data revealed mutations in all of these genes As a group, BCL2 mutations do not appear to be associated with except RHOH, but of these only BCL2 showed a significant clinical outcome; however, a larger sample size would be required to enrichment for mutations in the GCB subtype (P ¼ 0.004; Table 2). confirm this. Should a larger cohort become available for such an There are three possible causes for the BCL2 mutations observed: analysis, sequencing the regions rich in mutations in the amino SHM machinery could be targeting (a) the IGH locus, (b) the BCL2 terminus of the protein is likely to provide sufficient data. We locus---targeting of which our data does not support since we do also found that three cell lines previously described as sensitive to not observe mutations in normal germinal center B cells and ABT-737(ref. 38) harbor BCL2 mutations in the ABT-737-binding area, (c) IGH/BCL2 sequence as brought together by t(14;18). BCL2 may indicating that these mutations are not sufficient to affect sensitivity be the only gene preferentially mutated in the GCB subgroup to BH3 mimetics. Interestingly, the best clinical responses to ABT-737 because BCL2 translocations are known to be associated with GCB or ABT-263 were found in patients with CLL,52 alymphomasubtype classification.6 After BCL2, PIM1 showed the next highest number that does not harbor mutations of BCL2. of potentially functional mutations, with non-synonymous single In summary, we report that BCL2 is the most commonly nucleotide variants in 17 out of 89 DLBCL samples. Notably, the mutated gene in GCB-type DLBCL. It is difficult to predict the single tumor with the most mutations in PIM1 (4 non-synonymous, effect of mutations on BCL2 function due to its pleiotropism, with 11 total) was of the ABC subtype. It is possible that mutations known roles in autophagy, ER Ca2 þ storage,53 cell-cycle entry and reflect the outcome of a cascade of events in which cell type apoptosis, in which it can act as protector or killer. BCL2 function origin affects chromatin configuration and gene expression, which also varies depending on which genes are co-expressed, and influences the likelihood that a specific gene is available for whether those genes are also mutated.37,54 translocation and/or mutation. BCL2 is a cell fate master switch, and a large portion of its Our sequence analysis of BCL2 in over 400 samples revealed a decision-making may rest on its large FLD. Systematic functional distribution of non-synonymous mutations (see Figure 3) that characterization of BCL2 mutants will be necessary to understand was not apparent in smaller sample sets,8,32,39 --41 the largest of the effects on cell fate decisions, B-cell development, lymphoma- which sequenced B3 kb in 121 samples.8 We found very few non- genesis and potentially treatment response. synonymous mutations in the BH domains required for BCL2 to block pro-apoptotic family members, but did find many mutations in the FLD. The FLD can act as an auto-inhibitory domain reducing CONFLICT OF INTEREST 42 anti-apoptotic potential. It contains the caspase-mediated The authors declare no conflict of interest.

Leukemia (2012) 1383 --1390 & 2012 Macmillan Publishers Limited BCL2 mutations in DLBCL JM Schuetz et al 1389 ACKNOWLEDGEMENTS 15 Gurudutta GU, Verma YK, Singh VK, Gupta P, Raj HG, Sharma RK et al. Structural We thank Bruce Woolcock for laboratory work assistance; Martin Hirst, Yongjun Zhao, conservation of residues in BH1 and BH2 domains of Bcl-2 family proteins. Angela Tam, Richard Moore and Thomas Zeng for assistance with RNA-seq data; FEBS Lett 2005; 579: 3503 --3507. Martin Krzywinski for assistance with the design of Figure 2; and Douglas E Horsman 16 Sattler M, Liang H, Nettesheim D, Meadows RP, Harlan JE, Eberstadt M et al. for cytogenetics input. JMS was supported by the Lionel McLeod Award by the Structure of Bcl-xL-Bak peptide complex: recognition between regulators of Alberta Heritage Foundation for Medical Research and by the Canadian Institutes for apoptosis. Science 1997; 275: 983 --986. Health Research (CIHR). JMS was a PhD candidate at the University of British 17 Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying Columbia and this work was submitted in partial fulfillment of the requirement for mammalian transcriptomes by RNA-Seq. Nat Methods 2008; 5: 621 --628. the PhD. RDM was a Vanier Scholar of the CIHR and holds a Senior Graduate 18 Morin RD, Johnson NA, Severson TM, Mungall AJ, An J, Goya R et al. Somatic Studentship from the Michael Smith Foundation for Health Research (MSFHR). DWS mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas and KT were supported by the Terry Fox Foundation Strategic Health Research of germinal-center origin. Nat Genet 2010; 42: 181 --185. Training Program in Cancer Research at Canadian Institutes of Health Research (Grant 19 Morin RD, Mendez-Lago M, Mungall AJ, Goya R, Mungall KL, Corbett RD et al. No. TGT-53912). ARB-W was a Senior Scholar of the MSFHR. RDG and JMC are Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma. supported by a Program Project Grant for the Terry Fox Foundation (019001). This Nature 2011; 476: 298 --303. project was funded in part with Federal funds from the National Cancer Institute, 20 Savage KJ, Johnson NA, Ben-Neriah S, Connors JM, Sehn LH, Farinha P et al. MYC National Institutes of Health, under Contract No. HHSN261200800001E. gene rearrangements are associated with a poor prognosis in diffuse large B-cell lymphoma patients treated with R-CHOP chemotherapy. Blood 2009; 114: 3533 --3537. AUTHOR CONTRIBUTIONS 21 Shustik J, Han G, Farinha P, Johnson NA, Ben Neriah S, Connors JM et al. JMS and NAJ performed the experiments and wrote the paper. RDM performed the Correlations between BCL6 rearrangement and outcome in patients with diffuse analysis of whole-genome libraries. All authors participated in study design and large B-cell lymphoma treated with CHOP or R-CHOP. 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