EZH2MutationsinFollicularLymphomafromDifferentEthnic ... · Results: Our results showed similar...

Human Cancer BiologySee related article by Amengual and O'Connor, p. 3047

EZH2Mutations in Follicular Lymphoma fromDifferent EthnicGroups and Associated Gene Expression Alterations

Shuangping Guo1,6, John K.C. Chan2, Javeed Iqbal6, Timothy McKeithan6, Kai Fu6, Bin Meng3, Yi Pan4,Wah Cheuk2, Donglan Luo5, Ruian Wang1, Weiwei Zhang6, Timothy C. Greiner6, and Wing C. Chan6

AbstractPurpose:Gain-of-functionmutations of enhancer of Zeste homolog 2 (EZH2) occur frequently in diffuse

large B-cell lymphomas and in follicular lymphomas. However, the frequency of EZH2mutation inChinese

follicular lymphomas and the potential targets affected by this mutation are unknown.

Experimental Design: We determined EZH2 codon 641 mutations in Chinese follicular lymphomas

(n¼ 124) and compared themwithWestern follicular lymphomas (n¼ 70) using a sensitive pyrosequencingassay. Gene expression profiling (GEP)was performed to determine differential gene expression between the

mutated versus unmutated subgroups, and selected genes were validated using immunohistochemistry.

Results:Our results showed similar frequencies of EZH2 codon 641 mutations in Chinese and Western

follicular lymphoma cohorts (16.9% vs. 18.6%, c2 test, P ¼ 0.773), including all five reported mutationvariants. We observed significant association of EZH2 mutation with low morphologic grade follicular

lymphomas (grade 1–2, 23.6%vs. grade 3, 7.7%, c2 test, P¼ 0.02). EZH2mutations also showed significantassociation with BCL2 rearrangement in the Chinese cohort (26.8% vs. 8.8%, c2 test, P ¼ 0.008) andcombined cohorts (26.3% vs. 9.1%, c2 test, P ¼ 0.002). GEP analysis identified several genes, includingTCF4, FOXP1, TCL1A, BIK, and RASSF6P, with significantly lower mRNA expression (P < 0.01) in mutatedcases, and the potential target TCL1A showed consistent results at the protein level.

Conclusion: Similar prevalence of EZH2 mutation in two ethnic groups suggests shared pathogenetic

mechanisms. The much lower frequency of EZH2mutation in cases without BCL2 translocation suggests a

different pattern of evolution of this subtype of follicular lymphoma. GEP studies showed a set of

differentially expressed genes and suggested that EZH2 mutation may help to lock the tumor cells at the

germinal center stage of differentiation. Clin Cancer Res; 20(12); 3078–86. �2014 AACR.

IntroductionFollicular lymphoma is a common germinal center B-cell

(GCB)-derived non-Hodgkin lymphoma (NHL) exhibitinga spectrum of morphologies, immunophenotypes, andgenetic aberrations. It is the second most common type ofNHL representing about 30% of NHL in Western countries(1, 2); however, the incidence of follicular lymphoma islower in Asian countries (3, 4). A recent study of 98 Chinesefollicular lymphoma cases revealed a lower frequency of

BCL2 rearrangement in Chinese follicular lymphoma(58.5%) than in Western follicular lymphoma (80%–90%; ref. 5). The BCL2 rearrangement results in constitutiveexpression of the antiapoptotic BCL2 protein, which playsan important role in tumorigenesis of follicular lymphoma;however, BCL2 rearrangement alone is not sufficient formalignant transformationofB cells.Multiple genetic abnor-malities, including mutation of enhancer of Zeste homolog2 (EZH2), have recently been reported in follicular lym-phoma (6–9). The EZH2 gene encodes a histone methyl-transferase that constitutes the catalytic component of thepolycomb repressive complex 2 (PRC2) and is involved inrepressing gene expression through methylation of histoneH3 on lysine 27 (H3K27). EZH2 codon 641 mutation, themost common mutation hotspot, is a gain-of-functionmutation leading to enhanced trimethylation of histoneH3K27 and plays an important role in the tumorigenesis ofGCB-type diffuse large B-cell lymphoma (DLBCL) andfollicular lymphoma (6–11). Interestingly, highly selectiveEZH2 inhibitors have now been developed which is apotential therapeutic strategy for lymphomas with EZH2activating mutations (12). However, it is unclear whetherChinese follicular lymphoma utilizes similar pathogeneticpathways as their Western counterpart and the frequency of

Authors' Affiliations: 1Department of Pathology, Xi Jing Hospital, theFourth Military Medical University, Xi'an, Shaan Xi Province; 2Departmentof Pathology, Queen Elizabeth Hospital, Hong Kong; 3Department ofPathology, Shandong University School of Medicine, Jinan, ShandongProvince; 4Department of Pathology, Tianjin Cancer Hospital; 5Departmentof Pathology, Guangdong General Hospital and Guangdong Academy ofMedical Sciences, Guangzhou, China; and 6Department of Pathology andMicrobiology, University of Nebraska Medical Center, Omaha, Nebraska

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Wing C. Chan, City of Hope Medical Center,1500E Duarte Rd, Duarte, CA 91010. Phone: 626-218-9437; Fax: 626-301-8842; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-1597

�2014 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 20(12) June 15, 20143078

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Published OnlineFirst March 14, 2014; DOI: 10.1158/1078-0432.CCR-13-1597

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EZH2 mutation in Chinese follicular lymphoma is notknown. The aim of the present study is to identify thefrequency of EZH2 gene codon 641 mutation in Chinesefollicular lymphoma and the association with the status ofBCL2 expression and rearrangement. We also analyzed thegene expression profiling (GEP) data of follicular lympho-mas with and without EZH2 gene mutation to identifyunique associations with EZH2 mutation in follicularlymphoma.

Materials and MethodsPatient materialsOne hundred twenty-four Chinese follicular lymphomas

diagnosed between January 1999 and May 2010 wereobtained from Hong Kong and provinces of Shandong,Tianjing, and Shaan Xi, China, among which 109 cases hadBCL2 rearrangement data detected by interphase FISH. Wealso included 70 Western follicular lymphomas with BCL2rearrangement data detected by FISH from the University ofNebraska Medical Center (UNMC; Omaha, NE) for com-parison. As follicular lymphoma with or without t(14;18)may evolve through different molecular pathways, ourinitial hypothesis is that EZH2 mutation may be presentat different frequencies in follicular lymphoma with differ-ent t(14;18) status. We enriched both cohorts with a sub-stantial number of follicular lymphoma cases with no BCL2rearrangement (from previous cytogenetic and FISH stud-ies) to test the hypothesis as such cases are uncommonparticularly in Western follicular lymphoma. The caseswere reviewed by expert hematopathologists (J.K.C. Chan,

W.C. Chan, and B. Meng) and re-reviewed by W.C. Chanand S. Guo, and classified on the basis of the World HealthOrganization criteria. These cases showed at least 30% to40% of the sectional areas consisting of malignant follicleson hematoxylin and eosin (H&E) examination. This studywas approved by the Institution Review Boards of therespective institutions.

Construction of tissue microarray andimmunohistochemistry

Representative areas of follicular lymphoma tissue blockswere selected to prepare the tissue microarray (TMA) byexamination of the corresponding H&E-stained slides. Toensure accurate representation, three areas of each tumorwere selected for core preparation. Each target area on theselected blocks was punched to obtain a 1-mm diametertissue core to be placed consecutively on the recipientmaster blocks. TMAs containing representative tumor sec-tions from 124 Chinese and 70 Western follicular lympho-mas were used for this retrospective analysis. Sections fromthe TMAs were stained with anti-human BCL2 (clone 124),CD20 (clone L26), BCL6 (clone PG-B6p), (Dako Corp),and CD10 (clone 56C6), CD21 (clone EP3093), CD3(clone SP7), (Abcam), and anti-TCL1A antibody (clone1–21, Santa Cruz Biotechnology Inc). Of note, 5 mm thicktissue sections were deparaffinized with xylene, washedwith ethanol, and blocked with methanol with 3% hydro-gen peroxide. Sections were incubated with the aboveantibodies using Bond polymer refine detection kit andbond IHC Stainer (Leica Microsystems Inc.). For TCL1Astaining, GCB cells and Na€�ve B cells in the mantle zone ofreactive tonsil were used as positive controls. The per-centage of positive tumor cells and the staining intensitywere graded from 0 to 3, and the two scores were added toform a final score to divide the cases from negative tostrong TCL1A expression as described in SupplementaryTable S1A.

FISHFull sections and/or TMAs of 124 Chinese follicular

lymphomas and 70 Western follicular lymphomas wereassessed for the presence of BCL2 rearrangement on 4 mmformalin-fixed, paraffin-embedded (FFPE) sections usingthe Vysis LSI BCL2 dual color break-apart rearrangementprobe (Abbott Molecular). Cases were scored as BCL2 rear-ranged on the basis of detecting unambiguous probe sep-aration above the set threshold.

DNA extraction, PCR, and pyrosequencingFive to 10 mm thick tissue sections of representative

FFPE tissue blocks were prepared. Genomic DNA wasextracted using a Qiagen DNA FFPE Tissue Kit. GenomicDNA encompassing EZH2 codon 641 was PCR amplifiedusing forward primer 50-GGCTGGGGGATTTTTATCAA-30

and biotinylated reverse primer 50- GTTATCAGTGCCT-TACCTCT CCA-30. The PCR products were sequenced on aQiagen Pyromark Q24 instrument using a forward sequenc-ing primer 50-GAAAAATGAATTCATCTCAG-30. Genomic

Translational RelevanceFollicular lymphoma is an indolent, but incurable

subtype of non-Hodgkin lymphoma. These tumors har-bor t(14;18) translocation in at least 90% of patients.Recently, activating enhancer of Zeste homolog 2(EZH2) mutations have been found in a significantnumber of patients with follicular lymphoma. However,the reported incidence has been quite variable and hascome almost exclusively in Western countries. We havedeveloped a sensitive pyrosequencing assay for all muta-tion variants affecting codon 641. Our study has shownthat Chinese and Western patients with follicular lym-phoma exhibit EZH2 Y641mutation at similar frequen-cies, and the mutation is far more frequently associatedwith theBCL2 rearranged group inbothpopulations.Wehave alsodemonstrated by gene expression profiling thatfollicular lymphoma cases with this mutation showeda set of significantly downregulated genes in EZH2-mutant cases. The gene signature provided some insighton the mechanisms of action of abnormally enhancedEZH2 function. EZH2mutations may play a similar rolein lymphomagenesis in different ethnic groups and canserve as a biomarker for potential EZH2 targeted therapy.

EZH2 Mutations in Follicular Lymphoma

www.aacrjournals.org Clin Cancer Res; 20(12) June 15, 2014 3079




DNA of cell line SU-DHL-6, harboring an EZH2 codonY641N mutation, and normal tonsil lacking EZH2 muta-tion, was mixed with SU-DHL-6 DNA constituting 5%,10%, 20%, 30%, 50%, and 70% of the total DNA, respec-tively. The mixture was used as template to determinethe sensitivity of pyrosequencing to detect the mutant.The sensitivity of the assay was determined to be at 5%of the genomic DNA of the cell line SU-DHL-6. Accordingto the principle of pyrosequencing, the light emission inthe process of sequencing is proportional to the quantityof deoxynucleotide triphosphate (dNTP) incorporatedinto the template. dNTPs were dispensed in a novel orderof G, A, A, T, C, G, A, T, and C, based on the sequence ofwild-type EZH2 codon 641 and five mutation variants.With this order of addition, at least one addition results innucleotide incorporation for the mutant but not the wild-type allele, allowing all five mutations to be identified anddetected with maximal sensitivity (Table 1). All detectedmutations were validated by repeating the assay.

GEP data analysisThe GEP data from 68 cases of UNMC follicular lym-

phoma were analyzed to find genes differentially expressedbetween follicular lymphomaswith orwithout EZH2muta-tion. We used HG-U133 plus 2 arrays (Affymetrix Inc.) forour analysis, and the methods for isolation and processingof RNA and acquisition of GEP raw data have beendescribed previously (13). The raw data were uploaded inBRB-Array Tools (version 3.7.0) for normalization, and theanalysis was supervised by the EZH2 gene mutation status.Genes were selected at a significance of P < 0.01 and >1.5-fold difference in expression levels between the two groups.To enhance the identification of differential gene expressionin follicular lymphoma tumor cells, only follicular lym-phoma cases with a high B-cell content [enriched B-cellsignature of >2-fold above the mean of the B-cell signature(pan B-cell markers) in the entire follicular lymphoma

dataset] were chosen for analysis (Data available in theGEO database: GSE 55267.)

Statistical analysisStatistical significance of differences in EZH2 mutation

frequency between lymphoma groups with or withoutBCL2 rearrangement and expression was assessed by c2 test.Statistical significance was defined as P < 0.05.

ResultsPatient characteristics

One hundred and twenty-four Chinese follicular lym-phomapatients were included in this study. Themedian ageof these patients was 59.5 years (range, 26–89 years), andincluded 64 (51.6%) males and 60 (48.4%) females. Thesecases were enriched in BCL2 translocation negative cases sothat only 56 harbored a BCL2 rearrangement and 68 lackeda BCL2 rearrangement. Among the 70 Western follicularlymphomas, the median age was 60 (range, 35–88 years),and there were 33 (47.1%) male and 37 (52.9%) femalepatients. This cohort was also enriched in BCL2 wild-typecases, 39 harbored a BCL2 rearrangement and 31 lacked aBCL2 rearrangement. There were no significant differencesin clinical features between Chinese and Western follicularlymphomas. On histologic morphologic assessment, 72 of124 (58%) in Chinese cohort and 39 of 70 cases (55.7%) inthe Western cohort were of grade 1–2.

Frequencies of EZH2 codon 641 mutationsWeobserved EZH2 codon 641mutations in 16.9% (21 of

124) Chinese follicular lymphoma and 18.6% (13 of 70)Western follicular lymphomas using pyrosequencing andthe results are summarized in Table 2. No significant dif-ference in the frequency of EZH2 mutation in follicularlymphomas between Chinese and theWestern patients wasobserved [16.9% (21/124) versus 18.6% (13/70), c2 test,P ¼ 0.773]. Representative results of pyrosequencing of

Table 1. EZH2 codon 641mutation variants by adding dNTPs in the designated order (G,A,A,T,C,G,A,T,C)during pyrosequencing

Amino acid change Nucleotide change Signal strength by nucleotide

Y641 TAC W-AATACTW-AATACT___A4 T2 C0 G0 A2 T0 C2

Y641F TAC>TTC W-AATACTM-AATTCT___A4 T3 C1 G0 A1 T1 C1

Y641S TAC>TCC W-AATACTM-AATCCT___A4 T2 C2 G0 A1 T1 C1

Y641C TAC>TGC W-AATACTM-AATGCT___A4 T2 C0 G1 A1 T0 C2

Y641N TAC>AAC W-AATACTM-AAAACT___A6 T1 C1 G0 A1 T1 C1

Y641H TAC>CAC W-AATACTM-AACACT___ A4 T1 C1 G0 A2 T0 C2

Abbreviations: W, wild-type; M, mutant; Y, tyrosine.

Guo et al.

Clin Cancer Res; 20(12) June 15, 2014 Clinical Cancer Research3080




wild-type EZH2 codon 641 and five mutant variants areshown in Fig. 1. The five mutation variants resulted in theamino acid change from tyrosine (Y) 641 to phenylalanine(F), serine (S), cysteine (C), asparagine (N), and histidine(H) as shown in Table 1. These five variants were detected in

Chinese follicular lymphoma with frequencies similar tothose of Western follicular lymphoma. Y641F was the mostcommon mutation, accounting for 38% of mutants, withdecreasing frequencies from Y641S (24%), Y641H (14%),Y641C (14%) to Y641N (10%).

Normal tonsil, wild-type EZH2 codon Y641 (A4 T2 C0 G0 A2 T0 C2) FL60, EZH2 codon Y641N (A6 T1 C1 G0 A1 T1 C1)

FL51, EZH2 codon Y641F (A4 T3 C1 G0 A1 T1 C1)

E S G A A T C G A T C E S G A A T C G A T C5

E S G A A T C G A T C E S G A A T C G A T C

E S G A A T C G A T C E S G A A T C G A T C5

FL45, EZH2 codon Y641C (A4 T2 C0 G1 A1 T0 C2) FL62, EZH2 codon Y641H (A4 T1 C1 G0 A2 T0 C2)FE

CFL66, EZH2 codon Y641S (A4 T2 C2 G0 A1 T1 C1)

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Figure 1. Pyrosequencing of EZH2 codon 641 mutation in representative follicular lymphomas (FL). A, normal tonsil, wild-type EZH2 codon Y641 (A4, T2, C0,G0, A2, T0, C2). B–F, dotted red lines show the wild-type pattern. Filled arrows show dNTP additions that yield incorporation into the mutant but not the wild-type allele. B, FL60, EZH2 codon Y641N (A6, T1, C1, G0, A1, T1, C1). C, FL51, EZH2 codon Y641F (A4, T3, C1, G0, A1, T1, C1). D, FL66, EZH2 codon Y641S(A4, T2, C2, G0, A1, T1, C1). E, FL45, EZH2 codon Y641C (A4, T2, C0, G1, A1, T0, C2). F, FL62, EZH2 codon Y641H (A4, T1, C1, G0, A2, T0, C2).

Table 2. EZH2 mutation and BCL2 rearrangement and expression in follicular lymphomas

Classification EZH2 mutation BCL2 rearranged BCL2 expression

All Chinese FL (n ¼ 124) 21 (16.9%) 56 (45.2%) 76 (61.3%)Grade 1–2 (n ¼ 72)a 17 (23.6%) 47 (65.3%) 57 (79.2%)Grade 3 (n ¼ 52) 4 (7.7%) 9 (17.3%) 19 (36.5%)All Western FL (n ¼ 70) 13 (18.6%) 39 (55.7%) 51 (72.9%)Grade 1–2 (n ¼ 39)b 10 (25.6%) 30 (76.9%) 33 (84.6%)Grade 3 (n ¼ 31) 3 (9.7%) 9 (29%) 18 (58.1%)Abbreviation: FL, follicular lymphoma.aIncludes grades 1 (n ¼ 33) and 2 (n ¼ 39).bIncludes grades 1 (n ¼ 17) and 2 (n ¼ 22).






Association between EZH2 mutation and morphologicfollicular lymphoma grades

The EZH2 mutation frequency was related to the mor-phologic follicular lymphoma grades, with higher fre-quency in low morphologic grade follicular lymphomasin both Chinese (grade 1–2, 23.6% vs. grade 3, 7.7%, c2

test, P¼ 0.02) andWestern population (grade 1–2, 25.6%vs. grade 3, 9.7%, c2 test, P ¼ 0.09; Table 2).The distri-bution of EZH2mutation in different morphologic gradesof Western follicular lymphoma was similar to that ofChinese follicular lymphoma. The percentage of EZH2mutation in grade 1 (7/33; 21.2%) and grade 2 (10/39;25.6%) follicular lymphoma was quite similar in Chinesefollicular lymphomas, and similar trend was observed inWestern follicular lymphomas.

Association between EZH2 mutation and BCL2translocation

As expected from our case selection, BCL2 rearrange-ments were detected in 56 of 124 (45.2%) and 39 of 70(55.7%) in Chinese and Western follicular lymphomas,respectively. BCL2 expressions were observed in 76 of124 (61.3%) and 51 of 70 (72.9%) in Chinese andWesternfollicular lymphomas, respectively. The positivity of BCL2rearrangement and expression was higher in grade 1–2 thanin grade 3 follicular lymphomas in the two cohorts (Table2). The frequency of EZH2 mutation was significantlyhigher in follicular lymphomas with a BCL2 rearrangementthan in those lacking a BCL2 rearrangement in Chinesefollicular lymphoma (26.8% vs. 8.8%, c2 test, P ¼ 0.008).However, the difference observed in Western follicularlymphoma did not reach statistical significance (25.6% vs.9.7%, c2 test, P ¼ 0.163). EZH2mutation was significantly

associated with BCL2 rearrangement in the combined Chi-nese and Western cases (26.3% vs. 9.1%, c2 test, P ¼0.002; Fig. 2). EZH2 mutation was also significantly asso-ciated with the BCL2 protein expression in Chinese (22.4%vs. 8.3%, c2 test, P ¼ 0.042) and the combined follicularlymphoma cohorts (22.8% vs. 7.5%, c2 test, P ¼ 0.007,respectively). The percentage of EZH2 mutation wasenriched in BCL2 protein-positive cases in Western cohort;however, it did not reach statistical significance (23.5% vs.5.3%, c2 test, P ¼ 0.155; Fig. 2).

Differential gene expression analysisOur analysis focused on follicular lymphoma cases with

high B-cell contents to facilitate the detection of changesrelated to the tumor cells. A set of genes was significantlydownregulated in mutated cases. Among these downregu-lated genes, a number of them, such as TCF4, FOXP1,TCL1A, BIK, and RASSF6P were also marked repressedcompared with normal centroblasts (Fig. 3). There wasalso a set of genes such as PTPN22, which was upregulatedin mutant cases probably as secondary changes. We alsocompared our signature with the specific gene signaturederived previously using an EZH2-specific inhibitor(GSK126) on GCB cell lines with EZH2 mutation (12).Notably, this signature was significantly (P < 0.02) down-regulated in cases with EZH2 mutation compared withwild-type cases (Fig. 3).

Downregulation of TCL1A in EZH2-mutated follicularlymphomas

One of the target genes that were reported to be upregu-lated upon treatment of EZH2-mutant cell lines by a specificinhibitor of EZH2-mutant protein (GSK126) is TCL1A (12).

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BCL2 rearranged

Chinese FLs (n = 124)

Chinese and Western FLs(n = 194)

Western FLs(n = 70)

BCL2 wild-type

BCL2-positive

BCL2-negative

Figure 2. EZH2mutation in follicularlymphomas (FL) by BCL2 status.EZH2 mutations were significantlymore frequent in cases withrearrangements by FISH andexpression of BCL2 by IHC inChinese follicular lymphoma andcombined Chinese and Westernfollicular lymphoma.

Guo et al.





Consistent with this observation, we observed that this genewas downregulated in follicular lymphoma cases carryingEZH2 Y641 mutation compared with our wild-type. Therehave been reports suggesting variable expression of TCL1Ain follicular lymphoma. We evaluated TCL1A expression atthe protein level and observed that B cells in the mantlezone of reactive tonsil showed strong expression of TCL1A,whereas GCB cells showed moderate expression in bothcytoplasm and nucleus. Of the follicular lymphoma caseswithout EZH2 Y641mutation, themajority of the follicularlymphoma cases (67.2%, 39 of 58) showed strong homog-enous expression of TCL1A, whereas 10.3% (6 of 58) weremoderate, and 22.4% (13 of 58) were either weak ornegative. Interestingly, none of the follicular lymphomacases with EZH2 Y641mutation showed strong expression;it was generally variable frommoderate (6 cases) to weak (1case), or negative (3 cases) in 10 mutant cases. The resultsare summarized in Supplementary Table S1B, and repre-sentative images are shown in Fig. 4.

DiscussionEZH2 is the catalytic subunit of the PRC2 and is involved

in repressing gene expression throughmethylation of lysine27 of histone H3. The EZH2 codon 641 mutation is a gain-of-function mutation leading to enhanced trimethylationof histone H3K27 (6). EZH2-mediated epigenetic silencingin GCB cells contributes to proliferation and lymphoma-genesis (14).The frequency of EZH2 mutations varied from 7.2% to

22% (Supplementary Table S2) in prior studies (7, 9–10);the variation is probably due to the different assay methods

used. Pyrosequencingwas used in the present study becauseit is more sensitive than conventional Sanger sequencingand is widely used clinically to detect point mutations inFFPE tissues. The assay was able to detect 5% of the mutantgenomic DNA of the cell line SU-DHL-6, which harbors anEZH2 codon Y641Nmutation. Assays with lower sensitivitymay miss the mutation in cases with low tumor content,which are not uncommon in follicular lymphomas. Wedetected a similar frequency of EZH2 codon 641mutationsin Chinese follicular lymphoma [21 of 124 (16.9%)] and inWestern follicular lymphoma [13 of 70 (18.6%)]. All of thefive reported mutations, Y641F, Y641S, Y641C, Y641H,and Y641N, were detected in Chinese follicular lympho-ma with frequencies similar to those previously reportedin Western follicular lymphoma (7, 9–11). Y641F was themost common mutation, accounting for 38% of mutants.It has been reported that all of the five mutant proteinsconfer a gain-of-function phenotype and that Y641N isthe most potent at generating H3K27me3, whereasY641H is the least potent (8).

The BCL2 rearrangement is a genetic hallmark of follic-ular lymphomas, but a small percentage of follicular lym-phoma cases lack this rearrangement; the latter are muchmore frequent in the Chinese population (5). It is possiblethat cases with or without BCL2 rearrangement may havedifferent pathogenetic mechanisms that may be reflected indifferent profiles of genetic mutations. A recent study inWestern patients indeed found amarkeddifference inEZH2mutation frequency between the two subgroups (7). Weinvestigated the association of EZH2 mutation and thestatus of BCL2 rearrangement in follicular lymphoma,including a substantial number of cases with no BCL2

Relative level of expression (× median value)

Wild-typeEZH2

MutantEZH2 C

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EZH2 mRNA

CYSLTR1TOX2CCDC50TCF4SMAD1CCL17PTPN1FAIM3LOC221442DOPEY2MYO7ATFPI2CCL22PLCL2TRAM2P2RY10TCF4TCF4CNKSR2PVALBADD3TFPI2LOC374443FZD3MYO1DTCF4TCF4FOXP1TCF4RASSF6FOXP1RASGRP2IGHMTCL1ATCL1ABIKE2F5TAGAP

RRAS2SC4MOLCYP51A1PTPN22PTPN22SPAG1

DHCR24PTPN22TXLNBIRAK2

RANBP2GRAMD1C

SYT11BTNL9ILDR1TESK2BRIP1GATMKCNH8INPP5FUSP12HS2ST1

TEX9INPP5FINPP5FRRAS2

C2orf88BFSP2

KIAA1274

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EZH2 target gene signature (mean)*

0.25 0.5 1 2 4

Figure 3. Differential geneexpression between EZH2 mutantversus wild-type follicularlymphoma cases (P < 0.01;>1.5-fold change). Only cases withhigh B-cell content were includedin the analysis. �, EZH2 target genesignature was derived in GCB celllines with mutant EZH2 genetreated with EZH2-specificinhibitor (GSK126). The authorshave included only upregulatedgenes (35 probe set) in at least fourof five cell lines after inhibition ofmutant EZH2 (12). CB, centroblast,CC, centrocyte.






rearrangement. The frequency of EZH2 mutation was sig-nificantly associated with BCL2 rearrangement in Chinesefollicular lymphoma, but did not reach statistical signifi-cance in Western follicular lymphoma, probably due to thesmall number of cases. However, overall (combining bothcohorts) EZH2 mutation was significantly associated withBCL2 rearrangement. The frequency of EZH2 codon 641mutation in Chinese follicular lymphoma with BCL2 rear-rangement was very close to that reported in a previouslystudy (28%), using a sensitive detection technique (7). Themutation frequency in Chinese follicular lymphoma lack-ing BCL2 rearrangement was low and comparable withsimilar patients in Western countries but far higher thanreported previously (7). As EZH2 codon 641 mutation wasfirst identified in a grade 1 follicular lymphoma lackingBCL2 rearrangement (9), the reported lack of EZH2 muta-

tion in cases negative for BCL2 rearrangement (7) is prob-ably spurious and due to the low number of cases investi-gated. The low frequency of EZH2 mutation in follicularlymphomas without BCL2 rearrangement and in grade 3follicular lymphoma suggests the preferential use of differ-ent pathways to promote the malignant transformation ofGCB lymphocytes to follicular lymphoma.

Although it has been demonstrated that the EZH2 codon641 mutation enhances trimethylation of histone H3K27,the genomic targets that are affected by the mutation andthus promote lymphomagenesis have not been identified.Our GEP data analysis revealed a set of differentiallyexpressed genes; among them TCF4, FOXP1, RASSF6,TCL1A, and BIK were downregulated, compared with bothwild-type follicular lymphoma and normal centroblast.Both TCF4 and FOXP1 were consistently upregulated in

A B

C D

E

Figure4. Expressionof TCL1A in follicular lymphomas.A, expressionof TCL1A inGCandmantle zonecells in a reactive tonsil (�40magnification). B, a follicularlymphoma casewithout EZH2Y641mutation showing strong TCL1A expression (�40magnification). C, follicular lymphoma caseswith EZH2Y641mutationshowing moderate expression of TCL1A (�40 magnification). D, mutant with weak TCL1A expression (�20 magnification). E, mutant negative for TCL1A,scattered T cell and mantle zone B cells were strongly positive (�40 magnification).

Guo et al.





ABC-DLBCL compared with GBC-DLBCL (15). This obser-vation suggests that one of the functions of EZH2mutationmay be locking the cells at the GCB cell stage of differen-tiation by downregulating the expression of crucial genesfor further differentiation. TCL1A is also of particularlyinterest; it was initially identified as an oncogene in T-cellprolymphocytic leukemias, but may also be expressed in B-cell lymphomas, including follicular lymphoma (16).TCL1A serves as a coactivator of the AKT/mTOR pathwayand has also been reported as an inhibitor of de novo DNAmethylation in B-cell chronic lymphocytic leukemia (17),so downregulation of TCL1A may have diverse effects,including epigenetic alterations. We further confirmed thefrequent expression of TCL1A protein in follicular lympho-mas. It was revealed that follicular lymphomas displayedstriking variability in the intensity of TCL1A staining andthere is a correlationwithEZH2Y641mutation. Thirty-ninecases showing strong positivity for TCL1A harbored noEZH2 Y641 mutation, whereas the expression of TCL1A inthemutant cases varied frommoderate toweak, or negative,and none showed strong positivity. The immunohisto-chemical results are in keeping with the GEP analysis andmay explain the reported variability of TCL1A expression infollicular lymphoma. Interestingly, it was also noted thatthe expression of TCL1A increased in DLBCL cell linesharboring EZH2 mutation when treated with the EZH2-specific inhibitor GSK126 (12). This observation is consis-tent with our finding and suggests that TCL1A is likelytargeted by EZH2 mutation in follicular lymphoma. Wealso observed that specific gene signature generated byEZH2-mutant inhibitor (GSK126; ref. 12) showed expectedcorrelation inmutant follicular lymphoma cases, suggestingthat such inhibitors may provide a rational and viabletherapeutic approach for these patients. Downregulationof BIK (Bcl2-interacting killer), a proapoptotic gene, is alsoof interest as 40% of cases of multiple myeloma werereported tohave amethylatedBIKCpG island (18). Anotherdownregulated gene is RASSF6, a Ras-association family(RASSF) member that has been shown to be frequentlyepigenetically inactivated by promoter CpG island hyper-methylation in childhood leukemias (19).Among the upregulated genes, PTPN22 is intriguing. The

expression of PTPN22 in follicular lymphomas with EZH2

mutation is significantly higher than in normal B cells(na€�ve B cells, centrocytes, and centroblasts) and in follic-ular lymphomas without EZH2 mutation. PTPN22 is con-sidered to be one of the principal negative regulators ofantigen receptor signaling in normal B and T lymphocytes,but it can also positively regulate the kinase AKT, providinga powerful survival signal. B-cell receptor signaling in fol-licular lymphoma may need to be finely tuned to avoidfurther differentiation, and simultaneous activating theAKTpathway may optimize cell survival, perhaps compensatingfor the effects of TCL1 downregulation on the AKT/mTORpathway (20).

In conclusion, EZH2 codon 641 mutations occur atsimilar frequency in Western and Chinese follicular lym-phoma andmore often associatedwith low-grade cases. Themutations are far more frequent in the BCL2 rearrangedgroup. This mutation, probably plays a similar role in thepathogenesis of follicular lymphomas in both Chinese andWestern patients and may serve as a marker for potentialEZH2 targeted therapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Iqbal, W.C. ChanDevelopment of methodology: S. Guo, T. McKeithan, T.C. Greiner, W.C.ChanAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): J.K.C. Chan, J. Iqbal, K. Fu, B. Meng, Y. Pan,W. Cheuk, D. Luo, T.C. GreinerAnalysis and interpretation of data (e.g., statistical analysis): S. Guo,J.K.C. Chan, J. Iqbal, K. Fu, R. Wang, W. Zhang, T.C. GreinerWriting, review, and/or revision of the manuscript: S. Guo, J.K.C. Chan,J. Iqbal, T. McKeithan, K. Fu, T.C. Greiner, W.C. ChanAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): B.Meng, Y. Pan, D. Luo,W.C. Chan

AcknowledgmentsThe authors thank Martin Bast for the clinical data collection and Debra

Lytle for technical assistance.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 14, 2013; revised January 8, 2014; accepted February 2,2014; published OnlineFirst March 14, 2014.

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Guo et al.




2014;20:3078-3086. Published OnlineFirst March 14, 2014.Clin Cancer Res Shuangping Guo, John K.C. Chan, Javeed Iqbal, et al. Groups and Associated Gene Expression AlterationsEZH2 Mutations in Follicular Lymphoma from Different Ethnic

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