NK-cell neoplasms

Pathology (- 2016) -(-), pp. 1–16

H A E M ATO L O G Y

Aberrant differential expression of EZH1 and EZH2 in Polycomb repressive complex 2 among B- and T/NK-cell neoplasms LAMIA ABDALKADER1,2, TAKASHI OKA1, KATSUYOSHI TAKATA1, HIAKI SATO3, ICHIRO MURAKAMI1,4, ARIE P. OTTE5 AND TADASHI YOSHINO1 1

Department of Pathology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan; 2Department of Pathology, Faculty of Medicine, Mansoura University, Egypt; 3Department of Medical Technology, Graduate School of Health Sciences, Okayama University, Okayama, 4Department of Molecular Pathology, Tottori University Medical School, Japan; and 5Department of Biochemistry Swammerdam Institute for Life Sciences, University of Amsterdam, Netherlands

Summary The Polycomb repressive complex-2 members (EZH2, EED, SUZ12 and EZH1) are important regulators of haematopoiesis, cell cycle and differentiation. Overexpression of EZH2 has been linked to cancer metastases and poor prognosis. Detailed information on the expression of other members in normal and neoplastic lymphoid tissue remains to be elucidated. Immunohistochemical and immunofluorescent analyses of 156 samples from haematopoietic neoplasms patients and 27 haematopoietic cell lines were used. B-cell neoplasms showed a significant over-expression of EZH2, EED and SUZ12 in the aggressive subtypes compared to the indolent subtypes and normal tissue (p = 0.000–0.046) while expression of EZH1 was decreased in mantle cell lymphoma compared to normal tissue (p = 0.011). T/NK-cell neoplasms also showed significant overexpression of EZH2, EED and SUZ12 (p = 0.000–0.002) and decreased expression of EZH1 (p = 0.001) compared to normal cells. EZH2 and EZH1 have opposite expression patterns both in normal and neoplastic lymphoid tissues as well as an opposite relation to Ki-67. These results were supported by western blotting analyses. Immunofluorescent staining revealed a difference in the intracellular localisation of EZH1 compared to other members. These evidences suggest that EZH2 and EZH1 are important in the counter-balancing mechanisms controlling proliferation/resting of lymphoid cells. The disruption of the balanced EZH2/EZH1 ratio may play important roles in the pathogenesis of lymphomas. Key words: EZH2; EZH1; EED; SUZ12; Polycomb group. Received 2 February, revised 3 May, accepted 4 May 2016 Available online: xxx

INTRODUCTION The process of lymphomagenesis is a multi-step event comprising both genetic and epigenetic alterations and resulting in a large group of B- and T/NK-cell neoplasms.

These tumours show a wide spectrum of morphological features that vary from a nearly intact nodal architecture in low grade subtypes to a diffuse pattern in large B- and T/NKcell lymphomas. Prognosis and therapeutic approaches vary greatly between the subtypes. Early correct subtyping is of great importance in terms of clinical outcomes.1–5 Polycomb group (PcG) proteins are important regulators of haematopoiesis and cell cycle check points through epigenetic manipulation of gene expression.6,7 They form large multimeric complexes known as Polycomb repressive complexes (PRCs) that induce histone modifications. So far, two main PcG complexes have been identified in mammals: PRC1 and PRC2. PRC1 complexes include several complexes that differ in their localisation and function. Recently, six subgroups have been identified and numbered (PRC1.1–1.6).8 Mammalian PRC2 includes two paralogs of Enhancer of zeste homologue 1 and 2 (EZH1, EZH2), and the core subunits embryonic ectoderm development (EED), suppressor of Zeste 12 (SUZ12) and RBBP4/7 and Jarid2. The essential function of PRC2 is the methylation of histone H3 on lysine (K) 27 generating (H3-K27me3) which is considered a mark of gene silencing. The catalytic subunits of PRC1 and PRC2 complexes are RING1A/B and EZH1/ EZH2, respectively.8–10 This mechanism results in compaction of chromatin as well as silencing of important tumour suppressors and genes of development, differentiation, adhesion, cell cycle and proliferation.11–14 The process of H3-K27 methylation is achieved through a highly cooperative mechanism of the four core PRC2 subunits.15,16 The catalytic subunit EZH2/EZH1 bears a SET domain which contains the enzyme active site. However, the enzyme is basically inactive on its own and requires critical inputs from its partner subunits EED, SUZ12, and RBBP4.17,18 The exact role and extent of interactions of EED are not clear yet. EED protein exists in four isoforms (EED1, EED2, EED3 and EED4), which arise from alternate translation initiation sites in the same mRNA.19 Besides its interactions with EZH2, EED was also reported to interact with histone deacetylase (HDAC) proteins, in a highly specific manner, both in vitro and in vivo.20 EED knockout embryos die during early gestation in mice.21

Print ISSN 0031-3025/Online ISSN 1465-3931 © 2016 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved. DOI: http://dx.doi.org/10.1016/j.pathol.2016.05.002 Please cite this article in press as: Abdalkader L, et al., Aberrant differential expression of EZH1 and EZH2 in Polycomb repressive complex 2 among B- and T/NK-cell neoplasms, Pathology (2016), http://dx.doi.org/10.1016/j.pathol.2016.05.002

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ABDALKADER et al.

Table 1

Patient age, gender and tumour location of B- and T/NK-cell neoplasms tested

Malignant lymphoma

Gender

Age range (mean age)

Nodal/Total

M

F

BL DLBCL-GCB DLBCL non-GCB FLG3 MCL FLG1 PCM PC CLL/SLL MALT lymphoma

10 4 8 5 8 5 6 6 6 2

0 6 2 5 2 5 4 2 2 8

8–70 (32.56) 26–79 (57.40) 38–83 (64.00) 34–83 (66.10) 57–78 (68.11) 38–74 (58.30) 42–85 (63.90) 45–84 (63.37) 24–72 (59.71) 28–82 (61.80)

2/10 2/10 4/10 6/10 6/10 3/10 0/10 1/8 8/8 0/10

B-cell neoplasms

60

36

8–85 (59.63)

32/96

T-LBL ATLL PTCL ALCL AITL CTCL NK/T

8 7 4 6 4 3 6

2 3 6 4 1 2 4

17–77 44–83 42–78 20–80 58–90 35–72 40–79

6/10 1/10 4/10 7/10 5/5 0/5 2/10

T- and NK-cell neoplasms

38

22

17–90 (57.61)

(39.60) (64.90) (64.50) (54.70) (73.25) (59.40) (58.20)

25/60

AITL, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large cell lymphoma; ATLL, adult T-cell leukaemia/lymphoma; BL, Burkitt lymphoma; CLL/SLL, chronic lymphocytic leukaemia/small lymphocytic lymphoma; CTCL, cutaneous T-cell lymphoma; DLBCL-GCB, diffuse large cell B-cell lymphoma germinal centre type; DLBCL non-GCB, diffuse large B-cell lymphoma non-germinal centre type; FLG1, follicular lymphoma grade 1; FLG3, follicular lymphoma grade 3; MALT, mucosa associated lymphoid tissue lymphoma; MCL, mantle cell lymphoma; NK/T, natural killer/T-cell lymphoma; PCM, plasma cell myeloma; PC, plasmacytoma; PTCL, peripheral T-cell lymphoma; T-LBL, T-cell lymphoblastic lymphoma.

EZH2 overexpression has been reported in solid organ cancers, including breast, prostate and stomach cancer. It is associated with proliferation, poor prognosis and treatment failure.22–26 Somatic gain of function mutations in the SET domain of EZH2 were reported in follicular lymphomas (FL) and diffuse large B cell lymphoma (DLBCL).27,28 On the Table 2

other hand, inactivating mutations of both EZH2 and SUZ12 have been described in T-cell acute lymphoblastic leukaemia (T-LBL)29,30 as well as in myeloid malignancies.31 A lot remains unknown about the expression profiles of PRC2 complex members and their significance in haematopoietic tumours, particularly EED, SUZ12 and EZH1, as they are

Culture conditions and origin of cell lines

Cell origin

Cell line name

Culture conditions

T-cell acute lymphoblastic leukaemia (T-LBL)

Jurkat MOLT4 IWA1 MT4 MT2 ED50823 ATL16T ATL45T ED40515 ATL55T NK-L Ramos Dawdi Raji Scott DLBCL2 SU-DHL-4 FL218 FL318 FL18 FL18EB TW-EBV-LCL KMS-21 KMS-34 KMS-12-PE HeLa hMSCs

RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS, 20u/mL IL2 RPMI1640, 10%FCS RPMI1640, 10%FCS, 20u/mL IL2 RPMI1640, 10%FCS RPMI1640, 10%FCS, 20u/mL IL2 RPMI1640, 15%FCS,100u/mL IL2 RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS RPMI1640, 10%FCS DMEM, 5%FCS DMEM, 5%FCS

HTLV-1-immortalised T-cell

Adult T-cell leukaemic cell (ATLL) Natural killer/T cell (NK/T) Burkitt lymphoma (BL)

B-cell acute lymphoblastic leukaemia (B-LBL) Diffuse large B-cell lymphoma (DLBCL) Follicular lymphoma (FL)

EBV-immortalised normal B-cell (LCL) Plasma cell myeloma Cervical cancer cell Human mesenchymal stem cell EBV, Epstein–Barr virus; HTLV-1, human T-cell leukaemia virus-1.

Please cite this article in press as: Abdalkader L, et al., Aberrant differential expression of EZH1 and EZH2 in Polycomb repressive complex 2 among B- and T/NK-cell neoplasms, Pathology (2016), http://dx.doi.org/10.1016/j.pathol.2016.05.002

EZH CORRELATION WITH AGGRESSIVENESS

Table 3

3

EZH2 and EED expression in different subtypes of B-, T/NK-cell neoplasms and normal control

Malignant lymphoma normal control

Specimen number

EZH2

EED

0

1

2

3

Positivity score/max

Positivity score %

0

1

2

3

Positivity score/max

Positivity score %

BL DLBCL-GCB DLBCL non-GCB FLG3 MCL FLG1 PCM PC CLL/SLL MALT

10 10 10 10 10 10 10 8 8 10

0 0 1 1 4 6 8 7 7 10

0 0 0 0 3 1 1 1 0 0

4 4 5 9 3 3 1 0 1 0

6 6 4 0 0 0 0 0 0 0

26/30 26/30 22/30 18/30 9/30 7/30 3/30 1/24 2/24 0/30

86.7% 86.7% 73.3% 60.0% 30.0% 23.3% 10.0% 4.2% 8.3% 0%

1 0 2 1 5 6 8 7 7 8

1 0 0 2 4 1 2 1 0 1

3 6 5 7 1 3 0 0 1 1

5 4 3 0 0 0 0 0 0 0

22/30 24/30 19/30 16/30 6/30 7/30 2/30 1/24 2/24 3/30

73.3% 80.0% 63.3% 53.3% 20.0% 23.3% 6.7% 4.2% 8.3% 10.0%

B-cell neoplasms

96

44

6

30

16

114/288

39.6%

45

12

27

12

102/288

35.4%

T-LBL ATLL PTCL ALCL AITL CTCL NK/T

10 10 10 10 5 5 10

0 2 1 1 1 1 1

0 1 2 4 0 1 0

9 6 7 5 4 3 8

1 1 0 0 0 0 1

21/30 16/30 16/30 14/30 8/15 7/15 19/30

70.0% 53.3% 53.3% 46.7% 53.3% 46.7% 63.3%

1 5 4 3 2 2 4

5 3 2 3 1 2 3

4 2 4 3 2 1 2

0 0 0 1 0 0 1

13/30 7/30 10/30 12/30 5/15 4/15 10/30

43.3% 23.3% 33.3% 40.0% 33.3% 26.7% 33.3%

T/NK-cell neoplasms

60

7

8

42

3

101/180

56.1%

21

19

18

2

61/180

33.9%

10 10 10 10 10 10 10 10

10 0 0 10 10 10 10 10

0 0 5 0 0 0 1 1

0 10 5 0 0 0 0 0

0 0 0 0 0 0 0 0

0/30 20/30 15/30 0/30 0/30 0/30 1/30 1/30

0% 66.7% 50.0% 0% 0% 0% 3.3% 3.3%

10 0 1 10 10 10 8 10

0 2 9 0 0 0 2 1

0 8 0 0 0 0 0 0

0 0 0 0 0 0 0 0

0/30 18/30 9/30 0/30 0/30 0/30 2/30 1/30

0% 60.0% 30.0% 0% 0% 0% 6.7% 3.3%

6 6 4

0 5 4

0 1 0

6 0 0

0 0 0

12/18 1/18 0/12

66.7% 5.6% 0%

0 4 4

0 2 0

6 0 0

0 0 0

12/18 2/18 0/12

66.7% 11.1% 0%

RLH and tonsil Mantle zone GC dark zone GC light zone Post GC B-cells Plasma cells Interfollicular T-zone Follicular T-helper cells NK/T cells Normal thymus Cortical T-cells Medullary T-cells Cutaneous T-cells

Positivity includes three intensity scores 1, 2 and 3. Total positivity score of each subtype = the sum of intensity scores of the specimens. Percentage = the positivity score/max score (specimen number × intensity score 3). AITL, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large cell lymphoma; ATLL, adult T-cell leukaemia/lymphoma; BL, Burkitt lymphoma; CLL/SLL, chronic lymphocytic leukaemia/small lymphocytic lymphoma; CTCL, cutaneous T-cell lymphoma; DLBCL-GCB, diffuse large cell B-cell lymphoma germinal centre type; DLBCL non-GCB, diffuse large B-cell lymphoma non-germinal centre type; FLG1, follicular lymphoma grade 1; FLG3, follicular lymphoma grade 3; GC, germinal centre; MALT, mucosa associated lymphoid tissue lymphoma; MCL, mantle cell lymphoma; MZ, mantle zone; NK/T, natural killer/Tcell lymphoma; Norm NK/T, normal natural killer T-cell; Norm PC, normal plasma cells; PCM, plasma cell myeloma; PC, plasmacytoma; Post GC, post germinal centre B-cells; PTCL, peripheral T-cell lymphoma; T-LBL, T-cell lymphoblastic lymphoma.

Paraffin blocks of 156 B- and T/NK-cell neoplasm samples (obtained at diagnostic level) from the archives of the Department of Pathology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, were used. The patients’ demographic data are included in Table 1. Reactive lymphoid hyperplasia (RLH, 10 specimens), inflammatory non-neoplastic skin (5 specimens) and thymus (6 specimens) were used as normal control. This study was conducted under general consent from all patients and approved by the Institutional Review Board at Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University and related hospitals, in accordance with the Declaration of Helsinki.

(Dako, Denmark), BCL6 (Santa Cruz Biotechnologies, USA) and SUZ12 (Abcam, USA). Rabbit anti-EZH1 (Abcam) and anti-Ki-67 (Novocastra) were used. Staining was performed with Leica BOND-MAX autostainer (Leica Biosysytems, Australia). Each case was evaluated in 10 high-power fields by three investigators. Positivity was defined as 25% nuclear staining of tumour cells in accordance with previous reports 25 and further scored as 1, 2 or 3 based on the intensity of staining. The positivity score of each lymphoma subtype was defined as the sum of intensity scores of the specimens, then the positivity percentage was calculated as the positivity score/Max expected score (specimen number × intensity score). High Ki-67 labelling index was defined as nuclear staining of 20% of tumour cells. Classification and comparisons with the postulated cells of origin were carried out according to Revised European American Lymphoma (REAL)/World Health Organization (WHO) classification 2008. Distinction between aggressive and indolent variants was made according to guidelines from WHO and previous literature.5,26,34,26 DLBCL cases were divided into two groups, germinal centre type (DLBCL-GCB) and non-germinal centre type (DLBCL non-GCB) following Hans’ algorithm in accordance with other studies.35–37

Immunohistochemical staining

Double fluorescent immunohistochemical staining

The following mouse monoclonal antibodies were used: EED (M26), EZH2 (clone M18),32,33 CD3 and CD20 (Novocastra, UK), CD10, CD56 (Novocastra), CD4, CD8 (Nichirei Biosciences, Japan), CD68, CD138, MUM-1

Cells were washed with PBS then processed to cytospin (105 per slide) and fixed in cold acetone. Blocking was done by incubation in 5% skim milk for 5 h at room temperature. After washing, cells were treated with a mix of two

less studied in literature. This study investigated the expression patterns of EZH2, EED, SUZ12 and EZH1 in different subtypes of B- and T/NK-cell neoplasms.

MATERIALS AND METHODS Patient samples

Please cite this article in press as: Abdalkader L, et al., Aberrant differential expression of EZH1 and EZH2 in Polycomb repressive complex 2 among B- and T/NK-cell neoplasms, Pathology (2016), http://dx.doi.org/10.1016/j.pathol.2016.05.002

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Table 4

SUZ12 and EZH1 expression in different subtypes of B- and T/NK-cell neoplasms and normal control

Malignant lymphoma and normal control

Specimen number

SUZ12

EZH1

0

1

2

3

Positivity score/max

Positivity score %

0

1

2

3

Positivity score/max

Positivity score %

BL DLBCL-GCB DLBCL non-GCB FLG3 MCL FLG1 PCM and PC CLL/SLL MALT lymphoma

6 6 6 6 6 6 6 6 10

1 1 2 1 3 1 3 4 6

0 0 0 0 3 1 2 2 4

4 1 3 2 1 2 1 0 0

1 4 2 3 0 0 0 0 0

11/18 14/18 12/18 13/18 5/18 5/18 4/18 2/18 4/30

61.1% 77.8% 66.7% 72.2% 27.8% 27.8% 22.2% 11.1% 13.3%

4 5 6 6 5 3 2 5 6

2 1 0 0 1 2 2 0 2

0 0 0 0 0 1 2 1 2

0 0 0 0 0 0 0 0 0

2/18 1/18 0/18 0/18 1/18 4/18 6/18 2/18 6/30

11.1% 5.6% 0% 0% 5.6% 22.2% 33.3% 11.1% 20.0%

B-cell neoplasms

58

22

12

14

10

70/174

40.2%

43

10

6

0

22/174

12.6%

6 6 6 6 5 5 6

0 2 1 3 0 0 1

3 2 1 0 5 5 0

3 2 4 2 0 0 5

0 0 0 1 0 0 0

9/18 6/18 9/18 7/18 5/15 5/15 10/18

50.0% 33.3% 50.0% 38.9% 33.3% 33.3% 55.6%

5 3 5 5 4 3 6

0 1 0 0 1 2 0

1 1 1 1 0 0 0

0 1 0 0 0 0 0

2/18 6/18 2/18 2/18 1/15 2/15 0/18

11.1% 33.3% 11.1% 11.1% 6.7% 13.3% 0%

40

7

16

16

1

51/120

42.5%

31

4

4

1

15/120

12.5%

6 6 6 6 6 6 6 6

6 0 0 6 5 4 4 5

0 0 6 2 1 2 2 1

0 6 0 0 0 0 0 0

0 0 0 0 0 0 0 0

0/18 12/18 6/18 2/18 1/18 2/18 2/18 1/18

0% 66.7% 33.3% 11.1% 5.6% 11.1% 11.1% 5.6%

6 6 6 2 23 0 3 3

3 0 0 4 4 3 2 2

3 0 0 0 0 3 1 1

0 0 0 0 0 0 0 0

9/18 0/18 0/18 4/18 4/18 9/18 4/18 3/18

50.0% 0% 0% 22.2% 22.2% 50.0% 22.2% 22.2%

6 6 4

0 4 2

0 2 2

6 0 0

0 0 0

12/18 2/18 2/12

66.7% 11.1% 16.7%

6 4 0

0 0 2

0 2 2

0 0 0

0/18 4/18 6/12

0% 22.2% 50.0%

T-LBL ATLL PTCL ALCL AITL CTCL NK/T T-cell neoplasms RLH and tonsil Mantle zone GC dark zone GC light zone Post GC B-cells Plasma cells Interfollicular T-zone Follicular T-helper cells NK/T cells Normal thymus Cortical T-cells Medullary T-cells Cutaneous T-cells

Positivity includes three intensity scores 1, 2 and 3. Total positivity score of each subtype = the sum of intensity scores of the specimens. Percentage = the positivity score/max score (specimen number × intensity score 3). AITL, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large cell lymphoma; ATLL, adult T-cell leukaemia/lymphoma; BL, Burkitt lymphoma; CLL/SLL, chronic lymphocytic leukaemia/small lymphocytic lymphoma; CTCL, cutaneous T-cell lymphoma; DLBCL-GCB, diffuse large cell B-cell lymphoma germinal centre type; DLBCL non-GCB, diffuse large B-cell lymphoma non-germinal centre type; FLG1, follicular lymphoma grade 1; FLG3, follicular lymphoma grade 3; GC, germinal centre; MALT, mucosa associated lymphoid tissue lymphoma; MCL, mantle cell lymphoma; MZ, mantle zone; NK/T, natural killer/Tcell lymphoma; Norm NK/T, normal natural killer T-cell; Norm PC, normal plasma cells; PCM, plasma cell myeloma; PC, plasmacytoma; Post GC, post germinal centre B-cells; PTCL, peripheral T-cell lymphoma; T-LBL, T-cell lymphoblastic lymphoma.

primary antibodies for 3–5 h at room temperature. Washing was repeated, then incubation with a mix of anti-mouse and anti-rabbit-IgG for 2–3 h at room temperature. Primary antibodies included rabbit monoclonal anti-EZH2 (Cell Signaling, USA) and anti-EED. Second antibodies were anti-mouse IgG Alexa Fluor 555 and anti-rabbit IgG Alexa Fluor 488 (Invitrogen, USA). Slides were mounted with SlowFade Gold antifade reagent with DAPI (Invitrogen) and examined with laser scanning microscope LSM510 Version 3.2 (Carl Zeiss, Germany). Cell culture Cell lines and culture conditions are summarised in Table 2. Peripheral blood mononuclear cells (PBMC) and CD4+ T-cells from healthy donors were used as normal control. Immunomagnetic isolation was performed using EasySep Human CD4+ T-Cell Enrichment Kit (#19052; StemCell Technologies, Canada). Dynabeads human T-cell activator CD3/CD28 (Invitrogen, USA) was used to activate and expand human T-cells. Western blotting analysis The western blot analyses were performed as described previously.26 The same primary antibodies of EZH2, SUZ12, EED and EZH1 as in immunohistochemical staining, mouse anti-beta-actin (Sigma Aldrich, USA) antibody and HRP-labelled anti-rabbit or anti-mouse antibodies (NA9340V GE and NA9310V, respectively; GE Healthcare, UK) were used for the analyses.

Statistical analysis Statistical analysis was carried out with SPSS version 19 (IBM, USA) using Chi square to compare the difference in expression between tumour and normal lymphoid tissue as well as to compare between different B-cell neoplasms. Spearman and Pearson were used for correlation analyses of EZH2, EED, SUZ12 and EZH1 with Ki-67.

RESULTS Expression of EZH2, EED and SUZ12 in normal lymphoid tissues In RLH, the expression of EZH2, EED and SUZ12 showed a similar pattern; the three proteins were expressed in the nuclei of germinal centre (GC) cells; centroblasts showed stronger staining than centrocytes. The resting cells of the mantle zone were negative. CD138 + plasma cells, follicular T-helper cells, interfollicular CD3+ T cells and CD56+ natural killer/T (NK/T) cells were mostly negative for the three proteins (Tables 3 and 4, Fig. 1). Cutaneous CD3+ T-lymphocytes were negative for EZH2 and EED and negative/weakly positive for SUZ12. In normal thymus, the expression of EZH2, EED and SUZ12 was seen mostly in the cortex and

Please cite this article in press as: Abdalkader L, et al., Aberrant differential expression of EZH1 and EZH2 in Polycomb repressive complex 2 among B- and T/NK-cell neoplasms, Pathology (2016), http://dx.doi.org/10.1016/j.pathol.2016.05.002

EZH CORRELATION WITH AGGRESSIVENESS

5

Immunohistochemical staining of EZH2, EED and SUZ12 in reactive lymphoid hyperplasia. (A–C) Expression of Ezh2, EED and SUZ12, respectively, was detected mainly in the germinal centre (GC) while the mantle zone (MZ) was negative. (D–F) CD3+ T-lymphocytes were negative for EZH2, EED and SUZ12, respectively. (G–I) CD138+ plasma cells were negative to Ezh2, EED and SUZ12, respectively. (J–L) CD56+ NK/T-cells were negative for Ezh2, EED and SUZ12, respectively. Red colour, CD3, CD138 and CD56; brown colour, Ezh2, EED and SUZ12.

Fig. 1

decreased noticeably in the medullary CD3+ cells (Tables 3 and 4). High expression of EZH2 in B- and T/NK-cell neoplasms In B-cell neoplasms, the overall positivity score of EZH2 was 39.6% (Table 3). Burkitt lymphoma (BL) and DLBCL-GCB expressed EZH2 in 86.7%. DLBCL non-GCB showed a positivity score of 73.3%. Follicular lymphoma grade 3 (FLG3) score was 60% (Table 3, Fig. 2). In the four subtypes, the staining was moderate/strong and the percentage of positive tumour cells was above 70%. The positivity score in mantle cell lymphoma (MCL) was 30%. Follicular lymphoma grade 1 (FLG1) score of EZH2 was 23.3%. Plasma cell myeloma (PCM), chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL) and plasmacytoma (PC)

scores were 10%, 8.3% and 4.2%, respectively (Table 3, Fig. 2). Mucosa associated lymphoid tissue (MALT) lymphoma was negative (Table 3). The percentage of positive tumour cells in the last five entities (MCL, FLG1, PCM, PC, CLL/SLL and MALT lymphoma) was below 30%. The overall positivity score of EZH2 in T- and T/NK-cell lymphomas was higher (56.1%). T-cell lymphoblastic lymphoma (T-LBL) positivity score of EZH2 was 70%. NK/ T-cell lymphoma positivity score was 63.3%. Peripheral T-cell lymphoma (PTCL), adult T-cell lymphoma/leukaemia (ATLL) and angioimmunoblastic lymphoma (AITL) positivity score was 53.3%. Anaplastic large cell lymphoma (ALCL) and cutaneous T-cell lymphoma (CTCL) positivity score was 46.7% (Table 3, Fig. 2). The percentage of positive tumour cells ranged between 40% and 95%. Moderate staining intensity of EZH2 in T- and T/NK-cell lymphomas

Please cite this article in press as: Abdalkader L, et al., Aberrant differential expression of EZH1 and EZH2 in Polycomb repressive complex 2 among B- and T/NK-cell neoplasms, Pathology (2016), http://dx.doi.org/10.1016/j.pathol.2016.05.002

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Fig. 2 Immunohistochemical staining of EZH2, EED and SUZ12 in B- and T/NK-cell neoplasms. (A) H&E of diffuse large B-cell lymphoma (DLBCL); inset shows high magnification of tumour cells. (B–E) DLBCL showed high expression of EZH2, EED, SUZ12 and Ki-67, respectively. (F) H&E of chronic lymphocytic leukaemia/ small lymphocytic lymphoma (CLL/SLL); inset shows high magnification of tumour cells. (G–J) CLL/SLL showed very low expression of EZH2, EED, SUZ12 and Ki67, respectively. (K) H&E of adult T-cell lymphoma (ATLL), inset shows high magnification of tumour cells. (L–O) ATLL showed high expression of EZH2, EED, SUZ12 and Ki-67, respectively.

Please cite this article in press as: Abdalkader L, et al., Aberrant differential expression of EZH1 and EZH2 in Polycomb repressive complex 2 among B- and T/NK-cell neoplasms, Pathology (2016), http://dx.doi.org/10.1016/j.pathol.2016.05.002

EZH CORRELATION WITH AGGRESSIVENESS

7

Immunohistochemical staining of EZH1 in reactive lymphoid hyperplasia, B- and T/NK-cell neoplasms. (A) Reactive lymphoid hyperplasia showed expression of EZH1 in mantle zone and interfollicular T-zone; inset shows germinal centre showing low expression of EZH1. (B) Mucosa associated lymphoid tissue (MALT) lymphoma was positive for EZH1. (C) Diffuse large B-cell lymphoma was negative for EZH1. (D) Peripheral T-cell lymphoma was negative for EZH1.

Fig. 3

Table 5 Differences of expression of EZH2, EED, SUZ12 and EZH1 between lymphoid neoplasms and normal counterpart as well as among subtypes of B-cell neoplasms Malignant lymphoma

Postulated cell of origin

Pre-GC (MCL)

Mantle zone of RLH?

GC-lymphomas (BL, DLBCL-GCB and FL)

Germinal centre dark and light zone of RLH Post-GC B-cells and plasma cells of RLH T/NK-cells of RLH and skin

Post-GC (DLBCL non-GCB, PCM, PC, CLL/SLL and MALT lymphomas) Mature T/NK-cell lymphomas (ATLL, PTCL, PTCL,AITL, CTCL and NK/T-cell lymphomas)

EZH2

EED

SUZ12

c2[8.571 p[0.014 c2[21.610 p[0.001 c2=7.039 p=0.071 c2[37.020 p[0.000

c2[6.667

c2[4.000

p[0.036 c2[18.228 p[0.000 c2=7.726 p=0.052 c2[15.061 p[0.002

p[0.046 c2[14.672 p[0.002 c2=6.238 p=0.101 c2[14.925 p[0.002

EZH1

c2[9.00a

p[0.011 c2=2.800 p=0.274 c2=3.001 p=0.223 c2[16.431a p[0.001

Bold figures are statistically significant. AITL, angioimmunoblastic T-cell lymphoma; ATLL, adult T-cell leukaemia/lymphoma; BL, Burkitt lymphoma; CLL/SLL, chronic lymphocytic leukaemia/small lymphocytic lymphoma; CTCL, cutaneous T-cell lymphoma; DLBCL-GCB, diffuse large cell B-cell lymphoma germinal centre type; DLBCL non-GCB, diffuse large B-cell lymphoma non-germinal centre type; FL, follicular lymphoma; GC, germinal centre; MALT, mucosa associated lymphoid tissue lymphoma; MCL, mantle cell lymphoma; NK/T, natural killer/T-cell lymphoma; PCM, plasma cell myeloma; PC, plasmacytoma; PTCL, peripheral T-cell lymphoma; RLH, reactive lymphoid hyperplasia. a

These values decreased in malignant lymphoma cases compared to postulated cell of origin.

was noted in contrast to almost complete lack of expression in normal CD3+ and CD56+ cells of RLH and skin (Table 3, Fig. 1 and 2). High expression of EED in B- and T/NK-cell neoplasms In B-cell neoplasms, the overall positivity score of EED was 35.4% (Table 3). The highest positivity score was found in DLBCL-GCB (80%). BL, DLBCL non-GCB and FLG3 positivity scores were 73.3%, 63.3% and 53.3%, respectively. In the four subtypes, the staining was moderate/strong and the percentage of positive tumour cells was above 50%. The positivity score in MCL was 20%. FLG1 positivity score was 23.3%. EED positivity score in MALT lymphoma, CLL/

SLL, PCM and PC was 10%, 8.3%, 6.7% and 4.2%, respectively (Table 3, Fig. 2). The percentage of positive tumour cells in the last five entities (MCL, FLG1, PCM, PC, CLL/SLL and MALT lymphoma) was below 40%. The overall positivity score of EED in T- and T/NK-cell lymphomas was 33.9%. The score in T-LBL was 43.3% and in ALCL was 40%. In PTCL, AITL and NK/T lymphomas, EED score was 33.3%. CTCL and ATLL positivity scores for EED were 26.7% and 23.3%, respectively (Table 3, Fig. 2). In the positive cases, the average percentage of positive tumour cells ranged between 30% and 60%. Weak/ moderate staining intensity was noted in contrast to almost complete lack of expression of EED in normal CD3+ and CD56+ cells of RLH and skin (Table 3, Fig. 1 and 2).

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Pathology (2016), -(-), -

Fig. 4 Expression profiles of EZH2, EED, SUZ12 and EZH1 in lymphoid neoplasms and normal control based on immunohistochemical staining analyses. (A) B-cell neoplasms, (B) T/NK-cell neoplasms. AITL, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large cell lymphoma; ATLL, adult T-cell leukaemia/lymphoma; BL, Burkitt lymphoma; CLL/SLL, chronic lymphocytic leukaemia/small lymphocytic lymphoma; CTCL, cutaneous T-cell lymphoma; DLBCLGC, diffuse large cell Bcell lymphoma germinal centre type; DLBCLNG, diffuse large B-cell lymphoma non-germinal centre type; FLG1, follicular lymphoma grade 1; FLG3, follicular lymphoma grade 3; GC dark, germinal centre dark zone; GC light, germinal centre light zone; IF, interfollicular; MALT, mucosa associated lymphoid tissue lymphoma; MCL, mantle cell lymphoma; MZ, mantle zone; NK/T, natural killer/T-cell lymphoma; Norm NK/T, normal natural killer T-cell; Norm PC, normal plasma cells; PCM, plasma cell myeloma; PC, plasmacytoma; Post GC, post germinal centre B-cells; PTCL, peripheral T-cell lymphoma; T-LBL, T-cell lymphoblastic lymphoma.

High expression of SUZ12 in B- and T/NK-cell neoplasms In B-cell neoplasms, the overall positivity score of SUZ12 was 40.2% (Table 4). The highest score of positivity was found in DLBCL-GCB and FLG3 (77.8% and 72.2%, respectively). BL positivity score was 61.1% while DLBCL non-GCB was 66.7%. In the four subtypes, the staining was moderate/strong and the percentage of positive tumour cells was above 70%. The positivity score in FLG1 and MCL was 27.8%. PCM and PC positivity score was 22.2%. MALT lymphoma and CLL/SLL scores were 13.3% and 11.1%, respectively (Table 4, Fig. 2). The percentage of positive tumour cells in the last five entities (MCL, FLG1, PCM, PC, CLL/SLL and MALT lymphoma) was below 40%. The overall positivity score of SUZ12 in T- and T/NK-cell lymphomas was 42.5%. NK/T lymphoma positivity score of SUZ12 was 55.6%. T-LBL and PTCL score of SUZ12 was 50%. ALCL score was 38.9% while ATLL, AITL and CTCL scores were 33.3% (Table 4, Fig. 2). In the positive cases, the average percentage of positive tumour cells ranged between 40% and 80%. Weak/moderate staining intensity was noted

in contrast to low/no expression of SUZ12 in normal CD3+ and CD56+ cells of RLH and skin (Table 4, Fig. 1 and 2). Expression of EZH1 in normal lymphoid tissues In RLH, the mantle zone cells were moderately positive for EZH1. Expression in GC was limited to a small fraction of cells. CD138+ plasma cells, interfollicular CD3+ T-cells, follicular T-helper cells and CD56+ NK/T cells were positive (Table 4, Fig. 3). EZH1 was expressed both in the cytoplasm and nucleus. Normal thymus showed no expression of EZH1 in the cortex and low expression in the medulla. Cutaneous CD3+ T-lymphocytes were positive (Table 4). Low expression of EZH1 in B- and T/NK-cell neoplasms In B-cell neoplasms, the overall positivity of EZH1 was 12.6% (Table 4). The highest positivity score was found in PCM and PC (33.3%). Positivity scores in FLG1 and MALT lymphoma were 22.2% and 20%, respectively. The staining was weak/moderate and the percentage of positive tumour

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EZH CORRELATION WITH AGGRESSIVENESS

Table 6

9

Differences of expression of EZH2, EED, SUZ12 and EZH1 between aggressive and indolent type of lymphoma

Aggressive subtypes of B-cell neoplasms

BL, DLBCL-GCB, DLBCL non-GCB, FLG3 and MCL

Indolent subtypes of B-cell neoplasms

EZH2

EED

SUZ12

EZH1

FLG1, PCM, PC,CLL/SLL and MALT lymphomas

c2[52.531

c2[39.138

c2[23.323 p[0.001

c2[6.586a

p[0.000

p[0.000

p[0.037

Bold figures are statistically significant. BL, Burkitt lymphoma; CLL/SLL, chronic lymphocytic leukaemia/small lymphocytic lymphoma; DLBCL-GCB, diffuse large cell B cell lymphoma germinal centre type; DLBCL non-GCB, diffuse large B-cell lymphoma non germinal centre type; FLG1, follicular lymphoma grade 1; FLG3, follicular lymphoma grade 3; MALT, mucosa associated lymphoid tissue; MCL, mantle cell lymphoma; PC, plasmacytoma; PCM, plasma cell myeloma. a

This value decreased in aggressive subtypes of B-cell neoplasms compared to indolent subtypes of B-cell neoplasms.

Opposite expression profiles of EZH2 and EZH1 in lymphoid neoplasms and normal control based on immunohistochemical staining analyses. (A) B-cell neoplasms, (B) T/NK-cell neoplasms. EZH1 versus EZH2 ratio was calculated as the percentage of EZH1 or that of EZH2/total EZH (EZH1+ EZH2) positivity. AITL, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large cell lymphoma; ATLL, adult T-cell leukaemia/lymphoma; BL, Burkitt lymphoma; CLL/SLL, chronic lymphocytic leukaemia/small lymphocytic lymphoma; CTCL, cutaneous T-cell lymphoma; DLBCLGC, diffuse large cell B-cell lymphoma germinal centre type; DLBCLNG, diffuse large B-cell lymphoma non-germinal centre type; FLG1, follicular lymphoma grade 1; FLG3, follicular lymphoma grade 3; GC dark, germinal centre dark zone; GC light, germinal centre light zone; IF, interfollicular; MALT, mucosa associated lymphoid tissue lymphoma; MCL, mantle cell lymphoma; MZ, mantle zone; NK/T, natural killer/T-cell lymphoma; Norm NK/T, normal natural killer T-cell; Norm PC, normal plasma cells; PCM, plasma cell myeloma; PC, plasmacytoma; Post GC, post germinal centre B-cells; PTCL, peripheral T-cell lymphoma; T-LBL, T-cell lymphoblastic lymphoma.

Fig. 5

cells was around 50%. BL and CLL/SLL positivity score was 11.1%. DLBCL-GCB and MCL positivity score was 5.6%. DLBCL non-GCB and FLG3 were negative (Table 4, Fig. 3). The overall positivity of EZH1 in T- and T/NK-cell lymphomas was 12.5%. ATLL positivity score of EZH1 was 33.3%. CTCL score was 13.3%. T-LBL, ALCL and PTCL

expressed EZH1 in 11.1%. AITL score was 6.7% while NK/ T-cell lymphoma was negative. A weaker staining intensity was noted in the positive cases compared to the staining intensity in normal CD3+ cells and CD56+ NK/T cells of RLH (Table 4, Fig. 3).

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Double fluorescent immunohistochemical staining of EZH2 and SUZ12 in lymphoid neoplasms and reactive lymphoid hyperplasia. (A) Nuclei of germinal centre (GC) cells in RLH showed moderate expression of EZH2 with a homogenous pattern. (B) Interfollicular (IF) T-cell zone of RLH showed a faint nuclear staining of EZH2. (C) Diffuse large B-cell lymphoma germinal centre type (DLBCL-GCB) cells showed strong expression of EZH2 with homogenous nuclear staining. (D) Adult T-cell lymphoma/leukaemia (ATLL) showed strong expression of EZH2 with homogenous nuclear staining. (E) Nuclei of GC cells showed moderate expression of SUZ12 with a homogenous pattern. (F) IF T-cell zone showed a faint nuclear staining of SUZ12. (G) DLBCL-GCB cells showed strong expression of SUZ12 with a homogenous nuclear staining. (H) ATLL showed moderate expression of SUZ12 with homogenous nuclear staining. (I) GC cells, DAPI. (J) IF T-cell zone, DAPI. (K) DLBCL-GCB, DAPI. (L) ATLL, DAPI. (M) Merged image showed co-localisation of EZH2 and SUZ12 in GC cells. (N) Merged image showed co-localisation of EZH2 and SUZ12 in IF T-cells. (O) Merged image showed co-localisation of EZH2 and SUZ12 in DLBCL-GCB cells. The white arrow and inset show cytoplasmic localization. (P) Merged image showed co-localisation of EZH2 and SUZ12 in ATLL cells. The white arrow and inset show cytoplasmic localisation in a mitotic cell. EZH2, green; SUZ12, red; DAPI, blue.

Fig. 6

Significant differences in the expression of EZH2, EED, SUZ12 and EZH1 between lymphoid neoplasms and normal lymphoid tissues as well as among subtypes of B-cell neoplasms Increased expression of EZH2, EED and SUZ12 compared to the normal cell of origin was noted in several subtypes of lymphomas: pre-GC origin neoplasms (MCL), GC-origin neoplasms (BL, DLBCL-GCB, FL) and mature T/NK-cell neoplasms (p = 0.000–0.014 for EZH2, 0.000–0.036 for EED and 0.002–0.046 for SUZ12) (Table 5, Fig. 4). In postGC neoplasms (DLBCL non-GCB, CLL/SLL, PCM, PC and MALT lymphomas) the difference was not significant for the three proteins. EZH1 expression was found decreased compared to the normal counterpart in pre-GC and mature Tcell lymphomas (p = 0.011 and 0.001, respectively) (Table 5, Fig. 4).

Among the subtypes of B-cell neoplasms, high expression of EZH2, EED and SUZ12 was detected mainly in the aggressive subtypes (BL, DLBCL, FLG3 and MCL) while the less aggressive/indolent types (FLG1, PCM, PC, CLL/ SLL and MALT) showed low expression (p = 0.000, 0.000 and 0.001, respectively). Expression of EZH1 was decreased in the aggressive subtypes compared to the indolent subtypes (p = 0.037). Within the follicular lymphoma, EZH2 expression was significantly higher in FLG3 compared to FLG1 (p = 0.023) (Table 6, Fig. 4). Opposite expression profiles of EZH2 and EZH1 in normal lymphoid tissues as well as B- and T/NK-cell neoplasms Immunohistochemical staining of RLH showed that EZH2 presented 100% of the total EZH expression in the

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EZH CORRELATION WITH AGGRESSIVENESS

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Fig. 7 Double fluorescent immunohistochemical staining of EZH1 and EED in lymphoid neoplasms and reactive lymphoid hyperplasia (RLH). (A) Germinal centre

(GC) cells in RLH showed a faint cytoplasmic expression of EZH1. (B) Interfollicular (IF) T-cell zone of RLH showed a moderate strong cytoplasmic staining and a faint nuclear staining of EZH1. (C) Diffuse large B-cell lymphoma germinal centre type (DLBCL-GCB) cells showed faint expression of EZH1 with nuclear and cytoplasmic staining. (D) Adult T-cell lymphoma/leukaemia (ATLL) showed weak expression of EZH1 with nuclear and cytoplasmic staining. (E) GC cells showed a moderate nuclear expression of EED with a homogenous pattern. (F) IF T-cell zone showed a faint cytoplasmic/nuclear staining of EED. (G) DLBCL-GCB cells showed strong expression of EED with homogenous nuclear staining. (H) ATLL showed moderate expression of EED with homogenous nuclear staining. (I) GC cells, DAPI. (J) IF Tcell zone, DAPI. (K) DLBCL-GCB, DAPI. (L) ATLL, DAPI. (M) Merged image showed different localisation of EZH1 and EED in GC cells. (N) Merged image showed some differences in the localisations of EZH1 and EED in IF T-cells. (O) Merged image showed co-localisation of EZH1 and EED in several cells in DLBCL-GCB. The white arrow and inset show cytoplasmic localisation in few tumour cells. (P) Merged image showed co-localisation of EZH1 and EED in ATLL cells. The white arrow and inset show cytoplasmic localisation in a few tumour cells. EZH1, green; EED, red; DAPI, blue.

proliferating cells of the GC while EZH1 presented 100% of the total EZH expression in the resting cells of the mantle zone and interfollicular T zone (Fig. 4 and 5). In the thymus cortex, EZH2 presented 100% of the total EZH and dropped down to 20.1% in the thymus medulla (Fig. 5A,B). In the aggressive B-cell neoplasms, EZH2 ranged between 84.3% (MCL) and 100% in DLBCL non-GCB and FLG3 of the total EZH expression. In the indolent neoplasms, the situation was reversed; EZH1 expression was dominant ranging between 48.8% (FLG1) and 100% in MALT lymphoma (Fig. 5A). In T/NK-cell neoplasms, EZH2 expression was dominant in the different subtypes, ranging between 61.5% (ATLL) and 100% of the total EZH expression (Fig. 5B). Immunofluorescent staining analyses EZH2 was moderately expressed in the nuclei of GC cells and faintly expressed in those of the IF cells (Fig. 6A,B). Both DLBCL and ATLL showed strong nuclear expression

of EZH2 (Fig. 6C,D). SUZ12 showed a similar pattern of expression both in normal and neoplastic tissues (Fig. 6E–H). The same pattern was noted for EED (Fig. 7E–H). The expression profile of the three proteins was very similar regarding both localisation and intensity changes. EZH1 showed faint cytoplasmic expression in the GC cells while the IF cells showed moderate/strong cytoplasmic staining and faint nuclear staining (Fig. 7A,B). Both DLBCL and ATLL showed faint expression of EZH1 both nuclear and cytoplasmic (Fig. 7C,D). EZH2, EED and SUZ12 expression correlated with proliferation activity The Ki-67 labelling index showed strong positive correlation with EZH2 expression in B-cell neoplasms [correlation coefficient (Co) = 0.902, p = 0.000) and considerable correlation in T/NK-cell neoplasms (Co = 0.646, p = 0.000) for EZH2 (Fig. 8A,B). In addition, both EED and SUZ12

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Correlation between (EZH2, EED, SUZ12 and EZH1) with Ki-67 expression based on percentage of positive cells in both B- and T/NK-cell neoplasms. (A,C,E,G) B-cell neoplasms. (B,D,F,H) T/NK-cell neoplasms. Co, Pearson correlation coefficient (*correlation is significant at the 0.01 level; **correlation is strongly significant). (A,B) Correlation between EZH2 and Ki-67, (C,D) EED and Ki-67, (E,F) SUZ12 and Ki-67, and (G,H) EZH1 and Ki-67.

Fig. 8

showed considerable correlation with Ki-67 in B-cell neoplasms (Co = 0.770, p = 0.000 for EED; Co = 0.701, p = 0.000 for SUZ12) and in T/NK-cell neoplasms (Co = 0.376, p = 0.014 for EED; Co = 0.382, p = 0.017 for SUZ12) (Fig. 8C–F). EZH1 correlation with Ki-67 was negative in Bcell (Co = –0.271, p = 0.079) and T/NK-cell neoplasms (Co = –0.269, p = 0.159), however, p value was not significant (Fig. 8G,H). Western blotting analyses of haematopoietic cell lines Normal resting PBMCs were almost negative for EZH2, EED, SUZ12 and EZH1. CD4+ cells showed a faint signal of

the latter two proteins (SUZ12 and EZH1). Stimulation of normal T-cells with CD3/CD28 immuno-beads induced weak expression of EZH2, SUZ12, EED and faint EZH1 (Fig. 9C). EZH2 and SUZ12 showed higher expression levels in Ramos, Daudi and Raji cell lines compared to FL18, FL218 and Epstein–Barr virus (EBV)-immortalised normal B-cell line TW-LCL; while EED and EZH1 showed an opposite pattern. FL18EB showed a stronger expression of EZH2 and SUZ12 versus a weaker signal of EZH1 compared to the parent FL18 cells (Fig. 9A). ATLL patient-derived T-cell lines ATL16T, ATL45T, ED405151 and ATL55T showed high expression levels of EZH2 and SUZ12 and decreased levels of EZH1 and EED (Fig. 9B).

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EZH CORRELATION WITH AGGRESSIVENESS

Western blotting analyses for EZH2, EED, SUZ12 and EZH1 in malignant lymphoma cell lines and normal control. (A) EZH2 and SUZ12 showed higher expression in Ramos, Daudi and Raji cell lines compared to FL18, FL218 and FL318, while EED and EZH1 showed opposite pattern. EZH1 signal was notably weaker than the three proteins (EZH2, EED and SUZ12). Stronger EZH1 signal was detected in the FL cell lines. (B) Leukaemic ATLL-cell lines (ATL55T, ED40515) showed higher expression of EZH2 and SUZ12 than EED. EZH1 signal was notably weaker than the three proteins (EZH2, EED and SUZ12). Stronger EZH1 signal was detected in HTLV-1 immortalised cell lines MT2, MT4 and IWA1. (C) Normal resting PBMCs were negative for EZH2, EED, SUZ12 and EZH1. CD4+ cells showed a faint signal of the latter two proteins (SUZ12 and EZH1). Activation of normal T-cells with CD3/CD28 immuno-beads induced a weak expression of EZH2, SUZ12, EED and EZH1.

Fig. 9

Densitometry data showed that EZH2 expression ranged between 26.9% and 46.9% of the total EZH in healthy PBMC and increased noticeably to (52.9–97.5%) in the leukaemic and malignant lymphoma cell lines. Ramos, Daudi and Raji cell lines showed a higher EZH2/EZH1 ratio compared to that in FL18, FL218 and EBV-immortalised normal B-cell line TW-LCL (Fig. 10A–C).

DISCUSSION Consistent with previous reports, this study found that the PRC2 complex members EZH2, EED and SUZ12 showed the same staining pattern in RLH; the expression of the three proteins was found in the proliferating cells of the germinal centre and thymus cortex while the resting cells of thymus

13

medulla, mantle zone and interfollicular zone were negative.38,39 In B-cell neoplasms, the present study showed that EZH2, EED and SUZ12 expression was significantly stronger in preGC and GC neoplasms compared to the normal counterpart. In post-GC neoplasms, the differences in the expression of these proteins compared to the normal counterpart were not significant (most cases in this entity were of the indolent types). A significant increase in the expression of the three proteins (EZH2, EED and SUZ12) was also detected between T/NK-cell neoplasms and the normal counterpart. No significant differences among the subtypes of T/NK-cell lymphomas were noted. This could be explained by the fact that most T/NK-cell neoplasms showed a high Ki-67 proliferation index and were considered aggressive neoplasms (Table 5, Fig. 4). These findings suggest that the high expression levels of PRC2 complex members (EZH2, EED and SUZ12) are associated with aggressive subtypes (T/NK-cell, BL, DLBCL, FLG3 and MCL) in non-Hodgkin lymphomas. The differences in expression levels were statistically significant when compared to those in the indolent subtypes (FLG1, CLL/SLL PC, PCM and MALT lymphoma) (Table 6). Western blot analysis confirmed this finding for both EZH2 and SUZ12; the expression levels were higher in the aggressive B-cell lines compared to the more indolent (Fig. 9). This was also supported by the significant positive correlation of EZH2, EED and SUZ12 expression with Ki-67 in B- and T/NK-cell neoplasms (Fig. 8). This is consistent with previous studies reporting an association between the over-expression of EZH2 with metastasis and poor prognosis in breast, prostate, and stomach cancers.22–25 The correlation with Ki-67 was higher in the case of EZH2 than both EED and SUZ12. McCabe et al., reported that therapeutic inhibition of EZH2 resulted in decreased proliferation in lymphomas with activating EZH2 mutations.40 This suggests a key role of EZH2 and the partner subunits EED and SUZ12 in controlling the proliferative activity of lymphoid neoplasms. In this study, immunohistochemical staining revealed that EZH1 has an expression profile opposite to that of EZH2 both in RLH and lymphoid neoplasms. The expression of EZH2 was high in the proliferating cells of both RLH and aggressive lymphomas, while EZH1 was high in the resting populations of RLH and indolent subtypes of B-cell neoplasms. In normal GC cells of RLH, EZH2 level was higher than that of EZH1, and this difference became even more evident in GCneoplasms. In pre-GC and mature T/NK-cell neoplasms, a significant increase in the expression of EZH2 accompanied by a decrease in that of EZH1 lead to a reverse of the EZH2/ EZH1 ratio in the neoplastic state compared to the normal counterpart. Post-GC neoplasms maintained a higher EZH1 level compared to EZH2 although the difference was smaller than that seen in normal cells (Table 5, Fig. 4 and 5). Western blot analysis results were consistent with these data. EZH2/EZH1 ratio was reversed in the neoplastic cell lines compared to normal PBMCs. PBMC#3 showed a higher level of EZH2 compared to other PBMC samples; however, it was still less than 50% of the total EZH. This difference in densitometry can be explained by the very low expression levels as seen in Fig. 9C. The aggressive subtypes of B-cell lines showed a higher EZH2/EZH1 ratio compared to that in the indolent and EBV-immortalised normal B-cell lines. The higher EZH2/EZH1 ratio noted FL18EB cells, an

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Fig. 10 EZH2 versus EZH1 expression profiles revealed from western blot analysis. (A–C) EZH2 versus EZH1 ratio was calculated from the densitometry of EZH2 and

EZH1 western blotting in Fig. 9.

EBV-infected cell line, compared to the parent FL18 cells, suggesting that EBV infection induced some alteration in the epigenetic control (Fig. 10). These opposite expression profiles are consistent with previous studies reporting that EZH2 and EZH1 are inversely correlated during development. A high expression of EZH2 was found in embryonic stem cells whereas that of EZH1 was found in the adult resting tissues.41 In this study, EZH2 showed a strong positive correlation with Ki-67 proliferation index while EZH1 showed a negative correlation (Fig. 8). This is consistent with the report that EZH1 prevents active proliferation in human stem cells.9 Together, these data suggest that EZH2 and EZH1 are counter-balancing to control the proliferation and development in the physiological condition of normal cells. The disturbance and/or abnormality of the regulatory

mechanisms controlling EZH2/EZH1 balance may be implicated in the pathogenesis of malignant lymphomas. A previous study reported that EED displays a dynamic change in the intracellular localisation during early developmental stages in porcine embryo.42 Localisation at the inner side of the plasma membrane was also described.43 We have found a cytoplasmic localisation of EZH2, EED and SUZ12 in a small percentage of tumour cells in malignant Band T/NK-cell neoplasms but not in RLH. We also found a change in the intra-cytoplasmic localisation of EZH1 between normal and neoplastic lymphoid tissues. EZH1 staining in RLH was mostly cytoplasmic while in lymphoma the staining became more nuclear. These data suggest a dynamic change of the intracellular localisation involving all PRC2 complex members in malignant lymphomas.

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EZH CORRELATION WITH AGGRESSIVENESS

The enzymatic activity of EZH2/EZH1 depends on other core members EED, SUZ12 and RBBP4.17,18,41 We found that EZH2 expression profile as well as intracellular localisation matched that of SUZ12 and EED. Interestingly, western blotting data from cell lines revealed that SUZ12 expression profile was more similar to EZH2 than that of EED. A similar pattern was also found in the immunohistochemical staining data of T/NK-cell lymphoma patient specimens. The exact effect of the changes in the expression of both SUZ12 and EED on the functional activity of EZH2 remains to be elucidated in view of the wide attention given recently to different pharmacological trials of inhibiting EZH2 complex.44–48 In this study, we found that EZH1 showed a dominant cytoplasmic localisation in RLH while both EED and SUZ12 showed a nuclear localisation. In malignant lymphomas, EZH1 localisation became more nuclear; however, the expression level was noticeably lower compared to the normal counterpart (Fig. 6, 7, 9 and 10). This raises questions about the activity level and mechanism of action of EZH1 both in normal and neoplastic lymphoid tissues. In conclusion, a high expression of PRC2 complex members EZH2, EED and SUZ12, is found in proliferating/ aggressive subtypes of B- and T-cell neoplasms. EZH1 has a low expression in lymphoid neoplasms particularly the aggressive subtypes. EZH2 and EZH1 have opposite expression patterns both in RLH and lymphomas as well as opposite relation with proliferation, suggesting an important counter-balance mechanism controlling cellular proliferation both in normal state and neoplasia. Better understanding of the localisation, complex mechanism of action of PRC2 and exact relation with EZH2/EZH1 enzymatic activity level and tumour proliferative activity is of great importance in terms of prognosis and therapy options. Acknowledgement: The authors gratefully acknowledge Dr Michiyuki Maeda (Institute for Virus Research, Kyoto University, Japan), Dr M. J. Robertson (Indiana University School of Medicine, USA), Dr Hitoshi Oono (Kyoto University Medical School, Japan), Dr Yoshinobu Matsuo (Fujisaki Cell Center, Hayashibara Biochemical Labs, Inc., Okayama, Japan), and RIKEN BioResource Center (Tsukuba, Japan) for kindly providing the cell lines. We also thank Mr Hiroshi Okamoto and Mr Kosei Isomoto (Central Research Laboratory, School of Medicine, Okayama University), Ms Hiromi Nakamura, Ms Mai Bando and Ms Miyuki Shiotani (Department of Pathology, Okayama University Medical School) and Dr Tarek Abdelkader, [Postdoctoral Fellow at Advanced Research Center for Oral and Craniofacial Sciences (ARCOCS)] for technical support. Conflicts of interest and sources of funding: This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (TO) (#22590312). The authors state there are no conflicts of interest to disclose. Address for correspondence: Takashi Oka, Department of Pathology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-chou, Kita-ku, Okayama 700-8558, Japan. E-mail: [email protected]

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Please cite this article in press as: Abdalkader L, et al., Aberrant differential expression of EZH1 and EZH2 in Polycomb repressive complex 2 among B- and T/NK-cell neoplasms, Pathology (2016), http://dx.doi.org/10.1016/j.pathol.2016.05.002