Differential Expression of Enhancer of Zeste Homolog 2 (EZH2) Protein in Low and High Grade B-Cell Non-Hodgkin Lymphomas and Differential Regulation of EZH2 Expression By p-ERK and MYC in High Grade B Cell Lymphomas

Background: EZH2, a member of the polycomb protein group, is an important methyltransferase that is over-expressed in various carcinomas, some B and T cell lymphomas, as well as myeloid disorders. We investigated EZH2 expression in the range of low and high grade B cell neoplasms and correlated its expression with that of p-ERK, MYC, and p-STAT3, potential regulators of EZH2 expression, in high grade B cell lymphomas.

Methods: Immunohistochemical staining (IHC) for EZH2 was performed on a total of 162 low and high grade B cell non-Hodgkin lymphomas, using formalin fixed, paraffin-embedded tissue [Table 1]. We subsequently performed IHC for p-ERK, p-STAT3, and MYC on high grade B cell lymphomas.  Cases were scored for percentage positivity of neoplastic cells using the above antibodies.

Table 1. EZH2 expression in low grade and high grade B cell lymphomas

Lymphoma type	Cases (POS/Total)	EZH2 positive %
low grade
MM	10/12	10
LPL	9/12	5-10
CLL/SLL	15/18	10--15
MCL	8/9	30
MZL	20/21	15-20
FL(1-2)	8/10	20
HCL	13/14	5-10
HCL-V	9/10	30-40
High grade
DLBCL	29/33	90
BL	19/19	100
CLL-Richter	5/5	80-90
FL-3	6/6	80
DHL	22/22	90-100
PMLBCL	19/19	60-80
B-ALL	11/11	95-100

Abbreviations: MM - multiple myeloma; LPL - lymphoplasmacytic lymphoma; CLL/SLL - chronic lymphocytic leukemia/small lymphocytic lymphoma; MCL - mantle cell lymphoma; MZL - marginal zone lymphoma; FL(1-2) - follicular lymphoma grade 1-2/3; HCL - hairy cell leukemia; HCL-V - hairy cell leukemia-variant; DLBCL - diffuse large B cell lymphoma; BL - Burkitt lymphoma; CLL-Richter transformation of CLL; FL -3 - follicular lymphoma grade 3/3; DHL - double hit lymphoma; PMLBCL - primary mediastinal large B cell lymphoma; B-ALL - B lymphoblastic leukemia/lymphoma.

Results: In low grade lymphomas, 5-40% of neoplastic cells were positive for EZH2, with variable intensity of staining. Of note, there was a significant difference in EZH2 expression in hairy cell leukemia versus hairy cell leukemia-variant (p<0.01). In high grade lymphomas, including those transformed from low grade B cell lymphomas, 70-100% of tumor cells were positive for EZH2 expression,  a significant difference in EZH2 expression compared with low grade B cell lymphomas (p<0.01). Among the high grade B cell lymphoma, 40-80% of neoplastic cells in DLBCL (24 cases), were positive for p-ERK expression, but <30% of neoplastic cells were positive for p-ERK expression in BL (15 cases) and DHL (17 cases). In contrast, 80-100% and 50-90% of neoplastic cells were positive for MYC expression in BL (17 cases) and DHL (26 cases), respectively, but only 5-50% of neoplastic cells were positive for MYC expression in DLBCL (30 cases). There were significant differences in both MYC and p-ERK expression in BL and DHL versus DLBCL (p<0.01). None of the high grade B cell lymphomas showed significant p-STAT3 positivity in neoplastic cells.

Conclusion: EZH2 expression correlates with tumor grade in B cell neoplasms, and the high level of EZH2 expression in high grade B cell lymphomas suggests that this molecule may function as an oncogenic protein in these neoplasms. Furthermore, our findings show there are different signaling cascades in the regulation of EZH2 expression in different types of high grade B cell lymphomas. The p-ERK signaling cascade, but not MYC expression,  plays an important role in high EZH2 expression in DLBCL; while in BL and DHL, high MYC expression, but not p-ERK expression, is associated with increased EZH2 expression, possibly through miRNA regulation. These findings suggested that EZH2 and specific disease-related signaling cascades may serve as therapeutic targets for the treatment of high grade B cell lymphomas.