Several lines of evidences suggest that the biological

Several lines of evidences suggest that the biological features of PCs are different from other CLL/SLL PFI-1 in the lymph node: the tumor cells in the PCs have been shown to accumulate cytogenetic abnormalities [1, 4] and aberrant expression of oncogene and tumorsuppressor microRNAs (miRs) [5, 6]. The accumulation of molecular alterations in the tumor cells is highly consistent with the finding that extended PCs are associated with a more aggressive clinical behavior [3, 7]. Enhancer of zeste 2 histone methyltransferase (EZH2), the core member of the polycomb repressive complex 2 (PRC2) has a critical role in multiple biological processes via epigenetic regulation of gene transcription [8]. Normally EZH2 promotes gene silencing by catalyzing the methylation of histone H3 lysine 27 (H3K27) [8]. Expression of EZH2 is proved to be controlled by different molecules, including miR-26a, c-Myc, E2F1 and Rb [9, 10, 11, 12, 13, 14]. Aberrant EZH2 function has been linked to different solid tumors and hematological malignancies, especially with more aggressive behavior [15, 16]. Higher expression of EZH2 protein has been confirmed in high-grade germinal center (GC)-derived lymphomas, including diffuse large B-cell lymphoma (DLBCL) and high-grade follicular lymphoma (FL) [17]. Recently, gain-of-function mutations of EZH2 have also been described in several types of lymphomas, especially in GC-DLBCL and FL [18, 19]. Overexpression of EZH2 has been linked to poor prognosis in CLL, however, the function of EZH2 in the pathomechanism of CLL/SLL is poorly defined [11, 20, 21]. Downregulation of miR-101 in a subset of CLLs with unmutated IgHV gene has also been associated with overexpression of EZH2 protein and poor clinical prognosis [22, 23].
Materials and methods Formalin-fixed paraffin-embedded (FFPE) lymph node samples from 15 patients diagnosed with CLL/SLL in the 1st Department of Pathology and Experimental Cancer Research, Semmelweis University were selected. All samples were obtained at the time of initial diagnosis of the patients. Microscopically, all cases showed pseudofollicular pattern with many PCs. The cohort consisted of 7 female and 8 male patients, the median age at diagnosis was 58.47 years (from 40 to 78 years) (Table 1.). The results of fluorescent in situ hybridization examination of del13q, del17p, del11q and tri12 as well as the IgHV mutational status were available in all cases and are summarized in Table 1. None of the cases underwent high-grade transformation during the follow up period (data not shown). Permissions to use the archived tissue have been obtained from the Local Ethical Committee (TUKEB-1552012) and the study was conducted in accordance with the Declaration of Helsinki.
Results
Discussion Proliferation centers in CLL/SLL lymph nodes are in the focus of investigations targeting the pathomechanism and progression of the disease. There is evidence of accumulation of molecular alterations, including cytogenetic abnormalities [1, 4] as well as aberrant expression of oncoproteins [25, 26] and miRs [5, 6] in the tumor cells of PCs, which may at least partly explain why PCs are associated with a more aggressive clinical behavior [3, 7]. To expand our knowledge on PCs and their role in the pathogenesis of CLL/SLL, we evaluated the expression level of EZH2 protein in 15 lymph nodes of CLL/SLL patients with PCs using immunohistochemistry with digital image analysis. We found that EZH2 is significantly overexpressed in the PCs of CLL/SLL lymph nodes compared to IF areas, which is in line with a previous study of van Kemenade et al. who showed that EZH2 expression in low-grade non-Hodgkin lymphomas including SLL, is limited to large Ki67 positive cells [17]. Increasing body of data suggests that overexpression of EZH2 in CLL/SLL is associated with worse prognosis, however, the function of EZH2 in the pathomechanism of CLL/SLL is poorly understood [11, 20, 21]. Based on our findings, overexpression of EZH2 is related predominantly to the larger cells enriched in the PCs, further supporting the adverse role of PCs in the progression of CLL/SLL.