Study On Expression And Prognostic Significance Of Trimethylation Of Histone H3 At Lysine 27 And EZH2 In DLBCL Via TMA Technique

ObjectiveTo explore the expression of epigenetic markers like H3K27me3 and EZH2, and expression of C-myc in diffuse large B-cell lymphoma(DLBCL), and to investigate the association between their expression and clinicopathological variables, and their prognostic significance as well.Materials and methodsFrom 2006 through 2008, 102 DLBCL and 5 lymphadenosis patients from Some Provincial Cancer Hospital were envolved in our study. No therapy had been given before specimen collection. The area of cancerous cells was marked in each case under microscopy, and then a tissue cylinder at a diameter of 1.5 mm was punched and re-arrayed in a new recipient block(UNITMA, South Korea). Serial sections at a thickness of 4 ?m were cut from the newly-built block and then used for immunostaining for H3K27me3, EZH2 and c-myc.Their clinicopathologic data were carefully collected(the end of followed-up time was January 2015). The SPSS 18.0statistical software package(SPSS Inc, Chicago, IL, USA) was employed for all analyses. The association between tested markers, different clinicopathologic characteristics and prognosis of the patients were evaluated. A P-value less than 0.05 was considered statistically significant.Results(1) Eighty-nine microarrays were achieved from 102 DLBCL and 5 lymphadenosis.Because of inadequacy of tissue amount, the other 18 specimens were analyzed with routine manoeuvre. The quality of TMA was perfect and meet the standard of analysis.(2) Among all DLBCL patients, 59.8% were characterized with high expression of H3K27me3, correlated with high expression of its methytransferase EZH2(r=0.260,P=0.009). Furthermore, high expression of H3K27me3 was correlated with advance in age, ECOG?2, extranodular disease number ?2, elevation of LDH, medium-high risk IPI, C-myc/Bcl-2 coexpression, etc.(3) In comparison to patients with low H3K27me3 expression, those with high H3K27me3 expression manifested lower complete remission rate(CR) and overall remission rate(OR), i.e., 20% vs.57.5% and 41.8% vs.90%, respectively(P<0.001). In addition, patients with high H3K27me3 expression showed shorter median survival time,i.e.,21.50 month(P<0.0001). Multivariate analysis indicated that H3K27me3 was an independent prognostic factor for DLBCL patients(P=0.049). After further analysis on the substratified groups in accordance with stages, Hans classification, and C-myc/bcl-2coexpression, H3K27me3 was still an independent prognostic factor for worse outcome.(4) High expression of EZH2 were found among 61.8% patients, and was negatively correlated with staging(r=?0.207, P=0.037) and positively correlated with expression of Bcl-2(r=0.207, P=0.036), while showed no statistical association with median survival time, i.e., 41.50 month and 46.90 month, respectively(P=0.961). Among all DLBCL patients, 43.14% were found coexpression of H3K27me3/EZH2, indicating obviously shorter median survival time in comparison to those without coexpression,i.e., 18.20 month(P=0.001).(5) Around 11.8% patients showed high expression of C-myc, obviously correlated with advance in age(r=0.232, P=0.019). Compared to low expression individuals,patients with high expression of C-myc suffered lower overall remission rate, i.e.,27.3% vs.66.7%(P=0.018), and shorter OS, i.e., 13.20 month vs. 57.50 month,respectively(P=0.008).? Multivariate analysis indicated that ECOG score, Ann Arbor staging, LDH level,Bcl-2 expression, C-myc/bcl-2 coexpression were also independent prognostic factors for DLBCL patients.Conclusion(1) TMA technique is useful for the construction of DLBCL tissue chips, and experimental results from TMA can well standard for the result of original blocks. Sothis technique is reliable, and is able to save the experiment cost as well as reagents, and result in improvement of efficiency, and is worthy of being widely recommended in clinical research.(2) High expression of H3K27me3 and its methyltransferase EZH2 indicated little response to treatment, worse outcome and shorter survival time, and could be used as independent prognostic factor for DLBCL patients.(3) Albeit expression of EZH2 was strongly correlated with expression of H2K27me3,it's not an independent prognostic factor for patients with DLBCL.