EZH2 Overexpression In Primary Gastrointestinal Diffuse Large B Cell Lymphoma And Its Association With The Clinicopathological Features

BackgroundThe gastrointestinal tract is the most frequent site of extra-lymph node lymphoma,and the primary gastrointestinal tract lymphoma accounts for 30-40% of the lymphoma.MALT lymphoma and DLBCL are the most common types of primary gastrointestinal tract lymphoma.MALT lymphoma is a low-grade lymphoma with a better prognosis.DLBCL is a high grade lymphoma with a poor prognosis.At present,no genetic difference was identified between primary gastrointestinal tract DLBCL and the primary DLBCL of lymph nodes,in particular,the clinical pathological features,immunological typing,molecular typing and prognostic factors of gastrointestinal tract DLBCL lack systematic research.Because special immune microenvironment of gastrointestinal tract,primary gastrointestinal DLBCL tumorigenic mechanism is very complex,this may be related tothe chronic inflammation,such as H.pylori infection,chylous diarrhea,inflammatory bowel disease and autoimmune diseases.We think that the pathogenesis of primary gastrointestinal DLBCL is probably different from that of DLBCL in the lymph nodes.Although there are a few studies on the primary gastrointestinal DLBCL,the clinicopathological features have not been fully elucidated.Histone methyltransferase enhancer of zeste homolog 2(EZH2),a catalytic subunit of polycomb repressive complex 2(PRC2),trimethylates lysine 27 of histone H3(H3K27me3),silencing the target genes involved in tumorigenesis.EZH2 overexpression and hyperactivity have been implicated in the pathogenesis of several cancers,including prostate breast,and endometrial carcinoma,where EZH2 overexpression is associated with aggressive clinical behavior and poor outcome.EZH2 plays a significant role in DLBCL tumorigenesis,and a gain-of-function mutation of EZH2 at tyrosine(Y)641 was found in 21.7% of germinal center B cell like(GCB)DLBCLs.However,EZH2 protein expression and gene mutation in primary gastrointestinal DLBCL has not been studied.Objective1.(1)To understand the clinical features of patients with primary gastrointestinal DLBCL,LDH level,clinical stage,IPI,treatment and prognosis,etc.,and understand the histological characteristics of the primary gastrointestinal DLBCL.(2)To understand the expression level of EZH2 protein in patients of primary gastrointestinal DLBCL and its relationship with clinical features.(3)To understand the expression of H3K27me3 level,BCL2 and c-MYC protein expression,the level of Ki-67 proliferation index,H.pylori infectionin patients with primary gastrointestinal DLBCL and their relationship with EZH2.2.(1)To explore the mutation of EZH2 Y641 in patients with primary gastrointestinal DLBCL and compare the differences between primary gastrointestinal DLBCL and DLBCL of primary lymph node.(2)To detect the fusion of BCL2/Ig H gene and the breakapart of c-MYC gene in patients with primary gastrointestinal DLBCL.Methods1.(1)To collectthe general clinical data of 100 patients with primary gastrointestinal DLBCL,including LDH level,clinical stage,IPI index,treatment method,etc.,and the patients were followed up.Afterwards,the data were analyzed and summarized,and the clinical characteristics and prognosis of patients primary gastrointestinal DLBCL were summarized.(2)At the same time,the paraffin embedded tissue of 100 patients with primary gastrointestinal DLBCL were collected,and HE staining was performed to observe the histological features.(3)The expression levels of EZH2,BCL2 and c-MYC protein and H3K27me3 level,Ki-67 proliferation index level and H.pylori infection were detected by IHC.2.(1)The DNAs were extracted from the paraffin-embedded tissues of 100 primary gastrointestinal DLBCL and 102 DLBCL of primary lymphnode using the QIAGEN kit.PCR was used to amplify EZH2 gene exon 16,the products were used to perform the electrophoresis,purification and sequencing.(2)The translocation of BCL2/Ig H gene and c-MYC were detected in 100 primary gastrointestinal DLBCLs by using FISH.Results1.(1)Patients usually show typical gastrointestinal symptoms,and DLBCL in the small intestine and colon more frequently appears in high clinical staging and high IPI index than those of stomach.(2)The overexpression of EZH2 was detected in 50.0% of the cases,and EZH2 overexpression was significantly associated with higher stage(P=0.014),higher International Prognostic Index score(P=0.003),reduced overall survival rate(P=0.030).(3)High levels of H3K27me3,BCL2 positive,c-MYC positive,Ki-67 high-proliferation index were 48(48.0%),36(36.0%),11(11.0%)and 74(74.0%)respectively.16 patients(6.0%)had BCL2/ c-MYC double expression.EZH2 overexpression was significantly associated with the level of H3K27me3(P=0.001)and c-MYC expression(P=0.008).EZH2 overexpression was not associated with BCL2 expression and the Ki-67 proliferation index.In the 55 cases of gastric DLBCL,20 patients(36.4%)were positive for Helicobacter pylori,and there was a trend that H.pyloriinfection was associated with higher TNM staging(P=0.056).2.(1)We detected EZH2 Y641 mutations in 1/33(3.0%)DLBCL with a germinal center immunophenotype(GCB-DLBCL).We also detected 9 cases(9/43,20.9%)EZH2 Y641 mutations in GCB-DLBCL of primary lymph node.The frequency of EZH2 Y641 mutation with primary gastrointestinal was significantly lower than that of patients with DLBCL of primary lymph node(P=0.022).(2)FISH for detecting t(14;18)(q32;q21)/Ig H-BCL2 fusion probe and c-MYC break-apart probe was successful for 62 and 59 cases,respectively.BCL2 and c-MYC translocation was detected in 6.5% and 5.1% of patients,respectively,and BCL2 translocation occurred exclusively in the GCB subtype(P=0.019).There was no BCL2/c-MYC double-hit case.No association was found between BCL2 and c-MYC protein expression and gene translocation.Conclusion1.(1)The high clinical staging,high serum LDH and high IPI index in patients with primary DLBCL in gastrointestinal tract indicated poor prognosis.There was no significant difference in histological classification between different sites.(2)Overexpression EZH2 is associated with poor prognosis.(3)EZH2 overexpression was significantly correlated with high H3K27me3 level and c-MYC expression.2.(1)The low frequency mutation of EZH2 Y641 in the primary gastrointestinal DLBCL indicates that the pathological features of primary gastrointestinal DLBCL are different from that of DLBCL of primary lymph nodes.(2)The frequencies of t(14;18)(q32;q21)/Ig H-BCL2 fusion and c-MYC breakapart in primary gastrointestinal DLBCL were significantly lower than the previous literature reports,which may be the distinctive pathological features of primary gastrointestinal DLBCL.