Correlation Between Extranodal Involvement And Lymphoma-associated Driver Gene Abnormality In Patients With Diffuse Large B-cell Lymphoma

Background and ObjectiveDiffuse large B-cell lymphoma(DLBCL)is a malignant tumor derived from mature B lymphocytes.It is the most common type of non-Hodgkin’s lymphoma(NHL),accounting for 30% to 40% of adult NHL.DLBCL can occur in almost any part of the human body,including lymphoid organs(Waldeyer’ s ring,spleen,thymus and lymph nodes)and mucosa-associated lymphoid tissue.The common extranodal sites involved are the nasal cavity、sinus、soft palate、salivary gland and thyroid of the head and neck,breast、lung and pleura of the chest,stomach、intestine、liver、pancreas、kidney and adrenal gland of the abdomen,uterus、ovary and testicle of the pelvic,brain、meninges and spinal cord of the central nervous system,skin and soft tissue,bone and bone marrow and so on.International Prognostic Index(IPI)score is the most commonly used tool to evaluate the prognosis of patients with DLBCL,including age,stage,number of extranodal lesions,LDH level and ECOG behavior status.The number of multiple extranodal sites involved(≥ 2)is considered to be one of the poor prognostic indicators.More and more studies have shown that the specific site of involvement is also related to the prognosis.In the National Comprehensive Cancer Network(NCCN)IPI score,central nervous system、bone marrow、liver、gastrointestinal tract and lung involvement are considered to be adverse prognostic factors.Our previous study also found that the prognosis of Waldeyer’ s ring involved was better,while that of central nervous system,bone marrow,breast and adrenal gland were poor.However,poor prognosis of the molecular mechanism and potential accurate treatment of multiple extranodal involvement and different extranodal sites involved are not clear.Due to the high heterogeneity of DLBCL,the existing clinical、morphological and immunophenotype still can not meet the needs of clinical accurate diagnosis and treatment.With the development of high-throughput second-generation sequencing technology,some genes have been found to occur frequently in lymphoma,suggesting that these mutated genes may be involved in the occurrence and development of lymphoma and may be used in the diagnosis,classification and prognosis of lymphoma,and even to guide clinical treatment decisions.In this study,through the detection and analysis of lymphoma-related driver gene abnormalities,we preliminarily explored the correlation between extranodal involvement and lymphoma driver gene abnormalities in DLBCL patients,aiming to reveal the poor prognosis of the molecular mechanism and potential precision treatment of multiple extranodal involvement and different extranodal sites involved.Materials and MethodsThe tumor tissue samples of 244 patients with diffuse large B-cell lymphoma treated in our hospital of from April 2006 to September 2020 were collected.32 hot spot driving gene mutations were detected by the second generation sequencing method.The data were processed and analyzed by SPSS 22.0.,the counting data were expressed by frequency(%).Bilateral χ2 test or Fisher exact probability method was selected for inter-group analysis,and Phi coefficient was used to represent the intensity of association.Logistic regression method was used in multivariate analysis.Kaplan-Meier method was used to analyze the survival,and the survival curve was drawn and Log-rank test was performed.Statistical significance was defined as P <0.05.Results1、Among the 244 patients with DLBCL,extranodal involvement occurred in163 cases,accounting for 66.8% of the total(163/244).Among them,81 cases had no extranodal involvement(81/244,33.2%),118 cases had single extranodal involvement(118/244,18.4%),and 45 cases had multiple extranodal sites(≥ 2)(45/ 244,18.4%).The median follow-up time was 20.4 months.The median progression-free survival time(PFS)was 27.0 months in patients with no extranodal involvement,53.6 months in patients with single extranodal involvement,and 15.6 months in patients with multiple extranodal sites involvement.The median overall survival time(OS)was not reached in the groups with no extranodal involvement and single extranodal site involvement,and multiple extranodal sites were involved for 56.0 months(P=0.273).Although there was no statistical difference in PFS and OS among the three groups,patients with multiple extranodal involvement showed a significantly poor prognosis.There are 17 sites involved in extranodal involvement in 244 patients with DLBCL.Among them,there were 38 cases of intestine(38/244,15.6%),34 cases of stomach(34/244,13.9%),30 cases of bone(30/244,12.3%),17 cases of bone marrow(17/244,7.0%),14 cases of serosa(14/244,5.7%),,12 cases of breast(12/244,4.9%),11 cases of liver(11/244,4.5%),8 cases of central nervous system(8/244,3.3%),7cases of thyroid(7/244,2.9%),and 7 cases of female reproductive system(7/244,2.9%),7 cases of salivary gland(7/244,2.9%),7 cases of lung(7/244.2.9%),6 cases of testic(6/244,2.5%),6 cases of adrenal gland(6/244,2.5%),6 cases of pancreas(6/244,2.5%),5 cases of male urinary system(5/244,2.0%),4 cases of skin and muscle tissue(4/244,1.6%).Multiple extranodal sites are more likely to be involved in bone,bone marrow,female reproductive system,serosa,liver,adrenal gland,lung,skin and muscle.2.32 hot spot driving gene mutations were successfully detected in 244 patients with DLBCL,and all genes had mutations at different frequencies.Among them,233cases(233/244,95.5%)had gene mutations,the median number of mutations was 5.00(0-24).14 genes had mutation frequencies ≥ 10%,they are PCLO(38.5%),PIM1(32.4%),MLL/MLL2(28.3%),TP53(27.0%),CD79B(24.6%),MYD88(20.5%),SOCS1(19.3%),CREBBP(15.2%),TNFAIP3(14.8%),CARD11(13.9%),B2M(11.9%),IRF4(11.5%),CCND3(11.1%),PRDM1(11.1%).3.Analyze the correlation between the number of different extranodal sites involved with clinicopathological features and driver genes abnormality in 244 DLBCL patients.Univariate analysis showed that the three groups of no extranodal site involvement,single extranodal site involvement and multiple extranodal sites involvement at LDH level(LDH elevated ratio: 40.7% vs 31.4% vs 68.9%;χ2 =18.938,P= 0.000),Ann Arbor stage(III/IV stage ratio: 42.0% vs 30.9% vs 100%;χ2= 59.212,P=0.000),IPI score(3-5 score ratio: 14.8 vs 8.5% vs 88.9%;χ2=118.326,P=0.000).There was no significant differences in age,sex,ECOG score,Hans type,double expression and TP53 protein expression.There was significant differences in EZH2(3.7% vs 1.7% vs 15.6%,P=0.002),MEF2B(6.2% vs 5.1% vs17.8%,P= 0.021),STAT6(12.3% vs 4.2% vs 2.2%,P=0.033)and there was no difference among the other 29 driving genes.Multivariate analysis showed that high IPI score and EZH2 mutation were risk factors for multiple extranodal sites involvement.4.The correlation between common involved sites(stomach,intestine,bone,breast,central nervous system,testis,adrenal gland,bone marrow)and driving genes in 244 patients with DLBCL was analyzed.It was found that stomach was associated with STAT3 mutation(OR=6.741,P=0.009);intestine was associated with CD79 A mutation(OR=9.412,P=0.049);breast(OR=4.062,P=0.029)、center nervous system(n=8;OR=6.771,P=0.014)、testis(n=6;OR=20.312,P=0.008),adrenal gland(n=6,OR=8.125,P=0.021)were all associated with MYD88 mutation,in which central nervous system was associated with MYD88 L265P(OR=7.100,P=0.012)and PIM1(OR=6.000,P=0.035)mutation,testis involvement was also associated with MYD88L265P(OR=7.100,P=0.027)mutation;no related driving genes were found in bone and bone marrow.Conclusions1.Extranodal involvement is common in DLBCL patients,and multiple extranodal involvement is positively correlated with EZH2 mutation.2.The common extranodal sites with poor prognosis in DLBCL patients have different gene mutation characteristics.Breast,CNS,testis and adrenal gland are associated with MYD88 gene mutation,while stomach,intestine,bone and bone marrow are not associated with MYD88 mutation,among which there are hot spot mutation of MYD88 L265 P in CNS and testis.