Prognostic Factors In Diffuse Large B Cell Lymphoma, A Preliminary Study

Backgroud and ObjectivesDiffuse large B-cell lymphoma(DLBCL),the most common subtype of non-Hodgkin's lymphoma,accounting for about 30%-40%in adult's NHL.It has an obvious heterogeneity for a set of morphological,immunological phenotype,genetics and clinical manifestations.The incidence of DLBCL in non-Hodgkin's lymphoma (NHL) accounted for 31%in Europe and the United States,even more than 40%in some Asian countries.In recent years the population of DLBCL in China was increasing.About 40%of patients are respond well to current therapy and have prolonged survival,whereas the remainder succumb to the disease.Recently,IPI (International Prognostic Index,IPI) is widely used in clinic,but could not benefint for most patients.We plan to investigate more feasible and convenience index to guide our therapy during the clinical application.The gene expression of DLBCL makes a access to prognosis for patients.Many researcher have studied some immunity makers,such as CD2O,CD79a,CD10,CD30,Mum-1/IRF-4,Bcl-2,Bcl-6,CD138,to typing the DLBCL.Alizadeh divided DLBCL to two types,GCB(germinal enter B cell like,) and ABC(activated B cell like) in 2000.Rosenwald raised the third type in later by 152 cases.Hans applied gene array to detect three antibodies(CD10,Mum-1,Bcl-6) to group the DLBCL in 2004.The study have implied that the prognosis of GCB was better than non-GCB. However,some argued that there was no significance between them.In addition, Mum-1 also play an important role in the prognosis.Zinzani PL argued that Mum-1 is a germinal center-associated protein,a good prognosis;but Hans,Chang and other researchers advocated that Mum-1 is an activated B cell marker for a poor prognosis. At present,several antigens which used commonly by foreign reserchers help to judge the prognosis of DLBCL.However,whether they are enough for prognosis and applicable to the Chinese people,the large sample study still rare in China.We found some patients suffered in bone invovlement at early stage.However some patients are not even if many of primary lesions or at the advanced stage.Is this is a universal or accident phenomenon? Is there any regularity for the occurrence of bone invovlement.Is there any antigents play roles in this process? As we all know, CD44 is a important antigent in normal development of B cells,which express in post-germinal center.It has a a negative contro with the expression of Bcl-6,and has a a positive control with Stat-3.CD44 is a cell surface glycoprotein and the principle receptor for hyaluronan,a major glycosaminoglycan of the extracellular matrix CD44 is well known to have multiple splice forms,normal lymphocytes and lymphomas mainly express a "hematopoietic" isoform,CD44H,which lacks many variable exons. In addition,splice variant containing exon 6(CD44v6) may possess unique properties to further incrase the metastatic potential of lymphoma cells.In the immune system, CD44 is called a homing receptor because recirculating peripheral blood lymphocytes use CD44 to bind to the postcapillary venuels in the lymph nodes in order to extravasate.Interestingly,some patients were bone marrow involvement early,but some were not even primary lesion widely.Maybe High level expression of CD44 would imply correlate with extranodal and disseminated DLBCLs,and predicts inferior survival.Bcl-6 mater regulator of germinal center formation and the most frequently targeted proto-oncogene in all B cell lymphomas.It was originally cloned in 1993 based on its frequent involvement in 3q27 translocations.During normal B cell development,the initiation and maintenance of GC are absolutely dependent upon BCL6.In addition,within the GC,BCL6 plays a gate-keeper's role to prevent premature commitment to terminal differentiation.BCL6 achieves this by actively repressing activation markers,e.g.CD44,CD69,CD80,and Blimpl,the master regulator of plasma cell differentiation..Results from many groups indicate that GCB group expresses high levels of the transcription repressor BCL6 and tends to respond better to the CHOP or R-CHOP regimen,while the ABC group is associated with constitutively activated NF-κB and Stat-3,and tend to respond poorly.Some recent work showing STAT3 as a direct BCL6 target gene also provides additional insight into the role of Bcl-6 during GC exit.The anti-CD20 monoclonal antibody has brought a immuochemotheray for DLBCL.ASCO published a historical control study in 2007 showed that,R-CHOP could improve the OS regardless of GCB type.Farinha,Nyman,etc argeued that the two groups showed different respond to rituximab,which the Non-GCB group was better than the GCB group,gnosis.Our study will combine the antigen expression and Hans division,and observe the effect of Rituximab in DLBCL.The goal of the current study is to evaluate if using a panel of markers such as CD20,CD44H,CD44v6,Bcl-6,Mum-1 and Stat3 by immunohistochemistry defines prognosis in patients with diffuse large B-cell lymphoma(DLBCL).Studying the effectivity of Hans typing standards and the expression of Bcl-6/Stat3 gruoping. Moreover,to evaluate the some antigents with both bone involvement and the importance of Rituximab in DLBCL.We expect to find out some effective indexes for prognosis and bone marrow involvement. Materials and Methods1.There were 120 patients consecutively diagnosed with diffuse large B-cell lymphoma(DLBCL) between January 1998 - October 2008 in Nanfang Hospital. We set up follow-up data,analysis for clinical diagnosis and the efficacy of telephone follow-up of survival time and survival status.2.Review HE sections,immunohistochemical single-stains for CD20,CD44H,CD446,Bcl-6,Stat3 and Mum-1 such the markers were perform on paraffin-embedded tissues.3.Review the single-staining sections,select the cases which the expressions of CD44H and Stat3 were +～++,then immunohistochemical double-stains forCD44H/Stat3,such the markers were perform on paraffin-embedded tissues.4.All of the results were analyzed by Spss 13.0,such as survival curve,Cox Regession chi-square test,Binary logistic Regession.Results:1.Case imformation:There are male 67 cases(55.8%) and53 cases(44.2%) of female in the 120 cases of diffuse large B-cell lymphoma patients.The range aged 29 to 82 years old,the average age of 54.59years(median age 55 years).77 cases of primary lymph nodes(60%),mostly in the neck lymph nodes,followed by axillary, submandibular and inguinal lymph nodes;48 cases of primary lymph node outside (40%),mostly in the gastrointestinal tract,followed by tonsil,testis,liver and spleen, and other parts.Chemoradiotherapy remains the major treatment for patients,all most account for 90%.2.The results of IHC2.1 The overall frequency of expression forCD20,CD44H,CD44v6,Bcl-6,Stat3 and Mum-1 in the 120 cases studied were as follows:114 of 120(95.0%),78 of 120(66.0%),67 of 120(55.8%),65 of 120(54.2%),50 of 120(41.7%) and 56 of 120 (46.7%).2.2 The results of double staining for CD44H/Stat-3.There were 6 cases express (+),and 4 case express(++),which expressed in one single cell(Fromowitz).The OR rate and median survival time of the groups were diffirent.The OR rate and median survival time in(+) group were 66.7%and 13 month;and the(++) group were 50%and 20 month.The results of which CD44H and Stat-3 expressed in diffirent cells were only 2 cases.The cases were CD44H(+)/Stat-3(++)和CD44H (+)/Stat-3(+).Compared the median survival time with the others,repectively 12.5 month and 20 months.3.The Median follow-up of 120 patient was17 months,range to 6-148 months,the average surviving time is 27.275 months.4.Survival for single factor and multi-factor analysis:The single factor survival analysis(Kaplan-Meier analysis and Log-rank test)showed that Hans group, Bcl-6/Stat-3 group,chemotherapy±Rituximab,age,systemic symptoms,LDH level, bone marrow involvement,IPI score,number of extranodal involvement,Bcl-6, CD44H,CD44v6 on the survival of patients with DLBCL influential.In the Cox multivariate analysis,only chemotherapy±Rituximab,age,extranodal involvement and CD44v6 have a significant statistical significance on the survival of patients with DLBCL(P = 0.000,0.001,0.000and 0.009 respectively).5.1 The associativity with antigen expression and clinical factors(χ~2 test).It showed that the expression of CD44H had a positive correlation with bone marrow involvement(C=0.205,P=0.022),and implied apoor effect(C=0.261,P= 0.003);the expression of CD44v6 only had a negative correlation with effect(C= 0.341,P=0.000),but had no any correlation with bone marrow involvement(C =0.175,P = 0.052);the expression of Bcl-6 had a negative correlation with age and extranodal involvement(C=0.201,0.247,P = 0.025,0.005 );the expression of Stat-3 had a negative correlation with pathology stage(C=0.195,P = 0.030);the expression of Mum-1 had a positive correlation with age and extranodal involvement(C = 0.220,0.203,P = 0.014,0.023 ),but had a negative correlation with bone marrow involvement(C=0.197,P = 0.028).The rest had no statistics,C＜0.2,P＞0.05.5.2 The associativity with antigens expression(χ~2 test).It showed that the expressions of CD44H and CD44v6 had a positive correlation(C=0.345,P= 0.000);the expression of CD44v6 and Stat-3 also had a positive correlation(C= 0.180,P = 0.045);the expression of Bcl-6 with Stat-3,Mum-1 had a negative correlation(C = 0.220,0.647,P = 0.013,0.000 );the expression of Stat-3 and Mum-1 also had a positive correlation(C=0.237,P = 0.007).The rest had no statistics,C＜0.2,P＞0.05.6.The analysis for Logistic Regression:Single analysis showed that age,systemic symptoms,Ferr,IPI,extranodal involvement,pathology stage,CD44v6,chems±R, bone marrow involvement,LDH level,the expression of CD44H,had a significant statistic.Multiplicity analysis showed only extranodal involvement and CD44v6 had a significant statistic(P = 0.001 and 0.022).7.Analysis the efficient of antigen expression and groups(±rituximab)(χ~2 test):In accordance with the expression of the antigen level and treatment(±rituximab) divided to different groups,then compare the efficacy.The results have shown that rituximab can improve the efficacy obviously.Furthermore,the groups of CD44v6(+),Bcl-6(-) and Stat-3(+),which accepted rituximab,showed better effect than which not accepted rituximab,P=0.000,0.003和0.003.Conclusions1.Our grouping of Bcl-6/Stat-3 and Hans classification criteria showed the same tendency in prognosis in DLBCL,which the GCB type was better than non-GCB type.Moreover,our grouping was more effective than Hans in prognosis.2.Bcl-6,Mum-1 and Stat-3 were the important index for typing in DLBCL.3.CD44H implied bone marrow involvement;CD44H and CD44v6 could provide a help for prognosis.4.Bcl-6(-),CD44v6(+) and Stat-3(+) provide actively singal to choose rituximab for DLBCLpatients.5.Chems±Rimximab,Age,bone marrow involvement,IPI,extranodal involvement, pathology stage,LDH level are the main clinical factors,which affect the survival time.Ferritin,IPI,extranodal involvement,pathology stage,chems±Rituximab, systemic symptoms,serum LDH level are is age are significant effecity indicators.