GNA13 Is A New Marker Of Germinal Center Derived B Cell Lymphomas

Purpose:G?13 is a member of the G12 subfamily of heterotrimeric G protein,encoded by GNA13 gene and located on the human chromosome 17.Its functions include regulating cellular morphology,cell contraction,cell migration and cellular differentiation.GNA13 is the most frequently mutated gene in germinal center?GC?-derived B-cell lymphomas,and mutation mainly in about 15-33%of GC B-cell subtype diffuse large B cell lymphoma?GCB-DLBCL?and 15%Burkitt lymphoma,but have not been reported in follicular lymphoma?FL?,and GNA13 mutations are largely absent from all other the non-GC lymphoid neoplasm.GNA13 mutations may cause the protein to be reduced or absent,however,the loss of GNA13 could trigger the growth and dissemination of GC B cells,which is conducive to the development and progression of GC-derived B-cell lymphomas.This experiment is intending to study whether GNA13 protein?G?13?could be served as the new immunohisto--chemical marker of GC-derived B-cell lymphomas,and analyze the relationship between protein expression and gene mutation of GNA13 in GC-derived B-cell lymphomas?GCB-DLBCL,FL and Burkitt lymphoma?,at the same time,to detect the relativity of Ki67 and MCM2 and the clinical application value of MCM2.Methods:140 cases of B-cell non-Hodgkin lymphomas?B-NHLs?were collected from the Department of Pathology,Tongji Hospitalof Tongji Medical College,Huazhong University of Science and Technology from 2015 to 2017 including 61 DLBCL,42 FL,6 Burkitt lymphoma,12 mantle cell lymphoma?MCL?,8 small lymphocytic lymphoma?SLL?,and 11 marginal zone lymphoma?MZL?.At the same time,8 cases of Angioimmunocytogenic T cell lymphoma?AITL?which derived the germinal center follicular helper T cells?TFH?were also collected.Another 16 cases of chronic tonsillitis and lymph node reactive hyperplasia?follicular hyperplasia?were collected to serve as the control group.First of all,immunohistochemistry?EnVision method?was used to detect the expression of GNA13 in all of the above cases,and immunohistochemical staining for CD10,BCL-6,GCET1,MUM1,FOXP1,and LMO2 were performed on the cases of DLBCL,according to the Hans,Choi,and Tally algorithms,DLBCL can be divided into two subtypes:GCB type and non-GCB type?collecting the consistent cases with the three algorithms?.Secondly,choosing the all negative cases,weakly positive and positive expression of GNA13 in GC-derived B-cell lymphomas?GCB-DLBCL,FL and Burkitt lymphoma?,another choosing non-GCB lymphomas including 15 non-GCB-DLBCL,12 MCL,11 MZL,8SLL,and 10 cases of control group to sequence the gene exons group of GNA13.Finally,immunohistochemical staining for MCM2 and Ki67 were performed on all of the above-mentioned B-NHLs cases,observing the expression of them and analyzing the relativity of them with scientific statistical methods.Results:1.Immunohistochemistry results?1?In the 61 cases of DLBCL,?1?According to the Hans algorithm,the submitted cases were classified into 30 GCB and 31 non-GCB subtypes.?2?The submitted cases were divided into 26 GCB and 35 non-GCB subtypes based on the Choi algorithm.?3?According to the Tally algorithm,these cases were divided into 20 GCB and 41 non-GCB subtypes.Through the pairwise comparison among the 3algorithms,there were no significant differences between any 2 of the 3 algorithms,and there were high consistency between the three kinds of algorithms?Hans vs.Choi,k=0.803,P<0.001;Hans vs.Tally,k=0.670,P<0.001;Choi vs.Tally,k=0.793,P<0.001?.?2?The 129 cases of B-NHLs included:?1?A total of 50 cases with DLBCL?the consistent cases with the three algorithms,and the other 11 cases were not unified for the three algorithms?,among them,20 cases were GCB type,30 cases were non-GCB type,and GNA13 expressed in a total of 11 cases and only existed in GCB type,the total positive rate was about 22.0%?11/50?and was about 55.0%?11/20?in the GCB type.?2?In the 42 cases of FL,GNA13 expressed a total of 34 cases,the total positive rate was 81.0%?34/42?,among them,8 FLI,10 FLII,5 FLIIIA and 19 FLIIIB,and the positive rate of GNA13 was about 100%?8/8?,100%?10/10?,80%?4/5?and63.2%?12/19?,respectively.?3?The positive rate of GNA13 was about 50.0%?3/6?in6 cases of Burkitt lymphoma.?4?In the 8 cases of SLL,12 cases of MCL and 11 cases of MZL,GNA13 was negative expression or only remained expression in the germinal center.In addition,the positive rate of GNA13 was about 100%?8/8?in 8cases of AITL.In the 16 cases of control group,GNA13 was expressed in the germinal center and the positive rate was about 100%?16/16?.2.The exome sequencing results of GNA13 The 114 cases of B-NHLs?including 20 GCB-DLBCL,42 FL,6 Burkitt,15non-GCB-DLBCL,12 MCL,11 MZL,and 8 SLL?and 5 cases of lymph node reactive hyperplasia?follicular hyperplasia?and 5 cases of chronic tonsillitis,the latter is set to the control group,all the above cases were detected by exon sequencing method to detect the gene mutation in GNA13.The results showed that there were 5cases of GCB-DLBCL and 3 cases of FL with GNA13 gene mutation in the 18 cases of GCB-DLBCL and 29 cases of FL,mutations all occurred in exon 4 of GNA13 gene,the mutation rate was respectively about 27.8%?5/18?and 10.3%?3/29?,and that these examples of gene mutation,immunohistochemical staining for GNA13 almost showed weak positive or negative expression in addition to one case of FL grade I showing positive expression.GNA13 mutations were not detected in other cases,included 3 Burkitt,11 non-GCB-DLBCL,11 MCL,9 MZL,4 SLL and 10 control group,and the remaining cases have shown sequencing failure.3.The correlation between Ki67 and MCM2Ki67 and MCM2 in the 140 cases of B-NHLs and 16 cases of control group were tested by immunohistochemistry,the results showed that MCM2 and Ki67 mainly expressed in the germinal center of secondary follicle in the control group.The positive expression rate of MCM2 was approximately 5%-100%in the B-NHLs,and the correlation coefficients of MCM2 and Ki67 of MZL,SLL,MCL,FL,DLBCL and Burkitt lymphoma were respectively r_MZL=0.807?P=0.005?,r_SLL=0.964?P=0.036?,r_MCL=0.988?P=0?,r_FL=0.973?P=0?,r_DLBCL=0.937?P=0?and r_Burkitt=0.995?P=0?,they all have strong correlation,therefore,MCM2 can replace the Ki67 proliferation index and measure the proliferation of tumor cells.And MCM2 from B cell lymphoma?MZL,SLL and the FL grade I-II?,to the invasive B cell lymphoma?FL grade III,MCL and DLBCL?,and finally to the highly invasive Burkitt lymphoma,the positive expression rate increased with the increase of invasion degree,it is suggested that MCM2 expression was related to tumor invasion.Conclusions1.According to the Hans,Choi,and Tally algorithms,DLBCL can be divided into two subtypes,and there were no signifcant differences among the 3 algorithms.2.In 10 cases of control group,the GNA13 expressed in the germinal center.There were varying degrees of expression in both the GC-derived B-cell lymphomas?GCB-DLBCL,FL and Burkitt lymphoma?and germinal center follicular helper T cells?TFH?deriving from AITL.And GNA13 was negative expression or only remained expression in the germinal center in the non-GC-derived B-cell lymphomas?non-GCB-DLBCL,SLL,MCL and MZL?.The above suggests that GNA13 is not only a new valuable marker for GC-derived B-cell lymphomas,but also a marker of TFH derived lymphomas,which can be a new marker for GC cells.3.In the GC-derived B-cell lymphomas,The positive rate of GNA13 decreased in turn in the low grade FL?I-II?,high grade FL?IIIA/B?,GCB-DLBCL and Burkitt lymphoma,The above suggested that GNA13 deficiency or low expression may be related to the invasion degree of GC-derived B-cell lymphomas.4.In the GC-derived B-cell lymphomas,a weak or negative expression of GNA13 protein may indicate the genetic mutation.5.In the B-NHLs,Ki67 and MCM2 have strong correlation.