Assessment Of Tumor Infiltrating T Lymphocytes,Checkpoint Molecules PD-1/PD-L1,CD47 And MYC Oncogene In Diffuse Large B Cell Lymphoma:Association With Clinopathological Features And Their Impact On Prognosis

Objective Functional adjustments of T cell subsets in the immune editing processes during neoplastic progression in DLBCL still remains unclear.Here,we studied the expression of CD4/CD8 tumor infiltrating lymphocytes（TILs）in DLBCL.Besides,the expression of two immune checkpoint molecules:PD-1/PD-L1 which are believed to be capable of strongly modulating the adaptive arm of immune system and play a vital role in tumor immune escape,are also studied in this study and analyzed the correlation with the concomitant immune response expressed by CD4/CD8 lymphocytes in DLBCL microenvironment.We further assessed the prognostic importance of these molecules by associating the findings with disease outcome.Methods Expression of CD4,CD8,CD68,PD-1 and PD-L1 was examined on serialsections of Formalin Fixed Paraffin Embedded（FFPE）biopsy specimens of 66 newly diagnosed and pretreatment DLBCL cases using immunohistochemically（IHC）method.Simultaneously,multiplex Immunofluorescence（m IF）was also used to measure the expression patterns and co-localization of these markers.To further support the expression of the checkpoint molecules,flow cytometry analysis for markers CD19,CD20,PD-1,PD-L1was performed in the pretreatment DLBCL FFPE biopsy specimens.Epstein–Barr virus（EBV）-encoded RNAs（EBERs）were detected using In Situ Hybridization（ISH）.After biopsy-confirmed diagnosis,these patients received standard R-CHOP chemotherapy.Results 56.06%of the total cases had high CD8+TILs while 43.94%of the cases had low CD8+TILs.Significant high CD8+TILs was seen in patients whose age was<60 years and Ann Arbor stage I/II（P=0.015,P=0.013 respectively）.Tumor cells expressing positive PD-L1(PD-L1+_TC)and tumor cells expressing positive PD-1(PD-1+_TC)DLBCL had significantly low CD8+TILs（P=0.032,P=0.017 respectively）.Univariate analysis showed high CD8+TILs,age<60 year,stage I/II,ECOG PS<1,IPI<2,patients without B symptoms,Germinal center DLBCL,and PD-1-_TC had favourable prognosis（P<0.05）.Kaplan–Meier curve and log-rank test showed that high infiltration of CD8+lymphocytes was significantly associated with longer Progression Free Survival（PFS）and Overall Survival（OS）（log rank test,P=0.003 and P=0.001 respectively）.PD-1+_TC DLBCL patients were significantly associated with shorter PFS and poor OS（log rank test,P=0.006,P=0.005 respectively）while no significant association could be found with PD-L1+/-_TC DLBCL patients.Conclusion This study suggests that abundant CD4 and CD8 lymphocytes infiltration in DLBCL show pre-existing antitumor immune response and these patients responded well to R-CHOP therapy and had better survival.Nevertheless,this study also highlights new targeted therapeutic approaches in PD-L1+_TC/PD-1+_TC DLBCL.Objective Programmed Death Ligand-1（PD-L1）,usually expressed by cells in tumor microenvironment.It is commonly used biomarker for predicting immune checkpoint inhibitors.A transcription factor known as MYC,is overexpressed in cancers which play a crucial role in impeding immune cells from attacking tumor cells by inducing the expression of PD-L1.This present study observed the expression of c MYC in DLBCL cases,assessed its correlation with PD-L1 expression and further explored their association with the clinicopathological and prognostic features in DLBCL cases.Methods Expression of PD-L1,c MYC,CD 68 was examined on serial sections of 66 newly diagnosed and pretreatment DLBCL cases using immunohistochemically（IHC）and multiplex Immunofluorescence（m IF）method,evaluating their association with clinicopathological features and overall survival.MYC rearrangement status of 66 DLBCLs cases were also studied using fluorescence in situ hybridization（FISH）.All the patients were treated with rituximab-cyclophosphamide,doxorubicin,vincristine,prednisone（R-CHOP）chemotherapy after biopsy-confirmed diagnosis.The correlation between the expression of these markers with the clinicopathological features and the overall survival（0S）of the patients were analysed.Results c MYC protein was expressed in 26 patients（39.39%）and the expression of c MYC protein was exclusively nuclear in all the positive cases.MYC break positive was identified in 13 cases.Out of 13 MYC break positive cases,10 cases（76.92%）showed > 80% c MYC positive lymphoma nuclei by IHC.The difference between expression status of c MYC in GCB and non-GCB is statistically significant（P= 0.027）.Significant positive correlation between c MYC and PD-L1 expression in tumor cells was found while significant negative correlation was found between c MYC and PD-L1 expression in tumor infiltrating immune cells（r = 0.340,P = 0.005;r =-0.324,P= 0.008 respectively）.c MYC +/PD-L1+ group showed significantly shorter progression free survival and overall survival.Conclusion A significant correlation was found between c Myc and PD-L1 expression which suggests the role of c MYC in the regulation of the expression of PD-L1.Though the present study didn’t show statistically significant difference in survival between PD-L1+ DLBCL patients and PD-L1-DLBCL patients,c Myc+PD-L1+ double positive DLBCL patients predicts lower progression free survival and poor overall survival.Thus,we consider that more strategy such as MYC and PD-L1 targeted therapy may be beneficial for c MYC+ PD-L1+ double positive DLBCL cases.Objective The mechanism to evade host anti-tumor immunity,as shown in most of the solid tumors,is through the upregulation of the immune checkpoint PD-1/PD-L1 pathway.However,CD47,an antiphagocytic ligand expressed on tumor cells,represents another cell surface molecule promoting tumor immune evasion via targeting the innate immune system.No studies have been reported yet showing the concomitant expression of CD47 with PD-L1 expression in DLBCL.In this study,we investigated the immuno histochemical expression and correlation between CD47,PD-L1 together with nuclear MYC protein in DLBCL and further assessed the association of CD47 expression with the clinicopathological features and its impact on prognosis.Methods: Immunohistochemical analysis of the expression of CD47,PD-L1 and c MYC was performed on serial sections of 66 newly diagnosed and pretreatment DLBCL cases.MYC rearrangement status of 66 DLBCLs cases were also studied using fluorescence in situ hybridization.After biopsy-confirmed diagnosis,these patients received standard R-CHOP chemotherapy.Results: 46.97% of the total cases showed CD47 positive expression.The positive expression of CD47 showed significant difference between number of extranodal sites（P= 0.028）while rest of the clinicopathological features didn’t show significant difference with CD47 expression.The expression of CD47 is significantly associated with c MYC protein expression（P= 0.018）and is found to have positively correlated（r= 0.297;P= 0.015）.The Kaplan –Meier curve and log-rank test showed that GCB cases are associated with significantly better survival than non-GCB cases（log rank test,P= 0.006）.However,when the cases were stratified according to the cell of origin,we found that in non-GCB group,CD47+ DLBCL patients had poor overall survival than CD47-DLBCL patients and the difference is statistically significant（log rank test,P= 0.023）while no significant survival difference was found in GCB group（log rank test,P= 0.713）.Conclusion: CD47 expression indicates a poor prognosis in DLBCL.This study supports that non-GCB patients with positive CD47 expression are likely to benefit from combined treatment of rituximab with CD47-blocking antibodies.However,additional detailed studies of CD47 expression is required before treatment with CD47 blockade therapy.Objective Diffuse large B-cell lymphoma（DLBCL）is the most common lymphoid malignancy in adults.In DLBCL,unusual expression of T cell antigens has been reported in literature,however,CD4 expression in DLBCL is exceedingly rare.we report a case of CD4+ DLBCL which was identified during our previous study on “infiltrating lymphocytes on DLBCL” and review the relevant literature to find out the incidence and the possible mechanisms of unusual expression of T-cell antigen in DLBCL.Method Among 66 cases of DLBCL,a case of CD4 positive DLBCL was revealed during examining the expression of CD4 and CD8 tumor infiltrating lymphocytes（TILs）on serial sections of 66 DLBCL cases using immunohistochemically（IHC）method.To further support the diagnosis of CD4 positive DLBCL,flow cytometry analysis was also performed in the immunophenotypically detected CD4 positive DLBCL FFPE biopsy specimen.The patient was treated with rituximab-cyclophosphamide,doxorubicin,vincristine,prednisone（RCHOP）chemotherapy.Results Immunohistochemical findings showed that neoplastic cells expressed CD20（+）,BCL2（+）,BCL6（+）,MUM（+）,CD4（+）,CD10（-）,CD5（-）,CD30（-）,ALK（-）,EBER（-）with rare scattered CD3（+）T cells and few CD8（+）T cells.Flow cytometric dot plots also showed the neoplastic B cells with coexpression for CD4.The patient survived for 16 months from the date of disease diagnosis.Conclusion Even though the clinical significance of expression of T antigens remains unclear in DLBCL,it is utmost necessary to be conscious of such phenotypic plasticity in order to prevent flawed diagnosis as CD4+ DLBCL can be misdiagnosed as T cell lymphomas which behaves more aggressively and may be managed in a different manner.Objective Reverse Variant of Follicular Lymphoma（RVFL）is one of the rare morphological variants of FL,characterized by dark staining small centrocytes in the center with pale staining large centroblasts at the periphery of the neoplastic follicles.Only rare cases of RVFL have been described to date.The histological appearance of this little known variant of FL may be misinterpreted if pathologists are unaware of its existence.The main purpose of this study is to draw pathologists’ attention to such uncommon growth pattern of FL so that this variant can be accurately recognized and the clinical significance further studied in the future.Method Four cases of FL with unusual morphologic features were evaluated for the expression pattern of CD20,CD21,CD10,BCL6,BCL2,CD3,CD5,Cyclin D1,Ig D,CD23 and Ki67 by immunohistochemistry.Fluorescence in situ hybridization（FISH）with breakapart probes was performed to detect BCL2 gene rearrangement.Results All four cases possessed distinctive configuration of RVFL;in addition,each also exemplified unique morphological features.Immunohistochemical stains confirmed the follicular cells in both the central areas and the peripheral cuffs had the same immunophenotypic profiles,a clear contrast to the FL with marginal zone differentiation.FISH demonstrated BCL2 gene rearrangement in all cases.Conclusion The growth pattern of this rare FL variant may mimic FL with marginalzone differentiation and other entities including but not limited to marginal zone lymphoma（MZL）,progressive transformation of germinal centers（PTGC）and nodular lymphocyte predominant Hodgkin lymphoma（NLPHL）.Pathologists should be familiar with this unusual morphological variant to avoid diagnostic pitfalls.