Molecular Markers On Drug Resistance Of Activated B Cell Type Diffuse Large B Cell Lhympoma

Objective:According to the different cell origins,diffiise large B-cell lymphoma(DLBCL)is divided into activated B cell-like(ABC)-DLBCL and germinal center B cell-like(GCB)-DLBCL.The ABC-type has a poor prognosis and easily invades bone marrow.Over 30%of patients are converted to relapsed/refractory DLBCL(RR-DLBCL)after clinical standardized first-line treatment.Our aim is to identify bone marrow molecular markers that might be utilized to predict relapsed/refractory ABC-DLBCL patients.Meanwhile,we constructed a rituximab-resistant ABC-DLBCL cell line in vitro to provide an experimental basis for solving the clinical problem of ABC-DLBCL resistance to ntuximab.Methods:We recruited 202 patients with ABC-DLBCL at the National Cancer Center/Cancer Hospital,Chinese Academy of Medical Sciences from January 2009 to January 2016.We detected expression of bone marrow molecular marker proteins by immunocytochemistry.After completing standard first-line chemotherapy,they were followed up regularly every three months to evaluate whether recurrence or progression of the cancer occurred.The rituximab-resistant ABC-DLBCL cell line SU-DHL-2-R was constructed in vitro using a "gradient dosing".Flow cytometry was used to detect the expression of CD20,complement dependent cytotoxicity(CDC)and antibody-dependent cell-mediated cytotoxicity(ADCC)of parental and drug-resistant cells.Western blotting was used to detect the expression of apoptosis-related proteins.The differentially expressed proteins of drug-resistant cell line and parental cell line were identified by Label-free mass spectrometry,and subsequent bioinformatics analysis was performed using KEGG database.Results:We detected key molecules in multiple signaling pathways in bone marrow aspirate smears.Proteins with higher expression levels in bone marrow cells were STAT3(46.0%),NF-κB(38.6%),SYK(38.10),BTK(28.2%),Bcl-2(23.3%),and Pax5(21.2%).Two of the NF-κB,STAT3,Bcl-2,Pax5,SYK,and BTK proteins were expressed in numerous smear samples.Comparing the ratio of drug-resistant cases in each group,the difference between the double-positive or double-negative groups was significantly greater than the difference between samples with single-protein expression.There were significant differences in the two-year RFS rates between double-positive and double-negative protein expression groups of STAT3/NF-κB(P=0.005),Bcl-2/STAT3(P=0.009),Bcl-2/Pax5(P=0.003),and BTK/SYK(P<0.001).We have successfully constructed drug-resistant cell line(SU-DHL-2-R)through a series of phenotypic test results.Mass spectrometry results showed that compared with parental cell line(SU-DHL-2-WT),there were 45 up-regulated proteins and 77 down-regulated proteins in SU-DHL-2-R.GO analysis suggested that the biological behavior of differential proteins mainly focused on B cell differentiation,cadherin binding,DNA damage,and apoptosis.KEGG pathway analysis indicated that the differential proteins between drug-resistant cells and parental cells were mainly enriched in B-cell receptor signaling pathway and HIF-1 signaling pathway.Conclusion:The positive protein expression combination in the bone marrow smear of ABC-DLBCL patients can predict the relapse and resistance of Patients,and successfully construct Rituximab resistant cell lines in vitro.Our research provides further clinical evidence of ABC-DLBCL drug-resistant molecules and theoretical basis for rational second-line treatment after drug resistance.