Establishment And Characterization Of A Novel "Double-hit" Follicular Lymphoma Cell Line,FL-SJC

Background:Follicular lymphoma(FL)accounts for 20% to 25% of all non-Hodgkin's lymphoma cases.Approximately 5 % of cases of FL are so-called double-hit(DH)lymphomas,defined by a chromosomal translocation or rearrangement involving MYC(8q24.2)in combination with another recurrent breakpoint,usually BCL2(18q21.3).MYC/BCL2 double-hit lymphomas tend to present with advanced stage and often involve the bone marrow and the central nervous system.Double-hit follicular lymphoma(DH FL)cell lines would improve our understanding and drug development on FL.But there are only few DH FL cell lines.Here we established a new MYC/BCL2 DH FL cell line.Methods:Mononuclear cells were extracted from the pleural effusion of a patient diagnosed with follicular lymphoma in IV stage,and cultured in vitro to passage 140 times in 1640 medium containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin solution.We named it as FL-SJC cell line.Subsequently,series of characteristics of the cell line were tested: 1.Cell counting kit-8 was used to detect its proliferationcompared with the other two follicular lymphoma cell lines;2.Conventional R-banded karyotyping and fluorescence in situ hybridization(FISH)were used to detect its cytogenetic characteristics;3.Flow cytometry was used to test the immunophenotype of the the original FL cells and FL-SJC cells;4.Targeted gene sequencing;5.Xenograft of FL-SJC cells into the SCID mice;6.PCR was used to detect the presence of EBV infection of the FL-SJC cell lines;7.CCK8 kit was used to evaluate the FL-SJC cell line chemosensitivity;8.Quantitative RT-PCR and WB(Western Blot)were uesd to detect the expression of MLL2 in FL-SJC cells.Results:FL-SJC cells proliferated stably with a doubling time of 24 hours.Wright-Giemsa staining indicated that the nucleo-plasmic ratio was high,similar to the original cells.FL-SJC cells demonstrated complex immunophenotype: CD19++,CD20+,CD22++,HLA-DR+,CD10+,CD38+,Lambda+,CD23+,CD5+/-,and Kappa-.The chromosome karyotypic analysis confirmed the co-existence of t(8;22)(q24;q11)and t(14;18)(q32;q21),as well asadditional abnormalities involving chromosomes 2,and 3.Cytogenetically,FISH analysis showed IGH/BCL2 fusion gene and the MYC rearrangement.In addition,the FL-SJC cells displayed KMT2D/MLL2 and CREBBP gene mutations by target region sequencing.Importantly,the SCID mice developed solid tumor masses within 6-8 weeks after subcutaneously inoculation of FL-SJC cells.Cell line samples were proven to be free of Epstein-Barr(EB)virus infection and have drug-resistance.Quantitative RT-PCR revealed that mRNA expression of MLL2 in FL-SJC and two other follicular lymphoma cell lines(Dohh2 and SC1)was not significantly different from the normal control.WB authenticated that the expression of MLL2 protein in FL-SJC cells is reduced compared to normal control cells.Conclusion:In this article,we established a novel MYC/BCL2 double-hit follicular lymphoma cell line and designated it as FL-SJC.The cell line can be used as a potentially available tool for the basic research,together with the drug development for MYC/BCL2 DH FL.