Establishment and characterization of a novel ‘double‐hit’ follicular lymphoma cell line, FL‐SJC

Abstract About 5 per cent of follicular lymphoma (FL) cases are double‐hit (DH) lymphomas. Double‐hit follicular lymphoma (DHFL) cell lines can improve our understanding and drug development on FL. But there are only few DHFL cell lines. Here, we established a new MYC/BCL2 DHFL cell line, FL‐SJC. The cells were obtained from the hydrothorax of a patient with MYC/BCL2 DHFL and cultured for 140 passages in vitro. FL‐SJC cells demonstrated 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 as additional abnormalities involving chromosomes 2 and 3. Fluorescence in situ hybridization analysis (FISH) showed IGH/BCL2 fusion gene and the MYC rearrangement. In addition, the FL‐SJC cells displayed KMT2D/MLL2 and CREBBP gene mutations. After subcutaneous inoculation of FL‐SJC cells, the SCID mice developed solid tumour masses within 6‐8 weeks. FL‐SJC cells were proven to be free of Epstein‐Barr (EB) virus infection and be multidrug‐resistant. In a conclusion, the FL‐SJC cell line has been identified as a novel MYC/BCL2 double‐hit follicular lymphoma that can be used as a potentially available tool for the clinical and basic research, together with the drug development for MYC/BCL2 DHFL.


| INTRODUC TI ON
Follicular lymphoma (FL) which accounts for 20%-25% of all non-Hodgkin's lymphoma (NHL) cases is the second most common form of NHL. [1][2][3] Cytogenetically, 85%-90% of patients have a typical translocation t(14;18)(q32;q21) that leads to overexpression of BCL2 protein. 4,5 Although FL is considered as indolent, most patients are still incurable and 15% to 28% of cases will turn into an invasive phenotype, usually diffuse large B-cell lymphoma (DLBCL) within 10 years. 2 Recently, the concept of double-hit (DH) lymphoma has attracted considerable attention. DH lymphoma is defined as a chromosomal translocation between the MYC gene which locates in 8q24.2 and another recurrent oncogene, such as BCL2, BCL6 or rarely other genes. [6][7][8] MYC is a transcription factor that regulates the expression of several target genes related to the cell cycle, DNA damage repair, metabolism, protein synthesis and stress response. 9 BCL2 usually plays an anti-apoptotic role. 6 MYC/BCL2 DH lymphomas represent approximately 60%-85% of all cases of DH lymphoma. 7,8 DH lymphoma (all types) is considered to be 'high-grade' B-cell lymphoma, 6,10 has an aggressive clinical course, has poor prognosis, and often involves the bone marrow and the central nervous system with a median overall survival no more than 1-2 years. 10,11 The studies on the pathogenesis of DH lymphoma depend on well-validated DH lymphoma cell lines. 12 The major advantages of cell lines include the probability of unlimited supply, the global availability, the certainty of background and the infinite viable storability in liquid nitrogen. Until 2016, nearly 30 cell lines meet the diagnosis of DH lymphoma, bearing both MYC and BCL2 rearrangement. [13][14][15][16][17] Among them, most of them were derived from patients with DLBCL, or Burkitt lymphoma (or B-ALL), while only 4 cell lines were from patients with FL, 13 including FLK-1, 3 FL-18, 18,19 SC-1 20,21 and TAT-1. 22 FLK-1 holding t(2;8)(p12;q24) and t(14;18)(q32;q21), established in 2001, was found to depend on a follicular dendritic cells. When follicular dendritic cells were removed, FLK-1 cells stopped growing and eventually died. 3 So FLK-1 is unstable and inconvenient as a cell line. 3 FL-18 was established in 1985, in which the translocation [t(8;22)(q24;q13) and t(14;18)(q32;q21)] was not verified by fluorescence in situ hybridization (FISH), Southern blot, polymerase chain reaction (PCR) or other method, 13,18,19

| Patient
A 52-year-old male patient with cough, chest tightness, asthma, night sweat, weight loss and enlarged lymph nodes was admitted to our hospital in July 2016 (no fever or chill) (

| Cell culture
The pleural effusion was collected through thoracentesis in August 2016. The cells were harvested and cultured in RPMI 1640 medium with 10% foetal bovine serum and 1% penicillin/streptomycin without external growth factors or stimulatory cytokines. The cells passaged for at least 140 times were named as the 'FL-SJC'.

| Flow cytometry
The original FL cells from pleural effusion and FL-SJC cells were centrifuged, respectively, and tested for immunophenotypic analysis by flow cytometry. The panel of monoclonal antibodies, including those specific for CD45, CD19, CD20, HLA-DR, CD19, CD5, CD22, CD23, CD10, CD38, and Kappa and Lambda light chains, was bought from BD Biosciences Co.

| Cytogenetic analysis
Chromosome analysis was performed using conventional R-banding.
Peripheral blood mononuclear cells from healthy volunteers were served as a control. Reverse transcription was performed using

| Western blot
The FL-SJC cells were lysed using cell lysates. Peripheral blood mononuclear cells from healthy volunteers were served as a control. The boiled proteins were electrophoresed with 10% SDS-PAGE and then transferred onto PVDF membrane. Proteins were detected using the rabbit anti-MLL2 polyclonal antibody (sc-292359) purchased from Santa Cruz Biotechnology, Inc, and mouse anti-β-actin monoclonal antibody (mAbcam8226) purchased from Abcam, followed by HRP-conjugated secondary antibodies. Immunoreactive bands were detected by ECL. The signal intensity of the respective bands was measured by the NIH Image J software.

| Drug-resistance assay
The cell counting kit-8 was utilized to assess chemosensitivity of

| Statistical analysis
Statistical analyses were performed using the GraphPad Prism 5.0.
The measurement data were expressed as mean ± standard error.
The t test and the analysis of variance were used as appropriate.
P < .05 indicates that the difference is statistically significant. FL-SJC cells were small-to-medium sized and blast-like lymphocytes, and the cytoplasm was moderately rich and basophilic ( Figure 3A). The large nuclei were round to ovoid with rough chromatin and obvious nucleoli ( Figure 3A). The morphologic features of FL-SJC cells were similar to that of original FL cells ( Figure 2B).

| Establishment of the FL-SJC cell line
The viability of FL-SJC cells was high, while a proliferative rate was not as high as that of Dohh2 and SC1 cells ( Figure 3B).

90% cells had a MYC(8q24) gene rearrangement most likely involv-
ing the IGL gene in 22q11 region according to the result of conventional cytogenetic analysis. The proliferation of IGH was also detected ( Figure 3D). The detection rate of FISH was higher in FL-SJC cells than that in FL cells, probably due to the purification of long culture.

| mRNA level and protein expression of MLL2
The quantitative RT-PCR results showed that mRNA level of MLL2 was significantly up-regulated in K562 compared with that in the normal control cells (P < .05), consistent with the previous report. 26 However, mRNA expression of MLL2 in FL-SJC, as well as other two follicular lymphoma cell lines (Dohh2 and SC1), had no statistically significant difference (P > .05) compared with the normal control ( Figure 3E). The protein expression of MLL2 in FL-SJC cells was decreased compared with that in normal control cells ( Figure 3F).

| EBV status in FL-SJC cells
PCR analysis showed EBV negative in FL-SJC cells. As expected, the genomes of EBV1 (type 1) and EBV2 (type 2) were detected in the Raji cell line (as positive control), but not in the Jeko-1 cell line (as negative control) ( Figure 5A).

| Drug resistance
The chemosensitivity of FL-SJC, Dohh2 and SC1 to Ara-c, MTX and CTX is shown in Figure 5B, 5C and 5D. The inhibition rate of Ara-c in FL-SJC was lower than that in Dohh2 and SC1 and the inhibition rate of MTX in FL-SJC was lower than that in Dohh2, suggesting that FL-SJC demonstrated a high drug resistance.

| D ISCUSS I ON
Although therapeutic outcomes of FL have markedly improved in the last few decades, 15% to 28% of cases will transform into an aggressive phenotype, typically DLBCL. 1,2 The therapeutic approaches for FL are most often based on stage, FLIPI and burden of disease. 1,6 In the beginning, the disease was diagnosed as FL with grade II, and a high FLIPI score. This patient had high LDH, infiltration in the bone marrow, reproductive system and the central nervous system.
All these features were fulfilled in this patient. Most common transformation of DHFL is transformation into DLBCL 2 ; the biopsy from infiltrated mass near the pubic symphysis in this patient confirmed the DLBCL transformation at the late stage of disease. One of main limitations of the successful treatment of MYC/ BCL2 DH lymphoma is lack of comprehensive understanding of disease pathogenesis and mechanisms of chemotherapeutic resistance, as well as knowledge of potential therapeutic targets. Cell lines are critical for mechanism exploration and drug discovery. 28,29 However, as far as we know, there have been only a few published manuscripts describing the establishment and characterization of defined DHFL cell lines. 3,13,[18][19][20][21][22] Some of them are not in full detail in genetic messages. 13,18,19 In this report, we established a novel 'double-hit' follicular lymphoma cell line, FL-SJC, which we wish as a useful tool for the improved understanding of MYC/BCL2

TA B L E 1 Gene mutation of FL-SJC cells
'double-hit' FL.
The success rate for the establishment is low, and it is impossible to predict whether it is successful for specimens from source. 12 Those lymphoma cell lines which were established successfully were high-grade lymphomas 30  We then identified the cell line according to the published guidelines. 31,32 The clinical characteristics and cell culture data, as well as immunological and cytogenetic features were described.
The biopsy from initial lymph node fulfilled the immunophenotype of follicular lymphoma. 33 The biopsy from late infiltrated mass near the pubic symphysis confirmed FL transforming into DLBCL.
The positive rate of Ki67 gradually increased suggesting aggressive conversion. The FL-SJC cells expressed CD19, CD10, CD22, HLA-DR, CD38, Lambda and CD20, but did not express CD23, CD5 and Kappa, which was similar to that of the original FL cell from pleural effusion.
The FL-SJC cells were fully negative for CD23 and CD5 which were 8% and 5% positive, respectively, in primary FL cell, suggesting that the FL-SJC cells have a higher purity than original tumour cells.
Xenotransplantation of FL-SJC cells into SCID mice can cause the solid tumour mass, suggesting the high proliferative viability.
The immunohistology of tumour showed the loss of CD20. Pham LV et al ever reported that the RC cell line showed gradually reduced CD20 expression with the culture. 14 Denyssevych T et al also observed that the expression of the CD20 antigen on the surface of Tat-1 cells grown in mice gradually decreased [23]. It is possibly due to selection of more immature lymphoma cells for its growth advantage during the culture and inoculation, because CD20 is mainly ex- Additionally, recently some genes had been reported to be associated with the prognosis of lymphoma. 23 We tested the progno-  23 was not found in FL-SJC, while CREBBP (a histone modification enzyme) mutation, which was associated with poor prognosis, 23 was found in FL-SJC MLL2, also known as KMT2D or ALR, is another histone modification enzyme, gene mutations of which are mainly found in lymphomas (especially DLBCL and FL from indolent to aggressive) and myeloma. 34 MLL2 acts as a central tumour suppressor in FL. 35 The frameshift mutation of MLL2 gene, which has 54 exons, also was found in Exon14 in FL-SJC cells. By We had made a preliminary estimation of FL-SJC sensitivity to chemotherapeutic drugs, Ara-c, MTX and CTX. FL-SJC demonstrated a higher resistance to Ara-c than Dohh2 and SC1 did, and to MTX than Dohh2 did. This may reflect subtle changes in cell growth regulation.

| CON CLUS ION
We described the establishment and characterization of a new MYC/ BCL2 DHFL cell line, FL-SJC, which would be useful in studies on human DHFL, and therapeutic drug development.