The Study On The Relation Of IRF4 Expression And Prognostic Value In Patients With Diffuse Large B-cell Lymphoma

Background and objective:Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease and accounts for 30% to 40% in all adult non-Hodgkin's lymphomas. Patients with DLBCL usually have a good response to conventional anthracycline-based treatment, but about 50%-60% patients occurred relapse and resistance and die from this disease eventually. The mechanism of different response remains unclear. Because high-dosage and intension chemotherapy and other new approaches can be used to increase efficacy in high-risk group, it is more crucial to search for predictive markers. The International Prognostic Index (IPI) is a practicable tool to guide therapy in DLBCL, but it is inadequate to evaluate the prediction and does not reflect biological distinction. It is necessary to find new predictive indications. B cell proliferation and differentiation are accurately regulated by many transcription factors, which abnormal expression may be one reason for lymphomagenesis.Interferon regulation factor-4(IRF4/MUM1) is a myeloma-associated oncogene transcriptionally activated as a result of t(6;14)(p25,q32) chromosomal translocation. It is a transcription factor belongs to IRF family and specifically expresses in lymphocytes. Many studies have demonstrated that IRF4 overexpression contributed to tumorigenesis. Recent researches also showed IRF4 expression in 50%-70% patients with DLBCL, but the relation between expression and prognosis remained no consensus.The aims of this study were to investigate the value of IRF4 expression in DLBCL patients and explore new target and method to treat DLBCL. This study is divided into two sections, the first section is retrospectively clinical study, measuring IRF4 expression by immunohistochemistry and exploring the clinical value of IRF4 expression in DLBCL. The other is study in vitro, observing sensitivity to doxorubicin in different B lymphoma cell lines with yes or no IRF4 expression and proteasome inhibitor velcade effect on drug-resistant B cell line with IRF4 expression.Materials and Methods:Patients: 95 patients with DLBCL were enrolled in this study. All the patients were from Henan tumor hospital and Ruijin Affiliated Hospital, Shanghai Jiao Tong University, from 2001 to 2005. 58 male and 37 female were included in the study. The median age was 51 years (range from 18 to 55). These cases were divided into two groups according to IRF4 positive or negative. Disease-free survival (DFS) and overall survival (OS) were followed up.Cell lines: The B lymphoma cell lines, Namalwa and Daudi, were cultured in PRMI-1640 medium containing 10% FBS and incubated in 5% CO₂ at 37℃.MTT method: Cells were plated (in triplicate) at 2.0 x 104 cells/well in 200μl of RPMI1640 medium with 10% FBS and different concentration of doxorubicin (5,10, 20, 30, 50ng/ml) combined with or without velcade 15nM and incubated in 5% CO₂ at 37℃in a 96-well plate. After 48 hours treatment, 20μl MTT (5mg/ml) was added to each well and continued to culture 6 hours. 96-well plate was taken out and centrifuged for 10 min at 2000 rpm, the supernatant was discarded, then 200μl DMSO was added. OD values of each well were detected at 570nm spectrum by spectrophotometer. Annexin V: Different concentration of doxorubicin combined with or without velcade 15nM were used to treat B lymphoma cell lines .After 48 hours treatment, cells were collected, washed and stained with Annexin V^FITC, then apoptosis cell was detected with FACS.RNA extraction: Cells (3×10⁶cells/group) cultured after 12, 24, 48, 72 hours were collected. RNA was extracted with Trizal reagents. RNA was quantitated by spectrophotometer and its quality was measured by agarose gel electrophoresis.RT-PCR and semi-quantitative RT-PCR: Complementary DNA (cDNA) copy was made from total RNA using random hexamer primers and M-MLV reverse transcriptase. 2ul cDNA product was used in PCR amplification to detect the expression of IRF4.β-globin was used as control.IRF4 upstream primer and downstream primer is 5'-AGAACGAGGAGAAGAGCATC-3'and5'-CCTTTAAACAGTGCC CAAG-3' respectively.β-globin upstream primer and downstream primer is 5'-TGGTCTCCTTAAACCTGTCTTG-3' and 5'-ACACAACTGTGT TCACTAGC-3' respectively.Western blotting: Cells treated after 12, 24, 48, 72 hours were lysed with Laemmli buffer (62.5mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 5% p-mercaptoethanol). Western blotting was performed on cell lysates to analyze IRF4 protein expression.ResultsTranscriptional factors IRF4 expression in DLBCL patientsIRF4 were found in 61.1% (58/95) of DLBCL patients, IRF4 positive was related to adverse prognostic factors, such as age>60, advanced Ann Arbor stage, extranodal involvement site>1, increased LDH level and high-risk IPI. Clinical significance of IRF4 expression in DLBCL patientsAll patients received anthracycline-based chemotherapy for 6-8 cycles and were followed up 24 months (range from 6 to 44 months). Kaplan-Meier survival analysis showed that event-free survival (EFS) in IRF4 positive patients was significantly shorter than those IRF4 negative patients (P=0.0053). However the difference of OS between IRF4 positive and negative patients was not significant (P=0.0694).Transcriptional factors IRF4 expression in Daudi and Namalwa cell linesTranscriptional factors IRF4 and IRF4 protein were found in Namalwa cell lines by RT-PCR and Western blotting, but not in Daudi cell lines. The growth inhibition and apoptosis in two cell lines after doxorubicin treatment.We found that growth inhibition and apoptosis of Daudi cell line is more than that in Namalwa after 48 hours treatment with different concentration of doxorubicin by MTT and Annexin V method. These findings show that Namalwa cell line is more resistant to doxorubicin than Daudi cell line.Doxorubin combinated with velcade increased the growth inhibition and apoptosis in Namalwa cell line.The growth inhibition rate of cells treated with velcade plus different concentration of doxorubin (26%, 42%, 41.3%, 64.1%, 82% and 88.2%) was significantly higher than that in doxorubicin alone (0, 2.87%, 4.9%, 28.2%, 39.2% and 58.3%) (P<0.05). In addition, apoptosis rate induced by velcade plus doxorubicin was also higher than doxorubin alone (P<0.05). These findings show that velcade may overcome resistance to doxorubin in Namalwa cell line.Velcade and velcade combinated with doxorubin downregulated IRF4 expression in Namalwa cell lineWe observed the alteration of IRF4 gene and protein expression after 12,24,48,72 hours treatment with doxorubin(50ng/ml), velcade(15nM) and VELCADE combined with doxorubin by semi-quatitative RT-PCR and Western blotting. IRF4 gene and protein expression were not changed in doxorubin groups. IRF4 gene expression was downregulated at 48 hours and protein at 72 hours in velcade groups. IRF4 gene was down-regulated at 24 hours and protein at 48 hours subsequently in doxorubicin plus velcade groups. These findings show that downregulating IRF4 expression may be one mechanism of velcade overcoming doxorubicin resistance in Namalwa cell line.Conclusion1. IRF4 expressed in more than half of patients with DLBCL and predicted a unfavorable outcome.2. Transcriptional factor IRF4 may be associated with conventional chemoresistance in DLBCL.3. Proteasome inhibitor velcade(Bortezomib) might overcome conventional chemoresistance by downregulating IRF4 expression. Doxorubicin plus velcade may enhance this action.4. IRF4 may be a noval predictive marker and therapeutic target in DLBCL.