Expression In Diffuse Large B-cell Lymphoma Research Articles

Prognostic Implication of Semi-quantitative Immunohistochemical Assessment of CD20 Expression in Diffuse Large B-Cell Lymphoma.

Background:Immunohistochemical demonstration of CD20 in diffuse large B-cell lymphoma (DLBCL) is prerequisite not only for the diagnosis but also for assigning patients to rituximab-containing chemotherapy. However, little is known about the impact of abundance of CD20 expression assessed by immunohistochemistry on the clinical outcome of DLBCL. We performed a semi-quantitative immunohistochemical analysis of CD20 expression in DLBCL to examine the prognostic implication of the level of CD20 expression.Methods:Pre-treatment diagnostic tissue samples from 48 DLBCL patients who were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) regimen were represented in a tissue microarray and immunostained for CD20. The relative abundance of CD20 expression was semi-quantitatively scored using a web-based ImmunoMembrane plug-in. Receiver operating characteristic curve analysis was used to determine a prognostically relevant cut-off score in order to dichotomize the patients into CD20-high versus CD20-low groups.Results:The levels of CD20 expression were heterogeneous among the patients, with a wide and linear distribution of scores. Patients in CD20-low group showed significantly poor clinical outcome.Conclusions:The levels of CD20 expression in DLBCL are heterogeneous among the patients with DLBCL. A subgroup of the patients with CD20 expression levels below the cut-off score showed poor clinical outcome.

CD4-positive diffuse large B-cell lymphoma: A variant with aggressive clinical potential.

CD4 expression is rare in diffuse large B-cell lymphoma (DLBCL), with 4 previously reported cases. Its significance is uncertain. We report five patients with CD4+ DLBCL and one CD4+ primary mediastinal large B-cell lymphoma. Cases were identified by searching the electronic database of the department; each was reviewed. Average age was 56 years. Neoplastic cells expressed CD20 (5/6 tested cases). BCL2/BCL6 expression were seen in 3/3 tested cases, suggesting a germinal center origin. Additionally, expression of T-cell antigens CD2 and CD5 was noted in 2/2 and CD7 in 1/1 tested case. CD3 was negative in all. Lymph nodes were commonly involved (67%). Patients received chemotherapy +/- radiation (6/6) and bone marrow transplant (2/6). Average survival was 44.2 mo. CD4 expression in DLBCL raises questions of lineage commitment. CD4+ DLBCL is rare; care should be exercised not to diagnose these as T-cell lymphomas. A subset behaves aggressively.

Expression and prognostic value of CARD11 in diffuse large B cell lymphoma

目的了解CARD11在弥漫大B细胞淋巴瘤（DLBCL）中的表达情况及其与预后的关系。方法收集经福尔马林固定、石蜡包埋的DLBCL患者治疗前淋巴结活检病理标本及临床资料，对标本进行组织芯片制备，并采用EnVision两步法对CARD11进行免疫组化染色，同时利用Hans法（CD10、BCL6、MUM1）对DLBCL标本进行分型。分析CARD11表达与患者特征及预后的关系。结果共纳入79例初治DLBCL患者，采用2例反应性增生患者的淋巴结标本作为对照。对77例患者进行生存分析，75例患者可判定CARD11表达结果。在DLBCL组织的肿瘤细胞中，CARD11表达阳性率为65.33%（49/75）。CARD11与患者特征均无显著相关性。CARD11阳性组2年无事件生存（EFS）率低于阴性组（52.03％对86.12%，P=0.036）。在国际预后指数3~5分的患者中CARD11阴性组与阳性组的中位EFS率差异有统计学意义（未达到对18.7个月，P=0.033）。CARD11不是影响预后的独立危险因素。结论在DLBCL中，CARD11阳性患者EFS时间显著短于CARD11阴性患者，在国际预后指数高中危和高危的患者中，这种差异依然存在，提示CARD11有助于对高危DLBCL患者进一步进行预后分层。

FOXO1-p300-Txn Circuit Regulates Oxidative Stress Responses in Diffuse Large B-Cell Lymphomas Characterized By Enhanced Oxidative Phosphorylation

Diffuse large B-cell lymphoma (DLBCL) is a clinically and molecularly heterogeneous disease. The comparison of DLBCLs transcriptional profiles using multiple clustering algorithms led to the identification of distinct DLBCL subtypes reflecting tumor-intrinsic features. The OxPhos-DLBCL subtype is characterized by enhanced mitochondrial oxidative phosphorylation, a major source of potentially toxic reactive oxygen species (ROS). Therefore, we investigated the role of potential mechanisms attenuating ROS toxicity in this DLBCL subtype.We found significantly increased thioredoxin (TXN) mRNA abundance in DLBCLs classified as OxPhos subtype compared to other subtypes. The overall survival (OS) of patients with high TXN mRNA expression was significantly shorter than of those with low TXN mRNA expression, regardless of treatment regimen (R-CHOP and CHOP). TXN overexpression in OxPhos-DLBCLs was also confirmed in a cell line panel at protein level using immunoblotting. To explain transcriptional mechanisms responsible for differential TXN expression in different DLBCLs, we analyzed the TXN promoter and identified two BCL6 binding sites within 1kb upstream of TXN transcription start site. Unlike in other molecular subtypes, BCL6 does not exhibit a repressor activity in OxPhos-DLBCLs (PNAS, 2007; 104: 3207-12). Using luciferase reporter assays and shRNA-mediated gene expression knock-down, we demonstrated that relative differences in TXN abundance between DLBCL subtypes are at least in part caused by the lack of BCL6 transcription repressor activity.We next tested the consequences of TXN depletion in DLBCLs. We found that OxPhos cells with silenced TXN expression were uniformly more sensitive to apoptosis induced by ROS production than control cells. TXN inhibition sensitized all tested cell lines to doxorubicin, the fundamental drug used in DLBCL chemotherapy acting in part as a ROS inducer.In addition to its role in maintaining redox homeostasis, TXN also regulates transcriptional responses to ROS. For example, TXN reduces disulfide bonds between FOXO4 and acetyltransferase p300, which results in reduced FOXO4 acetylation and impaired proapoptotic signaling. For this reason, we assessed whether p300 and TXN are involved in acetylation of FOXO1, a major FOXO member expressed in DLBCLs. In cells with blocked TXN activity, FOXO1 acetylation was significantly higher compared to cells overexpressing wild-type TXN. TXN decreased p300-mediated FOXO1 acetylation, reduced its proapoptotic activity and expression of FOXO1-dependent genes (TRAIL, p27, BIM). We found that FOXO1 and p300 interact in redox and TXN-dependent manner and identified a conserved FOXO1's Cys612 to be responsible for FOXO1-p300 binding. We mutated FOXO1 C612 to Ala and found that when cotransfected with p300, C612A FOXO1 exhibited dramatically suppressed ROS-induced acetylation. Blockade of FOXO1 acetylation resulted in markedly lower expression of FOXO1 target genes, higher cellular proliferation and lower apoptosis. Furthermore, TXN inhibited FOXO1 nuclear translocation in response to oxidative stress in OxPhos-DLBCLs. Finally, silencing FOXO1 in OxPhos cells with knocked-down TXN expression markedly inhibited DLBCL cell line apoptosis in response to oxidative stress, suggesting that FOXO1 is an essential TXN-regulated sensor and effector of ROS toxicity in OxPhos-DLBCL cells.Taken together, these results demonstrate that TXN is overexpressed in a subset of DLBCLs, and high TXN mRNA abundance is related to shorter OS of DLBCL patients. TXN knock-down enhances oxidative stress toxicity in OxPhos cell lines at least in part by facilitating FOXO1 nuclear retention and increasing acetylation of this transcription factor, thus augmenting its proapoptotic activity. DisclosuresNo relevant conflicts of interest to declare.

Transcriptome Sequencing Reveals Thousands of Novel Long Non-Coding RNAs in B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of lymphoma. Roughly 40% of patients do not respond or relapse after current therapy. Gene expression profiling of DLBCL has revealed broad gene expression deregulation compared to normal B Cells and categorized DLBCLs into two main subtypes: germinal center B-cell like and activated B-cell like (GCB-DLBCL and ABC-DLBCL), both associated with distinct clinical outcomes. Recent RNAseq studies have demonstrated that large portion of the genome consists of long non-coding transcripts (lncRNAs) that exhibit cell type specific expression patterns and are shown to play critical roles in disease specific epigenetic gene regulation. We sought to discover and characterize novel lncRNAs expressed in DLBCL that are responsible for the disease phenotypes. We performed a systematic de novo lncRNA discovery analysis on RNAseq data obtained from 116 primary DLBCL tumors (dbGaP, phs000235.v6.p1), and 8 normal B-cell counterparts (4 Naïve B-cell and 4 Germinal Center B-cell samples). We identified 2,632 novel expressed lncRNAs in DLBCL. These novel lncRNAs exhibit characteristics similar to known lncRNAs, such as low coding potential, > 200 nt in length, and exon counts (>=2 exons). Further comparison between tumors and normal B-cells revealed that 942 lncRNAs were expressed in DLBCL only. We noticed that many of these lncRNAs were located nearby coding-genes that are important in B-cell biology, such as BCL6, IKZF1, IL10, IRF4, PRDM1, TNFAIP3, etc. Indeed, using a computational tool GREAT, we found that the coding-genes located nearby DLBCL-specific lncRNAs were significantly enriched in immune cell activation and differentiation pathways (FDR < 0.05), suggesting that novel DLBCL lncRNAs are preferentially located near key B-cell and lymphoma genes perhaps contributing to the expression regulation of these genes. Furthermore, through a systematic approach, we found that 88% of these lncRNAs correlated significantly with at least one coding gene expression (Spearman correlation, FDR < 0.2), and collectively these co-expressed coding genes were enriched in B-cell specific functional pathways, including CD40-regulated pathways. Interestingly, we observed that 81 lncRNA were located within 17 DLBCL locus control regions (LCRs or super enhancers), representing a significant enrichment of lncRNAs at LCRs (p < 0.001). This suggests that super-enhancers may coordinate the co-expression of lncRNAs and their neighborhood coding-genes. Moreover, through supervised analysis, we derived a lncRNA expression signature that can differentiate ABC and GCB subtypes of DLBCL. There were 465 lncRNAs significantly differentially expressed between ABC and GCB DLBCLs (FDR < 0.05). When we performed pathway analysis on the protein coding genes significantly co-expressed with these 465 subtype-specific lncRNAs, we found they were enriched in signatures that can define the two subtypes, including Germinal_center_B-cell_DLBCL and ABC_gt_GCB_Affy signatures.To understand the regulation of lncRNA expression in DLBCL, we examined the histone mark profiles at lncRNA loci. First, we found in any given DLBCL cell line, there was a significant overlap of lncRNA promoters and H3K4me3 peaks (p < 2.2e-16), suggesting like coding genes, lncRNA promoters are marked with H3K4me3. Second, we identified that expression of 323 lncRNAs were negatively correlated with BCL6 expression (FDR < 0.2) in this cohort of DLBCL patients, among which 104 exhibited increased expression upon BCL6 knocking down. BCL6 is a transcription repressor and a master regulator of germinal center transcription programming. Taken together, our data suggest that expression of novel DLBCL lncRNAs is potentially under the control of B-cell specific transcription factors.Finally, we performed single nucleotide variant analysis on these novel lncRNAs by using VarScan. We identified 9,713 SNVs within the exonic regions of these lncRNAs. We calculated the Minimum Free Energy Scores (MFEs) by using RNAfold to examine whether these SNVs would have an impact on the stability of the lncRNA secondary structure. We observed a significantly higher than background shift towards the stabilization of the secondary structure with the SNVs (p < 2.2e-16, Wilcoxon test), indicating a potential genetic mechanism of aberrant expression of lncRNA in DLBCL. DisclosuresMelnick:ROCHE: Other: Research; Genentech: Speakers Bureau; Janssen: Other: Research; Celgene: Consultancy; Eli Lilly: Consultancy; Epizyme: Consultancy.

Identification of Blut, a Novel Long-Noncoding RNA Differentially Expressed in Burkitt Lymphoma

The class of the so-called long non-coding RNAs (lncRNAs) is defined by their lacking coding potential and their length of more than 200 nucleotides. LncRNAs are readily detected in transcriptome analysis but have so far not been characterized or catalogued exhaustively. Although lncRNAs make up a major portion of the mammalian non-coding transcriptome, it is challenging to predict their functions due to their diversity in terms of size, sequence and biogenesis. However, several lncRNAs have already been shown to be involved in crucial cellular processes such as imprinting, regulation of cell proliferation and cellular differentiation. Their mode of action generally involves epigenetic modifications as well as transcriptional and post-transcriptional regulation.Within the ICGC (International Cancer Genome Consortium)-MMML-Seq (Molecular Mechanisms in Malignant Lymphoma by Sequencing) Consortium, which aims at fully characterizing a total of 250 germinal center derived B-cell lymphomas, including Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), we conducted a search for yet unidentified genes.We here report the identification of a novel transcript, which we have named BLUT (Burkitt Lymphoma Unknown Transcript). BLUT is highly expressed in BL, with only very low expression levels in FL and no detectable expression in DLBCL. It is around 1050 nucleotides long with splice evidence for three exons, whereby exons 1 and 3 are shared between all isoforms.BLUT represents a putative lncRNA since it lacks conserved protein domains and has a splice site conservation pattern typical of non-coding transcripts. Furthermore, it is predicted as non-coding by the software Coding Potential Calculator and there was not a single peptide match between a published peptide database (Human Proteome Map) and any potential BLUT peptide (all isoforms). Moreover, BLUT shares a high sequence identity with two (so far) uncharacterized non-coding RNAs (Pan Troglodyte, Chlorocebus sabeus).We tested a cell line panel (consisting of 16 different cell lines including non-lymphoma cell lines) for BLUT expression but could only detect it in the two BL cell lines Namalwa and Raji.Cellular fractionation experiments showed that BLUT is predominantly localized in the nucleus in both Namalwa and Raji.Overexpression of BLUT in SU-DHL-4 resulted in the identification of 222 differentially regulated genes (FDR < 0.05) with a logFC of ± 0.25, among which 49 genes had a logFC of ± 0.50. Several lymphoma relevant genes were part of this list including BIN1, CCL22, CCR7, EBI3, ID2, JAG1, NQO1, and RGS1.Given its nuclear localization, we currently perform chromatin isolation by RNA purification experiments to identify the genomic regions which BLUT binds to. In combination with our overexpression data this will allow further insights into the functions of BLUT.In summary, we have identified a novel long-noncoding RNA termed BLUT, which is differentially expressed between lymphoma subtypes with a high expression in Burkitt lymphoma. Overexpression of BLUT resulted in the deregulation of several genes with known roles in lymphomagenesis. Given its predominantly nuclear localization, we are currently investigating its functions on chromatin regulation.(Supported by BMBF through 01KU1002A-J and by the Duesseldorf School of Oncology (funded by the Comprehensive Cancer Center Duesseldorf/Deutsche Krebshilfe and the Medical Faculty HHU Düsseldorf)) DisclosuresTruemper:Amgen, roche, Mundipharma: Research Funding; Sandoz, Celgene, AMGEN, Nordic Nanovector: Other: Advisory board.

Prognostic Significance of EBV Association in Diffuse Large B-Cell Lymphoma in the Rituximab Era

Introduction: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of diseases in terms of morphology, immunohistochemistry and molecular features. The prognostic factors of DLBCL in the rituximab era are different from previously reported prognostic factors. Hans, et al. reported that patients with non-germinal center B-cell like (GCB)-type DLBCL had a significantly poorer prognosis than those with GCB-type DLBCL in the pre-rituximab era. In patients who received rituximab combination chemotherapy, there is a report that did not find a difference in survival ratio between those with GCB DLBCL or non-GCB DLBCL. On the other hand, there was a report that found a difference in survival ratio between patients with GCB DLBCL and those with non-GCB DLBCL. In addition, the previously reported that patients with Epstein-Barr virus (EBV)-positive DLBCL had a significantly poorer prognosis than those with EBV-negative DLBCL in the pre-rituximab era. In the present study, we considered the prognostic factors of DLBCL patients who received rituximab combined chemotherapy.Patients and Methods: The subjects were 209 DLBCL patients in whom immunohistochemical markers (CD20, CD5, CD10, BCL-2, BCL-6, MUM-1) could be analyzed. In addition, 86 patients were examined EBV-encoded RNA (EBER) in situ hybridization. Pathologic evaluation of the materials from each patient was performedat several central review meetings by six hematopathologists in the ALTSG pathology review board. Patients were treated with cyclophosphamide, vincristine, bleomycin, etoposide, doxorubicin, and prednisolone (CyclOBEAP) regimen or CHOP regimen. Rituximab was administered to all patients. One hundred forty-six DLBCL patients who underwent CHOP-like therapy were assumed as historical controls. The median follow-up period was 69 months (range, 46-88months).Results: In the R-chemotherapy and CHOP-like groups, patients with stage III or IV disease comprised 57% and 67%, respectively, patients with performance status≧2 comprised 32% and 23%, respectively, and patients with serum LDH >normal comprised 61% and 69%, respectively. There were no significant differences in clinical characteristics between the R-chemotherapy group and CHOP-like group. BCL2 was positive in 72 (55%) of the 131 patients who received the R-chemotherapy and in 71 (50%) of the 142 patients who received the CHOP-like regimen. When the 209 patients who received the R- chemotherapy group were divided into the GCB group and the non-GCB group, the GCB group consisted of 74 patients (35%) and the non-GCB group consisted of 135 patients. The relationships between immunohistological markers and outcome among the patients with DLBCL who received CHOP-like therapy were studied. CD5, CD10, and BCL6 had no prognostic impact on 5-year overall survival (OS) and progression-free survival (PFS) rates. When the patients were divided into the GCB DLBCL and non-GCB DLBCL groups, the 5-year PFS of the GCB group was 78% and that of the non-GCB group was 48% (p=0.0008). Next, the effect of rituximab with chemotherapy was examined. CD5, CD10, BCL2, BCL6, and MUM-1 had no prognostic impact on 5-year OS and PFS rates. When the patients were divided into the GCB DLBCL (n=74) and non-GCB DLBCL (n=135) groups, the 4-year PFS of the GCB group was 79% and that of the non-GCB group was 73%, showing no significant difference.We also evaluated the significance of EBER expression among patients by incorporating the EBV-positive DLBCL (n=14) and EBV-negative DLBCL (n=72).There were no significant differences in immunophenotyping analysis between the EBV-positive DLBCL and EBV-negative DLBCL. As for EBER expression in DLBCL, the 4-year PFS of the EBER-positive group was 37% and that of the EBER-negative group was 75% (P=0.007), indicating that the EBER-positive group showed significantly poorer prognosis. The 4-year OS of the EBER-positive group was 50% and the EBER-negative group was 86% (P=0.0005).Conclusions: The EBER may be an important prognostic factor in patients with DLBCL who underwent R-chemotherapy therapy. DisclosuresNo relevant conflicts of interest to declare.

MiR-31 and miR-17-5p levels change during transformation of follicular lymphoma

The 30% of patients whose indolent follicular lymphoma transforms to aggressive diffuse large B-cell lymphoma (DLBCL) have poor survival. Reliable predictors of follicular B-cell lymphoma transformation to DLBCL are lacking, and diagnosis of those that will progress is challenging. MicroRNA, which regulates gene expression, has critical functions in the growth and progression of many cancers and contributes to the pathogenesis of lymphoma. Using 5 paired samples from patients who presented with follicular lymphoma and progressed to DLBCL, we identified specific microRNA differentially expressed between the two. Specifically, miR-17-5p levels were low in follicular lymphoma and increased as the disease transformed. In contrast, miR-31 expression was high in follicular lymphoma and decreased as the lymphoma progressed. These results were confirmed in additional unpaired cases of low-grade follicular lymphoma (n = 13) and high-grade follicular lymphoma grade 3 or DLBCL (n = 17). Loss of miR-31 expression in DLBCL was not due to deletion of the locus. Changes in miR-17-5p and miR-31 were not correlated with immunophenotype, genetics, or status of the MYC oncogene. However, increased miR-17-5p expression did significantly correlate with increased expression of p53 protein, which is indicative of mutant TP53. Two pro-proliferative genes, E2F2 and PI3KC2A, were identified as direct messenger RNA targets of miR-31, suggesting that these may contribute to follicular lymphoma transformation. Our results indicate that changes in miR-31 and miR-17-5p reflect the transformation of follicular lymphoma to an aggressive large B-cell lymphoma and may, along with their targets, be viable markers for this process.

LincRNA-p21 predicts favorable clinical outcome and impairs tumorigenesis in diffuse large B cell lymphoma patients treated with R-CHOP chemotherapy.

Despite improved survival for the patients with diffuse large B cell lymphoma (DLBCL), the prognosis after relapse is poor. LincRNA-p21 is a long intergenic noncoding RNA, which is located on chromosome 17, approximately 15kb upstream from the Cdkn1a (p21) gene. However, its clinical importance and biological role in DLBCL prognosis are unknown. In this study, we conducted quantitative reverse-transcription polymerase chain reaction to investigate the lincRNA-p21 expression in DLBCL. We found that lincRNA-p21 levels were markedly decreased in DLBCL tissues compared with normal. Its expression level was significantly correlated with Ann Arbor stages, B symptoms, performance status, IPI score and serum LDH. Moreover, patients with high levels of LincRNA-p21 expression had a favorable overall survival and progression-free survival. Furthermore, ectopic expression of lincRNA-p21 inhibited cell proliferation, arrested cycle progression and modulated cyclin D1, CDK4 and p21 expression in DLBCL cell lines. These results demonstrated lincRNA-p21 can be identified as a potential novel prognostic biomarker for prognosis in DLBCL and regulate cell proliferation and cycle in vitro. Our findings highlight the value of integrated comprehensive analysis to identify prognostic markers and genetic driver events not previously implicated in DLBCL.

Prognostic and Clinicopathological Value of Survivin in Diffuse Large B-cell Lymphoma

Up to date, survivin, a well-known inhibitor of apoptosis, has attracted considerable attention as a potential biomarker and therapeutic target in diffuse large B-cell lymphoma (DLBCL). Nevertheless, there still remains no consensus on heterogeneous results. Herein, a meta-analysis was performed to clarify a convincing significance of survivin status on prognosis and clinicopathology of DLBCL patients.Eligible studies were identified by searching Medline, Embase, Scopus, CNKI, and Wanfang databases (last updated on November 30, 2014). Pooled hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Heterogeneity and sensitivity were also analyzed. Moreover, Begg, Egger test, and funnel plots were applied to evaluate the publication bias.We finally included 17 eligible studies with the total number of 1352 patients in the meta-analysis. The pooled results showed that positive survivin expression in DLBCL was associated with inferior overall survival (OS) (HR: 1.880, 95% CI: 1.550-2.270) in patients. Moreover, a significant association was revealed between survivin expression and advanced clinical stage (III + IV) (OR: 0.611, 95% CI: 0.452-0.827), higher International Prognosis Index (IPI) score (Score 3-5) (OR: 0.559; 95% CI: 0.410-0.761), elevated serum lactic dehydrogenase (LDH) (OR: 0.607, 95% CI: 0.444-0.831), presence of bone marrow involvement (OR: 2.127, 95% CI: 1.154-3.921) together with reduced complete remission (CR) rate (OR: 0.478, 95% CI: 0.345-0.662).The results suggest that survivin could be a useful prognostic biomarker, and a promising target for DLBCL therapeutic intervention. Considering limited HR data adjusted for standard prognostic variables could be retrieved, future high-quality studies will be needed in evaluating the independent prognostic value of survivin expression in DLBCL.

Loss of HLA-DR expression is related to tumor microenvironment and predicts adverse outcome in diffuse large B-cell lymphoma

The interaction between tumor cells and the tumor microenvironment is essential in the development and progression of diffuse large B-cell lymphoma (DLBCL). Loss of human leukocyte antigen DR (HLA-DR) in DLBCL is a robust adverse prognostic marker. We evaluated the immunohitochemical expression of HLA-DR in lymphoma and the biologic implications of the loss of HLA-DR. The loss of HLA-DR correlated with clinical stage (p < 0.05), International Prognosis Index (p < 0.05), soluble interleukin-2 receptor (p < 0.05) and poor outcome in patients with DLBCL, especially among elderly patients. Flow cytometry analysis of the infiltrating T-cells showed that the mean CD4 + CD25 +/CD8 ratio of the infiltrating T-cells was higher in the HLA-DR positive group than in the HLA-DR negative group (p < 0.05). These data suggest that loss of HLA-DR expression in DLBCL decreases the ratio of helper T-cell within the T-cell population in the tumor microenvironment and might contribute to escape from immunosurveillance.

The expression of CD30 based on immunohistochemistry predicts inferior outcome in patients with diffuse large B-cell lymphoma.

The prognostic value of CD30 expression indiffuse large B-cell lymphoma (DLBCL)remains controversial. Herein, we performed this retrospective study to investigate the clinical and prognostic significance of CD30 expression in patients with DLBCL.Among all the 146 patients, the expression of CD30 was observed in 23 cases (15.7%).The DLBCL patients with CD30 expression showed more likely to present B symptoms, bone marrow involvement, non-germinal centre B-cell-like (Non-GCB) DLBCL, BCL-2 and Ki-67overexpression(p<0.05). Patients with CD30 expression showed significantly poor overall and event-free survivalcompared with CD30 negative patients(p = 0.031 and 0.041, respectively), especially those with the high intermediate/high-risk international prognostic index (IPI)(p = 0.001 and 0.007, respectively). The prognostic value of CD30expression retained in DLBCL patients treated with eitherCHOP (cyclophosphamide, doxorubicin, vincristine,prednisone) or R-CHOP(rituximab+CHOP). The multivariate analysisrevealed that the expression of CD30 remained an unfavorable factor for both overall and event-free survival (p = 0.001 and 0.002, respectively).In conclusion, these data suggest that CD30 is expressed predominantly in Non-GCBDLBCL. The expression of CD30 implied poor outcomein DLBCL patientstreated with either CHOP or R-CHOP, especially those with the high intermediate/high-risk IPI, possibly indicating that anti-CD30 monoclonal antibody could be of clinical interest.

CD43 expression in diffuse large B-cell lymphoma, not otherwise specified: CD43 is a marker of adverse prognosis.

CD43 (leukosialin) is a transmembrane glycoprotein expressed in a variety of hematopoietic cells, including B lymphocytes, and a variety of malignancies including lymphoma, leukemia, and solid tumors. CD43 plays an important role in the development of many diseases, and coexpression of CD43 and CD20 on peripheral B cells is a predictive factor of hematopoietic malignancy. Although CD43 is expressed in approximately 25% of diffuse large B-cell lymphomas (DLBCLs), its prognostic significance remains unclear. To analyze CD43 expression in DLBCL, not otherwise specified (DLBCL, NOS), and assess its prognostic value, we analyzed clinical data from 160 patients with DLBCL, NOS. We observed that CD43 expression was detected in 47 (29.4%) of 160 cases. CD43 expression was positively correlated with old age (>60 years), high serum lactate dehydrogenase level, B symptoms, non-germinal center type, and DLBCL, NOS, mortality. Patients with CD43-positive DLBCL, NOS, had poorer overall survival (P < .001, log-rank test) and event-free survival (P < .001, log-rank test) than CD43-negative patients. Univariate analysis showed that CD43 expression, age, sex, Ann Arbor stage, International Prognostic Index category, and germinal center phenotype were prognostic factors for DLBCL, NOS, patient survival. Multivariate analysis showed that CD43 expression was an independent significant prognostic factor for event-free survival (P < .001) and overall survival (P < .001). Based on these data, we conclude that CD43 expression is a novel adverse prognostic factor for patients with DLBCL, NOS.

Expression of LMO3 and SNAP25 in Diffuse Large B-Cell Lymphoma Cells and Its Relation to Clinical Features

Diffuse large B-cell lymphoma (DLBCL) is a clinicopathologically heterogeneous group of lymphomas. Gene expression profiling (GEP) has identified two distinct forms of DLBCL: activated B-cell-like (ABC) type and germinal center B-cell-like (GCB) type. Approximately 10% of DLBCL are positive for CD5, and most of CD5-positive (CD5+) DLBCLs are classified as ABC DLBCL. CD5+ DLBCL is also characterized by aggressive clinical course, poor prognosis, and frequent central nervous system relapse (Yamaguchi M, et al. Blood 2002, Haematologica 2008; Miyazaki K, et al. Ann Oncol 2011).We previously analyzed 90 patients with DLBCL by GEP and identified a signature gene set that enabled categorization of DLBCLs into two groups: CD5+ group and CD5-negative (CD5-) group. Our analysis of samples from 78 patients with ABC DLBCL showed that the CD5+ ABC DLBCL signature gene set comprised many genes which are related to the neuronal system (Miyazaki K, et al. ASH2010, #4164). To clarify the clinical significance of protein expression of these genes not only in CD5+ DLBCL but also in whole DLBCL, we examined the protein expression of the genes in DLBCL cells and analyzed the relationship with clinical features.The study comprised 167 patients with DLBCL who were diagnosed between 1993 and 2010 at Mie University Hospital. Of the 90 patients who were previously analyzed by GEP, 60 patients were included in the present study. The histologic diagnosis of DLBCL was made according to the 2008 WHO classification. Thirty (18%) of 167 patients were diagnosed as having CD5+ DLBCL. We selected six genes which were included in the CD5+ ABC DLBCL signature gene set: LMO3, SNAP25, SYT5, SV2C, NEUROD1, and SYN2. Protein expression of these genes was analyzed by means of immunohistochemistry using frozen sections.We identified LMO3 and SNAP25 expressions in DLBCL cells. The other four proteins were not detected in DLBCL cells of the study. SNAP25 was expressed in the cytoplasm of DLBCL cells, whereas LMO3 was expressed in the nucleus of DLBCL cells. LMO3 was positive in 35% (58/164) and SNAP25 was positive in 45% (74/164) of the patients examined. Thirty-three patients were positive for both antigens. For validation of our GEP data, we separately analyzed the 60 patients who were previously analyzed by GEP, and found that CD5 expression in DLBCL showed significant correlation with LMO3 (68%, P = 0.0002) and SNAP25 (73%, P= 0.032) expression.Analyses of clinical features at diagnosis according to each protein expression revealed that the LMO3+ group was characterized by poor performance status (> 1) (LMO3+ group, 36% vs. LMO3- group, 11%; P= 0.0004). There was no significant difference in clinical features between SNAP25+ and SNAP25- groups.The most prevalent first-line treatment in the 167 patients was CHOP chemotherapy (142 patients). Forty-four patients received rituximab during first-line chemotherapy. Patients with LMO3+ DLBCL and SNAP25+ DLBCL who attained complete response with the initial therapy accounted for 74% (43/58) and 80% (59/74), respectively, and patients with LMO3- DLBCL and SNAP25- DLBCL for 86% (91/106) and 84% (76/90), respectively. At a median follow-up of 84 months, the LMO3+ group also showed a significantly worse OS than the LMO3- group (P = 0.034, 5-year OS; 46% and 64%, respectively). There was no significant difference in OS between SNAP25+ and SNAP- groups. As the two genes were included originally in the CD5+ ABC DLBCL signature gene set, we divided the patients into two groups by CD5 expression and analyzed the OS of each group. Each protein expression had no significant difference in OS in patients with CD5+ DLBCL; however, LMO3 expression had a significant correlation with a shorter OS (P= 0.048) in the CD5- DLBCL group.The present study revealed the expressions of LMO3 and SNAP25 in DLBCL cells and an association between LMO3 expression and prognosis in patients with DLBCL. Further studies on LMO3 in DLBCL are warranted. (Supported in part by the National Cancer Center Research and Development Funds: #21-6-3, #23-A-17, and #26-A-4) DisclosuresNo relevant conflicts of interest to declare.

Upregulation of Cereblon Expression By the DNA Methyltransferase Inhibitor Azacytidine Strongly Enhances Lenalidomide Cytotoxicity in Germinal Center B-Cell-like (GCB) and Activated B-Cell-like (ABC) Diffuse Large B-Cell Lymphoma (DLBCL)

▪INTRODUCTION: Lenalidomide monotherapy exerts clinical activity in relapsed/refractory Diffuse Large B-cell Lymphoma (DLBCL) with better response rate and progression-free survival being recorded in activated B-cell-like (ABC) rather than germinal center B-cell-like (GCB)-DLBCL. Reasons for such a difference are likely due to different expression of key molecules involved in mediating activity of Lenalidomide, such as Interferon regulatory factor 4(IRF4) and cereblon (CRBN). Evidences supporting the key role of DNA methylation and histone modifications in regulating genome stability and gene expression in DLBCL prompted us to investigate the capacity of Azacytidine in modulating Lenalidomide activity, thereby sensitizing GCB-DLBCL to Lenalidomide and enhancing Lenalidomide efficacy in ABC-DLBCL.METHODS: DLBCL cell lines with ABC (U-2932, RIVA) or GCB (SU-DHL4, SU-DHL6) genotype were used to investigate the effects of Lenalidomide and Azacytidine on cell growth and cell death. Western blotting (WB) and immunofluorescence analysis were used to assess modulating effects of the two-drug combination on molecular determinants of Lenalidomide activity. Additionally, we studied CRBN, IRF4 and CRBN binding proteins expression, such as Ikaros and Aiolos (IKZF1 and IKZF3) by real time polymerase chain reaction (RT-PCR) in response to drug treatment.RESULTS: Graded concentrations of Lenalidomide (0.1-100 µM) inhibited cell proliferation by 20% to 40% and increased cell death up to 30% to 40% in ABC-DLBCL cell lines, whereas had minimal effects on GCB-DLBCL cell lines. Untreated ABC-DLBCL but not GCB-DLBCL consistently showed a high expression of CRBN and IRF4. Upon Lenalidomide treatment (3 days) CRBN was significantly upregulated and IRF4 downregulated in ABC-DLBCL, but not GCB-DLBCL cells. Since DNA methylation regulates gene expression in DLBCL cell lines, we next examined whether Azacytdine could modulate CRBN and IRF4 expression and in turn enhance responsiveness to Lenalidomide. Exposure of both ABC- and GCB-DLBCL cell lines to Azacytidine (up to 72 hours) induced a marked increase of CRBN and IRF4 transcripts; addition of Lenalidomide strongly increased Azacytidine-induced increase of CRBN and significantly downregulated IRF4 expression; the combined treatment induced a marked downregulation of Ikaros and Aiolos protein levels. At the cellular level, the concomitant Azacytidine (10 μM)/Lenalidomide (10 μM) treatment inhibited in a synergistic manner the mean (±SEM) cell growth of both ABC-DLBCL (Lena: -16 ± 4%; AZA: -22 ± 2%; AZA/Lena: -70 ± 1%, P<0.001) and GCB-DLBCL (Lena: -17 ± 3%; AZA: -40 ± 4%; AZA/Lena: -82 ± 2%, P<0.001). Additionally, the two drug exposure was associated with a 3-fold decrease of S phase cells(Lena: 28 ± 2%; AZA: 22 ± 0.8%; AZA/Lena: 9 ± 1%, P<0.001); a marked p21 overexpression, and a 3- to 4-fold cell death increase (P<0.001) in both ABC- and GCB-DLBCL.CONCLUSIONS: Our results indicate that Azacytidine sensitizes GCB-DLBCL to the cytotoxic effects of Lenalidomide and enhances Lenalidomide efficacy against ABC-DLBCL resulting in synergistic anti-proliferative and pro-apoptotic effects in both ABC- and GCB-DLBCL cell lines. Cytotoxicity of the two drug combination is mediated by signaling events involving CRBN upregulation and IRF4 downregulation leading to CRBN-binding proteins downregulation. Azacytidine-dependent activation of CRBN and IRF4 expression allow to hypothesize a methylation-driven regulation of these genes. These results might provide a rationale for clinical studies using Azacytidine and Lenalidomide combination in ABC- and GCB-DLBCL. DisclosuresNo relevant conflicts of interest to declare.

BCL6 Overexpression – Regulated By AhR/ARNT and Wild-Type MEF2B – Drives Expression of Germinal Center Markers MYBL1 and LMO2

The evolution of tumor clones and the clonal architecture of tumors can be followed by the analysis of clone-specific mutations. The diffuse large B-cell lymphoma (DLBCL)-derived cell line U-2932 comprises two subclones (R1 and R2). Immunoglobulin gene hypermutation analysis showed that R1 and R2 represent subclones of the original tumor. Thus, the two U-2932 subclones seemed to be ideal to study the cellular consequences of clonal evolution. Both clones were derived from a presumptive mother clone with genomic BCL2 amplification, which acquired distinct sets of secondary rearrangements leading alternatively to the overexpression of BCL6 (R1) and MYC (R2) in the respective daughter clones. R2 carries t(8;14) a classical activating rearrangement of MYC in B-cells. R1 did not show any of the typical BCL6 translocations responsible for aberrant BCL6 expression. We applied a whole genome array to find out whether numerical aberrations might explain BCL6 expression in R1 and to investigate if subclone-specific gene expression might be attributable to numerical aberrations in general. More than 200 genes showed >10 fold expression differences between R1 and R2. Statistical analysis of results from copy number aberration and expression data analysis revealed that for 58/221 of the differentially expressed genes, numerical differences between the two subclones effectively predict differences in gene expression (sensitivity 0.64; specificity 0.94; accuracy 0.78). Thus, for a sizeable minority of genes numerical aberrations provided an explanation for the differences in gene expression between the U-2932 subclones. However, BCL6 was none of these genes. Thus, we searched for an alternative explanation for the R1-restricted overexpression of this germinal center oncogene. MEF2B point mutations occur in 11% of DLBCL contributing to the genesis of BCL6 positive lymphomas. The U-2932 subclones did not carry MEF2B mutations. Interestingly however, expression levels of MEF2B paralleled those of BCL6 in the U-2932 subclones. Knockdown experiments showed that wild-type MEF2B controlled BCL6 transcription. To test whether MEF2B and BCL6 showed coordinated expression in general, we analyzed the expression and the mutational status of these genes in 23 DLBCL cell lines. Confirming a positive correlation, independence of MEF2B and BCL6 expression levels could be rejected with a p-value according to Fisher´s exact test of 0.0001 against a level of significance of 0.05 (sensitivity 0.92, specificity 0.9, accuracy 0.91).The MEF2B promoter carries binding sites of the AhR/ARNT transcriptional complex. AhR inhibition and ARNT knockdown experiments with the U-2932 subclones revealed that MEF2B is a downstream target of AhR/ARNT signalling. A positive correlation between AhR and MEF2B expression levels could be shown for the majority of 23 DLBCL cell lines tested (sensitivity 0.61, specificity 1.0, accuracy 0.78). These results indicate that the AhR/ARNT-induced expression of wild-type MEF2B might be an independent regulator for BCL6 expression in DLBCL, besides canonical BCL6 translocations, BCL6 promoter hypermutation and MEF2B mutations. To find out the extent to which BCL6 contributed to the subclone-specific gene expression in the U-2932 subclones, we ectopically expressed BCL6 in subclone R2: 48/221 differentially expressed genes were affected. Interestingly, 28/48 genes were upregulated by BCL6 inducing the germinal center markers MYBL1 and LMO2, although BCL6 is believed to act as a transcriptional repressor.In summary, the two subclones of the DLBCL cell line U-2932 faithfully model tumor heterogeneity. Significant expression differences were shown for 221 genes, more than half of which were attributable to genomic copy number differences or to clone-specific expression of the signal transducer AhR and the oncogene BCL6. Moreover, we could show that BCL6 overexpression – regulated by AhR/ARNT and wild-type MEF2B – drives expression of subclone-specific germinal center markers MYBL1 and LMO2 in the DLBCL cell line U-2932. DisclosuresNo relevant conflicts of interest to declare.

Overexpression of BCL-2 Does Not Inhibit Autophagy in Human Follicular and Diffuse Large B-Cell Lymphomas

Background: Human follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the most common forms of indolent and aggressive NHL, respectively. The t(14;18) translocation characterizes approximately 85% of FL and 20% of DLBCL and results in constitutive overexpression of the anti-apoptotic protein BCL-2. It was previously reported that BCL-2 plays dual roles in preventing apoptosis and autophagy. Autophagy is a physical and pathological process whereby cells sequester portions of cytoplasm including organelles to form autophagosomes where they are degraded and recycled. Growing evidence demonstrates that autophagy plays important roles in tumorigenesis, tumor progression, and resistance to chemotherapy.Aims: The autophagy status in human B-cell lymphomas is unknown. We hypothesized that overexpression of BCL-2 could change autophagy status and aimed to determined expression of autophagy-related genes and proteins in FL and DLBCL primary samples by PCR array and tissue microarray. We aimed to evaluate whether expression of the autophagy-related proteins p62, Beclin-1 and LC3 individually and in combination with BCL-2 protein expression could risk-stratify FL and DLBCL patients at diagnosis.Patients and methods: Using PCR array, the autophagy-related gene expression profiles were determined in purified and unpurified reactive and malignant human lymph node tissue biopsies. Diagnostic tissues from FL (n=117) and DLBCL (n=109) patients were microarrayed and autophagy protein expression was evaluated using immunohistochemistry. Univariate and multivariate analyses on both continuous and categorical variables were conducted to measure overall survival (OS), disease specific survival (DSS), and progression-free survival (PFS).Results: Seven autophagy machinery genes were up-regulated in purified FL B-cells, namely ATG9A, ATG16L1, MAP1LC3A, GABARAPL1, ULK1, LAMP1 and HDAC6 compared with reactive B-cells. Two autophagy machinery genes, MAP1LC3A and DRAM1, were up-regulated in DLBCL B-cells. In unpurified tissue biopsies, 20 of 46 genes in FL and 2 of 5 genes in DLBCL with increased expression were autophagy machinery genes. CTSD (cathepsin D) and TGM2 (transglutaminase 2) genes and proteins were mainly up-regulated in DLBCL tumor-infiltrating macrophages. These results demonstrate that FL and DLBCL showed increased expression of autophagy-related genes, regardless of the heterogeneity of these diseases.p62, a selective autophagy substrate; LC3, an autophagosome membrane protein; and Beclin-1, an essential autophagy effector, are often used to evaluate autophagy activity in the cell. 91% FL samples were BCL-2 positive but significantly decreased expression of p62, LC3 and Beclin-1 in FL samples was observed in both intra-follicular and non-malignant inter-follicular areas. This suggests that increased basal autophagy activity in both malignant FL cells and surrounding tumor infiltrating cells, indicating that BCL-2 does not inhibit basal autophagy activity. DLBCL samples displayed heterogeneous expression patterns of BCL-2, p62, LC3 and Beclin-1.We found that decreased p62 expression confers worse OS (continuous P=0.015; and categorical P=0.003), DSS (continuous P=0.037; categorical P=0.014) and PFS (categorical P=0.002) in DLBCL patients. Decreased expression of Beclin-1 was also confers poor prognosis in both FL and DLBCL as conducted by categorical analysis, OS (DLBCL, P=0.015; FL, P=0.004), DSS (FL, P=0.006), and PFS (DLBCL, P=0.029). p62 retains prognostic significance after adjustment for the International Prognostic Index (IPI) score and levels of BCL-2, Beclin-1 and LC3 in multivariate analysis. Beclin-1 retains its prognostic significance in FL after adjusting for FLIPI scores. Low p62 plus high BCL-2 expression in DLBCL confers the worst OS (P<0.0001) and DSS (P=0.001) compared with other combinations.Conclusions: These results demonstrate that FL has increased basal autophagy activity, while it varies in DLBCL. p62 is a novel, independent prognostic biomarker for DLBCL but not for FL. Combining p62 with BCL-2 provides a more robust and reliable method to risk-stratify DLBCL patients at diagnosis. Importantly, we report for the first time that overexpression of BCL-2 in human NHL does not inhibit basal autophagy activity. We propose that increased autophagy activity could be a therapeutic target for treatment of NHL. DisclosuresGribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

Immunohistochemical expression of CD-10, BCL-6 and MUM-1 antibodies and immediate clinical response in patients of diffuse large B-cell lymphomas after six cycles of chemotherapy.

To determine the expression of CD-10, BCL-6 and MUM-1 in patients with diffuse large B-cell lymphoma (DLBCL) and its association with immediate clinical response after six cycles of CHOP chemotherapy. Analytical study. Armed Forces Institute of Pathology (AFIP), Rawalpindi in collaboration with Nuclear medicine, Oncology and Radiotherapy Institute (NORI), Islamabad from September 2010 to September 2011. CD-10, BCL-6 and MUM-1 antibodies were applied on cases diagnosed as DLBCL. Immediate clinical response was noted after 6 cycles of chemotherapy with the help of oncologist and divided into complete response, partial response, stable disease and relapse/ progression. Patient's age, results of expression of CD-10, BCL-6 and MUM-1 and results of immediate clinical response to chemotherapy were noted. Regarding analysis of prognostic markers (CD-10, BCL-6 and MUM-1), chi-square test was used for immediate clinical response to chemotherapy in DLBCL. CD-10 was positive in 40% cases, BCL-6 in 58.7% cases and MUM-1 was positive in 46.7% cases. About 41.3% of patients showed complete response, 10.6% partial response, 17.3% stable disease and 30.8% showed relapse/progression. CD-10 expression in DLBCL was associated with better immediate clinical response (p=0.011) whereas MUM-1 expression in DLBCL was associated with poor immediate clinical response (p<0.0001). However, there was no statistically significant association of BCL-6 with immediate clinical response (p=0.22). DLBCL shows expression of CD-10, BCL-6 and MUM-1 in nearly fifty percent of the cases. CD-10 is associated with good whereas MUM is associated with poor response. However, there was no association of BCL-6 with immediate clinical response.

Abstract 5582: A targeted molecular classifier of MYC activity and BCL-2 expression in aggressive B-cell lymphomas, designed for clinical practice

Abstract Background: High MYC and BCL2 co-expression as detected by immunohistochemical staining (IHC) of fixed biopsy samples identifies a sub-group of diffuse large B-cell lymphomas (DLBCLs) with inferior outcome among patients treated with standard chemotherapy, and the differential expression of MYC and BCL2 among DLBCL subtypes provides a biological basis for the prognostic value of the ‘Cell of Origin’ classification system. Thus quantifying MYC activity and BCL2 expression in formalin-fixed paraffin embedded (FFPE) biopsy specimens could help to identify patients who might benefit from more aggressive chemotherapy. Unfortunately, IHC is not a reproducibly quantitative test due to a number of pre- and post- analytical factors. In contrast, gene expression profiling (GEP) allows for the possibility of better standardization and quantitation of biomarkers in biopsy samples, but traditional GEP has required RNA isolated from frozen tissue. Design: We sought to develop a molecular classifier of MYC activity and BCL2 expression that is applicable to FFPE biopsy samples using the ‘NanoString nCounter’ platform in a 2-stage approach: 1. Discriminate between Burkitt Lymphoma (BL) and DLBCL using a selection of genes specific for each diagnostic category. 2. Quantify MYC and BCL2 expression using statistically justified ‘MYC target’ genes as well as other genes selected with an unbiased approach, those with significant differential expression between MYC IHC High and IHC Low cases. 3. Normalize data to selected housekeeping genes and assess the tissue microenvironment. The initial gene set was developed in silico based on the whole genome gene expression of 56 carefully selected de novo DLBCL that had companion MYC immunostaining. Results: An initial gene set comprising 200 genes was tested on a discovery cohort of FFPE biopsy samples of 42 aggressive B-cell lymphomas (12 Burkitt Lymphoma [BL] and 30 DLBCL). Differential analysis and prediction models were used to construct a classifier comprising 87 genes that resulted in the successful classification of these tumors. We next validated the approach using an independent cohort of FFPE tissue biopsies (12 BL, 7 genetic “double hit” lymphomas, and 38 DLBCL lacking MYC and BCL2 rearrangements). Targeted profiling and molecular classification correctly diagnosed 100% of tumors as either BL or DLBCL. For DLBCLs, the molecular classifier correctly predicted the MYC expression in 87% of cases when compared to a well-validated IHC assay. We conclude that a targeted gene expression profile, using the Nanostring nCounter platform, coupled with a validated molecular classifier can effectively distinguish DLBCL from BL and quantify MYC activity and BCL2 expression in DLBCL. This protocol will be useful for the routine diagnostic and prognostic stratification of aggressive B-cell lymphomas in clinical practice. Citation Format: Christopher D. Carey, Daniel Gusenleitner, Bjoern Chapuy, Heather Sun, Azra Ligon, Alexandra E. Kovach, Long P. Le, Aliyah R. Sohani, Margaret Shipp, Stefano Monti, Scott J. Rodig. A targeted molecular classifier of MYC activity and BCL-2 expression in aggressive B-cell lymphomas, designed for clinical practice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5582. doi:10.1158/1538-7445.AM2014-5582

Prognostic significance of PRAME expression based on immunohistochemistry for diffuse large B-cell lymphoma patients treated with R-CHOP therapy.

The preferentially expressed antigen of melanoma (PRAME), a tumor-associated antigen, is considered a prognostic marker for various human malignancies. The prognostic significance of PRAME expression for diffuse large B-cell lymphoma (DLBCL) patients treated with rituximab-containing chemotherapy has not been evaluated to date, and the ability of immunohistochemistry (IHC) to detect PRAME expression in these patients has not yet been studied, although IHC is simple to perform in clinical practice. We evaluated the prognostic significance of PRAME expression based on IHC analysis in 160 DLBCL patients treated with R-CHOP therapy. There was a significant association between higher PRAME expression and shorter progression-free survival (PFS), and a trend toward shorter overall survival (OS) in patients with higher PRAME expression than that in patients with lower PRAME expression (5-year PFS, 48.1 vs. 61.1 %; 5-year OS, 65.6 vs. 79.1 %). Patients with high PRAME expression tended to have lower chemotherapeutic responses. Thus, IHC is useful for detecting and assessing PRAME expression in DLBCL. Further, we found a positive correlation between IHC and quantitative real-time RT-PCR measurements of PRAME expression. Our findings indicate that IHC results of PRAME expression can be a novel prognostic maker in DLBCL patients treated with R-CHOP therapy.