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De novo CD5 positive diffuse large B cell lymphoma (CD5+ DLBCL) has poor survival in the era of immunochemotherapy. We conducted a multi-center retrospective study to explore the clinicopathologic characteristics, genomic profiling and prognostic elements of 61 CD5+ DLBCL and 60 CD5- DLBCL patients in China. In contrast with CD5- DLBCL, elderly onset, advanced stage, central nervous system (CNS) involvement, as well as MYC/BCL-2 and P53 overexpression were more prevalent in CD5+ DLBCL. In addition, most of CD5+ DLBCL patients were of non-germinal center B-cell-like (non-GCB) and activated B-cell-like (ABC) subtype according to immunohistochemistry and Lymph2Cx assay. Genetic analysis by next generation sequencing (NGS) showed the proportion of MCD subtype in CD5+ DLBCL was higher than that of CD5- DLBCL (50% vs 5%, p = 0.0007). Compared with CD5- cohort, CD5+ DLBCL patients showed poorer 5-year overall survival (OS) (70.9% vs 39.0%, p76 years, advanced stage, CNS involvement and hypoalbuminemia were independent factors associated with poor prognosis in CD5+ DLBCL. In conclusion, CD5+ DLBCL conveyed poor prognosis and showed distinctive clinicopathologic characteristics and predominant genetic features of ABC and MCD subtypes. Disclosures No relevant conflicts of interest to declare.

Hemophagocytic lymphohistiocytosis (HLH) is an immune disorder with rapid progression and poor survival. Individual treatment strategy is restricted, due to the absence of precise stratification criteria. In this multicenter retrospective study, we aimed to develop a feasible prognostic model for adult HLH in China. A total of 270 newly diagnosed patients of adult HLH were retrieved from Huaihai Lymphoma Working Group (HHLWG), of whom 184 from 5 medical centers served as derivation cohort, and 86 cases from 3 other centers served as validation cohort. X-Tile program and Maxstat analysis were used to identify optimal cut-off points of continuous variables; univariate and multivariate Cox analysis were used for variables selection, and Kaplan-Meier curve was used to analyze the value of variables on prognosis. The C-index, brier score and calibration curve were used for model validation. Multivariate analysis showed that age, creatinine, albumin, platelet, lymphocyte ratio and alanine aminotransferase were independent prognostic factors. By rounding up the hazard ratios from 6 significant variables, a maximum of 9 points was assigned. The final scoring model of HHLWG-HPI was identified with four risk groups: low risk (≤3 pts), low-intermediate risk (4 pts), high-intermediate risk (5-6 pts), and high risk (≥7 pts), with 5-year overall survival rates of 68.5%, 35.2%, 21.3%, and 10.8%, respectively. The C-indexes were 0.796 and 0.758 in derivation and validation cohort by using bootstrap resampling program. In conclusion, the HHLWG-HPI model provides a feasible and accurate stratification system for individualized treatment strategy in adult HLH. Disclosures No relevant conflicts of interest to declare.

Elevated Epstein-Barr virus (EBV) DNA load is common in lymphomas. However, it remains unclear whether the disparity in viral load and its prognostic value in lymphomas are correlated with EBER status. In this retrospective multicenter study, we collected the data of pretreatment whole blood EBV DNA (pre-EBV DNA) and EBER status and evaluated their disparity and prognostic values in lymphomas. A total of 454 lymphoma patients from December 2014 to August 2020 were retrospectively retrieved. Mann-Whitney U test, Kruskal-Wallis test, and Bonferroni's adjustment were used to explore the disparity of EBV DNA and EBER status in lymphomas. Time-dependent receiver operating characteristic analysis and MaxStat analysis were used to determine optimal cut-off points of pre-EBV DNA load. Univariable and multivariable Cox proportional hazards models were established for the estimation of prognostic factors. The positive rate of EBV-DNA in NKTL patients was higher than that in DLBCL, FL, and HL patients, and the median positive pre-EBV copy number of NKTL was also higher than that of FL and DLBCL. EBV-DNA could clearly distinguish the prognosis of DLBCL, NKTL, HL and PTCL, and the integration of EBER status and EBV DNA could differentiate the prognosis of HL patients. Multivariable results revealed that pre-EBV DNA load had an effect on the prognosis of NKTL, FL and DLBCL. The status of pre-EBV DNA and EBER were disparate. Whole blood pre-EBV DNA predicted the prognosis of lymphomas, and the integration of EBV and EBER status could differentiate the prognosis of HL Disclosures No relevant conflicts of interest to declare.

Diffuse large B cell lymphoma (DLBCL) is the most common type lymphoma and the standard therapy R-CHOP regimen has made most patients complete remission, however, 30-40% patients still remains be refractory or relapsed and have a dismal outcome, indicating standard cytotoxic therapy also has limits in these patients. Therefore, it is essential to identify novel therapeutic targets and agents to understand the depth molecular pathogenesis mechanism of DLBCL and overcome the relapsed/refractory. EHMT2 abnormally expression has been discovered in various kinds of malignant cells, and its higher expression may be concerned with poor prognostic of these cancers. BIX-01294 is a small molecule compound which specifically inhibits EHMT2 activity and induces demethylation of H3K9. However, the role of BIX-01294 and the involvement of EHMT2 in DLBCL is not well study now. In the present study, we first examined the expression of EHMT2 and found EHMT2 were downregulated both in protein and mRNA levels. To determine the effect of BIX-01294 on DLBCL cells growth status, CCK-8 assay was preformed to detect the viability. BIX-01294 exhibited notable proliferation suppression in a dose-dependent manner in DLBCL cells, no matter the ABC type or GCB type cells. Then flow cytometry analysis was carried out to investigate the cell cycle distribution when treated with BIX-01294. The cells in G1 phase were increased in a dose-dependent fashion both in U2932 and SUDHL2 cells, and accompanied by the population in S phase was decreased. Furthermore, we preformed flow cytometric assay to elucidate apoptotic effect and found that BIX-01294 treatment induced U2932 and SUDHL2 apoptosis. By western blot and RT-qPCR, we also showed that the expression of anti-apoptotic protein c-FLIP was decreased and the level of DR4 and DR5 was upregulated. Conformably, BIX-01294 down-regulated anti-apoptotic protein Mcl-1 expression and up-regulated pro-apoptotic protein Bax level, indicating BIX-01294 activates exogenous and endogenous apoptotic signaling pathway in human DLBCL cells. We also showed both protein and mRNA levels of LC3B increased when cells treated with BIX-01294 in DLBCL cells, proves BIX-01294 also triggers autophagy. To elucidate the mechanism of BIX-01294-induced apoptosis and autophagy in DLBCL cells, we studied the active stage of ER (endoplasmic reticulum) stress. We examined the expression of GRP78, CHOP, ATF3 and ATF4, which were regarded as important protein markers of ER stress and found that their expression were all enhanced in a dose-dependent fashion, indicate BIX-01294 activates ER stress. We then wondered whether ATF3 and ATF4 influenced apoptosis and autophagy induced by BIX-01294. We conducted shRNA to inhibit ATF3 or ATF4 expression and found inhibition of ATF3 or ATF4 upregulation decreased the expression of LC3B, indicating ATF3 and ATF4 were contributed to BIX-01294 induced autophagy. CCK8 assay showed that the viability was increased in ATF3 or ATF4 abrogated cells after exposure to BIX-01294. Consistently, the percentage of apoptosis was significantly decreased in ATF3 or ATF4 knockdown cells than control cells by Annexin-V staining flow cytometry. In our experiments, we also showed that suppressed ATF4 expression inhibited ATF3 and CHOP expression in DLBCL cells. In conclusion, we showed that with the increased concentration of BIX-01294, the cell survival rate of DLBCL was obviously inhibited and cell cycle was arrested in G1 phase. BIX-01294 induced DLBCL cells apoptosis and autophagy. Furtherly, we explored one of the underlying mechanisms is through activating the ER stress pathway. We speculated that BIX-01294 treatment induces ATF4 upregulation, and then promotes ATF3 and CHOP expression, subsequently contributes to BIX-01294-mediated autophagy and apoptosis. Disclosures No relevant conflicts of interest to declare.

Objective Chimeric antigen receptor T (CAR-T) cells therapy demonstrated remarkable efficiency in refractory and relapsed diffuse large B cell lymphoma (R/R DLBCL). Antigen-loss potentially leads to failure after single-target CAR-T cellss therapy. Aim to evaluate the efficiency and safety of double-target CAR-T cellss therapy, we performed a phase Ⅰ/Ⅱ clinical trial of combination anti-CD19 and anti-CD20 CAR-T cellss therapy for R/R DLBCL. Methods A total of 21 patients were enrolled, and patients were monitored for treatment response, toxicity and persistence. Patients received a conditioning regimen of fludarabine and cyclophosphamide followed by infusion of anti-CD19 and anti-CD20 CAR-T cellss. Results Of the 21 patients, 17 had objective response, and the ORR was 81.0% (95% CI, 58 to 95). 11 had CR, the CR rate was 52.4% (95% CI, 26 to 70). 4 of 9 patients in completed remission at 3 months remain in remission by 6 months, the CR rate was 44.4% (95% CI, 14 to 79). The median OS was 8.1 months (95% CI, 7 to 10) and the median PFS was 5.0 months (95% CI, 2 to 8). The median duration response was 6.8 months (95% CI, 4 to 10). Cytokine release syndrome (CRS) occurred in all patients. Of the 21 patients, 15 (71.4%) had grade 1-2 CRS, 6 (28.5%) had severe (≥grade 3) CRS, and no grade 5 CRS occurred. There were 5 patients with different degrees of neurotoxicity, namely CAR-T associated encephalopathy syndrome (CRES). There were 2 cases with grade 3 or above CRES, 5 of them were self-limited, and none of them died of severe CRS or CRES. There were significant differences in peak levels of IL-6 (P=0.004)、ferritin (P=0.008) and CRP (P=0.000) secretion between CRS 1-2 and CRS 3-4 patients within one month after CAR-T cell infusion. In terms of hematological toxicity, there were 11 cases of neutropenia above grade 3 (52.4%), 6 cases of anemia (28.6%) and 6 cases of thrombocytopenia (28.6%). After 12 patients with response and 1 patient without response received CAR-T cell therapy, CD19 cell subsets all disappeared after 2 weeks. The level of serum immunoglobulin in 14 patients with response decreased progressively after 1 week of treatment with CAR-T cells, and maintained at a relatively low level. Eight patients received intravenous immunoglobulin during CAR-T cell therapy. Conclusion Anti-CD19 combined with anti-CD20 CAR-T cell is effective in the treatment of R/R DLBCL patients.2. Anti-CD19 combined with anti-CD20 CAR-T cell therapy has the occurrence of CRS, CRES and hematological toxicity, and adverse reactions could be controlled. This is the first report to our knowledge of successful treatment of combination of anti-CD19 and anti-CD20 CAR-T cellss in R/R DLBCL. Our results provide strong support for further multiple-target CAR-T cells therapy, which could potentially resolve antigen-loss related failure. Disclosures No relevant conflicts of interest to declare.