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Background Although classic Hodgkin lymphoma (cHL) is highly curable with current treatment paradigms, there is still 20-30% of patients who will experience therapeutic failure (refractory or relapse). Tislelizumab, a monoclonal antibody targeting programmed cell death protein 1, is showing promise in the clinic with obvious response in relapsed/refractory cHL (RRcHL) patients on monotherapy. Further improvements in antitumor efficacy as salvage therapy may require exploration of a tislelizumab-based combination regimen. We conducted a phase II multi-center, open-label, non-randomized study (ChiCTR2000033441) to investigate the efficacy and safety of tislelizumab with gemcitabine plus oxaliplatin (GemOx) in patients with RRcHL. Methods Patients who had at least one previous therapy for cHL and were relapsed or refractory were eligible. Enrolled subjects received up to eight courses of gemcitabine (1g/m 2 on day 1) and oxaliplatin (100 mg/m 2 on day 1) combined with tislelizumab (200 mg on day 2) at 21-day intervals. Then, the cohort was divided into a tislelizumab maintenance group (every 2 months for 2 years) and an autologous hematopoietic stem cell transplantation group based on the choice of the investigators. The primary endpoint of this study was the best complete response rate (CRR), and secondary endpoints included overall response rate (ORR), progression-free survival (PFS) at 12 months, and safety profile. Results As of July 2021, a total of 22 patients (median age of 33 years old) were included. The predominant histologic subtype was nodular sclerosing cHL. Early-stage cHL was found in six patients and late-stage cHL in 16 (two of them with bulky disease). Correspondingly, 59.1%, 18.2%, and 22.7% of patients had 1, 2, or ≥ 3 prior therapies, respectively, and more than half of patients (59.1%) reported refractory to the latest treatment. The efficacy evaluable population comprised 20 patients. The disease control rate was 100% (95% confidence interval [95% CI]: 83-100%) and the ORR was 95% (95% CI: 75-100%). At the time of follow-up cutoff, the best CRR reached 90% (95% CI: 68-99%). Out of these, only one patient demonstrated stable disease and was switched to the other regimen. A total of 126 courses of immunochemotherapy were administered, with a median number of six courses per patient. The follow-up time averaged 191 days (range, 31-345 days), and the estimated PFS rate was 100% at 12 months. After 6-8 courses of tislelizumab plus GemOx, ten subjects received tislelizumab maintenance, and all remain in remission so far, with the longest follow-up of 345 days. Overall, the treatment was well tolerated with the majority of adverse events (AEs) being grade 1-2 in severity. Serious AEs were grade 3 anemia (n=1) and grade 3 thrombocytopenia (n=1). Conclusion Tislelizumab plus GemOx demonstrated promising antitumor activity with manageable toxicities as a salvage treatment for RRcHL. A longer follow-up is needed to demonstrate the durability of the remission after tislelizumab maintenance. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Objective: Diffuse large B-cell lymphoma (DLBCL) is a subtype of non-Hodgkin's lymphoma with a higher incidence rate and is highly heterogeneous.The current first-line treatment effect is limited. After entering the relapse/refractory treatment, the second-line treatment lacks an efficient solution. Explore DLBCL Potential therapeutic targets are imperative. The cGAS/STING pathway can promote or inhibit tumorigenesis. The specific tumor type,genetic background,pathway activation level and microenvironment may determine the tumor's response to STING agonists or inhibitors. The disorder of cGAS/STING pathway function can lead to many diseases such as tumors and autoimmune diseases, but it has not been reported in DLBCL. This study found for the first time that STING is heterogeneously expressed in DLBCL and is related to tumor cell proliferation. The STING inhibitor H-151 can kill DLBCL tumor cells. At the same time, multi-omics methods are used to further screen the specific DLBCL population sensitive to H-151, provide a theoretical basis for the improvement of the prognostic model of DLBCL and the choice of targeted therapy drugs. Methods Immunofluorescence staining to detect the expression of cGAS/STING on tissue microarrays in patients with DLBCL; Confocal microscopy imaging technology for localization and quantitative analysis of intracellular cGAS/STING expression; Whole-exome sequencing analysis of mutant genes related to drug sensitivity and pathway;The combination of quantitative proteomics and transcriptome sequencing to compare the biological characteristics of cell lines in different response groups, analyze gene expression differences and related activation pathways;Western Blotting to detect the expression of related proteins;CCK8 and flow cytometry were used to detect cell proliferation and apoptosis;NOD-SCID mice and Hu-PBMC mice were used to construct DLBCL tumor-bearing mouse models to explore the effect of STING inhibitors in vivo. Results The cGAS/STING pathway-related proteins are expressed heterogeneously in real-world clinical DLBCL samples and DLBCL cell lines, and the expression of cGAS/STING pathway-related proteins is positively correlated with the proliferation level of DLBCL cell lines(Figure A). Based on this, we speculate the increased expression of cGAS/STING pathway-related proteins may potentially promote the proliferation of tumor cells. The STING inhibitor H-151 can inhibit the proliferation and induced apoptosis of some DLBCL tumor cells in a concentration-dependent manner.The IC50 of H-151 has a certain positive correlation with the expression of cGAS/STING pathway-related proteins, which suggests that the up-regulation of cGAS/STING pathway-related proteins may be potentially related to the resistance of DLBCL to H-151(Figure B).Whole transcriptome sequencing combined with single-cell transcriptome sequencing (scRNA-seq) analyzes potential subgroups related to H-151 sensitivity and related activation pathways(Figure C-E).. We also use whole exome sequencing to screen potential targets for assessing H-151 sensitivity We found that celllines with ITIH6, NPIPA5, NPY2R and TUBGCP3 mutations are more sensitive to the treatment of H-151, while cell lines with SH2B2 and LILRB5 mutations were less responsive to H-151,while the related gene mutations in the C-MYC pathway may be potentially related to the resistance of DLBCL cells to H-151, so as to screen potentially sensitive target populations for the effective use of H-151(Figure F). Conclusion This study found that the cGAS/STING pathway is heterogeneously expressed in DLBCL and is related to tumor cell proliferation, and the STING inhibitor H-151 has anti-DLBCL effects. The anti-tumor mechanism of STING inhibitor H-151 was further explored by using multi-omics methods, and the sensitivity evaluation system of H-151 was established.Thus providing new ideas for precise and efficient treatment of DLBCL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Objective: Angioimmunoblastic T-cell lymphoma (AITL) is a common subtype of peripheral T-cell lymphoma (PTCL). AITL is an aggressive malignancy with a poor prognosis, and its clinical manifestations vary greatly among individuals. The current chemotherapy regimens based on anthracycline show limited efficacy, and there is no best rescue treatment for patients with relapsed and refractory (RR) AITL. In addition, the lack of optimal AITL models in vitro greatly limits the basic research on the mechanism of disease occurrence and progression, and also hinders the development of new drugs and preclinical trials. Our study aims to deeply analyze the tumor heterogeneity and clonal evolution of AITL, discovering key molecules of drug resistance and potential theraputic targets. Methods : We detected fresh lymph node samples from newly diagnosed and relapsed/ refractory AITL patients using single-cell RNA sequencing, combined with imaging mass cytometry (IMC) and whole exome sequencing. IMC was performed to analyze the spatial position relationship and protein expression characteristics of different subgroups in the tumor microenvironment of AITL. In addition, AITL patient-derived organoid model was established to study the regulatory role of YY1 and its inhibitors in relapsed and refractory AITL. Results : ScRNA-seq revealed the significant differences in the tumor microenvironment of newly diagnosed and RR-AITL patients (Fig A,B). B cells and myeloid subgroups may play important roles in the development of AITL (Fig C). Transcription factor YY1, highly expressed in follicular helper T cell (Tfh) of RR-AITL patients, promoted the proliferation and drug resistance of AITL cells (Fig E,F). The proportion of CD8+ T cells in the RR-AITL sample was reduced, while the proportion of Treg was increased, as well as the depletion of T cells (Fig G,H). Furthermore, the stemness of B cells in RR-AITL was enhanced and exhibits significant malignant characteristics (Fig C,I-K). We also found decreased interaction in RR-AITL samples (Fig L,M). Moreover, for the first time, we established AITL patient-derived organoid models that can be stablely cultured in vitro (Fig N). On this basis, we could further clarify the important roles of transcription factor YY1 in the drug resistance of AITL, evaluate the cytotoxic effect of YY1 inhibitor NP-001 on AITL tumor cells. Conclusion : In conclusion, our study revealed the differences between newly diagnosed and relapsed /refractory AITL in terms of immune microenvironment, single-cell transcriptomes, and signal pathway activation. YY1 may serve as an novel target for drug resistance for RR-AITL patients. These findings may provide a theoretical foundation for improving the clinical treatment of AITL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Purpose: Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated impressive responses in refractory and relapsed acute lymphoblastic leukemia (ALL) and non-hodgkin's lymphoma (NHL), however, the outcome among chronic lymphoblastic leukemia (CLL) seems to be inferior compared to other lymphoblastic malignancies, indicating that efficacy of CAR-T cell therapy may be attributed to inherent T cell defects that are characteristic of CLL which impaired their proliferative capacity and sustained persistence in vivo. Thereby, infusion of less-differentiated T cells which have the capacity to persist and engraft long-term in vivo may enhance the anti-tumor activity. Materials and methods: On day 0, cryopreserved PBMCs from healthy donors (HDs) and CLL patients were thawed and seeded on anti-CD3 antibody (0.1μg/ml vs 1μg/ml) in combination with anti-CD28 antibody (1μg/ml) coated 24-well plates. On day 3, activated T cells (ATCs) supplied with retroviral supernatant of the third-generation RV-SFG.CD19.CD28.4-1BBzeta vector were transferred into 24-well plates previously coated with retronectin. Transduction efficiencies and phenotypes of CAR-T cells were evaluated on days 7, 10 and 14 after transduction using flow cytometry analysis. On a functional level, chromium 51 (Cr-51) release assay and intracellular staining (ICS) analysis were performed to explore the altered cytotoxic capability of CAR-T cells. Results: We observed that the decrease of anti-CD3 antibody concentration (0.1μg/ml) showed no influence on viability, expansion, transduction efficiency of CAR-T cells generated from HDs or CLL patients compared to standard anti-CD3 antibody concentration (1μg/ml). Moreover, the decrease of anti-CD3 antibody (0.1μg/ml)-mediated T cell activation resulted in an enrichment of less-differentiated naïve (CD45RA +CCR7 +) and central memory (CD45RA -CCR7 +)-like T cells both among CD4 + and CD8 + CAR-T cells. Additionally, cytokines-production (TNF-α, IFN-γ) were dramatically increased evaluated with ICS analysis from HDs and CLL patients in two different concentrations (0.1μg/ml vs. 1μg/ml) . Notably, CAR-T cells derived from HDs displayed decreased cytotoxic capability while CLL patients-derived CAR-T cells demonstrated increased cytotoxicity with lower anti-CD3 antibody concentration (0.1μg/ml) in the assessment of Cr-51 release assay, indicating that the proliferative capacity and sustained persistence of CAR-T cells derived from CLL patients were obtained in vivo. Conclusion: Anti-CD3 antibody-mediated activation of T cells altered anti-tumor efficiency of CAR-T cells before the transduction of ACTs with virus vectors. Consequently, when exploring the strategies to improve the efficacy of CAR-T cells, especially among CLL patients with inherent T cell defects, improvement of the functionality of T cells has to be taken into account. Figure 1 Figure 1. Disclosures Schmitt: TolerogenixX: Current holder of individual stocks in a privately-held company; Apogenix: Research Funding; Hexal: Other: Travel grants, Research Funding; Kite Gilead: Other: Travel grants; MSD: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Travel grants, Research Funding; Bluebird Bio: Other: Travel grants.

Objective: During tumor development, energy constraints caused by malnourished microenvironments could exert selective pressure on cancer cells. Tumor cells are driven to metabolic reprogramming to meet the increased demand for energy and metabolites for their rapid proliferation and survival. Chronic lymphocytic leukemia (CLL) is a disease with about 1% of CLL cells proliferating every day which is highly than commonly thought. CLL cells were reported to maintain high levels of proliferation through metabolic changes, but extensive studies did not clearly explain the underlying mechanism of driving genes in CLL metabolism. Circular RNA (circRNA) has recently been shown to play an important role in cell metabolism through lipid accumulation. The purpose of this study is to explore the role of circRNA in lipid metabolism of CLL and provide novel therapeutic targets for CLL. Methods: To analyze circRNAs expression profiles and metabolism map in CLL, peripheral blood mononuclear cells (PBMC) from 53 treatment-naïve CLL patients were collected for transcriptome sequencing. Candidate circRNA circRIC8B in a larger cohort of patients was validated and the clinical characteristics were analyzed. Overexpression and knockdown virus were constructed to infect CLL cells, and untargeted metabolomics was used to find the key lipid metabolic pathway modulating by circRIC8B. The oncogenic functions of circRIC8B were further measured in CLL cell lines (MEC-1 and JVM-3) by performing CCK8 assay, flow cytometry, nile red staining and triglyceride detection. Moreover, we explored the molecular mechanisms of circRIC8B and verified the interactions among circRIC8B, miR-199b-5p and LPL by performing RNA-FISH, RIP, dual-luciferase reporter assay and Western blotting. The killing effects of lipid metabolism inhibitors on CLL cells were detected by CCK8 and flow cytometry. Results Transcriptome analysis showed that abnormal lipid metabolism was significantly related to the survival and prognosis of patients with CLL, and circRNAs could be involved in the regulation of lipid metabolism. Kaplan-Meier survival analysis confirmed that patients with higher fatty acid biosynthesis had a significantly lower OS (Figure 1A-B). circRIC8B which is positively correlated with the expression of lipoprotein lipase (LPL) was finally selected for further investigation. qRT-PCR analysis showed that circRIC8B was significantly higher expressed in CLL compared with healthy donors. Moreover, consistent with the sequencing results, circRIC8B was positively correlated with LPL and highly relevant to IGHV region mutation status, which has long been considered as an important prognostic indicator of CLL (Figure 1C). Patients with higher circRIC8B level are associated with worse survival and advanced disease progression (Figure 1D and E). LC-MS/MS results showed that circRIC8B are able to modulated lipid metabolism of CLL cells. Functional analysis demonstrated the promoting role of circRIC8B in cell proliferation. Nile red staining showed lipid accumulation in CLL cells with circRIC8B overexpression increased significantly, while lipid accumulation in circRIC8B knockdown cells decreased significantly, and the quantitative results of triglycerides were similar. Next, we unraveled an original mechanism in CLL that up-regulated circRIC8B was mainly enriched in the cytoplasm, acted as a "sponge" of miR-199b-5p. CCK8 assay, nile red staining showed that the cell viability and lipid accumulating of CLL cell lines were evidently decreased after RNAi of circRIC8B and this result could be reversed by miR-199b-5p inhibitor transfection (Figure 1F-H). In addition, ezetimibe, one of the inhibitors of lipid metabolism was found effectively inhibit the proliferation and promote apoptosis of CLL cells. Conclusions In conclusion, as an independent prognostic factor of CLL, circRIC8B was involved in the progress of CLL disease through the miR-199b-5p/LPL axis. In addition, circRIC8B is a key factor in regulating lipid accumulation in CLL, resulting in significant changes in cellular lipid storage, thus supporting the proliferation of CLL cells. Metabolic inhibitor Ezetimibe can effectively block this process and exert anti-tumor functions. This study provides new clues for the role of circRNA in abnormal lipid metabolism of CLL and novel therapeutic strategy for CLL patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: To evaluate the efficacy and safety of venetoclax, rituximab plus dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (VR-DA-EPOCH) in Richter's syndrome (RS), we conducted a single-arm, retrospective, observational, real-world study in our center. Methods: Patients who had history of CLL/SLL were diagnosed as RS by biopsy during treatment or watch and wait strategy. VR-DA-EPOCH was given as follow, venetoclax was administered with accelerated ramp-up from 20 mg per day to 400 mg per day, d1-10 during cycle 1, 400 mg daily on day1-10 of cycle 2-6, rituximab 375 mg/m 2 on day 0 of cycle 1 and 500 mg/m 2 on day 0 of cycle 2-6, plus etoposide (50 mg/m 2,day1-4), vincristine (0.4 mg/m 2 day 1-4) or vindesine 3 mg/m 2 day 1 , doxorubicin (10 mg/m 2 day 1-4), prednisone (60 mg/m 2, day 1-5), cyclophosphamide (750 mg/m 2 day 5), 21 days per cycle，dose adjustment on the basis of nadir ANC and platelet count are as previously reported by Wison WH. Response assessment was conducted after 2 or 3 cycles by enhanced CT or PET/CT and after 6 cycles (EOT) by PET/CT according to 2014 Lugano criteria. Minimal residual disease (MRD) of CLL cell in peripheral blood (PB) and bone marrow (BM) was detected after 2 or 3 and 6 cycles by flow cytometry. uMRD was defined as less than 1 CLL cell per 10 4 leukocytes. Results: 7 RS patients were enrolled in Pukou CLL Center from 10/2019 to 7/2021 and the last follow up was 07/25/2021. The median age was 52 years old. Unmutated IGHV, complex karyotype (CK) and TP53 deletion and/or mutation was detected in 100% (6/6), 20% (1/5) and 40% (2/5) patients, respectively. 5 patients received at least one prior line (range: 1-5) treatment for CLL/SLL, with 4 patients received ibrutinib as last prior therapy and one patient previously exposed to venetoclax. 2 patients were diagnosed as RS during watch and wait. The median duration from diagnoses or previous treatment for CLL/SLL to RS was 12 months (range: 3-14). All patients underwent lymph node (n=6) or bone biopsy(n=1) at the site of SUVmax or secondary SUV uptake (unaccessible for SUVmax) by PET/CT and was confirmed as transformed to non-GCB type of diffuse large B-cell lymphoma (DLBCL). Furthermore, 4 of 4 (100%) available patients were confirmed as clonal-related RS by detecting IGHV gene usage. 3 patients acquired CK, and 2 patients appeared BTK C481S mutation. 7 patients completed at least 2 cycles and were available for efficacy and safety assessment. Overall response rate (ORR) was 100% after 2 or 3 cycles, and CR rate (CRR) was 60% after 6 cycles in 5 patients who completed 6 cycles. 2 patients experience disease progression (PD) after cycle 2 and cycle 4 respectively, with one ceased after the addition of brentuximab and the other received CD20 UCAR-T and progressed 3 months later, transit to allo-hemapoietic stem cell transplant (allo-HCT). One patient received auto-hemapoietic stem cell transplant (auto-HCT) and CD19-CAR-T as consolidation and remains in CR, one patient experience PD after 6 cycles and attained CR with chidamide , programmed death-1 (PD-1) inhibitor Sintilimab and XPO1 inhibitor Selinexor, bridging to allo-HCT and remains in CR. Another patient who achieved CR after 6 cycles progressed 6 months later and ceased within one month. 2 patients transformed without previous treatment wait for final evaluation. 60% (3/5) patients attained MRD negativity both in the PB and BM after 6 cycles. The median tolerable dose was 60% (50%-70%) of standard EPOCH and the median dose of venetoclax was 400mg daily for 7 days each cycle. No tumor lysis syndrome (TLS) happened during venetoclax ramp-up. The most common grade 3 or 4 adverse effects (AEs) was agranulocytic fever (6/7, 85.7%), thrombocytopenia (3/7, 42.9%) and sepsis (2/7, 28.6%). 3 (42.9%) patients discontinued venetoclax due to severe AEs and the median duration of discontinuation was 3 days. 85.7% (6/7) had venetoclax dose reduction or interruption due to grade 3 or 4 neutropenia. Conclusion: VR-DA-EPOCH showed high response rate, impressive CR with uMRD and manageable toxicity in patients with RS, even with previous exposure to new target drugs. VR-DA-EPOCH could be recommended as an effective treatment choice for RS, CAR-T or allo-HCT should be considered as subsequent strategy for long term disease control. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Aims: Diffuse large B-cell lymphoma (DLBCL), the most frequent malignant lymphoma subtype, is a group of highly invasive diseases with great heterogeneity in genomic alterations, clinical characteristics, morphological manifestations, treatment response and prognosis. Although most DLBCL patients can be cured by immunochemotherapy, nearly 40% of DLBCL patients still develop drug resistance and relapse. For relapsed/ refractory (R/R) DLBCL patients, there is still no optimal treatment. The heterogeneity and clonal evolution of tumor cells are the core driving forces for the occurrence and development of DLBCL, and the root causes for their refractory, recurrence and drug resistance. In this study, we screened out a novel small molecule compound effectively killing DLBCL cells, and analyzed its potential mechanism of anti-tumor. Meanwhile, by using single cell sequencing technology, we try to further investigate the heterogeneity and clona evolution and drug resistance mechanism of DLBCL under different drug pressure, explore core driver factors of drug resistance, evaluate and develop new treatment strategies. Methods: In this study, GEXSCOPE microfluidic platform was used for single-cell transcriptome sequencing. Seruat software was used for cell type recognition and clustering analysis. In order to further investigate the molecular mechanism of LAQ824 inducing the apoptosis of DLBCL cells and explore the target of LAQ824, antibody chip was performed to detect the phosphorylation of related signaling pathway. Gene expression was detected by real-time qPCR and Western blot. ChIP, RNA interfering (RNAi) and dual-luciferase activity assay were performed to validate the potential drug resistance targets for LAQ824. Moreover, WES of 21 DLBCL cell lines were performed to map mutations and analyze the correlation between related mutations and LAQ824 resistance. In this study, we established DLBCL animal models using NOD SCID mice transplanted with DLBCL cell lines, by which we could evaluate the tumor inhibition efficiency of LAQ824 alone and/or combination with other small molecular inhibitors. Results: Using GDSC database, we screened out Dacinostat (LAQ824), a novel HDAC inhibitor, was highly sensitive that could effectively induce the apoptosis of most DLBCL cells at low concentrations. Functional assay showed that LAQ824 could inhibit cell proliferation and promote apoptosis of tumor cells. LAQ824 treatment could significantly upregulate the acetylation level of histone H3 within a certain concentration range, and the DNA damage repair function of DLBCL cells was supressed by inhibiting Chk2 expression, thus significantly inducing cell apoptosis and effectively killing DLBCL cells. Meanwhile, through single-cell sequencing analysis, it was found that c-Fos could be activated under certain drug pressure of LAQ824. As a potential drug-resistant core driver gene, the expression level of c-Fos is highly correlated with IC50 of LAQ824 and the prognosis of patients with DLBCL, which can be used as a sensitivity indicator of LAQ824. Treatment with c-Fos inhibitor combined with LAQ824 can significantly improve the tumor inhibition rate, validated both in vitro and in vivo, which is expected to alleviate the recurrence and drug resistance of DLBCL patients. Conclusions: In general, we explores potential therapeutic drugs for DLBCL parients, adjusts and explores new clinical treatment strategies on this basis, and provides theoretical basis and data support for the realization of individualized precise treatment and the solution of DLBCL recurrence and drug resistance. Disclosures No relevant conflicts of interest to declare.

Objective: Ibrutinib is currently the most widely used BTK inhibitor that approved for the treatment of both initially diagnosed and relapsed and refractory chronic lymphocytic leukemia (CLL) patients. Although ibrutinib shows high response rates in clinical practice, it has certain limitations. There are still a certain number of patients who have to discontinue treatment due to drug-resistance or side effects. The ibrutinib resistance of CLL patients has caused widespread concerns, necessitating the development of novel treatment strategies. Methods: Here, we examined the heterogeneity of peripheral blood mononuclear cells (PBMCs) from patients with ibrutinib-sensitive (IBS) and -resistant (IBR) CLL by analyzing bulk and single-cell level gene expression profiles, clinical features, biological properties, and phenotypes. Seven distinct ibrutinib-resistant subpopulations were identified and two candidate genes LGALS1 (galectin 1, Gal-1) and LAG3 (lymphocyte-activating gene 3, CD223) were screened that contribute toward ibrutinib-resistance and poor survival in CLL patients. These results were validated in primary cells from CLL patients and also in ibrutinib-resistant CLL cell line (MEC1-IR) which was generated by culturing the parental cell line in vitro with progressively increasing concentrations of ibrutinib. Marker-gene expression was detected using qRT-PCR, western blotting, and ELISA, while functional analyses including CCK8, flow cytometry and trypan blue staining were conducted with or without OTX008, a selective Gal-1 inhibitor. Results: ScRNA-seq revealed that cells from IBR and IBS samples were distributed in different clusters and suggested that IBR cells display a unique transcriptional pattern (Fig A). IBR-B cells have higher stemness scores and are enriched in some energy metabolism Pathways (Fig B). According to the proportion of B cells from IBR samples, we classified each B-cell cluster into three main subgroups, i.e., IBR, IBS, and shared cluster (Fig C). IBR-B cells displayed more interactions with monocytes, NK, T, and dendritic cells than IBS B cells, suggesting that IBR B cells may actively build connections with other immune cells to reshape the protective niche (Fig D). A close correlation between LGALS1 and LAG3 expression was observed and both of them were found to be highly expressed in IBR CLL patients (Fig E), their expression level gradually increased along the trajectory of B cells from IBS to IBR (Fig F). Diagnosis and prognosis stratification of CLL with receiver operating characteristic (ROC) curves revealed that patients with higher expression of both LGALS1 and LAG3 showed the poorest overall survival, indicating that LGALS1 and LAG3 are associated with ibrutinib-resistance and poor prognosis in CLL (Fig G). Concordantly, acquired resistance following chronic exposure to ibrutinib leads to upregulation of LGALS1 and LAG3 (Fig H). LGALS1 inhibitor OTX008 effectively inhibits the growth of ibrutinib-resistant CLL cells, particularly for IBR patients (Fig I). Conclusion: In conclusion, our findings demonstrate that ibrutinib-resistant CLL cells exhibit a unique transcriptional pattern. The combination of LGALS1 and LAG3 expression could serve as an indicator of the sensitivity of ibrutinib and prognosis of CLL patients. LGALS1 inhibitor OTX008 helps to overcome ibrutinib-resistance of CLL cells. Our findings may expand the current knowledge regarding ibrutinib-resistant CLL patients, identify improved biomarkers for patient selection, and offer a promising combinatorial therapeutic strategy for IBR CLL patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction Chronic lymphocytic leukemia(CLL), characterized by monoclonal CD5+ B cells appearing in blood, marrow and lymphoid tissues, is the most common chronic leukemia in western countries. Until now CLL remains incurable with a heterogeneous clinical course, some patients developing progression rapidly while the others presenting a relatively indolent course. It has been reported that higher percentage of prolymphocytes was associated with increasing refractoriness to treatment and worse prognosis. In this study we were trying to explore the effect of prolymphocytes on the prognosis of CLL. Methods Two hundred and fifty-one treatment naïve CLL patients in Jiangsu Province Hospital were retrospectively enrolled in our study from April 2014 to November 2019. The median time from diagnosis to peripheral-blood smear examination was 0.13 month and median follow-up time was 30.87 months. A total of 200 lymphocytes and prolymphocytes were counted. The percentage of prolymphocytes was collected. Basic clinical characteristics and other prognostic markers including age, sex, Rai and Binet stage, B2-microglobulin(B2MG), lactate dehydrogenase(LDH) level, CD38 and ZAP70 expression level, hemoglobulin(HB), platelets count(PLT), absolute lymphocyte count(ALC), thymidine kinase-1(TK-1), albumin(ALB), IGHV mutation status and TP53 status were also put in the analysis. The time to first treatment(TTFT)was defined as the time from sampling to first treatment and the overall survival(OS) time was the time from sampling to death. An X-tile analysis provided the optimal prolymphocyte cutoff point. Wilcoxon rank sum test was used to compare the distribution of prolymphocytes percentage in different subgroups. The Kaplan-Meier method was used to construct the survival curves and the log-rank test was used to compare the difference. Multivariate analysis was conducted based on the Cox-regression model. All tests were two-sided and P Results Among 252 patients, most of them were male(67.7%) and 35.9% were older than 65 years old. We found significantly different distribution of prolymphocytes percentage in patients with different B2MG, Rai stage, LDH, CD38 expression, TK1, IGHV mutation status and TP53 status(Table 1). The optimal cutoff of prolymphocytes percentage provided by X-tile analysis was 1%. Then all the patients were divided into two groups based on the prolymphocytes percentage and obvious survival difference between the two groups was shown in both TTFT(P Discussion In this study we found the optimal cutoff of prolymphocytes percentage was 1% which is different with previous study(Oscier et al. 2016). That may be due to that we choose TTFT as the endpoint. Besides, about prolymphocytes percentage was not statistically significant in multivariate analysis, we guess that may be attributed to that the sample was too little or there were only Chinese patients in our cohort. In conclusion, prolymphocytes percentage has certain prognostic significance in CLL patients and we think multivariate analysis would be different after we expanded our cohort. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction Peripheral T-cell lymphoma (PTCL) is a group of hematological malignancies originating from mature T/NK cells. Most of the subtypes are associated with aggressive clinical features and dismal outcomes. Routine first-line chemotherapy has low efficiency and a high recurrence rate, so there is an urgent need for new drugs. Monotherapy or combination therapy of epigenetic inhibitors have been shown to be effective in several hematologic malignancies. Here, we report the interim efficacy of an epigenetic priming regimen with azacytidine and chidamide prior to salvage chemotherapy for relapsed or refractory (R/R) PTCL. Methods The prospective phase II study (ChiCTR2000037232) enrolled pts were pathologically confirmed T/NK cell non-Hodgkin's lymphoma with at least one imaging measurable lesion. Pts needed to have received at least one systemic chemotherapy regimen including hematopoietic stem cell transplantation, radiotherapy, or a single epigenetic drug. Pts received AZA hypodermically at a dose of 100 mg on days 1 to 7, chidamide of 20 mg orally twice per week; the combined chemotherapy regimens included but were not limited to GemOx (gemcitabine, oxaliplatin); CPT (cyclophosphamide, prednisone, thalidomide) , etc. Treatment was performed for up to eight cycles of each 21 days. Pts who achieved partial response (PR) and better remission began maintenance therapy every two months with double epigenetic inhibitors for two years. The trial aimed to explore the efficacy and safety of AZA and chidamide combined chemotherapy in the treatment of R/R PTCL. The primary objective was investigator-assessed best overall response rate (ORR). Secondary objectives included duration of response (DOR), complete response rate (CRR), progression-free survival (PFS), overall survival (OS), and safety profiles. Results A total of 24 pts have been enrolled, baseline characteristics are shown in Table 1. Pathological subtypes included angioimmunoblastic T-cell lymphoma（AITL, n=15）, PTCL-not otherwise specified (PTCL-NOS, n=4), extranodal NK/T-cell lymphoma (ENKTCL, n=3) and mycosis fungoides(MF, n=2). The median age was 57 (range,38-72) years with male predominance. Ann Arbor Classification ≥ stage III in 20 pts. Twelve pts had B symptoms at the time of diagnosis, five pts had performance status ≥ 3 before treatment. The median number of previous systemic treatment regimens was two. Autologous hematopoietic stem cell transplantation in two pts, radiation in three pts and prior treatments containing chidamide in eight pts. At the time of data cutoff, the median number of treatments for all pts was four cycles (range,1-13). Among 16 pts evaluable for response, the best ORR was 68.8% (11/16) with five pts achieved CR, six achieved PR. In subgroup analysis, eleven AITL pts achieved an objective response. The best ORR was 72.7% (8/11) with four pts attained CR, four attained PR (Table 2). The median follow-up was 12.4 (range, 0.1-18.7) months. For all pts, the median PFS was 6.7 months (95% CI,5.8-7.6), the median OS was 8.4 months (95% CI,0.0-18.3) (Figure 1). And the median DOR was 10.2 months (95% CI,4.9-15.5). For AITL pts, the median PFS was 14.6 months (95% CI,3.6-25.6), and the median OS was not reached (Figure 2). The OS between AITL and other subtypes pts was statistically significant (1-year OS: 76.2% vs 13.9%; p=0.003, Figure 3). Almost all pts had experienced at least one adverse event (AE). The most common grade 3 or 4 AEs were anemia, leukopenia, neutropenia, thrombocytopenia, and infections. Conclusions Epigenetic priming regimen with azacitidine plus chidamide with salvage chemotherapy is effective and tolerable. The best ORR of all enrolled pts with AITL were 68.8% and 72.7%, respectively. Compare to other subtypes, patients with AITL subtype benefit more obviously from our regimen with durable remission. And further studies will focus on patients with AITL and follicular helper T-cell originated. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction To evaluate the safety and efficacy of zanubrutinib, lenalidomide plus R-CHOP (ZR 2-CHOP) as the first-line treatment for DLBCL patients, we conducted this single-arm retrospective observational study. Methods Treatment-naïve patients with DLBCL(including but not limited to double-hit, double expression) aged 18 to 75 years were enrolled.ZR 2-CHOP was given as follows, Oral zanubrutinib was given continuously (160 mg twice daily) from Day 0, lenalidomide 25 mg daily Day 1-7. Patients were administered intravenously rituximab (375 mg/m 2 Day 0), cyclophosphamide (750 mg/m 2 Day 1), doxorubicin (50 mg/m 2 Day 1), vincristine (1.4 mg/m 2 Day 1), and oral prednisone (100 mg Day 1-5). All patients were recommended to receive 6 cycles of ZR 2-CHOP (R-CHOP or R 2-CHOP were allowed in cycle 1-2 due to poor physical condition at treatment) and patients older than 70 years old were administered ZR 2-miniCHOP (Figure 1). CT or PET-CT scans were applied to mid-term efficacy and PET-CT scan was conducted after 6 cycles. ctDNA was dynamically detected before treatment, after 3 and 6 cycles to evaluate tumor mutational burden. The primary endpoint was complete response ratio (CRR) at mid-term and after 6 cycles. The secondary endpoint was overall response rate (ORR), ctDNA dynamics and adverse events (AE). AEs were graded based on CTCAE (version 5.0). Results 12 treatment-naïve DLBCL patients diagnosed in Pukou CLL Center were enrolled in this cohort between July 2020 and June 2021. The median age was 56 years old and all patients had ECOG-PS ≤2. 1 patient (1/12) was diagnosed as double-hit DLBCL and 9 patients (9/12) as double-expression. 10 patients were non-GCB and 2 were GCB. 7 patients were classified as high-intermediate and high-risk group according to NCCN-IPI (Table 1). At data cutoff (1st July, 2021), the median follow-up was 8 months (3-12 months) with all patients have completed at least 3 cycles and mid-term assessment has been conducted. The ORR was 100.0%, with 10 patients achieved CR and 2 patients achieved PR (both two patients received R-CHOP regimen in cycle 1/cycle 1-2 due to poor physical condition at initial treatment, Figure 1). 10 patients have received 6 cycles, all of them achieved CR (Figure 2). ctDNA was dynamically detected in six patients. The median number of detectable ctDNA mutation among six patients was 8 (0-12) with two patients classified as MCD subtype and one patients as EZB subtype. All six patients showed undetectable ctDNA after 3 cycles. During end of treatment follow-up, one patient (triple-hit, EZB) who scheduled to receive auto-SCT underwent disease progression 4 months later and reemergence of ctDNA showed previous homologous clones. The most common hematological toxicity events were lymphocytes count decreased, neutrophil count decreased, thrombocytopenia and anemia, with 3-4 level occurrence rate was 66.7%, 25.0%, 25.0% and 16.7%. The most common non-hematological toxicity events were nausea, fatigue and anorexia. One patients discontinued oral zanubrutinib and lenalidomide in last two cycles due to drug rash. Conclusion ZR 2-CHOP could attain high CR rate and ctDNA clearance in TN DLBCL, including patients with DEL and/or high-risk. The overall tolerability was manageable. ZR 2-CHOP could be one of the promising choice for TN DLBCL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Zanubrutinib was used in the initial treatment of high-risk DLBCL.

Introduction To evaluate the safety and efficacy of BTK inhibitor ibrutinib or zanubrutinib plus bendamustine and rituximab (iBR or zBR) as the initial treatment for chronic lymphocytic leukemia (CLL)/ small lymphocytic leukemia (SLL) patients, we conducted this single-arm retrospective observational study. Methods We enrolled patients over 18 years old with CLL/SLL who required treatment. iBR or zBR were administrated as following, ibrutinib 420 mg daily or zanubrutinib 160 mg twice daily from day 0, rituximab 375 mg/m 2 on day 0 of cycle 1 and 500 mg/m 2 on day 0 of cycle 2-6, bendamustine (70 mg/m 2, days 1-2), 28 days per cycle, ibrutinib or zanubrutinib was maintained at least 2 years and planned to discontinue in patients with undetectable minimal residual disease (uMRD) in both peripheral blood (PB) and bone marrow (BM). Response assessment was conducted after 3 cycles and 2 months after 6 cycles (EOT) according to 2018 iwCLL criteria in patients with CLL and 2014 Lugano criteria in patients with SLL. Minimal residual disease (MRD) in PB was detected after each cycle and MRD in BM was detected after 3 and 6 cycles by flow cytometry. uMRD was defined as less than 1 CLL cell per 10 4 leukocytes by flow cytometry. R esults At data cut-off (15 th June 2021), 10 treatment-naïve patients in the First Affiliated Hospital of Nanjing Medical University were enrolled, with 9 CLL and 1 SLL. All patients had completed planned six cycles of iBR(n=8) or zBR(n=2). The median age was 56 years old. Unmutated IGHV was detected in 30.0% (3/10) patients, one patient (1/8, 12.5%) had both del(17p) and TP53 mutation. Among 9 patients with CLL, 3 (33.3%) was classified as low-risk group according to CLL-IPI, 4(44.4%) in intermediate-risk, 1(11.1%) in high-risk group and 1(11.1%) in very-high risk group (Table 1). After 3 cycles, the overall response rate (ORR) was 100%, and complete remission (CR) rate was 20.0%, one patient achieved CRi (CR with incomplete bone marrow recovery). 50% (5/10) and 37.5% (3/8) patients achieved uMRD in PB and BM respectively. The ORR was 100% (10/10) and CR rate was 60.0%, 3 patients achieved CRi (30.0%) at EOT. 60.0% (6/10) and 50.0% (5/10) patients achieved uMRD in PB and BM respectively (Table 2, Figure 1). The most common hematological toxicity events were neutropenia and thrombocytopenia, and the non-hematological toxicity events were malaise, pruritus, nausea, vomiting and hematuria. The occurrence rate of grade 3 to 4 neutropenia and agranulocytic fever were 40.0% (4/10) and 10.0% (1/10) respectively. The most common ibrutinib-related AE were purpura, rash, diarrhea and hematuria. The occurrence rate of AE induced discontinued ibrutinib or zanubrutinib were 30.0% (3/10). Conclusion Here, we first reported the efficacy and tolerance of BTK inhibitor plus BR as initial treatment in a small cohort of CLL/SLL patients. BTK inhibitor plus BR could achieved high response rate and high proportion of undetectable MRD，with manageable toxicity. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Objective: Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that mainly affects the elderly and is characterized by the expansion of small mature B-cells. New targeted drugs, such as the BTK inhibitor ibrutinib, have greatly improved patient survival but have also posed the challenge of drug resistance. The three-dimensional (3D) spatial structure of chromatin is highly dynamic and varies greatly between cell types and developmental stages, with the maintenance of chromatin homeostasis being of major significance in disease prevention. Accumulating evidence has suggested that changes in 3D genomic structures play an important role in cell development and differentiation, disease progression, as well as drug resistance. Nevertheless, the characteristics and functional significance of chromatin conformation in the resistance of CLL to ibrutinib remain unclear. In this study, we aimed to investigate the mechanism underlying ibrutinib resistance through multi-omics profiling, including the study of chromatin conformation. Thus, we would be able to demonstrate the importance of chromatin spatial organization in CLL and highlight the oncogenic factors contributing to CLL development and mediating ibrutinib resistance. Methods: An ibrutinib-resistant cell line was established by exposing cells to increasing doses of ibrutinib. High-throughput chromosome conformation capture (Hi-C), assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), bulk RNA sequencing (RNA-seq), and Tandem Mass Tag (TMT) were performed to explore differences between ibrutinib-resistant and parental cells. Peripheral blood mononuclear cells (PBMCs) from 53 CLL patients were collected for RNA-seq. Mitochondrial respiration and glycolysis were assessed via Seahorse analysis. The growth-inhibitory effects of tested drugs were evaluated via a CCK8 assay, and the combination index (CI), indicating synergy, was calculated using CompuSyn software. Apoptosis was detected via annexin V staining. Results: Between ibrutinib-resistant and parental cells changes in some chromosomes, including chr11 were observed (Figure 1A). p21-activated kinase 1 (PAK1), which is located on chr11 and frequently overexpressed or excessively activated in almost all cancer types and involved in almost every stage of cancer progression, was first explored for its role in CLL progression and drug resistance. The oncogene PAK1 was observed locate in a region where B-to-A compartment switching occurred (Figure 1B). Consistent with the results of ATAC-seq, RNA-seq, and TMT, Hi-C analysis revealed a transcriptional upregulation of PAK1 in ibrutinib-resistant CLL cells (Figure 1C). Functional analysis demonstrated that PAK1 overexpression significantly promoted cell proliferation, while knockdown markedly suppressed cell viability (Figure 1D). Cell viability assays indicated that the depletion of PAK1 increased ibrutinib sensitivity (Figure 1E). In addition, PAK1 positively regulates glycolysis and oxidative phosphorylation in CLL cells (Figure 1F and G). To verify the results of sequencing and further explore the role of PAK1 in CLL, B-cells from healthy volunteers and PBMCs from CLL patients were collected. The level of PAK1 mRNA expression was significantly higher in CLL primary cells than in B-cells from healthy volunteers (Figure 1H). Kaplan-Meier survival analysis of qRT-PCR data confirmed that patients with high PAK1 expression had a significantly lower OS (Figure 1I). IPA-3, the small molecular inhibitor of PAK1 suppressed the proliferation of ibrutinib-resistant and parental CLL cells in a dose-dependent manner. The combination of IPA-3 and ibrutinib exerted potent cell growth inhibition (Figure 1J), and the combination index (CI) calculated using the CompuSyn software confirmed the synergistic effect (CI Conclusions: In the current study, we have provided a genome-wide view of alterations in 3D chromatin organization between ibrutinib-resistant and parental CLL cells and confirmed the oncogenic role of PAK1 in CLL. Most importantly, our research provides promising therapeutic targets for overcoming ibrutinib resistance. In particular, the treatment of CLL patients with a combination of IPA-3 and ibrutinib may improve clinical outcomes. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background The prognosis of R/R PTCL and ENKTL remains poor. Current therapeutic options are limited, highlighting the need for novel approaches. ATG-010 (selinexor) is a novel, oral selective inhibitor of nuclear export, which blocks exportin 1. It has demonstrated clinical activity in different hematological malignancies. Preclinical synergistic anti-tumor effects are confirmed when combined with gemcitabine, cisplatin, or etoposide. Method The aim of this study was to evaluate safety and efficacy of ATG-010 plus GemOx or ICE regimen followed by ATG-010 maintenance in patients (pts) with R/R PTCL or ENKTL in China. The study planned to enroll 30 pts with PTCL-NOS, AITL, ALCL, PTCL-TFH, FTL and ENKTL. Pts must have had prior exposure to an anthracycline-based regimen for PTCL or an asparaginase-based regimen for ENKTL, and were relapsed or refractory from the last therapy. ATG-010 was administered orally (60 mg day 1, 8) plus standard-dose of either ICE or GemOx regimen every 3 weeks as per investigator's choice. If response was achieved after initial 2-6 cycles, pts continued to receive ATG-010 maintenance at the dose of 60 mg weekly until disease progression, intolerability or withdrawal of consent. Primary endpoints included safety according to the NCI CTCAE 5.0 and overall response rate (ORR) evaluated by investigators according to the Lugano criteria (2014). Secondary endpoints included duration of response (DOR), progression-free survival (PFS) and overall survival (OS). This trial was registered at ClinicalTrials.gov (NCT04425070). Results From Aug 18, 2020 to June 25, 2021, 24 pts (20 in GemOx cohort, 4 in ICE cohort) were enrolled and received at least one cycle of treatment. In this abstract, we report results of the GemOx cohort with 9 (45%) PTCL-NOS, 6 (30%) ENKTL, 4 (20%) AITL, and 1 (5%) ALCL ALK-. At study entry, median age was 55 years (range 35-69); 17 (85%) had stage III/IV disease. Five (36%) pts with PTCL had IPI score ≥3, and 5 (83%) ENKTL pts with had PINK-E score ≥2. Median number of prior regimens was 3.5 (range 1-7) with 5.5 for ENKTL and 2 for PTCL. All pts were refractory from last therapy; 11 (55%) pts had received gemcitabine-based regimens. Median time from last-line therapy to this trial was 1.5 months (range 1.0-16.7). The most common treatment-emergent adverse events (TEAEs) were hematological toxicities. All grade hematological TEAEs were neutropenia (90%), thrombocytopenia (90%) and anemia (85%). Grade≥3 hematological TEAEs included neutropenia (85%), thrombocytopenia (80%) and anemia (40.0%). The most common (>30%) non-hematological TEAEs were nausea (70%), diarrhea (65%), decreased appetite (65%), asthenia/fatigue (60%), vomiting (50%), pyrexia (40%), and hyponatremia (35%). Grade≥3 non-hematological TEAEs (≥10%) were diarrhea (15%) and pyrexia (10%). Serious TEAEs occurred in 7 (35%) pts with the most common being thrombocytopenia (20%). Three (15%) pts discontinued treatment due to TEAEs. TEAEs with a fatal outcome occurred in 1(5%) patient, who experienced rapid disease progression before death. The majority of adverse events were manageable by dose modification and supportive care. Of 17 efficacy evaluable pts, ORR was 52.9% (9/17), and CR rate was 35.3% (6/17). ORR of PTCL-NOS, ENKTL, AITL, ALCL were 62.5% (5/8), 60% (3/5), 0 (0/3), 100% (1/1); CR rate were 37.5%, 40%, 0, 100%, respectively. At a median follow-up of 6.1months, median PFS, DOR and OS of the whole cohort was 2.9, 3.1 months, and not reached (6-month OS rate 68.9%), respectively. Patients with ENKTL enjoyed a relatively longer median PFS (4.7 months). Conclusions ATG-010 plus GemOx regimen showed a manageable safety profile, and favorable activity with impressive CR rate in refractory pts, potentially offering a new therapeutic option for heavily pretreated pts with refractory PTCL or ENKTL. Disclosures Lou: Antengene Therapeutics Ltd.: Current Employment. Fei: Antengene Therapeutics Ltd.: Current Employment. Wang: Antengene Therapeutics Ltd.: Current Employment. Lynch: Antengene Therapeutics Ltd.: Current Employment.

Purpose: To evaluate the safety and efficacy of ibrutinib plus fludarabine, cyclophosphamide, and rituximab (iFCR) as the treatment for chronic lymphocytic leukemia (CLL)/ small lymphocytic leukemia (SLL) patients, we conducted time-limited, minimal residual disease (MRD)-driven real world study in The First Affiliated Hospital of Nanjing Medical University Methods: We enrolled patients aged 18 to 65 years old with CLL/SLL who required treatment. Oral Ibrutinib was given continuously (420 mg/day) from day 0, and patients were administered intravenously rituximab (375mg/m2 in day 0 of cycle 1; 500mg/m2 in day 0 of cycle 2-3), fludarabine (25mg/m2, days 1-3) and cyclophosphamide (250mg/m2, days 1-3), every 28-day cycles. All the patients received 3 cycles of iFCR in the first stage. Interim analysis was done according to the 2018 iwCLL criteria and MRD was detected by flow cytometry. Patients who achieved complete response (CR) or partial response (PR) with low MRD level (L-MRD, 10-4-10-2) or undetectable MRD (uMRD) level (10-2) in PB or bone marrow (BM), or with diameter of residual lymph node larger than 3cm were administered another 3 cycles of iFCR (6 in all) and followed by ibrutinib maintenance. The primary endpoint was the proportion of patients who achieved uMRD in the PB after 3 cycles of iFCR. The secondary endpoint was overall response rate (ORR), complete response rate (CR) and the number of adverse events (AE). Results: Between May 2019 and June 2020, 29 CLL/SLL patients were enrolled in this cohort with 27 CLL and 2 SLL. 26 of patients were the previously untreated and 3 of patients were relapse or treated before. The median age of patients was 53 years old. Unmutated IGHV was detected in 14 (50.0%) of 29 patients, 4 patients had del(17p) and/or TP53 mutation (13.8%), 7 patients had del(11q) (24.1%), 7 had NOTCH1 mutation (24.1%) and 7 had MYD88 mutation (24.1%). 8 patients (30.8%) were classified as low-risk group according to CLL-IPI, with 9 in intermediate-risk group (34.6%), 5 in high-risk group (19.2%) and 4 in very-high risk group (15.4%). At data cutoff (1st July, 2020), the median follow-up and treatment time was seven months (1-13 months) with 23 patients had completed at least two cycles of iFCR. At interim analysis, the ORR was 100% with 8 patients achieved CR (34.8%), 3 achieved CRi (13.0%) and 12 achieved PR (52.2%). 57.1% (12/21) achieved uMRD in both PB and BM, among which 6 of them (28.6%) achieved CR/CRi and 6 achieved PR (28.6%). All four patients with TP53 abnormalities achieved PR and two of them (50.0%) achieved uMRD in both PB and BM. One of 6 patients with del(11q) achieved CR (16.7%) and five achieved PR (83.3%), with 2 achieved uMRD in both PB and BM (33.3%). Among 13 assessable patients with unmutated IGHV, four of them achieved CR (30.8%) and nine achieved PR (69.2%), with 5 achieved uMRD in both PB and BM (38.5%). 18 patients had turned to ibrutinib maintenance with 12 of them finished 4th cycle of iFCR or 3 additional cycles of rituximab and could be assessed further. 10 of the patients achieved CR/CRi (83.3%) and 2 achieved PR (16.7%), with 8 achieved uMRD in both PB and BM (66.7%). The most common hematological toxicity events were neutropenia and thrombocytopenia and the non-hematological toxicity events were nausea and vomiting. The occurrence rate of grade 3 to 4 neutropenia and agranulocytic fever were 51.9% (15/29) and 17.2% (5/29) respectively. The most common ibrutinib-related AE were purpura, rash and diarrhea. The occurrence rate of AE induced discontinued ibrutinib more than 7 days, discontinued ibrutinib fewer than 7 days and ibrutinib decrement were 20.7%(6/29), 17.2%(5/29) and 20.7%(6/29) respectively. Conclusion: MRD-driven Ibrutinib plus FCR could achieve high response rate with uMRD in yonger fit patients with CLL/SLL, with manageable toxicity. Ibrutinib plus FCR could be one of the most promising choice as a time-limited regimen in the new drug era. Disclosures No relevant conflicts of interest to declare.

Background: Tislelizumab, a humanized immunoglobulin 4 monoclonal anti-programmed cell death protein 1 (anti-PD-1) antibody, has an engineered Fc region that minimizes binding to Fcγ receptor (FcγR) on macrophages, thereby abrogating antibody-dependent phagocytosis (ADCP)-induced T-cell clearance. Tislelizumab demonstrated an overall response rate of 87.1% with a high complete response (CR) rate (62.9%) and was generally well tolerated in a phase 2 study in Chinese patients with relapsed/refractory (R/R) classical Hodgkin lymphoma (cHL) (BGB-A317-203 study; clinicaltrials.gov identifier: NCT03209973). The present study explored the underlying mechanism of action (MOA) of tislelizumab and its potential contribution to the high CR rate associated with it in patients with R/R cHL. Methods: Seventy patients with confirmed R/R cHL were included in this study. Tissue samples were collected at baseline for biomarker testing. Forty-one samples were evaluable for multiple immunohistochemistry (mIHC) assays, and 36 samples were evaluable for gene expression profiling (GEP). For programmed death-ligand 1 (PD-L1), CD8 (cytotoxic T-cell marker), CD68 (macrophage pan-marker), CD64 (FcγRΙ), and CD30, mIHC samples were stained using Opal 7-Color IHC kit. Spatial analysis for the markers was conducted using HALO software. GEP utilized the HTG EdgeSeq Precision Immuno-Oncology panel. Genes expressed differentially in CR and non-CR were identified using the Lima R Bioconductor package. Gene signature score was calculated by the gene set variation analysis method. Median value across the biomarker population was used as the cutoff to define biomarker high versus low group. Differential biomarker tests between CR and non-CR were conducted by Wilcoxon rank-sum test. Results: For anti-PD-1 antibodies with a functional Fc, the Fc/FcγR interaction resulted in macrophage-induced T-cell clearance and dampened anti-tumor activity, while tislelizumab activity was not affected by macrophages in the mouse cancer model (Zhang T, et al. Cancer Immunol Immunother. 2018;67:1079-1090). In cHL clinical samples, we observed a similar CR rate for tislelizumab in FcγRΙ+ macrophages (CD68+CD64+) high versus low group (71.4% vs 60%, P=0.85 by Fisher-test). In the CD8+ T-cell high microenvironment where ADCP-induced T-cell clearance is more likely, neither the total number of CD68+/CD64+ cells (CR rate 86.6% for high vs 85.7% for low, n.s. by Fisher-test) nor the average number of CD68+CD64+ cells within 30 µm of CD8+ T cells (85.7% vs 80%, n.s. by Fisher-test) were observed to affect the CR rate. Additional tumor microenvironment components that may contribute to the high CR rate were also explored. We found that FcγRΙ+ and CD8+ cell percentage by mIHC were higher in CR patients (P=0.04 and P=0.08, CR vs non-CR). GEP results show that the tumor inflammation gene signature (TIS) (eg, CD8A, CCL5, PD-L1, IDO1, IFNG, CXCL9) were higher in CR patients (CR vs non-CR, P=0.04). Specific gene/gene signatures were associated with CR for different histology subtypes. In the mixed cellular subtype, CD8+ T cells by mIHC (P=0.0004) and the TIS gene signature by GEP (P=0.007) showed a larger difference between CR and non-CR. In the nodular sclerosis subtype, the extracellular matrix and fibroblast-related gene signature (eg, ICAM, COL1As, ITGAs, PDGFRB) were higher in CR patients (P=0.04, CR vs non-CR). Conclusions: Tislelizumab demonstrated a high CR rate regardless of the FcγRΙ expressing macrophage abundance in the cHL tumor microenvironment, which may be a functional consequence of its engineered Fc region and may differentiate its MOA from the MOAs of other anti-PD-1 agents. CD8+ T-cell abundance and tumor inflammatory gene signatures in the microenvironment may be associated with higher CR rate for cHL patients treated with tislelizumab. Disclosures Wang: BeiGene Co., Ltd.: Current Employment, Current equity holder in private company. Zhang:BeiGene Co., Ltd.: Current Employment, Current equity holder in private company. Liu:BeiGene Co., Ltd.: Current Employment, Current equity holder in private company. Zhang:BeiGene Co., Ltd.: Current Employment, Current equity holder in private company. Shen:BeiGene Co., Ltd.: Current Employment, Current equity holder in private company. Wang:BeiGene Co., Ltd.: Current Employment, Current equity holder in private company. Yang:BeiGene Co., Ltd.: Current Employment, Current equity holder in private company. Guo:BeiGene Co., Ltd.: Current Employment, Current equity holder in private company.

Introduction: Patients with diffuse large B cell lymphoma (DLBCL) arising from the testis have a relatively poor outcome. Age, stage and use of radiation and surgery are important prognostic factors in patients with primary testicular DLBCL. Additionally, the study by Gundrum et al suggested that laterality was also an important predictor of outcomes in patients with primary testicular DLBCL, with left side involvement being associated poorer prognosis. However, most patients included in the study by Gundrum et al were diagnosed in the pre-rituximab era, therefore, the role of laterality in the prognostification of patients with primary testicular DLBCL remains to be explored. In this study, the Surveillance, Epidemiology, and End Results (SEER) database was used to evaluate the prognostic roles of laterality in patients with primary testicular DLBCL. Methods: Data from the SEER 18 Registries were used to conduct this study. Cases with newly-diagnosed testicular (International Classification of Diseases for Oncology, 3rd Edition [ICDO-3] codes 9823, sites: C62.0-C63.2) in the time period between 1973 and 2015 were included. Exclusion criteria included history of cancer, unknown laterality, unknown survival data and unknown cause of death. For each case we included age at the time of diagnosis, laterality (left, right, bilateral), SEER cause-specific death classification, survival months and vital status. Overall survival (OS) was defined as time from diagnosis to death or last follow-up and cancer-specific survival (CSS) was calculated as time from diagnosis to death from DLBCL or last follow-up. Survival curves were plotted by the Kaplan-Meier method and the log-rank test was used for comparison. P value was 2-sided and P Results: A total of 1213 patients were included in this analysis. The median follow-up was 43 months (interquartile range[IQR]: 13-90 months). Of these patients, 372 patients were diagnosed from 1973-2000 (pre-rituximab era) and 841 patients were diagnosed from 2001-2015 (rituximab era). We found that patients with bilateral testis involvement had a significantly decreased CSS (median CSS: 53 vs. 142 months, P=0.0035) and OS (median OS: 32 vs. 77 months, P=0.0008) compared with those with unilateral involvement. Patients with left-side involvement had a similar CSS (median CSS: 136 vs. 153 months, P=0.2997) and OS (median OS: 76 vs. 80 months, P=0.7360) compared to those with right-side involvement. For patients with left-side involvement, patients diagnosed in the rituximab era had a significantly longer CSS(hazards ratio[HR]: 0.4140, 95% confidence interval[CI]: 0.3065 to 0.5593, P Conclusion: our study demonstrated that laterality was not a prognostic factor for patients with primary testicular DLBCL. And the improvement in the prognosis from pre-rituximab era to rituximab era was more remarkable in primary testicular DLBCL patients with left-side involvement than those with right-side involvement. These data suggest primary testicular DLBCL from different sides had different responses to therapy and may have different biological characteristics. Figure 1 Disclosures No relevant conflicts of interest to declare.

Introduction : PI3Kδ, one of the class I PI3Ks, is found expressed primarily in leukocytes and plays an essential role in B-cell development and function. Here, we comprehensively evaluated the in vitro and in vivo antitumor activity and the underlying mechanism of SHC014748M, an oral selective inhibitor of PI3Kδ under Phase I clinical evaluation. Methods : Biochemical and cell-based assays were used to measure compound potency and selectivity in lymphoma cell lines as well as primary CLL cells, and PI3K/AKT pathway was measured by Western blot assay, Alphalisa and Elisa. Xenograft model was carried out to validate in-vivo antitumor potency of the compound. Besides, chemokines and cytokines derived from blood samples of patients were also detected. Results: SHC014748M was 125- to 306-fold more selective for PI3Kδ inhibition relative to other class I PI3K enzymes and showed in vitro activity in most of 23 B lymphoma cell lines. We identified that SHC014748M treatment resulted in a 3.1- to 5.5-fold increase in annexin V/7-ADD staining, indicating a significant apoptosis induction. SHC014748M inhibited phosphorylation of AKT, targets downstream of PI3Kδ, in lymphoma cells. Among the 15 primary CLL cells, the 50% inhibitory concentration (IC50) of SHC014748M varies from 850 nM to 37040 nM respectively and expression of phosphorylation AKT decreased to the normal levels in the presence of SHC014748M or positive control, Idelalisib. In-vivo study revealed that SHC014748M significantly reduced lymphoma cell growth in the treatment group compared with control mice. CCL4, CCL17, CCL22 and CXCL13 derived from patients decreased sharply after SHC014748M treatment. Conclusion: According to the results, SHC014748M appeared to be a novel promising compound in the treatment of B cell lymphomas and CLL. Disclosures Wang: Nanjing Sanhome Pharmaceutical Co., Ltd.: Employment. Wang:Nanjing Sanhome Pharmaceutical Co., Ltd.: Employment. Zhang:Nanjing Sanhome Pharmaceutical Co., Ltd.: Employment.

Background Circular RNAs (circRNAs), a novel family of non-coding RNAs, have crucial roles in cancer progression. Conventional research is mainly focus on nuclear genome derived circRNAs (nu-circRNAs). The biological and clinical characteristics of mitochondrial genome derived-circRNAs (mt-circRNA) remains largely unknown, especially in chronic lymphocytic leukemia (CLL), the most prevalent incurable B-cell neoplasm in western countries. Lack of convenient and reliable clinical biomarkers is a hindrance in monitoring the progression of CLL. It is in urgent need of screening effective new biomarkers and exploring potential therapeutic targets associated with CLL initiation and progression. Recent studies have shown that circRNAs are enriched and stable in serum exosomes. However, the biological mechanisms of exosomal circRNAs remain unclear. In this study, we attempted to identify the novel characteristics of a mt-circRNA mc-COX2, which was abundant in plasma exosomes and could be involved in the progression of CLL. Since CLL patients have specific expression features of exosomal circRNAs in plasma, the function of the circRNAs and their clinical significance is urged to be explored. Methods Firstly, to unveil circRNAs expression profiles in CLL, plasma samples from five treatment-naive CLL patients and five age-/sex-matched healthy donors (HDs) were collected for circRNA microarray analyses. Northern blot and RNA- FISH were conducted to verify the existence of circRNAs in mitochondria. qPCR and other functional analysis such as RNase R, actinomycin D and RIP experiments were carried out to demonstrated the clinical and biological characteristics of mc-COX2, one of the mt-circRNAs. Cell apoptosis ability was determined by FCM. Moreover, electron microscope, partical size analysis, FCM and Western blot were used to explore the existence of exosomes and q-PCR analysis was performed to detect the expression of mc-COX2. Results Mt-circRNAs were highly expressed in CLL patients plasma (Figure A, B). Herein, we reported a novel circRNA named as mc-COX2 which was generated from the COX2 gene on mitochondrial genome by back-splicing and closely related to prognosis of CLL patients (Figure C). The enrichment of mc-COX2 in the mitochondria was further confirmed by RNA-FISH (Figure D). Northern blot was performed using head-to-tail probe of mc-COX2 and the results showed that mc-COX2 was detectable within the splice sites (Figure E). Notably, obviously different from nu-circRNAs, mc-COX2 showed lower stability with lower tolerance to RNase R and actinomycin D, but it was much more stable compared with linear RNAs (Figure G, F). And mc-COX2 cannot bind to AGO2 protein, suggesting that it probably function via other mechanisms instead of acting as ceRNA (Figure H). Furthermore, the up-regulated expression of mc-COX2 was positively associated with leukemogenesis and worse survival of CLL patients (Figure I, J). CLL patients with TP53 deletion rather than mutation displayed higher expression of mc-COX2 (Figure K). The endogenous reduction of mc-COX2 could induce cell apoptosis (Figure L). In addition, we indicated that mc-COX2 was highly enriched in exosomes, by which circRNAs could enter the circulation and be readily measured in the serum (Figure M). Moreover, the existence of mc-COX2 in plasma suggests that mc-COX2 may serve as a potentially novel prognosis biomarker for CLL patients, guiding targeted therapy in clinic. Conclusions In summary, we demonstrated the existence and clinical significance of mc-COX2, a novel class of circRNA species abundant in CLL plasma exosomes for the first time, which was distinct from nu-circRNAs. Furthermore, the specific high expression of mc-COX2 in CLL plasma which was strongly correlated with P53 deletion, can indicate worse prognosis of CLL patients. Taken together, our study not only identifies a novel circRNA which may serve as a new "liquid biopsy" biomarker for CLL prognosis but also expands the current knowledge regarding molecular mechanisms of circRNAs, providing potential diagnostic and therapeutic implications for CLL. It would be of great interest to explore the biogenesis of mt-circRNAs and their impact on mitochondrial function in future studies. Figure Disclosures No relevant conflicts of interest to declare.

Introduction: Socioeconomic factors including insurance and marital status have impacts on the outcomes of cancer patients. Until now, there are few data regarding whether insurance status and marital status have effects on the outcomes of patients with chronic lymphocytic leukemia (CLL). In this study, the Surveillance, Epidemiology, and End Results (SEER) database was used to evaluate the prognostic roles of insurance and marital status in patients with CLL. Methods: Data from the SEER 18 Registries were used to conduct this study. Cases with newly-diagnosed CLL/small lymphocytic lymphoma (CLL/SLL) (International Classification of Diseases for Oncology, 3rd Edition [ICDO-3] codes 9823) in the time period between 2008 and 2015 were included. Exclusion criteria included history of cancer, unknown insurance status, unknown marital status, unknown survival data, unknown cause of death and survival months documented as 0. For each case we included age at the time of diagnosis, sex, marital status (married, divorced, single, widowed, unmarried or domestic partner, or separated), insurance status (Medicaid, insured, or uninsured), SEER cause-specific death classification, survival months and vital status. Survival curves were plotted by the Kaplan-Meier method and the log-rank test was used for comparison. P value was 2-sided and P Results: Atotal of 23,611 patients with CLL/SLL were included into the current analysis. The median follow-up was 34 months. We found that insurance status (uninsured or Medicaid) was significantly associated with decreased cancer-specific survival (CSS) (hazards ratio[HR]: 1.378, 95% confidence interval [CI]:1.251-1.664, P Conclusion: Insurance status and marital status have significant impacts on survival outcomes of patients with CLL/SLL. A 3-points prognostic index comprising insurance status, marital status and age could be used for risk stratification for patients with CLL/SLL. Figure 1 Disclosures No relevant conflicts of interest to declare.

Introduction: Through previous studies using Sanger sequencing and targeted gene sequencing, we found that the frequencies of certain genes in Chinese patients with chronic lymphocytic leukemia (CLL) were different from those in CLL patients from the western countries. However, until now, there are no published whole-exome sequencing (WES) studies in Chinese CLL patients. To better define the genomic landscape of CLL cases in China, we conducted WES studies of CLL cases in our center. Methods: This study include 43 CLL patients, of whom 40 were untreated at the time of sampling. The median age of the 43 patients were 61 years, the male-to-female ratio was 2:1. We isolated peripheral blood mononuclear cells, then CD19 positive cells were purified by using magnetic microbeads in some cases. The buccal cells or the leukocytes that were depleted of CD19 positive cells were used as germline controls. We purified genomic DNA and used the purified DNA for library construction, after which we sequenced the library using the Illumina HiSeq2500 platform. After sequencing, software including Burrows-Wheeler Aligner (BWA) and GATK were used for the bioinformatic analysis. Results: In the 43 Chinese patients with CLL, the median number of somatic mutations was 67 (range:10-128) and the median number of nonsynonymous mutations was 26 (range: 5-55). We found that the number of mutations in patients older than 60 was significantly higher than that in the younger patients. Somatic mutations were detected across 991 different genes, of which 24 genes were identified as drivers in the study from the Dana-Faber Cancer Institute (DFCI). The 24 genes included NOTCH1, SF3B1, MYD88, POT1, TP53 and so on. Seventy-nine genes were recurrently mutated and 13 genes were found to be mutated in 3 or more cases. The most frequently mutated gene was NOCTH1 (6 cases, 14.0%). FBXW7, another gene that is involved in the regulation of the NOTCH1 pathway, was also mutated in 3 cases. IGLL5, MUC4 and MYD88 were also among the most frequently mutated genes, with each of them being mutated in 5 cases (11.6%). We found that the frequencies of mutations in IGLL5 (11.6% vs. 2.2%, P=0.0056), MYD88 (11.6% vs. 3.0%, P=0.0147), BCOR (9.3% vs. 2.2%, P=0.0246), and SF3B1 (4.7% vs. 21.0%, P=0.0084) were significantly different between Chinese CLL patients and patients from western countries. BCOR-mutated patients had more frequent aberrations involving NOTCH1 pathway (NOTCH1 and/or FBXW7 mutation) than BCOR-unmutated patients (3/4 [75.0%] vs. 6/39 [15.4%], P=0.0242) (Figure 1A). Analysis of 10967 tumor samples from the Cancer Genome Atlas (TCGA) program revealed that tumors with BCOR mutations had a higher frequency of NOTCH1 aberrations (NOTCH1 and/or FBXW7 mutation) than those without BCOR mutations (116/349 [33.2%] vs. 722/10618 [6.8%]; odds ratio: 6.824, 95%CI: 5.393-8.634, P Conclusion: Patients with CLL in China and patients with CLL in the western countries have different mutational landscapes. BCOR gene mutations may cooperate with aberrations involving the NOTCH1 pathway in the pathogenesis of CLL. NFKBIA could be a potential tumor suppressor in the pathogenesis of CLL. Figure 1 Disclosures No relevant conflicts of interest to declare.

Background: Tislelizumab is a humanized IgG4 monoclonal antibody with high affinity/specificity for programmed cell death protein 1 (PD-1). Tislelizumab was specifically engineered to minimize binding to FcɤR on macrophages, thereby abrogating antibody-dependent phagocytosis, a potential mechanism of T-cell clearance and resistance to anti-PD-1 therapy (Dahan 2015). Results of tumor growth inhibition studies suggest that tislelizumab had superior antitumor activity compared with nivolumab in mice transplanted with human cancer cells and peripheral blood mononuclear cells. Favorable results with other PD-1 inhibitors in patients with relapsed or refractory (R/R) classical HL (cHL) provide a strong rationale to investigate tislelizumabin this disease. Methods: BGB-A317-203 (clinicaltrials.gov NCT03209973) is a single-arm, open-label, multicenter, phase 2 study of tislelizumab in Chinese patients with R/R cHL; all patients received tislelizumab 200 mg intravenously every 3 weeks until progression or unacceptable toxicity. Patients were eligible if they (a) failed to achieve a response or progressed after autologous stem cell transplant (ASCT) or (b) received ≥2 prior systemic chemotherapy regimens for cHL and were ineligible for ASCT. Diagnosis of cHL was confirmed in all patients by central pathologic review.The primary endpoint was overall response rate (ORR) determined using the Lugano criteria (Cheson, 2014) as assessed by an independent review committee (IRC). Key secondary endpoints included progression-free survival (PFS), duration of response, rate of complete response (CR), time to response, safety, and tolerability. Treatment emergent adverse events (TEAEs) were summarized according to NCI-CTCAE v4.03. Results: In total, 70 patients were enrolled from 11 Chinese centers; patient characteristics are shown in the Table. With a data cutoff date of 25 May 2018, the median follow-up was 7.9 months (range, 3.4 to 12.7). The IRC-assessed ORR was 85.7%, based on PET-CT scans. A total of 43 patients (61.4%) achieved CR, 38 of whom were in CR at the first on-study response assessment. At data cutoff, 53 patients remained on treatment and 17 had discontinued (11 for progressive disease [PD]; 4 for TEAEs; 1 withdrew consent; 1 due to pregnancy). The estimated 6-month PFS rate was 80%. The most frequently reported (≥15%) TEAEs due to any cause were pyrexia (52.9%), hypothyroidism (30.0%), increased weight (28.6%), upper respiratory tract infection (27.1%) and cough (17.1%). Grade ≥3 TEAEs reported in ≥2 patients were upper respiratory tract infection (2.9%) and pneumonitis (2.9%). Immune-related TEAEs were reported in 23 patients (32.9%); Grade ≥3 in 5 patients (7.1%): pneumonitis (n=2), organizing pneumonia, nephritis (focal segmental glomerulosclerosis) and increased creatine phosphokinase (each n=1). There were no Grade 5 TEAEs. TEAEs that led to treatment discontinuation in 4 patients (5.7%) included pneumonitis (n=2), organizing pneumonia (n=1), and focal segmental glomerulosclerosis (n=1). One patient died on study due to PD. Conclusions: In this study, tislelizumab therapy was shown to be highly active resulting in a high CR rate in patients with R/R cHL who had failed or were ineligible for ASCT. Tislelizumab was generally well-tolerated in Chinese patients with R/R cHL. The safety profile was generally consistent with that of other PD-1 inhibitors for the treatment of cHL. Disclosures Song: Peking University Cancer Hospital (Beijing Cancer Hospital): Employment. Liu:West China Hospital of Sichuan University: Employment. Guo:BeiGene (Shanghai) Co., LTD: Employment. Yang:BeiGene (Beijing) Co., Ltd.: Employment. Elstrom:BeiGene (Beijing) Co., Ltd.: Employment, Equity Ownership. Huang:BeiGene (Beijing) Co., Ltd.: Employment, Equity Ownership. Novotny:BeiGene (Beijing) Co., Ltd.: Employment, Equity Ownership. Wei:BeiGene (Beijing) Co., Ltd.: Employment, Equity Ownership. Zhu:Beijing Cancer Hospital: Employment.

Background: Immunoglobulin heavy chain variable region (IGHV) has been an important prognostic factor for chronic lymphocytic leukemia (CLL) for decades. 98% being a cut-off value for IGHV is a mathematical choice and researches on the best cut-off value have never been stopped. Chinese CLL patients are known to differ from Caucasian CLL patients on both clinical and genetical features. However, the optimal cutoff for IGHV mutational status has not yet been studied in this particular ethnic group. Method: We carried out a study on 595 Chinese CLL patients in order to find out whether 98% is the best cut-off value for IGHV in Chinese CLL patients. Genomic DNA from peripheral blood or bone marrow was subjected to PCR amplification following the IGH Somatic Hypermutation Assay v2.0 protocol (InVivoScribe). Sequences were aligned to ImMunoGeneTics/V-QUEry and Standardization (IMGT/-VQUEST) database. Result: 600 sequences were received after IGHV rearrangement sequencing. IGHV3-23, IGHV4-34, IGHV3-7, IGHV4-39 and IGHV1-69 were the most frequently used IGHV genes. 352 (58.7%) cases were IGHV-mutated while 248 (41.3%) cases were IGHV-unmutated if the classical 98% classification by ERIC was used. In order to determine the optimal cut-off value, we used 1% as the interval to divide the entire cohort into 7 groups according to the mutational rate, which were Conclusion: 98% is the optimal cutoff value for IGHV mutational status to predict the prognosis of CLL patients in China. Table 1. Table 1. Disclosures No relevant conflicts of interest to declare.

Objective: Determine efficacy and safety of PEMD (pegaspargase, etoposide, methotrexate, dexamethasone) in persons with newly-diagnosed advanced-stage extra-nodal NK/T-cell lymphoma. Subjects and methods: Twenty-seven consecutive subjects with newly-diagnosed advanced-stage (stage III-IV) extra-nodal NK/T-cell lymphoma were prospectively studied from July, 2010 to August, 2015. All subjects received PEMD (methotrexate, 3.0 g/m2 IV over 6 h on day 1, etoposide, 100 mg/m2 IV on days 2-4, dexamethasone, 40 mg IV on days 1-4 and pegaspargase, 2500 U/m2 IM on day 2). Courses were given every 3 week. Primary co-endpoints were response and survival. Secondary endpoints were proportion of subjects completing planned therapy (4 to 6 cycles of PEMD regimen) and frequencies of adverse events. Results: Median age was 46 y (range, 17-73 y). There were 21 males (78%). Nine subjects (33%) had non-nasal NK/T-cell lymphoma including 5 of the skin involvement, 1 of the testis involvement, 1 of the muscle involvement and 2 of the gastrointestinal tract involvement. Thirteen subjects (48%) had elevated serum LDH levels. Eleven subjects (41%) had higher level of EBV-DNA in blood (>5000 copies/mL). Twenty-one subjects (78%) had B-symptoms and 13 (48%) had an IPI score of 3-5. Subjects received a median of 4 courses of PEMD (range, 1-6). Median follow-up is 48 mo (range, 13-74 mo). Three patients had early death (within 3 mo after the diagnosis). Overall response rate (ORR) was 74% (95%CI 54%-89%) in the 27 subjects with advanced-stage disease including complete response (CR)/unconfirmed CR (CRu) in 12 (44% [95%CI 26%-65%]) and a partial response (PR) in 8 (30% [95%CI 14%-50%]). Four-year progression-free survival (PFS) was 44% (95%CI 25%-63%) and overall survival (OS) 51% (95%CI 32%-70%) (Figure 1). PFS and OS were not correlated between nasal and non-nasal types of ENKTL. There was no treatment-related death or serious allergic reactions. The most common grade-3/-4 hematologic complication was neutropenia (37% [95%CI 19%-58%]). The most common non-hematologic complications were infection (16% [95%CI 4%-34%]) and hypo-fibrinogenemia (12% [95%CI 2%-29%]). Conclusion: PEMD is effective and safe in persons with newly-diagnosed advanced-stage extra-nodal NK/T-cell lymphoma. Figure 1. PFS and OS for all the patients (N=27). Figure 1. PFS and OS for all the patients (N=27). Disclosures No relevant conflicts of interest to declare.

The circulating chronic lymphocytic leukemia (CLL) cells appear not to be overly utilizing aerobic glycolysis. However, CLL cells' recurrent contact with the stromal microenvironment leads to an increase of aerobic glycolysis and the cells' overall glycolytic capacity, which promotes cell survival and proliferation. TP53-induced glycolysis and apoptosis regulator (TIGAR) has been directly implicated in cellular metabolism in the control of glycolysis. TIGAR inhibits glycolysis and protects cells from intracellular reactive oxygen species (ROS)-associated apoptosis. Here, we investigated correlation between TIGAR expression and apoptosis in CLL primary cells, along with its relationship with the clinical characteristics and prognosis in 102 newly diagnosed CLL patients. Our data showed TIGARoverexpression correlated with protection from spontaneous apoptosis in CLL cells, and is strongly associated with advanced Binet stage, unmutated immunoglobulin heavy-chain variable region (IGHV) status, CD38 positivity, β2-microglobulin and p53 aberrations. Higher expression of TIGAR was associated with briefer treatment-free survival (median: 3 months vs. 51 months, p = 0.0108) and worse overall survival (median: 74 months vs. not reached, p = 0.0242), as well as their diverse responses to fludarabine based chemotherapy. TIGAR expression of the patients who were resistant to chemotherapy was significantly higher than those who were sensitive to chemotherapy (mean: 0.3859±0.1710 vs. 0.0974±0.0291, p = 0.0290). Taken together, our findings depict how bioenergetics characteristics could be therapeutically exploited in CLL. Disclosures No relevant conflicts of interest to declare.

Pericytes and vascular smooth muscle cells (VSMCs), which are recruited to developing blood vessels by platelet-derived growth factor BB, support endothelial cell survival and vascular stability. Here, we report that imatinib, a tyrosine kinase inhibitor of platelet-derived growth factor receptor β (PDGFRβ), impaired growth of lymphoma in both human xenograft and murine allograft models. Lymphoma cells themselves neither expressed PDGFRβ nor were growth inhibited by imatinib. Tumor growth inhibition was associated with decreased microvascular density and increased vascular leakage. In vivo, imatinib induced apoptosis of tumor-associated PDGFRβ(+) pericytes and loss of perivascular integrity. In vitro, imatinib inhibited PDGFRβ(+) VSMC proliferation and PDGF-BB signaling, whereas small interfering RNA knockdown of PDGFRβ in pericytes protected them against imatinib-mediated growth inhibition. Fluorescence-activated cell sorter analysis of tumor tissue revealed depletion of pericytes, endothelial cells, and their progenitors following imatinib treatment. Compared with imatinib, treatment with an anti-PDGFRβ monoclonal antibody partially inhibited lymphoma growth. Last, microarray analysis (Gene Expression Omnibus database accession number GSE30752) of PDGFRβ(+) VSMCs following imatinib treatment showed down-regulation of genes implicated in vascular cell proliferation, survival, and assembly, including those representing multiple pathways downstream of PDGFRβ. Taken together, these data indicate that PDGFRβ(+) pericytes may represent a novel, nonendothelial, antiangiogenic target for lymphoma therapy.

Purpose The aim of this study was to determine whether the sum of the SUVmax of all the nodal and extranodal lesions can predict prognosis in patients with extranodal natural killer/T-cell lymphoma (ENKTL). Methods We conducted a retrospective analysis of 54 patients with newly-diagnosed ENKTL who underwent pretreatment 18F-FDG PET/CT. Three new models (WB1SUVmax, WB2SUVmax, WB3SUVmax) on the basis of the whole-body SUVmax of 11 nodal lesions (waldeyer ring, neck, infraclvicular, axillary and pectoral, mediastinal, hilar, spleen, paraaortic, mesenteric, lilac, inguinal and femoral) and 10 extranodal lesions (upper aerodigestive tract, skin/subsutaneous, central nervous system and spinal canal, lung, myocardium, bone and bone marrow, bowel, renal and adrenal, liver and testis) were designed to predict the overall survival (OS) and progression-free survival (PFS) Results During follow-up period (median 33.2 months), 31 patients showed disease progression and 28 patients died from the disease. Receiver operating characteristics curve analysis showed cut-off values for SUVmax, WB1SUVmax, WB2SUVmax, WB3SUVmax of 9.9 (sensitivity 82.6%, specificity 50%, AUC 0.658, P =0.060), 16.0 (sensitivity 87.00%, specificity 68.20, AUC 0.742, P =0.002), 15.8 (sensitivity 86.96%, specificity 68.18%, AUC 0.800, P Conclusion Our results demonstrated the importance of the WBSUVmax, which was calculated on the basis of the sum of the SUVmax of 11 nodal and 10 extranodal lesions, as a prognostic factor in ENKTL patients. The use of WBSUVmax together with the IPI score may be useful for better prognostic discrimination in the future. Table 1. Univariate and multivariate Cox regression analysis for PFS and OS Univariate analysis (OS) Multivariate analysis (OS) Univariate analysis (PFS) Multivariate analysis (PFS) Characteristic HR (95%CI) P HR (95%CI) P HR (95%CI) P HR (95%CI) P WB3SUVmax£¾15.8 6.67 (2.48-17.92) 60 years 1.79 (0.32-1.95) 0.611 -- -- 1.72 (0.30-1.78) 0.489 -- -- ECOG PS>1 2.14 (0.73-6.21) 0.162 -- -- 1.89 (0.66-5.42) 0.239 -- -- LDH>ULN 7.64 (2.87-20.33) Figure 1. ROC curve analyses for identification of the optimal cut-off values of PET parameters predicting survival in patients with ENKTL Figure 1. ROC curve analyses for identification of the optimal cut-off values of PET parameters predicting survival in patients with ENKTL Disclosures No relevant conflicts of interest to declare.

Objection: Chronic lymphocytic leukemia (CLL) is a chronic lymphoproliferative disease characterized by highly clinical and biological heterogeneity. A number of biomarkers have been identified in predicting the overall survival (OS) over the last decades besides the traditional clinical staging. Recently, an international prognostic index (IPI) combing clinical staging and biomarkers was developed by the investigators of the Cochrane Haematological Malignancies Group. Due to genetic differences between Caucasic and Chinese CLL patients, our study was to validate the guiding function of IPI on Chinese CLL cases. Method: We performed a validation of the IPI proposed by the Cochrane Haematological Malignancies Group to stratify Chinese CLL patients prognostically in 225 CLL cases registered at our center. The five parameters (age, TP53 abnormalities, IGHV mutation status, b2-microglobulin and Binet stage) involved in the IPI were collected by clinical data, serum test, PCR and fluorescence in situ hybridization (FISH). Chi-square test, survival analysis, log-rank test and cox hazard regression analysis were utilized in the validation. Result: In the 225 Chinese CLL cases analysed in the validation, all five parameters involved in the IPI were associated with overall survival (OS) independently. The multivariate analysis demonstrated that age above 65 years old (HR 2.22; [1.15-4.30]; P=0.018), b2-microglobulin over 3.5 mg/L (HR 2.46; [1.22-4.94]; P=0.001), Binet staging B/C (HR 3.40; [1.02-11.33]; P=0.046), TP53 abnormalities (HR 2.72; [1.50-4.94]; P=0.012) and IGHV unmutation (HR 5.19; [2.51-10.77]; P Conclusion: Our results basically validated the IPI proposed by the Cochrane Haematological Malignancies Group to prognostically stratify CLL patients in China, which confirmed the value of the novel prognostic index externally. However, a slight adaption was made to accommodate the Chinese cases better via the combination of the low-risk and intermediate-risk groups. We considered that a universally recognized prognostic model would be utilized to predict the disease progression and guide the treatment when initially diagnosed. Disclosures No relevant conflicts of interest to declare.

Purpose The aim of this study was to examine the prognostic value of bone marrow involvement (BMI) assessed by PET-CT in treatment-naïve patients with diffuse large B-cell lymphoma (DLBCL). Patients and methods All patients from a single centre diagnosed as DLBCL between 2005 and 2014 had data extracted from staging PET-CT, bone marrow biopsy (BMB), and treatment records. The final diagnosis of BMI was defined as: (i) positive bone marrow biopsy; (ii) positive PET-BMI confirmed by guided biopsy or targeted MR imaging; (iii) concomitant disappearance of bone marrow uptake and uptake in other lymphoma lesions on PET-CT after R-chemotherapy. Results Of 169 patients, 20 patients (12%) had BMI on BMB, whereas 35 patients (21%) had positive BMI according to PET-CT findings (PET-BMI(+)). Thirty-three out of the 35 patients with PET-BMI(+) showed a focal pattern and 2 a diffuse pattern, respectively. In multivariate analyses, PET-BMI(+) remained significant for overall survival (OS) (HR 2.90, 95%CI 1.21−6.96, P=0.017) while progressive-free survival (PFS) was significant only in univariate analysis (P Conclusions Our data raised several important issues about the predictive significance of BMI assessed by PET-CT in DLBCL: (i) The bone marrow status assessed by PET-CT is an independent predictor of OS independent of IPI; (ii) For baseline PET-BMI(+) patients, the optimal cutoff value of SUVmax(BM) to predict the survival outcomes was 8.6; (iii) In patients with stage IV disease, worse survival outcomes were observed in patients with BMI than that without BMI; (iV) Patients with PET-BMI(+) in the intermediate risk-group should be managed as high-risk group patients. Table 1. Cox regression analysis for PFS and OS for all the patients with DLBCL (N=169) PFS OS Univariate analysis Multivariate analysis Univariate analysis Multivariate analysis HR (95%CI) P HR (95%CI) P HR (95%CI) P HR (95%CI) P PET-BMI+ 3.96 (2.38-6.59) 2 7.27 (4.19-12.63) 60 1.61 (0.98-2.64) 0.060 1.18 (0.61-2.27) 0.627 Stage III or IV 6.08 (2.77-13.36) ULN 4.68 (2.82-7.78) 1 3.15 (1.91-5.18) Figure 1. Survivals according to SUVmax in patients with PET-BMI(+) at diagnosis Figure 1. Survivals according to SUVmax in patients with PET-BMI(+) at diagnosis Figure 2. Survivals according to PET-BMI status in cases with stage IV Figure 2. Survivals according to PET-BMI status in cases with stage IV Figure 3. Survivals according to PET-BMI status in cases with IPI score of 2-3 Figure 3. Survivals according to PET-BMI status in cases with IPI score of 2-3 Disclosures No relevant conflicts of interest to declare.

Background : LEF1, lymphoid enhancer factor 1, is a key transcription factor of Wnt signaling pathways. In normal blood cells, the LEF1 mainly expresses in pre-B and T cells, involves in proliferation and differentiation. With mature of the cells, the expression of LEF1 reduces gradually. Its abnormal expression is associated with a variety of tumors, such as various kinds of leukemias and lymphomas. Previous studies have explored the relationship between LEF1 gene and tumorogenesis as well as tumor development, and there is little report on the diagnostic significance of LEF1. Recent studies have shown that the LEF1 protein expression in chronic lymphocytic leukemia (CLL) is significantly higher than other B-cell chronic lymphoproliferative disorders (B-CLPD) and normal controls. Our study aimed to compare the LEF1 gene mRNA expression and the LEF1 protein expression between different subtypes of patients with B-CLPD, then established the differential diagnostic value of LEF1 in B-CLPD. Methods: We determined the expression level of LEF1 protein by immunohistochemistry in bone marrow samples from 143 patients with leukemic phase B-CLPD including 78 cases of CLL, 17 cases of mantle cell lymphoma (MCL), 20 cases of Waldenström macroglobulinemia (WM), 20 cases of follicular lymphoma (FL), 5 cases of marginal zone lymphoma (MZL), 3 cases of B-cell chronic lymphoproliferative disorders-unclassified (B-CLPD-U). We used real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) to compare the level of LEF1 mRNA expression in malignant cells from 54 B-CLPD patients, including 44 cases of CLL, 3 cases of MCL, 2 cases of MZL, 3 cases of FL, one case of hairy cell leukemia (HCL), one case of B-cell prolymphocytic leukemia (B-PLL) and 5 cases of healthy donors. All statistical analysis of the data were performed using SPASS version 20.0. Mann-Whitney U test was used to compare the difference between the cohorts. P Results: The expression of LEF1 protein was positive in 68 of 78 patients with CLL and 2 of 20 patients with FL, both of whom were pathological low-grade. LEF1 expression of patients with WM, MCL, B-CLPD-U were all negative. The expression level of LEF1 protein in CLL was higher than other B-CLPD patients (including all FL, WM, MCL, B-CLPD-U, P=0.000). Positive expression of LEF1 protein can be used as novel differential diagnostic marker for patients with CLL with higher sensitivity and specificity (sensitivity=87%, specificity=97%).The expression of LEF1 protein was negative in 10 of 78 CLL patients. The 10 patients had atypical immunophenotype. The expression of CD20 was increased, even strong expression in 5 CLL patients. There were 9 CLL with CD200/CD148 Conclusions: The expression level of LEF1 protein was significantly higher than non-CLL in B-CLPD. We analyzed LEF1 protein may be a novel marker in the differential diagnosis of B-CLPD with higher sensitivity and specificity.The mRNA expression level of LEF1 gene in CLL patients was significant higher than non-CLL patients, but the expression of LEF1 mRNA in 25% CLL patients was lower than healthy controls and 33.3% CLL patients was lower than non-CLL patients. Disclosures No relevant conflicts of interest to declare.

Background CD8+ T cells from chronic lymphocytic leukemia (CLL) patients have been demonstrated to exhibit a number of alterations in global gene expression profiles when compared with healthy controls. It has been shown that CD8+ T cells from CLL patients have increased expression of T-cell exhaustion markers like PD-1. CLL-induced functional defects in T cells are thought to directly contribute to the failure of antitumor immunity and are considered a hallmark of this disease. Nevertheless, the molecular regulation of T-cell dysfunction in CLL patients still remains poorly understood. Methods In the present study, CD8+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of patients with CLL (n=10) and healthy donors (n=5), and analyzed by genome-wide DNA methylation profiling using Illumina Infinium 450K methylation array. The differentially methylated genes (KLRG1, CCR6 and TCRA) identified by the 450K array analysis were validated by bisulfite pyrosequencing in additional CLL and healthy control samples. DNA methylation in the first intron, distal upstream, and proximal promoter regions of PD-1 was also examined by pyrosequencing. Luciferase reporter assays were used to determine the effects of DNA methylation on the enhancer activity of a PD-1 upstream sequence. To investigate whether CLL cells can directly alter the methylation of the candidate genes in CD8+ T cells, healthy PBMCs were cultured alone or co-cultured with purified allogeneic CLL cells for 72 hours. In parallel, healthy PBMCs were cultured in CD3mAb-coated plates containing CD28mAb or treated with PMA/ionomycin for 72 hours. Cultured PBMCs were then harvested for flow cytometrc analysis and CD8+ T cells purification. Multicolor flow cytometry was used to characterize T-cell subsets and expression of PD-1, KLRG1 and TCRα/β. Bisulfite pyrosequencing was used to determine the methylation changes of KLRG1, CCR6, TCRA, and PD-1 in CD8+ T cells after co-culture with CLL cells or after T-cell activation. Results The Illumina 450K methylation array analysis identified 312 differentially methylated CpG sites (Student t-test, p0.25) between CD8+ T cells from CLL and healthy controls with 199 hypermethyated and 113 hypomethylated CpG sites that are associated with 206 genes. Interestingly, 4 out of the 7 most significant CpG sites (FDR Conclusion For the first time, our investigation demonstrates the genome-wide DNA methylation profiles of CD8+ T cells isolated from CLL patients and determined that recurrent epigenetic changes in PD-1, KLRG1, CCR6, and TCRA in CD8+ T cells occur in CLL patients. Our methylation data suggest that the exhaustion phenotype observed in CLL patient CD8+ T cells maybe associated with altered DNA methylation profiles, an event previously seen in antigen-specific CD8+ T cells that undergo chronic viral infection-induced epigenetic changes. Disclosures Awan: Boehringer Ingelheim: Consultancy; Lymphoma Research Foundation: Research Funding. Wang:NIH/NIMHD: Research Funding. Shi:NIH/NCI: Research Funding; Georgia Research Alliance: Research Funding.

Background T-cell large granular lymphocytic leukemia (T-LGLL) is a rare lymphoproliferative disorder of cytotoxic T cells (CTLs) and often associated with autoimmune disorders. The signal transducer and activator of transcription 3 (STAT3) is an oncogene, and its activation plays a key role in cell signaling transduction pathways in many types of cancer. The aim of the current study was to analyze the characteristics of T-LGLL. Methods We did this by determining the mutation status of STAT3 in 28 patients presenting with T-LGLL and evaluating STAT3 status in association with serum level of lacticdehydrogenase (LDH) and β2-microglobulin (β2-MG). Flow cytometric analysis for immunophenotype and TCR variable β-chain (Vβ) was performed in the patients. Results FC-Vβ analysis was performed in 26 patients, and 22 (84.6%) patients had a restricted Vβ reactivity pattern, with predominance of a single Vβ mAb reactivity. There was no significant difference between serum LDH levels, gender, age or symptoms at diagnosis (P=0.062), lymphocytosis, anemia (P=0.057), thrombocytopenia, splenomegaly, LGL count or STAT3 mutation status. However, high β2-MG levels (P=0.005), neutropenia (P=0.018) and pure red blood cell aplasia (PRCA) (P=0.001) all displayed a significant association with STAT3 mutations. In univariate analysis, treatment-free survival (TFS) was affected by STAT3 mutation status (P=0.008) and β2-MG (P=0.006). In multivariate analysis, only anemia (P Conclusions We found an incidence of STAT3 mutations of 21.4% in patients presenting with T-LGLL and two mutations, E616V and V671F, had not been previously reported. Our results further demonstrate the remarkable correlation of STAT3 mutation with anemia, neutropenia and β2-MG. β2-MG and LDH is commonly used laboratory indicators, and detecting of them is helpful in estimating clinical characteristics and guiding treatment. We conclude from our data that FC-Vβ analysis is a fast and relatively economical alternative approach that can be used as a powerful tool in screening patients with suspected T-LGLL. Our study not only confirmed some of the previously reported observations, but it also provided new information on clinical associations, laboratory findings, prognostic factors, therapy and outcome in these patients. Disclosures: No relevant conflicts of interest to declare.

Introduction Anthracycline-containing regimens (CHOP and CHOP-like regimens) are still the cornerstone of elderly patients with diffuse large B-cell lymphoma (DLBCL), although the therapeutic response is acceptable, high incidence of co-morbidities and treatment-related adverse effects including cardiac and hematologic toxicities are major obstacle in clinical practice, and management of elderly DLBCL patients remains a great challenge. We employed a platinum-based regimen (oxaliplatin-gemcitabine) which is widely used to treat solid tumors plus anti-CD20 antibody rituximab(R-GemOx) as first-line treatment in elderly patients with DLBCL. We have performed an interim analysis of this ongoing Phase 2 study and evaluated safety and efficacy of this regimen. Methods This study was a prospective, multicenter, single-arm phase 2 clinical trial (NCT01670370). Eligible patients included those with previously untreated, CD20 positive DLBCL, ≥70 years of age or ≥60 years with ECOG performance status score ≥2. Therapeutic protocol of R-GemOx regimen was as follows: rituximab 375 mg/m2 i.v. on day 1; gemcitabine 1 g/m2 i.v. on day 2; oxaliplatin 100 mg/m2 i.v. on day 2, each cycle of treatment was administered every 14 to 21 days according to recovery of toxicities. Interim evaluation was conducted after 3 cycles of treatment, patients who responded to the treatment and did not have disease progression received up to six cycles of R-GemOx treatment. Results At the time of interim analysis, 25 patients were enrolled between July 2010 and March 2013 and 22 patients were eligible to evaluate. Patients were 50.0% (11/22) male with median age 74 (range: 61-85) yrs, four patients (18.2%) were Conclusions R-GemOx immunochemotherapy regimen is highly active and well-tolerated in previously untreated elderly patients with DLBCL, gastrointestinal complications and hematologic toxicities can occur and should be carefully monitored, the encouraging interim data suggest R-GemOx is a promising first-line regimen for this subgroup of patients, Longer follow-up and further evaluation are required to evaluate efficacy and safety of this regimen. Disclosures: No relevant conflicts of interest to declare.

Mounting evidence indicates that immunoglobulin variable heavy-chain (IGHV) repertoire and mutational status in chronic lymphocytic leukemia (CLL) patients are prognostically relevant. However, rare data is available in Chinese CLL population. Our group investigated 270 Chinese CLL patients for their IGHV sequences and discovered significant differences between Chinese and European CLL patients. First of all, 169 (62.6%) patients in our group got mutated IGHV and 101 (37.4%) were unmutated, rendering a considerable higher percentage of mutated subgroup compared with European patients (55%) (Figure 1). While IGVH3 is still the most frequently used IGVH gene in Chinese CLL patients (135/270, 50%), discrepancy occurs in the usage of IGVH1 gene, which only presents in 13.7% (37/274) patients in our cohort whereas 23.79% for European (Figure 2). Regarding IGHV subgroups, IGHV3-23 and IGHV4-34 are more often used in Chinese CLL patients (10.7% and 10.4%, respectively). Remarkably, IGHV1-69, the most prevalent IGHV subgroup in European CLL patients (12.81%), only accounts for 5.2% (14/270) Chinese cases.Figure 1Higher percentage of mutated IGHV in Chinese CLL patientsFigure 1. Higher percentage of mutated IGHV in Chinese CLL patientsFigure 2Different IGHV gene usage between Chinese and European CLL patients, with IGVH1 gene accounts for 23.79% of European CLL patients and for only 13.70% of Chinese CLL patients.Figure 2. Different IGHV gene usage between Chinese and European CLL patients, with IGVH1 gene accounts for 23.79% of European CLL patients and for only 13.70% of Chinese CLL patients.Figure 3IGVH1-69 is the most prevalent IGHV gene among European CLL patients(12.81%), however, only 5.20% Chinese CLL patients use VH1-69. IGVH4-39 and IGVH4-59 are more often used in Chinese CLL patients (7.80% vs 3.73% and 5.60% vs 2.75%, respectively).Figure 3. IGVH1-69 is the most prevalent IGHV gene among European CLL patients(12.81%), however, only 5.20% Chinese CLL patients use VH1-69. IGVH4-39 and IGVH4-59 are more often used in Chinese CLL patients (7.80% vs 3.73% and 5.60% vs 2.75%, respectively). We further studied the distribution of stereotyped BCR in our cohort. Thirty-eight patients (14.07%) with stereotyped BCR that belonged to 21 subsets were identified, with 1 to 7 sequences contained each. Among them, subset 1 and subset 8 are the most common types with 6 and 7 cases respectively. Three new subsets were discovered (Table 1). Notably, only 1 case belonged to subset 2, the subset with largest group size in western world. Hence, we conclude that Chinese CLL patients show unique IGHV repertoire features compared to patients from western countries. While the mechanism within remains unknown, the discrepancy might due to antigenic difference in geographically remote areas.Table 1Three new subsets of BCR stereotypy in Chinese CLL patientsNO.IGHVIGHDIGHJM/UMIdentityHCDR3 AA sequenceLengthNovel 1NJ-15IGHV4-59*083-22*016*03UM100,00%ARGNYYDSSGYYYVGYYYYYMDV23NJ-31IGHV4-59*013-22*016*03UM99,65%ARGDYYDSSGYYYVGYYYYYMDV23Novel 2NJ-186IGHV3-23*013-22*014*02M96.60%AKGYRDNYDGDQSSVFDS18NJ-23IGHV3-23*012-21*014*02M96,53%AKGYRDNYDGDQSSVFDS18Novel 3NJ-36IGHV4-34*016-6*015*02M93,33%AKLMAGRPNWFDP13NJ-123IGHV4-34*016-6*015*02M91,67%AKLMAGRPNWFDP13 Disclosures: No relevant conflicts of interest to declare.

Introduction Hemophagocytic lymphohistiocytosis (HLH), also known as hemophagocytic syndrome (HPS) is characterized by cytokines storm and uncontrolled activation of macrophages. Secondary HLH in adults may occur in different occasions, including infections, autoimmune diseases, carcinomas and hematologic malignancies, in which Lymphoma-associated hemophagocytic lymphohistiocytosis(LA-HLH) has a high fatality rate and the worst outcome. The major cause of LA-HLH is aggressive non-Hodgkin's lymphoma (NHL), especially T/NKT cell lymphomas. Until now, there is no recommended therapeutic schedule for this fatal disease. Because of promising results of etoposide-containing regimen for HLH and high effective of continuous infusion chemotherapy regimen for aggressive NHL .The investigators therefore employed an intensive chemotherapy regimen--DA-EPOCH regimen (etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin) to treat non-Hodgkin's lymphoma with hemophagocytic lymphohistiocytosis and we have performed a preliminary analysis of this ongoing Phase 2 study and evaluated safety and efficacy of this regimen. Methods The clinical trial was designed as a prospective, multicenter, single-arm phase 2 clinical trial (NCT01818908). Enrolled patients included those with previously untreated NHL, and met the diagnostic criteria of either HLH2004 or ASH 2009 of HLH, patients with primary HLH and secondary HLH other than NHL were excluded. Therapeutic protocol of DA-EPOCH regimen was as follows: etoposide 50 mg/m2 CI 24h d1-d4, doxorubicin 10 mg/m2 CI 24h d1-d4, vincristine 0.4 mg/m2 CI 24h d1-d4, cyclophosphamide 750 mg/m2 IV d5, prednisone 60 mg/m2 oral d1-d5, If enrolled patient was histologically confirmed CD20+ B cell lymphoma, standard dose of rituximab will be recommended to combine with DA-EPOCH regimen. Drug dose was adjusted as previously described (Wyndham H. et al, 2002), each cycle of treatment was administered every 21 to 28 days according to recovery of toxicities. For patients who responded to the treatment and did not have disease progression, interim evaluation was conducted after 3 cycles of treatment. Results At the time of analysis 26 patients were enrolled and 20 patients were eligible to evaluate. Patients were 60.0% (12/20) male with median age 44(range: 14-60), the age-adjusted international prognostic score (aaIPI) enrolled to study was high/intermediate (2 score) 10/20, high risk group (3 score) 10/20. Histologies were four B-NHL, ten T-NHL and six NK-NHL. The median number of cycles of therapy was 3, patients who had discontinued study were mainly due to rapid disease progression. The overall response rate (ORR) among treated pts was 50.0%, with 35.0% (7/20) of patients achieving CR/CRu and 15.0% (3/20) achieving PR, and 50.0% of patients (10/20) had PD. With the median follow-up of 8 months, progression-free survival (PFS) rate was 46.3% and in subgroup analysis, PFS of B-NHL, T-NHL and NK-NHL was 75%, 45.7% and 25% respectively (Fig. 1); Overall survival (OS) rate was 52.4%(Fig. 2) and 75.0%, 58.3%, 22.2% in subgroup of B-NHL, T-NHL and NK-NHL(Fig. 3). The most common grade ≥3 treatment-related side effects were hematologic toxicities including neutropenia and thrombocytopenia. Conclusions Lymphoma-associated hemophagocytic lymphohistiocytosis progresses rapidly and has a high mortality rate, our preliminary results suggest DA-EPOCH regimen has acceptable toxicities and promising activity in NHL with HLH, especially for B-NHL and T-NHL, but prognosis of NK-NHL with HLH remains dismal, urgent need for novel therapeutic strategies may benefit the survival of patients with this disease. Disclosures: No relevant conflicts of interest to declare.

Abstract 3667 Introduction: Anthracycline-based regimen(CHOP and CHOP-like regimens) is still the most widely used treatment of elderly patients with diffuse large B-cell lymphoma (DLBCL), but because of low rates of the overall response, high incidence of co-morbidities and treatment-related adverse effects, the prognosis of these patients remain dismal. Our aim was to develop a non-anthracycline-based immunochemotherapy regimen, rituximab combination with GemOx (gemcitabine and oxaliplatin) for elderly patients with DLBCL and investigate the safety and preliminary efficacy of this regimen. Design and Methods: This study was a prospective, single-arm clinical trial. Eligible patients was previously untreated, CD20 positive DLBCL patients diagnosed by biopsy, ≥70 years of age or ≥60 years with ECOG PS of ≥2. Therapeutic protocol of R-GemOx regimen was as follows: rituximab 375 mg/m2 i.v. on day 1; gemcitabine 1 g/m2 i.v. on day 2; oxaliplatin 100 mg/m2 i.v. on day 2, each cycle of treatment was administered every 14 to 21 days according to recovery of toxicities. Patients who responded to the treatment and did not have disease progression received up to six cycles of R-GemOx treatment. Results: 15 patients were enrolled between July 2010 and April 2012 and 12 patients were eligible to evaluate. Patients were 41.7% (5/12) male with median age 73(range: 61–85 yrs), three patients (25.0%) were Conclusions: This is the first clinical study to investigate the safety and efficacy of R-GemOx regimen in elderly patients with DLBCL. Rituximab combination with GemOx appears highly active and favorable toxicity profiles in elderly patients with DLBCL requiring treatment, the encouraging preliminary data suggest R-GemOx is a promising first-line regimen for this subgroup of patients, and further evaluation is required. Disclosures: No relevant conflicts of interest to declare.

Abstract 4564 Objective Human homolog of murine double minute 4 (MDM4) belongs to murine double minute (MDM) family. Splicing variant of MDM4 (S-MDM4) is obtained from the deletion of exon 6, which results in an internal deletion of 68 bp. Murine double minute 2 (MDM2) is an analogy of MDM4 and shares highly similar structure with each other. The MDM4 gene plays a crucial role in regulating p53 activity and has been found to overexpress in chronic lymphocytic leukemia (CLL). The purpose of this study was to investigate the prognostic significance of MDM4, and characterize the role of MDM4 in p53 pathway. Methods Full-length MDM4 (FL-MDM4), S-MDM4 and MDM2 mRNA expressions were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in 140 Chinese patients with CLL. The correlation between those MDM expressions and CLL prognostic marker such as clinical stage, immunoglobulin heavy-chain variable region (IGHV) mutational status, ZAP-70, CD38, and chromosomal abnormalities were analyzed. Furthermore, primary CLL cells were treated in vitro with either fludarabine or Nutlin-3, to explore the interaction between p53 status and those MDMs. Results FL-MDM4 and S-MDM4 expressions were significantly increased with the del(17p13) (P=0.037 and P=0.006), p53 mutations (P=0.023 and P Conclusion S-MDM4 overexpression is an indicator of p53 aberrations in CLL patients, suggesting those patients have a poor prognosis. FL-MDM4 inhibitory effect on p53 can be removed by MDM2-p53 and saved by Nutlin-3, and S-MDM4 overexpression marks a potential therapeutic target in CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract 2848 Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. Alterations in microRNAs (miRNAs) expression have been proposed to play role in CLL pathogenesis. However, the reasons of miRNAs expression alterations observed in CLL have not been elucidated, one possible explanation suggested is deterioration of miRNA biogenesis machinery involves Dicer and Drosha. Dicer and Drosha are main regulators of miRNA biogenesis, and deregulation of their expressions have been observed in various cancers including breast cancer, ovarian cancer, prostate cancer, neuroblastoma tumors, et al. To investigate the role of Dicer and Drosha in CLL, we assessed the expression of Dicer and Drosha and their correlation with other prognostic factors, including Binet stages, immunoglobulin heavy chain variable gene (IGHV) mutation status, TP53 mutation status, ZAP-70 protein and CD38 expression level in 165 CLL patients by using real-time PCR methods. We found that Dicer expression was significantly higher in patients of Binet B and C stage compared with Binet A stage (mean ± SD, 0.017 ± 0.010 vs. 0.015 ± 0.019; p =0.001). Patients with unmutated IGHV genes had significant lower expression of dicer than patients with IGHV mutations (mean ± SD, 0.012± 0.006 vs 0.019± 0.013, p Disclosures: No relevant conflicts of interest to declare.

Abstract 3725 Growing evidence has implicated the tumor microenvironment, demarcated by neo-vessels, as an essential disease compartment which influences neoplastic growth and clinical outcome in human lymphoma. The mechanisms whereby stromal cells contribute to the pathogenesis and progression of lymphoma remain poorly understood. Pericytes / vascular smooth muscle cells (VSMC), regulated by platelet-derived growth factor receptor β (PDGFRβ) signaling, play important roles in endothelial cell survival and vascular stability. We hypothesized that the functionality and stability of an effective lymphoma-associated vascular network depends upon the integrity of vascular pericytes, and that biologic agents which selectively target vascular pericytes would induce endothelial dysfunction and impair lymphoma growth. We tested our hypothesis in SCID mice bearing human diffuse large B-cell lymphoma (DLBCL) cell lines by treating with selective PDGFRβ inhibitors including imatinib mesylate and monoclonal anti-PDGFRβ antibody. Treatment with imatinib impaired tumor growth of human lymphoma xenografts including Farage, Karpas422 and OCI-Ly7, while the tumor cells themselves neither expressed PDGFRβ nor were inhibited by imatinib directly. Confocal microscopic analysis of the xenograft tumor tissue showed decreased microvessel density, decreased vascular functional flow, as well as increased vascular leak and apoptosis in the imatinib-treated cohorts, compared to untreated controls. Imatinib targeted tumor-associated PDGFRβ+ pericytes in vivo by inducing apoptosis and disruption of the PDGFRβ+ perivascular network, and PDGFRβ+ VSMC in vitro by inhibition of proliferation. FACS analysis of mononuclear cell suspensions of xenograft tumor tissue revealed decreased numbers of mature pericytes and endothelia, as well as their progenitors, with imatinib treatment. Compared to imatinib, treatment with anti-PDGFRβ monoclonal antibody partially inhibited the growth of Farage lymphomas by depleting pericytes and attenuating angiogenesis. Lastly, microarray analysis of differentially expressed genes in PDGFRβ+ VSMC treated with imatinib showed significant down-regulation of genes implicated in proliferation, survival and angiogenesis, including those within the PI3K/AKT and MAPK/ERK1/2 pathways downstream of PDGFRβ signaling. Taken together, these data suggest that effective targeting of angiogenesis within the tumor microenvironment, without directly targeting tumor cells, can translate into therapeutic effects in DLBCL lymphoma models. PDGFRβ+ pericytes may represent a novel, non-endothelial, anti-angiogenic target for lymphoma therapy. Disclosures: Leonard: glaxosmithkline: Consultancy; EMD Serono: Consultancy; Sanofi Aventis: Consultancy; Pfizer: Consultancy; Immunomedics: Honoraria; Cell Therapeutics: Consultancy; Celgene: Consultancy; Johnson and Johnson: Consultancy; Calistoga: Consultancy; Cephalon: Consultancy; Biogen IDEC: Consultancy; Hospira: Consultancy; Millenium: Consultancy; Novartis: Consultancy; Abbott: Consultancy; Seattle Genetics: Consultancy.

Abstract 2424 Objective: A single nucleotide polymorphism (SNP) at position 309 in the promoter region of MDM2 leading to increased expression of MDM2 and attenuated function of p53 has recently been suggested as an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL) although this has been questioned. Because inactivation of p53 by deletion and/or mutations also impacts on the clinical course of CLL, we assessed the role of the SNP309 genotype in CLL among Chinese populations. Methods: The MDM2 SNP309 genotypes in 166 CLL patients and 260 healthy controls were detected by the PCR-RFLP method, which all CLL samples was confirmed by direct DNA sequencing. We correlated the results with established CLL prognostic factors, overall survival (OS) and treatment-free survival (TFS). In addition, the correlation of the MDM2 SNP309 genotype with the MDM2 mRNA expression level was evaluated by QPCR. Results: 1. The MDM2 T309G genotype frequencies were 22.9% (T/T), 48.2% (T/G), and 28.9% (G/G) among the cases, and 31.5% (T/T), 54.2% (T/G), and 14.2% (G/G) in the control subjects, and the difference was statistically significant (P=0.001). Logistic regression analyses revealed that the SNP309 G/G genotype instead of T/G heterozygote was associated with a significantly increased risk of CLL (adjusted OR = 2.8; 95% CI 1.57–4.98; P 0.05). 2. In the entire cohort, no correlation was shown between the MDM2 SNP309 genotypes and Binet stage, IGHV mutational status, p53 mutation/deletion and no association existed between any particular MDM2 SNP309 genotype, OS and TFS. 3. The frequency of MDM2 mRNA expression in GG genotype was significantly higher than that in T/G (P=0.026) and T/T genotypes (P=0.003). Furthermore, patients with p53 aberrations (mutated and deleted) MDM2 expression were higher than SNP 309 T/G (P=0.046) and T/T genotypes (P=0.001), but were similar with G/G genotype with p53 wild-type (P=0.532), which prompted us to study the role of the polymorphism in p53 wild-type individuals. 4. In the p53 wild-type groups, we also confirmed MDM2 expression levels with SNP 309 G/G (P Conclusions: The results suggest that MDM2 309G polymorphisms contribute to the risk of developing CLL. The unfavorable SNP309 G/G genotype was associated with a gene-dosage-dependent increase of MDM2 expression. MDM2 SNP309 was found to be associated with TFS in p53 wild-type Chinese populations. Disclosures: No relevant conflicts of interest to declare.

Abstract 4387 Chronic lymphocytic leukemia (CLL) is one of the most common lymphoid malignancies in the Western countries, however, infrequent in Asian populations. Although the median survival is around 10 years, CLL is a disease with an extremely variable clinical course with overall survival times ranging from months to decades; some patients never need treatment, while others require intensive treatment early after diagnosis. Some factors, such as clinical stages, IGHV mutational status, cytogenetic abnormalities, ZAP-70, and the expression of CD38 in leukaemic cells, were strong indicator of prognosis in CLL. However, the prognostic factors of Chinese patients with CLL compared with the Western countries have not yet been clarified. The aim of this study was to explore the influence of factors on the prognosis of Chinese patients with CLL. One hundred and twenty-nine patients with CLL were enrolled in this study. Multiplex PCR and sequencing, fluorescence in situ hybridization (FISH), and flow cytometry were used to detect IGHV mutational status, cytogenetic abnormalities, and the expression of ZAP-70 and CD38, respectively. A panel of FISH probes included 13q14 (D13S319), 17p13 (p53 gene), 11q23 (ATM gene), 6q23(MYB gene), the centromere of chromosome 12 (D12Z3) and 14q32 (IGHC/IGHV). In 129 CLL patients, according to the Binet clinical staging system, 65 (50.4 %) patients were in Binet A, 28 (21.7 %) in Binet B and 36 (27.9 %) in Binet C. Eighty-four (65.1%) patients had mutated IGHV, and 45 (34.9%) had unmutated IGHV. The most frequently expressed VH gene family was found to be VH3 (50.4%) followed by VH4 (32.6%), VH1 (10.9%), VH2 (2.3%), VH5(2.3%) and VH7 (1.6%), with no expression of VH6 gene families. VH1-69 and VH3-21 which commonly overused in Western CLL patients were very low in our cohort (0.8% and 3.1%, respectively). Molecular cytogenetic aberrations were found in 94 patients (72.9%) and 36 patients (27.9%) with more than two abnormalities. The most frequent abnormalities detected in our patients was del(13q14), with an incidence of 53.0%, followed by 14q32 translocation of 20.2%, +12 of 18.3%, del(11q23) of 10.8%, del(17p13) of 10.o%, and del(6q23) of 6.1%. Forty-one patients (31.8%) were positive for ZAP-70 (≥20%), and 51 patients (39.5%) were positive for CD38 (≥30%). With a median follow-up of 32 months (range, 4-58 months), eight patients (6.2%) died (CLL-related deaths). In univariate analysis for survival, advanced Binet stage (P=0.023), unmutated IGHV status (P=0.002), deletions of 17p13 or 11q23 (P=0.003), high expression of ZAP-70 (P=0.034), and high expression of CD38 (P=0.046) were poor prognostic factors. The prognostic factors with statistical significance were further used in a two-variables Cox analysis, which comparing unmutated IGHV status to other prognostic factors individually to show prognostic independence. The unmutated IGHV status were the independent prognostic factors and strongly associated with OS. This study demonstrates that the frequencies of IGHV gene families indicated significant difference in Chinese CLL patients compared with Western patients, suggesting involvement of ethnic and/or environmental factors in CLL disease initiation. The unmutated IGHV status, Binet clinical stages, Chromosomal aberrations of del(17p13) and del(11q23), high expression of ZAP-70 and CD38 have been shown highly predictive prognostic value for Chinese patients with CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract 3450 Poster Board III-338 Chronic lymphocytic leukaemia (CLL) is characterized by a progressive accumulation of monoclonal B lymphocytes in the bone marrow, lymphatic organs and blood. Even with the use of newer therapies, many patients with CLL develop progressive, treatment-resistant disease. Rituximab (RTX), a monoclonal antibody, targets CD20 on B lymphocytes and widely used in indolent B cell neoplasms. The lower density of CD20 on CLL cells may play a role, however, the causes of the lesser susceptibility of CLL to RTX remain poorly understood. Complement deficiencies have been identified in many CLL patients and appear to be more pronounced in patients with more advanced disease. Since complement mediated cell lysis is one of the major mechanisms of RTX action, we hypothesized that the therapeutic effect of RTX in CLL may be enhanced by the provision of complement through concurrent administration of fresh frozen plasma (FFP). Between August 2008 and April 2009, twelve patients with refractory advanced CLL were enrolled in this open clinical trial. All had been previously treated with fludarabine and two also failed treatment with RTX. The median age patients was 57 years (range, 48-82 years). According to the Binet staging system, 2 patients were in Binet B, 10 patients in Binet C. Florescence in situ hybridization (FISH) studies identified del(13q14) in 8 patients, and 4 patients with del(13q14) as the sole abnormality. Trisomy 12 was found in 3 patients, del(11q22.3) in 2 paitents, del(17p13) in 2 patients, del(6q23) in 1 patient, and IgH translocation in 1 patient, respectively. By flow cytometry, CD38 was positive in 9 patients, and ZAP-70 positive in 8 patients. Immunoglobulin heavy chain variable gene (IgVH) mutational status was detected by multiplex PCR, and six patients had unmutated VH. Two units of FFP followed with RTX 375 mg/m2 as a single agent, repeated every 1-2 weeks up to a total of 2-5 cycles. Complete response (CR) was achieved in 4 patients (33.3%), partial remission (PR) in 6 patients (50.0%). A major improvement of clinical signs and symptoms was achieved in all patients. Lymphocyte counts dropped markedly followed by shrinkage of lymph nodes and spleen and improvement of the anaemia and thrombocytopenia. This could be maintained over 9 months (range, 3-11 months) without progression. Toxicity was minimal and the treatment was well tolerated in all cases. It was show that RTX combining with FFP may provide a useful therapeutic option in patients with refractory advanced CLL. Disclosures No relevant conflicts of interest to declare.

Abstract 3941 Poster Board III-877 BACKGROUND Increases in the number of circulating endothelial cells (CECs) and endothelial progenitors (EPCs) have been reported in various pathological conditions including cancer. Both preclinical and clinical studies in a number of human malignancies have shown that the kinetics of CECs and EPCs correlate with clinical outcomes in patients who undergo anti-angiogenic treatment. Angiogenesis biomarkers in lymphoproliferative diseases have been undefined to date. We investigated the dynamics of circulating peripheral blood levels of endothelial cells and progenitors in lymphoma patients as potential biomarkers of disease status and treatment response. METHODS Circulating CD45-CD146+CD34+CD31+ mature endothelial cells (CECs) and CD45-/dimCD133+VEGFR2+ endothelial progenitors (EPCs) were measured by 4-color flow cytometry in 79 lymphoma patients with chronic lymphocytic lymphoma/leukemia (CLL, n=38) and mantle cell lymphoma (MCL, n=41). Normal volunteers served as controls (n=5). Non-parametric Kruskal-Wallis tests were performed to compare the difference in median marker values of different groups. Mann-Whitney tests were then used to compare marker values in pair-wise fashion between clinical groups. P-values were adjusted for multiple comparisons using the Bonferroni method. RESULTS In CLL, the CECs were 525 ± 656/mL in previously untreated patients (n=27), and 50 ± 49/mL in patients with remission following fludarabine-based therapy (n=11), compared to 81 ± 51/mL in normal controls (n=5). Significant differences in CECs were observed in patients with untreated disease compared to patients in remission (p=0.0006) and normal controls (p=0.01). EPCs trended down during remission following treatment from 350 ± 932/mL to 45 ± 84/mL (p=0.052). In MCL, CECs were 397 ± 433/mL in previously untreated patients (n=11), 73 ± 85/mL in patients with remission following R-CHOP-based therapy (n=9), and 110 ± 215/mL in patients with relapse (n=21). A significant decline of CECs was detected in remission compared to pretreatment values (p=0.03). In addition, a significant difference in EPCs was noted comparing pre-therapy baseline values (383 ± 445) to normal controls (86 ± 53/mL) (p=0.03). EPCs trended down in remission from 383 ± 445/mL to 103 ± 138/mL (p=0.052). No significant differences were observed for baseline pretreatment CEC and EPC values between CLL and MCL. CONCLUSIONS Dynamic levels of circulating endothelial cells and endothelial progenitors appear to correlate with disease status and treatment response in CLL and MCL. Large-scale studies are warranted to further evaluate the prognostic significance of these results, and to investigate whether circulating pro-angiogenic cell populations in selected lymphoma subtypes may be useful as biomarkers to select patients for and monitor response to anti-angiogenic therapy. Disclosures: No relevant conflicts of interest to declare.